On-target Off-tumor Toxicity Research Articles

FGFR4-specific CAR-T cells with inducible caspase-9 suicide gene as an approach to treat rhabdomyosarcoma.

Metastatic rhabdomyosarcoma is associated with poor survival and unsatisfactory treatment outcomes. Therefore, new immunotherapeutic methods are urgently required. Fibroblast growth factor receptor 4 (FGFR4), a new therapeutic target for rhabdomyosarcoma, plays a crucial role in its onset and development. This study aimed to generate FGFR4 single-chain variable fragment-based chimeric antigen receptor (CAR) T cells without causing evident toxicity and incorporating an inducible caspase-9 (iCasp9) suicide gene system to enhance their safety. FGFR4 antigen expression was evaluated in normal murine tissues, normal human tissues, and specimens from patients with rhabdomyosarcoma. Combined with a 4-1BB co-stimulatory domain, a CD3ζ signaling domain, and an iCasp9 suicide gene, CAR-T cells with an FGFR4-specific single-chain variable fragment were developed. The specific cytotoxic effects, T-cell proliferation, cytokine secretion, apoptosis induction by chemical dimerization (AP20187), and toxicity of FGFR4 CAR-T cells were investigated in vitro and in vivo. FGFR4 CAR-T cells generated a variety of immune-promoting cytokines, including tumor necrosis factor α, interleukin 2, and interferon γ, and displayed effective cytotoxic activity against FGFR4-overexpressing rhabdomyosarcoma cells in vitro. FGFR4 CAR-T cells were relatively effective against FGFR4-overexpressing rhabdomyosarcoma, with tumor regression and poor survival in a subcutaneous xenograft model. The iCasp9 gene was incorporated into FGFR4 CAR-T cells and it was demonstrated that effective and reliable suicide gene activity depends on the administration of AP20187. By making use of the cross-reaction of FGFR4 CAR-T cells with murine FGFR4 in a syngeneic tumor model, this study found that FGFR4 CAR-T cells could regulate the growth of tumors without evident toxicity. Our study demonstrates that FGFR4 is a prospective target for CAR-T cell therapy in rhabdomyosarcoma without serious on-target off-tumor toxicity. FGFR4 CAR-T cells with the iCasp9 suicide gene system as a safety switch to limit toxicity may broaden the clinical applications of cellular therapy.

CAR T cells in solid tumors and metastasis: paving the way forward.

CAR T cell therapy, hailed as a breakthrough in cancer treatment due to its remarkable outcomes in hematological malignancies, encounters significant hurdles when applied to solid tumors. While notable responses to CAR T cells remain sporadic in these patients, challenges persist due to issues such as on-target off-tumor toxicity, difficulties in their trafficking and infiltration into the tumor, and the presence of a hostile and immunosuppressive microenvironment. This review aims to explore recent endeavors aimed at overcoming these obstacles in CAR T cell therapy for solid tumors. Specifically, we will delve into promising strategies for enhancing tumor specificity through antigen targeting, addressing tumor heterogeneity, overcoming physical barriers, and counteracting the immune-suppressive microenvironment.

Efficacy and safety evaluation of cross-reactive Fibroblast activation protein scFv-based CAR-T cells.

Fibroblast activation protein (FAP) overexpression on cancer-associated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAP-positive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells.

Acute myeloid leukemia stem cell signature gene EMP1 is not an eligible therapeutic target.

