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Semaphorin 4D (SEMA4D or CD100) is a member of the semaphorin family of proteins and an important mediator of the movement and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. Blocking the binding of SEMA4D to its receptors can result in physiologic changes that may have implications in cancer, autoimmune, and neurological disease. To study the effects of blocking SEMA4D, we generated, in SEMA4D-deficient mice, a panel of SEMA4D-specific hybridomas that react with murine, primate, and human SEMA4D. Utilizing the complementarity-determining regions from one of these hybridomas (mAb 67-2), we generated VX15/2503, a humanized IgG4 monoclonal antibody that is currently in clinical development for the potential treatment of various malignancies and neurodegenerative disorders, including multiple sclerosis and Huntington's disease. This work describes the generation and characterization of VX15/2503, including in vitro functional testing, epitope mapping, and an in vivo demonstration of efficacy in an animal model of rheumatoid arthritis.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer; increased expression correlates with poor prognosis. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the tumor microenvironment (TME), SEMA4D is expressed strongly at the invasive margin and modulates the infiltration and spatial distribution of leukocytes, suppressing anti-tumor activity. Neutralization of SEMA4D was evaluated for effects on immune activity and tumor growth in preclinical models, incorporating single agent and combination treatments with other immunotherapies, including immune checkpoint blockade inhibitors. The safety and tolerability of humanized anti-SEMA4D antibody VX15/2503 was assessed in a Phase I clinical trial. RESULTS: SEMA4D restricts migration of myeloid cells expressing cognate PLXNB1/2 receptors, determined using trans-well migration assays and IHC of in vivo tumors. Antibody neutralization disrupted the SEMA4D gradient at the invasive margin, which correlated with recruitment of activated APCs and T lymphocytes into the TME, and significant shift toward increased Th1 cytokines (IFNg, TNFa) and CTL-recruiting CXCL9 chemokine, with concurrent reduction in Treg- and M2-macrophage promoting chemokines (CCL2, CXCL1, CCL17). Accordingly, an increase in Teff:Treg ratio (3x, p SEMA4D antibody treatment was well tolerated in nonclinical and clinical studies; including a Phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. Progression free survival strongly correlated with elevated baseline lymphocyte counts (r = 0.6133), supporting an immune mediated mechanism of action for VX15/2503. CONCLUSION: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. A phase 1b/2 trial of combination therapy with an immune checkpoint inhibitor is planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Crystal Mallow, Christine Reilly, Maria Scrivens, Ekaterina Klimatcheva, Laurie A. Winter, Renee Kirk, Alan Howell, Leslie Balch, John E. Leonard, Mark Paris, Terrence L. Fisher, Siwen Hu-Lieskovan, Antoni Ribas, Ernest S. Smith, Maurice Zauderer. Antibody blockade of semaphorin 4D breaks down stromal barriers to enhance tumoricidal immune infiltration, supporting rational immunotherapy combinations. [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 4888.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, we describe a novel immunomodulatory function of SEMA4D in regulation of immune cell infiltration and activity in the tumor microenvironment (TME). Purpose: Characterize immune-related and anti-tumor activity mediated by antibody neutralization of SEMA4D, as a single agent and in combination with other immunomodulatory therapies. Methods: Blockade of SEMA4D with monoclonal murine antibody was evaluated in subcutaneous models, as well as an orthotopic ERBB2+ breast carcinoma syngeneic model. Anti-tumor immune response in pre-clinical models was characterized by selective in vivo immune cell depletions, as well as immunohistochemistry, flow cytometry, and functional assays. The safety and tolerability of a humanized anti-SEMA4D antibody VX15/2503 was assessed in Phase I clinical trials in oncology. Results: SEMA4D restricts migration of macrophage cell lines in vitro. Strong expression of SEMA4D at the invasive margins of actively growing in vivo tumors modulates the infiltration and spatial distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient