Targeting Tumor Antigens Research Articles

Abstract A037: Enhanced anti-tumor activity through targeted immunotherapy combined with mutant KRAS inhibition in pancreatic cancer

Abstract Constitutively-active KRASG12D has become targetable via clinical-stage KRASG12D-selective small molecule inhibitors. Prior preclinical research has demonstrated that KRASG12D inhibition makes pancreatic ductal adenocarcinoma (PDAC) more amenable to immunotherapy. However, the impact of such inhibition on antigen presentation by tumor cells remains unclear. To elucidate this phenomenon, we employed a multi-omic profiling workflow to investigate alterations in the proteome and peptide MHC ligandome (immunopeptidome) following treatment of HPAC human cancer cells with a RAS(ON) G12D-selective inhibitor RM-044 (representative of investigational agent RMC-9805) and/or IFNɣ in vitro. Multi-omic profiling revealed significant changes in antigen presentation following KRASG12D inhibition. Pairwise comparisons of differentially expressed proteins in treated vs. untreated conditions showed increased expression of proteins associated with interferon signaling and replication stress. Notably, antigen processing and presentation proteins such as HLA-B and -C alpha chains showed some of the highest expression levels. Immunopeptidomic analysis revealed a 2-fold increase in ligand diversity and abundance in treated vs. untreated conditions. Sequence motif enrichment analysis depicted motifs consistent with HPAC’s HLA-B and -C but not -A alleles, aligning with proteomic findings and highlighting how the integration of these two data layers informs the shaping of the immunopeptidomic landscape. Building on these findings, we hypothesized that KRASG12D signaling inhibition in preclinical models could augment immune responses initiated by immunization targeting tumor antigens in vivo. Our hypothesis was that the release of antigens from KRASG12D-induced cell death, coupled with increased MHC-I expression in remaining tumor cells, would enhance interactions with T cells activated by immunization. To test this, we orthotopically inoculated C57Bl/6 mice with a murine PDAC-KRASG12D cell line engineered to express the model self/tumor-associated antigen gp100. Mice were immunized with either empty ionizable lipid nanoparticles (e-iLNPs) or iLNPs packaged with mRNA encoding full-length gp100 (gp100-iLNP). Following a priming and boost series, mice were treated with either KRASG12D inhibitior MRTX1133 or vehicle for two weeks. Remarkably, mice treated with the gp100 mRNA-iLNP immunization followed by KRASG12D inhibition experienced significant tumor regression compared to mice that received KRASG12D inhibition alone. These preclinical findings underscore the dynamic nature by which PDAC antigen presentation can be modulated by KRAS signaling inhibition and IFNɣ. The combination of allele-specific KRAS inhibition with tumor antigen immunization resulted in significant tumor regression in a stringent in vivo PDAC preclinical model, compared to either treatment modality alone. This rationalizes the combination of both approaches for enhanced anti-tumor activity, offering a strategy that is worth exploring clinically for improving outcomes in pancreatic cancer treatment. Citation Format: Amanda Creech, Hailey Lee, Khalid Rashid, Thuc Le, Alykhan Premji, Tony Luu, Ahmad Kassem, Weihang Zhao, Luyi Li, Nanping Wu, Norbert Pardi, James Wohlschlegel, Timothy Donahue, Caius Radu. Enhanced anti-tumor activity through targeted immunotherapy combined with mutant KRAS inhibition in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A037.

Industry Perspective on First-in-Human and Clinical Pharmacology Strategies to Support Clinical Development of T-Cell Engaging Bispecific Antibodies for Cancer Therapy.

T-cell-engaging bispecific antibodies (TCEs) that target tumor antigens and T cells have shown great promise in treating cancer, particularly in hematological indications. The clinical development of TCEs often involves a lengthy first-in-human (FIH) trial with many dose-escalation cohorts leading up to an early proof of concept (POC), enabling either a no-go decision or dose selection for further clinical development. Multiple factors related to the target, product, disease, and patient population influence the efficacy and safety of TCEs. The intricate mechanism of action limits the translatability of preclinical models to the clinic, thereby posing challenges to streamline clinical development. In addition, unlike traditional chemotherapy, the top dose and recommended phase II doses (RP2Ds) for TCEs in the clinic are often not guided by the maximum tolerated dose (MTD), but rather based on the integrated dose-response assessment of the benefit/risk profile. These uncertainties pose complex challenges for translational and clinical pharmacologists (PK/PD scientists), as well as clinicians, to design an efficient clinical study that guides development. To that end, experts in the field, under the umbrella of the American Association of Pharmaceutical Scientists, have reviewed learnings from published literature and currently marketed products to share perspectives on the FIH and clinical pharmacology strategies to support early clinical development of TCEs.

