Abstract 5896: Platform approach to develop antibodies specifically recognizing cancer-associated glycoforms

Background: Mature T cell lymphomas are aggressive, treatment resistant cancers that are associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy T cells. Unlike B cell depletion, pan T cell aplasia is prohibitively toxic. The mutually exclusive expression of TCR β chains (TRBC) 1 or 2 allows targeting the malignant T cell population while preserving T cell function. Notably, a two amino acid inversion is the only exposed and antibody-accessible feature differentiating the two isoforms, making the development of therapeutics targeting TRBC1 and TRBC2 challenging. We have previously described a TRBC1 specific antibody which has been incorporated into a Chimeric Antigen Receptor (CAR) (Maciocia et al, Nat Med, 2017). Here we describe the derivation and characterization of a TRBC2 specific binder and CAR. In addition, we investigate use of these selective binders as Antibody Drug Conjugates (ADCs) for the treatment of HTLV-1 associated leukemia/lymphoma for which CAR T cell therapy may not be well suited. Methods: Anti TRBC2 antibodies were derived through crystallography and structural engineering of the previously described TRBC1 specific antibody. Briefly, TRBC2 binders were obtained by screening of small libraries of the TRBC1 binder with randomizations in key residues identified by crystallographic data and in silico design. TRBC2 binder candidates were first optimized for CAR function. Subsequently, TRBC2 specific CAR T cells were evaluated in vitro and in vivo for anti-tumor activity and selectivity. TRBC1/TRBC2 targeting antibodies, were further characterized as ADCs for biophysical properties, antibody internalization and cytotoxic function. Results: In vitro testing of TRBC2 CARs showed comparable efficacy to the previously described TRBC1 CAR. TRBC2 CAR T cells were effective at killing TRBC2 target cells while sparing TRBC1 positive cells. In vivo mouse models demonstrated that both TRBC1 and TRBC2 directed CARs could target their respective antigens with a high degree of specificity. TRBC1 and TRBC2 specific antibodies were used to generate ADC molecules as a proof of concept. Anti-TRBC1/TRBC2 antibodies were internalized and showed potential as ADCs. Interestingly, we demonstrate that an optimal affinity window facilitates improved antibody uptake and have further engineered both our TRBC1 and TRBC2 antibodies to take advantage of this particularity. Conclusions: Following on from structural and library-based approaches to generate CAR T cells capable of specifically targeting TRBC2, we have further characterized TRBC2 specific CAR T cells in vitro and in vivo. We have shown that highly specific antibodies, engineered as targeting moieties for TRBC1 and TRBC2 CAR T cells, show promising characteristics for utility potentially also as ADCs, offering another modality through which this targeting paradigm can be exploited for the treatment of peripheral T cell lymphomas. Citation Format: Mathieu Ferrari, Vania Baldan, Priyanka Ghongane, Alex Nicholson, Reyisa Bughda, Zulaikha Akbar, Patrycja Wawrzyniecka, Paul Maciocia, Shaun Cordoba, Simon Thomas, Shimobi Onuoha, Martin Pule. Targeting TRBC1 and 2 for the treatment of T cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2183.

