Xia, Hong

Abstract Chimeric antigen receptor T (CART) cell therapy is potentially curative in certain hematological malignancies, resulting in several FDA approvals since 2017. However, widespread adoption is limited by 1) cost and complexity of an autologous platform, 2) severe toxicities, and 3) limited durable remissions. To better predict CART efficacy and toxicity, live CART imaging would allow the realtime assessment of in vivo CART expansion and trafficking. Incorporation of the sodium iodide symporter (NIS)in CART cells (NIS+ CART cells) allows the CART cells to be tracked via PET imaging at high sensitivity. PET signal correlates with CART expansion and toxicity in mouse models (PMID: 34244299). Building on this platform, we aimed to track xenogeneic CART cells to monitor for their rejection, utilizing dogs with spontaneous lymphoma. Canine diffuse large B-cell lymphoma (DLBCL) affects over 250,000 dogs a year and has shared pathologic, biologic, phenotypic, genetic, immunotherapeutic targets, and treatment responses with human DLBCL, providing strong rationale for studying canine lymphoma as a pre-clinical model. We therefore initiated a USDA-CVB regulated pilot study and treated companion dogs with naïve canine CD20 (cCD20)+ DLBCL with human-derived TRAC- and B2M-KO NIS+ CART cells which targeted cCD20. First, we tested and optimized a chemotherapy regimen to achieve optimal lymphodepletion in dogs. Healthy beagles were treated with pentostatin (pen) on days 2-4 (0.125, 0.25, or 0.5 mg/kg i.v.), cyclophosphamide (cy) on day 4 (400 mg/m2 i.v.), or combination cy/pen. The most robust decrease in lymphocytes was seen with combination cy/pen (all cy/pen doses vs all other treatments, p≤0.0001) and in neutrophils with cy or cy/pen (all cy/pen doses vs all pen doses, p≤0.0001; cy vs 0.25 mg/kg pen, p≤0.001; cy vs 0.5 mg/kg pen, p≤0.0001). All lymphodepletion was transient and recovered within two weeks. All regimens were well-tolerated, and we therefore used 0.5 mg/kg pen plus 300 mg/m2 cy for the companion dog pilot study. Next, we assessed the feasibility of generating cCD20-targeted NIS+ human CART cells (CART20-NIS) with double KO (DKO) of TRAC and B2M. Primary T cells from healthy human donors were transduced with bicistronic lentivirus encoding 41BB-costimulated cCD20-targeted CAR and NIS. Two days after transduction, cells were transfected with CRISPR Cas9-gRNA targeted to TRAC and B2M. The resulting CART20-NIS DKO cells were expanded until day 11 and then were further depleted for CD3 and cryopreserved. CART20-NIS DKO cells demonstrated high CAR expression (untransduced T cells (UTD) vs CART20-NIS DKO, p<0.0001) and KO efficiency (TCRαβ and HLA-ABC expression of UTD vs CART20-NIS DKO, p<0.0001). NIS functionality was confirmed via a qualitative in vitro I-125radioactive uptake assay. We then assessed antigen specificity and activity in vitro. CART20-NIS DKO or UTD were co-cultured with either wildtype or cCD20-overexpressing K562 cells for three days. Target cell killing was then quantified via flow cytometry. CART20-NIS DKO cells demonstrated significant cytotoxicity against cCD20+ K562 cells (UTD vs CART20-NIS DKO, p<0.0001). Lastly, we treated two companion dogs in a USDA-CVB regulated pilot study of CART20-NIS DKO cells in naïve cCD20+ DLBCL. Following lymphodepletion on days -5 to -3, on day 0, dogs were i.v. administered 1 x 108 CART20-NIS DKO cells and intratumorally administered 2.5 x 107 CART20-NIS DKO cells in two lymph nodes, with contralateral saline injection as controls. Dogs underwent 18F-TFB-PET imaging on days 1, 3, and 7, which showed evidence of CART20-NIS DKO cells both in the CART-injected lymph nodes and distal lymph nodes, as determined by SUVmax. Both dogs sustained clinical remission for the duration of the 21-day study, as determined by sum of longest diameters and accompanying x-ray radiology and abdominal ultrasound. Histology showed marked reduction of CD20 cells in the lymph nodes. Organ and hematological toxicities were minimal, and dogs remained in remission for 95 and 101 days post-CART. In summary, this study demonstrated that xenogeneic CART cells were well-tolerated, induced remission, and were visualized via PET imaging in companion dogs with lymphoma. These results are promising both for the treatment of companion dogs with novel cell therapies and for the translation of novel CART cell therapies into human clinical trials.

