αPD-1 Therapy Research Articles

PD1 blockade augments adoptive T cell therapy via endogenous T cells rather than direct enhancement of transferred T cells

Abstract Adoptive T cell therapy (ACT) has yielded promising clinical results but improvements are needed. Cell therapy efficacy may be improved by blockade of the inhibitory receptor PD1. Independent T cell models demonstrated cooperation between ACT and α-PD1. The cooperative effect of α-PD1 was abolished in three separate hosts devoid of polyclonal T cells (Rag1-KO, Trac-KO and irrelevant TCRtg hosts). Moreover, endogenous CD4 and CD8 T cells in the tumor increased and transferred T cells decreased in number following α-PD1 treatment. Efficacy of ACT was not improved when transferred cells were engineered with PD1-targeting miRNA or over-expression of PD1 without the cytoplasmic signaling domain. However, ACT with PD1-KO T cells was improved with the combination of α-PD1. Additionally, transferred T cells did not respond to α-PD1 when they were repeatedly stimulated until high co-expression of PD1+LAG3+TIM3+ (85% vs 34%) prior to transfer, when they were allowed prolonged in vivo tumor engagement prior to α-PD1 administration, or when they were targeting tumors with enforced expression of PDL1. Lastly, α-PD1 therapy showed no efficacy during the treatment of less immunogenic tumors, engineered with ubiquitin-tagged antigen constructs rather than RFP-antigen constructs. This finding established that PD1-sensitive endogenous T cells targeted immunogenic antigens within RFP. The role of endogenous T cells was further corroborated by TCR clonotype skewing when α-PD1 was administered. These data demonstrated that blocking the PD1 axis did not directly benefit adoptively transferred T cells. Rather, the benefit of α-PD1 was dependent on the presence of endogenous T cells specific for immunogenic tumor antigens.

Engineering ApoE3-incorporated biomimetic nanoparticle for efficient vaccine delivery to dendritic cells via macropinocytosis to enhance cancer immunotherapy

Efficient delivery of vaccines to dendritic cells (DCs) is critical for inducing sufficient immune response and realizing effective cancer immunotherapy. In the past decade, researchers have spent tremendous effort in delivering vaccines by using nanoparticles. However, most of the present strategies are designed based on receptor-mediated endocytosis to increase nanovaccines uptake by DCs, and underestimate the role of macropinocytosis in taking up exogenous antigen. Here, we proposed that macropinocytosis, an efficient pathway for DCs to internalize extracellular fluid-phase solutes, might be utilized as a highly-effective approach to facilitate nanovaccines uptake in DCs. Accordingly, we designed a biomimetic nanovaccine (R837-αOVA-ApoE3-HNP), composing of a poly-(D, l-lactide-co-glycolide) (PLGA) core to encapsulate adjuvant imiquimod (R837), a phospholipid membrane to load antigen peptide (αOVA), and apolipoprotein E3 (ApoE3), to boost the internalization of antigens into DCs. The nanovaccine exhibited highly efficient cellular uptake into DCs through the macropinocytosis pathway, and significantly promoted DCs maturation and antigen presentation. After subcutaneous injection, the nanovaccine was efficiently drained to lymph nodes. Strong T cell immune responses including the generation of antigen-specific CD8+ T cells, expansion of IFN-γ+ CD8+ T cells and the secretion of IFN-γ+ were observed after the vaccination of R837-αOVA-ApoE3-HNP. It also efficiently inhibited the formation of tumor metastasis in lung as a prevention vaccine, and exerted superior therapeutic efficiency on B16-OVA tumor-bearing mice when in combination with αPD-1 therapy. Overall, our work demonstrated that by utilizing the macropinocytosis pathway, ApoE3-incorporated biomimetic nanoparticle has great potential to function as a feasible, effective, and safe nanovaccine for cancer immunotherapy.