Relapse in pediatric acute myeloid leukemia (pedAML) patients is known to be associated with residual leukemic stem cells (LSC). We have previously shown that epithelial membrane protein 1 (EMP1) is significantly overexpressed in LSC compared to hematological stem cell fractions. EMP1 was also documented as part of the 17-gene stemness score and a 6-membrane protein gene score, both correlating high EMP1 expression with worse overall survival. However, its potential as a therapeutic target in pedAML is still unexplored. Association analyses of EMP1 expression with clinical and molecular AML characteristics were performed. Expression of EMP1 was evaluated in pedAML and cord blood samples. Expression in normal blood cells and tissues was evaluated by flow cytometry and immunohistochemistry, respectively. In silico analyses showed variable mRNA expression of EMP1 in multiple pedAML datasets, and a significant correlation between high EMP1 transcript levels and the presence of inv(16). Flow cytometry showed overexpression of EMP1 in pedAML samples, as well as expression in normal blood subsets. Importantly, immunohistochemistry revealed EMP1 expression in multiple normal tissues. Although EMP1 presents as an interesting membrane-associated target in pedAML, its abundant expression in normal blood cells and tissues will impede it from further exploration as a therapeutic target. EMP1 is highly expressed in multiple cancer types, but expression in acute myeloid leukemia (AML) and normal tissues is unexplored. As EMP1 is investigated in other cancer types, expression in normal tissues and blood cells is relevant in predicting the success of EMP1-targeted therapies. In this study, we showed expression of EMP1 in multiple tissues, predicting high on-target off-tumor toxicity, which will warn other researchers of possible toxicities when generating EMP1-targeted therapy. Finally, we showed that high EMP1 expression is associated with better overall survival of pediatric AML patients, reducing the need for EMP1-targeted therapy.

Grooved Microneedle Patch Augments Adoptive T Cell Therapy Against Solid Tumors via Diverting Regulatory T Cells.

The efficacy of adoptive T cell therapy (ACT) for the treatment of solid tumors remains challenging. In addition to the poor infiltration of effector T (Teff) cells limited by the physical barrier surrounding the solid tumor, another major obstacle is the extensive infiltration of regulatory T (Treg) cells, a major immunosuppressive immune cell subset, in the tumor microenvironment. Here, this work develops a grooved microneedle patch for augmenting ACT, aiming to simultaneously overcome physical and immunosuppressive barriers. The microneedles are engineered through an ice-templated method to generate the grooved structure for sufficient T-cell loading. In addition, with the surface modification of chemokine CCL22, the MNs could not only directly deliver tumor-specific T cells into solid tumors through physical penetration, but also specifically divert Treg cells from the tumor microenvironment to the surface of the microneedles via a cytokine concentration gradient, leading to an increase in the ratio of Teff cells/Treg cells in a mouse melanoma model. Consequently, this local delivery strategy of both T cell receptor T cells and chimeric antigen receptor T cells via the CCL22-modified grooved microneedles as a local niche could significantly enhance the antitumor efficacy and reduce the on-target off-tumor toxicity of ACT.

CRPA1A2: A novel TCR-based T-cell engager targeting MAGEA1-positive solid tumors.

e14589 Background: MAGEA1, identified as a cancer/testis antigen, is highly expression in various solid tumors, including hepatocellular carcinoma, non-small cell lung cancer, but absent in normal tissues except male germ cells and placenta, both of which lacking HLA class molecules. This selective expression of HLA-MAGEA1 epitope makes it a promising target for cancer therapy. However, its intracellular localization poses a challenge for conventional antibodies designed to target surface-expressed antigens. TCR-based T-cell engagers, such as Tebentafusp, overcome this hurdle by targeting intracellular protein-derived peptides presented on cancer cell surfaces via HLA, enabling precise cytotoxic T cell attacks. Notably, Tebentafusp has shown significant clinical benefits by improving survival rates in metastatic uveal melanoma, underscoring the efficacy of TCR-based T-cell engagers in treating MAGEA1-positive tumors. Methods: We have developed an advanced structural format, CR62, consisting of a anti-CD3 single chain fragment, a soluble modified TCR, and an Fc region. Additionally, the use of CorreGene's SMART-TCR system has facilitated the acquisition of TCRs with high affinities in the pico-molar range. By leveraging CR62 and a high-affinity TCR, we have conceptualized the TCR-based T-cell engager, CRPA1A2. Comprehensive preclinical assessments have been conducted to evaluate CRPA1A2's safety and efficacy. Results: CRPA1A2 is designed to specifically target the HLA-A*02:01-restricted MAGEA1 epitope, effectively inhibiting tumor growth. CRPA1A2 is characterized by its exceptional soluble expression in CHO cells, minimal homodimer miss-assembly and simplifying purification. It is strategically engineered with a high-affinity TCR and a low-affinity anti-CD3 scFv to optimize the efficacy and safety. In vitro studies, CRPA1A2 showed strong ability to eliminate tumor cells and induce IFN-γ release, effective even at low MAGEA1 epitope densities, with a therapeutic window that spans over three orders of magnitude, suggesting a 1000-fold greater activity against antigen-positive cancer cells compared to antigen-negative cells. In vivo, using a tumor-bearing mouse model, CRPA1A2 has been shown to recruit T cells into subcutaneous solid tumors, expand CD8+ T cell populations, and significantly reduce tumor mass. Notably, CRPA1A2 has an extended half-life of 120 hours and demonstrates preferential accumulation in tumor tissues, thereby enhancing its therapeutic potential. Moreover, the safety assessments for CRPA1A2 highlight its exceptional specificity, with in vitro studies showing no evidence of off-target toxicity, on-target off-tumor toxicity, or alloreactivity and favorable tolerance in mice during in vivo studies. Conclusions: CRPA1A2 has emerged as a superior TCR-based T-cell engager with exceptional antitumor efficacy and promising safety profile.

