Mutation of the TGN1412 anti-CD28 monoclonal antibody lower hinge confers specific FcγRIIb binding and retention of super-agonist activity.

The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγRs and agonistic action was consequentially lost, confirming the FcγR dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilizing mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies retained their super-agonistic ability, demonstrating that CD28- and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signaling.

Harnessing the immune system via FcγR function in immune therapy: a pathway to next-gen mAbs.

The human fragment crystallizable (Fc)γ receptor (R) interacts with antigen-complexed immunoglobulin (Ig)G ligands to both activate and modulate a powerful network of inflammatory host-protective effector functions that are key to the normal physiology of immune resistance to pathogens. More than 100 therapeutic monoclonal antibodies (mAbs) are approved or in late stage clinical trials, many of which harness the potent FcγR-mediated effector systems to varying degrees. This is most evident for antibodies targeting cancer cells inducing antibody-dependent killing or phagocytosis but is also true to some degree for the mAbs that neutralize or remove small macromolecules such as cytokines or other Igs. The use of mAb therapeutics has also revealed a "scaffolding" role for FcγR which, in different contexts, may either underpin the therapeutic mAb action such as immune agonism or trigger catastrophic adverse effects. The still unmet therapeutic need in many cancers, inflammatory diseases or emerging infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires increased effort on the development of improved and novel mAbs. A more mature appreciation of the immunobiology of individual FcγR function and the complexity of the relationships between FcγRs and antibodies is fueling efforts to develop more potent "next-gen" therapeutic antibodies. Such development strategies now include focused glycan or protein engineering of the Fc to increase affinity and/or tailor specificity for selective engagement of individual activating FcγRs or the inhibitory FcγRIIb or alternatively, for the ablation of FcγR interaction altogether. This review touches on recent aspects of FcγR and IgG immunobiology and its relationship with the present and future actions of therapeutic mAbs.

The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

Anti-EGFR Antibody-Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth.

PURPOSE: Anti-EGFR antibodies show limited response in breast cancer, partly due to activation of compensatory pathways. Furthermore, despite the clinical success of cyclin-dependent kinase (CDK) 4/6 inhibitors in hormone receptor-positive tumors, aggressive triple-negative breast cancers (TNBC) are largely resistant due to CDK2/cyclin E expression, whereas free CDK2 inhibitors display normal tissue toxicity, limiting their therapeutic application. A cetuximab-based antibody drug conjugate (ADC) carrying a CDK inhibitor selected based on oncogene dysregulation, alongside patient subgroup stratification, may provide EGFR-targeted delivery. EXPERIMENTAL DESIGN: Expressions of G1/S-phase cell cycle regulators were evaluated alongside EGFR in breast cancer. We conjugated cetuximab with CDK inhibitor SNS-032, for specific delivery to EGFR-expressing cells. We assessed ADC internalization and its antitumor functions in vitro and in orthotopically grown basal-like/TNBC xenografts. RESULTS: Transcriptomic (6,173 primary, 27 baseline, and matched post-chemotherapy residual tumors), single-cell RNA sequencing (150,290 cells, 27 treatment-naïve tumors), and spatial transcriptomic (43 tumor sections, 22 TNBCs) analyses confirmed expression of CDK2 and its cyclin partners in basal-like/TNBCs, associated with EGFR. Spatiotemporal live-cell imaging and super-resolution confocal microscopy demonstrated ADC colocalization with late lysosomal clusters. The ADC inhibited cell cycle progression, induced cytotoxicity against high EGFR-expressing tumor cells, and bystander killing of neighboring EGFR-low tumor cells, but minimal effects on immune cells. Despite carrying a small molar fraction (1.65%) of the SNS-032 inhibitor, the ADC restricted EGFR-expressing spheroid and cell line/patient-derived xenograft tumor growth. CONCLUSIONS: Exploiting EGFR overexpression, and dysregulated cell cycle in aggressive and treatment-refractory tumors, a cetuximab-CDK inhibitor ADC may provide selective and efficacious delivery of cell cycle-targeted agents to basal-like/TNBCs, including chemotherapy-resistant residual disease.

Anti-cancer pro-inflammatory effects of an IgE antibody targeting the melanoma-associated antigen chondroitin sulfate proteoglycan 4.

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

CDK Inhibition Primes for Anti-PD-L1 Treatment in Triple-Negative Breast Cancer Models.

