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.

Abstract On-target off-tumor toxicity (OTOT) is one of the major hurdles preventing tumor-associated antigen (TAA) targeted therapy from effectively treating patients with solid tumors due to the shared presence of the same TAA on normal tissues. We have previously demonstrated high expression of carbonic anhydrase IX (CAIX) in ccRCC patient samples and low-density expression on healthy bile duct epithelium. In 2006, a clinical trial was stopped due to OTOT bile duct toxicity observed in patients infused with anti-CAIX CAR T cell therapy. To address the issue of OTOT in the bile duct, we performed affinity/avidity fine-tuning of the anti-CAIX CAR G36 to specifically target ccRCC tumor cells with high CAIX expression while sparing cholangiocytes with low CAIX expression. We generated a complementarity-determining region (CDR) single variant mutagenesis library of G36 and displayed the library on yeast for screening. Using flow cytometry, we were able to identify six populations based on antigen-binding affinity: high, med-high, med, med-low, low, and loss of binding. These libraries were sequenced via PacBio, a next generation sequencing (NGS) platform that excels in long-read sequencing, providing a powerful tool to detect single mutations in the scFv. The results were analyzed to identify enriched mutations compared to the wild-type G36, and inspected using in silico modeling and docking. Furthermore, enriched mutants from med-low and low populations were tested in CAR-T format for their cytotoxic capacity on tumor cells and bile duct cholangiocytes, as well as their avidity. We demonstrate that mutations in CDR2 and CDR3 of the heavy chain successfully mitigate OTOT toxicity on cholangiocytes with physiological levels of CAIX expression while maintaining tumor-killing capacity, shedding light on CDR mutagenesis as a useful tool to mitigate OTOT toxicity. Citation Format: Stephen Hsien-Chi Yuan, Yufei Wang, Rabia Abbas, Peterson Mathenge, Charles Chien, Christian D. Coherd, Branson Bajoua, Matthew R. Chang, Nithyassree Murugan, Gabriella M. Kastrunes, Guilin Wang, Song-My Hoang, Wayne A. Marasco. Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4004.

Treatment for renal cell carcinoma (RCC) has improved dramatically over the last decade, shifting from high-dose cytokine therapy in combination with surgical resection of tumors to targeted therapy, immunotherapy, and combination therapies. However, curative treatment, particularly for advanced-stage disease, remains rare. Cell therapy as a “living drug” has achieved hematological malignancy cures with a high response rate, and significant research efforts have been made to facilitate its translation to solid tumors. Herein, we overview the cellular therapies for RCC focusing on allogeneic hematopoietic stem cell transplantation, T cell receptor gene-modified T cells, chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) cells, lymphokine-activated killer (LAK) cells, γδ T cells, and dendritic cell vaccination. We have also included perspectives for using other recent approaches, such as CAR macrophages, dendritic cell-cytokine induced killer cells and regulatory CAR-T cells to shed light on preclinical development of cell therapy and advancing cell therapy into clinic to achieve cures for RCC.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR cells targeting carbonic anhydrase IX (CAIX) to secrete anti -PD -L1 monoclonal antibody (mAb), termed mune -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 man leukocytes derived from fetal CD34* hematopoietic stem cells (HSCs) and bearing human ccRCC skrc59 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.

