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 Bcell malignancies. Interferon-alpha (IFN␣) 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 IFN␣. Fusion proteins had reduced IFN␣ activity in vitro compared with native IFN␣, 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 immunocom- IntroductionThe anti-CD20 antibody rituximab (C2B8/Rituxan; Genentech/ Biogen-IDEC) has substantially improved treatment outcomes in B-cell non-Hodgkin lymphomas (NHLs), achieving high response rates in low-grade B-cell lymphomas, 1 and improving survival in both indolent and aggressive lymphomas in combination with chemotherapy. 2,3 However, many tumors do not respond to or relapse after rituximab-based therapies. 4 Thus, new approaches are needed to improve anti-CD20 efficacy and overcome rituximab resistance.The in vivo antilymphoma effects of rituximab are believed to be mediated by antibody dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), induction of apoptosis in tumor cells, and recruitment of T cells responding to tumor antigens released upon antibody-mediated tumor lysis. [5][6][7] Clinical studies have suggested that ADCC plays a dominant role in rituximab action in humans. 8,9 Thus, attempts have been made to boost rituximab-mediated ADCC by activation of Fc receptorbearing natural killer (NK) cells, monocytes/macrophages, or granulocytes via systemic administration of cytokines such as interleukin-2, interleukin-12, or granulocyte-macrophage colonystimulating factor, 10-12 with limited efficacy. None of these trials involving systemic administration of cytokines offered a clear advantage over the expected efficacy of rituximab alone, likely due to the inability of systemically administered agents to achieve high concentrations within the tumor bed.Interferon-alpha (IFN␣), a member of the type I interferon family (␣, ␤, ), is a pleiotropic cytokine with attractive features for combination with rituximab in treating NHL. 13,14 Beneficial properties of IFN␣ against NHL and other cancers include direct antiproliferative and proapoptotic effects, 15-17 blockade of autocrine growth factor loops, 18 repression of c-myc oncogene expression, 19 down-regulation of telomerase activity, 20 and inhibition of angiogenesis. 21 Favorable immunologic effects of IFN␣ for lymphoma treatment...

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 (Rx) 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 (ROI) analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to asses 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 (RIT), and prediction of response to therapy.

Type I interferons (IFNα/β) are cytokines with a broad spectrum of anti-tumor activities including anti-proliferative, pro-apoptotic, and immunostimulatory effects, and are potentially useful in the treatment of B cell malignancies and other cancers. To improve anti-tumor potency and diminish the systemic side effects of IFN, we recently developed anti-CD20-IFNα fusion proteins with in vitro and in vivo efficacy against both mouse and human lymphomas expressing CD20. Since IFNβ binds more tightly to the IFNα/β receptor (IFNAR) and has more potent anti-tumor activities, we have now constructed an anti-CD20 fusion protein with murine IFNβ (mIFNβ). Anti-CD20-mIFNβ was more potent than recombinant mIFNβ and anti-CD20-mIFNα 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-mIFNβ required the physical linkage of mIFNβ to anti-CD20 antibody (Ab). Importantly, anti-CD20-mIFNβ 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 non-targeted mIFNβ fusion protein, yet treatment with anti-CD20-mIFNβ eradicated 83% of tumors. Anti-CD20-mIFNβ was also more potent in vivo against 38C13-huCD20 than anti-CD20-mIFNα, curing 75% versus 25% of tumors (p = 0.001). Importantly, while anti-CD20-mIFNα could not eradicate 38C13-huCD20 IFNARlo tumors, anti-CD20-mIFNβ treatment prolonged survival (p = 0.0003), and some animals remained tumor-free. Thus, Ab fusion proteins targeting mIFNβ to tumors show promise as therapeutic agents, especially for use against tumors resistant to the effects of mIFNα.

IntroductionAmong human cancers, B-cell lymphomas appear among the most susceptible to immunotherapeutic strategies, because of their high rate of response to monoclonal antibodies (mAbs) targeting the B-cell differentiation antigen CD20 and encouraging results from early phase clinical trials of tumor-specific therapeutic vaccines. 1 The availability of both passive and active immunotherapeutic agents against B-cell lymphomas has made them an important testing ground for the development of clinically effective immunotherapies in humans. [1][2][3] The best characterized target for active immunotherapy of B-cell lymphoma is tumor-specific immunoglobulin (idiotype, Id). 4 Immunization of patients with Id protein derived from their own tumors can elicit humoral and T cell-mediated immune responses associated with improvements in survival and tumor burden. [5][6][7][8] Traditional Id vaccines consist of Id protein chemically conjugated to the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH) and injected together with an immunologic adjuvant. 1 Because of their potent antigenpresenting properties, 9 dendritic cells (DCs) have been used to augment lymphoma vaccine effectiveness, and durable tumor regressions have been observed after immunization with Id-loaded DCs. 10,11 Granulocytemacrophage colony-stimulating factor (GM-CSF), a DC growth and maturation factor, has also been used as an effective adjuvant in Id-KLH vaccines. 4,7,12 However, despite the elegant nature of the Id vaccine approach, shortcomings of this strategy include the requirement of producing a custom-made protein for each patient and limitation of the antitumor response to a single antigen. In contrast, vaccines using whole tumor cells offer the opportunity to elicit immunity against the entire collection of antigens expressed by the tumor. Pulsed DC vaccination using apoptotic tumor cells or lysates has emerged as a popular strategy for immunization against tumors in a variety of preclinical and human studies. While killed tumor cells in the form of apoptotic bodies or freeze-thaw lysates alone display limited immunogenicity, DCs loaded with these preparations have been found to elicit antitumor immunity in a variety of preclinical models [13][14][15][16] and early clinical trials. [17][18][19][20][21] Other strategies using DCs to present the full repertoire of tumor antigens expressed by tumor cells include fusion with tumor cells 22 or pulsing with tumorderived RNA. 23 The goal of these approaches is to achieve processing and presentation of exogenous cell-derived antigenic peptides by professional antigen-presenting cells (ie, "crosspresentation"), thereby evoking a CD8 ϩ T-cell antitumor response. 24 One attractive strategy for increasing tumor antigen crosspresentation is the targeting of IgG-complexed antigens into DCs via Fc␥ receptors. 25 Antigen-antibody complexes internalized via Fc␥ receptors at the DC surface efficiently enter both the MHC class I 26-28 and class II 29,30 antigen-presentation pathways. Several investi...

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.