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.

Synthetic biology approaches for improving the specificity and efficacy of cancer immunotherapy.

Immunotherapy has shown robust efficacy in treating a broad spectrum of hematological and solid cancers. Despite the transformative impact of immunotherapy on cancer treatment, several outstanding challenges remain. These challenges include on-target off-tumor toxicity, systemic toxicity, and the complexity of achieving potent and sustainable therapeutic efficacy. Synthetic biology has emerged as a promising approach to overcome these obstacles, offering innovative tools for engineering living cells with customized functions. This review provides an overview of the current landscape and future prospects of cancer immunotherapy, particularly emphasizing the role of synthetic biology in augmenting its specificity, controllability, and efficacy. We delineate and discuss two principal synthetic biology strategies: those targeting tumor surface antigens with engineered immune cells and those detecting intratumoral disease signatures with engineered gene circuits. This review concludes with a forward-looking perspective on the enduring challenges in cancer immunotherapy and the potential breakthroughs that synthetic biology may contribute to the field.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Abstract LB122: JNJ-87890387, a novel ENPP3 bispecific T-cell redirector (ENPP3xCD3) with tumor selectivity through targeting apical surface antigens

Abstract Clinical success of T-cell redirection therapy for solid tumors has been limited potentially due to a lack of target specificity against cancer, leading to on-target off-tumor toxicity at sub-efficacious doses. ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3) is a transmembrane protein that was identified with the help of an artificial intelligence machine learning algorithm. ENPP3 is characterized by apically restricted expression in most normal tissues and elevated, depolarized expression in several solid tumors, thus potentially providing tumor selectivity. ENPP3 expression exhibits a high prevalence in several solid tumors including renal cell carcinoma (RCC) - clear cell (93%) and papillary (78%), lung adenocarcinoma (50%), endometrioid uterine (53%) and ovarian (47%) cancers, colorectal carcinoma (51%), and hepatocellular carcinoma (HCC, 47%). JNJ-87890387 is a fully human bispecific antibody with high affinity and specific binding to ENPP3 on tumor cells and lower affinity binding to cluster of differentiation (CD)3 on T-cells. In vitro, JNJ-87890387 demonstrated potent (pM) T-cell activation and cytotoxicity against tumor cell lines with different levels of endogenous ENPP3 membrane expression, and absence of killing against cells lacking ENPP3 expression, confirming JNJ-87890387 specificity. In vivo, JNJ-87890387 demonstrated ENPP3-expression-dependent potent anti-tumor activity in multiple cell line-derived and patient tumor-derived RCC and HCC xenograft models with complete tumor regressions, and evidence of dose-dependent CD4+ and CD8+ T cell infiltration into the tumors. Preclinical safety assessment in the cynomolgus monkey with a tool molecule using the therapeutic ENPP3 binding arm but with a cynomolgus CD3 cross-reactive arm only identified toxicities consistent with the modality. In summary, these data support JNJ-87890387’s clinical development with an ongoing first-in-human Phase I study to evaluate the safety and preliminary anti-tumor activity in advanced-stage solid tumors with a high prevalence of ENPP3 expression. Citation Format: Smruthi Vijayaraghavan, Pankaj Seth, Annmarie Winkis, Kristen Chevalier, Alexa Marthaler, Katrin Sproesser, Vince Torti, Nirav Shah, Eilyn Lacy, Anna-Lena Frisk, Heather Deutsch, Gerald Chu, Michael Sharp, Jonathan Pabalan, Cholpon Tilegenova, Katherine Thorton, Josh Lauring, Bethany Mattson, Ken Tian, Jacqueline Kinyamu-Akunda, Laurie Lenox, Wan Cheung Cheung, Sheri Moores, Sylvie Laquerre. JNJ-87890387, a novel ENPP3 bispecific T-cell redirector (ENPP3xCD3) with tumor selectivity through targeting apical surface antigens [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 LB122.

