Anti-mouse PD-1 Antibody Research Articles

3:54 PM Abstract No. 196 Molecularly targeted photothermal ablation enhances the effect of anti-PD1 therapy in a mouse model of hepatocellular carcinoma

Recent clinical studies have shown that immunotherapy approaches to exploit the full potential of the adaptive immune system to control hepatocellular carcinoma (HCC) progression need improvement. Here, we sought to assess the effect of combining immune checkpoint blockade strategies with a novel molecularly targeted photothermal ablation (MTPA) modality for the treatment of HCC using a mouse model. C3H mice were treated via oral gavage with CCl4 (20% vol/vol) for 12 weeks to induce liver cirrhosis. The mice then received implantations of syngeneic HCA-1 cells in the liver (5x106). 12 days after, the animals were randomly assigned to 4 different treatment groups: (1) untreated (n = 2), (2) MTPA only (n = 3), (3) anti-mouse PD1 antibody only (n = 4, 100μg per I.P. injection every 3 days), and (4) MTPA+anti-PD1 (n = 2). MTPA was performed following an I.V. injection of 0.5 mg/kg indocyanine green (ICG), and by illuminating the tumor with a 785nm near infrared laser coupled to a clinical fiberoptic catheter. The mice underwent T2 MRI on POD-13, and tissues were harvested on POD-14. Tumor sections were processed for histology and for characterization of tumor-infiltrating lymphocytes (TILs) through flow cytometry. Masson’s trichrome stain demonstrated extensive liver cirrhosis at the time of tumor implantation. MRI and histology data showed cases of nodular and diffuse infiltrative HCC. Characterization of TILs showed that MTPA increases the CD3+ population and, when used in combination with anti-PD1, together they increase the number of CD8+ cells while reducing the number of CD4+/FoxP3+ cells (Tregs). Our preliminary results indicate that even in the context of impaired liver function, the combination of anti-PD1 therapy with our novel MTPA has a synergistic effect whereby greater numbers of cytotoxic lymphocytes infiltrate the hepatic tumors, while at the same time decreasing the presence of immunosuppressive cells. Longer follow-up should reveal how these changes in the TILs impact disease progression in both nodular and diffuse infiltrative HCC subtypes.

Immunogenicity and antitumor efficacy of a novel human PD-1 B-cell vaccine (PD1-Vaxx) and combination immunotherapy with dual trastuzumab/pertuzumab-like HER-2 B-cell epitope vaccines (B-Vaxx) in a syngeneic mouse model

ABSTRACT Therapeutic blockade of PD-1/PD-L1 signaling with monoclonal antibodies (mAbs) has shown clinical success and activity across a broad set of cancer subtypes. However, monotherapy with PD-1/PD-L1 inhibitors are only effective in a subset of patients and ongoing studies show efficacy of treatment depends on a combinatorial approach. Contrary to mAbs chimeric B-cell cancer vaccines incorporating a “promiscuous” T-cell epitope have the advantage of producing a polyclonal B-cell antibody that can potentially induce memory B- and T-cell responses, while reducing immune evasion and suppression. Here, we describe a novel PD-1 B-cell peptide epitope vaccine (amino acid 92–110; PD1-Vaxx) linked to a measles virus fusion peptide (MVF) amino acid 288–302 via a four amino acid residue (GPSL) emulsified in Montanide ISA 720VG that aims to induce the production of polyclonal antibodies that block PD-1 signaling and thus trigger anticancer effects similar to nivolumab. In preclinical studies, the PD1-Vaxx outperformed the standard anti-mouse PD-1 antibody (mAb 29F.1A12) in a mouse model of human HER-2 expressing colon carcinoma. Furthermore, the combination of PD1-Vaxx with combo HER-2 peptide vaccine (B-Vaxx) showed enhanced inhibition of tumor growth in colon carcinoma BALB/c model challenged with CT26/HER-2 cells. The PD-1 or combined vaccines were safe with no evidence of toxicity or autoimmunity.

Abstract 582: Tumor immunoediting in a lung cancer mouse model harboring EGFR mutations

Abstract Background: Anti-Programed cell death 1 (PD-1) antibodies such as nivolumab or pembrolizumab show promising treatment effects in smoking-related lung cancer. However, anti-PD-1 antibodies have very little effect on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). EGFR mutations are the most frequent oncogenic drivers in non-smoking related NSCLC. Therefore, we aimed to explore the mechanism underlying tumor immunoediting in a lung cancer mouse model harboring EGFR mutations. Methods: We previously established two strains of transgenic lung cancer mouse (C57BL/6) harboring a mouse Egfr exon 19-deletion (mDEL) or a human EGFR L858R (hLR) mutation, driven by a surfactant protein (SP)-C promoter (Ohashi, Cancer Sci. 2008; Ohashi, Cancer Res. 2009). These mice developed EGFR-dependent multifocal lung adenocarcinomas from type II pneumocytes. We modified the two models by transplanting lung tumors subcutaneously into C57BL/6 (Ohashi, AACR. 2018). Evaluation of mice tumor lymphocyte profile was done through flow cytometry or immunohistochemistry (IHC). Effects of T cell depletion or anti-mouse PD-1 antibody (4H2) in vivo were also assessed. Comprehensive analysis of mRNA expression of immuno-checkpoint molecules in the tumors was performed using PanCancer Immune Profiling Panel (NanoString Technologies). Results: CD4+ T cells and/or CD8+ T cells were depleted in the subcutaneously transplanted tumor model followed by treatment with gefitinib (50 mg/kg/day, 5 days/week) for 2 weeks. As a result, after gefitinib discontinuation, rapid regrowth was seen in the tumors from groups with depletion of CD8+ T cells; depletion of CD4+ T cells had little effect on tumor regrowth, and tumor inhibition continued for 2 weeks after cessation of gefitinib. IHC showed relatively increased numbers of CD4+ T cells in the mice tumors whereas a decrease in the number of CD8+ T cells or Foxp3 positive cells was noted. PD-L1 expression was not observed in the tumors. Consistently, 4H2 showed little effect on tumor growth in vivo. The cancer immune panel showed increased expression of immune mediators such as Ccl6, Clec4n, and Lcn2. Conclusions: Our results suggest that CD8+ T cells can recognize mice tumors in vivo. New immuno-checkpoint molecules regulating CD8+ T cells from PanCancer Immune Profiling Panel were evaluated. Further, we plan to evaluate the efficacy of various immuno-checkpoint inhibitors in vivo. Citation Format: Kazuya Nishii, Kadoaki Ohashi, Kiichiro Ninomiya, Go Makimoto, Hiromi Watanabe, Hirohisa Kano, Naofumi Hara, Heiichiro Udono, Katsuyuki Kiura. Tumor immunoediting in a lung cancer mouse model harboring EGFR mutations [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 582.

