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Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, FcγRIIB (CD32B), is leading to the development of improved mAb treatments.

Background Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (Fc[gamma]R) and impaired by the single inhibitory Fc[gamma]R, Fc[gamma]RIIb. Methods We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. Results We report that TAMs are Fc[gamma]RIIb.sup.bright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate Fc[gamma]RIIb. This enhanced Fc[gamma]RIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human Fc[gamma]RIIb.sup.+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of Fc[gamma]RIIb can partially restore phagocytic function in human monocytes. Conclusion Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of Fc[gamma]RIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies. Keywords: Hypoxia, Hypoxia inducible factors, Fc[gamma]RIIb, Fc gamma receptors, Tumor-associated macrophages, Monocytes, Monoclonal antibody, Tumor microenvironment, Resistance, Cancer

Background Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. Methods We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. Results We report that TAMs are FcγRIIb bright relative to healthy tissue counterparts and under hypoxic conditions , mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb + / + transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. Conclusion Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Issue Title: Antibodies: Assembly to Secretion, and Receptors as Therapeutic Targets Proceedings from the 4th International Forum on Immunology Research Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, FcγRIIB (CD32B), is leading to the development of improved mAb treatments.

Complement-dependent cytotoxicity (CDC) is a primary mechanism-of-action of monoclonal antibody (mAb) immunotherapies used to treat haematological cancers, including rituximab and daratumumab. However, mAb efficacy may be limited by reduced bioavailability of complement C1q – which activates the complement classical pathway following interactions with mAb-opsonised target cells. C1q is secreted by phagocytes upon recruitment to sites of muscle damage to facilitate muscular repair, hence we hypothesised that muscle damaging exercise may increase C1q ‘spill-over’ into blood. Additionally, other complement proteins (e.g., C1s) have been reported to increase following ultra-endurance and resistance exercise. Taken together, we hypothesised that muscle damaging exercise could be harnessed to enhance mAb-mediated CDC. In this study, n  = 8 healthy males (28 ± 5-years) completed two 45-minute treadmill running protocols: (1) a flat running protocol at a speed 15% above anaerobic threshold, and (2) a downhill running protocol (− 10% slope) at the same speed. Blood samples were collected before, immediately after, and 1-hour, 24-hours, 2-days, and 4-days after exercise. Isolated serum was assessed for C1q by ELISA, and used to measure mAb (rituximab, daratumumab) mediated CDC against two haematological cancer cell lines (Raji, RPMI-8226) in vitro. Isolated plasma was assessed for markers of inflammation (C-reactive protein [CRP]), and muscle damage (creatine kinase [CK]) by turbidimetry. C1q and CDC activity were not different between running protocols and did not change over time ( p  > 0.05). Significantly greater perceived muscle soreness ( p  < 0.001) and fluctuations observed from baseline to 24-hours post-exercise in the downhill running trial in CK (+ 171%) and CRP (+ 66%) suggests some degree of muscle damage was present. It is possible that any increase in C1q post-exercise may have been masked by the increase and subsequent interaction with CRP, which utilises C1q to facilitate muscular repair. This is the first study to investigate whether exercise can increase circulating C1q and improve mAb-mediated CDC and our findings show that downhill running exercise does not increase circulating C1q nor improve CDC in vitro.

