anti-PD1 Blockade Research Articles

Docetaxel remodels prostate cancer immune microenvironment and enhances checkpoint inhibitor-based immunotherapy.

Background: Prostate cancer is usually considered as immune “cold” tumor with poor immunogenic response and low density of tumor-infiltrating immune cells, highlighting the need to explore clinically actionable strategies to sensitize prostate cancer to immunotherapy. In this study, we investigated whether docetaxel-based chemohormonal therapy induces immunologic changes and potentiates checkpoint blockade immunotherapy in prostate cancer.Methods: We performed transcriptome and histopathology analysis to characterize the changes of prostate cancer immune microenvironment before and after docetaxel-based chemohormonal therapy. Furthermore, we investigated the therapeutic benefits and underlying mechanisms of chemohormonal therapy combined with anti-PD1 blockade using cellular experiments and xenograft prostate cancer models. Finally, we performed a retrospective cohort analysis to evaluate the antitumor efficacy of anti-PD1 blockade alone or in combination with docetaxel-based chemotherapy.Results: Histopathology assessments on patient samples confirmed the enrichment of tumor-infiltrating T cells after chemohormonal therapy. Moreover, we found that docetaxel activated the cGAS/STING pathway in prostate cancer, subsequently induced IFN signaling, resulting in lymphocytes infiltration. In a xenograft mouse model, docetaxel-based chemohormonal therapy prompted the intratumoral infiltration of T cells and upregulated the abundance of PD1 and PD-L1, thereby sensitizing mouse tumors to the anti-PD1 blockade. To determine the clinical significance of these results, we retrospectively analyzed a cohort of 30 metastatic castration-resistant prostate cancer patients and found that docetaxel combined with anti-PD1 blockade resulted in better prostate-specific antigen progression-free survival when compared with anti-PD1 blockade alone.Conclusions: Our study demonstrates that docetaxel activates the antitumoral immune response and facilitates T cell infiltration in a cGAS/STING-dependent manner, providing a combination immunotherapy strategy that would improve the clinical benefits of immunotherapy.

CTIM-26. PHASE I/II STUDY OF THE COMBINATION OF PEMBROLIZUMAB (MK-3475) AND LASER INTERSTITIAL THERMAL THERAPY (LITT) IN RECURRENT GLIOBLASTOMA

Abstract BACKGROUND The blood brain barrier (BBB) remains a potential barrier to central nervous system (CNS) penetration of novel immunotherapies in recurrent glioblastoma (rGBM). Laser interstitial thermal therapy (LITT) was recently demonstrated to induce BBB disruption. When combined with anti-PD1 blockade, LITT may therefore potentiate host T-cell mediated cytotoxicity. This phase I/II study aims to evaluate the safety and efficacy of combining LITT and the PD-1 inhibitor pembrolizumab for rGBM. METHODS Phase I treated eligible bevacizumab-naïve high grade glioma patients with LITT and the anti-PD1 inhibitor pembrolizumab at 3 dose levels (100, 150, and 200 mg IV Q3W; 3 patients at each level), while phase II was restricted to rGBM patients only with the recommended phase II dose (RP2D) of pembrolizumab. Phase II was initially designed to randomize rGBM to either pembrolizumab or pembrolizumab+LITT but was later amended to receive only pembrolizumab+LITT after 16 patients were randomized (10 pembrolizumab+LITT arm, 6 pembrolizumab-alone arm). Progression-free survival (PFS) and overall survival (OS) were evaluated using the Kaplan Meier method, and adverse events (AE) documented. RESULTS Phase I enrolled 9 patients (7 rGBM and 2 anaplastic astrocytomas, 33% IDH-mut, 44% MGMTp-methylated) with no dose limiting toxicities (DLT), prompting selection of 200mg Q3W as the RP2D. Phase II interim analysis included 34 rGBM patients (9% IDH-mut, 50% MGMTp-methylated; 6 receiving pembrolizumab alone and 28 pembrolizumab+LITT) plus two Phase I rGBM patients who received RP2D. On per-protocol analysis, pembrolizumab+LITT cohort had improved PFS (median PFS 10.5 months vs 2.1 months) and OS (median OS 11.4 months vs 5.2 months) than pembrolizumab alone. A single treatment-related grade 3 AE was noted (respiratory infection). CONCLUSION: LITT may be safely combined with pembrolizumab for rGBM with promising clinical outcomes on interim analysis. Enrollment for Phase II, correlative studies, and continued AE documentation are ongoing. Clinical Trial ID NCT02311582.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

