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Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti–4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor–mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP–4-1BBL (RG7826) and CD19–4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen–mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP–4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer–bearing rhesus monkey. Combination of FAP–4-1BBL with tumor antigen–targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP– or CD19–4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP– and CD19–4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes—M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Therapeutic monoclonal antibodies contribute to elimination of tumor cells by different mechanisms including blocking of receptor signaling, induction of antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and induction of adaptive immunity. FcγR-expressing monocytes and macrophages have recently been identified as important immune effectors contributing to the activity of monoclonal antibodies in vivo. Therefore, we analyzed the FcγR-dependent effector functions mediated by both cell subsets upon treatment with glycoengineered (GE) antibodies (having enhanced FcγRIIIa (CD16a) binding affinity), wild-type (WT), and Fc-mutated antibodies that lack binding to FcγRs (P329G LALA mutation). Trastuzumab, cetuximab, rituximab and ofatumumab were used as WT antibodies, whereas GE trastuzumab, GA201 (targeting EGFR) and GA101 (targeting CD20) were used as GE antibodies. To better reflect the plasticity of myeloid lineage cells we evaluated the activities on classical and non-classical/intermediate monocytes, M1 and M2c macrophages. We found that GE and WT antibodies displayed comparable binding and induced similar monocyte/macrophage-mediated ADCC and ADCP in absence of unspecific endogenous IgGs. Interestingly, in their presence, i.e. in a situation that more closely mimics physiological conditions, GE antibodies displayed significantly superior binding and induced higher levels of ADCC/ADCP compared to WT antibodies. Data collected by imaging further indicated that the degree of phagocytized cells is higher and faster in presence of GE antibodies and that M2c macrophages perform serial phagocytosis by engulfing 2-3 tumor cells. Fc-mutated P329G LALA variant showed no binding and no functional activity. A direct comparison of the three anti-CD20 antibodies (GA101, rituximab and ofatumumab), pointed out that GA101 mediated significantly stronger elimination of tumor cells further underscoring the relevance of combination of both glycoengineering and cell death induction for monocyte/macrophage-mediated killing of tumor cells. These data show, for the first time that, in addition to enhancing NK cell-mediated cytotoxicity (ADCC), glycoengineering also enhances monocyte and macrophage cytotoxic and phagocytic activities through enhanced binding to FcγRIIIa under conditions that more closely resemble the physiological setting. Citation Format: Herter Sylvia, Christian Klein, Martina Birk, Thomas Weber, Pablo Umana, Marina Bacac. Modulation of monocyte- and macrophage-mediated antibody-dependent cell phagocytosis and cytotoxicity (ADCP/ADCC) by Fc engineering of therapeutic antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3631. doi:10.1158/1538-7445.AM2014-3631