Effect Of PD-1 Blockade Research Articles

Effect of PD-1 Blockade on Neonatal Sepsis and Septic Kidney Injury in Mice

Effect of PD-1 Blockade on Neonatal Sepsis and Septic Kidney Injury in Mice

Abstract 1195: Prospective study for the identification of nivolumab biomarkers via analyses of pre-treatment plasma exosome mRNAs from head and neck cancer patients (BIONEXT)

Abstract Nivolumab paved a new way in the treatment of recurrent or metastatic (RM) head and neck squamous cell carcinoma of (HNSCC). However, the limited rates of long-term survivors (< 20%) demand a robust prognostic biomarker. This nationwide multi-centric prospective study aimed to identify a plasma exosome (PEX) mRNA signature, which serves as a companion diagnostic of nivolumab and as well provides a biological clue to develop effective therapies for a majority of non-survivors. Pre-treatment plasmas (N = 104) were subjected to comprehensive PEX mRNA analyses for prognostic marker discovery and validation. In parallel, paired treatment naïve tumor and plasma samples (N = 20) were assayed to elucidate biological implications of the PEX mRNA signature. A combination of 6 candidate PEX mRNAs plus neutrophil-to-lymphocyte ratio precisely (P = 0.000124) distinguished non-survivors from >2-yr survivor (0% vs 57.7%) with a high hazard ratio (2.878; 95% confidence interval 1.639-5.055; P = 0.0002348). In paired samples, high PEX HLA-E mRNA (a non-survivor-predicting marker) reflected the increased HLA-E protein expression (P = 0.0191) and the dense population of tumor-infiltrating NK cells (P = 0.024) in the corresponding tumor. It is estimated that the effects of PD-1blockade is canceled by the HLA-E-NKG2A immune checkpoint in patients with high PEX HLA-E mRNA. The combination of NKG2A antibody (i.e., monalizumab) and nivolumab may be a promising strategy for non-survivors predicted by a RT-qPCR-based pre-treatment measurement of PEX mRNAs. A candidate companion diagnostic of nivolumab that indicates a possible treatment option is identified. Citation Format: Muneyuki Masuda, Satoshi Toh, Mioko Matsuo, Masashi Sugasawa, Keisuke Yamazaki, Ueki Yushi, Torahiko Nakashima, Hideoki Uryu, Takeharu Ono, Tsutomu Ueda, Hirohito Umeno, Satoshi Kano, Kiyoaki Tsukahara. Prospective study for the identification of nivolumab biomarkers via analyses of pre-treatment plasma exosome mRNAs from head and neck cancer patients (BIONEXT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1195.

Abstract 1875: Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy

Abstract Programmed death 1 (PD-1) is an immune checkpoint (IC) receptor that negatively regulates T-cell immune function. Blocking the interaction of PD-1 with its ligand PD-L1 unleashes the immune system and enhances anti-tumor responses. Immunotherapy using PD-1 blockade has led to a paradigm shift in cancer drug discovery, due to its durable effect against a wide variety of cancers. However, despite its tremendous clinical success in cancer treatment, a significant fraction of patients and tumor types remain unresponsive to this therapy. This has led to the development of combination therapies combining PD-1 checkpoint inhibitors with a broad range of clinically active treatments including those targeting other IC receptors. A major challenge in immunotherapy drug development is the lack of quantitative and reproducible functional assays as existing methods rely heavily on primary immune cells and are highly variable. Here, we report the development of a suite of cell line-based reporter bioassays for monoclonal antibody targeting human PD-1/PD-L1, or bispecific molecules target PD-1/PD-L1 and a co-stimulatory receptor (e.g., 4-1BB, OX40, ICOS) or an immune inhibitory receptor (e.g., CTLA-4, LAG-3, TIGIT). A mouse PD-1/PD-L1 assay and a human PD-1/PD-L2 assay are also developed and can be used in parallel with human PD-1/PD-L1 assay to address preclinical study and assay specificity. Each of these assays consist of two engineered cell lines: a T effector cell line that express a luciferase reporter driven by specific promoter/response elements responding to the intracellular signals mediated by the T cell receptor (TCR), PD-1 and the second IC receptor, and an artificial antigen presenting cells (aAPCs). Particularly, the combination bioassays are able to measure the synergetic effect of PD-1 blockade with a second IC inhibitor receptor blockade or a costimulatory receptor activation. These reporter-based assays reflect the mechanisms of action for the therapeutic drugs of interest and are sensitive and quantitative. They are prequalified according to ICH guideline and demonstrated the performance characteristics (specificity, precision, accuracy, and linearity) to be used as potency assay for product release and stability studies during immunotherapy drug development and manufacture. Citation Format: Jamison Grailer, Julia Gilden, Jun Wang, Denise Garvin, Pete Stecha, Jim Hartnett, Steven Edenson, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Novel cell-based bioassays for monoclonal antibody and bispecific molecules in PD-1 blockade monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1875.