CAR T cells can be engineered to overcome antigen loss (from Fig. 2C of Landoni et al., Cancer Immunol Res 2021)One factor limiting chimeric antigen receptor (CAR) T-cell therapy efficacy against solid tumors is CAR antigen loss. Johnson et al. show that engineering CAR T cells to express the RNA RN7SL1 increases their antitumor efficacy in multiple models. RN7SL1 enhances CAR T-cell expansion and effector-memory differentiation, and also is transferred in extracellular vesicles to other immune cells that then promote activation of endogenous T cells that can reject solid tumors with CAR antigen loss. By testing various combinations of CAR constructs, Hirabayashi et al. find that T cells engineered to be dual targeting—with each CAR signaling through a different costimulatory endodomain, only one of which includes a CD3ζ-chain—provide the most effective antitumor effects and prevent antigen loss.Johnson LR, …, Minn AJ. Cell 2021 Sep 16;184:4981–95.e14.Hirabayashi K, …, Dotti G. Nat Cancer 2021 Sep 23;2:904–18.Local delivery of cytokine-ending mRNA could improve immunotherapy efficacy (by Gwillhickers via Wikimedia Commons)How to effectively deliver immunotherapy locally to tumors remains a challenge. Hotz et al. test the intratumoral delivery of a mixture of mRNAs encoding four cytokines/cytokine fusion proteins (IL12 single chain, IFNα4, GM-CSF, and IL15 sushi) in multiple tumor models. They find the treatment effectively activates antitumor immunity and induces immune memory; therapeutic effects are observed for primary, distant, and metastatic tumors. Combining the mRNA mixture with immune checkpoint blockade further enhances therapeutic benefit. The data suggest that this treatment strategy has the potential to improve current immunotherapy regimens, and it is now being tested in clinical trials.Hotz C, …, Wiederschain D. Sci Transl Med 2021 Sep 8;13:eabc7804.Pancreatic adenocarcinoma frequently evades the immune system (by Min Yu via NIH Flickr)Pancreatic adenocarcinoma (PDAC) is largely refractory to immunotherapy. Freed-Pastor et al. have developed organoid models of the subset of PDACs they predict to have high-affinity neoantigens and find that some of these organoids evade the immune system after transplantation into immune-competent mice. Immune-evasive organoids and human PDACs are infiltrated with dysfunctional neoantigen-specific CD8+ T cells and have high CD155 expression. Blockade of CD155/TIGIT signaling, together with anti–PD-1 blockade and CD40 agonism, limited immune evasion and CD40-agonist resistance in the mice, identifying a potential combination therapeutic strategy for PDAC.Freed-Pastor WA, …, Jacks T. Cancer Cell 2021 Oct 11;39:1342–60.e14.A CRISPR screen links tumor suppressor genes and the adaptive immune system (by Ernesto del Aguila III via NIH Flickr)Genetic screens for drivers of tumorigenesis are often conducted using systems that do not account for the complexity of the tumor microenvironment, including pressure exerted by the immune system. By performing transcriptome-wide CRISPR screens in mouse tumor models using animals with and without functional adaptive immunity, Martin et al. find an enrichment for loss of tumor suppressor genes as a requirement for tumor growth in the presence of an adaptive immune system. Loss of one tumor suppressor gene, GNA13, increases recruitment of tumor-associated macrophages by enhancing CCL2 secretion. These data highlight potential new functions for tumor suppressor genes that could be harnessed for immunotherapy.Martin TD, …, Elledge SJ. Science 2021 Sep 17;373:1327–35.Engineering IL-2 to be a partial agonist can enhance adoptive cell therapy (by Emw from Wikimedia Commons)IL-2 is often used in the generation of T cells for adoptive cell therapy because it promotes T-cell expansion. However, it also induces terminal differentiation and expression of coinhibitory molecules, limiting therapeutic efficacy. Mo et al. identify an IL-2 partial agonist, H9T, that promotes CD8+ T–cell expansion but not terminal differentiation; rather, it drives transcriptional, epigenetic, and metabolic profiles that restrict T-cell exhaustion and maintain a stem cell–like state. T-cell receptor transgenic T cells and CAR T cells expanded with H9T show enhanced antitumor activity in mice compared with cells expanded in IL-2, highlighting the potential for engineering cytokines to increase adoptive cell therapy efficacy.Mo F, …, Leonard WJ. Nature 2021 Sep 23;597:544–8.A feedback loop facilitates tumor metastasis (by AleXXw via Wikimedia Commons)Tumor cells use various mechanisms to disseminate from the primary tumor to a metastatic site. Morrissey et al. show that tumor-derived exosomes drive polarization of macrophages in the premetastatic niche to an immune-suppressive phenotype, thereby promoting tumor metastasis. Macrophage interaction with tumor-derived exosomes leads to upregulation of PD-L1 and metabolic alterations, which act to further increase macrophage PD-L1 expression. The data highlight a feedback loop that potentiates immune suppression and identifies tumor-derived exosomes, macrophages, and metabolism-related factors as key players in the process.Morrissey SM, …, Yan J. Cell Metab 2021 Oct 5;33:2040–58.e10.

The ongoing mystery of renal cell cancer

Au et al. take an in-depth longitudinal look at tumor cells and T cells in patients with renal cancer undergoing anti-PD1 blockade.

