Death receptor 5 (DR5) is an attractive target for cancer therapy due to its broad upregulated expression in multiple cancers and ability to directly induce apoptosis. Though anti-DR5 IgG antibodies have been evaluated in clinical trials, limited efficacy has been attributed to insufficient receptor crosslinking. IGM-8444 is an engineered, multivalent agonistic IgM antibody with 10 binding sites to DR5 that induces cancer cell apoptosis through efficient DR5 multimerization. IGM-8444 bound to DR5 with high avidity and was substantially more potent than an IgG with the same binding domains. IGM-8444 induced cytotoxicity in a broad panel of solid and hematologic cancer cell lines but did not kill primary human hepatocytes in vitro, a potential toxicity of DR5 agonists. In multiple xenograft tumor models, IGM-8444 monotherapy inhibited tumor growth, with strong and sustained tumor regression observed in a gastric PDX model. When combined with chemotherapy or the BCL-2 inhibitor ABT-199, IGM-8444 exhibited synergistic in vitro tumor cytotoxicity and enhanced in vivo efficacy, without augmenting in vitro hepatotoxicity. These results support the clinical development of IGM-8444 in solid and hematologic malignancies as a monotherapy and in combination with chemotherapy or BCL-2 inhibition.

Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that activates the extrinsic apoptotic pathway when bound and multimerized by its ligand, TNF-related apoptosis inducing ligand (TRAIL). DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have been unsuccessful due to lack of efficacy or due to hepatotoxicity. We have developed IGM-8444, an engineered pentameric IgM with 10 binding sites specific for DR5, which is designed to multimerize DR5 to selectively and potently induce tumor cell apoptosis while sparing hepatocytes. Here, we describe the rationale behind the selection of IGM-8444 as our clinical candidate. A panel of agonistic DR5 antibodies were evaluated for DR5 binding affinity, epitope, and in vitro potency versus hepatotoxicity. Antibodies formatted as an IgM showed enhanced potency when compared to an IgG with the same binding domain. IGM-8444 binds an epitope on DR5 within cysteine-rich domain 1 (CRD1) that competes with TRAIL binding. While the binding affinities of the panel of anti-DR5 antibodies were comparable, IGM-8444 was selected from a subset of anti-DR5 IgM antibodies capable of potently killing tumor cells without exhibiting cytotoxicity of primary human hepatocytes in vitro. Further mechanistic studies examined the kinetics of apoptotic induction by IGM-8444 and other DR5 agonists. Interestingly, we noted that DR5 agonists with the fastest kinetics of tumor cell apoptotic induction also displayed the most hepatotoxicity in vitro. In spite of the kinetic differences, IGM-8444 has similar maximal cytotoxicity in vitro and comparable anti-tumor efficacy in xenograft mouse tumor models when compared with an IgM antibody targeting a different DR5 epitope. In cynomolgus monkeys, IGM-8444 showed no evidence of hepatotoxicity or other adverse events when dosed repeatedly up to 30 mg/kg, the highest dose tested. These preclinical properties of IGM-8444 provide an opportunity for enhanced tumor cytotoxicity without additional hepatotoxicity when combined with standard of care agents. Indeed, we have demonstrated enhanced anti-tumor efficacy by combining IGM-8444 with chemotherapies such as 5-FU and irinotecan in colorectal cancer models, as well as combining with Bcl-2 inhibitor ABT-199 in hematological malignancy models. In summary, we have evaluated the mechanism by which IGM-8444 agonizes DR5, which potently kills tumor cells without accompanying hepatotoxicity. IGM-8444 is currently being evaluated in a Phase 1 study as a single agent and in combination with chemotherapy-based regimens in patients with solid cancers and NHL (NCT04553692). Citation Format: Beatrice T. Wang, Tasnim Kothambawala, Kevin C. Hart, Xingjie Chen, Melanie Desbois, Susan E. Calhoun, Poonam Yakkundi, Rodnie A. Rosete, Yuan Cao, Katie Cha, Thomas J. Matthew, Ling Wang, Paul R. Hinton, Maya K. Leabman, Genevive Hernandez, Maya F. Kotturi, Eric W. Humke, Angus M. Sinclair, Bruce A. Keyt. Mechanistic evaluation of anti-DR5 IgM antibody IGM-8444 with potent tumor cytotoxicity, without in vitro hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 52.