Cannabis suppresses antitumor immunity by inhibiting JAK/STAT signaling in T cells through CNR2

The combination of immune checkpoint blockade (ICB) with chemotherapy significantly improves clinical benefit of cancer treatment. Since chemotherapy is often associated with adverse events, concomitant treatment with drugs managing side effects of chemotherapy is frequently used in the combination therapy. However, whether these ancillary drugs could impede immunotherapy remains unknown. Here, we showed that ∆9-tetrahydrocannabinol (THC), the key ingredient of drugs approved for the treatment of chemotherapy-caused nausea, reduced the therapeutic effect of PD-1 blockade. The endogenous cannabinoid anandamide (AEA) also impeded antitumor immunity, indicating an immunosuppressive role of the endogenous cannabinoid system (ECS). Consistently, high levels of AEA in the sera were associated with poor overall survival in cancer patients. We further found that cannabinoids impaired the function of tumor-specific T cells through CNR2. Using a knock-in mouse model expressing a FLAG-tagged Cnr2 gene, we discovered that CNR2 binds to JAK1 and inhibits the downstream STAT signaling in T cells. Taken together, our results unveiled a novel mechanism of the ECS-mediated suppression on T-cell immunity against cancer, and suggest that cannabis and cannabinoid drugs should be avoided during immunotherapy.

Effects of PD-1 blockade on ovarian follicles in a prepubertal female mouse.

Immunotherapy has emerged at the forefront of cancer treatment. Checkpoint inhibitor pembrolizumab (KEYTRUDA), a chimeric antibody which targets programmed cell death protein 1 (PD-1), has been approved by the Food and Drug Administration (FDA) for use in pediatric patients with relapsed or refractory classical Hodgkin's lymphoma. However, there is currently no published data regarding the effects of pembrolizumab on the ovary of female pediatric patients. In this study, prepubertal immunocompetent and immunodeficient female mice were injected with pembrolizumab or anti-mouse PD-1 antibody. The number of primordial follicles significantly decreased post-injection of both pembrolizumab and anti-mouse PD-1 antibody in immunocompetent mice. However, no changes in follicle numbers were observed in immunodeficient nude mice. Superovulation test and vaginal opening experiments suggest that there is no difference in the number of cumulus-oocyte complexes (COCs) and the timing of puberty onset between the control and anti-mouse PD-1 antibody treatment groups, indicating that there is no effect on short-term fertility. Elevation of pro-inflammatory cytokine TNF-α following COX-2 upregulation was observed in the ovary. CD3+ T-cell infiltration was detected within some ovarian follicles and between stromal cells of the ovaries in mice following treatment with anti-mouse PD-1 antibody. Thus, PD-1 immune checkpoint blockade affects the ovarian reserve through a mechanism possibly involving inflammation following CD3+ T-cell infiltration.

Abstract P062: PI3Kγδ inhibitor plus radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold', imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a PI3Kγδ inhibitor were then administered every other day for 2 weeks. Tumors from humanized patient-derived breast cancer xenograft (PDX) model was sequenced to identify immune-related pathways and to profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngenic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. According to FACS analysis, RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. There were significantly decreased tumor expressions of p-AKT, PD-L1, and HIF1α by PI3Kγδ inhibition. Triple combination therapy significantly delayed primary tumor growth in humanized PDX model as well and analyses of RNA sequencing data of humanized PDX samples showed decreased immune suppressive pathways with decreased and M2 macrophage and increased CD8+ T-cell by triple combination therapy. In the TCGA pan-cancer cohort, high Treg/CD8+T-cell and M2/M1 TAM ratios and poor overall patient survival was associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: In Ah Kim, Min Guk Han, Bum Sup Jang, Mi Hyun Kang. PI3Kγδ inhibitor plus radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer and humanized patient-derived xenograft model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P062.

