Programming human cells to perform desired therapeutic functions is the basis for the growing field of cell therapy. One key example is chimeric antigen receptor (CAR) T cell immunotherapy, in which patient-derived T cells are genetically modified to express artificial antigen-targeting receptors to allow redirection to attack tumors. While cell therapies have shown clinical promise, the field has navigated challenges relating to safety, barriers to activity due to tumor heterogeneity, immune cell exhaustion, and the immune-suppressive tumor microenvironment. In addition, the complex manufacturing of CAR-Ts and other cell-based therapies translates to high costs and places constraints on scale and accessibility.

In this interview (conducted by senior editor Fay Lin), we asked Geulah Livshits, Senior Research Analyst at Chardan, to assess the advances and challenges facing the cell therapy field. Livshits discusses how the field has progressed with advances in tumor-immune interactions, cell engineering technologies, as well as improvements in cell manufacturing and the logistics of cell therapy administration in the clinic.

(This interview has been edited for length and clarity.)

How would you describe the current state of CAR-T therapies? What are the challenges?

Livshits: Its been more than five years since the first approval of CAR-Ts targeting CD19 for B-cell leukemia and lymphoma. The field has learned a lot since then, both with respect to science and what it takes to make cell therapies work in the commercial setting. While these initial agents were approved in patients who had progressed in standard of care therapies (third-line or later), weve also seen some CAR-Ts outperform standard-of-care chemotherapy in second-line lymphoma over the past year and gain approval in additional lymphoma subsets. This has allowed these advanced therapies to be used in earlier line settings, as well as in larger numbers of patients.

Beyond CD19, there has been a lot of success with CAR-Ts for multiple myeloma, targeting B-cell maturation antigen (BCMA), where some agents have shown response rates of up to 95% and thus far have impressive durability. This has led to interest in developing engineered cell therapies for solid tumors, which account for close to 90% of cancer deaths in the United States.

CAR-T manufacturing is complex as they are living drugs. It involves cell collection, genetic engineering within appropriate specifications such as viability, and then infusion back into the patient. Approved CAR-Ts are autologous, meaning that theyre manufactured individually for each patient. As a result, you cant scale up by making a bigger batch of cells. You have to scale out by making more batches at once and have the equipment to run those batches as patients need them. Theres a lot of logistics involved in ensuring that cancer patients who are eligible to receive CAR-Ts can actually get them manufactured and delivered in a timeframe during which they can still benefit.

CAR-Ts are also not the only modalities that are being developed for their indications. For example, bispecific antibodies (bsAbs) and to some extent, antibody-drug conjugates are also being developed for similar targets. Those are off-the-shelf biologics. Data on cell therapies are being analyzed in the context of durability of response, safety, and convenience relative to what is also emerging on some of these other agents.

In principle, cell therapies can be engineered to have additional functionalities beyond the currently approved CAR-Ts. Some areas of active innovation include a few overlapping buckets. These strategies include expanding the use of cell therapies to solid tumors, enhancing the safety elements of cell therapy so that they could be used in earlier lines and in community and outpatient treatment settings, and addressing some of those logistical challenges that are associated with current cell therapies. Different groups are focusing on the identification of optimal targets and additional cargos that could be incorporated to drive activity, such as different cell sources and cell types that have distinct activity, safety profiles, and manufacturability.

How are currently approved CAR-Ts developed and administered to patients?

The current approved CAR-Ts, such as Kymriah, Yescarta, Abecma, Carvykti, etc. are patient specific. The process involves harvesting immune cells from the patient by apheresis, genetically modifying them with the vector to get the construct, and then expanding them over several days. While doses vary by product, several of them are above 1 million cells per kilogram. The engineered cells are typically frozen down, shipped to the patients hospital, and infused into the patient. The turnaround time is often around three weeks, sometimes more.

Once the cells are infused, they rapidly expand and reach peak levels within two weeks of infusion and then decline. The long-term persistence and kinetics are often variable between products, patients, and indications, but CAR-Ts can persist at detectable levels for months to years due to the long lifespan of T cells.

To promote the expansion and activity of CAR-Ts, patients typically undergo lymphodepletion conditioning, typically with chemo drugs, such as fludarabine and cyclophosphamide. This transiently depletes a patients own lymphocytes and removes the sinks for cytokines to allow them to be available to support CAR-T expansion. Those expanded CAR-Ts then circulate around the body and kill cells with their respective targets, such as CD19, BCMA, and others. That includes both cancer cells as well as normal B cells.

How effective are these CAR-T therapies?

Responses tend to be pretty high for CD19 cancers and multiple myeloma. Many of these patients achieve a complete response or partial clearance within four weeks. In many cases, 40% of lymphoma patients and high numbers of multiple myeloma patients can remain responsive up to two years post-treatment. However, the durability numbers vary.

While thats encouraging, theres still room for improvement, particularly in the third-line setting. The activity has been quite encouraging in these cancer types but the answer is [to be determined] in other indications as we start to look beyond the lowest hanging fruits. Durability, toxicity, and safety are other relevant metrics that people use to evaluate these therapies.

