787 Natural killer cell engagers activate innate and adaptive immunity and show synergy with proinflammatory cytokines

BackgroundNatural Killer cell Engagers (NKEs) are multifunctional molecules that target activating receptors on the surface of NK cells, bind to tumor associated antigens and engage Fc gamma receptors expressed on effector cells of the immune system. NKEs promote tumor cell lysis by redirecting NK cells to their targets, and drive activation and proliferation of NK cells. Engagement of NK cells, an effector cell population of the innate immune system, provides an opportunity to target cancers with reduced expression of MHC molecules that are less responsive to therapies targeting the adaptive immune system. Therefore, NKEs have a potential to provide an additional treatment option to patients who respond poorly to T cell tailored immunotherapies.MethodsExpanding on Xencor's XmAb bispecific Fc platform, we developed NKE molecules targeting NKG2D, an activating receptor expressed on cytotoxic immune cells, B7H3, a pan tumor antigen, while simultaneously engaging Fc gamma receptors. Functional activity of NKEs was evaluated via assessing anti-tumor cytotoxicity and activation of NK and T cells in co-culture studies with human cancer cell lines.ResultsNKEs were engineered for synergistic effects on NK cells by the simultaneous engagement of NKG2D and Fc gamma receptors. Additionally, the NKG2D variable domains were selected for their ability to provide a co-stimulatory signal to T cells in the presence of TCR-mediated signaling. Developed NKEs showed cytotoxic activity and immune cell activation in co-culture studies of human cancer cell lines with either PBMCs, T cells or NK cells. Combination of NKEs with proinflammatory cytokines, such as IL15, showed enhancement of the cytotoxic activity against tumor cells and augmented NK cell activation.ConclusionsXmAb bispecific NKEs engineered to engage innate and adaptive immunity show encouraging tumor cell killing activity and synergistic cytotoxicity in combination with proinflammatory cytokines. These data have identified several promising candidate NKEs for future in vivo efficacy studies in mouse tumor models expressing B7H3.

Fc Gamma Receptors as Regulators of Bone Destruction in Inflammatory Arthritis

Bone erosion is one of the primary features of inflammatory arthritis and is caused by excessive differentiation and activation of osteoclasts. Fc gamma receptors (FcγRs) have been implicated in osteoclastogenesis. Our recent studies demonstrate that joint-deposited lupus IgG inhibited RANKL-induced osteoclastogenesis. FcγRI is required for RANKL-induced osteoclastogenesis and lupus IgG-induced signaling transduction. We reviewed the results of studies that analyzed the association between FcγRs and bone erosion in inflammatory arthritis. The analysis revealed the dual roles of FcγRs in bone destruction in inflammatory arthritis. Thus, IgG/FcγR signaling molecules may serve as potential therapeutic targets against bone erosion.

Blockage of Fc Gamma Receptors Alleviates Neuronal and Microglial Toxicity Induced by Palmitic Acid

Background: Palmitic acid (PA) promotes brain pathologies including Alzheimer’s disease (AD)-related proteins, neuroinflammation, and microglial activation. The activation of neurons and microglia via their Fc gamma receptors (FcγRs) results in producing inflammatory cytokines. Objective: To investigate the expression of FcγRs, FcγR signaling proteins, AD-related proteins, proinflammatory cytokines, and cell viability of neurons and microglia in association with PA exposure as well as the effects of FcγR blockade on these parameters in response to PA. Methods: 200 and 400μM PA-conjugated BSA were applied to SH-SY5Y and HMC3 cells for 24 h. For FcγR blockage experiment, both cells were exposed to FcγR blocker before receiving of 200 and 400μM of PA-conjugated BSA for 24 h. Results: PA significantly increased AD-related proteins, including Aβ and BACE1, as well as increasing TNFα, IL-1β, and IL-6 in SH-SY5Y and HMC3 cells. However, the p-Tau/Tau ratio was only increased in SH-SY5Y cells. These results were associated with an increase in FcγRs activation and a decrease in cell viability in both cell types. FcγRs blockage diminished the activation of FcγR in SH-SY5Y and HMC3 cells. Interestingly, blocking FcγRs before PA exposure reduced the increment of AD-related proteins, proinflammatory cytokines caused by PA. FcγRs blocking also inhibits cell death for 23%of SH-SY5Y cells and 64%of HMC3 cells, respectively. Conclusion: These findings suggest that PA is a risk factor for AD via the increased AD-related pathologies, inflammation, FcγRs activation, and brain cell death, while FcγR blockage can alleviate these effects.

