Human PD-1 Research Articles

1218P - Antitumor activity, immunogenicity and safety of a novel PD-1 vaccine in combination with two chimeric HER-2 peptide vaccine in syngeneic Balb/c, C57Bl/6 models and in beagle dogs

BackgroundAnti-PD-1 blocking monoclonal antibodies (mAbs) such as nivolumab or pembrolizumab have shown potent anti-tumor effects in different metastatic cancers. Similarly, anti-HER-2 mAbs such as Herceptin and Perjeta are approved for breast cancer. These mAbs are only effective in 10-25% of cancer patients, and are limited by high costs, side effects and development of resistance. Contrary to treatment with monoclonal antibodies, chimeric B-cell cancer vaccines incorporating a “promiscuous” T cell epitope have the advantage of safer and cheaper alternatives, producing a specific immune response that induces memory B & T cell responses, while reducing immune evasion, suppression and resistance. MethodsWe have translated two HER-2 combination peptide vaccines (B-Vaxx) to the clinic in a Phase 1/2b trial to safely deliver curative and transformative cancer immunotherapies to advanced cancer patients. We have created and established the development of a novel B-cell peptide vaccine (Key-Vaxx) with high immunogenicity that binds to human PD-1 and produces tumor inhibition in vivo in two animal models of colon cancer. We will describe the CT-26, CT26-HER2 and MC38 tumor models in Balb/c and C57/Bl6 used to test for synergistic effects of anti-PD1 immunization therapy in combination with anti-HER2 immunization therapy. The immunogenicity/optimal immunologic dose and toxicity profile was investigated in beagle dogs. ResultsWe show robust HER-2 and PD-1 antibody responses in mice, beagle dogs and non-human primates. The individual (PD-1) or combined vaccination (B-Vaxx)) was effective in reducing tumor growth in a Balb/c and C57Bl/6 syngeneic model of colon carcinoma and exhibited superior activity compared to the positive gold control anti-mouse PD-1 (CD279) monoclonal antibody. ConclusionsDevelopment of PD-1 B-cell vaccine was successful, showing no evidence of toxicity or autoimmunity in mice, rabbits and beagle dogs. The combination vaccines could provide improved outcomes in early stage disease sparing patients the toxicity of chemotherapy. A phase 1 clinical trial with the PD-1 vaccine is under planning. Legal entity responsible for the studyThe authors. FundingNIH; Imugene. DisclosureAll authors have declared no conflicts of interest.

A small-sized protein binder specific for human PD-1 effectively suppresses the tumour growth in tumour mouse model

Immune checkpoint inhibitors have drawn a consider attention as an effective cancer immunotherapy, and several monoclonal antibodies targeting the immune checkpoint receptors, such as human programmed cell death-1 (hPD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), are clinically used for treatment of various cancers. Here we present the development of a small-sized protein binder which specifically binds to hPD‐1. The protein binder, which is composed of leucine-rich repeat (LRR) modules, was selected against hPD-1 through phage display, and its binding affinity was maturated up to 17 nM by modular evolution approach. The protein binder was shown to be highly specific for hPD-1, effectively inhibiting the interaction between hPD-1 and its ligand, hPD-L1. The protein binder restored T-cell function in vitro, and exhibited a strong anti-tumour activity in tumour mouse model, indicating that it acts as an effective checkpoint blockade. Based on the results, the developed protein binder specific for hPD-1 is likely to find a potential use in cancer immunotherapy.

Recombinant Adenovirus Expressing a Soluble Fusion Protein PD-1/CD137L Subverts the Suppression of CD8+ T Cells in HCC.

Oncolytic viruses are an excellent platform for developing effective strategies in cancer immunotherapy. Several challenges remain in the use of viro-immunotherapy for cancer, such as the lack of costimulatory signals and negative regulation of immune checkpoints. In this study, we designed a novel adenovirus expressing a soluble fusion protein, programmed cell death protein 1 (PD-1)/CD137L, which contains the extracellular domains of PD-1 and CD137L at each terminus (Ad5-PC). Ad5-PC preserved the costimulatory activity of CD137L and facilitated the persistence of activated CD8+ Tcells. Ad5-PC induced strikingly increased antitumor activity in both ascitic and subcutaneous hepatocellular carcinoma (HCC) tumor models, with 70% and 60% long-term cure rates, respectively. The improved antitumor effect of Ad5-PC was attributed to the sustained high-level lymphocyte activation and interferon (IFN)-γ production in the tumor microenvironment, and was essentially dependent on CD8+ Tcells rather than natural killer (NK) cells. Moreover, Ad5-huPC-expressing human soluble PD-1/CD137L fusion protein was effective in suppressing tumor growth and improving survival in a humanized mouse model. We confirmed that Ad5-PC induced tumor-specific and systematic protection against tumor rechallenges at both in situ and distant sites. Thus, Ad5-PC harnesses several distinct functions to efficiently overcome several major hurdles of viro-immunotherapy.

