The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fc; receptors (Fc;R). Although an early goal has been enhanced Fc;RIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as Fc;RIIa. Here, we describe a set of engineered Fc variants with diverse Fc;R affinities, including a novel substitution G236A that provides selectively enhanced binding to Fc;RIIa relative to Fc;RIIb. Variants containing this substitution have up to 70-fold greater Fc;RIIa affinity and 15-fold improvement in Fc;RIIa/Fc;RIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual Fc;R to effector functions mediated by NK cells and macrophages. These experiments show that Fc;RIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor Fc;RIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complement-enhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.

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IntroductionIn the past decade, monoclonal antibodies directed against B-cell antigens have proven beneficial for patients with B-lineage neoplasms. Approved immunotherapeutics include the anti-CD52 antibody alemtuzumab (Campath-1H), 1 2 anti-CD20 antibodies, rituximab (Rituxan) 2 and most recently ofatumumab (Arzerra), 3 and 2 anti-CD20 conjugates carrying radioactive isotopes, 90 Yibritumomab tiuxetan (Zevalin) 4 and 131 I-tositumomab (Bexxar). 5 In particular, when used as a single agent or in combination with chemotherapy, rituximab has shown efficacy and relative safety in many B-cell non-Hodgkin lymphomas (NHLs) and some B-cell leukemias. 2,6 However, some patients do not respond to rituximab, and many of those that do will relapse. 2 In addition, some B-cell tumors either lack CD20 expression or lose it during rituximab treatment. 7 Thus, antibodies targeting other B-cell antigens are warranted, and several are being explored, including those targeting CD40, a 45-to 50-kDa transmembrane glycoprotein belonging to the tumor necrosis factor receptor family.CD40 is broadly expressed on hematologic cells, including B cells and on several types of epithelial and endothelial cells. [8][9][10] The ligand of CD40, CD40L (CD154), is preferentially expressed on activated T cells but has also been found on other hematologic cell types. Signaling via the CD40 pathway plays a key role in defining cellular and humoral immune responses. 10 CD40 is overexpressed by the malignant counterparts to Blineage cells and is found on most mature B-cell malignancies, including NHL, Hodgkin disease, and chronic lymphocytic leukemia (CLL), and on some early acute lymphocytic leukemias (ALL) 8,11,12 ; it is also expressed on multiple myeloma (MM) cells. 13 Many solid tumors express CD40, including melanoma and carcinomas of the kidney, bladder, lung, pancreas, colon, prostate, ovary, breast, head, and neck. 14 CD40 agonists have been shown to trigger immune responses against various tumors 15,16 and to inhibit the growth of different neoplastic cells, both in vitro and in vivo. 17,18 Broad stimulation of the immune system and expression on a wide range of malignancies make CD40 an attractive target for antibody-based immunotherapy. Over the past 2 decades, several CD40-specific antibodies have been tested against B-lineage malignancies in vitro and in animal models, yielding various degrees of success. [19][20][21][22][23] Recently, 2 humanized antibodies have emerged for further development: a mild CD40 agonist, 20,24,25 and a CD40 antagonist, HCD122 (CHIR-12.12). 21,23 These agents were evaluated in phase 1 and 2 trials in patients with relapsed MM, NHL, and CLL. Thus far, they have demonstrated a favorable safety profile and have shown some antitumor activity in NHL and MM. [26][27][28] In addition, a strongly agonistic anti-CD40 antibody, CP-870,893, evaluated in phase 1 trials in patients with melanoma and other solid tumors, was found to be well tolerated and to have antitumor activity. 29 Therefore, preclinical and early clinic...

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.