MATURE-NK by Blazon Publishing and Media Ltd

Photograph displaying splitting and feeding of cells in the cell culture laboratory. © Fraunhofer IZI

Natural killer cells to target cancer Natural killer cells have the ability to kill tumour cells, now researchers are looking to harness their potential as a means of treating different forms of cancer. The Mature NK project provides training to early stage researchers, helping them develop the skills that could lead to the development of more effective anti-cancer immunotherapies in future, as Dr Erhard Hofer explains on behalf of Dr. Ulrike Köhl, the coordinator of the project. A type of

lymphocyte, natural killer (NK) cells are an important part of the innate immune system, with the ability to kill harmful cells. Researchers in the Mature NK project are exploring the possibility of manufacturing these cells for use in treating certain types of cancer, representing an attractive alternative to other options like T-cell therapies, which can lead to toxic side-effects. “We would not have this problem with NK cells. It has been shown that NK cells are usually very welltolerated when infused into patients,” explains Dr Erhard Hofer, part of the project’s scientific management team. The project brings together research groups and biotech companies from across Europe to provide training to early stage researchers (ESRs), who are conducting research into several different aspects of NK cells. “Some of the laboratories in the project are active in basic immunology, while others are more clinically-related. The project consortium includes several research institutions and biotech companies that are active in the development of NK cell therapy,” says Dr Hofer.

Mature-NK project The wider aim here is to help translate research advances into effective treatments, with the partners in the project investigating several

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different means of modifying NK cells and then manufacturing them for use in treating certain types of cancer. There are various methods by which NK cells can be manufactured, one of which starts with taking cells from a blood donor. “The primary white blood cells of donors can be taken and then amplified. You can select the NK cells using antibodies and amplify them in special cell growth media to get a limited number of batches for NK cell

of iPSC, that then can be frozen and stored. “Therefore off-the-shelf-strategies for NK cell products are possible,” says Dr Hofer. NK cells can then be genetically modified to enhance their effectiveness in terms of identifying and then killing tumour cells. “These NK cells can be equipped with artificial effectors that would help them directly identify and kill targets like tumour cells more effectively,” explains Dr Hofer. “For

Some of the laboratories in the project are active in basic immunology, while others are more clinicallyrelated. The project consortium includes several research institutions and biotech companies that are active in the

development of natural killer cell therapy.

infusions in patients. Another method involves using induced pluripotent stem cells (iPSCs). These can be prepared from small numbers of various cell types including fibroblasts or various blood cells The advantage is that the generated iPSC cells can be grown in an unlimited way and then can be triggered to differentiate into NK cells,” outlines Dr Hofer. This will allow repeated production of large numbers of NK cells from the same batch

example, antibodies have been prepared that target tumour cells, helping NK cell receptors bind to them more effectively.” Normally, these NK cells have a number of different surface receptors, proteins that bind to both normal cells and also tumour cells. One of the ways by which NK cells kill their targets – which differentiates them from T-cells – is that they look for the presence of normal, selfidentifying receptors on the surface of normal

appropriate tumour antigen on the cancer cells, using CAR technology or NK cell engagers, the tumour could be specifically targeted, NK cells would properly invade and then the right level of reactivity would develop. Further preventing inhibitory interactions of tumour cells with NK cells will be required.” he says. “A large number of clinical trials are ongoing into the use of NK cells. Most are on leukemia, but there are also some looking at solid cancers.”

Modify cells MATURE-NK group photo taken at the first Winter School.

cells. “When they see these kinds of receptors, the activity of NK cells is blocked. These are surface molecules from the family of the socalled major histocompatibility complex. Where a cell doesn’t have these normal surface receptors, the NK cells would react by killing all cells that do not display this molecule,” says Dr Hofer. The nature of these surface receptors, that vary between individuals in a population, is an important factor in matching the donor for the most reactive NK cells to the right recipient, and Dr Hofer says tools are available now to help achieve this. “If you take cells from a donor, they need to be selected in such a way that their reactivity towards the patient’s cell is as high as possible. There are now reagents around to do this,” he outlines. Furthermore, a number of different methods are available to enhance the reactivity of the cells. The easiest way is simply to activate cells using cytokines, natural triggers that increase the general activity of the cell, while Dr Hofer says there are also other options. “You could genetically modify the cell – one possibility is using so-called Chimeric Antigen Receptor (CAR) constructs, that would endow the NK cells to bind specifically to tumour antigens and to get activated for killing. Another possibility is to use soluble, multi-valent cross-linkers,” he explains. These cross-linkers would be constructed from single chain antibodies that bind to the tumour surface and NK cell receptors (NK cell engagers). “These are soluble reagents that could be infused into the bloodstream for example, or they could be bound to therapeutic NK cells before infusion. Then they could be used to increase the chance that the NK cell would find the target cells - in that case the tumour cells,” continues Dr Hofer. “The NK cells would then bind – via this reagent – to the tumour cell, and at the same time get activated by the agent.” Researchers in the project are looking to use these kinds of methods to develop treatment for acute myeloid leukemia, a type of cancer which affects white blood cells, while a lot

of attention is also being paid to multiple myeloma. The hope is that the project’s research will lead to new treatments against these and other types of leukemia, and also open up new possibilities in the treatment of solid cancers, which are currently quite difficult to treat using immunotherapies. “There has been progress recently with solid cancers, but a big obstacle is that you would need to get the cells on the one hand to properly invade the tumour mass and on the other hand to prevent them from being inactivated by the tumour,” explains Dr Hofer. Different approaches would be required for different kinds of cancer, a topic on which Dr Hofer hopes progress will be made. “The hope is that by linking the NK cells to an

