Enhancing the antitumor activity of CD19/BCMA CAR-T cells in vitro with a PD1IL7R chimeric switch receptor.

Advancing together and moving forward: Combination gene and cellular immunotherapies

B-cell maturation antigen chimeric antigen receptor T-cell re-expansion in a patient with myeloma following salvage programmed cell death protein 1 inhibitor-based combination therapy.

Chimeric antigen receptor (CAR) T-cell therapy has encountered limited success in solid tumors. The lack of dependable antigens and the immunosuppressive tumor microenvironment (TME) are major challenges. Within the TME, tumor cells along with immunosuppressive cells employ an immune-evasion mechanism that upregulates programmed death ligand 1 (PD-L1) to deactivate effector T cells; this makes PD-L1 a reliable, universal target for solid tumors. We developed a novel PD-L1 CAR (MC9999) using our humanized anti-PD-L1 monoclonal antibody, designed to simultaneously target tumor and immunosuppressive cells. The antigen-specific antitumor effects of MC9999 CAR T-cells were observed consistently across four solid tumor models: breast cancer, lung cancer, melanoma, and glioblastoma multiforme (GBM). Notably, intravenous administration of MC9999 CAR T-cells eradicated intracranially established LN229 GBM tumors, suggesting penetration of the blood-brain barrier. The proof-of-concept data demonstrate the cytolytic effect of MC9999 CAR T-cells against immunosuppressive cells, including microglia HMC3 cells and M2 macrophages. Furthermore, MC9999 CAR T-cells elicited cytotoxicity against primary tumor-associated macrophages within GBM tumors. The concept of targeting both tumor and immunosuppressive cells with MC9999 was further validated using CAR T-cells derived from cancer patients. These findings establish MC9999 as a foundation for the development of effective CAR T-cell therapies against solid tumors.

ObjectiveColorectal cancer (CRC) presents significant treatment challenges, including immune evasion and tumor microenvironment (TME) suppression. Chimeric antigen receptor (CAR) T-cell therapy has shown promise in hematologic malignancies, but its effectiveness against solid tumors is hampered by the detrimental effects of the TME. This article aims to explore the potential of bispecific CAR T cells targeting programmed death-ligand 1 (PD-L1) and cancer-associated fibroblasts (CAFs) in CRC treatment.Material and MethodsDual-targeted CAR-T cells against PD-L1 and CAF were engineered using the GV400 lentiviral vector. Programmed death-1 (PD-1)/nanobody (Nb) and fibroblast activation protein (FAP)/Nb-encoding lentiviral vectors were generated, and CAR T cells were produced through a three-plasmid system in 293T cells. Human peripheral blood mononuclear cells (PBMCs) were separated, transduced with these vectors, and then expanded. Functional characterization of CAR-T cells was performed through enzyme-linked immunosorbent assay (ELISA), Western blot analysis, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and cell counting kit-8 (CCK-8) assay. Migration and invasion assays were conducted using Transwell chambers to assess the ability of FAP-PD-1/Nb CAR-T cells to migrate toward tumor cells and invade the extracellular matrix.ResultsWe developed dual-targeted CAR-T cells incorporating PD-L1 and CAF Nbs, which continuously secreted PD-1/Nb. Western blot confirmed PD-1/Nb expression in PD-1/Nb and FAP-PD-1/Nb CAR-T cells, with no expression in the untreated (UTD) group (P < 0.01). Flow cytometry showed a significantly higher cluster of differentiation (CD)25 and CD69 expression in FAP-PD-1/Nb CAR-T cells upon stimulation with FAP-positive target cells compared with the other groups (P < 0.01). TUNEL, flow cytometry, and CCK-8 assays revealed that FAP-PD-1/Nb CAR-T cells exhibited superior cytotoxicity and proliferation inhibition against FAP-positive HCT116 cells (P < 0.01). ELISA demonstrated increased interferon-gamma and tumor necrosis factor-alpha levels and reduced interleukin-10 (P < 0.01), suggesting enhanced cytokine modulation and antitumor immunity. Compared with single-target CAR-T cells and UTD, FAP-PD-1/Nb CAR-T cells showed notably enhanced Matrigel penetration and invasion (P < 0.01). Safety tests confirmed minimal cytotoxicity to normal PBMCs, indicating favorable safety.ConclusionThis study successfully developed dual-targeted CAR-T cells against PD-L1 and CAF and demonstrated their superior antitumor activity and immunomodulatory effects on CRC treatment. This novel therapeutic strategy was established using CAR T-cell technology for the treatment of CRC.