Abstract 1504: Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1

Abstract The human checkpoint targets PD-1 and PDL-1 continue to demonstrate great promise in the clinic. With much of the current focus turning toward combination regiments, an animal model using the human clinical antibodies will be beneficial for evaluating new combination strategies. Here we continue characterization of a C57BL/6 transgenic mouse model expressing both the human PD-1 and PD-L1 combined with a modified murine MC38 colon tumor cell line expressing human PD-L1. Both PD-1 and PD-L1 expression of the transgenic mice were verified in ex vivo stimulated splenocytes by flow cytometry analysis. Expression of PD-L1 on the genetically modified MC38 cells was also demonstrated by flow cytometry. For in vivo efficacy evaluation, tumor-implanted mice were treated with the clinical agents nivolumab and pembrolizumab at 100 µg and atezolizumab at 1 mg on Days 3, 7, 10, and 14 post implant. Treatment with nivolumab and pembrolizumab initiated tumor regression by Day 17. Complete tumor regressions were seen at Day 28 in nivolumab, 62.5% complete regression, and pembrolizumab, 71.4% complete regression. Growth inhibition was 83.9%, 69.7%, and 95.0% on Day 28 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To compare the human anti-PD-1 and PD-L1 clinical agent specificity, the non-transgenic parent C57BL/6 mouse strain was implanted with the unmodified MC38 colon tumor cells. Treatment with pembrolizumab and atezolizumab was conducted as with the transgenic animals. Through 28 days no growth inhibition, tumor size 105.9% of control, was seen in the pembrolizumab treated group, demonstrating lack of cross reactivity of the human therapeutic in the standard mouse model. Atezolizumab did demonstrate a 56.9% growth inhibition compared to controls and is consistent with the known cross reactivity of atezolizumab between human and mouse. We have shown a genetically modified MC38 colon tumor expressing human PD-L1 in transgenic mice expressing both human PD-1 and PD-L1 to be a suitable model for checkpoint inhibitor evaluation of the human form of the particular checkpoint therapeutic. Further research will involve combining each of the checkpoint antibodies with various chemotherapeutic agents. Citation Format: Michael Koratich, Ted Green, Jie Liu, Charlotte Hammond, LaJuana Durbin, Jerry Zhou, Anna Chen, Murray Stackhouse. Characterization of a MC38 mouse syngeneic tumor model expressing human PD-L1 in the transgenic C57BL/6 mouse system expressing human PD-1 and PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1504.

Abstract LB-063: Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics

Abstract Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents. Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.

Abstract 4121: Preclinical characterization of CS1003, a novel clinical-stage PD-1 monoclonal antibody

Abstract Background: The programmed cell death protein 1 receptor (PD-1) is an immune-checkpoint protein that negatively regulates the immune system to avoid collateral damage to self-tissues. Tumors hijack this mechanism as a way to avoid immune detection and destruction. Presently, the FDA has approved three anti-PD-1 antibodies for the treatment in more than ten cancer types. CS1003 is a novel anti-PD-1 mAb recognizing a unique epitope developed to disrupt the PD-1 interaction with PD-L1/PD-L2 to restore or improve T-cell function as stand-alone therapy or in combination with other approaches. Methods: A panel of rat PD-1 mAbs were generated through conventional hybridoma technology. Several mAbs with favorable characteristics were further humanized. CS1003, a humanized, hinge-stabilized IgG4 mAb, was selected as our lead candidate for further characterization including antigen binding, biophysical properties as well as functional characterization. Results: CS1003 binds mouse, cynomolgus monkey and human PD-1 with similar affinity. CS1003 bound PD-1-expressing cell lines and chronically-activated T cells, blocked PD-1 interactions with PD-L1/PD-L2, resulting in inhibition of PD-1 signaling, and enhanced T cell cytokine secretion and proliferation to levels comparable to those observed with nivolumab or pembrolizumab reference molecules. CS1003 showed significantly anti-tumor activity in conventional MC38 as well as in hPD-1 knock-in mice. CS1003 showed no unexpected cross-reactivity in human tissues, with specific staining observed primarily in lymphocytes of lymphoid organs. In a pharmacokinetic (PK) study in cynomolgus monkeys following single intravenous administration at multiple dose levels, PK properties were linear with the proportionally increasing exposures from 2-18 mg/kg. In a repeated-dose toxicity study, CS1003 was well tolerated in cynomolgus monkeys and the major finding was mononuclear infiltration in multiple organs, which was consistent with its pharmacologic activity. CS1003 demonstrated a favorable safety profile with the highest non-severely toxic dose (HNSTD) of 100 mg/kg. Conclusion: CS1003 is a novel anti-PD-1 mAb with favorable preclinical characteristics and safety profile in cynomolgus monkeys. The unique feature of CS1003 is its cross-reactivity with both human and murine PD-1. This should greatly facilitate further evaluation its potential for combination therapy in various syngeneic mouse tumor models. A phase I study for CS1003 is being conducted to evaluate the safety, tolerability, PK and anti-tumor activity in patients with advanced tumors [NCT03475251]. Citation Format: Fu Li, Jingrong Li, Ke Yin, Juan Zhang, Zhenhu Li, Liang Lu, Yuanwu Bao, Dan Pu, Zhen Qin, Yong Zheng, Baotian Yang, Jing Li, Xinzhong Jon Wang. Preclinical characterization of CS1003, a novel clinical-stage PD-1 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4121.

