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Purpose: mAbs blocking immune checkpoints have emerged as important cancer therapeutics, as exemplified by systemic administration of the IgG1 anti-CD47 mAb that blocks the “don't eat me” pathway. However, this strategy is associated with severe toxicity. Experimental Design: To improve therapeutic efficacy while reducing toxicities for ovarian cancer, we engineered an oncolytic herpesvirus (oHSV) to express a full-length, soluble anti-CD47 mAb with a human IgG1 scaffold (OV-αCD47-G1) or IgG4 scaffold (OV-αCD47-G4). Results: Both IgG1 and IgG4 anti-CD47 mAbs secreted by oHSV-infected tumor cells blocked the CD47–SIRPα signal pathway, enhancing macrophage phagocytosis against ovarian tumor cells. OV-αCD47-G1, but not OV-αCD47-G4, activated human NK-cell cytotoxicity and macrophage phagocytosis by binding to the Fc receptors of these cells. In vivo, these multifaceted functions of OV-αCD47-G1 improved mouse survival in xenograft and immunocompetent mouse models of ovarian cancer when compared with OV-αCD47-G4 and a parental oHSV. The murine counterpart of OV-αCD47-G1, OV-αmCD47-G2b, also enhanced mouse NK-cell cytotoxicity and macrophage phagocytosis and prolonged survival of mice bearing ovarian tumors compared with OV-αmCD47-G3. OV-αmCD47-G2b was also superior to αmCD47-G2b and showed a significantly better effect when combined with an antibody against PD-L1 that was upregulated by oHSV infection. Conclusions: Our data demonstrate that an oHSV encoding a full-length human IgG1 anti-CD47 mAb, when used as a single agent or combined with another agent, is a promising approach for improving ovarian cancer treatment via enhancing innate immunity, as well as performing its known oncolytic function and modulation of immune cells.

OV-αmCD47-G2b treatments promote macrophage M1 polarization and NK cell activation in ID8 immunocompetent ovarian cancer metastasis mouse model.

OV-Q1, OV-αCD47-G1 and OV-αCD47-G4 treatments increase immune cell infiltration in the ID8-hCD47 immunocompetent ovarian cancer metastasis mouse model.

The effect of αCD47-G1 on the expression of the genes markering macrophage polarization in vitro.

OV-Q1, OV-αmCD47-G2b and OV-αmCD47-G3 treatments increase immune cell infiltration in the ID8 immunocompetent ovarian cancer metastasis mouse model.

Schematic map of OV-αCD47 used in this study.

Primers used in this study.

The effect of αCD47-G1 or αCD47-G4 on the expression of NK cell markers.

OV-αCD47-G1 treatments promote macrophage M1 polarization and NK cell activation in ID8-hCD47 immunocompetent ovarian cancer metastasis mouse model.

OV-αCD47-G1 improves the therapeutic efficacy against ovarian cancer in a xenograft model.

Anti-viral immune responses after OV-αmCD47-G2b.

OV-αmCD47-G2b is efficacious to treat ovarian cancer in an ID8 immunocompetent metastasis mouse model.

The concentration of αmCD47 antibody in the bloodstream and at the local tumor sites.

OV treatments increase NK cell and T cell infiltration in the ID8-hCD47 immunocompetent ovarian cancer metastasis mouse model.

OV-αCD47-G1 enhances the therapeutic efficacy in an immunocompetent ovarian cancer metastasis mouse model.

All supplementary data: supplementary figures s1-s13 and table s1

Purpose:mAbs blocking immune checkpoints have emerged as important cancer therapeutics, as exemplified by systemic administration of the IgG1 anti-CD47 mAb that blocks the “don't eat me” pathway. However, this strategy is associated with severe toxicity.Experimental Design:To improve therapeutic efficacy while reducing toxicities for ovarian cancer, we engineered an oncolytic herpesvirus (oHSV) to express a full-length, soluble anti-CD47 mAb with a human IgG1 scaffold (OV-αCD47-G1) or IgG4 scaffold (OV-αCD47-G4).Results:Both IgG1 and IgG4 anti-CD47 mAbs secreted by oHSV-infected tumor cells blocked the CD47–SIRPα signal pathway, enhancing macrophage phagocytosis against ovarian tumor cells. OV-αCD47-G1, but not OV-αCD47-G4, activated human NK-cell cytotoxicity and macrophage phagocytosis by binding to the Fc receptors of these cells. In vivo, these multifaceted functions of OV-αCD47-G1 improved mouse survival in xenograft and immunocompetent mouse models of ovarian cancer when compared with OV-αCD47-G4 and a parental oHSV. The murine counterpart of OV-αCD47-G1, OV-αmCD47-G2b, also enhanced mouse NK-cell cytotoxicity and macrophage phagocytosis and prolonged survival of mice bearing ovarian tumors compared with OV-αmCD47-G3. OV-αmCD47-G2b was also superior to αmCD47-G2b and showed a significantly better effect when combined with an antibody against PD-L1 that was upregulated by oHSV infection.Conclusions:Our data demonstrate that an oHSV encoding a full-length human IgG1 anti-CD47 mAb, when used as a single agent or combined with another agent, is a promising approach for improving ovarian cancer treatment via enhancing innate immunity, as well as performing its known oncolytic function and modulation of immune cells.

