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Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in leukemia and lymphomas but limited responses in solid tumors. We conducted a phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.ICD9), demonstrating feasibility and safety of administering GD2 CAR-Ts in children and young adults with neuroblastoma and, for the first time, osteosarcoma. 15 patients aged 8-28 years were enrolled on four dose levels, of which 13 patients were infused. No dose-limiting toxicities were observed, and administration of up to 1x107 GD2-CAR-T/kg was feasible and safe for children and young adults with neuroblastoma and osteosarcoma. At Day 28 following GD2 CAR-T infusion, 23.1% (3/13) of evaluable patients had progressive disease and 76.9% (10/13) had stable disease (SD). 3/10 SD patients remained stable at 60 days post-infusion of GD2 CAR-T, but all patients eventually progressed. Since a major barrier to CAR-T efficacy is inadequate CAR-T expansion, we evaluated CAR-T levels and found that patients stratified into good and poor expander groups, observed across dose levels and associated with pro-inflammatory cytokine signatures in patients. To understand the immune cell contributors to CAR-T expansion, patient pre-treatment apheresis, CAR-T product, and post-infusion samples were evaluated by high-dimensional proteomic (CyTOF), transcriptomic (RNAseq), and epigenetic (ATACseq) analyses. In patient apheresis, good CAR-T expansion associated with more open chromatin and with both proteomic and transcriptomic enrichment of naïve T cells, while poor CAR-T expansion associated with increased levels of T effector memory (TEMRA) cells and enrichment of myeloid derived suppressor cell (MDSC) transcriptomic signatures. CAR-T products across patients, regardless of CAR-T expansion, demonstrated increased T cell activation proteomic signatures, with enhanced exhaustion transcriptomic signatures in poor expanders compared to good. The most robust cellular correlate to good CAR-T expansion was a population of CXCR3-expressing monocytes in pre-treatment apheresis. Interestingly, this CXCR3+ monocyte population reduced in post-infusion timepoints of good expanders, resembling levels found in poor expanders. Our findings were validated in TARGET-OS patient data in The Cancer Genome Atlas, where high CXCR3 expression was found to be associated with survival benefit in osteosarcoma patients. CXCR3 has been extensively studied on T cells, but its function on myeloid populations is yet to be fully explored. These results are the first to demonstrate that the peripheral immune environment prior to CAR-T administration may effectively predict and modulate CAR-T expansion in patients. Citation Format: Tara Murty, Sabina Kaczanowska, Ahmad Alimadadi, Cristina Contreras, Caroline Duault, Priyanka Balasubrahmanyan, Warren Reynolds, Norma Gutierrez, Reema Baskar, Catherine Wu, Franziska Michor, Jennifer Altreuter, Yang Liu, Aashna Jhaveri, Vandon Duong, Hima Anbunathan, Radim Moravec, Joyce Hong, Roshni Biswas, Stephen Van Nostrand, James Lindsay, Mina Pichavant, Elena Sotillo, Bita Sahaf, Sean Bendall, Holden Maecker, Steven Highfill, David Stroncek, Melinda Merchant, John Glod, Catherine Hedrick, Crystal Mackall, Sneha Ramakrishna, Rosandra Kaplan. Immune determinants of CAR-T expansion in solid tumor patients receiving GD2 CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2142.

Squamous cell lung cancer (SqCLC) is a type of non-small cell lung cancer strongly associated with cigarette smoking and with known targetable mutations, however some patients do not have matching targeted drugs. The S1400I Phase III randomized LungMap sub-study of nivo+ipi versus nivo accrued 275 (252 eligible) previously-treated patients with stage IV SqCLC and absence of matched mutations (NCT02154490).Here, to investigate the longitudinal (baseline, C2 week 3, C4 week 7, C5 week 9) serum protein changes associated with treatment or response, we performed Olink proximity extension assay in 160 patients (561 samples) using an immuno-oncology panel of 92 analytes. We utilized mixed linear and joint models to identify differentially expressed proteins between treatment arms (nivo vs. nivo+ipi), response and quantify the effect of other potential prognostic factors. This approach quantifies the effect of covariates such as smoking status, demographics, prior radiation therapy, and metastases. We jointly modeled expression and survival to identify changes in proteins over time. The thresholds for significance in differentially expression tests were a log fold change of at least 0.5 and a false discovery rate of under 0.05 (FDR as a multiple testing adjustment method). The joint models used thresholds of log fold change of more than 1 and hazard ratio of more than 1. Results revealed increases in 40 serum proteins after treatment with either nivo alone or combined with ipi, including CXCL9, CXCL10, CXCL11, CXCL13, IL6, IL8, IL10, IFNg, and soluble PD-L1 and PDCD1. nivo+ipi treatment showed greater increases in IL8 and CXCL13 post-treatment compared to nivo alone. In addition, circulating IL6, CXCL13, and MIC-A/B were higher in patients with stable or progressive disease compared to those with objective response after treatment. The joint model revealed a worse hazard ratio with increases in 10 soluble proteins, including IL6, IL8, and CXCL13. Finally, for patients with similar values of IL8, those treated by combination had a better survival outcome than those treated by nivo alone. Using a data-modeling approach, we identified significant longitudinal changes in 40 serum proteins out of 92 tested in patients with SqCLC treated with nivo or nivo+ipi. Increases in serum inflammatory markers IL6 and CXCL13 were associated with worse disease. Although overall survival was not statistically different between arms, our modeling approaches suggested that IL-8 monitoring may help identify patients benefiting more from the combination vs. nivo alone. Citation Format: Edgar Gonzalez-Kozlova, Hsin-Hui Huang, Mary Redman, Roy Herbst, Scott Gettinger, Luda Bazhenova, Hui Xie, Manishkumar Patel, Kai Nie, Jocelyn Harris, Kimberly Argueta, Karen Kelly, Ethan Cerami, James Lindsay, Joyce Yu, Roshni Biswas, Stephen Van Nostrand, Radim Moravec, Diane Marie Del Valle, Seunghee Kim-schulze, Sacha Gnjatic. Dynamic changes in circulating protein levels reveal an association between ipilimumab and nivolumab combination treatment (SWOG Lung-MAP S1400I trial) with outcomes in squamous cell lung cancer [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 5026.