OP 8.4 - HIV persistence despite reservoir decay during combinatorial immunotherapy including therapeutic conserved elements (CE) DNA vaccination, αPD-1 therapy, GS-986 TLR7-agonism, and CCR5 gene-edited CD4 T cell infusion in rhesus macaques

OP 8.4 - HIV persistence despite reservoir decay during combinatorial immunotherapy including therapeutic conserved elements (CE) DNA vaccination, αPD-1 therapy, GS-986 TLR7-agonism, and CCR5 gene-edited CD4 T cell infusion in rhesus macaques

Single-Cell Deconvolution Identifies T-Cell Correlates of Response to PD-1 Blockade Treatment in AML

Background The combination of the αPD-1 (nivolumab) and hypomethylating agent azacytidine demonstrated encouraging response rate and overall survival in relapsed/refractory acute myeloid leukemia (AML) patients, compared to azacytidine alone1 (NCT02397720). However, the percentage of the patient who achieved an IWG 2016 response to such therapy was still limited (overall response rate = 33%), so it is desirable to have early predictive biomarkers to facilitate patient stratification and selection for future trials. A better understanding of T cells (primary targets of αPD-1 therapy) from bone marrow (BM) and peripheral blood (PB) in AML pre-therapy and on-therapy should yield valuable insights on the treatment-induced anti-tumor response. Methods We performed whole transcriptomic profiling (RNA-sequencing, RNA-seq) on T cells from a cohort of AML patients who were enrolled in the clinical trial mentioned above, and treated with azacytidine and nivolumab (Table 1). Sixty-four FACS-sorted patient-derived T cell samples from cryopreserved peripheral blood (PB) or bone marrow (BM) aspirates, which were collected pre-therapy (T0) and after the first round of treatment (end of cycle one, EOC1), were evaluated. By leveraging subset definitions based on scRNA-seq results from T cells of cancer patients, we implemented deconvolution of our bulk T-cell RNA-seq data to obtain the relative abundance of different T-cell subsets (in-silico dissection without physical isolation). Results We performed ex vivo cDNA library preparation on 2,000-100,000 sorted T cells and yielded a minimal of 17 million sequencing reads per BM T-cell library and 2.6 million sequencing reads per PBMC T-cell library. We compared the gene expression profile of peripheral blood T cells from AML patients (CD4: n = 16; CD8: n = 15) and healthy donors (HD, n = 8)2,3 to validate our methodology. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients4,5 (Figure 1). In comparison with HDs, circulating CD4 T cells from AML patients consisted of a higher frequency of Treg (AML versus HD: 5.1% versus 0.6%, P<0.0001)4. Peripheral blood CD8 T cells from AML patients were with a significantly lower frequency of naïve (AML versus HD: 22.1% versus 55.9%, P<0.0001), a higher frequency of effector phenotype (AML versus HD: 35.6% versus 6.7%, P =0.0011), and an increasing frequency of exhausted phenotype (AML versus HD: 19.1% versus 0.0%, P<0.0001)5. Independent of the clinical responses attained on the azacytidine and nivolumab, comparison of the pre-therapy CD8 T cells from BM and PB from all AML patients using both gene set enrichment analysis and deconvolution indicated that the BM CD8 T cells were more activated/differentiated compared with PBMC CD8 T cells, likely reflective of an ongoing immune response against the AML. We also found treatment-induced gene expression profile changes in the AML circulating CD8 T cells, characterized by increased cell metabolism and cell proliferation. Deconvolution identified that pre-therapy relative abundance of exhausted (CD3+CD8+PD-1+CD45RO+) and effector (CD3+CD8+CD45RA+Tbet+PD-1lo) CD8 T cells (plasticity) could serve as subpopulations relevant for patient stratification (Figure 2). We further validated these results using CyTOF wherein these same subpopulations were differentially abundant between responders and non-responders at the pre-therapy time-point. Conclusions Collectively, the analyses of RNA-seq of CD8 T cells from AML patients on the azacytidine with nivolumab trial revealed that (1) the PD-1 blockade-based treatment-induced gene expression profiling changes (increased cell proliferation and metabolism) of CD8 T cells are detectable as early as EOC1 in the peripheral blood; (2) specific subpopulations of plastic CD8 T cells identified may have the potential to serve as an actionable biomarker to select AML patients most likely to benefit from such immune checkpoint therapies. These findings need to be confirmed in larger studies, planned or currently being conducted, with αPD-1 based therapies in AML and MDS.

