Books

by William A. Haseltine

AffordableExcellence:theSingaporeHealthcareStory;WilliamA Haseltine(2013)

ImprovingtheHealthofMotherandChild:SolutionsfromIndia; Priya Anant, Prabal Vikram Singh, Sofi Bergkvist, William A. Haseltine&AnitaGeorge(2014)

Modern Aging:APractical GuideforDevelopers,Entrepreneurs, and Startups in the Silver Market; Edited by Sofia Widén, StephanieTreschow,andWilliamA.Haseltine(2015)

Aging with Dignity: Innovation and Challenge is Sweden-The VoiceofCareProfessionals;SofiaWidenandWilliamA.Haseltine (2017)

Every Second Counts: Saving Two Million Lives. India’s EmergencyresponseSystem.TheEMRIStory;WilliamAHaseltine (2017)

VoicesinDementiaCare;AnnaDirksenandWilliamAHaseltine (2018)

AgingWell;JeanGalianaandWilliamA.Haseltine(2019)

World Class. Adversity, Transformation and Success and NYU LangoneHealth;WilliamA.Haseltine(2019)

ScienceasaSuperpower:MyLifelongFightAgainstDiseaseAnd TheHeroesWhoMadeItPossible;WilliamA.Haseltine(2021)

Livingebooks

A Family Guide to Covid: Questions and Answers for Parents, GrandparentsandChildren;WilliamA.Haseltine(2020)

ACovidBackToSchoolGuide:QuestionsandAnswersforParents andStudents;WilliamA.Haseltine(2020)

CovidCommentaries:AChronicleofaPlague,VolumesI,II,III, IV,V,andVI;WilliamA.Haseltine(2020)

MyLifelongFightAgainstDisease:FromPolioandAIDStoCovid19;WilliamA.Haseltine(2020)

Variants!: The Shape-Shifting Challenge of Covid-19 Vaccine Evasion&Reinfection;WilliamA.Haseltine(2021)

CovidRelatedPost-traumaticStressDisorder(CV-PTSD):WhatIt IsAndWhatToDoAboutIt;WilliamA.Haseltine(2021)

NaturalImmunityAndCovid-19:WhatItIsAndHowItCanSave YourLife;WilliamA.Haseltine(2022)

Omicron: From Pandemic to Endemic; William A. Haseltine (2022)

Welcome to Monoclonal Antibodies: The Once and Future Cure forCovid-19!

Here, we investigate many of the questions raised by the discovery, fall, and resurrection of monoclonal antibodies for Covid-19. These questions are of vital importance to understanding the future of the pandemic. The stories included here were written in the heat of the moment as the news of antibody treatments emerged throughout the pandemic. As such, these stories should be considered to be snapshots in time of what we knew and when we knew it. Each story is followed by a link to the original publication, which may include more detailed figures. For the latest information about Covid-19 and new variants, please visit www.accessh.org/covid-19/

The format of this book is something that I have dubbed a living ebook a format suitable for a rapidly evolving pandemic such as COVID-19. I will continue to update Monoclonal Antibodies: The Once and Future Cure for Covid-19 as we learn more. You may download these updates at no additional cost by visiting www.williamhaseltine.com/antibody Password: antibody

Thank you for your interest.

Foreword

It was late 2020. The President was deathly ill. For a few days, amid a pandemic, he had severe Covid symptoms and refused to admit them. At long last, it became clear that he would shortly expire unless he was treated immediately. He was helicoptered with haste to Walter Reed Hospital for emergency treatment.

Within days, he had returned to the White House, spotted inside the presidential limousine waving to the crowds. What happened to cause such a remarkable turnaround? He was treated with a combination of monoclonal antibodies directed against a virus and another antiviral to quell virus production.

What were these drugs responsible for such rapid improvement? Where did they come from? Why did they work? More importantly, why have they recently stopped working? Will a time come when drugs of this sort work again?

This book answers these questions. Following the introduction, this book chronicles the rise, implementation, fall, and rebirth of monoclonal antibodies for treating Covid-19 in real-time. Each entry is accompanied by the original publication date, providing a unique lens through which to observe monoclonal antibodies throughout the Covid pandemic.

As the book approaches the present day, we outline why we believe the future for monoclonal antibodies is bright, not only for Covid19 but also for other infectious diseases.

