The PIM kinase family is critically involved in tumorigenesis, yet its role in primary T cells is understudied. We reported that PIM2, distinct from the other 2 isoforms, inhibits T cell responses to alloantigen. Here, we further established PIM2 as a key negative regulator in antitumor immunity. Pim2 deficiency in tumor antigen-specific or polyclonal T cells enhanced their ability to control tumor growth in murine breast cancer, melanoma, and leukemia models. Pim2 deficiency enhanced cytokine production and metabolic activities in tumor-infiltrating CD8 T cells. Pim2 deficiency increased TCF1 expression and memory-like phenotype in CD8 T cells from lymphoid organs. Mechanistically, PIM2 facilitated LC3 lipidation, P62 degradation, and autophagic flux in T cells, leading to impaired glycolysis and effector cytokine production. Furthermore, through modulating VPRBP kinase phosphorylation, PIM2 inhibited histone methyltransferase activity of EZH2 in CD8 T cells, causing disrupted memory-like phenotype. Notably, the PIM2 inhibitorJP11646 markedly enhanced antitumorT cell response. The immunosuppressive role of PIM2 was validated in human T cells, where inhibition of PIM2 enhanced antitumor responses in engineered human T cells, including melanoma-specific TCR T cells and CD19 CART cells. Collectively, PIM2 represents a promising target for improving cancer immunotherapy through enhancing effector differentiation and persistence of CD8 T cells.

The recent FDA approval of various CAR, TCR, and TIL therapies has only increased the excitement over T cell based adoptive transfer therapies. We have manufactured TCR and CAR T cells to treat a total of 29 patients with melanoma (N=7), clear cell renal cell carcinoma (N=15), and non-Hodgkins lymphoma (N=7). While there were objective clinical responses in the three patient populations with the NHL patients treated with CD19 CAR T cells had the best outcomes. What is clear is that the genetically modified T cells can persist for years but they become nonfunctional, probably due to the host environment. Results from our trials support this notion and will be discussed with potential solutions to overcoming the immune suppressive tumor microenvironment.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy and unique long-term outcomes in treating relapsed/refractory hematological malignancies in both pediatric and adult patients. However, a subset of patients remains unresponsive, with disease progression continuing despite treatment. Moreover, CAR-T therapy has shown limited success in treating solid tumors. Accumulating evidence on the role of various lymphocyte subpopulations in antitumor immunity suggests that cytokine-induced killer (CIKs) cells represent a promising alternative for adoptive cell therapy. A key advantage of CIKs is their low toxicity and the presence of unrestricted MHC cytotoxicity against both hematological and solid malignancies. In our study, we hypothesize that modifying CIKs with CARs would enhance their antitumor efficacy compared to unmodified CIKs. Using a unique retroviral vector containing the CD34 or CD14 marker gene, we successfully generated CD19 CAR-expressing lymphocyte populations, including both classically activated T lymphocytes and CIKs. We then compared their functional properties by analyzing lymphocyte subpopulations based on markers such as CD3, CD4, CD8, CD56, CD62L, and CD45RA, as well as their ability to specifically recognize CD19-positive targets. Additionally, we assessed cytokine production, including IFN-γ (via ELISA and intracellular staining), IL-2, TNF-α, and IL-10 (via intracellular staining). Our findings indicate that CIKs can be efficiently modified using a retroviral vector while maintaining functional activity. CD19 CAR-CIK populations contained higher numbers of NKT (CD3⁺CD56⁺) and CD3⁺CD8⁺ lymphocytes compared to CD19 CAR-T cells. However, the proportion of naïve-like T cells (CD62L⁺CD45RA⁺) and effector memory RA⁺ T cells (CD62L⁻CD45RA⁺) remained similar in both CAR-modified populations. Additionally, CD19 CAR-CIKs exhibited a slightly increased ability to produce IFN-γ and TNF-α in response to CD19⁺ tumor cells. Despite that, key questions remain regarding the functionality of CD19 CAR-CIKs in an immunosuppressive tumor microenvironment, particularly in the presence of high levels of TGF-β, IL-10, and PD-L1, which may inhibit their activity or lead to exhaustion. Our future research will focus on overcoming these challenges to enhance the therapeutic potential of CAR-CIKs.