Immune checkpoint protein and cytokine expression by T lymphocytes in pleural effusion of cancer patients receiving anti–PD-1 therapy

ObjectivesPleural effusion (PE) occasionally develops in cancer patients during treatment with antibodies to programmed cell death–1 (PD-1) or to its ligand PD-L1 (hereafter, αPD-1 therapy). Such effusion often contains infiltrated mononuclear cells, although the types of immune cell present as well as the outcome of such patients have remained unclear. Materials and methodsWe performed a multi-institutional, observational study to examine the clinical outcome of patients who develop PE after the onset of αPD-1 therapy. We compared the immune cell profiles and the immune status of lymphocytes in PE as determined by flow cytometry between nine patients who developed effusion during αPD-1 therapy (αPD-1 group) and 15 patients who developed PE during treatment with other anticancer agents (control group). ResultsMost mononuclear cells in PE were lymphocytes in both the αPD-1 and control groups. The frequency of both CD4+ and CD8+ T lymphocytes expressing the immune checkpoint proteins TIM-3 or TIGIT as well as that of CD8+ T lymphocytes expressing PD-L1 were increased in the αPD-1 group compared with the control group. αPD-1 therapy continued for a substantial period after the emergence of PE in six of the nine patients in the αPD-1 group, and the frequency of CD4+ T lymphocytes in PE expressing the immune checkpoint protein LAG-3 or the cytokine interkeukin-17 was lower for these patients than for those who did not receive a sustained treatment benefit. ConclusionOur results suggest a clinical benefit of continuing αPD-1 therapy in some patients who develop PE. We found that infiltrating T lymphocytes in PE manifest a more exhausted phenotype during αPD-1 therapy than during treatment with other cancer drugs, with subpopulations of these cells characterized by specific immune checkpoint protein and cytokine expression profiles possibly contributing to the antitumor immune response.

Abstract 2270: Recruitment of CD103+DCs via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy

Abstract Checkpoint inhibitor antibody (CPI) therapy has demonstrated significant clinical benefit in a number of tumor types. Unfortunately, certain tumor characteristics, such as the lack of immune cell infiltration, often correlate with poor responses to CPI therapy. Studies have identified C-C Motif Chemokine Ligand 4 (CCL4) as a key molecule necessary for the recruitment of cross-presenting, CD103+dendritic cells (DCs) to the tumor; tumors lacking CCL4 expression exhibit a “cold tumor” phenotype and respond poorly to immunotherapy (Spranger and Gajewski, Nat. Rev. Cancer, 2018). Based on these results, we hypothesized that tumor-targeted CCL4 could enhance immune cell infiltration into the tumor and synergize with CPI therapy. We generated a fusion protein comprised of CCL4 and a collagen binding domain (CBD) derived from von Willebrand factor. Utilizing exposure of collagen in leaky tumor vasculature due to its disordered structure, we observed that that i.v. infusion of CBD-CCL4 fusion proteins, but not native CCL4, can enhance infiltration of CD103+DCs, CD8+T cells, and natural killer cells and slow B16F10 tumor growth when combined with CPI therapy consisting of anti-cytotoxic T-lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death-ligand 1 antibody (αPD-L1). Further analysis showed strong correlations between the presence of CD103+DCs and CD8+ T cells and tumor regression. Similarly, in the EMT6 breast cancer model, tumor-targeted CCL4 in combination with CPI, but not native form CCL4, enhanced recruitment of CD103+ DCs, CD8+ T cells, and led to a reduction in tumor growth. To confirm the importance of CD103+DCs in mediating anti-tumor responses, we utilized Batf3 knockout mice bearing B16F10 tumors; in this instance, anti-tumor efficacy of CPI + CBD-CCL4 was completely lost. Preliminary results also showed that CBD-CCL4 can synergize with αPD-1 antibody therapy, slowing tumor growth in CT26 and MC38 colon cancer models compared to αPD-1 therapy alone. Importantly, CBD-CCL4 treatment did not lead to any detectable levels of systemic cytokine elevation or serum liver damage markers, suggesting that this therapy was well-tolerated following systemic administration. These results highlight the utility of recruiting CD103+DCs to the tumor to improve the efficacy of CPI therapy. This engineered chemokine delivery strategy demonstrates significant translational potential by targeting the tumor stroma following systemic administration. Citation Format: John-Michael Williford, Jun Ishihara, Ako Ishihara, Aslan Mansurov, Melody A. Swartz, Jeffrey A. Hubbell. Recruitment of CD103+DCs via tumor-targeted chemokine delivery enhances efficacy of checkpoint inhibitor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2270.