Background: In the Phase III randomized trial (SWOG Lung-MAP S1400I non match trial), adding ipilimumab (I) to nivolumab (N) did not improve survival of patients with advanced, pretreated, immune checkpoint inhibitor naive lung squamous cell carcinoma (SCC). The I+N combination demonstrated superiority in patients with a tumor mutation burden (TMB) ≥10 mt/MB and PD-L1 expression Methods: RNA extracted from baseline FFPE samples from 66/252 patients enrolled in the S1400I trial was received from the SWOG bank with 28 samples considered inadequate due to low input RNA. 38 samples were run on the ncounter platform using the PanCancer Immune Profiling panel using the manufacturer’s instructions with the addition of Human Reference RNA control. Data were processed and normalized using nSolver. All samples passed the post run QC with no batch effect. Using a log-rank test on dichotomized normalized data, we performed time to event analysis to identify genes that correlated with overall survival (OS) and PFS (Progression Free Survival) for all 38 samples and for each arm (23 N and 15 I+N). We ran a Cox model using significant genes identified independently and in association with TMB≥10 and PD-L1 ≥5 thresholds identified in the clinical studies. TIMER and nSolver advanced analysis were used to infer immune cells that correlated to clinical outcomes. Results: We observed that BLNK, CD163, FCGR2A associated with increased OS (p Conclusion: Despite a small dataset, this analysis shows a potential advantage in PFS and OS with increased presence of immune cells including exhausted CD8+ T cells and genes associated with myeloid cells. Validation of these findings is warranted and may refine patient populations that would benefit from this combination strategy. Acknowledgments: Scientific and financial support: U10CA180888, U10CA180819, U24CA224285, U24CA224316, PACT, PPP and FNIH Citation Format: Dzifa Y. Duose, Jiexin Zhang, Mary Redman, Baili Zhang, Ethan Cerami, James Lindsay, Joyce Yu, Roshni Biswas, Stephen Van Nostrand, Radim Moravec, Rajyalakshmi Luthra, Gheath Al-Atrash, Karen Kelly, Roy Herbst, Ignacio Wistuba, Scott Gettinger, Lyudmila Bazhenova, J. Jack Lee, Jianjun Zhang, Cara Haymaker. Immune gene expression signatures associated clinical benefit from nivolumab and ipilimumab for previously treated patients with stage IV squamous cell lung cancer: An immune biomarker analysis of phase III SWOG LungMAP S1400I trial [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 1974.