The following introduction briefly describes monoclonal antibodies: how they work, why they failed, and how they could make their resurgence. Then we will embark on our journey through time.

Introduction

What isanAntibody?

Antibodies are small proteins crucial to our immune system’s defense against invading pathogens. Their primary role is identifying and neutralizing foreign objections and protecting the host’s cells and vital organs from infection and disease. Each antibody features a hand and glove fit, a definite shape of atoms to lock onto the pathogen it is designed to counteract, down to the resolution of one-tenth of an atomic radius. Billions of antibodies are continuously produced within human and nonhuman hosts, protecting us from common diseases such as the cold or deadly pathogens such as Covid-19.

NaturalImmunity

Most vaccines work by inducing long-lived antibodies and memory. Upon infection, there is an immediate reaction to the virus, the innate immune response, which mobilizes intracellular defenses before the development of antibodies. These defenses hold off the virus as well as they can until the immune system can initiate antibody development.

Source:ACCESSHealthInternational

Upon contact with a pathogen, the immune system sends macrophages and dendritic cells to capture and deconstruct the pathogen, presenting the invader to B cell lymphocytes. The B cell lymphocyte then carves a unique key on a blank antibody that locks solely to the captured pathogen. Therefore, every antibody is specifically tailored to the captured pathogen for which it was created. This is known as somatic hypermutation and is the bedrock of the adaptive immune response. The encoded antibody is cloned millions of times and released into the bloodstream to defend against a repeat invader. However, these antibodies fade after about a year, but lasting memory allows for long-term protection against the worst severe progression, explaining why those who have received the vaccine are more protected long-term than those who do not. Were that same pathogen to return, the memory B cells with the imprinted key would produce a wave of protective antibodies that would lock onto the pathogen's surface and either disable it themselves or signal other immune cells to come to neutralize the invader.

Monoclonal antibody treatments are a way of loading up the body system with the most effective antibodies that exist, in the most ideal case, to prevent and treat the viral disease.

Structure

Roughly 10nm long, an antibody presents in a ‘Y’ shape, consisting of four polypeptide chains. The four chains consist of two identical light chains and two identical heavy chains.

Source:ACCESSHealthInternational

The two light chains consist of a variable and constant domain, whereas the heavy chains consist of one variable and three constant domains. The chains are held together by a series of disulfide bonds.

The key region of the antibody that locks to the pathogen is known as an antigen-binding fragment. These are the two arms of the ‘Y’ shape consisting of the light chains and the variable and one constant domain of the heavy chains. The B cell lymphocyte encodes the variable domains with the antigen-binding site to the pathogen.

The trunk of the ‘Y’ shape is the Fc region, comprised of the other two constant domains of the heavy chains. This region enables effector molecule binding and half-life modification, which we will discuss later. While most antibodies we discuss in the ‘Y’ configuration are immunoglobulin G (IgG), there are also IgE and IgD antibodies in a similar configuration. Additionally, IgM and IgA are present in their unique shapes.

Source:ACCESSHealthInternational

The germline characteristics of antibodies are inherited, selected over many millions of years to approximate pathogens we are likely to encounter. Before being matched to a specific pathogen, antibodies are close but imperfect matches to an invader, much like an off-the-rack suit compared to a tailored one. An antibody is tailored to that specific target only once a pathogen is introduced. Both off-the-rack and tailored antibodies are derived from B cells, which begin in the body’s lymph nodes. The lymphatic system involves the spleen, tonsils, etc. They may become inflamed during viral infection due to B cell activation. The outer portion of lymph nodes consists of groups of inactivated B cells called follicles. Once activated, these cells produce tailored antibodies to a specific antigen.

The off-the-rack antibodies are extrafollicular and can recognize pathogens, but to a lesser extent, creating the first line of defense. Aggressive symptoms such as cytokine storms or thyroid complications may arise due to extrafollicular activation upon pathogen entry. Microorganisms will not activate all extrafollicular antibodies, only the ones that approximate, akin to a smaller man only pulling smaller suits off the rack. However, it seems some microorganisms use the approximation to their advantage, only activating extrafollicular antibodies that are least harmful to themselves. The attacker is choosing which defense our body employs.