Preclinical development of combination therapy targeting the dominant cytokine interleukin-1β for renal cell carcinoma.

e14237 Background: Targeting myeloid-derived suppressor cells (MDSCs), key mediators of intrinsic and adaptive immune resistance, remains a significant challenge in kidney cancer immunotherapy. One potential driver of MDSC mediated tumor immunosuppression is the cytokine interleukin-1 beta (IL-1β). Although large cancer prevention studies show that targeting IL1 decreases cancer risk, the exact mechanism of anti-IL-1 mediated anti-tumor efficacy is not yet defined. To understand the immunogenic effects of IL-1β blockade and effects on MDSCs, we used the RENCA model of RCC. Methods: We inoculated BALB/c mice with 1x106 RENCA cells. On Day +10 we treated mice (n = 8/group) with vehicle, αPD1, αIL1β, cabozantinib, combination αPD1/αIL1β, or cabozantinib/αIL1β. We harvested day +18 tumors to quantify tumor size and immune cells by flow cytometry. We then isolated immune cells for differential gene expression profiling by RNAseq. Results: Treatment with αIL1β significantly decreased tumor size compared to vehicle or αPD1 monotherapy. Combining αPD1/αIL1β or cabozantinib/αIL1β potentiated the anti-tumor effects of αPD1 or cabozantinib monotherapy respectively. Anti-IL1β therapy decreased polymorphonuclear (PMN)-MDSC infiltration (αIL1β: 0.76% +/- 0.21 vs. vehicle: 1.89% +/- 0.37, P = 0.014). Relative to PD1 monotherapy, combining αPD1 and αIL1β decreased PMN-MDSCs (αPD1: 2.14% +/- 0.56 vs αPD1/αIL1β: 0.91% +/- 0.15, P = 0.033) and increased M1-like tumor associated macrophages (TAM) (αPD1: 0.798% +/- 0.21 vs. αPD1/αIL1β: 12.04% +/- 2.9, P = 0.0008). Gene expression profiling from sorted immune cells from αIL1β treated tumors revealed multiple differentially regulated cytokines including interleukin-6, IFN-γ, and IL-10. Conclusions: Blockade of IL-1β depletes intratumoral PMN-MDSCs and increases M1-like TAM with minimal effects observed on the T cell compartment. Treatment with cabozantinib or αPD1 in combination with αIL1β led to sustained control of tumor growth. IL-1β represents a promising target for kidney cancer immunotherapy. Based on the preclinical data, a clinical trial of neoadjuvant αPD1 and αIL1β therapy is planned for patients with high risk RCC.

Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity

ABSTRACTMetastatic melanoma is a significant clinical problem with a 5-year survival rate of only 15–20%. Recent approval of new immunotherapies and targeted inhibitors have provided much needed options for these patients, in some cases promoting dramatic disease regressions. In particular, antibody-based therapies that block the PD-1/PD-L1 checkpoint inhibitory pathway have achieved an increased overall response rate in metastatic melanoma, yet durable response rates are reported only around 15%. To improve the overall and durable response rates for advanced-stage melanoma, combined targeted and immune-based therapies are under investigation. Here, we investigated how the natural products called schweinfurthins, which have selective anti-proliferative activity against many cancer types, impact anti-(α)PD-1-mediated immunotherapy of murine melanomas. Two different compounds efficiently reduced the growth of human and murine melanoma cells in vitro and induced plasma membrane surface localization of the ER-resident protein calreticulin in B16.F10 melanoma cells, an indicator of immunogenic cell death. In addition, both compounds improved αPD-1-mediated immunotherapy of established tumors in immunocompetent C57BL/6 mice either by delaying tumor progression or resulting in complete tumor regression. Improved immunotherapy was accomplished following only a 5-day course of schweinfurthin, which was associated with initial tumor regression even in the absence of αPD-1. Schweinfurthin-induced tumor regression required an intact immune system as tumors were unaffected in NOD scid gamma (NSG) mice. These results indicate that schweinfurthins improve αPD-1 therapy, leading to enhanced and durable anti-tumor immunity and support the translation of this novel approach to further improve response rates for metastatic melanoma.