Pathway signatures derived from on-treatment tumor specimens predict response to anti-PD1 blockade in metastatic melanoma

Both genomic and transcriptomic signatures have been developed to predict responses of metastatic melanoma to immune checkpoint blockade (ICB) therapies; however, most of these signatures are derived from pre-treatment biopsy samples. Here, we build pathway-based super signatures in pre-treatment (PASS-PRE) and on-treatment (PASS-ON) tumor specimens based on transcriptomic data and clinical information from a large dataset of metastatic melanoma treated with anti-PD1-based therapies as the training set. Both PASS-PRE and PASS-ON signatures are validated in three independent datasets of metastatic melanoma as the validation set, achieving area under the curve (AUC) values of 0.45–0.69 and 0.85–0.89, respectively. We also combine all test samples and obtain AUCs of 0.65 and 0.88 for PASS-PRE and PASS-ON signatures, respectively. When compared with existing signatures, the PASS-ON signature demonstrates more robust and superior predictive performance across all four datasets. Overall, we provide a framework for building pathway-based signatures that is highly and accurately predictive of response to anti-PD1 therapies based on on-treatment tumor specimens. This work would provide a rationale for applying pathway-based signatures derived from on-treatment tumor samples to predict patients’ therapeutic response to ICB therapies.

DNA Aptamer Against Anti- Programmed Cell Death-1 (Anti-PD1-Apt) Induces Robust Anti-Leukemic Activity In Vitro and In Vivo Humanized NSG Mice with Myeloid Leukemia Xenografts

Background: Cancer cells have an extraordinary ability to escape immune response by modulating proteins that regulate immune checkpoints and their cognate ligands. Programmed cell death protein (PD-1), a cell surface receptor expressed on T cells is one such immune checkpoint receptor when bound to its ligands- PDL1 or PDL2 transmits an inhibitory signal. Such modulation often leads to inhibition of T-cell activation allowing tumors to escape immune surveillance. Recently, several antibodies that inhibit the signaling from PD-1/PD-L1 axis were developed that showed enhanced anti-cancer immune reponse leading to their FDA approval for therapeutic use. Aptamers are synthetic small molecule ligands composed of short, single-stranded RNA or DNA oligonucleotides with specific 3D conformation. Aptamers mimic antibodies to recognize and bind to their targets with high affinity. The nucleic acid component has several advantages over the protein counterparts- such as ease of production under less stringent conditions, long shelf life and low cost. FDA has approved several aptamers for therapeutic use that include Macugen, a RNA aptamer that inhibits VEGF signaling to treat age-related macular degeneration, Vitravene to treat cytomegalovirus (CMV) retinitis and Kynamro to treat homozygous familial hypercholesterolemia. There are several other potentially promising aptamers in ongoing preclinical and clinical tests.Methods: Here, we report the development of several PD-1 specific aptamers by systematic evolution of ligands by exponential enrichment (SELEX) technology against endogenous immunoprecipitated PD-1 protein using DNA library with a complexity of 1014. Following several rounds of SELEX, the selected aptamers sequenced by high throughput Next-Generation Sequencing (NGS) were found to have highly conserved regions. Six PD-1 specific aptamers (Anti-PD1-Apt) were then assessed for target validation using leukemic cell lysates (cell lines and primary patient samples) and were found to bind to the PD-1 in its native state.Results: The selected Anti-PD1-Apt were able to specifically pull down the PD-1 protein from the lysates mimicking PD-1 antibody. The specific interaction of the Anti-PD1-Apt was also demonstrated by flow cytometry and fluorescent microscopy. As expected, Anti-PD1-Apt was able to bind to PD-1 with Kd of ~ 500 pM as assessed by Bio-Layer Interferometry. Furthermore, we also characterized and confirmed Anti-PD1-Apt biological activity using an PD-1/PD-L1 cell-based assay using PD-1/NFAT reporter-Jurkat cells. We have observed several fold induction of NFAT luciferase reporter activity (Relative Luciferase Units) in PD-1/NFAT reporter-Jurkat cells co-cultured with HEK293 cells overexpressing PD-L1 and TCR activator in the presence of Anti-PD1-Apt compared to control. Our preliminary data also demonstrate robust Anti-PD1 blockade in Mixed Lymphocyte Reaction (MLR) along with induction of Th1 cytokines Interferon-gamma and IL-2 from different donor sets of PBMCs. In vitro cytotoxicity T- cell assays demonstrated the ability of PD-1 aptamer to potently enhance T-cell response and cytokine production in MLR. Our findings from pharmacokinetics in vivo studies demostrated that PEG-conjugation of PD-1 aptamer has improved the stability of DNA in mouse plasma. A PEGylated form of PD-1 DNA aptamer has demonstrated the ability to block PD-1:PD-L1 interaction and significantly reduced the growth of HEL92.1.7 leukemic tumor cells in vivo in humanized NSG mice with a potency equivalent to an antagonistic anti-PD-1 therapeutic agents. Tumor growth inhibition in humanized NSG mice treated with PD-1 DNA aptamer was 22.59% and comparable to PD-1 antibody over the control group.Conclusions: Overall, our findings demonstrate a comprehensive preclinical characterization of PD-1 aptamer for which antitumor activity was demonstrated in mouse studies. DisclosuresIyer:Genentech: Research Funding; Takeda: Research Funding.

