Antigen-specific Cytotoxicity Research Articles

Abstract IA023: Dual-drug ADC platform for effective and safe cancer treatment

Abstract Breast tumor heterogeneity presents significant treatment challenges, particularly in overcoming drug resistance and tumor relapse driven by heterogeneous gene expression variability. Antibody-drug conjugates (ADCs) have demonstrated therapeutic success, yet intratumor heterogeneity remains a hurdle. We investigated dual-drug ADCs as a promising approach to address this clinical challenge. Our conjugates achieved potent and antigen-specific cytotoxicity, favorable pharmacokinetic properties, and high tolerability. In xenograft models with heterogeneous HER2 expression, our anti-HER2 dual-drug ADC significantly outperformed co-administration of two separate single-drug ADCs, demonstrating enhanced capacity to surmount tumor heterogeneity and resistance. Additionally, we developed a novel glutamic acid-glycine-citrulline (EGCit) linker, which enhances ADC hydrophilicity and stability in circulation. This EGCit linker also resists neutrophil protease degradation, a factor implicated in ADC-associated neutropenia. Compared to currently approved ADCs, our EGCit-based ADC exhibited reduced blood and liver toxicity, alongside superior antitumor efficacy in preclinical xenograft studies. These findings underscore the potential of our technology platform for advancing safer, more effective treatments for breast and other refractory cancers. Citation Format: Kyoji Tsuchikama. Dual-drug ADC platform for effective and safe cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr IA023.

Stage-specific CAR-mediated signaling generates naïve-like, TCR-null CAR T cells from induced pluripotent stem cells.

Genetically modified, induced pluripotent stem cells (iPSCs) offer a promising allogeneic source for the generation of functionally enhanced, chimeric antigen receptor (CAR) T cells. However, the signaling of CARs during early T cell development and the removal of the endogenous T cell receptor required to prevent alloreactivity pose significant challenges to the production of mature conventional CAR T cells from iPSCs. Here, we show that TCR-null, CD8αβ CAR T cells can be efficiently generated from iPSCs by engineering stage-specific onset of CAR expression and signaling to both permit conventional T cell development and to induce efficient positive selection. CAR T cells produced using this approach displayed a uniform, naïve T cell phenotype and demonstrated superior antigen-specific cytotoxicity compared to iPSC-derived effector memory CAR T cells. Multimodal sequencing revealed CAR-mediated positive selection induced the persistent upregulation of key transcription factors involved in naïve T cell development. Achieving precise control of CAR expression and signaling in developmentally sensitive T precursors will be critical to realizing the full potential for "off-the-shelf", iPSC-derived cellular therapies.

Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

Despite the central role of T cells in tumor immunity, attempts to harness their cytotoxic capacity as a therapy have met limited efficacy, partially as a result of the suppressive microenvironment which limits their migration and activation. In contrast, myeloid cells massively infiltrate tumors and are well adapted to survive these harsh conditions. While they are equipped with cell-killing abilities, they often adopt an immunosuppressive phenotype upon migration to tumors. Therefore, the questions of how to modify their activation programming against cancer, and what signaling cascades should be activated in myeloid cells to elicit their cytotoxicity have remained unclear. Here, we found that activation of IgM-induced signaling in murine myeloid cells results in secretion of lytic granules and massive tumor cell death. These findings open venues for designing novel immunotherapy by equipping monocytes with chimeric receptors that target tumor antigens and consequently, signal through IgM receptor. Nonetheless, we found that myeloid cells do not express the antibody-derived portion used to recognize the tumor antigen due to the induction of an ER stress response. To overcome this limitation, we designed chimeric receptors that are based on the high-affinity FcγRI for IgG. Incubation of macrophages expressing these receptors along with tumor-binding IgG induced massive tumor cell killing and secretion of reactive oxygen species and Granzyme B. Overall, this work highlights the challenges involved in genetically reprogramming the signaling in myeloid cells and provides a framework for endowing myeloid cells with antigen-specific cytotoxicity.

Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity.