Abstract 1554: PI3Kγδ inhibitor combined with radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer model and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold', imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade (10 mg/kg) and a PI3Kγδ inhibitor (IPI145; 15 mg/kg) were then administered every other day for 2 weeks. We analyzed mRNA-sequencing data from humanized patient-derived xenograft (PDX) model to identify differentially activated immune-related pathways. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the ‘xCell' deconvolution algorithm was used to profile immune cellular distributions at certain levels of PIK3C and PIK3CD expression. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngeneic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. Triple combination therapy significantly delayed tumor growth in humanized PDX model as well. Analyses of RNA sequencing data of humanized PDX samples showed triple combination increased immune response pathways and increased CD8+ T-cell and decreased Treg and M2 TAM. In the TCGA pan-cancer cohort, higher tumor purity-adjusted Treg/CD8+T-cell and M2/M1 TAM ratios and worse overall patient survival were associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: Min Guk Han, Bum-Sup Jang, Mi Hyun Kang, In Ah Kim. PI3Kγδ inhibitor combined with radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer model and humanized patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1554.

Camrelizumab plus famitinib for advanced renal cell carcinoma or unresectable urothelial carcinoma: Updated results from a phase II trial.

4550 Background: Considering the synergistic/additive effect of PD-1 blockade and angiogenesis inhibition, we conducted an open-label, multi-center phase II study of camrelizumab (an anti-PD-1 antibody) plus famitinib (a TKI against VEGFR-2, PDGFR, c-kit, and FGFR) in pts with heavily treated advanced renal cell carcinoma (RCC) and unresectable urothelial carcinoma (UC). Methods: Based on an adaptive two-stage design, 22 pts were enrolled in the RCC and UC cohort, respectively, at stage 1; if there were ≥7 responders of the 22 pts, enrollment would be extend to 33 at stage 2. Pts received famitinib 20 mg orally QD plus camrelizumab 200 mg IV Q3W. Primary endpoint was ORR per RECIST v1.1. Preliminary data at stage 1 were reported at ASCO 2020 annual meeting (#5085). More than 7 pts in each cohort had CR/PR during stage 1; enrollment of stage 2 had been completed, and the data are firstly reported here. Results: 74 pts (38 advanced RCC and 36 unresectable UC) were enrolled from Jan 23, 2019 to Dec 14, 2020. In RCC cohort, 65.8% of pts had received ≥1 prior VEGFR inhibitors. In UC cohort, all pts had progressed on or relapsed after a platinum-containing chemotherapy. As of Jan 10, 2021, median time from enrollment to data cutoff was 18.8 mos (range, 8.5-22.7) for RCC cohort and 7.0 mos (range, 0.9-23.6) for UC cohort. In RCC cohort, ORR was 63.2% (95% CI, 46.0-78.2; 24 PRs), DCR was 89.5% (95% CI, 75.9-95.8), median DOR was not reached (range 2-19+ mos), mPFS was not reached, and 12-mo OS rate was 88.0%. Most pts (92.1%) had reduction in target lesions, and median reduction was 47% from baseline. ORR was 84.6% (95% CI, 57.8-97.3; 11/13) for untreated RCC pts and 52.0% (95% CI, 31.3-72.2; 13/25) for pre-treated pts; DCR was 100% (95% CI, 77.2-100.0) and 84.0% (95% CI, 65.3-93.6), mPFS was not reached and 13.4 mos (95% CI: 4.1-21.0), respectively. In UC cohort, of the 33 pts with post-baseline efficacy evaluation, ORR was 33.3% (95% CI, 19.8-50.4; 1 CR, 10 PRs), DCR was 60.6% (95% CI, 43.7-75.3), median DOR was not reached (range 1.0+-16.7+ mos), mPFS was 6.4 mos (95% CI, 2.1-11.8), and mOS was not reached. ORR trended to be higher for bladder cancer (43.8%, 7/16) than upper tract urothelial carcinoma (25.0%, 4/16). Treatment-related AEs (TRAEs) occurred in 97.3% of 74 pts. Grade 3 or 4 TRAEs occurred in 63.5% of pts, mainly hypertension (23.4%), decreased platelet count (21.3%), proteinuria (17.0%), anemia (14.9%), and palmar-plantar erythrodysesthesia syndrome (14.9%). 1 pt died of TRAE (multiple organ dysfunction syndrome). Conclusions: Camrelizumab plus famitinib showed potent anti-tumor activity in pts with advanced RCC and UC with no new safety concerns. Additionally, promising ORR and durable DOR were observed in pts with heavily-treated RCC. These results support further investigation in these settings. Clinical trial information: NCT03827837.

