Biomarker correlates with response to NY-ESO-1 TCR T cells in patients with synovial sarcoma

Abstract

Autologous T cells transduced to express a high affinity T-cell receptor specific to NY-ESO-1 (letetresgene autoleucel, lete-cel) show promise in the treatment of metastatic synovial sarcoma, with 50% overall response rate. The efficacy of lete-cel treatment in 45 synovial sarcoma patients (NCT01343043) has been previously reported, however, biomarkers predictive of response and resistance remain to be better defined. This post-hoc analysis identifies associations of response to lete-cel with lymphodepleting chemotherapy regimen (LDR), product attributes, cell expansion, cytokines, and tumor gene expression. Responders have higher IL-15 levels pre-infusion (p = 0.011) and receive a higher number of transduced effector memory (CD45RA- CCR7-) CD8 + cells per kg (p = 0.039). Post-infusion, responders have increased IFNγ, IL-6, and peak cell expansion (p < 0.01, p < 0.01, and p = 0.016, respectively). Analysis of tumor samples post-treatment illustrates lete-cel infiltration and a decrease in expression of macrophage genes, suggesting remodeling of the tumor microenvironment. Here we report potential predictive and pharmacodynamic markers of lete-cel response that may inform LDR, cell dose, and strategies to enhance anticancer efficacy.

Conflict of interest statement

A.G. is an employee of and holds stocks/shares in GSK and holds stocks/shares in Amgen. St.Z., E.A.H., J.K., M.N., T.F., M.W., D.C.T. and I.E. are employees of and hold stocks/shares in GSK. Y.F.C. is an employee of GSK. Sh.Z. is a former employee of and holds stocks/shares in GS.K. A.N.H. is a former employee of and holds stocks/shares in GSK and receives patents and royalties from Atara Biotherapeutics. J.G. is protocol investigator for SARC and PBTC sponsored studies. R.N.K. and D.M.A. have no disclosures. S.P.D.A. participated on advisory boards for GSK, Nektar, Adaptimmune, and Merck; reports consulting fees from EMD Serono, Amgen, Nektar, Immune design, GSK, Incyte, Merck, Adaptimmune, and Immunocore, and received support for travel expense by Adaptimmune, EMD Serono, and Nektar. WAC reports grant and honoraria received from GSK. M.D. has received consulting fees from Adaptimmune and honoraria from Deciphera. GDD reports leadership roles with Blueprint Medicines, Merrimack Pharmaceuticals (ended Oct 2019), and Translate Bio (ended Sept 2021); stocks/options/shares in Blueprint Medicines, G1 Therapeutics, Caris Life Sciences, Erasca Pharmaceuticals, RELAY Therapeutics, Bessor Pharmaceuticals, CellCarta, IKENA Oncology, Kojin Therapeutics, and IDRX; paid consulting fees from Bayer, Pfizer, Novartis, Roche/Genentech, GSK, PharmaMar, Daiichi Sankyo, GSK, EMD-Serono, MEDSCAPE, Mirati, WCG/Arsenal Capital, MJ Hennessey/OncLive, C4 Therapeutics, Synlogic, McCann Health, G1 Therapeutics, Caris Life Sciences, RELAY Therapeutics, CellCarta, IKENA Oncology, Kojin Therapeutics and IDRX; royalties, patents or licenses from Novartis via Dana Farber for “use patent” of imatinib in GIST; and non-financial interests in AACR Science Policy and Government Affairs Committee and Alexandria Real Estate Equities summit conference series. BAVT received consulting fees from ADRx, Ayala Pharmaceuticals, Cytokinetics Inc, and Bayer; has honoraria from Adaptimmune Ltd, GSK, Bionest Partiners, and Intellisphere LLC; has received payment for expert testimony from Hinshaw & Culbertson LLP, Rodney Law, CRICO Risk Management Foundation, and Tracey & Fox Law Firm; has received research funding from GSK, Merck, Pfizer, and Tracon; has received travel, accommodations, and expenses from GSK and Adaptimmune; has participated on advisory board meeting with Adaptimmune Ltd, Apexigen Inc, Boehringer Ingelheim, Daiichi Sankyo, Deciphera Pharmaceuticals Inc, Epizyme, GSK, Novartis, PTC Therapeutics, and Lilly, and is a board member for Polaris; and holds royalties or licenses for work performed with Accuronix Therapeutics. S.A.G. has received grants for study support from Novartis, Jazz Pharmaceuticals, Kite, Vertex, and Servier; has received consulting fees from Novartis, Roche, GSK, Vertex/CRISPR, CBMG, and Janssen/JnJ; has received payment for expert testimony from Irwin Mitchell and Jones Day; has patents managed according to the University of Pennsylvania patent policy; has participated on advisory boards for Novartis, Jazz Pharmaceuticals, Adaptimmune, Cellectis, Juno, Vertex, Allogene, and Cabaletta. G.D.F. is a former employee of GSK and has stock options in GSK and PACT Pharma, and holds stocks in Bluebird Bio, Agios, and BioMarin.

© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.

Figures

Fig. 1. Standard LDR of fludarabine and cyclophosphamide forms supportive environment for T cells and highlights impact of cell dose.

a, b Endogenous lymphocyte (a) and monocyte (b) cell counts across cohorts on day of infusion (n = 43 biologically independent samples). All responders except two in Cohorts 3 and 4 had <10 lymphocytes/μL prior to T-cell infusion. c,d, IL-15 levels prior to T-cell infusion across cohorts (c) and association with response (d) (n = 34 biologically independent samples). Values are log-transformed for consistency with other cytokine analyses. ANOVA and t-test were performed in c and d to be consistent with remaining cytokine analyses; Kruskal–Wallis p-value = 0.005 for (c) and Wilcoxon p-value = 0.050 for d. e Impact of transduced cell dose/kg on reduction in tumor size (n = 22 biologically independent samples) for patients in cohorts 1 and 2. Box plots depict median as horizontal line within box, with box bounds as the first and third quartiles. Dots represent individual data points. Lower whisker is the minimum value of the data within 1.5 times the interquartile range below the 25th percentile. Upper whisker is the maximum value of the data within 1.5 times the interquartile range above the 75th percentile. Two-sided p-values were calculated via bootstrapped median regression (10,000 bootstraps) to adjust for cohort or responder status (a, b), ANOVA (c), t-test (d), and standard test for Spearman correlation (e). CR complete response, IL interleukin, LDR lymphodepleting chemotherapy regimen, PD progressive disease, PR partial response, SD stable disease, SLD sum of longest diameter.

Fig. 2. Lete-cel expansion post-infusion associated with response, cell dose/kg, and LDR.

a Association of peak cell expansion (Cmax) with response (n = 43 biologically independent samples). b,c, Relationship of Cmax with weight-normalized cell dose with line of best fit in blue (b) and LDR (c) (n = 43 biologically independent samples). d Persistence of lete-cel at Week 4 post-infusion in all 4 cohorts, stratified by response (n = 35 biologically independent samples). e Relationship between Cmax and PFS (n = 43 biologically independent samples). PFS was defined as the time from T-cell infusion to the earliest documentation of disease progression or death from any cause or surgical resection or start of prohibited medication. Box plots depict median as horizontal line within box, with box bounds as the first and third quartiles. Dots represent individual data points. Lower whisker is the minimum value of the data within 1.5 times the interquartile range below the 25th percentile. Upper whisker is the maximum value of the data within 1.5 times the interquartile range above the 75th percentile. Two-sided p-values were calculated via Wilcoxon test (a, c, d) or linear (b) regression, and Cox proportional hazards model (e). CR complete response, Cy cyclophosphamide, LDR lymphodepleting chemotherapy regimen, PD progressive disease, PFS progression-free survival PR partial response, SD stable disease.

Fig. 3. T-cell product enriched with activated, effector memory CD8 cells is associated with response.

a Number of CD8 + Pentamer+ cells/kg infused per memory phenotype in non-responders vs responders (n = 36 biologically independent samples). b, c Association of number of infused cells/kg per memory phenotype with peak cell expansion (b) and peak IFNγ post-infusion (c) (n = 34 and 31 biologically independent samples for parts b and c respectively). Associations with EM cells are statistically significant with outlier at lowest infused cell counts removed—EM with Cmax (R = 0.42, p = 0.016) and EM with IFNγ (R = 0.41, p = 0.027). d Association of EM phenotype between apheresis and product (n = 34 biologically independent samples). Box plots depict median as horizontal line within box, with box bounds as the first and third quartiles. Dots represent individual data points. Lower whisker is the minimum value of the data within 1.5 times the interquartile range below the 25th percentile. Upper whisker is the maximum value of the data within 1.5 times the interquartile range above the 75th percentile. Nominal two-sided p-values based on the Wilcoxon rank sum test or log-rank test for PFS are presented and correlations are based on Spearman method. Line of best fit shown in blue for significant associations and gray area represents 95% confidence interval around the regression line. CM central memory (CD45RA-CCR7+), CR complete response, EM effector memory (CD45RA-CCR7-), Naïve (CD45RA + CCR7+), PFS progression-free survival, PD progressive disease, PR partial response, SD stable disease, TEMRA T effector memory RA (CD45RA + CCR7−), TSCM T stem cell memory (CD45RA + CCR7 + CD45RO-CD95 + CD127+).