Description MSLN is highly expressed in solid tumors but weakly in few normal tissues, making it a promising target for CAR T cell therapy. Early MSLN CAR T trials using the SS1 anti-MSLN binder with 41BB-CD3z showed clinical safety, but lacked efficacy. A recent report describing patient lung toxicity with a MSLN CAR T using a different binder underscores the urgent need for safer CAR T with enhanced tumor specificity. We previously developed a KIR-CAR, SynKIR-110 comprised of a split-chain design: a SS1 MSLN binding domain, and DAP12 T cell activation domain. This design mimics natural multichain immunoreceptors and demonstrates greater potency vs CD3z-based CAR T in vivo. To investigate SynKIR-110 tumor specificity we compared it to the clinically tested SS1-41BBz. T cells were stimulated with MSLN-positive (MSLN+) or negative (MSLN-) targets. T cell growth, phenotype, co-culture cytokines, and longitudinal tumor lysis were assessed. SynKIR-110 showed 4-fold higher growth, 4-fold lower CAR MFI expression and 6-fold lower 41BB+% vs SS1-41BBz at baseline. Both eliminated MSLN+ targets similarly. However, SS1-41BBz also showed higher basal killing and secreted higher cytokine levels upon MSLN- target co-culture, while SynKIR-110 did not. Overall SynKIR-110 demonstrates improved tumor specificity without the constitutive activation seen in SS1-41BBz. Further studies will explore the molecular pathways linked to enhanced tumor specificity, potency and exhaustion resistance of SynKIR-110. Funding Sources Verismo Therapeutics Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Background: Gastric cancer ranks among the top 5 cancer types in terms of both incidence and mortality, with a 5-year overall survival rate of only 29%. CD44 variant 6 (CD44v6) is an isoform of CD44 and is identified in 70% of gastric cancer cases. CD44v6 is considered a marker of poor prognosis, and its expression varies among different types of gastric cancer. CD44v6 has a role in apoptosis resistance, metastasis, and tumor progression, and is shown to be enriched in recurrent tumors. CD44v6 expression in normal tissue is very low and is restricted to certain cell types. Therefore, CD44v6 is a promising target for targeted therapies against gastric cancer. In this study, we aim to develop a chimeric antigen receptor (CAR) T cell therapy targeting CD44v6 and evaluate its functionality, cytotoxic activity and antitumoral effect. The scFv was derived from the monoclonal antibody (mAb) known as bivatuzumab (BIWA-4), a humanized mAb that has been used in clinical studies as radiotherapy and as antibody drug conjugates. Methods: Plasmid containing the CAR sequence with the BIWA-4 scFv was synthesized, and Jurkat T cell line and primary human T cells were transduced with lentivirus containing the CAR sequence. Jurkat cell line was used to measure binding to recombinant protein and assess CD69 expression after antigen exposure. Primary T cells transduced with CAR were co-cultured at different E:T ratios with gastric cancer cell lines (AGS, SNU 638, and MKN45) with different levels of expression of CD44v6. Target cell killing was measured by flow cytometry, bioluminescence, and fluorescence microscopy. T cell exhaustion and exhaustion phenotype were evaluated prior killing assays. For in vivo experiments, NSG mice will be engrafted with gastric cancer cell lines and tumor progression will be evaluated by IVIS. Results: While high affinity variants of bivatuzumab have been used to construct CAR (BIWA-8), ours is the first study that used medium affinity variant known as BIWA-4. Jurkat cells transduced with the car construct demonstrated specific binding to the recombinant monomeric CD44v6, which triggered surface expression of early T cell activation marker CD69. With varying concentrations of monomeric CD44v6, the affinity of CAR to CD44v6 was determined to be ∼100 nM KD. Transduction efficiency of primary T cells remained stable, reaching ∼60% after 10 days of expansion. Most CAR T cells exhibited effector memory phenotype, with low levels of PD1. CD44v6 CAR T killing in vitro was correlated with the E: T ratio and the level of antigen expression, demonstrating specificity of CAR and avoiding off-target effects with CD44v6 negative cells. Conclusion: We have successfully developed CAR T cells possessing medium affinity against CD44v6 and confirmed CAR-specific killing of CD44v6 positive gastric cancer cells. Citation Format: gonzalo gonzalez, Moonsoo Jin, Yanping Yang, Elizabeth Ortiz, Yogindra Vedvyas. Cytotoxic and antitumoral effect of an anti-CD44v6 CAR-T against gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6113.

Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy can provide durable remission in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) after failure of chemoimmunotherapy. However, patients who are refractory or relapsing after CAR-T therapy have poor outcomes. Multiple mechanisms of CAR-T therapy failure have been proposed but management of these patients remains a challenge. As CAR-T therapy moves earlier in the treatment of DLBCL, we urgently need trials focused on patients with relapse after CAR-T therapy. Recent advances in novel immunotherapies such as bispecific antibodies, antibody-drug conjugates and next-generation CAR-T therapies may provide avenues for treatment. Here we review the available data on using these drugs after failure of CAR-T therapy and provide a framework for the ideal sequencing of these novel agents.

Chimeric antigen receptor (CAR) T-cell therapy has been acclaimed as a revolution in cancer treatment following the impressive results in hematologic malignancies. Unfortunately, in patients with solid tumors, objectives responses to CAR T cells are still anecdotal, and important issues are driven by on-target but off-tumor activity of CAR T cells and by the extremely complex biology of solid tumors. Here, we will review the recent attempts to challenge the therapeutic impediments to CAR T-cell therapy in solid tumors. We will focus on the most promising strategies of antigen targeting to improve tumor specificity and address the tumor heterogeneity, efforts to circumvent the physical barriers of the tumor architecture such as subverted tumor vasculature, impediments of CAR T-cell trafficking and immune suppressive microenvironment.

Antibody-based therapies have revolutionized cancer treatment but have several limitations. These include downregulation of the target antigen, mutation of the target epitope, and, in the case of antibody-drug conjugates (ADC), resistance to the chemotherapy warhead. As TROP2-targeted therapy with ADCs yields responses in TROP2+ solid tumors, but the responses lack the durability observed with other immunotherapy-based approaches, we developed TROP2-targeting chimeric antigen receptor (CAR) T cells as an alternative. The TROP2-directed CAR T cells showed high potency against multiple solid tumor models. Moreover, TROP2-directed CAR T-cell therapy preserved high potency in models of ADC resistance and could be further engineered to prevent cell therapy resistance. This was achieved by leveraging fully human single-domain (VH-only) binder discovery to rationally engineer dual epitope binding-based (biparatopic) CARs. This work highlights the potency of CAR T-cell therapies and how rational engineering leveraging dual-VH targeting domains can overcome resistance pathways to current therapies. In future work, the CAR engineering approaches presented here can serve as a platform to be partnered with other strategies to address the suppressive tumor microenvironment.

Introduction: Antibody Drug Conjugates (ADCs) represent a new category of therapeutic molecular targeting for solid tumors. To date 6 ADCs have been approved in solid tumors against 5 targets (HER2/ERBB2, Folate alpha, Tissue Factor, Nectin-4, and Trop-2). Thus far, no ADCs have been approved for head and neck cancers. Methods: We queried protein expression data from a publically available consortium of datasets (TNMPlot.com) containing protein expression data from over 56,000 tumor and normal samples from GEO, GTex, TCGA, and TARGET databases. Genes examined for protein targets, included those with ADC targets of both presently approved and investigational drugs from clinical trials.The level of overexpression was determined as the percent of tumors over the 75th percentile of expression in normal tissue. Results: Protein expression data were available for 99 HNSCC samples. Among targets with ADCs available, increased protein expression was seen in HNSCC for 0% of ERBB2 (n=0/99), 38% of FOLR1 (n=38/99), 0% of TACSTD2 (n=0/99), and 42% of F2 (n=42/99) genes.Among gene targets with investigational ADCs, increased protein expression was seen in HNSCC for 35% of of AXL (n=35/99), 13% of B7H3/CD27 (n=12/99), 50% of EGFR (n=50/99), 63% of VTCN1 (n=63/99), 10% of MSLN (n=10/99), 77% of CDH3 (n=76/99), and 39% of PDGFRA (n=39/99). Conclusion: Increased protein expression for approved ADC targets were infrequent in HNSCC. However, increased protein expression is seen for several ADCs under clinical investigation supporting the potential therapeutic approaches for investigational ADC targets. Citation Format: Justin Tyler Moyers, Seth L. Smith, Miranda M. Mora, Roberto Carmagnani Pestana. Analysis of protein expression in head and neck cancers as therapeutic targets for antibody drug conjugates [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 5093.

