CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease by Access-Health

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

William A. Haseltine PhD and Amara Thomas ACCESS Health Press

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Copyright © 2023 by William A. Haseltine, PhD Cover art by Kim Hazel All rights reserved. No part of this book may be used or reproduced by any means, graphic, electronic, or mechanical, including photocopying, recording, taping, or by any information storage retrieval system, without the written permission of the publisher except in the case of brief quotations embodied in critical articles and reviews. All author proceeds from the sale of this book will be donated to the nonprofit global think tank ACCESS Health International.

Recent Books by William A. Haseltine Affordable Excellence: the Singapore Healthcare Story; William A Haseltine (2013)

Improving the Health of Mother and Child: Solutions from India; Priya Anant, Prabal Vikram Singh, Sofi Bergkvist, William A. Haseltine & Anita George (2014)

Modern Aging: A Practical Guide for Developers, Entrepreneurs, and Startups in the Silver Market; Edited by Sofia Widén, Stephanie Treschow, and William A. Haseltine (2015)

Aging with Dignity: Innovation and Challenge is Sweden-The Voice of Care Professionals; Sofia Widen and William A. Haseltine (2017)

Every Second Counts: Saving Two Million Lives. India’s Emergency response System.The EMRI Story; William A Haseltine (2017)

Voices in Dementia Care; Anna Dirksen and William A Haseltine (2018)

Aging Well; Jean Galiana and William A. Haseltine (2019) World Class. Adversity, Transformation and Success and NYU Langone Health; William A. Haseltine (2019) Science as a Superpower: My Lifelong Fight Against Disease And The Heroes Who Made It Possible; William A. Haseltine (2021) The Future of Medicine: Healing Yourself | Regenerative Medicine; William A Haseltine (2023)

Living ebooks

A Family Guide to Covid: Questions and Answers for Parents, Grandparents and Children; William A. Haseltine (2020) A Covid Back To School Guide: Questions and Answers for Parents and Students; William A. Haseltine (2020) Covid Commentaries: A Chronicle of a Plague, Volumes I, II, III, IV, V, and VI; William A. Haseltine (2020) My Lifelong Fight Against Disease: From Polio and AIDS to Covid19; William A. Haseltine (2020) Variants!: The Shape-Shifting Challenge of Covid-19 Vaccine Evasion & Reinfection; William A. Haseltine (2021) Covid Related Post-traumatic Stress Disorder (CV-PTSD): What It Is And What To Do About It; William A. Haseltine (2021) Natural Immunity And Covid-19: What It Is And How It Can Save Your Life; William A. Haseltine (2022) Omicron: From Pandemic to Endemic; William A. Haseltine (2022)

Monoclonal Antibodies: The Once and Future Cure for Covid-19; William A. Haseltine and Griffin McCombs (2023)

The Future of Medicine: Healing Yourself: Regenerative Medicine Part One, William A. Haseltine (2023) Viroids and Virusoids: Nature’s Own mRNAs, William A. Haseltine and Koloman Rath (2023

William Haseltine, PhD To my wife, Maria Eugenia Maury, my children Mara and Alexander, my stepdaughers Karina, Manuela and Camila, my grandchildren Pedro, Enrique and Carlos, and last but not least our three dogs, Sky, Luna and Ginger.

Amara Thomas: To my loving parents, siblings and friends, whose belief in me fuels my determination.

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Welcome to CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease. This work explores an ever-changing sector of cell therapy research called CAR T therapy—an investigative field that has celebrated recent triumphs and may usher more to come. Through genetic engineering and other brilliant, novel modalities, this therapy will transform the way we treat cancer, autoimmune diseases and genetic disorders. The collection of stories found here was written in the heat of the moment as new discoveries emerged. As such, consider these pieces as snapshots of time, a reflection of what we know and when we know it. Each story is followed by a link to the original publication, which may include more detailed figures. The format of this book is something that I have dubbed a living ebook— a format suitable for a rapidly evolving advance such as CAR T therapy. I will continue to update CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease as we learn more. You may download these updates at no additional cost by visiting: www.williamhaseltine.com/car-t-therapy Thank you for your interest.

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Contents Introduction To CAR T Therapy.............................................. 1 The Immune System, Cancer and CAR T Therapy .................... 3 Adaptive Immunity and CAR T Therapy....................................... 4 What About Cancer? ..................................................................... 5 From Killer T Cells to CAR T Cells.............................................. 2 Preparing for Infusion.................................................................... 6

Proof of Principle ............................................................................ 8 Clinical Success for B Cell Cancers ........................................... 10 Design Improvements, Overcoming Limitations ....................... 12 Improving Lack of Persistence and Efficacy................................. 12 Improving Limited Cytotoxicity (for Solid Tumors) .................... 15 Improving Limited Proliferation.................................................. 18 Improving Control and Safety ..................................................... 18 Improving Limited Targeting Specificity ..................................... 21 Improving T Cell Exhaustion...................................................... 24 Improving Manufacture: FasT CAR T cell and Off-the-Shelf CAR T Therapy ................................................................................... 25 Exploring Novel Immune Cell Niches ........................................ 27

Potential Future Applications ...................................................... 32 Solid Tumors .............................................................................. 32 Autoimmunity ............................................................................. 35 T Cell-Derived Cancers .............................................................. 35 Cellular Senescence.................................................................... 37 HIV/AIDS ................................................................................... 38

CAR T’s Downsides ...................................................................... 39 Adverse Effects ............................................................................ 40 Risks of Lymphodepleting Chemotherapy ................................... 42 What if CAR T therapy fails? ....................................................... 43

Barriers to Access: Time and Cost ............................................... 44 A Multi-faceted Solution: mRNA Technology ........................... 46 ix

Impact on Inherited Diseases....................................................... 46

The Future of CAR T Therapy .................................................... 48 Chapter I: CAR T Therapy For B Cell Cancers ...................... 49 From Lymphoma To Lupus And Beyond: The Remarkable Research Of CAR T Therapy ....................................................... 49 The “T’ of CAR T Therapy .......................................................... 50 The Remarkable Research Of CAR T Therapy: B Cell Cancers .............................................................................. 59 CAR T Therapy: A New Direction for Multiple Myeloma Treatment....................................................................................... 67 CAR T Therapy Dramatically Reduces Risk Of Relapse For Multiple Myeloma ........................................................................ 73 Hope For Universal, Ready-Made CAR T Therapy For Multiple Myeloma ........................................................................................ 79 Altered CAR T Therapy Shrinks Ovarian Tumors In Mice ...... 86 A Different Kind Of Cancer Killer: Improving CAR NK Cell Therapy .......................................................................................... 95 Chapter II: CAR T THERAPY FOR AUTOIMMUNITY ..... 102 CAR T Therapy: From Cancer To Autoimmune Disease, The Lupus Example ............................................................................ 102 CAR T Therapy To Treat And Cure Rheumatoid Arthritis ....106 CAR T Therapy, A Promising New Therapy For Multiple Sclerosis? ...................................................................................... 111 Early Success: mRNA & CAR T Therapy To Treat Rare Autoimmune Disease Myasthenia Gravis ................................. 117 Chapter III: New Wave CAR T Therapy: Works In Progress ............................................................ 123

Researchers Control Cancer Treatment With New Innovation: CAR T Switch(blade) ................................................................. 123 CAR T Cell-Like Therapy To Treat T Cell Leukemia (T-ALL).......................................................... 130 Treating Troubling Tumors: CAR T Therapy For Aggressive Childhood T Cell Leukemia ..................................................... 136 CRISPR Technology To Simplify And Enhance CAR T Cancer Treatment (Part 1)....................................................................... 141 Teaming Up Two Biotech Winners to Fight Cancer: CRISPR and CAR T (Part 2) ..................................................................... 149 CAR T Cells Derived From Stem Cells Open Door To Universal Donor Cell Lines ....................................................... 155 Synthetic Gene Circuit To Hone CAR T Therapy ................. 160 Engineering Cells For Medical Use: Learning The Language Cells Use To Communicate With One Another ..................... 165 New Generation Of Flexible And Controllable CAR T Therapies (Part 1) ........................................................................ 172 SynNotch, A New Generation Of Flexible And Controllable CAR T Therapies (Part 2) .......................................................... 180 Adapted SynNotch, A New Generation Of Flexible And Controllable CAR T Therapies (Part 3) .................................... 183 Chapter IV: The Future Of CAR T Therapy ............................... 188 The Future Of Cancer Treatment? Treating Multiple Myeloma With mRNA-CAR T Technology .............................................. 188 CAR T Therapy For Cardiac Fibrosis: A New Way Forward . 193 How mRNA Could Reinvent Blood Stem Cell Transplant Preparations (Part 1).................................................................... 202 How mRNA Could Cure Sickle Cell Gene Mutations (Part 2) ....................................................................... 209 xi

How mRNA Could Safely Replace Blood Stem Cell Transplantation (Part 3) .............................................................. 217 Acknowledgments ................................................................. 224 References ............................................................................. 225

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INTRODUCTION TO CAR T THERAPY

Ancient Greek mythology depicts a chimera as a hybrid creature—oftentimes a fire-breathing monster with the head and body of a lion and a snake’s head for a tail. This patchwork element resonates with a chimeric antigen receptor's mix of artificial and natural components.

he field of cancer treatment has long nurtured a profound hope: to harness the body’s innate ability to heal itself. This aspiration gained traction in the late 1980s when scientists discovered that specific immune cells could counter cancer in mice with leukemia. Research intensified over the years with the immune system—the body’s safeguard against foreign invaders and internal abnormalities—at its center. Today, this dream finally begins to bear fruit, albeit through diverging paths.Immune checkpoint inhibitors 1

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

represent one avenue in the chase for a cure. This budding immunotherapy releases a switch in certain immune cells so that they can kill cancer cells. While significant, this book will focus on another developing medical innovation called CAR T therapy. Chimeric Antigen Receptor T cell therapy, commonly referred to as CAR T therapy, is an intervention that uses a patient’s own cells to fight their cancer. With this gene therapy, cells can precisely identify and eliminate a programmed target. For some patients, this translates to a long-term remission or cure of their disease. The CAR T horizon is ever-expanding. The therapy first received US Food and Drug Administration (FDA) approval in 2017. Six approved CAR T products are now on the market, and hundreds of clinical trials are in progress. Market projections suggest that this burgeoning industry, CAR T therapy for cancer, will grow significantly by 2032. And yet CAR T has the potential to do much more. The groundwork paved by rudimentary forms of CAR T therapy for cancer may combat conditions such as heart disease and rheumatoid arthritis in the future. CAR T cells provide an adaptable vehicle to rewire fundamental immune processes—one that can synergize with other contemporary technologies to become more accurate, potent, or accessible. To help you navigate these shifting waters, the pages ahead will detail how CAR T cell therapy works, describe its current uses to treat cancer and explore ongoing efforts that adapt this system to treat other illnesses.

William A. Haseltine, PhD

The Immune System, Cancer and CAR T Therapy Researchers develop potential cancer treatments by learning more about how the body protects itself and how cancer tampers with these security measures. Learning about the immune system is necessary to grasp the magic of CAR T therapy. The immune system's role is to protect the body against illness and disease (Lawrence, 2018). The body engages a complex network of organs, tissues, cells and proteins to combat foreign intruders (e.g., bacteria, viruses) and irregular host cells that threaten our delicate internal balance. This intricate system branches into several arms, each with a distinct function. The most pertinent component for CAR T therapy is a specialized branch called adaptive immunity.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 1: The immune system is composed of several organs, tissues and cells. SOURCE: Access Health International

Adaptive Immunity and CAR T Therapy Adaptive immunity is a tailored defense system that memorizes invader signs. If the same attack should occur again, the adaptive immune system can quickly recognize it and prepare an adequate response. This branch of immunity relies on white blood cells— antibody-producing B cells, CD4+ “helper” T cells and CD8+ “killer” T cells—to function. Conversations around CAR T therapy highlight the killer T cell in particular. While helper T cells aid other immune cells, the killer 4

William A. Haseltine, PhD

T cells naturally destroy irregular and cancerous cells. CAR T therapy borrows this cytotoxic ability and bolsters it through gene editing, transforming the killer T cell into a CAR T cell.

FIGURE 2: Adaptive immunity forms a specialized immune response by concerting B cells and T cells. Two main T cell subsets exist—CD4 “helper” T cells and CD8+ “killer” T cells. CAR T therapy alters a patient’s killer T cells to improve their cancer detection abilities. SOURCE: Access Health International

What About Cancer? Cancer skews normal immune processes. Cells must grow and die in a controlled manner to prevent worn-out, malfunctioning cells from accumulating in the body. With cancer, the genes in these cells mutate, and the cells lose the indispensable ability to trigger their death (Michaud et al., 2015). These abnormal, rapidly 5

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

growing cells crowd out healthy cells and form tissue masses called tumors on the organs or in the blood. Above all, cancer has an uncanny ability to evade and neutralize the immune system (Kim & Cho, 2022). Cancer cells can change their cell surface, becoming unrecognizable to T cells. They can create toxic environments that prevent white blood cell activity. Cancerous cells can even release chemicals to exhaust T cells and render them ineffective, just as a worn pair of shoes loses functionality. CAR T therapy is not exempt from these challenges. Instead, the treatment enhances T cell abilities and helps the immune system fight back despite these setbacks. Let's go into more detail.

CAR T Therapy Can Change Lives For Some

Scott McIntyre tried everything to fight his cancer (Six years, et al. 2023). After being diagnosed with diffuse large B cell lymphoma (DLBL) in 2013 and a life expectancy of six months, Scott began an unending series of treatments. Several rounds of chemotherapy, a stem cell transplant, targeted radiation therapy, and even two clinical trial treatments could not keep his cancer away long—although it teased him with temporary remission. The “R” word— remission—became taboo. By 2016, cancer had spread to Scott’s lungs. He was running out of options (Parrott et al., 2017). It was that year he started an experimental treatment called CAR T therapy. This experimental therapy delivered what other treatments could not. Six weeks after receiving the treatment, Scott’s scans showed a “remarkable reduction of cancer.” The second scan a week later suggested his cancer was gone (Guy et al., 2016). Six years later, in 2021, Scott happily reported that he remains cancer-free. He feels “like a walking miracle.”

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

How CAR T Therapy Works Described simply, CAR T cells improve the body’s own defenses, pitting a patient’s immune cells against cancer. For an eligible patient, the journey begins in the hospital. The first step involves leukapheresis, a process that separates a portion of white blood cells from circulated blood; the remaining components are returned to the body (Boyiadzis et al., 2018). The collected white blood cells are then sent to a lab for further isolation and modification.

From Killer T Cells to CAR T Cells In the lab, the white blood cells are isolated and purified to extract a specific type of immune cell: cytotoxic CD8+ T cells, also known as “killer” T cells. CAR T therapy borrows this cell’s natural ability to destroy infected and cancerous cells. Normal cytotoxic T cells require a two-step activation process to kill. First, an antigen must be presented to the T cell’s receptor; eventually, the T cell will identify any cell with that antigen as an enemy. Second, the T cell must receive a co-stimulatory signal at a distinct receptor. Naïve T cells require at least two signals for activation (Alberts et al., 2022). Both are provided by an antigenpresenting cell, which is usually a dendritic cell. Signal 1 is provided by MHC-peptide complexes binding to T cell receptors, while signal 2 is mainly provided by B7 costimulatory proteins binding to CD28 on the T cell surface. Activation now complete, the T cell gains the ability to pinpoint and bind to a target cell. The bound and activated T cell releases a set of killer proteins and other chemicals that destroy the target cell without stoking excessive inflammation.

William A. Haseltine, PhD

FIGURE 3: Cytotoxic T cells must activate in two steps. An antigen-presenting cell (APC) such as a dendritic cell brings an antigen to a molecule called the class I major histocompatibility complex (MHC I) found on the T cell’s surface; this is the first signal. Simultaneously, the T cell’s CD28 coreceptor receives a costimulatory signal that helps activate the cell. With both signals received, the T cell can then identify and bind to matching antigens present on the surface of other cells. SOURCE: Access Health International

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 4: When cytotoxic T cells encounter and bind to their cell target, the T cell releases chemicals such as perforin and granzyme that bore holes into the target cell and promote apoptosis, a form of cell death that does not trigger excessive inflammation. The T cell then disengages from the withering target cell. SOURCE: Access Health International

CAR T technology takes advantage of the killer T cell’s anticancer abilities and improves on them by removing the requirement for a secondary signal. Scientists use viral vectors to deliver new genes to change the T cell receptor. The resulting hybrid protein recognizes target antigens on cells and activates the T cell in the same step. The simplest version of this hybrid protein has three parts, as illustrated in Figure 2. The protein on the outside of the cell is a modified monoclonal antibody that sticks to the target protein (Brandt et al., 2020). This exterior, antibody-like receptor is joined to a linker that penetrates the cell membrane. The linker, in turn, is connected to the native intracellular activation domain of the killer 4

William A. Haseltine, PhD

T cell receptor. When the exterior protein binds to the target cell, it activates the CAR T cell program. The “C” in CAR T reflects the receptor's chimera-like nature.

FIGURE 5: Schematic of basic CAR T cell design. A chimeric antigen receptor is composed of natural and artificial components. The receptor combines the T cell signal machinery with the precision targeting of an antibody. SOURCE: Access Health International

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 6: Schematic of CAR T cell components. At its most basic form, the native T cell receptor signaling machinery (CD3ζ) is combined with a protein derived from antibodies called a single chain variable fragment (scFv). The single chain variable fragment can precisely recognize and bind to an antigen target such as CD19 or BCMA (B-cell maturation antigen). SOURCE: Access Health International

The killer T cells, now equipped with new receptors, must be multiplied. Exposing the cells to a culture medium and specific cytokines—immune-signaling chemicals found in humans— stimulates the cells to expand to large numbers (Wang & Rivière, 2016). The final product is then cryopreserved and returned to the patient via infusion.

Preparing for Infusion A few days before infusion, a patient will undergo a short course of preparatory chemotherapy using drugs such as fludarabine and 6

William A. Haseltine, PhD

cyclophosphamide. This step does not treat the cancer, but instead decreases the number of other white blood cells in the body. The resulting slightly suppressed immune system is less likely to reject the influx of CAR T cells (Mehrabadi et al., 2022); similarly, the space formed leaves room for the thousands to millions of engineered T cells to grow and multiply. The final step, the CAR T cell infusion, often takes less than an hour to complete (DeMarco, 2023).

FIGURE 7: Summary of the CAR T therapy process. T cells are isolated from the blood and genetically modified to express a new chimeric antigen receptor. The modified and expanded cells are returned to the body via infusion.

SOURCE: From CAR T Cells: Engineering immune cells to treat cancer. National Cancer Institute (2022). https://www.cancer.gov/about-cancer/treatment/research/car-t-cells

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Proof of Principle CAR T Therapy first demonstrated its feasibility with B cell-derived blood cancers, including certain leukemias and lymphomas, and multiple myeloma Burger & Wiestner, 2018. These cancers involve the uncontrollable growth of B cells, white blood cells that create antibodies when mature. Early forms of B cells have no specific function and must gain a specific purpose through a process called differentiation (see Figure 8).

FIGURE 8: General B cell lineage. B cells can be found in various stages of development. Similarly to T cells, B cells start as stem cells and gain a specific function through a process of differentiation. Plasma B cells are a mature type of B cell which produce essential antibodies needed to tag threats to the immune system. Cancer can occur if B cells, matured or not, grow erratically. SOURCE: Access Health International

Most B cells, cancerous or not, share similar antigens on their cell surface. CAR T therapies are designed to detect common B cell antigens such as CD19 or B cell maturation antigen (BCMA).

William A. Haseltine, PhD

Once a patient receives a CAR T cell infusion, the treatment begins eliminating any B cells it encounters—regardless of whether they are malignant. This can decimate healthy B cells. Interestingly, B cell deficiency is often an expected outcome and can imply the therapy’s success (Halim & Maher, 2020). Post-procedure, a patient may need to replenish the lost antibodies with immunoglobulin replacement therapy.

FIGURE 9: Anti-CD19 CAR T cell for certain B cell cancers. Most federally approved CAR T therapies use single chain variable fragments (scFv) that target antigen CD19 or B cell maturation antigen (BCMA); these antigens commonly reside on the surface of B cells, whether cancerous or noncancerous. The CAR T cell binds to any encountered cell with CD19 or BCMA and releases signaling molecules to eliminate it. This process can often leave a patient with B cell aplasia, otherwise known as a deficiency of healthy B cells. SOURCE: Access Health International

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

The field of cancer treatment has long nurtured a profound hope: to harness the body’s innate ability to heal itself. This aspiration gained traction in the late 1980s when scientists discovered that specific immune cells could counter cancer in mice with leukemia. Research intensified over the years with the immune system—the body’s safeguard against foreign invaders and internal abnormalities—at its center. Today, this dream finally begins to bear fruit, albeit through diverging paths. Immune checkpoint inhibitors represent one avenue in the chase for a cure. This budding immunotherapy releases a switch in certain immune cells so that they can kill cancer cells. While significant, this book will focus on another developing medical innovation called CAR T therapy. Chimeric Antigen Receptor T cell therapy, commonly referred to as CAR T therapy, is an intervention that uses a patient’s own cells to fight their cancer. With this gene therapy, cells can precisely identify and eliminate a programmed target. For some patients, this translates to a long-term remission or cure of their disease. The CAR T horizon is ever-expanding. The therapy first received US Food and Drug Administration (FDA) approval in 2017. Six approved CAR T products are now on the market, and hundreds of clinical trials are in progress. Market projections suggest that this burgeoning industry, CAR T therapy for cancer, will grow significantly by 2032. And yet CAR T has the potential to do much more. The groundwork paved by rudimentary forms of CAR T therapy for cancer may combat conditions such as heart disease and rheumatoid arthritis in the future. CAR T cells provide an adaptable

William A. Haseltine, PhD

vehicle to rewire fundamental immune processes—one that can synergize with other contemporary technologies to become more accurate, potent, or accessible. To help you navigate these shifting waters, the pages ahead will detail how CAR T cell therapy works, describe its current uses to treat cancer and explore ongoing efforts that adapt this system to treat other illnesses. Clinical Success for B Cell Cancers CAR T therapy shines in some cases where other cancer treatments have failed. It can reduce signs of cancer and extend life expectancy, giving patients another chance at life. While the recovery process, as for any cancer treatment, is physically and mentally demanding, a study published in Blood Advances noted that patient quality of life typically improves around six months after the procedure (Johnson, 2023). For the therapy to succeed, it must cause some form of remission, meaning a reduction in cancer signs or symptoms. Complete remission occurs if cancer can no longer be detected under a microscope. In a best-case scenario, a patient may be considered ‘cured’ if their remission lasts more than five years. Initial remission rates for CAR T therapy can range as high as 98% (Martin et al., 2021). For a minority of patients, the treatment can send cancer into complete remission. For example, one clinical study found that CAR T therapy achieved remission for more than 82% of young patients with aggressive acute lymphoblastic leukemia (Kymriah, 2022). Of that group, however, less than half of the

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

patients achieved complete remission and lived without relapse for more than five years. Design Improvements, Overcoming Limitations CAR T therapy has improved over the years, with each roadblock ushering a new opportunity to fine-tune the design. The simplest form of chimeric antigen receptors emerged in the late 1980s (Mitra et al, 2023). Although the preclinical tests demonstrated anticancer activity, these initial CAR T cells struggled to survive and multiply once infused in humans (Moritz et al., 1994; Kershaw et al., 2006). The cells also demonstrated poor efficacy due to T cell exhaustion, a phenomenon where T cells lose their ability to respond to a stimulus after repeated antigen exposure.

Improving Lack of Persistence and Efficacy The basic receptor design demonstrated limited persistence and efficacy in humans. To remedy this problem, proteins called costimulatory molecules were added to bolster the T cells’ activation signal. Activation signals widely influence the T cells’ fate. According to elegant research, while the antigen binding domain is crucial for target finding, it is the resulting signaling cascade that defines the nature of the cell’s response (Stevens et al., 2023). How long the T cell survives and grows, and how well it destroys cancer, depends on the cell’s signal transduction. The number, combination, and position of the molecules yield different results. Two standard options include molecules 4-1BB, which can increase T cell memory and persistence, and CD28, which is associated with effective killing but reduced persistence. 12

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Overall, adding one costimulatory molecule produces longer halflives, more durable immune responses, and more potent cytotoxicity compared to the basic design. Chimeric receptors sporting two auxiliary costimulatory molecules display even better persistence and proliferation, but it is uncertain whether the efficacy exceeds that of a single costimulatory molecule (Sterner & Sterner, 2021).

FIGURE 10: Basic CAR T cell design gained additional costimulatory signaling molecules to improve T cell expansion after infusion and T cell survival once in circulation. SOURCE: Access Health International

All CAR T products approved by the FDA use one costimulatory molecule. Kymriah was the first to be approved for clinical use in 2017. Since then, five more CAR T therapies have joined the ranks. Table 1 lists all available products and highlights the differences in their construction.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease List of FDA Approved CAR T Cell Therapies Brand Name

Kymriah

Yescarta

Date Antigen Approved Target

8/2017

10/2017

CD19

Antigen Recognition Domain

Intracellular Signaling Domain

Diseases Targeted/ Line of Therapy

scFV

B cell precursor adult lymphoblastic leukemia (3rd line) Diffuse large B cell 4-1BB—CD3ζ lymphoma (3rd line) Follicular lymphoma (3rd line)

scFV

Diffuse large B cell lymphoma (2nd line) CD28—CD3ζ == Follicular lymphoma (3rd line)

Tecartus

7/2020

CD19

scFV

Mantle cell lymphoma (3rd line) CD28—CD3ζ B cell precursor adult lymphoblastic leukemia (3rd line)

Breyanzi

2/2021

CD19

scFV

4-1BB—CD3ζ

Large B cell lymphoma (2nd line)

Abecma

3/2021

BCMA

scFV

4-1BB—CD3ζ

Multiple myeloma (5th line)

Carvykti

2/2022

BCMA

VHH

4-1BB—CD3ζ

Multiple myeloma (5th line)

TABLE 1: Chart of all FDA-approved CAR T cell therapies. Abbreviations: BCMA, B cell maturation antigen | scFV, single chain variable fragment | VHH, single variable domain on a camelid heavy chain antibody (i.e., nanobody). SOURCE: Access Health International

William A. Haseltine, PhD

Improving Limited Cytotoxicity (for Solid Tumors) Costimulatory molecules alone could not improve the therapy’s inefficacy against solid tumors. Researchers pivoted to a new strategy to boost CAR T cell cytotoxicity and reduce immune suppression in the solid tumor environment in response. The new approach typically integrates a cytokine-producing protein into the receptor design. When the receptor activates, this protein—a transcription factor known as nuclear factor of activated T cells or NFAT—allows the T cell to secrete cytokines such as interleukin-12 to encourage other immune cells to fight the tumor (Mehrabadi et al., 2022). Other possible cytokines include interleukin-7, -15, -18 and -23 (Brookens & Posey Jr, 2023; Hawkins et al., 2021; Chmielewski & Abken, 2020). These CAR T cells are referred to as “T Cells Redirected for Universal Cytokine-mediated Killing,” or “TRUCKS.” Cytokine-releasing CAR T cells exhibit encouraging cytotoxic activity in mice models of lymphoma and advanced solid tumors (Hu et al., 2017; Kueberuwa et al., 2018; Chmielewski & Abken, 2017). Clinical data, however, remains sparse. One paper reports that two multiple myeloma patients experienced complete remission over 12 months after TRUCK CAR T cell infusion (Duan et al., 2021). The CAR here secretes IL-17, a cytokine that influences T cell proliferation and homeostasis, and CCL19, a cytokine that attracts CCL19+ T cells to the tumor site.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 11: One method to improve CAR T therapy’s efficacy against solid tumors is fold cytokine secretion into the receptor design. T Cells Redirected for Universal Cytokine-mediated Killing (TRUCKs) accomplish this with addition of a nuclear transcription factor known as Nuclear Factor of Activated T cells (NFAT). This protein drives cytokine secretion once the receptor is engaged. SOURCE: Access Health International

Removing a gene may also enhance CAR T cell cytotoxicity. A report published in Nature Immunology describes how expression of a membrane protein called sushi domain containing 2 or SUSD2 can inhibit CD8+ T cell antitumor activity (Zhao et al., 2022). This protein’s role in antitumor immunity is largely unknown, but the authors observed that CD8+ T cells that lacked SUSD2 more effectively slowed tumor growth in several mice tumor models. The 16

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protein inhibited antitumor activity by suppressing the binding of CD8+ T cells with interleukin (IL)-2, a growth factor that drives the expansion of activated CD8+ T cells; in other words, SUSD2 inhibits IL-2 signaling, which then dulled the antitumor function of the killer T cells. Eliminating this protein’s expression in CAR T cells also elicited a strong anti-tunmor response in mice, demonstrating the potential of SUSD2 as a target for cancer immunotherapy. It may take a synergy of two novel immunotherapies—immune checkpoint inhibitors and CAR T therapy—to overcome solid tumors. Here’s how immune checkpoint inhibitors work. The body depends on immune checkpoints to limit dangerously strong immune responses. The checkpoints bind with partner proteins to shut down T cell activity. Some cancers hijack these checkpoints to their advantage, forcibly shutting down T cell activity to evade immune system detection. Checkpoint inhibitors are antibody drugs that block proper binding with partner proteins, allowing T cell activation to continue. Akin to releasing a dam, these drugs ultimately promote T cell proliferation and antitumor responses by neutralizing roadblocks (i.e., immune checkpoints). Checkpoint inhibitors alone can treat a wide variety of cancers, including melanoma, breast cancer and non-small cell lung cancer (Shiravand et al., 2022). The hope is that coadministering checkpoint inhibitors alongside CAR T cells will elicit stronger antitumor responses than CAR T cells by themselves, thus overcoming the challenges of solid tumors. Mouse models of cancer suggests that checkpoint inhibitors can potentially reduce CAR T cell exhaustion (Shin et al., 2012; Cherkassky et al., 2016), but 17

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

clinical evidence remains mixed, and is currently developing on a small-scale.

Improving Limited Proliferation CAR T cell proliferation is essential for the therapy’s success. Proliferation ensures that a sufficient number of CAR T cells circulate the body to maintain their therapeutic effect and persist over time. One way to improve proliferation is to introduce truncated versions of a cytokine receptor in the intracellular domain of the CAR (Mehrabadi et al., 2022). The new addition activates a JAK-STAT signaling cascade—a series of signals that can improve cell proliferation, survival and antitumor responses. Preclinical research suggests that activating this signal pathway can encourage CAR T cell proliferation and prevent the cells from losing their functions over time (Kagoya et al., 2018).

Improving Control and Safety Several chimeric receptor concepts attempt to address safety issues. Prominent shared features include the ability to turn the treatment off/on or to control the strength of the response. One method to manipulate CAR T cells is to integrate an inducible suicide gene into the receptor as a safety switch. This brake system allows clinicians to selectively eliminate the synthetic cells before a dangerous immune response is stoked; the patient permanently loses the CAR T cells in the process. Two oft-used suicide genes include herpes simplex virus thymidine kinase (HSC-TK) and inducible caspase 9 (Beltinger et al., 1999; Diaconu et al., 2017). The viral kinase gene reacts to certain antiviral drugs. The gene activates the drug, thus preventing CAR T 18

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cell DNA synthesis and slowly triggering cell death signaling pathways. Inducible caspase 9, a gene that encodes for an inactive enzyme, works by administering specific small molecules to activate the enzyme and subsequently initiate CAR T cell apoptosis, or controlled cell death. Caspase 9 may soon outperform other suicide gene systems, as it is human-derived (and thus safer for human use) and can thoroughly eliminate CAR Ts in a prompt, dose-dependent and nontoxic manner (Di Stasi et al., 2011; Diaconu et al., 2017; Gargett & Brown, 2014). Some studies use monoclonal antibodies as safety switches instead. For example, monoclonal drug Rituximab can bind to CD20 surface receptors found naturally on CAR T cells (Philip et al., 2014). The interaction attracts immune cells called natural killer cells to the area. The natural killer cells then release toxic chemicals to destroy the targeted cell. Integrating the safety switch into the chimeric receptor—a construct known as Cubi-CAR—can also streamline the cells’ manufacture (Valton et al., 2018). Another potential solution to runaway immune responses is to equip CAR T cells with a bi-specific antibody switch. CAR T cells with such a switch only respond if the switch is bound to both the chimeric receptor and the target antigen on the cancer cell. An antibody switch could provide a clinically feasible means to activate or deactivate CAR T cells already inside the body, with the immune response stopping if the switch is no longer present. Clinical research is currently underway and highly anticipated to improve a particular adverse effect called cytokine release syndrome (Rodgers et al., 2016; Arcangeli et al., 2016). One CAR T cell design elicits fewer toxicities by selectively eliminating multiple myeloma cells while sparing healthy cells 19

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(Vander Mause et al., 2023). The secret to its preclinical success relies on identifying an ideal protein sequence to optimize the synthetic receptor. Researchers at the University of Maryland School of Medicine crafted a CAR T cell which hones in on CD229 antigens on myeloma cells; however, the engineered cells also attacked healthy blood cells that express the antigen at lesser amounts. To prevent this unwanted crossfire, the authors developed an approach called affinity tuning to systematically identify a CAR protein sequence that readily binds to myeloma cells alone. They swapped out small protein units called amino acids, ultimately testing 305 protein sequence variations before landing on the most favorable binding domain. The final product—the optimized receptor paired with additional copies of c-Jun, a protein naturally present in CAR T cells—displays similar cytotoxic activity to standard CD229 CAR T cells without harming healthy blood cells. This study suggests affinity tuning could be widely adopted to optimize chimeric receptor binding and reduce adverse effects. Rather than destroying the CAR T cells or restricting their binding, gene circuits can exert control by modulating chimeric receptor expression. The gene components in the circuit respond to specific stimuli and produce a tailored output. One study relied on synthetic zinc fingers, a type of protein that recognizes specific DNA motifs and helps convert DNA into genetic material called RNA (Li, Israni, et al., 2022). Introducing a small molecule drug turns the gene on, and removing the molecule turns the gene off; CAR expression, as a result, can be influenced by the dosage of small molecules. With gene circuits, researchers can choose when to activate receptor expression in mice models of cancer. They can also create 20

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a dual signal circuit, where Signal 1 encourages the immune cells to proliferate and Signal 2 instructs the cells to express CAR. This two-punch mechanism significantly lowers tumor burden in mice—much more than those treated with plain CAR T cells or CAR T cells activated with a single signal.

Improving Limited Targeting Specificity A major difficulty with tackling cancer is pinpointing a target amongst its numerous changing faces. Ever the elusive foe, cancer cells can undergo a process called antigen escape, where they downregulate the expression of their surface proteins to avoid immune system detection. Once hidden, the cancer cells can proliferate anew. Immune evasion by cancer cells presents a distinct problem for single-target CAR T therapy. If a blood cancer cell downregulates antigen CD19, for example, conventional anti-CD19 CAR T cells will no longer be able to recognize their programmed target. Antigen escape can render the therapy ineffective over time, resulting in cancer relapse. A recent study published in Nature illustrates this phenomenon. The researchers discovered that multiple myeloma cells can mutate to resist CAR T therapies tailored against highly expressed myeloma antigens B cell maturation antigen (BCMA) and/or orphan G-protein-coupled receptor (GPRC5D) (Lee, Ahn, et al., 2022). In a parallel vein, solid tumors express a wide variety of antigens, many of which can be found in healthy tissues, as well. The dilemma lies with securing a single antigen target that is only highly expressed in solid tumors. Shared antigen expression in healthy tissues leads to toxic “on-target” but “off-tumor” effects (Sun et al.,

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

2018); this means that the T cell successfully attacks its target on pathogenic and nonpathogenic cells. Both scenarios highlight an issue with CAR T cell targeting specificity. To properly recognize and counter cancer cells, it may be necessary to break away from standard receptor designs that hone in on a single antigen. If one is not enough, how about two? There are several potential methods to target multiple tumor-associated antigens at once. Two distinct mono-specific CAR T cell products could be co-infused into the patient; each product could be separately controlled to decrease the risk of adverse effects (Van der Schans et al., 2020). A single T cell could co-express two chimeric receptors, one for each antigen target; the synthetic, genetic material could be delivered to the T cell by two separate viral vectors or by a single, combinatorial or bicistronic vector. Another option is to create a CAR T cell that expresses two single chain variable fragments (scFv) on the same receptor, otherwise known as tandem CARs. Research by the New York Memorial Sloan Kettering's Cellular Therapeutics Center suggests that between the pooled monospecific CAR T cells, dual co-expressed CAR T cells and tandem CAR T cells, the dual coexpressed CAR T cells provide superior efficacy while avoiding the trouble of manufacturing two separate CAR T infusions (Fernández de Larrea et al., 2020). An antibody adaptor could also be used to achieve multiple targeting. With this system, the CAR T cell does not bind directly to the tumor; the CAR T cell first binds to the adaptor, and then the adaptor binds to the tumor. Just as a universal travel adaptor allows a single plug to be used around the world, this platform allows a mono-targeting CAR T cell to interact with several tumor antigens. 22

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As an added benefit, flooding, withdrawing or repeatedly infusing the adaptor can vary the strength of the therapeutic response. Scientists at the University of Pittsburgh demonstrated proof-ofconcept in mouse models of cancer with their universal adaptor, SNAPtag (Ruffo et al., 2023). The adaptor is composed of an antibody and a tag made from a synthetic molecule called benzylguanine (BG). The tag irreversibly bonds to the unique chimeric receptor, while the antibody can be interchanged to detect a wide range of tumor antigens.

