Chimeric antigen receptor (CAR)-T cell therapies are changing the way that many cancers are treated. The approval of several products around the world reflects the potential of this novel approach to immuno-oncology. However, side effects, particularly those related to cytokine storms, remain an issue. Fortunately, increased knowledge and understanding of cancer biology and immune responses are being applied to the development of next-generation CAR therapies with greater safety and efficacy against even the most difficult-to-treat solid tumors.

Improving CAR-T Cell Therapies

Cellular immunotherapy, also known as adoptive cell therapy, leverages immune cells to target and eliminate tumors. Chimeric antigen receptor (CAR)-T cell therapy is the most widely known adoptive cell therapy, with five products approved in the United States alone at the time of writing. In this approach, T cells harvested from patients are genetically modified to express the CAR protein that binds to tumor cells with specific surface markers.

While CAR-T cell therapies can be very successful for treating liquid tumors (e.g., blood-related cancers), they have not yet been proven effective for the treatment of solid tumors. In addition, they present a risk of serious side effects, such as cytokine storm syndrome, which involves a severe immune reaction that can be life-threatening to the severely ill patients receiving these therapies. In some cases, CAR-T cells can be indiscriminate, killing both cancer and healthy cells.

Manufacturing autologous CAR-T cell therapies is also a challenge. There is no way to know how successful viral transduction of the CAR gene will be for each patients’ cells or how well the modified cells will grow in culture or behave once administered to patients.¹ CAR-T cells also have a limited life span in the body that varies from patient to patient. Despite these challenges, the promise of CAR-T cell therapy continues to drive development. As of mid-September 2021, over 630 clinical trials for CAR-T cell therapies were registered on ClinicalTrials.gov as either recruiting or ongoing.¹

Many of these trials involve second-, third-, or possibly even later-generation therapy designs that attempt to address many of the concerns associated with first-generation CAR technology. One new approach is to engineer CAR-T cells with switches that control when they are activated to ensure that they only target tumor cells. Examples include switches that are activated when exposed to blue light or ultrasound radiation, both of which can be focused on the tumor.² Many of the most promising advances in CAR-T cell design allow spatial and temporal control over CAR expression or T cell activation.³

Other researchers are designing CAR-T cells that are only activated in the presence of multiple proteins, rather than just one.² Second-generation CARs contain one co-stimulatory domain, while third-generation CARs contain two co-stimulatory domains.⁴ Clinical comparison of first- and second-generation CAR-T cell therapies has shown that T cells expressing a second CD28 co-stimulation domain had greater persistence once administered to patients. Incorporation of 4-1BB co-stimulation has also been shown to prevent T cell exhaustion.⁴ The challenge in this case is to avoiding adding too much complexity, which increases the risk for complications.²

To increase the lifetimes and efficacy of CAR-T cells and enable them to survive the tumor microenvironment associated with solid tumors, some researchers are engineering them to be resistant to tumor-signaling proteins such as TGF-b — and in some cases to be activated rather than deactivated in the presence of such proteins.² Others are developing CARs that can be turned on and off so that they do not become exhausted as quickly as first-generation CAR-T cells.

Meanwhile, universal CARs utilize mAbs, which gives the CARs their antigen specificity, allowing the same cells to target multiple antigens using a combination of mAbs.⁴ This approach is attractive, because it allows the use of different, already approved tumor-specific antibodies.

The expression of co-stimulatory ligands in combination with CARs allows stimulation of other immune cells.⁴ Similarly, the expression of cytokine genes can lead to the production of IL-12 and other cytokines that have been shown to impact the tumor microenvironment. In addition, CAR-T cells that are designed to also express a chemokine receptor that binds chemokines present in the tumor microenvironment exhibit enhanced tumor targeting.

Bispecific CARs that require binding to two separate tumor antigens, meanwhile, have been shown to afford greater safety and increased efficacy.⁴ Incorporating “suicide genes” within the CAR design creates safety switches that enable CAR-T cell activity to be halted. A recent example is the inclusion of monomeric caspase-9 subunits that, when exposed to a dimerizing agent, drive rapid clearance of the CAR-T cells.

A recent example of a new approach to CAR-T cell therapy was announced in late December by Novartis.⁵ The company launched its next-generation CAR-T platform T-Charge™, which it will use in the development of various investigational CAR-T therapies based on positive early data from first-in-human dose-escalation trials with YTB323 in DLBCL (73% complete response rate at month three) and PHE885 in multiple myeloma (100% best overall response). The platform preserves T cell stemness and leverages new simplified processes and streamlined quality control for greater manufacturing efficiency. Specifically, CAR-T cell expansion occurs primarily in vivo rather than in a bioreactor. The result of these advances, according to Novartis, is higher-quality products with reduced risk of adverse responses combined with a more durable response.

