Abstract Background: Despite improvements in outcome for patients treated with triplet and quadruplet based regimens, multiple myeloma is still a relapsing and ultimately fatal disease. We previously reported that targeting an investigational attenuated form of IFNα, or Attenukine™, to multiple myeloma (MM) tumor cells via direct fusion to an anti-CD38 antibody (TAK-573) has direct anti-proliferative activity on MM cancer cells in vitro and induces robust and durable responses in MM xenograft tumor models, alone and in combination with standard-of-care (SOC) agents. Here, we further elucidate the broad immunomodulatory impact and antitumor activity of a murine reactive version of TAK-573, anti-mouse CD38-attenuated murine IFNα (mCD38-mATT), alone and in combination with SOC agents in immunocompetent murine tumor models of MM. Methods: Murine CD38 receptor expression and direct sensitivity to murine IFNα (mIFNα) of 2 murine MM cell lines were characterized. Murine MM cell line-derived tumor models were established in immunocompetent mice and antitumor activity of mCD38-mATT treatment was evaluated alone and in combination with approved SOC agents for MM. Pharmacodynamic changes in the tumor immune microenvironment post-treatment were determined using multicolor flow cytometry. Results: Both MM murine tumor cell lines were insensitive to direct mIFNα treatment, indicating a reliance on immune-mediated antitumor activity when tested in vivo. Immunoprofiling analyses post-administration with single-agent mCD38-mATT indicated increased prevalence and activation of NK cells in peripheral blood and increased intratumoral CD8+ T: Treg and CD8+ T: CD4+ T cell ratios, compared to anti-mCD38 antibody or murine IFNα alone. Follow-on experiments evaluating single-agent mCD38-mATT indicated modest activity on tumor growth in vivo. Combination administration of mCD38-mATT with SOC agents had increased anti-tumor activity compared to either single-agent administration alone. Conclusion: Administration of mCD38-mATT induced broad immunomodulation and antitumor responses, alone and in combination with SOC agents in immunocompetent mouse models, highlighting the impact of this agent on CD38+ cells, both tumor and immune. These results support further investigation of combination therapies in the ongoing clinical evaluation of TAK-573 in a Phase 1 trial in patients with relapsed refractory MM. Citation Format: Tomoya Hara, Christina Wong, Jie Yu, Sabrina Collins, Haiqing Wang, Hiroshi Sugimoto, Hong Zhang, Pia Bjorck, Michael D. Curley. A murine reactive version of TAK-573 (anti-CD38 attenuated IFNα fusion protein) shows immunomodulatory and antitumor activity, alone and in combination with standard-of-care agents, in IFNa-insensitive, immunocompetent murine multiple myeloma tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5546.

Even with transformative therapies, multiple myeloma is a relapsing and ultimately fatal disease. We previously reported that targeting an attenuated form of IFNα, or Attenukine™, to MM tumor cells via direct fusion to an anti-CD38 antibody (TAK-573) has direct anti-proliferative activity on MM cancer cells in vitro and induces robust and durable responses in MM xenograft tumor models. Here we demonstrate the anti-tumor impact of TAK-573, alone and in combination with SOC agents in MM xenograft tumor models in vivo.

Despite recent improvements in survival outcome for multiple myeloma (MM) patients, most will eventually relapse and require additional treatment. We previously reported that targeting an attenuated form of IFNα, or Attenukine™, to MM tumor cells via direct fusion to an anti-CD38 antibody (TAK-573) has direct anti-proliferative activity on MM cancer cells in vitro and induces robust and durable responses in MM xenograft tumor models. Additional mechanism of action (MOA)-based analyses in MM xenograft tumor models pointed to a role for M1 macrophage and NK cells in propagating this anti-tumor response. Here we demonstrate the anti-tumor and broad immunomodulatory impact of a murine reactive version of TAK-573 in an immunocompetent murine lymphoma tumor model.

Abstract The success of immune checkpoint inhibitors validates the concept that immunotherapy is an effective approach for the treatment of solid tumors. In addition to reversing tumor-induced immune suppression, immune activating antibodies are being explored as the next generation of immuno-oncology therapeutics. CD40, which is expressed on antigen presenting cells such as dendritic and B cells initiates and regulates both innate and adaptive immunity and is essential for the activation of antigen-specific T cells. APX005M is a humanized IgG1 CD40 agonistic antibody developed using Apexigen's APXiMAB™ discovery platform. APX005M binds with high affinity to human (Kd = 0.12nM) and monkey (Kd = 0.37nM) CD40. It recognizes a unique epitope that overlaps with the CD40 ligand binding sites and thus blocks the binding of CD40 to CD40L. APX005M is a potent CD40 agonistic antibody capable of activating antigen presenting B cells (EC50 = 12pM) and dendritic cells (EC50 = 0.49nM). Its CD40 agonistic activity requires crosslinking by Fc-gamma receptors since F(ab)’2 fragment of APX005M loses the agonistic activity. Upon binding to CD40 expressing tumor cells APX005M induces antibody-dependent cellular phagocytosis (ADCP) and apoptosis. In vivo APX005M completely eradicates CD40+ lymphoma tumors and inhibits the growth of rituximab-resistant tumors. The data suggest that a CD40 agonistic mAb such as APX005M, that activates CD40 through binding to the CD40L binding site while being dependent on crosslinking via Fc-gamma receptors, may represent an ideal immune activating antibody drug candidate for stimulating effective immune responses against tumors. APX005M is currently being evaluated in clinical trials for the treatment of patients with solid tumors. Citation Format: Pia Björck, Erin Filbert, Xiaodong Yang, Ovidiu C. Trifan. APX005M, a humanized anti-CD40 antibody with strong immune-modulatory activities capable of tumor eradication in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5004.