Despite the central role of T cells in tumor immunity, attempts to harness their cytotoxic capacity as a therapy have met limited efficacy, partially as a result of the suppressive microenvironment which limits their migration and activation. In contrast, myeloid cells massively infiltrate tumors and are well adapted to survive these harsh conditions. While they are equipped with cell-killing abilities, they often adopt an immunosuppressive phenotype upon migration to tumors. Therefore, the questions of how to modify their activation programming against cancer, and what signaling cascades should be activated in myeloid cells to elicit their cytotoxicity have remained unclear. Here, we found that activation of IgM-induced signaling in murine myeloid cells results in secretion of lytic granules and massive tumor cell death. These findings open venues for designing novel immunotherapy by equipping monocytes with chimeric receptors that target tumor antigens and consequently, signal through IgM receptor. Nonetheless, we found that myeloid cells do not express the antibody-derived portion used to recognize the tumor antigen due to the induction of an ER stress response. To overcome this limitation, we designed chimeric receptors that are based on the high-affinity FcγRI for IgG. Incubation of macrophages expressing these receptors along with tumor-binding IgG induced massive tumor cell killing and secretion of reactive oxygen species and Granzyme B. Overall, this work highlights the challenges involved in genetically reprogramming the signaling in myeloid cells and provides a framework for endowing myeloid cells with antigen-specific cytotoxicity.

Abstract LB071: Weaponizing CAR T-cells to attack PD-L1 presenting cells in solid tumors

Abstract 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.

Abstract 6311: From bench to bedside: GMP manufacturing and testing of an LGR5-targeting CAR-T against colorectal cancer

Abstract Colorectal cancer is the third most diagnosed cancer worldwide, and is responsible for the second largest number of cancer related deaths, warranting the need for new treatments. The successful use of chimeric antigen receptor T (CAR-T) cells against B-cell malignancies has inspired the search for CAR-T cell therapies against solid tumours such as colorectal cancer. Cancer stem cells (CSC) are a small population of cells within a tumour with the capacity to self-renew, differentiate, and initiate new tumours. CSC are resistant to chemotherapy and radiotherapy and are enriched in residual disease. Targeting CSC is a strategy to reduce the tumours’ ability to generate new cells. Leucine-rich G protein-coupled receptor 5 (LGR5) is a marker of CSC and LGR5+ cancer cells are implicated in tumour progression and metastasis in colorectal cancer. We designed and tested a chimeric antigen receptor (CAR) targeting the extracellular domain of LGR5. LGR5-targeting CAR-T cells showed significant antigen-specific cytotoxicity and IFNγ release against colorectal cancer (CRC) cell lines in vitro, and in vivo efficacy in a CRC xenograft mouse model. We confirmed the specificity and safety of the LGR5 targeting CAR-T cells by testing against normal human tissues. A GMP ready protocol for clinical scale manufacture of LGR5-targeting CAR-T cells have been developed using CD3+ T-cells from healthy donors. We have successfully tested this protocol using CRC patient-derived CD3+ T-cells and were able to generate clinically relevant cell numbers (80-fold expansion) with high CAR expression. Cells manufactured using this GMP protocol expressed markers of self-renewing naïve-like and central memory phenotype and showed very low percentage of cells that were triple-positive for PD1, TIM3 and LAG3. This protocol has been successfully tested and validated in a GMP manufacturing facility for the production of LGR5 targeting CAR-T cells, in preparation for an FDA approved first in man clinical trial against metastatic colorectal cancer, starting in 2023 (NCT05759728). Citation Format: Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Jade Foeng, Dylan McPeake, Timona Tylis, Emma Thompson, Stuart Mills, Allison Cowin, Claudine Bonder, Timothy Sadlon, Shaun McColl, Simon Barry. From bench to bedside: GMP manufacturing and testing of an LGR5-targeting CAR-T against colorectal 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 6311.

Abstract 723: Reduction of immunosuppressive function of regulatory T cells enhances antigen-specific T cell mediated cytotoxicity against tumor cells