Death receptor 5 (DR5) is a tumor necrosis factor receptor (TNF) superfamily member that requires multimerization to activate the extrinsic apoptotic pathway and is broadly expressed on solid and hematologic cancers. IGM-8444 is a multivalent IgM DR5 agonist that efficiently multimerizes DR5 to induce tumor cell apoptosis while maintaining a favorable in vitro and in vivo safety profile. The universal mechanism of apoptotic cell death and the safety profile of IGM-8444 makes it an attractive combination partner with standard of care treatment regimens. Here we describe the characterization of single agent and combinatorial cytotoxicity with different classes of chemotherapeutic agents. IGM-8444 was first evaluated as a monotherapy across a panel of human solid tumor cell lines in vitro and xenograft tumor models in vivo. IGM-8444 responses ranged from highly sensitive to resistant. In a sensitive Colo205 model IGM-8444 additionally showed rapid intratumoral pharmacodynamic (PD) activity, inducing maximal caspase-3 cleavage at 6 hours post-dose. Next a panel of solid tumor (including colorectal, gastric, non-small cell lung cancer, and pancreatic) cell lines were selected to evaluate IGM-8444 in combination with standard of care chemotherapeutic agents. Synergistic cytotoxicity was observed when IGM-8444 was used in combination with certain classes of chemotherapeutic agents including topoisomerase inhibitors, microtubule inhibitors, nucleoside analogs, and platinum-based agents. Enhanced anti-tumor activity was also observed in xenograft mouse models where IGM-8444 was dosed in combination with these classes of chemotherapeutic compounds. Mechanistically, this synergistic combinatorial cytotoxicity may be explained by the reported increase in DR5 expression on tumor cells following treatment with many of these classes of chemotherapeutic agents. In summary, IGM-8444 shows in vitro cytotoxicity and in vivo PD and anti-tumor efficacy responses in preclinical models, with enhanced activity in combination with several classes of chemotherapies reported to upregulate DR5 expression. IGM-8444 combination with FOLFIRI standard of care is currently under evaluation in a Phase 1 study in patients with metastatic colorectal cancer (NCT04553692). Citation Format: Beatrice T. Wang, Thomas J. Matthew, Poonam Yakkundi, Miho Oyasu, Mélanie Desbois, Susan E. Calhoun, Ling Wang, Tasnim Kothambawala, Devinder K. Ubhi, Marvin S. Peterson, Eric W. Humke, Maya F. Kotturi, Bruce A. Keyt, Angus M. Sinclair. Characterization of the synergistic tumor cytotoxicity of agonistic DR5 IgM antibody IGM-8444 with chemotherapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6123.

Background: Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that multimerizes when bound to its ligand, TNF-related apoptosis inducing ligand (TRAIL), to activate the extrinsic apoptotic pathway. DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have been unsuccessful due to lack of efficacy or hepatotoxicity. We have developed IGM-8444, a pentameric IgM with 10 binding sites specific for DR5, that multimerizes DR5 to selectively and potently induce tumor cell apoptosis while maintaining tolerability. We have previously presented the in vitro and in vivo efficacy of IGM-8444 in solid tumor models, demonstrating low picomolar potency across multiple tumor cell lines, strong tumor regressions in cell line and patient derived xenograft mouse tumor models, and dose-dependent increases in apoptotic biomarkers. Here, we evaluate the activity of IGM-8444 in hematologic malignancies in combination with chemotherapy or targeted agents including Bcl-2 inhibitors targeting the intrinsic apoptotic pathway. Methods: Human hematologic cancer cell lines and primary human hepatocytes were evaluated in vitro for dose-dependent IGM-8444-induced cytotoxicity. Cell lines were further evaluated using IGM-8444 in combination with chemotherapy or targeted agents including Bcl-2 inhibitor ABT-199. In vivo efficacy was evaluated using IGM-8444 in combination with ABT-199 in cell line-derived xenograft mouse tumor models. Results: In a previous cancer cell line screen profiling single agent IGM-8444 cytotoxicity across 190 solid and hematologic cell lines, 25 (13%) were classified as highly responsive and 75 (39%) as moderately responsive to IGM-8444 induced cell death. Here the in vitro activity of IGM-8444 was evaluated across a subset of 32 NHL and AML cell lines. 