Our aim is to find antibodies that have been activated that cause the most damage to an invading pathogen, countering its effort to activate the least damaging. Different antibody types may be activated in some cases, such as IgM or IgA. In others, there may be broadly neutralizing antibodies with a very low affinity that need scientific modification to optimize.

AntibodyVariability

From the off-the-rack antibodies, how does our body produce antibodies with such incredible specificity? Some estimates suggest a human produces roughly 10 billion distinct antibodies; all copied countless times. From the ancestral germ line cells that adapted over many years, the immune system uses several tricks and methods to re-engineer antibodies in various ways to create an army of pathogen-specific antibody soldiers.

Our immune systems have overcome this hurdle by evolving B cell antibody generation to include a swathe of complex processes such as V(D)J recombination.

V(D)J recombination is the removal and recombination of gene segments in antibodies to yield vast arrays of antibody combinations from a limited building supply. The variable domains of antibodies are comprised of gene segments categorized as variable (V), diversity (D), and joining (J). These gene segments can be rearranged significantly without altering the antigen-binding site. Take the alphabet, for example. In the English language, there are 26 letters from ‘A’ to ‘Z.’

Abcdefghijklmnopqrstuvwxyz

V(D)J combination removes unnecessary clutter from an antibody blueprint. For instance, we may not need the letters ‘C’ through ‘F’ and ‘J’ through ‘P’ AB-GHI-QRSTUVWXYZ

This process takes a set number of inputs, the letters A to Z, and yields a countless number of outputs depending on the blueprint drawn from the pathogen in question.

The result is a broad range of antibodies that bind and neutralize a wide swathe of pathogens.

How to Make Monoclonal Antibodies

Monoclonal antibodies are tailor-fitted antibodies that can be harvested, cloned, and produced in bulk by pharmaceutical companies for the purpose of antiviral treatment and prevention. We can take the tailor-fitted antibody response our bodies create against varying pathogens, identify the most effective antibodies at neutralizing those pathogens, and create a drug based on that antibody.

Monoclonal antibodies are among our greatest assets in preventing and treating moderate to severe cases of Covid-19. As new variants of SARS-CoV-2 arise, new monoclonal antibody candidates must be discovered and examined to overcome the more immune-evasive virus versions. Here we dive deeper into the discovery process and describe how our antibody drugs are isolated and examined.

InfectedHumanSeraIsolation

Throughout the pandemic, most monoclonal antibodies have been discovered by analyzing the blood samples of previously infected Covid-19 patients. When we are infected, our immune systems naturally develop antibodies against an invading pathogen to prevent the same pathogen from infecting us in the future. However, the antibodies we develop are variable, slightly differing in binding epitope, neutralization effectiveness, etc.

Sera samples are extracted from previously infected patients, and the variable SARS-CoV-2 antibodies are located and isolated for further examination. This allows for precise data on individual antibodies’ neutralization capacity, binding affinity, etc.

The hundreds, if not thousands, of antibodies are typically sorted by binding affinity, meaning the strength to which antibodies latch onto the virus target. Those that bind poorly or not at all are discarded from contention.

Next, antibody candidates are analyzed for neutralization capacity. In the case of SARS-CoV-2, antibodies are often tested against the wild-type virus and the latest emerging variants of SARS-CoV-2. An antibody that neutralizes the Wuhan virus of 2020 may not neutralize the newest Omicron versions. Those with the most robust neutralizing capacity are then closely examined by cryo-electron microscopy to determine the exact makeup of the antibody and whether it should advance to further trial.Many of the FDAapproved antibodies in the pandemic have been found using this discovery method, though most lose neutralization of later SARSCoV-2 variants.

VaccinatedHumanSeraIsolation

Since Pfizer/BioNTech and Moderna's wide release of mRNA vaccines in the Spring and Summer of 2021, roughly 68% of the US population has been fully vaccinated with two doses. When a pathogen invades a patient, and the immune system reacts by creating antibodies tailored to the pathogen, a similar process occurs with vaccination. The Covid-19 mRNA vaccines are essentially a weakened form of the SARS-CoV-2 spike protein, the portion of the virus that binds to host cells. The vaccine teaches our immune system to fight against the virus in a lower-stakes setting than an infection.

Vaccinated human sera isolation is the same process as infected human sera isolation, though the target patient is those vaccinated instead of infected. While less common, this method yields similar results to the first method. However, antibodies isolated from vaccinated sera may be less effective against later SARS-CoV-2 variants, as the vaccines were designed using the wild-type spike protein.

