Human Fcγ Receptors Research Articles

Cellular Membrane-Derived Nanovesicles Expressing hCD64 for Targeting Prostate Cancer.

Extracellular vesicles are ideal therapeutic potentiators for various diseases. However, they commonly lack targeting capability and are rapidly cleared by phagocytes. This requires appropriate administration at high doses, which can lead to toxic and adverse reactions. To overcome these limitations, we developed bleb nanovesicles containing human Fcγ receptor I (hCD64), known for their strong affinity to monomeric IgG. In this study, we focused on prostate cancer, which has a specific membrane antigen. We have utilized the hCD64-expressing bleb nanovesicles attaching anti-prostate-specific membrane antigen (PSMA) antibodies and confirmed their targeting ability in PSMA-related cell lines and prostate cancer xenograft models. Our findings underscore the promising potential of nanovesicle Fcγ receptor-IgG as a platform for cancer diagnosis and therapy systems, inspiring further research.

Site-Specific Glycosylation Analysis of Murine and Human Fcγ Receptors Reveals High Heterogeneity at Conserved N-Glycosylation Site.

Fc γ-receptors (FcγRs) on leukocytes bind immunoglobulin G (IgG) immune complexes to mediate effector functions. Dysregulation of FcγR-mediated processes contributes to multiple inflammatory diseases, including rheumatoid arthritis, lupus, and immune thrombocytopenia. Critically, immunoregulatory N-glycan modifications on both FcγRs and IgGs alter FcγR-IgG binding affinity. Rapid methods for the characterization of N-glycans across multiple Fcγ receptors are needed to propel investigations into disease-specific contributions of FcγR N-glycans. Here, we utilize nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) to characterize FcγR glycosylation and report quantitative and site-specific N-glycan characterization of recombinant human FcγRI, FcγRIIIA V158, and FcγRIIIA F158 from CHO cells and murine FcγRI, FcγRIII, and FcγRIV from NS0 cells. Data are available via ProteomeXchange with identifier PXD043966. Broad glycoform distribution (≥30) was observed at mouse FcγRIV site N159 and human FcγRIIIA site N162, an evolutionarily conserved site. Further, mouse FcγRIII N-glycopeptides spanning all four predicted N-glycosylation sequons were detected. Glycoform relative abundances for hFcγRIIIA V/F158 polymorphic variants are reported, demonstrating the clinical potential of this workflow to measure differences in glycosylation between common human FcγRIIIA allelic variants with disease-associated outcomes. The multi-Fcγ receptor glycoproteomic workflow reported here will empower studies focused on the role of FcγR N-glycosylation in autoimmune diseases.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p < 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p < 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p < 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.

Initial findings from a first-in-human, multicenter, open-label study of ATOR-1017, a 4-1BB antibody, in patients with advanced solid malignancies.

