The ideal CAR-T cell target antigen is expressed exclusively on cancer cells, with minimal or absent expression on normal cells to mitigate unwanted on-target off- tumor toxicities. Four of the five FDA-approved CAR-T cells target CD19, a membrane- bound protein expressed on both normal and malignant B cells. Patients treated with these therapies develop B cell aplasia and hypogammaglobulinaemia, though this adverse effect is tolerable with periodic intravenous immunoglobulin replacement. CARs have been designed to target many different tumor antigens, including additional targets in hematologic malignancies and novel targets in solid tumors. At the time of this publication, there are over 500 active clinical trials investigating CAR-T cell therapies for cancer registered with the United States National Library of Medicine.
Hematologic Malignancies
CAR-T cell design to date has primarily focused on hematologic malignancies. In addition to the five FDA approved CAR-T cells, there have been a number of trials focused on improving the function and expanding the use of CAR-T cells targeting CD19. Additional clinical trials for CD19-targeted CAR-T cells are underway in patients with multiple myeloma and mantle cell and follicular lymphoma. Additional B cell malignancy targets are being explored to overcome CD19 antigen escape that occurs after CAR-T cells are administered. CAR-T cells targeting both CD19 and CD20 or CD22 are being investigated as individually transduced CAR-T cells that are co-administered or in tandem CAR designs. Another tandem CAR showing promising results in preclinical models targets CD79b in addition to CD19.
CAR-T cells targeting B-cell maturation antigen (BCMA) hold great promise in the treatment of multiple myeloma. BCMA expression is restricted to terminally differentiated B cells and plasma cells but is also found on multiple myeloma cells. Two CAR-T cells targeting BCMA, bb2121 (ide-cel) and JNJ-4528, were granted breakthrough therapy designations by the FDA and ide-cel was approved for treatment of relapsed or refractory multiple myeloma in 2021. Despite this success, evidence of antigen escape has been observed with BCMA-negative relapses, and immune rejection of the mouse scFV of BCMA-targeted CAR-T cells has also been a therapeutic obstacle. To overcome these limitations, CAR-T cells targeting dual antigens or using alternative antigen binding domains have been developed for multiple myeloma. In addition to BCMA, transmembrane activator and CAML interactor (TACI) can be targeted by CARs using a natural ligand that binds both BCMA and TACI, which has demonstrated anti-tumor activity in preclinical models. BCMA-targeting CAR-T cells with camelid VHH or fully human antigen binding domains have also been developed. Additional targets being explored for multiple myeloma include CD38, CD138, CS1 (SLAMF7), immunoglobulin kappa light chain, CD44v6, CD56, Lewis Y, CD229, and others. Many of these are also being explored for the treatment of other B cell malignancies; however, most have overlapping expression on normal tissues, which may limit their therapeutic potential. Another target, G protein-coupled receptor, class C group 5 member D (GPRC5D), is expressed both on hair follicles and myeloma cells but has been safely targeted by CAR-T cells, eradicating multiple myeloma in a BCMA antigen escape model without significant off-tumor effects.
Individual targets are also being explored in T cell malignancies and acute myeloid leukemia (AML). CAR-T cells targeting CD37, an antigen expressed by B and T cell malignancies, have shown efficacy in xenograft mouse models, and a phase I trial of CAR37 cells is ongoing (NCT04136275). Other targets for T cell malignancies include CD5 and CD7. CAR-T cell development for AML has been difficult due to the lack of surface target antigens unique to tumor cells. The most common targets used in clinical trials for AML are CD33, also expressed on hematopoietic stem and progenitor cells (HSPCs) and in the lung, skin, and prostate, and CD123, with lower HSPC expression. CD33-targeting antibody-drug conjugates caused significant toxicities when administered in patients, whereas a CD33-targeted CAR-T cell reduced marrow blasts in one patient for 9 weeks with only minor toxicities. More recently, a unique approach of knocking out CD33 in HSPCs and performing a bone marrow transplant along with the CD33-CAR was successful in a preclinical model. A similar approach has been taken with CD5 and CD7, to prevent fratricide of T cells when targeting a T cell antigen. The first few patients treated with CD123 CAR-T cells experienced severe toxicities, but there are still ongoing clinical trials (NCT03672851, NCT04265963, NCT04014881).