Abstract Regulatory T cells (Tregs) maintain immune homeostasis by suppressing abnormal immune response to self and non-self-antigens. Tregs are also involved in tumor development and progression by inhibiting anti-tumor immunity. Infiltration of Tregs in the tumor microenvironment (TME) inhibit anti-tumor immunity by suppressing tumor antigen-specific T cell responses. This results in tumor progression and poor prognosis in patients with various types of cancers. Therefore, depletion of Tregs could be a promising immunotherapy target to augment the anti-tumor immune response. Molecules highly expressed by Tregs such as CD25, immune checkpoint molecules, and chemokine receptors have been targeted by antibodies or small molecules to deplete Tregs and its immunosuppressive function has been tested in the clinic. However, a thin line exists between augmenting effective immunity and inducing autoimmunity, posing difficulties in harnessing Tregs modulation in cancer treatment. Thus, additional strategies to selectively control Tregs are urgently needed for the effective treatment of cancer. In this study we evaluated the role of Tregs in suppressing the effector function of antigen-specific T cells and targeted killing of cancer cells in vitro. We observed Tregs mediated suppression of antigen-specific cytotoxicity against cancer cells. When Tregs were treated with cyclophosphamide, CD25 depleting antibody and anti-CTLA4 antibody alone or in combination, we observed enhanced killing of cancer cells in vitro as a result of potentiated effector function of antigen-specific T cells. This study provides a path forward to fine-tune and optimize strategies to target various molecules that are highly expressed on Tregs and augment anti-tumor immunity by antigen-specific T cells to achieve therapeutic outcome. Citation Format: Arif Azam Khan, Jaehyung Park, Nate LaVoy, Chris Tompkins, Jeff Bellinder, Tisha San Miguel, Neal Lawrence, Brianna Schoen. Reduction of immunosuppressive function of regulatory T cells enhances antigen-specific T cell mediated cytotoxicity against tumor cells [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 723.

Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model

AbstractAntibody-based immunotherapies have revolutionized leukemia and lymphoma treatment, with animal studies being crucial in evaluating effectiveness and side effects. By targeting the evolutionary conserved Slamf7 immune receptor, which is naturally expressed by the murine multiple myeloma cell line MPC-11, we have developed a syngeneic mouse model for direct comparison of 3 immunotherapies: monoclonal antibodies (mAb), bispecific T-cell engagers (BiTE), and chimeric antigen receptor (CAR) T cells (CART), all targeting Slamf7. Slamf7-BiTE is a bispecific single-chain antibody consisting of α-Slamf7 and α-CD3 Fv fragments joined through a Gly-Ser linker, and Slamf7-CART comprises the α-Slamf7 Fv fragment fused to the msCD8α transmembrane and msCD28, 4-1BB, and CD3ζ intracellular signaling domains. Slamf7-BiTE and Slamf7-CART effectively killed MPC-11 cells in vitro, independently of Slamf7-mediated inhibitory signaling by self-ligation. After chimerizing the constant region of the rat–anti-mouse Slamf7 antibody to mouse Fc-immunoglobulin G2a for enhanced effector functions, Slamf7-mAb triggered antigen-specific antibody-dependent cellular cytotoxicity by binding to Fcγ receptor IV. In vivo, all 3 immunotherapies showed antitumor effects against Slamf7-expressing targets. Unlike Slamf7-mAb, Slamf7-BiTE led to considerable side effects in test animals, including weight loss and general malaise, which were also observed to a lesser extent after Slamf7-CART infusion. In allogeneic transplant, Slamf7-BiTE and Slamf7-CART maintained activity compared with the nontransplant setting, whereas Slamf7-mAb displayed enhanced antimyeloma activity. In summary, our model faithfully replicates treatment efficacy and side effects detected after human immunotherapy. It aids in developing and improving immunotherapies and may help devise novel approaches to mitigate undesired effects in steady state and allogeneic stem cell transplantation.

A chimeric antigen receptor-based cellular safeguard mechanism for selective in vivo depletion of engineered T cells.

