Gendler, Sandra

Abstract Purpose: Naturally occurring tumor-specific T cells derived from peripheral blood (PB) offer a clinically accessible source for adoptive immunotherapy. However, the expansion of these T cells from patients with cancer remains a challenge. We hypothesized that mimicking innate immune activation could optimally stimulate antigen-driven T-cell expansion in vitro, unlocking the therapeutic potential of PB-derived T cells. Patients and Methods: We previously developed an ex vivo culture system that selectively expands tumor antigen–activated T cells from PB mononuclear cells (PBMC), generating multiclonal effector and central memory T cells. In this hypothesis-generating study, we evaluated the therapeutic potential of MUC1-activated T cells in patients with relapsed and/or refractory multiple myeloma (r/rMM) in a phase I clinical trial (NCT05411497). MUC1 is an oncoprotein overexpressed in r/rMM. We translated our small-scale culture into GMP-compliant, large-scale manufacturing, which achieved expansion of T cells from heavily pretreated patients’ PBMCs without exhaustion. Five patients were treated with escalating doses of up to 1 × 1010 T cells. Results: Although the cell infusions were well tolerated, no objective responses occurred. One patient, who received the highest dose, has had stable disease for 2 years after infusion. This patient exhibited transient dermatitis with localized MUC1 and CD3 staining, as potential evidence of on-target, off-tumor T-cell reactivity, perhaps contributing to disease stabilization. T-cell receptor sequencing revealed the T-cell product in four of the patients’ blood, which correlated with the product’s degree of polyfunctionality and MUC1 reactivity. Conclusions: These findings demonstrate the feasibility, safety, and biological activity of PB-derived, MUC1-specific T cells as adoptive immunotherapy. Significance: Expanding tumor antigen–specific T cells for clinical use from the blood of heavily pretreated patients with cancer remains a significant hurdle. We scale up a novel culture method that integrates innate immune signals to stimulate T-cell expansion and demonstrate safety, feasibility, and a case of long-term disease stabilization upon treatment of patients with multiple myeloma in a phase I clinical trial.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown clinical successes in B cell hematological malignancies. Two B cell maturation antigen (BCMA)-directed CART cell therapies treating multiple myeloma (MM) were approved by FDA but most patients relapse within 1-2 years. Ex vivo-expanded antigen specific T cells as cancer treatment rely on TCR-mediated killing of tumors. We hypothesized that arming ex vivo enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions. First, we expanded mucin 1 (MUC1)-activated T cells from patients with MM through ex vivo stimulation by MUC1 peptides-loaded autologous PBMCs for 19 days. Then, we engineered MUC1-activated T cells with 4-1BB-co-stimulated BCMA CAR to generate MUC1 TCR+BCMA CAR+ T cells. Baseline T cells without MUC1 stimulation were engineered with BCMA CAR as control. Baseline T cells, MUC1 T cells, baseline BCMA CART cells, or MUC1 BCMA CART cells were cocultured with luc+ RPMI-8226, a MUC1+BCMA+ MM cell line, for 3 days. MUC1 BCMA CART cells showed stronger tumor killing than BCMA CART or MUC1 T cells in all 3 patients (p<0.0001). MUC1 BCMA CART showed improved proliferation than BCMA CART or MUC1 T cells (p=0.0765, p<0.0001, respectively) after a 5-day stimulation by RPMI-8226. To exclude possibility of metabolic alterations by 19-day PBMC incubation leading to better T cell functions in MUC1 BCMA CART, baseline and MUC1-stimulated T cells from 3 healthy donors were processed for the same assays. Day-19 BCMA CART cells showed inferior tumor killing compared to baseline BCMA CART in all 3 donors (p<0.0001). Lower T cell proliferation was also observed in day-19 BCMA CART (p=0.0838). Our data indicate that improved T cell functions in MUC1 BCMA CART were unlikely due to metabolic changes and required stimulation by tumor antigens prior to CAR engineering likely through MUC1 TCR. Additionally, MUC1 T cells, BCMA CART, or MUC1 BCMA CART cells from tested patients were stimulated by RPMI-8226 for bulk RNA sequencing. Both BCMA CART and MUC1 T cells had distinct transcriptomic features from MUC1 BCMA CART cells. Differential gene expression analysis showed upregulation of T cell function-associated genes (RYR2, ITGA1, GNLY, GZMA, etc.) in MUC1 BCMA CART vs BCMA CART. Enrichment of T cell function-associated pathways was also observed in MUC1 BCMA CART. Analysis between MUC1 T cell and MUC1 BCMA CART showed upregulation of T cell function-associated genes (CSF2, CRTAM, IFNG, GZMB, etc.) and enrichment of immune response pathways in MUC1 BCMA CART. Our findings suggest that equipping ex vivo-enriched tumor-specific T cells with CARs targeting a different antigen improves T cell functions in vitro. Importantly, this provides a rationale for a therapeutic regimen where patients receive ex vivo-enriched tumor-induced T cells followed by CAR engineering. Persistence of the engineered T cells is being investigated Citation Format: Kun Yun, R. Leo Sakemura, Ismail Can, Omar Gutierrez Ruiz, Ekene J. Ogbodo, Truc N. Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli M. Stewart, Jennifer M. Feigin, Grace E. DeFranco, Sophia Y. Goldberg, Dominic A. Skeele, Mariam Stein, Dara Missan, Latha Pathangey, Long Mai, Hong Xia, Brooke Kimball, Lionel A. Kankeu Fonkoua, Mehrdad Hefazi, Michael W. Ruff, Elizabeth L. Siegler, Leif Bergsagel, Michael Gustafson, Sandra Gendler, Saad S. Kenderian. BCMA CAR engineering arms T cells with superior functions post ex vivo stimulation by MUC1 peptide-loaded autologous PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6103.