Abstract 4103: Blockade of intrinsic PD-1 promotes human colon cancer cells survival, chemo resistance and in vivo tumor growth of human colon cancer cells

Abstract A considerable proportion of colorectal cancer patients develops local recurrence and distant metastasis within 5 years after surgical treatment. The anti-PD-1, Nivolumab, is indicated for treatment of patients with metastatic colorectal cancer carrying DNA mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) whose disease has progressed on fluoropyrimidine, oxaliplatin, and irinotecan. Thus only a minority of patients with CRC will benefit from the immunotherapy. Beyond immune cell expression, recent evidence demonstrates that PD-1 is expressed on human cancer cells, such as melanoma and NSCLC cells. It is possible to speculate that colon cancer immune resistance could partially reside on intrinsic colon cancer cells PD-1 overexpression. PD-1 expression was evaluated through RT-PCR and flow cytometry in human colon cancer cell lines (HT29, HCT116, SW620, Colo205, LOVO). PD-1 was robustly expressed in HT29 (74,4%), in HCT116 (57,5%) and in SW620 (62,9%) while the ligand PD-L1 was weakly expressed in these cell lines. Recombinant PD-L1 fusion protein significantly decreased HT29 and HCT116 proliferation, while Nivolumab protects the cells by PD-L1 effect. To investigate on the effect of PD-1 on chemo sensitivity, cytotoxicity assays were conducted with Oxaplatinum (OXAL) or 5-Fluoruracil (5-FU) in the presence of Nivolumab. Unexpectedly, Nivolumab significantly protected cancer cells from OXAL and 5-FU (HT29 cells were 2.02- and 1.77-fold more resistant to 5FU and OXAL in the presence of Nivolumab and HCT116 were 1,7- and 2,1-fold more resistant to 5FU and OXAL in the presence of Nivolumab). Moreover, Nivolumab protected HT29 cells from apoptosis 5-FU-OXAL induced at 72 hrs. In vivo, Nivolumab accelerated the growth of subcutaneous HT29 tumor growth and reduced the efficacy of chemotherapy. Mechanistically, preliminary results demonstrated that Nivolumab increased the mTOR target p4EBP1, pErk and p38 signaling. Taken together, these results demonstrate that, while intrinsic PD-1 axis activation decreases human colon cancer cell proliferation, Nivolumab protects the human colon cancer cells PD-1 overexpressing. These effects need to be considered in the evaluation of immunotherapy efficacy in colon cancer. Citation Format: Caterina Ieranò, Crescenzo D'Alterio, Maria Napolitano, Luigi Portella, Giuseppina Rea, Gelsomina Monaco, Piera Maiolino, Antonio Luciano, Antonio Barbieri, Giuseppe Palma, Claudio Arra, Roberto Pacelli, Stefania Scala. Blockade of intrinsic PD-1 promotes human colon cancer cells survival, chemo resistance and in vivo tumor growth of human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4103.

Abstract LB-018: Orally active small molecule PD-L1 inhibitor demonstrating similar efficacy as Durvalumab in human knock-in MC38 model

Abstract Cancer immunotherapy with PD-1/PD-L1 antibody drugs has made tremendous progress in clinical application. It was also recognized by the scientific community with awarding of the 2018 Nobel Price. However, the discovery of orally active, small molecule drugs targeting PD-1/PD-L1 pathway remains highly challenging. We wish to report for the first time the similar efficacy as measured by TGI between small molecule inhibitor Max-10181 and Durvalumab in the head to head test of human PD-L1 knock-in MC38 cell line in human PD-1 knock-in mice. In addition, we would also like to report the superior efficacy of Max-10181 to mouse anti-PD-1 Ab in PD-L1 expression low syngeneic mouse 4T1 model. Our data indicated that small molecule drugs may have different dependency of PD-L1 expression level. As a result, they may have different therapeutic application in clinics. Furthermore, the data related to the mechanistic aspect of these observed results will also be discussed. Citation Format: Yuguang Wang, Nong Zhang, Feilan Wang, Qiang Zhao, Zenggang Li. Orally active small molecule PD-L1 inhibitor demonstrating similar efficacy as Durvalumab in human knock-in MC38 model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-018.

