Alan Howell

Cutting Edge: Selective Requirement for the Wiskott-Aldrich Syndrome Protein in Cytokine, but Not Chemokine, Secretion by CD4+ T Cells

The mechanism of cytokine secretion is not well understood, but cytokines appear to be synthesized and released in a polarized fashion toward an Ag-specific target cell. In this study, we demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an essential component of the cytokine secretory pathway in CD4+ T cells. Murine WASp-deficient CD4+ T cells fail to polarize cytokines toward a target and show an unexpected and striking block in cytokine secretion. In contrast, chemokine secretion and trafficking of plasma membrane proteins, transported via the constitutive secretory pathway, are unaffected by the lack of WASp. These results suggest that CD4+ T cell cytokines require a specialized, WASp-dependent pathway for cellular traffic and/or vesicle release that is distinct from that required for chemokine release. We propose that the use of different secretory pathways for cytokines and chemokines enables CD4+ T cell activity to be further fine-tuned to serve specialized effector functions.

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.

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.

Early BAFF receptor blockade mitigates murine Sjögren's syndrome: Concomitant targeting of CXCL13 and the BAFF receptor prevents salivary hypofunction.

Sjögrens syndrome (SS) is a debilitating autoimmune disease. Patients with SS may develop xerostomia. This process is progressive, and there are no therapeutics that target disease etiology. We hypothesized BAFF receptor (BAFFR) blockade would mitigate SS disease development, and neutralization of CXCL13 and BAFF signaling would be more efficacious than BAFFR blockade alone. We treated NOD/ShiLtJ SS mice with soluble BAFF receptor (BAFFR-Fc) or anti-CXCL13/BAFFR-Fc in combination, prior to the development of clinical disease. Our results show treatment with BAFFR-Fc reduced peripheral B cell numbers and decreased sialadenitis. In addition, this treatment reduced total serum immunoglobulin as well as IgG and IgM specific anti-nuclear autoantibodies. NOD/ShiLtJ mice treated with BAFFR-Fc and anti-CXCL13 antibody were protected from salivary deficits. Results from this study suggest blockade of CXCL13 and BAFFR together may be an effective therapeutic strategy in preventing salivary hypofunction and reducing autoantibody titers and sialadenitis in patients with SS.

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.

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.

Maximizing immune responses: the effects of covalent peptide linkage to beta-2-microglobulin.

Major histocompatability molecules (MHC) are involved in presentation of peptide antigens for recognition by the immune system. The density and stability of presented peptides is a critical parameter in determining the magnitude of the immune response. Increasing the half-life and density of an MHC class I-peptide complex should promote a stronger cytotoxic T lymphocyte (CTL) response to clinically important peptides, including those that exhibit low or suboptimal MHC class I binding affinity. We hypothesized that the covalent linkage of a known tumor antigen peptide to beta-2-microglobulin (beta2m) would increase peptide immunogenicity and, therefore, in vivo effectiveness as an antitumor vaccine in BALB/c mice. The iL3 peptide fusion protein (iL3-L12-hbeta2m) was developed based on the mutant iL3 peptide, derived from the L3 ribosomal protein, and expressed in the mutagenized murine fibroblastic tumor cell line, BCA34. The iL3-L12-beta2m and a negative control fusion protein utilizing the H-2K(d)-restricted NP(147-155) influenza peptide (NP-L12-hbeta2m) were both produced in E. coli for exogenous antigen presentation by dendritic cells. In vitro, the iL3-L12-hbeta2m protein was found to stabilize H-2K(d) over time on the surface of H-2K(d)-expressing target cells and sensitized them to peptide-specific CTL-mediated lysis. Furthermore, mice immunized with dendritic cells pulsed with the iL3-L12-hbeta2m protein rejected a challenge with BCA34 cells significantly more so than mice immunized with dendritic cells pulsed with free peptide and hbeta2m. We conclude that vaccines incorporating peptides covalently linked to beta2m may have future potential in the specific targeting of human malignancy.

Abstract 3661: Breaking down the barrier restricting infiltration and differentiation of APC in the tumor microenvironment with a first-in-class antibody targeting Semaphorin4D, and rational combination therapies

Purpose: We expand mechanistic findings in preclinical studies to demonstrate that antibody blockade of Semaphorin 4D (SEMA4D, CD100) reduces expansion of MDSC and shifts the balance of myeloid cells within the TME to facilitate tumor rejection. Efficacy is further enhanced when combined with various immunotherapies. Design of Phase 1b/2 combination trials of VX15/2503, a humanized IgG4 antibody targeting SEMA4D, with immune checkpoint inhibition will be presented. Methods: Anti-SEMA4D antibodies were evaluated alone and in combination with other immunotherapies in various preclinical models. Anti-tumor activity and immune response was characterized by immunohistochemistry, flow cytometry, functional assays, and cytokine, chemokine and gene expression analysis. A Phase I trial for single agent VX15/2503 was completed, and several 1b/2 combination immunotherapy trials are planned. Results: SEMA4D restricts migration of monocytes and promotes expansion of suppressive myeloid cells in vitro. Strong expression of SEMA4D at the invasive margins of actively growing tumors in vivo restricts the infiltration and modulates polarization of leukocytes in the TME. Antibody blockade of SEMA4D facilitated recruitment of activated DCs and T lymphocytes in preclinical models. M-MDSCs were significantly reduced in tumor and blood following treatment. A significant shift towards increased Th1 cytokines (IFNγ, TNFα) and CTL-recruiting chemokine CXCL9, with concurrent reduction in Treg-, MDSC- and M2-macrophage promoting chemokines (CCL2, CXCL1, CXCL5) was observed. Accordingly, Teff:Treg ratio (3x, p Conclusions: SEMA4D blockade represents a novel mechanism to promote functional immune infiltration into the tumor and enhance immunotherapy. VX15/2503 treatment was well tolerated in a Phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Phase 1b/2 trials of combination therapy with avelumab in NSCLC patients who are immunotherapy naive, and combinations with anti- anti-PD-1 and/or anti-CTLA-4 in melanoma and HNSCC patients who are refractory to PD1 inhibitors are planned in 2017. Citation Format: Elizabeth E. Evans, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Maria Scrivens, Cathie Foster, Alan Howell, Stephen R. Comeau, Leslie Balch, Alyssa Knapp, John E. Leonard, Terrence L. Fisher, Siwen Hu-Lieskovan, Antoni Ribas, Ernest S. Smith, Maurice Zauderer. Breaking down the barrier restricting infiltration and differentiation of APC in the tumor microenvironment with a first-in-class antibody targeting Semaphorin4D, and rational combination therapies [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 3661. doi:10.1158/1538-7445.AM2017-3661

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.