Chimeric Antigen Receptor Construct Research Articles

Abstract PO4-06-08: ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER

Abstract Triple negative breast cancer (TNBC) has the worst prognosis of BC subtypes, largely due to lack of approved targeted therapies. Chimeric antigen receptor (CAR) T cells have demonstrated great success in certain hematological malignancies; however, in the treatment of solid cancers, CAR T cells are faced with a variety of obstacles that limit their efficacy, including on-target/off-tumor toxicity. To minimize these toxicities, the ideal CAR T cell target must be overexpressed by the tumor and absent from vital organs. Cancer/germline (CG) antigens satisfy this need, as their expression is restricted to male or female germ cells. Using a bioinformatic pipeline to identify novel CG antigens expressed in TNBC and other solid cancers, we have identified zona pellucida glycoprotein 4 (ZP4) as a candidate CG antigen target for CAR T cell therapy. This protein is a component of the human zona pellucida matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. We have identified ZP4 expression in approximately 10% of TNBC cases from available RNAseq and proteomic databases and have confirmed ZP4 expression in several TNBC PDX models. The overall purpose of this study is to redirect T cells against ZP4 by engineering and validating a second generation ZP4-specific CAR. We have generated a library of monoclonal antibody (mAb) candidates against the extracellular portion of human ZP4. Antibody clones were screened, and their specificity validated via ELISA and flow cytometry, and the three strongest binders (z108, z128, and z164) were selected for further investigation. By IHC, we confirmed that the ZP4 mAb clones stained the area surrounding normal oocytes and did not show binding to any of the 23 normal tissues screened. The variable regions of the antibody heavy and light chains were sequenced using 5’RACE and used to assemble second generation CAR constructs composed of the variable heavy and light chain fragments separated by a linker, a CH2/CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3z signaling domain. Activated T cells transduced with retroviral supernatant express the ZP4 CAR constructs at high levels. While all ZP4 CAR T cells demonstrate antigen-specific cytotoxic killing of ZP4+ target tumor cells in short-term in vitro assays, z128 ZP4 CAR T cells demonstrate superior persistence and efficacy in long-term repeat tumor challenge in vitro assays and orthotopic in vivo mouse models. In summary, ZP4 is a promising target for TNBC and we have successfully engineered ZP4 specific CAR T cells. ZP4 CAR T cells effectively target and kill ZP4+ cells in in vitro cytotoxicity assays but perform differently in repeat tumor challenge assays and in vivo. Future studies will elucidate the precise differences between our three candidate CAR constructs and further test the effector function, trafficking, proliferation, and persistence of z128 ZP4 CAR T cells in vivo using ZP4+ PDX models. This work has exciting potential to bring a novel CAR T cell therapy to patients with TNBC, without concerns for on-target off-tumor toxicity. Citation Format: Lauren Somes, Jonathan Lei, Paul Shafer, Xinpei Yi, Diego Chamorro, Emily Madaras, Michael Lewis, Bing Zhang, Valentina Hoyos. ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-08.

Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo.

Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

Immunotherapy with genetically engineered T cells holds promise for the treatment of nonmalignant diseases in the dog.

The ability to genetically redirect the antigenic specificity of T cells using chimeric antigen receptors (CAR) has led to unprecedented durable clinical remissions in human patients with relapsed/refractory hematological malignancies. This remarkable advance in successful immune cell engineering has now led to investigations into the application of CAR-T-cell technology to treat nonmalignant diseases. The use of CAR-T cells to target and eliminate specific cell subsets involved in the pathogenesis of autoimmunity, fibrosis, senescence, and infectious disease represents a new direction for adoptive cell therapies. While the use of CAR-T cells for nonmalignant disease is still in its infancy, early reports of dramatic clinical responses to CAR-T cells targeting CD19+ B cells in patients with severe autoimmune disease raise the possibility that this approach could lead to durable remissions, eliminating the need for ongoing conventional immunosuppressive therapies. Excitingly, nonmalignant disease processes that may be addressed by CAR-T-cell therapy in humans also occur in our canine populations. Given that technologies for developing canine CAR constructs are now available, robust protocols have been described for generating canine CAR-T cells, and experience is being gathered with their clinical use in oncology, it is anticipated that CAR-T cells will soon enter the veterinary clinics for the treatment of debilitating nonmalignant diseases. Here, we provide a broad overview of CAR-T-cell therapies for nonmalignant diseases and extrapolate these advances into the veterinary space, highlighting areas in which canine CAR-T cells are poised to enter the clinics for the treatment of nonmalignant disease.