2529 Background: ATOR-1017 is a human agonist Fcγ-receptor cross-linking dependent IgG4 antibody targeting the co-stimulatory receptor 4-1BB (CD137). ATOR-1017 activates T cells and natural killer cells in the tumor environment, leading to immune-mediated tumor cell killing. Methods: In this first-in-human, dose escalation, multicenter, phase 1 study (NCT04144842), adult patients with solid tumors refractory to standard therapy were enrolled in single patient cohorts for doses up to 40 mg, and thereafter in cohorts of 3-6 patients, to receive ATOR-1017 in a 21-day cycle. Intra-patient dose escalation of ATOR-1017 is allowed depending on the occurrence of Dose-Limiting Toxicities (DLTs) during cycle 1. ATOR-1017 is administered intravenously as monotherapy every three weeks until unacceptable toxicity, progressive disease, or withdrawal of consent. The primary objectives of the study are to determine the maximum tolerated dose, assess adverse events (AEs), evaluate DLTs, and to determine the recommended phase 2 dose. Secondary objectives include pharmacokinetics (PK), immunogenicity, and clinical efficacy, assessed with CT scans using Immune Response Evaluation Criteria in Solid Tumors (iRECIST). Exploratory objectives include assessment of pharmacodynamic (PD) biomarkers. Results: 22 patients (18 females), median age 55 years (34-76) with solid tumors, previously treated with median of 4 (0-6) lines of chemotherapy and/or 2 (0-3) lines of immunotherapy, were included and received at least one cycle ATOR-1017. Nine dose levels have been evaluated; 0.38 mg, 1.5 mg, 5 mg, 15 mg; 40 mg, 100 mg, 200 mg, 360 mg, and 600 mg and dose escalation is ongoing. Treatment-related (TRAEs) were reported in 12 out of 22 patients (54.5%); most common (≥10%) were fatigue (13.6%) and neutropenia (13.6%). Five patients experienced a grade 3-4 TRAE; neutropenia (n = 2), febrile neutropenia (n = 1), chest pain (n = 1), increased liver enzymes (n = 1) and leukopenia/thrombocytopenia (n = 1). No patients discontinued due to TRAEs, no DLTs were observed, and the maximum tolerated dose has not been reached. The median time for patients in the study was 12 weeks (range 4-67). As per data cut-off, January 18, 2022, four patients remained on treatment and 18 patients had discontinued treatment. The reasons for discontinuation include confirmed disease progression (n = 10), clinical deterioration (n = 4), withdrawal of consent (n = 1), death due to disease progression (n = 3). Preliminary PK data showed dose-proportional kinetics. Dose-dependent increase in PD biomarkers demonstrated target-mediated biological activity and proof-of-mechanism. The best response was stable disease observed in a total of 10 patients (45%). Conclusions: ATOR-1017 is safe and well-tolerated at doses up to 600 mg and has shown biologic activity. No DLTs have been reported so far and dose escalation continues. Clinical trial information: NCT04144842.

An Afucosylated Anti-CD32b Monoclonal Antibody Induced Platelet-Mediated Adverse Events in a Human Fcγ Receptor Transgenic Mouse Model and Its Potential Human Translatability.

To assess the safety and tolerability of NVS32b, a monoclonal, afucosylated, anti-CD32b (FCGR2B) antibody, we used a humanized transgenic (Tg) mouse model that expresses all human Fc gamma receptors (FCGRs) while lacking all mouse FCGRs. Prior to its use, we extensively characterized the model. We found expression of all human FCGRs in a pattern similar to humans with some exceptions, such as low CD32 expression on T cells (detected with the pan CD32 antibody but more notably with the CD32b-specific antibody), variation in the transgene copy number, integration of additional human genes, and overall higher expression of all FCGRs on myeloid cells compared to human. Unexpectedly, NVS32b induced severe acute generalized thrombosis in huFCGR mice upon iv dosing. Mechanistic evaluation on huFCGR and human platelets revealed distinct binding, activation, and aggregation driven by NVS32b in both species. In huFCGR mice, the anti-CD32b antibody NVS32b binds platelet CD32a via both Fc and/or complementarity determining region (CDR) causing their activation while in human, NVS32b binding requires platelet preactivation and interaction of platelet CD32a via the Fc portion and an unknown platelet epitope via the CDR portion of NVS32b. We deemed the huFCGR mice to be overpredictive of the NVS32b-associated human thrombotic risk.

SAR442085, a novel anti-CD38 antibody with enhanced antitumor activity against multiple myeloma