PI3Kγδ inhibitor combined radiation enhances the antitumor immune effect of PD-1 blockade in syngenic triple negative breast cancer models.

e13041 Background: Breast cancer is generally viewed as immunologically cold, imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent BALB/c mice, humanized patient-derived xenograft (PDX) were established, and both were irradiated by 3 fractions of 24 Gy. A PD-1 blockade (10 mg/kg) and a PI3Kγδ inhibitor (IPI145; 15 mg/kg) were then administered every other day for 2 weeks. Tumors from humanized PDX was sequenced to identify immune-related pathways and to profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the deconvolution algorithm was used to profile immune cellular distributions at certain levels (high vs. low) of PIK3Cand PIK3CD expression. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival. According to FACS analysis, RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. There were significantly decreased tumor expressions of p-AKT, PD-L1, and HIF1α by PI3Kγδ inhibition. Triple combination therapy significantly delayed primary tumor growth in humanized PDX model as well and analyses of RNA sequencing data of humanized PDX samples showed decreased immune suppressive pathways with decreased and M2 macrophage and increased CD8+ T-cell by triple combination therapy. In the TCGA pan-cancer cohort, high Treg/CD8+T-cell and M2/M1 TAM ratios and poor overall patient survival was associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusions: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer.

Anti-PD-1/PD-L1 Based Combination Immunotherapy to Boost Antigen-Specific CD8+ T Cell Response in Hepatocellular Carcinoma.

Simple SummaryThe cytotoxic T cell response against hepatocellular carcinoma antigens is exhausted and fails in its task of deleting tumoral cells. These cells are featured by the expression of negative immune checkpoints that can be modulated to restore T cell function. The blockade of the PD-1/PD-L1 pathway has shown promising results in rescuing hepatocellular carcinoma-specific CD8 T cells but only a reduced group of cases is sensitive to this treatment and the effect is usually temporary. Therefore, new anti-PD-1 based combinatory strategies are underway to increase the response by adding the effect of blocking neo-angiogenesis and other negative immune checkpoints, boosting positive immune checkpoints, blocking suppressive cytokines, or inducing the expression of tumoral neoantigens. The restoration of T cell responses with these anti-PD-1 based combinatory therapies will change the outcome of advanced hepatocellular carcinoma.Thirty to fifty percent of hepatocellular carcinomas (HCC) display an immune class genetic signature. In this type of tumor, HCC-specific CD8 T cells carry out a key role in HCC control. Those potential reactive HCC-specific CD8 T cells recognize either HCC immunogenic neoantigens or aberrantly expressed host’s antigens, but they become progressively exhausted or deleted. These cells express the negative immunoregulatory checkpoint programmed cell death protein 1 (PD-1) which impairs T cell receptor signaling by blocking the CD28 positive co-stimulatory signal. The pool of CD8 cells sensitive to anti-PD-1/PD-L1 treatment is the PD-1dim memory-like precursor pool that gives rise to the effector subset involved in HCC control. Due to the epigenetic imprints that are transmitted to the next generation, the effect of PD-1 blockade is transient, and repeated treatments lead to tumor resistance. During long-lasting disease, besides the TCR signaling impairment, T cells develop other failures that should be also set-up to increase T cell reactivity. Therefore, several PD-1 blockade-based combinatory therapies are currently under investigation such as adding antiangiogenics, anti-TGFβ1, blockade of other negative immune checkpoints, or increasing HCC antigen presentation. The effect of these combinations on CD8+ T cells is discussed in this review.

Abstract PO-043: Radiation combined with PI3Kγδ inhibitor enhances the antitumor immune effect of PD-1 blockade in syngeneic murine breast cancer model and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold’, imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade (10 mg/kg) and a PI3Kγδ inhibitor (IPI145; 15 mg/kg) were then administered every other day for 2 weeks. We analyzed mRNA-sequencing data from humanized patient-derived xenograft (PDX) model to identify differentially activated immune-related pathways. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the ‘xCell’ deconvolution algorithm was used to profile immune cellular distributions at certain levels (high vs. low) of PIK3Cand PIK3CD expression. Results: In the immune-competent syngeneic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngeneic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. According to FACS analysis, RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Treg cells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. Triple combination therapy significantly delayed primary tumor growth in humanized PDX model as well and analyses of RNA sequencing data of humanized PDX samples showed triple combination decreased immune suppressive pathways with decreased M2 macrophage and increased CD8+ T-cell. In the TCGA pan-cancer cohort, higher tumor purity-adjusted Treg/CD8+T-cell and M2/M1 TAM ratios and worse overall patient survival were associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: Min Guk Han, Bum-Sup Jang, Mi Hyun Kang, In Ah Kim. Radiation combined with PI3Kγδ inhibitor enhances the antitumor immune effect of PD-1 blockade in syngeneic murine breast cancer model and humanized patient-derived xenograft model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-043.