Integrins have long been sought as therapeutic targets in cancer, but there are no FDA approved drugs to target this gene family. Since integrins require the dimerization between an alpha and beta subunit to function, we developed a CRISPR library targeting all 26 alpha and beta subunits to identify which pair is the most important. We found a strong correlation between ITGAV and ITGB5 (Integrin Beta 5), which form a known heterodimer, in solid tumors. CRISPR targeting either ITGAV or ITGB5 led to attenuated cell proliferation with increased cell cycle stalling and apoptosis. As the more essential partner, we designed a CRISPR tiling scan targeting the entire coding region of ITGAV and determine the essential nature of each amino acid. We found a sub-domain within the beta propeller to be important for ITGAV function, the CRISPR identified pocket (CIP). Genetic targeting of this pocket disrupted the ability to detect the heterodimer signal of ITGAV-ITGB5 by a specific antibody. Furthermore, we confirmed that essential amino acids within the CIP responsible for the heterodimer stability by the NanoBRET energy transfer system. Knowing the role of the CIP in heterodimer stability we searched for a small molecule to bind in the CIP to phenocopy our genetic findings. A combined in-silico and in-vitro high throughput drug screen started tested compounds for cell killing and disruption of the ITGAV-ITGB5 heterodimer. “Compound 2” (cpd_2) showed both strong cell killing and dissociation of the heterodimer. Disruption was measured by detection by flow cytometry and through the NanoBRET system. Cpd_2’s binding was confirmed with thermal shift studies with purified ITGAV beta propeller, validating that this compound is behaving and binding as we expect at the CIP. Next, we sought to understand how the ITGAV-ITGB5 heterodimer controls cancer cell survival. From the Dependency Map Public database (DepMap) we found that Rac1 dependency is significantly correlated only with ITGAV and ITGB5. We hypothesized that this meant that ITGAV-ITGB5 regulated Rac1 activity. Loss of Rac1 phenocopies knock out of ITGAV or ITGB5 and loss of either Rac1 or ITGAV led to dysregulation of the actin cytoskeleton, a known downstream feature of Rac1. A constitutively Rac1 (CA_Rac1) rescues the effects of targeting of ITGAV or treatment with cpd_2. Altogether this data suggests that ITGAV-ITGB5 signals to Rac1 and that attenuation of their function can be reversed by an active Rac1 In summary, our study highlights a novel strategy of targeting the integrin heterodimer ITGAV-ITGB5 with a small molecule binding to a novel subdomain, CIP, of ITGAV. The CIP is responsible for the heterodimer stability of ITGAV and ITGB5. Disruption of this heterodimer led to the dysregulation of Rac1 and destabilization of the actin cytoskeleton. This mechanism highlights a novel strategy for targeting integrins as a therapeutic target in cancer. Citation Format: Nicole Mattson, Kazuya Miyashita, Anthony Chan, Lu Yang, Sheela Pangeni Pokharel, Wei Liu, Ming li, Qiao Liu, Xiaobao Xu, Priyanka Singh, Leisi Zhang, Mingye Feng, Chun-Wei Chen. Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression [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 6234.