Adoptive immunotherapy based on chimeric antigen receptor (CAR)-engineered T cells has exhibited impressive clinical efficacy in treating B-cell malignancies. However, the potency of CAR-T cells carriethe potential for significant on-target/off-tumor toxicities when target antigens are shared with healthy cells, necessitating the development of complementary safety measures. In this context, there is a need to selectively eliminate therapeutically administered CAR-T cells, especially to revert long-term CAR-T cell-related side effects. To address this, we have developed an effective cellular-based safety mechanism to specifically target and eliminate the transferred CAR-T cells. As proof-of-principle, we have designed a secondary CAR (anti-CAR CAR) capable of recognizing a short peptide sequence (Strep-tag II) incorporated into the hinge domain of an anti-CD19 CAR. In in vitro experiments, these anti-CAR CAR-T cells have demonstrated antigen-specific cytokine release and cytotoxicity when co-cultured with anti-CD19 CAR-T cells. Moreover, in both immunocompromised and immunocompetent mice, we observed the successful depletion of anti-CD19 CAR-T cells when administered concurrently with anti-CAR CAR-T cells. We have also demonstrated the efficacy of this safeguard mechanism in a clinically relevant animal model of B-cell aplasia induced by CD19 CAR treatment, where this side effect was reversed upon anti-CAR CAR-T cells infusion. Notably, efficient B-cell recovery occurred even in the absence of any pre-conditioning regimens prior anti-CAR CAR-T cells transfer, thus enhancing its practical applicability. In summary, we developed a robust cellular safeguard system for selective in vivo elimination of engineered T cells, offering a promising solution to address CAR-T cell-related on-target/off-tumor toxicities.

Dual Split-Signaling TIM3+CLEC12a Targeting CAR T-Cells with Optimized Signaling As a Safe Potential Therapy for Acute Myeloid Leukemia

Background: The success of chimeric antigen receptor (CAR) T-cell therapies targeting B cell maturation antigen (BCMA) in multiple myeloma and CD19 in B-cell malignancies has demonstrated the immense potential of this immunotherapeutic strategy. However, the translation of CAR T-cell therapy to acute myeloid leukemia (AML) patients has encountered challenges due to the absence of surface antigens specifically or preferentially expressed on AML cells. To overcome this hurdle, we explored the feasibility and safety of simultaneously targeting the AML-associated antigens CLEC12a and TIM3 using an AND-gate strategy in which the essential stimulatory and co-stimulatory T-cell activation signals are segregated into separate CARs. To improve the safety of our strategy we also attenuated signaling through the stimulatory anti-CLEC12a CAR via affinity-optimization or through targeted mutations in its immunoreceptor tyrosine-based activation motif (ITAM) regions. Methods: To identify the most optimal dual-split CAR T-cell configuration, we designed several chimeric antigen receptor constructs in which we targeted CLEC12a with stimulatory CARs (sCARs) with high (C hi) or low (C low) affinities. In addition, the high affinity C hi stimulatory CAR was mutated in the second and third ITAM regions (denoted as C hixx) to attenuate the CD3ζ stimulatory signal. TIM3 was targeted by a costimulatory CAR (cCAR) containing both CD28 and 41BB signaling domains. The dual-split CARs were generated using two separate retroviral constructs. After the assessment of the CAR expression and specific surface markers associated with memory phenotype and exhaustion, dual-split CAR T-cells were functionally tested using an isogenic cell line model for specific lysis against dual CLEC12a/TIM3 positive or single CLEC12a or TIM3 positive isogenic target cells as well as for dual antigen-specific cytokine secretion. The dual-split CAR T-cells were also tested for their efficiency to specifically eliminate leukemic stem/progenitor cells by the colony formatting unit (CFU) assay. Moreover, anti-AML activity and persistence of the dual-split CAR T-cells was assessed using an AML PDX mouse model in which human primary AML cells expressing TIM3 and CLEC12a were injected. Results: Using the isogenic cell line model, we revealed that the dual-split CAR T-cells with the combination of the high affinity CLEC12a sCAR and TIM3 cCAR (C hi+T) show antigen-specific cytokine secretion and cytotoxicity, and that this specificity was enhanced when the CLEC12a sCAR was attenuated through ITAM mutations (C hixx+T). In CFU assays, all tested CLEC12a/TIM3 dual-split CAR T-cell combinations exhibited potent inhibition (up to 95%) of leukemia blast viability and leukemia stem/progenitor self-renewal. In vivo, although AML did not engraft in all mice, the best-performing dual-split C hixx+T1 CAR T-cells displayed potent anti-leukemic activity, as they prevented engraftment in two additional mice compared to control groups (n=6 each group). Furthermore, in contrast to control groups dual-split CAR T-cells exhibited prolonged persistence in the blood up to 20 weeks after injection. Conclusion: We successfully generated highly specific dual-targeting CAR T-cells by splitting the T-cell activation signals into two simultaneously expressed CARs, and using a novel approach of attenuating the sCAR through mutations in ITAM regions. Overall, we show that dual-split CAR T cells targeting CLEC12a and TIM3, particularly the combination C hixx+T CAR T-cells, have specific and promising anti-AML activity. Our findings underscore the potential of innovative dual-split CAR T-cell therapies as a novel and effective immunotherapeutic approach for combating AML.

Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells

NK cells armed with chimeric antigen receptors (CAR) enable NK cells specifically to target cancer cells by recognizing tumor associated antigens. Previous studies have proven that human iPSC-derived CAR-NK cells have enhanced anti-tumor activity. Targeted B-cell maturation antigen (BCMA) therapy for relapsed refractory MM (rrMM) has been widely used with antibody-drug conjugate (ADC), bispecific antibodies, and CART cells, which has produced a rapid response, but relapse is common. Recent work has revealed GPRC5D as an alternative target in MM for immunotherapy and patients who have previously received anti-BCMA therapy have responded to GPRC5D CART cells. To assess target GPRC5D therapy with CAR-NK cells, which may in future serve as allogeneic immunotherapy, we transduced anti-GPRC5D CAR into an iPSC line derived from a healthy donor using a piggybac transposon system. More than 97% of CAR inserted-iPSCs (CAR-iPSCs) expressed anti-GPRC5D scFV with a similar expression level identified in CAR-iPSC-derived-NK cells (CAR-iNK, 92.7%). The anti-GPRC5D CAR-iNK demonstrated high purity (>99.9% for CD45 + and CD56 +) and expressed a high level of CD16 (63.6%), the NK cell activating receptor NKG2D (96.3%) and NKp30 (98.7%), and the co-stimulatory receptors CD244 (99.6%) and CD226 (97.8%). Nonetheless expression of TCR⍺β and TCRƳ- was relatively low (<2%). Cytotoxicity assay revealed that anti-GPRC5D CAR-iNK had similar cytotoxicity against K562 cells (No GPRC5D antigen) to cord blood-derived NK cells (CB-NK), wide type (WT) iNK and anti-BCMA CAR-iNK. Cytotoxicity was dose-dependent indicating that these NK cells had similar innate anti-cancer cytotoxicity. When incubated at a ratio of 4:1 with NCI-H929 cells (MM) for 4 hours, which express a high level of both BCMA and GPRC5D, anti-GPRC5D CAR-iNK demonstrated an overwhelming advantage (approximately 90% killing rate) over the CB-NK (no killing) and WT iNK (around 10% killing rate). Furthermore, the anti-GPRC5D CAR-iNK also showed superior cytotoxicity to anti-BCMA CAR-iNK (Figure 1), indicating that GPRC5D may be a more efficient target for CAR-NK-based immunotherapy in MM. We further confirmed that anti-GPRC5D CAR-NK have acceptable cytotoxicity to NCI-H929 and OPM-2 cells, even when transported over a long distance (more than 2000 km) and following cryopreservation. The lower expression by OPM-2 cells of BCMA and GPRC5D compared with NCI-H929 indicates that both fresh and cryopreserved anti-GPRC5D CAR-iNK can specifically target MM with a higher and lower expression of GPRC5D. In addition, cryopreserved anti-GPRC5D CAR-iNK cells maintain comparable antigen-specific cytotoxicity in vitro, and demonstrate the ability to reduce tumor burden in an antigen-specific manner in an OPM-2 xenograft model. Our results demonstrate that genome-engineered human iPSCs can provide an unlimited source for manufacturing scalable, off-the-shelf, and cost-effective CAR-NK cells, and GPRC5D is a potent immunotherapeutic target in multiple myeloma.

Translational development of a novel BAFF-R CAR-T therapy targeting B-cell lymphoid malignancies.