Abstract 41: Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning

Abstract Purpose: Mesothelin (MSLN) is a surface antigen associated with tumor invasion and is overexpressed in mesothelioma and lung, pancreatic, ovarian, and other cancers. Chimeric antigen receptor (CAR) T cells targeting MSLN have been tested in patients with MSLN-positive solid tumors. Despite reported patient fatalities in trials sponsored by PENN, Memorial Sloan Kettering/Atara Bio, and MD Anderson/TCR2, the design approach for MSLN CAR remains constructing high affinity CARs to maximize target cell killing. In this study, we designed MSLN CARs with two different antibodies: one targeting human MSLN specifically and another binding both human and mouse MLSN with comparable affinities. The objectives are to 1) elucidate the risk of fatal on-target off-tumor toxicity associated with high affinity MSLN CAR, and 2) demonstrate how optimizing the affinity of CARs can render CAR T cells to spare normal tissues while retaining cytotoxicity against tumors. Methods: To asses on-target off-tumor toxicity in animal models, we developed a CAR using a nanobody isolated from an immune alpaca library with nanomolar affinity for both human and mouse MSLN (labeled as hmMSLN CAR). For comparison, we constructed a CAR using a scFv specific only to human MSLN, designated hMSLN CAR. Each CAR is designed to co-express somatostatin receptor 2 to enable PET/CT imaging of CAR T cells with 18F-NOTA-Octreotide. Anti-tumor activities and toxicities were assessed in NSG mice using MSTO-211H cell line expressing human MSLN. Results: In NSG mice bearing subcutaneous MSTO-211H tumors, hMSLN CAR T cells eliminated tumors rapidly without inducing any overt toxicity. In contrast, hmMSLN CAR T cells not only failed to control tumor growth but also caused severe toxicity, resulting in significant body weight loss and necessitating euthanasia. PET/CT imaging revealed while hMSLN CAR T cells expanded exclusively within the tumor and subsequently contracted after tumor elimination, hmMSLN CAR T cells exhibited extensive infiltration and expansion in the lungs, liver, and bone marrow, indicating off-tumor, normal tissue-driven proliferation of CAR T cells. To test if affinity-tuned hmMSLN CAR T cells can selectively target tumors, we used a yeast display system to screen for affinity variants of the hmMSLN CAR. We identified one micromolar affinity CAR that binds approximately 1,000 times weaker to MSLN than the parental CAR. In stark contrast to the onset of severe toxicity soon after the administration of the parental hmMSLN CAR T cells, animals treated with micromolar affinity hmMSLN CAR T cells exhibited tumor regression without overt toxicity. Conclusions: Our findings underscore the risks of using high affinity CAR against MSLN, which may cause unpredictable on-target, off-tumor toxicities. Fine tuning the affinity of MSLN CAR offers a promising strategy for developing a potent and safe CAR for the treatment of MSLN-expressing solid tumors. Citation Format: Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Ju Y. Son, Niloufar Salehi, Irene M. Min, Moonsoo M. Jin. Mitigating on-target off-tumor toxicity of mesothelin-targeting CAR T cells by affinity-tuning [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 41.

