Mark Paris

SEMA4D compromises blood-brain barrier, activates microglia, and inhibits remyelination in neurodegenerative disease

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by immune cell infiltration of CNS, blood-brain barrier (BBB) breakdown, localized myelin destruction, and progressive neuronal degeneration. There exists a significant need to identify novel therapeutic targets and strategies that effectively and safely disrupt and even reverse disease pathophysiology. Signaling cascades initiated by semaphorin 4D (SEMA4D) induce glial activation, neuronal process collapse, inhibit migration and differentiation of oligodendrocyte precursor cells (OPCs), and disrupt endothelial tight junctions forming the BBB. To target SEMA4D, we generated a monoclonal antibody that recognizes mouse, rat, monkey and human SEMA4D with high affinity and blocks interaction between SEMA4D and its cognate receptors. In vitro, anti-SEMA4D reverses the inhibitory effects of recombinant SEMA4D on OPC survival and differentiation. In vivo, anti-SEMA4D significantly attenuates experimental autoimmune encephalomyelitis in multiple rodent models by preserving BBB integrity and axonal myelination and can be shown to promote migration of OPC to the site of lesions and improve myelin status following chemically-induced demyelination. Our study underscores SEMA4D as a key factor in CNS disease and supports the further development of antibody-based inhibition of SEMA4D as a novel therapeutic strategy for MS and other neurologic diseases with evidence of demyelination and/or compromise to the neurovascular unit.

CXCL13 antibody for the treatment of autoimmune disorders

BackgroundHomeostatic B Cell-Attracting chemokine 1 (BCA-1) otherwise known as CXCL13 is constitutively expressed in secondary lymphoid organs by follicular dendritic cells (FDC) and macrophages. It is the only known ligand for the CXCR5 receptor, which is expressed on mature B cells, follicular helper T cells (Tfh), Th17 cells and regulatory T (Treg) cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune disorders (e.g. Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosis). We, therefore, hypothesized that antibody-mediated disruption of the CXCL13 signaling pathway would interfere with the formation of ectopic lymphoid follicles in the target organs and inhibit autoimmune disease progression. This work describes pre-clinical development of human anti-CXCL13 antibody MAb 5261 and includes therapeutic efficacy data of its mouse counterpart in murine models of autoimmunity.ResultsWe developed a human IgG1 monoclonal antibody, MAb 5261 that specifically binds to human, rodent and primate CXCL13 with an affinity of approximately 5 nM and is capable of neutralizing the activity of CXCL13 from these various species in in vitro functional assays. For in vivo studies we have engineered a chimeric antibody to contain the same human heavy and light chain variable genes along with mouse constant regions. Treatment with this antibody led to a reduction in the number of germinal centers in mice immunized with 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Keyhole Limpet Hemocyanin (NP-KLH) and, in adoptive transfer studies, interfered with the trafficking of B cells to the B cell areas of mouse spleen. Furthermore, this mouse anti-CXCL13 antibody demonstrated efficacy in a mouse model of Rheumatoid arthritis (Collagen-Induced Arthritis (CIA)) and Th17-mediated murine model of Multiple Sclerosis (passively-induced Experimental Autoimmune Encephalomyelitis (EAE)).ConclusionsWe developed a novel therapeutic antibody targeting CXCL13-mediated signaling pathway for the treatment of autoimmune disorders.

C35 (C17orf37) is a novel tumor biomarker abundantly expressed in breast cancer

Identification of shared tumor-specific targets is useful in developing broadly applicable therapies. In a study designed to identify genes up-regulated in breast cancer, a cDNA clone corresponding to a novel gene C35 (C17orf37) was selected by representational difference analysis of tumor and normal human mammary cell lines. Abundant expression of C35 transcript in tumors was confirmed by Northern blot and real-time PCR. The C35 gene is located on chromosome 17q12, 505 nucleotides from the 3′ end of the ERBB2 oncogene, the antigenic target for trastuzumab (HerceptinTM) therapy. The chromosomal arrangement of the genes encoding C35 and ERBB2 is tail to tail. An open reading frame encodes a 12-kDa protein of unknown function. Immunohistochemical analysis detected robust and frequent expression of C35 protein, including 32% of grade 1 and 66% of grades 2 and 3 infiltrating ductal carcinomas of the breast (in contrast to 20% overexpressing HER-2/neu), 38% of infiltrating lobular carcinoma (typically HER-2/neu negative), as well as tumors arising in other tissues. C35 was not detected in 38 different normal human tissues, except Leydig cells in the testes and trace levels in a small percentage of normal breast tissue samples. The distinct and favorable expression profile of C35 spanning early through late stages of disease, including high frequency of overexpression in various breast carcinoma, abundant expression in distant metastases, and either absence or low level expression in normal human tissues, warrants further investigation of the relevance of C35 as a biomarker and/or a target for development of broadly applicable cancer-specific therapies. [Mol Cancer Ther 2006;5(11):2919–30]