Anti-CD38 monoclonal antibodies (mAbs) represent a breakthrough in the treatment of multiple myeloma (MM), yet some patients fail to respond or progress quickly with this therapy, highlighting the need for novel approaches. In this study we compared the preclinical efficacy of SAR442085, a next-generation anti-CD38 mAb with enhanced affinity for activating Fcγ receptors (FcγR), with first-generation anti-CD38 mAb daratumumab and isatuximab. In surface plasmon resonance and cellular binding assays, we found that SAR442085 had higher binding affinity than daratumumab and isatuximab for FcγRIIa (CD32a) and FcγRIIIa (CD16a). SAR442085 also exhibited better in vitro antibody-dependent cellular cytotoxicity (ADCC) against a panel of MM cells expressing variable CD38 receptor densities including MM patients' primary plasma cells. The enhanced ADCC of SAR442085 was confirmed using NK-92 cells bearing low and high affinity FcγRIIIa (CD16a)-158F/V variants. Using MM patients' primary bone marrow cells, we confirmed that SAR442085 had an increased ability to engage FcγRIIIa, resulting in higher natural killer (NK) cell activation and degranulation against primary plasma cells than preexisting Fc wild-type anti-CD38 mAbs. Finally, using huFcgR transgenic mice that express human Fcγ receptors under the control of their human regulatory elements, we demonstrated that SAR442085 had higher NK cell-dependent in vivo antitumor efficacy and better survival than daratumumab and isatuximab against EL4 thymoma or VK*MYC myeloma cells overexpressing human CD38. These results highlight the preclinical efficacy of SAR442085 and support the current evaluation of this next-generation anti-CD38 antibody in phase I clinical development in patients with relapsed/refractory MM.

Simultaneous Engagement of Tumor and Stroma Targeting Antibodies by Engineered NK-92 Cells Expressing CD64 Controls Prostate Cancer Growth.

Metastatic castration-resistant prostate cancer (mCRPC) has been largely resistant to immunotherapy. Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill transformed cells without prior sensitization, and their infiltration into prostate tumors corresponds with an increased overall survival among patients with mCRPC. We sought to harness this knowledge to develop an approach to NK-cell based immunotherapy for mCRPC. We engineered an NK cell line (NK-92MI) to express CD64, the sole human high-affinity IgG Fcγ receptor (FcγR1), and bound these cells with antibodies to provide interchangeable tumor-targeting elements. NK-92MICD64 cells were evaluated for cell-activation mechanisms and antibody-dependent cell-mediated cytotoxicity (ADCC). A combination of mAbs was used to target the prostate tumor antigen tumor-associated calcium signal transducer 2 (TROP2) and the cancer-associated fibroblast marker fibroblast activation protein alpha (FAP). We found that CD64, which is normally expressed by myeloid cells and associates with the adaptor molecule FcRγ, can be expressed by NK-92MI cells and mediate ADCC through an association with CD3ζ. Cytotoxicity from the combination approach was two-fold higher compared to treatment with NK-92MICD64 cells and either mAb alone, and seven-fold higher than NK-92MICD64 cells alone at an effector-target cell ratio of 20:1. The cytotoxic effect was lost when using isotype control antibodies, indicating a selective targeting mechanism. The combination approach demonstrated efficacy in vivo as well and significantly reduced tumor growth compared with the saline control. This combination therapy presents a potential approach for treating mCRPC and could improve immunotherapy response.

Evaluation and use of an anti-cynomolgus monkey CD79b surrogate antibody-drug conjugate to enable clinical development of polatuzumab vedotin.