Abstract 4087: Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes

Abstract Human checkpoint genes PD-1 and PD-L1 are the target of extensive research for treating human cancers. Checkpoint inhibitor therapy shows great promise in the clinic. Combining checkpoint inhibitors with standard chemotherapeutic agents is an important for these inhibitors. Animal models that use human clinical antibodies and chemotherapeutic agents will be beneficial for evaluating new therapies. A novel transgenic mouse model is described that expresses human PD-1 and PD-L1 in place of the murine genes. Additionally, a murine MC38 colon tumor cell line was modified to express human PD-L1. We initially evaluated early treatment efficacy of human check point antibodies nivolumab, pembrolizumab, and atezolizumab in the transgenic animal model with modified MC38 colon cancer cells. Five hundred thousand transgenic MC38 cells were implanted in the C57BL/6 PD-1/PD-L1 knock in mice and allowed to grow for three days. Tumor-implanted mice were treated with nivolumab and pembrolizumab at 100 µg per animal and atezolizumab at 1 mg per animal on Days 3, 7, 10, and 14. Treatment with nivolumab and pembrolizumab caused tumor regression by Day 17. Growth inhibition was 84%, 58%, and 94% on Day 17 for nivolumab, atezolizumab, and pembrolizumab, respectively, compared to the control animals. There was no significant body weight loss and no signs of toxicity in any of the treated animals. To extend the utility of the model we implanted mice and allowed the tumors to grow to 63 to 126 mg before treatment initiation (Day 10). Similar results were observed in established tumors as for early treatment with the PD-1 inhibitors nivolumab and pembrolizuman. In contrast there was little activity from the PD-L1 inhibitor atezolizumab in established tumors. To prepare for combination studies, we also tested cisplatin, irinotecan, gemcitabine, decitabine, rucaparib, and thiarabine near the MTD for each agent with various treatment schedules against the established tumors. The doses for cisplatin, irinotecan and decitabine resulted in excess animal deaths and require reduced doses for combination therapies. Gemcitabine, decitabine, and thiarabine (in house development drug) were too active with complete regressions observed; the doses for combination treatment will be reduced. Due to the effectiveness of the PD-1 checkpoint inhibitors the number of doses will be reduced from four to two treatments in combination studies; no change for atezolizumab. We have shown that PD-1 inhibitors are effective in treating genetically modified MC38 colon tumors in transgenic mice in early treatment and in established tumors while the PD-L1 inhibitor was only effective in early treatment. We also have identified doses and schedules of five chemotherapeutic agents to use in future combination studies in this model. Citation Format: Murray A. Stackhouse, Ted Green, Jay Liu, Charlotte Hammond, LaJuana Durbin, Ana Chen, Jerry Zhou, Mike Koratich. Efficacy of three checkpoint Inhibitors, five chemotherapeutic agents, and the experimental drug thiarabine against MC38 colon cancer expressing human PD-L1 in transgenic C57BL/6 mice expressing human PD-1 and PD-L1 checkpoint genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4087.

Abstract CT085: Pharmacodynamic correlates in a phase I study of INCMGA00012, a PD-1 antagonistic monoclonal antibody

Abstract Background: The role of the PD-1 / PD-L1 axis in limiting T cell activity has been well established. Effective blockade of this pathway has been demonstrated to lead to an increase in T cell activity and to yield clinical activity in cancer patients. INCMGA00012 is a humanized IgG4 monoclonal antibody that binds to human PD-1 and blocks its interaction with PD-L1/PD-L2. This therapeutic antibody has demonstrated acceptable tolerability with evidence of clinical activity in a phase 1 study in patients with solid tumors (NCT03059823). Pharmacodynamic markers demonstrating biological activity of INCMGA0012 were assessed in samples collected during the study. Methods: Blood samples were collected at baseline and at various time points following treatment from patients receiving doses of either 3mg/Kg (Q2W) in the tumor-specific expansion cohort or a flat-dose of 500 or 750mg (Q4W) in the tumor-agnostic expansion cohort. Receptor occupancy was measured on circulating T cells by flow cytometry and serum cytokine levels were measured using either a proximity extension assay or immunoassays. In addition, circulating immune cell phenotyping was analyzed using flow cytometry and PBMC functionality evaluated using ex-vivo re-stimulation. Results: All tested doses of INCMGA0012 evaluated demonstrated a full saturation of the PD-1 receptor at trough on circulating CD4 and CD8 T cells. In all tested cohorts, an increase in serum CXCL9 and CXCL10 was observed following infusion with INCMGA0012. Moreover, an increase in the proliferation of circulating T cells was observed on treatment compared to baseline. Concurrently, following ex vivo re-stimulation, T cells isolated from peripheral blood mononuclear cells demonstrated an increased capacity to secrete cytokines. Conclusion: These results provide evidence that INCMGA0012 is biologically active and leads to an increase in IFNγ-related protein levels (i.e. CXCL9 and CXCL10) and in T cell proliferation. The clinical activity of INCMGA0012 is undergoing further evaluation in several indications, including endometrial cancer, Merkel cell carcinoma and squamous carcinoma of the anal canal. Several combination studies have also been initiated. Citation Format: Thomas Condamine, Sherry Owens, Patricia Feldman, Ross La Motte-Mohs, John Muth, Bradley Sumrow, Paul Moore, Robert Newton. Pharmacodynamic correlates in a phase I study of INCMGA00012, a PD-1 antagonistic monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT085.