FIGURE 12: SNAPtag adaptor schematic. At its essence, the molecular adaptor relies on a tag, an antibody, and the chimeric antigen receptor. The adaptor is made from conjugating a tag made from benzylguanine (BF) NHS ester with a chosen antibody. The complete adaptor covalently bonds to the unique chimeric receptor (SNAP-CAR). SOURCE: Access Health International

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 13: The SNAPtag adaptor, composed of a molecular tag and antibody, allows for multiple antigen targeting. The CAR T cell covalently binds to the tag (BG-antibody conjugate), while the antibody binds to the target cell. Tagging different antibodies expands the CAR T cell’s targeting range. SOURCE: Access Health International

Improving T Cell Exhaustion CAR T therapy is often dubbed “a living drug,” partly because some CAR T cells transform into memory T cells. Memory CAR T cells patrol for and recognize cancer signs years after the initial infusion, translating to lasting remission for some patients. This prized longevity can be interrupted by T cell exhaustion, a dysfunctional state caused by continuous exposure to target antigens. These chronically overstimulated T cell receptors become less responsive 24

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to target antigens over time, causing weaker antitumor responses and less proliferation. Understanding and reducing T cell exhaustion is essential for developing more effective CAR T therapies. Recent research points to a possible solution: addressing a particular group of proteins in the cell nucleus called mSWI/SNF complexes (Battistello et al., 2023). By restructuring chromatin, a subunit of chromosomes, these protein complexes can activate or repress the expression of genes involved in T cell activation and exhaustion. Cell culture and mouse experiments show how restricting the protein complexes with small molecule inhibitors or CRISPR-Cas9 gene editing can prevent CAR T cell exhaustion; CAR T cell persistence increases in response. With additional testing, this avenue may fruitfully extend the life and anti-cancer power of CAR T therapy.

Improving Manufacture: FasT CAR T cell and Off-the-Shelf CAR T Therapy CAR T therapy is a personalized medicine. Each patient receives a unique infusion made of their own bioengineered immune cells. While currently a standard industry practice, this specialized approach is resource-intensive and time-consuming. Early-stage research is largely conducted in large (often urban) academic centers for this reason, as they possess the necessary infrastructure, equipment and personnel necessary to execute this complex process. Contributions from the private sector are sensitive to recent market downturns (Fidler, 2022); so, while many biotech companies may continue to prioritize cell therapy and CAR T

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

therapy efforts, they operate with a diminished workforce due to funding setbacks and layoffs (Jensen, 2023; Pagliarulo, 2023). Whether to reduce time from extraction to infusion, decentralize and simplify procedures or increase profit margins, manufacturing will be a major bottleneck for CAR T therapy. Above all, improving manufacturing would give more people the opportunity to use this revolutionary treatment—especially those suffering from rapidly progressive disease who otherwise cannot receive the therapy. FasT CAR T cell manufacturing promises to address this concern. The platform expedites weeks-long CAR T cell production into a 24-hour process. Rather than activate the T cells before viral transduction as usual, the platform combines T cell activation, transduction and expansion into a single step (FasTCAR, n.d.). The resting T cells are simultaneously activated and tranduced using XLenti vectors derived from human immunodeficiency (HIV) viruses that cannot transmit the disease to the patient. A phase I clinical trial of 25 patients with relapsed/refractory B cell acute lymphoblasic leykemia (B-ALL) demonstrated encouraging early efficacy data using FasT CAR T cells, although larger scale testing is still needed (Yang et al., 2022). Notwithstanding, this system’s innate potential has not been overlooked. This platform received the Biotech Innovation Award as part of the 2022 Fierce Life Sciences Innovation Awards, which demonstrates solutions “that have the greatest potential to save money, engage patients, or revolutionize the industry” (Questex LLC, n.d.). Another popular solution under investigation is “off-the-shelf” or allogeneic CAR T therapy. To create this ready-made treatment, large batches of T cells would be extracted from healthy donors instead of patients. CAR T cell manufacture could then be 26

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centralized and more large-scale, ultimately bypassing the need to create a custom batch of CAR T cells for each patient. This would mean that more patients could be treated in a shorter time timeframe; patients, too, could easily receive repeat doses if necessary. The prevailing roadblock to adopting allogeneic CAR T therapy is host rejection. The donor CAR T cells express different proteins on their cell surface than the host cells. The body detects this protein mismatch, recognizes the CAR T cells as foreign and attacks. This complication, graft-versus-host-disease (GvHD), is also commonly observed with other cell and tissue transplants. A singular solution to overcoming host rejection for ready-made CAR T therapies does not yet exist. For now, research is branching into several arenas to solve this issue. Potential solutions include “hiding” the CAR T cells from the immune system by cutting out problematic genes, attaching chimeric receptors to different immune cells, and combining other novel technologies with CAR T therapy.

Exploring Novel Immune Cell Niches Cytotoxic T cells are not the only cells that can sport chimeric receptors. Early research suggests enginering other immune cells may not only be viable, but potentially preferable to standard CAR T therapy. Natural killer (NK) cells are one such example (Li, Song, et al., 2022). They kill cancerous and infected cells just as cytotoxic CD8+ T cells do, albeit through a distinct mechanism. This fundamental difference lends natural killer cells an advantage over killer T cells regarding CAR T therapy safety and manufacturing. 27

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Natural killer cells must act quickly as part of the innate immune system and the body’s first response to threats. To accomplish this, these cells forgo antigen-specific binding—they do not need antigen exposure to find their target. CAR NK cells retain this sweeping cytotoxicity even as precise, chimeric receptors are attached to their cell surface, allowing the cell to eliminate a broader spectrum of targets. Natural killer cells also demonstrate a favorable safety profile. CAR NK cells are not as prone to common adverse effects of CAR T therapy such as cytokine release syndrome (CRS) or neurotoxicity. The therapy also has a shorter lifespan, as natural killer cells do not convert into memory cells as cytotoxic T cells do. In essence, the treatment thus trades long-lasting protection to lower the risk of ontarget, off-tumor toxicity to normal tissues. Additionally, natural killer cells possess a lower risk of graft-versus-host-disease than cytotoxic T cells, making them ideal candidates for ready-made CAR therapy (Simonetta et al., 2017). Some researchers believe that CAR NK therapy’s limited lifespan can be remedied. One method under investigation is to give a repeat dose of the treatment. Another solution is to integrate IL-15 cytokine secretion into the receptor design (Li, Mohanty, et al., 2023). Mice with lymphoma live longer when given an infusion of IL-15expressing CAR NK cells than those without. Combining both strategies or introducing CAR NK therapy with other immunotherapies, such as checkpoint inhibitors, may be necessary to enhance the therapy’s limited expansion and persistence in vivo or bolter its antitumor activity.

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TABLE 2: Comparison between natural killer cells and cytotoxic CD8+ T cells. While both cells kill, they operate under distinct mechanisms. Abbreviations: HLA, human leukocyte antigen; MHC I, major histocompatibility complex class I. SOURCE: Access Health International

There are also alternatives within the T cell lymphocyte family— notably, γδ T cells, CD4+ T cells and regulatory T cells.

γδ T cells are a diverse subgroup of T cells with unique receptors. Unlike classic T cell receptors (e.g., receptors found on CD4+ helper T cells and CD8+ killer T cells) that contain an alpha and beta protein chain, this minority carries a gamma and delta protein chain in its place (Vantourout & Hayday, 2013). The composition and function of these cells varies depending on location. Small populations of γδ T cells can be found in certain tissue and mucosal surfaces, such as the skin, intestines and lungs. Although much is still unknown about γδ T cell function, researchers have identified a few key characteristics that set them apart from their conventional counterparts. Similarly to innate immune cells, γδ T cells do not require major histocompatibility protein complexes (MHC) to activate. This should translate to a 29

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

lower risk of graft-vs-host disease if turned into a universal, donorderived CAR therapy (Capsomidis et al., 2018). γδ T cells may also be more equipped than traditional T cells to infiltrate some solid tumors (Mabuchi et al., 2013). They are already positioned in mucosal tissues and possess accessory receptors to selectively home in on inaccessible tumor sites (Mirzaei et al., 2016; McCarthy & Eberl, 2018).

FIGURE 14: Comparison of αβ and γδ T cell receptors (TCR). Only 0.5–5% of all T cell lymphocytes carry gamma and delta protein chains in their T cell receptor (Ou et al., 2021). SOURCE: Access Health International

CD4+T cells are another area of interest for CAR T therapy. While CD8+ T cells compose the majority of a CAR T product, genetically edited CD4+ T cells may also be present in variable 30

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ratios. How do CD4+ T cells, known “helpers” of the adaptive immune system (Alberts et al., 2002), contribute to the resulting antitumor effect? Recent studies suggest that these helpers can kill, too (Alizadeh et al., 2023). Researchers at the Institut Pasteur in France discovered that CD4+ CAR T cells can attack certain tumor cells by producing an apoptotic cytokine called interferon-gamma (IFNγ), although they can also kill using perforins and granzymes as CD8+ T cells do (Boulch et al., 2023). Cell models demonstrate that CD4+ cells can trigger apoptosis in tumor cells that are intrinsically sensitive to this cytokine. Their analysis of 63 CAR T cell patients with diffuse large B cell lymphoma also revealed that CAR T cell products with high CD4+ to CD8+ CAR T cells show strong correlations between serum interferon-gamma induction and positive clinical outcomes such as progression-free survival. Future CAR T products thus may benefit from optimizing CD8+ to CD4+ T cell ratios. And what about modifying cells that do not kill? Regulatory T cells are a subset of CD4+ T cells that do not promote antitumor activity. Rather, these cells maintain immune system balance by suppressing overreactive immune responses. This mechanism is particularly important in preventing autoimmunity, in which the body attacks its own tissues. CAR Treg therapy could be used to prevent graft-vs-house disease during transplantations. For example, the T cell could be modified to target a surface protein called HLA-A2 (Arjomandnejad et al., 2022). This protein is a member of a group of proteins called human leukocyte antigens, which the body uses to distinguish its own cells from foreign entities. The body attacks any cell that does not have the correct protein, causing the organ and cell transplantation 31

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rejection associated with graft-vs-host-disease. CAR Treg cells can target the grafted cells with HLA-A2 and should prevent the immune system from rejecting the graft. One study reveals that these CAR Treg cells can prevent this rejection in mice transplantations more effectively than ordinary regulatory T cells (MacDonald et al., 2016). Other tests using skin allografts on mice suggest that CAR Treg cells can prevent transplant rejection for over 40 days (Noyan et al., 2017). The immune suppression exerted by CAR Treg therapy may also benefit chronic autoimmune diseases, according to mice models of autoimmunity (Imura et al., 2020). Potential Future Applications CAR T therapy’s modular and malleable nature stirs the imagination for treating diseases beyond B cell-derived blood cancers. While much of this work is still in its infancy, the field maintains an optimistic forward momentum.

Solid Tumors Blood cancers, the primary target for CAR T therapy today, represent a mere 10% of all diagnosed cancer cases in the United States (Siegel et al., 2023). Translating CAR T therapy to treat solid tumors—including prostate, lung, and pancreatic cancer—would address a significant therapeutic gap. Three obstacles block the way: CAR T cell antigen targeting, tumor infiltration, and persistence. Antigens found on solid tumors vary greatly and often overlap with antigens found on healthy tissues and organs; if the CAR T cells destroy healthy tissue, it cannot be repaired and supplemented as B cell deficiency can. It is also 32

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arduous for a CAR T cell to reach the tumor’s center due to the tissue barrier. Lastly, the solid tumor environment actively suppresses immune responses, thus blunting T cell efficacy. One potential solution involves using CRISPR technology to create a chimeric receptor with multiple antigen targets (Foy et al., 2023). Another possibility involves editing the T cell receptor genes to activate more efficiently. A study of mice with ovarian cancer found that mutating the CAR T cells’ signaling domain reduced tumor burden (Schoutrop et al., 2023). This suggests that streamlined T cell activation could significantly impact the cells’ persistence and antitumor response—two factors needed to fight solid tumors. The search for an ideal antigen target will differ from cancer to cancer. Cancers that impact the central nervous system—think nerves and other cells in the spinal cord and brain—may benefit from antigen GD2 targeting. This antigen is highly expressed in most neuroblastomas and certain gliomas (Heczey et al., 2023; Majzner et al, 2022); the former are aggressive cancers found in early nerve cells, while the latter are cancerous cells that support nerve function. In contrast, CAR T studies for pancreatic cancer, ovarian cancer (among others) may rely on antigens such as mesothelin or TAG72 instead (Good et al., 2021; Schoutrop et al., 2023 Lee, Murad, et al., 2023). Changing CAR T cell delivery could also improve issues with cell trafficking and tumor penetration, but this concept is still under active investigation. Given that CAR T cells struggle to travel to and penetrate solid tumors, the idea is to deliver the cells directly to the tumor site by repurposing existing chemotherapy techniques (Sagnella et al., 2022). For example, CAR T cells targeting tumors in the central nervous system could be distributed using an 33

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Ommaya reservoir, a small, soft plastic device that is surgically implanted under the scalp. The device carries a dome-shaped space for holding liquid and a thin, flexible tube to distribute the contents. This process starkly contrasts conventional methods, where the CAR T infusion enters the bloodstream systemically via an intravenous line (IV). Locoregional CAR T cell delivery may be especially beneficial for treating head and neck cancers by circumventing the blood-brain barrier. A clear disadvantage to this involved approach would be the anticipated cost.

FIGURE 15: An Ommaya reservoir is a device that is surgically inserted under the scalp to collect cerebrospinal fluid or deliver fluid medicines. The device is composed of a dome-shaped soft plastic reservoir and a catheter. SOURCE: Adapted from “Schematische Darstellung eines implantierten Ommaya-Reservoirs” by A. Kübelbeck and P.J Lynch, 2008, March 23, Wikipedia Commons. Copyright 2006 by Patrick J. Lynch.

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Autoimmunity Another burgeoning area for CAR T cells is the treatment of autoimmune diseases such as lupus, multiple sclerosis, and rheumatoid arthritis. Current treatments for these conditions only manage symptoms, but CAR T therapy could potentially treat these autoimmune diseases at their source. For example, lupus is an autoimmune disease that stems from the overproduction of malignant antibodies. A small study revealed that anti-CD19 CAR T cells could achieve complete remission for patients with severe and treatment-resistant forms of the disease (Mackensen et al., 2022). One study in a mouse model of rheumatoid arthritis tested cells wielding a chimeric antigen receptor that recognizes and eliminates errant helper T cells, the immune cells responsible for that disease’s development (Whittington et al., 2022). The intervention successfully delays the onset and severity of rheumatoid arthritis in mice; with more adjustments, the intervention could potentially cure the condition altogether. An alternative design for multiple sclerosis (MS) also attacks selfreactive helper T cells (Yi et al., 2022). The researchers posit that implementing CAR T cells at different stages could prevent disease flare-ups or mitigate symptoms in mice—a mechanism that would benefit patients once translated clinically.

T Cell-Derived Cancers It is surprisingly difficult to use CAR T cells to treat T cell leukemia, which originates from the uncontrolled growth of T cells. The main challenge lies in finding an antigen target. Targeting antigens in the T cell lineage would destroy healthy T cells, cancerous T cells, and 35

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even CAR T cells themselves. An additional risk is T cell deficiency, a potentially life-threatening condition. Making a CAR T therapy that targets a common T cell antigen may be possible. A small study published in The New England Journal of Medicine created a product that sent two pediatric T cell leukemia patients into remission (Chiesa et al., 2023). The CAR T cells targeted CD7, an antigen found on cancerous T cells, healthy T cells, and CAR T cells alike. The team sourced the cells from donors instead of the patients—who likely have few T cell counts as is—and then precisely cut three genes with CRISPR gene editing; these changes prevented the transplanted CAR T cells from attacking each other and from being rejected (a common issue for any transplantation). Although this procedure holds promise, it can also spark significant complications. The third patient experienced a fatal infection due to these overlapping adverse effects. The same company is also clinically testing CAR T cells with four base edits instead of three, but results are not yet available (Beam Therapeutics, 2023). CAR T therapy alone may not be enough. One study developed an immunotherapy that, rather than replacing a receptor on T cells, modifies them to secrete a special antibody (Jiménez-Reinoso et al., 2022). The antibody is designed to bind to cancerous T cells on one side and healthy T cells on the other, thus encouraging nearby T cells to target the liquid tumor. The authors described this interaction as “the bystander effect.” The results from the in vivo assays suggest that the bystander effect can augment CAR T therapy or even be used effectively alone.

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Cellular Senescence It may be possible to treat a wide range of aging-related conditions with CAR T therapy. The theory is to use the intervention to wipe out cells in a state called senescence. When cells normally experience damage, disease or stress, they are removed from the immune system through apoptosis. With senescence, a dysfunctional cell does not undergo apoptosis as it should (McHugh & Gil, 2018). It stops growing and multiplying, but continues to release inflammatory chemicals. While this can be beneficial in the short term (eg. wound healing), if not eventually cleared away by other immune cells, the chronic inflammation triggered by a few lingering senescent cells can harm healthy cells nearby, just as sparks in a dry forest can transform into wildfire. Senescent cells accumulate as the immune system becomes less efficient at clearing dysfunctional cells away with age. The damage caused by senescence weakens the body and contributes to agerelated diseases such as osteoarthritis, diabetes, liver and lung fibrosis, glaucoma, cancer and more (He & Sharpless, 2017). Selectively eliminating these cells may alleviate symptoms and even promote longevity (Baker et al., 2011). CAR T therapy can viably remove senescent cells in mouse and nonhuman primate studies of age-associated organ decline. The therapy’s cytotoxic power has been directed at two targets in particular: urokinase-type Plasminogen Activator Receptor (uPAR) and Natural Killer Group 2 member D (NKG2D). Both antigens are highly expressed in senescent cells. Mice with liver fibrosis demonstrate reduced scarring without notable toxicity after treatment with uPAR-targeting CAR T cells (Amor et al., 2020); the CAR T cells do wane rapidly after infusion, which could be 37

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attractive as a short-term, repeat treatment. Similarly, NKG2D-CAR T cells successfully kill senescent cells in aged mice and nonhuman primates without observable adverse effects; aged mice even exhibit improved physical performance with this treatment (Yang, Sun, et al., 2023). The next step for this line of research is to understand the therapy’s short-term and long-term safety profile in humans.

HIV/AIDS CAR T therapy could one day treat HIV/AIDS. HIV, or human immunodeficiency virus, is a virus that primarily infects CD4+ T cells and macrophages and weakens the immune system (Simon et al., 2006). It spreads through contact with certain bodily fluids such as blood, semen and vaginal fluids. The virus remains in the body for life. Over time, people with HIV become vulnerable to diseases, infections and cancers. Some patients develop acquired immunodeficiency syndrome (AIDS), the most advanced form of the disease. HIV was once considered a fatal disease, but with the development of antiretroviral medicines, it has become a manageable, nontransmissible chronic condition. Prompt and consistent treatment can suppress viral replication to low, often undetectable, levels. However, this method cannot completely eradicate the virus due to latent infection, where HIV hides its DNA in memory CD4+ T cells and persists long term. People with HIV must take this medicine daily for the rest of their lives to keep the virus from rebounding. CAR T therapy could change this landscape by achieving a functional or sterilizing HIV cure; the former refers to lasting remission, and the latter refers to complete eradication of the virus.

William A. Haseltine, PhD

In an ideal world, the engineered cells would target and eliminate the stubborn HIV-infected cell reservoirs with a single infusion. If any reservoirs linger, the long-lasting CAR T cells should be able to contain any small outbreaks and maintain remission. Progress on this front is mixed yet hopeful. CAR T cells failed to control HIV viral loads in early trials, but were present and functional in patients a decade after infusion—a positive sign of the therapy’s safety (Scholler et al., 2012). While not the first, a CAR T clinical trial started this year that plans to recruit up to 18 participants with HIV (Connolly, 2023). To qualify, the patients take at least 12 months of antiretroviral medication before participation; HIV viral loads must also be undetectable for at least 12 months. This treatment is paused after administering the experimental CAR T cell infusion; this should, in theory, force latently infected T cells from hiding. The therapy uses two distinct chimeric receptors at once to target gp120, a protein HIV-infected T cells express when infecting other T cells (Yoon et al., 2010). The infected cell attaches to the CAR T cell’s CD4 domain, thinking it will enter and infect a CD4+ T cell as usual. The gp120 protein then changes shape in preparation; this shift, however, reveals a previously hidden binding site that the second chimeric receptor latches onto, thus initiating the cytotoxic cascade. This clever design overcomes the flaw of previous CAR T cells that only targeted CD4 and were vulnerable to HIV infection. The study results are anticipated for 2025 (Carvalho, 2023). CAR T’s Downsides CAR T therapy, effective as it can be, is not a miracle cure-all. Despite the innovative solutions in progress today, the current 39

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reality is that CAR T therapy as a field operates on uncharted waters. The clinical data available now is relatively new and often conducted on a small scale. These factors cast a shadow of doubt over the treatment’s long-term durability and effect on survival. For example, currently approved CAR T products for multiple myeloma only possess response rates from around 18 to 28 months after initial infusion ( Longer-term Data, 2022; Abecma, 2023); CAR T critics speculate that these trials will need half a decade to gather reliable durability and survival information (Abid, 2023). Another consideration is that early-phase clinical trials on novel CAR T constructs may underreport non-relapse mortalities—“any death without relapse or progression of the underlying disease after CAR T therapy”—and other clinically relevant toxicities (Abid, 2023). Even if the numbers are correct, many known associated risks can jeopardize a patient’s health and well-being. Clinicians and prospective patients should consider all drawbacks carefully to prioritize safety.

Adverse Effects CAR T cells must produce strong and durable immune responses to eliminate cancer cells. While these responses are beneficial to an extent, adverse events occur if the immune system becomes overly stimulated from the T cell infusion. Doctors monitor patients for around a month after infusion, watching for common complications such as cytokine release syndrome (CRS) and a neurotoxic condition known as immune effector cell-associated neurotoxicity syndrome (ICANS) (The CAR T-Cell Therapy , 2021). While generally reversible, both conditions can manifest symptoms ranging from mild to fatal.

William A. Haseltine, PhD

Cytokine release syndrome, also referred to as a cytokine storm, results when stimulated white blood cells release inflammatory chemicals called cytokines (Song & Milone, 2021). These chemicals can activate other white blood cells and perpetuate a cycle of inflammation. Fever usually manifests first, often accompanied by headaches, muscle/joint pain, and more. Severe cytokine storms can lower blood pressure and oxygen levels, leading to eventual organ failure and death. The condition can usually be reversed within 5 to 17 days with antihistamines, oxygen therapy, or immunosuppressive medications. Antibody-drug tocilizumab and corticosteroid drug dexamethasone are examples of two immunosuppressive medicines often used to control cytokine storms (Chen et al., 2019). Neurotoxicity can also occur, although usually less frequently (Brandt et al., 2020; Gust, 2017). Unlike cytokine syndrome, the mechanism behind why neurotoxicity occurs is poorly understood. Some believe the process involves cytokine release disrupting the blood-brain barrier or stoking fluid buildup around the brain. Patients may experience confusion, headaches, tremors, and hallucinations. Symptoms can even, on rare occasions, cause severe delirium, seizure, or coma. Immunosuppressive medicines and corticosteroids can alleviate symptoms just as with cytokine storms. The condition, when addressed, typically resolves within 21 days of infusion. CAR T patients may experience other adverse effects besides cytokine release syndrome and neurotoxicity. The therapy can lower blood cell counts to dangerous levels. These conditions, called cytopenias, cause symptoms such as fatigue and weakness (Schubert et al., 2021). B cell deficiency or aplasia occurs from the

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

treatment’s on-target, off-tumor effect—from the successful elimination of CD-19-carrying B cells. Tumor lysis syndrome (TLS) is an uncommon complication of CAR T therapy. It can occur in response to bridging chemotherapy, a course used to prevent disease progression as patients wait for their CAR T product to be manufactured. As tumor cells die and break down, they can release their contents into the bloodstream and imbalance a patient’s electrolyte and metabolite levels (Tosi et al., 2008). Organ failure results if the condition is unchecked, so patients are closely monitored and receive preventative medicines if necessary (Mahadeo, 2019). People can also develop an allergic reaction to the CAR T cell infusion on rare occasions (Maus et al., 2013).

Risks of Lymphodepleting Chemotherapy Most prospective CAR T patients will undergo chemotherapy before infusion (Kansagra et al., 2019). While brief and standard, the procedure increases the risk of several complications. The immune system, already compromised from cancer, weakens further with CAR T therapy. This risk for bacterial, viral and fungal infections only grows with preparatory chemotherapy (Korell et al., 2021). Notably, these infections are usually detected and appropriately managed. Patients with low red or white blood cell counts may need to forgo chemotherapy to avoid life-threatening cell depletion (Sharma et al., 2022). Chemotherapy can also catch healthy cells in its toxic crossfire. Permanent tissue damage and organ dysfunction could potentially occur as a result.

William A. Haseltine, PhD

What if CAR T therapy fails? People turn to CAR T therapy when other treatments have proven ineffective. The CAR T cells should ideally send their cancer into a complete and lasting remission, but this is a reality for only a fraction of patients. Unfortunately, there is currently no way to anticipate how well a patient will respond to the treatment—although investigating gene expression patterns in patients with persistent CAR T cells may eventually lead us to a potential marker of success (Anderson et al., 2023). So what happens if cancer returns after taking the chance on CAR T therapy? There are no conclusive answers at the moment. Treatment plans will vary on a case-by-case basis, as decided by the patient and their healthcare team. Salvage chemotherapy or radiation therapy are potential alternatives (Imber et al., 2020). It could be more appropriate to try other CD19directed therapies depending on prior treatment. CD19-directed drug loncastuximab tesirine elicited antitumor responses in six of 13 patients who previously underwent anti-CD19 CAR T therapy (Caimi et al., 2022). Another route is to administer a bispecific monoclonal antibody. Glofitamab and Epcoritamab are monoclonal antibodies that simultaneously target CD3 and CD20 antigens and may work for CAR T cell patients. Glofitamab elicits a 35% complete response rate in a study of 52 B-cell lymphoma patients who previously underwent CAR T therapy (Dickinson et al., 2022); Epcoritamab achieves a similar complete response rate in previous CAR T patients (Thieblemont et al., 2023).

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Some people may benefit from a second CAR T cell infusion. A study of 44 patients with relapsed or refractory B cell-derived cancers suggests that undergoing lymphodepleting chemotherapy before the second infusion may increase the therapy’s durability (Gauthier et al., 2021). Outcomes may also improve if the components in the second batch of CAR T cells come from a different source (mouse vs. human origins), thereby limiting the chances of immune rejection (Maude et al., 2017). T cell engaging therapies such as CAR T therapy or bispecific antibodies appear to benefit patients with relapsed multiple myeloma, according to a study published in Blood this year (Van der Schans., 2020); bispecific antibodies encourage T cell activity by binding to both T cells and cancer cells, bringing the T cell in close enough proximity to act. On the other hand, checkpoint inhibitors do not elicit durable responses in patients who relapse from CAR T therapy (Chong et al., 2022). The theory is that checkpoint inhibitors could combat T cell exhaustion and the immunosuppressive tumor microenvironments contributing to CAR T failure, thus reinvigorating CAR T cells. However, a large-scale analysis published in Blood Advances reports a 19% overall response rate for patients who receive a checkpoint inhibitor regime after CAR T cell therapy fails to treat their relapsed/refractory B cell lymphomas (Major et al., 2023). And of course, after exhausting several options, the next best step may involve clinical trials or palliative care. Barriers to Access: Time and Cost The greatest hurdle for CAR T therapy is its inaccessibility. CAR T cell production is labor intensive because it is customized for each 44

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patient; it requires high-tech facilities and well-trained personnel, often located at academic hospitals in urban areas ( Car t-cell program, n.d.). In addition, it can take several weeks to cultivate the retroviruses necessary to deliver new genes to the T cells. Overall demand for viral vectors has increased dramatically due to vaccine production and cell therapy research, causing vector supply shortages and bottlenecks in CAR T cell production during the height of the pandemic (Zamecnik, 2022). As a consequence of these factors, the price of the therapy is steep. A single infusion costs between $373,000 and $475,000 in the US depending on the treatment (Kite’s yescarta, 2017); Skinner, 2017). Including other services such as hospital stay, imaging and medicine, the total cost of care can easily exceed $500,000 without health insurance (Tice, 2018). Medicare approved the intervention for coverage in 2019, but it’s clear that CAR T products remain exorbitantly expensive (“Ncd,” 2019; Sahli et al., 2021). Minimizing manufacturing costs and streamlining production will be necessary to solve issues of time and price. One possibility is to use CRISPR technology to edit the genes, a process that is safer and more streamlined than the retroviral alternative. Several studies have successfully manufactured CAR T cells using this method, but the procedure has yet to become an industry standard (Foy et al., 2023). Some place their hopes in an off-the-shelf version of CAR T therapy. These T cells would be derived from donors instead of directly from the patient, and therefore would be cheaper to manufacture in large quantities. It would also be faster to administer the treatment with a stock of ready-made cells at hand. Despite the heightened risk of tissue rejection, clinical trial results published in Nature Medicine 45

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

demonstrate that the concept could be implemented safely for patients with multiple myeloma (Benjamin et al., 2022). A Multi-faceted Solution: mRNA Technology An even more cost-effective solution would be to temporarily create CAR T cells inside the body instead of in the lab. This could potentially be achieved by unifying CAR T with another highly adaptable advance: mRNA technology. Lipid nanoparticles would deliver the mRNA code for the desired chimeric receptor; as mRNA does not integrate into the genome, the T cells would take up the nanoparticles and transiently express a new receptor. This process mirrors the mRNA technology used in current COVID-19 vaccines, which can be manufactured in the US for less than $3.00 a dose (Light & Lexchin, 2021). This method could potentially slash prices, but only if proven just as or more effective than standard CAR T therapy. Luckily, preliminary research in mice with cardiac fibrosis successfully used this approach to reduce heart damage (Rurik et al., 2022). As an additional benefit, the transient nature of mRNA technology means it can be used as an on-and-off switch, thus exerting a more precise level of control than standard CAR T therapies. The study raises hope for CAR T to become more accessible and widely applicable, although further investigation is still needed to test the concept’s clinical feasibility.

Impact on Inherited Diseases The allure of mRNA is rippling into other cell-based gene therapies, as well. Researchers at the University of Pennsylvania successfully used mRNA-carrying lipid nanoparticles to genetically modify 46

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blood stem cells (rather than killer T cells) in culture and in mouse models (Breda, 2023). This technique almost perfectly corrected the genetic code of early sickle cells and allowed the team to selectively deplete specific stem cells—a potentially less toxic alternative to preconditioning chemotherapy. Patients suffering from sickle cell disease, hemophilia and other genetic blood disorders may one day benefit from this safer alternative to stem cell transplantation. These initial achievements for CAR T therapy and stem cell modification foreshadow a future of gene therapy that is ever more entwined with mRNA technology.

FIGURE 16: CAR T cells made with mRNA. Lipid nanoparticles (LNP) carry the desired genetic information and are absorbed by T cells with CD5 glycoproteins. The nanoparticle degrades once inside, releasing the mRNA for the T cell to take up temporarily. The new chimeric receptor targets fibroblast activation protein (FAP), an antigen found on cells responsible for fibrosis. SOURCE: Access Health International

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

The Future of CAR T Therapy The world of CAR T therapy is steadily expanding—and thankfully so. Although this long-awaited technology allows the immune system to fight cancer in unprecedented ways, its use is limited and there is ample room for improvement. These synthetic cells can currently only be used for certain patients with certain B cell cancers, and can cause potentially life-threatening complications. Remission is threatened by factors such as T cell exhaustion and antigen escape. Most importantly, the benefits of CAR T therapy will only reach a minority of patients due to the intervention’s inaccessibility. Despite the steep challenges, the field continues to attract researchers, especially in academia, who see the platform’s potential. There are hundreds of ongoing trials investigating strategies to fine-tune CAR T cell design or reimagine it all together (Barros et al., 2022). We may see breakthroughs with solid tumors with newly developed safety mechanisms or by altering different immune cells altogether, as seen with NK cells, γδ T cells and more. Excitement hums for CAR T therapy to treat illnesses beyond cancer that also lack a simple solution, such as autoimmunity, aging and inherited diseases. The integration of mRNA technology, in particular, may pave the way for versions of CAR T that exceed current standards on all counts. This momentum suggests that CAR T therapy advances towards a bright future, albeit at a marathon pace.

CHAPTER I

CAR T THERAPY FOR B CELL CANCERS

From Lymphoma To Lupus And Beyond: The Remarkable Research Of CAR T Therapy

his is a series on the advances in CAR T, a remarkable immunotherapy treatment dubbed a “living drug.” This first installment will lay the foundation for understanding how CAR T works. Future installments will focus on CAR T applications and recent innovations that further the field. One of the dreams of cancer therapy is to use the power of the body to heal itself. This dream, long in the making, is becoming a reality thanks to deep and fundamental understandings of the immune system, the primary means by which we protect ourselves from external and internal threats. The immune system recognizes and eliminates threats, whether from viruses and bacteria from outside the body or by cells behaving abnormally within the body. Using the body’s own immune cells as anticancer agents has long been part of this dream. I was an early pioneer in creating one of the first proven cell therapies using dendritic macrophages to treat prostate cancer. Today, immune cell therapy offers hope to people with cancer and other previously untreatable diseases.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

This series will explain a recent and revolutionary cell therapy called CAR T, delving into current successes and future opportunities. The “T’ of CAR T Therapy CAR T is short for Chimeric Antigen Receptor T cells. Essential to understanding this therapy is an understanding of T cells and cellmediated immunity.

Adaptive Immunity Adaptive immunity allows humans to form a tailored defense to foreign invaders. Adaptive immune cells memorize the telltale signs of enemies and trigger defensive mechanisms if the signs are detected in the future. This branch of immunity concerts two separate arms—humoral immunity driven by antibody-producing B cells, and cell-mediated immunity driven by “helper” T cells and “killer” T cells. CAR T technology alters the typical functioning of cytotoxic cells. Instead of indirectly aiding antiviral processes as CD4+ helper T cells do, CAR T borrows the cytotoxic power of CD8+ killer T cells to destroy infected or abnormal host cells, thus transforming into a “living drug.” A typical cytotoxic T cell eliminates threats using the following process:

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FIGURE 1: A T cell activates once it encounters an antigen presenting cell (APC) with its corresponding antigen. An antigen presenting cell (APC) breaks down foreign proteins into smaller fragments within the cell. In the case of killer T cells, a major histocompatibility complex sits on the cell surface of the APC and presents the antigen. The T cell receptor binds to the antigen; simultaneously, a co-stimulator signal is received. The result is an activated T cell. The result is a cytotoxic T cell with the targeting power of antibodies. This design can be altered further to increase precision targeting. SOURCE: Access Health International

The process (as illustrated in Figure 1) begins with the differentiation of an inactive T cell—in essence, any T cell without a specified purpose. As if waiting for the right key, a T cell does not activate unless it encounters an antigen presenting cell (APC) with

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

its corresponding antigen. In order for an interaction between the two cells to occur, several steps must occur. Firstly, the antigen presenting cell must process the antigen, the enemy components, into smaller peptides. Then, these peptides must be carried to the antigen presenting cell’s surface by major histocompatibility complexes (MHC). Immature CD8+ T cells require MHC Class I molecules to facilitate this translocation. Around this time, a secondary signal such as CD80 or CD86 must also be received by the T cell. In the final step, the antigen presenting cell releases a protein signal called CD40 and cooperates with helper T cells to finalize the differentiation process. How Killer T Cells Kill Killer T cells destroy infected and abnormal cells by inducing apoptosis, a form of controlled cell death which does not spark inflammation. Pockets of enzyme within the T cell must make contact with the target cell to trigger its death. When a cytotoxic T cell recognizes its target, it binds to the class I MHC molecule on the surface of the target cell (see Figure 2) to create a bridge. With the bridge completed, the T cell can then release the enzymes. One enzyme drills pores in the target cell’s membrane, thus ruining its integrity. The other travels through these newly made tunnels, tipping an enzyme cascade inside the target cell which accelerates its degradation. The crumbling target cell mimics the imagery of bricks falling from castle walls. Nearby phagocytes recognize the “crash of bricks”— more accurately, sense a change in the membrane—and begin ingesting the target cell. The target cell breaks down to nothing

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inside the phagocyte without stimulating inflammation or other side effects.

FIGURE 2: Cytotoxic T cell activation and subsequent apoptosis of cancer/infected cell. SOURCE: Access Health International

Construction of a Chimeric Antigen Receptor Killer T cells are clearly useful in clearing irregular host cells. Researchers recognized this and sought to harness this natural design to eliminate cancer cells through CAR T. Chimeric antigen receptors are engineered to detect a specific antigen and trigger the destruction of a target cell. The most basic CAR T design accomplishes this by manipulating antigen binding sites normally intrinsic to antibodies—single chain variable fragments (scFV)—to lend cytotoxic T cells higher antigen specificity. The CAR T cell recognizes specific antigens thanks to this domain. Next comes the flexible hinge region. This region simultaneously stabilizes the CAR while its length provides grants easier access to

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

specific antigens. The transmembrane domain anchors the antibody and hinge structure. The intracellular domain describes receptors lying within the T cell. Basic CAR T design employs CD3 here, a T cell receptor needed for T cell differentiation (see Figure 3). Second and third generation CAR models included secondary signal receptors such as CD28 to improve target cell elimination and cell signaling (Figure 4). More recent research developments in CAR design deviate from this foundational model to finetune precision and function. For example, T cell receptor fusion construct (TRuC) CAR tethers the scFV region to the several intracellular CD3 subunits, thereby reducing secondary signaling hypothesized to be unnecessary. Universal CAR (uCAR), on the other hand, augments antibody specification by fusing biotin to the transmembrane domain and the endodomain. Other research efforts incorporate cytokines (signaling molecules) and other molecules to improve T cell expansion and persistence, as well as synthetic control switches to minimize the therapy’s toxic side effects. The groundwork model inspires many alternative CAR designs beyond those demonstrated here. The beauty of this science lies in the melding of two previously separate abilities. CAR T therapy replaces the T cell receptor with an antibody-like structure, all while maintaining the transduction machinery of a T cell. Like this, MHC class I binding becomes irrelevant and a response can be immediately triggered.