Background and PurposePolatuzumab vedotin is an antibody–drug conjugate (ADC) being developed for non‐Hodgkin's lymphoma. It contains a humanized anti‐CD79b IgG1 monoclonal antibody linked to monomethyl auristatin E (MMAE), an anti‐mitotic agent. Polatuzumab vedotin binds to human CD79b only. Therefore, a surrogate ADC that binds to cynomolgus monkey CD79b was used to determine CD79b‐mediated pharmacological effects in the monkey and to enable first‐in‐human clinical trials.Experimental ApproachPolatuzumab vedotin, the surrogate ADC, and the corresponding antibodies were evaluated in different assays in vitro and in animals. In vitro assessments included binding to peripheral blood mononuclear cells from different species, binding to a human and monkey CD79b‐expressing cell line, binding to human Fcγ receptors, and stability in plasma across species. In vivo, ADCs were assessed for anti‐tumour activity in mice, pharmacokinetics/pharmacodynamics in monkeys, and toxicity in rats and monkeys.Key ResultsPolatuzumab vedotin and surrogate ADC bind with similar affinity to human and cynomolgus monkey B cells, respectively. Comparable in vitro plasma stability, in vivo anti‐tumour activity, and mouse pharmacokinetics were also observed between the surrogate ADC and polatuzumab vedotin. In monkeys, only the surrogate ADC showed B‐cell depletion and B‐cell‐mediated drug disposition, but both ADCs showed similar MMAE‐driven myelotoxicity, as expected.Conclusions and ImplicationsThe suitability of the surrogate ADC for evaluation of CD79b‐dependent pharmacology was demonstrated, and anti‐tumour activity, pharmacokinetics/pharmacodynamics, and toxicity data with both ADCs supported the entry of polatuzumab vedotin into clinical trials.

Human Fcγ receptors compete for TGN1412 binding that determines the antibody's effector function.

The first-in-human clinical trial of the CD28-specific monoclonal antibody (mAb) TGN1412 resulted in a life-threatening cytokine release syndrome. Although TGN1412 was designed as IgG4, known for weak Fc:Fcγreceptor (FcγR) interactions, these interactions contributed to TGN1412-induced T-cell activation. Using cell lines (TFs) expressing human FcγRI, -IIa, -IIb, or -III, we show that TGN1412 and TGN1412 as IgG1 and IgG2 are bound by FcγRs as it can be deduced from literature. However, upon coculture of TGN1412-decorated Tcells with TFs or human primary blood cells, we observed that binding capacities by FcγRs do not correlate with the strength of the mediated effector function. FcγRIIa and FcγRIIb, showing no or very minor binding to TGN1412, mediated strongest Tcell proliferation, while high-affinity FcγRI, exhibiting strong TGN1412 binding, mediated hardly any T-cell proliferation. These findings are of biological relevance because we show that FcγRI binds TGN1412, thus prevents binding to FcγRIIa or FcγRIIb, and consequently disables T-cell proliferation. In line with this, FcγRI- FcγRII+ but not FcγRI+ FcγRII+ monocytes mediate TGN1412-induced T-cell proliferation. Collectively, by using TGN1412 as example, our results indicate that binding of monomeric IgG subclasses does not predict the FcγR-mediated effector function, which has major implications for the design of therapeutic mAbs.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

A recombinant human IgG1 Fc multimer designed to mimic the active fraction of IVIG in autoimmunity.

The antiinflammatory effects of i.v. Ig (IVIG) in the treatment of autoimmune disease are due, in part, to the Fc fragments of Ig aggregates. In order to capitalize on the known antiinflammatory and tolerogenic properties of Ig Fc aggregates, we created a recombinant human IgG1 Fc multimer, GL-2045. In vitro, GL-2045 demonstrated high-avidity binding to Fc receptors, blocked the binding of circulating immune complexes from patients with rheumatoid arthritis to human Fcγ receptors (FcγRs), and inhibited antibody-mediated phagocytosis at log order-lower concentrations than IVIG. In vivo, administration of GL-2045 conferred partial protection against antibody-mediated platelet loss in a murine immune thrombocytopenic purpura (ITP) model. GL-2045 also suppressed disease activity in a therapeutic model of murine collagen-induced arthritis (CIA), which was associated with reduced circulating levels of IL-6. Furthermore, GL-2045 administration to nonhuman primates (NHPs) transiently increased systemic levels of the antiinflammatory cytokines IL-10 and IL-1RA, reduced the proinflammatory cytokine IL-8, and decreased surface expression of CD14 and HLA-DR on monocytes. These findings demonstrate the immunomodulatory properties of GL-2045 and suggest that it has potential as a treatment for autoimmune and inflammatory diseases, as a recombinant alternative to IVIG.

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3ζ and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fcγ receptors (FcγRs) by different monoclonal anti-CD20 antibodies in clinical use.