Abstract Modulation of co-stimulatory and co-inhibitory molecules on immune cells has become a promising approach for cancer immunotherapy. CD40 engagement on antigen presenting cells (APCs) by its ligand CD154 leads to maturation and expression of co-stimulatory molecules such as CD80, CD86, OX-40L, and 4-1BBL that are requisite for optimal antigen-specific T-cell activation, an essential component of the anti-tumor immune response. To evaluate the therapeutic potential of targeting the CD40-CD154 pathway, Apexigen has developed APX005M - a humanized IgG1 CD40 agonistic antibody that binds with high affinity to human CD40. APX005M mimics CD154, has potent CD40 agonistic activity and promotes activation of APCs. Monocyte-derived dendritic cells treated with APX005M display a mature phenotype characterized by upregulation of CD80, CD86 and HLA-DR and increased secretion of IL-12. In mixed lymphocyte reaction and viral antigen recall assays, APX005M significantly enhances proliferation of and IFN-ã secretion from both naïve and memory T cells. Since CD40-CD154 signaling is involved in the priming phase of T cell activation and acts upstream of many critical costimulatory pathways, CD40 agonistic antibodies may represent an essential component of combination immunotherapy. In support of this, we show that APX005M synergizes with checkpoint inhibitors to promote antigen-specific T-cell responses. Importantly, in an ex vivo tumor assay APX005M induces T-cell proliferation and cytokine secretion. These data demonstrate that APX005M binds to APCs and induces their activation and maturation to ultimately drive a potent tumor-specific T-cell response. T cell activation in cancer patients receiving APX005M will be assessed in current and future clinical studies. Citation Format: Erin L. Filbert, Pia Björck, Xiaodong Yang, Ovidiu C. Trifan. The CD40 agonistic antibody APX005M ‘licenses’ antigen presenting cells to promote tumor-specific T-cell responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4867.

Meeting abstracts The co-stimulatory receptor CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and plays an important role in the control and regulation of immune activation, especially in crosstalk between T cells and antigen presenting cells (APCs). The natural ligand for

Background IDO1 inhibition with a 1st-generation inhibitor has shown promising clinical benefit in melanoma patients, as well as exacerbated toxicity, when combined with ipilimumab. We have identified a novel class of IDO1 inhibitors (IDO1i), which surpass the potency, selectivity and pharmacokinetic parameters of 1st-generation IDO1i. Methods IDO1 inhibition was assessed from kynurenine (kyn) levels produced by stimulated HeLa or dendritic cell cultures or recombinant IDO1. T-cell proliferation was measured using eFluor®670 dye. CT26 tumours were grown SC in Balb/c mice. IDO1i and kyn levels were measured by LC/MS/MS. Assessment of immune parameters was performed on tumour-infiltrating leucocytes by FACS. Findings These IDO1i potently inhibit cellular IDO1 with IC50 ≤ 10 nM, retain their potency in human serum (IC50 ≤ 10 nM) and restore T-cell proliferation, which is inhibited by allogeneic IDO1 + DC (EC50 = 2–5 nM). They exhibit PK characteristics consistent with once-daily dosing in humans. One of these molecules, selected for clinical evaluation, has undergone extensive characterisation, including the relationship between plasma levels of compound and kyn, and its impact on tumour growth and immune parameters in mouse syngeneic tumours, as single agent and in combination with various immune checkpoint inhibitors. Discussion This study provides the preclinical basis for clinical evaluation of a 2nd-generation IDO1i in combination with other immunotherapies. IDO1 inhibition with a 1st-generation inhibitor has shown promising clinical benefit in melanoma patients, as well as exacerbated toxicity, when combined with ipilimumab. We have identified a novel class of IDO1 inhibitors (IDO1i), which surpass the potency, selectivity and pharmacokinetic parameters of 1st-generation IDO1i. IDO1 inhibition was assessed from kynurenine (kyn) levels produced by stimulated HeLa or dendritic cell cultures or recombinant IDO1. T-cell proliferation was measured using eFluor®670 dye. CT26 tumours were grown SC in Balb/c mice. IDO1i and kyn levels were measured by LC/MS/MS. Assessment of immune parameters was performed on tumour-infiltrating leucocytes by FACS. These IDO1i potently inhibit cellular IDO1 with IC50 ≤ 10 nM, retain their potency in human serum (IC50 ≤ 10 nM) and restore T-cell proliferation, which is inhibited by allogeneic IDO1 + DC (EC50 = 2–5 nM). They exhibit PK characteristics consistent with once-daily dosing in humans. One of these molecules, selected for clinical evaluation, has undergone extensive characterisation, including the relationship between plasma levels of compound and kyn, and its impact on tumour growth and immune parameters in mouse syngeneic tumours, as single agent and in combination with various immune checkpoint inhibitors. This study provides the preclinical basis for clinical evaluation of a 2nd-generation IDO1i in combination with other immunotherapies.