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.

3595 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 generally been unsuccessful due to toxicity or lack of efficacy. We have developed a multivalent IgM DR5 agonist, IGM-8444, that multimerizes DR5 to selectively and potently induce tumor cell apoptosis while maintaining tolerability. Methods: IGM-8444 is an engineered, pentameric IgM antibody with 10 binding sites specific for DR5. Human tumor cell lines or hepatocytes were evaluated in vitro for dose dependent IGM-8444 induced cytotoxicity. The efficacy of IGM-8444 was evaluated with or without chemotherapy, in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse tumor models, with IGM-8444 administered at various dose levels and schedules when tumors reached approximately 100 mm3. Sera and tumors were analyzed for biomarkers of tumor apoptosis. Results: In vitro cytotoxicity assays identified IGM-8444 activity across cell lines from 18 solid and hematologic malignancies. In IGM-8444 partially resistant cell lines, combination with chemotherapy or a Bcl2 inhibitor enhanced in vitro cytotoxicity. IGM-8444 was efficacious as a monotherapy in CDX and PDX tumor models including colorectal, lung, and gastric indications. In a gastric PDX model, IGM-8444 induced complete and durable dose-dependent tumor regressions. In vivo, combination of IGM-8444 with standard-of-care chemotherapies, such as irinotecan, led to enhanced efficacy. IGM-8444 administration increased markers of tumor apoptosis, identifying potential clinical pharmacodynamic biomarkers. At doses several log-fold higher than efficacious doses, IGM-8444 demonstrated a favorable single agent in vitro safety profile, with little to no in vitro cytotoxicity observed using primary human hepatocytes from multiple donors. Conclusions: These data support the clinical development of IGM-8444 in both solid and hematological malignancies as a single agent and in combination with standard of care therapy. IGM-8444 is projected for IND filing in 2020.

The structural dynamics of two nucleosomes, one was H3 containing and other was CENP-A containing nucleosome, were characterized with molecular dynamics simulations and the findings were experimentally confirmed. The simulations showed that histone proteins of both, which is the core structure of nucleosome, are structurally stable and maintain the structure determined by x-ray crystallography, while the wrapped DNAs are highly flexible at the entry or exit region and largely deviate from the crystal structures. In particular, about 20-25 bp DNAs of entry or exit of the CENP-A containing nucleosomes showed several times of open and close conformational changes within 100ns simulations, which was not observed on the H3 containing nucleosomes. The detailed analysis clarifies that this dynamics difference is due to the difference in two basic amino acids at the a-N helix, two Arg residues of H3 are mutated to Lys residue at the corresponding sites. The difference in ability of forming hydrogen-bond to the DNA controls the flexibility of the nucleosomal DNA at entry or exit region. This increase in flexibility was confirmed with a nuclease susceptibility assay of a nucleosome that contains H3 mutant with the two Arg residues replaced with Lys.

Immunostimulatory cytokines are a promising immunotherapy for the treatment of advanced malignancies, but generally have been associated with severe toxicities when administered systemically. The recent development of antibody-cytokine fusion proteins, or immunocytokines, aims to localize cytokine activity to the tumor microenvironment and thus improve their therapeutic index. We have developed IGM-7354, a high affinity, high avidity anti-PD-L1 pentameric IgM antibody with an IL-15Rα chain and IL-15 fused to the joining (J) chain. The IGM-7354 immunocytokine was designed to deliver IL-15-mediated stimulation of NK and CD8+ T cells to PD-L1-expressing tumors and antigen-presenting cells, to enhance anti-tumor immune responses. The multivalent binding of IGM-7354 to PD-L1 provided a stronger binding avidity for human PD-L1 than the monovalent binding of IL-15 to IL-15Rb as confirmed in kinetic binding assays. In vitro IGM-7354 induced the proliferation of a cytotoxic T cell line responsive to IL-15 stimulation and enhanced the proliferation of NK and CD8+ T cells from healthy donor human PBMCs. In cytotoxicity assays with human PBMC and PD-L1+ cancer cell lines, IGM-7354 enhanced cancer cell killing through NK and CD8+ T cell expansion and cytotoxic activity, evidenced by Ki67 and Granzyme B upregulation in these cell populations. Next, in vivo pharmacodynamic studies were performed in two humanized mouse models: non-tumor-bearing BRGSF-HIS mice engrafted with human CD34+ cells, and PD-L1+ MDA-MB-231 tumor-bearing MHC-/- NSG mice engrafted with human PBMCs. In the BRGSF model, IGM-7354 increased NK cell activation and Granzyme B expression as well as NK and CD8+ T cell proliferation. In the tumor-bearing mouse model, IGM-7354 dose-dependently increased NK and CD8+ T cell proliferation in blood and infiltration of lymphocytes into the tumor. This pharmacodynamic activity correlated with IGM-7354 anti-tumor activity in the MDA-MB-231 model. Lastly, IGM-7354 increased the proliferation of NK and CD8+ T cells in cynomolgus monkeys and particularly induced the expansion of effector memory CD8+ T cells in the periphery. In summary, IGM-7354 induces NK and CD8+ T cell proliferation in both in vitro and in vivo preclinical models, resulting in the killing of PD-L1+ tumor cells. The strong avidity of IGM-7354 for PD-L1 may enhance IL-15 delivery to tumors and antigen-presenting cells and thus provide a more favorable safety profile. A Phase 1 clinical trial is planned. Citation Format: Thierry D. Giffon, Melanie Desbois, Poonam Yakkundi, Susan Calhoun, Keerthana Sekar, Carolyn Denson, Tasnim Kothambawala, Alexander Pearson, Sivani Pandey, Deepal Pandya, Rodnie Rosete, Daniel Machado, Pat Raichlen, Dean Ng, Abhinav R. Jain, Roel Funke, Eric Humke, Paul R. Hinton, Beatrice Wang, Bruce A. Keyt, Maya F. Kotturi, Angus M. Sinclair. IGM-7354, an immunocytokine with IL-15 fused to an anti-PD-L1 IgM, induces NK and CD8+ T cell mediated cytotoxicity of PD-L1-positive tumor cells. [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 5660.