Cytokine Release Research Articles

Abstract 3192: A novel long acting pegylated T cell engager targeting CD3 and cd19 with effective tumor killing and no potential CRS risk in CD19+ hematologic malignancies

Abstract The development of bispecific T cell engagers represents a highly effective immunotherapy strategy for the treatment of hematologic malignancies. However, Blinatumomab, a bispecific T cell engager of CD3/CD19, has disadvantages in clinical setting due to the adverse pharmacokinetics (PK), which resulted in less than 2 hours of in vivo elimination half-life, neurotoxicity, and cytokine release syndrome. In this study, a bispecific antibody JY108 was developed to target CD19 and CD3e with much reduced affinities towards both targets (revealed by Biacore assay results). JY108 was prepared by a patented pegylation bispecific linker technology, which could address the limitations of current therapeutic approaches. In vitro cytotoxicity assay results demonstrated that the EC50s of the drug specific cytotoxicity of T cells mediated by JY108 are in the concentrations of ng/ml, while blinatumomab mediated cytotoxicity to the same CD19+ cells are in the concentrations of pg/ml. Despite the weaker in vitro cytotoxicity than Blinatumomab, JY108 presents comparable tumor inhibition effects with Blinatumomab in different in vivo studies, which include the PBMC reconstructed models with established MEC-1 or Farage tumors, the delayed drug administration on human PBMCs/tumor co-transplanted model of Raji. In addition, JY108 prolonged the survival of animals with Nalm6 brain metastasis more significantly than Blinatumomab at the same dosage. Furthermore, the PK study revealed that at the different dosages, the circulation T1/2s of JY108 in mice are above 20 hours, which is expected to result in more significant efficacious benefits in clinical setting. Meanwhile, the completed package of GLP-toxicity study did not observe any drug related tox to the model animals (including no cytokine release in vivo, no neurotoxicity, no respiratory toxicity, etc.), and JY108 did not induce significant cytokine release than the blank control to the whole blood or PBMCs from healthy donor in vitro. In the efficacious concentrations of exerting cytotoxicity to CD19+ tumor cells in the presence of PBMC, JY108 did not induce significant cytokine release than the blank control, while blinatumomab always induced significant cytokine release. We postulated that the lower CD3 affinity of JY108 most probably resulted in the decoupling of the signaling for activating the gene expression of cytokines from the secretion of the polarized lytic granules by T cells, which resulted in a much milder T cell activation by JY108. Currently, JY108 is a phase I trial candidate and awaits further evaluations. Citation Format: Shumin (Sam) Liu, Yu (Yvonne) Wen, Weidong Lyu, Shuqiang Yin, Yang Lei, Dechun Wu, Zibin Wu, Qiudong Zhuo. A novel long acting pegylated T cell engager targeting CD3 and cd19 with effective tumor killing and no potential CRS risk in CD19+ hematologic malignancies [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 3192.

Abstract 2374: Bispecific and trispecific GPRC5D antibodies with potent cell-killing activity against multiple myeloma

Abstract Background: GPRC5D is a G protein-coupled receptor that is expressed on multiple myeloma cells but absent from most healthy tissues except for hair follicles. It is an attractive target for anti-tumor modalities including T cell-engaging bispecific antibodies. Recent clinical data demonstrate that combination therapy of T cell-engaging molecules individually targeting GPRC5D and BCMA on tumor cells offers unprecedented therapeutic benefits in relapsed/refractory multiple myeloma patients. A trispecific antibody that simultaneously engages these molecules while activating T cells is expected to provide comparable and additional benefits as a monotherapy. Methods: Multipass membrane proteins are valuable therapeutic targets but are largely inaccessible for antibody discovery due to structural complexity and high conservation. We developed an antibody discovery platform (MPS) tailored for membrane proteins that utilizes advanced immunization techniques including DNA, mRNA, and Lipoparticles (virus-like particles) and evolutionarily divergent host species. From the parental antibodies isolated, we engineered panels of GPRC5DxCD3 bispecific and GPRC5DxBCMAxCD3 trispecific antibodies using multiple formats and CD3 arms that encompass different geometries and binding stoichiometries. GPRC5D multispecific antibodies were tested for target specificity using Integral Molecular’s Membrane Proteome Array (MPA), and epitope residues were defined using alanine scanning mutagenesis. Both bispecific and trispecific antibodies were tested for in vitro potency and cytokine release using MM.1R and RPMI 8226 multiple myeloma cell lines. In vivo efficacy for a subset of antibodies was determined using xenograft models in PBMC-engrafted mice. Results: We immunized chickens with GPRC5D to obtain high-titer immune responses and configured resulting antibodies as T cell-engaging bispecific molecules with a CD3 binding arm. A subset of GPRC5DxCD3 bispecific antibodies displayed potent T cell-mediated cytotoxicity with picomolar potency on multiple myeloma cell lines. Antibody specificity profiling showed high specificity binding, and epitope mapping helped to explain why only some GPRC5D antibodies were active as multispecific antibodies. Bispecific molecules were then re-engineered as trispecific antibodies with additional binding to BCMA, a well-validated multiple myeloma target. GPRC5DxBCMAxCD3 trispecifics showed improved cytotoxic activity against multiple myeloma cells. Both bispecific and trispecific antibodies demonstrated a large window between cell killing and cytokine release. We will present in vivo efficacy data for a panel of bi- and trispecific molecules against MM.1R xenografts in PBMC-engrafted mice. Citation Format: Hayley Roth, Daniel Rogers, Ileine Sanchez, Breanna Tyrell, Trevor Barnes, Ami Snyder, Nenna Nowgu, Kyle Doolan, Benjamin J. Doranz, Ross Chambers, Joseph B. Rucker. Bispecific and trispecific GPRC5D antibodies with potent cell-killing activity against multiple myeloma [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 2374.

MRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions.

A small fraction of people vaccinated with mRNA-lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech's Comirnaty and Moderna's Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1β, and TNF-α, suggesting C-dependence of these cytokines' induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.

