Tracking The Latest Developments In CAR T Safety And Efficacy

By Francisco Conesa Buendía

CAR T cell therapy, a form of immunotherapy, has transformed cancer treatment by genetically modifying patients' own immune cells (T-lymphocytes) to seek out and destroy cancer cells.¹ While CAR-T therapy has demonstrated remarkable effectiveness in treating hematological cancers, it also carries significant risks, including cytokine release syndrome (CRS) and neurological toxicities (ICANS, immune effector cell-associated neurotoxicity syndrome), which can be life-threatening.² Furthermore, the manufacturing process is complex and lengthy, making it challenging to produce CAR T cells in large quantities and at a rapid pace.³ This process demands highly skilled personnel, cutting-edge facilities, and stringent maintenance in compliance with current good manufacturing practices (cGMP), along with sophisticated equipment that requires careful control and upkeep.³

To tackle these challenges, clinicians and researchers are exploring various strategies and technologies aimed at enhancing the safety and efficacy of CAR T cell therapy. These efforts include developing safer CAR T cell products, optimizing the design of CAR T cells, and employing advanced manufacturing techniques. Together, these approaches hold great promise for improving the safety and effectiveness of CAR-T therapy, ultimately leading to better patient outcomes in the battle against cancer.

Number of clinical trials conducted for more than a decade with CAR-T or T-cell receptor(TCR) therapies as of April 2024. Source: Beacon by Hanson Wade.

Researchers are optimizing the design of CAR T cells to enhance their ability to target and destroy cancer cells. This includes engineering CAR T cells with more persistent anti-tumor activity, while at the same time trying to reduce the adverse effects and toxicity associated with these cell therapies. This involves engineering CAR T cells to produce fewer toxic cytokines or incorporating suicide genes that can be activated to eliminate CAR T cells in case of severe toxicity.

Remote-Controlled CAR T cells

A novel idea involves "remote-controlled" CAR T cells, which would allow for the modulation of their activity and depletion when needed. Labanieh et al.⁴ introduced a high-throughput drug-regulatable system known as signal neutralization by an inhibitable protease (SNIP), which is controlled by an FDA-approved small molecule (grazoprevir) that has favorable pharmacokinetics in humans. These CAR-T SNIP cells offer a reliable safety switch, enabling the prevention of severe toxicity by simply stopping the drug after toxicity begins. Additionally, CAR-T SNIP cells show enhanced efficacy in various models, demonstrating better functionality and less depletion, along with higher memory levels compared to those treated with conventional CAR T cells.

Another intriguing study showed that creating compact synthetic transcriptional regulators, primarily derived from human proteins, could serve as an effective tool for CAR T cell design⁵ As proof of concept, researchers developed gene switches and circuits that enable precise, user-defined control over therapeutically relevant genes in primary T cells using FDA-approved orthogonal small molecule inducers. These circuits can instruct T cells to sequentially activate various cellular programs, such as proliferation and antitumor activity, leading to synergistic therapeutic effects. This platform facilitates the creation of compact gene switches that allow for dose- and time-dependent regulation of important genes both in vitro and in vivo.

Additionally, from an innovative standpoint, enhancing the programmability and safety of universal CAR T cells is achieved by engineering off-switch adapters that can conditionally regulate CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to small molecules or light stimuli.⁶ Researchers have developed novel CAR T cells using the biotin-binding domain of affinity-enhanced monomeric streptavidin 2 (mSA2). When expressed in T cells, these can target cancer cells coated with biotinylated antibodies. CAR mSA2 T cells were effective in mediating cancer cell lysis and producing IFNγ in a dose-dependent manner based on antibody concentration.⁶ When exposed to UV light or a small molecule, the biotin tag is cleaved, preventing the adaptor from facilitating interactions between CAR mSA2 T cells and target cells.⁶ This approach allows CAR mSA2 to be combined with tumor-specific biotinylated antibodies, potentially targeting various tumor types.

Humanized Targeting Domains

Aside from remote control or the use of physical, biological, or chemical agents to activate or inhibit CAR T cells, another strategy involves enhancing safety by employing humanized target domains.^7,8 Researchers globally have found that humanized CAR T cells demonstrate lower immunogenicity and fewer off-target effects compared to traditional CAR T cells. Various factors influence the effectiveness of CAR T cell therapies, including CAR T cell quality, differentiation status, metabolic profile, and, crucially, CAR design.^7,8 Given the issues of limited persistence and immunogenicity associated with murine scFv-engineered CAR T cell products, efforts to create humanized versions that maintain affinity, specificity, and sensitivity could further improve the therapeutic potential of CARs targeting tumor antigens. For instance, several iterations of CD19-specific humanized CAR T cells are currently undergoing testing in clinical trials.⁹ Additionally, affinity tuning of scFvs is not the sole method for enhancing CAR T cell function; a combination of other strategies — including scFv structure, hinge length, choice of co-stimulatory domain, and strength of the activation domain — should also be considered to optimize the performance of CAR T cells in cancer immunotherapy.^7,8,9

Stealth CAR T Cells And Precision Targeting

Currently, four of the six FDA-approved CAR T cell products use variable fragments derived from a murine monoclonal antibody as the extracellular binding domain.¹⁰ Clinical studies demonstrate that patients develop humoral and cellular immune responses to non-self-CAR components of autologous CAR T cells or to donor-specific antigens of allogeneic CAR T cells, which is believed to be able to limit CAR T cell persistence and success of repeated dosing.¹¹ A recent paper describes how to prevent rejection of modified T cells by simultaneously reducing antigen presentation and surface expression of both classes of major histocompatibility complex (MHC).¹⁰. Expression of viral inhibitors of the antigen processing-associated transporter (TAPi) in combination with a transgene encoding an shRNA targeting the MHC class II transactivator (CIITA) provides this phenotype to the cells.¹⁰

In line with the above, Precision BioSciences Inc. is working on the candidate PBCAR19B stealth cell therapy. This CAR-T product is an allogenic anti-CD19 targeting cell product designed to evade immune rejection. The treatment goal of this product is to potentially displace autologous CAR-T in the second-line DLBCL (diffuse large B cell lymphoma) setting.