The cell surface proteome provides a pivotal interphase that connects the intracellular signaling with external environments, which may harbor molecular targets for therapeutic development. To identify essential cell surface proteins in cancers, we established a CRISPR library that contains 2,910 sgRNAs targeting 582 genes encoding cell surface receptors (to cytokines, extracellular matrices [ECM], etc.), molecular channels, and signaling components. We then screened this library in 3 solid cancer (MDA-MB-231, SW620, PANC1) and 3 liquid cancer (MV4-11, HUT78, CCRF-CEM) cell models expressing the Cas9 endonuclease, and observed an essential role of ITGAV (Integrin Alpha V) specifically in the solid cancer. As integrins require the dimerization between an alpha and beta subunit to function as cell surface ECM receptors, we further developed a focused CRISPR library with 650 sgRNAs targeting all 26 integrin alpha and beta subunits. This validation screen revealed a strong correlation between ITGAV and ITGB5 (Integrin Beta 5) in solid tumors. CRISPR targeting either ITGAV or ITGB5 led to attenuated cell proliferation with increased cell cycle stalling and apoptosis. Knockout of ITGAV-ITGB5 also suppressed the capacity of tumor cells to migrate and invade through the ECM, highlighting the therapeutic benefit via blocking the ITGAV-ITGB5 heterodimer. To identify novel compounds that can disrupt ITGAV-ITGB5 dimerization, we first modeled a 3D crystal structure of ITGAV (PDB ID: 3IJE) using AutoSite (Scripps) to reveal the protein surface pockets amenable to the small molecular binding. This analysis revealed an AVB pocket at the N-terminal extracellular domain of ITGAV, which is in close contact with the beta subunit. CRISPR targeting this AVB pocket abolished the ITGAV-ITGB5 heterodimers (detected by an AV-B5-complex antibody) and induced rapid cell death, rationalizing this pocket for further therapeutic development. Based on these observations, we computationally docked >100,000 compounds from the NCI DTP Open Chemicals Repository using AutoDock Vina (Scripps), subsequently obtaining the top 500 binder compounds for in-lab validation, and identified a lead AVB pocket inhibitor “Cpd_2” (C32H40N4). Treatment of Cpd_2 killed tumor cells and diminished the AV-B5-complex antibody stain. Furthermore, we confirmed the capacity of Cpd_2 to dissociate ITGAV and ITGB5 on the cell surface by a NanoBRET energy transfer system. Of note, while the traditional ITGAV inhibitor cilengitide (an RGD peptide mimic) blocked the ECM interaction and detached the cells from TC dishes, they failed to dissociate the ITGAV-ITGB5 heterodimer nor induce rapid cell death. In summary, our study highlights a novel strategy of targeting the ITGAV-ITGB5 heterodimer stability by the small molecular inhibitor Cpd_2 for future anti-tumor treatment. Citation Format: Nicole Mattson, Kazuya Miyashita, Anthony Chan, Lu Yang, Sheela Pangeni Pokharel, Wei Lu, Mingli Li, Qiao Liu, Xiaobao Xu, Mingye Feng, Chun-Wei Chen. Targeting integrin alpha V beta 5 heterodimer stability using a novel small molecular inhibitor for tumor suppression [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 2920.

Immune checkpoint (IC) blockade for programmed cell death 1 (PD1) and PD-Ligand 1 (PD-L1) has shown promising and durable therapeutic outcomes in cutaneous T-cell lymphoma (CTCL), but resistance and/or relapses are common. While research focused on improving adaptive immune functions in CTCL, we investigated the role of 1) the “do not eat me signal” (CD47), and PD-L1, as a dual (innate and adaptive IC)-targeting strategy in CTCL. RNA sequencing and flow cytometric analysis showed that CTCL tumor cells and CTCL cell lines (MyLa, Hut78, HH, and H9 cells) overexpressed CD47 and PD-L1 compared with healthy control, and CD47 positively correlated with PD-L1 in CTCL patients. Overexpression of CD47 and PD-L1 was induced by high MYC expression in CTCL tumor cells lines. In addition, the signal-regulatory protein (SIRP)α receptor for CD47 and PD-L1 were significantly more abundant on macrophages, dendritic cells and natural killer cells in CTCL patients than healthy control by flow cytometric analysis. Furthermore, the RNA sequencing data indicated that the M2 macrophages, immature dendritic cells, and inhibitory receptors expressed natural killer cells in CTCL patients were higher than in healthy control. Notably, TTI-621 (SIRPαFc) treatment decreased M2 macrophage, immature dendritic cells, and inhibitory receptors expressed natural killer cells in CTCL patients at the end of the treatment, compared to baseline. In vitro, TTI-621 treatment increased the macrophage phagocytic activity compared to untreated control. Moreover, TTI-621 synergized with an anti-PD-L1 antibody (durvalumab) to reprogram M2-like TAMs, induced by MyLa supernatant, to M1-like phenotypes. The simultaneous blockade of both CD47 and PD-L1 inhibited growth of CTCL cell lines more potently than each single antibody alone or blank control in vitro. Enhanced CD8+ T cell mediated killing was detected using a chromium-release assay following dual blockade of in CTCL cell lines, suggesting anti-CD47 and anti-PD-L1 may synergistically facilitate elimination of CTCL tumor cells through specific pathways. RNA-sequencing analysis indicated that these effects were mediated by cell death related pathways such as apoptosis, autophagy, and necroptosis. Collectively, our findings demonstrated that CD47 and PD-L1 are critical regulators of innate and adaptive immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy to improve tumor control in CTCL. Citation Format: Zhen Han, Mingye Feng, Xiwei Wu, Chingyu Su, Yate-Ching Yuan, Hanjun Qin, James F. Sanchez, Jasmine Zain, Steven T. Rosen, Christiane Querfeld. CD47 Blockade potentiates immunotherapy of durvalumab against cutaneous T cell lymphoma [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 5196.