Abstract LB-268: Assessment of flat dosing strategy for INCMGA00012 in patients with advanced tumors

Abstract Background: INCMGA00012, a humanized, hinge-stabilized, IgG4κ monoclonal antibody that recognizes human PD-1, is being developed for the treatment of multiple solid tumor types, both as monotherapy and in combination with other potentially immunoactive agents. Flat dosing is preferred to weight-based dosing for multiple reasons, including convenience of preparation and administration, reduced errors in preparation calculation, and minimization of drug waste. A modeling and simulation approach was employed to support flat doses for later phase studies. Patients and Methods: INCMGA00012 has been administered to approximately 200 participants in a Phase 1 study (Study INCMGA 0012-101) at weight-based doses ranging from 1 to 10 mg/kg Q2W or Q4W and at flat doses of 500 mg Q4W and 750 mg Q4W. PK data from weight-based doses were used for population PK model development and data from flat doses were used for model validation. Simulations were performed to support the selection of RP2 flat dose. Results: The concentrations of INCMGA00012 were adequately described by a 2-compartment model with first-order elimination. Higher clearance was estimated for 1 mg/kg (23.5 mL/h) than the other dose groups (13.5 mL/h). Body weight dependence of clearance and volume of distribution for central compartment were characterized by power relationships with exponents of 0.911 and 0.493, respectively. The model was validated using PK data from participants who received INCMGA00012 500 mg Q4W and 750 mg Q4W, respectively, in the cohort expansion phase. Median steady state trough concentrations of 500 mg Q4W and 750 mg Q4W exceeded the target value. Conclusion: Modeling and simulation provide justification for 500 mg Q4W for further clinical development of INCMGA00012 based upon favorable safety, PK characteristics and presumed flat exposure-response relationships for efficacy. Citation Format: Xuejun chen, Phil Wang, Sanjeev Kaul, Bradley Sumrow, Swamy Yeleswaram. Assessment of flat dosing strategy for INCMGA00012 in patients with advanced tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-268.

Abstract 1453: Development of a novel PD-1 vaccine and in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth in a syngeneic Balb/c model challenged with CT26/HER-2 carcinoma cell line

Abstract Background: Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer and demonstrated impressive activity across a broad set of cancer subtypes, even at advanced and metastatic stages of disease. Contrary to treatment with monoclonal antibodies, chimeric B-cell cancer vaccines have the advantage of producing a specific immune response that can potentially induce memory B & T cell responses, while reducing immune evasion and suppression. Methods: We have translated two HER-2 combination peptide vaccines (B-Vaxx) to the clinic in a Phase 1/2b trial to safely deliver curative and transformative cancer immunotherapies to advanced cancer patients. We have created and established the development of a novel B-cell peptide vaccine (Key-Vaxx) with high immunogenicity that binds to human PD-1 and produces tumor inhibition in vivo in an animal model of colon cancer. Here, we describe the transferable CT-26-HER2 neu tumor model in Balb/c was used to test for synergistic effects of anti-PD1 immunization therapy in combination with anti-HER2 immunization therapy to determine whether this combination could increase immunogenicity, enhance anti-tumor responses and provide synergistic benefit in inhibiting tumor growth. Results: We show robust HER-2 and PD-1 antibody responses in all vaccinated mice indicating that the combined vaccination is effective in reducing tumor growth in a Balb/c syngeneic model of colon carcinoma versus either the PD-1 vaccine or more importantly the positive control gold standard that is anti-mouse PD-1 monoclonal antibody. The vaccine combination was found to be safe and did not appear to exhibit toxicity or autoimmunity. Conclusion: Development of a novel PD-1 vaccine in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth with no evidence of toxicity or autoimmunity. A clinical trial with the combination is under planning. Citation Format: Tanios Bekaii-Saab, Jay Overholser, Yuhong Yang, Manuel Penichet, Pravin Kaumaya. Development of a novel PD-1 vaccine and in combination with two Chimeric HER-2 peptide vaccine provides synergistic inhibition of tumor growth in a syngeneic Balb/c model challenged with CT26/HER-2 carcinoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1453.