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-βRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4–6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.

Comparative functional and pharmacological characterization of Sandoz proposed biosimilar adalimumab (GP2017): rationale for extrapolation across indications

ABSTRACTBackground: Biosimilars are approved biologics that match reference medicine in quality, safety, and efficacy. The development of Sandoz proposed biosimilar adalimumab (SPBA; GP2017) involved a target-directed, iterative state-of-the-art quality-by-design development program. Here, we describe the functional and pharmacological characterization of SPBA and its proposed mechanism of action in immune-mediated inflammatory diseases.Methods: Sensitive in vitro binding and functional characterization studies, and nonclinical evaluations (pharmacokinetics, pharmacodynamics, and safety/toxicology) were performed as part of a stepwise approach to confirm the biosimilarity of SPBA with reference adalimumab.Results: Matching values were reported for SPBA and reference adalimumab in binding assays involving tumor necrosis factor (TNF)-α, complement 1q and human immune effector cell Fcγ receptor subtypes in cell-based bioassays for Fc receptor function (complement- and antibody-dependent cytotoxicity), and in apoptosis inhibition. Furthermore, SPBA and reference adalimumab were equivalent in terms of membrane TNF binding and induction of reverse signaling. Pharmacokinetics of SPBA and reference adalimumab were comparable in rabbits, and the two biologics were equally effective in a human TNF transgenic mouse model of polyarthritis.Conclusion: SPBA matches reference adalimumab with regards to target binding, functional, pharmacokinetic, and pharmacodynamic properties at the nonclinical level supporting its approval in all indications of the reference adalimumab.

Abstract 3813: Development of JNJ-64164711, a low fucose anti-GITR antibody for enhanced depletion of tumor regulatory T cells (Tregs)

Abstract Background: Checkpoint blockade of PD-1/PDL-1 or CTLA-4 has demonstrated clinical benefit in a subset of cancer patients (pts). To further improve the outcome, approaches to deplete immunosuppressive Tregs are being developed. Emerging evidence supports that the TNF receptor superfamily member 18 (TNFRSF18 or GITR) is highly expressed on intratumoral (i.t.) Tregs with limited expression on effector T cells or circulating immune cells (1,2). This differential expression allows for development of monoclonal antibody (mAb) therapies that eliminate this population while sparing other immune subsets. Herein we characterize JNJ-64164711 (JNJ-711), a fully human anti-GITR mAb being developed to selectively deplete GITR+ Tregs and expand the population of pts that may respond to immunotherapy. Methods: Treg frequency and GITR expression on human cancer specimens was performed by IHC and multicolor flow cytometric analysis (FC). JNJ-711 was assessed for binding to GITR (human and cyno) and human Fcγ receptors, and inhibition of hGITRL binding to hGITR. The JNJ-711 binding epitope was mapped by hydrogen-deuterium exchange. JNJ-711 functional activity was determined by 1) an NF-kB reporter assay, 2) antibody dependent cellular cytotoxicity (ADCC) on GITR+ cell lines, and 3) JNJ-711 mediated Treg depletion in primary pts tumor samples. Results: GITR expression prevalence was evaluated by IHC in multiple solid tumors. Immune cell GITR expression was observed in head and neck (HN), esophageal (ESO), non-small cell lung carcinoma (NSCLC), colorectal (CRC) and prostate cancers. GITR was moderately expressed on HN and ESO tumor cells. GITR expression (by FC) was higher on tumor Tregs versus other T cell populations in the tumor and periphery across NSCLC, CRC, ovarian and renal cell carcinoma samples. GITR was differentially higher on i.t. effector Tregs (eTreg) whereas CCR4 was highest on CD4+ T cells in circulation. JNJ-711 binds to hGITR on cells with high affinity [KD= 180 pM], has a log greater affinity to FcγRIIIa [KD= 37 nM on 158V; KD= 180 nM on 158F], a slower dissociation rate than most GITR agonist (Ag) mAb tested [Koff= 5.47 x 10-4 s-1 for Fab], and binds to the CRD1 of GITR. Unlike other GITR Ag mAbs tested, JNJ-711 does not block GITR interaction with GITRL. JNJ-711 demonstrates higher ADCC mediated cell depletion than other GITR Ag mAbs in clinical development on 1) GITR+ hematologic tumor cell lines (HuT102, KMS-12-BM, RPMI 8226, JJN-3), 2) primary activated GITR+ T cells, and 3) in vitro expanded Tregs. Using dissociated primary pts samples, JNJ-711 selectively depleted i.t. eTregs. Conclusion: JNJ-64164711 selectively depletes hGITR+ cells including Tregs and has potential to enhance antitumor immune response. This antibody is being evaluated for clinical development.