Abstract 1239: SAIL66, a next generation T cell engager targeting CLDN6, potentiates efficacy by binding to CD3/CD137

Abstract Background: Claudin-6 (CLDN6), one of over 20 known human CLDN family members, is upregulated in many solid tumors but shows minimal or no expression in healthy adult tissues. This makes it an ideal target for cancer therapy. Although several therapeutic modalities targeting CLDN6 are currently under investigation—including T cell engagers (TCE), CAR-T, and antibody drug conjugates—a more potent therapeutic option is still desirable. TCEs are a promising avenue within cancer immunotherapy, but their current efficacy and safety must be improved, particularly against solid tumors. Methods and Results: To enhance immune activation and achieve successful clinical outcomes against solid tumors expressing CLDN6, we generated SAIL66, a CLDN6-targeting next generation TCE, using a new technological format called Dual-Ig®. SAIL66 is a tri-specific monoclonal antibody designed to bind to CLDN6 on cancer cells with one Fab arm and to both CD3 and CD137 with the other Fab arm. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3. SAIL66 demonstrated high specificity for CLDN6 despite its similarity to CLDN3, CLDN4, and CLDN9, suggesting a lack of off-target toxicity in patients. Using a Jurkat cell system harboring NFAT or NF-κB reporter genes, we confirmed that SAIL66 exerts CD3 and CD137 signal induction activity depending on CLDN6 expression. SAIL66 induced activation of T cells, release of cytokines, and lysis of CLDN6-positive cancer cells. In vivo studies with a humanized mouse (huNOG) model showed that SAIL66 had better anti-tumor efficacy in eliminating CLDN6-positive cancer cells than a conventional TCE. Flow cytometry analysis in the huNOG model revealed that SAIL66 also more significantly increased CD3+ T cell infiltration of tumor tissue and resulted in a smaller percentage of exhausted T cells in tumor. Moreover, transcriptome analysis in the huNOG model showed that both SAIL66 and the conventional TCE increase expression of genes associated with immune activation, with a durable increase observed especially with SAIL66. Further, we clarified that T cells repeatedly stimulated with SAIL66 had a greater ability to inhibit the growth of CLDN6-positive cancer cells than T cells stimulated with the conventional TCE in vitro. Conclusion and Discussion: Our study shows that SAIL66 has better anti-tumor efficacy against CLDN6-positive cancer cells than a conventional TCE. This is due to effective immune activation by SAIL66 which triggers CD3 signaling (signal 1) combined with CD137 co-stimulatory signaling (signal 2) in T cells. Thus, SAIL66 represents a promising therapeutic option for solid tumors expressing CLDN6. A clinical trial for CLDN6-positive cancer patient is now ongoing (NCT05735366). Citation Format: Moe Yoshimoto, Naoki Kimura, Takayuki Kamikawa, Shinya Ishii, Masaru Muraoka, Kenji Taniguchi, Ryo Uchikawa, Momoko Okuda-Miura, Sho Akai, Tatsushi Kodama, Hirofumi Sakumoto, Shigeto Kawai, Mei Shimada, Mika Kamata-Sakurai, Hiroyuki Aburatani, Takehisa Kitazawa, Tomoyuki Igawa. SAIL66, a next generation T cell engager targeting CLDN6, potentiates efficacy by binding to CD3/CD137 [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 1239.

Abstract 2838: ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer

Abstract Immunomodulatory agents are an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviruses (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA, activating stimulator of interferon (IFN) genes (STING). We reported that DNMTis in combination with PARPis induce STING-dependent signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but are not fully understood in cancer. Our studies of the little-known gene, NFX1-type zinc finger-containing 1 (ZNFX1), show that it acts as a master nucleic acid (dsRNA/DNA) sensor for mt gateway function. Bioinformatics analysis in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, shows that increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high-grade and endometrial serous carcinoma (OC) cell lines (N=3), transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib [TAL]) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane. Functional studies of mt dysfunction in OC cells, show that dsRNA/DNA as well as above viral mimicry drugs increase mt reactive oxygen species (ROS) as measured by mitosox flow cytometry. AZA and TAL treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured by long range PCR and 8-oxoguanine (8-oxoG) ELISA assays. These drug treatments also increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. Notably, ZNFX1 knockout (KO) attenuates these dynamics, including in bioinformatics of RNA seq data, thus defining this gene as essential for IFN/inflammasome signaling induced by mtDNA damage. Further pathway analysis of ZNFX1 KO vs wild-type (WT) RNAseq data suggest increased proliferation and epithelial to mesenchymal transition (EMT) that was validated by in vitro and in vivo assays. Not only is ZNFX1 a master regular of the above mt dynamics, but it is a tumor suppressor in OC. Abrogating ZNFX1 expression in OC cells triggers increased cell proliferation, invasive capacity and, in-vivo, increased tumor growth. This work then suggests novel approaches to improve immune therapy responses in OC through manipulating ZNFX1. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael Topper, Stephen Baylin, Kenneth Nephew, Feyruz Rassool. ZNFX1 is a master regulator and tumor suppressor in epigenetically induced pathogen mimicry, mitochondrial dysfunction and STING-dependent signaling in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2838.

Abstract 6712: LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia

Abstract Background: Acute myeloid leukemia (AML) is a malignant clonal disorder linked to a broad spectrum of molecular alterations. It is still an incurable cancer, the prognosis is even worse in elderly patients, where overall survival (OS) at 1 year is approximately 10-15%. Now, several CD3 bispecific immunotherapies that harness T cells against AML are at various stages of preclinical and clinical development, but most of them are hampered by the lack of specific targets to distinguish tumor cells from normal hematopoietic stem cells (HSC). Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an immune checkpoint inhibitory receptor, which is highly expressed on FAB M4 and M5 AML cells with a lack of expression on normal HSC and progenitor cells. LILRB4 expression level inversely correlates with OS of patients with M4 and M5 AML. All of these indicate that LILRB4 represents a promising target for developing a T cell redirecting bispecific antibody to treat patients with AML, especially for more aggressive M4 and M5 subtype. Herein a novel bispecific antibody targeting LILRB4 and CD3, LBL-043, is developed to specifically kill LILRB4 positive tumor cells by engaging T cells. Methods: LBL-043 was constructed using our proprietary LeadsBodyTM T cell engager platform, which is a 2:1 format bispecific antibody with two VHH arms targeting LILRB4 with high affinity and one scFv arm targeting CD3 with fine-tuned low affinity. The binding affinity of LBL-043 to LILRB4 and CD3 was determined with Fortebio, while the activity was measured using several cell based assays including reporter gene and TDCC assays. The anti-tumor activity of LBL-043 was investigated in B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 (human AML-M5a) tumor model. Results: The binding affinity of LBL-043 to LILRB4 and CD3 protein was 0.724 nM and 35.7 nM, respectively. In CD3 reporter gene assays, LBL-043 could activate the NFAT reporter signaling through LILRB4 binding dependent CD3 cross-linking with an EC50 value of 2.459 nM. LBL-043 was shown a potent TDCC activity in different LILRB4 expression level MOLM-13 cells and HL-60 cells with T cell activation and cytokine release. In B-NDG/hu-PBMC reconstructed mice implanted with MOLM-13 tumor cells, LBL-043 was shown significant anti-tumor activity. Simultaneously, LBL-043 can effectively improve the survival rate and maintain relatively stable body weight in the study compared to the vehicle group. Conclusion: LBL-043, a novel bispecific antibody targeting CD3 and LILRB4 with affinity differentiation, induces T cell killing of AML cells by simultaneously binding LILRB4 expressed on tumor cells and CD3 on T cells, redirecting T cells to efficiently killing of AML cells. It is also shown a great anti-tumor efficacy in CDX mouse models. These data support LBL-043 as a novel therapeutic bispecific antibody for the treatment of LILRB4 positive AML and CMML leukemia patients. Citation Format: Yujia Dang, Xiao Huang, Yurong Qin, Xiaoya Liu, Min Chen, Duqing Jiang, Guojin Wu, Mi Ye, Jianming Sun, Baohui Wang, Jing Guan, Tingting Li, Jordan Zhu, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-043, a novel LILRB4xCD3 T cell engager, for the treatment of LILRB4 positive leukemia [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 6712.