Abstract 4110: Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy

Abstract Immune checkpoint (ICP) blockade has become a major focus in cancer immunotherapy, and the application of monoclonal antibodies (mAbs) as ICP inhibitors shows impressive results in the therapy against cancers like melanoma, renal cell carcinoma or non-small cell lung cancer. Active immunization with B cell epitopes/mimotopes of ICP inhibitors rather than application of the corresponding mAbs for passive immunotherapy may, however, provide advantages such as induction of antibodies by the patient’s own immune system and overcoming the costly treatment of the mAbs. Applying a platform for bacterial surface-display of overlapping peptides spanning the extracellular domains of human PD1 (hPD1) and mouse PD1 (mPD1), as well as in vitro solid phase-based ELISA assay and cellular-based assays involving human Jurkat T cells expressing human/mouse PD1 and/or PDL1, B cell epitopes/mimotopes of Nivolumab and an anti-mPD1 mAb (with a blocking capacity) were identified. The mimotopes were not only shown to inhibit the binding of the corresponding mAbs but also to revert the PD1-PDL1 blocking inhibitory capacity of the respective mAbs. More importantly, to proof the antitumor activity of antibodies against PD1 mimotopes in vivo, rabbits were immunized with the mimotope of anti-mPD1 mAb to generate mimotope-specific IgGs. The specific IgGs not only inhibited the interaction of mPD1 and mPDL1 in vitro, but also significantly reduced tumor growth in a syngeneic model of mice grafted with cells expressing the breast cancer-associated tumor antigen Her-2/neu. Our results show systematic identification and characterization of mimotopes of anti-PD1 ICP inhibitor B cell mimotopes conferring in vivo anti-tumor activity, and also indicate the potential of such mimotopes for active immunization in cancer therapy. Based on the results, enhancement of our second generation of anti-Her-2/neu vaccine (HerVAXX; Tobias et al, 2017*) by combining with mimotopes of ICP inhibitors is also proposed, which may potentially pave the way to a paradigm change in active immunotherapy against Her-2/neu overexpressing cancers using an effective multi-level vaccine. The study is granted by Imugene Limited, Australia, and Medical University of Vienna. * Tobias J. et al. BMC Cancer, 2017, 17:118. Citation Format: Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Baier Karin, Katharina AMbroye, Mirjana Drinic, Erika Garner-Spitzer, Christoph Zielinski, Michael Kundi, Peter Steinberger, Ursula Wiedermann. Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4110.

Abstract 3219: In vivo efficacy and safety evaluation of anti-human PD-1 and CD40 mAbs using double humanized PD-1/CD40 mouse model

Abstract In recent years, monoclonal antibodies have been successfully used in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA4, PD-1, PD-L1, CD40 and others. Combination therapy can promote antigen release and T cell priming, T cell activation and homing, which helps to overcome tumor immune-evasive mechanisms and maximize efficacy, ultimately benefitting most patients. However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially to test combination therapy using two IO antibodies. The transmembrane protein receptor CD40 is a member of the tumor necrosis factor (TNF) receptor super family and is involved in co-stimulation of immune cells. CD40 is expressed by antigen-presenting cells (APCs) including dendritic cells (DCs), B-cells, macrophages, and monocytes. It has the ability to "wake" DCs to prime effective cytotoxic T-cell responses. Thus, effective anti-CD40 antibodies provide an ideal therapy alternative in combination with other IO antibodies. To evaluate the in vivo efficacy and safety (immune-related adverse events) of CD40 and PD-1 combination antibodies, we developed a double humanized B-hPD-1/hCD40 mouse model. In this model, mouse PD-1 gene exon 2, and CD40 gene exons 2-7 were replaced with their human counterparts. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hCD40 mice. We compared the effect among single hPD-1 antibody Keytruda, single hCD40 antibody Selicrelumab and combination of the two. Combination treatment of Selicrelumab and Keytruda show higher inhibitory effects than single antibody treatments. The blood chemistry assays showed that the CD40 antibody had no toxicity effects compared with the control group. Based on these findings, we conclude that the B-hPD-1/hCD40 double-humanized mouse model is a powerful tool for in vivo efficacy and safety evaluation of hPD-1 and hCD40 antibodies for combination therapy. Citation Format: Yanan Guo. In vivo efficacy and safety evaluation of anti-human PD-1 and CD40 mAbs using double humanized PD-1/CD40 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3219.

Tumor mutational burden identifies chemorefractory gastric cancer with overall survival advantage after receiving toripalimab, a PD-1 antibody.

4021 Background: Tumor mutational burden (TMB) is correlated with enhanced objective response rate (ORR) and progression-free survival for certain cancers receiving immunotherapy. This study aimed to investigate the safety and activity of toripalimab, a humanized PD-1 antibody, in advanced gastric cancer (AGC), and the efficacy predictive value of biomarkers including TMB and PD-L1. Methods: This study was a part of phase Ib/II trial evaluating the safety and activity of toripalimab as a single agent therapy or in combination with chemotherapy in chemo-refractory or treatment-naïve AGC, esophageal squamous cell carcinoma, nasopharyngeal carcinoma and head and neck squamous cell carcinoma. This report focused on the chemo-refractory AGC cohort receiving toripalimab (3 mg/Kg d1, Q2W) as a single agent therapy. Primary endpoint was ORR. Biomarkers including tumor PD-L1 expression, TMB, microsatellite instability (MSI) and Epstein-Barr virus (EBV) infection status were evaluated for their correlation with clinical efficacy as preplanned. Tumor PD-L1 expression was assessed with the SP142 immunohistochemistry assay, and the other biomarkers were assessed with whole exome sequencing based on tumor samples. Results: There were 58 subjects included in this cohort. The ORR was 12.1% and the disease control rate was 39.7%. Only 1 subject was MSI-H and achieved partial response. One out of 4 EBV positive subjects achieved partial response. Significant higher ORR was observed in subjects with positive PD-L1 expression (ORR 37.5%, 3/8) or TMB ≥12 Mutations/Mb (ORR 33.3%, 4/8) than those with negative PD-L1 expression (ORR 8.5%) or TMB < 12 Mutations/Mb (ORR 7.0%). The TMB-high subgroup showed significant superior OS than the TMB-low subgroup (HR = 0.48 [96% CI 0.24 to 0.96], p = 0.038), while PD-L1 expression status failed to differentiate OS. Conclusions: Toripalimab demonstrated promising anti-tumor activity in chemo-refractory AGC patients. TMB might serve as a better predictive marker for OS than PD-L1 expression for chemo-refractory AGC patients receiving PD-1 blockade immunotherapy. Clinical trial information: NCT02915432.