Abstract 4009: Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy

Abstract Century Therapeutics is developing NECTIN4 targeted induced Pluripotent Stem Cell (iPSC) derived Chimeric Antigen Receptor (CAR) T cell therapy. NECTIN4 targeted antibody drug conjugate enfortumab vedotin, while generally well tolerated, causes severe skin adverse events in some patients, thought to be driven by on-target off-tumor toxicity against NECTIN4 displaying skin keratinocytes. We include a safety switch in our first clinical-stage iPSC-derived CAR-NK cell therapy, CNTY-101, which provides an antibody target for elimination of the engineered cells should that be necessary. An additional method for reducing the incidence and severity of this type of adverse event is through the incorporation of an inhibitory CAR, also known as a NOT-gate, directed against antigen expressed on normal skin keratinocytes, but the optimal surface protein to target with such an inhibitory CAR for a NECTIN4 targeted therapy is unknown. Here, we identify DSG1 (Desmoglein-1) as a top inhibitory CAR target for NECTIN4 targeted CAR-T cell therapy by conducting a multi-omics analysis of public single-cell RNAseq, bulk RNAseq, and protein microarray immunohistochemistry datasets. We find that DSG1 is constitutively and uniformly displayed on the surface of skin keratinocytes in the layers of epidermis where they display NECTIN4, and DSG1 gene expression in normal skin is uniformly high across individuals. Conversely, DSG1 mRNA and protein are rarely expressed in NECTIN4 expressing cancer indications including bladder, breast, non-small cell lung, ovarian, and pancreatic. Further, autoimmune antibodies specific to DSG1 have been described, suggesting that DSG1 is targetable by a CAR in situ. Finally, DSG1 is not expressed by our iPSC-derived CAR-T cells. While NECTIN4 expression is greatest in skin keratinocytes, epithelial cells in other tissues express NECTIN4 at lower levels. In this study, we compare the expression levels of NECTIN4 to targets associated with tissue and cell-type specific on-target off-tumor toxicity in primary CAR-T cell clinical trials. Our findings support the strategy of DSG1-directed inhibitory CAR incorporation into NECTIN4 specific CAR-T cell therapeutic candidates. Citation Format: Matthew S. Hall, Christopher M. Dower, Michael Miller, John Wheeler, Jill Carton, Ohad Manor, Hyam Levitsky. Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived 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 4009.

Abstract 744: Novel conditionally active biologic (CAB) tetravalent T-cell engagers targeting solid tumors

Abstract The use of T-cell engagers (TCEs) has great therapeutic potential in oncology. This potential, however, is diminished due to severe toxicity (cytokine release effects, as well as on-target off-tumor toxicities). We leveraged BioAtla’s Conditionally Active Biologic (CAB) technology (1) to develop bispecific antibodies which have no or very little binding to CD3 and to the tumor target antigen (TAA) in healthy tissue (normal physiological conditions), yet have strong binding in diseased tissues (i.e. tumor microenvironment, TME) derived from glycolytic tumor metabolism, which supports tumor growth (Warburg effect). The conditional activity in the TME is achieved by optimizing the sequences of the binding domains and does not require masking and subsequent activation of the binding domain as used in pro-drugs. We have developed dual-CAB T-cell engagers targeting serval well-established tumor associated antigens. Data demonstrating superior potency with reduced off-target binding through in vitro and in vivo characterization of three new DualCAB TCEs, targeting PSMA, Trop2, and Tissue Factor (TF) will be presented.(1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ and Short, JM Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118. Citation Format: Ana Paula Cugnetti, Haizhen Liu, Patricia McNeeley, Charles Xing, Kathryn Woodard, Hwai Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active biologic (CAB) tetravalent T-cell engagers targeting 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 744.

Abstract 4004: Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma

Abstract On-target off-tumor toxicity (OTOT) is one of the major hurdles preventing tumor-associated antigen (TAA) targeted therapy from effectively treating patients with solid tumors due to the shared presence of the same TAA on normal tissues. We have previously demonstrated high expression of carbonic anhydrase IX (CAIX) in ccRCC patient samples and low-density expression on healthy bile duct epithelium. In 2006, a clinical trial was stopped due to OTOT bile duct toxicity observed in patients infused with anti-CAIX CAR T cell therapy. To address the issue of OTOT in the bile duct, we performed affinity/avidity fine-tuning of the anti-CAIX CAR G36 to specifically target ccRCC tumor cells with high CAIX expression while sparing cholangiocytes with low CAIX expression. We generated a complementarity-determining region (CDR) single variant mutagenesis library of G36 and displayed the library on yeast for screening. Using flow cytometry, we were able to identify six populations based on antigen-binding affinity: high, med-high, med, med-low, low, and loss of binding. These libraries were sequenced via PacBio, a next generation sequencing (NGS) platform that excels in long-read sequencing, providing a powerful tool to detect single mutations in the scFv. The results were analyzed to identify enriched mutations compared to the wild-type G36, and inspected using in silico modeling and docking. Furthermore, enriched mutants from med-low and low populations were tested in CAR-T format for their cytotoxic capacity on tumor cells and bile duct cholangiocytes, as well as their avidity. We demonstrate that mutations in CDR2 and CDR3 of the heavy chain successfully mitigate OTOT toxicity on cholangiocytes with physiological levels of CAIX expression while maintaining tumor-killing capacity, shedding light on CDR mutagenesis as a useful tool to mitigate OTOT toxicity. Citation Format: Stephen Hsien-Chi Yuan, Yufei Wang, Rabia Abbas, Peterson Mathenge, Charles Chien, Christian D. Coherd, Branson Bajoua, Matthew R. Chang, Nithyassree Murugan, Gabriella M. Kastrunes, Guilin Wang, Song-My Hoang, Wayne A. Marasco. Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma [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 4004.