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FIGURE 3: The basic design of a chimeric antigen receptor (CAR) synthetically combines the structure and function of T cells and B cells. It borrows the single chain variable fragment (scFv) from antibodies and the CD3 co-receptor from T cells. The result is a cytotoxic T cell with the targeting power of antibodies. This design can be altered further to increase precision targeting. SOURCE: Access Health International

FIGURE 4: Modifications to CAR design. Many of the changes improve antigen targetability, CAR T cell function and applicability. Abbreviations: dual chain CAR (dcCAR), the T cell receptor fusion construct (TRuC). SOURCE: From The Evolving Protein Engineering in the Design of Chimeric Antigen Receptor T Cells by Hughes-Parry HE, Cross RS, Jenkins MR (2020). https://doi.org/10.3390/ijms21010204

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

The CAR T Therapy Process What does the CAR T therapy process look like? Figure 2 illustrates the progression clearly. For a patient receiving CAR T therapy, the process may begin with a medical professional drawing their blood and separating T cells from that sample using apheresis; this would be an autologous treatment, as the cells used originate from the same patient. T cells can also be isolated from a healthy donor’s blood sample, otherwise known as allogeneic transplantation. The cells must then be genetically altered to recognize a particular target in a cell processing center. To do this, the cells are “expanded”—a process which stimulates T cell proliferation. The new plethora of T cells must be purified and then genetically modified with a gene that encodes the desired chimeric antigen receptor. CRISPR technology can be used here to accomplish the task. The cells are now ready for infusion. The cells are frozen and sent back to the treatment center. The patient preps for infusion with a lymphocyte-depleting chemotherapy; the chemotherapy reduces the number of white blood cells in the blood to reduce competition for the CAR T cells, thus helping them multiply. With success, the engineered T cells will recognize the antigen on cancerous cells, bind to it, and mark it for destruction via apoptosis. The infusion takes between 30 to 90 minutes to complete, but the patient will be closely monitored for days, weeks or months to watch for any adverse side effects and to receive additional treatments.

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FIGURE 2: Chimeric Antigen Receptor T Cell production and infusion, broken down into five steps, as follows: (1) Isolation of T cells (2) Incorporation of a gene encoding chimeric antigen receptor in the T cells (3) T cells gain a specific target antigen (4) Engineered T cells proliferate in cell culture, and (5) Infusion of engineered cells into patient. SOURCE: From CAR T-cell therapy by Reyasingh56, (2018, July 4), Wikipedia Commons. https://en.wikipedia.org/wiki/CAR_T_cell#/media/File:CAR_Tcell_Therapy.svg

Side effects can occur if the “living drug” multiplies too actively, the most common being cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Also commonly known as “cytokine storm,” CRS occurs when proteins called cytokines flood the immune system and send it into overdrive. Symptoms tend to be mild—fever, nausea, headache, rash, and more—and resolve within a couple of days, but they can also be severe or life-threatening. ICANS refers to a neurotoxic condition that appears within one to three weeks after T cell infusion. Early signs, such as tremor and lethargy, can lapse into stupor, seizures or coma if untreated. More on managing side effects to come in later installments in this series. The long-term side effects of CAR T are unknown. As a result, the FDA stipulates that gene editing treatments such as CAR T therapy should be monitored for up to 15 years—five years of annual follow57

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

ups, followed by ten years of questionnaires and/or other queries (Long Term Follow-Up, 2020). What Illnesses Can CAR T Treat? CAR T therapy is FDA-approved to treat B cell-derived lymphomas—cancers caused when B cells (not T cells) grow too rapidly—as well as multiple myeloma, cancer of plasma cells found in the bone marrow. These treatments tailor chimeric antigen receptors to target an antigen called CD19 found only on the tumor cells of lymphoma patients. Another target is BCMA, a B cell maturation antigen-specific to multiple myeloma. CAR T therapies may be federally approved, but they are not used as first or second-line cancer treatments; usually CAR T therapy is considered after receiving standard chemotherapy treatment and other alternatives. And as a newer treatment, it may be more expensive than other therapies or may not be fully covered by health insurance. But this field is ever-growing. Several hundred clinical trials are in progress to test the boundaries of this mechanism and enhance its design. The next installations in this series will cover some of the most recent discoveries in the CAR T circuit, such as treatment advances in B cell lymphomas, lupus and heart disease, as well as innovations in CAR T precision.

This article originally appeared in Forbes on October 3, 2022, and can be read online here: From Lymphoma To Lupus And Beyond: The Remarkable Research Of Car T Therapy

William A. Haseltine, PhD

The Remarkable Research Of CAR T Therapy: B Cell Cancers

This is a series on the advances in CAR T, a remarkable immunotherapy treatment dubbed a “living drug.” The first installment lays the foundation for understanding how CAR T works. This second piece delves into the use of CAR T to treat B cell cancers. CAR T is an effective treatment for some hard-to-treat cancers. This “living drug” is made by extracting killer T cells from the body, manipulating them to target cancer cells, multiplying the newly engineered cells and infusing them back into the body. Development over the last forty years has evolved the precision, efficiency and safety of this technology. Arguably the best example is the treatment of B cell cancers.

B cells to B cell cancers

FIGURE 1: B cells gain function through differentiation. Plasma B cells are a type of B cell which produce essential antibodies needed to tag threats to the immune system. SOURCE: Acces Health International

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Figure 1 illustrates the development of antibody cells. B cell maturation begins with stem cells in the bone marrow and is completed with the antibody-producing plasma B cells. Typically, threats to the body leave trails of foreign antigen which can be followed. B cells detect these antigens and proliferate to eliminate pathogens, but these numbers quickly subside. This is done by design. The body regulates this process to ensure the bloodstream is not flooded with too many antibodies to prevent normal function. However, this system can go awry at any point. B cell precursors, intermediate cells or plasma cells can mutate and grow uncontrollably, causing damage to the body rather than shielding from it. When this happens, the immune system weakens and B cell cancers result.

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FIGURE 2: Overview of the lymphatic system. This system maintains fluid balance in the body, absorbs fat from the digestive tract, removes waste products and abnormal cells, and protects the body from foreign invaders. Lymphoma is a cancer of the lymph organs (ex: lymph nodes), while leukemia concentrates in the bone marrow and blood. SOURCE: Access Health International

B cell lymphomas originate from the lymphatic system organs, vessels and tissues, such as the lymph nodes or the spleen. In contrast, leukemias circulate in the bone marrow and blood instead of the lymph organs. Although multiple myeloma is also a cancer of

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

the bone marrow, it entails the abnormal growth of plasma B cells in particular.

Treating B cell cancers Chemotherapy and radiation most successfully reduce the size and quantity of B cell tumors. Partial remission is very achievable, but complete remission—the total absence of cancer— is much more difficult to attain. For many, the cancer may temporarily recede for months or years after treatment before recurring. And when the cancer recurs, it can be resistant to treatment. CAR T cell therapy addresses this problem by transforming patient immune cells into an anti-cancer drug. Cells are taken from the body and modified to detect the tumor cells. CAR T cells are fitted with a fusion protein (scFV, Figure 3) made from antigenrecognizing regions of antibodies. This component is typically engineered to target CD19, a B cell antigen known for its role in B cell signaling. This protein is found in B cells of all stages and is present on the surface of many B cell cancers. CD19 is not found on hematopoietic stem cells—those which have yet to mature and gain purpose; as a result, the therapy is less likely to target noncancerous immune cells, an ideal quality in a therapeutic target.

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FIGURE 3: CD19 is an antigen expressed on cancer cells. CAR T cells are fitted with an antigen recognition domain, a single chain variable fragment (scFV), to target the CD19 on the surface of these cancer cells. Once the antigen domain binds to the cancer cell, the CAR T cell can induce apoptosis to eliminate the tumor cell. SOURCE: From Interaction between anti-CD19 CAR-T cell receptor and CD19 antigen-presenting tumor cell by Britten, O., Ragusa, D., Tosi, S., & Mostafa Kamel, Y. (2019). https://www.mdpi.com/2073-4409/8/11/1341#

Once the CAR T cell binds to CD19 on the tumor cell, several signals are released from the endodomain that trigger cell death of the tumor cell through apoptosis. The co-stimulatory molecules found in the interior of the CAR T cell allow it to multiply and persist in the body. Normal T cells from the body lack the precision of this antigenrecognizing protein and usually require specific proteins—major histocompatibility complexes—to present the antigen and facilitate similar binding. CAR T cells forgo these steps, producing superior hybrid molecules which combine antibody detection with T cell signal transduction. This synthetic engineering defines the chimeric nature of Chimeric Antigen Receptor T cells. 63

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Why CAR T therapy? As of publication, CAR T is only considered after standard cancer treatments have run their course. Why, then, do people turn to CAR T therapy if it is only considered after several other lines of treatment? For those who have B cancers which are unresponsive to alternative anti-cancer treatments, CAR T can deliver lasting remission and extend life expectancy by several years—sometimes without additional treatment. For example, one study revealed that 44% of young patients with acute lymphoblastic leukemia (ALL) live at least five years without relapse after CAR T therapy (Kymriah, the First CAR T-Cell Therapy, 2022). This is especially remarkable given how difficult it can be to treat the condition and the less than 10% five-year survival rate. Approved CAR T therapies also exist for patients with diffuse large B cell lymphoma (DLCL), follicular lymphoma, mantle cell lymphoma and multiple myeloma.

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FIGURE 4: Antigen escape hinders the long-term efficacy of CAR T therapy. Antigen escape occurs when a cancer cell alters or downregulates or a target antigen, rendering them “invisible” to the immune system. CAR T cells modified to detect and bind to antigen CD19 cannot act upon cancer cells that do not possess CD19; binding and subsequent elimination does not occur. SOURCE: Access Health International

There is a caveat—it is possible to experience relapse after CAR T therapy. One contributing factor is CD19 antigen escape, a type of CAR T resistance. As illustrated in Figure 4, patients with antigen escape develop cancer cells which no longer express CD19 and thus escape recognition by CAR T cells. So while CD19 targeting has proven effective, this phenomenon highlights the need to find alternative antigen targets to improve the drug’s efficiency. 65

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

One possible solution is dual targeting CAR T cells. By engineering T cells which detect more than one antigen on cancer cells, the therapy has a greater chance of attacking tumor-only cells and overcoming antigen escape. Current contenders include dual targeting of antigens CD19 and CD22, as well as CD19 and CD20 (Baird et al., 2021).

Summary CAR T shines best in solving what other therapies cannot. When other lines of cancer treatments such as chemotherapy or radiation cause relapse, CAR T therapy often provides a more lasting remission. There’s promise for these engineered T cells to become even more effective in the future with the advent of dual-targeting CAR T cells. And while none of the six FDA-approved CAR T therapies are currently used as first-line treatment, developments are underway to establish this innovative technology as a primary line of defense. This is a major step forward for treating B cell cancers, and we can anticipate more to come.

This article originally appeared in Forbes on October 19, 2022, and can be read online here: The Remarkable Research Of CAR T Therapy: B Cell Cancers

William A. Haseltine, PhD

CAR T Therapy: A New Direction for Multiple Myeloma Treatment

This is the third installment in a series on the advances CAR T, a remarkable immunotherapy treatment dubbed a “living drug.” This third installment highlights recent advances in treating multiple myeloma. Multiple myeloma is a relatively uncommon yet serious disease estimated to impact more than 30,000 US citizens this year. Although several treatment options exist, the illness is considered incurable as most treatments do not resolve the condition permanently—including the most recent advancements with CAR T cells. Here, we describe an approach using a different variant of CAR T cells for multiple myeloma that holds promise for those with treatment-resistant forms of the disease. What is Multiple Myeloma? Multiple myeloma (MM) or myeloma is a cancer of the plasma B cells found in the bone marrow. Although these white blood cells typically produce antibodies, for people with multiple myeloma, the plasma cells multiply faster than the body can handle, produce abnormal antibodies, and set the body out of balance. The illness can spread to other organs through the bloodstream, and masses of plasma cells may form in the bone marrow or soft tissues, as well. The illness usually occurs to people 60 years and older, and is unlikely to develop in individuals under 40 years of age. The symptoms can be widely varying—some even report having no symptoms at all—but most with this disease experience bone pain and fatigue. Other common complications include anemia, kidney problems, or thickened blood. 67

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Without treatment, the prognosis is poor. However, with the advent of chemotherapy and more advanced medicines, survival is usually four to five years. If diagnosed early, the five-year survival rate exceeds 77%. Patients with active myeloma first receive a combination of drugs to target the abnormal cells. Another alternative is chemotherapy. For example, I contributed to the creation of Velcade, a chemotherapy medicine which slows or stops the growth of myeloma cells. Stem cell transplants, steroids and even CAR T therapy—a newer medical technology which alters patient cells in the lab and infuses them back into the body to fight the cancer—may be tried as other potential options. Unfortunately, once a therapy fails, the body typically becomes resistant to its reintroduction and thus loses efficacy. The Current Reality of CAR T Therapy CAR T therapy has recently been approved to treat multiple myeloma, but it is only considered after four or more refractory lines of treatment—in other words, when other four or more options fail to achieve lasting remission. The two existing CAR T therapies on the market target B cell maturation antigen (BCMA), an antigen expressed on the surface of malignant plasma cells; in this piece, the antigen will be referred to as Target 1. A Chimeric Antigen Receptor T cell derives its name from the synthetic combination of T cell and antibody properties. Patient T cells are taken from the body and modified to detect Target 1 through an antibody-based fusion receptor (scFv). The lysing process relies on signaling from the T cell. When the CAR T

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antigen receptor binds with Target 1 on the cancer cell, the CAR T cell releases chemicals to trigger the cancer cell’s death. Clinical trials have confirmed these CAR T therapies as safe to use and capable of producing results. A study of Ide-cel found 73% of participants had a decrease in their cancer (Munshi et al., 2021). Even more successfully, a study of Cilta-cel saw a 98% response rate, with 78% of patients showing no signs of cancer in their bone marrow. The outstanding issue with both treatments, however, is that relapse does eventually occur—around 8.8 months later for Idecel, and 22 months later with Cilta-cel. With relapse and treatment resistance a prevailing concern for multiple myeloma treatments—not just CAR T—researchers are seeking new ways to sustain longer remission and increase survivability when other alternatives are exhausted. One possible method is to enhance current CAR T protocols with a new therapeutic target. Methods In their study, Mailankody et al. consider the safety of an alternative antigen target. The target is known as G protein-coupled receptor, class C, group 5, member D (GPRC5D), but shall be referred to as Target 2 for simplicity. Despite its unknown function in tissues, it poses as a promising CAR T antigen target due to its presence in several myeloma cell lines and in bone marrow plasma cells. The team chose a second generation CAR T design for their product. Second generation CAR T cells contain a single costimulatory domain (shown in blue in Figure 1) inside the T cell to extend the life of the cell once in the body. The chimeric antigen

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

receptor in this study is tailored to find cancer cells that express Target 2 (denoted in green in Figure 1). As depicted in Figure 3, the researchers first collected patient T cells through leukapheresis. They modified the T cells, expanded them to large numbers, and then infused the CAR T cells back into the body after completing preparatory chemotherapy. The patients received an escalating dose of the trial CAR T infusion, totaling to four doses.

FIGURE 1: Later generations of CAR T therapy include co-stimulatory signaling domains to improve T cell expansion after infusion and T cell survival once in circulation. SOURCE: Access Health International

Results A total of 17 participants received CAR T therapy, all who have previously tried five different kinds of multiple myeloma treatment. 70

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The majority of participants developed cancer resistance to their last line of treatment; this includes a group of individuals who previously received Target 1 CAR T therapy. On the whole, this study successfully confirms Target 2 CAR T therapy as safe and effective, particularly for individuals who have already received Target 1 CAR T cell therapy or have run through several other therapeutic options. Around 78% of patients had a partial response or better, and 59% had a very good partial response or better. The therapy was effective even ten months after infusion for some individuals. Common CAR T therapy side effects include cytokine release syndrome and immune effector cell-associated neurotoxicity (ICANS). While both conditions can be reversed with prompt treatment, the severity of both side effects can range from mild to life-threatening. Most participants experienced milder cytokine release, with the exception of one patient who experienced lifethreatening side effects. All with side effects were treated. Future Directions Mailankody et al. demonstrate that Target 2 CAR T therapy can effectively treat multiple myeloma. If Target 1 CAR T therapy fails, Target 2 appears to be a viable alternative. The results also suggest that using Target 1 and Target 2 CAR T therapies in succession could lead to positive outcomes. A third alternative is to enhance the T cell design further to allow for tandem targeting; an ideal synergy could be attained if T cells were fitted with both Target 1 and Target 2 receptors, hopefully resulting in longer remission periods and increased survival.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

This article originally appeared in Forbes on October 21, 2022, and can be read online here: CAR T Therapy [Part III]: A New Direction for Multiple Myeloma Treatment

William A. Haseltine, PhD

CAR T Therapy Dramatically Reduces Risk Of Relapse For Multiple Myeloma More than 35,000 Americans this year will be diagnosed with multiple myeloma, a cancer with an average five year survival rate of 58% (Key statistics for multiple myeloma, 2023; Survival rates for multiple myeloma, 2023). Preliminary study results by Johnson & Johnson and Legend Biotech suggest that their CAR T cell product may benefit multiple myeloma patients who received one to three previous lines of treatment. The clinical trial abstract has since been removed from the internet, as it appears to have been prematurely released. Nonetheless, the data appears promising and worth early discussion (MarketScreener, 2023). CAR T Therapy for Multiple Myeloma Chimeric Antigen Receptor T cell therapy uses bioengineering and a patient's own immune cells to treat their cancer. Genetic modification heightens the cancer-killing ability of a specific white blood cell called cytotoxic CD8+ T cells, aptly nicknamed “killer” T cells. The therapy received approval from the Food and Drug Administration to treat several kinds of blood cancers, including B cell lymphomas, certain leukemias, and multiple myeloma. CAR T therapy for multiple myeloma, a cancer of the plasma cells, is a newer development. The FDA approved the intervention’s use for the first time in 2021, but under the condition that a patient tries at least five lines of treatment prior. This means that standard treatments such as a regimen of chemotherapy drugs (proteasome inhibitors, immunomodulatory drugs), an antiCD38 monoclonal antibody or a stem cell transplant must fail before CAR T therapy becomes an option. 73

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Research is underway to push CAR T therapy earlier in the queue. Bristol-Meyer Squibbs released Phase 3 clinical trials results in February this year demonstrating that their product Abecma reduced the risk of disease progression and death by 51% (compared to standard treatments) for patients who tried two to four prior treatments (Abecma, 2023). J&J and Legend Biotech announced the unblinding of their own Phase 3 clinical trial CARTITUDE-4 in January, but for patients who received one to three prior lines of treatment (Janssen Announces, 2023). The preliminary results from this trial, as referenced here, should have been presented in May or June but instead was accidentally released last week.

Different CAR Structures Is there a difference between the two therapies? In short, yes. Both CAR T cell products target a biological tag commonly found on the surface of plasma cells—B cell maturation antigen (BCMA)—but the method differs slightly. The exterior portion of Abecma’s chimeric receptor contains a single chain variable fragment. This fusion protein connects an antibody heavy chain fragment and a light chain fragment by a flexible linker. It’s also commonly used in CAR T cell products for other blood cancers, albeit targeting a different antigen. In contrast, Carvykti uses a structure called a nanobody, also known as a single domain antibody (VHH) (see Figure 1). A nanobody is derived from camelid animals such as llamas. It is nearly half the size of a single chain variable fragment, but retains a similar function and superior stability (Asaadi et al., 2021). It is also easier to manufacture in bulk. Carvykti uses two tandem nanobodies in their product, which allows the CAR T cell to bind 74

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to two regions of BCMA instead of one, as with Abecma. There is currently no evidence to suggest this mechanism has any clinical effect (About carvykti, 2021). Figure 2 compares the chimeric receptor structure of both products.

FIGURE 1: Size and structure comparison between a single chain variable fragment (scFV) and a camelid single domain antibody fragment (VHH), otherwise known as a nanobody. Nanobodies are derived from animals such as camels, llamas, and alpacas. Despite its small size, a nanobody can bind just as specifically as other antibodies. [Abbreviations: CH1, heavy chain constant domain 1; CH2, heavy chain constant domain 2; CH3, heavy chain constant domain 3; CL, light chain constant domain; IgG, immunoglobulin G; VH, heavy chain variable domain; VHH, single variable domain on a heavy chain; VL, light chain variable domain] SOURCE: Adapted from Exploring cellular biochemistry with nanobodies by Cheloha, R. W., Harmand, T. J., Wijne, C., Schwartz, T. U., & Ploegh, H. L. (2020). https://doi.org/10.1074/jbc.REV120.012960

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 2: Chimeric antigen receptor (CAR) comparison. The antigen binding domain differs between the two CAR T cell products. Carvykti specifically uses dual nanobodies in this region. SOURCE: Adapted from Development of CAR T Cell Therapy in Children— A Comprehensive Overview by Boettcher M, Joechner A, Li Z, Yang SF, Schlegel P. (2022). https://doi.org/10.3390/jcm11082158

Promising Results We already know that Carvykti performs well for patients whose cancer returns after four or more standard treatments. The first clinical trial for Carvykti illustrated how the intervention had an effect in almost all patients, and completely removed signs of cancer in around 78% of patients (Berdeja et al., 2021). Eventually this number increased to 83% according to a 28-month follow-up (Longer-term Data, 2022). 76

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How, then, does Carvykti perform when administered as an earlier treatment option? The abstract suggests that Carvykti can elicit robust immune responses when administered earlier, as well. More than 400 patients were enrolled; around half received lymphodepletion chemotherapy (a preparatory measure) and CAR T therapy, while the other half received standard care. All underwent between one and three lines of treatment prior to the study before their cancer returned. The study’s primary endpoint was met. A median 16 months of follow-up revealed that Carvykti reduced the risk of disease progression/death by 74% compared to standard treatment—a major reduction in risk of relapse. Median progression-free survival (PFS) for the Carvykti group had not been reached; the researchers could not calculate this statistic because more than half of the group is still living, which is a positive sign. This is compared to a progression-free survival of 12 months for the patients under standard treatment. In addition, the CAR T therapy elicited more robust responses than the control intervention. In fact, around 73% of patients saw a complete reduction in their cancer with CAR T therapy versus 22% under standard care (Trader, 2023). The CAR T therapy and control safety profiles seem comparable, with 97% and 94% of patients (respectively) reporting serious adverse events. More than a third of Carvykti patients experienced cytokine release syndrome, a common adverse reaction to CAR T therapies. Notably, this adverse effect was not life-threatening. Neurotoxicity, another expected reaction, occurred in 5% of the CAR T therapy patients. 77

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Future Implications The study results deliver positive news. The CARTITUDE-4 trial demonstrates that anti-myeloma CAR T cells, applied sooner than later, can reduce the risk of relapse better than typical anti-myeloma treatments. This is not the first CAR T therapy moving its way to earlier lines of treatment. Research is proving that CAR T therapies, although previously considered a final resort for cancer patients, have the potential to enter the rotation of standard care. As shown here, efficacy and safety pose less of an issue than expected in achieving this dream.

This article originally appeared in Forbes on April 27, 2023, and can be read online here: CAR T Therapy Dramatically Reduces Risk Of Relapse For Multiple Myeloma

William A. Haseltine, PhD

Hope For Universal, Ready-Made CAR T Therapy For Multiple Myeloma A hallmark characteristic of CAR T therapy is that it is individually crafted. Each patient receives a unique cancer-fighting infusion made from their own immune cells. The crucial process of genetically modifying the cells, however, is lengthy and costly. Early study results from the UNIVERSAL clinical trial test a promising solution: a ready-made version of CAR T therapy for multiple myeloma (Mailankody et al., 2023). The treatment shows encouraging safety and efficacy profiles, bringing the concept of offthe-shelf CAR T therapy one step closer to clinical translation. Multiple Myeloma and CAR T Therapy Multiple myeloma (MM) is a cancer of the plasma cells in the bone marrow. This cancer initially responds well to chemotherapy, targeted therapy and other first-line therapeutics. Unfortunately, the disease often returns and grows resistant to prior treatments. When other options are no longer effective, CAR T cells can achieve a durable response. There are currently two FDA-approved CAR T cell products for multiple myeloma: ide-cel and cilta-cel. Ide-cel impressively decreases signs of multiple myeloma in 72% of patients, and ciltacel 98% of patients. Both require the extraction and modification of patient cells to target B cell maturation antigen (BCMA), a biological tag found on the surface of mature plasma cells. Note that while this antigen is a common target for anti-myeloma treatments, research is underway to identify more potential targets.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Shortcomings of CAR T Therapy The process of extracting, genetically modifying, proliferating and infusing cells for each patient is resource-intensive and expensive, with a delay of one month or more. A majority of patients also require bridging therapy—treatments to control their disease during the wait. For this reason only a minority of patients proceed with CAR T. One possible solution is to have pre-prepared CAR T cells that many patients could use instead of cells tailored to a single individual. Many have described this as “off-the-shelf.” Researchers believe that a ready-made CAR T product could be administered within days and reduce costs associated with manufacturing and bridging therapy. Here, we describe clinical trial results which use already prepared CAR T cells to treat patients with multiple myeloma. Off-the-Shelf CAR T Cells To make off-the-shelf CAR T cells, Mailankody first-line T cells from three healthy donors and engineered them with new chimeric antigen receptors (CARs) in place of a patient’s cells. The risk with this method is that the body may reject the donor cells, as often occurs with tissue transplantation. In consequence, the researchers’ CAR T design and study protocol address major hurdles such as graft-vs-host disease (GvHD) and CAR T cell rejection. The researchers employed three safety features to reduce tissue rejection. First, they modified a familiar CAR T cell design used for myeloma. Typical components include an anti-BCMA antibody fragment to detect the plasma cells; a 4-1BB costimulatory molecule to support the cell’s survival and proliferation; and a CD3γ signaling domain to release cancer-killing chemicals. The notable new 80

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addition is the off-switch illustrated in Figure 1. This off-switch allows the team to deactivate the CAR T cells if needed by implementing a monoclonal antibody called rituximab. Next, the team stopped the expression of a gene called T cell receptor alpha constant (TRAC) in the CAR T cells. They used gene-editing enzymes called Transcription Activator-Like Effector Nucleases—TALENs for short—which can recognize and cut out this gene associated with graft-vs-host-disease. The knockout reduces the expression of T cell receptor complexes (Figure 2) on the CAR T cell surface. With fewer receptors to communicate with, host T cells are less likely to recognize the CAR T cells and eliminate them. Lastly, the researchers altered common preparation procedures. Patients usually begin with an immunosuppressive therapy called lymphodepletion prior to CAR T cell infusion. In addition to this standard practice, the team incorporated a new monoclonal antibody to remove threats before they turned into enemies. The monoclonal antibody targets host immune cells with glycoprotein CD52 on their surface. These host cells can mediate graft-vs-host disease and would counter the CAR T infusion if left alone. With this looming threat eliminated, the CAR T cells can proliferate freely.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 1: This study’s second generation CAR T design features familiar components: anti-BCMA single chain variable fragment (scFV), a CD8 hinge, a 4-1BB costimulatory molecule and a CD3γ signaling domain. New to the design is the unique addition of two peptides. These proteins allow the CAR T cell to essentially turn off in the presence of an anti-CD20 monoclonal antibody called rituximab. SOURCE: Access Health International

William A. Haseltine, PhD

FIGURE 2: Structure of a T cell receptor alpha-beta (TCR αβ) complex. The alpha and beta chains allow T cells to recognize antigens presented by major histocompatibility complexes (MHC). The presence of this structure is also associated with graft-vs-host disease. The researchers knocked out the gene which encodes for the constant region of the alpha chain (TRAC, highlighted in red) to prevent CAR T cell rejection. SOURCE: Adapted from T cell receptor—Illustration from Anatomy & Physiology. Wikipedia Commons (2013, June 19). http://cnx.org/content/col11496/1.6/

Encouraging Safety and Tolerability Results A total of 43 patients were given a lymphodepletion regime and escalating doses of the experimental CAR T cell infusion. These patients failed at least three prior lines of treatment—none with prior exposure to BCMA-directed CAR T therapy. Lymphodepletion was given up to five days before infusion. The 83

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patients did not need treatment to manage their cancer between enrollment and CAR T cell infusion.

Adverse Events The trial produced promising safety profile results. None of the patients experienced graft-vs-host disease. The authors attribute this success to the knockout of the T cell receptor gene knockout (TRAC). This does not mean, however, that there were no negative responses to the therapy. All 43 patients experienced some adverse event, but none were sufficient to terminate the trial. The most common CAR T side effects are cytokine release syndrome and neurotoxicity. Both conditions are caused by the abundant release of immune chemicals, and the severity of the side effects increases on a scale of one to four, with one representing mild symptoms to four at life-threatening. Half of patients experienced mild to moderate cytokine release syndrome and 14% percent experienced mild to moderate neurotoxicity. Only one person experienced Grade 3 or higher neurotoxicity, which is a slightly lower but notable difference from rates of other antimyeloma CAR T therapies. The use of a new monoclonal antibody during the lymphodepletion regime did not seem to increase the number of severe infections compared to other custom-made myeloma CAR T therapies. Notwithstanding, the authors do recommend viral monitoring of cytomegalovirus (CMV), a common virus that many carry but does not impact healthy people. The monoclonal antibody increases the risk of reactivating this virus, and the infection will have to be treated with prophylaxis.

William A. Haseltine, PhD

Response Rates The CAR T therapy was well-tolerated. The trial yielded an overall response rate of 56%. The group that received a 320 million CAR T cell dose had a promising 71% response rate with a median duration of response of 8.3 months. Six people in this cohort had undetectable signs of their multiple myeloma. Looking Forward A universal, ready-made CAR T therapy may not be a distant dream. The early results from the UNIVERSAL trial demonstrate that offthe-shelf CAR T cells can perform safely if the cell design and lymphodepleting regime accurately address possible donor rejection. These cells pose one possible solution to reducing the labor, manufacture and time costs associated with alreadyestablished CAR T therapy for multiple myeloma. In spite of this, off-the-shelf CAR T therapy may still be vastly prohibitive to most who need it. This underlying problem of access may require other improved technologies such as the use of mRNA to modify cells in vivo, as well.

This article originally appeared in Forbes on February 23, 2022, and can be read online here: Hope For Universal, Ready-Made CAR T Therapy For Multiple Myeloma

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Altered CAR T Therapy Shrinks Ovarian Tumors In Mice Cancer treatments are rapidly evolving with the advent of cell therapy. Within the last five years alone, CAR T therapy has become widely recognized for its ability to harness the immune cell’s natural abilities to clear cancer and enhance them with genetic modification. Unfortunately, this major achievement is currently restricted to clinical treatment of leukemia and similar blood cancers. Could this standard design be changed to treat solid tumors, too? A study published in The Journal for ImmunoTherapy of Cancer attempts to answer this question in a mouse model of ovarian cancer (Schoutrop et al., 2023). The researchers tweak this oft-used design to improve cell signaling and create a CAR T cell which is more capable of shrinking solid tumors than its standard counterparts. A Unique Challenge: CAR T Therapy & Solid Tumors Cancer arises when cells malfunction and lose their ability to stop growing. The uncontrollable and detrimental growth of these cells could manifest either liquid tumors or solid tumors. Liquid tumors refer to cancers that stem from the bone marrow and circulate the blood. Leukemia rests within this group, alongside lymphoma and multiple myeloma. Solid tumors, in contrast, form a mass of cells and constitute around 90% of all adult cancers (Common Cancer Types, 2023). For now, CAR T therapy can only be used clinically to treat blood cancers. The reasoning comes down to the cell’s design and the unique nature of solid tumors.

William A. Haseltine, PhD

Fundamental CAR T Cell Structure The principle behind Chimeric Antigen Receptor T cell (CAR T) therapy involves extracting a patient’s immune cells and altering them to fight cancer once reinfused. The most basic CAR T cell contains two fundamental components: a cancer-detecting region found on the cell surface, and a signaling region found naturally within the cell (see Figure 1). The synthetic, antibody-like fragment detects any cells which have a particular antigen or biological tag. The signaling machinery releases chemicals to kill the target. This combination of natural and unnatural elements in the cell’s receptor underlines the therapy’s chimeric nature. The antibody-like fragment (single chain variable fragment, scFV) is programmed to hone in on a single target. This means that CAR T therapy indiscriminately clears cells regardless if they’re healthy or cancerous; all that matters is if a particular tag is detected. This is fine for patients with blood cancers, as the affected cells can be supplemented with additional treatments. This strategy, however, is not effective against solid tumors.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 1: A CAR T cell’s most basic form attaches a synthetic cancerdetecting structure onto existing T cell signaling machinery. Variations in this basic model yield first, second and third generation designs, among others. SOURCE: From Building Potent Chimeric Antigen Receptor T Cells With CRISPR Genome Editing. by Liu J, Zhou G, Zhang L and Zhao Q (2019). https://doi.org/0.3389/fimmu.2019.00456

Inhospitable Solid Tumors Solid tumors present distinct challenges that current iterations of CAR T cells struggle to overcome. First, unlike for liquid tumors, it can be very difficult to pinpoint an antigen target that is unique to solid tumors. Many potential targets overlap with antigens found on normal tissues; unlike with supplemental antibody treatments, it is not possible to replace this tissue if attacked. Due to the additional tissue barrier, it is physically more difficult for CAR T cells to travel into solid tumors. The T cells need to

William A. Haseltine, PhD

penetrate the cancerous tissue to be effective. In comparison, liquid tumors circulate the blood and thus are much easier to access. Lastly, solid tumors possess an extensively inhospitable microenvironment for the T cells. The tumor environment is hypoxic, meaning it lacks oxygen needed for metabolic processes. Additionally, these tumors actively suppress immune responses which CAR T cells need to eliminate the cancer. Scientists believe that CAR T cell design could potentially meet these challenges—but not without changes first. A team from the Karolinska Institutet in Sweden tests a potential version of CAR T therapy to treat ovarian cancer in mice. Ovarian Cancer and CAR T Cell Signaling The ovaries are small organs responsible for producing eggs and female hormones. As seen in Figure 2, cancerous tumor masses can form on this pair of organs and disrupt normal functioning. More than 19,000 women will receive a new diagnosis for ovarian cancer this year ( Ovarian cancer , 2023). The 5 year survival rate hovers around 49%. Most treatments entail surgery, radiation or chemotherapy, but perhaps CAR T therapy could eventually be added to the mix. In their study, Schoutrop, et al. created three types of CAR T cells for ovarian cancer. Figure 3 illustrates the differences in design. All three of the therapies targeted human mesothelin, a surface protein which is overexpressed in many ovarian tumors. They all contained a costimulatory molecule used in keeping with second generation design. All second generation CAR T cells contain a version of this molecule, usually either CD28 or 4-1BB (see Figure 1). It releases a secondary signal that bolsters the cell’s performance 89

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

or survival, strengthening the main signal as a wifi booster would. Notably, the six CAR T therapies on the market for blood cancers also use a second generation design, but target different antigens: either CD19 or BCMA.

Mutating CD3 ζ Molecule The important distinction lies not in the costimulatory molecules, but with activation of the main signal. For one of the CAR T cells, the team mutated DNA sequences in the CD3ζ molecule of the chimeric antigen receptor (see Figure 3). They specifically reduced the number of ITAMs (short for immunoreceptor tyrosine-based activation motifs) sequences from three to two, and juxtaposed its performance with other second generation CAR T cells. While these motifs are certainly involved in T cell signaling, it is unknown whether all motifs are needed to send a clear signal. Based on background research, the team predicted that the mutation would finetune the cell’s activation signaling cascade and improve cell persistence.

William A. Haseltine, PhD FIGURE 2: The ovaries are female reproductive glands which produce eggs. For people with ovarian cancer, the cells of one or both of ovaries begin to multiply uncontrollably and can cause tumors. SOURCE: Image by brgfx on Freepik

FIGURE 3: The researchers constructed second generation CAR T cells with an anti-mesothelin antibody fragment, a CD28 costimulatory molecule and a mutated CD3ζ signaling domain (in red). The mutation removes two DNA sequences—immunoreceptor tyrosine-based activation motifs (ITAMs) 2 and 3. The mutated CAR T cell is then compared to similar second generation CAR T cells with either a 4-1BB or CD28 costimulatory domain (MBBz and M28z respectively). SOURCE: From Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models by Schoutrop E, Poiret T, ElSerafi I, et al. Copyright 2023 by BMJ Publishing Group Ltd. https://doi.org/10.1136/jitc-2022-005691

Study Results The researchers tested their three CAR T cell designs in cell cultures and mouse models of ovarian cancer. The cell culture experiments entailed repeatedly exposing ovarian cancer cells to one of the three CAR T cells in test tubes (and a control). All three mesothelin-related T cells displayed multifunctionality and similar tumor-killing potential. 91

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

To test the synthetic cells in vivo, mice received a transplantation of ovarian tumor cells and received a CAR T cell treatment 21 days after. The team then tracked the tumor burden for the control and all three cell treatments using bioluminescent monitoring. Although all three CAR T therapies decreased tumor burden, the mutated CAR T treatment outperformed the rest. The tumor size comparison seen in Figure 3 and 4 clearly demonstrates this significant antitumor response. In fact, more than 50% of mice treated with the mutated T cells had undetectable signs of tumor. The team did a parallel analysis to analyze further how the mutations in CD3ζ influence tumor burden. The CAR T cell which shared the same costimulatory molecule but lacked the CD3ζ mutations reduced tumor growth in mice injected with ovarian cancer cells. However, it did not surpass the survival spurred by the CD3ζ mutated T cells. Based on these observations, the researchers concluded that removing the two DNA motifs calibrated the CAR T cell’s activation. This, in turn, improved the CAR T cell’s persistence and antitumor responses—two qualities that are needed to confront the unique challenges presented by solid tumors.