Abstract 3996: Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy

Abstract Background: Success of Chimeric Antigen Receptor (CAR) T cells therapy for solid tumors are limited by the suppressive solid tumor microenvironment (TME), T cell exhaustion, lack of persistence, and poor trafficking to tumors. Strategies to improve this include therapeutic combinations, such as checkpoint inhibition (CPI) or cytokine support. However, CPI therapy has largely failed because of refractory and resistant tumors and cytokine administration can lead to lethal toxicities. To this end, we propose that enabling CAR T cells to secrete bi-functional fusion proteins consisting of cytokine modifiers (ie: IL12, IL15, or TGFβ-trap) combined with checkpoint inhibition (αPDL1 scFv) can provide tumor PDL1 sequestered cytokine activity and local tumor-immune modulation to boost solid tumor CAR T cell efficacy, enhance CPI impacts, and safely improve durable outcomes. Methods: Mouse T cells dual transduced to express surface CAR targeting prostate or ovarian solid tumors in addition to secretable cytokine fused to an αPDL1 scFv were generated. In vitro, the PDL1 blockade capacity and tumor-surface cytokine presentation of supernatants from dual transduced CAR T cells was assessed on PDL1 induced tumor cells. Further, secreted fusion protein transduced CAR T cells were assessed for function, phenotype, and cytokine release in a repetitive tumor rechallenge assay. In vivo, mouse CAR T cells dual transduced with fusion proteins and appropriate controls were assessed for anti-tumor efficacy, survival kinetics, and toxicity in immunocompetent solid tumor mouse models of prostate cancer and intraperitoneal disseminated ovarian cancer. Results: CAR T cells were shown to be successfully dual transduced, and secreted αPDL1-cytokine fusion proteins exhibiting functional PDL1 binding characteristics in vitro. CAR T cells engineered with αPDL1-IL12 fusion protein had greater anti-tumor activity and CAR specific expansion compared to other tested fusion proteins in in vitro co-culture assays. In a syngeneic prostate tumor model, mice receiving CAR T cells with αPDL1-IL12 fusion T cells out competed other cytokine fusion combinations. In a syngeneic ovarian tumor model we safely achieved 100% curative response rate with CAR secreting αPDL1-IL12 fusion in contrast to CAR with αPDL1mutIL12 control along with tumor regional PDL1 blockade on myeloid subsets within the TME. Conclusions: Our findings suggest that CAR T cells engineered to secrete αPD-L1-IL12 fusion protein show improved tumor control, less systemic toxicity, and enhanced expansion which promotes eradication of disease in two independent in vivo tumor models with two unique solid tumor CAR targets. We believe this strategy has the potential to improve solid tumor CAR T-cell efficacy and enhance durable innate immune responses. Citation Format: Lea Christian, John P. Murad, Lupita Lopez, Anthony Park, Jason Yang, Eric Lee, Candi Trac, Stephen Forman, Saul J. Priceman. Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (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 3996.

Abstract 6340: Novel conditionally active tetravalent B7-H3 x CD3 T-cell engagers targeting solid tumors

Abstract B7-H3, also known as CD276, is an immune-checkpoint molecule and a type 1 transmembrane protein that exerts a variety of biological functions. In cancer biology, the overexpression of B7-H3 is correlated with tumor cell invasion and metastasis, decreased T-cell infiltration and function, poor prognosis, and resistance to therapy. As B7-H3 is expressed in multiple solid tumors and plays an important role in modulating anti-tumor immune response as well as promoting cancer cell formation, it became an important target for the development of antibody-based immunotherapy. Many forms of bispecific antibodies targeting B7-H3 have shown antitumor response in preclinical studies. However, bispecific T-cell engagers (TCE) targeting B7H3 and CD3 have been correlated with cytokine release syndrome and hepatic adverse events, a common toxicity observed using TCEs which has limited the development of this immunotherapeutic in clinic. We leveraged BioAtla’s Conditionally Active Biologic (CAB) platform (1) to develop a DualCAB T-cell engager targeting B7-H3 and CD3. This DualCAB-TCE molecule, BA3142, was engineered to bind with high affinity to both recombinant B7-H3 and CD3 receptor/antigens under conditions that mimic the acidic tumor microenvironment, but with low affinity in alkaline physiological conditions. In vitro and in vivo efficacy and toxicity data for the clone BA3142 will be presented. Our data demonstrated that BA3142 promotes cytotoxicity of B7-H3-expressing cancer cells in vitro and induces significant tumor regression in vivo. In Non-Human Primates BA3142 bispecific antibody was tolerated up to 25 mg/kg with minimal cytokine release and no clinical and histopathological signs of toxicities. The CAB technology offers the opportunity to expand the number of targets suitable for drug development in a new class of DualCAB TCEs with superior safety margin and potency in the clinic. (1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ, and Short, J.M. 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, PhD, Haizhen Liu, Patricia McNeeley, Kathryn Woodard, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active tetravalent B7-H3 x CD3 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 6340.