Abstract IA17: Molecular mechanisms of T-cell exhaustion and implications for immunotherapy

Abstract T-cell exhaustion is common during chronic infections and cancer and prevents optimal immunity. Exhausted CD8 T cells (TEX) are defined by the loss of ability to perform effector functions efficiently, low proliferative capacity, and poor survival following antigen stimulation. TEX also co-express multiple inhibitory receptors that negatively regulate their function. Indeed, receptors such as PD-1 have become major targets of clinical immunotherapies in cancer and infectious disease aimed at reinvigorating TEX. Despite the clinical success of PD-1-based therapies in human melanoma and other cancers, the majority of patients do not have durable clinical benefit from anti-PD-1 monotherapy. A major challenge remains identifying which patients will respond to anti-PD-1 therapy and defining the underlying mechanisms for successful, durable response versus treatment failure. Detailed immune profiling of peripheral blood from stage IV melanoma patients before and after pembrolizumab (pembro) identified reinvigoration in circulating TEX. These data indicated that nearly 80% of patients experienced a strong, on-target immunologic effect of PD-1 blockade. However, the objective clinical response rate in this cohort was less than 40% and reinvigoration of circulating TEX alone did not predict clinical outcomes. We are currently now using neoadjuvant clinical trial designs and preclinical models to dissect the molecular and mechanistic features of effective and ineffective responses to checkpoint blockade and to understand the underlying molecular program of exhausted CD8 T cells. Our emerging data not only provide a framework for dissecting distinct types of treatment failures in melanoma and have implications for stratifying patients into additional immunotherapeutic treatment approaches, but also point to key underlying molecular pathways involved in programming and controlling the developmental biology of exhausted T cells. The implications of these findings for clinical therapies will be discussed. Citation Format: E. John Wherry. Molecular mechanisms of T-cell exhaustion and implications for immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA17.

Immuno-Stimulatory Effects of Mechanical and Thermal High Intensity Focused Ultrasound

Abstract INTRODUCTION While immunotherapy through anti-PD1 checkpoint inhibition has demonstrated impressive clinical efficacy against a number of tumor types, its application has been ineffective against glioblastomas METHODS We wished to determine whether high intensity focused ultrasound (HIFU) augment the efficacy of anti-PD1 checkpoint inhibition against glioblastomas. HIFU can be administered through low-duty cycle to induce subcellular fragmentation without causing temperature elevation (termed mechanical HIFU). Alternatively, HIFU delivered through high-duty cycle can induce foci of temperature elevation and thermal ablation (termed thermal HIFU). We tested the impact of mechanical and thermal HIFU on the anti-glioblastoma effects of the immune checkpoint inhibitor, ipilimumab RESULTS In an in Vivo murine glioblastoma model, sites targeted by mechanical HIFU exhibited a 10 to 100 fold increase in accumulation in tumor infiltrating lymphocytes and interferon-gamma in the presence of PD-1 blockade. At least 75% of mice engrafted with glioblastomas achieved remission when treated with mechanical HIFU and PD-1 blockade. In contrast, none of the mice treated with single therapies achieved durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (P < .001) and IFN-gamma levels (P = .001). The synergy observed between mechanical HIFU and PD-1 blockade was not seen when combining thermal HIFU and PD-1 blockade, suggesting thermal protein denaturation is prohibitive to immune-stimulatory effects of HIFU CONCLUSION Mechanical HIFU, but not thermal HIFU, augments the anti-glioblastoma effects of PD-1 blockade. Our findings inform the utility of HIFU as an immune-adjuvant in glioblastoma therapy and provide a paradigm for conceptualizing the immune effects of laser thermal ablation

Programmed Death-1 Restrains the Germinal Center in Type 1 Diabetes.