Fighting Pancreatic Cancer with a Vaccine-Based Winning Combination: Hope or Reality?

Pancreatic adenocarcinoma (PDA) represents the fourth leading cause of cancer-related mortality in the USA. Only 20% of patients present surgically resectable and potentially curable tumors at diagnosis, while 80% are destined for poor survival and palliative chemotherapy. Accordingly, the advancement of innovative and effective therapeutic strategies represents a pivotal medical imperative. It has been demonstrated that targeting the immune system represents an effective approach against several solid tumors. The immunotherapy approach encompasses a range of strategies, including the administration of antibodies targeting checkpoint molecules (immune checkpoint inhibitors, ICIs) to disrupt tumor suppression mechanisms and active immunization approaches that aim to stimulate the host's immune system. While vaccines have proved effective against infectious agents, vaccines for cancer remain an unfulfilled promise. Vaccine-based therapy targeting tumor antigens has the potential to be a highly effective strategy for initiating and maintaining T cell recognition, enhancing the immune response, and ultimately promoting cancer treatment success. In this review, we examined the most recent clinical trials that employed diverse vaccine types to stimulate PDA patients' immune systems, either independently or in combination with chemotherapy, radiotherapy, ICIs, and monoclonal antibodies with the aim of ameliorating PDA patients' quality of life and extend their survival.

Contemporary Management of Renal Cell Carcinoma: A Review for General Practitioners in Oncology.

Renal cell carcinoma accounts for a significant proportion of cancer diagnoses in Canadians. Over the past several years, the management of renal cell cancers has undergone rapid changes in all prognostic risk categories, resulting in improved oncologic outcomes. Novel strategies for metastatic disease make use of the synergy between checkpoints and angiogenesis inhibition. Moreover, combination checkpoint inhibition has demonstrated durable efficacy in some patients. Adjuvant immunotherapy has recently shown a survival benefit for the first time in select cases. Significant efforts are underway to explore new compounds or combinations for later-line diseases, such as inhibitors of hypoxia-inducible factors and radiolabeled biomolecules targeting tumor antigens within the neoplastic microenvironment for precise payload delivery. In this manuscript, we provide a comprehensive review of the available data addressing key therapeutic areas pertaining to systemic therapy for metastatic and localized disease, review the most relevant prognostic tools, describe local therapies and management of CNS disease, and discuss practice-changing trials currently underway. Finally, we focus on some of the practical aspects for general practitioners in oncology caring for patients with renal cell carcinoma.

NK Cell Degranulation Triggered by Rituximab Identifies Potential Markers of Subpopulations with Enhanced Cytotoxicity toward Malignant B Cells.

A promising strategy in cancer immunotherapy is to restore or enhance the cytotoxicity of NK cells, among others, by activating the mechanism of antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies targeting tumor antigens, such as rituximab (targeting CD20), induce NK cell-mediated ADCC and have been used to treat B cell malignancies, such as non-Hodgkin lymphoma, but not always successfully. The aim of this study was to analyze the gene expression profile of the NK cells involved in the cytolytic response stimulated by rituximab. NK cells were co-cultured with rituximab-opsonized Raji cells. Sorting into responder and non-responder groups was based on the presence of CD107a, which is a degranulation marker. RNA-seq results showed that the KIT and TNFSF4 genes were strongly down-regulated in the degranulating population of NK cells (responders); this was further confirmed by qRT-PCR. Both genes encode surface proteins with cellular signaling abilities, namely c-KIT and the OX40 ligand. Consistent with our findings, c-KIT was previously reported to correlate inversely with cytokine production by activated NK cells. The significance of these findings for cancer immunotherapy seems essential, as the pharmacological inhibition of c-KIT and OX40L, or gene ablation, could be further tested for the enhancement of the anti-tumor activity of NK cells in response to rituximab.