AnimalSeraIsolation

The third method for antibody isolation comes not from human patients, but from animals. Animal sera isolation follows the same steps as infected human sera isolation but in nonhuman hosts such as mice, hamsters, camelids, and monkeys.

There are two main benefits of animal sera isolation for antibody discovery. The first, more practical benefit is the increased flexibility of animal subjects in laboratory settings. Humans have variable schedules, lifestyles, etc.; relying on human subjects for experimentation and scientific pursuits can be more taxing than necessary. Animal subjects, however, are highly controlled, both in their availability for experimentation and lifestyles outside of the lab, as their health is closely and easily monitored. Many animals, such as mice, hamsters, camelids, and monkeys, have similar immune systems to humans and can be substituted for human subjects.

Modified germline sequencing is the second, more theoretical benefit of animal sera isolation. Throughout the pandemic, the SARS-CoV-2 virus has developed mutations to evade and overcome our immune defenses, fueling the search for new monoclonal treatments to which the virus may evolve to overcome once again. This game of cat and mouse may be avoided using animal sera isolation. The viruses circulating today have been selected against human immune responses, not animals.

For instance, a recently isolated antibody from macaque monkeys displays an inherited germline sequence not found in antibodies developed from human hosts. The core sequential structure of the antibody is different than human antibodies. This may be the key to enabling isolated antibodies to overcome rapidly mutating SARSCoV-2 variants.

PhageDisplay

The final popular method of antibody discovery is phage display. This technique involves encoding proteins on a phage coat gene, which displays the protein outside the gene. The protein can then be introduced to various other molecules to detect interactions. In simpler terms, phage display can show which proteins interact with which molecules in vitro in a very concise and organized manner.

Figure 4.Phagedisplaycycle.1)fusionproteinsfora viralcoatprotein + thegene to beevolved(typicallyanantibodyfragment)areexpressedin bacteriophage.2)thelibraryofphageis washedover an immobilized target.3)theremaininghigh-affinitybindersare usedto infectbacteria.

4)thegenesencodingthehigh-affinitybindersare isolated.5)those genesmay haverandommutations introducedandusedtoperform anotherroundofevolution.Theselectionandamplificationsteps can be performedmultipletimes atgreaterstringency to isolatehigher-affinity binders.

Source:WikipediaPublicDomain

In the early 1990s, researchers at the Scripps Research Institute reported the first use of phage display to detect and isolate human antibodies which bound tetanus toxin. Since then, laboratories and pharmaceutical companies worldwide have used phage libraries containing millions of antibodies to isolate monoclonal antibodies for human use.

CamelidNanobodies

One particular antibody-deriving animal group of note is the camelid. Camelids consist of camels, llamas, alpacas, and other members of the Camelidae family. Single-domain antibodies, or nanobodies, are fragments of a sole variable antibody domain. The first nanobodies were engineered from camelid heavy chain antibodies. They contain a single variable domain in the arms of the ‘Y’ shape and no light chain.

The main therapeutic advantage of camelid antibodies is that they are more easily administered in drug form via inhalation. Most antibody therapies require at least an intramuscular shot or, more likely, an intravenous administration, which mandates a professional to administer. They are also often as, or more specific than, regular antibodies, creating an opportunity for a highly effective treatment.

Researchers Koenig et al. isolated a panel of nanobodies from alpacas immunized with wild-type SARS-CoV-2 spike protein. Four nanobodies effectively bound and neutralized live SARS-CoV-2: E, U, V, and W. Two of these, E and V, worked well together to lock the receptor-binding domain. This region of the spike shifts between the down and up positions based on the stage of infection. By locking the receptor-binding domain in the “up” position, the virus cannot release from the host cell upon ACE2 binding, neutralizing it by preventing further spread.

In conjunction, the V and E nanobodies display a 62-fold increase in neutralization and a 22-fold increase in binding compared to the nanobodies in isolation. This trend could also be duplicated in other pathogens, as nanobodies are more effective than their IgG counterparts. Their smaller stature could lead to a more densely packed drug in the latter stages of development. More nanobodies per mL could lead to more effective moderate to severe disease treatment.