Abstract 2984: Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse

Abstract The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting CTLA-4 and PD-1. Blocking either checkpoint relieves the negative regulation of T-cells resulting in significant responses in patients with cancer. Data has now begun to emerge regarding differences between these two therapies. While αPD-1 therapy has a greater response rate (~30% vs. 11%) according to RECIST criteria, recent reports have suggested responses to αPD-1 may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 3-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. Previous studies in bacterial or chronic LCMV infectious models have shown that αCTLA-4 can increase CD8+ memory T-cell formation, whereas genetic ablation of PD-1 on T-cells often promotes the terminally differentiated exhausted CD8+ T-cell phenotype, while attenuating memory T-cell formation. However, the mechanism which leads to relapse following αPD-1 treatment in tumor models is not clear. The goal of this project is to understand how immunotherapies shape memory T-cell formation and how that relates to the mechanism of tumor relapse. To test whether αCTLA-4 or αPD-1 can induce a better memory T-cell response, mice were vaccinated with irradiated B16F10 murine melanoma cells and treated αCTLA-4 or αPD-1. Mice were re-challenged with B16F10 80 days after vaccination. Although both αCTLA-4 and αPD-1 improved tumor rejection compared with controls, αCTLA-4 treated mice exhibited superior tumor control compared to αPD-1 (p<0.0005) suggesting the memory T-cell response mediated by αCTLA-4 is more durable. In order to test whether this memory T-cell response is antigen-specific, mice were re-challenged with unrelated MC38 or 3LL cancer cells. The antibody treated groups did not show improved antitumor effect compared with vaccine control (p>0.5). To test whether the frequency of memory T-cells recruited to the re-challenged tumor could affect memory T-cell response, antigen-specific pmel-1 T-cells were infused to mice following vaccination with αCTLA-4 or αPD-1. Our result suggested that there were more tumor-infiltrating pmel-1 T-cells in the αCTLA-4 treated group compared to the αPD-1 treated group (p<0.05). In order to augment the durability of αPD-1 treatment, αPD-1 was combined with αCTLA-4 following vaccination. The combined treatment group has superior antitumor response compared to that with αPD-1 (p<0.000003) and overlapped with the αCTLA-4 treated group (P>0.05) during re-challenge, indicating that the effect of the combined treatment is dominated by αCTLA-4. Collectively, our studies facilitate the design of combination immunotherapy treatments that enhance both response rates and generation of memory T-cells to prevent relapse. Citation Format: Stephen Mok, Colm R. Duffy, James P. Allison. Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse [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 2984.