Several CD19-targeting CAR-T cells are used to treat leukemias and lymphomas; however, relapsed and/or refractory (R/R) disease is still observed in a significant number of patients. Additionally, the success of CD19-CAR-T cell therapies is not uniform across hematological malignancies, particularly in chronic lymphocytic leukemia (CLL). In this study, we present the development of a novel CAR-T cell therapy targeting B-cell activating factor receptor (BAFF-R), a key regulator of B-cell proliferation and maturation. A new monoclonal antibody against BAFF-R was generated from a hybridoma clone and used to create a novel MC10029 CAR construct. Through a series of in vitro and in vivo models using the Nalm-6 cell line for leukemia and the Z138 cell line for lymphoma, we demonstrated the antigen-specific cytotoxicity of MC10029 CAR-T cells against tumor cells. Additionally, MC10029 CAR-T cells exhibited potent antitumor effects against CD19 knockout tumor cells, mimicking CD19-negative R/R disease. MC10029 CAR-T cells were specifically targeted to CLL, in which BAFF-R is nearly always expressed. The cytotoxicity of MC10029 CAR-T cells was first shown in the MEC-1 CLL cell line, before we turned our efforts to subject-derived samples. Using healthy donor-engineered MC10029 CAR-T cells against enriched primary tumor cells, followed by subject-derived MC10029 CAR-T cells against autologous tumor cells, we showed the efficacy of MC10029 CAR-T cells against CLL subject samples. With these robust data, we have advanced to the production of MC10029 CAR-T cells, using GMP lentivirus, and obtained an IND approval in preparation for a Phase 1 clinical trial.

Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy targeting nfP2X7

Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel treatment that genetically modifies the patients’ own T cells to target and kill malignant cells. However, identification of tumour-specific antigens expressed on multiple solid cancer types, remains a major challenge. P2X purinoceptor 7 (P2X7) is a cell surface expressed ATP gated cation channel, and a dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from multiple tissues, while being undetectable on healthy cells. We present a prototype -human CAR-T construct targeting nfP2X7 showing potential antigen-specific cytotoxicity against twelve solid cancer types (breast, prostate, lung, colorectal, brain and skin). In xenograft mouse models of breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibit robust anti-tumour efficacy. These data indicate that nfP2X7 is a suitable immunotherapy target because of its broad expression on human tumours. CAR-T cells targeting nfP2X7 have potential as a wide-spectrum cancer immunotherapy for solid tumours in humans.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

Chimeric antigen receptor T cells targeting FcRH5 provide robust tumour-specific responses in murine xenograft models of multiple myeloma

BCMA-targeting chimeric antigen receptor (CAR) T cell therapy demonstrates impressive clinical response in multiple myeloma (MM). However, some patients with BCMA-deficient tumours cannot benefit from this therapy, and others can experience BCMA antigen loss leading to relapse, thus necessitating the identification of additional CAR-T targets. Here, we show that FcRH5 is expressed on multiple myeloma cells and can be targeted with CAR-T cells. FcRH5 CAR-T cells elicited antigen-specific activation, cytokine secretion and cytotoxicity against MM cells. Moreover, FcRH5 CAR-T cells exhibited robust tumoricidal efficacy in murine xenograft models, including one deficient in BCMA expression. We also show that different forms of soluble FcRH5 can interfere with the efficacy of FcRH5 CAR-T cells. Lastly, FcRH5/BCMA-bispecific CAR-T cells efficiently recognized MM cells expressing FcRH5 and/or BCMA and displayed improved efficacy, compared with mono-specific CAR-T cells in vivo. These findings suggest that targeting FcRH5 with CAR-T cells may represent a promising therapeutic avenue for MM.

Translational development of BAFF-R-specific chimeric antigen receptor T-cell therapy targeting B-cell lymphoid malignancies.

e19501 Background: Relapsed and/or refractory (R/R) disease is observed in patients who receive CD19 targeting chimeric antigen receptor (CAR)-T cells to treat B-cell lymphoid leukemias and lymphomas. We generated a novel CAR, MC10029 that targets B-cell activating factor receptor (BAFF-R) to address this unmet medical need. Methods: We engineered a second-generation BAFF-R CAR (MC10029 CAR). The construct includes the single chain variable fragment (scFv) of our newly developed BAFF-R antibody with CD28 costimulatory and CD3ζ signaling domains. We confirmed MC10029 CAR-T cell antigen-specific activation with in vitro degranulation assays and a direct cytolytic assay. We used BAFF-R positive leukemia and lymphoma cell lines in both in vitro and in vivo models to further characterize MC10029 CAR-T cells, and finally, we enriched tumor primary cells from chronic lymphocytic leukemia (CLL) patients as a target for MC10029 CAR-T cells. In preparation for our Phase I clinical trial, we have manufactured three batches of CAR-T cells using a MC10029-encoding lentiviral vector produced under GMP conditions. The production batches of MC10029 CAR-T cells were evaluated for product quality with Viability, Identity and Potency as well as Safety from adventitious viral agents. Antigen specific cytotoxicity of the MC10029 CAR-T cells was confirmed using our standard degranulation assay. Results: MC10029 CAR-T cells elicited in vitro antigen-specific cytotoxicity against Nalm-6 cells, Z138 cells, MEC-1 cells, and a CD19 knock out Nalm-6 model designed to mimic CD19-negative R/R disease. We demonstrated in vivo antitumor effects of MC10029 CAR-T cells in NSG mice challenged with these same cell lines. We showed cytotoxicity of MC10029 CAR-T cells against CLL patients’ tumors. We also showed our quality control results to highlight our clinical trial trajectory. Conclusions: Novel MC10029 CAR-T cells showed broad utility against BAFF-R positive B-cell lymphoid malignancies in a series of in vitro, in vivo, and ex vivo models. With these robust data, we have moved forward to generate clinical grade MC10029 CAR-T cells using cGMP manufacturing and quality protocols in preparation of a MC10029 CAR-T cell Phase I clinical trial.