Abstract P66: Off-The-Shelf Epstein Barr Virus Specific T Cells Armored with B7-H3-Targeting Chimeric Antigen Receptors Is an Effective and Safe Tumor- Agnostic Therapy For B7-H3 Positive Solid Malignancies

Abstract In contrast to its limited expression in normal tissues, the B7 homolog 3 protein (B7-H3) is over-expressed in many human tumors, making it an attractive target for chimeric antigen receptor (CAR) T cell therapy. To address the technical challenges limiting broader patient access to autologous CAR T cell therapy, we armoured Epstein-Barr Virus Specific T Cells (EBVSTs) with CARs as an off-the-shelf therapy against solid tumors. We identified and utilised a nanobody as the domain specifically targeting B7-H3 in our CAR construct to reduce the stability issues associated with scFv-based domains. Our B7H3.CAR armored EBVSTs demonstrated potent in-vitro and in-vivo activity against multiple B7-H3 positive tumor and patient-derived xenografts models. Analysis of human healthy tissues and tumors microarrays indicated that B7-H3 expression, while elevated in patient tumors, was differentially low in healthy tissues. Consistent with this, mouse T cells expressing a murine equivalent of our B7H3.CAR displayed good on-target off-tumor safety in immunocompetent mice bearing syngeneic tumors. Further in-vitro evaluation revealed that while human T, B and NK cells were unaffected by B7H3.CAR EBVSTs targeting, monocytes were readily targeted due to the upregulation of B7-H3. Consequent to this targeting and depletion of B7-H3 positive human myeloid cells which are key mediators of CRS, a low incidence of cytokine release syndrome was observed in humanized mice after B7H3.CAR EBVST treatment. Lastly, we showed that B7H3.CAR EBVSTs can target myeloid-derived suppressor cells (MDSCs) that express B7-H3 and in doing so, alleviate MDSC-driven immune suppression. Our findings demonstrate that our nanobody-based B7H3.CAR EBVSTs present not only an effective and safe off-the-shelf approach for targeting any B7-H3-positive solid tumor while also addressing challenges that hindered the clinical development of CAR T cells in such indications. Citation Format: Siok Ping Yeo, Lindsay Kua, Jin Wei Tan, Joanna Kristyn Lim, Fiona Wong, May Delos Santos, Chek Meng Poh, Angeline Goh XH, Xin Yu Koh, Xiao Hua Zhou, Rajarethinam R, Qing Feng Chen, Zhisheng Her, Ivan D Horak, Lionel Low, Kar Wai Tan. Off-The-Shelf Epstein Barr Virus Specific T Cells Armored with B7-H3-Targeting Chimeric Antigen Receptors Is an Effective and Safe Tumor- Agnostic Therapy For B7-H3 Positive Solid Malignancies [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P66.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [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 LB070.

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Abstract 3993: A tunable safety switch for solid tumor CAR T-cell therapy