Abstract 3767: Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing

Abstract Cancer therapy has been reshaped by checkpoint inhibitors (CPIs), making it possible for durable responses against cancers with traditionally low cure rates. Current efforts are focused on combination therapies in the hopes of evading resistance to CPIs and improving overall response. One escape mechanism attributed to acquired resistance to CPIs includes defective antigen presentation, namely a loss in MHC class I expression. This leads to loss of CD8 T cell-mediated tumor killing and disease relapse. This recent revelation has stimulated a need for therapies that activate other cytotoxic effector cells such as NK cells to kill tumors. Imprime PGG (Imprime) is a soluble, systemically delivered yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern) capable of triggering innate immune cell function leading to a cascade of immune activation and enhanced tumor killing. Imprime activates the innate immune system via dectin-1, eliciting production of a variety of chemokines and cytokines, including type I IFN, leading to the mobilization and stimulation of innate cell types including dendritic cells and monocytes. Unlike other PAMPs which systemic administration often leads to toxic side effects, Imprime has been administered safely by intravenous infusion to >400 human subjects. Currently, Imprime PGG is being evaluated in combination with αPD1 therapy in multiple clinical trials. Previously we have shown that combination therapy of anti-Trp1 antibody and Imprime leads to a significant reduction in both number and size of lung metastases in the B16F10 metastatic melanoma tumor model over anti-Trp1 antibody alone. This reduction of metastases is highly dependent on NK cells but not CD8 T cells. To explore the impact of Imprime on NK-mediated cytotoxicity, we further evaluated in vivo killing of MHC class I deficient TapKO cells after intravenous administration of Imprime. In these experiments Imprime was able to enhance the NK cytotoxic killing of the targets. All NK cell killing observed was dependent on type I IFN, phagocytic cells and dectin 1. Imprime treatment increased cytokines that drive enhanced NK activation and effector phenotype. Significant increases were observed in the cytokines IL15/IL15rα, IL18, IL12p70 in lymph node lysates as well as increases in the effector molecules CD69, GrB, and CD107a on splenic NK cells. The upregulation of all of these molecules during Imprime treatment was dependent on dectin 1. Additionally, IL15/IL15rα production was also dependent on type I IFN, and phagocytic cells. Interestingly, Ly6c hi monocytes, which are increased after Imprime treatment, also show enhancement of IL15rα expression. Collectively, these data demonstrate that Imprime contributes to enhanced NK functionality and killing which may provide a unique immunotherapeutic approach to complement existing therapies. Citation Format: Kathryn A. Fraser, Takashi Kangas, Ross B. Fulton, Steven M. Leonardo, Ben Harrison, Yumi Yokoyama, Nandita Bose, Jeremy R. Graff, Mark Uhlik, Keith B. Gorden. Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing [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 3767.

Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse

Abstract Clinical studies have shown that αPD-1 therapy mediates a greater response rate compared to αCTLA-4 according to RECIST criteria. However, it may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 10-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. The goal of this study is to understand the relationship between memory T-cell formation in tumor models mediated by checkpoint antibodies and tumor relapse. Our results have shown that αCTLA-4 could induce a superior memory antitumor response during tumor rechallenge on mice compared to that treated with αPD-1 after 80 days. We also showed that this memory antitumor response was antigen-specific. These antigen-specific memory T-cells in mice that were treated with αCTLA-4 had better proliferative potential with cytokine producing function and more CD62L+CD44+ memory T-cells were converted back to CD62L-CD44+ effectors during rechallenge. We found that αCTLA-4 and αPD-1 facilitated the development of CD4 and CD8 memory T-cells respectively. We also demonstrated that the memory antitumor activity mediated by the combination of αCTLA-4 and αPD-1 overlapped with αCTLA-4 alone suggesting the effect of the combined treatment is dominated by αCTLA-4.

Abstract 976: ImmunoMap: a novel bioinformatics tool for immune cell repertoire analysis

