Maria M. Dasovich

Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice

T cell–derived cytokines are important in the development of an effective immune response, but when dysregulated they can promote disease. Here we identify a four-helix bundle cytokine we have called interleukin 31 (IL-31), which is preferentially produced by T helper type 2 cells. IL-31 signals through a receptor composed of IL-31 receptor A and oncostatin M receptor. Expression of IL-31 receptor A and oncostatin M receptor mRNA was induced in activated monocytes, whereas epithelial cells expressed both mRNAs constitutively. Transgenic mice overexpressing IL-31 developed severe pruritis, alopecia and skin lesions. Furthermore, IL-31 receptor expression was increased in diseased tissues derived from an animal model of airway hypersensitivity. These data indicate that IL-31 may be involved in promoting the dermatitis and epithelial responses that characterize allergic and non-allergic diseases.

MOR209/ES414, A Novel Bispecific Antibody Targeting PSMA For The Treatment of Metastatic Castration-Resistant Prostate Cancer

Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ϵ to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155–65. ©2016 AACR.

Abstract 597: Bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecules for redirected T-cell cytotoxicity in hematological malignancies

Introduction: CD123 is a component of the IL-3 receptor expressed in several hematological malignancies including AML, ALL, HCL, and MDS. CD123 is a compelling target in AML due to its overexpression on AML blasts as well as leukemic stem cells, which are thought to be resistant to chemotherapy and may be responsible for relapse of disease following treatment. While CD123 is expressed by some normal leukocyte populations in circulation and hematopoietic progenitor cells in the bone marrow, the low frequency of expression on normal cell types provides a therapeutic window for targeting CD123 in tumor settings with the potential for durable response and reversible side effects. We have developed bispecific anti-CD123 x anti-CD3 ADAPTIR molecules APVO436 and APVO437 for redirecting T-cell cytotoxicity to CD123-expressing tumor cells. Results are presented that examine the in vitro and in vivo activity of these molecules in preclinical models of AML. Methods: APVO436 and APVO437 proteins were expressed in CHO cells. Affinity SPR studies were performed using recombinant CD123-ectodomain. In vitro functional studies were conducted with CD123+ AML tumor cell lines and primary human and cynomolgus macaque T-cell populations. Cytotoxic activity was determined using chromium release assays. On-cell binding, T-cell activation and proliferation were assessed using multi-color flow cytometry. Pharmacokinetic parameters were determined in BALB/c mice using a single IV dose of approximately 10 mg/kg. In vivo studies to examine tumor growth inhibition activity were performed with NOD/SCID mice co-implanted subcutaneously with AML tumor cells and human T-cells followed by treatment with APVO436 or APVO437. Tumor growth was assessed by measuring tumor volume and Bioluminescent Imaging. Results: APVO436 and APVO437 bound human CD123 protein with high affinity and binding to CD123 and CD3 expressing cell lines was confirmed by flow cytometry. Both APVO436 and APVO437 induced concentration-dependent lysis of CD123+ AML cell lines with primary human effector T-cells, accompanied by T-cell activation and proliferation. Comparable redirected T-cell cytotoxicity function was observed using primary cynomolgus macaque T cells. These activities were dependent on the expression of CD123 by the tumor target cells. APVO436 and APVO437 demonstrated an extended elimination half-life in mouse serum, typical of molecules capable of binding the neo-natal Fc receptor. In vivo, growth of AML tumor cells was inhibited by treatment with low doses of APVO436 and APVO437, significantly improving host survival. Conclusion: Taken together these data demonstrate potent in vitro and in vivo activity of APVO436 and APVO437 against CD123 expressing tumor cells and are supportive of further investigation of this approach as a potential treatment option for AML and other hematological malignancies. Citation Format: Michael R. Comeau, Danielle Mitchell, Rebecca Gottschalk, Lynda Misher, Mollie Daugherty, Lara Parr, Peter Pavlik, Brian Woodruff, Hang Fang, Megan Aguilar, Jeannette Bannink, Starrla Johnson, Gary Li, Robert E. Miller, Robert Bader, Nicole Zhang, Toddy Sewell, Maria Dasovich, Gabriela H. Hoyos, John W. Blankenship, Catherine McMahan, David Bienvenue, Jane A. Gross. Bispecific anti-CD123 x anti-CD3 ADAPTIR™ molecules for redirected T-cell cytotoxicity in hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2017-597

Abstract 4995: anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer

Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC. Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume. Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival. Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies. Citation Format: John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer. [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 4995.