Abstract 2258: Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) potentiates anti-PD-1 therapy in TNBC syngeneic mouse model

Abstract Background : Anti-PD-1 immunotherapy, although efficient in several cancer indications; has shown some limitations due to the appearance of resistance mechanisms. More specifically, the overexpression of A2A receptor (adenosine receptor) on the surface of infiltrated CD8 T cells following anti-PD-1 treatment blunts the immune response in an adenosine-rich tumor microenvironment (TME). In addition, increased evidence of immune escape has been described through gene hypermethylation processes. Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) is an innovative experimental therapy designed to directly reduce systemic levels of L-methionine, an amino-acid nutrient needed for tumor growth and metastasis. Erymethionase is expected to indirectly decrease adenosine levels in TME and to reduce hypermethylation reactions. This bimodal action makes of erymethionase a promising agent to combine with immune checkpoint inhibitors. The benefit of such combination (erymethionase and PD-1 blockade agent) was investigated in a TNBC-like syngeneic mouse model. Methods : Mice bearing orthotopic EMT-6 syngeneic breast carcinoma were intravenously injected once weekly for 4 consecutive weeks with vehicle or mouse erymethionase at 30 or 60 U/kg alone or in combination with anti-mouse PD-1 antibody (RMP1-14 clone, intraperitoneal route, 10 mg/kg, twice weekly for 3 consecutive weeks) from D7 (D0 referring to injection of tumor cells). Erymethionase treatment was associated to daily oral vitamin B6 (pyridoxine, PN) uptake to maintain enzyme activity. The body weight, the length and width of the tumor were measured twice a week. Animal behavior was checked every day. Satellite animals receiving 60 U/kg of erymethionase or vehicle were sacrificed to collect 500-1000 m3 size-tumors to perform immunophenotyping, measure metabolites and/or assess biomarkers. Results : Analysis of health parameters revealed that all treatments were well tolerated along the experiment. A delayed start of exponential growth was reported for the combination at the highest dose of erymethionase vs single agents leading to a significant tumor growth inhibition and an increased survival (median survival time of 35 days for the combination vs 23 days for anti-PD-1 or erymethionase 60 U/kg alone; statistically significant). The antitumor effects were less pronounced at the lower dose level of erymethionase in combination with anti-PD1. Analysis of collected tumors to investigate on the mechanism(s) of action is ongoing. Conclusion : To our knowledge, this is the first in vivo demonstration of anti-PD-1 therapy potentiation using a L-methionine-restricting agent. The data are supportive of the planned erymethionase first-in-human study. Citation Format: Karine Sénéchal, Sylvie Maubant, Marie Leblanc, Séverine Ciré, Fanny Gallix, Aurély Andrivon, Olivier Duchamp, Fabrice Viviani, Françoise Horand, Alexander Scheer, Vanessa BOURGEAUX. Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) potentiates anti-PD-1 therapy in TNBC syngeneic 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 2258.

Abstract A08: Combination with a novel STING agonist significantly improves efficacy of anti-PD1 therapy in mouse syngeneic tumor models

Abstract Activated STING (STimulator of INterferon Genes) bound to its natural ligand 2,3-cGAMP (cyclic guanosine-adenosine monophosphate), initiates type I interferon (IFN) and pro-inflammatory cytokine production. IFN upregulation is essential to promote antigen-specific CD8+ T-cell priming and leads to potent anti-tumor activity. To exploit this mechanism we synthesized a new STING agonist, MSA-1, that potently activates both mouse and human STING. Intratumoral (IT) administration of MSA-1 to MC38 syngeneic tumor-bearing mice increased tumor and plasma cytokine levels and was effective at driving complete responses (CRs) in 100% of the animals. Most surviving animals developed tumor-specific adaptive immune memory as demonstrated by robust protection against re-challenge with the same tumor type. Mechanistic studies in immune-deficient mice suggested that the initial antitumor activity is in part due to cytokine-driven cytotoxicity and/or other innate immune mechanisms, which may have contributed to some animals not developing an adaptive immune memory. Importantly, MSA-1 caused long-term tumor regressions or CRs in CT26 and B16-F10 tumor models, both of which are intrinsically resistant to single-agent therapy with a fully murinized anti-mouse PD-1 antibody (muDX400). The antitumor immune response in these models was further enhanced when treating the animals with MSA-1 in combination with muDX400. This combination restored T-cell responses in both blood and tumors of the treated mice and provided long-lived immunologic memory in a majority of the animals. Taken together, these data strongly support the development of STING agonists in combination with Keytruda (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single agent anti-PD-1 therapy. Citation Format: Samanthi A. Perera, Johnny E. Kopinja, Yanhong Ma, Jason Laskey, Kalyan Chakravarthy, Long Cui, Yiping Chen, Jeremy Presland, Sharad Sharma, Shuxia Zhao, Jennifer Piesvaux, Ellen C. Minnihan, Heidi Ferguson, Hyun Chong Woo, Ian Knemeyer, Ilona Kariv, Archie Tse, Saso Cemerski, Jared Cumming, B. Wesley Trotter, Bo-Sheng Pan, George H. Addona, Brian J. Long. Combination with a novel STING agonist significantly improves efficacy of anti-PD1 therapy in mouse syngeneic tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A08.