Abstract 2613: Treatment of solid tumors with antibody drug conjugates targeting aberrant O-glycoproteins

Abstract The lack of antibodies with sufficient cancer selectivity is currently limiting the treatment of solid tumors by immunotherapies. Many solid tumors display aberrant glycosylation that results in the expression of tumor-associated carbohydrate antigens (TACAs) distinct from healthy tissues. Targeting aberrantly glycosylated glycopeptide epitopes provides the cancer selectivity needed for immunotherapy of solid tumors. However, only a few such glycopeptide epitopes have been targeted to date. We have developed a strategy to predict, design, and produce cancer-specific antibodies targeting novel O-glycoprotein epitopes with exquisite cancer specificity. Our mAbs avoid on-target off-tumor toxicity due to the specificity of aberrant glycosylation, and cover a broad range of cancer indications, including breast, lung, colon, pancreatic, and ovarian cancer. Additionally, our antibodies can be used as an IHC companion diagnostic and can be functionalized across a number of modalities, including next-generation CAR-T, T-cell bispecifics, and antibody-drug-conjugates (ADCs).Here, we present two novel antibodies, GO-08 and GO-013, selectively targeting cancer-specific Tn-glycosylated proteins in a site-specific manner with low nanomolar affinities. GO-008 and GO-013 were collectively shown to be highly cancer-selective by immunohistochemistry of sections from a large array of different squamous and adenocarcinomas, including triple-negative breast cancer, lung cancer, gastric cancer, ovarian cancer, pancreatic, and colorectal cancer. The two antibodies were functionalized utilizing ConjuAllTM technology, conjugating the microtubule disrupting payload MMAE via a site-specific beta-glucuronidase cleavable linker. ConjuAllTM functions as a plasma stable, tumor labile linkage, and due to the minimal expression pattern of beta-glucuronidase in healthy tissues, reduces both off- and on-target toxicity due to the minimization of release of free payload in non-tumor tissues. The ADCs based on GO-08 and GO-013 (LCB45A and LCB22A) demonstrated target-specific cytotoxicity invitro and in vivo with tumor elimination in breast and lung PDX models. GO-013 conjugated to MMAE (LCB22A) was well tolerated in cynomolgus monkey toxicity studies. Moreover, the ADCs selectively kill target cells in a human mixed organotypic cancer model without affecting healthy cells, indicating tolerability and safety. The combination of specificity of TACA targeting to differentiate ADC binding from healthy tissue and ConjuAll’s tumor selective payload release promises to enable an excellent anti-cancer therapy. Citation Format: Aaron C. Groen, Nisha Shrestha, Boris Klebanov, Byeongjun Yu, BeomYong Lee, Chul-Woong Chung, Changsik Park, Mikkel K. Aasted, Sally Dabelsteen, Hans H. Wandall. Treatment of solid tumors with antibody drug conjugates targeting aberrant O-glycoproteins [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 2613.

