Kristopher Steward

Targeted delivery of interferon-alpha via fusion to anti-CD20 results in potent antitumor activity against B-cell lymphoma

The anti-CD20 antibody rituximab has substantially improved outcomes in patients with B-cell non-Hodgkin lymphomas. However, many patients are not cured by rituximab-based therapies, and overcoming de novo or acquired rituximab resistance remains an important challenge to successful treatment of B-cell malignancies. Interferon-alpha (IFNalpha) has potent immunostimulatory properties and antiproliferative effects against some B-cell cancers, but its clinical utility is limited by systemic toxicity. To improve the efficacy of CD20-targeted therapy, we constructed fusion proteins consisting of anti-CD20 and murine or human IFNalpha. Fusion proteins had reduced IFNalpha activity in vitro compared with native IFNalpha, but CD20 targeting permitted efficient antiproliferative and proapoptotic effects against an aggressive rituximab-insensitive human CD20(+) murine lymphoma (38C13-huCD20) and a human B-cell lymphoma (Daudi). In vivo efficacy was demonstrated against established 38C13-huCD20 grown in syngeneic immunocompetent mice and large, established Daudi xenografts grown in nude mice. Optimal tumor eradication required CD20 targeting, with 87% of mice cured of rituximab-insensitive tumors. Gene knockdown studies revealed that tumor eradication required expression of type I IFN receptors on the tumor cell surface. Targeting type I IFNs to sites of B-cell lymphoma by fusion to anti-CD20 antibodies represents a potentially useful strategy for treatment of B-cell malignancies.

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8(+) T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.

Anti-CD20-interferon-β fusion protein therapy of murine B cell lymphomas

Type I interferons (IFN&agr;/&bgr;) are cytokines with a broad spectrum of antitumor activities including antiproliferative, proapoptotic, and immunostimulatory effects, and are potentially useful in the treatment of B-cell malignancies and other cancers. To improve antitumor potency and diminish the systemic side effects of IFN, we recently developed anti-CD20-IFN&agr; fusion proteins with in vitro and in vivo efficacy against both mouse and human lymphomas expressing CD20. As IFN&bgr; binds more tightly to the IFN&agr;/&bgr; receptor (IFNAR) and has more potent antitumor activities, we have now constructed an anti-CD20 fusion protein with murine IFN&bgr; (mIFN&bgr;). Anti-CD20-mIFN&bgr; was more potent than recombinant mIFN&bgr; and anti-CD20-mIFN&agr; in inhibiting the proliferation of a mouse B-cell lymphoma expressing human CD20 (38C13-huCD20). Growth inhibition was accompanied by caspase-independent apoptosis and DNA fragmentation. The efficacy of anti-CD20-mIFN&bgr; required the physical linkage of mIFN&bgr; to anti-CD20 antibody. Importantly, anti-CD20-mIFN&bgr; was active against tumor cells expressing low levels of IFNAR (38C13-huCD20 IFNARlo). In vivo, established 38C13-huCD20 tumors were largely insensitive to rituximab or a nontargeted mIFN&bgr; fusion protein, yet treatment with anti-CD20-mIFN&bgr; eradicated 83% of tumors. Anti-CD20-mIFN&bgr; was also more potent in vivo against 38C13-huCD20 than anti-CD20-mIFN&agr;, curing 75% versus 25% of tumors (P=0.001). Importantly, although anti-CD20-mIFN&agr; could not eradicate 38C13-huCD20 IFNARlo tumors, anti-CD20-mIFN&bgr; treatment prolonged survival (P=0.0003), and some animals remained tumor-free. Thus, antibody fusion proteins targeting mIFN&bgr; to tumors show promise as therapeutic agents, especially for use against tumors resistant to the effects of mIFN&agr;.

ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20. Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivo. Results:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells. Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo. Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242–52. ©2017 AACR.

Antibody-CD20-interferon-alpha fusion protein has superior in vivo activity against human B cell lymphomas compared to Rituximab, and enhanced complement-dependent cytotoxicity in vitro

Meeting abstracts We previously reported an anti-CD20-interferon-alpha (IFNα) fusion protein able to induce apoptosis and promote in vivo eradication of a human CD20-expressing mouse B cell lymphoma [Xuan et al, Blood 2010]. We now report the activity of a recombinant anti-CD20-human IFNα fusion

Abstract B182: IGN004 is an antibody-interferon-alpha fusion protein against a novel tumor-associated antigen with both direct anti-tumor and immunostimulatory effects