Abstract 3917: Potentiating effect of reovirus in anti-PD1 therapy in colorectal cancer

Abstract Background: Microsatellite instability (MSI) high colorectal cancers (CRCs) have a deficiency in mismatch repair (MMR) and increased levels of PD-L1, LAG-3, and IDO, and respond positively to anti-programmed death (PD) therapy. MSI low or microsatellite stable (MSS) CRCs that make up majority of tumors in clinical practice have not seen any benefit with PD inhibition. MSS CRC have higher proportion of KRAS oncogenic mutations as compared to MSI CRC. Reovirus, a naturally occurring oncolytic double-stranded RNA virus, has intrinsic preference for replication in KRAS mutant cells causing apoptosis in CRC. Current study was designed to investigate if reovirus could potentiate a beneficial effect of anti-PD therapy in MSS CRC. Methods: An array of CRC cell lines were screened for sensitivity to reovirus by MTT assay and expression of stem cell markers by RNA-seq and FACS. Based on MSI and KRAS status, four cell lines were explored further. Cells were treated with 5MOI reovirus for 48hr and expression of PD-L1 and PD-L2 with and without the potentiating effect of IFN-γ was assayed using FACS and qPCR. Combinatorial effect of reovirus with anti-PD-1 agent was studied in syngeneic models of BALB/c (CT26; KRASmut, MSS) and C57BL/6 (MC38; KRASwt, MSI) mice. The mice were grouped as control (PBS/IgG2A isotype control), reovirus, anti-mouse PD-1 antibody, and combination. Reovirus was used at a dose of 10 million/100 uL daily and anti PD-1 antibody was given i.p 200 ug/100 uL twice a week. Survival data and tumor volumes were recorded. HCT116 cells were xenografted in nude mice and treated with equivalent dose of reovirus. At the end point IHC was performed on excised paraffin-fixed tumor tissue with CD8 and granzyme antibodies. Results: Administration of reovirus was found to be most effective in cell lines tested. HCT116 (MSI & KRASmut), SW620 (MSS, KRASmut), LIM2405 (MSI, KRASwt) and HT29 (MSS, KRASwt) were chosen based on expression of CD133, CD44 and CD24. HCT116, LIM2405 and SW837 revealed increased and HT29 reduced expression of PD-L1 upon treating with reovirus. Survival data and tumor volume measurements showed better potentiating effect of reovirus on anti-PD-1 therapy in CT26 syngeneic model when compared with MC38. While single-agent therapy did not increase survival, the combination did improve survival with significance vs. control in both BALB/c (median 42 vs. 16 days, p=0.003) and C57BL/6 (median 24 vs. 17 days, p=0.02). The reovirus-treated xenografted tumor tissue showed a higher infiltration of T lymphocytes as confirmed by CD8-positive and intensified granzyme staining. Conclusion: Reovirus as single agent is more potent in KRASmut CRC. Syngeneic mice models proved synergistic anticancer effect of reovirus and anti-PD1 agent combination. Reovirus administration increased PD-L1 expression in CRC cells; possible mechanistic rationale for synergistic efficacy. Citation Format: Titto A. Augustine, Radhashree Maitra, Peter John, Sanjay Goel. Potentiating effect of reovirus in anti-PD1 therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3917.

Abstract 3856: Antitumor activity of ABI-009 (nab-rapamycin) in combination with anti-PD1 antibody in a syngeneic mouse model of B16 melanoma

Abstract Background: mTOR pathway has been implicated in cell survival and proliferation and is an attractive target for cancer therapy. Loss of PTEN, a negative regulator of mTOR pathway, is frequently observed in multiple cancer types. Recent studies indicate that loss of PTEN promotes resistance to T cell-mediated immunotherapy. Since PTEN loss can result in downstream mTOR activation, we investigated the safety and efficacy of ABI-009 (nab-rapamycin, a novel mTOR inhibitor) in combination with anti-PD1 antibody in a syngeneic mouse model of B16 melanoma. ABI-009 (nab-rapamycin) is a nanoparticle form of human albumin-bound rapamycin with a mean particle size of approximately 100 nm developed with a proprietary nanoparticle albumin-bound (nab®) technology. ABI-009 is currently in phase 1 and 2 clinical studies for the treatment of malignant perivascular epithelioid cell carcinoma (PEComa), severe pulmonary arterial hypertension (PAH), nonmuscle-invasive bladder cancer, soft-tissue sarcomas, and various childhood cancers. Methods: Syngeneic B16 melanoma tumors were implanted in immunocompetent C57BL/6 mice. ABI-009 was administered IV at 5 mg/kg 3 times weekly. Monoclonal anti-mouse PD-1 antibody (RMP1-14, BioXcell, West Lebanon, NH, USA) was administered IP at 250 μg every 2 days for 3 times. The study drugs were either administered as single agent or in combination with 3 different dosing schedules: concurrent (A), ABI-009 one week before anti-PD1 (B), or anti-PD1 one week before ABI-009 (C). Results: Overall, all treatments were well tolerated with no significant body weight loss in any group. All treatment groups showed significant antitumor effect and longer survival compared with the saline control. Addition of ABI-009 simultaneously or after anti-PD1 significantly improved tumor growth suppression and survival compared with anti-PD1 alone. Tumor volumes were reduced 35.5% for group A and 44.8% for group C when compared with anti-PD1 alone (p < 0.05). Median survival was 26 days for group A and 26 days for group C vs. 20 days for anti-PD1 alone (p < 0.05). On the other hand, group B failed to significantly improve antitumor effect and survival over anti-PD1 alone. The combination treatment regimen of anti-PD1 given before ABI-009 (group C) was the best schedule among the 3 schedules tested. Conclusions: The combination of ABI-009 and anti-PD1 antibody was well tolerated. Results from this study support the treatment regimens of anti-PD1 immunotherapy, concurrent or followed by ABI-009. Pretreatment with ABI-009 followed by anti-PD1 is not recommended. A phase 1b investigation of safety/efficacy of nivolumab and ABI-009 in patients with advanced sarcoma has been initiated. Citation Format: Shihe Hou, Berta Grigorian, Anita Schmid, Neil Desai. Antitumor activity of ABI-009 (nab-rapamycin) in combination with anti-PD1 antibody in a syngeneic mouse model of B16 melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3856.