Cytokine release syndrome in a patient with colorectal cancer after vaccination with BNT162b2

Patients with cancer are currently prioritized in coronavirus disease 2019 (COVID-19) vaccination programs globally, which includes administration of mRNA vaccines. Cytokine release syndrome (CRS) has not been reported with mRNA vaccines and is an extremely rare immune-related adverse event of immune checkpoint inhibitors. We present a case of CRS that occurred 5 d after vaccination with BTN162b2 (tozinameran)—the Pfizer-BioNTech mRNA COVID-19 vaccine—in a patient with colorectal cancer on long-standing anti-PD-1 monotherapy. The CRS was evidenced by raised inflammatory markers, thrombocytopenia, elevated cytokine levels (IFN-γ/IL-2R/IL-18/IL-16/IL-10) and steroid responsiveness. The close temporal association of vaccination and diagnosis of CRS in this case suggests that CRS was a vaccine-related adverse event; with anti-PD1 blockade as a potential contributor. Overall, further prospective pharmacovigillence data are needed in patients with cancer, but the benefit–risk profile remains strongly in favor of COVID-19 vaccination in this population.

A MyD88/IL1R Axis Regulates PD-1 Expression on Tumor-Associated Macrophages and Sustains Their Immunosuppressive Function in Melanoma.

Macrophages are critical mediators of tissue homeostasis, cell proliferation, and tumor metastasis. Tumor-associated macrophages (TAM) are generally associated with tumor-promoting immunosuppressive functions in solid tumors. Here, we examined the transcriptional landscape of adaptor molecules downstream of Toll-like receptors in human cancers and found that higher expression of MYD88 correlated with tumor progression. In murine melanoma, MyD88, but not Trif, was essential for tumor progression, angiogenesis, and maintaining the immunosuppressive phenotype of TAMs. In addition, MyD88 expression in myeloid cells drove melanoma progression. The MyD88/IL1 receptor (IL1R) axis regulated programmed cell death (PD)-1 expression on TAMs by promoting recruitment of NF-κBp65 to the Pdcd1 promoter. Furthermore, a combinatorial immunotherapy approach combining the MyD88 inhibitor with anti-PD-1 blockade elicited strong antitumor effects. Thus, the MyD88/IL1R axis maintains the immunosuppressive function of TAMs and promotes tumor growth by regulating PD-1 expression. SIGNIFICANCE: These findings indicate that MyD88 regulates TAM-immunosuppressive activity, suggesting that macrophage-mediated immunotherapy combining MYD88 inhibitors with PD-1 blockade could result in better treatment outcomes in a wide variety of cancers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2358/F1.large.jpg.

NLRC5/CITA expression correlates with efficient response to checkpoint blockade immunotherapy

Checkpoint blockade-mediated immunotherapy is emerging as an effective treatment modality for multiple cancer types. However, cancer cells frequently evade the immune system, compromising the effectiveness of immunotherapy. It is crucial to develop screening methods to identify the patients who would most benefit from these therapies because of the risk of the side effects and the high cost of treatment. Here we show that expression of the MHC class I transactivator (CITA), NLRC5, is important for efficient responses to anti-CTLA-4 and anti-PD1 checkpoint blockade therapies. Melanoma tumors derived from patients responding to immunotherapy exhibited significantly higher expression of NLRC5 and MHC class I-related genes compared to non-responding patients. In addition, multivariate analysis that included the number of tumor-associated non-synonymous mutations, predicted neo-antigen load and PD-L2 expression was capable of further stratifying responders and non-responders to anti-CTLA4 therapy. Moreover, expression or methylation of NLRC5 together with total somatic mutation number were significantly correlated with increased patient survival. These results suggest that NLRC5 tumor expression, alone or together with tumor mutation load constitutes a valuable predictive biomarker for both prognosis and response to anti-CTLA-4 and potentially anti-PD1 blockade immunotherapy in melanoma patients.

Heat Shock Protein-90 Inhibition Alters Activation of Pancreatic Stellate Cells and Enhances the Efficacy of PD-1 Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC), or cancer-associated fibroblasts (CAF). Targeting inflammatory pathways present within the stroma may improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90) is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC/CAF has not been explored in detail. We hypothesized that Hsp90 inhibition would limit inflammatory signals, thereby reprogramming the PDAC tumor microenvironment to enhance sensitivity to PD-1 blockade. Treatment of immortalized and primary patient PSC/CAF with the Hsp90 inhibitor XL888 decreased IL6, a key cytokine that orchestrates immune changes in PDAC at the transcript and protein level in vitro XL888 directly limited PSC/CAF growth and reduced Jak/STAT and MAPK signaling intermediates and alpha-SMA expression as determined via immunoblot. Combined therapy with XL888 and anti-PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors. Tumors from mice treated with both XL888 and anti-PD-1 had a significantly increased CD8+ and CD4+ T-cell infiltrate and a unique transcriptional profile characterized by upregulation of genes associated with immune response and chemotaxis. These data demonstrate that Hsp90 inhibition directly affects PSC/CAF in vitro and enhances the efficacy of anti-PD-1 blockade in vivo.