[Purpose] Immunosuppressive tumor microenvironment (TME) is the major hurdle to cancer immunotherapy. Advanced hepatocellular carcinoma (aHCC) is one of the representative cold tumors with immunosuppressive TME and presents obvious correlation between immune check point expression, such as programmed cell death ligand 1 (PD-L1), and poorer prognosis. In aHCC patients, cancer cells and stellate cells in TME appeared to express high level of PD-L1, which should suppress activities of antitumor immune cells. In this context, the combination of PD-1/PD-L1 inhibitors and other therapeutic modalities are tried to treat aHCC. The chimeric antigen receptor (CAR) T cell therapy is a potent way of eradicating cells expressing targetable antigens. Removal of PD-L1 expressing cells in the aHCC TME will improve the clinical outcomes of current therapies against aHCC. In the present study, we developed an autologous anti-PD-L1 CAR T strategy and show high efficacy of tumor suppression using an HCC xenograft model. [Result] We constructed second generation CAR lentiviral vector expressing an unique anti-PD-L1 scFv manifesting moderated affinity. The scFv was specifically generated by phage display technique using human B cell-derived naïve cDNA library, which targets PD-L1 with intermediate affinity in solid tumor TME. As the secondary signaling motif, CD28 was chosen and tested along with CD3 zeta chain. The effector CAR T, named VPC1, cells specifically recognized PD-L1 expressing HCC cells and exhibited rapid and robust cytotoxic activity in 2D and 3D culture experiments. In an HCC xenograft NSG mouse model, VPC1 CAR T cells significantly suppressed the tumor growth. More importantly, VPC-1-treated animals have well tolerated the CAR T therapy with no significant adverse manifestations during one month observation period and exhibited gradual weight gains since 3 days after CAR T cell injection. [Conclusion] We hereby report that a new autologous PD-L1 CAR-T therapeutic is capable of eradicating solid tumors expressing PD-L1, which would significantly potentiate efficacies of current conventional and immunotherapeutic strategies. Citation Format: Kyung-Sik Lee, Hye-Seong Park, Guk-Yeol Park, Gi-Chan Lee, Eun-Ji Choi, Jae-in Yu, Seung-Joo Yang, Mihwa Kim, Yoonjoo Choi, Je-Jung Lee, Joon Haeng Rhee, Bum Chan Park, Jae Eun Park. Development of an anti hepatocellular carcinoma CAR T strategy targeting PDL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6189.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

CRG-023 Is a Novel Tri-Specific CAR T Product Candidate Engineered to Prevent Antigen Escape and Sustain Durable Anti-Tumor Functionality Against B-Cell Malignancies

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Shortening the ex vivo culture of CD19-specific CAR T-cells retains potent efficacy against acute lymphoblastic leukemia without CAR T-cell-related encephalopathy syndrome or severe cytokine release syndrome.

CAR-T Cells Targeting BAFF-Receptor for B-Cell Malignancies: A Potential Alternative to CD19

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of 9%. Major obstacles to successful treatment of pancreatic cancer are the immunosuppressive tumor microenvironment (TME) and antigenic complexity or heterogeneity. Programmed death-ligand 1 (PD-L1) is expressed on PDAC and immunosuppressed cells within the TME, providing suitable immunotherapy targets. We applied a chimeric antigen receptor (CAR) strategy to target immune checkpoint programmed death-1 (PD-1)/PD-L1 interactions. Lentiviral vectors were used to express the extracellular domain of human PD-1 (PD-1-CD28-4-1BB activating chimeric receptor [PD1ACR]) or the single-chain variable fragment (scFv) region of anti-PD-L1 (PDL1CAR) that binds to PD-L1, and each was fused to intracellular signaling domains containing CD3 zeta, CD28, and 4-1BB (CD137). Both engineered CAR T cells recognized and eliminated PD-L1-overexpressing CFPAC1 cells efficiently at approximately 80% in vitro. Adoptive transfer of both CAR T cells enhanced T cell persistence and induced specific regression of established CFPAC1 cancer by >80% in both xenograft and orthotopic models. Ki67 expression in tumors decreased, whereas proinflammatory cytokines/chemokines increased in CAR T cell-treated mouse sera. PD1ACR and PDL1CAR obtained a similar therapeutic efficacy. Thus, these armed third-generation PD-L1-targeted CAR T cells confer antitumor activity and the ability to combat T cell exhaustion, providing a potentially new and innovative CAR T cell immunotherapy against pancreatic cancers.