Susceptibility to Plasmodium falciparum Malaria: Influence of Combined Polymorphisms of IgG3 Gm Allotypes and Fc Gamma Receptors IIA, IIIA, and IIIB

The binding of immunoglobulin (Ig) to Fc gamma receptors (FcgR) at the immune cell surface is an important step to initiate immunological defense against malaria. However, polymorphisms in receptors and/or constant regions of the IgG heavy chains may modulate this binding. Here, we investigated whether polymorphisms located in FcgR and constant regions of the heavy chain of IgG are associated with susceptibility to P. falciparum malaria. For this purpose, a clinical and parasitological follow-up on malaria was conducted among 656 infants in southern Benin. G3m allotypes (from total IgG3) were determined by a serological method of hemagglutination inhibition. FcgRIIA 131R/H and FcgRIIIA 176F/V genotypes were determined using the TaqMan method and FcgRIIIB NA1/NA2 genotypes were assessed by polymerase chain reaction using allele-specific primers. Association analyses between the number of malaria infections during the follow-up and polymorphisms in IgG G3m allotypes and FcgR were studied independently by zero inflated binomial negative regression. The influence of combinations of G3m allotypes and FcgRIIA/FcgRIIIA/FcgRIIIB polymorphisms on the number of P. falciparum infections, and their potential interaction with environmental exposure to malaria was assessed by using the generalized multifactor dimensionality reduction (GMDR) method. Results showed that individual carriage of G3m24 single allotype and of G3m5,6,10,11,13,14,24 phenotype was independently associated with a high risk of malaria infection. A risk effect for G3m6 was observed only under high environmental exposure. FcgRIIIA 176VV single genotype and combined carriage of FcgRIIA 131RH/FcgRIIIA 176VV/FcgRIIIB NA1NA2, FcgRIIA 131HH/FcgRIIIA 176FF/FcgRIIIB NA1NA1, FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA2NA2 and FcgRIIA 131HH/FcgRIIIA 176VV/FcgRIIIB NA1NA2 genotypes were related to a high number of malaria infections. The risk was accentuated for FcgRIIIA 176VV when considering the influence of environmental exposure to malaria. Finally, the GMDR analysis including environmental exposure showed strengthened associations with a malaria risk when FcgRIIA/FcgRIIIA/FcgRIIIB genotypes were combined to G3m5,6,11,24 and G3m5,6,10,11,13,15,24 phenotypes or G3m10 and G3m13 single allotypes. Our results highlight the relevance of studying IgG heavy chain and FcgR polymorphisms, independently as well as in combination, in relation to the individual susceptibility to P. falciparum infection. The intensity of individual exposure to mosquito bites was demonstrated to impact the relationships found.

Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies

A variety of Fc domain engineering approaches for abrogating the effector functions of mAbs exists. To address some of the limitations of the current Fc domain silencing approaches, we are exploring a less commonly considered option which relies on the deletion of the hinge. Removal of the hinge domain in humanized IgG1 and IgG4 mAbs obliterates their ability to bind to activating human Fc gamma receptors I and IIIA, while leaving their ability to engage their target antigen intact. Deletion of the hinge also reduces binding to the Fc neonatal receptor, although Fc engineering allows partial recovery of affinity. Engineering of the CH3 domain, stabilizes hinge deleted IgG4s and prevents Fab arm exchange. The faster clearing properties together with the pacified Fc make modality of the hinge deleted mAb an appealing solution for therapeutic and diagnostic applications.

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

ABSTRACTCancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fc gamma receptors or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. Yet how these competing signals are integrated is poorly understood mechanistically, raising questions about how to effectively titrate immune responses. Here we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand on targets over a broad range of absolute molecular densities. Using endogenous as well as chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering this ratio reduces FcγR phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα in vitro, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.