Abstract On-target off-tumor toxicity, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS) are severe immune-related adverse events (irAEs) that are frequently associated with Chimeric Antigen Receptor (CAR) T-cell therapy. Current efforts to manage such therapy-related toxicities involve incorporation of an inducible suicide agent within CAR constructs, such as iCaspase-9 or herpes simplex virus type 1 thymidine kinase that can be selectively activated to produce toxic effects within CAR T cells and attenuate their activity. However, while activation of these agents helps to mitigate or overcome such unwarranted toxicities, the therapeutic benefit of CAR T cells anti-tumor activity is also compromised. Therefore, to continue maintenance of CAR T cells’ therapeutic function while minimizing irAEs, an ideal safety switch should 1) rapidly inhibit the activation and proliferation of CAR T cells exposed to the target antigen, 2) reversibly inhibit activity without inducing CAR T cell elimination and 3) be clinically translatable for safe application in patients. Our laboratory investigated one such safety switch to inhibit CAR T-cell activity while maintaining their therapeutic function. We showed that incorporating a mutant variant of c-KIT D816V (KITv) in the intracellular domain of mesothelin-targeting second-generation CAR T cells (M28z-KITv) improved efficacy in solid tumors with low antigen, or an immunosuppressive environment. Herein, we evaluate the use of Dasatinib, a clinically available multitarget (BCR, SRC, c-KIT) tyrosine kinase inhibitor (TKI), as a tunable safety switch to reversibly inhibit M28z-KITv CAR T-cell functional activity. In cohorts of mice established with lung adenocarcinoma, daily administration of Dasatinib starting on day 1 or day 3 after CAR T-cell administration stabilized tumor growth, which otherwise continued to regress in untreated mice, indicating inhibition of CAR T-cell functional activity. Upon discontinuation of Dasatinib, tumors regressed, indicating reversal of CAR T-cell functional activity. In an experiment conducted to investigate functional persistence of CAR T cells upon long-term exposure to Dasatinib (1 month), we noted uninhibited activity of CAR T cells to rechallenged tumors. Dasatinib, thus may act as a tunable safety switch to regulate M28z-KITv CAR T-cell activity without compromising its therapeutic function. Citation Format: Kyohei Misawa, Meriem Taleb, Srijita Banerjee, William-Ray Vista, Navin K. Chintala, Prasad S. Adusumilli. A tunable safety switch for solid tumor CAR T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3993.

Abstract 5250: Chimeric antigen receptor macrophages (MOTO CARs) driving repolarization of macrophages in solid tumors

Abstract Introduction: Though chimeric antigen receptor (CAR) T cells have been effective in treating hematological cancers, they have lacked in solid tumors. Due to the nature of solid tumors and how macrophages are localized there, we propose that macrophages are a better cell type for targeting solid tumors. In tumors, most of the macrophages are Tumor Associated Macrophages (TAMs) which are M2 polarized and promote tumor growth, angiogenesis, and metastases. If these macrophages could be repolarized to an anti-tumor, M1 phenotype, they could alter the tumor immune microenvironment and facilitate destruction of the tumor by immune cells. Methods: We hypothesize that CAR macrophages (MOTO-CARs) could infiltrate solid tumors, and affect the tumor immune microenvironment to repolarize (TAMs) from an M2 to M1 phenotype. Over 150 signaling domains from published sequences were cloned into a CAR constructs, comprised of an anti-mesothelin scFv, CD34 hinge, and transmembrane and activation domains from various immune cells. Functional activation assays were preformed in reporter HEK cells lines. Functional constructs were then transduced into THP-1 cells. The function of these MOTO-CARs were evaluated using a flow cytometry based phagocytosis assay (indication of an M1 phenotype) and RT-qPCR repolarization assay. Results: Of the constructs tested in HEK reporter cell lines, around one third demonstrated significant activation, that is 20% higher than the background control. The phagocytosis assay performed on the functional constructs showed that around one third of these constructs exhibited higher phagocytosis compared with a control construct with no signaling domain. However, it is noteworthy that this no signaling domain control had significantly higher rates of phagocytosis than untransduced controls. The RT-qPCR demonstrated that M2-like THP-1 cells transduced with the CAR constructs exhibited a significant shift in expression towards M1 characteristic genes when stimulated with mesothelin beads, with the TLR4 construct exhibiting the greatest shift. Conclusion: Several of the MOTO-CAR constructs, including TLR4, showed significant activation, phagocytosis, and ability to repolarize macrophages from M2 to M1 phenotype. Further analysis hopes to prove the functionality of these MOTO-CAR constructs with primary cells as well as in vivo, making MOTO-CARs a valuable treatment option for patients with solid tumors. Citation Format: Michael Boyer, Michelle Townsend, Timothy Phares, Adam Blaszczak, Rachel Garlick, Kenneth Katschke, Gaju Shrestha, Toni Mortimer, Ashlin Cowager, McCall Jensen, Abigail Cheever, Kim O'Neill. Chimeric antigen receptor macrophages (MOTO CARs) driving repolarization of macrophages in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5250.