William A. Haseltine, PhD

FIGURE 4: The researchers monitored tumor burden through weekly bioluminescence monitoring. In red, the mutated CD3ζ CAR T cell displayed significantly less tumor burden than its un-mutated counterparts. SOURCE: From Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models by Schoutrop E, Poiret T, ElSerafi I, et al. Copyright 2023 by BMJ Publishing Group Ltd. https://doi.org/10.1136/jitc-2022-005691

FIGURE 5: The resulting tumor burden differed significantly between interventions. Of the group, the CAR T cell infusion with the mutated CD3ζ chain demonstrated the least tumor growth in the mice.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease SOURCE: From Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models by Schoutrop E, Poiret T, ElSerafi I, et al. Copyright 2023 by BMJ Publishing Group Ltd. https://doi.org/10.1136/jitc-2022-005691

Looking Forward The researchers in this study adjusted CAR T cell signaling and achieved notable success in shrinking ovarian tumors in mice. They show that, despite the success CAR T therapy has already achieved, there is still room yet to expand its potential applications. In particular, a focus on streamlining CD3ζ may be an important key in efforts to treat solid tumors with CAR T therapy.

This article originally appeared in Forbes on March 17, 2023, and can be read online here: Altered CAR T Therapy Shrinks Ovarian Tumors In Mice

William A. Haseltine, PhD

A Different Kind Of Cancer Killer: Improving CAR NK Cell Therapy Recent cancer advance CAR T therapy relies on the killing power of cytotoxic CD8+ T cells to destroy a patient’s cancer, but they are not the only cells that can kill. Natural killer cells also target cancerous cells and could be used instead of CD8+ T cells to address several CAR T therapy limitations. On the road to translating this vision into a therapeutic reality, a study published in Scientific Advances uncovers a critical insight into why natural killer cells with chimeric receptors can lose efficacy over time (). They also propose a means to overcome this setback. This study and others venture into a new realm of chimeric receptor technology that may lead to more ideal cancer therapy. Killer T Cells vs. Natural Killer Cells Chimeric Antigen T cell therapy (CAR T therapy) involves extracting a patient’s cytotoxic CD8+ T cells—also known as “killer” T cells—and boosting their natural abilities to fight cancer with bioengineering. Chimeric antigen receptors are fitted to the cell surface, allowing T cells to precisely target and eliminate cancer cells. Then, the edited cells are returned to the body via infusion. The chimeric receptors activate faster than the receptors T cells naturally equip. Typically, an antigen-presenting cell must first show the T cell the exact antigen, or a protein target, to search for. Then, a second costimulatory signal must follow to complete the activation. Now activated, the T cell can recognize and bind to specific protein complexes found on cancer cells; the complex comprises a target antigen and a molecule called major histocompatibility complex (MHC) class I. 95

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

In comparison, CAR T cells bind directly to the target antigen and activate in one fell swoop—no prior exposure needed—which allows them to act more swiftly in comparison (Harris & Kranz, 2016).

Figure 1: Activation comparison between cytotoxic T cells and CAR T cells. A) Cytotoxic T cells require a two-step activation process involving antigenpresenting cells (APCs). The T cell receptor (TCR) binds a protein complex made from a major histocompatibility complex class I (MHC I) molecule and an antigen peptide. Meanwhile, costimulatory molecule CD28 receives a secondary signal. Now activated, the TCR can recognize cancer cells with the same peptide-MHC class I protein complex. B) In contrast, CAR T cells bind to the target antigen on tumor cells and activate in the same step. SOURCE: From Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations by Weinkove, R., George, P., Dasyam, N. and McLellan, A.D. (2019). Copyright 2019 by John Wiley and Sons. https://doi.org/10.1002/cti2.1049

William A. Haseltine, PhD

Natural Killer Cells Conventional CAR T therapy can effectively treat patients with certain blood cancers, including lymphomas, leukemias and multiple myeloma. However, manufacturing this customized treatment is time-consuming, resource-intensive and costly. While some look to allogeneic or donor-derived CAR T cells to solve this issue, another potential solution is to use a different immune cell as the therapy’s base. Natural killer cells are a particularly prime alternative. Natural killer cells can also cull infected and cancerous cells, but the process differs from cytotoxic T cells. These cells earned the title of “natural” killers because they do not rely on prior exposure to specific antigens to find their target. Instead, they only attack stressed cells or cells that downregulate the expression of major histocompatibility complex class I molecules, as cancer cells often do to avoid immune system detection. This generalized response is characteristic of innate immune cells, our body’s first-line responders, while cytotoxic T cells operate under a slower and more tailored adaptive immune response. Once activated, natural killer cells wipe out their target in one of three main ways: they can release perforin and granzymes—the same molecules cytotoxic T cells release—to destroy cancer (Prager & Watzl, 2019); they can release chemicals called cytokines to recruit other cells to attack; or they can bind to a specific molecule on the target cell to trigger its cell death pathways.

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

FIGURE 2: NK cells attack based on the presence of major histocompatibility complex class I (MHC I) molecules on the target cell, known as “missing self” recognition. MHC class I molecules are a sign of the “self” because they are present on almost all cells in the body with a nucleus. A) The NK cell’s inhibitory receptor binds to MHC class I molecules on healthy cells with normal levels of the molecule; this tells the NK cell to hold back its attack. B) The NK cell activates if the molecule is not there, releasing cytokines and cytotoxic chemicals to destroy the target. SOURCE: Access Health International

CAR NK Cells: A Promising Alternative Natural killer cells may be an apt candidate for chimeric antigen receptor therapy (Xie et al., 2020). CAR NK cells can eliminate a broader range of tumor cells than CAR T cells, as they can kill via their chimeric receptor or their antigen-independent mechanisms. CAR NK cells also demonstrate a favorable safety profile. These cells express different cytokines than cytotoxic T cells, and therefore are not as prone to common adverse effects of CAR T therapy such as cytokine release syndrome (CRS) or neurotoxicity. In addition, natural killer cells do not convert into memory cells as T cells do. 98

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Although this translates to a shorter lifespan, it also lowers the risk of on-target/off-tumor toxicity to normal tissues. Most importantly, making an allogeneic, “off-the-shelf” CAR therapy from NK cells may be more advantageous. Their natural ability to distinguish between healthy and cancerous cells lowers the risk of graft-vs-host-disease (GvHD), a complication where the immune system recognizes a donor transplant as harmful and rejects it. CAR NK and Metabolic Fitness CAR NK therapy is a developing concept, and much is still unknown. To this end, University of Texas MD Anderson Cancer Center researchers closely monitored how NK cells behave in different settings. They tested NK cells that either express nothing (control), anti-CD19 antigen CAR, a cytokine that encourages NK cell proliferation and survival called interleukin-15 (IL-15), or a combination of CAR and IL-15. Analyzing how the cells express genes revealed a previously unknown mechanism behind why CAR NK therapy can weaken over time—loss of metabolic fitness—and a possible means to combat this resistance.

FIGURE 3: Schematic of vectors used to create the CAR NK cells. The CAR construct includes a suicide gene to turn off the therapy if necessary (iCasp9) and interleukin-15 (IL-15) to armor the receptor. SOURCE: From Loss of metabolic fitness drives tumor resistance after CARNK cell therapy and can be overcome by cytokine engineering by Li et al. Copyright 2023 by Science Advances. https://doi.org/10.1126/sciadv.add6997

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

All cells require energy to carry out their functions. Metabolic fitness here refers to a natural killer cell’s ability to produce enough energy for optimal functioning—to proliferate, to kill cancer cells, and to sustain their activity over time (Bittman et al., 2022). CAR19/IL-15 NK cells show the most robust enrichment for metabolic pathways among the four products. This means that integrating IL-15 expression into the synthetic receptor may help NK cells fight cancer. The team confirmed this with mouse models of lymphoma. Mice treated with CAR19/IL-15 NK cells demonstrate significantly lower tumor burden and improved survival compared to controls or CAR19 NK cells, but these mice still eventually die. With more experimentation, the researchers observe that tumor relapse occurs despite the higher metabolic activity and persistence of IL-15-expressing CAR NK cells. The tumor cells appear to activate the NK cells; the NK cells initially exhibit increased function and metabolism for a time; then, the NK cells become dysfunctional and antitumor responses decline.

Possible Mitigation Strategy: Double Dosing If IL-15 expressing CAR NK cells lose steam over time, could a second infusion improve overall performance? The team tested this hypothesis by giving mice with lymphoma two infusions of the CAR19/IL-15 NK cells two weeks apart. Mice with two infusions achieve a 60% survival rate—a much better response than mice dosed with two control infusions or a single infusion of IL-15expressing CAR NK cells. While the first infusion begins to dysfunction, an additional infusion may increase the population of functional NK cells ready to combat the blood tumors. 100

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CAR NK Cells in Humans How do IL-15-expressing CAR NK cells perform in humans? To understand this, CAR NK cells were extracted from two participants with relapsed/refractory lymphoma seven and 14 days after infusion. Each patient received a single infusion of CAR-NK cells. The CAR NK cells did not work for one patient, but cleared all signs of cancer for the second patient within 30 days of infusion. The NK cells from the responder patient showed higher expression of genes associated with NK cell activation and cytokine activity. This gene expression pattern is even more pronounced in Day 14 CAR NK cells. The results suggest that the CAR NK cells in the responder patient likely possess better metabolic fitness and gene expression than those of the nonresponder patient. Future Implications Natural killer cells possess attractive qualities for chimeric receptor therapy, but more research is needed to understand potential roadblocks. This study demonstrates that gradual loss of metabolic fitness may limit this concept’s clinical feasibility. Future strategies may benefit from addressing this resistance mechanism, such as with IL-15 expression or repeat dosing.

This article originally appeared in Forbes on September 7, 2023, and can be read online here: A Different Kind Of Cancer Killer: Improving CAR NK Cell Therapy

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CHAPTER II

CAR T THERAPY FOR AUTOIMMUNITY

CAR T Therapy: From Cancer To Autoimmune Disease, The Lupus Example

revious installments in this series have focused on CAR T therapy as a cancer treatment. A recent study published in the journal Nature Medicine highlights the potential of CAR T therapy beyond this realm—specifically for lupus and other autoimmune diseases (Mackensen et al., 2022). What is Lupus? Lupus (systemic lupus erythematosus) is an autoimmune disease that affects women approximately ten more than men, and is characterized by the overproduction of antibodies that attack the body's own tissues. Lupus symptoms—ranging from mild to lifethreatening—often come and go, making the condition hard to diagnose. Characteristic signs such as fatigue, muscle pains, joint pains and fever also coincide with symptoms of other diseases. Current Lupus Treatments Although lupus has no cure, modern-day symptomatic treatments ensure a normal life expectancy for 80-90% of people with lupus. One of our successes at Human Genome Sciences, a company I founded and served as Chair and CEO, was the use of genomics to 102

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discover and bring to market the first drug to treat lupus: Benlysta. Although the medicine proved to be effective, for some with lupus even the strongest drugs offer no relief. CAR T Therapy for Lupus In their study, Mackensen et al. test the effectiveness of CAR T therapy for treatment-resistant forms of lupus. The theory derives itself from CAR T cells’ ability to kill cells. In lupus, B cells produce antibodies that attack the body and trigger inflammation. Using CAR T therapy, the researchers aimed to purge the B cell lineage, allowing the body to restore B cells de novo. To do this, the researchers first collected patients’ white blood cells. The patients then underwent lymphodepletion, the use of chemotherapy drugs (i.e. fludarabine and cyclophosphamide) to preferentially kill B cells. This drug regimen leaves room for the later infusion of engineered T cells, but can be very dangerous if the immune system is too thoroughly depleted. The team altered patient T cells with new genetic information. The new, chimeric T cell products contained a new receptor—a singlechain variable (scFv) fragment poised to detect CD19-expressing cells—a 4-1BB costimulatory domain and a CD3 intracellular domain. The antibody-derived receptor and additional costimulatory structure do not naturally occur on T cells, lending the chimeric nature the therapy is coined after (Chimeric Antigen Receptor T cells).

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FIGURE 1: A second generation chimeric antigen receptor T cell combines the signaling machinery of a T cell with an antibody-derived receptor. SOURCE: Adapted from Chimeric antigen receptor signaling: Functional consequences and design implications. Copyright 2020 by Science Advances.

Results Five patients with severe, treatment-resistant lupus (four women and one man) participated in the study. Lupus impacted several of their organs, including the kidney, heart, lungs, and joints. In addition, these patients did not respond to steroids, antimalarial drugs and other immunosuppressive medicines. Each of the patients received a transfusion of modified T cells after chemoablation treatment. The chemoablation successfully depleted patient B cells while T cell numbers remained within normal range. Moreover, the team could no longer detect malignant autoantibodies (ie. anti-double-stranded DNA antibodies). The participants’ responses to vaccines also remained largely unchanged, suggesting that the CAR T therapy correctly 104

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targeted detrimental B cells without damaging the entirety of the immune system. Three months later, prior symptoms including kidney inflammation, arthritis, fatigue, and heart fibrosis disappeared, and all other immunosuppressive drugs could be discontinued. The symptoms did not return even when B cells began to reconstitute months later. Remission was defined by DORIS, a standardized criteria used to measure lupus symptom severity. Future Possibilities for CAR T This study demonstrates how CAR T can send treatment-refractory lupus to remission. This is a first hopeful step. The search is now on for ways to improve CAR T-induced remission for prior B cell ablation using a cocktail of cytotoxic drugs. The study also opens the door to the possibility of applying CAR T to other difficult-to-treat autoimmune diseases.

This article originally appeared in Forbes on November 1, 2022, and can be read online here: CAR T Therapy: From Cancer To Autoimmune Disease, The Lupus Example

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CAR T Therapy To Treat And Cure Rheumatoid Arthritis A study published in the Journal of Immunology advances work towards a cure for rheumatoid arthritis via cell-mediated therapies (Whittington et al., 2022). Around 1.3 million people in the US have this autoimmune disease. The condition can severely impair quality and can reduce life expectancy by 3 to 10 years. The study builds upon a recent cancer innovation to effectively target the cells associated with disease progression. Although the results have not yet been verified in humans, the findings demonstrate the burgeoning potential of this treatment to address autoimmunity. Autoimmunity and Rheumatoid Arthritis Rheumatoid arthritis (RA) differs from the “wear and tear” arthritis most recognize. Rheumatoid arthritis occurs when the immune system wrongly attacks the body’s own joint tissues and leaves widespread inflammation in its wake. Figure 1 clearly illustrates how the injured joint tissue swells and erodes the bone. The affected joints often become painful, stiff and red as a result. Further side effects include fever, fatigue and weight loss. Existing treatment options usually involve medicines to suppress the immune system and slow down disease progression. However, these treatments do not address the underlying cause: the errant and selfreactive immune cells.

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FIGURE 1: With rheumatoid arthritis, the immune system attacks the synovial membrane, the thin tissue encapsulating the joint; the membrane becomes inflamed and causes bone erosion. SOURCE: Diagram showing rheumatoid arthritis in a hand by brgfx by Freepik

Haywire Immune Mechanism Current understandings of autoimmune arthritis suggest that the disease relates to a set of genes called human leukocyte antigen (HLA) class II. Many versions of this gene exist. In general, reading these genes produces a variety of molecules that can present biological tags to nearby helper T cells, also known as CD4+ T cells. The system works similarly to a flag and pole. The biological tag— formally known as an antigen (Ag)—acts as a flag; the flag cannot be read unless a major histocompatibility (MHC) class II molecule 107

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lifts it up for helper T cells to see. Corresponding helper T cells can then recognize their “flag,” bind to it, and trigger an immune response. Considering that particular helper T cells react and progress rheumatoid arthritis, targeting these haywire CD4+ T cells could cease the undesired inflammatory response. Innovative CAR T Cell Targeting Researchers at the Memphis Veterans Affair Medical Center sought to target the pathogenic helper T cells responsible for rheumatoid arthritis. To accomplish this, the team designed a CAR T cell that autoreactive helper T cells can bind to. A typical Chimeric Antigen Receptor (CAR) T cell artificially combines an antigen-detecting region derived from antibodies with a signaling domain derived from killer T cells (see Figure 3). Once infused into the blood, the CAR T cells detect antigens on the surface of cancer cells and subsequently eliminate the cancer. As this method does not work against pathogenic CD4+ cells, the researchers developed their own chimeric antigen receptor in response. Figure 2 illustrates the team’s innovative design. The receptor maintains the same activation and costimulation domains seen in typical CAR T cells. The antibody component, however, is replaced with something new: a synthetic, rheumatoid arthritis-specific protein complex. The complex mimics a specific, rheumatoidassociated “flag and pole” (MHCII and antigen) pair that autoreactive helper T cells in mice can recognize. Once bound, the CAR T cell releases chemicals to kill the bound helper T cell.

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FIGURE 2: This study uses a chimeric antigen receptor that contains CD3ζ T cell machinery alongside CD28 costimulatory molecules. The most notable departure from cancer-based CAR T cell design is the major histocompatibility class II/antigen domain, which enables the cell to interact with pathogenic CD4+ T cells in mice. Abbreviations: TM, transmembrane; costim., costimulatory molecule; TCR, T cell receptor. SOURCE: Access Health International

Study Results The researchers found that the new CAR T cells could indeed be recognized by rheumatoid-associated helper T cells when tested in the lab; the helper T cells could only be stimulated if the corresponding antigen on the CAR T cell was present. The synthetic T cells also successfully eliminated the pathogenic helper T cells. The researchers then used the CAR T cells on mice immunized with collagen type II (CII). This induced autoimmunity similar to 109

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that of human rheumatoid arthritis. The team removed killer T cells from the mice, genetically modified them to express the DR1 chimeric antigen receptor, and reinfused them into the body. Overall, this new CAR T cell design seemed to effectively eliminate the target CD4+ T cells. The mice treated with the CAR T therapy had significantly reduced helper T cell responses—in some cases completely eliminating them. The administration of CAR T therapy also reduced autoantibody responses linked to rheumatoid tissue damage. In addition, the treatment delayed the onset and severity of rheumatoid arthritis in mice when given early on in disease development. The eventual arrival of arthritis signaled that the CAR T cells, effective as they may be, circulated in the body for a shorter time than expected. The CAR T cell design may need further adjustments to support CAR T cell expansion inside the body. Future Directions This study illustrates that CAR T therapy can indeed translate from cancer to autoimmune treatment. The CAR T cells successfully targeted and eliminated self-reactive CD4+ cells, as well as reduced autoantibody production. While the concept proves feasible in mice, the hope is to translate this work for human use. The results broaden the scope of possibility for treating and potentially curing rheumatoid arthritis and other autoimmune diseases.

This article originally appeared in Forbes on December 19, 2022, and can be read online here: CAR T Therapy To Treat And Cure Rheumatoid Arthritis

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CAR T Therapy, A Promising New Therapy For Multiple Sclerosis? A study of mice by the researchers at the Washington University School of Medicine suggests that CAR T therapy offers a new opportunity to treat and possibly cure multiple sclerosis (Yi et al., 2022). Around 1 million people in the United States have this autoimmune disease. The symptoms that arise—numbness, double vision, and tremor among others—impact the brain and nerves, and can potentially disable. Available treatment options only patch the problem and do not cure it. CAR T therapy may help piece together some missing clues. While the therapeutic is best known for its uses against cancer, it has shown recent promise to treat other autoimmune diseases including lupus and rheumatoid arthritis. Here, the researchers extend this potential to multiple sclerosis in their animal model of the disease—a hopeful step towards eventual success in humans. What is Multiple Sclerosis? Multiple sclerosis (MS) occurs when the immune system wrongfully perceives the body’s own cells as a threat. White blood cells called helper T cells or CD4+ cells go rogue and target the outer covering of the nerves. This covering, similar to insulation on wires, protects the nerve fiber inside (see Figure 1). When damaged, the nerves cannot properly send electrical signals to other parts of the body and thus impedes communication between the brain, spinal cord and other parts of the body. The degradation of the nerve sheath, otherwise known as demyelination, manifests signs variably from person to person. 111

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Multiple sclerosis-related nerve damage commonly causes numbness and tingling, fatigue or impaired vision among other symptoms. Although treatment options such as steroids or physical therapy can ease MS flare-ups and relapses, a cure has yet to surface.

FIGURE 1: With multiple sclerosis, the immune system targets the outer sheath covering the nerve and exposes the nerve fiber. This can cause a range of symptoms including numbness, tingling and pain.

SOURCE: From Damaged myelin of the human neuron by brgfx by Freepik CAR T Cells Target Autoimmunity A recent study published in Science Immunology builds towards a possible cure for multiple sclerosis by using CAR T Therapy (Yi et al., 2022). The therapy relies on Chimeric Antigen Receptor T cells to detect and destroy malignant cells inside the patient’s body. To create CAR T cells, scientists attach new receptors onto a patient’s extracted killer T cells—usually this meshes the detection power of an antibody onto killer T cell machinery. The CAR T cell, 112

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once reinfused into the body, possesses a heightened ability to recognize and destroy cells with a specific biological tag, otherwise known as an antigen. This CAR T design has proven effective against cancer, but cannot be applied to autoimmunity without first changing the chimeric antigen receptor. Instead of targeting antigens on the surface of cancer cells, the CAR T cell must now target the self-reactive helper T cells which perpetuate the autoimmune disease.

CAR T Cell Design for Multiple Sclerosis For this study, the researchers created a chimeric antigen receptor that can bind to MS-related helper T cell receptors in mice. The signaling molecules remained the same as usual. However, a protein complex replaced the oft-seen antibody-derived antigen detection region. Figure 2 illustrates this design. It utilizes a major histocompatibility class II (MHC II) molecule, a structure not found on killer T cells, alongside a peptide attached by a flexible linker. The team found that they could easily change the presentation—and thus the targeting—of this protein complex, pointing to the adaptable nature of the design. They programmed the CAR T cells to initially target MOG, a protein found on the surface of myelin that pathogenic helper T cells react to in mice models of multiple sclerosis.

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FIGURE 2: Schematic of a peptide/major histocompatibility class II chimeric antigen receptor (pMHC II CAR). The signaling domain consists of a CD3ζ molecule and a CD28 costimulatory molecule. The pMHC II domain can be changed and personalized depending on the desired allele. SOURCE: Access Health International

Success for CAR T Therapy The researchers tested the efficiency of this design in several stages. They first confirmed that the basic CAR T cell design seen in Figure 2 could target helper T cells with specific receptors in mice. Even without standard practices such as lymphodepletion, the CAR T cells eliminated almost all of the T cells they were programmed to target. The therapy only found partial success in mice induced with an autoimmune disease to mimic multiple sclerosis in humans. The

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CAR T cells did not efficiently eliminate their first target: 2D2 T cells known for inducing multiple sclerosis in animals. The authors surmised that, due to low affinity between these harmful helper T cells and the MOG CAR T cells, the cells fail to bind strongly to each other and thus influences the response. Fascinatingly, the therapy significantly limited the severity of disease when injected at the first sign of illness. So while the CAR T cells failed to efficiently deplete low affinity pathogenic helper T cells, they reacted more readily to alternate T cells which can cause disease. The team then altered the CAR T cell design to further support the cells’ survival. In doing this, the CAR T cells gained higher sensitivity to both low and high affinity MOG-specific helper T cells. Through this process, the researchers discovered a two-part mechanism that drives multiple sclerosis in animals. Based on the responses from the CAR T therapy, the team realized that higher affinity pathogenic T cells seem to initiate the disease, while their lower affinity counterparts seem to perpetuate the condition. As a result, the most comprehensive use of CAR T cells takes into consideration different stages of disease. Implications This CAR T therapy mouse model leaves room to hope for a longsought cure for multiple sclerosis. The researchers successfully crafted CAR T cells which can target either low and high affinity helper T cells responsible for multiple sclerosis in mice. From these observations, CAR T therapy could be strategically used to either prevent disease or mitigate existing symptoms. If translated to 115

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humans, CAR T therapy could be administered to either prevent MS flare-ups or to thoroughly address already-existing symptoms.

This article originally appeared in Forbes on December 21, 2022, and can be read online here: CAR T Therapy, A Promising New Therapy For Multiple Sclerosis?

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Early Success: mRNA & CAR T Therapy To Treat Rare Autoimmune Disease Myasthenia Gravis Medical innovation Chimeric Antigen Receptor T cell (CAR T) therapy has attracted attention in recent years for its ability to send difficult-to-treat blood cancers into remission—even curing cancer for a minority. Now, research suggests that the therapy could potentially be adapted to treat a rare autoimmune disease called myasthenia gravis (Granit et al., 2023). The small clinical trial delivered a favorable safety profile and may even cause a long-lasting reduction in symptoms. A larger-scale randomized control study is in progress and anticipated to finish around December of this year (Cartesian Therapeutics, 2023a; Cartesian Therapeutics, 2023c;). What is Myasthenia Gravis?

Myasthenia gravis is a rare, lifelong autoimmune disease estimated to impact 20 out of every 100,000 Americans (Cea et al., 2013). As with other autoimmune diseases, the immune system mistakenly attacks the body’s tissues. Myasthenia gravis is partly mediated by autoantibodies damaging communication between the neurons and the muscles, in particular. The disruption prevents the skeletal muscles which control the eyes, face, arms, legs and more from contracting as they should. This mix of Latin and Greek words translates to “grave muscle weakness.” As the name states, people with myasthenia gravis lose control of certain muscles, manifesting tell-tale signs that include drooping eyes, unstable walking, as well as trouble talking and swallowing. Notably, physical activity worsens symptoms while rest usually improves them. Symptom management will usually entail

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medication to improve muscle weakness and suppress the immune symptom (Alhaidar et al., 2022). CAR T Therapy for Cancer vs Autoimmune Disease CAR T therapy is a novel cell-based treatment which extracts and genetically modifies a patient’s immune cells to fight their cancer. Synthetic DNA is delivered to a set of immune cells called cytotoxic “killer” T cells. The DNA encodes a receptor which merges the T cell’s natural ability to destroy cancerous cells with honing abilities associated with antibodies. These permanently modified T cells become powerful, but hard to control, cancer killers upon infusion. This model has yielded optimistic and long-lasting results for many patients with certain lymphomas, leukemias, and multiple myeloma. Tantalized by this early success, researchers hope to tweak the platform to treat other illnesses, too. Examples of other efforts currently underway include CAR T therapy for lupus, cardiac fibrosis, and rheumatoid arthritis.

Autoimmune Context Despite its promise, traditional CAR T therapy for cancer carries risks that are particularly unfavorable for people with myasthenia gravis. Conventional CAR T cells cannot be controlled once infused. The engineered cells often overstimulate the immune system, causing two common yet potentially fatal adverse events: cytokine release syndrome (CRS) and neurotoxicity. This is disadvantageous to people with myasthenia gravis, who already have an overactive immune system and neurological symptoms; these effects can exacerbate already present complications. 118

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Additionally, preparatory chemotherapy is usually required before an infusion of conventional CAR T cells. The short course of chemotherapy drugs destroys some immune cells to make room for the infused CAR T cells to proliferate later on. Further immunosuppression on top of already prescribed medication for myasthenia gravis could lead to an increased risk of infections and imbalance an already delicate immune system.

Descartes-08: CAR T Therapy for Myasthenia Gravis In their study, Granit et al. turn to an alternative and temporary CAR T therapy called Descartes-08 to treat patients with myasthenia gravis. The therapy was originally designed for multiple myeloma patients but was later translated (Haseltine, 2023d). This is possible because both diseases share a potential therapeutic target: B cell maturation antigen (BCMA) found on the surface of mature plasma cells. Cancerous plasma cells drive myeloma progression, and pathogenic plasma cells produce toxic antibodies that contribute to myasthenia gravis. The most important feature, however, is the therapy’s half-life. It relies on synthetic mRNA, an inherently unstable molecule, to deliver the genetic information needed to encode the chimeric receptor. Unlike DNA, this information is not integrated into the cell genome. The synthetic mRNA naturally degrades in the T cell after a week, and receptor expression follows. Theoretically, mRNA-based CAR T cells could mitigate several risks associated with traditional CAR T therapy. Each infusion of T cells may elicit a weaker response, but they can be controlled through dose repetition; the slow and gradual introduction of T cells should prevent immune system overload. Likewise, 119

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lymphodepletion would not be needed to make space for these CAR T cells. Study Details The promising design needed to be tested. The researchers in this study sought to determine how safe the therapy was, and what dose would be ideal for treatment. To accomplish this, 14 participants with generalized myasthenia gravis were recruited. All relied on immunosuppression drugs for their condition. Symptoms were graded from a scale of 0 to 36, and all participants had a score of six or higher. They did not undergo a lymphodepletion regime, and most continued their previous medication course throughout the study (pyridostigmine for muscle weakness, and corticosteroids to suppress the immune system). The maximum tolerated dose was determined by giving three participants an escalating dose of mRNA-CAR T cells once a week, for three weeks. Once established, the other eleven participants were sorted into three groups with different treatment schedules: 1. Two infusions weekly for three weeks, 2. One infusion weekly for six weeks, or 3. One infusion per month, for six months.

The intravenous infusion lasted 15 to 30 minutes each time, and the patients were monitored up to an hour afterwards. Evaluations occurred on weeks 8, 12, 16 and 20, and then on months 6, 9 and 12. Changes in symptoms were measured using four different and validated scales for measuring myasthenia gravis severity.

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Encouraging Results Interestingly, the therapy did not produce any dose-limiting toxicities or treatment-related serious adverse events. As a result, the highest tested CAR T cell dose became the maximum tolerated dose by default. One infusion per week, for six weeks, was established as the ideal dosing schedule. Participants in this group experienced the most significant and most prolonged decrease in disease severity; some experienced minimal symptom expression even months after the initial dosing regimen. It is encouraging to see that symptom reduction persisted up to 12 months for certain individuals. The CAR T therapy also demonstrated a favorable safety profile. None of the patients experienced cytokine release syndrome or neurotoxicity, the two most common adverse effects associated with traditional CAR T therapy. The adverse events that did occur were resolved within 24 hours of the infusion. This included headache, nausea, vomiting and fever. Future Implications CAR T therapy is a needed alternative for many patients who do not respond to current cancer treatments. The platform, with certain adjustments, could also provide a safe and lasting reduction in symptoms for people who struggle with myasthenia gravis. The ability to target pathogenic plasma cells is especially novel in the realm of myasthenia gravis treatment. The authors posit that Descartes-08 may be most useful as an infrequent, as-needed treatment. It is early days for mRNA-based CAR T therapy to treat rare autoimmune diseases. This study demonstrates the promise for this 121

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prototypical therapy to accomplish what other treatments could not. It will be crucial to see how the therapy performs in the larger, randomized control trial currently in progress.

This article originally appeared in Forbes on July 7, 2023, and can be read online here: Early Success: mRNA & CAR T Therapy To Treat Rare Autoimmune Disease Myasthenia Gravis

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CHAPTER III

NEW WAVE CAR T THERAPY: WORKS IN PROGRESS

Researchers Control Cancer Treatment With New Innovation: CAR T Switch(blade)

ere we describe a recent research advancement that fine-tunes CAR T therapy.

People with treatment-resistant blood cancers turn to CAR T therapy, a recent medical innovation, for long-sought respite but can suffer potentially fatal side effects. Early study results from Scripps Research shine light on a possible solution: controlling the cells with a molecular switch (Calibr reports, 2022). With this innovation CAR T cells can activate or deactivate as needed, simultaneously improving the therapy’s safety and versatility. CAR T Therapy and Toxic Side Effects Chimeric Antigen Receptor T cell (CAR T) therapy entails extracting, modifying and increasing one’s own immune cells to counter cancer. While white blood cells cannot effectively attack cancer cells on their own, with genetic modification these T cells obtain a new receptor that can target antigen CD19, a biological tag found on the surface of cancerous and noncancerous B cells. The modified T cells, primed with new chimeric receptors and expanded to large numbers, can then bind and kill cancer cells once

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infused into the body. Figure 1 describes the design of the CAR T cell in more detail.

FIGURE 1: Typical CAR T design contains an antigen recognizing domain composed of a single chain variable fragment, an antibody-derived component which targets CD19 or BCMA on the surface of B cells. The inner cell is composed of co-stimulatory molecules which help the T cells persist in the blood and a CD3 protein subunit which stabilizes and transmits T cell activation signals. SOURCE: From Interaction between anti-CD19 CAR-T cell receptor and CD19 antigen-presenting tumor cell by Britten, O., Ragusa, D., Tosi, S., & Mostafa Kamel, Y. (2019). https://www.mdpi.com/2073-4409/8/11/1341#

The beauty of CAR T therapy lies in its ability to treat difficult blood cancers such as lymphoma, leukemia and multiple myeloma. These B cell cancers become resistant or unresponsive to previous lines of treatment—typically chemotherapy, targeted drug therapy, or radiation therapy. When these options are exhausted, CAR T cells attack the cancer anew and can even linger in the body to provide longer-term protection.

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#1 Major Side Effect: Cytokine Release Syndrome (CRS) This health innovation can come with a price. Almost all CAR T patients experience a side effect known as cytokine release syndrome (CRS) or cytokine storm syndrome (CSS). As a result of CAR T cells continually stimulating the immune system, white blood cells may release inflammatory chemicals called cytokines. The cytokines can activate other white blood cells and perpetuate a cycle of inflammation. The widespread inflammation manifests a gamut of symptoms ranging from mild to life-threatening. Mild to moderate symptoms include fluctuating fever, fatigue and muscle/joint pain. More severe cases experience low blood pressure and oxygen levels which can result in organ failure and death. Cytokine release syndrome can usually be reversed within five to 17 days with treatments such as antihistamines, oxygen therapy or immunosuppressive medicines as needed.

#2 Major Side Effect: Neurotoxicity CAR T therapy can also cause neurotoxic effects alongside cytokine release syndrome. Referred to as immune effector cell-associated neurotoxicity syndrome (ICANS), this potentially life-threatening complication impacts cognitive function—likely due to cytokines disrupting the blood-brain barrier. Common symptoms include confusion, tremors, and hallucinations. Symptoms can escalate, albeit more rarely, to delirium, seizure or coma. Supportive care can resolve these neurotoxic side effects within 21 days of CAR T therapy.

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Why and How to Make the “Switch” Although the negative effects of CAR T therapy can be reversed, the risk of fatality raises questions on possible ways to control the treatment. If CAR T cells could pause or mobilize when prompted, this could prevent complications from worsening; once side effects have stabilized, cancer-fighting activity could restart again.

CAR T “Switch” Design Akin to a switchblade, researchers have developed a method to manipulate CAR T cells on and off to produce more precise results by using an antibody switch. Traditional CAR T therapy alters T cells to detect the cancer cell directly. Rather than the cancer cell, switchable CAR T cells (sCAR T) target the antibody switch. As seen in Figure 3, the switch acts as a bridge, binding to the switchable CAR T cell on one side and the cancer cell on the other to trigger a cytotoxic response. Figure 3 highlights the deviations from typical CAR T design. Researchers create the molecular switch by grafting a region called a peptide neoepitope (PNE) onto an anti-CD19 antibody clone; the protein neoepitope does not naturally occur in humans, making it a clear target for the therapy. Unlike traditional CAR T cells, sCAR T cells do not target CD19 but the peptide neoepitope on the switch. The desired response is therefore controlled in vivo by the presence and dosage of the switch.

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FIGURE 2: Schematic, antibody acts as activating bridge between sCAR T cell and cancer cell. CREDIT: Access Health International

Results Calibr, the nonprofit translational research institute of Scripps Research, recently reported preliminary results from their sCAR T clinical trial. The Phase I study tested the safety and optimal dosage of their sCAR T treatment on nine patients with B cell malignancies. The participants underwent a median of five prior treatments for their condition.

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Of the nine participants, seven responded to the therapy (78%) and six experienced a complete response (67%), meaning all detectable signs of cancer disappeared. A single infusion of CAR T cells and a single injection of molecular switches elicited most responses in participants, while further switch injections hinted at deepening responses over time. The lower dosing seemed to achieve promising early results, with some doses reaching higher amounts of CAR T cell in the peripheral blood over the first 90 days than other approved CAR T therapies. Importantly, the switchable therapy successfully minimized adverse side effects. Cytokine release syndrome and neurotoxicity associated with CAR T therapy typically resolves within five to 17 days when treated traditionally. However, by holding or reducing the switch dosage after observing early signs of side effects, the CAR T cells could essentially halt their activity; as a result, the patients experienced side effects for a shorter duration of time (between two to three days). The Future of sCAR T The future of CAR T therapy continues to brighten. The early study result from Scripps Research suggests that switchable CAR T cells are not only safe to use for patients with B cell cancers, but comparatively safer and more effective than some CAR T therapies currently on the market. This also bolsters confidence in the universal molecular switch design. Using this basis, CAR T therapy could likely target any therapeutic antigen by altering the molecular switch. Further down the line, perhaps mRNA and sCAR T technology could combine to create the most ideal form of CAR T therapy—one that forgoes the lab entirely to create a potent and controllable “living drug” inside the body. 128

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This article originally appeared in Forbes on November 15, 2022, and can be read online here: Researchers Control Cancer Treatment With New Innovation: CAR T Switch(blade)

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CAR T Cell-Like Therapy To Treat T Cell Leukemia (T-ALL) As part of our series in cellular therapy, we previously described how CAR T therapy for treatment of B cell leukemia achieved early success in the field (Haseltine, 2022a). T cell leukemia, however, engages a different part of the immune system and cannot be treated using similar cellular therapies. There is a need for more therapeutic options, as around 15% percent of people with T cell leukemia experience treatment-resistant relapse. Here we discuss a study published in the journal BMJ investigates a promising new approach inspired by CAR T therapy on mouse models (JiménezReinoso et al., 2022). T Cell Acute Lymphoblastic Leukemia (T-ALL) T Cell Acute Lymphoblastic Leukemia (T-ALL) comprises up to 15% of acute leukemia cases in children and up to 25% of cases in adults. The condition arises when DNA in a bone marrow stem cell mutates spontaneously. Immature forms of T cells, important white blood cells for immunity, then begin to grow and divide at alarming rates. The abnormal, undeveloped cells ultimately crowd out other healthy white blood cells and impede normal function. Chemotherapy and stem cell transplants drastically improved prognoses for people with this disease in recent years; the five-year event-free survival rate now hovers around 85%. However, if a treatment fails, oftentimes it cannot be repeated due to treatment resistance. This leaves few remaining options for people with relapsed and resistant leukemia. In their study, Jiménez-Reinoso et al. pursue a possible alternative evolved from CAR T therapy.