Background Renal cell carcinoma (RCC) represents approximately 3% of adult cancers and about 70–80% of RCC cases have clear cell histology (ccRCC), with 30% of patients developing metastatic ccRCC. Despite a significant improvement in advanced ccRCC therapy, a curative treatment remains rare. Chimeric Antigen Receptor (CAR) T cell therapy as a 'living drug' has achieved hematological malignancy cures with a high response rate, and significant research efforts have been made to facilitate its translation to solid tumor treatment. Methods Here, we constructed a series of anti-carbonic anhydrase IX (CAIX) CARs with various affinities and assessed the avidity and cytotoxicity of those CAR-T cells using CAIX-high skrc-59 cells and CAIX-low MMNK-1 cholangiocytes. A tetracycline (Tet)-On inducible CAIX expressing system was established, providing different CAIX levels on the cell surface covering the range from the density on tumor cells to the one on cholangiocytes quantified using direct stochastic optical reconstruction microscopy (dSTORM). To evaluate the therapeutic effect of CAR-T on patient samples, we generated both advanced RCC patient derived organotypic spheroids (PDOTS) ex vivo cultures which recapitulates ccRCC patient tumor microenvironment (TME), and tested CAR-T cell migration and cytokine release using these miniature tumors. Results We identified a low affinity, high avidity anti-CAIX CAR G9, which only kills CAIX high tumor cells but not CAIX-low normal tissues in vitro. G9 demonstrated a CAIX density dependent response on Tet-On inducible CAIX expressing cell lines, with a wider therapeutic window compared to G250 that caused severe adverse events in the first anti-CAIX CAR-T clinal trial.1–3. G9 exhibited superior efficacy ex vivo on PDOTS 3D cultures derived from advanced ccRCC, as well as mitigated toxicity on cholangiocyte spheroids. In an orthotopic RCC mouse model, G9 showed enhanced tumor control compared to G250. Conclusions In summary, affinity/avidity fine-tuned CAR-T cell therapy holds the promise to achieve cures of advanced RCC by maintaining killing on tumor cells and mitigating toxicity on normal tissues. References Lamers CH, Klaver Y, Gratama JW, Sleijfer S & Debets R. Treatment of metastatic renal cell carcinoma (mRCC) with CAIX CAR-engineered T-cells-a completed study overview. Biochem Soc Trans. 2016 Jun 15; 44: 951–959, doi:10.1042/bst20160037 Lamers CH, Sleijfer S, Vulto AG, Kruit WH, Kliffen M, Debets R, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol. 2006 May 1; 24: e20–22, doi:10.1200/jco.2006.05.9964 Lamers CH, Willemsen R, van Elzakker P, van Steenbergen-Langeveld S, Broertjes M, Oosterwijk-Wakka J, et al. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells. Blood. 2011 Jan 6; 117: 72–82, doi:10.1182/blood-2010-07-294520

Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug” that has proven to be a powerful immunotherapy for hematologic malignancies. To date, there are six CAR-T products approved by the FDA for hematologic malignancies, four targeting CD19, and two targeting B-cell maturation antigen (BCMA). However, this success has not yet been transferred to solid tumors. A major hurdle is the on-target off-tumor toxicities due to the shared expression of target antigen on normal tissues. Carbonic anhydrase IX (CAIX) is highly expressed in clear cell renal cell carcinoma (ccRCC); however, it is also expressed on bile duct at a lower physiological level leading to off-tumor toxicity of CAIX targeted therapies. The first anti-CAIX CAR-T studies, using the 1st generation G250 CAR-T cells plus IL-2 to treat patients with metastatic ccRCC, caused severe liver enzyme abnormalities in the treated patients after CAR-T cell infusions. To understand CAIX expression on tumor and normal tissues, we quantified CAIX expression on ccRCC patient samples and healthy bile duct tissues using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution. Tet-On inducible CAIX expressing cell lines were established to mimic various CAIX densities on normal tissue and tumor samples. Using biolayer interferometry (BLI) and avidity analyzer, we identified a low-affinity, high-avidity anti-CAIX CAR G9. G9 CAR-T cells only kill CAIX high ccRCC tumor cells but not CAIX low normal cholangiocytes, and exhibited a CAIX density dependent response to Tet-On inducible CAIX expressing cell lines. Compared to high-affinity G250 CAR-T cells, G9 showed a better safety profile and a wider therapeutic window. G9 