Abstract Background: There has been a dramatic increase in T-cell Receptor (TCR) sequencing spurred, in part, by the clinical demand in Immuno-oncology and technological advances in TCR sequencing. However, there has been little in the way of approaches to parse the data in a biologically meaningful fashion. The ability to parse this data to understand the T-cell repertoire in a structurally relevant manner has the potential to open new discoveries about how the immune system responds to insults such as cancer and infectious diseases. Methods: Here we describe a novel method to visualize and quantify TCR repertoire sequence diversity. This method includes metrics such as visualization of repertoire with: 1) weighted phylogenetic trees that display relatedness and frequency of the sequences; 2) dominant motif analyses identifying clusters of highly homologous sequences that contribute significantly to response and; 3) TCR diversity score measuring the average relatedness (by sequence homology) of all TCR’s in a sample. To demonstrate the power of the approach, we have applied it to understanding the CD8 T Cell response to model self (TRP2) and foreign (SIY) antigens in naïve and tumor-bearing (B16 melanoma) B6 mice. Additionally, this method was applied to tumor infiltrating lymphocytes, TIL, taken pre- and on-therapy, from patients undergoing Nivolumab (α-PD1) therapy for metastatic melanoma. Results: Analysis of the naïve CD8 response demonstrated a highly conserved (measured by the TCR diversity score) and less clonal response to SIY whereas the response to TRP2 was less conserved and highly clonal. Dominant motif analysis demonstrated highly rich motifs consisting of many homologous sequences in the SIY response but few sequences per motif in the TRP2 response. This may reflect the outcome of tolerance mechanisms to self-antigens. Presence of tumor demonstrated differential immune pressure on the TRP2 vs SIY response. Tumor primed novel SIY motifs but constricted the number of dominant motifs in the TRP2 response while additionally altering the sequence of the motifs. In patients undergoing α-PD1 therapy, we identified signatures in pre- and post-therapy TCR repertoires that correlated with clinical outcome response. Prior to therapy, patients whose dominant motifs were rich with many sequences responded favorably to checkpoint inhibition over those with less rich motifs. After four week on therapy, patients whose TCR repertoires became more conserved responded more favorably to PD1 treatment while those who did not respond had no change in their TCR diversity score. Conclusions: In summary, we have developed and demonstrated a novel method to meaningfully parse and interpret TCR repertoire data and have applied it to yield a novel insight of CD8 T Cell responses to different types of antigens in model systems as well as key characteristics of TIL repertoires from patients who respond clinically to α-PD1 therapy. Citation Format: John-William Sidhom, Catherine A. Bessell, Jonathan J. Havel, Timothy A. Chan, Jonathan P. Schneck. ImmunoMap: a novel bioinformatics tool for immune cell repertoire analysis [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 976. doi:10.1158/1538-7445.AM2017-976

Abstract 1685: Effects of anti-ctla-4 and anti-pd-1 on memory T-cell differentiation and resistance to tumor relapse

Abstract The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting CTLA-4 and PD-1. Blocking either checkpoint relieves the negative regulation of T-cells resulting in significant responses in patients with cancer. Data has now begun to emerge regarding differences between these two therapies. While αPD-1 therapy has a greater response rate (~30% vs. 11%) according to RECIST criteria, recent reports have suggested responses to αPD-1 may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 3-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. Previous studies in bacterial or chronic LCMV infectious models have shown that αCTLA-4 can increase CD8+ memory T-cell formation, whereas genetic ablation of PD-1 on T-cells often promotes the terminally differentiated exhausted CD8+ T-cell phenotype, while attenuating memory T-cell formation. However, the mechanism which leads to relapse following αPD-1 treatment in tumor models is not clear. The goal of this project is to understand how immunotherapies shape memory T-cell formation and how that relates to the mechanism of tumor relapse. To test whether αCTLA-4 or αPD-1 can induce a better memory T-cell response, mice were vaccinated with irradiated B16F10 murine melanoma cells and treated αCTLA-4 or αPD-1. Mice were re-challenged with B16F10 80 days after vaccination. Although both αCTLA-4 and αPD-1 improved tumor rejection compared with controls, αCTLA-4 treated mice exhibited superior tumor control compared to αPD-1 (p<0.0005) suggesting the memory T-cell response mediated by αCTLA-4 is more durable. In order to test whether this memory T-cell response is antigen-specific, mice were re-challenged with unrelated MC38 or 3LL cancer cells. The antibody treated groups did not show improved antitumor effect compared with vaccine control (p>0.5). To test whether the frequency of memory T-cells recruited to the re-challenged tumor could affect memory T-cell response, antigen-specific pmel-1 T-cells were infused to mice following vaccination with αCTLA-4 or αPD-1. Our result suggested that there were more tumor-infiltrating pmel-1 T-cells in the αCTLA-4 treated group compared to the αPD-1 treated group (p<0.05). In order to augment the durability of αPD-1 treatment, αPD-1 was combined with αCTLA-4 following vaccination. The combined treatment group has superior antitumor response compared to that with αPD-1 (p<0.000003) and overlapped with the αCTLA-4 treated group (P>0.05) during re-challenge, indicating that the effect of the combined treatment is dominated by αCTLA-4. Collectively, our studies facilitate the design of combination immunotherapy treatments that enhance both response rates and generation of memory T-cells to prevent relapse. Citation Format: Stephen Mok, Colm R. Duffy, James P. Allison. Effects of anti-ctla-4 and anti-pd-1 on memory T-cell differentiation and resistance to tumor relapse [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 1685. doi:10.1158/1538-7445.AM2017-1685