Addition of anti-TIM3 or anti-TIGIT Antibodies to anti-PD1 Blockade Augments Human T cell Adoptive Cell Transfer

ABSTRACT PD1 blockade to reinvigorate T cells has become part of standard of care for patients with NSCLC across disease stages. However, the majority of patients still do not respond. One potential mechanism of resistance is increased expression of other checkpoint inhibitory molecules on T cells leading to their suppression; however, this phenomenon has not been well studied in tumor-reactive, human T cells. The purpose of this study was to evaluate this compensatory mechanism in a novel model using human effector T cells infiltrating and reactive against human lung cancer. Immunodeficient mice with flank tumors established from a human lung cancer cell line expressing the NYESO1 antigen were treated with activated human T cells expressing a TCR reactive to NYESO1 (Ly95) with or without anti-PD1 alone and with combinations of anti-PD1 plus anti-TIM3 or anti-TIGIT. A month later, the effect on tumor growth and the phenotype and ex vivo function of the TILs were analyzed. Anti-PD1 and Ly95 T cells led to greater tumor control than Ly95 T cells alone; however, tumors continued to grow. The ex-vivo function of PD1-blocked Ly95 TILs was suppressed and was associated with increased T cell expression of TIM3/TIGIT. Administering combinatorial blockade of PD1+ TIM3 or PD1+ TIGIT with Ly95 T cells led to greater tumor control than blocking PD1 alone. In our model, PD1 blockade was suboptimally therapeutic alone. The effect of TIM3 and TIGIT was upregulated on T cells in response to PD1 blockade and anti-tumor activity could be enhanced when these inhibitory receptors were also blocked with antibodies in combination with anti-PD1 therapy.

Neoadjuvant anti-CTLA4 and anti-PD1 blockade in advanced urothelial cancer.

Neoadjuvant anti-CTLA4 and anti-PD1 blockade in advanced urothelial cancer.

Abstract 3166: The degree of conserved interferon gamma signaling guides response to immune checkpoint blockade therapy

Abstract The long-lasting responses to immune blockade therapy in a subset of patients with melanoma remain largely unexplained. We assessed baseline and on-therapy biopsies from 118 patients with advanced melanoma treated with the anti-PD-1 antibody nivolumab alone (N = 82) or in combination with the anti-CTLA-4 antibody ipilimumab (N = 36) (CheckMate 038 study). There were 46 progressive disease (PD) cases, 26 stable disease (SD) cases, and 46 complete or partial response (CRPR) cases. Tumors were analyzed by RNA sequencing. RNAseq bulk immune cell deconvolution revealed that the largest immune cell change on-treatment was an increase in CD8+ T cell infiltration for patients regardless of treatment or clinical response (P < 0.04, 0.02, 2e-4 for PD, SD, CRPR). In CRPR, there was a corresponding increase in CD4+ T cells (P < 2e-4), Tregs (P < 9e-4), NK cells (P < 3e-5) and macrophages (P < 3e-5). Principal component analysis showed that T cell infiltration was the best organizer of the RNAseq biopsies; 3694 genes were correlated or negatively correlated with T cell infiltration. Analysis of 58 human melanoma cell lines exposed in vitro to interferon-gamma (IFNγ) for 6 hours showed a conserved response to this cytokine in all cell lines able to signal through the IFNγ receptor (N = 46), including an increase in antigen presentation machinery, an increase in expression of IFNγ signaling molecules representing a signal amplification, and production of chemokines that attract other immune cells, while there was a decrease in cell cycle and WNT signaling pathways. These same gene sets are present in the list of genes correlated and negatively correlated with T cell infiltration in the patient data, as well as the list of genes significantly up- or down-regulated in CRPR cases. Only cell lines with JAK1 or JAK2 mutations (N = 12) did not exhibit this conserved response; in fact, these mutants had no genes with at least a 2-fold change. Analysis of the patient biopsies using the IFNγ regulated gene sets revealed that in CRPR cases there was a large increase in HLA expression (P < 0.04, 4e-3, 8e-4 for HLA-A, HLA-B, HLA-C), along with a corresponding large decrease in WNT signaling (P < 3e-4), a driver of immune exclusion. We did not find genomic alterations (in the subset of cases that we exome sequenced) or gene expression differences pre-treatment to explain the greater response in the CRPR cases, including no JAK1/2 mutations. The lack of naturally occurring mutations disabling IFNγ signaling in our baseline biopsies is likely selected for by the role IFNγ plays in up-regulating PD-L1 expression necessary for establishing the anti-PD1 immune blockade. Using RNAseq immune cell deconvolution, the 36 CRPR cases (16 monotherapy, 20 combination) had significantly larger CD8+ T cell infiltration pre-treatment (P < 8e-4 compared to PD cases). Therefore, our data showed that the strength of the antitumor T cell response and corresponding downstream IFNγ signaling is the main driver of response or resistance to immune checkpoint blockade therapy. Citation Format: Catherine Grasso, Jennifer Tsoi, Mykola Onyshchenko, Gabriel Abril-Rodriguez, Ameya Champhekar, Egmidio Medina, Davis Torrejon, Daniel Shin, Michael Quist, Petra Ross-Mcdonald, Megan Wind-Rotolo, Daniel Speiser, Anusha Kalbasi, Anthony Ribas. The degree of conserved interferon gamma signaling guides response to immune checkpoint blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3166.