Abstract 4015: Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Overall survival rates are low for metastatic RMS, emphasizing the urgent need of improved therapies. Chimeric Antigen Receptor (CAR) Ts have shown remarkable results against hematological malignancies, but CAR T cell therapy remains challenging in solid tumors, mainly due to a lack of ideal tumor specific targets, and of the immunosuppressive tumor microenvironment (TME). To maximize antitumoral activity against RMS and to address optimal CAR T persistence in vivo, novel CAR constructs were investigated against upregulated RMS targets. By surfaceome profiling, we recently identified L1CAM as promising candidate for RMS CAR T cell therapy, and confirmed CD276 and FGFR4 expression. CD276- (CD276.CD28HD.28TM.28CSD.3z) and Dual-CAR Ts targeting both CD276 and FGFR4 (F8-FR4.CD28HD.28TM.28CSD.3z) showed tumor eradication in an orthotopic RMS model, whereas FGFR4-CAR Ts exhibited only a partial response in vivo. Moreover, CD276-CAR Ts showed a limited activity against RMS tumors expressing lower amounts of CD276, eradicating the tumor in 1/5 mice, underlying the need of improved CAR T cell potency against low-density target-expressing tumors. First, to improve FGFR4-CAR Ts activity, we optimized the recognition of FGFR4 epitope by modifying the CAR moiety “hinge”. We replaced the CD28-derived hinge with various hinges of different length: no hinge, IgG4 (12aa), IgG4CH3 (119aa), and IgG4CH2CH3 (229aa). Luciferase assays showed effective in vitro killing capacity of FGFR4-CAR Ts expressing an IgG4-derived hinge, with a tumor cell survival less than 10% at Effector to Target (E:T) ratio of 5:1. They also released the double amount of IFN-γ (50pg/ml) compared to the other constructs, when co-incubated with Rh4 cells. Next, to verify the potential of L1CAM as CAR T target for RMS, we generated a panel of L1CAM-CARs expressing either a long (IgG4CH2CH3, 229aa) or a short (CD28, 35aa) Hinge, and a CD28- or 41BB-derived costimulatory domain (CSD). L1CAM.IgG4CH2CH3Hinge.28TM.28CSD.3z CAR Ts outperformed the other constructs, killing ~60% of Rh30 at the E:T ratio of 1:1 and more than 90% at the E:T ratio of 5:1. They also exhibited a 3-fold higher IFN-γ release (120 pg/ml) compared to the other constructs. However, although our examination of patient-derived TMA revealed a strong immunoreactivity for L1CAM, more expressed in aggressive RMS subtypes, the selected L1CAM-CAR Ts against Rh30 cells, exhibiting ~10’000 L1CAM copies, showed limited activity in an in vivo pilot study. Based on these results and on the RMS TME investigation, which revealed consistent collagen (COL4A1, COL4A2 and COL18A1) deposition in RMS-derived mice tumor xenografts, we hypothesize that co-expressing a collagenase, able to partially degrade the dense extracellular matrix surrounding tumors, will lead to an improved CAR T cell infiltration and activity in vivo. Citation Format: Caroline Piccand, Andrea Timpanaro, Stenija Anton-Joseph, Michele Bernasconi, Jochen Karl Rössler. Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4015.