demonstrated a superior ex vivo efficacy on ccRCC patient derived organotypic tumor spheroids (PDOTS) 3D cultures which recapitulate ccRCC patient tumor microenvironment (TME), as well as low toxicity on cholangiocyte derived organotypic spheroids (CDOS). In summary, affinity/avidity fine-tuned CAIX targeted CAR-T cell therapy holds promise to achieve cures of ccRCC by efficaciously killing tumor cells and mitigating on-target off-tumor toxicity on normal tissues. Citation Format: Yufei Wang, Alicia Buck, Gabriella Kastrunes, Rabia Abbas, Michael Lynch, Zhou Zhong, Song-My Hoang, Andras Miklosi, Kun Huang, Jae-Won Cho, Marion Grimaud, Cecile Razimbaud, Matthew Chang, Atef Fayed, Audrey Apollon, Nithyassree Murugan, Ze-Hua Li, Tran Thai, Luann Zerefa, Brandon Piel, Elena Ivanova, Amy Cameron, Quang-De Nguyen, Zhu Zhu, Kevin Wei, Yasmin Nabil Laimon, Aseman Bagheri Sheshdeh, Sabina Signoretti, David A. Braun, Catherine J. Wu, Toni K. Choueiri, Jon Wee, Cloud P. Paweletz, Martin Hemberg, Aedin C. Culhane, David A. Barbie, Gordon J. Freeman, Wayne A. Marasco. Fine-tuned CAIX targeted CAR-T cells exhibit superior efficacy and mitigate on-target off-tumor side effects [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 886.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug” that has proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. To date, there are five CAR-T products approved by FDA, four CD19 targeted CAR-T cells, and one targeting B-cell maturation antigen (BCMA). However, this success has not yet been transferred to solid tumors. A major challenge is the immunosuppressive tumor microenvironment (TME). Development of immunotherapies has traditionally been hampered by discrepancies observed between in vitro and in vivo studies and actual clinical trial outcomes. The lack of clinically relevant mouse models for human immunotherapy testing is often seen as the primary cause. Existing clear cell renal cell carcinoma (ccRCC) in vivo models poorly recapitulate the tumor microenvironment (TME). Here we report a ccRCC orthotopic humanized NSG-SGM3 mouse model (ccRCC-hNSG-SGM3) with reconstituted human lymphocytes and bearing human ccRCC skrc-59 cells under the kidney capsule. Human leukocyte antigen (HLA) matched CD34+ human stem cells were used for the humanization to reduce T cell alloreactivity against skrc-59 human ccRCC cells. Tumors were harvested, sorted for CD45+ tumor infiltrating leukocytes (TILs) and single cell RNA sequencing (scRNAseq) was performed to profile the TME in ccRCC-hNSG-SGM3. By comparing to patient data from prospective clinical trials of the anti-PD-1 monoclonal antibody (mAb) nivolumab in advanced ccRCC, the results demonstrated that CD45+ TILs from ccRCC-hNSG-SGM3 reconstitute most CD45+ cell types, including dendritic cells, exhausted CD8 T cells, and regulatory T cells (Tregs), that are observed in advanced ccRCC patient TME. Furthermore, we generated HLA matched Immune Restoring (IR) CAR-T cells which can secrete anti-PD-L1 monoclonal antibody (mAb) locally to restore active antitumor immunity, and we assessed the efficacy and safety of IR CAR-T cells in this model. Anti-CAIX CAR-T cells armored with anti-PD-L1 mAb showed superior efficacy in tumor regression and significantly decreased TIL exhaustion compared to irrelevant CAR or irrelevant payload in this model. In addition, in situ hybridization (ISH) results showed CAR-T cells infiltration in tumor but no CAR-T cells were observed in normal tissues. In summary, the ccRCC-hNSG-SGM3 system is able to model the advanced ccRCC TME and provides a powerful tool for ccRCC TME study and immunotherapy assessment. IR CAR-T cells exhibited superior tumor regression and reversed immunosuppressive TME in humanized mice. Citation Format: Yufei Wang, Alicia Buck, Marion Grimaud, Aedin Culhane, David Braun, Sreekumar Kodangattil, Cecile Razimbaud, Matthew Chang, Atef Fayed, Audrey Apollon, Gabriella Kastrunes, Luann Zerefa, Brandon Piel, Elena Ivanova, Dennis Bonal, Kristen Jones, Quang-De Nguyen, Zhu Zhu, Kevin Wei, Nicholas Hayden, Madison O'Donnell, Ying Huang, Rebecca Jenning, Miriam Ficial, Maura Aliezah Sticco-Ivins, Sabina Signoretti, Catherine Wu, Toni Choueiri, Jon Wee, Cloud Paweletz, David A. Barbie, Gordon Freeman, Wayne A. Marasco. Anti-CAIX Immune Restoring (IR) CAR-T cells display superior antitumor activity and reverse immunosuppressive TME in a humanized ccRCC orthotopic mouse model [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 2814.

