Chimeric Antigen receptor-T cell Therapy for Solid Tumors with Antigen Heterogeneity.

Xing-Ning Li¹, Chunfeng Qu^1*

¹Immunology Department, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

Abstract

Chimeric antigen receptor-T (CAR-T) cell therapy in the treatment of solid tumors remains limited, though it has demonstrated remarkable success for hematological malignancies. One of the major restrictions is immune escape from the attack mediated by primary targeted CAR-T cells, driven by the heterogeneous cell components of solid tumors, including heterogeneous expression of tumor antigens. Here, we reviewed the specified challenges and corresponding strategies which are being developed, including the use of multi-targeted CAR-T cells and the activation of endogenous immune responses.

Introduction

The adoptive cell therapy (ACT), an administration of tumor-reactive immune cells, has demonstrated significant antitumor efficacy. Administration of genetic modification of a patient’s own T cells to express a single chain fragment variable regions (scFv) of an antibody is one of ACT, that enables T cells to specifically recognize and attack the tumor-associated antigens expressed on the surface of tumor cells without the restriction of the major histocompatibility complex (MHC). Currently, CAR-T product has demonstrated remarkable success and approved for treating hematological malignances. CAR-T therapeutic efficacy is challenged due to distinct microenvironment of solid tumors, such as the limited infiltration of CAR-T cells into tumor sites, CAR-T cell exhaustion in the tumor beds, and the other obstacles. Regarding the challenges and corresponding strategies of CAR-T cell therapy in solid tumors, these have been extensively reviewed in previously published comprehensive reviews^{1, 2}. However, currently available effective solutions for tackling the heterogeneity of tumor antigens are still scarce. In this review, we discuss key strategies for targeting multiple antigens and activating endogenous immune responses, aiming to enhance the therapeutic efficacy of CAR-T cells in solid tumors characterized by antigen heterogeneity.

The spatiotemporal heterogeneity of tumor antigens in solid tumors

CAR-T cells possess the capability to specifically recognize target antigens and subsequently eliminate tumor cells. Ideally, the target antigen should be uniformly and highly expressed on the surface of all tumor cells while remaining absent in normal tissues and cells. Nonetheless, the expression of target antigens in solid tumors at baseline often exhibits significant heterogeneity in both intensity and distribution. A previous study involving 52 colon cancer tissues demonstrated that only 28 tissue sections showed high expression levels of carcinoembryonic antigen (CEA) although all cancers expressed CEA³. Furthermore, antigen heterogeneity is also presented after the treatment due to the interaction between tumor cells and immune cells (Figure1). For instance, a clinical study revealed that following the infusion of epidermal growth factor receptor variant III (EGFRvIII)-CAR T cells, five out of seven glioblastoma (GBM) patients exhibited decreased expression levels of the EGFRvIII. This decline was associated with diminished immune responses and reduced therapeutic efficacy⁴. Immune escape of target antigen or antigen heteogeneity in tumor cells at baseline can both lead to tumor recurrence due to the outgrowth of target antigen-negative tumor cells after the clear of target antigen-positive tumor cells by CAR-T cells.

Figure 1: Spatial and temporal heterogeneity of tumor antigens in solid tumors

Dual-targeted CAR-T cell therapy overcoming antigen heterogeneity

To address antigen heterogeneity, one of the approaches is targeting two distinct tumor antigens, either of which can activate T cell responses (Figure2). Preclinical studies have shown that bivalent tandem CAR-T cells targeting combinations such as CD70 and B7-H3, or CD44 and CD133, exhibit enhanced anti-tumor functionality in lung cancer, melanoma, and glioblastoma multiforme (GBM)^{5, 6}. The function of tandem CAR is effected by the sequence of the variable region of light chain and heavy chain in each scFv, the sequence of two scFvs and so on. Bispecific T cell engagers (BiTEs) redirect T cells by linking two scFvs: one targeting a tumor antigen and the other binding to the CD3 molecule on T cells. For instance, Muc16-CAR T cells producing ESK1-BiTEs targeting the WT1-derived epitope RMFPNAPYL (RMF) demonstrate improved antitumor activity against epithelial ovarian cancer cells with low Muc16 expression⁷. However, broadening tumor recognition through dual-targeting CAR-T cells also raises concerns about on-target, off-tumor toxicity (OTOT) due to low-level expression of target antigens in normal tissues. The cytokine storm and pulmonary toxicity occurred in a patient with colon cancer following the administration of ERBB2-CAR T cells due to the location to the lung and the recognition of low levels of ERBB2 on lung epithelial cells⁸. It’s necessary to assess the safety of CAR-T cell therapy in clinical trials. The synNotch-CAR circuit, a sophisticated ‘IF-THEN’ logical circuit, initiates CAR-mediated cytotoxicity exclusively upon prior activation by the synNotch ligand. The synNotch-CAR circuits enable CAR-T cells recognize tumor-specific but heterogeneous neoantigen EGFRvIII and subsequently induces the expression of tandem CAR that can recognize GBM-associated target antigens, including ephrin type A receptor 2 (EphA2) and interleukin 13 receptor α2 (IL13Rα2), which have imperfect specificity. CAR-T cells engineered with synNotch-CAR circuits spatially restrict the CAR-driven cytotoxicity to the tumor site, thereby avoiding OTOT⁹. Furthermore, introducing exogenous antigens into tumor cells presents another strategy to tackle antigen heterogeneity while simultaneously avoiding the risk of OTOT. For example, fluorescein isothiocyanate (FITC)-CAR T cells have been shown to effectively target solid tumors following intratumoral delivery of a FITC-conjugated lipid-poly(ethylene)-glycol amphiphile, which inserts itself into tumor cell membranes, leading to tumor regression. This approach is both independent of endogenous antigen expression and the tissue of origin¹⁰.