Dissecting FcγR Regulation through a Multivalent Binding Model.

Many immune receptors transduce activation across the plasma membrane through their clustering. With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing affinities that are in turn bound to multivalent antigen. As a consequence of this activation mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is complicated by, among other factors, differing affinities between FcγR species and changes in the valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand binding can effectively account for the contribution of IgG-FcγR affinity and immune complex valency. This model in turn enables us to make specific predictions about the effect of immune complexes of defined composition. In total, these results enable both rational immune complex design for a desired IgG effector function and the deconvolution of effector function by immune complexes.

Human IgG subclass cross-species reactivity to mouse and cynomolgus monkey Fcγ receptors

In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies.

Backbone 1H, 13C, and 15N assignments of the extracellular region of human Fcγ receptor IIIb.

Fcγ receptor (FcγR) promotes various immune responses through interactions with the Fc portion of immunoglobulin G (IgG). FcγRIIIb is a glycosylphosphatidylinositol-linked protein expressed on neutrophils and triggers degranulation and opsonic phagocytosis. The extracellular region of FcγR is composed of two Ig-fold domains and can be cleaved as a soluble form (sFcγRIIIb), which is also reactive with complement receptor type 3. Although structure and Fc interaction of sFcγRIIIb have been characterized by X-ray crystallography, little has been known about its structure in solution. We herein report the backbone NMR assignments of human sFcγRIIIb to gain basic understanding of functional IgG-FcγRIII interactions of immunological and biopharmaceutical interest regarding the structural investigation.

Alga-Made Anti-Hepatitis B Antibody Binds to Human Fcγ Receptors.

Microalgae are unicellular eukaryotic organisms which represent an emerging alternative to other cell biofactories commonly used to produce monoclonal antibodies. Microalgae display several biotechnological advantages such as their rapid growth rate and their phototrophic lifestyle allowing low production costs as protein expression is solar-fueled. Recently, a fully assembled recombinant IgG antibody directed against Hepatitis B surface antigen is produced and secreted in the culture medium of the diatom Phaeodactylum tricornutum. A biochemical characterization of this recombinant antibody demonstrated that the Asn-297 is N-glycosylated by oligomannosides. In the immune system, antibodies interact with effector molecules and cells through their Fc part and the recognition of Fcγ receptors (FcγR) which are important for inducing phagocytosis of opsonized microbes. Interactions between IgG and FcγR are influenced by the N-glycan structures present on the Asn-297. In this study, the authors characterized the binding capacity of the anti-hepatitis B recombinant IgG produced in P. tricornutum to two human Fcγ receptors (FcγRI and IIIa) using a cellular binding assay and surface plasmon resonance (SPR). This allowed us to demonstrate that the alga-made antibody is able to bind FcγRI with a reduced affinity and engages FcyRIIIa with 3-times higher affinity compared to a control human IgG1.

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody‐dependent cellular cytotoxicity towards cancer cells

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti-cancer antibodies act through different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47-SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab-coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa-131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa-131R. Furthermore, ADCC was consistently enhanced by targeting CD47-SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47-SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47-SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.