Abstract Understanding the critical relationship between cancer and the immune system has led to novel immune therapies including chimeric antigen receptor (CAR) T-cell therapy. In the clinic, CAR T cells have been most effective against hematological malignancies. CD19 is expressed in almost all stages of B cell development as well as in most B cell malignancies, making it an ideal antigen to target using CAR T cells. Despite the promising efficacy of CD19 CAR T cell therapy, there remains a subset of patients who are resistant to therapy. At Loyola University Chicago, we initiated a CD19 CAR T cell clinical trial in 2020. A total of 6 patients were treated on protocol, with a 50% durable complete response rate. Importantly, no treated patients experienced severe side effects. Unique to the CD19 CAR T cells, is the incorporation of a CD34 marker gene (CD34t) in the CAR retroviral construct used for T cell transduction. The use of CD34t allows us to track infused CAR T cells in the blood. With the ability to distinguish CD19 CAR T cells from the endogenous T cell population, we have developed an immunofluorescence staining panel of 39 functional cell surface proteins to study the treated patient blood samples. We found that CD19 CAR T cells persist regardless of clinical outcome. We are studying the changing CAR T cell phenotype in patients with varying clinical outcomes to identify phenotypic trends that may have clinical implications and could serve as relevant clinical biomarkers.

CD19-specific chimeric antigen receptor (CAR) T cells have demonstrated impressive responses in patients with relapsed and refractory B cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T cells involve T cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T cells in the absence of activation. In these studies, we used IL-7 to generate CD19 CAR T cells without stimulating CD3. Nonactivated and IL-7 cultured (NICE) CD19 CAR T cells were enriched with the T memory stem cell population, retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B cell lymphoma models. These results suggest that NICE CD19 CAR T cells may improve outcomes for B cell malignancies and warrant clinical evaluation.

Abstract The role of tumor microenvironment mediated disruption of Golgi architecture and function,termed Golgi stress, in the regulation of T cell survival and function are largely unknown. Here weshow that the disruption of Golgi architecture, identified by the decreased expression of GM130,was reverted upon treatment with hydrogen sulfide (H2S) donor GYY4137, or over-expressingcystathionine β-synthase (Cbs), an enzyme involved in the biosynthesis of endogenous H2S -that promoted stemness, antioxidant capacity and exhibited increased protein translationmediated in part by ER-Golgi shuttling of Peroxiredoxin-4. In in vivo models of melanoma andlymphoma, anti-tumor T cells conditioned ex vivo with exogenous H2S or overexpressing Cbsdemonstrated superior tumor control upon adoptive transfer. Further, Golgihi T cells, with highGolgi content, exhibited unique metabolic and glycation signature with enhanced anti-tumorcapacity. These data suggest that strategies to mitigate Golgi network stress or using Golgihitumor-reactive T cells can improve tumor control upon adoptive transfer. Citation Format: Nathaniel Oberholtzer, Paramita Chakraborty, Mohamed Faisal Kassir, James Dressman, Zacharia Hedley, Gina Scurti, Monika Gooz, Lauren E. Ball, Elizabeth Hill, Anand S. Mehta, Eduardo N. Maldonado, Michael I. Nishimura, Besim Ogretmen, Shikhar Mehrotra. Golgihi T cells exhibit reduced susceptibility to exhaustion and improved tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB343.

The role of tumor microenvironment (TME)-associated inadequate protein modification and trafficking due to insufficiency in Golgi function, leading to Golgi stress, in the regulation of T cell function is largely unknown. Here, we show that disruption of Golgi architecture under TME stress, identified by the decreased expression of GM130, was reverted upon treatment with hydrogen sulfide (H2S) donor GYY4137 or overexpressing cystathionine β-synthase (CBS), an enzyme involved in the biosynthesis of endogenous H2S, which also promoted stemness, antioxidant capacity, and increased protein translation, mediated in part by endoplasmic reticulum-Golgi shuttling of Peroxiredoxin-4. In in vivo models of melanoma and lymphoma, antitumor T cells conditioned ex vivo with exogenous H2S or overexpressing CBS demonstrated superior tumor control upon adoptive transfer. Further, T cells with high Golgi content exhibited unique metabolic and glycation signatures with enhanced antitumor capacity. These data suggest that strategies to mitigate Golgi network stress or using Golgihi tumor-reactive T cells can improve tumor control upon adoptive transfer.