Abstract Even though it is known that the immune response is highly relevant in ovarian cancer (OC), as demonstrated by the strong association between tumor infiltration by CD8 T cells and overall survival in high grade serous ovarian cancer, immunotherapy has not yet benefited patients with this disease. Therefore, there is a critical unmet need to identify new therapeutic targets and approaches that will improve outcomes for women with resistant disease. We used a novel culture system that uses a two-step activation process to elicit tumor-specific Type 1 (tumor-killing) T cells from the peripheral blood of OC patients. Briefly, the T cells are stimulated over the first two days with GM-CSF, MUC1 peptide antigens, and Toll-like receptor agonists (TLRa). After the second day in culture, the cells are washed and cultured in media containing IL-7 for the next 17 days. Using peripheral blood mononuclear cells (PBMCs) from 4 ovarian cancer patients, who had undergone between 1-3 prior lines of therapy, we have shown that all 4 were successfully activated and expanded with MUC1 antigens. Overall, the CD3+ cell population expanded from 5.7 – 12.6 fold over the 19 day culture, which was consistent with the sample from a healthy donor. Intracellular cytokine staining for IFNγ was used to determine MUC1-specific T cell responses at day 19. All OC patient T cells had increased reactivity to the MUC1-peptides as compared to the negative control peptide. Total MUC1-specificity ranged from 9.6 – 30.1% in CD4+ T cells and 6.6 – 16.8% in CD8+ T cells. We additionally performed high-parameter mass cytometry by time of flight (CyTOF) analysis using 34 antibodies targeting surface and intracellular markers. The FlowSOM algorithm followed by Marker Enrichment Modeling were used to identify major clusters of T cells and their associated phenotypes. Using this approach 12 major clusters of T cells were identified and overlayed on a tSNE plot. Of the 12 clusters, 7 represented CD8+ T cells and 5 represented CD4+ T cells. The day 19 clusters primarily represented T effector cells, T effector memory, and T central memory cells. The majority of day 19 cells expressed markers of effector function, including T-bet, EOMES, GRANZ-B, and GRANZ-K, which were present in both CD8+ and CD4+ populations. Exhaustion and regulatory markers were found to be low or absent in the day 19 cells. These results demonstrate that highly polyfunctional T cells with memory markers are generated from our innovative culture system using PBMCs from OC patients. These results provide preclinical evidence that will support future clinical trials of adoptive cell therapy using this culture system in OC. Citation Format: Brenda Ernst, Latha Pathangey, Michael Gustafson, Sandra Gendler, Marion Curtis. Rapid expansion of natural, MUC1-activated T cells from the peripheral blood of ovarian cancer patients for adoptive cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A086.