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.

Novel TROP2-targeted CAR T cells with high specificity and sustained efficacy for solid tumors in animal models.

e14563 Background: CAR T cell therapy, successful in hematological malignancies, encounters challenges against solid tumors due to immune exhaustion and senescence within the tumor microenvironment. The imperative need for an optimal tumor target antigen is evident in advancing CAR T therapy. TROP2, a membrane-associated glycoprotein, is highly expressed in diverse cancers, including cervical, bladder, esophageal, ovarian, and endometrial cancers. TROP2 overexpression, approaching 100% in cervical and hormone-resistant prostate cancer, correlates inversely with prognosis and survival, presenting TROP2 as a promising CAR T therapy target. This study engineered the novel TROP2 antibody R4702, featuring a distinct binding epitope from Sacituzumab and Datopotamab, to develop TROP2-specific CAR T cells and evaluated their efficacy against a spectrum of solid tumors. Methods: Expansion of CAR T Cells and Cytotoxicity: CAR T cells were created through lentivirus-mediated genetic engineering with the TROP2 antibody, followed by 10 days of expansion. T cell differentiation was evaluated, and luciferase-encoding target cells incubated with varying CAR T cell ratios overnight were quantified luciferase activity. In Vivo Efficacy and Persistence: CAR T cells were adoptively transferred into ASID mice with tumors. In vivo tumor imaging used luciferase activity, and calipers monitored tumor size weekly. CAR T cell persistence assessed their ability to suppress rechallenged tumor growth. Results: R4702, a novel TROP2-specific antibody, displayed high binding affinity to a distinct epitope. Post- ex vivo expansion, R4702 CAR T cells showed a substantial increase (100 to 200-fold) over 10 days, with a high expression level (50~80%) of R4702 CAR on T cells. Over 60% of R4702 CAR T cells displayed a less differentiated status, characterized by a CD62L+ population comprising TSCM and TCM. In vitro cytotoxicity revealed potent and specific efficacy against various TROP2-positive cancer cell lines, with no observed killing activity against TROP2-negative cells. In vivo efficacy of R4702 CAR T cells demonstrated 100% tumor growth inhibition within 30 days post-treatment across diverse xenograft models. Importantly, R4702 CAR T cells exhibited enduring anti-tumor efficacy against successive tumor challenges in xenograft models of pancreatic, gastric, NSCLC, TNBC, and ovarian cancers. In a head-to-head comparison with Sacituzumab and Datopotamab CAR T, R4702 demonstrated superior anti-tumor efficacy to Sacituzumab and comparable efficacy with Datopotamab in the BxPC-3 CDX model of pancreatic cancer. Conclusions: A novel TROP2-specific R4702 CAR T cell, developed, showcased robust tumor-killing capabilities. The anti-tumor activity persisted upon repeated tumor challenges across various xenograft models, underscoring the potential of R4702 CAR T cells as a promising therapeutic strategy.

A phase 1/2, first-in-human, open-label, dose-escalation study of TAK-280, an investigational B7-H3 x CD3ε conditional bispecific redirected activation (COBRA) T-cell engager, in adult patients with unresectable, locally advanced, or metastatic solid tumors.