Abstract 3212: Development of a Trop2/CD3 bispecific antibody with modulated Trop2 binding affinity as a novel immunotherapy for advanced gastrointestinal cancer treatment

Abstract Trophoblast cell surface antigen 2 (Trop2) is considered as a potential therapeutic target because its over-expression in tumors is found to be associated with poor prognosis of patients with different cancers. Trop2-directed antibody-drug conjugates have been approved for the treatment of solid tumors, however, the expression of Trop2 in a broad range of epithelial tissues, even in low expression, would limit their clinical benefits. The present work generated a novel Trop2/CD3 bispecific antibody with its binding affinity towards Trop2 being well-modulated to mitigate the chance of on-target off-tumor effect. By linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv, a Trop2/CD3 bispecific antibody was produced. In order to adjust the bispecific antibody's affinity to the tumor target, ProABC-2 was used for in-silico paratope prediction (Ambrosetti F et al., 2020). A complementarity-determining residue (CDR) variant was produced and its binding to Trop2 antigen was analyzed using biolayer interferometry. The binding of both wild type (WT) and variant antibodies to Trop2-positive cancer cells, the activation of T cells upon target engagement and the redirection of T cell-mediated killing on cancer cells expressing high level of Trop2 were examined. The binding affinity of CDR variant to Trop2-expressing tumor cells was significantly reduced, which led to a slower induction of cytokines and T-cell proliferation, less acute T-cell activation, and a milder cytotoxic effect. This allowed the bispecific antibody to have a higher level of safety by restricting the cytotoxicity only to cancer cells that expressed high level of Trop2. Our novel Trop2/CD3 bispecific antibody not only showed potent anti-tumor activity, but also demonstrated a lower risk of on-target off-tumor toxicity. Monkey toxicity and other IND-enabling studies are beginning to further bring the drug candidate into clinical trial. Table showing EC50 values of Trop2/CD3 bispecific WT and CDR variant antibodies in different assays WT CDR variant Binding affinity (M) Trop2 4.62E-11 2.14E-10 Cytotoxicity (M) AsPC1 (low Trop2 level) 1.24E-11 No killing effect SW480 (medium Trop2 level) 1.25E-12 2.25E-11 NCI-H596 (high Trop2 level) 5.65E-12 5.89E-10 Receptor occupancy (%) Trop2 0.65 2.75 CD3 0.08 5.83 Cytokine induction (M) IL-2 4.82E-11 5.42E-11 IFNγ 6.46E-13 2.59E-11 T cell activation (M) CD25+ marker 2.02E-12 2.39E-10 CD69+ marker 3.81E-13 7.40E-11 T cell proliferation (M) Ki-67 marker 3.54E-12 8.80E-11 Citation Format: Chui Yee O, Po Yee Wong, Kronos Chow, Kwan Wa Wong, Dennis Wong, John Moon Luk, Kwong Fai Wong. Development of a Trop2/CD3 bispecific antibody with modulated Trop2 binding affinity as a novel immunotherapy for advanced gastrointestinal cancer treatment [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 3212.

Abstract 3213: A novel CDH17/PD-1 bispecific antibody for treatment of advanced gastrointestinal cancers

Abstract Targeting immune checkpoints like programmed death receptor 1 (PD-1) with monoclonal antibodies has achieved durable responses in a range of cancers, however, immune-related adverse events may occur in a subset of patients. As such, we developed a bispecific antibody targeting cadherin-17 (CDH17) and PD-1 with a hope to rejuvenate specifically the exhausted T cells within tumor microenvironment. In healthy individuals, CDH17 is expressed at a restricted level in intestinal epithelial junction that is not accessible to biologics, however, in gastrointestinal cancers, CDH17 is overexpressed and can be targeted by antibodies. Our new CDH17/PD-1 bispecific antibody would reactivate anti-tumor immunity with reduced risk of on-target off-tumor toxicity. Different bispecific antibody formats were designed, cloned, and expressed. Strong binders were first identified using CDH17 and PD-1 ELISA. The binding affinities to CDH17 and PD-1 of the selected candidate were examined using biolayer interferometry. The effect of these bispecific antibodies on blocking PD-1/PD-L1 axis in T cells was demonstrated in a cell-based reporter assay. T-cell mediated killing on CDH17-positive GI cancer cells and induction of T cell activation markers upon antibody treatment were also studied. A total of 7 CDH17/PD-1 bispecific antibodies of different formats were screened. Of these candidates, one, named ARB204, was selected because of its potent binding to both antigens in ELISA (i.e., EC50 values: CDH17, 1.6nM; PD-1, 0.4nM) and biolayer interferometry (i.e., KD values: CDH17, 2.4E-09M; PD-1, 6.02E-11M). ARB204 blocked PD-1/PD-L1 axis in the reporter assay with EC50 value 4.4nM. Notably, ARB204 redirected T cells to eradicate CDH17-positive pancreatic AsPC1 cells with an EC50 value of 3.5nM. No cytotoxicity against CDH17-negative cancer cells were seen. ARB204 effectively bound CDH17 on GI cancer cells and PD-1 on T cells. This bispecific design allowed selective T-cell mediated killing on cancer cells expressing CDH17, mitigating the risk of off-tumor toxicity. ARB204 warrants further studies on its immune checkpoint inhibition in animal models. Citation Format: Kwan Wa Wong, Po Yee Wong, Kronos Chow, Chui Yee O, Dennis Wong, John Moon Luk, Kwong Fai Wong. A novel CDH17/PD-1 bispecific antibody for treatment of advanced gastrointestinal cancers [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 3213.