Programmed death-1 (PD-1) inhibits T and B cell function upon ligand binding. PD-1 blockade revolutionized cancer treatment, and although numerous patients respond, some develop autoimmune-like symptoms or overt autoimmunity characterized by autoantibody production. PD-1 inhibition accelerates autoimmunity in mice, but its role in regulating germinal centers (GC) is controversial. To address the role of PD-1 in the GC reaction in type 1 diabetes, we used tetramers to phenotype insulin-specific CD4+ T and B cells in NOD mice. PD-1 or PD-L1 deficiency, and PD-1 but not PD-L2 blockade, unleashed insulin-specific T follicular helper CD4+ T cells and enhanced their survival. This was concomitant with an increase in GC B cells and augmented insulin autoantibody production. The effect of PD-1 blockade on the GC was reduced when mice were treated with a mAb targeting the insulin peptide:MHC class II complex. This work provides an explanation for autoimmune side effects following PD-1 pathway inhibition and suggests that targeting the self-peptide:MHC class II complex might limit autoimmunity arising from checkpoint blockade.

Abstract 529: A PI3Kαδ inhibitor combined with irradiation enhance the effect of PD-1 blockade in syngenic triple negative breast cancer model

Abstract Background:Breast cancer has been showing relatively poor response to immune checkpoint blockades potentially due to low immunogenicity of tumor and immunosuppressive tumor microenvironment. Radiation (RT) has been showing immune stimulatory effect and PI3Kδhas immune suppressive effect via modulation of regulatory T cell (Treg). Therefore, we hypothesize that in situ tumor vaccination via RT and suppression of immune tolerance via PI3Kαδ inhibition could enhance the clinical efficacy of immune checkpoint blockade. As PI3Kα controls a key oncogenic signaling pathway, we evaluated whether RT or novel PI3Kαδ inhibitor or both could enhance the efficacy of antitumor effect of a PD-1 blockade in immune competent syngenic triple negative breast cancer (TNBC) model. Materials & Methods:4T1 murine breast cancer cells were grown subcutaneously in the hind limb of BALB/c mice. The mice were grouped as following seven groups: control, RT, PI3Kαδ inhibitor, PI3Kαδ inhibitor + RT, PD-1 blockade, PD-1 blockade + RT, and triple combination group. Tumors were irradiated using 24 Gy/3 fractions. PD-1 blockade (10 mg/kg) and PI3Kαδ inhibitor (4 mg/kg) were administered every other day for two weeks, respectively. Tumor size was measured periodically using a Vernier caliper and bioluminescence imaging to evaluate efficacy of each modality and combination therapy. Immune-modulatory function was evaluated using FACS and immunohistochemical (IHC) study. Results:Triple combination of RT, PD-1 blockade, and PI3Kαδ inhibitor significantly delayed tumor regrowth whereas PD-1 inhibitor alone showed only modest effect in 4T1 syngenic TNBC model. FACS and IHC study for immune repertoire using tumor samples showed that RT and PD-1 blockade modestly increased the proportion of cytotoxic CD8+ T cells and PI3Kαδ inhibitor led to decrease the proportion of Treg. Triple combination showed remarkable increase of cytotoxic CD8+ T cells suggesting synergistic immune modulatory effect of RT, PD-1 blockade and PI3Kαδ inhibitor. Triple combination led to significant upregulation of c-GAS/STING pathway in the tumor and increased IFN-γ level in blood as well compared to each modality alone. Conclusions:Taken together, combination of RT and PI3Kαδ inhibitors maximized immune stimulatory effect in immune competent syngenic TNBC model and enhanced the response of PD-1 blockade via non-redundant synergistic immune modulatory effect. This study provides a preclinical rationale for the combination of PI3Kαδ inhibitor and RT with PD-1 blockade to overcome the immune tolerance of breast cancer. (Work supported by the grants from NRF #2017R1A2B4002710 & #2017M2A2A7A01018438 to In Ah Kim) Citation Format: Ji Min Park, Joo Ho Lee, Danhyo Kim, Eric Eunsik Kim, Jun Hyung Bae, In Ah Kim. A PI3Kαδ inhibitor combined with irradiation enhance the effect of PD-1 blockade in syngenic triple negative breast cancer 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 529.