Background Chimeric Antigen Receptor (CAR) T cell therapy is a new type of "living drug" that has proven to be a powerful immunotherapy for hematologic malignancies. To date, there are six CAR-T products approved by FDA, four CD19 targeted CAR-T cells, and two targeting B-cell maturation antigen (BCMA).1-8 However, this success has not yet been transferred to solid tumors. A major hurdle is the on-target off-tumor toxicities due to the shared expression of target antigen on healthy tissues. Methods Here, we assessed the in vitro cytotoxicity of carbonic anhydrase IX (CAIX) targeted CAR-T cells generated from a series of single chain fragment variables (scFvs) that have various affinities against CAIX. In addition, we studied the avidity of CAR-T cells using a cell avidity analyzer. We established a tetracycline (Tet)-On inducible CAIX expressing system that provides different CAIX levels on the cell surface covering the range from the density on CAIX-high skrc-59 cells to the one on CAIX-low MMNK-1 cholangiocytes. To assess the therapeutic effect of CAR-T on patient samples, we generated patient derived organotypic spheroids (PDOTS) ex vivo cultures and tested CAR-T cell migration and cytokine release using these miniature tumors. Results We identified a low affinity, high avidity anti-CAIX CAR G9, which only kills CAIX high tumor cells but not CAIX-low normal tissues in vitro. G9 demonstrates a CAIX density dependent response on Tet-On inducible CAIX expressing cell lines. G9 CAR has a wider therapeutic window compared to G250 that caused serious adverse events in the first anti-CAIX CAR-T clinal trial.9-11 G9 exhibits superior efficacy ex vivo on ccRCC PDOTS 3D cultures which recapitulates ccRCC patient tumor microenvironment (TME), as well as mitigating toxicity on cholangiectasis spheroids. Conclusions In summary, affinity fine-tuned CAR-T cell therapy holds the promise to achieve cures of ccRCC by killing ccRCC tumor cells and mitigating on-target off-tumor toxicity on normal tissues. References Maude, S. L., Laetsch, T. W., Buechner, J., Rives, S., Boyer, M., Bittencourt, H. et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018; Feb 1; 378: 439-448, doi:10.1056/NEJMoa1709866. Schuster, S. J., Bishop, M. R., Tam, C. S., Waller, E. K., Borchmann, P., McGuirk, J. P. et al. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma.N Engl J Med. 2019; Jan 3; 380: 45-56, doi:10.1056/NEJMoa1804980 Wang, M., Munoz, J., Goy, A., Locke, F. L., Jacobson, C. A., Hill, B. T. et al. KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma.N Engl J Med. 2020; Apr 2; 382: 1331-1342, doi:10.1056/NEJMoa1914347. Abramson, J. S., Palomba, M. L., Gordon, L. I., Lunning, M. A., Wang, M., Arnason, J. et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet. 2020; Sep 19; 396: 839-852, doi:10.1016/s0140-6736(20)31366-0 Neelapu, S. S., Locke, F. L., Bartlett, N. L., Lekakis, L. J., Miklos, D. B., Jacobson, C. A. et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.N Engl J Med. 2017; Dec 28; 377: 2531-2544, doi:10.1056/NEJMoa1707447. Locke, F. L., Ghobadi, A., Jacobson, C. A., Miklos, D. B., Lekakis, L. J., Oluwole, O. O. et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1-2 trial.Lancet Oncol. 2019; Jan; 20: 31-42, doi:10.1016/s1470-2045(18)30864-7. FDA.FDA Approves First Cell-Based Gene Therapy for Adult Patients With Multiple Myeloma. 2021; Mar 27. Available at: https://www.fda.gov/news-events/press-announcements/fda-approves-first-cell-based-gene-therapy-adult-patients-multiple-myeloma.: Berdeja, J. G., Madduri, D., Usmani, S. Z., Jakubowiak, A., Agha, M., Cohen, A. D. et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study.Lancet. 2021; Jul 24; 398: 314-324, doi:10.1016/s0140-6736(21)00933-8 Lamers, C. H., Klaver, Y., Gratama, J. W., Sleijfer, S. & Debets, R. Treatment of metastatic renal cell carcinoma (mRCC) with CAIX CAR-engineered T-cells-a completed study overview. Biochem Soc Trans. 2016; Jun 15; 44: 951-959, doi:10.1042/bst20160037 Lamers, C. H., Sleijfer, S., Vulto, A. G., Kruit, W. H., Kliffen, M., Debets, R. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; May 1; 24: e20-22, doi:10.1200/jco.2006.05.9964 Lamers, C. H., Willemsen, R., van Elzakker, P., van Steenbergen-Langeveld, S., Broertjes, M., Oosterwijk-Wakka, J. et al. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells.Blood. 2011; Jan 6; 117: 72-82, doi:10.1182/blood-2010-07-294520 Ethics Approval The study obtained DFCI Office for Human Research Studies (OHRS) approval (IRB protocol #19-194).