and facilitates recruitment of potent antigen presenting cells and T lymphocytes into the TME, shifting the balance of cytokines toward increased Th1 and reduced immunosuppressive cytokines. This orchestrated change in the tumor architecture was associated with durable tumor rejection and immunologic memory in preclinical models. Immune-mediated tumor rejection may enhance the disruption of ERBB2 transactivation with SEMA4D receptors, which has been reported for ERBB2 and Met oncogenes. Importantly, the immunomodulatory activity of anti-SEMA4D antibody can also be further enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival, with significant 58% increase in tumor regression and maximal increase in survival, as compared to monotherapy. Treatment with anti-SEMA4D antibodies was well tolerated in nonclinical and clinical studies, including completion of a Phase I prospective multiple ascending dose trial in patients with advanced refractory solid tumors. Weekly doses of between 0.3 and 20 mg/kg were administered; no MTD was determined. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. Progression free survival correlated with elevated baseline lymphocyte counts, supporting an immune mediated mechanism of action for VX15/2503. Conclusion: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. A phase 1b/2a trial of combination therapy with immune checkpoint inhibition is planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Crystal Mallow, Laurie A. Winters, Christine Reilly, Ekaterina Klimatcheva, Janaki Veeraraghavan, Alan S. Jonason, Maria Scrivens, Renee Kirk, Alan Howell, Leslie Balch, John E. Leonard, Mark Paris, Terrence L. Fisher, Ernest S. Smith, Maurice Zauderer. Antibody blockade of Semaphorin 4D neutralizes barrier to immune infiltration and facilitates immune-mediated tumor rejection. [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 A119.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, effects on cellular movement mediated by SEMA4D/PLXNB1 interactions have been described in relation to vascular stabilization and transactivation of ERBB2. Herein, we describe a novel function of SEMA4D in regulation of immune cell infiltration and activity in the tumor microenvironment (TME). PURPOSE: Characterize immune-related and anti-tumor activity mediated by antibody neutralization of SEMA4D, as a single agent or in combination with other immunomodulatory therapies. METHODS: Blockade of SEMA4D with monoclonal murine antibody was evaluated in subcutaneous Colon26 and orthotopic ERBB2+ breast carcinoma syngeneic models. Growth kinetics were assessed following selective in vivo immune cell depletions. Anti-tumor immune response was characterized by immunohistochemistry, FACS, and functional assays. RESULTS: Strong expression of SEMA4D at the invasive margins of actively growing tumors modulates the infiltration and spatial distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient and enhances recruitment of activated antigen presenting cells and lymphocytes into the TME, shifting the balance of cytokines toward increased Th1 and reduced immunosuppressive cytokines. This orchestrated change in the tumor architecture was associated with durable tumor rejection and immunologic memory in Colon26 and ERBB2+ mammary carcinoma models. Efficacy was dependent on both active adaptive and innate immune cell responses. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival, with significant 58% increase in tumor regression and >295% increase in survival, as compared to monotherapy. CONCLUSION: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. Humanized anti-SEMA4D therapeutic antibody, VX15/2503, has completed a Phase I prospective multiple ascending dose trial in 42 adult patients with advanced refractory solid tumors, in which the highest doses were well tolerated. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. A phase 1b/2a trial of combination therapy is planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Crystal Mallow, Laurie A, Winters, Christine Reilly, Katya Klimatcheva, Janaki Veeraraghavan, Alan S. Jonason, Maria Scrivens, Renee Kirk, Sue Giralico, Alan Howell, John E. Leonard, Mark Paris, Terrence L. Fisher, Ernest S. Smith, Maurice Zauderer. Antibody blockade of semaphorin 4D promotes infiltration of activated tumor infiltrating leukocytes and reverses tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 278. doi:10.1158/1538-7445.AM2015-278