TPS2684 Background: TAK-280 is a novel COBRA T-cell engager that targets the B7 homolog 3 protein (B7-H3), an antigen highly expressed in a range of solid tumors including metastatic castration-resistant prostate cancer (mCRPC) and non–small cell lung cancer. In its prodrug form, TAK-280 binds to B7-H3, but not to CD3ε. Once in the protease-rich tumor microenvironment, TAK-280 undergoes protease-mediated activation through formation of CD3ε-binding active dimers, which stimulate CD3 T-cell activation and cytotoxic antitumor response against co-engaged B7-H3–expressing cells. In preclinical studies, TAK-280 demonstrated cleavage-dependent conditionality, engagement of tumor target antigen, and induction of T-cell mediated tumor killing. TAK-280 has the potential to target solid tumors that are not amenable to treatment with conventional first- or second-generation T-cell engagers, while limiting toxicity to normal tissues. Methods: This phase 1/2 study is enrolling adult patients with unresectable, locally advanced, or metastatic solid tumors described in the literature to have enhanced B7-H3 expression, who are ineligible or intolerant to standard therapies. Other eligibility criteria include Eastern Cooperative Oncology Group performance status ≤1 and measurable disease per RECIST v1.1. Key exclusion criteria include history of autoimmune disease, major surgery ≤8 weeks before first dose of TAK-280, and history of clinically significant cardiac or gastrointestinal disorders. During phase 1 dose escalation, TAK-280 is administered as an intravenous infusion, once weekly, in 28-day cycles at a pre-defined dose level range. Patients who do not experience dose-limiting or other unacceptable toxicities may receive up to 14 treatment cycles depending on response; patients are treated until disease progression, unacceptable toxicity, or withdrawal from study. The dose-escalation phase will determine two recommended doses for expansion to be assessed during the cohort-expansion phase. The primary endpoint is incidence of dose-limiting or other toxicities during dose escalation; secondary endpoints include pharmacokinetic parameters, response assessment per RECIST v1.1, overall response rate, duration of response, progression-free survival, overall survival, disease control rate, and prostate-specific antigen response in patients with mCRPC. Cytokine release syndrome is closely managed and reported per American Society for Transplantation and Cellular Therapy consensus grading. Approximately 182 patients are planned to be enrolled (42 in dose escalation and 100–142 in cohort-expansion); as of the data cutoff date of Jan 10, 2024, 18 patients have received the study drug. Clinical trial information: NCT05220098 .

Immune profiling of dedifferentiated liposarcoma and identification of novel antigens for targeted immunotherapy

Dedifferentiated liposarcoma (DDLS) is an aggressive, recurring sarcoma with limited treatments. T-cell immunotherapies selectively target malignant cells, holding promise against DDLS. The development of successful immunotherapy for DDLS requires a thorough evaluation of the tumor immune microenvironment and the identification and characterization of targetable immunogenic tumor antigens. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens, we used the nCounter NanoString platform, analyzing gene expression profiles across 29 DDLS and 10 healthy adipose tissue samples. Hierarchical clustering of tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumors and 14 non-inflamed tumors, demonstrating tumor heterogeneity within this sarcoma subtype. Among the identified antigens, PBK and TTK exhibited substantial upregulation in mRNA expression compared to healthy adipose tissue controls, further corroborated by positive protein expression by IHC. This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS, and provides a novel targetable antigen in DDLS. The results of this study lay the groundwork for the development of a novel immunotherapy for this highly aggressive sarcoma.

Identifying MAGE-A4-positive tumors for TCR T cell therapies in HLA-A∗02-eligible patients

T cell receptor (TCR) Tcell therapies target tumor antigens in a human leukocyte antigen (HLA)-restricted manner. Biomarker-defined therapies require validation of assays suitable for determination of patient eligibility. For clinical trials evaluating TCRTcell therapies targeting melanoma-associated antigen A4(MAGE-A4), screening in studies NCT02636855 and NCT04044768 assesses patient eligibility based on: (1) high-resolution HLA typing and (2) tumor MAGE-A4 testing via an immunohistochemical assay in HLA-eligible patients. The HLA/MAGE-A4 assays validation, biomarker data, and their relationship to covariates (demographics, cancer type, histopathology, tissue location) are reported here. HLA-A∗02 eligibility was 44.8% (2,959/6,606) in patients from 43 sites across North America and Europe. While HLA-A∗02:01 was the most frequent HLA-A∗02 allele, others (A∗02:02, A∗02:03, A∗02:06) considerably increased HLA eligibility in Hispanic, Black, and Asian populations. Overall, MAGE-A4 prevalence based on clinical trial enrollment was 26% (447/1,750) across 10 solid tumor types, and was highest in synovial sarcoma (70%) and lowest in gastric cancer (9%). The covariates were generally not associated with MAGE-A4 expression, except for patient age in ovarian cancer and histology in non-small cell lung cancer. This report shows the eligibility rate from biomarker screening for TCR Tcell therapies and provides epidemiological data for future clinical development of MAGE-A4-targeted therapies.