Abstract 5243: CAR B cells to accelerate antigen presentation in cancer

Abstract Engineered B cells hold promise for multiple therapeutic applications. Walking Fish Therapeutics is engineering B cells to produce replacement proteins for deficiency diseases, antibodies of defined specificity and effector function, and Chimeric Antigen Receptor (CAR) B cells with and without therapeutic payloads for use in the oncology setting. B cells are professional antigen presenting cells (APCs) with the ability to present antigen to CD4+ T cells. Recent studies have found the presence of tumor infiltrating B cells are associated with favorable outcomes across multiple cancer types. Adoptive transfer of B cells has demonstrated antitumor activity in a syngeneic mouse model. B cells from OVA-vaccinated transgenic luciferase mice were adoptively transferred to mice bearing EG7.OVA-tumors. We observed B cells migrating to the tumor and tumor draining lymph node (TDLN), locations where B cells can engage with antigen. Further, we observed a strong anti-tumor response in mice receiving B cells from OVA-vaccinated donors. Based on these results, we hypothesized that B cells engineered to target tumor specific antigens may engage and activate the immune system leading to an enhanced antitumor immune response. We generated CAR B cells expressing synthetic BCRs with tumor-antigen specific scFvs fused to designated B cell signaling motifs. Expression of different CAR constructs were confirmed in both human and mouse B cells. Upon antigen engagement CAR B cells release pro-inflammatory cytokines. Importantly, we demonstrate in vitro that antigen-specific CAR B cells can recognize cognate cell surface antigen, leading to uptake, processing, and presentation on MHCII as detected by activation of peptide-specific CD4+ T cells. When administered in vivo by IV administration, we observed a significant increase in mouse CAR B cells at the tumor site compared to unengineered B cells. We are currently investigating additional CAR formats that may lead to enhanced anti-tumor activity. Citation Format: Mary Ellen Molloy, Jessica Balero, Ana Contreras-Sandoval, Mark Selby, Hangil Park, Srinivas Kothakota, Kathleen Boyle, Rosa Romano, Smrithi Rajendiran, Moises Hernandez, Ariel Pios, Rusty Williams. CAR B cells to accelerate antigen presentation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5243.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6330.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Abstract 4005: HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC

Abstract Approximately 3% of non-small cell lung cancer (NSCLC) cases harbor ERBB2/HER2 mutations. Notably, the majority of actionable HER2 mutations in NSCLC are in-frame insertions in exon 20. Current HER2-targeting approaches, including antibody-drug conjugates (ADCs) such as trastuzumab deruxtecan, have shown promising response rates. To explore novel strategies for targeting HER2-positive NSCLC, we investigated chimeric antigen receptor (CAR)-based therapies to enhance the anti-tumor activity of immune effector cells. We engineered a series of HER2 CAR constructs utilizing scFvs derived from trastuzumab, pertuzumab, and FRP5, recognizing distinct domains of HER2. These scFvs were integrated into second-generation BB3z (with 4-1BB and CD3z intracellular domains) and third-generation 28BB3z (with CD28, 4-1BB, and CD3z intracellular domains) CAR backbones, which were subsequently transduced into activated T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. High transduction efficiencies were achieved, with over 80% in CAR-T cells and over 50% in CAR-NK cells. We next evaluated their activity against a panel of NSCLC cell lines expressing varying levels of HER2 in vitro using firefly luciferase assays. All HER2 CAR-T cells demonstrated more than 90% killing against H2170 (HER2 high) and H322 (HER2 medium) at an effector:target (E:T) ratio of 10:1, whereas HER2 CAR-T cells yielded a killing effect of 35% against A549 (HER2 low) cells. HER2-targeting CAR-NK cells also displayed potent cytotoxicity against HER2-expressing NSCLC cells. Moreover, we observed that treatment of HER2 mutant and HER2 amplified NSCLC cells with low doses of HER2 tyrosine kinase inhibitors (TKIs) such as poziotinib enhanced the expression of HER2 on the cell surface. This upregulation resulted in enhanced activation of HER2 CAR-T and CAR-NK cells in coculture experiments as measured by CD107a expression on effector cells. Additionally, treatment with the HER2 TKI poziotinib potentiated the killing activity mediated by HER2 CAR-T and CAR-NK cells. Using a HER2 YVMA duplication patient-derived xenograft (PDX) NSCLC model, we observed that the in vivo combination of poziotinib with HER2 CAR-NK cells exhibited superior anti-tumor activity as compared to poziotinib treatment alone. In conclusion, HER2 CAR-T and CAR-NK cells effectively target HER2-mutant and HER2-positive NSCLC, and their combination with HER2 TKIs enhances vulnerability to HER2-targeting strategies. Citation Format: Yan Yang, Monique B. Nilsson, Alissa Poteete, Junqin He, Qian Huang, John V. Heymach. HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4005.