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CAR T Therapy Limitations Cancer researchers in Barcelona and Madrid, Spain collaborated to determine if a derivative of CAR T therapy can potentially treat relapsed and refractory forms of T cell acute leukemia. Important to this research is an understanding of CAR T therapy, and why it cannot be applied to T cell acute leukemia without alteration. In CAR T therapy, a practitioner extracts a patient’s T cells, genetically modifies the cells to express new machinery, and infuses the cells back into the patient’s body to fight the cancer. An infused Chimeric Antigen Receptor T cell can specifically recognize and kill all B cells—cancerous or healthy—by identifying a particular biological tag (antigen) found on their cell surface (see Figure 1). This approach does not translate well for T cell acute leukemia. The major issue lies with the antigen target. An antigen target that attacks all immune cells in the lineage—as CAR T cell therapy often does for all B cells—would cause life-threatening immunodeficiency. The CAR T cells would attack cancerous T cells, cancer-fighting CAR T cells and healthy T cells alike. This is not a problem for patients who receive CAR T therapy to treat B cell cancers; they receive a supply of antibodies to compensate for the diminished amount of B cells in their body. However, a similar therapy to replete T cells does not yet exist.

A Novel Target: Antigen CD1a A different biological tag is needed—one rarely found on healthy cells, but consistently found on malignant T cells. Jiménez-Reinoso et al. previously made CAR T cells which eliminated cells with an antigen called CD1a. This tag is found in many cancerous T cells and scarcely on any others, making it an ideal therapeutic target. 131

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The team used this key discovery to derive a more precise cell therapy.

FIGURE 1: Illustration of CAR T cell design. Most important here is the singlechain variable fragment (scFV), which is derived from antibodies to give the T cell improved targeting ability. The fragment detects antigens on the surface of other cells. An anti-CD19+ antibody fragment is used to eliminate B cells for cancers such as B Cell Acute Lymphoblastic Leukemia (B-ALL). The researchers here altered the design to target cortical T cell leukemia cells using an anti-CD1a antibody fragment. SOURCE: Access Health International

The Next Stage: STAb Cell Therapy A novel target acquired, the researchers created a new therapy: STAb-T immunotherapy, short for Secreting T cell-redirecting Antibodies. This method modifies T cells to secrete special antibodies. Illustrated in Figure 2, the antibodies target CD1a (the tumor 132

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antigen) on one side and CD3 (a common antigen found on T cells) on the other. The team believed that the resulting antibody bridge (see Figure 3) would encourage healthy T cells nearby to target the tumor, as well. This bystander effect doesn't occur with CAR T cells, which alone engage in tumor elimination.

Results When tested in vitro and in vivo animal models, the antibodysecreting T cells performed comparatively—if not better—than CAR T therapy. The team discovered that the bispecific antibodies can successfully bind to neighboring T cells. In cell culture, the bystander effect observed contributed to the stronger killing power observed for STAb cells compared to CAR T cells. The authors note that STAb cells achieve this superior cytotoxicity without releasing as many immune chemicals. This suggests that STAb cells have a reduced risk of a common side effect associated with CAR T therapy called cytokine release syndrome, and thus pose as a safer alternative. To test the in vivo effect short term the team injected T cell leukemia cells into mice and followed three days later with normal and activated T cells, CAR T cells, or STAb cells. The mice with normal T cells experienced unhampered disease progression, while the mice treated with CAR T and STAb cells displayed similarly controlled levels of leukemia. CAR T cells and STAb cells performed comparably in a longer, two week model as well; the engineered cells could remove and reduce tumor burden when initially challenged and when the disease is artificially relapsed. STAb T cells may hold a technical advantage over CAR T therapy. In vitro, STAb cells demonstrated killing capacity even at low ratios; 133

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the CAR T cells, in contrast, required higher concentrations to elicit significant cytotoxicity. STAb cells may require lower doses to achieve effective results—a factor that could reduce manufacturing costs and allow the therapy to reach more patients once clinically translated.

FIGURE 3: The STAb T cell is designed to produce a bispecific antibody. The antibody binds to antigen CD1a on tumor cells and antigen CD3 on nearby T cells like a bridge. This allows unmodified T cells to contribute to antitumor activity. SOURCE: Access Health International

Looking Forward In their study, Jiménez-Reinoso et al. demonstrate the promise of STAb-secreting T cells as an alternative to CAR T therapy. Clinical 134

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adaptation would provide a much-needed therapeutic alternative for those struggling with treatment-resistant T cell leukemia. The paper also exemplifies how bispecific antibodies can bring additional precision to CAR T and similar cell therapies. Another innovative use of antibody bridges can be found here: Researchers Control Cancer Treatment With New Innovation: CAR T Switch(blade).

This article originally appeared in Forbes on February 13, 2023, and can be read online here: CAR T Cell-Like Therapy To Treat T Cell Leukemia (T-ALL)

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Treating Troubling Tumors: CAR T Therapy For Aggressive Childhood T Cell Leukemia In recent years, CAR T therapy has become an established treatment for patients suffering from blood cancers derived from B cells; this includes B cell lymphomas, leukemias and multiple myeloma. However, the therapy has yet to achieve similar success for T cell-derived cancers. A small study published in the New England Journal of Medicine (NEJM) suggests that highly precise gene editing could be used to confront this therapeutic gap (Chiesa et al., 2023). Their method demonstrates proof-of-concept and opens doors for CAR T therapy to potentially treat children with aggressive T cell leukemia. T Cell Acute Lymphoblastic Leukemia T cell acute lymphoblastic leukemia (T-ALL) is an aggressive, rapidly forming blood cancer borne from white blood cells called T cells. Healthy T cells normally target abnormal or infected cells and aid other immune cells; with this kind of cancer, the immature T cells overcrowd and weaken the immune system. People with T cell leukemia often experience persistent infections, bruising, and symptoms such as anemia and bone pain. Treatment usually includes chemotherapy and, if that fails, stem cell transplantation. These measures successfully extend survival, but unfortunately relapse can still occur afterwards. When this happens, previous lines of treatment lose their efficacy (Raetz & Teachey, 2016). Chimeric Antigen Receptor T Cell (CAR T) therapy, a recent medical innovation, can achieve durable remission for patients with

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relapsed B cell-derived cancers. With modification, perhaps CAR T therapy could be used to treat T cell cancer patients, too. CAR T Therapy Challenges for T Cell Leukemia The current standard for CAR T therapy involves extracting a patient’s T cells, genetically modifying them to improve their cancer detection, and then returning the strengthened cells to the patient to wipe out their cancer. The cells identify a specific biological tag, or antigen, found on the surface of a lineage of white blood cells; this wipes out cancerous and healthy cells in the process. Although effective for patients with B cell-derived cancers, this unabashed killing poses challenges for patients with T cell leukemia. CAR T therapy relies on T cells. Therefore, a CAR T cell designed to attack T cell antigens would inevitably cause CAR T cell fratricide, a phenomenon where CAR T cells attack each other. This would decrease the efficacy of the treatment. Additionally, the CAR T cells may attack too many healthy T cells and lead to T cell deficiency. This deficiency is hard to supplement and compromises the patient’s ability to fight off viral and fungal infections (Vaillant & Qurie, 2022). Adapting CAR T Therapy for T Cell Leukemia Researchers from the University College of London and National Health Services Trust collaborated to confront these unique CAR T therapy challenges. They used healthy donor T cells and bioengineered them to express chimeric receptors against a T cell antigen called CD7. Unlike traditional CAR T therapy, this procedure does not alter the patient’s own immune cells.

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Most interestingly, the team used CRISPR-Cas9 gene editing to simultaneously and precisely target three genes in the donated T cells. The genes were base-edited, a process which converts a single base of DNA code into another base (ie. C to T, cytosine changed to thymine). This change is read as a stop codon and turns off the gene. The first gene change involved deactivating T cell receptor genes such as TRBC1 and TRBC2. Disrupting T cell receptor expression means that host T cells will not be able to find and destroy the CAR T cells. This should prevent the donor CAR T cells from being rejected upon infusion as a tissue transplant would, a phenomenon commonly known as graft-versus-host disease (GVHD). The second gene change terminated CD7 antigen expression on the T cells. Without CD7 on their cell surface, the CAR T cells should be able to ignore each other. Finally, a gene called CD52 was silenced to prevent T cell depletion. Prospective CAR T therapy candidates usually undergo preparatory chemotherapy to kill some immune cells and make space for the CAR T cell infusion to proliferate. This gene change helps the CAR T cell evade alemtuzumab, a chemotherapy drug that targets CD52. Results Demonstrate Proof-of-Concept The researchers enrolled three children with relapsed leukemia in their study. Each child underwent a chemotherapy regime of alemtuzumab, fludarabine, and cyclophosphamide before they received a single infusion of ready-made CAR T cells. A follow-up was made 28 days after the initial infusion to determine the therapy’s safety and measure its ability to achieve remission. 138

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The CAR T therapy sent two patients into remission—a result consistent with CAR T therapy for B cell derived cancers. The third patient responded to the therapy, but also suffered fatal adverse effects. The first patient, a 14-year-old girl, had complete remission of cancer signs at Day 28. Afterwards, she received a stem cell transplant from her original donor and continued to maintain cancer remission. Similarly, one 15-year-old boy successfully went into remission and continued on to receive stem cell transplantation. The last patient, a 13-year-old boy, had a starkly different experience. This patient responded to the therapy, but later developed fatal fungal lung disease on top of already pre-existing complications.

Significant Adverse Events The CAR T therapy elicited significant adverse effects—some align with other CAR T therapies and others may be exacerbated by T cell leukemia challenges. All patients experienced symptoms of cytokine release syndrome (CRS) within a week of the infusion. This is a common yet potentially fatal effect of CAR T therapy. Neurotoxicity, another known CAR T effect, was also observed. Patients with T cell leukemia may be at higher risk for infections caused by T cell deficiency. All three children had low white blood cell counts; this likely left them vulnerable to viral (cytomegalovirus) reactivations and fungal infections. Overlapping infections can burden the body severely, as seen with the third patient. Previous studies suggest this deficiency may be attributed to the CAR T cells’ anti-CD7 activity. 139

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It would be useful to control the CAR T cells before they triggered adverse events. The authors mention a possible solution: integrating suicide genes into the CAR T cells. These genes could be activated externally, allowing the engineered cells to self-destruct before they become overly dangerous. Toxic genes could also be used to terminate CAR T cells that mutate and turn cancerous. These cells would otherwise grow unchecked by the immune system due to reduced T cell receptor expression. Future Implications There may be hope yet for patients with difficult-to-treat T cell leukemia. CAR T therapy, an established treatment for certain B cell-derived cancers, could potentially achieve remission—but not without substantial changes. The universal CAR T cells in this study are designed to prevent host rejection, T cell deficiency and CAR T cell fratricide. The infusion achieved remission for two out of three patients, but not without significant risks. It will be compelling to see how this ready-made concept performs with further investigation and a larger study cohort.

This article originally appeared in Forbes on July 18, 2023, and can be read online here: Treating Troubling Tumors: CAR T Therapy For Aggressive Childhood T Cell Leukemia

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CRISPR Technology To Simplify And Enhance CAR T Cancer Treatment (Part 1)

Here we describe the integration of CRISPR gene-editing technology to improve CAR T therapy design. Researchers find that combining novel gene-editing CRISPR technology with CAR T therapy could simplify and improve CAR T therapy in one fell swoop. Traditional CAR T Therapy A remarkable feat in cancer care, today people with difficult-to-treat blood cancers can receive CAR T therapy, a personalized “drug” made from their own immune cells. Chimeric Antigen Receptor T cell (CAR T) therapy relies on extracting a patient's immune cells and modifying them in the lab with a new, synthetic receptor. The new receptor allows the white blood cell to target and destroy cancer cells once re-infused back in the bloodstream. Evoking the patched image of a mythical chimera, these receptors merge signaling machinery typical of a T cell with an antibody-derived detection region to create a powerful “living drug” which continually expands inside the body. Figure 1 highlights the basic design of a CAR T cell, while Figure 2 illustrates the step-by-step process in more depth.

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FIGURE 1: A T cell and a chimeric antigen receptor combine to create a CAR T cell. Most approved CAR T therapies employ a similar CAR design. The receptor usually consists of an antigen-recognition domain (scFv), a costimulatory molecule(s) to help the cell expand and persist inside the body, and a CD3 T cell signaling subunit to activate the cell upon binding. SOURCE: Access Health International

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FIGURE 2: In CAR T therapy, 1) T cells are first isolated from the patient’s collected blood. 2) In the lab, these T cells are genetically modified to equip the desired chimeric antigen receptor. 3) Scientists multiply or expand the number of CAR T cells to millions before 4) the lymphodepleted patient receives the CAR T infusion. 5) The modified T cells circulate the blood, targeting and eliminating encountered cancer cells. SOURCE: From CAR T Cells: Engineering immune cells to treat cancer. National Cancer Institute (2022). https://www.cancer.gov/aboutcancer/treatment/research/car-t-cells

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Gene Editing with Viral Vectors To craft CAR T cells, the very genes of the T cells must be altered to express the chimeric antigen receptor. Gene editing, therefore, provides the foundation for the therapy. Integrating CAR genes normally requires the use of a viral vector. Retroviruses in particular have the unique ability to insert and meld their own foreign genetic material into human cells permanently. This allows viruses to use host machinery to produce viral proteins. Scientists have repurposed this strength to deliver CAR genes into T cells. An inactivated form of the virus is filled with genetic material which encodes for CAR. The desired genes are then transferred from the virus into the T cells through a process called transduction. As if reading biological instructions, the T cell uses the genetic information to construct the receptor before expressing it onto the cell surface.

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The industry standard may depend on viral vectors, but the procedure lacks in some aspects. This stage of the CAR T process is the most time-consuming and expensive; it can take a year or longer to produce a batch of viral vectors, and can cost up to $50,000 per dose. For these reasons researchers now hope to turn to CRISPR technology, a recent scientific breakthrough in gene editing, to resolve these issues. Enter CRISPR/Cas9 Gene Editing CRISPR originates from organisms such as bacteria and plays a major role in their defense. The acronym CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats—in essence, they are short, repeating DNA sequences that read the same forwards or backwards, similarly to words such as “MADAM” or “DEED.” Sandwiched between these repeats are protospacers, a genetic history of viruses the bacteria encounters (see Figure 4). When a virus tries to insert its genetic information into the bacteria, the bacteria can recognize the sequence from its protospacer catalog. The bacteria transcribes the protospacer DNA into RNA; this RNA guides enzymes such as Cas9 to the viral DNA to cut and deactivate it. The same CRISPR/Cas9 interface can also snip human DNA. As seen in Figure 6, an RNA guide can be made to cut DNA at a

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specific site. The broken DNA, eager to repair itself, can easily adopt a new DNA sequence in that location. Translating this concept to CAR T therapy, researchers could modify T cell DNA directly to express a new receptor. Synthesizing an RNA guide is cheaper and more efficient than cultivating retroviral vectors. If successful, CRISPR could simply solve two major drawbacks associated with CAR T therapy: price and time-todelivery.

FIGURE 4: CRISPR consists of spacers—unique, virus-derived DNA sequences—sandwiched between short, repeating sequences of DNA. SOURCE: Access Health International

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FIGURE 5: CRISPR/Cas9 editing mechanism. Researchers leverage CRISPR/Cas9 to cut and insert genes at a desired site on the genome. The guide RNA directs the Cas9 enzyme to snip the DNA at a specific location. SOURCE: From Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy by Rasul, M. F. et al., (2022). https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943021-01487-4

Conclusion CAR T therapy, although a triumph of human engineering in its own regard, still has room for improvement. There is potential to propel CAR T design forward by integrating contemporary innovations such as CRISPR/Cas9 technology. Although this 147

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method still requires T cell manipulation outside the body, this change could streamline the process while becoming more accessible. The most critical step now is to test the feasibility of this concept. The next installment in the series will explore the latest clinical results from PACT Pharma and the University of California, Los Angeles on their CRISPR/CAR T dual interface.

This article originally appeared in Forbes on November 15, 2022, and can be read online here: CRISPR Technology To Simplify And Enhance CAR T Cancer Treatment

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Teaming Up Two Biotech Winners to Fight Cancer: CRISPR and CAR T (Part 2)

Here we describe early clinical trial results on combination CRISPR and CAR T therapy, a sequel to an earlier, introductory piece. CAR T therapy can treat blood cancers by inserting new genes into a patient’s own immune cells using viruses. Early clinical trial results present an alternative that forgoes viral gene transfer: CRISPR technology. Such integration of CRISPR gene editing could improve the precision, speed and cost-effectiveness of CAR T cell production. In addition, researchers hope CRISPR will broaden CAR T therapy applications from blood cancers to solid tumors, which the engineered T cells notoriously have failed to target. Inserting Genes into CAR T Cells Chimeric Antigen Receptor T cell (CAR T) therapy genetically alters a patient’s T cells to recognize cancer cells and subsequently kill them. This engineered recognition relies on hybrid T cell receptors with antibody components to detect antigens, or biological tags, found on the surface of cancer cells (see Figure 1).

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FIGURE 1: Illustration of a chimeric antigen receptor. The structure utilizes an antibody-derived domain to detect specific antigens, all while leveraging a T cell CD3ζ complex for its signal machinery. SOURCE: Access Health International

Researchers typically incorporate hybrid receptor genes into a CAR T cell via viral gene insertion. Despite its regard as a staple in cell therapy, retroviral gene transfer comes with several drawbacks. Viral vector manufacturing is expensive and time-consuming. The method lacks precision and could potentially allow an unwanted gene entry. Perhaps most limiting, it cannot be personalized to detect uncommon antigens. For this reason, all approved CAR T therapies in circulation target blood cancers that share a common antigen (usually CD19 or BCMA) rather than solid tumors, which greatly vary in antigen presentation. Standardizing a new means to insert genes would improve the accessibility, efficiency and usage of CAR T therapy. 150

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Innovating with CRISPR Gene Editing In their Phase I clinical trial, the researchers at PACT Pharma and the University of California, Los Angeles explore the possibility of a different type of CAR T therapy—one that creates a hybrid receptor with CRISPR gene editing. With CRISPR, the team selectively removed native T cell receptor genes and replaced them with new, cancer-fighting alternatives. The researchers began by searching and isolating a novel T cell receptor from the patient’s own immune system. First, they screened the patients by sequencing DNA from healthy blood samples and tumor biopsies; this step identified mutations that the tumor cells share but cannot be found in normal tissue. Algorithms then predicted which antigens would be present on the tumor. Next, the team copied the antigens and mixed them with different versions of HLA, a type of molecule needed to present antigens to T cells. This process revealed specific T cells which could react to this particular combination of antigen-HLA. Researchers copied up to three of the highly personalized receptor genes to be integrated into the T cells using CRISPR/Cas9. Figure 2 illustrates the subsequent process. The CRISPR/Cas9 interface knocked out two T cell receptor genes, TRCα and TRCβ (see Figure 3), and replaced them with three new receptor genes in a single step—decidedly more efficient than sourcing and cultivating retroviruses for gene transfer, as is currently standard in CAR T therapy. The researchers multiplied the T cells to great numbers. Finally, the patients underwent lymphodepletion chemotherapy before

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receiving up to three doses of their personalized CRISPR/CAR T cell infusion.

FIGURE 2: Overview of the CAR T therapy using CRISPR technology. Genes for two native T cell receptors, TRCα and TRCβ, are removed and replaced with genes for new and personalized t cell receptors. SOURCE: Adapted from CAR T Cells: Engineering immune cells to treat cancer. National Cancer Institute (2022). https://www.cancer.gov/aboutcancer/treatment/research/car-t-cells

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FIGURE 3: The structure of a T cell receptor (TCR), composed mainly of dimer TCRα and TCRβ and accompanied by a CD3ζ protein complex. Note the Human Leukocyte Antigen (HLA) molecule present on the antigen presenting cell, denoted in purple; this structure is necessary for the T cell receptor to receptor to recognize the antigen peptide. SOURCE: Access Health International

Results The researchers assessed the safety and dosage of combination CRISPR/CAR T therapy to treat 16 people with various kinds of solid tumors, including breast and lung tumors. All of the patients

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experienced drug resistance, and had already received five or more prior lines of therapy. The CRISPR/CAR T infusion did not prove overly dangerous. All the patients experienced typical side effects of lymphodepleting chemotherapy. One patient in particular experienced mild cytokine release—an expected side effect of CAR T therapy. Another experienced severe encephalitis. The combination CRISPR/CAR T therapy did not cure any patients. Through biopsies, the team found that the CAR T cells successfully multiplied and traveled into the tumors of eight participants. Four weeks after infusion, five participants had stable disease, meaning their condition did not change. The other eleven patients’ cancer worsened. Future Implications Gene integration via viral vectors establish the current standard for CAR T therapy, but could soon be replaced with cheaper and more efficient CRISPR gene editing. The clinical results demonstrate that tumor-specific CAR T cells can be made and used safely; that these CRISPR-edited immune cells can recognize solid tumor masses; and that this method holds potential to be effective against drug-resistant solid tumors. While this realm of research still warrants room for improvement, especially with more uniform tumors (ex: lung tumors only), this foundation sets an excellent springboard for advancements to come.

This article originally appeared in Forbes on December 6, 2022, and can be read online here: Teaming Up Two Biotech Winners to Fight Cancer: CRISPR and CAR T 154

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CAR T Cells Derived From Stem Cells Open Door To Universal Donor Cell Lines What if an effective cancer treatment became cheaper and easier to produce? One study published in the journal Nature Biomedical Engineering builds on this vision to improve medical innovation CAR T therapy by using stem cells and extensive gene editing (Wang et al., 2021). The results demonstrate that the experimental CAR T cells—derived from stem cell donors instead of patients— can be used to effectively treat mice models of cancer. Looking forward, clinical translation of this model could lower the cost, time and labor barriers that make CAR T therapy prohibitive for most. The High Cost of CAR T Therapy Patients who undergo Chimeric Antigen Receptor T cell (CAR T) therapy receive a treatment that is personalized to their cancer. The process involves extracting a patient’s immune cells, altering them in the lab to express a new receptor, and reinfusing them to fight their difficult-to-treat cancer. The procedure can achieve a longsought reduction in symptoms or even remission, but this success comes at a high price—up to $475,000 per infusion, depending on the product (Novartis, 2017). This price takes into account all of the time and labor required to genetically modify each patient’s cells for the treatment. A ready-made CAR T product would theoretically be easier to produce at a large scale; this would bypass the need to create a custom batch of CAR T cells for each patient. An example effort to create an “off-the-shelf” version of CAR T therapy uses healthy donor T cells as the basis for the therapy. Here, we describe an

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alternative method that relies on stem cell-grown immune cells— not patient cells or donor T cells—to establish its universal source. Testing a Solution: Stem Cell-Derived CAR T Cells In their study, researchers Wang et al. sought to grow a universal source of cytotoxic T cells to use in cell therapies such as CAR T. These cells would be allogeneic, or not derived from the patient. The team turned to induced pluripotent stem cells in particular to accomplish this task. Induced pluripotent stem cells (iPSC) are adult cells that have been reprogrammed to revert into an immature state. Similar to embryonic stem cells, induced pluripotent cells can thus develop into different cell types. This unique ability means that, in theory, an unlimited number of antigen-specific T cells could be grown from these cells. Expanding these cells can also create a sea of clones with the same genetic characteristics. This is ideal for testing genetic changes; researchers can make multiple clones and reliably compare the effects caused with each gene edit.

Confronting Host Rejection The use of stem cells always incurs the risk of host rejection. This phenomenon known as Graft-vs-Host Disease (GvHD) occurs when a patient’s body does not recognize a cell transplant as its own. The immune system may engage immune cells such as cytotoxic T cells, helper T cells, or natural killer cells to eliminate the graft. To overcome this, several genes were knocked out from the stem cellderived T cells using CRISPR-Cas9 gene editing. First, gene B2M was knocked out to eliminate the expression of a cell surface molecule called human leukocyte antigen I (HLA-I). 156

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This removal prevents host cytotoxic T cells from mounting an unwanted immune response and killing the stem cell T cells. Second, gene CIITA was inactivated to prevent human leukocyte antigen II (HLA-II) expression. Without this antigen present, helper T cells did not target and eliminate the stem cell T cells as they normally would. The first modification could lead to an attack from natural killer cells. To counter this, gene PVR was eliminated. This downregulated expression of a ligand that natural killer cells normally recognize and destroy. With this ligand gone, the stem cell-derived T cells could escape scrutiny. Results: Slowed Tumor Growth The heavily edited T cells were then retrovirally modified to carry different chimeric antigen receptors and tested on mouse models of cancer. Their performance was compared to stem cell-derived T cells that carried a chimeric receptor, but lacked the extensive gene modifications to prevent host rejection. In the first test, the T cells carried an anti-CD19 chimeric receptor. CD19 is a common therapeutic target for conventional CAR T therapies against certain lymphomas and leukemias. The mice here carried either leukemia or lymphoma tumor cells before receiving an injection of edited or unedited CAR T cells. The results show that the heavily gene-edited CAR T cell injection slowed tumor growth and prolonged the survival of the mice in both groups. This suggests that the edited CAR T cells were effective against the tumors, while harmless against normal cells. Next, the T cells were modified to carry an anti-CD20 chimeric receptor. CD20 is mostly used as an experimental target for CAR T 157

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therapy, an alternative to antigen CD19. The mice here carried CD-20-expressing leukemia or lymphoma cells, and were given multiple cycles of either edited or unedited CAR T cells. The edited CAR T cells immediately slowed tumor growth, while the unedited alternative took 12 days to show a similar effect. The delay could be attributed to the host cytotoxic cells expanding to oppose the tumor. After each injection, the edited CAR T cells proved more stable than their unedited counterparts. The authors posit that the body rejected the unedited T cells much more. Overall, the extensively edited CAR T cells survived and inhibited tumor growth in both tests.

Possible Complications The universal donor T cells here are designed to be invisible to the immune system. Notably, such cells may eventually become a source of new tumors and cancers as a result of mutations that arise from uncontrolled cell growth. To analyze this risk, the authors performed whole-genome sequencing on the cytotoxic T cell lines and their parent stem cell lines to determine the possibility of permanent mutations occurring in the coding regions of the genome. The analysis yielded several single nucleotide variations with seven or more mismatched base pairs compared to all three genomic RNA-targeting sequences. Coupled with the fact that mice injected with edited CAR T cells did not develop tumors, it seemed unlikely that unwanted genome mutations would occur. It may also be necessary to integrate inducible suicide genes into the chimeric receptors to further reduce the possibility of developing tumors. This would allow the cells to be intentionally 158

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destroyed if they ever posed a danger to the patient. Examples of inducible toxic genes include molecules such as Fas or Cas9, herpes simplex virus thymidine kinase, and truncated epidermal growth factor receptor (Hashem Boroojerdi et al, 2020). Future Implications This study challenges conventional methods of sourcing cytotoxic T cells. CAR T therapy traditionally uses a patient’s own cells, but this method is costly and resource-intensive. In comparison, induced pluripotent stem cells can be expanded and cloned in a way that patient cells and donor T cells cannot. The authors demonstrate that their stem cell-derived cytotoxic T cells can survive allogeneic transplant and suppress tumor growth in mouse models of cancer. This optimistic combination of stem cell technology and CAR T therapy may bring the cancer treatment closer to a more ideal, ready-made form.

This article originally appeared in Forbes on July 10, 2023, and can be read online here: CAR T Cells Derived From Stem Cells Open Door To Universal Donor Cell Lines

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Synthetic Gene Circuit To Hone CAR T Therapy While CAR T therapy represents one of the most impressive innovations in cancer care, the treatment can cause autoimmunelike side effects. Many have contended with the dilemma of maximizing the therapy’s benefits while minimizing its side effects. A recent advance published in Science shows great promise in improving control of CAR T therapy with the hope to eventually benefit patients (Li, Israni, et al., 2022). Challenges of CAR T therapy Chimeric Antigen Receptor T cell therapy relies on engineering synthetic receptors onto a patient’s immune cells to recognize and eliminate a programmed target. The treatment has proven effective in targeting antigens, or biological tags, found on the surface of some blood cancer cells. Although successful in this regard, the modified T cells cannot be controlled once injected back into the body. This factor, coupled with other obstacles such as its inability to target more than one antigen at once, leaves space to improve the treatment.

Possible Solutions There are several budding research initiatives seeking possible solutions. One previously discussed method of controlling CAR T therapy involves creating CAR T cells that bind to antibody switches. Researchers found that a single infusion of antibody switches could help mitigate the worst of CAR T therapy’s toxic side effects. Although this method could potentially fight solid tumors by altering the antibody target, it has yet to be tested in animal or human models. 160

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A recent study from scientists at Boston University offers an alternative and versatile platform that could transform CAR T therapy implementation. The team turns to synthetic gene circuits for answers and receives encouraging results. A New Alternative: Synthetic Gene Circuits In a gene circuit, the process of turning an input into a desired output can be controlled through the use of a synthetic regulator (see Figure 1). The synthetic regular can, in theory, tailor a cell’s gene expression to produce a desired result. The researchers in this study designed such a circuit using synthetic proteins and small molecule switches. To establish the circuit, the team relied on synthetic versions of proteins called transcription factors. Transcription factors can recognize certain DNA motifs and help convert that DNA into RNA, genetic instructions for protein synthesis. They leverage synthetic zinc fingers (SynZiFTR) in particular due to its compact size and human origin; Figure 2 illustrates the structure. Both factors together allow the protein to move efficiently in human cells while minimizing unwanted side effects. Additionally, several zinc fingers can be joined together to create a structure capable of recognizing potentially unique human DNA sequences in the genome, as seen in Figure 3. This circuit must be controlled using a gene switch. The team experimented with three clinically approved small molecule inducers to accomplish this task. The unique combination of zinc fingers and small molecule inducers allowed genes to be turned “on” with the introduction of the inducer, and turned “off” with its removal. 161

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FIGURE 1: A synthetic gene circuit can alter a network of input signals to produce a custom response. Using this underlying base mechanism, CAR T therapy could be controlled through the use of synthetic zinc fingers to produce a more precise result.

FIGURE 2: Representation of the Cys2His2 zinc finger motif. The zinc ion is represented in green. SOURCE By Splettstoesser, T. (2007) Zinc finger [Image] Wikipedia. https://commons.wikimedia.org/wiki/File:Zinc_finger_rendered.png

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Combining Synthetic Gene Circuits with CAR T Therapy How do these synthetic gene circuits fare when used on CAR T cells in vitro and in vivo? The team investigated this question in several stages using cell and animal models. The researchers first tested the efficacy of a gene circuit that controlled the expression of chimeric antigen receptors. They found that in a xenograft liquid tumor model, the engineered receptor’s expression could indeed be controlled in a drug-dependent manner. The treatment could be reinforced using the same stimuli and secondary infusion of chimeric antigen receptors with a different antigen target, demonstrating the adaptable premise of the platform. The platform produced similar results when tested on blood tumor models in mice, as well. The mice treated with drug inducer or a drug-increasing cocktail could clear the tumor, while those without had high tumor burden. This phase illustrated how gene circuits can be influenced in a drug-dependent manner in living creatures. One of the most fascinating findings of this study is the creation of a dual-switch gene circuit. Researchers crafted a gene circuit which impacted cytokines known to influence cellular proliferation. This cytokine circuit could successfully prime dual-switch CAR T cells in mice with leukemia; a secondary signal to encourage CAR expression sparked their anti-tumor activity. The sequential and synergistic effect of the dual-switch circuit can be seen in Figure 4. This duo reduced tumor burden in mice more successfully than untreated CAR T cells or CAR T cells induced with anti-tumor stimuli only.

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Looking Forward The researchers here show how synthetic gene circuits can feasibly improve CAR T cell proliferation and anti-tumor activity in animal models. This promising platform could prove clinically viable once translated to humans. However, the implications of this study stretch beyond the rapidly growing field of CAR T therapy. The underlying mechanism could be customized to improve other gene and cell therapies, or combined with other powerful technologies such as CRISPR-Cas9 to achieve more bespoke results.

This article originally appeared in Forbes on December 29, 2022, and can be read online here: Synthetic Gene Circuit To Hone CAR T Therapy

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Engineering Cells For Medical Use: Learning The Language Cells Use To Communicate With One Another Cells are the building blocks of life. How cells recognize other cells and external signals can lead to various biological fates, including cell growth, death and mobility. Researchers seek to understand cell-cell communication, reverse engineer it, and ultimately sculpt cell interactions that surpass natural capabilities. Although cell therapies already exist, the future of such cell therapy will likely involve deeper modification of patient’s cells to treat a gamut of diseases and to repair tissues. In a previous article, we reviewed a study that modularly substitutes the extracellular portion of a protein to recognize different ligands; this “reassembled” protein transduces the same signal pathway as long as the transmembrane and intracellular portions are kept intact. Here, we discuss a paper which emphasizes the intracellular portion of the cell instead (Daniels et al., 2022). The researchers from the University of California, San Francisco theoretically recompose the signaling domains of CAR T cells and explore possible impacts on cell-cell communication. Making a Chimeric Antigen Receptor Chimeric Antigen Receptors (CAR) require genetic modification to express new, synthetic components. Figure 1 illustrates the three main regions of a CAR T cell: the antigen-binding domain, the transmembrane domain, and the signaling domain. Scientists often fixate on the binding domain and tailor it for a specific therapeutic target (ex: proteins found on cancer cells). The researchers here, however, focus on the composition of the signaling domain and its influence on CAR T cell performance. 165

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Costimulatory Molecules The signaling domain of a CAR T cell usually contains a CD3ζ T cell receptor (TCR) molecule and any combination of costimulatory molecules. Costimulatory molecules contain multiple signal motifs, or short peptides which bind to specific downstream signaling molecules. These molecules influence T cell signal transduction to varying effects. Two examples include 4-1BB, which can increase T cell memory and persistence, and CD28, which is associated with effective T cell killing but reduced T cell persistence.

FIGURE 1: A chimeric antigen receptor design can be split into three main regions: an antigen binding domain; a transmembrane domain; and a signaling domain. The signaling domain is most relevant to this study, as the researchers investigate possible combinations of costimulatory molecules to improve the performance and survivability of the CAR T cell. SOURCE: Adapted from Chimeric antigen receptor structure by Liu, J., Zhou, G., Zhang, L., & Zhao, Q. (2019). https://doi.org/10.3389/fimmu.2019.00456

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Expanding Possibilities with Machine Learning The researchers at the Wendell Lim lab sought to find unspoken rules governing costimulatory signaling and thereby optimize CAR T cell characteristics. They wielded a synthetic signal motif library, machine learning, and a unique conceptual approach to discover combinations beyond what naturally occurs.

From Words, to Sentences, to Language The researchers looked to natural signaling proteins, pulled signal motifs from them, and synthetically assembled combinations of signaling motifs to form unique signaling programs. This approach can be conceptualized as a sentence-building exploration. Figure 2 illustrates this rearrangement of various “words”— signaling motifs—into distinct “sentences” or signaling programs. To understand and predict the “language” of these combinations, the team then used machine learning algorithms called neural networks to detect the underlying “grammar” of the datasets. This revealed the importance of word order, word meaning, and word combinations in the final product—otherwise reframed as the impact of signal motif identity, function and arrangement on T cell phenotype. The team curated a library of anti-CD19 CAR T cells with assorted costimulatory domains. Each cell contained either one, two or three signal motifs taken from natural signal proteins (see Figure 3). The team randomly inserted 12 native signal motifs alongside one spacer motif into positions i, j and k to yield a total of 2,379 distinct motif configurations, as seen in Figure 3. Next, researchers screened random subsets from the library to classify the T cells’ cytotoxicity and ability to proliferate (stemness). 167

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This process formed unique and unusual combinations, including combinations comparable to costimulatory molecule 4-1BB (ex: M10-M1-M1-ζ).

FIGURE 2: The procedure used in this study can be understood through a “language forming” lens. Different combinations of “words,” or signaling motifs from costimulatory molecules, create “sentences.” Machine learning algorithms and neural networks unearth the “grammar” in the sentences—that is to say, the associations between the arrangement of certain signaling domains and the resulting characteristics. Abbreviations: scFv, single-chain variable (antibody) fragment. SOURCE: Access Health International

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FIGURE 3: The team created a total of 2,379 different motif combinations. All cells equipped the same anti-CD19 antibody fragment and CD8α hinge/transmembrane domain. The signaling domain contained a CD3ζ molecule alongside one, two or three synthetic costimulatory molecules (highlighted in pink). Abbreviations: scFv, single-chain variable (antibody) fragment; TMD, transmembrane domain. SOURCE: Access Health International

Decoding “Language” Using Predictive Neural Networks The signaling motif sequences possessed varying levels of cytotoxicity and stemness, according to experimental analysis. The team then leveraged this data to understand the invisible rules surrounding costimulatory molecule design. An artificial neural network proved crucial to this investigation. As seen in Figure 4, the data was split to either train or test the algorithm to predict a chimeric antigen receptor’s cytotoxicity or stemness. This process elucidated several associations, such as the ability of 4-1BB-like costimulatory domains to enhance cytotoxicity and stemness.