Abstract 6343: AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS

Abstract Background: CD3+ T cell engaging bispecific antibodies (TCE) redirect T cells to attack targeted tumor cells by simultaneously binding TAA on cancer cells and CD3 complex on a T cell to form a TCR-independent immune synapse. Multiple TCEs have demonstrated promising therapeutic efficacy in the treatment of hematological malignancies. In contrast, suboptimal efficacy in the treatment of solid tumors and the risk of inducing cytokine release syndrome (CRS) continue to be major challenges. Several factors, such as format, affinity, and valency of the antibody, TAA copy number, and antigen size and epitope, can impact the therapeutic efficacy and safety of a TCE. In this study, we developed a novel “2+1” TCE platform, AnTenGagerTM, which enables conditional T cell binding and activation upon TAA-crosslinking, potent anti-tumor activity, and reduced risk of CRS. Methods: Humanized CD3 antibodies were derived from mouse hybridoma, followed by affinity optimization utilizing Computer Aided Drug Design. The Fab-scFv-Fab format was used as the backbone structure of AnTenGagers. The anti-CD3 scFv sequences and the length of peptide linkers between VH/VL, scFv/CH1, and scFv/CH2 were screened and compared in different preclinical assays. LALA mutations (L234A, L235A) were introduced to abolish the Fc receptor binding capability. In a series of in vitro and/or in vivo models, the efficacy, safety and developability of AnTenGagers targeting multiple TAAs, such as Her2, GD2, CDH6, LILRB4, FLT3 and GPRC5D, were evaluated. Results: A library of humanized parental CD3 antibodies with a broad spectrum of affinities were generated, some of which cross react with monkey CD3. AnTenGagers exhibited limited binding to CD3-positive cells before TAA-crosslinking, suggesting a reduced risk of CRS that is caused by systemic CD3+T cell activation. Four CD3 sequences with varying affinities were utilized to construct AnTenGagers with identical TAA-targeting arm. Compared to clinical benchmarks targeting the same TAA, all AnTenGagers demonstrated substantially lower binding capacity to CD3+ cells in the absence of TAA-crosslinking, while inducing increased cytotoxicity against target-positive tumor cells. In addition, they induced significantly less ex-vivo cytokine release by human PBMC than benchmarks. Notably, all AnTenGagers demonstrated enhanced in vivo efficacy compared to the benchmark, exhibiting complete response (CR) in PBMC humanized MM1S mouse CDX model while the serum concentration of proinflammatory cytokines was substantially lower in groups treated with AnTenGagers. AnTenGagers also demonstrated good developability with high expression yield, good thermostability and high stability under different stress conditions. Conclusions: These results suggested that AnTenGagerTM is a promising platform for developing the next generation of TCEs with enhanced efficacy and safety. Citation Format: Bing Hou, Hui Yuwen, Tengteng Li, Yijing Ren, Ao Sun, Zaoshun Hu, Jay Mei, Bo Shan. AnTenGagerTM, a novel “2+1” T cell engager platform, enables conditional T cell activation with reduced risk of CRS [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 6343.

Abstract 6705: A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1

Abstract Background: Wilms' tumor gene 1 (WT1) is considered an oncogenic factor, with high expression observed in several hematological malignancies and solid tumors. However, its expression is rarely found in normal adult tissue. WT1 can be degraded by the proteasome, processed and presented on the cell surface as major histocompatibility complex (MHC) I epitopes and recognized by T cell receptors (TCR). Here, we described the development of WT1-TOPAbody, a novel tri-specific T cell engager (TCE), that activated T cells by both CD3 and 4-1BB signaling upon recognition of WT1 in the context of HLA-A02, leading to superior anti-tumor efficacy. Materials and Methods: WT1-TOPAbody was evaluated for its antigen binding activity and target specificity by a series protein/peptide and cell-based assays. In vitro cytotoxicity and T cell activation mediated by WT1-TOPAbody was further assessed in a panel of WT1+ tumor cell lines in coculture with effector cells. Anti-tumor activity was investigated in a humanized syngeneic mice model bearing WT1/HLA-A02 expressing tumors. Safety assessment including cytokine release assay (CRA) and preliminary CMC developability was also performed. Results: WT1-TOPAbody exhibited superior binding affinity to the WT1/HLA-A02 complex while displaying lower binding to off-target peptides and CD34+ HSC from healthy donor than clinical benchmark WT1-CD3 bispecific antibody, RG-6007. WT1-TOPAbody effectively induced tumor lysis in multiple WT1+ tumors including leukemia, ovarian, breast, and colon cancer cells. Moreover, serial killing of tumor cells were observed in WT1-TOPAbody treated T cells, accompanied by lower exhaustion markers expressed, suggesting a prolonged anti-tumor inhibition effect of WT1-TOPAbody. In a humanized CD3e/4-1BB/HLA-A2.1 mouse model, WT1-TOPAbody significantly inhibited tumor growth in a dose-dependent manner and the efficacy was superior than that of the benchmark. Additionally, IL-6 release was minimal in PBMC upon treatment of the antibody, indicating a reduced risk of inducing cytokine release syndrome (CRS). Drug developability assessment indicated that the leading WT1-TOPAbody was a reasonably developable molecule to be taken forward. Conclusion: WT1-TOPAbody is a differentiated WT1/HLA-A02 targeted T cell engagers that displayed potent and durable anti-tumor activity. These results collectively support the potential of WT1-TOPAbody as a novel therapeutic agent against WT1/HLA-A02+ cancers and warrant its further development. Citation Format: Shan Gao, Liu Yang, Jun Du, Wenqing Jiang, Lei Fang. A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1 [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 6705.

Abstract 3739: Different combination regimens for OX40 agonists and PD-1 inhibitors exert different antitumor effects in OX40 humanized mice