Antibody Blockade of Semaphorin 4D Promotes Immune Infiltration into Tumor and Enhances Response to Other Immunomodulatory Therapies

Evans and colleagues describe a novel immunomodulatory function of semaphorin 4D (SEMA4D) and show that blocking SEMA4D enhances immune infiltration into tumor and increases antitumor activity in synergy with other immunomodulatory therapies. Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 (PLXNB1) are broadly expressed in murine and human tumors, and their expression has been shown to correlate 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, SEMA4D–PLXNB1 interactions have been reported to affect vascular stabilization and transactivation of ERBB2, but effects on immune-cell trafficking in the tumor microenvironment (TME) have not been investigated. We describe a novel immunomodulatory function of SEMA4D, whereby strong expression of SEMA4D at the invasive margins of actively growing tumors influences the infiltration and distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient of expression, enhances recruitment of activated monocytes and lymphocytes into the tumor, and shifts the balance of cells and cytokines toward a proinflammatory and antitumor milieu within the TME. This orchestrated change in the tumor architecture was associated with durable tumor rejection in murine Colon26 and ERBB2+ mammary carcinoma models. 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. Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the TME and inhibit tumor progression. Cancer Immunol Res; 3(6); 689–701. ©2015 AACR.

Generation and preclinical characterization of an antibody specific for SEMA4D.

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 Huntingtons 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.

Immunomodulation of the tumor microenvironment by neutralization of Semaphorin 4D.

Semaphorin 4D is highly expressed at the invasive tumor margin and acts as a guidance molecule, restricting movement of tumoricidal immune cells into the tumor microenvironment. We recently showed that antibody neutralization of SEMA4D augmented activated monocyte and anticancer T-cell tumor penetration and that anti-SEMA4D antibody potentiated other immunomodulatory therapies in murine tumor models.

Antibody blockade of semaphorin 4D breaks down barriers to enhance tumoricidal immune infiltration and supports rational immunotherapy combinations

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 regulating immune cell infiltration and anti-tumor activity. Activity is enhanced in preclinical studies when combined with other immunotherapies, including immune checkpoint blockade inhibition.

Abstract B023: Antibody blockade of Semaphorin 4D enhances infiltration of APC and CD8 T cells and reduces immune suppression to facilitate immune-mediated tumor rejection

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. We have reported a novel role for SEMA4D in modulating the tumor microenvironment (TME) to exclude activated antigen presenting cells and cytotoxic T lymphocytes so as to promote tumor growth; this effect can be reversed by antibody blockade. Purpose: Characterize immune-related anti-tumor activity mediated by antibody neutralization of SEMA4D, as a single agent and in combination with other immunomodulatory therapies, including immune checkpoint inhibition. Methods: Blockade of SEMA4D with monoclonal murine antibody was evaluated in murine melanoma and colon cancer models. Immune response in pre-clinical models was characterized by immunohistochemistry, flow cytometry, functional assays, as well as cytokine, chemokine and gene expression analysis. Therapeutic activity was evaluated in various preclinical models. A Phase I trial in patients with advanced solid tumors was completed. Results: SEMA4D restricts migration of macrophage cell lines and promotes expansion of suppressive tumor associated macrophage (TAM) and myeloid derived suppressor cells (MDSC) in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo modulates the infiltration and polarization of leukocytes in the TME. In preclinical models, antibody neutralization disrupted the SEMA4D gradient at the invasive margin, which correlated with recruitment of activated APCs and T lymphocytes into the TME. This was accompanied by a significant shift towards increased Th1 cytokines (IFNΓ, TNFA) and CTL-recruiting CXCL9 chemokine, with concurrent reduction in Treg-, MDSC- and M2-macrophage promoting chemokines (CCL2, CXCL1, CXCL5). Accordingly, an increase in Teff:Treg ratio (3x, p Conclusion: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. Phase 1b/2a trials of combination therapy with immune checkpoint inhibition are planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Ekaterina Klimatcheva, Maria Scrivens, Cathie Foster, Alan Howell, Leslie Balch, Alyssa Knapp, John E. Leonard, Mark J. Paris, Terrence L. Fisher, Siwen Hu-Lieskovan, Antoni Ribas, Ernest S. Smith, Maurice Zauderer. Antibody blockade of Semaphorin 4D enhances infiltration of APC and CD8 T cells and reduces immune suppression to facilitate immune-mediated tumor rejection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B023.

Abstract A119: Antibody blockade of Semaphorin 4D neutralizes barrier to immune infiltration and facilitates immune-mediated tumor rejection

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.

Abstract 4888: Antibody blockade of semaphorin 4D breaks down stromal barriers to enhance tumoricidal immune infiltration, supporting rational immunotherapy combinations

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.