Semaphorin4D (SEMA4D) normally functions to regulate, adhesion, motility and activation state of multiple cell types including those of the nervous, vascular and immune systems. In the setting of cancer, SEMA4D has been shown to promote tumor progression and metastasis. SEMA4D and its high affinity receptor plexin B1 are broadly expressed in human and murine tumors, and expression levels correlate with invasive disease in several human tumors. The interaction of plexin B1 with MET and ERBB2 can lead to SEMA4D-mediated transactivation of these membrane receptor kinases with a direct effect on tumor cell migration and invasive growth. Moreover, SEMA4D is highly expressed by inflammatory cells that are recruited to the tumor microenvironment. Purpose: Determine the role(s) of SEMA4D in shaping the tumor microenvironment, and investigate the anti-tumor response mediated by antibody neutralization of SEMA4D as a single agent or in combination with other immunomodulatory therapies. Methods: Subcutaneous colon and orthotopic ERBB2+ breast carcinoma models were employed to investigate treatment with a monoclonal antibody to SEMA4D. Anti-tumor response and tumor infiltrating immune cell profiles were characterized by in vivo growth kinetics, immunohistochemistry, FACS, and functional assays. Results: We describe an immunomodulatory function of SEMA4D within the tumor microenvironment, whereby SEMA4D influences the recruitment and activity of cytotoxic CD8+ T cells, the density of B cells within the tumor, and regulates the balance and localization of inflammatory M1 and tolerance-inducing M2 macrophage in tumor stroma. Antibody blockade of SEMA4D modifies the balance of immune cells and production of pro-inflammatory cytokines within the tumor microenvironment, promotes tumor rejection, and induces ∼30% tumor growth delay (TGD) in Colon26 and >70% TGD in Tubo breast tumor models. Furthermore, combination of SEMA4D antibody with immune checkpoint blockade inhibitor antibodies to CTLA4 and PD1, significantly improves frequency of complete tumor rejection (58% and 37%) and increases survival (>295% and >55% TGD respectively) as compared to monotherapy with these targeted agents. Similar combinatorial effects were observed when anti-SEMA4D antibody is administered with immunomodulatory chemotherapy, such as cyclophosphamide, which increases efficacy (81% TGD) and frequency of complete rejection (40%) in tumor engrafted mice. Conclusion: SEMA4D represents a novel immunological target and antibody neutralization of SEMA4D may be a novel therapeutic option for cancer treatment. The humanized anti-SEMA4D antibody, VX15/2503, has successfully completed IND-enabling toxicology testing and the dose escalation phase of a Phase I trial, in which the highest doses were well tolerated in adult patients with advanced solid tumors. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Janaki Veeraraghavan, Alan S. Jonason, Michael A. Doherty, Christine Reilly, Crystal Mallow, Jennifer Seils, Mark Paris, Terrence L. Fisher, William J. Bowers, Maria Scrivens, Leslie Balch, Renee Kirk, Alan Howell, Valerie Iddison, John E. Leonard, Ernest S. Smith, Maurice Zauderer. Antibody blockade of Semaphorin 4D promotes tumor rejection and improves response to immune checkpoint blockade and chemotherapy. [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 5030. doi:10.1158/1538-7445.AM2014-5030

Semaphorin 4D (SEMA4D; CD100) has been implicated in several key mechanisms of tumor progression, including transactivation of several oncogenes, metastasis, tumor invasion, and neovascularization. Expression of SEMA4D and its receptor plexin-B1 (PLXNB1) correlates with invasive disease in humans. SEMA4D is over-expressed in a wide array of tumor types and is also produced by inflammatory cells recruited to the tumor microenvironment. SEMA4D binding to PLXNB1 on endothelial cells activates RhoA and AKT signaling pathways, which promotes formation of new blood vessels and tumor growth in vivo. In addition to its effects on endothelial cells, the interaction of PLXNB1 with MET and ERBB2 can lead to SEMA4D-mediated transactivation of these membrane receptor kinases with a direct effect on tumor cell migration and invasive growth. It is well known that tumor growth and metastasis involve a complex process of cross talk amongst the tumor cells, stroma and immune infiltrate, as well as the endothelial cells and vasculature. Our understanding of the role of SEMA4D in