OC-0151 Radiation and PI3K-αδ inhibitor enhanced anti-tumor effect of PD-1 blockade in syngeneic tumor model

OC-0151 Radiation and PI3K-αδ inhibitor enhanced anti-tumor effect of PD-1 blockade in syngeneic tumor model

PS02.124: NOVEL APPROACHES AND CONSIDERATIONS FOR IMMUNOTHERAPY IN OBESITY-ASSOCIATED OESOPHAGEAL ADENOCARCINOMA

Abstract Background Immunotherapies are transforming cancer treatment for inoperable and advanced disease. However, the incidence of obesity-associated cancers, including oesophageal adenocarcinoma (OAC) continue to increase. Treatment with immune-based therapies present a unique challenge for immunologists, as they need to enhance anti-tumour immunity without exacerbating pre-existing carcinogenic inflammation. Therefore, we examined the effect of PD-1 blockade in omentum and liver of OAC patients, sites of activated inflammatory T cells, which play a key role in obesity-associated inflammation. In addition, we examined novel ways to reduce the infiltration of inflammatory T cells to these sites in a bid to reduce adipose tissue inflammation. Methods Blood, omentum and liver samples were obtained from consenting OAC patients and treated with anti-PD-1 antibody. CD4+ and CD8+ T cell activation, cytokine expression and cytotoxic potential were assessed by flow cytometry. To identify potential chemokine pathways to alter T cell trafficking to the omentum and liver, chemokine receptor expression was examined, along with levels of secreted chemokines using flow cytometry and ELISAs. Pre-treatment of T cells with chemokine receptor antagonists was performed prior to chemotaxis assays using an in vitro transwell system. Results In addition to OAC tumour, omentum and liver were found to be enriched with substantial populations of PD-1 expressing T cells. Treatment of omental and hepatic T cells with anti-PD-1 did not enhance inflammatory cytokine expression or proliferation, but transiently increased CD107a expression by CD8+ T cells. MIP-1α, MIP-1β and IP-10 mediate T cell trafficking to the omentum and liver in OAC. OAC-derived T cells preferentially migrate to the adipose and liver tissue conditioned media of obese OAC patients and this can be significantly reduced using a MIP-1α receptor antagonist. Conclusion This study provides evidence that anti-PD-1 treatment is unlikely to exacerbate pre-existing T cell-mediated inflammation outside the tumour in obesity-associated cancers. Furthermore, novel CCR1 antagonists may be used to attenuate pathological inflammation in obesity. This dual targeting approach aims to enhance anti-tumour immunity through the use of immune checkpoint inhibitors, and has identified a novel immunotherapeutic approach to reduce obesity-associated inflammation by targeting key chemokine pathways. In vivo testing is required to determine the efficacy of this approach. Disclosure All authors have declared no conflicts of interest.

Association of changes in T regulatory cells (Treg) during nivolumab treatment with melanoma outcome.

3031 Background: PD-1 blocking antibodies have significant efficacy in the treatment of melanoma; however, many patients fail to respond and resistance mechanisms remain unknown. We addressed the role of Tregs, an immunosuppressive T-cell population, in patient outcome after treatment with nivolumab. Methods: Peripheral blood mononuclear cells (PBMC) were obtained from patients on trials with nivolumab as adjuvant therapy for resected disease or as treatment for metastatic melanoma. To measure suppression, Tregs were flow-sorted from PBMC and evaluated in allogeneic mixed lymphocyte reactions. Tregs and conventional CD4 T-cells were evaluated for gene expression changes by RNA-sequencing. Treg percentages and phosphorylated STAT3 (pSTAT3) expression were evaluated by flow cytometry. The effects of PD-1 blockade with nivolumab were evaluated in vitro using T-cells from baseline patient PBMC samples. Results: Tregs from responding patients or adjuvant patients without evidence of disease (NED) had reduced suppressive function post-nivolumab (p < 0.05), but no changes were observed in relapsing/non-responding patients; their Tregs were more suppressive than NED/responding Tregs (p < 0.001). NED Tregs had unique gene expression changes and associated pathways post-nivolumab compared to relapsing patient Tregs and conventional CD4 T-cells, including up-regulation of proliferation pathways (q < 8e-19) and downregulation of oxidative phosphorylation (q < 7e-5). NED Tregs had upregulation of pSTAT3 expression post-nivolumab (p < 0.05), which was not observed in relapsing patients. Evaluation of Tregs from patients with active disease also showed upregulation of pSTAT3 in responders (p < 0.05) but not non-responders. The relative increase in Treg pSTAT3 was associated with increased overall survival (R2= 0.49, p < 0.05). In vitro assays using PD-1 blocking antibodies recapitulated the increase in pSTAT3 (p < 0.05) and Treg percentages (p < 0.001), which were diminished with the addition of a STAT3 inhibitor (p < 0.01). Conclusions: These results demonstrate previously unknown roles of decreased Treg suppressive function and induction of STAT3 as biomarkers of patient’s outcome to nivolumab therapy.