Abstract IA18: Interrogating anti-PD1 immunotherapy resistance mechanisms

Abstract Background: The response rate of 15-20% with anti-PD1 targeting in head and neck squamous cell carcinoma (HNSCC) highlights the need for strategies to overcome resistance. We have focused on delineating mechanistic aspects of immune checkpoint response and resistance using novel clinical trial approaches coupled with analyses in high-fidelity immunocompetent murine oral carcinoma (MOC) model to study HNSCC immunobiology. Methods: Using the MOC HNSCC model, we generated anti-PD1 resistant models. Whole-exome sequencing and RNA-Seq were used to compare mutational burden and expression signatures. Time of flight mass cytometry (CyTOF) was used to analyze tumor microenvironment (TME) remodeling in response to checkpoint inhibition. 2-D coculture system was used to assess tumor recognition by MHC class I restricted cytotoxic T cells. Tumor cells were loaded with SIINFEKL peptide and OT1 T cells were used as effectors in T-cell cytotoxicity assays. The anti-PD1 resistant MOC model overexpressing CAS9 with high editing efficiency was generated for a genome-scale CRISPR screen. The Broad Institute Genomic Perturbation Platform Brie lentiviral library, including 78,637 gRNAs targeting 19,674 with 1000 controls, was transduced into this line to define modifiers of CD8+ T cell mediated killing. Ezh2 inhibitors and knockout lines were used in characterizing the role of EZH2 in antigen presentation and antitumor immunity. Results: To examine immunotherapy resistance we generated 2 independent anti-PD1 adaptive resistant cell line models, MOC1-esc1 and MOC1-esc2, from their isogenic MOC1 parental line. RNA-Seq showed that MOC1-esc1 upregulated Myc and E2F targets and lost inflammatory signatures whereas -esc2 tumors have upregulated DNA damage and unfolded protein response. When MOC1-esc tumors were retransplanted into naive mice, they exhibited resistance to anti-PD1, while remaining responsive to anti-CTLA4 treatment. To gain comprehensive insights into the tumor microenvironment (TME) as a contributor to adaptive resistance, we analyzed tumor-infiltrating lymphocytes (TIL) in naive MOC1 and MOC1-esc1 tumors using CyTOF with a panel of 38 markers. MOC1-esc1 tumors were highly infiltrated with regulatory T cells (Tregs) and M2-like tumor-associated macrophages (TAMs), while MOC1 tumors have more M1-like TAMs and neutrophils. Furthermore, we observed that both anti-PD1 and anti-CTLA4 treatment dramatically expanded CD8+ T cells and decreased neutrophils in MOC1-esc1 tumors. In responding MOC1-esc1 tumors, anti-CTLA4 treatment resulted in Treg depletion, decreased M2-like TAMs and neutrophils, as well as a striking increase in M1-like TAMs compared with isotype control treated tumors. In contrast, anti-PD1 treated MOC1-esc1 tumors showed decreased M1-like TAMs, while M2-like TAMs were increased compared with control. Depletion of Tregs, neutrophils, or repolarization of TAM using monoclonal antibodies confirmed their contribution in immunotherapy resistance. As the MOC1-esc1 line showed in vitro resistance to T-cell cytotoxicity compared to MOC1 and to define tumor cell intrinsic modulators of T-cell cytotoxicity, we then completed a genome-scale CRISPR screen in MOC1-esc1. This screen identified 355 candidate genes whose loss of function regulated T-cell cytotoxicity. Chromatin modifiers emerged as a major class of genes regulating T-cell recognition of tumor cells. We validated Ezh2 as a therapeutic target and showed that inhibition of this pathway enhanced Class I expression and sensitized MOC1-esc1 cells to T-cell killing in vitro. Analysis of human HNSCC cell line models and TCGA data revealed conservation of Ezh2 impact on Class I expression. Combination therapy with Ezh2 inhibitors and anti-PD1 blockade resulted in enhanced therapeutic efficacy compared to either agent alone in the anti-PD1 resistant MOC1-esc1 model. Conclusions: In summary, this study identified (1) immune modulators within TME involved in adaptive immunotherapy resistance of HNSCC and remodeling with checkpoint therapy and (2) tumor cell intrinsic candidate pathways regulating T-cell recognition. Findings from these studies will advance our understanding of HNSCC immunotherapy resistance and will accelerate the discovery of new combination therapeutic targets and biomarkers in adaptive resistance. Citation Format: Ravindra Uppaluri. Interrogating anti-PD1 immunotherapy resistance mechanisms [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA18.

Effects of B7-H3 expression on tumour-infiltrating immune cells and clinicopathological characteristics in non–small-cell lung cancer

PurposeB7-H3 has emerged as a promising target for cancer immunotherapy. We assessed the role of B7-H3 expression in tumour-infiltrating immune cells in non–small-cell lung cancer (NSCLC). MethodsTumour-infiltrating immune cell characterisation was performed by flow cytometry in a prospective cohort, whereas the relationship between B7-H3 expression and clinicopathological features was explored in a retrospective cohort. ResultsB7-H3 expression was detected in tumour/epithelial cells and immune cells, including macrophages, monocytes, dendritic cells (DCs) and myeloid-derived suppressor cells. B7-H3 was expressed at higher levels in cells within the tumour than in cells within non-neoplastic tissues. B7-H3 expression score in tumour cells positively correlated with the amount of CD45+ immune cells (rho = 0.305, P = 0.010), CD8+ T-cells (rho = 0.330, P = 0.005), and the percentage of CD8+/CD3+ T-cells (rho = 0.403, P < 0.001). Patients with high tumoural B7-H3 expression showed increased numbers of immune cells (P = 0.002), CD8+ T-cells (P = 0.011), natural killer cells (P = 0.073) and plasmacytoid DCs (P = 0.015). Tumoural B7-H3 expression was higher in males, smokers, squamous cell carcinomas, tumours with wild-type EGFR, poor differentiation, larger size and nodal metastasis (P < 0.05, all). Tumoural B7-H3 expression was associated with PD-L1 expression (P = 0.001), shorter 5-year overall survival (P = 0.012) and poor survival after anti–PD-1 blockade (P = 0.026). ConclusionsTumoural B7-H3 overexpression was associated with increased tumour-infiltrating cytotoxic lymphocytes and poor prognosis in NSCLC. Thus, B7-H3 is a promising prognostic biomarker and immunotherapeutic target in NSCLC.