Speaking of toxicity, are there any toxicities associated with these CAR-T therapies?

Years of clinical experience have shown some clear patterns on that front. CAR-Ts have been associated with certain toxicities including cytokine release syndrome, macrophage activation syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS), as well as infections. The infections are often related to the lymphodepletion component because youre eliminating a persons immune cells, but other components arise from different elements of the CAR-T expansion.

Clinicians who have been administrating these therapies over the past several years have been increasingly adept at navigating the safety issues and treating cytokine release syndrome by using tocilizumab, an IL6 antibody. ICANS has been managed using corticosteroids. The field is seeing increasing assessment of CAR-T administration in outpatient settings. It is also important to keep in mind that all of this is learned from blood cancers. The situation could be different in solid tumors, where cell therapy strategies have also been in the clinic for quite a while.

What is the history behind cell therapy in oncology?

Years before CAR-Ts were [developed], there was work from Steve Rosenbergs group back in the 1980s and 1990s at the National Cancer Institute that showed immune cells could be harnessed to mediate rejection of a tumor. Much of that work focused on tumor infiltrating lymphocytes (TILs). TILs could be isolated from a patients tumor and then grown up in culture to doses of billions of cells and then infused back into that patient. That approach led to durable regressions of late-stage solid tumors. The idea behind TILs is that T cells that are found within a tumor might be likely to be enriched for T cells that are reactive to that tumor. Expanding and reinvigorating those cells outside the body can help them mount a more successful attack against that tumor.

Historically in those studies, TILs werent engineered and there was much less understanding about how that worked. Now we know that T cells, via their T-cell receptors (TCRs), can recognize tumor cells and kill them, particularly when theyre expressing proteins that are derived from mutations. That approach has been quite heavily studied, particularly in cancers that have a high number of mutations like melanoma. Companies that are operating in that space, such as Achilles Therapeutics, Instil Bio, Iovance Biotherapeutics, KSQ Therapeutics, and Lyell Immunopharma, are working on professionalizing elements of isolation of the TILs, their expansion, as well as looking at different strategies to boost their activity, either by reducing exhaustion or increasing cell killing capabilities.

How does research in engineered TCRs compare with CAR-T research?

The major difference between CARs and TCRs is that TCRs can recognize protein targets that have been processed into short fragments and then are presented on the cell surface in the context of the major histocompatibility complex (MHC). Both cell-surface and intracellular proteins are presented this way, meaning TCRs can target a wide range of potential targets. However, these MHC complexes vary from person to person. What that functionally means from a therapeutic development standpoint is that separate TCR products do need to be produced to target distinct alleles of the human leukocyte antigen (HLA) system.

The prevalence of different alleles varies across geographies. For example, in the United States, its estimated that around 40% of the Caucasian population has the most common HLA allele. There are other frequencies for less common variants. A series of products would need to cover additional segments of that population. This is distinct from CAR-Ts, where the CAR comprises an antibody-like domain that then recognizes unprocessed protein targets on the surface of tumor cells. Its not restricted by these HLA allele specificities, but they are limited to targeting proteins on the surface of the cell. They have a narrower potential antigen target pool, but a broader potential patient coverage by not being HLA restricted.

The antibody-like binders of CAR-T constructs are restricted to recognizing cell surface proteins. Are they able to distinguish between tumor and normal cells bearing their target? What are some solutions for off-target effects?

The currently approved CAR-Ts cant distinguish between cancer cells and normal cells. They kill cells that have enough of the target, and that can lead to on-target off-tumor toxicity. In the case of B-cell cancers, that translates to depletion of normal B cells along with the cancer cells. While thats not ideal, its an acceptable trade-off given the activity profile, considering that a patient can get by without B cells, especially with supportive treatment. In other tumor types, this can pose a bigger safety risk. For example, a patient died during an early study of CAR-Ts targeting HER2 days after administration, potentially because the T cells recognized low levels of their target in the lung tissue, which led to high inflammation in the lung.

Companies and academic groups are looking at safety switches or regulatable CARs or logic gates to try to reduce those off-tumor toxicities. Theyre focusing on targets that are highly expressed in cancers and have more limited expression in health tissues. This is also where the TCR T cells come into play. Theres a larger target space of intracellular proteins used to select targets that are overexpressed in cancer or neoantigen peptides that arise from mutated proteins. Targets that are overexpressed in cancer, such as NY-ESO-1, MAGE-A4, and PRAME, are being explored by companies like Adaptimmune, Immatics, and Tscan Therapeutics, while others such as Affini-T Therapeutics are targeting neoantigens.

While the TCR T space has lagged behind CAR-Ts, there are several biotech-driven programs that are now in the clinic. Adaptimmune expects to complete a rolling BLA submission for its MAGE-A4 TCR T program for synovial sarcoma in the middle of this year. A key technological area of focus among programs that are either pre-clinical or early development is screening T-cell receptors to have that optimal binding affinity to their target, but also screening to make sure that they dont cross-react with other proteins in healthy cells. There are also strategies to functionalize different types of T cells, for example, not just CD8 cells but also CD4 cells. There are some programs being advanced in conjunction with histological markers to allow their use in patients who express the target and would be more likely to benefit from the treatment.