Background: Antibody-IFNα fusion proteins represent a cancer therapeutic with properties of an antibody-drug conjugate and an immunotherapeutic agent, having both direct anti-tumor and immune-activating effects. These powerful molecules have the potential to target the anti-tumor cytotoxic effects of IFN to the tumor and to change the immunosuppressive tumor microenvironment to activate anti-tumor immunity. We previously reported the activity of IGN002, a recombinant anti-CD20-IFNα fusion protein, against human non-Hodgkin B-cell lymphomas (Yamada et al, JCO 2013). We now report the in vitro and in vivo anti-tumor activity of IGN004, an antibody-IFNα fusion protein against a novel tumor associated antigen expressed by many solid and liquid tumors. Methods: IGN004 was evaluated against a panel of human non-small cell lung cancer (NSCLC), melanoma, multiple myeloma (MM), and acute myeloid leukemia (AML) cell lines. Antigen expression was assessed by flow cytometry and immunohistochemistry (IHC). Anti-proliferative activity was measured by MTS assay. MHC, costimulatory, and coinhibitory molecule expression was assessed by flow cytometry. T-cell killing of tumor cells by TALL-104 effector cells was assessed by MTS assay. Human tumor cell line and patient-derived xenografts were grown in immunodeficient mice. Results: IGN004 antibody bound to the majority of tumor cell lines investigated, including melanoma, NSCLC, AML, and MM. In addition, IGN004 antibody also bound to nearly 100% of the primary solid tumor samples tested by IHC. IGN004 induced stronger growth inhibition than the unfused antibody and IFNα. Incubation of tumor cells with IGN004 caused an increase in surface expression of MHC class I, PD-L1, and OX-40L. In an in vitro T-cell killing assay using TALL-104 cells as effectors and NSCLC cells as targets, treatment of tumor cells with IGN004 led to increased effector cell killing of tumor targets (69.2% killing without IGN004 vs. 100% killing with IGN004; p = 0.001). The enhanced T-cell killing was observed even with concentrations at the sub-picomolar level (EC50 = 0.87 pM). Importantly, IGN004 demonstrated robust in vivo efficacy against MM, AML, melanoma, and NSCLC xenografts, including patient-derived tumors. Against U266 MM xenografts, IGN004 fusion protein caused complete regression of all established tumors and achieved long-term survival in 62.5% of mice. IGN004 unfused antibody caused a significant delay in tumor progression (p=0.002 vs. vehicle) but did not achieve long-term survival (p=0003 vs. IGN004 fusion protein). When efficacy was tested against a panel of patient-derived NSCLC xenografts, IGN004 fusion protein demonstrated efficacy against 7/11 tumor models, including tumor regression in 3. Against a patient-derived xenograft model of melanoma, IGN004 unfused antibody was ineffective while IGN004 fusion protein inhibited the growth of the tumors. In a patient-derived xenograft model of AML, IGN004 treatment caused a reduction in the percentage of AML cells in the blood, spleen and bone marrow, compared to PBS treated animals. Conclusions: IGN004 demonstrated robust in vitro and in vivo anti-tumor activity against both solid and liquid tumors. Treatment of tumor cells with IGN004 caused an increase in surface expression of MHC class I, PD-L1, and OX-40L. IGN004 demonstrated the ability to enhance the effector T-cell-mediated killing of NSCLC cells in an in vitro assay using the TALL-104 cell line as effectors and NSCLC cell lines as targets. Against both cell line and patient-derived human xenograft tumors IGN004 had robust in vivo anti-tumor efficacy. These results support the further development of IGN004 as a potent targeted cancer immunotherapeutic agent that also has direct anti-tumor effects. Citation Format: Kristopher Steward, Michael Gresser, Raj Sachdev, Sanjay Khare. IGN004 is an antibody-interferon-alpha fusion protein against a novel tumor-associated antigen with both direct anti-tumor and immunostimulatory effects. [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 B182.

IGN004 is an antibody-interferon-alpha fusion protein against a novel tumor-associated antigen with both direct anti-tumor and immunostimulatory effects

Results IGN004 unfused antibody bound to the majority of tumor cell lines and primary tumors assessed. Against tumor antigen-positive cells in anti-proliferation experiments, IGN004 demonstrated enhanced potency compared to unfused IFNa while reduced potency was observed in cells lacking antigen expression. IGN004 treatment upregulated MHC class I, PD-L1, and OX-40L on tumor cells. In an in vitro T cell killing assay using TALL-104 cells as effectors and A549 NSCLC cells as targets, the addition of IGN004 led to enhanced effector cell killing of tumor (69.2% killing without IGN004 vs. 100% killing with IGN004; p = 0.001). Importantly, IGN004 demonstrated robust in vivo efficacy against MM, NSCLC, AML, and melanoma xenografts, including patient-derived xenografts (PDX). Against U266 MM xenografts, IGN004 fusion protein caused complete regression of all tumors and achieved long-term survival in 62.5% of mice. Efficacy was tested against a panel of 14 NSCLC PDX tumors and IGN004 had a response rate of 64%, including tumor regression in 29%. In an AML PDX model, IGN004 treatment caused a reduction in AML cells in the blood, spleen and bone marrow. Against a PDX model of melanoma, IGN004 unfused antibody was ineffective while IGN004 fusion protein inhibited tumor growth.