Fig. 4. Characterization of transduced T cells post infusion.

a Association of % and number of CD 8+  Pentamer+EM cells/kg with PFS.PFS was defined as the time from T-cell infusion to the earliest documentation of disease progression or death from any cause or surgical resection or start of prohibited medication (n = 36 biologically independent samples). Blue and red shaded areas represent 95% confidence interval for the survival curve. b Levels of CD8 + Pentamer+ cells at Week 4. c Levels and composition of memory phenotypes of CD8 + Pentamer+ cells post-infusion. Product analyses consist of 36 patients. Number of patients for post-infusion analyses are listed on figures. Box plots depict median as horizontal line within box, with box bounds as the first and third quartiles. Dots represent individual data points. Lower whisker is the minimum value of the data within 1.5 times the interquartile range below the 25th percentile. Upper whisker is the maximum value of the data within 1.5 times the interquartile range above the 75th percentile. Nominal two-sided p-values based on the Wilcoxon rank sum test or log-rank test for PFS are presented and correlations are based on Spearman method. CM central memory (CD45RA-CCR7+), CR complete response, EM effector memory (CD45RA-CCR7-), Naïve (CD45RA + CCR7+), PFS progression-free survival, PD progressive disease, PR partial response, SD stable disease, TEMRA T effector memory RA (CD45RA + CCR7-), TSCM T stem cell memory (CD45RA + CCR7 + CD45RO-CD95 + CD127+).

Fig. 5. Increase in proinflammatory cytokines in responders post lete-cel infusion and correlation to peak cell expansion.

a Heatmap of Responder vs Nonresponder ratios in cytokine levels over time obtained from linear mixed effects (LME) model. T-cell infusion (TCI) occurred on Day 0. Nominal p-values from linear effects models were calculated via Wald test and t-distribution and are shown as one dot for 0.01 < p ≤ 0.05 and two dots for p ≤ 0.01. b Time courses of IFNγ, IL-6, IL-17A, and GM-CSF, cytokines differentially upregulated by responders. Geometric means and 95% confidence intervals from LME model (accounting for left-censoring where appropriate) are plotted. Dashed, horizontal lines represented lower limit of quantification. c Time course of soluble IL-2Rα levels showing increase in responders and correlation to peak cell expansion. Line of best fit shown in black and gray area represents 95% confidence bands, both from standard least-squares regression. Sample size varied across timepoints and cytokines, with an overall median of 32 patients (range, 15–38 depending on cytokine/timepoint). For c, Spearman correlation and corresponding two-sided p-value are presented. DY day, GM geometric mean, GM-CSF granulocyte-macrophage colony-stimulating factor, IFN interferon, IL interleukin, WK week.

Fig. 6. Tumor remodeling post lete-cel infusion.

a Macrophage markers CD163, CD68, and CD84 pre-infusion (n = 10 biologically independent samples) and at progression (n = 5 biologically independent samples). b CD163 expression in brown by IHC in patient (Subject ID 36) in baseline and at progression biopsy (Day 125) from the same lung lesion (representative region of tissue from an IHC run). c, d Average expression and heatmap of IFN downstream genes pre-infusion (n = 10 biologically independent samples). e Association between CD163 (left) and CD68 (right) and IFN downstream genes. Line of best fit shown in blue and gray area represents 95% confidence bands. Pre-infusion samples from seven archival screening samples (~1 year pre-infusion) and three fresh baseline samples (pre-lymphodepletion). At progression, samples consist of five samples. Box plots depict median as horizontal line within box, with box bounds as the first and third quartiles. Dots represent individual data points. Lower whisker is the minimum value of the data within 1.5 times the interquartile range below the 25th percentile. Upper whisker is the maximum value of the data within 1.5 times the interquartile range above the 75th percentile. Heatmap show z-scores per gene. Nominal two-sided p-values obtained from linear mixed effects models (a), limma models (c), and standard test for Spearman correlation coefficient (e). IFN interferon, IHC immunohistochemistry, NR non-responder, R responder.

Fig. 7. Decreased expression of HLA-A and antigen-presenting genes at progression.

a Change in gene expression of CTAG1 (NY-ESO-1) and HLA-A between pre-infusion (n = 10 biologically independent samples) and at progression (n = 5 biologically independent samples). b Average expression and heatmap of antigen presentation genes at pre-infusion (n = 10 biologically independent samples) and progression (n = 5 biologically independent samples). The following genes had background expression across all samples: KLRC2, KIR3DL2, KIR3DL1, KIR2DS1, and KR2DL1. c Persistence of lete-cel in blood of patient 32. d Characterization of patient 32 biopsy taken 919 days post-infusion. RNAScope results show CD3 cells in blue and lete-cel in red (left) (representative region of tissue from an IHC run). PDL1 and LAG3 staining in brown by IHC (middle and right images). Tumor samples were primarily from lung metastases. Pre-infusion samples from seven archival screening samples (~1 year pre-infusion) and three fresh baseline samples (pre-lymphodepletion). At progression, samples consist of five samples. Box plots depict median as horizontal line within box, with box bounds as the first and third quartiles. Dots represent individual data points. Lower whisker is the minimum value of the data within 1.5 times the interquartile range below the 25th percentile. Upper whisker is the maximum value of the data within 1.5 times the interquartile range above the 75th percentile. Heatmaps show z-scores per gene. Nominal two-sided p-values obtained from linear mixed effects models (a, b). HLA human leukocyte antigen, IHC immunohistochemistry, NR non-responder, R responder.