The glycoprotein Mucin 16 (MUC16) has been previously investigated as a targetable tumor antigen. Crawford et al. now report on a bispecific antibody that binds CD3 and MUC16, which could potentially be used in ovarian cancer treatment. The antibody induced T cell activation and ovarian cancer cells killing both in vitro and in mouse models. This antibody worked well with checkpoint blockade and was shown to be safe when administered to nonhuman primates. A clinical trial using this antibody is already underway.

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.

Because of the potency of a bispecific antibody, it is essential to identify a tumor target antigen with sufficient tumor selectivity to limit toxicity. Mucin 16 (MUC16) is a large integral membrane glycoprotein that is highly expressed in a number of epithelial cancers ( 16 ) but at low abundance in epithelial cells of several tissues including trachea, eye, and female reproductive organs, as well as the serosal epithelial lining of the peritoneal and thoracic cavity. MUC16 is thought to provide a barrier function in some of these organs ( 17 , 18 ). Proteolytic cleavage of cell surface MUC16 results in the shedding of the extracellular portion of MUC16 [known as cancer antigen 125 (CA-125)] into the bloodstream and a short, membrane-associated C-terminal domain that remains on the cell surface. Circulating CA-125 is a biomarker of ovarian cancer ( 17 – 19 ). Agents using several MUC16-targeted approaches are in early phases of clinical development, such as chimeric antigen receptor (CAR) T cells ( 20 ) and antibody-drug conjugates ( 21 ). MUC16 can be detected in around 80% of ovarian carcinomas and is expressed on all ovarian subtypes (serous, mucinous, endometrioid, and clear cell), although expression is heterogeneous ( 22 , 23 ). Because of its up-regulation in tumors and apical expression on normal tissue where it may be less accessible to therapeutic antibodies, MUC16 is a viable and attractive ovarian tumor target.

CD3-engaging bispecific antibodies are emerging as a potent immunotherapy for the treatment of cancer because they can broadly activate CD3-expressing T cells in the presence of tumor cells. The only clinically approved CD3 bispecific molecule is blinatumomab, a CD19×CD3-targeting molecule for acute lymphoblastic B cell leukemia ( 15 ). Given that the bispecific therapeutic approach induces polyclonal T cell activation, they may be efficacious in a low neoantigen burden disease such as ovarian cancer.

Evidence suggests that ovarian cancer may be amenable to some forms of immunotherapy ( 8 ). For example, patients with ovarian cancer whose tumors were positive for intraepithelial CD8 + T lymphocyte infiltration had notably better overall and progression-free survival than patients without intraepithelial CD8 + T lymphocyte infiltration, suggesting that T cells can play a role in controlling tumor growth ( 9 , 10 ). Moreover, some patients have shown evidence of immune response to their tumors, demonstrated by detection of tumor-reactive T cells and antibodies in the blood, tumor, or ascites of patients with advanced disease ( 11 – 14 ). Blockade of the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) checkpoint pathway has shown some benefit in ovarian cancer; PD-1 blockade monotherapy resulted in an overall response rate of about 10 to 15% in early clinical trials ( 9 ). Thus, blockade of this pathway alone has shown only modest clinical responses.

Ovarian cancer is the most lethal of the gynecologic malignancies ( 1 , 2 ), partially because it is frequently diagnosed at an advanced stage ( 3 ). The current standard of care for ovarian cancer is surgery, followed by chemotherapy with a combination of platinum agents and taxanes ( 4 ). Although most patients respond to initial treatment, most experience a recurrence of the disease, resulting in a cycle of repeated surgeries and additional rounds of chemotherapy. Despite recent advances in therapy such as poly(adenosine 5′-diphosphate–ribose) polymerase inhibitors for patients carrying BRCA or other homologous recombination deficiency mutations, advanced ovarian cancer remains a disease of high unmet need ( 5 – 7 ).