Abstract PR05: Peripheral blood immune profiling of anti-PD-1 therapy in human melanoma reveals a link between T cell re-invigoration and tumor burden that predicts response

Abstract Despite the clinical success of PD-1 based therapies in human melanoma patients, the majority of patients do not have durable clinical benefit from anti-PD-1 monotherapy. A major challenge remains identifying which patients will respond to anti-PD-1 therapy and defining the underlying reasons for successful response versus treatment failure. Pre-existing T cell infiltration and/or PD-L1 expression in tumors may predict clinical responses; however, the use of blood-based profiling to understand the immunologic mechanism of PD-1 blockade has been less explored. Here we used detailed immune profiling of peripheral blood from stage IV melanoma patients before and after pembrolizumab (pembro), and identified pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (TEX). Robust induction of Ki67 in this subset of circulating CD8 T cells post-therapy (re-invigoration) occurred in 78% of patients indicating strong, on target immunological effects of PD-1 blockade in most patients studied here. Despite this high immunological response rate, the objective clinical response rate in this cohort was less than 40%. Ki67 in CD8 T cells alone did not predict clinical outcomes and, in fact, higher systemic immune activation at baseline was associated with lower overall survival. Rather, the magnitude of re-invigoration of circulating TEX in relation to pre-treatment tumor burden correlated with clinical response. We identified a TEX re-invigoration to tumor burden ratio which could be used to predict clinical response and overall survival as early as 6 weeks post therapy. Consistent observations were found in a second independent cohort and suggest that clinical failure of PD-1 blockade in many patients may not solely be due to an inability to induce immune re-invigoration but rather, an imbalance between T cell re-invigoration and tumor burden. Thus, by focused profiling of a mechanistically relevant circulating T cell subpopulation calibrated to pre-treatment disease burden, we identify a clinically accessible predictor of response to PD-1 blockade. These findings also provide a framework for dissecting distinct types of treatment failures in melanoma and have implications for stratifying patients into additional immunotherapeutic treatment approaches. Citation Format: Alexander Huang, Michael A. Postow, Robert J. Orlowski, Rosemarie Mick, Bertram Bengsch, Sasi Manne, Wei Xu, Shannon Harmon, Matthew Adamow, Deborah Kuk, Katherine Panangeas, Cristina Carerra, Phillip Wong, Felix Quagliarello, Kristen E. Pauken, Ramin S. Herati, Suzanne McGettigan, Shawn Kothari, Sangeeth M. George, Brandon Wenz, Kurt D'Andrea, Xiaowei Xu, Ravi K. Amaravadi, Giorgos Karakousis, Lynn M. Schuchter, Katherine L. Nathanson, Jedd D. Wolchok, Tara C. Gangadhar, John Wherry. Peripheral blood immune profiling of anti-PD-1 therapy in human melanoma reveals a link between T cell re-invigoration and tumor burden that predicts response [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR05.

PD-1 blockade enhances the vaccination-induced immune response in glioma.

DC vaccination with autologous tumor lysate has demonstrated promising results for the treatment of glioblastoma (GBM) in preclinical and clinical studies. While the vaccine appears capable of inducing T cell infiltration into tumors, the effectiveness of active vaccination in progressively growing tumors is less profound. In parallel, a number of studies have identified negative costimulatory pathways, such as programmed death 1/programmed death ligand 1 (PD-1/PD-L1), as relevant mediators of the intratumoral immune responses. Clinical responses to PD-1 pathway inhibition, however, have also been varied. To evaluate the relevance to established glioma, the effects of PD-1 blockade following DC vaccination were tested in intracranial (i.c.) glioma tumor- bearing mice. Treatment with both DC vaccination and PD-1 mAb blockade resulted in long-term survival, while neither agent alone induced a survival benefit in animals with larger, established tumors. This survival benefit was completely dependent on CD8+ T cells. Additionally, DC vaccine plus PD-1 mAb blockade resulted in the upregulation of integrin homing and immunologic memory markers on tumor-infiltrating lymphocytes (TILs). In clinical samples, DC vaccination in GBM patients was associated with upregulation of PD-1 expression in vivo, while ex vivo blockade of PD-1 on freshly isolated TILs dramatically enhanced autologous tumor cell cytolysis. These findings strongly suggest that the PD-1/PD-L1 pathway plays an important role in the adaptive immune resistance of established GBM in response to antitumor active vaccination and provide us with a rationale for the clinical translation of this combination therapy.