Recombinant humanized anti-PD-1 monoclonal antibody (JS001) in patients with refractory/metastatic nasopharyngeal carcinoma: Interim results of an open-label phase II clinical study.

6017 Background: Metastatic nasopharyngeal cancer (NPC) patients progressed afterstandardtherapy have limited treatment options. Toripalimab, also known as JS001, a humanized IgG4 antibody specific for human PD-1, has been approved for 2nd line treatment of metastatic melanoma in China. Here we report the results from a phase IIstudy in metastatic NPC patients treated with toripalimab.(Clinical trial ID: NCT02915432). Methods: This multi-center, open-label, phase II registration study is designed to evaluate the safety and efficacy of toripalimab in metastaticNPC patients who have failed systemic treatment. Toripalimabis given at 3 mg/kg IV Q2W until disease progression or intolerable toxicity.Tumor PD-L1 expression, plasma EBV DNA level and other biomarkerswill be correlatedwith clinical response. Results: Enrollment of 190chemo-refractory metastatic NPCpatients was completed by Feb 2019 from 17 participating centers. The median age was 46 years, with 89.5% patients received at least 2 lines of prior systemic therapies. Treatment related adverse events (TRAE)occurred in 92% patients, which were mostly grade 1 or 2.Common TRAE includedanemia, hypothyroidism, AST increased, proteinuria, pyrexia, cough, constipation, ALT increased, hypoalbuminemia and pruritus.Grade 3 or higherTRAEoccurred in 25% patients.By the cut-off date of Jan 7 2019, among 135 evaluable patients, 3 complete responses, 31 partial responses and 40 stable diseaseswere observed for an objective response rate (ORR) of 25.2% and a disease control rate of 54.8%. PD-L1 expression results were obtained from 125 patients and 45.6% (57/125) were PD-L1+.PD-L1+ patientsachieved slightly higherORR than PD-L1- patients, 29.8% versus 22.1%. In addition, an average drop of 47-fold plasma EBV DNA copy number was observed in responding patients, which typically proceeded the radiographic identification of clinical benefit. Conclusions: Toripalimab has demonstrated a manageable safety profileand encouraging clinical activity in the largest check-point blockade study in NPC to date. A change in plasma EBV DNA copy number might serve as a predictive marker for favorable clinical response. Patients will be continuously monitored for additional safety and survival readouts. Clinical trial information: NCT02915432.

Humanized PD-1/PD-L1 (HuPD) mice facilitate the direct functional comparison of immune checkpoint inhibitors in vivo

Abstract Though anti-PD-1/L1 antibody therapies have generated unprecedented responses in a limited number of patients, the biological and immunological parameters that define their effective use are still largely unknown. The absence of appropriate pre-clinical models has limited our ability to compare the mechanistic differences between the approved PD-1/L1 antibodies in vivo and to determine the context in which each might be most effective. To address this critical need, we have produced humanized PD-1 and PD-L1 mice in which the extracellular domains (antibody binding sites) of mouse PD-1 and PD-L1 are replaced with the sequences of human PD-1 and PD-L1. The PD-1 and PD-L1 in the resulting “HuPD” mice can be recognized by FDA-approved antibodies, while the intracellular domains are kept intact for normal downstream signals. Following characterization of the new mouse model, in our first in vivo treatment studies, HuPD mice were immunized with OVA/Poly I:C in the context of FDA-approved PD-1/L1 antibodies and antigen-specific T cell production was quantified. Nivolumab, Pembrolizumab, and Atezolizumab all increased effector CD8+T cell production against the OVA antigen as expected given their immune stimulating function. Surprisingly, OVA-tetramer staining was unable to detect antigen-specific CD8+T cells in the spleens of immunized mice treated with Avelumab. This may reflect the unique ability of Avelumab to induce ADCC, and indicates that our model is capable of distinguishing key differences among the antibodies in vivo. Ongoing studies will identify how these differences might be exploited to render the most effective results for patients in a variety of tumor types and immune environments.