The HIF‐1 and β‐catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine Mucin 1 (Muc1) protects kidney function and morphology in a mouse model of ischemia‐reperfusion injury (IRI) by stabilizing HIF‐1α, enhancing HIF‐1 downstream signaling and thereby preventing metabolic stress (Pastor‐Soler et al. 2015 Am. J. Physiol. Renal Physiol. 308, F1452‐F1462). We asked if Muc1 regulates the β‐catenin protective pathway during IRI as published data indicate that (i) β‐catenin is found in co‐immunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, (ii) β‐catenin nuclear targeting is MUC1‐dependent in cultured human cells, and (iii) MUC1 prevents β‐catenin phosphorylation by glycogen synthase kinase 3β (GSK3b) and thereby β‐catenin degradation. Using our same mouse model of IRI, and by using immunohistochemistry and immunoblotting of kidney tissue, we found that levels of active GSK3β were significantly lower in kidneys of control mice compared to Muc1 KO mice. Consequently, β‐catenin was significantly increased at 24 and 72 h recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both β‐catenin induction and nuclear targeting were absent in Muc1 KO mice. In accord with these data, we also found downstream induction of β‐catenin pro‐survival factors (activated Akt, survivin, transcription factor TCF4 and its downstream target cyclin D1) and repression of pro‐apoptotic factors (p53, active Bax and cleaved caspase 3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during AKI proceeds by enhancing both the HIF‐1 and β‐catenin protective pathways. Support or Funding Information This work was supported by National Institutes of Health (NIH) Grants DK‐099345 (to T. A. Sutton), DK‐084184 (to N. M. Pastor‐Soler), CA‐64389 (to S. J. Gendler), DK‐62277, DK‐100287, and DK‐095498 (to S. P. Monga), DK‐097889 (to M. M. Al‐bataineh), DK107632, DK‐079307 (Pilot Project) and Samuel and Emma Winters Foundation grant (to R. P. Hughey), and P30‐DK079307 (to the Microscopy Core N. M. Pastor‐Soler and S. Bastacky).

The preventive efficacy of MUC1-specific DNA immunization on inflammation-driven colon carcinogenesis in human MUC1 transgenic (MUC1.Tg) mice was investigated. Mice were vaccinated with MUC1 DNA mixed with autologous bone-marrow-derived dendritic cells (BMDCs), and then colonic tumors were induced by azoxymethane (AOM) injection and oral administration of dextran sulfate sodium (DSS). Two types of tumors, squamous metaplasia and tubular adenoma, were observed. Both expressed high levels of MUC1 as indicated by the binding of anti-MUC1 antibodies with different specificities, whereas MUC1 expression was not detected in normal colonic mucosa. When mice were immunized with MUC1 DNA + BMDCs, tumor incidence, tumor number, and tumor size were significantly reduced. In contrast, vaccination with MUC1 DNA alone or BMDCs alone was ineffective in reducing tumor burden. Inflammation caused by DSS was not suppressed by the MUC1 DNA + BMDCs vaccination. Furthermore, MUC1 protein expression levels, as judged by anti-MUC1 antibody binding in tumors grown after vaccination, did not significantly differ from the control. In conclusion, an inflammation-driven carcinogenesis model was established in MUC1.Tg mice, closely resembling human colon carcinogenesis. In this model, vaccination with MUC1 DNA + BMDCs was effective in overriding MUC1 tolerance and reducing the tumor burden by a mechanism not affecting the level of colonic inflammation.

Abstract Regulation of complement activation is essential in the prevention of damage to autologous tissue. This activity is mediated by the presence of specific complement regulatory proteins on the surface of host cells. In humans, one molecule involved in this regulation is a 70-kDa glycoprotein that has been designated decay-accelerating factor (DAF). We present the full-length cDNA sequence and chromosomal localization of the mouse genetic homologue of the human DAF gene. Interestingly, two classes of cDNA clones were obtained that, rather than representing alternately spliced mRNAs, were derived from two separate but closely related linked genes. Both genes encoded proteins with an amino-terminal signal sequence, followed by four short consensus repeats and a domain rich in serine and threonine. Hydrophilicity plots and alignment with human DAF predicted that one gene encoded a glycosylphosphatidylinositol-anchored form of mouse Daf with 64% nucleotide and 47% amino acid identity to human DAF. The product encoded by the second gene was predicted to have an alternate amino-terminal signal sequence and carboxyl-terminal membrane-spanning and cytoplasmic domains. The two mouse Daf genes share 85% nucleotide and 78% amino acid identities, and have been designated Daf-glycosylphosphatidylinositol and Daf-transmembrane to reflect the two alternate mechanisms of membrane attachment. mRNA expression analysis indicated that the two mouse Daf genes were differentially expressed in the adult mouse. Chromosome localization studies mapped the mouse Daf genes to chromosome 1, where they segregated with the C4-binding protein (C4bp) gene.