this process is evolving. We selected a humanized IgG4 antibody, VX15/2503, that blocks SEMA4D interaction with the high affinity receptor PLXNB1 and a lower affinity receptor, CD72, expressed on immune cells. The antibody binds with between 1-5 nM affinity to rat, mouse, primate, and human recombinant SEMA4D. Affinity to native cell-associated SEMA4D on primary human T cells was, however, determined to be 0.5 nM. We demonstrate that antibody-mediated SEMA4D neutralization delays tumor growth in several primary and metastatic in vivo models. Inhibition of SEMA4D regulates angiogenesis and vascular permeability in these models. Additionally, tumor growth delay results from modulation of the tumor microenvironment, such as infiltrating immune cells. We also demonstrate direct effects of SEMA4D/PLXNB1 interaction acting as a guidance signal for tumors, as previously described for axons, whereby tumor migration is affected ∼80%. Antibody blockade restores these functional activities. Moreover, using a genetic fingerprinting approach, we identified a unique gene signature that is related to changes in PLXNB1 expression and sensitivity to existing targeted therapies. This signature sheds light on combination therapies with anti-SEMA4D antibodies that may produce additive anti-tumor effects. In summary, blockade of SEMA4D reduces tumor growth through effects on tumor microenvironment, angiogenesis and vascular permeability, as well as direct effects on tumor. Therefore, antibody neutralization of SEMA4D represents a new therapeutic strategy for cancer treatment. The humanized antibody, VX15/2503, has successfully completed IND-enabling toxicology testing and a Phase I trial is currently being conducted in adult patients with advanced solid tumors. Citation Format: Elizabeth E. Evans, Alan S. Jonason, Mark Paris, Terrence L. Fisher, Sebold Torno, Holm Bussler, Jessica Decker, Maria Scrivens, He Huang, Laurie A. Winter, Tracy Pandina, Leslie Balch, Michael A. Doherty, Renee Kirk, Alan Howell, Jennifer Seils, Christine Reilly, Maurice Zauderer, John E. Leonard, Ernest S. Smith. Reduction of tumor growth and metastasis by a humanized IgG4 monoclonal antibody to SEMA4D (VX15/2503). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2013-1245 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

It has been demonstrated that semaphorin 4D (SEMA4D; CD100) is a potent pro-angiogenic molecule. SEMA4D binding to its receptor plexin-B1 (PLXNB1) on endothelial cells transactivates c-MET and promotes formation of new blood vessels and tumor growth in vivo. SEMA4D is over-expressed in a wide array of tumor types, and is also produced by inflammatory cells present in the tumor microenvironment. It has recently been demonstrated that in an environment lacking SEMA4D, the ability of mouse breast cancer cells to originate tumor masses and metastases is severely impaired, and that SEMA4D produced by tumor associated macrophages is required for tumor angiogenesis and growth. In addition to its effects on endothelial cells, SEMA4D binding to PLXNB1 on tumor cells results in MET transactivation and migration of tumor cells. It has been further reported that overexpression of PLXNB1 and MET in breast and ovarian cancers is a negative prognostic factor. Tumors co-expressing PLXNB1 and MET were characterized as having a higher grade and an increased frequency of metastases. Collectively, these results suggest that expression of SEMA4D, either by tumor cells or by tumor associated macrophages, may represent a mechanism of tumor neovascularization, and that expression of the SEMA4D receptor in tumors may further induce tumor invasion, thereby enhancing their growth, survival, and metastatic potential. Antibody neutralization of SEMA4D thus may represent a new therapeutic strategy for cancer treatment. We selected a high affinity human IgG4 antibody that is specific for both mouse and human SEMA4D, and utilized several in vitro functional assays to demonstrate that this antibody blocks SEMA4D - PLXNB 1 interactions. Using both transplanted and orthotopic tumor models we demonstrated that antibody mediated SEMA4D neutralization in vivo inhibits tumor growth and tumor angiogenesis. This human antibody is currently in IND-enabling toxicology testing and we anticipate the initiation of human clinical trials in 2H 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-36.

Meeting abstracts Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the