Abstract 2618: BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models

Abstract EGFR is a favored target in various cancer therapies. However, EGFR-based treatments have been challenged by drug resistance and cytotoxicity. Targeting EGFR with an antibody-drug conjugate (ADC) is a promising new therapeutic strategy to address the drug resistance, due to the potent killing effects of the payload. Furthermore, we hypothesized that development of a bispecific ADC (bsADC) targeting EGFR and another tumor-associated antigen (TAA) might improve the tumor selectivity, thereby limiting the on-target off-tumor toxicity. One such TAA candidate is PTK7, which belongs to the same receptor tyrosine kinase family as EGFR, and is co-expressed with EGFR in multiple types of solid tumors, including lung, esophageal, head and neck, and colon cancers. Herein, we developed a fully human anti-human PTK7 EGFR bsAb using our proprietary common light chain RenLite® mouse platform and knobs-into-holes technology. The anti-PTK7 EGFR bsAb demonstrates good purity by SEC-HPLC, and can cross-react with human and cynomolgus monkey antigens with favorable affinity. The PTK7 x EGFR bsAb demonstrated higher binding to various types of cancer cell lines, as well as enhanced internalization in vitro compared with PTK7 parental antibody. Next, the bsAb was conjugated with monomethyl auristatin E (MMAE) to generate an anti-PTK7 x EGFR bsADC, BCG017, with a drug-to-antibody ratio of approximately 4. BCG017 demonstrated superior anti-tumor activity compared to benchmarks and its PTK7 parental ADC in cell-derived non-small-cell lung cancer (NSCLC) xenografts, and showed enhanced anti-tumoractivity when compared to benchmark ADCs and parental ADCs in patient-derived breast and pancreatic ductal adenocarcinoma xenograft models. These results suggest that BCG017 have the potential to be a novel therapeutic alternative for PTK7 and EGFR co-expressing tumors. Citation Format: Sufei Yao, Chengzhang Shang, Zhuolin Li, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models [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 2618.

Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Engineering a Programmed Death-Ligand 1-Targeting Monobody Via Directed Evolution for SynNotch-Gated Cell Therapy.

Programmed death-ligand 1 (PD-L1) is a promising target for cancer immunotherapy due to its ability to inhibit T cell activation; however, its expression on various noncancer cells may cause on-target off-tumor toxicity when designing PD-L1-targeting Chimeric Antigen Receptor (CAR) T cell therapies. Combining rational design and directed evolution of the human fibronectin-derived monobody scaffold, "PDbody" was engineered to bind to PD-L1 with a preference for a slightly lower pH, which is typical in the tumor microenvironment. PDbody was further utilized as a CAR to target the PD-L1-expressing triple negative MDA-MB-231 breast cancer cell line. To mitigate on-target off-tumor toxicity associated with targeting PD-L1, a Cluster of Differentiation 19 (CD19)-recognizing SynNotch IF THEN gate was integrated into the system. This CD19-SynNotch PDbody-CAR system was then expressed in primary human T cells to target CD19-expressing MDA-MB-231 cancer cells. These CD19-SynNotch PDbody-CAR T cells demonstrated both specificity and efficacy in vitro, accurately eradicating cancer targets in cytotoxicity assays. Moreover, in an in vivo bilateral murine tumor model, they exhibited the capability to effectively restrain tumor growth. Overall, CD19-SynNotch PDbody-CAR T cells represent a distinct development over previously published designs due to their increased efficacy, proliferative capability, and mitigation of off-tumor toxicity for solid tumor treatment.