RESULTS

REGN4018 binds to MUC16-expressing tumor cells and CD3 on human and cynomolgus monkey T cells CD3-binding bispecific antibodies that redirect T cells to kill MUC16-expressing ovarian cancer cells were generated using VelocImmune mice (24, 25), which express human immunoglobulin genes. REGN4018 resulted from an anti-MUC16 antibody and an anti-CD3 antibody, using the light chain from the MUC16 antibody (Fig. 1A). The constant region of REGN4018 is based on a hinge-stabilized, effector function–minimized version of the immunoglobulin G4 (IgG4) isotype as previously described (16, 26) and shown in Fig. 1A. Cell surface binding of REGN4018 to human and cynomolgus monkey T cells was assessed by flow cytometry using peripheral blood mononuclear cells (PBMCs). REGN4018 bound to both human and cynomolgus T cells. The binding to cynomolgus T cells was weaker than that to human T cells (Fig. 1B). To assess binding to MUC16, tumor cell lines expressing a range of MUC16 abundance were selected. The antibody binding capacity for REGN4018 on endogenously expressing cells, defined as the number of antibody molecules bound to the cell surface under saturating conditions, ranged from 26,000 on CFPAC-1 cells (pancreatic cancer cells) to 300,000 on OVCAR-3 cells (ovarian cancer cells), demonstrating that REGN4018 binds tumor cells expressing a range of MUC16 (Fig. 1C). REGN4018 specifically bound to all MUC16-expressing cell lines tested with half maximal effective concentration (EC 50 ) values ranging from 1.7 nM for the endogenously expressing CFPAC-1 cells to 2.15 nM for ID8 mouse ovarian tumor cells engineered to express a membrane-proximal portion of human MUC16 up to the fifth SEA (sea urchin sperm protein, enterokinase, and agrin) domain [ID8–vascular endothelial growth factor (VEGF)/huMUC16Δ] (denoted in Fig. 1D). The nonbinding control antibody did not bind to any tested cell lines (Fig. 1E), and REGN4018 did not bind the ID8-VEGF parental cells, which were included as a negative control cell line (Fig. 1E). Fig. 1 REGN4018 binds to human and monkey CD3 and MUC16. (A) Schematic of bispecific antibody format. Heavy-chain variable regions of different specificities are in red and green. Red asterisk indicates the Fc mutation used for purification. (B) REGN4018 binding to CD3 on human and cynomolgus monkey T cells (CD2+CD16−) shown by flow cytometry (two replicates). (C) The number of MUC16 epitopes per cell on the cell lines is reported as antibody binding capacity. (D) Schematic of MUC16 including the “membrane-proximal region” where the antibody binds. (E) Binding of REGN4018 to human and cynomolgus monkey MUC16 on a range of tumor cell lines shown by flow cytometry. Isotype control, green; REGN4018, blue (two replicates).