5/21 (24%) of NHL cell lines and 5/11 (45%) of AML cell lines tested were classified as highly responsive or moderately responsive to IGM-8444-induced cytotoxicity. The DOHH-2 and JEKO1 NHL cell lines were amongst the most sensitive, with growth-normalized EC50 values as low as 0.03 ng/mL (0.03 pM) for JEKO1. Combinations with chemotherapy including cytarabine and doxorubicin or targeted agents such as Bcl-2 inhibitor ABT-199 resulted in synergistic in vitro cytotoxicity in multiple cell lines, as determined by Bliss synergy scores. IGM-8444 demonstrated minimal to no in vitro cytotoxicity to primary human hepatocytes at doses several log-fold higher than efficacious doses, and this favorable in vitro safety profile was maintained in combination with chemotherapeutic agents and ABT-199. Combination of IGM-8444 with ABT-199 also resulted in synergistic in vivo efficacy. In a DOHH-2 NHL model, IGM-8444 and ABT-199 showed modest tumor growth inhibition as single agents. However the combined treatment regimen led to tumor regressions during the first 2 weeks of treatment, with 3 of 10 animals showing a partial response and 2 of 10 animals achieving a complete response. The combined treatment also extended median overall survival compared to the control group, which was a significant improvement compared to either agent alone. Collectively, these results provide a strong rationale for simultaneously targeting the extrinsic and intrinsic apoptotic pathways to achieve enhanced efficacy. Conclusions: These data support the clinical development of IGM-8444 in hematological malignancies as a single agent, in combination with standard of care chemotherapy, and in combination with targeted agents that impact the intrinsic signaling pathway such as Bcl-2 inhibitor ABT-199. Initiation of a Phase I clinical study evaluating the safety of IGM-8444 is anticipated in 2020. Disclosures Wang: IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Matthew:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Wang:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Kothambawala:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Calhoun:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Humke:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Sinclair:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company. Keyt:IGM Biosciences Inc: Current Employment, Current equity holder in publicly-traded company.

Apoptosis is induced through extrinsic and intrinsic signaling pathways. Extrinsic apoptosis can be activated through multimerization of death receptor 5 (DR5), a tumor necrosis factor (TNF) receptor family member highly expressed in many cancers. However, cellular resistance mechanisms within the intrinsic pathway may limit DR5 activity, including inhibitor of apoptosis proteins (IAPs) that block caspase activity or promote pro-survival NFκB signaling. Second mitochondria-derived activator of caspases (SMAC) is an endogenous bivalent IAP antagonist. Birinapant, a bivalent SMAC mimetic that binds and degrades IAPs, has been evaluated through Phase 2, demonstrating good safety and on target activity but minimal efficacy as a monotherapy. We hypothesized that simultaneously targeting the extrinsic apoptotic pathway with IGM-8444, an anti-DR5 multivalent IgM agonist, and the intrinsic apoptotic pathway with birinapant could enhance tumor cell apoptosis. Human cancer cell lines including triple negative breast cancer (TNBC), head and neck, ovarian, colorectal, lung, and sarcomas, were screened for sensitivity to IGM-8444 and birinapant combination. Strong synergistic cytotoxicity was observed in vitro in 36/45 (80%) cancer cell lines, as measured by both Bliss synergy score and maximal killing. IGM-8444 and birinapant combination also induced synergistic cytotoxicity in cells with acquired resistance to DR5 agonist antibodies. By contrast, IGM-8444 and birinapant did not kill primary human hepatocytes in vitro, demonstrating the potential clinical safety for this combination. In vivo, the IGM-8444 and birinapant combination dose-dependently reduced tumor growth in a MDA-MB-231 TNBC model, with 8/10 tumor-free mice at the highest dose of birinapant tested. By comparison, birinapant combination with an anti-DR5 IgG agonist showed a modest response. IGM-8444 and birinapant also showed significant anti-tumor responses in additional cell line and patient-derived xenograft models, including 7/9 tumor-free animals in a HT-1080 fibrosarcoma model and 9/10 complete responses in a EBC-1 lung squamous cell lung carcinoma model. Lastly, pharmacodynamic biomarkers including cIAP1 degradation, caspase activation, and caspase-cleaved cytokeratin 18 in tumor and serum correlated with anti-tumor response. In summary, combined targeting of the extrinsic and intrinsic apoptotic pathways with IGM-8444 and birinapant respectively enhances tumor cytotoxicity in multiple preclinical models. The combination of IGM-8444 with birinapant is currently under evaluation in a Phase 1 study in patients with relapsed and/or refractory solid cancers (NCT04553692). Citation Format: Beatrice T. Wang, Melanie Desbois, Susan E. Calhoun, Thomas J. Matthew, Poonam Yakkundi, Ling Wang, Xingjie Chen, Tasnim Kothambawala, Miho Oyasu, Maya F. Kotturi, Genevive Hernandez, Xiaohan Liu, Marvin S. Peterson, Eric W. Humke, Bruce A. Keyt, Angus M. Sinclair. Anti-DR5 agonist IgM antibody IGM-8444 combined with SMAC mimetic birinapant induces strong synergistic tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1068.