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Successful Prediction with M1 Costimulatory Molecule Could the neural network accurately foretell the fate of a T cell with a particular costimulatory combination? The team tested the waters by adding costimulatory molecule M1 to 4-1BB-like versus CD28like signaling domains. The neural network predicted that adding M1 motifs would demonstrate enhanced cytotoxicity and stemness in 4-1BB-like domains whilst having no effect in the CD28-like counterpart. In an in vitro model, the CAR T cells with 4-1BB-like domains and M1 motifs effectively killed tumor cells and maintained T cell stemness; on the other hand, the addition of M1 motifs caused no change for the CD28-like derivatives. This correct prediction translated to mouse model results, as well. The 4-1BB/M1 CAR T cells delayed the growth of tumor cells for two weeks longer than 41BB-only CAR T cells. These observations demonstrate how a neural network can be used to accurately forecast T cell characteristics depending on involved synthetic signaling motifs. Possibilities for CAR T Therapy It can be difficult to predict how a synthetic receptor component will influence the resulting cell’s characteristics. This study unscrambles part of this mystery with signal motif libraries and machine learning. By analyzing CAR T cell costimulatory domain combinations, the team created a neural network that successfully predicts T cell phenotype based on the costimulatory molecules present. This, in turn, revealed rules of CAR T costimulatory signaling that can be used to design better synthetic signaling domains. Similar libraries and subsequent analyses could be applied to improve other modular regions of a CAR T cell. 170

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This article originally appeared in Forbes on January 25, 2023, and can be read online here: Engineering Cells For Medical Use: Learning The Language Cells Use To Communicate With One Another

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New Generation Of Flexible And Controllable CAR T Therapies (Part 1) Chimeric Antigen Receptor T cell (CAR T) therapy is one of the exciting new modalities of cancer treatment. The therapy modifies a patient’s own immune cells to treat specific tumors. This is traditionally accomplished by bioengineering hybrid receptors and attaching them to a set of killer T cells taken from the patient. CAR T therapy has yielded exciting results for those with certain blood cancers, but as with many early, first-wave cancer therapies, there’s substantial room for improvement. One previously discussed solution used a peptide neo-antigen (PNE) adaptor administered through an injection to potentially reduce adverse effects. Reducing or halting the adaptor dosage prevented the CAR T cells from overreacting. Here, we discuss a new and distinct adaptor system published in the journal Nature Communications which also holds promise (Ruffo et al., 2023). Why Use Adaptors? Chimeric Antigen Receptor T cell (CAR T) therapy is an effective cell treatment against certain blood cancers. However, it also carries distinct and intrinsic limitations.

Single Antigen Targeting One major limitation of traditional CAR T therapy is that the engineered cell can only target a single antigen. To treat multiple myeloma, for example, the CAR T cell binds to BCMA found on the myeloma cell surface. It would be wonderful to integrate multitargeting instead, thus expanding the therapy’s potential applications. 172

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Multitargeting is a potential solution to antigen escape, a phenomenon that occurs when tumor cells in patients in remission express less of the target antigen over time. When this happens, CAR T cells can no longer recognize their enemies, and cancer returns as a result. Rather than repeating the already expensive and timely procedure from scratch, engineering a multitargeting CAR T cell right from the start would allow the therapy to remain effective for longer. This would particularly benefit patients with solid tumors. These tumors express multiple varied antigens on their cell surface, rendering traditional CAR T therapy ineffective.

Lack of Control Another common issue is lack of control. Sometimes, CAR T cells elicit an overly robust immune response and trigger adverse effects such as cytokine release syndrome or neurotoxicity. Although a weaker CAR T response could prevent these effects, apart from adjusting the infusion dosage, there is no way to precisely control how the CAR T cells react once inside the body. There is, as a result, a begging need to be able to turn the therapy on and off.

Adaptor Has Potential to Overcome Current Limitations Enter the “universal” adaptor. With this system, the CAR T cell does not bind directly to the tumor antigen as usual. Instead, the cell binds to an antibody adaptor, and the antibody adaptor binds to the tumor cell. Changing or including multiple targets can be achieved by infusing several different kinds of antibodies at once or sequentially. Additionally, altering the adaptor dosage can control the nature of the immune response.

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Designing SNAPtag — a Universal Adaptor System In their study, Ruffo et al. designed a universal adaptor using engineered enzymes called SNAPtag. Although this adaptor demonstrated potential beyond CAR T therapy, here we focus on progress specific to CAR T cells.

Making the Adaptor System The universal adaptor is made of two fused parts: an antibody and a tag (Figure 1). The antibody end binds to a specific antigen, while the tag on the other end binds to the CAR T cell; this tag is made from a synthetic molecule called benzylguanine (BG). As illustrated in Figure 2, this adaptor requires a unique kind of chimeric antigen receptor. While the intracellular and transmembrane regions remain the same, the extracellular region adopts a new molecule instead of the usual single-chain variable fragment. Importantly, this new, engineered protein attaches strongly via irreversible covalent bonding to any molecule with benzylguanine (Figure 3). Conceptually this means that, so long as the benzylguanine tag is present, a variety of antibodies can be used in this universal adaptor system.

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FIGURE 1: The universal adaptor is made by fusing an antibody to a benzylguanine motif (BG). This BG-antibody conjugate bonds covalently to the chimeric antigen receptor to elicit a response. SOURCE: Access Health International

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CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease FIGURE 2: A traditional CAR T cell typically uses a fusion antibody (single chain variable fragment, scFV) in its extracellular domain. A different design must be used to work with a universal adaptor. While the adapted CAR T cell contains similar intracellular components, the extracellular domain wields a new protein: SNAPtag. Abbreviations: TM, transmembrane; cyto, cytoplasm. SOURCE: Access Health International

FIGURE 3: The new molecule covalently binds to the universal adaptor, and the adaptor binds to the target antigen. This activates the CAR T signal cascade. SOURCE: Access Health International

How Does the Adaptor Work? The presence or absence of the universal adaptor allows a single pool of T cells to complete several functions. The strength of the therapy can grow stronger or weaker depending on the adaptor concentration. Administering several types of antibodies (with the 176

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same tag) allows the T cells to target several tumor antigens at once (Figure 4). The antigen target can also be switched by sequentially administering different antibodies. The therapy can be stopped by flooding the system with the tag alone—essentially creating an off switch; without the complete adaptor, the CAR T cell binds to the tag without sparking a response.

FIGURE 4: The SNAPtag universal adaptor should allow for multiple antigen targeting. The chimeric receptor can respond to different antibodies bearing the same tag. This concept would be particularly beneficial for avoiding antigen escape and for treatment of solid tumors, which notoriously express a variety of antigens on their surface. SOURCE: Access Health International

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Proof-of-Concept in Mouse Models How might this adaptor and CAR T cell pairing fair therapeutically? To test this, mice were injected with anti-HER2 tumor cells to initiate a tumor graft. After four days, the mice underwent in vivo imaging before receiving one of four treatments: HER2 adaptor alone, SNAP-CAR T cells alone, SNAP-CAR cells with an antiHER2 adaptor, or antiHER2 CAR T cells (a traditional CAR T cell with a single chain variable fragment). The adaptor injections were given every three days—totaling six injections—while the CAR T infusions were only given once. Previous testing revealed that the adapted CAR T cells would require antibody supplements to encourage the T cell engraftment. The authors posit that adapted T cells lack antibody protection in these mouse models, and are prone to attack from innate immune or stromal cells. In response, an intravenous antibody supplement was given with the initial SNAP-CAR T cell infusion and any following adaptor injections.

Antitumor Effect of SNAP-CAR T Cells The mice underwent in vivo imaging every five days until Day 40 or Day 60. The imaging showed rapid tumor growth for the mice treated with the adaptor alone or the SNAP-CAR T cells alone. In comparison, the mice treated with traditional CAR T therapy or the adapted CAR T cells demonstrated significant tumor growth inhibition. By Day 60, the majority of mice in this group showed zero signs of tumor growth. The researchers repeated this experiment with a new antigen target: CD20. Similarly to before, the mice were inoculated with tumor cells and categorized into four distinct treatment groups: anti-CD20 178

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adaptor only, SNAP-CAR T cells only, SNAP-CAR T cells with the adaptor, and traditional SNAP-CAR cells. The mice with adaptor or SNAP-CAR T cells only experienced rapid tumor growth. However, unlike previously, the remaining mice exhibited partial tumor growth and tumor relapse. This likely is not the result of faulty CD20 targeting; the tumor cells lost CD20 expression over time and therefore could avoid CAR T cell detection. Future Directions A universal adaptor extends the use of a single plug and allows it to be used around the world. Similarly, universal adaptors provide a possible solution to CAR T therapy’s many restrictions. This study highlights a particularly versatile design and its ability to shrink tumors in preclinical models. But this universal adaptor has more to offer. In our next installment, we describe how this platform can be combined with a different cell treatment called SynNotch—a duo which may prove even more customizable than its CAR T therapy alternative.

This article originally appeared in Forbes on May 23, 2023, and can be read online here: A New Generation Of Flexible And Controllable CAR T Therapies

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SynNotch, A New Generation Of Flexible And Controllable CAR T Therapies (Part 2) Chimeric Antigen Receptor T cell (CAR T) therapy is a cell-based treatment that has garnered success in treating blood cancers like large B cell lymphoma, leukemia, and multiple myeloma. However, certain limitations prevent the therapy’s adaptation to other diseases. Recent research suggests an alternative receptor called synNotch may address these problems (Ruffo et al., 2023). What is SynNotch? SynNotch is a synthetically engineered version of Notch, a receptor protein present in a variety of animals. This receptor is crucial for cell development. When a Notch receptor receives a signal, it activates a mechanical response that determines the identity and differentiation of unspecialized immune cells. Interestingly, Notch dysfunction is associated with cancers such as leukemia. Notch activates simply. First, the extracellular portion of the receptor senses and binds to a particular family of proteins found on nearby cells. Binding mechanically pulls the receptor up, exposing a cleavage site to enzymes inside of the cell. The enzymes cut and release a transcription factor into the cytoplasm. The transcription factor then travels to the nucleus, where it attaches to a particular DNA sequence and ultimately influences the expression of certain genes. With synNotch, the regulatory domain found in the original receptor is kept. However, as illustrated in Figure 1, both the extracellular and intracellular components are customized to achieve a desired response. A new antigen target can be assigned by changing the fusion antibody (single chain variable fragment, scFv) 180

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found in the extracellular region. Similarly, fusing a different transcription factor to the receptor can positively or negatively affect any gene of interest.

FIGURE 1: SynNotch is a synthetic version of a Notch receptor. SynNotch is more customizable than its natural counterpart; changing the extracellular and intracellular receptor components allows researchers to decide to assign a novel input and novel output. SOURCE: Access Health International

Benefits of SynNotch This modular design has great potential for cell therapy. With this system, researchers can determine who the receptor binds to and what effect is caused—essentially crafting the input and output of a T cell response. CAR T cell appears limited in comparison; even if the antigen target is changed, the cell can only trigger the same signal pathway. Another major difference is that CAR T cells produce cytokines and do not influence gene expression as Notch or synNotch receptors do.

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Limitations and Implications SynNotch cells, while flexible by design, possess similar limitations to CAR T cells. To this end, the researchers at the University of Pittsburgh developed a universal adaptor that worked not only with CAR T cells, but with synNotch T cells, as well. Ideally, the adaptor would grant synNotch cells the ability to target multiple antigens at the same time or sequentially; to switch targets by introducing a new antibody with the same tag; or to stop or start cell activity by altering the adaptor concentration or introducing tags as a competitive inhibitor. In the final installment of this series, we will describe how the authors developed their adaptor to work for synNotch cells.

This article originally appeared in Forbes on October 3, 2022, and can be read online here: SynNotch, A New Generation Of Flexible And Controllable CAR T Therapies (Part 2)

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Adapted SynNotch, A New Generation Of Flexible And Controllable CAR T Therapies (Part 3)

This is the final installment in our series on alternate CAR T therapies. Part one describes a version of CAR T which uses a universal adaptor. Part two introduces SNAPtag, an alternative CAR T therapy. Visit the website www.williamhaseltine.com for more on CAR T therapies. In a previous installment, we described an alternative cell treatment that relies on synNotch receptors. These synthetic receptors react to a specific input and produce a desired cell response. This modular design promises greater therapeutic flexibility than its more wellestablished counterpart, CAR T therapy. The design is further improved when integrated with a universal adaptor called SNAPtag. Here, we describe how researchers at the University of Pittsburgh developed this synNotch-adaptor duo and how it works (Morsut et al., 2016). SynNotch and the Universal Adaptor SynNotch cells depend on transcription factors to change a gene, while CAR T cells trigger T cell signal cascades. Despite relying on distinct mechanisms, the two therapies share similar limitations: they are both hard to control and can only target one antigen at a time. To this end, Ruffo and colleagues sought to develop an adaptor that worked not only with CAR T cells, but with synNotch T cells, as well. The adaptor should, in theory, create a bridge between the receptor and the target antigen. In this way, using an adaptor on synNotch cells would allow the therapy to target multiple antigens at the same time or sequentially. The antigen target could also be switched by 183

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introducing a new antibody with the same tag. And importantly for control, the therapy could be halted or intensified by altering the adaptor concentration or introducing tags as a competitive inhibitor.

Universal Adaptor As demonstrated previously, the universal adaptor is composed of two key elements: an antibody and a benzylguanine (BG) tag (Figure 1). The tag permanently fastens to the extracellular portion of the synNotch receptor via covalent bonding. Meanwhile, the antibody tail senses and binds to a specific antigen on a target cell. Antigen binding stretches the synNotch receptor and reveals a cleavage site on its core protein. As Figure 2 shows, enzymes will cut the cleavage site and release a transcription factor into the intracellular domain of the cell. The transcription factor should travel to the nucleus, where it influences the expression of a target gene.

FIGURE 1: The universal adaptor depends on two components: an antibody and a benzylguanine (BG) tag. The tag covalently binds to the synNotch receptor, while the antibody binds to a specific antibody target. The same tag

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FIGURE 2: SynNotch receptors trigger a different pathway than CAR T cells in the presence of a universal adaptor. When the adaptor tag is bound to the receptor (and the adaptor antibody to the target antigen), mechanical pulling forces stretch the receptor and expose the cleavage sites on the core protein. Cleaving releases the transcription factor and activates a target gene once inside the nucleus. SOURCE: Access Health International

Does Adapted SynNotch Work? SynNotch receptor function was tested with four different types of adaptors, each composed of a distinct and clinically relevant antibody (see Table 1). The synNotch cells were co-incubated with 185

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corresponding tumor cells and exposed to differing concentrations of the adaptor. After 48 hours, the cells were assayed using flow cytometry to measure gene expression. Target gene expression should increase if the adaptor molecules successfully bind to a tumor cell on one side and a synNotch receptor on the other.

TABLE 1: List of antibodies used to test the universal adaptor. Each antibody targets a specific antigen found on tumor cells. SOURCE: Access Health International

Results The synNotch cells successfully activated target genes when exposed to the universal adaptor. Notably, the synNotch cells could regulate the expression of a gene called IL-7 which promotes T cell proliferation—an important component to bolster a patient’s immunity. Receptor activation proved sensitive at low concentrations of the adaptor. Fascinatingly, once the adaptor concentration exceeded 0.25µg/mL, target gene expression no longer increased. In fact, a “hook effect” was observed; although the adaptor concentration increased, target gene expression continually decreased. Gene expression was completely inhibited in adaptor concentrations of 10µg/mL. 186

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The authors posit that the “hook effect” occurred due to oversaturation. Likely at these concentrations, the adaptor molecules are binding to the target tumor or synNotch cells without creating a bridge. Without binding on both sides of the adaptor, the synNotch receptor cannot be activated. Implications SynNotch T cells have the potential to exceed CAR T cell performance with their flexibility. The design allows researchers to determine the input and output of a cell response. Integrating a universal adaptor further extends the possibilities of the treatment to respond to multiple antigen targets or turn on/off at will. While presently in early stages of testing, clinical translation of this platform would greatly benefit patients with solid tumors and other hard-to-treat diseases.

This article originally appeared in Forbes on May 31, 2023, and can be read online here: Adapted SynNotch, A New Generation Of Flexible And Controllable CAR T Therapies (Part 3)

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The Future Of Cancer Treatment? Treating Multiple Myeloma With mRNA-CAR T Technology

t the frontier of cancer treatment development, there is hope for two modern technologies to combine and achieve unprecedented results: messenger RNA and Chimeric Antigen Receptor T Cell Therapy. Biopharm company Cartesian Therapeutics is determined to use this unique platform to benefit patients with a particular blood cancer called multiple myeloma. The company released preclinical trial results in the journal Nature Leukemia, and has a clinical trial underway (Lin et al., 2021; Cartesian Therapeutics, 2023b). Here, we will explore the appeal behind joint mRNA-CAR T therapy for cancer and describe key findings from the preclinical trial. Multiple Myeloma and CAR T Therapy Multiple myeloma is a cancer caused by the irregular growth of a white blood cell called plasma cells. These cells, found primarily in the bone marrow, typically protect the body from infections by producing antibodies. However, when these cells turn cancerous, they interfere with other essential cells in the blood and bones. Chemotherapy, targeted therapy and stem cell transplantation are three common treatment options for myeloma patients. In recent years, CAR T therapy has joined the ranks, albeit as a later stage 188

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alternative. Patients who turn to CAR T therapy after trying and failing five or more previous cancer treatments may finally see a significant, and sometimes complete, reduction in cancer signs.

How CAR T Therapy Works CAR T therapy works by extracting and permanently strengthening a pool of immune cells—killer T cells—to eliminate a patient’s cancer upon reinfusion. In the lab, the cells are bioengineered with DNA that encodes a new receptor. The antibody-like structure on the receptor called a single variable chain fragment (scFv) gives the T cell a heightened ability to detect cancer cells. The fragment attaches to B cell maturation antigen, or BCMA, on the cancer cell before triggering a signal cascade in the T cell. The T cell can then release its usual bevy of chemicals to destroy the cancerous target.

Shortcomings of CAR T Therapy Although CAR T therapy can benefit patients with difficult-to-treat myeloma, there lies a major drawback: its toxic effects. The therapy is unrelentingly rigid. The engineered cells are permanently set to detect and destroy myeloma cells. The covert disadvantage here is that the cells cannot be stopped if they go too far. Overstimulating the immune system like this can lead to potentially fatal side effects such as cytokine release syndrome (CRS) or neurotoxicity. It would be ideal to slowly stimulate the immune system, instead. Adverse events can occur prior to the CAR T infusion, as well. Patients undergo a preparatory course of chemotherapy to eliminate some native immune cells. This practice makes space for the T cell 189

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infusion to proliferate once infused. As chemotherapy is notoriously hard on the body, it would be helpful to bypass this step, if possible. Benefits of mRNA Technology Enter mRNA technology. This innovation could single-handedly transform CAR T therapy into a safer and more flexible cancer treatment. The crux here is to temporarily instruct a patient’s T cells to fight cancer. The treatment can therefore be controlled in a repeated, dose-dependent manner. If less CAR T cells are infused at once, preparatory chemotherapy might also be avoided altogether. Traditional CAR T therapy changes a T cell’s DNA. The new genetic information is folded into the genome and kept intact even as the cell replicates and multiplies. In contrast, messenger RNA is inherently unstable genetic information. As the mRNA inevitably degrades, receptor expression should likewise recede. Optimistic Study Results How feasible is this concept? Researchers Lin et al. tested this in their preclinical study using mice models of multiple myeloma. Mice with humanized models of myeloma received an injection of humanized T cells each week for four weeks. The mice were then split into three groups, receiving either: 1) non-active substance (vehicle) to control for potential injection effects, 2) a control of killer T cells, and 3) Descartes-08, a CAR T therapy which modifies T cells to express anti-myeloma chimeric receptors for only a week. The mRNA encoded a single chain variable fragment (scFV); a CD28 molecule to support cell signaling; and a CD3 signaling domain responsible for killer signal cascade. 190

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The control T cell and CAR T cell infusions were administered once a week after Day 7. Both the CAR T product and the control T cells relied on CRISPR gene editing to decrease expression of native T cell receptors. This prevents the intervention from being rejected by the host. Additionally, all of the mice were preconditioned with a chemotherapy drug called cyclophosphamide. Of the three groups, the CAR T cell mice demonstrated the most prolonged survival; the median survival was significantly higher at 69 days. Comparatively, the vehicle-treated and killer T cell control mice began to lose weight and show signs of myeloma; the median survival lingered at 43 and 44 days respectively. This suggests that the transient CAR T therapy, when administered intermittently, can successfully control tumor growth and extend survival.

Patient Example The transient CAR T therapy also improved tumor burden for a 76year-old patient suffering from plasma cell leukemia. Similarly to multiple myeloma, plasma cell leukemia involves the uncontrollable growth of plasma cells. Both illnesses share the same therapeutic target—B cell maturation antigen on plasma cells. First, the patient received preparatory chemotherapy. This was followed by three CAR T cell infusions given over the span of two weeks. The patient did not experience any adverse effects from the infusions. After twelve weeks, the patient had no residual signs of cancer. This is an encouraging example of what transient CAR T cells can accomplish for cancer patients. The next step is to test with a larger study population. 191

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News on the Clinical Trial A clinical trial is currently in progress. The study will see how advanced myeloma patients fare when given increasing doses of the aforementioned CAR T product. In Phase I of the study, none of the myeloma patients experienced cytokine release syndrome or neurotoxicity (Cartesian therapeutics initiates, 2021). This suggests that the treatment was well-tolerated and could potentially circumvent unwanted effects. The estimated trial completion date is set for next year. Looking to the Future Standard CAR T cell therapy, while effective, invokes its own risks. Integrating mRNA technology could be the solution to these prevailing issues. By transiently expressing the desired chimeric receptor, the therapy could be simplified and the safety could be improved all at once. Given the promising preclinical trial and case study results, we highly anticipate the clinical trial completion date.

This article originally appeared in Forbes on June 27, 2023, and can be read online here: The Future Of Cancer Treatment? Treating Multiple Myeloma With MRNA-CAR T Technology

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CAR T Therapy For Cardiac Fibrosis: A New Way Forward CAR T therapy, a “living drug,” traditionally involves isolation and purification of T cells outside the body. The cells are then modified with a synthetic receptor and then re-infused into the body for treatment of cancers. Researchers have now successfully demonstrated that T cells can be modified in vivo by mRNA technology, bypassing the need for extraction, chemotherapy and reinfusion (Rurik et al, 2022). Although this method proves effective in treating mice with scarred hearts, considering fibrosis contributes to over 800,000 deaths worldwide, the study contains great potential for human treatment. A Damaged Heart The heart, flexible yet strong, circulates blood through the body by pumping blood through its chambers. Aging and injury tamper with this function, creating scarred and thickened tissue called fibrosis. Although fibrosis occurs normally when healing, a highly fibrotic heart loses its elasticity; the stiffened tissues and interrupted electrical signaling prevent proper contractions of the heart (see Figure 1). Cardiac fibrosis is highly associated with heart disease and heart failure. Cardiac fibrosis has no “cure-all” treatment. Early detection improves prognosis, but options dwindle as damage progresses irreversibly. People with advanced cardiac fibrosis may take drugs that antagonize overstimulation of the heart or might even require heart valve replacement.

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FIGURE 1: Comparison of the cells in a healthy heart and the cells seen in cases of cardiac fibrosis. Note the thickened extracellular matrix, which impedes on normal heart function in several ways.

SOURCE: Adapted from Cardiac Fibrosis by Hinderer, S., & Schenke-Layland, K. (2019). https://doi.org/10.1016/j.addr.2019.05.011 How CAR T Cells Work In their study, Rurik et al. explore a new method to directly counter cardiac fibrosis. This method builds upon the basics of CAR T: the use of T cells with a synthetically engineered receptor to target and kill specific cells. CAR T is approved to treat people with certain lymphomas, leukemias, and multiple myeloma. Figure 2 illustrates this process. In these cases, the desired T cells are extracted from the patient’s body. Synthetic mRNA is inserted into the cell with a retrovirus, a virus commonly used in gene therapy to permanently change other 194

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cells’ genomes. The altered and expanded cells are then infused back into the body after preparatory chemotherapy. These T cells target either CD19 or BCMA, two antigens found on malignant B cells.

FIGURE 2: The CAR T process has several steps. T cells must be extracted from the blood, then genetically modified with a new receptor and expanded to great numbers. The patient prepares with chemotherapy before the CAR T cells are introduced into the bloodstream. SOURCE: From CAR T Cells: Engineering immune cells to treat cancer. National Cancer Institute (2022). https://www.cancer.gov/aboutcancer/treatment/research/car-t-cells

The benefit of inserting genetic information with a retrovirus lies in its permanence. The CAR T cells can expand and persist in the 195

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body for a long time after infusion, continually fighting the cancerous cells they encounter. However, this is of no benefit to researchers hoping to fight cardiac fibrosis. If T cells continuously target fibrotic cells, they would impair normal healing processes and potentially induce autoimmunity. Rurik et al. employ an elegant solution that shortens the CAR T cells’ active duty, thereby circumventing the extraction process altogether. New CAR T Cell Design The team adapted mRNA delivery technology seen in current COVID-19 vaccines and applied it to basic Chimeric Antigen Receptor design. The mRNA does not integrate into the T cell genome, allowing for temporary transcription of the mRNA and transient expression of the new receptor.

CD5 Lipid Nanoparticles (LNP) The authors adopted a strategy to introduce the chimeric receptor to T cells in the body rather than extracting and purifying them outside the body. To accomplish this aim, they first synthesized mRNA that encodes a receptor against fibroblast activation protein (FAP), a protein expressed on activated fibroblasts responsible for fibrosis. They purified the mRNA and packaged the engineered mRNA into standard lipid nanoparticles (LNP). The team then decorated the lipid nanoparticle surface with CD5targeting antibodies to direct lipid uptake. The integration of CD5 antibodies allowed the lipid nanoparticles to target antigen CD5 naturally expressed by T cells once injected into the body; the CAR T cells are made after a single shot.

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Chimeric Antigen Receptor The chimeric antigen receptor contains a single chain variable fragment (scFv) derived from fibroblast activation protein monoclonal antibodies; this recognition domain enables the CAR T cell to target cells which express fibroblast activation protein. The CAR design also includes CD28 and CD3z signaling domains in the cytoplasm. All three components are mouse-specific. Not illustrated in Figure 3 is an added small peptide that prevents immune suppression.

FIGURE 3: The mRNA encoded for a chimeric antigen receptor composed of a fibroblast activation protein (FAP) antigen recognition domain and a CD28 and CD3ζ signaling domain in the cytoplasm. The team also included an additional peptide to prevent immune suppression. SOURCE: Access Health International

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Genetic Integration In Vivo The team found that lipid nanoparticles could successfully deliver the mRNA package to T cells, as seen in Figure 4. The killer T cell absorbs the lipid nanoparticle by endocytosis. The lipid particle then degrades and the synthetic mRNA releases into the cell. Finally, the cellular machinery reads the genetic instruction and briefly produces the receptor against fibroblast activation protein. This is possible with both animal and human T cell cultures.

FIGURE 4: To create a CAR T cell with transient CAR expression, a lipid nanoparticle (LNP) with the desired genomic information is absorbed by the T cells through endocytosis. Once inside the T cell, the lipid nanoparticle degrades and releases the mRNA which encodes for the desired receptor. The expressed receptor allows the T cell to detect fibroblast activation protein located on the surface of many activated fibroblasts. SOURCE: Access Health International

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Transitory CAR Expression Unlike traditional CAR T cells that carry a chimeric receptor encoded by DNA inserted into the genome, these CD5+ T cells carry mRNA only transiently. The mRNA is not integrated into the cell’s genome and remains stuck in the T cell cytoplasm before degrading. This is ideal; fibroblast activation protein receptors must be expressed briefly as longer expression may harm other tissues. Results The research team assessed the efficacy of the CAR T cells in different conditions. When they treated the cells in tissue culture, more than 80% of T cells expressed the chimeric antigen receptor and could effectively kill target cells with fibroblast activation protein. The team then tested this model on mice with cardiac fibrosis. The mice received medication to injure the heart and induce scarring. After one week, the team administered the lipid-mRNA injection. Consistent CAR expression was noted 48 hours after injection, and disappeared after one week. The results were impressive. The function of the heart’s largest chamber improved, in some cases returning to uninjured levels . Similarly, the amount of blood filling the heart normalized to safe volumes. The therapy notably reduced the thickness of the heart. Finally, although the mass of the largest chamber did not normalize, it trended toward improvement. One caveat in lipid-CAR T cell delivery is that some cells, perivascular fibroblasts, do not express fibroblast activation protein. In consequence, these cells were not impacted by CAR T cells and some fibrosis persisted. No overly toxic side effects were noted. 199

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Trogocytosis A key observation of effective CAR T therapy is the ability of the modified T cells to take small bites of the target cell—a phenomenon known as trogocytosis. Deriving “trogo” from the Greek word “to bite,” trogocytosis entails one cell nibbling another and, in the process, transferring the surface molecules from one to the other. The researchers found evidence of CAR T cells “nibbling” the activated fibroblasts and retaining the stolen antigens (illustrated in Figure 5), suggesting that the T cells successfully adopted the chimeric antigen receptors in vivo.

FIGURE 5: Trogocytosis occurs when a cell ingests small “bites” of another cell, thus taking the surface molecules from one cell and expressing them on its own cell surface. Here, the T cell detects the activated fibroblast, ingests parts of its surface, and then expresses the ingested surface molecules. SOURCE: Access Health International

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Future Implications CAR T therapy revolutionized cancer treatment with its efficacy and innovation. Combining mRNA technology to this therapy creates a temporary version of this “living drug” that does not sacrifice on quality. The therapy is well tailored to heal mice with damaged and scarred hearts, and widens the possibilities to treat other non-cancerous human ailments. If translated to clinical settings, transient CAR T therapy may be less expensive and more readily available than its traditional counterpart.

This article originally appeared in Forbes on November 9, 2022, and can be read online here: CAR T Therapy For Cardiac Fibrosis: A New Method

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How mRNA Could Reinvent Blood Stem Cell Transplant Preparations (Part 1)

This story is part of a series on the current progression in Regenerative Medicine. This piece discusses advances in stem cell gene therapy. In 1999, I defined regenerative medicine as the collection of interventions that restore to normal function tissues and organs that have been damaged by disease, injured by trauma, or worn by time. I include a full spectrum of chemical, gene, and protein-based medicines, cell-based therapies, and biomechanical interventions that achieve that goal. mRNA technology is ushering waves in stem cell gene therapy. A preclinical study published in Science showcases three exciting findings demonstrating how mRNA-based innovations could make treating and curing genetic blood diseases simpler and safer than the current treatment standard (Breda et al., 2023). As part of a three-part series, this installment will explore how mRNA could replace toxic chemotherapy and radiation procedures. Future installments will describe how the researchers corrected problematic gene mutations at their source and genetically altered blood stem cells in vivo with the same platform. The Current Treatment Standard Genetic blood disorders encompass many diseases that impact the blood, including sickle cell anemia and thalassemia. Such conditions arise when the genes responsible for producing essential proteins in blood cells mutate or become abnormal. These protein abnormalities affect blood cell development and are typically

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hereditary—meaning they are passed down from parent to child through genetic inheritance. One way to correct the irregular blood cell production is to address the source: hematopoietic stem cells. All blood cells in the body, functioning or otherwise, develop from these immature blood stem cells (Figure 1). Replacing the aberrant, disease-causing stem cells with healthy ones allows the body to repopulate with healthy blood cells. This procedure is known as hematopoietic stem cell transplantation or bone marrow transplant (Khaddour et al., 2019).

FIGURE 1: All blood cells originate and develop from hematopoietic stem cells. Hematopoeitic stem cell transplantation replaces abnormal, diseasecausing hematopoietic stem cells with healthy ones to replenish the blood. SOURCE: Access Health International

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Risks of Stem Cell Transplantation Stem cell transplantation can treat and cure, but the process incurs certain risks. For example, high-dose chemotherapy or radiation may be used to suppress the patient’s immune system, leaving space for the transplanted cells to engraft and establish themselves (Lanzkowsky, 2011). These conditioning methods are toxic to healthy cells, increasing the risk of long-term complications such as organ damage or failure (Bhatia, 2011). Damage often targets the lungs, liver and reproductive organs. Researchers hope to develop a new ablation method that significantly reduces this associated toxicity. mRNA: a Less Toxic Alternative Researchers at the University of Pennsylvania turned to mRNA technology as a potentially less toxic solution to deplete stem cells. mRNA-based innovations offer a favorable safety profile, precise targeting and a general flexibility that could benefit a variety of treatments—be it vaccines, CAR T therapy or blood stem cell transplantation, as shown through this study.

Attuned for Ablation In basic terms, mRNA technology is a delivery system for genetic information (Figure 2). Lipid nanoparticles encapsulate and transport the desired mRNA to a specific cellular destination. Upon arrival, the target cell will engulf the nanoparticle; the nanoparticle will degrade and release the mRNA cargo; and the target cell will produce proteins based on the released genetic instructions. Several key changes were needed to adapt the platform for blood stem cell ablation. mRNA technology relies on antibodies to direct to the vehicle to the right cell target. The study authors determined 204

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that antibodies against surface protein CD117 worked better than anti-CD47 antibodies at targeting hematopoietic stem cells. AntiCD117 antibodies were then attached to the lipid nanoparticles via SATA-maleimide chemistry. The process alters the nanoparticles to have maleimide groups and the antibodies with sulfhydryl groups. The two groups act as a molecular hook and loop, easily connecting together to form a strong link. Next, the nanoparticles were designed to carry synthetic mRNA that promotes controlled cell death in stem cells; the encoded gene is called PUMA, short for p53 up-regulated modulator of apoptosis. If the delivery is successful, the mRNA should initiate a selfdestruction pathway in the cells, thus culling functional stem cell numbers in the bone marrow.

FIGURE 2: mRNA technology relies on antibody-decorated lipid nanoparticles to deliver the desired mRNA to the target cell (here, a hematopoeitic stem cell).

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CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease The target cell engulfs the nanoparticle, creating a pocket-like space called an endosome. Next, the endosome begins to break down, exposing the nanoparticle to degradation. This frees the desired mRNA into the cell cytoplasm, where it can be translated to create proteins necessary to activate apoptosis. SOURCE: Access Health International

Mouse Model Results Researchers tested their CD117-targeted, mRNA-carrying lipid nanoparticles in cell culture and mice models. The series of experiments illustrates this platform’s potential to safely target and deplete hematopoietic stem cells. The team treated one set of bone marrow cells with CD117targeting PUMA lipid nanoparticles and left another set untreated. These cells were injected at equal or increasing ratios into mice with a reduced number of bone marrow cells. Within two weeks, the mice that received high nanoparticle ratios die from transplantation from bone marrow failure—a positive sign for stem cell depletion. In contrast, the mice injected with higher proportions of untreated bone marrow cells live up to the four-month endpoint. They do not undergo thorough ablation and maintain higher blood cell counts. This result was echoed with in vivo testing. Blood stem cell numbers reduce significantly six days after mice receive a small dose of the PUMA-carrying lipid nanoparticles. Could the nanoparticles be used to prep mice for stem cell transplantation? To test this, one group of mice received a unique and precise lipid nanoparticle injection. The mRNA inside included PUMA, the apoptotic gene, and miR-122, a microRNA that should help downregulate PUMA expression in liver cells.

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These mice received a stem cell transplantation seven days later. The mice in Group Two only received stem cell transplantation. In the following four to 16 weeks, Group Two mice fail to engraft donor cells. Comparatively, the Group One mice readily accept the transplantation—likely due to the cells having enough space to engraft. To further validate this observation, the researchers extracted the successfully engrafted cells from Group One mice and transplanted the cells into another set of mice. Remarkably, these mice do not need pre-conditioning to engraft the donor cells. This result suggests that the initial experimental treatment paved the way for the donor cells to outcompete any remaining blood stem cells in this new set of mice.

Caveats: Possible Toxic Effects Although the nanoparticle system can accurately target CD117 in hematopoietic stem cells, there is a chance for the nanoparticles to impact unintended cells. One method to prevent this off-target toxicity is to integrate microRNA binding sites into the mRNA, such as miR-122. This can limit the expression of the delivered mRNA to specific cell types. Here, miR-122 prevents liver cell depletion, but more experimentation will be needed to refine this effect in other organs. The lipid nanoparticles could also accidentally target healthy, CD117-carrying hematopoietic stem cells. Introducing inducible suicide genes in the synthetic mRNA may help mitigate this unwanted effect (Falcon et al., 2022). The genes would activate with a trigger (ex: administering a small molecule drug) and instruct the lipid nanoparticle to self-destruct, thus curbing off-target effects. 207

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Future Implications This study addresses major toxicity concerns for prospective stem cell transplant patients. The lipid nanoparticles in these mouse models accurately and safely deliver apoptotic genes to blood stem cells, thus depleting their numbers. An additional benefit is that mRNA-carrying nanoparticles can deliver more than just proapoptotic genes. The lipid nanoparticles described in upcoming installations carry different synthetic mRNAs to produce striking results. To conclude, mRNA technology may one day replace dangerous, one-trick-pony conditioning regimes such as chemotherapy. However, further testing will be necessary to confirm this method’s clinical feasibility.