AntibodyModification

BispecificMonoclonalAntibodies

Bispecific antibodies have two binding sites directed at two antigens or two different epitopes on the same antigen. In baseball, most pitchers are only pitchers, but some are two-way players, capable of pitching and hitting, making them incredibly valuable. Bispecific antibodies are equally as valuable but uncommon. They are more commonly produced in a lab than isolated naturally. Despite their difficulties, the clinical therapeutic effects of bispecific antibodies are superior to those of monoclonal antibodies.

Immunotoxins

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Immunotoxins are a relatively novel but effective treatment in the oncology field, potentially filling a large void for accessible drugs. Two such drugs approved or in development are Lumoxiti for hairy cell leukemia, approved in the United States in 2018, and Oportuzumab monatox for bladder cancer, pending review for approval in the US.

Intrabodies

An intrabody is an antibody that works within the cell to bind to an intracellular protein. Due to the lack of a reliable mechanism for bringing antibodies into a living cell from the extracellular environment, these antibodies are modified for intracellular localization. Since 2007, intrabodies have been developed for several therapeutic applications, including hepatitis B, bird flu, prion disease, inflammation, Parkinson’s, and Huntington’s.

ArtificialIntelligence

Antibodies, while highly effective and relatively inexpensive compared to some treatments, require a time-intensive development process. Much of the time spent developing antibodies is finding ones that work. Our bodies produce seemingly countless antibodies in reaction to infection with a pathogen. When infected with something new, our immune systems go into overdrive, producing waves of new antibodies from B cells.

The antibody researcher is tasked with isolating highly effective antibodies against a given pathogen from the sera of an infected or vaccinated patient. Many of the antibodies analyzed will have little to no effect, but some could be potently neutralizing. The trick is isolating the winners. While human researchers could take days, weeks, or even months to test a large panel of antibody candidates, what if there were an artificially intelligent program to do the work in a fraction of the time?

In recent months, new and exciting advances in artificial intelligence biotechnology may pave the way for antibody discovery in the near future. Designed by researchers Parkinson etal.from the University of California - San Diego, the new AI mechanism, RESP, finds and tests antibody binding at a highly accelerated rate.

Antibody discovery typically means isolating antibodies from the convalescent sera of a patient, testing a panel of the isolated antibodies against the pathogen, then focusing on those that produce the most robust binding and neutralization. RESP does this process at a much faster rate and more.

A particularly compelling aspect of RESP is its testing of acutely mutated antibodies. It takes the antibodies isolated from sera, tests those antibodies against the pathogen, then tests the same antibodies with slightly mutated structures to see if improvements can be made to the isolated antibodies. The antibody is fine-tuned by slightly modifying the structure to be as highly binding and neutralizing as possible.

For instance, RESP found a slightly mutated version of the approved cancer immunotherapy Atezolizumab that displayed a 17fold increase in binding affinity. RESP can be used for all sorts of pathogens, Covid-19 notwithstanding. There is only one approved monoclonal antibody drug for Covid-19: Tocilizumab. This drug yields a modest 4-8% reduction in death compared to a placebo control in clinical studies.

If RESP can increase the effectiveness of the cancer therapy

Atezolizumab by 17-fold, it is not out of the question to assume similar results can be seen with Covid-19 drugs. An anti-Covid monoclonal treatment with a 68+% reduction in death rate sounds much more appealing than 4-8%. That is what makes RESP and AI antibody identification all the more special. It can improve the drugs we already produce.

If we can harness the full potential of RESP or an AI mechanism similar, it could lead to a new wave of highly effective antiviral treatments and prophylactics.

AntibodyOptimization

A critical issue with monoclonal antibodies is that they fade. Similar to how protection from severe disease fades over time with most vaccines, monoclonal antibody treatments are not permanent shields against their given pathogen. Researchers have recently developed a few methods to extend and optimize monoclonal antibodies.

FcRegions

As described in a previous section, the Fc region is the trunk of an antibody's “Y” shape. The Fc binds the Fc receptor on an effector cell. When an antibody binds a pathogen, it either neutralizes it directly or signals effector cells to neutralize it. Effector cells could be B lymphocytes, macrophages, killer cells, etc. Modifying the Fc interaction of monoclonal antibodies may yield an increased halflife for therapeutic and prophylactic use.