Abstract A203: Generation of human TIM3 knock-in mice for preclinical efficacy assessment of therapeutic antibodies

Abstract Immune checkpoint inhibitors, i.e., PD1 and CTLA4 antibody therapeutics, have led to long-term survival in many late-stage solid tumor patients. Despite this revolutionary clinical impact, the overall response rate is still low. One major roadblock is that compensatory immune inhibitory pathways were turned on to protect tumor cells from being attacked by T cells. T cell immunoglobulin- and mucin domain-containing molecule 3 (Tim-3) is a type I transmembrane protein expressed in T cells, monocytes, macrophages, and dendritic cells. PD-1 and Tim-3 coexpression is often associated with an exhausted phenotype of tumor-infiltrating lymphocyte (TIL) in various tumor types. TIM-3 expression has been reported as a compensatory mechanism in the PD1 nonresponsive patients. Simultaneously targeting PD1 and TIM3 is now being actively tested in clinical trials. However, we still do not have an animal model that can efficiently evaluate the efficacy of therapeutic TIM3 antibodies or combinations. Here we report generation of a human TIM3 knock-in C57BL/6 mouse model, in which we replaced mouse exon 2-5 of HAVCR2 gene with human counterparts. The engineered knock-in mice express a chimeric TIM3 where its extracellular and transmembrane domains are human sequence, while the signal peptide and intracellular domain are kept intact. We confirmed that homozygous knock-in mice only express this chimeric TIM3, recognizable by human TIM3 antibody in NK cells, macrophages, and stimulated T cells. In an efficacy study, we inoculated the TIM3 knock-in mice with MC38 syngeneic tumor cells. Treating the mice with 5 mg/kg of human TIM3 antibody resulted in modest tumor growth inhibition (TGI), while in combination with anti-mouse PD1 antibody, the TGI improved to 62% following 2 weeks of treatment. We are now breeding the human PD1 and human TIM3 double knock-in mice to test the combinatory therapeutic antibodies. We have also developed human PD-L1 expressing MC38 cells, so that when inoculated in human TIM3 knock-in mice, PD-L1 and TIM3 therapeutic combination can be evaluated. Citation Format: Lei Zheng, Xuesong Huang, Wenyi Ouyang, Gang Chen, Annie Xiaoyu An, Xin Dong, Jay Liu, Jean Pierre Wery, Qian Shi, Davy Xuesong Ouyang. Generation of human TIM3 knock-in mice for preclinical efficacy assessment of therapeutic antibodies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A203.

Abstract 1071: Efficacy of trifluridine/tipiracil + anti-mouse PD-1 antibody combination on mouse colorectal cancer model and related tumor immunomodulatory effects

Abstract Background: Trifluridine/tipiracil (FTD/TPI) is an oral nucleoside antitumor agent that is composed of trifluridine and tipiracil hydrochloride at a molecular ratio of 1:0.5. Checkpoint-blockade immunotherapies are particularly effective in patients with tumor T cell infiltrations. In this study, the antitumor effects of FTD/TPI + anti-mouse PD-1 antibody combination were studied in a syngeneic mouse model and the tumor-infiltrating lymphocyte (TIL) subsets were evaluated. Method: The mouse colorectal cancer cell line CMT-93 was subcutaneously implanted into C57BL/6 mice. Vehicle (0.5% Hydroxypropyl methylcellulose, 10 mL/kg, p.o.), FTD/TPI (75, 100, and 150 mg/kg/day, twice daily, days 1-14, p.o.), anti-mouse PD-1 antibody (clone RMP1-14; 0.1 mg/body; once daily, days 1, 5, and 9; i.p.), and FTD/TPI + anti-mouse PD-1 antibody combination were administered, and inhibitory activity was evaluated according to tumor-volume changes. Single-cell suspensions were prepared from collected tumors. Based on cell marker expression, CD4+ T cells, CD8+T cells, and regulatory T cells (Tregs) were identified using antibodies against CD4, CD8, CD25, and Foxp3, and subsets of TILs were evaluated by flow cytometry. Results: Both anti-mouse PD-1 antibody and FTD/TPI monotherapies were effective in vivo. Tumor-growth inhibition by anti-mouse PD-1 antibody was 81.7% at 0.1 mg/kg/day and that by FTD/TPI was 33.4%, 46.1%, and 59.5% at 75, 100, and 150 mg/kg/day, respectively. FTD/TPI + anti-mouse PD-1 antibody combination using FTD/TPI (75, 100, and 150 mg/kg/day) with anti-mouse PD-1 antibody (0.1 mg/kg/day) inhibited tumor growth at significantly higher rates (P < 0.05; 91.8%, 95.7%, 98.4%, respectively) than monotherapy. Remarkably, the 150-mg/kg/day FTD/TPI + 0.1-mg/kg/day anti-mouse PD-1 antibody combination caused complete tumor regression in four/five mice without body-weight reduction or drug-related deaths; however, none of the monotherapies caused complete tumor regression. Flow cytometric analysis revealed a higher CD8+ T cell percentage among total lymphocytes and a lower Treg percentage in CD4+ T cells after combination therapy compared with the controls. Conclusion: FTD/TPI + anti-mouse PD-1 antibody combination was synergistically effective on CMT-93 mouse colon tumor. This combination increased CD8+ T cell percentages in whole lymphocyte and decreased Treg percentages in CD4+ T cell. This suggests that FTD/TPI + anti-mouse PD-1 antibody combination modulates tumor T cell populations and improves their antitumor activity. Citation Format: Norihiko Suzuki, Hiroshi Tsukihara, Fumio Nakagawa, Takashi Kobunai, Teiji Takechi. Efficacy of trifluridine/tipiracil + anti-mouse PD-1 antibody combination on mouse colorectal cancer model and related tumor immunomodulatory effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2017-1071