Solid Tumors
Identifying CAR-T cell targets for solid tumors is challenging, in part due to tumor antigen heterogeneity and simultaneous expression on normal tissues. Many targets are being explored for numerous types of solid tumors. We will discuss a subset of antigens shared across different tumor types that have been or are currently being tested in clinical trials.
One very promising target, EGFRvIII, is uniquely expressed on tumor cells as a mutated form of the epidermal growth factor receptor (EGFR) lacking the ligand binding domain. It is a neoantigen expressed on several tumor types including glioblastoma, medulloblastoma, non-small cell lung carcinoma, and breast, colon, ovarian, head and neck, and metastatic prostate cancer. EGFRvIII-targeted CAR-T cells have unfortunately not shown promising response rates in phase I trials in glioblastoma patients. Post-treatment tumor biopsies in these patients demonstrated infiltration of suppressive regulatory CD4+ T cells and a decrease in EGFRvIII protein expression, suggesting TME-mediated immunosuppression and antigen escape as mechanisms of treatment failure. Given that EGFR is over expressed in glioblastoma and other cancers, CAR-T cell targeting EGFRvIII were designed to secrete a T cell engager specific to EGFR and showed promising results with no off-tumor toxicity after injection locally in a xenograft mouse model.
Another member of the EGFR family, HER2, has also been explored as a CAR-T cell target. While HER2 is overexpressed in multiple tumor types, including glioblastoma, breast cancer, and GI malignancies, it is also widely expressed on normal tissues. A clinical trial of CAR-T cells targeting HER2 in glioblastoma patients demonstrated safety, without dose-limiting toxicity, in a trial of 17 patients, but limited anti-tumor effect was observed. Another studied target in glioblastoma is IL-13Rα2, which is overexpressed in tumors but also found in normal tissues. CAR-T cells targeting IL-13Rα2 were engineered with an IL-13 zetakin as the antigen binding domain and showed impressive tumor killing in a patient after direct tumor injection and intrathecal infusion. CARs targeting HER2 and IL-13Ra2 in tandem or in a tricistronic CAR-T cell also targeting ephrin-A2 (EphA2) have been developed to overcome tumor heterogeneity and antigen escape, showing promising results in preclinical models. HER2 and IL-13Ra2 are also being targeted by CAR-T cells in other types of tumors, including sarcoma, colorectal cancer, and melanoma.
Other promising targets for CAR-T cells in solid tumors are mesothelin, B7-H3, and MUC1. Mesothelin, a cell-surface antigen highly expressed in lung, pancreatic, ovarian, and other cancers, has been used as a target in multiple CAR-T cell pre- clinical studies and showed promising efficacy. Several clinical trials of mesothelin-targeting CAR-T cells are ongoing. B7-H3 is an immune checkpoint molecule with low surface expression that has been studied as a CAR-T cell target. To minimize on-target off-tumor toxicity, B7-H3 CARs were designed with an scFv recognizing only high concentration of target antigen, a strategy that showed safe anti-tumor activity in pre-clinical models of pediatric brain tumors and sarcomas. MUC1, a glycoprotein expressed in many normal tissue types, is also expressed as a unique isoform in certain cancers and represents a potential neoantigen target. CAR-T cells targeting Tn-MUC1 have demonstrated anti-tumor efficacy in xenograft mouse models and there are several clinical trials under way for CAR-T cells targeting MUC1 in esophageal, breast, and non-small cell lung cancer (NCT03706326, NCT03525782, NCT04020575).
Though CAR-T cells are designed to target antigens on the surface of cancer cells and initiate a T cell response in a non-MHC restricted manner, there are many intracellular antigens that are unique to tumor cells but are out of reach for CAR-T cells. One way to overcome this is to design a CAR with an antigen binding domain based on an antibody that binds to a specific peptide epitope of a tumor antigen presented on an MHC. For example, NY-ESO-1 is a cancer testis antigen that is expressed in the cytoplasm of a wide range of tumors but is not expressed in normal somatic cells. CAR-T cells specific to an NY-ESO-1 peptide in the context of HLA-A*0201 have shown efficacy in mouse models of melanoma and multiple myeloma. This approach has the potential to vastly expand the range of target antigens for CAR-T cells by making them accessible to more neoantigens and opening new avenues for cancer immunotherapy.