A costimulatory chimeric antigen receptor targeting TROP2 enhances the cytotoxicity of NK cells expressing a T cell receptor reactive to human papillomavirus type 16 E7

Immune cells modified to express a tumor-reactive T cell receptor (TCR) have shown limited efficacy as stand-alone therapy against solid tumors. Genital and oropharyngeal carcinomas induced by human papillomavirus (HPV) type 16 express constitutively its E6 and E7 oncoproteins, which makes them convenient targets for adoptive cell immunotherapy. However, viral antigen presentation by tumor cells is low and limits the anti-tumor efficacy of CD8+ T cells. To enhance the functionality of immune effector cells, we have devised a strategy combining a costimulatory chimeric antigen receptor (CAR) with a TCR. We used a clinically tested TCR specific to E7 (E7-TCR) of HPV16 and a newly constructed CAR targeting the trophoblast cell surface antigen 2 (TROP2), which carried the intracellular costimulatory domains CD28 and 4-1BB, but was devoid of the CD3ζ domain. Flow cytometry analyses showed a notable upregulation of activation markers and of cytolytic molecule release by NK-92 cells genetically engineered to express CD3, CD8 and both E7-TCR and TROP2-CAR, after co-incubation with HPV16+ cervical cancer cells. Furthermore, the E7-TCR/TROP2-CAR NK-92 cells demonstrated enhanced antigen-specific activation and augmented cytotoxicity against tumor cells compared with NK-92 cells expressing the E7-TCR alone. A costimulatory TROP2-CAR can synergistically cooperate with the E7-TCR in NK cells thereby enhancing their signaling strength and antigen-specific cytotoxicity. This approach might improve the outcome of adoptive cell immunotherapies for HPV16+ cancer patients that are currently under investigation.

Regeneration of T cells from human-induced pluripotent stem cells for CAR-T cell medicated immunotherapy.

Background: Chimeric antigen receptor (CAR) T cell treatment involves in vitro production of T cells from patient blood with synthetic receptors specific to a cancer antigen. They circumvent the major histocompatibility complex to recognize the tumor antigen, reducing hematologic malignancy remission rates by 80%. Considering the efficacy of CAR-T treatment, the present work aimed at generating functional clusters of differentiation (CD)8 + T cells from human induced pluripotent stem cells (hiPSC) and to generate hiPS-CAR-T cells with high antigen-specific cytotoxicity. Methods: The Alkaline phosphatase assay and MycoEasy rapid mycoplasma detection kit was implemented for detection of hiPSCs and mycoplasma, respectively. The CD34+ HSPCs were harvested in AggreWellTM 400 using a 37-micron reversible strainer. Likewise, the lymphoid progenitor and CD4+CD8+ DP T cells were also harvested. The Cell Counting Kit-8 (CCK-8) assay was used to mark cytotoxicity and ELISA was used to detect IFN-γ secretion. Further, flow cytometry and transwell chambers were used to assess cell cycle, and migration and invasion. Finally, the in vivo antitumor effects of the CAR-T cells were evaluated using experimental animals (mice). Results: Results revealed that a serum-free, feeder layer-free differentiation system significantly yielded hiPSC-based T cell immunotherapy with interleukin-2, interleukin-15, and activators at the differentiation stage to promote the maturation of these cells into human induced pluripotent stem (hiPS)-T cells. The infection of hiPSCs with the CD19 CAR lentivirus resulted in the production of the hiPSC-CAR-T cells. We validated the function of hiPS-CAR-T cells in vivo and in vitro experimentation which revealed no significant differences in cell morphology and function between hiPSC-derived hiPS-CAR-T cells and peripheral blood-derived CAR-T cells. Conclusion: This study developed a culture method that is efficient and clinically useful to make functional CD8+ T cells from hiPSC and to get hiPS-CAR-T cells with high antigen-specific cytotoxicity that are not very different from CAR T cells found in peripheral blood. As a result, our findings may open the way for the clinical use of hiPSC to create functional CD8+ T and hiPS-CAR-T cells cells for use in cell-based cancer therapy.