An open-label, single-arm, phase II clinical trial of RP1, an enhanced potency oncolytic herpes virus, combined with nivolumab in four solid tumor types: Initial results from the skin cancer cohorts.

e22050 Background: RP1 is an oncolytic HSV that encodes a fusogenic GALV-GP R- protein and GM-CSF. RP1 demonstrated tolerable safety and tumor regression alone and with nivolumab (nivo) in ph 1 in patients (pts) with a number of tumor types. To further define the efficacy of the combination, ph2 cohorts of 30 pts with 4 tumor types were then opened. Initial data from the melanoma (mel) and the non-mel skin cancer (NMSC) cohorts will be presented. Methods: Unresectable stage IIIb-IV mel pts for whom anti-PD-1 was indicated or who were refractory to 1 prior standard therapy including anti-PD-1 or ipi/nivo were enrolled. NMSC pts were anti-PD1 naïve. Pts received up to 8 doses of RP1 (&lt;=10 mL/visit based on tumor diameter) Q2W (first dose 106 PFU/mL then 107 PFU/mL). From the second RP1 dose pts also received nivo (240 mg IV Q2W for 4 mos then 480 mg IV Q4W up to 2 yrs in the absence toxicity or confirmed progressive disease (PD)). Imaging was done every 8 wks and response assessed by RECISTv1.1 (with confirmation required for PD). Results: As of Jan 22nd 2020, 30 mel pts and 9 NMSC pts had been enrolled with follow up between &lt;1 and 7mo. Of the mel pts 21 were cutaneous, 5 were mucosal and 4 were ocular. Of the NMSC pts, 6 had squamous cell, 1 had basal cell, 1 had Merkel cell carcinomas and 1 had angiosarcoma. Recruitment of the mel cohort is complete, with recruitment into the NMSC cohort ongoing. Based on initial data in melanoma, a further cohort of 125 pts with anti-PD1 refractory cutaneous mel has been opened. Adverse events (AEs) in the ph2 cohorts have been consistent with those in ph1, with RP1 side effects of in general Grade 1/2 constitutional and related symptoms, self-limiting within 72hrs of RP1 injections, with no exacerbation of the side effects expected for nivo. With currently short follow up, multiple objective responses have been observed in treatment naïve mel, anti-PD1 refractory mel (including ipi/nivo refractory and mucosal), and NMSC. Of note 3 of the first 4 anti-PD1 refractory mel pts treated are responding to treatment, as are 5 of the first 6 CSCC pts, including 3 CR. Tumor biopsies routinely showed immune activation, including robust recruitment of CD8+ T cells and increased PD-L1 expression. Treatment remains ongoing in the majority of patients, and current data will be presented. Conclusions: RP1 and nivo has continued to be well tolerated, with promising signs of efficacy in patients with skin cancers, including with anti-PD1 refractory disease. These data support the further development of RP1 combined with anti-PD1 blockade. Clinical trial information: NCT03767348 .

A phase II trial of atezolizumab and bevacizumab in patients with relapsed/refractory and unresectable malignant peritoneal mesothelioma.

9013 Background: Malignant peritoneal mesothelioma (MPeM) is an orphan malignancy. No recommended/FDA approved therapies exist for salvage treatment beyond first-line platinum and pemetrexed based chemotherapy. While immune checkpoint inhibition has shown preliminary efficacy in mesotheliomas, data and efficacy is limited in MPeM patients (pts) [objective response rate (ORR) ~ 11%; median progression-free survival (mPFS) ~ 4 months (m); median overall survival (mOS) ~ 11 m]. We aimed to prospectively assess the safety and efficacy of combined anti-PD1 (atezolizumab) and VEGF (bevacizumab) blockade (AtezoBev) in pts with MPeM. Methods: In this phase 2 study, eligible pts with histologically confirmed MPeM, ECOG PS 0-1, and prior platinum and pemetrexed treatment were treated with 1200 mg of atezolizumab and 15 mg/kg of bevacizumab IV every 21 days until disease progression, unacceptable toxicity, or withdrawal. Primary endpoint was confirmed ORR by RECIST 1.1 by independent radiology review. Duration of response (DOR), PFS and OS were pre-specified secondary endpoints. Results: Among 20 enrolled pts (3/2017 - 2/2019), median age was 63 (range, 33-87) years, 12 (60%) were female, 12 (60%) had PS 0, and 2 (10%) had biphasic MPeM. Among 20 evaluable pts (median cycles 14), confirmed ORR was 35% (7 pts; 95% CI: 15.4-59.2) (median DOR 8.8 m). Responses were ongoing in 5/7 (71.4%) pts at data cutoff. The median follow-up was 20.5 months. Six deaths were observed during follow-up, and the 1-year OS was 79% (95% CI: 52 – 91) (median OS ~ NR). Median PFS was estimated as 17.6 m (95% CI: 9.1 – NR). The 1-year PFS was 54% (95% CI: 28 – 74). Grade 3 (no grade 4/5) treatment-emergent adverse events occurred in 10 (50%) pts; most common being hypertension (40%) and anemia (10%). Two (10%) pts had grade 3 immune-related adverse events. Translational studies are ongoing. Conclusions: AtezoBev showed promising and durable efficacy in relapsed/refractory MPeM with acceptable safety profile. Ongoing multiomic analyses of pre and on-treatment tissue/liquid biopsies obtained on all these pts will provide additional insight into mechanisms and biomarkers of response and resistance. Clinical trial information: NCT03074513.