Background We discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T cell-mediated killing. Here, we described the cloning of a novel T cell receptor (TCR) targeting a CT-RCC HERV-E-derived antigen specific to ccRCC and characterized antitumor activity of HERV-E TCR-transduced T cells (HERV-E T cells).Methods We isolated a CD8+ T cell clone from a patient with immune-mediated regression of ccRCC post-allogeneic stem cell transplant that recognized the CT-RCC-1 HERV-E-derived peptide in an HLA-A11-restricted manner. We used 5’Rapid Amplification of cDNA Ends (RACE) to clone the full length HERV-E TCR and generated retrovirus encoding this TCR for transduction of T cells. We characterized HERV-E T cells for phenotype and function in vitro and in a murine xenograft model. Lastly, we implemented a good manufacturing practice-compliant method for scalable production of HERV-E T cells.Results The HLA-A11-restricted HERV-E-reactive TCR exhibited a CD8-dependent phenotype and demonstrated specific recognition of the CT-RCC-1 peptide. CD8+ T cells modified to express HERV-E TCR displayed potent antitumor activity against HLA-A11+ ccRCC cells expressing CT-RCC HERV-E compared with unmodified T cells. Killing by HERV-E T cells was lost when cocultured against HERV-E knockout ccRCC cells. HERV-E T cells induced regression of established ccRCC tumors in a murine model and improved survival of tumor-bearing mice. Large-scale production of HERV-E T cells under good manufacturing practice conditions generated from healthy donors retained specific antigen recognition and cytotoxicity against ccRCC.Conclusions This is the first report showing that human ccRCC cells can be selectively recognized and killed by TCR-engineered T cells targeting a HERV-derived antigen. These preclinical findings provided the foundation for evaluating HERV-E TCR-transduced T cell infusions in patients with metastatic ccRCC in a clinical trial (NCT03354390).

In vivo expansion of genetically modified T cells in cancer patients following adoptive transfer has been linked to both anti-tumor activity and T cell-mediated toxicities. The development of digital PCR has improved the accuracy in quantifying the in vivo status of adoptively infused T cells compared to qPCR or flow cytometry. Here, we developed and evaluated the feasibility and performance of nanoplate-based digital PCR (ndPCR) to quantify adoptively infused T cells engineered with a T cell receptor (TCR) that recognizes a human endogenous retrovirus type E (HERV-E) antigen. Analysis of blood samples collected from patients with metastatic kidney cancer following the infusion of HERV-E TCR-transduced T cells established the limit of detection of ndPCR to be 0.3 transgene copies/mu L of reaction. The lower limit of quantification for ndPCR was one engineered T cell per 10,000 PBMCs, which outperformed both qPCR and flow cytometry by 1 log. High inter-test and test-retest reliability was confirmed by analyzing blood samples collected from multiple patients. In conclusion, we demonstrated the feasibility of ndPCR for detecting and monitoring the fate of TCR-engineered T cells in adoptive cell therapy.

Abstract Gene-modified TCR transduced T cells and CAR T cells are used clinically and display a range of efficacy across multiple cancers. Many therapies have successfully controlled patient’s tumors; however, some have shown off-target adverse events which are harmful to patients’ overall outcomes. T cell-based therapies have high variation in patient outcome due to multiple factors, such as transduction efficiency and T cell phenotypes. To reduce product variability, our lab developed a selectable marker gene for use in cellular constructs. This selectable marker is a truncated, non-signaling CD34 molecule (CD34t), which can be used to remove the non-transduced T cells that function as bystanders. Our clinical trials have used two TCRs (TIL 1383I and HERV-E) and one CAR (CD19 CAR). Following transduction with these CD34t constructs, we determined transduction efficiency by expression of CD34t. CD34t expression correlates with transgene, allowing us to select high CD34t expressors, and thus high transgene expressors. The selected population produces higher levels of inflammatory cytokines and CD107a expression compared to the nonselected. This improved functionality is likely due to the selected population having higher levels of transgene across the population, whereas the nonselected has a broader range of transgene expressions with fewer expressors total. This selectable marker contributes to a more functional product, which may in turn improve patient response clinically.