A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus.

Invasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the single-chain variable fragment domain of monoclonal antibody AF-269-5 and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited a potent release of cytotoxic effectors and type 1 T cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL-2, IL-2 + IL-21, or IL-7 + IL-15) significantly suppressed mycelial growth of AF-293 after 18 hours of co-culture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 post-infection (P < 0.001) and significantly improved 7-day survival (P = 0.049) compared with mice receiving non-targeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo. Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR T-cell therapy after further refinement and thorough preclinical evaluation.IMPORTANCEInvasive aspergillosis (IA) remains a formidable cause of morbidity and mortality in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation. Despite the introduction of several new Aspergillus-active antifungals over the last 30 years, the persisting high mortality of IA in the setting of continuous and profound immunosuppression is a painful reminder of the major unmet need of effective antifungal immune enhancement therapies. The success of chimeric antigen receptor (CAR) T-cell therapy in cancer medicine has inspired researchers to translate this approach to opportunistic infections, including IA. Aiming to refine anti-Aspergillus CAR T-cell therapy and improve its feasibility for future clinical translation, we herein developed and validated a novel antibody-based CAR construct and lentiviral transduction method to accelerate the production of CAR T cells with high targeting efficacy against Aspergillus fumigatus. Our unique approach could provide a promising platform for future clinical translation of CAR T-cell-based antifungal immunotherapy.

Mesothelin antigen density influences anti-mesothelin chimeric antigen receptor T cell cytotoxicity

Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Mutation-specific CAR T cells as precision therapy for IGLV3-21R110 expressing high-risk chronic lymphocytic leukemia.

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.

Current advances in experimental and computational approaches to enhance CAR T cell manufacturing protocols and improve clinical efficacy

Since the FDA’s approval of chimeric antigen receptor (CAR) T cells in 2017, significant improvements have been made in the design of chimeric antigen receptor constructs and in the manufacturing of CAR T cell therapies resulting in increased in vivo CAR T cell persistence and improved clinical outcome in certain hematological malignancies. Despite the remarkable clinical response seen in some patients, challenges remain in achieving durable long-term tumor-free survival, reducing therapy associated malignancies and toxicities, and expanding on the types of cancers that can be treated with this therapeutic modality. Careful analysis of the biological factors demarcating efficacious from suboptimal CAR T cell responses will be of paramount importance to address these shortcomings. With the ever-expanding toolbox of experimental approaches, single-cell technologies, and computational resources, there is renowned interest in discovering new ways to streamline the development and validation of new CAR T cell products. Better and more accurate prognostic and predictive models can be developed to help guide and inform clinical decision making by incorporating these approaches into translational and clinical workflows. In this review, we provide a brief overview of recent advancements in CAR T cell manufacturing and describe the strategies used to selectively expand specific phenotypic subsets. Additionally, we review experimental approaches to assess CAR T cell functionality and summarize current in silico methods which have the potential to improve CAR T cell manufacturing and predict clinical outcomes.

CAR-T cell therapy: a game-changer in cancer treatment and beyond.

In recent years, cancer has become one of the primary causes of mortality, approximately 10 million deaths worldwide each year. The most advanced, chimeric antigen receptor (CAR) T cell immunotherapy has turned out as a promising treatment for cancer. CAR-T cell therapy involves the genetic modification of T cells obtained from the patient's blood, and infusion back to the patients. CAR-T cell immunotherapy has led to a significant improvement in the remission rates of hematological cancers. CAR-T cell therapy presently limited to hematological cancers, there are ongoing efforts to develop additional CAR constructs such as bispecific CAR, tandem CAR, inhibitory CAR, combined antigens, CRISPR gene-editing, and nanoparticle delivery. With these advancements, CAR-T cell therapy holds promise concerning potential to improve upon traditional cancer treatments such as chemotherapy and radiation while reducing associated toxicities. This review covers recent advances and advantages of CAR-T cell immunotherapy.

In vitro re-challenge of CAR T cells.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.