Abstract LB-113: Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models

Abstract Background: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine-based inhibitors including JQ1, I-BET762, and OTX015. Phase I clinical trials have now been initiated with this agent based on its potent anti-tumor activity in various in vitro and in vivo models of hematologic malignancies and solid tumors. In view of the recent publications implicating a role for BET protein BRD4 in the suppression of PD-L1 expression, an immune checkpoint ligand for PD-1, we sought to evaluate ODM-207 for its effect on immune-mediated anti-tumor efficacy in pre-clinical models. Methods and Results: Mouse splenocytes were stimulated with anti-CD3 and anti-CD28 in the presence or absence of ODM-207 for four days and changes in immune cell population were analyzed by FACS. Results revealed an increase in the level of activated cytotoxic CD8+ T cells as indicated by increased intracellular IFNγ and granzyme B with ODM-207 treatment. After confirming the lack of direct anti-proliferative activity on the mouse colon carcinoma cell line CT26, in vivo evaluation of ODM-207 was carried out in the syngeneic CT26 subcutaneous tumor model established in BALB/c mice. Daily oral administration of ODM-207 at 30 mg/kg was well tolerated in this model and resulted in a statistically significant inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ODM-207 was comparable to that with a commercially available anti-mouse PD1 antibody. Studies to characterize the immune changes in the tumor and anti-tumor activity of ODM-207 in combination with an anti-mouse PD1 antibody are currently underway and the results will be presented. Conclusions: In summary, these studies demonstrate the anti-tumor activity of BET inhibitor in a syngeneic model of colon carcinoma in the absence of a direct anti-proliferative activity on tumor cells. Observed tumor growth inhibition correlated with the in vitro activation of cytotoxic CD8+ T cells supporting the immune-mediated effect leading to tumor growth inhibition. In view of the remarkable success with the immune-based therapeutic approaches, these findings are relevant in devising appropriate strategies for the continued clinical development of ODM-207. Citation Format: Pratima Deshpande, Ravi Krishna Babu, Prashant Yallappa Vadnal, Mahaboobi Jaleel, Murali Ramachandra, Chandrasekhar Abbineni, Susanta Samajdar, Anu-Maarit Moilanen, Pekka Kallio. Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2017-LB-113

Abstract 1017: Lipid metabolic reprogramming drives resistance to PD1 blockage

Abstract The mechanisms underlying immunosuppression and resistance to PD1 inhibitors in cancer are not well understood. We attempted to fill this gap with an integrated analysis of mRNA, microRNA, and protein expression in an anti-PD1-resistant lung adenocarcinoma mouse model. The model was created by in vivo passage of 344SQ murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host repeatedly dosed with anti-mouse PD1 antibodies. Anti-PD1-resistant 344SQ (344SQ_R) and 344SQ parental (344SQ_P) cells were then inoculated into syngeneic 129Sv/ev mice, which were then dosed twice with anti-PD1 or control IgG antibodies. Tumor tissues were collected and analyzed as follows: transcriptome with Affymetrix; protein levels by reverse phase protein array analysis; signature enrichment by gene set enrichment analysis; metabolome by mass spectrometry; and lipid content with fluorescent probes Oil O rad and BODIPY. We also isolated tumor-infiltrating immune cells for flow cytometry and gene expression analyses. We identified lipid-related metabolic pathways as being the most highly enriched in anti-PD1-resistant tumors (344SQ_R) vs. their 344SQ_P counterparts; the resistant cells also had more lipid droplets than the 344SQ_P cells. The anti-PD1-resistant tumors overexpressed several genes involved in lipogenesis and fatty acid pathways (e.g., fatty acid binding proteins [FABPs], fatty acid synthase, acetyl-coA-acyltransferase 2, fatty acid elongases). Specifically, FABP overexpression promoted fatty acid uptake and lipid-droplet accumulation in resistant tumors. Lipid-sensitive targets linked to inflammation and insulin signaling (e.g,. stress-activated kinases such as JNK and NFκB) were altered in 344SQ_R vs. 344SQ_P tumors. Mechanistically, JNK downregulation by NFκB-regulated microRNAs protected PD1-resistant tumors from lipotoxicity caused by FABPs upregulation and fatty acid uptake. FABP levels were higher in plasma from 344SQ_R than from 344SQ_P tumors. Tumor-infiltrating macrophages from 344SQ_R tumors had 4 times the amount of FABP mRNA than parental tumors and a correspondingly higher percentage of M2-like macrophages. 344SQ_R tumors promoted immune suppressive cells by upregulating FABPs expression in M2-like macrophages, marked by increased fatty acid intake and fatty acid oxidation. Conversely, percentages of CD4+ and CD8+ tumor-infiltrating lymphocytes were reduced in the resistant tumors. These results suggest that lipid metabolic rewiring drives resistance PD1 inhibitors supporting the accumulation of immunosuppressive cells, including M2-like macrophages, preventing type I immune responses elicited by T cells. Collectively, these findings reveal new potential lipid-related targets for drug development or new treatments combining inhibitors of these targets with anti-PD1 therapy. Citation Format: Maria A. Cortez, Sharareh Niknam, Efrosini Cuko, Jonathan E. Schoenhals, Hampartsoum Barsoumian, Ahmed I. Younes, Ailin Li, Jody V. Vykoukal, Cristina Ivan, George A. Calin, Patrick Hwu, James W. Welsh. Lipid metabolic reprogramming drives resistance to PD1 blockage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2017-1017

Fatty-acid-binding proteins as a novel target for the treatment of anti-PD-1-resistant tumors.