BackgroundImmune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of patients with cancer including those with poorly immune infiltrated ‘cold’ tumors are resistant to currently available ICB therapies. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is one of few clinically validated targets for ICB, but toxicities linked to efficacy in approved αCTLA-4 regimens have restricted their use and precluded full therapeutic dosing. At a mechanistic level, accumulating preclinical and clinical data indicate dual mechanisms for αCTLA-4; ICB and regulatory T cell (Treg) depletion are both thought to contribute efficacy and toxicity in available, systemic, αCTLA-4 regimens. Accordingly, strategies to deliver highly effective, yet safe αCTLA-4 therapies have been lacking. Here we assess and identify spatially restricted exposure to a novel strongly Treg-depleting, checkpoint-blocking, vectorized αCTLA-4, as a highly efficacious and potentially safe strategy to target CTLA-4.MethodsA novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for monoclonal antibodies (mAbs) and targets associated with superior Treg-depleting activity. A tumor-selective oncolytic vaccinia vector was then engineered to encode this novel, strongly Treg-depleting, checkpoint-blocking, αCTLA-4 antibody or a matching surrogate antibody, and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (VVGM-αCTLA-4).ResultsThe identified 4-E03 antibody showed significantly stronger Treg depletion, but equipotent checkpoint blockade, compared with clinically validated αCTLA-4 ipilimumab against CTLA-4-expressing Treg cells in a humanized mouse model in vivo. Intratumoral administration of VVGM-αCTLA-4 achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion, which elicited antigen cross-presentation and stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody therapy. Efficacy correlated with FcγR-mediated intratumoral Treg depletion. Remarkably, in a clinically relevant mouse model resistant to systemic ICB, intratumoral VVGM-αCTLA-4 synergized with αPD-1 to reject cold tumors.ConclusionOur findings demonstrate in vivo proof of concept for spatial restriction of Treg depletion-optimized immune checkpoint blocking, vectorized αCTLA-4 as a highly effective and safe strategy to target CTLA-4. A clinical trial evaluating intratumoral VVGM-αhCTLA-4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has commenced.

BackgroundBI-1808 is a human IgG1 monoclonal antibody targeting TNFR2 by blocking the interaction of TNFR2 with its ligand TNF-α, confering FcγR-dependent depletion of intratumoral Tregs and mediating expansion of intratumoral CD8+ T cells. Upon co-administration of BI-1808 and anti-PD-1 surrogate antibodies to immunocompetent tumor-bearing mice, with partial sensitivity to checkpoint blockade, complete cures were observed in all treated mice, indicating a potentially synergistic activity.MethodsSafety and tolerability of BI-1808 as a single agent and in combination with pembrolizumab is currently investigated in the Phase 1/2a trial 19-BI-1808–01 in patients with advanced malignancies or cutaneous T-cell lymphoma (CTCL). The trial consists of Phase 1 Parts A and B (dose escalation with single agent and combination with pembrolizumab, respectively), and Phase 2a Parts A and B (dose expansion with single agent and combination therapy, respectively). Dose escalation uses a modified toxicity probability interval-2 protocol (mTPI-2), investigating ascending dose levels of 25–1000 mg every three weeks (Q3W). Dose escalation aims to select both single agent RP2D and combination RP2D of BI-1808 for Phase 2a.Patients are sampled for pharmacokinetics (PK) of BI-1808, antidrug-antibodies and pharmacodynamics including lymphocyte subsets, regulatory T cells, memory T-cells, soluble TNFR2 serum concentration (sTNFR2) and BI-1808 receptor occupancy (RO).ResultsAs of June 19th, 2023, 24 subjects with various advanced solid malignancies received doses of up to 1000 mg BI-1808 as single-agent treatment, and 7 subject received 225 mg doses of BI-1808 with pembrolizumab.Across the completed monotherapy arm, no Grade 3/4 AEs, AEs related to BI-1808 and no DLTs were observed. No MTD was defined. The number of potentially related AEs of Gr 1/2 are evenly distributed across the dose range, with no target system organ class of special notice identified. Best clinical response recorded are stable disease (SD) in 7/19 evaluable patients in the monotherapy arm. The first dose cohort for BI-1808 at 225 mg in combination with pembrolizumab is currently ongoing.BI-1808 exhibits a non-linear PK. At doses > 675 mg Q3W, t½ was approximately 1 week resulting in accumulation of drug, with complete RO throughout the dosing interval.ConclusionsPreliminary data from the BI-1808 monotherapy arm from the clinical trial 19-BI-1808–01 is promising. BI-1808 has a favorable safety profile, with no DLTs observed. SD was observed in 7/19 evaluable patients. Doses of 675 mg and higher are expected to provide complete RO throughout the dose interval, and will be further explored in Ph2a.