The idea here is to modify the NK cells in such a way that they would specifically target tumour cells and get similar reactivity as has been achieved with CAR T-cells, another type of recently successful immunotherapy. If this can be achieved, then the prospect of applying NK cells more widely in cancer treatment would move a step closer, and Dr Hofer believes they would be more cost-effective than many alternatives. “NK cells would be a lot easier and cheaper to develop than T-cells, and also off-the-shelf NK cell products for the use in different individuals would be possible,” he stresses. While advanced biological knowledge and technical expertise are essential to the development of these types of treatments, business skills are also required to bring them to clinical application, a topic that is addressed in the project. “The students get training in business management. They are trained in patents, and they are taught

how to write at least an initial stage patent application,” outlines Dr Hofer. “While some of the companies involved in the consortium could commercialise the project’s research, the intention is to help students develop their skills so that they would also be prepared to found their own companies in future.” There are positive early signs in terms of potential applications arising from the project’s research, with Dr Koehl and her colleagues looking into conducting an initial stage clinical trial protocol on one of the most promising reagents. This could then provide the basis for a follow-up clinical trial with one of the project’s clinical partners, while Drs Koehl and Hofer are also looking into the possibility of a successor

project. “It might also be possible to apply for a follow-up project. A future project could be more focused on clinical and commercial partners,” he says. The ESRs in Mature-NK also have the opportunity to share their findings with the different groups in the consortium, which is stimulating further research and opening up new avenues of investigation. “A satellite workshop to the NK2022 meeting will be held in May, at which all our students will present their data. The NK2022 meeting is a conference of the society of natural immunity, where all the major players in the field come together,” continues Dr Hofer. “We hope that all of the ESRs in the project will take the opportunity to participate.”

MATURE-NK MAnufacturing TUmor-REactive Natural Killer cells

Project Objectives

The MATURE-NK project is designed as a research training network for 13 early stage researchers (ESRs, PhD students) to perform research encompassing basic, translational and clinical aspects of tumor-reactive natural killer (NK) cells. Improved procedures for NK cell manufacturing for clinical application are developed. In a wider context the general objectives are i) to fortify NK cell anti-tumor cytotoxicity, ii) to develop NK cell-engineering technology and an improved manufacturing process for NK cell products and iii) to prepare for follow-up clinical trials.

Project Funding

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie Actions.

Project Partners

CAR (chimeric antigen receptor)-modified NK cells.

http://www.mature-nk.eu/partners.html

The principal of the CAR technology to target NK cells towards cancer cells is displayed. The NK cells are modified by transduction with a viral CAR vector so that the NK cells express an artificial activating CAR on their cell surface that has the capacity to guide the cells to the cancer cell and to activate the NK cell for killing when it binds to the tumor antigen on the cancer cell. Depending on the cancer type CAR constructs mediating binding to different tumor antigens preferentially expressed on different cancer types are already available or can be generated.

Contact Details

Professor Erhard Hofer Medizinische Universität Wien Schwarzspanierstrasse 17 A-1090 Wien, Austria T: +43 676 5581928 E: erhard.hofer@meduniwien.ac.at W: www.mature-nk.eu/ : @MATURE-NK : MATURE-NK Erhard Hofe

Ulrike Köhl

Erhard Hofer has a focus of research in molecular immunology and vascular biology. He has been a professor at the Medical University of Vienna (MUW) from 1997 to 2014, when he retired. Since then he has been involved in the management of the EC-funded cancer immunotherapy projects NATURIMMUN and more recently MATURE-NK. Ulrike Köhl is Director of the Fraunhofer Institute for Cell Therapy and Immunology (IZI) in Leipzig and of the Institute of Cellular Therapeutics at Medical School Hannover since 2012 and 2017, respectively, and holds professorships at University of Leipzig and Medical School Hannover. Previously, she worked at MD-Anderson-Cancer-Centre (Houston/ USA) and at the University Hospital Frankfurt. Her goals are focused on the manufacturing of ATMPs and the development of cell-based immunotherapies.

Manufacturing of GMP (good manufacturing practice) grade natural killer (NK) cells: A schematic overview of the automated process steps for the isolation of NK cells from peripheral donor blood and their 2 weeks expansion in ex vivo cell culture using CliniMACS Prodigy® equipment is presented. In short, leukapheresis (LA) products (PBMNCs - peripheral blood mononuclear cells) from healthy donors are depleted for CD3+ T lymphocytes followed by immunomagnetic enrichment of CD56+ NK cells (PF - positive fraction). Expansion of NK cells is then performed over 14 days using NK MACS growth medium supplemented with various interleukins (IL) as displayed. Samples are collected over the whole process (IPC - in process samples; QC - quality control samples) to establish cell number and several quality parameters. After 3 days of culture the NK cells are transduced with the CAR (chimeric antigen receptor) vectors and CAR expression on the surface of NK cells is separately monitored. Finally, the cytotoxic activity of the CAR NK cells towards the cancer cells of interest is tested. (Adapted from Oberschmidt et al., 2019, Human Gene Therapy Methods 30, DOI: 10.1089/hgtb.2019.039)

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