11562 Background: The mechanisms underlying immunosuppression and resistance to PD1 inhibitors in cancer are not well understood. We attempted to fill this gap with an integrated transcriptome analysis in an anti-PD1-resistant lung adenocarcinoma mouse model. The model was created by in vivo passage of 344SQ murine lung cancer cells (p53R172HΔg/+K-rasLA1/+) in a syngeneic host repeatedly dosed with anti-mouse PD1 antibodies. Anti-PD1-resistant 344SQ (344SQ_R) and 344SQ parental (344SQ_P) cells were then inoculated into syngeneic 129Sv/ev mice, which were then dosed twice with anti-PD1 or control IgG antibodies. Methods: Tumor tissues were collected and analyzed as follows: transcriptome with Affymetrix; protein levels by reverse phase protein array analysis; signature enrichment by gene set enrichment analysis; metabolome by mass spectrometry; and lipid content with fluorescent probes Oil O and BODIPY. We also isolated tumor-infiltrating immune cells for flow cytometry and gene expression analyses. Results: We identified lipid-related metabolic pathways as being the most highly enriched in anti-PD1-resistant tumors (344SQ_R) vs. their 344SQ_P counterparts; the resistant cells also had more lipid droplets than the 344SQ_P cells. The anti-PD1-resistant tumors overexpressed several genes involved in lipogenesis and fatty acid pathways, including fatty acid binding proteins (FABPs). Specifically, FABP overexpression promoted fatty acid uptake and lipid-droplet accumulation in resistant tumors. 344SQ_R tumors promoted immune suppressive cells by upregulating FABPs expression in M2-like macrophages, marked by increased fatty acid intake and fatty acid oxidation. Conversely, percentages of CD4+ and CD8+ tumor-infiltrating lymphocytes were reduced in the resistant tumors. Conclusions: These results suggest that lipid metabolic rewiring drives resistance PD1 inhibitors supporting the accumulation of immunosuppressive cells, including M2-like macrophages, preventing type I immune responses elicited by T cells. Collectively, these findings reveal new potential lipid-related targets for drug development or new treatments combining inhibitors of these targets with anti-PD1 therapy.

Suppression of Type I IFN Signaling in Tumors Mediates Resistance to Anti-PD-1 Treatment That Can Be Overcome by Radiotherapy.

Immune checkpoint therapies exhibit impressive efficacy in some patients with melanoma or lung cancer, but the lack of response in most cases presses the question of how general efficacy can be improved. In addressing this question, we generated a preclinical tumor model to study anti-PD-1 resistance by in vivo passaging of Kras-mutated, p53-deficient murine lung cancer cells (p53R172HΔg/+K-rasLA1/+ ) in a syngeneic host exposed to repetitive dosing with anti-mouse PD-1 antibodies. PD-L1 (CD274) expression did not differ between the resistant and parental tumor cells. However, the expression of important molecules in the antigen presentation pathway, including MHC class I and II, as well as β2-microglobulin, were significantly downregulated in the anti-PD-1-resistant tumors compared with parental tumors. Resistant tumors also contained fewer CD8+ (CD8α) and CD4+ tumor-infiltrating lymphocytes and reduced production of IFNγ. Localized radiotherapy induced IFNβ production, thereby elevating MHC class I expression on both parental and resistant tumor cells and restoring the responsiveness of resistant tumors to anti-PD-1 therapy. Conversely, blockade of type I IFN signaling abolished the effect of radiosensitization in this setting. Collectively, these results identify a mechanism of PD-1 resistance and demonstrate that adjuvant radiotherapy can overcome resistance. These findings have immediate clinical implications for extending the efficacy of anti-PD-1 immune checkpoint therapy in patients. Cancer Res; 77(4); 839-50. ©2016 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 569: MicroRNA-mediated resistance to anti-PD1 therapy in lung cancer

Abstract Immunotherapies targeting programmed cell death receptor-1 (PD-1) have shown some clinical success, validating the role of immune modulation in the treatment of cancer. However, only 18% of non-small cell lung cancer patients have responded to anti-PD1 treatment thus far, the remaining 82% have not. This raises fundamental questions about mechanisms of resistance to anti-PD1 therapy and potential strategies to overcome resistance. Previously, our group generated an anti-PD-1-resistant preclinical tumor model after sequential in vivo passage of the murine lung cancer cell line 344SQ (p53R172HΔg/+KrasLA1/+) into syngeneic mice, previously treated with anti-mouse PD-1 antibodies. In this study, we analyzed global gene expression of microRNAs and correlated with our previous mRNA microarray data to validate potential target candidates in order to identify mechanisms of resistance to anti-PD1 therapy. For the microRNA microarray studies, 344SQ_P (parental) or 344SQ_R (resistant) cells were inoculated into the right flank of 129Sv/ev mice (female mice, 12-14 weeks old, 5 mice per group). Anti-PD1 or control IgG (10mg/kg) antibodies were given on days 4 and 7 after tumor inoculation. On day 11, tumor tissues were collected and immediately frozen in liquid nitrogen. RNAs from three independent biologic replicates per group were used for GeneChip miRNA 4.0 Array (Affymetrix). Significant differences in microRNA gene expression levels between groups were defined as an adjusted P value <0.05 and absolute fold change of > 2. Heatmaps were created by using the heatmap.2 function in the gplots R package. Our previous studies demonstrated that major histocompatibility complex (MHC) class I and II and antigen presentation pathway, were significantly downregulated in the anti-PD-1-resistant tumors compared with parental tumors. Here, we found that microRNAs significantly upregulated in the anti-PD1 resistant model directly regulates MHC class I and II and antigen presentation machinery genes, including B2M, CIITA, PSMB8, PSMB8, TAP2 and TAPBP, promoting resistance to anti-PD1 therapy. Because activated CD8+ tumor-infiltrating lymphocytes must bind to MHC I proteins to kill target tumor cells, one way in which tumors can evade destruction by the immune system is through downregulating expression of MHC I on their surfaces. Therefore, our study identified upregulation of microRNAs that modulates the antigen presentation pathway as an underlying mechanism by which some tumors do not respond to anti-PD1 therapy. Our future goal is to validate these findings on our ongoing clinical studies. Citation Format: Maria A. Cortez, Xiaohong Wang, Cristina Ivan, Jonathan E. Schoenhals, Sharareh Niknam, Ailin Li, David Valdecanas, James P. Allison, Padmanee Sharma, Willem W. Overwijk, Daniel Gomez, Joey Y. Chang, Stephen Hahn, George A. Calin, James W. Welsh. MicroRNA-mediated resistance to anti-PD1 therapy in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 569.

