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Most antibody–drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

Supplementary Data from Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054

In vivo processing

Supplementary Figure S5 shows expression of human PD-1 and human LAG-3 in PD-1xLAG-3 mice

In the tumor microenvironment, multiple inhibitory checkpoint receptors can suppress T-cell function, thereby enabling tumor immune evasion. Blockade of one of these checkpoint receptors, PD-1, with therapeutic antibodies has produced positive clinical responses in various cancers; however, the efficacy of this approach can be further improved. Simultaneously targeting multiple inhibitory checkpoint receptors has emerged as a promising therapeutic strategy. Here, we report the development and characterization of REGN3767, a fully human IgG4 antibody targeting LAG-3, another inhibitory receptor on T cells. REGN3767 binds human and monkey LAG-3 with high affinity and specificity and blocks the interaction of LAG-3 with its ligand, MHC class II. In an engineered T-cell/antigen-presenting cell bioassay, REGN3767 alone, or in combination with cemiplimab (REGN2810, human anti-PD-1 antibody), blocked inhibitory signaling to T cells mediated by hLAG-3/MHCII in the presence of PD-1/PD-L1. To test the in vivo activity of REGN3767 alone or in combination with cemiplimab, we generated human PD-1xLAG-3 knockin mice, in which the extracellular domains of mouse Pdcd1 and Lag3 were replaced with their human counterparts. In these humanized mice, treatment with cemiplimab and REGN3767 showed increased efficacy in a mouse tumor model and enhanced the secretion of proinflammatory cytokines by tumor-specific T cells. The favorable pharmacokinetics and toxicology of REGN3767 in nonhuman primates, together with enhancement of antitumor efficacy of anti-PD-1 antibody in preclinical tumor models, support its clinical development.

Supplementary Table S2 shows PK data for REGN3767 in monkeys

CRISPR RNA gene list

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Supplementary figure legends and methods

Purpose:Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy.Experimental Design:Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors.Results:In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling.Conclusions:Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

Glucocorticoids (GCs) are widely used to treat a variety of autoimmune and inflammatory diseases; however, systemic delivery of GCs is associated with side effects that affect essentially every organ system, reflecting the nearly ubiquitous expression of the glucocorticoid receptor (GR). Targeted delivery of GCs to diseased tissues using antibody-glucocorticoid conjugates (GC-ADCs) offers a therapeutic alternative to overcome these adverse effects. Herein, we describe novel classes of GCs that exhibited greater potency than dexamethasone and budesonide, a 100-fold selectivity toward the GR over other nuclear receptors, and no in vitro safety liability in pharmacology assays (hERG, AMES) and that demonstrated a substantial reduction in tumor necrosis factor-α (TNF-α) release in mice challenged with lipopolysaccharide (LPS). The site-specific conjugated GC-ADCs via cathepsin-cleavable linkers were highly stable in plasma and specifically released GCs in antigen-positive cells, suggesting that these novel GCs can serve as ADC payloads to treat autoimmune and inflammatory diseases.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%–5% of lung cancers), and acquired resistance limits response durability. An antibody–drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

The recognition of antigenic peptide-MHC (pMHC) molecules by T-cell receptors (TCR) initiates the T-cell mediated immune response. Structural characterization is key for understanding the specificity of TCR-pMHC interactions and informing the development of therapeutics. Despite the rapid rise of single particle cryoelectron microscopy (cryoEM), x-ray crystallography has remained the preferred method for structure determination of TCR-pMHC complexes. Here, we report cryoEM structures of two distinct full-length α/β TCR-CD3 complexes bound to their pMHC ligand, the cancer-testis antigen HLA-A2/MAGEA4 (230–239). We also determined cryoEM structures of pMHCs containing MAGEA4 (230–239) peptide and the closely related MAGEA8 (232–241) peptide in the absence of TCR, which provided a structural explanation for the MAGEA4 preference displayed by the TCRs. These findings provide insights into the TCR recognition of a clinically relevant cancer antigen and demonstrate the utility of cryoEM for high-resolution structural analysis of TCR-pMHC interactions.

The interleukin-6 (IL-6) family cytokines signal through gp130 receptor homodimerization or heterodimerization with a second signaling receptor and play crucial roles in various cellular processes. We determined cryo–electron microscopy structures of five signaling complexes of this family, containing full receptor ectodomains bound to their respective ligands ciliary neurotrophic factor, cardiotrophin-like cytokine factor 1 (CLCF1), leukemia inhibitory factor, IL-27, and IL-6. Our structures collectively reveal similarities and differences in the assembly of these complexes. The acute bends at both signaling receptors in all complexes bring the membrane-proximal domains to a ~30 angstrom range but with distinct distances and orientations. We also reveal how CLCF1 engages its secretion chaperone cytokine receptor–like factor 1. Our data provide valuable insights for therapeutically targeting gp130-mediated signaling.

Purpose: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. Experimental Design: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. Results: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. Conclusions: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.