Abstract OX40, also known as CD134 or TNFRSF4, is a member of the tumor necrosis factor receptor (TNFR) superfamily which mainly expressed on the surface of activated T cells including Treg cells. Engagement of OX40 with its ligand, TNFSF4, leads to effector T cell expansion and cytokine release, inhibits activation-induced cell death (AICD) and promotes the survival of antigen-specific memory T cells. In contrast to the massive hit of immune checkpoint inhibitors like PD-1 and PD-L1, the benefit of agonistic antibodies in clinical trials has not been completely demonstrated to date. On one hand, higher doses could leads to T cell exhaustion or AICD, paradoxically, with no efficacy improvement. Another hand, the limited understanding of the biology of immune checkpoints underlying “accelerator and brake” should account for the slow clinical progress of OX40-targeted biologics. It seems that the dosing sequence or timing of anti-OX40 and anti-PD-1 is an important consideration in combination therapies. Given this, we firstly evaluated the antitumor effects of two different anti-human OX40 agonists in syngeneic MC38 and Pan02 tumor models, respectively, in hOX40 knockin mice. We observed tumor regressions upon treatment of OX40 agonistic Ab1 in a dose-dependence way in the homozygous hOX40 knockin mice engrafted with MC38 tumors. The anti-hOX40 Ab1 at high-dose (10 mg/kg) led to ~60% TGI, but no response at very low dose (0.1 mg/kg). Furthermore, the homozygous hOX40 knockin mice inoculated with Pan02 pancreatic tumors were treated with a novel strong anti-hOX40 agonist (Ab2) and surrogate anti-mPD-1. Notably, Anit-hOX40 (Ab2) alone at low dose (1 mg/kg, Q3D*9 doses) exerted strong anti-tumor potency (TGI=59.3%, p&amp;lt; 0.005) and no statistical significant antitumor difference was observed between high-dose group (at 5mg/kg) and low-dose group (at 1 mpk) as monotherapy, though anti-hOX40 (Ab2) at 5mg/kg showed the best therapeutic response (TGI=72%, p=0.0002) among all treatment groups. In contrast, the second therapeutic response was observed in the combo group treated with first dosing of 5mg/kg anti-hOX40 (Ab2) combining delayed dosing of 10mg/kg anti-mPD-1 (TGI=64%, p&amp;lt; 0.005) and the concurrent dosing group of Ab2 (5 mg/kg, Q3D × 9 doses) plus anti-mPD-1 (2 mg/kg, Q3D × 9 doses) (TGI=62.3%, p= 0.001). In conclusion, different human-specific OX40 agonists displayed different anti-tumor activity as monotherapy, and different dose led to different antitumor potency for the same agonist. In terms of different combination regimens, although no advanced benefit was observed for the combination of anti-mPD-1/anti-OX40 (Ab2) in comparison to strong anti-OX40 agonist (Ab2) alone, it demonstrated that the dosing timing and sequence of either anti-PD-1 or anti-hOX40 did has impact on the therapeutic outcome. Citation Format: Yi Li, Yanlei Zhang, Xiaolei Qiu, Dongxiao Feng, Ruilin Sun, Daniel X. He. Different combination regimens for OX40 agonists and PD-1 inhibitors exert different antitumor effects in OX40 humanized mice [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 3739.

Abstract 6716: DLL3 TriTCE Co-Stim: A next generation trispecific T cell engager with integrated CD28 costimulation for the treatment of DLL3-expressing cancers

Abstract Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer with a poor prognosis and high unmet medical need. Current standards of care, including chemotherapy, targeted therapy and immunotherapy, are associated with limited duration of response. Furthermore, SCLC is characterized by an immunosuppressive microenvironment and poor T cell infiltration which present additional treatment challenges. Delta-like ligand 3 (DLL3) is an inhibitory Notch ligand that is aberrantly overexpressed in SCLC and other neuroendocrine tumors, but minimally expressed in normal tissues, and is a new therapeutic target. Bispecific T cell engagers (TCE) targeting DLL3 have entered the clinic and demonstrated promising anti-tumor activity in SCLC patients; however, we believe there is room to improve on the rate and depth of response. To address treatment challenges and enhance the durability and sustainability of T cell activation, we have engineered a trispecific co-stimulatory T cell engager (TriTCE Co-stim) molecule that optimally engages CD3 and CD28 and redirects and enhances cytotoxic T cell responses to DLL3-expressing tumor cells while maintaining a desired safety profile. A panel of DLL3xCD3xCD28 trispecific antibodies (Abs) was engineered with a conventional anti-CD28 agonist paratope in a variety of Ab formats, geometries, paratope affinities and specificities. Geometry and design features of the tri-specific Abs were informed by prior TriTCE Co-stim molecule optimization1. AzymetricTM and EFECTTM platforms were used to facilitate heterodimeric multi-chain antibody assembly with silenced Fc gamma function. Lead TriTCE Co-stim Abs were selected for enhanced DLL3-dependent antitumor cytotoxic activity and increased T cell function including T cell activation, proliferation and cytokine production, compared to competitor bispecific TCEs. DLL3-independent activity and cytokine release were assessed with human PBMCs in solid and solution phase assays to evaluate the potential for cytokine release induction. In summary, we have identified multiple TriTCE Co-Stim Ab formats with improved DLL3-dependent cytotoxicity and T cell activation over competitor bispecific TCEs across multiple DLL3-expressing tumors. By screening various Ab formats, geometries and paratope affinities, we have selected lead DLL3 TriTCE Co-stim Abs displaying target-dependent activation of T cells with potent anti-tumor activity which may translate to improved and more durable antitumor responses in the treatment DLL3-expressing cancers. Citation Format: Peter Repenning, Desmond Lau, Diana C. Hernaez, Alec Robinson, Diego P. Escanda, Mariana Rocha, Begonia S. Moreno, John Zhang, Polly Shao, Nichole Escalante, Lisa Newhook, Purva Bhojane, Chayne L. Piscitelli, Paul A. Moore, Thomas Spreter Von Kreudenstein, Nina E. Weisser. DLL3 TriTCE Co-Stim: A next generation trispecific T cell engager with integrated CD28 costimulation for the treatment of DLL3-expressing 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 6716.

Abstract 13: Discovery and development of T cell receptors targeting MAGE family antigens for adoptive T cell therapy against solid tumors