CD8+ T cell targeting of tumor antigens presented by HLA-E.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Abstract 2479: Discovery of agonist CD28-specific single domain antibodies from alpaca

Abstract Upon immune activation, in addition to the first signal driven by the TCR-peptide/MHC interactions, the costimulatory signals are indispensable to optimal T-cell activation, the absence of which will trigger naive T cells to an anergic state. The CD28-CD80/CD86 pathway is widely known as the most classical and critical costimulatory signal for the initial activation of T cells. Bispecific T-cell engagers targeting CD3e can activate cytotoxic T-cells and have shown remarkable tumor regressions in clinical trials. However, the absence of a costimulatory signal through CD28 results in insufficient T-cell activation and early exhaustion. Combination of both CD3e and CD28 targeting antibodies offers attractive approach to optimally boost T-cell activity. Development of superagonistic CD28-targeting antibody has been paused after TeGenero‘s TGN1412 Phase 1 trial in 2006 due to life-threatening cytokine release syndrome. Unlike superagonist TGN1412, which activates T cells and induce cytokine storm, tumor targeting antigen and cross-linking dependent CD28 bispecific antibody is supposed to display little or no toxicity. In the present study, we explored Alpaca immunized phage library approach to generate cross-linking dependent agonist CD28 single domain antibody for bispecific antibody development. Alpacas were immunized subsequently with recombinant human CD28 extracellular domain and 293F cells stably expressing human CD28. Serum samples after immunizations showed decent immune response against CD28. At the end of Boost 2 and 3, peripheral blood was collected and phage display libraries were constructed using VHH specific primers. Phage display libraries were panned against recombinant CD28 ECD protein and CHOK1 cells overexpressing CD28. After one round of panning, both protein and cell panning tracks showed robust enrichment in output titer and repeating sequences. Next, 600 clones were randomly picked for screening by the binding to U266B1 cells endogenously expressing CD28. Of the 326 clones showed positive binding to U266B1, 79 unique sequences were identified. Secondary screening by ELISA and FACS using periplasmic extract confirmed 69 unique sequences that bound to both CD28 protein and cells expressing CD28. Seventeen unique sequences were selected for VHH-Fc production. All purified VHH-Fc showed sub-nanomolar affinity by ELISA and bind to primary T cells by flow cytometry. Three VHH-Fc induced IL-2 production in a primary T cells activation assay only when cross-linked and in the presence of CD3 co-stimulation and are suitable for further bispecific T cell engager development. Citation Format: Yunyun Chen, Yannan Ding, Xiaoyan Liu, Qianqian Zhu, Wenfang Xin, Hu Liu, Teddy Yang, Yikai Qiu. Discovery of agonist CD28-specific single domain antibodies from alpaca [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 2479.

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

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

Abstract 243: Organoid co-cultures with allogenic T cells to assess toxicity and efficacy of bispecific antibodies

Abstract Recent advances in cancer immunotherapy positively impacted the life expectancy of patients for an extensive range of clinical indications. With new treatment strategies, druggable targets and biomarkers being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. For example, the emerging Bispecific T cell Engagers (BiTEs) designed to simultaneously target CD3 and tumor-specific antigens and promote T cell cytotoxicity have seen recent clinical success. While BiTEs have been approved for the treatment of hematologic malignancies, promising therapeutic developments for solid tumors face hurdles in translating preclinical findings into therapy since conventional two-dimensional cancer models fail to recapitulate the immune cell interactions in the tumor microenvironment and therefore hold low clinical predictive value. We developed an innovative alternative, building on the discovery that adult stem cells proliferate and organize into three-dimensional organotypic structures that maintain complex characteristics of the original parental tissue, including molecular heterogeneity and morphological and functional traits. Here, we show the development of a patient-relevant preclinical platform for immunotherapy development in which colorectal (CRC) and non-small cell lung cancer (NSCLC) organoids are co-cultured with allogenic or autologous T cells to evaluate the activity and assess the cytotoxic potential of a BiTEs. The expression of tumor-specific BiTE target antigens is determined in patient-derived organoids (PDOs) by flow cytometry. Following this assessment, T cell-induced organoid death is quantified and characterized in real-time by image-based analysis of fluorescent live/dead dyes. Additionally, we measure cytokine secretion as a read-out of T cell activation and function. In our co-cultures, T cell-induced PDO death was increased upon BiTEs treatment. Similarly, IFNγ secretion correlated with expression levels of the targeted tumor antigen. In addition, PDO apoptosis and IFNγ secretion increased in a dose-dependent fashion in the presence of the TA-targeting TCB. In summary, our organoids and T cell co-cultures offer a powerful platform for developing and validating immunotherapies such as T cell-bispecific antibodies and will significantly contribute to our understanding of the critical factors determining successful immunotherapies. Citation Format: Cesar Oyarce, Virginia Bruno, Lorenzo Spagnuolo, Claudia Beaurivage, Javier Frias Aldeguer, Farzin Pourfarzad, Robert G. Vries, Sylvia F. Boj. Organoid co-cultures with allogenic T cells to assess toxicity and efficacy of bispecific antibodies [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 243.