Immunovirotherapy with measles virus strains in combination with anti-PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment.

Glioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome. In vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy. In vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells. This report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

Synergy of radiotherapy and PD-1 blockade in Kras-mutant lung cancer.

Radiation therapy (RT), a critical modality in the treatment of lung cancer, induces direct tumor cell death and augments tumor-specific immunity. However, despite initial tumor control, most patients suffer from locoregional relapse and/or metastatic disease following RT. The use of immunotherapy in non-small-cell lung cancer (NSCLC) could potentially change this outcome by enhancing the effects of RT. Here, we report significant (up to 70% volume reduction of the target lesion) and durable (up to 12 weeks) tumor regressions in conditional Kras-driven genetically engineered mouse models (GEMMs) of NSCLC treated with radiotherapy and a programmed cell death 1 antibody (αPD-1). However, while αPD-1 therapy was beneficial when combined with RT in radiation-naive tumors, αPD-1 therapy had no antineoplastic efficacy in RT-relapsed tumors and further induced T cell inhibitory markers in this setting. Furthermore, there was differential efficacy of αPD-1 plus RT among Kras-driven GEMMs, with additional loss of the tumor suppressor serine/threonine kinase 11/liver kinase B1 (Stk11/Lkb1) resulting in no synergistic efficacy. Taken together, our data provide evidence for a close interaction among RT, T cells, and the PD-1/PD-L1 axis and underscore the rationale for clinical combinatorial therapy with immune modulators and radiotherapy.

Evaluation of innate and adaptive immunity contributing to the antitumor effects of PD1 blockade in an orthotopic murine model of pancreatic cancer

ABSTRACTDespite the clinical success of anti-PD1 antibody (α-PD1) therapy, the immune mechanisms contributing to the antineoplastic response remain unclear. Here, we describe novel aspects of the immune response involved in α-PD1-induced antitumor effects using an orthotopic KrasG12D/p53R172H/Pdx1-Cre (KPC) model of pancreatic ductal adenocarcinoma (PDA). We found that positive therapeutic outcome involved both the innate and adaptive arms of the immune system. Adoptive transfer of total splenocytes after short-term (3 d) but not long-term (28 d) PD1 blockade significantly extended survival of non-treated tumor-bearing recipient mice. This protective effect appeared to be mostly mediated by T cells, as adoptive transfer of purified natural killer (NK) cells and/or granulocyte receptor 1 (Gr1)+ cells or splenocytes depleted of Gr1+ cells and NK cells did not exhibit transferrable antitumor activity following short-term PD1 blockade. Nevertheless, splenic and tumor-derived CD11b+Gr1+ cells and NK cells showed significant persistence of α-PD1 bound to these cells in the treated primary recipient mice. We observed that short-term inhibition of PD1 signaling modulated the profiles of multifunctional cytokines in the tumor immune-infiltrate, including downregulation of vascular endothelial growth factor A (VEGF-A). Altogether, the data suggest that systemic blockade of PD1 results in rapid modulation of antitumor immunity that differs in the tumor microenvironment (TME) when compared to the spleen. These results demonstrate a key role for early immune-mediated events in controlling tumor progression in response to α-PD1 treatment and warrant further investigation into the mechanisms governing responses to the therapy at the innate-adaptive immune interface.

Melanoma-specific MHC-II expression to predict response to α-PD-1 therapy.

9041 Background: αPD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma (MEL) patients (pts). While promising, many pts do not benefit clinically. Predictive biomarkers to...