We recently demonstrated that capturing human monoclonal antibodies (hmAbs) using high affinity anti-human Fc (AHC) antibodies allows reliable characterization of antibody-antigen interactions. Here, we characterized six human Fc specific mouse monoclonal antibodies (mAbs) and compared their binding profiles with three previously characterized goat AHC polyclonal antibodies (pAbs), exhibiting properties of a good capture reagent. All six mouse AHC mAbs specifically bound with high affinity to the Fc region of hIgG1, hIgG2, hIgG4 and to 43 different hIgG variants, containing substitutions and/or mutations in the hinge and/or Fc region, that have been reported to exhibit modified antibody effector function and/or pharmacokinetics. Biacore sensor surfaces individually derivatized with mouse AHC mAbs exhibited2.5-fold higher hIgG binding capacity compared to the three goat AHC pAb surfaces and reproducibly captured hIgG over 300 capture-regeneration cycles. The results of the capture kinetic analyses performed on 31 antibody-antigen interactions using surfaces derivatized with either of the two highest affinity AHC mAbs (REGN7942 or REGN7943) were in concordance with those performed using goat AHC pAb surfaces. Our data demonstrate that AHC mAbs such as REGN7942 and REGN7943 that have properties superior than the three goat AHC pAbs are highly valuable research reagents, especially to perform capture kinetic analyses of antibody-antigen interactions on optical biosensors.

In the tumor microenvironment, multiple inhibitory checkpoint receptors can suppress T-cell function, thereby enabling tumor immune evasion. Blockade of one of these checkpoint receptors, PD-1, with therapeutic antibodies has produced positive clinical responses in various cancers; however, the efficacy of this approach can be further improved. Simultaneously targeting multiple inhibitory checkpoint receptors has emerged as a promising therapeutic strategy. Here, we report the development and characterization of REGN3767, a fully human IgG4 antibody targeting LAG-3, another inhibitory receptor on T cells. REGN3767 binds human and monkey LAG-3 with high affinity and specificity and blocks the interaction of LAG-3 with its ligand, MHC class II. In an engineered T-cell/antigen-presenting cell bioassay, REGN3767 alone, or in combination with cemiplimab (REGN2810, human anti-PD-1 antibody), blocked inhibitory signaling to T cells mediated by hLAG-3/MHCII in the presence of PD-1/PD-L1. To test the in vivo activity of REGN3767 alone or in combination with cemiplimab, we generated human PD-1xLAG-3 knockin mice, in which the extracellular domains of mouse Pdcd1 and Lag3 were replaced with their human counterparts. In these humanized mice, treatment with cemiplimab and REGN3767 showed increased efficacy in a mouse tumor model and enhanced the secretion of proinflammatory cytokines by tumor-specific T cells. The favorable pharmacokinetics and toxicology of REGN3767 in nonhuman primates, together with enhancement of antitumor efficacy of anti-PD-1 antibody in preclinical tumor models, support its clinical development.

Recently, de novo peptide sequencing has made it possible to gain new insights into the human immunopeptidome without relying on peptide databases, while identifying peptides of unknown origin. Many recent studies have attributed post-translational proteasomal splicing as the origin of those peptides. Here, we describe a peptide source assignment workflow to rigorously assign the source of de novo sequenced peptides and find that the estimated extent of post-translational splicing in the immunopeptidome is much lower than previously reported. Our approach demonstrates that many peptides that were thought to be post-translationally spliced are likely linear peptides, and many peptides that were thought to be trans-spliced could be cis-spliced. We believe our approach furthers the understanding of post-translationally spliced peptides and thus improves the characterization of immunopeptidome which plays a critical role in the immune response to antigens in cancer, autoimmune disease, and infections.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

Background Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. Objectives This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response. Methods Bet v 1–specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model. Results REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1– and birch pollen extract–induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1. Conclusions These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.

Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.

Epitopes of a native pollen allergen protein, birch Bet v1, against four of the noncompeting anti-Bet v1 antibodies individually or in combination, were identified by solution-phase amide backbone H/D exchange (HDX) coupled with high-resolution Q-TOF or Orbitrap mass spectrometry. The HDX results indicates that the four anti-Bet v1 antibodies protected specific regions of Bet v1, explaining the difference in their blocking efficiency of each antibody against Bet v1 binding to polyclonal IgEs in Bet v1 allergic patients. An in-house HDX-MS system was further developed to explore the surface protection of Bet v1 in the presence of all four antibodies with 100% sequence coverage and high redundancy. The data demonstrated that four anti-Bet v1 antibodies were able to simultaneously bind to Bet v1 in solution to provide the most effective blocking for 9 of 10 tested IgE donors in an in vitro antibody-blocking assay. For the first time, we have applied HDX to elucidate the therapeutic advantage of combination antibodies compared with individual antibodies in treating Bet v1 induced allergy.

Background For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/ I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO- REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53–5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of ~14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR.