As of May 30, 2023, in Phase 1 (NCT04649112) results they observed an acceptable safety profile with high overall response rates among all evaluable subjects with evidence of molecular remission and preliminary durability. Among the results obtained, the following should be highlighted: 71% ORR (overall response rate, generally the sum of complete responses) and 43% CR (complete response) rate; there were also “no Grade 3 or greater cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), infection, or graft versus host disease was observed, [and] PBCAR19B stealth cell achieved proof of concept and appeared to be effective in delaying recovery of host T- and NK-cells,” according to a Precision news release.

Logic Gate Systems And Cell Surface Receptors

Emerging strategies are focused on enhancing tumor specificity and safety through dual antigen targeting and logic gates (such as or, and, not, if-then, and if-better), which can be combined (e.g., or-not gate).¹² Multispecific CARs, co-expressed CARs, or reversible CARs enable the targeting of multiple antigens. The goal of these logic gates is to optimize and improve the efficacy of CAR T cell responses and actions.¹² For instance, cytolytic activity can be triggered when either target molecule A or B is present (or gate) or only when both are present (and gate). Alternatively, the action can be inhibited if both targets are detected (not gate). Additionally, an if-then gate could be employed to stimulate the expression of a missing receptor when tumor cells are contacted — specifically, if A is present but B is not, then B is needed. Lastly, the if-better gate aims to enhance the CAR-T receptor's sensitivity to activation by refining binding, sensitivity, and affinity. However, early clinical trials involving tan-CARs and dual-CARs in B-cell neoplasms highlight the challenges associated with multi-targeting.^13,14,15

Enhanced Persistence And Overcoming Immunosuppression

Battram et al. studied the effects of IL-2, IL-15, and a combination of IL-15/IL-7 on the phenotype and function of a BCMA-targeted CAR T cell currently used to treat multiple myeloma (MM) patients.¹⁶ Their results revealed that IL-15 was superior to both IL-2 and the IL-15/IL-7 combination in enhancing the quality, efficacy, and persistence of anti-BCMA CAR T cells.¹⁶ This suggests that incorporating IL-15 into the clinical production of CAR T cells could extend the duration of treatment responses. Another study showed that anti-CD19 CAR T cells engineered to secrete IL-18 increased cytokine production, improved CAR T cell persistence, induced long-term B-cell depletion, and boosted mouse survival, even without prior preconditioning.¹⁷

Cancer has a remarkable ability to develop mechanisms that help it evade treatments.¹⁸ Despite the promise of immunotherapies and vaccines, solid tumors, like prostate cancer, have resisted treatment by employing immunosuppressive strategies. Prostate tumor cells, for example, secrete transforming growth factor β (TGF-β), which suppresses immune responses and facilitates tumor progression.¹⁸ Inhibiting TGF-β signaling in T cells enhances their infiltration, proliferation, and antitumor activity in prostate cancer models.¹⁸ Researchers found that expressing a dominant-negative TGF-β receptor in CAR T cells led to increased lymphocyte proliferation, enhanced cytokine secretion, resistance to depletion, prolonged in vivo persistence, and tumor eradication in aggressive prostate cancer mouse models.¹⁸

Outlook And Future Perspectives

CAR T cell therapy is continually advancing, with ongoing research focused on enhancing both the safety and efficacy of this groundbreaking treatment. As these innovative approaches move through clinical trials, CAR-T therapy is set to significantly improve the lives of cancer patients by offering more personalized and effective treatment options. Looking ahead, several future perspectives and challenges are worth noting. The development of off-the-shelf allogeneic CAR T cells presents a promising opportunity for a universal treatment that is not only less expensive but also more widely accessible. Current clinical trials are investigating the safety and efficacy of these allogeneic CAR-T products, which could revolutionize the treatment landscape.

Moreover, the application of CRISPR-Cas9 gene editing technology holds the potential for more precise modifications of CAR T cells, enhancing their safety profiles and therapeutic effectiveness. This technological advancement could lead to CAR T cells that are better equipped to target cancer cells while minimizing adverse effects on healthy tissues.

Finally, the exploration of novel delivery systems, such as nanoparticles, could enable targeted and controlled release of CAR T cells. This approach aims to improve the localization of CAR T cells to tumors while reducing off-target toxicity, thereby maximizing therapeutic benefits. By addressing these challenges and leveraging cutting-edge technologies, the future of CAR T cell therapy looks promising, with the potential to transform cancer treatment and patient outcomes significantly.

References:

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9. Myers RM, Li Y, Barz Leahy A, et al. 2021. Humanized CD19-Targeted Chimeric Antigen Receptor (CAR) T Cells in CAR-Naive and CAR-Exposed Children and Young Adults With Relapsed or Refractory Acute Lymphoblastic Leukemia. J Clin Oncol. 39(27):3044-3055. DOI: 10.1200/JCO.20.03458.
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About The Author:

Francisco Conesa Buendía, Ph.D., has been working in the field of advanced therapies since 2020 in Spain and the U.S. He has focused his research and work on the development of ATMPs based on stem cells and chondrocytes. Currently, he is a cell manufacturing assistant working on manufacturing and process optimization of cell and gene therapies based on CAR-T and T cells at Memorial Sloan Kettering Cancer Center in New York City. Connect with him on LinkedIn.