Source:WikipediaPublicDomain

Extended Half-Life

Using structure-guided design, researchers from Visterra Inc. extended the half-life of monoclonal antibodies over nine-fold in transgenic mice models. They observed Fc binding interactions between the Fc region and effector cell Fc receptor that mutational alterations could enhance.

They engineered a panel of unique Fc variants that could enhance Fc binding while maintaining a threshold of binding affinity and overall structural integrity. Their panel yielded several mutational sets in the Fc region that increased the half-life of monoclonal antibodies in a mouse mode. The most prominent were YTE (M252Y/S254T/T256E) and LS (M428L/N434S).

Including these mutations in monoclonal antibodies used for therapeutic use may extend their half-life by weeks, which could be crucial in critical cases of Covid-19 and Long Covid patients.

FcClassSwitching

Antibody class is determined by the heavy chain constant region. Antibodies may switch classes if the variable region is maintained and the constant region is modified. In an irreversible Fc class switching process, various enzymes create nicks in the DNA sequence at switch regions, allowing the constants to be excised. A repair enzyme joins the remaining variable segment onto a new constant region.

The differing antibody classes, IgG, IgM, and IgA, often have variable affinities and neutralizing capabilities. In many cases, IgG antibodies neutralize invading pathogens, though IgM antibodies may yield a stronger response in some cases, such as with Zika Virus.

Recent studies even indicate that IgG3 antibodies, which have a longer Fc fragment than IgG1, more efficiently activates Fc-related immune functions, leading to stronger recovery and protection for those infected with SARS-CoV-2.

EffectorFunctions

An antibody has four main methods by which antibodies exert their effects, known as effector functions: neutralization, opsonization, complement activation, and antibody-dependent cellular cytotoxicity (ADCC).

Neutralization, as we have discussed, is the process of stopping a pathogen from infecting healthy host cells.

Opsonization is a process by which the Fc region of antibodies is recognized by phagocytic cells. Instead of the antibody directly neutralizing a pathogen, it latches onto the pathogen and signals phagocytic cells to neutralize and clear the invader.

ADCC occurs when the Fc portion of an antibody bound to a cell's surface interacts with the Fc receptor on immune cells such as macrophages, NK cells, and neutrophils. This triggers the immune cell to target the antibody-coated cell for lysis.

Complement is a system of plasma proteins activated by antibodies bound on a cell. The complement cascade leads to the formation of a protein complex in bacteria that can kill the bacteria. The complement system can also activate phagocytes to destroy bacteria that would otherwise not be recognized by the immune system.

FcsthatBindComplement

In addition to binding Fc receptors on effector cells, Fc regions also bind complement proteins. The complement system is a segment of the innate immune system that enhances antibody clearance of microbes and damaged cells from the host. Complement activation promotes inflammation and pathogen cell membrane targeting.

The system is a series of fragments and subunits signaled by a bound antibody. These fragments and subunits are also called the complement cascade. Once signaled, the initial C1 complex fragments and fragments until small subunits of the initial complex recombine into the cylindrical membrane attack complex, which infiltrates and destroys the infected cell.

Source:WikipediaPublicDomain

Complement activation is not nearly as common as Fc receptor binding, as the complement system can damage host tissue and present additional negative health effects. However, its availability is a valuable tool for the antibody.

Sugar(Glycosylation)Modifications

Another antibody optimization method involves glycosylation. In the molecules of most microorganisms, there are chain-like structures composed of single sugar molecules linked together by chemical bonds. These are glycans, which are involved in the structural integrity of a molecule, as well as energy storage and systemic regulatory processes. Glycosylation is the process of covalent attachment between the glycan and the targeted macromolecule.

One macromolecule that attracts these glycans is antibodies, specifically the Fc portion of the antibody. All antibodies, including IgG, IgM, IgD, IgE, and IgA, bear N-linked glycosylation sites in their Fc heavy chain, each with varying levels of complexity due to differing Fc structures.

Recent advances in microbiological technologies have enabled us to look closely at antibody glycosylation. Firstly, it seems antibody glycosylation acts as a biomarker for disease severity. Antibody glycosylation is directly correlated to autoimmune and chronic disease severity, most notably high inflammation.

Secondly, and more directly, antibody glycosylation improves the immune response to pathogen infection. In both HIV and tuberculosis cases, patients with higher levels of glycosylated antibodies in their immune responses yielded stronger neutralizing reactions. Harnessing glycosylation in monoclonal antibody treatments could prove fruitful.

