Bindu Varghese

Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

Lymphoma Immunotherapy with CpG Oligodeoxynucleotides Requires TLR9 Either in the Host or in the Tumor Itself

Established widely metastatic tumor was cured in a transplanted mouse B cell lymphoma model, by the combination of chemotherapy plus intratumoral injection of oligodeoxynucleotides containing unmethylated C-G motifs (CpG). This therapeutic effect required that the CpG be injected directly into the tumor and was dependent on CD8 T cells. Although the efficacy of CpG oligodeoxynucleotides has been thought to depend on the expression of TLR9, we unexpectedly found that tumor rejection did not require host expression of TLR9. By using a TLR9-deficient tumor and a TLR9KO host, we demonstrate that TLR9 expression either by the host or the tumor is required. These results indicate that activation of Ag presentation by cells within the tumor via TLR9 stimulation can be an effective form of immunotherapy. This study forms the basis of an ongoing clinical trial in patients with lymphoma.

Toll-like receptor-3 as a target to enhance bioactivity of cancer immunotherapy.

OBJECTIVE The purpose of this study was to explore the potential of toll-like receptor-3 stimulation, with polyI:C(12)U (poly[l].poly[C(12),U]; rintatolimod [Ampligen; Hemispherx Biopharma, Philadelphia, PA]) to enhance bioactivity of cancer immunotherapies. STUDY DESIGN Several models of immune activation were assessed with polyI:C(12)U at concentrations that were achieved clinically. Dendritic cell maturation and antigen-specific immune responses were evaluated in vitro and in a murine model. The potential for polyI:C(12)U to enhance antibody-dependent cellular cytotoxicity against tumor was also evaluated. RESULTS Dendritic cells are matured and T-cell stimulation is enhanced in the presence of polyI:C(12)U. In addition, polyI:C(12)U induced the release of proinflammatory chemokines and cytokines. Prostate-specific antigen-specific T-cell and antibody responses were enhanced significantly in a BALB/c prostate-specific antigen transgenic mouse model. Finally, rituximab-mediated antibody-dependent cellular cytotoxicity against tumor targets was improved significantly by the addition of polyI:C(12)U. CONCLUSION PolyI:C(12)U shows promise as a potential agent for selective enhancement of effect with currently available and future cancer immunotherapies.

Adoptive Cell Therapy for Lymphoma with CD4 T Cells Depleted of CD137 Expressing Regulatory T Cells

Adoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (T(reg)). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding T(regs). In a murine model of B-cell lymphoma, only CD137(neg)CD44(hi) CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137(pos)CD44hi CD4 T cells consisted primarily of activated T(regs). Notably, this CD137(pos) T(reg) population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137(pos) cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137(pos)CD44(hi) CD4 cells to CD137(neg)CD44(hi) CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated T(regs) that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137(pos) CD4 T cells that were predominantly CD25(pos)FoxP3(pos) T(regs). In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory T(reg) population.

Generation of CD8 + T cell–mediated immunity against idiotype-negative lymphoma escapees

We investigated the ability of CpG-oligodeoxynucleotide to generate an anti-tumor CD8+ T-cell immune response and to synergize with passive antibody therapy. For these studies, we generated an antibody against the idiotype on the A20 B-cell lymphoma line. This antibody caused the regression of established tumors, but ultimately the tumors relapsed. The escaping surface IgG-negative tumor cells were resistant to both antibody-dependent cellular cytotoxicity and signaling-induced cell death. Addition of intratumoral CpG to antibody therapy cured large established tumors and prevented the occurrence of tumor escapees. The failure of the combination therapy in mice deficient for CD8+ T cells demonstrates the critical role of CD8+ T cells in tumor eradication. When mice were inoculated with 2 tumors and treated systemically with antibody followed by intratumoral CpG in just one tumor, both tumors regressed, indicating that a systemic immune response was generated. Although antibody therapy can eliminate tumor cells bearing the target antigen, it frequently selects for antigen loss variants. However, when a poly-specific T-cell response was generated against the tumor by intratumoral CpG, even large established tumors were cured. Such an immune response can prevent the emergence of antibody selected tumor escapees and provide long-lasting tumor protection.

Abstract 2440: CD47 is a prognostic factor and an antibody target that synergizes with rituximab to eradicate non-Hodgkin lymphoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative, and must be combined with cytotoxic chemotherapy for clinical benefit. Here we identify CD47 as an attractive monoclonal antibody target in NHL. A major function of CD47 is to inhibit phagocytosis, through binding its receptor, SIRP-alpha, on phagocytes. We hypothesize that NHL cells over-express CD47 to evade immune phagocytosis and that blockade of CD47 signaling by a monoclonal antibody can eliminate NHL cells by enabling phagocytic engulfment. Here we investigate the therapeutic potential of an anti-CD47 antibody alone and in combination with rituximab for the treatment of NHL. We predict that the combination of anti-CD47 antibody and rituximab through utilization of two independent antibody mechanisms will result in synergistic phagocytosis and elimination of NHL cells. We found that CD47 protein is highly expressed on primary human NHL cells compared to normal B cell counterparts. Higher CD47 gene expression predicted an inferior outcome in several cohorts of NHL patients, providing independent prognostic value. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells but not normal cell counterparts in vitro while the combination of anti-CD47 antibody and rituximab resulted in synergistic phagocytosis of NHL cells. In vivo, anti-CD47 antibody treatment of human NHL-engrafted mice reduced lymphoma burden and prolonged survival compared to IgG control treatment. Furthermore, anti-CD47 antibody treatment in combination with rituximab led to elimination of lymphoma and cure of NHL-engrafted mice compared to either antibody treatment as a single agent. The above in vivo results were observed in both a disseminated and localized human NHL cell line mouse model as well as in primary human lymphoma xenotransplants. These antibodies synergized through a novel mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers. Together, these data provide the rationale for utilizing an anti-CD47 antibody either alone or in combination with rituximab in treating human NHL. 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 2440.