Neuropilin-1 Amplifies Regulatory T cell Function in Human Cancer

Abstract Regulatory T cells (Tregs) maintain peripheral tolerance but also contribute to cancer progression by dampening anti-tumor immunity. Knocking out neuropilin-1 (NRP1) on murine Tregs attenuates tumor growth by rendering intratumoral Tregs non-suppressive. Whereas NRP1 is constitutively expressed on all mouse Tregs, NRP1 is infrequently expressed on Tregs in the peripheral blood of healthy humans. We hypothesized that (1) NRP1 expression is upregulated in cancer patients, (2) NRP1 marks highly suppressive human Tregs, and (3) sustained Treg activation initiates NRP1 expression. NRP1+ Tregs are enriched in tumors across numerous cancer types but largely devoid in non-cancerous inflamed and healthy tissues. Intratumoral NRP1 expression negatively correlates with disease-free survival in head and neck squamous cell carcinoma (HNSCC; p = 0.03; Hazard ratio = 5.4). Intratumoral NRP1+ Tregs upregulate a suppressive module indicated by co-expression of TIGIT, CCR8, and multiple TNF receptor super family members. Correspondingly, NRP1+ Tregs potently suppress CD8+ T cell proliferation in vitro, which can be disrupted with NRP1-specific blockade. Furthermore, Treg activation in vitro drives NRP1 expression, and sustained TCR signals are required to maintain NRP1 expression. We conclude that NRP1+ Tregs are selectively enriched in human cancer and oppose anti-tumor immunity. Therefore, destabilizing intratumoral Tregs using NRP1 blockade may complement other T cell therapies such as anti-PD1 blockade.

Beyond Tumor PD-L1: Emerging Genomic Biomarkers for Checkpoint Inhibitor Immunotherapy.

Despite the success of immune checkpoint blockade as a strategy for activating an antitumor immune response and promoting cancer regression, only a subset of patients have durable clinical benefit. Efforts are ongoing to identify robust biomarkers that can effectively predict treatment response to immune checkpoint inhibitors (ICIs). Although PD-L1 expression is useful for stratifying patients, it is an imperfect tool. Comprehensive next-generation sequencing platforms that are readily used in clinical practice to identify a tumor's potentially actionable genetic alterations also reveal tumor genomic features, including tumor mutation burden (TMB), that may impact the response to ICIs. High TMB enhances tumor immunogenicity through increased numbers of tumor neoantigens that may promote an immune response. Defective DNA repair, leading to microsatellite instability, is an endogenous mechanism for increased tumor TMB that augments response to anti-PD-1 blockade. Alternatively, DNA damage from exogenous factors is responsible for high TMB seen in melanoma, lung cancer, and urothelial carcinoma, among tumor subtypes with higher response rates to ICIs. In this review, we summarize data supporting the use of TMB as a biomarker as well as its known limitations. We also highlight specific tumor suppressor genes and oncogenes that are under investigation as biomarkers for ICI response and resistance. Efforts are ongoing to delineate which genomic tumor characteristics can eventually be utilized in clinical practice to ascertain the benefit of ICIs for an individual patient.

Alternative macrophage polarisation associated with resistance to anti-PD1 blockade is possibly supported by the splicing of FKBP51 immunophilin in melanoma patients

BackgroundFKBP51 immunophilin is abundantly expressed by immune cells. Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s. Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response. An expansion of FKBP51s+PD-L1+ monocytes was measured in a group of non-responding patients to anti-CTLA-4. The aim of this work was to confirm the predictive value of response of FKBP51s+Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s.MethodsCo-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s. PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s+Treg and FKBP51s+PD-L1+ monocytes in 22 advanced melanoma patients treated with anti-PD1. Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages’ behaviour.ResultsFKBP51s+Tregs count was increased in responders and had a prognostic value. Non-responders showed an early increase in FKBP51s+ PD-L1+ monocytes during anti-PD1 treatment. Manipulation of FKBP51s modulated the macrophage–phenotype, with forced protein expression promoting aspects associated with tolerance.ConclusionsFKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy. Manipulation of FKBP51s may overcome resistance.