This article originally appeared in Forbes on August 15, 2023, and can be read online here: How mRNA Could Reinvent Blood Stem Cell Transplant Preparations

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How mRNA Could Cure Sickle Cell Gene Mutations (Part 2)

This story is part of a series on the current progression in Regenerative Medicine. In 1999, I defined regenerative medicine as the collection of interventions that restore to normal function tissues and organs that have been damaged by disease, injured by trauma, or worn by time. I include a full spectrum of chemical, gene, and protein-based medicines, cell-based therapies, and biomechanical interventions that achieve that goal. This piece discusses advances in stem cell gene therapy via mRNA technology. Part One describes how mRNA could reinvent how we prepare patients for bone marrow transplants. The final installment will discuss how to alter blood stem cells with the same system. Sickle cell disease is the most common genetic blood disorder in the United States and affects 100,000 Americans (CDC, 2022). A bone marrow or blood stem cell transplantation remains the only curative therapeutic option, but it can be challenging to qualify for the treatment or risky to undergo. Researchers at the University of Pennsylvania investigated a new, potentially less toxic method that corrects the pathogenic genes at their source (Breda et al., 2023). The promising preclinical results build anticipation for an enhanced future treatment to come. Treating Sickle Cell Disease Sickle cell disease encompasses several inherited blood disorders that arise from mutations in a protein called hemoglobin. Hemoglobin is essential for red blood cell function. Oxygen attaches to this protein inside red blood cells and then is carried throughout the body.

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People with sickle cell disease inherit a gene from each parent that encodes for irregular hemoglobin. Abnormal hemoglobin deforms the flexible, round-shaped red blood cells typically have. The cells turn rigid, sticky and crescent or sickle-shaped. In turn, the blood cells more readily clump together and block blood flow. The resulting lack of oxygen to vital organs and tissues manifests complications such as infection and stroke.

The Cost of a Cure This lifelong condition can only be cured through a bone marrow or blood stem cell transplant. Sickle red blood cells develop from hematopoietic stem cells that carry the mutated hemoglobin gene. The procedure rids the patient of these diseased blood stem cells and replaces them with healthy ones, allowing the body to produce healthy red blood cells.

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FIGURE 1: All blood cells originate and develop from hematopoietic stem cells. Hematopoietic stem cell transplantation replaces abnormal, diseasecausing hematopoietic stem cells with healthy ones to replenish the blood. SOURCE: Access Health International

This permanent solution may sound tempting, but few can withstand the possible hazards (Acharya et al., 2023). As with other organ transplants, the body may reject the donated cells—even if they originate from close relatives—and stir up potentially lifethreatening immune responses. Another alternative is to source the cells from the patient and correct the genes in the lab. In either case, the patient cannot avoid the toxicities of chemotherapy or radiation used to prime the bone marrow before transplantation.

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Correcting Gene Mutations with mRNA Is it possible to cure sickle cell disease without the high stakes? Researchers at the University of Pennsylvania probed an innovative avenue: correcting the genetic mutation at its source with mRNA technology. mRNA technology depends on lipid nanoparticles to deliver genetic information to a programmed target—in this case, pathogenic hematopoietic stem cells. As discussed in a previous article, the team decorated the lipid nanoparticles with an antibody that detects a protein called antigen CD117 on the surface of blood stem cells. After the blood stem cell encircles the lipid nanoparticle, the nanoparticle breaks down and releases the desired mRNA. The cell then reads the mRNA and produces proteins based on its instructions, thus activating a cellular response.

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William A. Haseltine, PhD FIGURE 2: mRNA technology relies on antibody-decorated lipid nanoparticles to deliver the desired mRNA to the target cell (here, a hematopoietic stem cell). The target cell engulfs the nanoparticle, creating a pocket-like space called an endosome. Next, the endosome begins to break down, exposing the nanoparticle to degradation. This frees the desired mRNA into the cell cytoplasm, where it can be translated to create proteins necessary to activate apoptosis. SOURCE: Access Health International

In this experiment, the anti-CD117 lipid nanoparticles contained either 1) gene-editing mRNA that encodes a Cas9 adenine base editor fusion protein or 2) a single-guide RNA that should direct the mRNA to the hemoglobin sickle cell mutation. Cas9 is a recognizable component of the CRISPR-Cas9 gene editing system. Here, the protein converts a single DNA base (adenine) to another base (guanine) (Newby et al., 2021). The A to G conversion turns pathogenic hemoglobin DNA into a harmless, nonpathogenic variant. The nonpathogenic variant is not the typical hemoglobin seen in non-sickle cell patients; even though this variant does not naturally occur in humans, it is still functional (Chu et al., n.d.).

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FIGURE 3: In this study, the Cas9 adenine base editor fusion protein, along with the single-guide RNA, target and convert pathogenic hemoglobin DNA into a harmless variant. The base editor specifically converts an adenine:thymine (A:T) base pair into a guanine:cytosine (C:T) base pair. The change produces a different protein, alanine instead of valine, and turns the sickle cell hemoglobin into a nonpathogenic version. SOURCE: Access Health International

Four sickle cell specimens from separate donors simultaneously received the two types of nanoparticles. The researchers then observed the cell cultures, noting the number of pathogenic and nonpathogenic hemoglobins to see if the DNA was successfully corrected. Applying an excess of guide RNA to mRNA led to efficient base editing. In these cases, nonpathogenic hemoglobin

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numbers increase by more than 90%; comparatively, pathogenic hemoglobin decreases. The cell cultures were also exposed to hypoxic conditions, mimicking the low-oxygen environments sickle cells create when blocking the blood vessels. The nonpathogenic hemoglobin should behave normally and resist sickling red blood cells if adequately corrected. The team found that editing levels and the increase in nonpathogenic hemoglobin were directly correlated. In addition, even the highest dose of lipid nanoparticles did not inhibit the growth and function of the blood stem cells. The results combined suggest that base-editing mRNA, when paired with single-guide RNA, can neutralize the threat of sickle red blood cell mutations. Looking Forward mRNA’s potential applications continue to expand. Not only can mRNA-carrying lipid nanoparticles deplete stem cells, they can also accurately edit the human genome as demonstrated in this study. Although undoubtedly in its early stages, this finding holds immense therapeutic potential if clinically translated. A single injection of mRNA lipid nanoparticles could cure patients with sickle cell diseases—a potentially safer and simpler alternative to bone marrow transplants. The final installation of this mRNA series will explore another developing use for mRNA: editing hematopoietic stem cells in vivo.

To read more of this series, please visit www.williamhaseltine.com

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This article originally appeared in Forbes on August 18, 2023, and can be read online here: How mRNA Could Cure Sickle Cell Gene Mutations

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How mRNA Could Safely Replace Blood Stem Cell Transplantation (Part 3)

This story is part of a series on the current progression in Regenerative Medicine. In 1999, I defined regenerative medicine as the collection of interventions that restore to normal function tissues and organs that have been damaged by disease, injured by trauma, or worn by time. I include a full spectrum of chemical, gene, and protein-based medicines, cell-based therapies, and biomechanical interventions that achieve that goal. As part of a trio of stories on advances in stem cell gene therapy, this piece discusses how to alter blood stem cells using mRNA technology. Previous installments describe how the same platform could reinvent how we prepare patients for bone marrow transplants and correct pathogenic DNA. At present, the only way to cure genetic blood disorders such as sickle cell anemia and thalassemia is to reset the immune system with a stem cell transplantation. Only a fraction of patients elects this procedure, as the process is fraught with significant risks, including toxicity and transplant rejection. A preclinical study published in Science explores a solution that may be less toxic yet equally effective: mRNA technology (Breda et al., 2023). The cell culture and mouse model experiments offer a compelling avenue for future research to enhance or replace current stem cell transplantations altogether. The Risks of Transplantation All blood cells in the body, healthy or diseased, originate from hematopoietic stem cells. These long-term stem cells continuously regenerate and produce blood cells throughout a person's lifetime. 217

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Replacing diseased stem cells with healthy ones can cure people of their genetic blood condition by rebuilding the immune system.

There are two methods to replenish a person’s stem cells, each with its own complications. The first and most common method is to source healthy stem cells from a donor. Sourcing stem cells from others, or allogenically, is difficult due to the risk of transplant rejection. If the transplant is not well matched to the host, the body will view the new cells as foreign intruders and attack the graft. This phenomenon is known as graft-vs-host disease (GvHD). The associated risks decrease if a 218

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close relative, such as a brother or sister, offers to be a donor. However, only some patients have an eligible relative who can donate, and even then, rejection could still occur. The other alternative is to use the patient’s stem cells instead. The diseased stem cells are corrected in the lab using gene addition or gene editing techniques before transplantation. While this solves donor-matching or tissue rejection issues, it is costly to extract and gene edit a batch of stem cells for each patient (Aiuti et al., 2022). The expense of specialized, small-scale manufacturing limits this option’s accessibility—a problem faced by other personalized gene therapies (e.g., CAR T therapy). Lastly, it is difficult to avoid the toxic risks of chemotherapy or radiation, which may be used to prepare the patient’s bone marrow for either type of transplantation. A Novel Solution: Editing via mRNA Technology Researchers at the University of Pennsylvania explored the possibility of mRNA technology to enhance how we treat genetic blood diseases. The technology relies on antibody-covered lipid nanoparticles to deliver genetic information (mRNA) to a target cell; the antibodies establish the cell target. Upon arrival, the nanoparticle is encircled by the target cell and slowly broken down once inside the cell. Eventually, the genetic material is released into the cell cytoplasm, where cell machinery can read it to initiate immune responses. The hope is to deliver gene-editing instructions to a patient’s stem cells via a single mRNA injection, circumventing the need to extract and edit the cells in a specialized lab.

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FIGURE 2: mRNA technology relies on antibody-decorated lipid nanoparticles to deliver the desired mRNA to the target cell (here, a hematopoietic stem cell). The target cell engulfs the nanoparticle, creating a pocket-like space called an endosome. Next, the endosome begins to break down, exposing the nanoparticle to degradation. This frees the desired mRNA into the cell cytoplasm, where it can be translated to create proteins necessary to activate apoptosis. SOURCE: Access Health International

mRNA-Edited Stem Cell Transplant Could antibody-covered lipid nanoparticles successfully edit hematopoietic stem cells? To test this, lipid nanoparticles decorated with anti-CD117 antibodies were used to treat a batch of bone marrow cells. These antibodies effectively detect CD117 antigen on hematopoietic stem cells. Inside the nanoparticles lay a modified mRNA called Cre, short for cyclic AMP response element. If the mRNA is successfully delivered, the reporter gene inside the bone 220

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marrow cells should activate in response to the Cre-mRNA, turning the cell culture from red to green fluorescent. The researchers can then assess for gene editing through this color change. Mice received either the edited bone marrow cells or bone marrow cells treated with a control intervention. The team noted reporter gene expression for four months. The results demonstrate nearcomplete gene editing for mice treated with CD117-decorated, Crecarrying lipid nanoparticles. Red blood cells, white blood cells and other immune cells show evident reporter gene expression, while long-term blood stem cells exhibit high levels of gene editing. The editing rates appear dose-dependent, with higher doses resulting in higher editing percentages. When these bone marrow cells are transplanted again into another group of mice, the stem cells maintain their ability to generate different types of blood cells.

In Vivo Editing in Mice The ultimate goal is to gene-edit blood stem cells in vivo using a single injection of mRNA-carrying lipid nanoparticles. To mimic this, the investigators attempted to alter the hematopoietic stem cells inside the mice instead of editing the bone marrow cells first. Mice received an injection of either CD117 lipid nanoparticles with Cre mRNA or control lipid nanoparticles. Reporter gene expression was monitored for four months. Impressively, the intervention mice experience significantly higher editing in peripheral blood cells and long-term blood stem cells than control mice. Editing levels increased with larger doses of intervention treatment. The green color change persists even when bone marrow cells from the first set of mice are transplanted into a secondary set

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of mice, validating the successful editing of long-term blood stem cells.

Targeting Other Tissues? The CD117 lipid nanoparticles demonstrate spleen and bone marrow uptake—a positive sign of accurate targeting and mRNA delivery. This is because blood stem cells largely rest in the bone marrow; in turn, the spleen supports the maturation of blood cells derived from these stem cells. The team also checked to see if other tissues were affected by the lipid nanoparticles. They find reporter gene expression in the liver for both the control and intervention nanoparticles. The result points to a known phenomenon in which the liver interacts with receptors on the lipid nanoparticles. In the lung, the intervention nanoparticles elicited significantly higher reporter gene expression levels than controls. This may be attributed in part to the lung cells carrying CD117, the desired antigen target for the nanoparticles. The treatment does not impact the testis, according to this study. Cells from the testis in intervention mice do not stray from baseline. Moreover, the children from intervention and control male mice do not express the reporter gene. Noting tissue uptake is essential for gauging off-target effects. The lipid nanoparticle system used here may need further refinement to prevent gene editing in unwanted organs. Looking Ahead mRNA technology possesses an expansive potential to improve gene therapy. The study authors here demonstrate how the platform can successfully target and edit stem cells in cell culture and inside 222

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mice. The researchers also show how the same platform could therapeutically correct the human genome and eliminate preparatory chemotherapy. Building upon these findings could lead us to a simple treatment that replaces current stem cell transplantation procedures with a single injection.

To read more of this series, please visit www.williamhaseltine.com This article originally appeared in Forbes on August 23, 2023, and can be read online here: How mRNA Could Safely Replace Blood Stem Cell Transplantation

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Acknowledgments

would like to thank Amara Thomas for her unflagging effort and enthusiasm. I could not have attempted this book without the crucial support of the ACCESS Health US team: Courtney Biggs, Griffin McCombs, Koloman Rath, Kim Hazel and Robert Patarca. This work is supported by ACCESS Health International (www.accessh.org).

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References

Abecma (Idecabtagene vicleucel) reduced the risk of disease progression or death by 51% versus standard regimens in earlier lines of therapy for relapsed and refractory multiple myeloma based on results from phase 3 karmma-3 study . (2023, February 10). Bristol Myers Squibb. https://news.bms.com/news/details/2023/Abecmaidecabtagene-vicleucel-Reduced-the-Risk-of-DiseaseProgression-or-Death-by-51-Versus-Standard-Regimens-inEarlier-Lines-of-Therapy-for-Relapsed-and-Refractory-MultipleMyeloma-Based-on-Results-from-Phase-3-KarMMa-3Study/default.aspx Abid, M. B. (2023). The denominator in early phase CAR T-cell trials examining novel target antigens. The Lancet. https://www.thelancet.com/journals/lancet/article/PIIS01406736(23)00872-3/fulltext

About carvykti® | carvykti® (Ciltacabtagene autoleucel) hcp. (2021, June 3). CARVYKTI® (Ciltacabtagene Autoleucel). https://www.carvyktihcp.com/about-carvykti? Acharya, B., Mishra, D. P., Barik, B., Mohapatra, R. K., & Sarangi, A. K. (2023). Recent progress in the treatment of sickle cell disease: an up-to-date review. Beni-Suef University Journal of Basic and Applied Sciences, 12(1), 38. https://doi.org/10.1186/s43088-023-00373-w

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Aiuti, A., Pasinelli, F., & Naldini, L. (2022). Ensuring a future for gene therapy for rare diseases. Nature Medicine, 28(10), 19851988. https://doi.org/10.1038/s41591-022-01934-9 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Helper t cells and lymphocyte activation. In Molecular Biology of the Cell. 4th edition. Garland Science. https://www.ncbi.nlm.nih.gov/books/NBK26827/ Alhaidar, M. K., Abumurad, S., Soliven, B., & Rezania, K. (2022). Current treatment of myasthenia gravis. Journal of Clinical Medicine, 11(6), 1597. https://doi.org/10.3390/jcm11061597 Alizadeh, D., Wang, D., & Brown, C. E. (2023). Uncovering the Role of CD4+ CAR T Cells in Cancer Immunotherapy. Cancer Research, 83(17), 2813-2815. https://doi.org/10.1158/0008-5472.CAN-23-1948 Amor, C., Feucht, J., Leibold, J., Ho, Y. J., Zhu, C., AlonsoCurbelo, D., ... & Lowe, S. W. (2020). Senolytic CAR T cells reverse senescence-associated pathologies. Nature, 583(7814), 127-132. https://doi.org/10.1038/s41586-020-2403-9 Anderson, N. D., Birch, J., Accogli, T., Criado, I., Khabirova, E., Parks, C., ... & Ghorashian, S. (2023). Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia. Nature Medicine, 29(7), 1700-1709. https://doi.org/10.1038/s41591-023-02415-3 Arcangeli, S., Magnani, C. F., Tettamanti, S., & Biagi, E. (2016). Switchable chimeric antigen receptor T cells: a novel universal chimeric antigen receptor platform for a safe control of T-cell activation. Transl Cancer Res, 5(12), 174-7. https://doi.org/10.21037/tcr.2016.07.23 226

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Arjomandnejad, M., Kopec, A. L., & Keeler, A. M. (2022). CAR-T regulatory (CAR-Treg) cells: engineering and applications. Biomedicines, 10(2), 287. https://doi.org/10.3390/biomedicines10020287 Asaadi, Y., Jouneghani, F. F., Janani, S., & Rahbarizadeh, F. (2021). A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomarker Research, 9, 1-20. https://doi.org/10.1186/s40364-021-00332-6 Baird, J. H., Frank, M. J., Craig, J., Patel, S., Spiegel, J. Y., Sahaf, B., ... & Muffly, L. (2021). CD22-directed CAR T-cell therapy induces complete remissions in CD19-directed CAR– refractory large B-cell lymphoma. Blood, The Journal of the American Society of Hematology, 137(17), 2321-2325. https://doi.org/10.1182/blood.2020009432 Baker, D. J., Wijshake, T., Tchkonia, T., LeBrasseur, N. K., Childs, B. G., Van De Sluis, B., ... & Van Deursen, J. M. (2011). Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature, 479(7372), 232-236. https://doi.org/10.1038/nature10600 Barros, L. R. C., Couto, S. C. F., da Silva Santurio, D., Paixão, E. A., Cardoso, F., da Silva, V. J., ... & Rocha, V. (2022). Systematic review of available CAR-T cell trials around the world. Cancers, 14(11), 2667. https://doi.org/10.3390/cancers14112667 Battistello, E., Hixon, K. A., Comstock, D. E., Collings, C. K., Chen, X., Hernaez, J. R., ... & Kadoch, C. (2023). Stepwise activities of mSWI/SNF family chromatin remodeling complexes direct T cell activation and exhaustion. Molecular

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CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

cell, 83(8), 1216-1236. https://doi.org/10.1016/j.molcel.2023.02.026

Beam Therapeutics Announces First Patient Dosed in Phase 1/2 Trial of BEAM-201 in Relapsed, Refractory T-ALL/T-LL. (2023, September 5). Beam Therapeutics. Retrieved October 3, 2023, from https://investors.beamtx.com/news-releases/newsrelease-details/beam-therapeutics-announces-first-patientdosed-phase-12-trial Beltinger, C., Fulda, S., Kammertoens, T., Meyer, E., Uckert, W., & Debatin, K. M. (1999). Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligandindependent death receptor aggregation and activation of caspases. Proceedings of the National Academy of Sciences, 96(15), 8699-8704. https://doi.org/10.1073/pnas.96.15.8699 Benjamin, R., Jain, N., Maus, M. V., Boissel, N., Graham, C., Jozwik, A., ... & Vekhoff, A. (2022). UCART19, a first-in-class allogeneic anti-CD19 chimeric antigen receptor T-cell therapy for adults with relapsed or refractory B-cell acute lymphoblastic leukaemia (CALM): a phase 1, dose-escalation trial. The Lancet Haematology, 9(11), e833-e843. https://doi.org/10.1016/S2352-3026(22)00245-9 Berdeja, J. G., Madduri, D., Usmani, S. Z., Jakubowiak, A., Agha, M., Cohen, A. D., ... & Jagannath, S. (2021). Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study. The Lancet, 398(10297), 314-324. https://doi.org/10.1016/S0140-6736(21)00933-8

228

William A. Haseltine, PhD

Bhatia, S. (2011). Long-term health impacts of hematopoietic stem cell transplantation inform recommendations for follow-up. Expert review of hematology, 4(4), 437-454. https://doi.org/10.1586/ehm.11.39 Bittman, K. S. (2022). Immune Cell Metabolic Fitness for Life. Antibodies, 11(2), 32. https://doi.org/10.3390/antib11020032 Boettcher, M., Joechner, A., Li, Z., Yang, S. F., & Schlegel, P. (2022). Development of CAR T Cell therapy in Children—A Comprehensive Overview. [Image of Carvyki nanobody CAR] Journal of Clinical Medicine, 11(8), 2158. https://doi.org/10.3390/jcm11082158 Boulch, M., Cazaux, M., Cuffel, A., Guerin, M. V., Garcia, Z., Alonso, R., ... & Bousso, P. (2023). Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-γ is a major determinant of CD4+ CAR T-cell antitumor activity. Nature Cancer, 1-16. https://doi.org/10.1038/s43018-023-00570-7 Boyiadzis, M. M., Dhodapkar, M. V., Brentjens, R. J., Kochenderfer, J. N., Neelapu, S. S., Maus, M. V., ... & Bishop, M. R. (2018). Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance. Journal for immunotherapy of cancer, 6(1), 1-12. https://doi.org/10.1186/s40425-018-0460-5 Brandt, L. J. B., Barnkob, M. B., Michaels, Y. S., Heiselberg, J., & Barington, T. (2020). Emerging approaches for regulation and control of car t cells: A mini review. Frontiers in Immunology , 11. https://doi.org/10.3389/fimmu.2020.00326 Breda, L., Papp, T. E., Triebwasser, M. P., Yadegari, A., Fedorky, M. T., Tanaka, N., ... & Parhiz, H. (2023). In vivo 229

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

hematopoietic stem cell modification by mRNA delivery. Science, 381(6656), 436-443. https://doi.org/10.1126/science.ade6967 Britten, O., Ragusa, D., Tosi, S., & Mostafa Kamel, Y. (2019). MLL-Rearranged Acute Leukemia with t (4; 11)(q21; q23)— Current Treatment Options. Is There a Role for CAR-T Cell Therapy?. [Image of Anti CD19 CAR T Cell] Cells, 8(11), 1341. https://www.mdpi.com/2073-4409/8/11/1341# Brookens, S. K., & Posey Jr, A. D. (2023). Chimeric Antigen Receptor T-Cell Therapy: Current Perspective on T Cell– Intrinsic, T Cell–Extrinsic, and Therapeutic Limitations. The Cancer Journal, 29(1), 28-33. https://doi.org/10.1097/PPO.0000000000000636 Burger, J. A., & Wiestner, A. (2018). Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nature Reviews Cancer, 18(3), 148-167. https://doi.org/10.1038/nrc.2017.121 Burnett, F. M. (1957). Cancer: a biological approach. Brit. med. J, 1, 779-786. DOI: https://doi.org/10.1136/bmj.1.5022.779 Caimi, P. F., Ardeshna, K. M., Reid, E., Ai, W., Lunning, M., Zain, J., ... & Hamadani, M. (2022). The antiCD19 antibody drug immunoconjugate loncastuximab achieves responses in DLBCL relapsing after antiCD19 CAR-T cell therapy. Clinical Lymphoma Myeloma and Leukemia, 22(5), e335e339. https://doi.org/10.1016/j.clml.2021.11.005

Calibr reports promising results from first-in-human clinical trial of switchable CAR-T (Clbr001 + swi019), a next-generation universal CAR-T platform designed to enhance the versatility 230

William A. Haseltine, PhD

and safety of cell therapies. (2022, September 21). Scripps Research. https://www.scripps.edu/news-and-events/pressroom/2022/20220921-calibr-cart.html Capsomidis, A., Benthall, G., Van Acker, H. H., Fisher, J., Kramer, A. M., Abeln, Z., ... & Anderson, J. (2018). Chimeric antigen receptor-engineered human gamma delta T cells: enhanced cytotoxicity with retention of cross presentation. Molecular Therapy, 26(2), 354-365. https://doi.org/10.1016/j.ymthe.2017.12.001

CAR T Cell therapy market size, share, trends and forecast (20222035). (n.d.). Roots Analysis. Retrieved August 6, 2023, from https://www.rootsanalysis.com/reports/car-t-cell-therapymarket/269.html CAR T cell. (2023). In Wikipedia. https://en.wikipedia.org/w/index.php?title=CAR_T_cell&oldid =1177105135

Car t-cell program—Search results | bmt infonet. (n.d.). Retrieved September 21, 2023, from https://www.bmtinfonet.org/car-tcell-therapy-center-directorysearch?display_name_for_the_car_t_direc_705=

CAR-T Cell therapy market share, size, trends, industry analysis report, by target antigen (CD19/CD22, BCMA, and others); by indication; by region; segment forecast, 2023—2032. (2023). Polaris; Polaris Market Research. https://www.polarismarketresearch.com/index.php/industryanalysis/car-t-cell-therapy-market

Cartesian therapeutics initiates phase 2 clinical trial of first rnaengineered cell therapy for frontline cancer. (2021, February 231

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

22). Cartesian. https://www.cartesiantherapeutics.com/2021/02/22/cartesiantherapeutics-initiates-phase-2-clinical-trial-of-first-rnaengineered-cell-therapy-for-frontline-cancer/ Cartesian Therapeutics. (2023a). Autologous t-cells expressing a

chimeric antigen receptor directed to b-cell maturation antigen (Bcma) in patients with generalized myasthenia gravis(Mg) (Clinical Trial Registration NCT04146051). clinicaltrials.gov. https://clinicaltrials.gov/study/NCT04146051 Cartesian Therapeutics. (2023b). Phase i safety study of descartes-

11 in patients with relapsed/refractory multiple myeloma (Clinical Trial Registration NCT03994705). clinicaltrials.gov. https://clinicaltrials.gov/study/NCT03994705 Cartesian Therapeutics. (2023c, January 31). Cartesian therapeutics announces dosing of first participant in a phase 2b randomized clinical trial for generalized myasthenia gravis. Cartesian. https://www.cartesiantherapeutics.com/2023/01/31/cartesiantherapeutics-announces-dosing-of-first-participant-in-a-phase2b-randomized-clinical-trial/ Carvalho, T. (2023). First two patients receive CAR T cell therapy for HIV. Nature Medicine, 29(6), 1290–1291. https://doi.org/10.1038/d41591-023-00042-6 CDC. (2022, May 2). Data & statistics on sickle cell disease | cdc. Centers for Disease Control and Prevention. https://www.cdc.gov/ncbddd/sicklecell/data.html Cea, G., Benatar, M., Verdugo, R. J., & Salinas, R. A. (2013). Thymectomy for non‐thymomatous myasthenia gravis. 232

William A. Haseltine, PhD

Cochrane Database of Systematic Reviews, (10). https://doi.org/10.1002/14651858.CD008111.pub2 Cheloha, R. W., Harmand, T. J., Wijne, C., Schwartz, T. U., & Ploegh, H. L. (2020). Exploring cellular biochemistry with nanobodies. Journal of Biological Chemistry, 295(45), 1530715327. https://doi.org/10.1074/jbc.REV120.012960 Chen, H., Wang, F., Zhang, P., Zhang, Y., Chen, Y., Fan, X., ... & He, A. (2019). Management of cytokine release syndrome related to CAR-T cell therapy. Frontiers of medicine, 13, 610617. https://doi.org/10.1007/s11684-019-0714-8 Cherkassky, L., Morello, A., Villena-Vargas, J., Feng, Y., Dimitrov, D. S., Jones, D. R., ... & Adusumilli, P. S. (2016). Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition. The Journal of clinical investigation, 126(8), 3130-3144. https://doi.org/10.1172/JCI83092 Chiesa, R., Georgiadis, C., Syed, F., Zhan, H., Etuk, A., Gkazi, S. A., ... & Qasim, W. (2023). Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia. New England Journal of Medicine. https://doi.org/10.1056/NEJMoa2300709 Chmielewski, M., & Abken, H. (2017). CAR T cells releasing IL18 convert to T-Bethigh FoxO1low effectors that exhibit augmented activity against advanced solid tumors. Cell reports, 21(11), 3205-3219. https://doi.org/10.1016/j.celrep.2017.11.063 Chmielewski, M., & Abken, H. (2020). TRUCKS, the fourth‐ generation CAR T cells: current developments and clinical

233

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

translation. Advances In Cell And Gene Therapy, 3(3), e84. https://doi.org/10.1002/acg2.84 Chong, E. A., Alanio, C., Svoboda, J., Nasta, S. D., Landsburg, D. J., Lacey, S. F., ... & Schuster, S. J. (2022). Pembrolizumab for B-cell lymphomas relapsing after or refractory to CD19directed CAR T-cell therapy. Blood, The Journal of the American Society of Hematology, 139(7), 1026-1038. https://doi.org/10.1182/blood.2021012634 Chong, E. A., Melenhorst, J. J., Lacey, S. F., Ambrose, D. E., Gonzalez, V., Levine, B. L., ... & Schuster, S. J. (2017). PD-1 blockade modulates chimeric antigen receptor (CAR)– modified T cells: refueling the CAR. Blood, The Journal of the American Society of Hematology, 129(8), 1039-1041. https://doi.org/10.1182/blood-2016-09-738245 Chu, S. H., Ortega, M., Feliciano, P., Winton, V., Xu, C., Haupt, D., McDonald, T., Martinez, S., Liquori, A., Marshall, J., Lam, D., Olins, J., Rinaldi, C., Rehberger, K., Lazarra, C., Decker, J., Gantzer, B., Sun, M., Bohnuud, T., … Ciaramella, G. (n.d.). Conversion of HbS to Hb G-Makassar by adenine

base editing is compatible with normal hemoglobin function [Beam Therapeutics]. https://beamtx.com/media/aukjxapx/202112-ash-conversion-ofhbs-to-hb-g-makassar.pdf College, O. (n.d.-a). Illustration from Anatomy & Physiology,

Connexions Web site. Http://cnx.org/content/col11496/1.6/, Jun 19, 2013. Anatomy & Physiology, Connexions Web site. http://cnx.org/content/col11496/1.6/, Jun 19, 2013. Retrieved October 9, 2023, from

234

William A. Haseltine, PhD

https://commons.wikimedia.org/wiki/File:2215_AlphaBeta_T_Cell_Receptor.jpg College, O. (n.d.-b). Illustration from Anatomy & Physiology,

Connexions Web site. Http://cnx.org/content/col11496/1.6/, Jun 19, 2013. Anatomy & Physiology, Connexions Web site. http://cnx.org/content/col11496/1.6/, Jun 19, 2013. Retrieved October 10, 2023, from https://commons.wikimedia.org/wiki/File:2215_AlphaBeta_T_Cell_Receptor.jpg

Common cancer types. (2023, March 7). National Cancer Institute. https://www.cancer.gov/types/common-cancers Connolly, L. (2023, April 19). Clinical trial begins using CAR T cells to potentially cure HIV. UC Davis Health. https://health.ucdavis.edu/news/headlines/clinical-trial-beginsusing-car-t-cells-to-potentially-cure-hiv/2023/04 Daniels, K. G., Wang, S., Simic, M. S., Bhargava, H. K., Capponi, S., Tonai, Y., ... & Lim, W. A. (2022). Decoding CAR T cell phenotype using combinatorial signaling motif libraries and machine learning. Science, 378(6625), 1194-1200. https://doi.org/10.1126/science.abq0225 DeMarco, C. (2023, January 20). Need CAR T cell therapy? Here’s what to expect. MD Anderson Cancer Center. Retrieved March 6, 2023, from https://www.mdanderson.org/cancerwise/need-car-t-celltherapy--here-is-what-to-expect-what-happens-during-car-t-celltherapy.h00-159615489.html Di Stasi, A., Tey, S. K., Dotti, G., Fujita, Y., Kennedy-Nasser, A., Martinez, C., ... & Brenner, M. K. (2011). Inducible apoptosis 235

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

as a safety switch for adoptive cell therapy. New England Journal of Medicine, 365(18), 1673-1683. https://doi.org/10.1056/NEJMoa1106152 Diaconu, I., Ballard, B., Zhang, M., Chen, Y., West, J., Dotti, G., & Savoldo, B. (2017). Inducible caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells. Molecular Therapy, 25(3), 580-592. https://doi.org/10.1016/j.ymthe.2017.01.011 Dickinson, M. J., Carlo-Stella, C., Morschhauser, F., Bachy, E., Corradini, P., Iacoboni, G., ... & Hutchings, M. (2022). Glofitamab for relapsed or refractory diffuse large B-cell lymphoma. New England Journal of Medicine, 387(24), 22202231. https://doi.org/10.1056/NEJMoa2206913 Duan, D., Wang, K., Wei, C., Feng, D., Liu, Y., He, Q., ... & Gao, J. (2021). The BCMA-targeted fourth-generation CAR-T cells secreting IL-7 and CCL19 for therapy of refractory/recurrent multiple myeloma. Frontiers in Immunology, 12, 609421. https://doi.org/10.3389/fimmu.2021.609421 Falcon, C., Smith, L., Al-Obaidi, M., Abu Zaanona, M., Purvis, K., Minagawa, K., ... & Di Stasi, A. (2022). Combinatorial suicide gene strategies for the safety of cell therapies. Frontiers in immunology, 13, 975233. https://doi.org/10.3389/fimmu.2022.975233

FasTCAR | Gracell. (n.d.). Gracell. Retrieved October 4, 2023, from https://www.gracellbio.com/fastcar/ Fidler, B. (2022, February 7). ‘The music stopped’: Biotech rout leaves drug startups grounded as demand slumps for IPOs. BioPharma Dive. 236

William A. Haseltine, PhD

https://www.biopharmadive.com/news/biotech-ipo-venturestartup-investors-market-downturn/618205/ Fernández de Larrea, C., Staehr, M., Lopez, A. V., Ng, K. Y., Chen, Y., Godfrey, W. D., ... & Smith, E. L. (2020). Defining an optimal dual-targeted CAR T-cell therapy approach simultaneously targeting BCMA and GPRC5D to prevent BCMA escape–driven relapse in multiple myeloma. Blood cancer discovery, 1(2), 146-154. https://doi.org/10.1158/26433230.BCD-20-0020 Foy, S. P., Jacoby, K., Bota, D. A., Hunter, T., Pan, Z., Stawiski, E., ... & Mandl, S. J. (2023). Non-viral precision T cell receptor replacement for personalized cell therapy. Nature, 615(7953), 687-696. https://doi.org/10.1038/s41586-022-055311 Gargett, T., & Brown, M. P. (2014). The inducible caspase-9 suicide gene system as a “safety switch” to limit on-target, offtumor toxicities of chimeric antigen receptor T cells. Frontiers in pharmacology, 5, 235. https://doi.org/10.3389/fphar.2014.00235 Gauthier, J., Bezerra, E. D., Hirayama, A. V., Fiorenza, S., Sheih, A., Chou, C. K., ... & Turtle, C. J. (2021). Factors associated with outcomes after a second CD19-targeted CAR T-cell infusion for refractory B-cell malignancies. Blood, The Journal of the American Society of Hematology, 137(3), 323-335. https://doi.org/10.1182/blood.2020006770 Good, C. R., Aznar, M. A., Kuramitsu, S., Samareh, P., Agarwal, S., Donahue, G., ... & June, C. H. (2021). An NK-like CAR T

237

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

cell transition in CAR T cell dysfunction. Cell, 184(25), 60816100. https://doi.org/10.1016/j.cell.2021.11.016 Granit, V., Benatar, M., Kurtoglu, M., Miljković, M. D., Chahin, N., Sahagian, G., ... & Jewell, C. (2023). Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1b/2a study. The Lancet Neurology, 22(7), 578-590. https://doi.org/10.1016/S1474-4422(23)00194-1 Greenberg, P. D., Kern, D. E., & Cheever, M. A. (1985). Therapy of disseminated murine leukemia with cyclophosphamide and immune Lyt-1+, 2-T cells. Tumor eradication does not require participation of cytotoxic T cells. The Journal of experimental medicine, 161(5), 1122-1134. https://doi.org/10.1084/jem.161.5.1122 Gust, J., Hay, K. A., Hanafi, L. A., Li, D., Myerson, D., GonzalezCuyar, L. F., ... & Turtle, C. J. (2017). Endothelial activation and blood–brain barrier disruption in neurotoxicity after adoptive immunotherapy with CD19 CAR-T cells. Cancer discovery, 7(12), 1404-1419. https://doi.org/10.1158/21598290.CD-17-0698 Guy, K. (2016, August 23). Scott mcintyre’s medical bills, organized by kent guy. GoFundMe. https://www.gofundme.com/f/kua7bkhm Halim, L., & Maher, J. (2020). CAR T-cell immunotherapy of Bcell malignancy: The story so far. Therapeutic Advances in Vaccines and Immunotherapy, 8, 251513552092716. https://doi.org/10.1177/2515135520927164

238

William A. Haseltine, PhD

Harris, D. T., & Kranz, D. M. (2016). Adoptive T cell therapies: a comparison of T cell receptors and chimeric antigen receptors. Trends in pharmacological sciences, 37(3), 220-230. https://doi.org/10.1016/j.tips.2015.11.004 Haseltine, W. A. (2022a, October 14). The remarkable research of car t therapy [part ii]: B cell cancers . Forbes. https://www.forbes.com/sites/williamhaseltine/2022/10/14/theremarkable-research-of-car-t-therapy-part-ii-b-cell-cancers/ Haseltine, W. A. (2022b, October 21). Car t therapy for drug resistant multiple myeloma. Forbes. https://www.forbes.com/sites/williamhaseltine/2022/10/21/car-ttherapy-for-drug-resistant-multiple-myeloma/ Haseltine, W. A. (2022c, November 9). Car t-mrna therapy for cardiac fibrosis: A new way forward. Forbes. https://www.forbes.com/sites/williamhaseltine/2022/11/09/car-ttherapy-for-cardiac-fibrosis-a-new-method/ Haseltine, W. A. (2022d, November 15). Researchers control

cancer treatment with new innovation: Car t switch(Blade). Forbes. https://www.forbes.com/sites/williamhaseltine/2022/11/15/resea rchers-control-cancer-treatment-with-new-innovation-car-tswitchblade/ Haseltine, W. A. (2022e, November 15). Researchers control

cancer treatment with new innovation: Car t switch(Blade). Forbes. https://www.forbes.com/sites/williamhaseltine/2022/11/15/resea rchers-control-cancer-treatment-with-new-innovation-car-tswitchblade/