Despite its success in treating blood cancers, chimeric antigen receptor (CAR) T-cell therapy has not yielded anticipated clinical outcomes in patients with solid tumors. This discrepancy is primarily attributed to the lack of dependable antigens and the immunosuppressive nature of the tumor microenvironment (TME). A pivotal evasion mechanism employed by tumor to counter antitumor immunity involves the upregulation of programmed death ligand 1 (PD-L1), which interacts with PD1 on T cells, deactivating effector T cells. A variety of immunosuppressive cells within the TME employ similar mechanisms to inhibit T-cell functions. These findings provide a rationale for targeting PD-L1 in both tumors and the immunosuppressive cells. Using a humanized anti-PD-L1 monoclonal antibody, we engineered a second-generation PD-L1 CAR, namely MC9999. MC9999 CAR T-cells exhibited antigen-specific cytotoxicity in vitro and elicited potent in vivo antitumor effects against PD-L1-expressing MDA-MD-232 breast cancer cells. The effectiveness of MC9999 CAR T-cells was confirmed across three other solid tumor models: non-small cell lung cancer, melanoma and glioblastoma (GBM). Notably, intravenous administration of MC9999 CAR T-cells eradicated intracranially established LN229 GBM tumors, suggesting the potential to penetrate the blood-brain barrier. In addition to targeting tumor cells, MC9999 CAR T-cells exhibited cytotoxicity against three immunosuppressive cell models, including HMC3 microglia cells, M2 macrophages, and importantly, primary tumor-associated macrophages (TAMs) isolated from GBM. Furthermore, we engineered patient-derived MC9999 CAR T-cells and demonstrated their effectiveness against two primary tumor cell lines derived from GBM tumors and HMC3 cells. Collectively, these proof-of-principle study findings substantiate the viability of targeting PD-L1 against both tumors and their immunosuppressive microenvironment. This prototype MC9999 CAR lays the groundwork for further development of clinically applicable CAR T-cell therapy against solid tumors. Citation Format: Yaqing Qie, Emiliano Sanchez Garavito, Maria J. Ulloa Navas, Tanya Hundal, Alfredo Quinones-Hinojosa, Hong Qin, Yan Luo. Weaponizing CAR T-cells to attack PD-L1 presenting cells in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB071.

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

The successful outcomes of chimeric antigen receptor (CAR) T-cell therapy in treating hematologic cancers have increased the previously unprecedented excitement to use this innovative approach in treating various forms of human cancers. Although researchers have put a lot of work into maximizing the effectiveness of these cells in the context of solid tumors, few studies have discussed challenges and potential strategies to overcome them. Restricted trafficking and infiltration into the tumor site, hypoxic and immunosuppressive tumor microenvironment (TME), antigen escape and heterogeneity, CAR T-cell exhaustion, and severe life-threatening toxicities are a few of the major obstacles facing CAR T-cells. CAR designs will need to go beyond the traditional architectures in order to get over these limitations and broaden their applicability to a larger range of malignancies. To enhance the safety, effectiveness, and applicability of this treatment modality, researchers are addressing the present challenges with a wide variety of engineering strategies as well as integrating several therapeutic tactics. In this study, we reviewed the antigens that CAR T-cells have been clinically trained to recognize, as well as counterstrategies to overcome the limitations of CAR T-cell therapy, such as recent advances in CAR T-cell engineering and the use of several therapies in combination to optimize their clinical efficacy in solid tumors.

The success of chimeric antigen receptor (CAR) T-cell therapy in treating hematologic malignancies has generated widespread interest in translating this technology to solid cancers. However, issues like tumor infiltration, the immunosuppressive tumor microenvironment, and tumor heterogeneity limit its efficacy in the solid tumor setting. Recent experimental and clinical studies propose local administration directly into the tumor or at the tumor site to increase CAR T-cell infiltration and improve treatment outcomes. Characteristics of the types of solid tumors that may be the most receptive to this treatment approach remain unclear. In this work, we develop a spatiotemporal model for CAR T-cell treatment of solid tumors, and use numerical simulations to compare the effect of introducing CAR T cells via intratumoral injection versus intracavitary administration in diverse cancer types. We demonstrate that the model can recapitulate tumor and CAR T-cell data from imaging studies of local administration of CAR T cells in mouse models. Our results suggest that locally administered CAR T cells will be most successful against slowly proliferating, highly diffusive tumors, which have the lowest average tumor cell density. These findings affirm the clinical observation that CAR T cells will not perform equally across different types of solid tumors, and suggest that measuring tumor density may be helpful when considering the feasibility of CAR T-cell therapy and planning dosages for a particular patient. We additionally find that local delivery of CAR T cells can result in deep tumor responses, provided that the initial CAR T-cell dose does not contain a significant fraction of exhausted cells.