Engineered T Cell Receptor Therapy

Genetically modifying T cells with CARs is not the only approach to developing adoptive cell therapies based on T cells. T cell receptor (TCR) therapy involves genetically engineering T cells to target tumor antigens regardless of whether they are on the cell surface (a requirement for CAR T cells) or within the cell.¹ As a result, they could be applicable for the treatment of more types of cancer. They function by binding to proteins (surface or intracellular) produced by major histocompatibility complexes (MHCs) that mark pathogens and tumor cells and thus enhance the innate immune response.^6,7

TCR therapies in development (approximately 280 clinical trials were recruiting or ongoing as on mid-September 2021), like most CAR therapies, are autologous and patient-specific.¹ The added receptors are complimentary to the cancer antigens of the patient, personalizing the treatment and enhancing the immune response.⁶

The production of TCR therapies is similar to that of CAR therapies. T cells are collected from a patient and, once isolated and purified, are expanded and then modified using viral vectors to deliver the relevant genetic material. As such, they suffer from the same limitations as CAR therapies with respect to manufacturing.⁶ One advantage is that, because the antigens that mark cancer cells are known, programming the receptors for TCR therapies is easier. On the other hand, TCR can only be used for cancers that the body recognizes, while CAR therapies can treat cancers the body does not recognize or has not yet generated T cells to fight.

One hope for TCR therapy is that it will provide a means for fighting solid tumors, something for which CAR therapies are not well suited, because the antigens on the surfaces of solid tumor cells often closely resemble those of healthy cells. The nature of TCR therapies and the fact that they leverage natural mechanisms enable them to distinguish blood, lung, breast, prostate, colon, bone, skin, kidney, ovarian, and cervical cancer cells from healthy cells, at least in preclinical studies.^7,8

Current clinical trials involving this type of adoptive cell therapy include patients with leukemias, melanoma, sarcoma, oropharyngeal and nasopharyngeal cancer, other head and neck cancers, Merkel cell carcinoma, cervical cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer, and others.⁹ Notably, fewer issues with cytokine release syndrome (CRS) and neurotoxicity have been observed compared with CAR-T cell therapy.

Tumor Infiltrating Lymphocyte Therapy

Another therapeutic approach based on T cells leverages tumor infiltrating lymphocytes (TILs). The advantage of TIL cell therapy is that no genetic modification of the cells is required, because they already recognize tumor antigens, and their purpose is to infiltrate tumors.¹ They have also been demonstrated to have low off-target toxicity.¹⁰ In addition, TILs are able to recognize multiple tumor antigens, affording them the ability to overcome issues with tumor heterogeneity in solid tumors, which is not possible with CAR- or TCR-T cell therapies. Indeed, TILs have demonstrated superior clinical efficacy in solid tumors compared with CAR therapies.¹⁰

The aim of TIL cell therapy is to provide patients with larger numbers of TILs to boost their immune responses. The TILs are collected, isolated and purified, expanded, and then administered to the patient. The challenge with TIL cell therapy is the collection of the initial cells, which can only be isolated from tumor tissue that has been removed from the patient during a biopsy procedure.¹ The tissue must be dissected, plated, and digested before the TILs can be collected. The entire process takes six to eight weeks and requires operators with specialized technical expertise. In addition, TILs do not reproduce in the body, so repeated treatments are required to replenish the TILs, which can not only be costly and difficult for patients but can also lead to exhaustion of the immune response.¹¹

Despite these challenges, approximately 300 TIL cell therapy clinical trials were enrolling or active as of mid-September 2021.¹ The majority of trials involve patients with melanoma. Other popular targets include non-small cell lung, ovarian, head/neck, colorectal, liver, and breast cancers.¹⁰

Some researchers are looking to improve on TIL therapy performance (e.g., reduced need for high doses of IL-2, increased in vivo survival, a wider range of applicable tumor targets) through genetic modification, either using viral transduction to overexpress certain genes or gene editing technologies, such as CRISPR and TALEN, to knock out (KO) specific genes.¹⁰ Much work still needs to be done here, because TILs are difficult to manipulate, likely due to their varied cellular composition and growth rates. Next-generation TILs in the clinic are mainly designed to overexpress cytokines such as IL-2 and IL-12.

