A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies.

The clinical use of interleukin-2 (IL-2) for cancer immunotherapy is limited by severe toxicity. Emerging IL-2 therapies with reduced IL-2 receptor alpha (IL-2Rα) binding aim to mitigate toxicity and regulatory T cell (Treg) expansion but have had limited clinical success. Here, we show that IL-2Rα engagement is critical for the anti-tumor activity of systemic IL-2 therapy. A "non-α" IL-2 mutein induces systemic expansion of CD8+ T cells and natural killer (NK) cells over Tregs but exhibits limited anti-tumor efficacy. We develop a programmed cell death protein 1 (PD-1)-targeted, receptor-masked IL-2 immunocytokine, PD1-IL2Ra-IL2, which attenuates systemic IL-2 activity while maintaining the capacity to engage IL-2Rα on PD-1+ T cells. Mice treated with PD1-IL2Ra-IL2 show no systemic toxicities observed with unmasked IL-2 treatment yet achieve robust tumor growth control. Furthermore, PD1-IL2Ra-IL2 can be effectively combined with other T cell-mediated immunotherapies to enhance anti-tumor responses. These findings highlight the therapeutic potential of PD1-IL2Ra-IL2 as a targeted, receptor-masked, and "α-maintained" IL-2 therapy for cancer.

VSIG4 interaction with heparan sulfates inhibits VSIG4-complement binding.

V-set and immunoglobulin domain-containing 4 (VSIG4) is a complement receptor of the immunoglobulin superfamily that is specifically expressed on tissue resident macrophages, and its many reported functions and binding partners suggest a complex role in immune function. VSIG4 is reported to have a role in immune surveillance as well as in modulating diverse disease phenotypes such as infections, autoimmune conditions, and cancer. However, the mechanism(s) governing VSIG4's complex, context-dependent role in immune regulation remains elusive. Here, we identify cell surface and soluble glycosaminoglycans, specifically heparan sulfates, as novel binding partners of VSIG4. We demonstrate that genetic deletion of heparan sulfate synthesis enzymes or cleavage of cell-surface heparan sulfates reduced VSIG4 binding to the cell surface. Furthermore, binding studies demonstrate that VSIG4 interacts directly with heparan sulfates, with a preference for highly sulfated moieties and longer glycosaminoglycan chains. To assess the impact on VSIG4 biology, we show that heparan sulfates compete with known VSIG4 binding partners C3b and iC3b. Furthermore, mutagenesis studies indicate that this competition occurs through overlapping binding epitopes for heparan sulfates and complement on VSIG4. Together these data suggest a novel role for heparan sulfates in VSIG4-dependent immune modulation.

CD22-targeted CD28 bispecific antibody enhances antitumor efficacy of odronextamab in refractory diffuse large B cell lymphoma models.

Although many patients with diffuse large B cell lymphoma (DLBCL) may achieve a complete response to frontline chemoimmunotherapy, patients with relapsed/refractory disease typically have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through the activation of the T cell receptor/CD3 complex, has exhibited early, promising activity for patients with highly refractory DLBCL in phase 1 trials. However, not all patients achieve complete responses, and many relapse, thus representing a high unmet medical need. Here, we investigated whether adding a costimulatory "signal 2" by engaging CD28 receptors on T cells could augment odronextamab activity. We demonstrate that REGN5837, a bispecific antibody that cross-links CD22-expressing tumor cells with CD28-expressing T cells, enhances odronextamab by potentiating T cell activation and cytolytic function. In preclinical DLBCL studies using human immune system-reconstituted animals, REGN5837 promotes the antitumor activity of odronextamab and induces intratumoral expansion of reprogrammable T cells while skewing away from a dysfunctional state. Although REGN5837 monotherapy shows limited activity and no toxicity in primate studies, it augments T cell activation when dosed in combination with odronextamab. In addition, analysis of non-Hodgkin lymphoma clinical samples reveals an increase in CD28+CD8+ T cells after odronextamab treatment, demonstrating the presence of a population that could potentially be targeted by REGN5837. Collectively, our data demonstrate that REGN5837 can markedly enhance the antitumor activity of odronextamab in preclinical NHL models, and the combination of these two bispecific antibodies may provide a chemotherapy-free approach for the treatment of DLBCL.

Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054.

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.

Interference in a Neutralizing Antibody Assay for Odronextamab, a CD20xCD3 Bispecific mAb, from Prior Rituximab Therapy and Possible Mitigation Strategy.

A cell-based assay was developed to detect neutralizing anti-drug antibodies (NAbs) against odronextamab, a CD20xCD3 bispecific monoclonal antibody (mAb) under investigation for treatment of CD20+ B cell malignancies. In this assay, odronextamab bridges between two cell types, CD20-expressing HEK293 cells and CD3-expressing Jurkat T cells that generate a luciferase signal upon CD3 clustering. Patient samples containing NAbs directed to either arm of the bispecific drug block the odronextamab bridge formation between the cell lines thus preventing the generation of the luciferase signal. We determined that other anti-CD20 therapeutics also block bridge formation, resulting in false-positive results. In patient samples from odronextamab clinical trials, approximately 30% of baseline samples had a strong false-positive NAb signal that correlated with the presence of prior rituximab (anti-CD20) therapy. We determined that rituximab interference can be minimized by the addition of anti-rituximab antibodies in the NAb assay. Understanding and mitigating the impact of prior biologic exposure is increasingly important for implementing a successful bioanalytical strategy to support clinical drug development, especially in the immuno-oncology field. Odronextamab neutralizing antibody assay, interference, and mitigation. A Design of the odronextamab neutralizing antibody (NAb) assay where anti-CD20xCD3 drug bridges between CD20-expressing HEK293 cells and Jurkat T cells expressing an NFAT response element and luciferase reporter. True NAb prevents odronextamab from bridging between target and effector cells, thus preventing the expression of luciferase. B Interference with odronextamab from other anti-CD20 therapeutic antibodies (e.g., rituximab) from prior disease treatment generates a false-positive NAb result. Assay interference can be mitigated with an anti-idiotypic antibody against the interfering therapy.

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

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.

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail.

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.

Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy.

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.

A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies.

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.

Preclinical Development of the Anti-LAG-3 Antibody REGN3767: Characterization and Activity in Combination with the Anti-PD-1 Antibody Cemiplimab in Human PD-1xLAG-3-Knockin 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.

Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice.

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.

Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal.

Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide (CNO). The engineered receptor and small-molecule ligand form an orthogonal pair: The receptor does not respond to native ligands, and the inert drug does not bind to native cells. CNO-responsive migration can be engineered into a variety of cell types, including neutrophils, T lymphocytes, keratinocytes, and endothelial cells. The engineered cells migrate up a gradient of the drug CNO and transmigrate through endothelial monolayers. Finally, we demonstrate that T lymphocytes modified with the engineered receptor can specifically migrate in vivo to CNO-releasing beads implanted in a live mouse. This technology provides a generalizable genetic tool to systematically perturb and control cell migration both in vitro and in vivo. In the future, this type of migration control could be a valuable module for engineering therapeutic cellular devices.