Abstract Chronic inflammation in the aged closely resembles tumor-induced immune suppression, manifested as a progressive T1→T2 shift and an increased presence of MDSCs. We hypothesize that reversing these age-related phenomena should reduce the elderly’s heightened susceptibility to malignancy. We have now optimized real-time analyses of phosphorylated STATs in murine T cells and MDSCs in order to correlate STAT activation with immune suppression. Both young and old T cells responded to IL-4 stimulation with STAT6 activation and to IFN-γ stimulation with STAT1 activation. Uniquely, however, young T cells also phosphorylated STAT1, STAT3 and STAT5 in response to IL-4, and responded to IL-6 with STAT3 activation, all consistent with a greater susceptibility to T1/T17 differentiation. Regarding MDSCs, both old and young displayed constitutive STAT3 activation which could be enhanced by G-CSF or IL-6 treatment. Both young and old MDSCs activated STAT1 briskly in response to IFN-γ, STAT5 in response to GM-CSF and STAT6 in response to IL-4. However, younger MDSCs, like younger T cells, displayed more diverse responsiveness to IL-4, activating not only STAT6 but also STAT1 and STAT3. While chronic in vivo exposure to T2 cytokines may explain the decreased responsiveness of older MDSCs and T cells to IL-4, paradoxically IL-4 still robustly activated STAT6 in these cells. Such selective responsiveness may be required to preserve the immune suppressive potential of older MDSCs.

Abstract Recent guidelines by the American Society of Clinical Oncologists recommend that obese individuals receive chemotherapy based on their actual body weight (ABW) without dose reduction. Contrary to prior reports, our own data demonstrate that ABW-dosed chemotherapy or immunotherapy is as effective and well tolerated by obese mice as by lean mice. Nonetheless, obese mice develop significantly greater weight loss in a dose-dependent manner during the entire period that they receive therapy, with corresponding quantifiable reductions in visceral fat depots. This disparity is not explained by altered food intake, as both lean and obese mice experience less than a week of anorexia which does not recur after Cycle 1 of treatment. Furthermore, malabsorption is absent. These observations are relevant to multiple classes of chemo agents. Altered pharmacokinetics of chemotherapy agents in adipose tissue have been reported, yet the pharmacodynamics of obesity remain speculative. Fasting blood glucose monitoring demonstrates glucose level variances which are temporally linked to treatment, rendering it likely that chemotherapy and immunotherapy provoke adipose tissue lipolysis, or alternatively cytoreduction or immunomodulation of adipocyte progenitor cells. Understanding these effects could contribute to improved treatment regimens in the setting of obesity.

Abstract IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.

Abstract Although tumors from melanoma patients can often provide a source of therapeutically active T cells for adoptive therapy, it has historically proved challenging to employ peripheral blood as the T cell source for any type of cancer. We have observed nonetheless that HER2-specific T cells can be Ag-driven and enriched in culture when PBMCs from HER2-vaccinated breast cancer patients are treated with exogenous IL12. We hypothesized that stimulating innate immunity could further improve PBMC cultures by activating the DC subpopulation more effectively than adding exogenous rIL12. Bulk PBMC exposure to optimized TLR agonists produced vast quantities of IL12 and IL23, and upregulated HLA-DR, B7.1 and CD40 in the DC subpopulation, effects not produced by exogenous IL12. Subsequent IL7 exposure emulated homeostatic proliferation, selectively causing the Ag-driven T cell subset both to proliferate faster and to strongly resist apoptosis. Ag-specificity within two weeks approached 80-100% of both CD4+ and CD8+ T cells for recall Ags and 1-15% for a wide array of MUC1- and HER2-derived peptides, including the ability to distinguish glycoforms. Culture-expanded T cells retained a young CD28+/CD56- phenotype, uniformly expressed ROR-γ during culture, variably produced IL-2 and/or IL-17, and uniformly expressed T-bet and secreted IFN-γ when reexposed to Ag. Such T1/T17 bipotency may be ideal for anti-tumor adoptive therapy and highly efficient for use in Ag discovery.