γδ-T Cell Therapy

Gamma delta (γδ)-T cells represent yet another type of T cell that researchers are hoping to exploit as immunotherapeutic agents.^12,13 They account for approximately 1–10% of CD3⁺ T cells in human blood and tend to concentrate in the intestine and other barrier sites.¹² These T cells are challenging to leverage, however, because, while they have broad antigen specificity and high cytotoxicity for tumor cells, certain subsets — notably those expressing IL-17 — also exhibit immunosuppressive and tumor growth–promotion behaviors.¹³

Despite the difficulties, γδ-T cells have attracted attention because they act independently of the MHC-like CAR-T cells and recognize and kill transformed cells independent of human leukocyte antigen (HLA) restriction.¹¹ Some investigative approaches leveraging γδ-T cells include induction of endogenous γδ-T cell activation, adoptive transfer of expanded cells ex vivo, and genetic modification with CAR receptors.¹²

In vivo activation of γδ-T cells has most often been achieved with aminobisphosphonates (usually zoledronic acid, or ZOL), but in rare cases a phosphoantigen such as bromohydrin pyrophosphate (BrHPP) plus low-dose IL-2 have been used to the same effect.¹² In vitro expansion for adoptive cell therapy also usually involves γδ-T cell expansion using ZOL or BHP phosphoantigens. Clinical studies in renal cell carcinoma, lung cancer, hepatocellular carcinoma, breast cancer, prostate cancer, and multiple myeloma revealed some positive results, but there is much room for improvement, including the development of allogeneic therapies using allogeneic γδ-T cells obtained from healthy donors.

Use of γδ-T cell therapy, in combination with other immunotherapy treatments, such as antibodies and checkpoint inhibitors; chemotherapeutic agents and epigenetic regulators; local-dose gamma irradiation; and other strategies for enhancing both infiltration into solid tumors and inhibitors of the immunosuppressive tumor microenvironment (TME) could improve the performance of γδ-T cell therapy approaches used independently.¹²

CAR-NK Cell Therapy

Adoptive cell therapies are not limited to T cells. Research in recent years has focused on leveraging other immune cells. One approach that has received significant attention leverages natural killer (NK) cells, lymphocytes that specifically target tumor cells, releasing chemokines and cytokines that activate the adaptive immune system.¹ Genetic modification of NK cells to express the CAR protein enables them to overcome the inhibitory response of tumor cells, increasing their ability to fight cancer.

Perhaps most notably, CAR-NK therapies may not need to be autologous and patient-specific. Allogeneic, unmodified NK cells have been shown to be safe. Therefore, it is hoped that off-the-shelf genetically modified CAR-NK cell therapies will be, too. That would eliminate the time and cost challenges associated with collecting patient cells, transporting them, and processing them in a one-product-per-one-batch manner.¹ Researchers at MD Anderson, for instance, are isolating NK cells from donated umbilical cord blood (UCB) for the development of CAR-NK cell therapies as off-the-shelf products.¹⁴ In addition, CAR-NK cell therapies have been shown to be safer than CAR-T cell therapies with respect to CRS and graft-versus-host disease (GVHD) issues.^15,16

Manufacturing NK cell therapies isn’t exactly easy,¹ as expanding NK cells in bioreactors has proven to be difficult. In addition, NK cells are not known to be that effective at infiltrating tumor cells, so, like CAR-T cell therapies, CAR-NK cell therapies may be less suitable for the treatment of solid tumors. CAR-NK cells also have the disadvantage of shorter life spans in the body compared with CAR-T cells. Even so, more than 300 clinical trials involving NK cell therapies were enrolling patients or ongoing in mid-September 2021.¹

Some of these trials are targeting solid tumors, as evidence suggests that NK cells play a role in the modification of extravascular tumor growth, oncogenesis, and various cell modulation mechanisms.¹⁵ In addition, while initial CAR-NK cell therapies used CAR constructs that were originally developed for use in CAR-T cell therapies, more recent efforts have involved the use of CARs specifically designed for NK cells.¹³ As is the case in the CAR-T cell therapy field, some researchers are seeking to develop CAR-NK cell therapies that are engineered to co-express other molecules, such as cytokines, antibodies, and proteases that promote NK proliferation, trafficking, and tumor penetration.^16,17 Combination therapies are also being explored.

The sources of NK cells vary widely and include not only UCB but peripheral blood (PB) (NK cells account for 5–15% of human peripheral blood leukocytes¹⁶) and derivatives of stem cells, such as hematopoietic stem cells (HSCs) and human pluripotent stem cells (hiPSCs), the latter of which tend to enable NK cell production in high numbers and are more suitable for genetic modification.¹⁵ Owing to their immaturity, UCB-NK cells present some safety issues and typically require irradiation before injection, impacting their ability to persist once administered. Established cell lines are preferred over all of these options, because they have been designed to be easy to maintain and expand.¹⁷ The lymphoma-derived NK-92 cell line is most widely used. It can be easily genetically modified using both viral and non-viral methods.

