Quantification of CD3+CD8+ (CD8 T-cells) and (B) CD3+CD4+ (CD4 T-cells) in both tumors and spleens in YUMM2.1; gating strategy after exclusion of dead cells of CD3+CD8+ and CD3+CD4+ cells after gating for CD3+ cells; quantification of CD3+CD8+ (CD8 T-cells) in MC38 and YUMM2.1 spleens; CD3+CD4+ (CD4 T-cells) in MC38 and YUMM 2.1 spleens CD8 T-cells in both tumors and spleens in YUMM1.1.

IGV plot of RNA-Seq from YUMM1.1, YUMM1.7 and YUMM2.1; western blot analysis of cytoplasmic and nuclear beta-catenin in YUMM1.7 and YUMM2.1 cell lines with or without exposure to 10 uM 4HT for 48 hours; top-flash activity of total beta-catenin in YUMM1.7 and YUMM2.1 with or without exposure to 10uM 4HT for 48 hours; representative immunofluorescence of beta-catenin stained non-treated tumors.

Supplementary figure legends

Gating strategy of CD11c+B220-, CD11c+B220+, CD11c+B220-CD8+ and CD11c+B220-CD103+ cells; gating strategy of CD11b+MHC-IIhigh DCs in CD11c+ cells; gating strategy of CD11b+F4/80+TAMs, CD11b+F4/80+MHC-IIlow TAMs and CD11b+F4/80+MHC-IIhigh TAMs; gating strategy of MO-MDSC (CD11b+Ly6ChighLy6Glow) and PMN-MDSC (CD11b+Ly6ClowLy6Ghigh); gating strategy of Tregs (CD4+CD25+FoxP3+); corresponding normalized enrichment scores (NES), P values and false discovery rates (FDR) of the GSEA plots for YUMM2.1 versus YUMM1.1 enriched pathways involved in immune response, cytokine production and inflammatory response.

Supplementary Figure 1. Exome sequencing and copy number changes in M431 and corresponding whole-tumor biopsy. Supplementary Figure 2. Mutations in antigen presentation machinery from anti-PD1 treated melanoma cohort. Supplementary Figure 3. Immunohistochemistry (IHC) analysis of biopsies from melanoma metastases with a JAK1 loss of function mutation. Supplementary Figure 4. Selection of PD-L1 flow cytometry antibody and impact of phosphatase inhibitor as well as temperature on measuring surface PD-L1 expression and pAKT. Supplementary Figure 5. Flow cytometry gating strategy and dose response curve of interferon alpha, beta and gamma to determine the optimal concentrations. Supplementary Figure 6. Time course of PD-L1 surface expression upon interferon alpha, beta and gamma treatment for selected cell lines to determine the optimal time point for the screening. Supplementary Figure 7. Time course of PD-L1 expression upon interferon alpha, beta or gamma treatment for the cell lines with poor or no up-regulation upon 18 hours exposure. Supplementary Figure 8. Interferon signaling pathway in good and poorly responding cell lines. Supplementary Figure 9. PD-L1 expression upon interferon alpha and beta exposure. Supplementary Figure 10. Predicted functional consequences of M368 JAK2 D313 splice site mutation. Supplementary Figure 11. Immunohistochemistry (IHC) analysis of biopsies from melanoma metastases with a JAK1 loss of function mutation. Supplementary Figure 12. JAK1 wild-type lentiviral vector transduction. Supplementary Figure 13. Mutations in antigen presentation machinery from anti-PD1 treated colorectal cohort. Supplementary Figure 14. DNA damage repair gene mutations in endometrial cancer cell lines with JAK1/2 mutations. Supplementary Figure 15. Frequency of JAK1 and JAK2 alterations and their association with overall survival in additional TCGA datasets. Supplementary Figure 16. Functional effect of genetic loss of reactive PD-L1 on responses to PD-1 blockade. Supplementary Database 1. Whole exome sequencing of 23 baseline biopsies from patients analyzed in Fig. 1, and whole exome sequencing of the M431 cell line (Accession code: SRP067938).