Abstract Background: T cell receptors (TCRs) recognize epitopes from intracellular and cell surface antigens, enabling targeting of diverse tumor antigen classes including overexpressed, differentiation, cancer-testis antigens, as well as those from driver mutations, frameshift mutations, splice variants, and human endogenous retroviral elements (HERV). Melanoma-associated antigens (MAGE) are highly expressed in various solid tumor types, including melanoma, non-small cell lung cancers (NSCLC), bladder, and head and neck cancers, representing promising targets for TCR-T therapy. However, discovering robust and specific MAGE-reactive TCRs remains challenging due to limited throughput of TCR screening methodologies, and the inherently low frequency of tumor-reactive T cells in tumor samples and peripheral blood. Methods: We employ a proprietary mass-cytometry-based multiplexed tetramer staining approach, TargetScape®, to screen and characterize CD8+ T cells in healthy donors and patients’ samples. Our screening panels contain multiple peptides for MAGE family antigens including A1, C2, and A10. Subsequent single cell sequencing or single cell PCR (scPCR) of identified tumor antigen-specific T cells with high sensitivity enables comprehensive analysis of T cell phenotype, transcriptome, TCR specificity, and paired TCR sequence. TCRs identified through this method are transduced for expression in Jurkat reporter cell lines and their reactivity to corresponding peptides is assessed. Selected TCR candidates are then transduced into primary T cells and evaluated for activation and effector function using a variety of cancer cell lines expressing the corresponding endogenous antigens. Specificity assessment is performed through alanine scans and identification of potential off-target peptides and screening for reactivity. Results: We utilized this approach to discover natural TCRs that target several MAGE family antigens. Through Jurkat reporter screening, we observed specific activation of multiple TCRs in the presence of antigen presenting cells and the peptide initially employed to identify the TCRs. Primary T-cells expressing promising TCR candidates exhibited killing activities and cytokine release when confronted with multiple solid tumor cell lines expressing endogenous levels of the MAGE antigen. Specificity assessment is currently in progress, with preliminary data indicating overall favorable specificity profiles for selected TCRs. Conclusions: Our Deep Immunomics platform enables us to detect, characterize, and validate TCR candidates against highly clinically relevant shared cancer antigens. Leveraging this platform, we identified various natural tumor specific TCRs exhibiting activities against a range of solid tumor cell lines expressing MAGE antigens. Promising candidates are currently undergoing in vivo efficacy studies. Citation Format: Juliana Velez Lujan, Hannah Fields, Kan Xing Wu, Yovita Purwanti, Emily Xin Zi Tang, Michael Cordeau, Kathleen Mendez, Melissa Wirawan, Michael Fehlings, Katja Fink, Zilei Liu, Daniel MacLeod. Discovery and development of T cell receptors targeting MAGE family antigens for adoptive T cell therapy against 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 13.

Abstract 6359: Development of PSMA x CD3 T-cell engagers using an integrated, functional approach

Abstract In this study, we used a functional approach to generate CD3 T-cell engager (TCE) molecules targeting prostate-specific membrane antigen (PSMA). We present data on the robust in vitro characterization of T-cell activity and selection of molecules for further assessment. TCE function is dictated by the interplay between multiple factors that form the immune synapse, including binding kinetics, binding geometry, and epitopes of the CD3- and tumor-binding arms. To diversify immune synapse parameters, we engineer hundreds of bispecific molecules from highly diverse parental antibodies, and employ a high-throughput process to select molecules with desired functional properties. We applied this approach to engineer CD3 TCEs targeting PSMA. PSMA is an attractive target for the treatment of castration-resistant prostate cancer due to its high expression in prostate cancer cells and low relative expression in other tissues. We discovered hundreds of diverse, fully human PSMA-binding antibodies using high-throughput single B-cell screening and selected human/cynomolgus cross-reactive binders with a broad range of affinities and epitopes. We paired these with human/cynomolgus cross-reactive CD3-binding antibodies from our TCE platform to generate 180 bispecific PSMA x CD3 TCEs. CD3-binding antibodies covered several orders of magnitude of affinity (nanomolar to micromolar) and included binders specific for different CD3 subunits. We used high-throughput T-cell dependent cellular cytotoxicity (TDCC) and cytokine release assays to identify molecules with desired functional profiles. Bispecifics selected for further assessment had functional profiles spanning a range of properties that have been observed in clinical molecules, including potent killing and low cytokine release. To profile T-cell properties that are associated with anti-tumor immune responses, we subjected selected molecules to a battery of additional in vitro functional assessments, including proliferation, cytokine and chemokine production, and TDCC using cynomolgus T cells. Results demonstrate that a functional approach that begins with highly diverse CD3- and tumor-binding antibodies can generate promising TCEs with minimal need for protein engineering and optimization. Citation Format: Valentine de Puyraimond, Matt Mai, Alaa Amash, Nathalie Blamey, Gabrielle Conaghan, Jessica Fernandes Scortecci, Allison Goodman, Ahn Lee, Irene Yu, Franziska von Bank, Kate Caldwell, Lauren Clifford, Ingrid Knarston, Kelly Bullock, Melissa Cid, Cindy-Lee Crichlow, Lindsay Devorkin, Fiona Dickson, Patrick Farber, Stefan Hannie, Courteney Lai, Vivian Li, Stephanie K. Masterman, Iwona Niemietz, Philippe Pouliot, Ping Xiang, Bryan C. Barnhart, Raffi Tonikian. Development of PSMA x CD3 T-cell engagers using an integrated, functional approach [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 6359.

Abstract 21: Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive cancers

Abstract Introduction: Anaplastic Lymphoma Kinase (ALK) tyrosine kinase inhibitors (TKIs) have extended the survival of patients with ALK-rearranged cancers, including non-small cell lung cancer (NSCLC). Unfortunately, acquired resistance develops within 2-3 years, highlighting the urgent need for novel and effective therapeutic strategies for these patients. Here, we aimed to identify T cell receptor (TCR) clonotypes against two human ALK immunogenic peptides we previously identified by mass spectrometry in biopsies from patients with ALK-rearranged NSCLC (PMID:37430060) and to develop TCR-T cell therapies for ALK-positive NSCLCs. Methods: BALB/c mice were vaccinated with the murine ALK peptide PGPGRVAKI and transgenic mice expressing human HLA-B*07:02 were vaccinated with human ALK peptides RPRPSQPSSL and IVRCIGVSL. All mice received two priming injections (days 1 and 14) followed by two boosters, and activated CD8+ T cells were sorted and subjected to single-cell sequencing. Results: As proof of concept, we first vaccinated BALB/c mice with the ALK peptide PGPGRVAKI and identified 381 unique ALK-specific clonotypes. Among these, we cloned the most expanded 5 clonotypes and confirmed that these clonotypes were functional as evidenced by the release of significant amounts of IFN-γ and IL2, and potent in vitro killing activity following co-culture with murine ALK-positive lung cancer cells. To identify TCR clonotypes for the development of TCR-T therapy in patients with ALK-positive cancers, we next vaccinated transgenic mice expressing human HLA-B*07:02 with human ALK peptides RPRPSQPSSL and IVRCIGVSL. We again identified multiple unique ALK-specific TCR clonotypes among CD8+/CD137+ cells from these transgenic mice vaccinated with RPRPSQPSSL peptide (N=353 TCR clonotypes) and IVRCIGVSL peptide (N=742 TCR clonotypes). Single-cell RNA sequencing of the expanded clonotypes (TCR clonotype frequency ≥4) versus non-expanded clonotypes (TCR clonotype frequency &amp;lt;4) revealed significant upregulation of Ccl3, Ccl4, Ccl1, Ifng, Xcl1, and Il13 across mice vaccinated with RPRPSQPSSL and IVRCIGVSL (p &amp;lt;0.05). Gene set enrichment analysis (GSEA) confirmed significant upregulation of multiple pathways of adaptive immune response, including T cell activation, IFN-γ signaling and production, cytokine release, and T cell proliferation (adjusted p &amp;lt;0.05), suggesting functional activity of these TCR clonotypes against ALK. Conclusion: Here, we first demonstrated the feasibility of isolating ALK-specific TCR clonotypes by mice vaccination that exerted anti-tumor activity. Additionally, we identified ALK-specific TCR clonotypes from transgenic mice expressing human HLA-B*07:02 vaccinated with two human ALK peptides. This discovery lays the basis for the successful development of TCR-T cell therapies for ALK-positive NSCLCs, and possibly other ALK-positive cancers. Citation Format: Carmen Mecca, Ana Azambuja, Luca Alessandrí, Elisa Bergaggio, Simone Piane, Marcos Simoes-Costa, Ellis L. Reinherz, Rafael Blasco-Patiño, Roberto Chiarle. Discovery of ALK-specific TCR clonotypes for the development of TCR-T cell therapies against ALK-positive 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 21.