BACKGROUND:Evolutionary pressure has led to the emergence of SARS-CoV-2 variants, with the most recent Omicron variant containing an unparalleled 30 mutations in the spike protein. Many of these mutations are expected to increase immune evasion, thus making breakthrough cases and re-infection more common.METHODS:From June 2020 to December 2021 serial blood samples (initial post recovery, 6 months, 12 months) were collected from a COVID-19 convalescent cohort in Boston, MA. Plasma was isolated for use in Mesoscale Discovery based antibody binding assays. Unvaccinated donors or those vaccinated prior to the primary blood draw were excluded from this analysis, as were those who did not have at least two blood draws. Wilcoxon signed rank tests were used to compare pre- and post-vaccination titers and antibody response against different variants, while McNemar tests were used to compare the proportions of achieving ≥ 4 fold increases against different variants.FINDINGS:Forty-eight COVID convalescent donors with post-infection vaccination (hybrid immunity) were studied to evaluate the levels of cross-reactive antibodies pre- and post- vaccination against various SARS-CoV-2 Spike and receptor binding domain (RBD) proteins. Vaccination with BNT162b2, mRNA-1273 or Ad26.COV2.S led to a 6·3 to 7·8 fold increase in anti-Spike antibody titers and a 7·0 to 7·4 fold increase in anti-WT, Alpha and Delta RBD antibody. However, a lower response was observed for Beta and Omicron RBDs with only 7/48 (15%) and 15/48 (31%) donors having a ≥4 fold increase in post-vaccination titers against Beta and Omicron RBDs. Structural analysis of the Beta and Omicron RBDs reveal a shared immune escape strategy involving residues K417-E484-N501 that is exploited by these variants of concern.INTERPRETATION:Through mutations of the K417-E484-N501 triad, SARS-CoV-2 has evolved to evade neutralization by the class I/II anti-RBD antibody fraction of hybrid immunity plasma as the polyclonal antibody response post-vaccination shows limitations in the ability to solve the structural requirements to bind the mutant RBDs.FUNDING:Massachusetts Consortium on Pathogen Readiness (280870.5116709.0016) and the National Institute of Allergy and Infectious Diseases (1R01AI161152-01A1).

Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. ccRCC is characterized by the loss of chromosome 3p, where the von-Hippel-Lindau (VHL) gene is found. Its gene product, pVHL suppresses the hypoxia inducible factor (HIF) transcription factors and the deletion of VHL leads to HIF hyperactivation, upregulating the expression of many downstream genes involved in angiogenesis, metabolism, and cell-cycle regulation. Two gene products, carbonic anhydrase IX (CAIX) and CD70 which we are pursuing as therapeutic targets are downstream genes of the HIF-1α pathway and thus commonly overexpressed in ccRCC. Despite the development of checkpoint blockade inhibitors (CBIs) for the treatment of this disease, curative therapies are rare. Chimeric Antigen Receptor (CAR)-T cells are a new type of “living drug”, in which T cells are engineered to express a single chain variable antibody fragment (scFv) linked to an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB (second generation), or both (third generation) costimulatory domains. CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment (TME), and on-target off-tumor toxicities resulting from the sharing of CAR-T target epitopes on healthy tissues. To translate CAR-T cell therapy to ccRCC, we developed dual-targeted CAR-T cells against both CAIX and CD70, in which dual CAR can target double positive (CAIX+ CD70+) and single positive (CAIX+ CD70-, CAIX- CD70+) tumor cell populations to address this heterogeneity. By IHC staining, we demonstrated that these two target antigens are overexpressed in ccRCC and represent circa 90% of the tumor cell population regardless of disease stages. Here we build on our previous findings and demonstrate that 41BB CAR and CD4/CD8 CAR-T cell mixtures exhibit the superior efficacy and persistence in our ccRCC orthotopic NSG-SGM3 mouse model compared to other CAR constructs and CD8 alone. A panel of dual-targeted CARs have been encoded in our 41BB CAR construct and assessed in vitro and in vivo by using Celigo imaging cytometry assay and humanized ccRCC orthotopic NSG-SGM3 mouse model respectively. Dual-targeted anti-CAIX/CD70 CD4/8 CAR-T cells outperformed singly-targeted CAR-T cells with elevated efficacy. In summary, anti-CAIX/CD70 CAR-T cells hold a great promise to achieve ccRCC cures. Citation Format: Yufei Wang, Marion Grimaud, Alicia Buck, Atef Fayed, Matthew Chang, Rebecca Jennings, Maura Sticco-Ivins, Miriam Ficial, Leo L. Chan, Sabina Signoretti, Quan Zhu, Wayne A. Marasco. Develop dual-targeted CAR-T cells to achieve RCC cures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6606.