Supplemental Figure 1: Hypothetical model of peripheral blood TCR-transgenic cell repopulation. Supplemental Figure 2: GLP team organizational chart. Supplemental Figure 3. Bone marrow transplant (BMT) optimization studies in HLA-A2/Kb transgenic mice. Supplemental Figure 4. Body weight and hematology assessment at day 5 and 3 months after BMT. Supplemental Figure 5. Spleen and bone marrow cellularity at day 5 and 3 months after BMT. Supplemental Figure 6. Serum chemistry at 3 months after BMT. Supplemental Figure 7. Flow cytometry gating strategy for bone marrow and splenocytes phenotype characterization. Supplemental Figure 8. Survival and hematology 3 months after BMT with Lin- cells transduced with LV-empty, LV-NY-ESO-1 TCR or LV-NY-ESO-1 TCR/sr39TK. Supplemental Figure 9. Differentiation of NYESO TCR/sr39TK-engineered T cells in ATOs (artificial thymic organoids). Supplemental Table 1. Certificate of Analysis of the GMP-comparable LV-NY-ESO-1 TCR/sr39TK (RRL-MSCV-optNYESO-optsr39TK-WPRE, production volume: 20L). Supplemental Table 2. Certificate of Analysis of the Clinical Grade Lentivirus LV-NY-ESO- 1 TCR/sr39TK (RRL-MSCV-optNYESO-optsr39TK-WPRE, production volume 60L). Supplemental Table 3. Certificate of Analysis of the clinical grade RV-NY-ESO-1 TCR (MSGV1-A2aB-1G4A-LY3H10) (Production volume 18L)*. Supplemental Table 4. Certificate of Analysis of the GMP comparable RV-NY-ESO-1 TCR (MSGV1-A2ab-1G4A-Ly3H10, Production volume: 3L). Supplemental Table 5. Co-Administration of NY-ESO-1 TCR Genetically Modified T cells and Hematopoietic Stem Cells (HSCs) in HLA-A2.1/Kb mice. GLP studies cohort distribution. Supplemental Table 6. Cell manufacturing acceptance criteria. Supplemental Table 7. List of protocol-specific organs. Supplemental Table 8. Manufacturing validation runs. Supplemental Table 9. Comparison between fresh and cryopreserved product at 1, 30, 90 and 180# days.

Background Overcoming the suppressive tumor microenvironment (TME) remains an important unmet challenge for chimeric antigen receptor (CAR) T cell therapies. A key suppressive factor, transforming growth factor β (TGF-β), is a primary driver of T cell suppression reducing T cell receptor (TCR)-mediated cytotoxicity and driving the differentiation of immunosuppressive regulatory T cells (Tregs) in the TME. Methods To overcome TGF-β-specific immunosuppression, we employed a TGF-β-targeting CAR co-expressed in T cells with a human papillomavirus type 16 (HPV16) E711-19-specific, HLA-A*02:01-restricted TCR (E7 TCR). Results In comparison to T cells expressing E7 TCR alone or E7 TCR with an irrelevant CD19-targeting CAR, T cells co-transduced with E7 TCR and TGF-β CAR showed enhanced proliferation and cytokine production, while maintained cytotoxicity throughout repeat antigen challenge assays with HPV16+ Ca Ski tumor cells. The inclusion of TGF-β CAR also reduced PD1+ expression and Treg differentiation after repeat antigen challenges. Transcriptional analysis further confirmed reduced FOXP3 expression as well as enhanced proinflammatory genes such as TNF and IFNG. Conclusions In combination, these data clearly show that a TGF-β CAR can enhance TCR function and limit Treg differentiation and is therefore likely to improve the function and persistence of TCR therapies in the TME.

IGV plot exome sequencing from YUMM1.1, YUMM1.7 and YUMM2.1, tumor growth curve of YUMM1.7 and B16 with 4 mice in each group, analysis of the non-synonymous mutational load compared to a strain-matched normal with known dbSNP variants excluded.

There is a critical need for more effective therapy for acute myelogenous leukemia (AML). Although many patients achieve remission, most relapse with poor outcomes. Even after allogeneic Stem Cell Transplantation (SCT), 30-50% of patients relapse due to the persistence of residual disease.

Interferon-independent MHC class I induction restores T cell immunity.

Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti–PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti–PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non–T cell–inflamed TME.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells.

Abstract Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti–PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti–PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy. Significance: The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy. This article is highlighted in the In This Issue feature, p. 1079

Abstract Purpose: To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. Experimental Design: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. Results: TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. Conclusions: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Abstract NKTR-214 is a CD122-biased cytokine agonist designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand CD8 T and natural killer cells (NK) over Tregs in the tumor. We evaluated the tumor immunology, biodistribution and anti-tumor activity of NKTR-214 combined with ACT in the pmel-1 ACT/B16F10 melanoma tumor model. NKTR-214+ACT provides a robust and durable anti-tumor response compared to IL-2+ACT with less frequent dosing in the aggressive B16F10 model. NKTR-214+ACT led to significant tumor growth inhibition at day 14 compared with IL-2+ACT, 174mm3 vs 484mm3 tumor volume, respectively (p<0.05, n=12). Tumors of animals receiving IL-2+ACT grew to the endpoint of 1,500 mm3 18 days after treatment, while NKTR-214+ACT significantly improved the survival to 35 days (p<0.0001). Bioluminescence imaging (BLI) showed that NKTR-214+ACT treatment significantly increased T cells expansion in the spleen from day 5 to day 9 compared to IL-2+ACT (p<0.0001, n=5). At day 5, quantification of BLI of serial images with region of interest analysis at the site of spleen revealed an average radiance 14 folds higher in NKTR+ACT than in IL-2+ACT treated mice (1.3*10^7 vs 9.5*10^5 p/s/cm²/sr). BLI showed a stronger peak of activity of tumor-infiltrating effector T cells in the ACT+NKTR-214 group versus the ACT+IL-2 from day 5 to day 7 (p<0.0001, n =5). The second dose of NKTR-214 at day 9 triggered a second expansion of effector T cells in the spleen and tumor from day 12 to day 17, while no effect in the group treated with 3 doses IL-2 was observed (p<0.0001, n =5). The peak of signal was reached at day 14 with an average radiance 10 folds higher in the NKTR-214+ACT group compared to the IL2+act group (4.7*10^6 vs 4.5*10^5 p/s/cm²/sr). These data are supported by immuno-PET imaging using minibody (Mb) targeting CD8 in vivo. Ex vivo biodistribution analysis showed a signal in the spleen 5-folds higher in the ACT+NKTR-214 group compared with the ACT+IL-2 group on day 5 after treatment, 87% and 17% injected dose per gram, respectively (p<0.05, n=3). Flow cytometry analysis performed at the same time point showed that NKTR-214 treatment significantly increased pmel-1 CD8 T cells and amplified the CD8/Treg ratio compared to IL-2 both in spleen and tumor (3 and 6 folds, respectively, p<0.05, n=3). In conclusion, NKTR-214 + ACT is well tolerated and robustly mobilizes T cells into tumors where they durably persist, supporting NKTR-214 potential use in combination with cell-based therapeutics. Citation Format: Giulia Parisi, Justin Saco, Felix Bergara, Paige Krystofinski, Ruixue Zhang, Cristina Puig Saus, Siwen Hu-Lieskovan, Begonya Comin-Anduix, Anna Wu, Deborah H. Charych, Antoni Ribas. Enhanced expansion and tumor targeting of adoptively transferred T cells with NKTR-214 [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 3566.

Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy.

Abstract Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti–PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair–deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti–PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy. Significance: A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188–201. ©2016 AACR. See related commentary by Marabelle et al., p. 128. This article is highlighted in the In This Issue feature, p. 115

Abstract T cell receptor (TCR) engineered adoptive T cell transfer (ACT) has shown remarkable antitumor efficacy in several clinical trials. However, low persistence of modified cells limits long-term clinical responses. To overcome this hurdle, we propose a clinical trial co-administering genetically modified T cells and stem cells both expressing an NY-ESO-1 TCR such that the engrafted stem cells generate a source for constant renewal of modified T cells. Here we report a pre-clinical IND-enabling study performed at UCLA under Good Laboratory Practice (GLP) compliance to assess whether co-administration impacts (I) safety; (II) engraftment and cell lineage differentiation of gene modified stem cells; and (III) persistence of adoptively transferred T cells and stem cell-derived progeny. We performed 12 optimization studies to define the optimal conditions for TCR gene modified ACT and TCR gene modified hematopoietic stem cell (HSC) bone marrow transplantation (BMT). Sixty-four HLA-A2/kb transgenic mice were myelodepleted and received syngeneic BMT with Lineage depleted bone marrow (Lin-) cells transduced with the LV-NYESO-1 TCR/sr39TK and ACT with T cells transduced with the RV-NYESO-1 TCR. Control groups were as follows: untreated mice, mice receiving mock transduced Lin- cells and T cells, mice receiving transduced Lin- cells and mock transduced T cells, and mice receiving mock transduced Lin- cells and transduced T cells (n = 16 per group). Overall survival at 3 months was 87.5%; no significant differences in survival were observed among cohorts. After BMT we observed a decrease in body weight, elevation in creatinine kinase and transaminases, and gonadal germ cell ablation in all cohorts. Three months after BMT, all blood cell lineages were reconstituted in surviving mice. Using digital droplet PCR and flow cytometry, we confirmed that transduced stem cells engrafted and their progeny persisted long term. In the bone marrow, NY-ESO-1 TCR was expressed intracellularly among progenitor cells (Lin-, LSK and HSC) as well as all hematopoietic cell lineages within the spleen (CD8+ T cells, CD4+ T cells, NKT cells, B cells and granulocytes). Co-administration with gene modified T cells and stem cells did not affect engraftment, cell lineage differentiation or persistence of the gene modified stem cells. Moreover, co-administration with stem cells did not affect persistence of adoptively transferred T cells. These data demonstrate that 1) NY-ESO-1 TCR genetically modified stem cells engraft and differentiate into all hematopoietic cell lineage progeny, which persists at 3 months; 2) adoptively transferred NY-ESO-1 TCR T cells persist at 3 months; 3) co-administration of stem cells and T cells genetically modified to express an NY-ESO-1 TCR is safe and does not negatively impact stem cell engraftment, lineage differentiation and progeny persistence or T cell persistence. Citation Format: Cristina Puig-Saus, Giulia Parisi, Paige Krystofinski, Angel Garcia-Diaz, Salemiz Sandoval, James McCabe, Ruixue Zhang, Gardenia Cheung-Lau, Nhat Truong, Justin Saco, Sara Komenan, Agustin Vega-Crespo, Mignonette H Macabali, Begoña Comin-Anduix, Beata Berent-Maoz, Donald Kohn, Paula Kaplan-Lefko, Antoni Ribas. IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR [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 3765. doi:10.1158/1538-7445.AM2017-3765

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.