Abstract 6727: QL335, a novel T cell engager with enhanced safety profile targeting MSS colorectal cancer

Abstract Colorectal cancer (CRC) is the fourth leading cause of death in the world with a global mortality rate of over 700,000 annually. While recent advancement in diagnosis and prevention has reduced the overall number of deaths, the lack of effective and durable treatment has not significantly improved survival rate. Emerging modalities in immunotherapy such as T cell engagers have paved new ways for disease management. However, a common challenge to advancing T cell engagers in the clinic is poor target specificity and high toxicity associated with non-specific T cell activation and cytokine release. To overcome these shortcomings, we have developed QL335, a bispecific antibody bridging LY6G6D antigen on tumor cells and CD3 on T cells. LY6G6D or Lymphocyte Antigen 6 Family Member G6D, is a GPI-anchored membrane protein that, according to TCGA RNAseq data, is highly expressed in microsatellite stable (MSS) colorectal cancer but not in normal tissues. We analyzed LY6G6D expression by IHC in over 200 CRC patient tumors and 40+ normal tissue samples from different organs, confirming its highly restricted CRC expression pattern. We constructed our bispecific antibody in a 2+1 format, comprising two high affinity LY6G6D binding domains and one reduced affinity CD3 binding domain. Such format allows for higher tumor accumulation while mitigating non-specific T cell activation in the periphery. In vitro evaluation showed that QL335 induced potent yet highly specific T cell activation and killing of only target cells expressing LY6G6D. QL335 demonstrated robust anti-tumor efficacy in vivo against human colon cancer xenograft models with human PBMC reconstitution. Importantly, QL335 was well tolerated in cynomolgus monkeys dosed up to 50 mg/kg, supporting the enhanced safety profile by design of this therapeutic candidate. QL335 is currently being evaluated in a phase 1 clinical trial. Citation Format: Shenda Gu, Guangjie Guo, Helena Yee, Hieu V. Tran, Shihao Chen, Chuanzeng Cao, Ke Liu, Chunyan Jin, Weihua Lun, Ruixue Ma, Lele Ren. QL335, a novel T cell engager with enhanced safety profile targeting MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6727.

Abstract 6719: TriTCE Co-Stim: A next generation trispecific T cell engager platform with integrated CD28 costimulation, engineered to improve responses in the treatment of solid tumors

Abstract Bispecific T cell engagers (TCEs) have exhibited clinical successes in the treatment of hematological cancers, while treatment of solid tumors remains a challenge. Treatment of solid tumors with conventional CD3-engaging TCEs can result in limited T cell proliferation and recruitment to the tumor site, and treatment related T cell anergy, thus restricting the ability of bispecific TCEs to inhibit growth of these poorly infiltrated tumors and rapidly growing tumors. Next generation tumor-targeting, trispecific T cell engagers with integrated costimulation (TriTCE Co-Stim) have the potential to provide more durable responses and re-invigorate T cell responses by mediating integrated signaling through CD3 (signal 1) and co-stimulation through CD28 (signal 2). Superagonist anti-CD28 antibodies activate T cells but resulted in clinical toxicities with severe cytokine release syndrome (CRS). Therefore, achieving a balance between signal 1 and 2 is critical for optimal T cell activation and proliferation. Using our AzymetricTM and EFECTTM platforms, we generated heterodimeric TriTCE Co-Stim antibody formats with various geometries and affinities to optimize T cell response and widen the therapeutic window for the treatment of solid tumors. We have previously described screening and interrogation of different TriTCE Co-stim formats and paratope affinities, and the selection of a lead format with optimized CD3 and CD28 T cell activation. Here, we have further expanded our validation of the TriTCE Co-stim platform with our lead CLDN18.2 TriTCE co-stim format, to include a tolerability assessment in nonhuman primates.Our lead TriTCE Co-Stim format exhibited a favorable safety profile in vitro and mediated target-dependent induction of T cell activity, with enhanced proliferation, survival, and T cell-mediated cytotoxic potency compared to bispecific TCEs. In a predictive in vivo model of CRS, our lead TriTCE Co-Stim did not exhibit systemic toxicity or peripheral cytokine release. TriTCE Co-Stim mediated enhanced tumor growth regression in vivo with an increase of T cells within the tumor and no increase of T cells within the periphery. Repeat doses of the lead TriTCE Co-Stim molecule at 3 mg/kg were well-tolerated in nonhuman primates with no abnormal clinical signs. Minor decreases in body weight and food consumption were observed. Clinical pathology changes were consistent with a mild systemic inflammatory response. In summary, our lead TriTCE co-stim molecule has enhanced target-dependent antitumor activity vs. bispecific benchmarks and is tolerated a murine CRS model and repeat dose non-human primate study. These data suggest TriTCE Co-stim may provide tolerable and more durable antitumor responses and contribute to improved clinical outcomes. Citation Format: Lisa Newhook, Purva Bhojane, Kurt Stahl, Nichole E. Escalante, Peter Repenning, Diego Perez Escanda, Polly Shao, Maya C. Poffenberger, Alec Robinson, Kesha Patel, Alexandra Livernois, Chayne L. Piscitelli, Nicole Afacan, Paul A. Moore, Nina E. Weisser, Thomas Spreter von Kreudenstein. TriTCE Co-Stim: A next generation trispecific T cell engager platform with integrated CD28 costimulation, engineered to improve responses in the treatment of 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 6719.