REGN4018 induces target-dependent T cell activation and cytotoxicity in the presence of CA-125 The ability of REGN4018 to activate T cells was investigated in a nuclear factor of activated T cells–Luciferase (NFAT-Luc) reporter bioassay using engineered cells expressing human or cynomolgus (cyno) CD3 (Jurkat NFAT-Luc or Jurkat NFAT-Luc/cynoCD3, respectively). REGN4018 activated human CD3-mediated NFAT signaling in the presence of human OVCAR-3 cells, as well as monkey CD3-mediated NFAT signaling in the presence of monkey MUC16-expressing ID8-VEGF cells (Fig. 2A). The cytotoxic potency of REGN4018 was assessed in a flow cytometry–based cell killing assay. REGN4018 induced human T cells and cynomolgus T cells to kill OVCAR-3 cells (EC 50 , 1.36 × 10−11 M and 3.06 × 10−11 M, respectively) (Fig. 2B). OVCAR-3 cells were used in this assay because they express the large natural MUC16 glycoprotein, validating that REGN4018 can induce killing of MUC16-expressing cells in the presence of full-length MUC16. The cytotoxic activity of human and cynomolgus T cells to OVCAR-3 cells was accompanied by up-regulation of the T cell activation marker PD-1 (Fig. 2C). Comparison of CD69 and PD-1 up-regulation in response to anti-CD3/anti-CD28 stimulation confirms the ability of REGN4018 to potently activate T cells (fig. S2). The ability of REGN4018 to induce cytokine release in the presence of human T cells and OVCAR-3 cells was also examined. REGN4018 induced interferon-γ (IFN-γ), interleukin-2 (IL-2), and IL-10 (Fig. 2D). Furthermore, REGN4018 induced human T cells to kill ID8-VEGF cells expressing the membrane-proximal region of human or cynomolgus MUC16 (ID8-VEGF/huMUC16Δ or ID8-VEGF/cynoMUC16Δ, respectively). The cytotoxic activity was MUC16 dependent because no killing was observed when T cells were incubated with the ID8-VEGF line (Fig. 2E). These results demonstrate that REGN4018 is able to induce CD3-mediated activation of human and cynomolgus monkey T cells and killing of tumor cells expressing MUC16. Fig. 2 REGN4018 activates T cells in the presence of target and induces redirected T cell–mediated cytotoxicity in the presence of soluble CA-125 in vitro. (A) Jurkat/NFAT-Luc cells were incubated with OVCAR-3 cells (left), or Jurkat/NFAT-Luc cells expressing cynoCD3 (human CD3 deleted) were incubated with ID8-VEGF/cynoMUC16 cells (right) and serial dilutions of REGN4018 (blue squares) or a CD3-binding control (gray triangles). Relative luminescence units (RLU) are plotted. T cells and target cells were incubated with various concentrations of REGN4018 or CD3-binding control for 48 hours. Human (left) and monkey (right) T cell killing of OVCAR-3 cells are shown. (B) Assays performed in triplicate wells and plotted as means ± SD (n = 3). (C) At 48 hours, human and monkey T cells (CD2+CD16− and then CD4+ or CD8+) were examined for PD-1 expression by flow cytometry. (D) Cytokines were measured at 48 hours with anti-CD3/anti-CD28 used as a positive control. Assays performed in triplicate and plotted as means ± SD (performed twice). Significance measured by unpaired test in comparison to CD3-binding control (*P < 0.05, **P < 0.01, and ***P < 0.001). (E) Human T cell killing of ID8-VEGF/huMUC16Δ (left), ID8-VEGF/cynoMUC16 (middle), or ID8-VEGF cells (right). (F) Flow cytometry measurements of REGN4018 (blue) or anti-MUC16 clone 3A5 (black) binding alone or in the presence of CA-125 or a truncated MUC16. (G) Killing of OVCAR-3 cells by human PBMCs in the presence of REGN4018 with increasing concentrations of CA-125 (left) or truncated MUC16 (right) (performed twice). CA-125 is increased in the serum of most patients with ovarian cancer (27), and circulating CA-125 could affect any MUC16-targeted therapy by acting as an antigen sink. We therefore examined whether the functional activity of REGN4018 is affected by the presence of soluble CA-125. We performed flow cytometry to examine binding and cytotoxicity assays in the presence of high amounts of CA-125 purified from ascites of patients with ovarian cancer. The concentrations of CA-125 used in the assay (10,000 U/ml) greatly exceed the median published serum concentration of 656.6 U/ml found in patients with serous ovarian cancer (28). Excess CA-125 had minimal impact on REGN4018 binding to OVCAR-3 cells, suggesting minimal binding to CA-125. In contrast, CA-125 inhibited the ability of a comparator anti-MUC16 antibody by 70% (in-house version of antibody clone 3A5) (Fig. 2F). On the other hand, a soluble MUC16 construct containing the membrane-proximal region of MUC16 (MUC16∆) substantially inhibited binding of REGN4018, demonstrating that REGN4018 binds this membrane-proximal region (Fig. 2F). Minimal binding to CA-125 was confirmed by enzyme-linked immunosorbent assay (fig. S3). The limited binding to the CA-125 portion contrasts with many anti-MUC16 antibodies such as OC-125–like and M11-like antibodies (22, 23). In alignment with the binding studies, REGN4018 could still induce T cell–mediated killing in the presence of CA-125 but not in the presence of a high concentration of MUC16∆ (Fig. 2G). Thus, REGN4018 can bind to MUC16 and induce T cell–redirected killing even in the presence of high concentrations of CA-125.

