The antiangiogenic protein endostatin showed considerable preclinical antitumor activity, but limited efficacy in phase I/II trials. Prior studies using an anti-HER2 antibody-murine endostatin fusion showed enhanced antitumor activity compared to anti-HER2 antibody or endostatin given alone, or in combination. We have generated two anti-HER2 human endostatin fusion proteins by fusing either wildtype or a mutant human endostatin (huEndo-P125A) to the 3' end of a humanized anti-HER2 IgG3 antibody. Antitumor efficacy was examined in murine and human breast tumor models. HuEndo-P125A antibody fusion protein (aHER2-huEndo-

Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [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 2654.

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B lymphocytes may play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms by which B cells facilitate tumor growth are poorly understood. Murine EMT-6 mammary tumors grow readily in wild type mice (WT) but are rejected or grow very poorly in B-cell deficient μ-/- BALB/c mice (BCDM). Relative to BCDM, B cell reconstituted BCDM demonstrate enhanced tumor growth, marked expansion of Tregs in response to EMT-6 implantation, increased infiltration of both B cells and Tregs into tumor, and a markedly reduced cytolytic T cell response. Markedly decreased levels of CD49b+ NK cell and CD8+ T cell infiltration into the tumor bed were seen in WT or in B cell reconstituted BCDM, compared to levels of infiltration seen in BCDM. Adoptive transfer of either IL-10-/- B cells or MHC-II-/- B cells into BCDM also promoted tumor growth and expansion of Treg and suppressed cytotoxic CD8+ T cell anti-tumor activity and IFN-γ production in vivo. We hypothesized that tumor infiltrating B cells promote Treg expansion but also may directly suppress T cell activation and proliferation due to acquired regulatory function. In vitro co-culture of EMT-6 tumor cells with naïve B cells generated B cells of a distinct phenotype (PD-L1hiCD86hiIAdhiCD62LhiLAP+CD44lo but CD25-CD69-CD80-). EMT-6 co-cultured B cells markedly suppressed naïve CD4 T cell proliferation. Flow cytometric analysis of tumor infiltrating B cells (TIL-B) revealed that at day 21 post tumor implantation, TIL-B cells are LAPhiCD62LhiCD86hiIAdhiPD-L1hiCD44+CD25-. LAP/TGF-β expression on TIL-B cells was gradually upregulated as tumor burden increased, from 2∼3% on day 8 to ∼50% of infiltrating B cells by day 21. In contrast, splenic B cells at day 21 did not express LAP/TGF-β. TIL-B cells also markedly suppressed CD4+ T cell proliferation in-vitro when compared to splenic B cells. Our studies indicate that the T cell anti-tumor response is enhanced in the absence of B cells, due to acquisition of a suppressive immmunoregulatory phenotype by migrating B cells following tumor infiltration. We are currently looking for evidence of human correlates of B regulatory activity observed in murine models. Citation Format: Yu Zhang, Richard Morgan, Seung-uon Shin, Hyun-Mi Cho, Ahmed Albayati, Augustin Pimentel, Joseph D. Rosenblatt. Tumor educated B cells acquire immune suppressive function and promote tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1672. doi:10.1158/1538-7445.AM2014-1672

B lymphocytes play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms are poorly understood. EMT-6 mammary tumors grow well in wild type (WT) mice but show reduced growth in B-cell deficient μ-/- BALB/c mice (BCDM). WT mice demonstrate B cell infiltration into the tumor bed and a reduced cytolytic T cell response relative to BCDM. Expression of LAP/TGF-β1, CD80, CD86 and PDL-1 is increased in tumor infiltrating B cells (TIL-B) relative to splenic B cells. LAP/TGF-β1 expression progressively increased from 5.4±1.7% on d8 to 43.1±6.1% of TIL-B by d21 post tumor implantation. Co-culture of EMT-6 tumor cells with naïve B cells generated B cells (EMT6-B) with a similar immunophenotype to TIL-B. Purified TIL-B or in vitro generated EMT6-B markedly suppressed CD4+CD25- T cell proliferation, and Th1 cytokine secretion compared to naïve splenic B cells. Acquired B regulatory function required direct tumor cell: B cell contact, and was partially reversed by antibody to TGF-β1 or PDL-1, leading to tumor rejection in vivo. B cell acquisition of a suppressive phenotype following tumor infiltration may result in profound inhibition of T cell anti-tumor response. Similar B cell mediated immunosuppression may be operative in human solid tumors. Citation Format: Yu Zhang, Richard Morgan, Chuan Chen, Yancheng Cai, Seung-Uon Shin, Hyun-Mi Cho, Ahmed Al Bayati, Augustin Pimental, Joseph D. Rosenblatt. Tumor-educated B cells acquire LAP/TGF-β1 and PD-L1 expression and suppress antitumor immune response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A120.