Glioblastoma Multiforme (GBM) is a highly aggressive cancer with few specific molecular targets and poor therapeutic outcome. Epidermal Growth Factor Receptor variant III (EGFRvIII) is a promising target for the treatment of GBM due to its exclusive expression in GBM. However, prior therapeutics have failed in part due to the development of EGFRvIII negative tumors cells from heterogenous tumor populations. We therefore developed a potent antibody-drug conjugate with bystander killing capabilities to target heterogeneous EGFRvIII-expressing GBM tumors. REGN3124 is a fully human EGFRvIII-selective antibody that also demonstrates some binding to amplified wild-type EGFR. REGN3124 was conjugated to the pyrrolobenzodiazepine (PBD) linker-payload SG-3249 to form REGN3124-PBD, and the cytotoxicity and cellular bystander killing activity was characterized in vitro. Initial in vivo activity of REGN3124-PBD was assessed in subcutaneous (s.c.) U251/EGFRvIII and U87/EGFRvIII cell line xenografts and then in s.c. EGFRvIII-positive patient derived xenograft (PDX) models (GBM6 and GBM59). Immunohistochemistry demonstrated heterogenous expression of EGFRvIII in GBM59 tumors. Lastly, efficacy was assessed in animals with established intracranial GBM6 or GBM59 tumors to examine the activity of REGN3124-PBD in an orthotopic setting. REGN3124-PBD demonstrated sub-nM cytotoxicity in vitro and clear bystander killing of EGFRvIII negative U251 cells following targeting of U251/EGFRvIII cells. A single dose of 0.38 mg/kg REGN3124-PBD (3.4 drug: antibody ratio) induced sustained regression of both s.c. U251/EGFRvIII and U87/EGFRvIII xenografts. A single dose of 0.53 mg/kg REGN3124-PBD induced complete regression of s.c. GBM6 PDX tumors and sustained regression of GBM59 tumors. Single dose of 0.53 mg/kg REGN3124-PBD significantly prolonged survival of mice with established intracranial GBM6 or GBM59 tumors, with 5/8 and 7/8 animals surviving >90 days post-treatment, respectively. The high unmet need for effective therapies combined with the potent anti-tumor activity observed, including in those with heterogenous expression of EGFRvIII, support continued assessment of REGN3124-PBD as a novel therapy for treatment of GBM. Citation Format: Marcus P. Kelly, Sosina Makonnen, Carlos Hickey, Shu Mao, Feng Zhao, Arthur Kunz, Frank Delfino, Thomas Nittoli, Dangshe Ma, William C. Olson, Gavin Thurston, Jessica R. Kirshner. Novel EGFRvIII-selective antibody-drug conjugate REGN3124-PBD is strongly efficacious against orthotopic glioblastoma multiforme patient derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P134.

G-protein-coupled receptors (GPCRs) are activated by a diverse range of ligands, from large proteins and proteases to small peptides, metabolites, neurotransmitters and ions. They are expressed on all cells in the body and have key roles in physiology and homeostasis. As such, GPCRs are one of the most important target classes for therapeutic drug discovery. The development of drugs targeting GPCRs has therapeutic value across a wide range of diseases, including cancer, immune and inflammatory disorders as well as neurological and metabolic diseases. The progress made by targeting GPCRs with antibody-based therapeutics, as well as technical hurdles to overcome, are presented and discussed in this Review. Antibody therapeutics targeting C-C chemokine receptor type 4 (CCR4), CCR5 and calcitonin gene-related peptide (CGRP) are used as illustrative clinical case studies.

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti–PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1–dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861–70. ©2017 AACR.

The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681–93. ©2017 AACR.

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.

Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.

Deficiency in interleukin-36R (IL-36R) antagonist caused by loss-of-function mutations in IL-36RN leads to DITRA (deficiency of IL-36 receptor antagonist), a rare inflammatory human disease that belongs to a subgroup of generalized pustular psoriasis (GPP). We report a functional genetic mouse model of DITRA with enhanced IL-36R signaling analogous to that observed in patients with DITRA, which provides new insight into our understanding of the IL-36 family of molecules in regulating barrier integrity across multiple tissues. Humanized DITRA-like mice displayed increased skin inflammation in a preclinical model of psoriasis, and in vivo blockade of IL-36R pathway using anti-human IL-36R antibody ameliorated imiquimod-induced skin pathology as both prophylactic and therapeutic treatments. Deeper characterization of the humanized DITRA-like mice revealed that deregulated IL-36R signaling promoted tissue pathology during intestinal injury and led to impairment in mucosal restoration in the repair phase of chronic dextran sulfate sodium (DSS)-induced colitis. Blockade of IL-36R pathway significantly ameliorated DSS-induced intestinal inflammation and rescued the inability of DITRA-like mice to recover from mucosal damage in vivo. Our results indicate a central role for IL-36 in regulating proinflammatory responses in the skin and epithelial barrier function in the intestine, suggesting a new therapeutic potential for targeting the IL-36R axis in psoriasis and at the later stages of intestinal pathology in inflammatory bowel disease.

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.