REGN4018 mediates potent T cell–mediated killing of human ovarian cancer cells (OVCAR-3) growing intraperitoneally in mice To assess the in vivo antitumor activity of REGN4018, we established a tumor xenograft ascites model using OVCAR-3 ovarian cancer cells. We generated OVCAR-3 cells stably expressing the firefly Luc reporter enzyme to monitor tumor growth using in vivo bioluminescence imaging (BLI). To evaluate redirected killing induced by REGN4018, human T cells were provided by transferring human PBMCs to mice before tumor cell implantation. OVCAR-3/Luc cells were then intraperitoneally implanted into mice, and treatment began on day 6 (Fig. 3A, left) or day 7 (Fig. 3A, right) after tumor implantation. Mice treated with REGN4018 (0.05 mg/kg or higher) once tumor growth was established had significantly reduced tumor burden compared to mice treated with either a nonbinding control antibody or a CD3-binding control antibody (Fig. 3, A and B). REGN4018 treatment did not result in any changes in weight (fig. S4, A and B). Changes in weight and body condition scoring were also used as a readout for graft-versus-host disease (GvHD), which is a common phenomenon in this xenogenic model. Experiments were completed before GvHD was evident. Circulating REGN4018 was detected in the serum at all assessed time points for the two higher dose groups (fig. S4C). Tumor cells remaining in the peritoneal cavity maintained MUC16 expression (fig. S5). Fig. 3 REGN4018 activates T cells and reduces tumor burden in a xenogenic tumor model in a dose-dependent manner. (A) OVCAR-3/Luc cells were implanted into NOD SCID gamma (NSG) mice pre-engrafted with PBMCs and intraperitoneally treated with the indicated doses of REGN4018 or control antibodies on days 6, 10, 13, 16, and 21 after tumor implantation (four to five mice per group; left) and day 7 (five mice per group; right). Values represent group median with the associated confidence interval (range). *P < 0.05 and **P < 0.01 compared to nonbinding control by Mann-Whitney test. (B) BLI showing tumor burden in mice. (C) Serum from day 23 after tumor implantation was examined for CA-125 concentrations. (D) Cytokines from serum were examined at 4 hours after first dose. **P < 0.01 compared to nonbinding control by Mann-Whitney test. (E) CD8 T cells were examined for CD25, PD-1, and granzyme B 48 hours after administration of CD3-binding control or REGN4018 by flow cytometry; dotted line shows fluorescence minus one (FMO). MFI, median fluorescence intensity. (F) Spleen and ascites were examined for human T cells at end of the study by flow cytometry. Upon treatment, BLI abundance similar to those in nontumor-bearing mice (baseline) should correspond to complete tumor clearance. This was confirmed using flow cytometry; in mice with BLI that had returned to baseline after therapy, no tumor cells were detected at the end of the study. In contrast, mice treated with the CD3-binding control bispecific maintained high BLI and contained OVCAR-3/Luc cells in the peritoneal cavity (fig. S6, A and B). To enable examination of tumor burden for a longer period of time, mice were depleted of T cells to avoid development of GvHD and followed for 57 days after treatment stopped. No tumor growth was detected, and mice did not gain weight because of ascites in the REGN4018-treated group (fig. S6C). Elevated serum CA-125 can be detected in mice with high tumor burden in this model. CA-125 concentrations were significantly higher in control-treated mice compared to those treated with the two higher doses of REGN4018, and the amount of CA-125 in the serum correlated with tumor burden (BLI), showing that serum CA-125 can be used as a readout of therapeutic response (Fig. 3C). To assess in vivo T cell activation by REGN4018 in mice bearing OVCAR-3 tumors and human PBMCs, serum cytokines from tumor-bearing mice were measured. Serum samples were collected 4 hours after the first antibody dose in the REGN4018 (0.5 mg/kg), CD3-binding control, and nonbinding control groups. Treatment with REGN4018 activated T cells as determined by induction of IFN-γ, TNFα (tumor necrosis factor–α), IL-2, IL-6, IL-8, and IL-10, compared to the nonbinding control and the CD3-binding control (Fig. 3D). REGN4018 does induce a substantial serum cytokine response, and the concentrations of cytokines are higher when the mice are administered anti-CD3, which induces a systemic T cell activation via Fc receptor cross-linking (fig. S7A). Cell types other than T cells are unlikely to contribute to the human cytokine production because only T cells are maintained in the NSG mice in any appreciable frequency, with a slight skewing toward CD8+ T cells over CD4+ T cells (fig. S7B). Antitumor efficacy required the presence of human T cells (fig. S8A), and REGN4018-induced cytokine response required the presence of both human T cells and OVCAR-3/Luc cells (fig. S8B). Examination of T cells in the peritoneal cavity 48 hours after dosing showed that REGN4018 induced expression of CD25, PD-1, and granzyme B—all markers of T cell activation (Fig. 3E). REGN4018 did not deplete T cells because T cells were still present in the spleen and ascites of mice treated with REGN4018 (Fig. 3F).