What are the challenges posed by the tumor microenvironment on the effectiveness of these therapies? How are these challenges addressed?

The tumor microenvironment is definitely seen as a major barrier for cell therapy, particularly in solid tumors. There are several immune suppressive signals from different cell types that can promote T-cell exhaustion or other elements of T-cell dysfunction. Over the past decade, there have been many academic and industry studies teasing out how these tumor microenvironment mechanisms work and what characteristics are associated with functionality vs dysfunctionality (e.g. using CRISPR screens to functionally identify genes whose disruption can promote T-cell activity).

Other approaches identify additional components, such as cytokines or switch receptors, that can convert negative signals into positive or co-stimulatory signals to further boost activity in these immune suppressive microenvironments. These different approaches are expected to move through clinical trials in the coming years. Companies are also looking at combining T-cell therapies with approved checkpoint inhibitors as another way to boost activity.

How can off-the-shelf strategies simplify the manufacturing workflow of these therapies?

An advantage of off-the-shelf approaches is that the complex manufacturing and engineering does not have to be done individually for each patient. As a result, there is no 3-4 week lag to treatment. Also, in some cases, patients who have gone through multiple rounds of therapy may not have immune cells of sufficient quality or quantity to make a great autologous cell product.

The challenge is that immune systems are good at recognizing and eliminating foreign cells. Donor T-cells may attack a patients body (graft-versus-host disease, GvHD) or the bodys own immune cells can recognize donor cells as foreign and attack those. GvHD can be avoided by eliminating the endogenous TCR and T cells using tools such as gene editing, or by using other cell types that dont have this GvHD property. The issue with GvHD seems to be solvable with current technologies, but the immune invasion part is a bit more complicated. There are several strategies that are in development and the optimal approach remains to be seen at present. Companies such as Allogene Therapeutics, Beam Therapeutics, Caribou Biosciences, CRISPR Therapeutics, Precision BioSciences, and Sana Biotechnologies, are using different strategies to evade rejection from the patients T or NK cells.

There are several companies using NK cells, a cell type expected to have a shorter persistence. NK cells can be given at doses of over 1 billion cells in multiple administrations and potentially multiple cycles. One of the other attractive features of NK cells is that there seems to be a lower propensity towards cytokine release syndrome. That, in addition to engineering them with a CAR, can work in conjunction with some therapeutic antibodies to kill by multiple methods.

Different sources of NK cells are being explored by companies in the space. What is the impact of the starting material on the manufacturing process, costs, and attributes of the final product?

For NK cells, there have been a few different cell-sourcing strategies. Some groups, such as Nkarta, are developing donor-derived NK cells that are taken from other people and expanded. Others such as Fate Therapeutics, Century Therapeutics, and Shoreline Biosciences, are advancing NK cells that are derived from induced pluripotent stem cells (iPSCs). Companies like Takeda are advancing NK cells that are isolated from cord blood, which might have distinct properties. Donor-derived processes might yield expansion of NK cells several thousand-fold over a 2-3 week period and generate hundreds to thousands of doses per round. As this isnt bespoke, companies are able to scale up for commercial use. What that translates to in terms of cost per dose will depend on the dose paradigms that might end up being effective. Its still early days for the space, and the optimal dose regimens that would be used in commercial settings are not yet defined.

The difference with iPSC-derived therapies is that they can be expanded theoretically indefinitely and thus could be used to generate a clonally derived cell bank. This means that you can do all of the genetic engineering and have those associated costs upfront, freeze down a working cell bank, run a manufacturing batch, take cells from the bank and adjust culture conditions to differentiate them into NK cells. As its off-the-shelf, a batch can be hundreds or thousands of doses, depending again on manufacturing scale and dose levels and the exact process. Each of these approaches has its advantages and open questions.

What are the prospects for CRISPR, base and prime editing in the CAR-T field?

Various gene editing approaches are being used in cell therapy, particularly in the immune evasion setting. Were also starting to see use in autologous settings. For example, disruption of PD-1 in TILs from Iovance is an example of gene editing being used in cell-based therapies. There is interest in other approaches, such as base and prime editing that dont cause double-strand DNA breaks. Those approaches can make it easier to edit multiple genes at once while avoiding translocations. While current approved CAR-Ts use viral vectors to deliver the CAR construct, there is interest in using other approaches to insert the CAR or TCR construct, or other elements using non-viral approaches to target them in a particular location.

Geulah Livshits, PhD, ([emailprotected]) is a senior research analyst with Chardan in New York City, covering biotech companies.

GEN Biotechnology, published byMary Ann Liebert, Inc., is the new, marquee peer-reviewed journal publishing outstanding original research and perspectives across all facets of the biotech industry. Thisarticlewas originally published in the February 2023 issue of GEN Biotechnology,Volume 2, Issue 1.

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