Genetic modification of NK cells can be achieved by viral transduction using retroviral- and lentiviral-based vectors, as well as non-viral approaches, although the latter tend to result in only transient CAR expression.^15,17 Transfection with naked plasmid DNA and transposase-mediated DNA integration, as well as electroporation of mRNA, have also been used.¹⁷ In general, selecting the right transfection approach is crucial to the successful development of CAR-NK cell therapies.

Gene editing is another approach to engineering NK cells. One company pursuing this path is ONK Therapeutics, who in February 2022 announced a collaboration with Intellia Therapeutics, Inc. to develop CRISPR-edited NK cell therapies using Intellia’s ex vivo genome editing tools (guide RNAs) and lipid nanoparticle (LNP) delivery technology with ONK’s engineered NK cell therapies that have been optimized for cytotoxic potential, persistence, and metabolic health.¹⁸

For the CAR-NK cell therapies in clinical trials, HER2 (expressed on a subset of breast cancer cells) and the CD19 antigen (B cell malignancies) are the most popular targets for solid tumors and hematological cancers, respectively.¹⁷

CAR-M Cell Therapy

Macrophages are another type of innate immune cell, one that is known for infiltrating the TME.¹⁹ In particular, tumor-associated macrophages (TAMs) interact with most cellular components of the TME and are able to infiltrate solid tumor tissue. They destroy tumor cells via phagocytosis. Consequently, researchers are seeking to use CAR-modified macrophage (CAR-M) therapies to treat patients with solid tumors.

One of the first challenges in developing macrophage-based therapies is overcoming the ability of tumor cells to hide from them and/or trick them into contributing to tumor growth. It is also known that macrophages do not operate on their own; they require signals to guide their activity against tumors.¹⁹ CAR-M therapies enable the development of macrophage-based cell therapies that address these issues. They also present a lower risk of non-tumor toxicity and can enhance the toxicity of T cells toward tumors.

Current examples include CAR-phagocytes (CAR-P) that guide macrophages to devour specific targets, including ovarian and breast cancer cells, as well as cancerous Raji B cells with CD19 and HER2 antigens.¹⁹ Sources for the CAR-M therapies include iPSCs, primary monocytes, and different types of macrophages. Preclinical results in animal models have been promising.

The company leading the charge in the CAR-M space is Carisma Therapeutics, which is leveraging technology that was originally developed at the University of Pennsylvania by hematologist Saar Gill and graduate student Michael Klichinsky.²⁰ The company dosed the first HER2-overexpressing solid tumor patients with its HER2-targeted CAR-M candidate CT-0508 in a phase I trial in March 2021. CT-0508 was granted Fast Track status in solid tumors by the U.S. FDA in September of that year.²¹ In January 2022, Carisma and Moderna announced a partnership to develop CAR-M therapeutics for different cancers, leveraging Carisma’s engineered macrophage technology with Moderna’s mRNA and LNP technologies.²²

Penn Medicine researchers reported preliminary findings from the ongoing first-in-human clinical trial examining the safety, tolerability, and feasibility of CT-0508 at the Society for Immunotherapy of Cancer (SITC) 2021 Annual Meeting.^23,24 This therapy is a personalized treatment produced from primary monocytes isolated from each patient’s blood. The preliminary results suggest that CT-0508 has the ability to alter the solid tumor microenvironment and change the composition of myeloid cells and T cells while exhibiting no safety concerns. The tumors of two patients were biopsied, and analysis revealed broad activation of the tumor microenvironment, with signs of an adaptive immune response based on T cell expansion, activation, and proliferation.

The Ideal Solution: Synthetic Cells

Some researchers believe that the best approach for designing optimum immunotherapies is to create wholly synthetic cells that combine the best features of different natural immune cells. The first step in that direction is being taken by CoImmune, which has developed synthetic cytokine-induced killer (CIK) cells.² The CIK cells are generated from white blood cells, which are exposed to cytokines during their growth and can be designed to combine the features of different immune cells.

Completely artificial cells designed as cancer immunotherapies are further down the road. The ideal, as imagined by one scientist, is not to start from any type of human cell but instead to create wholly synthetic and minimal cells that only have the ability to kill cancer cells, cannot evolve or mutate, and can self-destruct when their job is finished.² The technology to create such cells does not yet exist, but could someday in the future.