Background: Lung squamous cell carcinoma (SCC) is the second most common type of non-small cell lung cancer. Immunotherapy is a promising treatment for SCC. However, only a small proportion of unselected SCC patients are able to achieve durable clinical benefit. We leveraged the precious specimens from S1400I trial and sought to explore whether the levels and spatial distributions of tumor-associated immune cells (TAICs) in screening samples are associated with PFS, and OS. Methods: We utilized paraffin tumor tissue provided by the SWOG bank from screening on LungMAP. 82/252 eligible SCC sample from patients treated with nivolumab plus ipilimumab (nivo+ipi; n = 35) or with nivolumab alone (nivo; n = 47) were studied. Image analysis tissue immunoprofiling was conducted using 9 markers in 2 multiplex immunofluorescence panels against cytokeratin, CD3, CD8, granzyme B, CD45RO, and FOXP3, PD1, PD-L1, and CD68. Densities of cells phenotypes (cells/mm2) were assessed and dichotomized as being high (> median) or low (≤ median) in tumor, stroma, and total compartments. Distances from malignant cells (MCs) to cell populations were measured and dichotomized as close from MCs (≤ median) and faraway from MCs (> median) and associated with clinical variables. Clinicopathologic features and data on PFS, and OS were retrieved from the CIDC portal as provided by the LungMap team. Non-parametric tests where utilized to assessment associations of cell phenotypes versus continue or categorical variables and log-rank test for survival analysis. Results: Univariate analysis showed that higher densities of memory T cells in total compartment (HR:0.63, CI:0.40-1.00, P=0.041), antigen-experienced T cells in the stroma (HR:0.60, CI:0.37-0.96, P=0.024) and total compartment (HR:0.60, CI:0.38-0.95, P=0.023) and activated cytotoxic T cells in the tumor compartment (HR:0.55, CI:0.35-0.87, P=0.011) were associated with significative better PFS. In the nivo+ipi arm, higher ratios of cytotoxic T cells to regulatory T cells in the stroma were associated with better PFS. The spatial analysis of these TAICs showed that the presence of activated cytotoxic T cells close to the MCs (median, ≤19.27 µm) was associated with better PFS (P=0.037). Conclusions: Our findings suggest that patients who have a detectable immunosuppressive tumor axis (PD-L1/PD1) with adequate activated cytotoxic T cells close to the tumor cells are the ideal patients for immune therapy to be targetable with these types of treatments. Acknowledgments: Scientific and financial support for: U10CA180888, U10CA180819U24CA224285, U24CA224316, PACT, PPP and FNIH. Citation Format: Edwin Roger Parra, Jiexin Zhang, Mary Redman, Rossana Lazcano, Piubelli Mario, Caddie Laberiano Fernandez, Renganayaki K. Krishna, Shanyu Zhang, Hong Chen, Ganiraju Manyam, Radim Moravec, Ethan Cerami, James Lindsay, Joyce Yu, Roshni Biswas, Stephen Van Nostrand, Dzifa Y. Duose, Jianjun Zhang, Roy Herbst, Gheath Al-Atrash, Kasthuri Kannan, Ignacio I. Wistuba, Scott Gettinger, Lyudmila Bazhenova, Jack Lee, Jianhua Zhang, Cara Haymaker. Infiltration and spatial distribution of immune cells are associated clinical benefit from Nivolumab and Ipilimumab for previously treated patients with stage IV squamous cell lung cancer: an immune biomarker analysis of Phase III SWOG LungMAP S1400I trial [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 2580.

9044 Background: While Immune checkpoint blockade (ICB) is standard treatment for lung cancer there are limited biomarkers that predict benefit, pharmacokinetic on-treatment activity or explain progression. S1400I was a randomized Phase III trial of nivolumab(N)+ipilimumab(I) versus N (NCT02154490, PMID 34264316) for ICB naïve, previously treated stage IV or recurrent squamous cell lung cancer. We performed circulating serum protein analysis of serial blood specimens from patients enrolled in S1400I to evaluate if serum proteins levels changed over time, changes differed by treatment arm, and if they were associated with overall survival. Methods: 561 serial blood specimens (baseline, weeks (wk) 3, 7, 9, and progression [PD]) from 160 of 252 eligible patients enrolled to S1400I were analyzed for 92 immuno-oncology analytes with the Olink proximity extension assay. Protein levels were normalized with use of internal controls and quantified as log2 protein expression (denoted as NPX). Linear mixed models evaluated change in expression from baseline at each time point (wks 3,7,9 and PD), and NPX differences at baseline, wk3, and PD depending on best objective response. A Cox model was used to evaluate the association between baseline NPX and survival. Overall survival and longitudinal cytokine expression were jointly modeled using a linear mixed model to estimate dynamic biomarker changes in NPX and a Cox model for survival. The joint models for time-varying NPX values included a random intercept and modeled time using a natural spline with three knots. Significance was defined as P < 0.05. Results: Serum proteins PCDC1, CXCL9, and CXCL10 were increased from baseline at wks 3,7,9 and at PD. CCL19 was increased at wks 3 and 7 but not at wk 9 and PD. IL10 and IFNγ were increased at wk 3 but subsequently returned to baseline. Change in CXCL13 from baseline to PD was larger for N+I versus N. Baseline CCL23, CSF-1, IL6, and MUC-16 were associated with shorter survival (HR > 1). Joint modeling of survival with cytokines showed an increased risk of death (HR > 1) with higher longitudinal serum levels of CXCL13, MMP12, CSF-1, and IL8. Patients achieving objective response had higher IL4 and LAMP3 and lower IL6 and IL8 at baseline and wk 3 compared to non-responders. Conclusions: Measurements of blood circulating soluble proteins represent easily accessible biomarkers that may be useful as indicators of outcome, and that will need to be prospectively confirmed. Clinical trial information: NCT02154490.