Mice expressing human MUC16 and CD3 have a normal T cell compartment, and REGN4018 induces no inflammation in MUC16-expressing tissues To investigate the antitumor efficacy of REGN4018 in a mouse with a fully intact immune system, mice were genetically engineered to express both human CD3 on T cells and the membrane-proximal region of MUC16 in the endogenous murine loci (knockin mice) (Fig. 4A). To validate these mice, MUC16 expression was examined by both reverse transcription polymerase chain reaction and immunohistochemistry (IHC). RNA expression was detected in the trachea and at low abundance in the lung, heart, ovary, pancreas, and bladder, similar to murine MUC16 expression (Fig. 4B) (29). To assess MUC16 protein expression, IHC was performed on OVCAR-3 cells and selected tissues using an antihuman MUC16 antibody that recognizes a membrane-proximal region of MUC16 (Fig. 4C). MUC16 protein expression was confirmed in the surface epithelium of the ovary and stomach, the tracheal lining/epithelium, and the submucosal glands, as described in humans (Fig. 4C, right) (17, 29). Fig. 4 REGN4018 does not induce any systemic cytokine response or histopathology in genetically engineered mice. (A) Schema of humanization of membrane-proximal region of MUC16. (B) Expression of the human MUC16 in various tissues from HuT mice (left) and mouse MUC16 (right), relative to expression in the trachea, was measured by TaqMan. (C) Left: IHC analysis of MUC16 expression on OVCAR-3 and HT29 cells. Right: IHC analysis of MUC16 expression in the trachea, stomach, and ovary from wild-type (WT) or HuT mice. (D) Flow cytometry analysis of TCR Vß usage in CD4 (left) and CD8 (right) T cells of wild-type (white) and huMUC16huCD3 (gray striped) mice (five mice per group, two replicates). (E) Total numbers of T cells from spleens of wild-type (white) or HuT (gray striped) mice were determined by flow cytometry. To examine activation of T cells, mice were dosed with REGN4018 (10 mg/kg; four to five mice per group, three replicates). T cell numbers in blood were examined 4 hours after dosing with phosphate-buffered saline (PBS; black), CD3-binding control (gray), REGN4018 (blue), or OKT3 (red). (F) T cells were identified as CD45+/CD90.2+CD19−/CD4+ or CD8+. (G) Serum cytokines were examined 4 hours after intravenous administration of CD3-binding control (gray), REGN4018 (blue), or OKT3 (red) by meso scale discovery (MSD). (H) Hematoxylin and eosin (H&E)–stained sections of the trachea, stomach, and ovary from wild-type or HuT mice 5 days after administration of REGN4018. The T cells in these mice express human CD3, are polyclonal as assessed by T cell receptor (TCR) Vß usage (Fig. 4D), and are present in similar numbers to wild-type mice (Fig. 4E). To determine whether REGN4018 induced any T cell activation or effects on normal tissues in these animals, nontumor-bearing mice were injected with a high dose of REGN4018 (10 mg/kg), and T cell numbers in blood, serum cytokines, and histopathology were examined. It has previously been described that, upon stimulation through CD3, a transient loss of T cells from the blood is observed. Although T cells can be activated by an antihuman CD3 antibody (OKT3) as measured by loss of T cells from the blood and increased serum cytokines, REGN4018 did not induce any such effects, suggesting limited accessibility of the MUC16 target (Fig. 4, F and G). To determine whether REGN4018 induced any microscopic changes in MUC16-expressing tissues, MUC16 and CD3 humanized target (HuT) mice received two doses of REGN4018 at 10 mg/kg on days 0 and 3. On day 5, several MUC16-expressing tissues (trachea, stomach, and ovary) were examined, and no cellular infiltration or necrosis was seen in these tissues (Fig. 4H).