Abstract 6782: 3D-EXpress - an ex vivo platform using viably cryopreserved tumoroids for rapid assessment of targeted therapeutic outcomes

Abstract Background: it has been a significant challenge to select, combine, and sequence the multiple FDA-approved IO therapies and pharmaceutical agents, highlighting the need to develop a precision oncology platform to explore novel combination treatments. 3D-EXpress is a unique ex vivo therapeutics testing platform using a biorepository of fresh patient tumoroids that were never dissociated, propagated, or reassembled to retain an intact microenvironment. Therefore, these specimens provide an excellent ex vivo platform to investigate the efficacy of therapeutics targeting the tumor microenvironment. Here, we employed the 3D-EXpress platform to compare the efficacy of different therapeutic modalities targeting tumor antigens and immune checkpoint ex vivo. Methods: Tumoroids measuring 150 μm in size retaining tumor cell heterogeneity, tumor-resident immune cells, stromal components, and extracellular matrix interaction were prepared from human endometrial and colorectal tumors. Tumoroids were cryopreserved and were selected based on tumor antigen status. Samples were treated for 72 hours ex vivo with antibody-drug conjugates, chimeric antigen receptor (CAR)T cells, or bispecific antibodies alone and in combinations. Treatment-mediated changes in tumor cell killing and tumor immune microenvironment were investigated. Results: 3D high throughput confocal imaging and Nilogen Oncosystem’s proprietary algorithm were used to detect and analyze treatment-induced tumor cell killing activity in tumoroids. The impact of ex vivo treatment by different modalities in combination with chemotherapeutic agents upon tumor resident immune cell populations was monitored by deep immune phenotyping and multiplex cytokine release assay. Based on the ex vivo responses tumors were assigned to treatment sensitive and resistant groups. Correlative analysis was performed and compared with detailed clinicopathologic data that is readily available for Biorepository tumors. Conclusions: The 3D-EXpress platform using cryopreserved 3D tumoroids with intact tumor microenvironment is an effective tool for the pre-clinical assessment of rational drug combinations for treatment of solid tumors. Furthermore, the 3D-EXpress platform provides unique insight into the microenvironment of both treatment responsive and non-responsive tumors and can aid in the development of patient-centered therapeutic regimens. Citation Format: Vanessa Garrido, Michelle Ataya, Nisha R. Dhanushkodi, Seth Currlin, Alliyah Humphrey, Jared Ehrhart, Rikhia Chakraborty. 3D-EXpress - an ex vivo platform using viably cryopreserved tumoroids for rapid assessment of targeted therapeutic outcomes [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 6782.

