Introduction: Chimeric antigen receptor (CAR) T cells are capable to be activated directly when in contact with a specific tumor antigen, becoming a new potent strategy against cancer. The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 CAR T cells in doses equivalent to ≅10 8 CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses for ccRCC in adjuvant settings and metastatic scenarios, becoming an important pillar treatment.Objectives: This project tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR T cells releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Methodology:Lentiviruses containing the different CAR constructions to be tested were produced, concentrated, and the transduction efficiency was determined by flow cytometry and IgG secretion. The cytotoxic effects of anti-CAIX CAR T were analyzed by flow cytometry and lactate dehydrogenase activity. The secretion of IL-2 and IFNγ was determined by ELISA. The exhaustion status was determined by flow cytometry. Alanine (ALT) and aspartate (AST) transaminases activity was determined by spectrophotometry.

Renal cell carcinoma (RCC) represents up to 90% of all kidney tumors, with 30% of patients presenting metastasis at diagnosis while 20-50% of those treated for curable localized disease experience recurrence. A better understanding of RCC biology is necessary to define the most efficient and personalized treatment or develop better antineoplastic drugs. Patient-derived xenografts (PDX) have emerged as one of the most promising approaches for that. This tool was generated by using surgical specimens of 87 patients implanted in immunodeficient NOD/SCID/gamma (NSG) mice, 17 of those subcutaneously and 70 at the renal subcapsular space. A total of 19 PDX developed only after orthotopic implantation and included 15 cases of clear cell RCC subtype, 3 cases of papillary subtype, and one unclassifiable tumor. One case of a PDX of a clear cell RCC recapitulated the phenotype of vena cava tumor thrombus extension that had been diagnosed in the source patient. The overall take rate was 27%, and the time to observed tumor growth varied from 5 to 13 months. An association between tumor growth and take rate was suggested by increasing take rates of 18%, 36%, and 100% among pT1 (pT1a + pT1b), pT3a, and pT3b stage tumors, respectively. PDX take rate was unrelated to tumor size (p =0.071). Patients whose tumor fragments engrafted experienced worse overall survival (OS) than those whose tumor fragments did not engraft (p = 0.003). Data also suggested a similar trend of association with metastasis-free survival (MFS) (p = 0.063). The median MFS for patients with successful PDX was 21.6 months (95% CI: 14.9, 28.3;), compared to a median of 34.4 months (95% CI: 30.4, 38.5 p = 0.003) among PDX engraftment-negative patients. PDX characterization by immunohistochemistry and targeted sequencing of the 21 most frequently mutated genes in the kidney indicated that all PDXs preserved RCC identity and major molecular alterations. From 19 PDX, the sequences of 13 patients’ tumor samples were compared with their respective PDX. In most patients (92% - 11/12), at least one mutation in patients’ tumor was identified in the PDX and in one case, no alterations were mapped. The most frequently mutated genes were VHL (50% - 6/12) and PBRM1 (41.7% - 5/12), followed by SETD2 (25% - 3/12), BAP1 and KDM5C (both 16.7% - 2/12), and ARID1A (8.3% - 1/12), indicating that all PDXs preserved RCC identity and major molecular alterations. These findings suggest that tumor engraftment capacity can identify patients at increased risk of relapse or death. Furthermore, pT1 stage tumors with the ability to engraft could also facilitate the identification of risk factors related to the rare pT1 cases with disease progression. These results suggest that the orthotopic xenograft model of RCC represents a suitable tool to study RCC biology, identify biomarkers, and test therapeutic candidates. Citation Format: Vilma Regina Martins, Adriano Beserra, Ethiene C. Estevan, Stephania Bezerra, Giovana T. Torrezan, Amanda Ikegani, Dellê Humberto, Isabela V. Cunha, Isabella T. Meira, Dirce M. Carraro, Primo N. Lara, Stenio Zequi, Tiago G. Santos. Patient-derived renal cell carcinoma xenografts engraftment identifies patients at risk of disease relapse, progression and death [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 3097.

BACKGROUND: Patient-derived xenografts (PDX) have emerged as one of the most promising model systems to study cancer biology and to develop new antineoplastic drugs. Renal cell carcinoma (RCC) represents up to 90% of all kidney tumors, exhibits aggressive behavior, and has a propensity for metastasis. At diagnosis, 30% of patients with RCC have metastases, while up to 50% of those with localized disease treated with curative protocols experience recurrence. OBJECTIVE: This study aimed to establish an RCC PDX platform to identify novel clinical and molecular biomarkers of recurrence risk in order to facilitate precision medicine. METHODS: Tumor samples were obtained from surgical specimens of 87 RCC patients; fragments were implanted in immunodeficient NOD/SCID/gamma (NSG) mice. Seventeen fragments were implanted subcutaneously in an initial group while a second group of 70 samples were implanted orthotopically in the subcapsular space. RESULTS: A total of 19 PDX developed only after orthotopic implantation, and included 15 cases of clear cell RCC subtype, 3 cases of papillary subtype, and one unclassifiable tumor. One PDX of clear cell RCC recapitulated the phenotype of vena caval tumor thrombus extension that had been diagnosed in the source patient. PDX characterization by immunohistochemistry and targeted sequencing indicated that all PDXs preserved RCC identity and major molecular alterations. Moreover, the capacity of tumor engraftment was a strong prognostic indicator for patients with locally advanced disease. CONCLUSION: Taken together, these results suggest that the orthotopic xenograft model of RCC represents a suitable tool to study RCC biology, identify biomarkers, and to test therapeutic candidates.