Abstract 549: Immune profiling uncovers neo-antigens and TCR repertoire responding to WX-mPD-1 and WX-mPD-L1 immune checkpoint inhibitors in syngeneic mouse xenograft models

Abstract Introduction: PD-1 and PD-L1antibodies have shown impressive clinical benefits to cancer patients. However, still a significant number of patients fail to respond to those checkpoint inhibitors. To understand the underlying mechanism, we performed genomic and immune profiling of both tumor tissues and cytotoxic T cells isolated from syngeneic mouse xenograft models treated with in-house WuXi anti-mouse PD-1(WX-mPD-1) and anti-mouse PD-L1 (WX-mPD-L1) antibodies. Material and methods: Syngeneic mouse xenograft models from melanoma cell lines, CloudmanS91 and B16-F10, were used to evaluate the anticancer efficacy of WX-mPD-1 and WX-mPD-L1 antibodies. Cancer cells and xenograft tumors before and after treatment were collected for RNASeq profiling, from which mRNA expression and DNA mutations were called and neo-antigens were predicted. FACS analysis and TCR sequencing were also performed to examine the tumor infiltrating lymphocytes (TILs) population and TCR repertoire diversity, respectively. Results: Among forty-eight established syngeneic models, nine had been tested with WX-mPD-1 and WX-mPD-L1 antibodies as single agent or in combination. More importantly, we have investigated the expression, mutation loads, neo-antigens for responsive model from CloudmanS91 and partially responsive model from B16-F10. CloudmanS91 model had 8 fold increases in mutation loads compared with B16-F10 model. Neo-antigens identified in CloudmanS91 cells were further knocked out to dissect their roles in activating immune response to WX-mPD-1 and WX-mPD-L1 antibodies in vivo. Conclusions: WX-mPD-1 and WX-mPD-L1 antibodies as well as syngeneic tumor models are useful tools for pre-clinical combination and MoA studies for cancer immunotherapy. Analysis of Neo-antigen and TCR repertoire helps us better understand immune response to these antibodies in preclinical settings, and suggest novel approaches to enhance the current immunotherapeutic interventions. Citation Format: Hua Dong, Yan Lu, Qiyao Zhang, Lingling Zhang, Xiaoyue Chen, Guizhu Yang, Shuqun Yang, Miao He, Xuzhen Tang, Yuxin Qin, Yong Zheng, Jiexing Cai, Yong Cang, Shaoyu Yan, Jing Li, Qunsheng Ji. Immune profiling uncovers neo-antigens and TCR repertoire responding to WX-mPD-1 and WX-mPD-L1 immune checkpoint inhibitors in syngeneic mouse xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 549.

Abstract 262: Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models

Abstract The PD-1/PD-L1 (programmed cell death protein 1; programmed death-ligand 1) pathway plays a key role in immunological tolerance and autoimmunity. PD-1 is primarily expressed on activated T and B cells while its receptor, PD-L1, is expressed in a variety of cell types including resting lymphocytes, DCs, and macrophages. Recently, multiple human solid tumor types (melanoma, NSCLC, RCC, ovarian, and colorectal cancer) have been found to overexpress PD-L1 within its tumor microenvironment. More importantly, PD-1/PD-L1 blocking agents (pembrolizumab, nivolumab, lambrolizumab, MPDL3280A) have been found active as a single agent or in combination with chemotherapy and some have produced durable clinical responses. On the other hand, human epithelial tumors are also known to overexpress the high-affinity folate receptor (FR)-alpha that is capable of bringing folate-linked drugs into the cell cytosol via endocytosis. Using flow cytometry, we assessed PD-L1 expression in various FR-positive syngeneic mouse tumor models (M109, Renca, L1210A, 4T1-Cl2, ID8-Cl15). PD-L1 expression was found to vary significantly among different tumor types and could be found not only on CD45- malignant cells but also on tumor stromal cells such as F4/80+CD11b+ tumor-associated macrophages. Using a rat anti-mouse PD-1 antibody (clone RMP1-14), we studied in-vivo efficacy of a folate-targeted chemotherapy in combination with PD-1/PD-L1 blockage in PD-L1-expressing tumor models. In syngeneic models with high PD-L1 expression and tumor-infiltrating lymphocytes, a synergistic and curable anti-tumor effect was observed and the animals developed tumor-protective immunity against rechallenge. Thus, our preliminary results suggest that folate-targeted chemotherapeutics may also be enhanced by PD-1/PD-L1 blockage by means of overcoming tumor-mediated immune suppression Citation Format: Yingjuan June Lu, Leroy W. Wheeler, Vicky Cross, Elaine Westrick, Alex Lloyd, Christopher P. Leamon. Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 262.