Triple-negative breast cancers (TNBC) expressing PD-L1 qualify for checkpoint inhibitor immunotherapy. Cyclin E/CDK2 is a potential target axis in TNBC; however, small-molecule drugs at efficacious doses may be associated with toxicity, and treatment alongside immunotherapy requires investigation. We evaluated CDK inhibition at suboptimal levels and its anti-tumor and immunomodulatory effects. Transcriptomic analyses of primary breast cancers confirmed higher cyclin E/CDK2 expression in TNBC compared with non-TNBC. Out of the three CDK2-targeting inhibitors tested, the CDK 2, 7 and 9 inhibitor SNS-032 was the most potent in reducing TNBC cell viability and exerted cytotoxicity against all eight TNBC cell lines evaluated in vitro. Suboptimal SNS-032 dosing elevated cell surface PD-L1 expression in surviving TNBC cells. In mice engrafted with human immune cells and challenged with human MDA-MB-231 TNBC xenografts in mammary fat pads, suboptimal SNS-032 dosing partially restricted tumor growth, enhanced the tumor infiltration of human CD45+ immune cells and elevated cell surface PD-L1 expression in surviving cancer cells. In tumor-bearing mice engrafted with human immune cells, the anti-PD-L1 antibody avelumab, given sequentially following suboptimal SNS-032 dosing, reduced tumor growth compared with SNS-032 alone or with avelumab without prior SNS-032 priming. CDK inhibition at suboptimal doses promotes immune cell recruitment to tumors, PD-L1 expression by surviving TNBC cells and may complement immunotherapy.

Tumor-Infiltrating B Lymphocyte Profiling Identifies IgG-Biased, Clonally Expanded Prognostic Phenotypes in Triple-Negative Breast Cancer.

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20+CD27+IgD- isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

The Human FcγRII (CD32) Family of Leukocyte FcR in Health and Disease.

FcγRs have been the focus of extensive research due to their key role linking innate and humoral immunity and their implication in both inflammatory and infectious disease. Within the human FcγR family FcγRII (activatory FcγRIIa and FcγRIIc, and inhibitory FcγRIIb) are unique in their ability to signal independent of the common γ chain. Through improved understanding of the structure of these receptors and how this affects their function we may be able to better understand how to target FcγR specific immune activation or inhibition, which will facilitate in the development of therapeutic monoclonal antibodies in patients where FcγRII activity may be desirable for efficacy. This review is focused on roles of the human FcγRII family members and their link to immunoregulation in healthy individuals and infection, autoimmunity and cancer.

The Rare Anaphylaxis-Associated FcγRIIa3 Exhibits Distinct Characteristics From the Canonical FcγRIIa1.

FcγRIIa is an activating FcγR, unique to humans and non-human primates. It induces antibody-dependent proinflammatory responses and exists predominantly as FcγRIIa1. A unique splice variant, we designated FcγRIIa3, has been reported to be associated with anaphylactic reactions to intravenous immunoglobulins (IVIg) therapy. We aim to define the functional consequences of this FcγRIIa variant associated with adverse responses to IVIg therapy and evaluate the frequency of associated SNPs. FcγRIIa forms from macaque and human PBMCs were investigated for IgG-subclass specificity, biochemistry, membrane localization, and functional activity. Disease-associated SNPs were analyzed by sequencing genomic DNA from 224 individuals with immunodeficiency or autoimmune disease. FcγRIIa3 was identified in macaque and human PBMC. The FcγRIIa3 is distinguished from the canonical FcγRIIa1 by a unique 19-amino acid cytoplasmic insertion and these two FcγRIIa forms responded distinctly to antibody ligation. Whereas FcγRIIa1 was rapidly internalized, FcγRIIa3 was retained longer at the membrane, inducing greater calcium mobilization and cell degranulation. Four FCGR2A SNPs were identified including the previously reported intronic SNP associated with anaphylaxis, but in only 1 of 224 individuals. The unique cytoplasmic element of FcγRIIa3 delays internalization and is associated with enhanced cellular activation. The frequency of the immunodeficiency-associated SNP varies between disease populations but interestingly occurred at a lower frequency than previously reported. None-the-less enhanced FcγRIIa3 function may promote a proinflammatory environment and predispose to pathological inflammatory responses.