Abstract 5226: A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology

Abstract We developed a screening platform for immune oncology using an assay based on automated image guided injection of tumoroids in wells of multi-well plates preloaded with a collagen-rich ECM. The identical x-y-z position and size of the ECM-embedded tumoroids in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. The model is used to generate quantitative data for T cell recruitment to tumoroids and killing of tumoroids by T cells. We have applied this to screening a panel of CD3:Her2 bispecific antibodies (BsAb) binding with different affinities to CD3 or Her2 or displaying high affinity interactions with different epitopes on Her2. Exposure to fresh, non-activated peripheral blood mononuclear cell (PBMC) derived T cells shows an initial phase of random T cell movement throughout the ECM followed by a BsAb-dependent phase of active T cell recruitment to tumoroids (day 2-4) and a subsequent phase of tumoroid killing (day 4-6). We show that the wave of T cell recruitment following initial T cell-tumoroid contact involves chemotactic signaling. Decreased affinity at the Her2 or CD3 arm can be compensated for by increasing BsAb concentrations. However, we detect major differences in efficacy for different high affinity epitopes. I.e., of two BsAbs interacting with high affinity with distinct Her2 epitopes and each causing effective tumor cell killing in 2D co-culture, only one was able to trigger a wave of T cell recruitment and subsequent tumoroid killing in 3D. We have applied the same setup to testing the efficacy of T cells expressing engineered TCRs aimed at application in adoptive T-cell therapy. Kinetics of experiments using these activated engineered T cells are considerably shorter (24 hours instead of ~6 days) but show a similar pattern of T cell recruitment and subsequent tumoroid killing. We demonstrate successful generation of quantitative data for T cell recruitment and tumoroid killing for engineered T cells targeting tumor antigens expressed on TNBC and uveal melanoma tumoroids. Citation Format: Erik H. J. Danen. A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology [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 5226.

Abstract 5230: A modified FcγRI expressing T cell (SolidT) exhibits profound anti-tumor activity followed by maturation into effector memory t cells that can be reactivated to kill a secondary tumor

Abstract We previously described a subset of cytotoxic FcγRI (CD64)-expressing T cells in tumors and inflamed tissues (PMID 31449054). We subsequently expressed an engineered modified CD64 into T cells (“SolidT”) and demonstrated that these cells possessed greater specificity for tumor over normal tissues, reduced exhaustion, and attenuated cytokine secretion compared to other engineered cell therapies. SolidT activation correlates with surface tumor antigen density enabling the differentiation between tumor cells and normal cells by use of a specific antibody targeting tumor antigens. We previously showed that SolidT + mAb has anti-tumor activity in vitro and in vivo in several tumor models, including immunodeficient HER2 xenografts and an immunocompetent syngeneic B16-TYRP1 model (PMID 37070661). In this study, we further assessed the effect of SolidT on HER2+ tumors in a rechallenge model and extended the platform to CD20+ lymphoma models by combining SolidT with rituximab. In an in vivo experiment using HER2+, NCI-N87 cells in immunodeficient mice, several mice in the SolidT + trastuzumab group experienced complete tumor clearance. These mice were monitored until day 84, then rechallenged with NCI-N87 cells along with a new control group. After tumor establishment, mice were treated only with trastuzumab weekly for two weeks. While tumors in the control group progressed, the tumors in the rechallenged mice were completely cleared with a trastuzumab boost only, suggesting a SolidT cell memory response. Bone marrow from these rechallenged mice was analyzed and revealed the presence of effector memory SolidT cells. The SolidT model was then studied in a human, CD20+ lymphoma model in vitro and in vivo in immunodeficient mice. SolidT + rituximab was more potent in-vivo against CD20+ lymphoma cells than rituximab. In vivo, mice treated with SolidT + rituximab had significantly greater survival, lower tumor burden, and fewer sites of metastases than mice treated with rituximab alone. Bone marrow from these mice was analyzed and revealed the presence of effector memory SolidT cells in mice treated with SolidT + rituximab. We demonstrated that marrow resident SolidT cells developed an effector memory phenotype that positively correlated with outcome in HER2+ and CD20+ xenograft models, consistent with earlier data from a syngeneic B16 melanoma model. We further demonstrated that SolidT cells can be reactivated in a rechallenge model with only the addition of tumor targeting mAb, resulting in the eradication of the recurring tumors. This data further confirms that the SolidT platform can be targeted against different tumor antigens by using different tumor targeting mAbs. The SolidT platform could represent a paradigm shift in the approach to engineered cellular therapy for hematological malignancies and solid tumors. Citation Format: Shahar Dotan, Noam Pilpel, Peleg Rider, Hana Shpilt, Diana Rasoulouniriana, Yaron Carmi, James E. Wooldridge. A modified FcγRI expressing T cell (SolidT) exhibits profound anti-tumor activity followed by maturation into effector memory t cells that can be reactivated to kill a secondary tumor [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 5230.