Simple SummaryThis review summarizes the current knowledge on mesothelin’s function, its role in cancer, and opportunities for immunotherapeutic targeting of mesothelin. Immunotherapies including monoclonal antibodies, antibody–drug conjugates, chimeric antigen receptor T and NK-cells, targeted alpha therapies, and bispecific T cell engaging molecules are reviewed. We show future directions for mesothelin targeting in hematological malignancies, including acute myeloid leukemia.Modern targeted cancer therapies rely on the overexpression of tumor associated antigens with very little to no expression in normal cell types. Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein that has been identified in many different tumor types, including lung adenocarcinomas, ovarian carcinomas, and most recently in hematological malignancies, including acute myeloid leukemia (AML). Although the function of mesothelin is widely unknown, interactions with MUC16/CA125 indicate that mesothelin plays a role in the regulation of proliferation, growth, and adhesion signaling. Most research on mesothelin currently focuses on utilizing mesothelin to design targeted cancer therapies such as monoclonal antibodies, antibody–drug conjugates, chimeric antigen receptor T and NK cells, bispecific T cell engaging molecules, and targeted alpha therapies, amongst others. Both in vitro and in vivo studies using different immunotherapeutic modalities in mesothelin-positive AML models highlight the potential impact of this approach as a unique opportunity to treat hard-to-cure AML.

Double-negative prostate cancer (DNPC) and triple-negative breast cancer (TNBC) are two challenging malignancies characterized by frequent metastasis and resistance to current therapies. Among them, DNPC may share similar traits with basal-like prostate cancer. These aggressive cancer subtypes demand new treatment strategies. This study investigates CDH3 (P-cadherin), a cell adhesion molecule, as a new target for antibody-drug conjugate (ADC) and chimeric antigen receptor (CAR) T-cell immunotherapeutics. We conducted comprehensive in silico analysis and confirmed the enriched expression of CDH3 in DNPC and metastatic TNBC. We hypothesize that CDH3-positive (CDH3+) prostate and breast cancer cells will be effectively targeted and eradicated using anti-CDH3 immunotherapeutics in vitro and in vivo. Both anti-CDH3 ADC and CAR-T cell therapies were developed and they both demonstrated significant target-specific cytotoxicity against CDH3high DU145 and MDA-MB-468 cell lines, while sparing CDH3low and CDH3-/- cell lines, highlighting their selectivity in vitro. In vivo efficacy was further validated using NOD-SCID mice engrafted with prostate and breast cancer cell lines in an experimental metastasis setting, where treatment with ADC or CAR-T therapy significantly reduced metastasis burden. Notably, CAR-T therapy, in combination with anti-PD-1 therapy, exhibited enhanced therapeutic potential.This study establishes the robust target-specific cytotoxicity of anti-CDH3 ADC and CAR-T therapies against the most virulent subtypes of metastatic prostate and breast cancers in vitro and in vivo, paving the way for potential clinical development of these therapeutic agents in patients with metastatic DNPC and TNBC. Citation Format: Guoqiang Liu, Shiyu Wang, Loan Duong, Xin Lu. CDH3-targeted ADC and CAR-T immunotherapies for metastatic double-negative prostate cancer and triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4823.