Abstract 1435: Utilization of CLDN18.2 syngeneic mouse models to study chimeric antigen receptor T cells in immunocompetent mice

Abstract CLDN18.2 expression has been observed in pancreatic, gastric and esophageal cancers, whereas normal tissue expression of CLDN18.2 is mostly restricted to differentiated gastric mucosal epithelial cells. This expression profile renders CLDN18.2 an ideal tumor associated antigen (TAA) for CAR-T targeting and an armored targeted CAR-T, AZD6422, is in clinical development. While CAR-T are beginning to show clinical promise in solid tumors, a consistent challenge is to overcome the immunosuppressive tumor microenvironment (TME) where immune and stromal cells secrete high levels of TGFb. Since the majority of pre-clinical CAR-T efficacy data is generated utilizing immunocompromised mouse models, which lack a proper TME, the team utilized syngeneic xenograft models to circumvent this challenge. ​ In vitro studies showed that CLDN18.2 targeted murine CAR-T cells generated by retroviral transduction induced efficient cytolysis of target positive mouse tumor cells accompanied by cytokine release. To model the clinical scenario, tumor-bearing mice were lymphodepleted with total body irradiation (TBI) to enhance CAR-T engraftment. The dosage of TBI used in these studies was predetermined to have caused leukopenia and minimal impact on tumor growth. We observed substantial tumor control and extended tumor efficacy in mice treated with mCAR-T cells compared to those infused with untransduced T cells or in non-target bearing control tumors in the absence of gross toxicities. ​ This syngeneic mouse model platform will additionally allow us to investigate how mCAR-T cells perform within a fully competent TME in combination with other therapeutic agents targeting cancer cells via altered mechanism of action. ​ Citation Format: Shannon Breen, Rosa Carrasco, Kelly McGlinchey, Brianna Janocha, Peter Zanvit, Lorenzo Ortiz, Benjamin Clark, Aleksandra Toloczko, Mark Cobbold, Gordon Moody, Emily Bosco, Allison Barrett. Utilization of CLDN18.2 syngeneic mouse models to study chimeric antigen receptor T cells in immunocompetent mice [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 1435.

Abstract 2913: Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model

Abstract Introduction: The tumor extracellular matrix (ECM) is a physical barrier which obstructs trafficking of CAR T cells to tumors. We hypothesize that co-expressing ECM remodeling/degrading enzymes heparanase (HPSE) or PH-20 with a CAR directed against folate receptor 1 (FolR1) will overcome the physical tumor microenvironment barrier and improve anti-tumor efficacy of anti-FolR1 CAR in a human ovarian cancer model. Methods: ECM CARs were constructed by combining anti-FolR1 ScFv with a CD8 hinge and transmembrane domain, 4-1BB, and CD3ζ endodomains, followed by a P2A element and full-length HPSE or GPI-anchored PH-20 sequence. CAR T cells were prepared from CD4+ and CD8+ T cells of healthy donors by lentiviral transduction. CAR expression was evaluated by flow cytometry; HPSE and PH-20 expression were measured via ELISA and Western blotting, respectively. CAR-mediated killing and cytokine release were observed in overnight co-culture assays with FolR1-expressing tumor cells. ECM CARs’ migration through CultrexTM and sodium hyaluronate matrices were assessed for HPSE and PH-20 function, respectively. ECM CARs’ efficacy was evaluated in vivo using a FolR1-expressing subcutaneous OV90 ovarian cancer xenograft model in NOD SCID Gamma (NSG) mice, and representative OV90 tumors were harvested at day 7 post-CAR T treatment for immunohistochemical staining. Results: FolR1 expression was confirmed in ovarian cancer cell lines and substrates targeted by HPSE (perlecan, glypican 3, CD138) and PH-20 (hyaluronan) were detected by immunofluorescence in human ovarian tumors’ sections. Lentiviral transduced primary T cells yielded robust expression of FolR1 CAR and ECM enzymes. Anti-FolR1 CAR and ECM CARs secreted pro-inflammatory cytokines IL-2, TNFα, IFNƳ and lysed OVCAR3 tumor cells with similar efficacy. ECM CARs with HPSE or PH-20 exhibited enhanced migration through ECM-rich matrices as compared to anti-FolR1 CAR alone. In vivo, all anti-FolR1 CAR T cells rejected OV90 tumors and extended survival compared to tumor alone or untransduced T cells (UTD). However, ECM CARs co-expressing HPSE or PH-20 accelerated tumor regression as compared to anti-FolR1 CAR alone. Tumor histology revealed CAR T cell infiltration (CD3+, CD8+) in mice treated with anti-FolR1 CARs but not UTD, indicating that anti-FolR1 CARs can effectively infiltrate OV90 tumors. Imaging studies are underway to evaluate the function of ECM CARs in the tumor microenvironment. Conclusions: The anti-FolR1 CARs boosted with ECM enzymes HPSE or PH-20 demonstrated specific lysis of FolR1-expressing cell lines, improved penetration through ECM-rich matrices in vitro, and accelerated tumor regression in an ovarian cancer model in vivo. Therefore, ECM CARs may help improve clinical outcomes in patients with ovarian tumors. Citation Format: Brittany L. Steimle, Peirong Hu, Ngoc Tran, Jia-Jye Lee, Kathleen Baysac, Kun Luo, Xu Wang, Genqing Liang, Saule Nurmukhambetova, Tri Tran, Pradyot Dash, Dina Schneider. Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model [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 2913.

Abstract 2057: Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay

Abstract The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon its generation via cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, it binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given its significant role as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. Credible evidence suggests that the cGAS–cGAMP–STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of Protein Complementation Assay (PCA) using nanoluciferase (nanoluc) complementation technology where a small peptide (SmBiT) derived from nanoluc linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. We have optimized the assay for higher sensitivity down to less than 10 nM cGAMP and less than one ng of cGAS and thus, and it is suitable for testing compounds that modulate cGAS activity. We also improved cell lysis condition to increase recovery of cGAMP that is generated intracellularly using several cGAS activators and correlating its concentration with cytokine release. We envision using this assay to monitor cGAMP presence in sera and spinal fluids of patients who have ALS and Alzheimer diseases. The assay is homogenous, with minimal or no false hits using LOPAC screening and HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, kevin Hsiao, Nate Murray, dareen Mikheil, Matt Larsen, Hui Wang. Monitoring cGAMP Biochemically and in cell using homogenous HTS formatted bioluminescent assay [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 2057.