Abstract 3211: PD-1-targeted Probody therapeutics provide anti-tumor efficacy and a 10-fold dose protection against systemic autoimmunity in preclinical studies

Abstract Immuno-oncology approaches have transformed the treatment of advanced melanoma, NSCLC and an increasing number of other cancers. Antibodies targeting the T cell checkpoint molecules CTLA-4 and PD-1 can reverse tumor suppression of T cell activation that prevents effective anti-tumor immunity. However, blockade of checkpoint molecules, particularly in combination, in non-tumor tissues can result in autoimmune side effects that can limit therapeutic utility. Combination of PD-1 and CTLA-4 blockade provides increased response rates but with concomitant increases in immune-related adverse events. Here we demonstrate that Probody™ therapeutics targeting PD-1 synergize with a CTLA-4 checkpoint inhibitor to eradicate established tumors in a mouse model while protecting against PD1-mediated systemic autoimmunity. Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs that have the potential to meaningfully widen therapeutic index by minimizing interactions with normal tissue while retaining anti-tumor activity. Probody therapeutics take advantage of protease activities that are abundant in tumors but suppressed in normal tissues. An extension of the antibody light chain with a masking peptide blocks binding of the antibody to antigen in normal tissue. Tumor-associated proteases cleave and release the mask, enabling the active antibody to bind antigen preferentially in the tumor. The feasibility of the Probody approach to checkpoint targets is supported by preclinical and clinical studies in which intratumoral delivery of low dose immune modulators enables anti-tumor responses despite negligible systemic exposure. For preclinical assessment of PD-1 as a Probody target, a family of Probody therapeutics derived from the anti-mouse PD-1 antibody J43 was developed. The intact Probody therapeutics demonstrated reduced binding to mouse PD-1 relative to the parental antibody that was completely restored following proteolytic activation. The anti-tumor efficacy of the J43 antibody and Probody therapeutics was assessed following systemic administration as single agents or in combination with an anti-CTLA-4 antibody to mice bearing established MC38 syngeneic tumors. As single agents, the PD-1 antibody and Probody therapeutics reduced tumor growth relative to an isotype antibody control and synergized with CTLA-4 blockade. Consistent with their reduced ability to bind target in the absence of proteolytic activation, the PD-1 Probody therapeutics were up to 10 times less potent than the parental anti-PD-1 antibody in inducing autoimmune diabetes in NOD mice. Our preclinical findings demonstrate that PD-1 Probody therapeutics retain anti-tumor efficacy with improved safety profiles and therefore have the promise to enable better tolerated PD-1 combination immunotherapies. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Kimberly A. Tipton, Chanty Chan, Kenneth R. Wong, Victoria Singson, Jennifer H. Richardson, W Michael Kavanaugh, Bryan A. Irving, James W. West. PD-1-targeted Probody therapeutics provide anti-tumor efficacy and a 10-fold dose protection against systemic autoimmunity in preclinical studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3211.

Abstract B97: Response to checkpoint inhibition by GEMM breast cancer allograft

Abstract Background. Cancer patient's response to immunotherapy, e.g. checkpoint inhibitors, differ greatly, likely due to their genetic composition (e.g. neo-mutation) or immunological profile of tumors. Experimental models may facilitate the identification of responders or of the approach to enhance response. Syngeneic mouse tumor models have been widely used as experimental model for testing surrogate immunotherapy, but limited by the fact that only a few are available and respond to the current checkpoint inhibitors. Allografts of mouse spontaneous tumors (MuPrimeTM) may be used as new type of I/O model with advantages: 1) its primary natures with “stem cell diseases” and relevant tumor microenvironment as seen in PDX1-5; 2) diverse cancer types and deriving from wider range of available spontaneous mouse tumors. Method. We created a MuPrime, MBR6004, by allografting the breast adenocarcinoma derived from GEMM-MMTV-PyVT in FVB/N mice1. We then characterized the model by examine it subcutaneous and orthotopic growth, and its ability to metastasis. We also transcriptome-sequenced and immune-profiled the tumor, and assessed its response to different treatments, including I/O therapy. Results. MBR6004 was found to over-express HER2 but negative for ER and PR. It maintains original primary tumour histopathology, grows robustly, and is confirmed to express its original transgene. It is also resistant to docetaxel. The orthotopic implantation resulted in lung metastasis. We can detected and quantitatively confirmed tumor-infiltrating immune cells, e.g. TIL, CTL, Treg, immune-suppression macrophage, NK, etc. We confirmed that the tumor cell expressed relatively low PD-L1, but inducible, per flow analysis. Our preliminary data indicated that it partially respond to anti-mouse PD1 and anti-mouse CTLA-4 antibodies when mice are preconditioned. Currently, we are investigating whether the responses are associated with the reduced Treg and increased CD8+ TIL in tumors, and also whether the combination therapy can enhance its tumor response synergistically. Conclusion. Our data seem to suggest that MuPrime could be another type of I/O model alternative to commonly used syngeneic cell line derived models at present.

Generation and characterization of an anti-PD1-resistant lune cancer line (TUM2P.1040)

Abstract Immunotherapies targeting PD1/PDL1 have shown unprecedented rates of durable clinical responses in patients with melanoma and lung cancer. However, there is a substantial portion of patients who do not respond to these therapies. The goal of this study is to generate an anti-PD1-resistant tumor line and identify mechanisms of resistance, which should provide novel insights to extend the benefit of immunotherapies. A preclinical lung cancer model was generated by inoculation of a murine lung cancer cell line 344SQ (containing p53 and kras mutations) into syngeneic 129SV/ev mice. Anti-mouse PD1 antibodies were administrated at day 4 post tumor inoculation for a total of 5 doses. The anti-PD1 resistant line was successfully generated after sequential in vivo passaging of a nonresponsive tumor followed by anti-PD1 treatment. Upon the anti-PD1 treatment, the parental line showed 70% tumor reduction, whereas the resistant line showed only 12% tumor reduction. PDL1 expression did not alter in the resistant line. Instead, anti-PD1 treatment remarkably increased MHC I expression in the parental line, whereas the resistant line showed significantly lower MHC I and II expression and failed to upregulate MHC I upon anti-PD1 treatment. The parental and resistant lines showed distinct profiles of tumor-infiltrating immune cells and costimulatory molecule expression in vivo. Next we will study if inducing MHC expression through radiation or HDAC inhibitors will overcome anti-PD1 resistance.