Building upon preclinical advances, we are uncovering immunotherapy strategies that are translating into improved outcomes in tumor subsets. Advanced pediatric solid tumors carry poor prognoses and resultant robust efforts to apply immunotherapy advances to pediatric solid tumors are in progress. Here, we discuss recent developments in the field using mAb and mAb-based therapies including checkpoint blockade and chimeric antigen receptors (CARs). The pediatric solid tumor mAb experience targeting the diganglioside, GD2, for patients with neuroblastoma has been the most compelling to date. GD2 and alternative antigen-specific mAbs are now being incorporated into antibody-drug conjugates, bispecific antibodies and CARs for treatment of solid tumors. CARs in pediatric solid tumors have not yet achieved comparative responses to the hematologic CAR experience; however, novel strategies such as bispecific targeting, intratumoral administration and improved understanding of T-cell biology may yield enhanced CAR-efficacy. Therapeutic effect using single-agent checkpoint blocking antibodies in pediatric solid tumors also remains limited to date. Combinatorial strategies continue to hold promise and the clinical effect in tumor subsets with high antigenic burden is being explored. Pediatric immunotherapy remains at early stages of translation, yet we anticipate that with advanced technology, we will achieve widespread, efficacious use of immunotherapy for pediatric solid tumors.

e20561 Background: Chimeric antigen receptor (CAR) technology has advanced cancer treatment, but predominantly hematopoietic malignancies. CAR T progress in solid tumors is more challenging due to target availability, antigen heterogeneity, and the tumor microenvironment. Checkpoint inhibitor-based immune therapy or antibody-drug conjugates play a critical role in lung cancer, yet the prognosis is still dismal in advanced, relapsed, or refractory disease. The significant unmet need in lung cancer has prompted new CAR T studies with various antigen targets. This review examines the preliminary clinical experience in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) treated with CAR T. Methods: A systematic review examined publications, abstracts, and clinical trials of CAR T therapy patients with NSCLC and SCLC, as of January 7, 2025. Studies of CAR T therapy that included ≥ 3 patients with lung cancer were included. Results: We identified 48 NSCLC and 5 SCLC patients treated with CAR T in 5 publications. More clinical trials are underway; 18 trials were identified for CAR T in NSCLC ± SCLC and another 3 for SCLC only. Of 21 trials, 11 (52%) were classified as “recruiting” or “active not recruiting,” 2 were “not yet recruiting,” 2 were terminated, and 6 had an “unknown” status. Two published trials (NCT03182816, NCT01869166) administered EGFR-CAR T products to treat 9 and 11 patients, respectively. Three patients achieved a partial response (PR), 10 had stable disease (SD), and the remainder had progressive disease (PD). Survival was reported for one EGFR study, with a median overall survival (OS) of 15.53 months (range 8.8-22.0) and median progression-free survival (PFS) of 7.1 months (range 2.7-17.1). Studies of MUC-1-CAR T (NCT03525782) and ROR1-CAR T (NCT02706392) in 28 NSCLC patients showed similar outcomes. For MUC1-CAR T in 20 patients, 11 had SD, and 9 developed PD. In the ROR1-CAR T study with 8 patients, one achieved a PR after receiving a second CAR infusion but had PD after 6 months. The majority (16/18) of patients in the study had SD by day +28, but all 16 patients developed PD by 6 months after CAR infusion. A study of 5 SCLC patients treated with DLL3-directed CAR T AMG 119 (NCT03392064) also showed PR in 1 patient, SD in 2, PD in 1, and not evaluable in 1. All products demonstrated favorable safety profiles, with the most frequent events being low-grade cytokine release syndrome (CRS), fever, chills, and dyspnea. One patient with NSCLC and extensive disease at baseline died of respiratory failure on day +17 after an ROR1-CAR-T. Conclusions: This systemic review of CAR T in advanced lung cancer shows brief, limited responses but with acceptable safety data. The best response has been PR in < 10% of reported patients; this is not encouraging, but the details of individual patients are unclear for a meta-analysis. The results of ongoing clinical trials will be important to inform the direction of future investigations.