239

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Haseltine, W. A. (2022f, November 23). Crispr technology to simplify and enhance car t cancer treatment. Forbes. https://www.forbes.com/sites/williamhaseltine/2022/11/23/crisp r-technology-to-simplify-and-enhance-car-t-cancer-treatment/ Haseltine, W. A. (2022g, December 19). Car t therapy to treat and cure rheumatoid arthritis . Forbes. https://www.forbes.com/sites/williamhaseltine/2022/12/19/car-ttherapy-to-treat-and-cure-rheumatoid-arthritis/ Haseltine, W. A. (2023a, January 17). Progress in tissue engineering: Controlling cell-cell signaling. Forbes. https://www.forbes.com/sites/williamhaseltine/2023/01/17/prog ress-in-tissue-engineering-controlling-cell-cell-signaling/ Haseltine, W. A. (2023b, February 23). Hope for universal, readymade car t therapy for multiple myeloma. Forbes. https://www.forbes.com/sites/williamhaseltine/2023/02/23/hope -for-universal-ready-made-car-t-therapy-for-multiple-myeloma/ Haseltine, W. A. (2023c, May 25). Synnotch, a new generation of flexible and controllable car t therapies(Part 2). Forbes. https://www.forbes.com/sites/williamhaseltine/2023/05/25/synn otch-a-new-generation-of-flexible-and-controllable-car-ttherapies-part-2/ Haseltine, W. A. (2023d, July 27). The future of cancer treatment? Treating multiple myeloma with mrna-car t technology. Forbes; Forbes. https://www.forbes.com/sites/williamhaseltine/2023/06/27/thefuture-of-cancer-treatment-treating-multiple-myeloma-withmrna-car-t-technology/

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William A. Haseltine, PhD

Hashem Boroojerdi, M., Rahbarizadeh, F., Safarzadeh Kozani, P., Kamali, E., & Safarzadeh Kozani, P. (2020). Strategies for having a more effective and less toxic CAR T-cell therapy for acute lymphoblastic leukemia. Medical Oncology, 37, 1-15. https://doi.org/10.1007/s12032-020-01416-3 Hawkins, E. R., D’Souza, R. R., & Klampatsa, A. (2021). Armored CAR T-cells: the next chapter in T-cell cancer immunotherapy. Biologics: Targets and Therapy, 95-105. https://doi.org/10.2147/BTT.S291768 Hinderer, S., & Schenke-Layland, K. (2019). Cardiac fibrosis–A short review of causes and therapeutic strategies. [Figure] Advanced drug delivery reviews, 146, 77-82. https://doi.org/10.1016/j.addr.2019.05.011 He, S., & Sharpless, N. E. (2017). Senescence in health and disease. Cell, 169(6), 1000-1011. https://doi.org/10.1016/j.cell.2017.05.015 Heczey, A., Louis, C. U., Savoldo, B., Dakhova, O., Durett, A., Grilley, B., ... & Brenner, M. K. (2017). CAR T cells administered in combination with lymphodepletion and PD-1 inhibition to patients with neuroblastoma. Molecular therapy, 25(9), 2214-2224. https://doi.org/10.1016/j.ymthe.2017.05.012 Heczey, A., Xu, X., Courtney, A. N., Tian, G., Barragan, G. A., Guo, L., ... & Metelitsa, L. S. (2023). Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results. Nature Medicine, 1-10. https://doi.org/10.1038/s41591-023-02363-y Hernandez, I., Prasad, V., & Gellad, W. F. (2018). Total costs of chimeric antigen receptor T-cell immunotherapy. JAMA 241

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

oncology, 4(7), 994-996. https://doi.org/10.1001/jamaoncol.2018.0977 Hu, B., Ren, J., Luo, Y., Keith, B., Young, R. M., Scholler, J., ... & June, C. H. (2017). Augmentation of antitumor immunity by human and mouse CAR T cells secreting IL-18. Cell reports, 20(13), 3025-3033. https://doi.org/10.1016/j.celrep.2017.09.002 Hughes-Parry HE, Cross RS, Jenkins MR. The Evolving Protein Engineering in the Design of Chimeric Antigen Receptor T Cells. International Journal of Molecular Sciences. 2020; 21(1):204. https://doi.org/10.3390/ijms21010204 Imber, B. S., Sadelain, M., DeSelm, C., Batlevi, C., Brentjens, R. J., Dahi, P. B., ... & Yahalom, J. (2020). Early experience using salvage radiotherapy for relapsed/refractory non‐Hodgkin lymphomas after CD19 chimeric antigen receptor (CAR) T cell therapy. British journal of haematology, 190(1), 45-51. https://doi.org/10.1111/bjh.16541 Imura, Y., Ando, M., Kondo, T., Ito, M., & Yoshimura, A. (2020). CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD. JCI insight, 5(14). https://doi.org/10.1172/jci.insight.136185

Janssen Announces Unblinding of Phase 3 CARTITUDE-4 Study of CARVYKTI® (cilta-cel) as Primary Endpoint Met in Treatment of Patients with Relapsed and Refractory Multiple Myeloma. (2023, January 27). Johnson & Johnson. https://www.jnj.com/janssen-announces-unblinding-of-phase-3cartitude-4-study-of-carvykti-cilta-cel-as-primary-endpoint-metin-treatment-of-patients-with-relapsed-and-refractory-multiplemyeloma

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Jensen, K. (2023, October 11). Cell therapy developer Sana turns again to layoffs. BioPharma Dive. https://www.biopharmadive.com/news/sana-layoffsrestructuring-cell-therapy/696283/ Jiménez-Reinoso, A., Tirado, N., Martinez-Moreno, A., Díaz, V. M., García-Peydró, M., Hangiu, O., ... & Martínez, D. S. (2022). Efficient preclinical treatment of cortical T cell acute lymphoblastic leukemia with T lymphocytes secreting antiCD1a T cell engagers. Journal for Immunotherapy of Cancer, 10(12). https://doi.org/10.1136/jitc-2022-005333 Johnson, P. C., Dhawale, T., Newcomb, R. A., Amonoo, H. L., Lavoie, M. W., Vaughn, D., ... & El-Jawahri, A. (2023). Longitudinal patient-reported outcomes in patients receiving chimeric antigen receptor T-cell therapy. Blood Advances, 7(14), 3541-3550. https://doi.org/10.1182/bloodadvances.2022009117 Kagoya, Y., Tanaka, S., Guo, T., Anczurowski, M., Wang, C. H., Saso, K., ... & Hirano, N. (2018). A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nature medicine, 24(3), 352-359. https://doi.org/10.1038/nm.4478 Kansagra, A. J., Frey, N. V., Bar, M., Laetsch, T. W., Carpenter, P. A., Savani, B. N., ... & Hashmi, S. K. (2019). Clinical utilization of Chimeric Antigen Receptor T-cells (CAR-T) in B-cell acute lymphoblastic leukemia (ALL)–an expert opinion from the European Society for Blood and Marrow Transplantation (EBMT) and the American Society for Blood and Marrow Transplantation (ASBMT). Bone marrow

243

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

transplantation, 54(11), 1868-1880. https://doi.org/10.1038/s41409-019-0451-2 Kershaw, M. H., Westwood, J. A., Parker, L. L., Wang, G., Eshhar, Z., Mavroukakis, S. A., ... & Hwu, P. (2006). A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clinical cancer research, 12(20), 6106-6115. https://doi.org/10.1158/1078-0432.CCR-06-1183

Key statistics for multiple myeloma. (2023, January 12). American Cancer Society. https://www.cancer.org/cancer/types/multiplemyeloma/about/key-statistics.html Khaddour, K., Hana, C. K., & Mewawalla, P. (2019). Hematopoietic stem cell transplantation. https://www.ncbi.nlm.nih.gov/books/NBK536951/ Kim, S. K., & Cho, S. W. (2022). The evasion mechanisms of cancer immunity and drug intervention in the tumor microenvironment. Frontiers in Pharmacology, 13, 868695. https://doi.org/10.3389/fphar.2022.868695

Kite’s yescartaTM (Axicabtagene ciloleucel) becomes first car t therapy approved by the fda for the treatment of adult patients with relapsed or refractory large b-cell lymphoma after two or more lines of systemic therapy. (2017, October 18). Gilead. https://www.gilead.com/news-and-press/press-room/pressreleases/2017/10/kites-yescarta-axicabtagene-ciloleucelbecomes-first-car-t-therapy-approved-by-the-fda-for-thetreatment-of-adult-patients-with-relapsed-or-refrac Korell, F., Schubert, M. L., Sauer, T., Schmitt, A., Derigs, P., Weber, T. F., ... & Schmitt, M. (2021). Infection complications after lymphodepletion and dosing of chimeric 244

William A. Haseltine, PhD

antigen receptor T (CAR-T) cell therapy in patients with relapsed/refractory acute lymphoblastic leukemia or B cell non-Hodgkin lymphoma. Cancers, 13(7), 1684. https://doi.org/10.3390/cancers13071684 Kruse, B., Buzzai, A. C., Shridhar, N., Braun, A. D., Gellert, S., Knauth, K., ... & Tüting, T. (2023). CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours. Nature, 1-8. https://doi.org/10.1038/s41586-023-06199-x Kübelbeck, A., & Lynch, P. J. (2008). Schematische Darstellung

eines implantierten Ommaya-Reservoirs. https://commons.wikimedia.org/wiki/File:Ommaya_01.png Kueberuwa, G., Kalaitsidou, M., Cheadle, E., Hawkins, R. E., & Gilham, D. E. (2018). CD19 CAR T cells expressing IL-12 eradicate lymphoma in fully lymphoreplete mice through induction of host immunity. Molecular Therapy-Oncolytics, 8, 41-51. https://doi.org/10.1016/j.omto.2017.12.003

Kymriah, the first CAR T-cell therapy, works for nearly half of blood cancer patients. (2022, July 12). Alliance for Cancer Gene Therapy. https://acgtfoundation.org/news/kymriahsurvival-data-shows-promise-of-car-t-cell-therapy/

KYMRIAH® (tisagenlecleucel) Package Insert. Food and Drug Administration (2017). (2023, May). https://www.fda.gov/media/107296/download Lanzkowsky, P. (2011). Chapter 29—Hematopoietic stem cell transplantation. In P. Lanzkowsky (Ed.), Manual of Pediatric Hematology and Oncology (Fifth Edition) (pp. 806–838). Academic Press. https://doi.org/10.1016/B978-0-12-3751546.00029-X 245

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Lawrence, D. A. (2018). 11. 01—Overview of the immune system and immunotoxicology. In C. A. McQueen (Ed.), Comprehensive Toxicology (Third Edition) (pp. 1–28). Elsevier. https://doi.org/10.1016/B978-0-08-100601-6.95626-6 Lee, E. H. J., Murad, J. P., Christian, L., Gibson, J., Yamaguchi, Y., Cullen, C., ... & Priceman, S. J. (2023). Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting. Nature Communications, 14(1), 4737. https://doi.org/10.1038/s41467-023-40115-1 Lee, H., Ahn, S., Maity, R., Leblay, N., Ziccheddu, B., Truger, M., ... & Bahlis, N. J. (2023). Mechanisms of antigen escape from BCMA-or GPRC5D-targeted immunotherapies in multiple myeloma. Nature Medicine, 1-12. https://doi.org/10.1038/s41591-023-02491-5 Li, H. S., Israni, D. V., Gagnon, K. A., Gan, K. A., Raymond, M. H., Sander, J. D., ... & Khalil, A. S. (2022). Multidimensional control of therapeutic human cell function with synthetic gene circuits. Science, 378(6625), 1227-1234. https://doi.org/10.1126/science.ade0156 Li, H., Song, W., Li, Z., & Zhang, M. (2022). Preclinical and clinical studies of CAR-NK-cell therapies for malignancies. Frontiers in Immunology, 13, 992232. https://doi.org/10.3389/fimmu.2022.992232 Li, L., Mohanty, V., Dou, J., Huang, Y., Banerjee, P. P., Miao, Q., ... & Rezvani, K. (2023). Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering. Science Advances, 9(30), eadd6997. https://doi.org/10.1126/sciadv.add6997

246

William A. Haseltine, PhD

Light, D. W., & Lexchin, J. (2021). The costs of coronavirus vaccines and their pricing. Journal of the Royal Society of Medicine, 114(11), 502-504. https://doi.org/10.1177/01410768211053006 Lin, L., Cho, S. F., Xing, L., Wen, K., Li, Y., Yu, T., ... & Tai, Y. T. (2021). Preclinical evaluation of CD8+ anti-BCMA mRNA CAR T cells for treatment of multiple myeloma. Leukemia, 35(3), 752-763. https://doi.org/10.1038/s41375-020-0951-5 Lindner, S. E., Johnson, S. M., Brown, C. E., & Wang, L. D. (2020). Chimeric antigen receptor signaling: Functional consequences and design implications. [Image of CAR] Science advances, 6(21), eaaz3223. https://doi.org/10.1126/sciadv.aaz3223 Liu, J., Zhou, G., Zhang, L., & Zhao, Q. (2019). Building potent chimeric antigen receptor T cells with CRISPR genome editing. Frontiers in immunology, 10, 456. https://doi.org/10.3389/fimmu.2019.00456

Long Term Follow-Up After Administration of Human Gene Therapy Products: Guidance for Industry. (2020). Food and Drug Administration. https://www.fda.gov/media/113768/download

Longer-term Data from CARTITUDE-1 Study Demonstrate Continued Deep and Durable Responses to CARVYKTI™ (ciltacabtagene autoleucel) in Heavily Pretreated Patients with Relapsed or Refractory Multiple Myeloma. Johnson & Johnson. (2022, June 4). Retrieved August 12, 2023, from https://www.jnj.com/longer-term-data-from-cartitude-1-studydemonstrate-continued-deep-and-durable-responses-to-carvykti-

247

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

ciltacabtagene-autoleucel-in-heavily-pretreated-patients-withrelapsed-or-refractory-multiple-myeloma Mabuchi, T., Singh, T. P., Takekoshi, T., Jia, G. F., Wu, X., Kao, M. C., ... & Hwang, S. T. (2013). CCR6 is required for epidermal trafficking of γδ-T cells in an IL-23-induced model of psoriasiform dermatitis. Journal of Investigative dermatology, 133(1), 164-171. https://doi.org/10.1038/jid.2012.260 MacDonald, K. G., Hoeppli, R. E., Huang, Q., Gillies, J., Luciani, D. S., Orban, P. C., ... & Levings, M. K. (2016). Alloantigenspecific regulatory T cells generated with a chimeric antigen receptor. The Journal of clinical investigation, 126(4), 14131424. https://doi.org/10.1172/JCI82771 Mackensen, A., Müller, F., Mougiakakos, D., Böltz, S., Wilhelm, A., Aigner, M., ... & Schett, G. (2022). Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nature medicine, 28(10), 2124-2132. https://doi.org/10.1038/s41591022-02017-5 Mahadeo, K. M., Khazal, S. J., Abdel-Azim, H., Fitzgerald, J. C., Taraseviciute, A., Bollard, C. M., ... & Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network. (2019). Management guidelines for paediatric patients receiving chimeric antigen receptor T cell therapy. Nature Reviews Clinical Oncology, 16(1), 45-63. https://doi.org/10.1038/s41571-018-0075-2 Mailankody, S., Matous, J. V., Chhabra, S., Liedtke, M., Sidana, S., Oluwole, O. O., ... & Kumar, S. K. (2023). Allogeneic BCMA-targeting CAR T cells in relapsed/refractory multiple myeloma: phase 1 UNIVERSAL trial interim results. Nature

248

William A. Haseltine, PhD

Medicine, 29(2), 422-429. https://doi.org/10.1038/s41591-02202182-7 Major, A., Yu, J., Shukla, N., Che, Y., Karrison, T. G., Treitman, R., ... & Kline, J. (2023). Efficacy of checkpoint inhibition after CAR-T failure in aggressive B-cell lymphomas: outcomes from 15 US institutions. Blood advances , 7(16), 4528-4538. https://doi.org/10.1182/bloodadvances.2023010016 Majzner, R. G., Ramakrishna, S., Yeom, K. W., Patel, S., Chinnasamy, H., Schultz, L. M., ... & Monje, M. (2022). GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature, 603(7903), 934-941. https://doi.org/10.1038/s41586-022-04489-4 MarketScreener. (2023, April 21). Abstract titled “First Phase 3

Results From CARTITUDE-4: Cilta-cel versus standard of care (Pvd or dpd) in lenalidomide-refractory multiple myeloma” - Form 6-K -April 21, 2023 at 08:40 am EDT | MarketScreener . Market Screener. https://www.marketscreener.com/quote/stock/LEGENDBIOTECH-CORPORATIO-107863240/news/Abstract-titledFirst-Phase-3-Results-From-CARTITUDE-4-Cilta-cel-versusstandard-of-care-PVd-or-43603023/ Martin, T., Usmani, S. Z., Berdeja, J. G., Jakubowiak, A., Agha, M., Cohen, A. D., ... & Jagannath, S. (2021). Updated results from CARTITUDE-1: phase 1b/2Study of ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T cell therapy, in patients with relapsed/refractory multiple myeloma. Blood, 138, 549. https://doi.org/10.1182/blood-2021-146060

249

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Maude, S. L., Hucks, G. E., Callahan, C., Baniewicz, D., Fasano, C., Barker, C., ... & Grupp, S. A. (2017). Durable remissions with humanized CD19-targeted chimeric antigen receptor (CAR)-modified T cells in CAR-naive and CAR-exposed children and young adults with relapsed/refractory acute lymphoblastic leukemia. Blood, 130, 1319. https://doi.org/10.1182/blood.V130.Suppl_1.1319.1319 Maus, M. V., Haas, A. R., Beatty, G. L., Albelda, S. M., Levine, B. L., Liu, X., ... & June, C. H. (2013). T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer immunology research, 1(1), 26-31. https://doi.org/10.1158/2326-6066.CIR-13-0006 McCarthy, N. E., & Eberl, M. (2018). Human γδ T-cell control of mucosal immunity and inflammation. Frontiers in immunology, 9, 985. https://doi.org/10.3389/fimmu.2018.00985 McHugh, D., & Gil, J. (2018). Senescence and aging: Causes, consequences, and therapeutic avenues. Journal of Cell Biology, 217(1), 65-77. https://doi.org/10.1083/jcb.201708092 Mehrabadi, A. Z., Ranjbar, R., Farzanehpour, M., Shahriary, A., Dorostkar, R., Hamidinejad, M. A., & Ghaleh, H. E. G. (2022). Therapeutic potential of CAR T cell in malignancies: A scoping review. Biomedicine & Pharmacotherapy, 146, 112512. https://doi.org/10.1016/j.biopha.2021.112512 Michaud, D. S., Houseman, E. A., Marsit, C. J., Nelson, H. H., Wiencke, J. K., & Kelsey, K. T. (2015). Understanding the role of the immune system in the development of cancer: New opportunities for population-based research. Cancer

250

William A. Haseltine, PhD

epidemiology, biomarkers & prevention, 24(12), 1811-1819. https://aacrjournals.org/cebp/article/24/12/1811/70668/Underst anding-the-Role-of-the-Immune-System-in-the Mirzaei, H. R., Mirzaei, H., Lee, S. Y., Hadjati, J., & Till, B. G. (2016). Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy. Cancer letters, 380(2), 413-423. https://doi.org/10.1016/j.canlet.2016.07.001 Mitra, A., Barua, A., Huang, L., Ganguly, S., Feng, Q., & He, B. (2023). From bench to bedside: the history and progress of CAR T cell therapy. Frontiers in Immunology, 14, 1188049. https://doi.org/10.3389/fimmu.2023.1188049 Moritz, D., Wels, W., Mattern, J., & Groner, B. (1994). Cytotoxic T lymphocytes with a grafted recognition specificity for ERBB2-expressing tumor cells. Proceedings of the National Academy of Sciences, 91(10), 4318-4322. https://doi.org/10.1073/pnas.91.10.4318 Morsut, L., Roybal, K. T., Xiong, X., Gordley, R. M., Coyle, S. M., Thomson, M., & Lim, W. A. (2016). Engineering customized cell sensing and response behaviors using synthetic notch receptors. Cell, 164(4), 780-791. https://doi.org/10.1016/j.cell.2016.01.012 Munshi, N. C., Anderson Jr, L. D., Shah, N., Madduri, D., Berdeja, J., Lonial, S., ... & San-Miguel, J. (2021). Idecabtagene vicleucel in relapsed and refractory multiple myeloma. New England Journal of Medicine, 384(8), 705-716. https://doi.org/10.1056/NEJMoa2024850

251

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Ncd—Chimeric antigen receptor (Car) t-cell therapy(110.24). (2019, August 7). Centers for Medicare & Medicaid Services. https://www.cms.gov/medicare-coveragedatabase/view/ncd.aspx?ncdid=374&bc=CAAAAAAAAAAA Newby, G. A., Yen, J. S., Woodard, K. J., Mayuranathan, T., Lazzarotto, C. R., Li, Y., ... & Liu, D. R. (2021). Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature, 595(7866), 295-302. https://doi.org/10.1038/s41586-021-03609-w Noyan, F., Zimmermann, K., Hardtke-Wolenski, M., Knoefel, A., Schulde, E., Geffers, R., ... & Jaeckel, E. (2017). Prevention of allograft rejection by use of regulatory T cells with an MHCspecific chimeric antigen receptor. American Journal of Transplantation, 17(4), 917-930. https://doi.org/10.1111/ajt.14175

Novartis sets a price of $475,000 for car t-cell therapy. (2017, August 31). OncLive. https://www.onclive.com/view/novartissets-a-price-of-475000-for-car-tcell-therapy Ou, L., Wang, H., Liu, Q., Zhang, J., Lu, H., Luo, L., ... & Xu, X. (2021). Dichotomous and stable gamma delta T-cell number and function in healthy individuals. Journal for Immunotherapy of Cancer, 9(5). https://doi.org/10.1136/jitc2020-002274

Ovarian cancer statistics | how common is ovarian cancer . (2023, October 3). American Cancer Society. https://www.cancer.org/cancer/types/ovarian-cancer/about/keystatistics.html

252

William A. Haseltine, PhD

Overview—T cell therapy—Cell therapy research—Areas of interest—Scientific resources. (n.d.). StemCell Technologies. https://www.stemcell.com/technical-resources/area-ofinterest/cell-therapy-research/t-cell-therapy/overview.html Pagliarulo, N. (2023, April 10). Biotech layoffs gather pace as industry downturn persists. BioPharma Dive. https://www.biopharmadive.com/news/biotech-layoffs-job-cutsindustry-consolidation/647164/ Parrott, J. (2017, September 3). South Bend man a “walking miracle” after cancer treatment breakthrough. South Bend Tribune. https://www.southbendtribune.com/story/news/local/2017/09/0 3/south-bend-man-a-walking-miracle-after-cancer-treatmentbreakthrough/45708161/ Philip, B., Kokalaki, E., Mekkaoui, L., Thomas, S., Straathof, K., Flutter, B., ... & Pule, M. (2014). A highly compact epitopebased marker/suicide gene for easier and safer T-cell therapy. Blood, The Journal of the American Society of Hematology, 124(8), 1277-1287. https://doi.org/10.1182/blood-2014-01545020 Prager, I., & Watzl, C. (2019). Mechanisms of natural killer cellmediated cellular cytotoxicity. Journal of leukocyte biology , 105(6), 1319-1329. https://doi.org/10.1002/JLB.MR0718-269R Questex LLC (n.d.). 2022 Fierce Life Sciences Innovation Awards Winners. Fierce Life Sciences. Retrieved October 3, 2023, from https://fiercelifesciencesinnovationawards.fiercelifesciences.co m/fiercelifesciencesinno/winners

253

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Raetz, E. A., & Teachey, D. T. (2016). T-cell acute lymphoblastic leukemia. Hematology 2014, the American Society of Hematology Education Program Book, 2016(1), 580-588. https://doi.org/10.1182/asheducation-2016.1.580 Rasul, M. F., Hussen, B. M., Salihi, A., Ismael, B. S., Jalal, P. J., Zanichelli, A., ... & Taheri, M. (2022). Strategies to overcome the main challenges of the use of CRISPR/Cas9 as a replacement for cancer therapy [Image]. Molecular Cancer, 21(1), 64. https://doi.org/10.1186/s12943-021-01487-4 Rodgers, D. T., Mazagova, M., Hampton, E. N., Cao, Y., Ramadoss, N. S., Hardy, I. R., ... & Young, T. S. (2016). Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proceedings of the National Academy of Sciences, 113(4), E459-E468. https://doi.org/10.1073/pnas.1524155113 Ruffo, E., Butchy, A. A., Tivon, Y., So, V., Kvorjak, M., Parikh, A., ... & Lohmueller, J. (2023). Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting. Nature Communications, 14(1), 1-16. https://doi.org/10.1038/s41467-023-37863-5 Rurik, J. G., Tombácz, I., Yadegari, A., Méndez Fernández, P. O., Shewale, S. V., Li, L., ... & Epstein, J. A. (2022). CAR T cells produced in vivo to treat cardiac injury. Science, 375(6576), 91-96. https://doi.org/10.1126/science.abm0594 Sagnella, S. M., White, A. L., Yeo, D., Saxena, P., van Zandwijk, N., & Rasko, J. E. (2022). Locoregional delivery of CAR-T cells in the clinic. Pharmacological Research, 182, 106329. https://doi.org/10.1016/j.phrs.2022.106329

254

William A. Haseltine, PhD

Sahli, B., Eckwright, D., Darling, E., Gleason, P. P., & Leach, J. (2021, April 13). Chimeric Antigen Receptor T-cell (CAR-T)

Therapy Real-World Assessment of Total Cost of Care and Clinical Events for the Treatment of Relapsed or Refractory Lymphoma among 15 Million Commercially Insured Members. Academy of Managed Care Pharmacy (AMCP) Annual Meeting, National Virtual Conference. https://www.primetherapeutics.com/wpcontent/uploads/2021/09/SP_AMCP_S21_CAR-T.pdf Scholler, J., Brady, T. L., Binder-Scholl, G., Hwang, W. T., Plesa, G., Hege, K. M., ... & June, C. H. (2012). Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Science translational medicine, 4(132), 132ra53132ra53. https://doi.org/10.1126/scitranslmed.3003761 Schoutrop, E., Poiret, T., El-Serafi, I., Zhao, Y., He, R., Moter, A., ... & Mattsson, J. (2023). Tuned activation of MSLN-CAR T cells induces superior antitumor responses in ovarian cancer models. Journal for Immunotherapy of Cancer, 11(2). http://dx.doi.org/10.1136/jitc-2022-005691 Schubert, M. L., Schmitt, M., Wang, L., Ramos, C. A., Jordan, K., Müller-Tidow, C., & Dreger, P. (2021). Side-effect management of chimeric antigen receptor (CAR) T-cell therapy. Annals of oncology, 32(1), 34-48. https://doi.org/10.1016/j.annonc.2020.10.478 Sharma, N., Reagan, P. M., & Liesveld, J. L. (2022). Cytopenia after CAR-T cell therapy—a brief review of a complex problem. Cancers, 14(6), 1501. https://doi.org/10.3390/cancers14061501

255

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Shin, J. H., Park, H. B., Oh, Y. M., Lim, D. P., Lee, J. E., Seo, H. H., ... & Choi, K. (2012). Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor models. Blood, The Journal of the American Society of Hematology , 119(24), 5678-5687. https://doi.org/10.1182/blood-2011-09-380519 Shiravand, Y., Khodadadi, F., Kashani, S. M. A., Hosseini-Fard, S. R., Hosseini, S., Sadeghirad, H., ... & Kulasinghe, A. (2022). Immune checkpoint inhibitors in cancer therapy. Current Oncology, 29(5), 3044-3060. https://doi.org/10.3390/curroncol29050247 Siegel, R. L., Miller, K. D., Wagle, N. S., & Jemal, A. (2023). Cancer statistics, 2023. Ca Cancer J Clin, 73(1), 17-48. https://doi.org/10.3322/caac.21763 Simon, V., Ho, D. D., & Karim, Q. A. (2006). HIV/AIDS epidemiology, pathogenesis, prevention, and treatment. The Lancet, 368(9534), 489-504. https://doi.org/10.1016/S01406736(06)69157-5 Simonetta, F., Alvarez, M., & Negrin, R. S. (2017). Natural killer cells in graft-versus-host-disease after allogeneic hematopoietic cell transplantation. Frontiers in immunology , 8, 465. https://doi.org/10.3389/fimmu.2017.00465

Six years after CAR T-cell therapy for lymphoma, patient still cancer-free. (n.d.). Retrieved April 10, 2023, from https://www.uchicagomedicine.org/forefront/cancer-articles/awalking-miracle-car-t-cell-therapy Skinner, G. (2017, September 3). Kymriah, the first gene therapy, arrives with a $475,000 price tag. Consumer Reports. 256

William A. Haseltine, PhD

https://www.consumerreports.org/drug-prices/kymriah-firstgene-therapy-costs-475000-dollars-childhood-cancer/ Song, E. Z., & Milone, M. C. (2021). Pharmacology of Chimeric Antigen Receptor–Modified T Cells. Annual Review of Pharmacology and Toxicology, 61, 805-829. https://doi.org/10.1146/annurev-pharmtox-031720-102211 Splettstoesser, T. (2007). Cartoon representation of the zinc-finger

motif of proteins. The zinc ion (Green) is coordinated by two histidine and two cysteine amino acid residues. Based on the X-ray structure of PDB 1A1L. self-made, based on PDB structure 1A1L, the open source molecular visualization tool PyMol and Cinema 4D. https://commons.wikimedia.org/wiki/File:Zinc_finger_rendere d.png Sterner, R. C., & Sterner, R. M. (2021). CAR-T cell therapy: current limitations and potential strategies. Blood cancer journal, 11(4), 69. https://doi.org/10.1038/s41408-021-00459-7 Stevens, A. J., Harris, A. R., Gerdts, J., Kim, K. H., Trentesaux, C., Ramirez, J. T., ... & Lim, W. A. (2023). Programming multicellular assembly with synthetic cell adhesion molecules. Nature, 614(7946), 144-152. https://doi.org/10.1038/s41586022-05622-z Sun, S., Hao, H., Yang, G., Zhang, Y., & Fu, Y. (2018). Immunotherapy with CAR-modified T cells: toxicities and overcoming strategies. Journal of immunology research, 2018. https://doi.org/10.1155/2018/2386187

257

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Survival rates for multiple myeloma. (2023, March 2). American Cancer Society. https://www.cancer.org/cancer/types/multiplemyeloma/detection-diagnosis-staging/survival-rates.html

The CAR T-Cell Therapy Clinical Trial Journey: A Guide for Patients and Carers. (2021). Advanced Therapy Treatment Centeres. https://www.theattcnetwork.co.uk/car-t-patient-guide Thieblemont, C., Phillips, T., Ghesquieres, H., Cheah, C. Y., Clausen, M. R., Cunningham, D., ... & Lugtenburg, P. J. (2023). Epcoritamab, a novel, subcutaneous CD3xCD20 bispecific T-cell–engaging antibody, in relapsed or refractory large B-cell lymphoma: dose expansion in a phase I/II trial. Journal of Clinical Oncology, 41(12), 2238. https://doi.org/10.1200/JCO.22.01725 Tice, J., Walsh, J., Otuonye, I., Kumar, V., Chapman, R., Seidner, M., Ollendorf, D., & Pearson, S. (2018). Chimeric Antigen

Receptor T-Cell Therapy for BCell Cancers: Effectiveness and Value: Final Evidence Report. Institute for Clinical and Economic Review. https://icer.org/wpcontent/uploads/2020/10/ICER_CAR_T_Final_Evidence_Rep ort_032318.pdf Tosi, P., Barosi, G., Lazzaro, C., Liso, V., Marchetti, M., Morra, E., ... & Tura, S. (2008). Consensus conference on the management of tumor lysis syndrome. haematologica, 93(12), 1877-1885. https://doi.org/10.3324/haematol.13290 Trader, On. (2023, April 19). Legend biotech: Leaked eha abstract

shows outstanding cartitude-4 results (Nasdaq:legn) | seeking alpha. https://seekingalpha.com/article/4594992-legend-

258

William A. Haseltine, PhD

biotech-leaked-eha-abstract-shows-outstanding-cartitude-4results Vaillant, A. A. J., & Qurie, A. (2022). Immunodeficiency. In StatPearls [Internet]. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK500027/ Valton, J., Guyot, V., Boldajipour, B., Sommer, C., Pertel, T., Juillerat, A., ... & Poirot, L. (2018). A versatile safeguard for chimeric antigen receptor T-cell immunotherapies. Scientific reports, 8(1), 8972. https://doi.org/10.1038/s41598-018-27264-w Van der Schans, J. J., Van de Donk, N. W., & Mutis, T. (2020). Dual targeting to overcome current challenges in multiple myeloma CAR T-cell treatment. Frontiers in Oncology, 10, 534747. https://doi.org/10.3389/fonc.2020.01362 Van Oekelen, O., Nath, K., Mouhieddine, T. H., Farzana, T., Aleman, A., Melnekoff, D. T., ... & Parekh, S. (2023). Interventions and outcomes of patients with multiple myeloma receiving salvage therapy after BCMA-directed CAR T therapy. Blood, The Journal of the American Society of Hematology, 141(7), 756-765. https://doi.org/10.1182/blood.2022017848 Vander Mause, E. R., Baker, J. M., Dietze, K. A., Radhakrishnan, S. V., Iraguha, T., Omili, D., ... & Luetkens, T. (2023). Systematic single amino acid affinity tuning of CD229 CAR T cells retains efficacy against multiple myeloma and eliminates on-target off-tumor toxicity. Science Translational Medicine, 15(705), eadd7900. https://doi.org/10.1126/scitranslmed.add7900

259

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

Vantourout, P., & Hayday, A. (2013). Six-of-the-best: unique contributions of γδ T cells to immunology. Nature Reviews Immunology, 13(2), 88-100. https://doi.org/10.1038/nri3384

Wang, B., Iriguchi, S., Waseda, M., Ueda, N., Ueda, T., Xu, H., ... & Kaneko, S. (2021). Generation of hypoimmunogenic T cells from genetically engineered allogeneic human induced pluripotent stem cells. Nature biomedical engineering, 5(5), 429-440. https://doi.org/10.1038/s41551-021-00730-z Wang, X., & Rivière, I. (2016). Clinical manufacturing of CAR T cells: foundation of a promising therapy. Molecular TherapyOncolytics, 3. https://doi.org/10.1038/mto.2016.15 Whittington, K. B., Prislovsky, A., Beaty, J., Albritton, L., Radic, M., & Rosloniec, E. F. (2022). CD8+ T cells expressing an HLA-DR1 chimeric antigen receptor target autoimmune CD4+ T cells in an antigen-specific manner and inhibit the development of autoimmune arthritis. The Journal of Immunology, 208(1), 16-26. https://doi.org/10.4049/jimmunol.2100643 Xie, G., Dong, H., Liang, Y., Ham, J. D., Rizwan, R., & Chen, J. (2020). CAR-NK cells: A promising cellular immunotherapy for cancer. EBioMedicine, 59. https://doi.org/10.1016/j.ebiom.2020.102975 Yang, D., Sun, B., Li, S., Wei, W., Liu, X., Cui, X., ... & Zhao, X. (2023). NKG2D-CAR T cells eliminate senescent cells in aged mice and nonhuman primates. Science Translational Medicine, 15(709), eadd1951. https://doi.org/10.1126/scitranslmed.add1951

260

William A. Haseltine, PhD

Yang, J., He, J., Zhang, X., Li, J., Wang, Z., Zhang, Y., ... & Lu, P. (2022). Next-day manufacture of a novel anti-CD19 CAR-T therapy for B-cell acute lymphoblastic leukemia: first-inhuman clinical study. Blood cancer journal, 12(7), 104. https://doi.org/10.1038/s41408-022-00694-6 Yi, J., Miller, A. T., Archambault, A. S., Jones, A. J., Bradstreet, T. R., Bandla, S., ... & Hsieh, C. S. (2022). Antigen-specific depletion of CD4+ T cells by CAR T cells reveals distinct roles of higher-and lower-affinity TCRs during autoimmunity. Science immunology, 7(76), eabo0777. https://doi.org/10.1126/sciimmunol.abo0777 Yoon, V., Fridkis-Hareli, M., Munisamy, S., Lee, J., Anastasiades, D., & Stevceva, L. (2010). The GP120 molecule of HIV-1 and its interaction with T cells. Current medicinal chemistry, 17(8), 741-749. https://doi.org/10.2174/092986710790514499 Zamecnik, A. (2022, March 16). A perfect storm: Covid-19 viral vector manufacturing adds further burden to CAR-T development. Pharmaceutical Technology. https://www.pharmaceutical-technology.com/features/a-perfectstorm-covid-19-viral-vector-manufacturing-adds-further-burdento-car-t-development/ Zhao, B., Gong, W., Ma, A., Chen, J., Velegraki, M., Dong, H., ... & Wen, H. (2022). SUSD2 suppresses CD8+ T cell antitumor immunity by targeting IL-2 receptor signaling. Nature immunology, 23(11), 1588-1599. https://doi.org/10.1038/s41590-022-01326-8 Zheng, J. Y., Mixon, A. C., & McLarney, M. D. (2021). Safety, precautions, and modalities in cancer rehabilitation: an

261

CAR T: A New Cure for Cancer, Autoimmune and Inherited Disease

updated review. Current Physical Medicine and Rehabilitation Reports , 9(3), 142-153. https://doi.org/10.1007/s40141-02100312-9

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