PD-L2 generates a signal through the PD-1 co-receptor distinct from that of PD-L1

Abstract Immune checkpoint blockade of the PD-1/PD-L1/PD-L2 signalling axis yields potent anti-tumor responses by disinhibiting T cell effector function. Mouse and human data demonstrating an inhibitory role for PD-L1 has resulted in FDA approval of anti-PD-L1 antibodies; however, PD-L2 remains sparsely studied despite being the higher affinity of the two ligands for PD-1. Recent findings demonstrate that PD-L2 is widely expressed by human malignancies. PD-L1 and PD-L2 have also been shown to induce distinct conformational changes on engaging human PD-1. We utilized a Jurkat T cell based bioluminescent assay of T cell activation and PD-1 inhibition to determine the unique mechanism of action of human PD-L2. In this system, human PD-L2 generates a solely co-inhibitory signal, but at reduced inhibitory potential relative to PD-L1. We find that unlike murine PD-L2, human PD-L2 appears to have a dedicated co-inhibitory function as we find no significant engagement of the human homolog of its murine co-stimulatory partner RGMb. Reverse phase protein array analysis of human PD-L1 and PD-L2 engagement of PD-1 on Jurkat T cells revealed significant differences in downstream T cell signalling generated by each ligand. We also find that human PD-L1 and PD-L2 have disparate impacts on proliferation wherein PD-L2 supports a preferential arrest of T cells in the S-phase of the cell cycle. We observed that combination blockade of PD-L1 and PD-L2 improves on the blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our findings address critical gaps in our knowledge about the basic function of PD-L2 and will inform future attempts to manipulate the PD-1 co-inhibitory circuit.

Ex vivo Hsp70-Activated NK Cells in Combination With PD-1 Inhibition Significantly Increase Overall Survival in Preclinical Models of Glioblastoma and Lung Cancer.

Heat shock protein 70 (Hsp70) which is expressed on the plasma membrane of highly aggressive tumors including non-small cell lung carcinoma and glioblastoma multiforme serves as a target for Hsp70-targeting NK cells. Herein, we aimed to investigate the antitumor effects of a combined therapy consisting of ex vivo Hsp70-peptide TKD/IL-2-activated NK cells in combination with mouse/human anti-PD-1 antibody in a syngeneic glioblastoma and a xenograft lung cancer mouse model. Mice with membrane Hsp70 positive syngeneic GL261 glioblastoma or human xenograft A549 lung tumors were sham-treated with PBS or injected with ex vivo TKD/IL-2-activated mouse/human NK cells and mouse/human PD-1 antibody either as a single regimen or in combination. Tumor volume was assessed by MR scanning and tumor-infiltrating CD8+ T, NK, and PD-1+ cells were quantified by immunohistochemistry (IHC). We could show that the adoptive transfer of ex vivo TKD/IL-2-activated mouse NK cells or the inhibition of PD-1 resulted in tumor growth delay and an improved overall survival (OS) in a syngeneic glioblastoma mouse model. A combination of both therapies was well-tolerated and significantly more effective with respect to both outcome parameters than either of the single regimens. A combined treatment in a xenograft lung cancer model showed identical effects in immunodeficient mice bearing human lung cancer after adoptive transfer of TKD/IL-2-activated human effector cells and a human PD-1 antibody. Tumor control was associated with a massive infiltration with CD8+ T and NK cells in both tumor models and a decreased in PD-1 expression on immune effector cells. In summary, a combined approach consisting of activated NK cells and anti-PD-1 therapy is safe and results in a long-term tumor control which is accompanied by a massive tumor immune cell infiltration in 2 preclinical tumor models.

Immune-Checkpoint Blockade Opposes CD8+ T-cell Suppression in Human and Murine Cancer.

Immune-checkpoint blockade enhances antitumor responses against cancers. One cancer type that is sensitive to checkpoint blockade is squamous cell carcinoma of the head and neck (SCCHN), which we use here to study limitations of this treatment modality. We observed that CD8+ tumor-infiltrating lymphocytes (TILs) in SCCHN and melanoma express excess immune checkpoints components PD-1 and Tim-3 and are also CD27-/CD28-, a phenotype we previously associated with immune dysfunction and suppression. In ex vivo experiments, patients' CD8+ TILs with this phenotype suppressed proliferation of autologous peripheral blood T cells. Similar phenotype and function of TILs was observed in the TC-1 mouse tumor model. Treatment of TC-1 tumors with anti-PD-1 or anti-Tim-3 slowed tumor growth in vivo and reversed the suppressive function of multi-checkpoint+ CD8+ TIL. Similarly, treatment of both human and mouse PD-1+ Tim-3+ CD8+ TILs with anticheckpoint antibodies ex vivo reversed their suppressive function. These suppressive CD8+ TILs from mice and humans expressed ligands for PD-1 and Tim-3 and exerted their suppressive function via IL10 and close contact. To model therapeutic strategies, we combined anti-PD-1 blockade with IL7 cytokine therapy or with transfer of antigen-specific T cells. Both strategies resulted in synergistic antitumor effects and reduced suppressor cell function. These findings enhance our understanding of checkpoint blockade in cancer treatment and identify strategies to promote synergistic activities in the context of other immunotherapies.