Generation and Characterization of Ex Vivo Expanded Tumor-infiltrating Lymphocytes From Renal Cell Carcinoma Tumors for Adoptive Cell Therapy.

Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance antitumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma.

Renal cell carcinoma with sarcomatoid differentiation (sRCC) is associated with poor survival and a heightened response to immune checkpoint inhibitors (ICIs). Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsies due to spatial heterogeneity. Herein, we characterized the epigenomic landscape of sRCC by profiling 107 epigenomic libraries from tissue and plasma samples from 50 patients with RCC and healthy volunteers. By profiling histone modifications and DNA methylation, we identified highly recurrent epigenomic reprogramming enriched in sRCC. Furthermore, CRISPRa experiments implicated the transcription factor FOSL1 in activating sRCC-associated gene regulatory programs, and FOSL1 expression was associated with the response to ICIs in RCC in two randomized clinical trials. Finally, we established a blood-based diagnostic approach using detectable sRCC epigenomic signatures in patient plasma, providing a framework for discovering epigenomic correlates of tumor histology via liquid biopsy.

Perioperative nivolumab versus observation in patients with renal cell carcinoma undergoing nephrectomy (PROSPER ECOG-ACRIN EA8143): an open-label, randomised, phase 3 study.

BACKGROUND: The standard of care for patients with intermediate-to-high risk renal cell carcinoma is partial or radical nephrectomy followed by surveillance. We aimed to investigate use of nivolumab before nephrectomy followed by adjuvant nivolumab in patients with high-risk renal cell carcinoma to determine recurrence-free survival compared with surgery only. METHODS: In this open-label, randomised, phase 3 trial (PROSPER EA8143), patients were recruited from 183 community and academic sites across the USA and Canada. Eligible patients were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-1, with previously untreated clinical stage T2 or greater or Tany N+ renal cell carcinoma of clear cell or non-clear cell histology planned for partial or radical nephrectomy. Selected patients with oligometastatic disease, who were disease free at other disease sites within 12 weeks of surgery, were eligible for inclusion. We randomly assigned (1:1) patients using permuted blocks (block size of 4) within stratum (clinical TNM stage) to either nivolumab plus surgery, or surgery only followed by surveillance. In the nivolumab group, nivolumab 480 mg was administered before surgery, followed by nine adjuvant doses. The primary endpoint was investigator-reviewed recurrence-free survival in patients with renal cell carcinoma assessed in all randomly assigned patients regardless of histology. Safety was assessed in all randomly assigned patients who started the assigned protocol treatment. This trial is registered with ClinicalTrials.gov, NCT03055013, and is closed to accrual. FINDINGS: Between Feb 2, 2017, and June 2, 2021, 819 patients were randomly assigned to nivolumab plus surgery (404 [49%]) or surgery only (415 [51%]). 366 (91%) of 404 patients assigned to nivolumab plus surgery and 387 (93%) of 415 patients assigned to surgery only group started treatment. Median age was 61 years (IQR 53-69), 248 (30%) of 819 patients were female, 571 (70%) were male, 672 (88%) were White, and 77 (10%) were Hispanic or Latino. The Data and Safety Monitoring Committee stopped the trial at a planned interim analysis (March 25, 2022) because of futility. Median follow-up was 30·4 months (IQR 21·5-42·4) in the nivolumab group and 30·1 months (21·9-41·8) in the surgery only group. 381 (94%) of 404 patients in the nivolumab plus surgery group and 399 (96%) of 415 in the surgery only group had renal cell carcinoma and were included in the recurrence-free survival analysis. As of data cutoff (May 24, 2023), recurrence-free survival was not significantly different between nivolumab (125 [33%] of 381 had recurrence-free survival events) versus surgery only (133 [33%] of 399; hazard ratio 0·94 [95% CI 0·74-1·21]; one-sided p=0·32). The most common treatment-related grade 3-4 adverse events were elevated lipase (17 [5%] of 366 patients in the nivolumab plus surgery group vs none in the surgery only group), anaemia (seven [2%] vs nine [2%]), increased alanine aminotransferase (ten [3%] vs one [<1%]), abdominal pain (four [1%] vs six [2%]), and increased serum amylase (nine [2%] vs none). 177 (48%) patients in the nivolumab plus surgery group and 93 (24%) in the surgery only group had grade 3-5 adverse events due to any cause, the most common of which were anaemia (23 [6%] vs 19 [5%]), hypertension (27 [7%] vs nine [2%]), and elevated lipase (18 [5%] vs six [2%]). 48 (12%) of 404 patients in the nivolumab group and 40 (10%) of 415 in the surgery only group died, of which eight (2%) and three (1%), respectively, were determined to be treatment-related. INTERPRETATION: Perioperative nivolumab before nephrectomy followed by adjuvant nivolumab did not improve recurrence-free survival versus surgery only followed by surveillance in patients with high-risk renal cell carcinoma. FUNDING: US National Institutes of Health National Cancer Institute and Bristol Myers Squibb.

Impact of renal cell carcinoma molecular subtypes on immunotherapy and targeted therapy outcomes.

Saliby et al. show that a machine learning approach can accurately classify clear cell renal cell carcinoma (RCC) into distinct molecular subtypes using transcriptomic data. When applied to tumors biospecimens from the JAVELIN Renal 101 (JR101) trial, a benefit is observed with immune checkpoint inhibitor (ICI)-based therapy across all molecular subtypes.

Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.