A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

ABSTRACT Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

Abstract 1153: IL-4 depletion leads to the improvement of CART cell therapy

Abstract While chimeric antigen receptor T-cell therapy targeting CD19 (CART19) has shown remarkable success in the treatment of hematological malignancies, the durable response rates remain approximately 40% and there are limited solutions for CART cell therapy in the treatment of solid tumors. To further understand mechanisms of resistance, including CART cell exhaustion, we employed three independent approaches: 1) RNA and ATAC sequencing on unstimulated vs. exhausted healthy donor CART19 cells by utilizing an in vitro model for exhaustion, 2) RNA and ATAC sequencing on pre-infusion CART19 cell products from responders and non-responders in the Zuma-1 clinical trial that led to the FDA approval of axi-cel CART19 therapy, and 3) a genome-wide CRISPR knockout screen in healthy donor CART19 cells using our in vitro model for exhaustion. In each of these approaches, IL-4 was identified as a regulator of CART cell dysfunction. In approach 1, ingenuity pathway analysis of genes that were both differentially accessible and expressed in exhausted compared with unstimulated CART19 cells revealed IL-4 as a top upstream regulator (p = 5E-6). In approach 2, IL-4 was one of two genes that were both upregulated and more accessible in CART19 cell products from non-responders (p < 5E-2). Finally, in approach 3, gene ontology enrichment analysis of genes that were positively selected during the genome-wide CRISPR knockout screen, revealed regulation of the IL-4 pathway as one of the top affected pathways (p = 1E-4). Together, our data indicates a role for IL-4 in CART cell dysfunction caused by exhaustion. Investigating this mechanism further, we saw an increase in the production of IL-4 as CART cells became exhausted (p = 4E-3). Treatment of CART19 cells with human recombinant IL-4 (hrIL-4) resulted in dysfunction as evident by a decrease in antigen specific cytotoxicity (p = 4E-3) and proliferative ability (p= 6.5E-2), as well as exhaustion-specific signs of dysfunction such as an increase in the expression of the inhibitory receptor, TIM-3 (p = 3E-3) and an increase in the transcription of the exhaustion-related transcription factor EOMES (p = 1E-2). Finally, we tested whether IL-4 neutralization enhances CART19 cell functions. Using a CD19+ JeKo-1 xenograft mouse model, we compared the combination treatment of CART19 cells and an IL-4 neutralizing monoclonal antibody (10 mg/Kg, clone # MP4-25D2) to CART19 cells and an IgG control. IL-4 neutralization in combination with CART19 cells resulted in reduced tumor burden (p = 4.6E-2), increased CART cell proliferation (p = 8E-3), and prolonged overall survival (p= 5E-2). In summary, our data indicates that 1) IL-4 induces CART cell dysfunction through a state of exhaustion and 2) IL-4 neutralization with a monoclonal antibody enhances CART cell therapy. As such, this novel combination therapy holds the potential to be translated to the clinic to improve durable responses from CART cell therapy. Citation Format: Carli Stewart, Michelle J. Cox, Reona Sakemura, Ekene J. Ogbodo, Ismail Can, Claudia Manriquez Roman, Kun Yun, Olivia Sirpilla, James H. Girsch, Truc Huynh, Elizabeth L. Siegler, Jenny J. Kim, Mike Mattie, Nathalie Scholler, Simone Filosto, Saad S. Kenderian. IL-4 depletion leads to the improvement of CART cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1153.