435 Background: Human endogenous retrovirus type E (HERV-E) is specifically expressed in ccRCC providing a safe target for T cell-based therapies. We investigated T cells transduced with a TCR targeting HERV-E (HERV-E T cells) for the treatment of mccRCC. Methods: This first-in-human study assessed the safety & efficacy of escalating doses of HERV-E T cells and manufacturing/clinical endpoints correlative analysis. HLA-A*11+ mccRCC patients (pts) were treated with a conditioning regimen, infusion of HERV-E T cells & IL-2. (NCT03354390). Results: Nineteen of185 pts tested were found to express HLA-A*11. 17 HLA-A*11 + pts enrolled on the study: 3 pts on each DL1-3 & 6 pts on DL4. 2 pts did not receive HERV-E T cells given disease progression during manufacturing period. Median age was 57 years. 86% received ≥ 3 prior systemic treatment (range 1-8). The manufacturing failure rate after first apheresis was 12% (n=2); both met target dose after second apheresis and repeat in vitro expansion. All HERV-E T cell products met release criteria for infusion including INF-γ production in response to HERV-E/HLA-A11-expressing tumor cells. Median HERV-E vector copy number (VCN) was 1.9. No dose-limiting toxicities (DLT), off-target toxicities or treatment-related deaths occurred. Pt#17 is on DLT monitoring period. 7 pts completed the planned 14 doses of IL-2 and all received at least 8 doses. Reasons for IL-2 discontinuation: hemodynamic (57%), cardiovascular (28%), pulmonary (28%), renal (14%) criteria & pts decision (14%). The best response was partial response in 7% & stable disease at least 8 weeks in 29% pts. HERV-E mRNA expression was detected in 5 primary & 9 metastatic specimens. HERV-E T cells were measurable in circulation post-dosing, with peak concentrations in the peripheral blood mononuclear compartment on day(D)+7. [DL1: 0.3 %, DL2: 1.2%, DL3: 0.5%, DL4: 12.3%]. Median HERV-E T cell VCN showed no correlation with HERV-E T cells peak concentration on D+4 & D+7. Conclusions: Proof of concept that HERV-T cells can induce tumor regression without evidence of causing off-target toxicities has been established by this trial. Infused HERV-E T cells were detectable transiently in vivo and induce effector cytokine production. Our initial results support the further development of HERV-E-directed therapies that focus on methods to improve in vivo persistence of TCR engineered T-cells and to target HERV-E antigens expressed on more commonly expressed HLA alleles.[Table: see text]

Legends for Supplementary Figures 1 and 2

Supplementary Materials and Methods

Supplementary Figure 3. Rapamycin-preconditioned CD8+ T cells upregulate the levels of antioxidant molecules

<p>Supplementary Figure Legend</p>

Marie Skłodowska-Curie Symposia on Cancer Research and Care (MSCS-CRC) promote collaborations between cancer researchers and care providers in the United States, Canada and Central and Eastern European Countries (CEEC), to accelerate the development of new cancer therapies, advance early detection and prevention, increase cancer awareness, and improve cancer care and the quality of life of patients and their families. The third edition of MSCS-CRC, held at Roswell Park Comprehensive Cancer Center, Buffalo, NY, in September 2023, brought together 137 participants from 20 academic institutions in the US, Poland, Ukraine, Lithuania, Croatia and Hungary, together with 16 biotech and pharma entities. The key areas of collaborative opportunity identified during the meeting are a) creating of a database of available collaborative projects in the areas of early-phase clinical trials, preclinical development, and identification of early biomarkers; b) promoting awareness of cancer risks and efforts at cancer prevention; c) laboratory and clinical training; and d) sharing experience in cost-effective delivery of cancer care and improving the quality of life of cancer patients and their families. Examples of ongoing international collaborations in the above areas were discussed. Participation of the representatives of the Warsaw-based Medical Research Agency, National Cancer Institute (NCI) of the United States, National Cancer Research Institutes of Poland and Lithuania, New York State Empire State Development, Ministry of Health of Ukraine and Translational Research Cancer Center Consortium of 13 cancer centers from the US and Canada, facilitated the discussion of available governmental and non-governmental funding initiatives in the above areas.

Supplementary Figure 2. Pretreatment of T cells with N-acetyl cysteine prevent TCR restimulation induced ROS accumulation.