Despite the discoveries of numerous agents including next generation proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, multiple myeloma (MM) remains an incurable disease. The field of myeloma treatment in refractory or relapsed patients after standard therapy entered a new era due to the B-cell maturation antigen (BMCA) targeted approach. BCMA is a member of the tumor necrosis factor receptor family with high expression in mature B-lymphocytes and plasma cells. Given the understanding of BCMA mechanism of action in MM, BCMA plays a promising role as a therapeutic target. Several clinical trials are underway to evolve the current BCMA targeted treatment concept such as antibody-drug conjugates (ADCs), bispecific T cell engagers (BITEs) and chimeric antigen receptor (CAR) T cell therapy. Current results of representative BCMA trials may close the gap of the unmet clinical need to further improve the outcome of heavily pretreated MM patients with the potency to change the paradigm in newly diagnosed and refractory MM. This comprehensive review will give an update on various BMCA targeted treatment modalities (ADCs, BITEs, CAR T cell therapy) and its existing results on efficacy and safety from preclinical and clinical trials.

<div>Abstract<p>Chemoimmunotherapy with antibody–drug conjugates (ADC) is emerging as a promising therapy for solid tumors, whereas radioimmunotherapy (RAIT) of solid tumors has been relatively ineffective because of their resistance to radiation. We developed antibody–SN-38 conjugates that have significant antitumor activity in xenograft models at nontoxic doses. The goal of this study was to determine if an ADC could be combined with RAIT to enhance efficacy without a commensurate increase in host toxicity. Nude mice bearing human pancreatic cancer xenografts (Capan-1 and BxPC-3) were treated with a single dose of <sup>90</sup>Y-labeled antimucin antibody (hPAM4; clivatuzumab tetraxetan) alone or in combination with an anti-Trop-2–SN-38 conjugate, typically administered twice weekly over 4 weeks. The combination, even at RAIT's maximum tolerated dose, controlled tumor progression and cured established xenografts significantly better than the individual treatments without appreciable toxicity. The ADC could be started 1 week after or up to 2 weeks before RAIT with similar enhanced responses, but delaying RAIT for 2 weeks after the ADC was less effective. A nonspecific ADC provided additional benefit over using free drug (irinotecan), but the response was enhanced with the specific ADC. When targeting Capan-1 with ample mucin, hPAM4 could be used as the RAIT and the ADC agent without losing effectiveness, but in BxPC-3 with less mucin, targeting of different antigens was preferred. These studies show the feasibility of combining ADC and RAIT for improved efficacy without increased toxicity. <i>Mol Cancer Ther; 10(6); 1072–81. ©2011 AACR</i>.</p></div>

CD70 CAR NK Cells in the Treatment of Multiple Myeloma

Immune checkpoints (ICs) are molecules implicated in the fine-tuning of immune response via co-inhibitory or co-stimulatory signals, and serve to secure minimized host damage. Targeting ICs with various therapeutic modalities, including checkpoint inhibitors/monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and CAR-T cells has produced remarkable results, especially in immunogenic tumors, setting a paradigm shift in cancer therapeutics through the incorporation of these IC-targeted treatments. However, the large proportion of subjects who experience primary or secondary resistance to available IC-targeted options necessitates further advancements that render immunotherapy beneficial for a larger patient pool with longer duration of response. B7-H3 (B7 Homolog 3 Protein, CD276) is a member of the B7 family of IC proteins that exerts pleiotropic immunomodulatory effects both in physiologic and pathologic contexts. Mounting evidence has demonstrated an aberrant expression of B7-H3 in various solid malignancies, including tumors less sensitive to current immunotherapeutic options, and has associated its expression with advanced disease, worse patient survival and impaired response to IC-based regimens. Anti-B7-H3 agents, including novel mAbs, bispecific antibodies, ADCs, CAR-T cells, and radioimmunotherapy agents, have exhibited encouraging antitumor activity in preclinical models and have recently entered clinical testing for several cancer types. In the present review, we concisely present the functional implications of B7-H3 and discuss the latest evidence regarding its prognostic significance and therapeutic potential in solid malignancies, with emphasis on anti-B7-H3 modalities that are currently evaluated in clinical trial settings. Better understanding of B7-H3 intricate interactions in the tumor microenvironment will expand the oncological utility of anti-B7-H3 agents and further shape their role in cancer therapeutics.