In vivo immuno-PET imaging and biodistribution of REGN4018 show targeting to secondary lymphoid organs through CD3 binding and MUC16-driven targeting to the tumor The in vivo localization of REGN4018 and the expression of MUC16 protein were assessed in wild-type and genetically HuT mice using positron emission tomography (PET) imaging. The biodistribution of the 89Zr-labeled anti-MUC16 parental antibody was similar in both wild-type and HuT mice, suggesting low expression/availability of the HuT MUC16 protein to the antibody. In contrast, when mice were administered therapeutically relevant doses of 89Zr-labeled REGN4018 (the bispecific antibody), distribution to the spleen (yellow arrow) and lymph nodes (LNs; green arrow) was evident, likely because of recognition of CD3+ T cells in these lymphoid organs (Fig. 5A). Ex vivo biodistribution analyses in individual tissues confirmed the localization to LNs and spleen (Fig. 5B). To assess whether REGN4018 can accumulate in MUC16-expressing tumors, 89Zr-labeled REGN4018 was administered to mice bearing ID8-VEGF-huMUC16Δ tumors. Although REGN4018 still accumulated in the spleen (yellow arrow) and LNs (green arrow) because of CD3 targeting, tumor distribution was also evident (red arrow), indicating that REGN4018 can localize to MUC16-expressing tumors in animals with an intact immune system and endogenous expression of CD3 and MUC16 (Fig. 5C). The addition of the MUC16 parental antibody to block MUC16 availability at the time of dosing with 89Zr-labeled REGN4018 reduced tumor targeting, demonstrating that at least some of the targeting to the tumor is MUC16 driven. The addition of a CD3-binding control antibody reduced targeting to spleen and LN to almost baseline, and targeting to the MUC16-expressing tumor was enhanced (Fig. 5, C and D). Fig. 5 REGN4018 accumulates in target-bearing tissues including the spleen, LNs, and MUC16-expressing tumors. WT mice or HuT mice for MUC16 and CD3 were administered with 89Zr-labeled REGN4018 (0.5 mg/kg) or anti-MUC16 parental antibody to track binding in vivo (n = 3 mice per group). (A) Images show analysis from 6 days after dosing. 89Zr-labeled nonbinding control antibody or REGN4018 was administered at 0.5 mg/kg to mice bearing ID8-VEGF/huMUC16Δ tumors implanted 20 days previously. (C) Anti-MUC16 parental antibody (10 mg/kg; second from the right) or CD3-binding control antibody (10 mg/kg; right) was administered at same time as 89Zr-labeled REGN4018 (n = 4 mice per group). Images show analysis from 6 days after dosing. (B and D) Graphs show the percentage of injected dose per gram of tissue (% ID/g), shown as means ± SD.

REGN4018 is efficacious in two syngeneic tumor models in HuT mice To investigate the antitumor efficacy of REGN4018 in an immunocompetent mouse model, we used the genetically engineered mice described above and the syngeneic tumor cell line ID8-VEGF/huMUC16Δ. All mice in these studies were given a total of five doses of REGN4018 or a CD3 binding control. REGN4018 significantly inhibited subcutaneously implanted ID8-VEGF/huMUC16∆ tumors compared to the CD3-binding control when treatment began on the day of tumor implantation or was delayed until day 10 after tumor implantation (Fig. 6A). In an ascites model, REGN4018 resulted in a markedly longer median survival time than control-treated mice (Fig. 6B). Expression of huMUC16∆ was maintained (although slightly reduced) on the tumor cells at end of study as measured by flow cytometry (Fig. 6C). REGN4018 induced activation of T cells in the peritoneal cavity (but not the spleen) early after administration. There was evidence of some endogenous T cell activation as indicated by the increased granzyme B expression in T cells in the peritoneal cavity of tumor-bearing mice compared to splenic T cells, and REGN4018 further increased granzyme B expression on both T cell subsets. In addition, REGN4018 resulted in a significant increase in Ki67 expression, a marker of proliferation (Fig. 6D). Fig. 6 REGN4018 induces antitumor efficacy in immunocompetent HuT mice. (A) HuT mice implanted with ID8-VEGF/huMUC16Δ cells subcutaneously were treated intraperitoneally with 100 μg of REGN4018 or CD3-binding control on days 0, 4, 7, 10, and 13 after implantation (left; n = 5 mice per group, two individual studies) or days 10, 14, 17, 21, and 24 after implantation (right). Mean tumor volume shown ± SEM (n = 5 to 6 mice per group, two individual studies). Significance measured by two-way analysis of variance (ANOVA) (**P < 0.01). HuT mice implanted with ID8-VEGF/huMUC16Δ cells were intraperitoneally treated with REGN4018 (5 mg/kg) or CD3-binding control from day 3. (B) Survival curves represent euthanasia upon 20% weight gain (n = 5 to 12 mice per group, four replicates). Significance measured by Gehan-Breslow-Wilcoxon test (****P < 0.0001). (C) Histograms show expression of MUC16 on tumor cells at the end of the study (blue, anti-MUC16; green, secondary alone; and gray, no antibody). (D) CD4 and CD8 T cells were examined for Ki67 and granzyme B (GZM B) 48 hours after administration of CD3-binding control or REGN4018 by flow cytometry (n = 4 mice per group, two individual studies). Significance measured by ANOVA (***P < 0.001, ****P < 0.0001 between ascites samples and #P < 0.05 between spleens and ascites samples in control groups). n.s., not significant.