Figure 2: Broad antigen recognition through multi-target therapy

Activation of endogenous immune cells boost the efficacy of CAR-T cells

Reprogramming the tumor microenvironment is another strategy by stimulating the endogenous immune responses to eradicate target antigen-negative tumor cells. Inducing endogenous T-cell responses against neoantigens can address antigen heterogeneity. The application of BiTEs not only activate CAR-T cells but also promote endogenous T cell responses further. Professional antigen-presenting cells, particularly dendritic cells (DCs), can capture and present the neoantigens released from cancer cells to naive T cells, thereby initiating and activating T cell responses specific to the neoantigens. Upon activated T-cell-mediated elimination of cancer cells, additional tumor antigens are released, enhancing the breadth and depth of the subsequent cancer-immunity cycle (Figure3a)^{11, 12}. Combining CAR-T cell therapy with DC-activating treatments can overcome antigen heterogeneity through priming endogenous T cell responses, such as vaccine, pattern recognition receptor agonists, and STING agonists (Figure3b)^13-16. Preclinical data revealed that combining a CAR ligand vaccine with CAR-T cells can recruit and activate DCs, thereby initiating the priming of endogenous antitumor T cells. This combination effectively controls solid tumors with preexisting antigen heterogeneity, even when the initial tumor burden was 50% target antigen-negative¹⁵.

Figure 3: a. The important role of DCs in antigen-spreading; b. Strategies to engage DCs to promote endogenous T cell priming and enhance the effector function.

Given the crucial role of DCs in the cancer-immunity cycle and antigen spreading, enhancing DC recruitment and function emerges as another strategy to augment CAR-T cell efficacy, particularly in solid tumors with limited DC infiltration (Figure3b). Previous research has demonstrated that engineering CAR-T cells to express chemokines such as IL-7 and CCL19 or IL-7 and CCL21 — crucial for maintaining T-cell zones within lymphoid organs via interactions with the chemokine receptor CCR7 — can significantly improve CAR-T cell responses against solid tumors exhibiting heterogeneous antigen expression. This is achieved by facilitating the infiltration of both DCs and T cells^{17, 18}. Conventional DCs (cDCs) are subdivided into two main subsets: CD11b⁺ cDC2s, CD8α⁺ lymphoid organ-resident cDC1s or CD103⁺ migratory cDC1s¹⁹. cDC1s can cross-present antigens to naïve CD8⁺ T cells and promote endogenous CD8⁺ T cell responses to tumor neoantigens. Studies revealed that the antitumor efficacy of CAR-T cells overexpressing CD40L is impaired in Batf3^-/- mice due to the absence of CD103⁺ cDC1s, underscoring the critical role of cDC1s in mediating antitumor responses²⁰. A previous study demonstrated that engineering CAR-T cells to secrete the dendritic cell growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) can expand intratumoral cDC1s and endogenous T cells to promote antigen spreading over target antigen in solid tumors²¹. One challenge for Flt3L therapies is that Flt3L expands not only cDC1s but also Treg cells, which may inhibit overall immune response²². CD103⁺ migratory cDC1s not only facilitate T-cell cross-priming within lymph nodes but also recruit CD8⁺ effector T cells back to the tumor site through the production of CXCR3 ligands²³. And the cDC1 subset is uniquely characterized by the expression of chemokine receptor XCR1 which responds to chemokine lymphotactin XCL1. Considering the crucial role of migratory cDC1s, we engineered CEA-CAR T cells to express IL-7 and XCL1 in our recent study, which can mobilize migratory cDC1s via XCR1 directly from peripheral blood, thereby promoting the generation of antigen-specific T cells against neoantigens released by CEA-positive tumor cells and resisting CEA-negative tumor cells²⁴.

Conclusion

The spatiotemporal heterogeneity of tumor antigens poses a significant challenge in the application of CAR-T cell therapy to solid tumors, contributing to limited efficacy. An attractive strategy to address this issue is optimizing the structure of CAR or employing combination approaches targeting two distinct tumor antigens. However, it is crucial to be mindful of on-target, off-tumor toxicities due to the low expression of target antigens in normal tissues. The tumor microenvironment plays a pivotal role in influencing the effectiveness of CAR-T cells. Combining CAR-T cell therapy with other immunotherapies can stimulate host immune cells and provoke endogenous T cell responses against neoantigens, thereby mitigating tumor recurrence driven by target antigen-negative cells. Additionally, various modification strategies complicate the manufacturing process of CAR-T cells, underscoring the need for standardized protocols to ensure safety and consistency. Prior to implementing CAR-T cell therapy, a careful risk-benefit assessment balancing efficacy and potential toxicities is essential, accompanied by rigorous post-treatment monitoring and symptomatic interventions. Despite these challenges, CAR-T cell therapy retains immense potential to revolutionize solid tumor treatment through innovative and multifaceted strategies.

Acknowledgements

This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-021), the Prospective Research Program of Changzhou Xitaihu Development Foundation for Frontier Cell-Therapeutic Technology (2024-P-003), National Natural Science Foundation of China (32100756).

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