SARS-CoV-2 Monoclonal Antibodies

ApprovedAntibodyTreatments

While antibodies represent our strongest tools against the virus, we lack approved and effective antibodies against the latest variants. Because the virus constantly mutates to evade antibody neutralization, treatments that may have worked against earlier versions of the virus are greatly reduced against more recent variants. As of May 2023, there are only two FDA-authorized monoclonal antibody treatments Evusheld and Tocilizumab, which is fully approved for use in hospitalized adults.

Evusheld is the combination therapy of the Tixagevimab and Cilgavimab antibodies. It was first isolated from Covid-19 patients infected in 2020, likely with the D614G SARS-CoV-2 virus. While the emergency use authorization still stands, it was revised in early 2023 to limit its use to when the “combined frequency of nonsusceptible SARS-CoV-2 variants nationally is less than or equal to 90%.” If later virus variants comprise more than 90% of infections, Evusheld cannot be used, rendering the treatment obsolete.

Tocilizumab, while approved, only modestly improves severe Covid outcomes. In the clinical studies that led to the drug’s FDA approval, cohorts who received Tocilizumab had reduced mortality rates of 4-8%.

Drugsagainst

Source:ACCESSHealthInternational

BroadlyNeutralizingMonoclonalAntibodies

While currently available treatments are somewhat limited, several monoclonal antibody candidate drugs are in development, many of which are effective against Omicron. Those in development are focused on broad neutralization by targeting conserved regions of the spike protein. Many older monoclonals target the virus at positions mutated in later strains, whereas newer antibody candidates specifically target spike residues that are conserved across many viral variants.

For instance, a number of broadly neutralizing monoclonal antibodies currently in development target portions of the S2 subunit of the spike. Mutations that occur in later variants of SARSCoV-2 are far more common in the receptor-binding and Nterminal domains of the S1 subunit. By targeting S2, antibodies are more likely to neutralize a wider range of variants. A notable example target is the fusion peptide, which plays a crucial role in membrane fusion post-contact with the host ACE2 receptor. An antibody may impede the fusion peptide, therefore impeding fusion and halting infection via a different avenue.

A particularly exciting antibody in development is AZD3152, a combination antibody therapy by AstraZeneca. A press release by the company claims the cocktail neutralizes all variants of SARSCoV-2, combining a new antibody candidate with cilgavimab, a crucial component of the successful Evusheld treatment. We eagerly await new details about this exciting drug.

These antibodies will be discussed in great detail in later sections of this book. Broadly neutralizing antibodies represent one of the better tools we could forge against the virus. Combining two or three broadly neutralizing monoclonal antibodies into a single treatment could be a particularly effective and perhaps even post-exposure prophylactic. We need only to continue their development in the coming months and years.

AlternateCovidAntibodies

Another effective strategy for Covid neutralization is cross-trimer binding. The SARS-CoV-2 spike protein attacks a host cell in sets of three, or a trimer. While most antibodies bind the epitope of a single spike monomer, a select few bind across the spike trimer, locking the entire mechanism and preventing infection. A notable example are the camelid nanobodies described by Koenig et al. early in the pandemic. This class of antibody often demonstrates a strong neutralization capacity thanks, in large part, to the cross-trimer strategy.

Further removed from traditional antibodies, a number of recent antibody candidates have targeted aspects of the human immune system. One such example is the anti-ACE2 described by scientists at Rockefeller and Stanford Universities. The antibody, h11b11, targets the host cell ACE2 receptor, blocking the virus from binding and infecting the cell. Another example is the T cell antibody

Foralumab developed by Tiziana Life Sciences. Foralumab modulates immune reactions brought about by infection, reducing disease severity. The concern with antibodies that bind host cells is the possibility of autoimmune reactions, but those have not occurred in these cases.

MonoclonalAntibodiesforNon-CovidPathogens

A positive dividend from the emergence of SARS-CoV-2 monoclonal antibody discovery is the reinvigorated effort to develop antibodies for other problematic pathogens as well. In just the past few years, highly effective antibody candidates have been developed for some of the world’s deadliest diseases, including Ebola, Lassa Virus, Malaria, Zika, and Yellow Fever, among others.