PD-1 blockade enhances antitumor activity of REGN4018 The PD-1 pathway is a negative regulator of T cell responses. To evaluate whether blockade of the PD-1 pathway can enhance the antitumor effects of REGN4018, we tested antitumor efficacy of REGN4018 alone or in combination with an anti-mouse PD-1 antibody. As shown earlier, REGN4018 induces PD-1 up-regulation on human and cynomolgus T cells in vitro. In addition, a subset of T cells in the peritoneal cavity of mice bearing ID8-VEGF/huMUC16∆ ascites expresses PD-1 (Fig. 7A), and the ID8-VEGF/huMUC16∆ cells express mouse PD-L1 ex vivo (Fig. 7B), validating this model for testing the combination of anti–PD-1 with REGN4018. Intraperitoneal administration of REGN4018 further increased PD-1 on T cells (Fig. 7C). REGN4018 administered alone showed significant antitumor efficacy in this model, and the addition of an anti-mouse PD-1 antibody enhanced the activity of REGN4018; the combination of REGN4018 and anti–PD-1 led to complete tumor clearance in four of the nine mice treated with the combination (Fig. 7D). Blockade of the PD-1 pathway alone did not show any antitumor efficacy alone, as previously described for the ID8 model (26, 30). The ability of PD-1 blockade to enhance antitumor efficacy was also demonstrated in the OVCAR-3 with a suboptimal dose of REGN4018 (fig. S9). These results demonstrate that PD-1 blockade can provide an added benefit to treatment with REGN4018. Fig. 7 The addition of PD-1 blockade enhances REGN4018 efficacy. (A) PD-1 expression on CD4 and CD8 T cells in the peritoneal cavity of tumor-bearing mice is shown by flow cytometry. T cells were examined 32 days after implantation when tumor growth was evident (n = 5 mice per group with three replicates). (B) Flow cytometry of PD-L1 expression on ID8-VEGF/huMUC16Δ cells ex vivo. PD-L1 was examined at the end of the study in four separate studies. (C) HuT mice implanted with ID8-VEGF/huMUC16Δ cells were treated with REGN4018 (5 mg/kg) or CD3-binding control, and T cells in the peritoneal cavity were examined for PD-1 expression 48 hours later (four mice per group, two replicates). (C) Significance measured by ANOVA (**P < 0.01 between ascites samples and ###P < 0.001 between spleens and ascites samples in control groups). HuT mice implanted with ID8-VEGF/huMUC16Δ cells were intraperitoneally treated with REGN4018 (5 mg/kg) or CD3-binding control in combination with anti–PD-1 (5 mg/kg, each). Survival curves represent euthanasia upon 20% weight gain (10 to 12 mice per group). (D) Significance measured by Gehan-Breslow-Wilcoxon test compared to CD3-binding control + isotype (***P = 0.0005, ****P < 0.0001, ##P = 0.0252). Three studies were completed.