Early Days Hold Great Promise

While most of the next-generation CAR therapies in development today face challenges, progress is being made at a rapid pace.¹ Knowledge of the immune system is expanding at a rapid rate, and, as new discoveries are made, further advances in CAR immunotherapies become realized. Importantly, regulatory agencies support efforts to develop adoptive cell therapies; the U.S. FDA in particular has taken steps to clarify its expectations through the issuance of several guidance documents.

CAR-based cell therapy is still quite immature. The early successes achieved to date hold great promise for the development of even more effective therapies in the near future. Preclinical development and in silico modeling will allow advances in the functional and structural design of CARs.⁴ For instance, future CAR-based therapies may have sensor and effector functions that allow the collection and processing of information from tumor cells and the TME in order to enable increased potency and specificity. The increased performance of next-generation CAR-based therapies will also be boosted even further by administering them in combination with one another and other cancer treatments.

References

1. Alsarraj, Marwan. “The Four Most Promising Adoptive Cell Therapies.” The Medicine Maker. 12 Sep. 2021.
2. Chen, Angus. “Scientists are making CAR-T cells more clever. Here’s what the next generation could look like.” STAT News. 14 2022.
3. Patrick, Chris. “Equipping next generation CAR T cells against cancer.” AIP Scitation. 8 Jan. 2022.
4. Abate-Daga, Daniel and Marco L Davila. “CAR models: next-generation CAR modifications for enhanced T-cell function.” Molecular Therapy Oncolytics. 3: 16014 (2016)..
5. Novartis announces with first-in-human data at ASH 2021. 13 Dec. 2021.
6. “TCR Vs. CAR-T: What is CAR-T Cell, TCR Therapy, and What are They Used For?” Arkadeum. Nov. 2020.
7. Withers, Nikki. “TCR therapy – an attractive alternative to CAR T.” Drug Target Review. 24 Mar.
8. “Immunotherapy for solid tumors.” Moffitt Cancer Center. n.d.,
9. Amorosi, Drew. “TCR T cells ‘poised to play a central role’ in cancer immunotherapy.” Healio News - In Practice. 1 Oct. 2021.
10. Wang, Shuhang et al. “Perspectives of tumor-infiltrating lymphocyte treatment in solid tumors.” BMC Medicine. 19: 140 (2021).
11. “What is Tumor-Infiltrating Lymphocyte (TIL) Therapy?” Arkadeum. Nov. 2020.
12. Kabelitz, Dieter et al. “Cancer immunotherapy with γδ T cells: many paths ahead of us.” Cellular & Molecular Immunology. 17: 925–939 (2020).
13. Park, Jang Hyun and Heung Kyu Lee. “Function of γδ T cells in tumor immunology and their application to cancer therapy.” Experimental & Molecular Medicine.53: 318–327 (2021).
14. Carter, Devon. “CAR NK therapy offers experimental treatment option for blood cancers.” MD Anderson Cancerwise. 5 Feb. 2020.
15. Marofi, Faroogh et al. “CAR-NK Cell: A New Paradigm in Tumor Immunotherapy.” Oncol. 10 Jun. 2021.
16. Xie, Guozhu et al. “CAR-NK cells: A promising cellular immunotherapy for cancer.” The Lancet. 59: 102975 (2020)..
17. Gong, Ying et al. “Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy.” Journal of Hematology & Oncology.14: 73 (2021).
18. Intellia and ONK Therapeutics Announce Collaboration to Advance Allogeneic CRISPR-Edited NK Cell Therapies for the Treatment of Patients with Cancer. ONK Therapeutics. 15 Feb. 2022.
19. Chen, Yizhao et al. “CAR-macrophage: A new immunotherapy candidate against solid tumors.” Biomedicine & Pharmacotherapy. 139: 111605 (2021).
20. Mast, Jason. “CAR-M era begins as Carisma doses first patient.” Endpoints News. 18 Mar. 2021.
21. Tucker, Nichole. “FDA Grants Fast Track Designation to Novel HER2-Targeted CAR-M Therapy in Solid Tumors.” Targeted Onocology. 22 Sep. 2021.
22. Moderna and Carisma Establish Collaboration to Develop in vivo Engineered Chimeric Antigen Receptor Monocytes (CAR-M) for Oncology. Moderna. 10 Jan. 2022.
23. First-in-Human Trial with CAR Macrophages Shows the Cell Therapy May Be Safe, Feasible for Solid Tumors. Penn Medicine. 11 Jan. 2022.
24. Doyle, Chase. “Phase I Trial Investigates HER-Targeted CAR-M for HER2-Overexpressing Solid Tumors.” The ASCO Post. 1 Dec. 2021.