Many of these pathogens are disproportionately active in low income nations in tropical regions and Sub-Saharan Africa. As they become available for public use, it will be critical to ensure the populations that need these interventions receive them, regardless of potential costs.

EconomicsofAntibodies

One of the major barriers to the widespread use of monoclonal antibody treatments is the cost. Throughout the pandemic, the United States government footed the bill for millions of vaccine doses and some treatments for specfic populations, shielding the patient from that out-of-pocket expense. With monoclonal antibody treatments, however, the cost to use the drug would fall on the patient and their insurer, particularly with the end of public health emergency declarations globally. Bebtelovimab, an monoclonal antibody that was previously used to treat Covid-19 but is now ineffective against Omicron, costs an eye-watering $2,100 per dose. Almost six in ten Americans do not have $1,000 to spare in case of an unexpected medical bill, much less $2,100 per dose. If a family of four sought treatment with Bebtelovimab, they would need $8,400, pending insurance contributions. That is over 10% of the median household income. Meanwhile, most antibodies only cost between $100-200 to produce per gram. Granted, this does not include the cost of research and development, but the profit margin per dose of monoclonal antibody treatment is significant. The cost could be brought down spectacularly with sufficient government intervention and regulation. Whether that occurs remains to be seen.

AZD3152:ANewHopeOnTheHorizon

A new Covid-19 antibody may be the renewed hope for SARS-CoV2 monoclonal therapies we desperately need in the ongoing Omicron era. For the past three years, emerging variants of SARSCoV-2 mutated to overcome neutralization by monoclonal antibodies. As new treatments were authorized by the FDA, new variants would circulate that evaded these treatments. In due course, antibody treatments lost the luster they once demonstrated.

At the time of writing, only Evusheld (tixagevimab + cilgavimab) and Actemra (tocilizumab) and authorized for use against Covid19. Over the past several months, we have written about the potential of many antibody candidates in development. Many of these antibodies target conserved regions of the SARS-CoV-2 spike protein that are less likely to mutate, while others may have different targets than the virus altogether, such as the anti-ACE2 monoclonal antibody.

Recently, a new antibody candidate has entered the ring: AstraZeneca’s AZD3152. While data on the antibody is limited and efficacy trials are ongoing, AZD3152 shows as much promise as any potential treatment we have discussed.

Structurally, AZD3152 is similar to many of the antibodies that have come and gone over the past three years. It is an IgG1 antibody, the most common of the four IgG subtypes. AZD3152 even has a duplicate constant region of that found in Evusheld, with only the Fab fragment antigen-binding domain replaced.

AZD3152 also does not bind an uncommon epitope. Whereas some recent antibodies we described target the S2 subunit or the fusion peptide, AZD3152 binds the often-targeted receptor-binding domain. The vast majority of the epitope is highly conserved, more than 99.9% of the time in observed sequences, but other binding residues mutate as much as 50% of the time.

However, what AZD3152 lacks in creativity, it makes up for in neutralizing capacity. Unlike any antibody we have observed, AZD3152 potently neutralizes all known variants of SARS-CoV-2, both dormant and current. At 100 ng/mL, every variant from D614G at the start of the pandemic to the many iterations of Omicron is neutralized by greater than 80%. At 1,000 ng/mL, that percentage jumps to over 95%.

We would have liked to analyze this antibody in far greater detail, but preprint or published research on AZD3152 is unavailable, as of yet. The above data was derived from a poster first displayed at the 33rd European Congress of Clinical Microbiology & Infectious Diseases in Copenhagen in mid-April.

AstraZeneca has communicated that efficacy trials are expected to be completed later this year, and early indications are that the drug will be highly successful. We eagerly anticipate any further news on this exciting treatment, as it could be the silver bullet we so desperately need to protect and treat at-risk populations from a disease that is very much still thriving.

MovingForward

There is a genuine opportunity with monoclonal antibodies to create tools that will prevent Covid-19 from further devastating communities worldwide without disrupting our daily lives. Many thousands continue to die weekly as a direct result of and due to complications from Covid-19. Only by accelerating and consistently funding research into these monoclonal antibodies will we find our way out of the Covid-19 crisis once and for all. We shall see how the United States and other countries worldwide choose to proceed with monoclonal treatments. We recommend they be pursued with great haste.