Abstract While chimeric antigen receptor T (CART) cell therapy has shown remarkable success, the development of exhaustion limits durable response. We identified a role for interleukin (IL)-4 in the development of CART cell exhaustion through three independent approaches including: 1) a genome-wide CRISPR knockout screen using healthy donor CART cells in an in vitro model for exhaustion, 2) RNA and ATAC sequencing on freshly produced and chronically stimulated healthy donor CART cells, and 3) RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the Zuma-1 clinical trial that led to the FDA approval of axi-cel. Further, in vitro validation studies revealed that CD19 directed CART (CART19) cells chronically stimulated in the presence of human recombinant IL-4 (hrIL-4) displayed signs of exhaustion such as 1) decreased proliferation (p = 0.01), 2) increased coexpression of inhibitory receptors (p = 0.01), and 3) decreased production of IL-2 and interferon (IFN)-γ (p= 0.02, p = 0.002). Encouragingly, CART19 cells combined with an IL-4 monoclonal antibody improved antitumor activity (p = 0.045) and expansion (p = 0.01) while also decreasing the co-expression of inhibitory receptors (p = 0.02) in a mantle cell lymphoma xenograft mouse model. Building on these results, we asked if IL-4 driven exhaustion results from a direct impact of IL-4 on CART cells. To test this, we used a tumor-free assay where CART19 cells were chronically stimulated with CD19 beads in the presence of hrIL-4 or diluent. CART cells treated with hrIL-4 displayed an exhausted phenotype characterized by increased co-expression of inhibitory receptors (p = 0.04) and decreased production of IL-2 (p = 0.01). Next, we asked if IL-4 driven CART cell exhaustion is dependent on the costimulatory domain. We tested the impact of IL-4 on both CD28ζ and 41BBζ costimulated CART19 cells. Similar to our previous studies with CART19-28ζ cells, chronic stimulation of CART19-BBζ cells in the presence of hrIL-4 enhanced the exhausted phenotype as seen by increased co-expression of inhibitory receptors (p = 0.04) and decreased production of IL-2 and IFN-γ (p = 0.08 and p = 0.007). Finally, we asked if IL-4 induces exhaustion independently of its classic role in Th2 polarization of CD4 CART cells. Following CART production, we isolated CD8 cells and chronically stimulated them in the presence of hrIL-4 or diluent. CD8 CART cells treated with hrIL-4 displayed an enhanced exhausted profile as seen by 1) decreased proliferative ability (p < 0.0001), 2) increased co-expression of inhibitory receptors (p = 0.01), and 3) decreased production of IL-2 and IFN-γ (p < 0.0001, p = 0.004). Together, our data indicates a novel role for IL-4 in the development of CART cell exhaustion that is independent of tumor cells, costimulatory domain, and CD4 cells. As such, we believe IL-4 neutralization may be a widely applicable and actionable approach to improve the durable response to CART cell therapy. Citation Format: Carli M. Stewart, Elizabeth L. Siegler, Truc N. Huynh, R. Leo Sakemura, Brooke Kimball, Long Mai, Kun Yun, James H. Girsch, Jennifer Feigin, Omar Gutierrez Ruiz, Makena Rodriguez, Ekene Ogbodo, Ismail Can, Claudia Manriquez Roman, Olivia Sirpilla, Hong Xia, Jenny Kim, Justin Budka, Mike Mattie, Nathalie Scholler, Simone Filosto, Saad S. Kenderian. IL-4 drives CART cell exhaustion in a CD4 independent manner [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 37.

Abstract Introduction: Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell therapy (CART19), durable remissions remain low, especially in chronic lymphocytic leukemia (CLL). Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase and is a first line therapy for CLL. Preclinical and early clinical data indicate that ibrutinib plus CART19 may be synergistic through inhibition of IL2-inducible T cell kinase. To systematically study the effects of prior ibrutinib treatment on subsequent CART functions, we utilized blood samples from the pivotal clinical trial E1912 (NCT02048813). E1912 was a phase 3 randomized trial comparing treatment with ibrutinib/rituximab (IR) to fludarabine/cyclophosphamide/rituximab (FCR) in previously untreated patients with CLL, which led to the FDA approval of ibrutinib as a first-line therapy in CLL. Methods: We evaluated the ex vivo expansion and effector functions of T cells and CART19 generated from patients (n=12) with CLL progression following treatment with IR or FCR. We generated CART19 from longitudinal samples obtained at baseline (pretreatment), 6 months post-treatment, 12 months post-treatment, and disease progression. Isolated T cells were lentivirally transduced to express CD28- or 41BB-costimulated CAR19. To assess antigen specific killing and proliferation, untransduced T cells or CART19 were co-cultured at a 1:1 ratio with the CD19+ cell line, JeKo-1, as a surrogate for CLL cells. Results: Ex vivo T cell expansion increased from an average of 1.5-fold at baseline to 6.7-fold at 6 months, 11.6-fold at 12 months, and 16.9-fold at progression, following ibrutinib-based therapy (p = 0.02, 0.04, and 0.04, respectively). Improved T cell expansion was independent of disease burden (p = 0.89, 0.73, and 0.54 for expansion vs B cell count at 6 months, 12 months, and progression). T cell expansion significantly worsened following treatment with FCR-based therapy compared to ibrutinib-based therapy (0.6- vs 11.6-fold at 12 months and 0.9- vs 16.9-fold at progression; p = 0.031 and 0.039, respectively). Starting at 6 months post-IR, CART19 demonstrated improved antitumor activity and antigen-specific proliferation against JeKo-1. There was no significant difference in the expansion of 41BB- vs CD28-costimulated CART19 following ibrutinib treatment. However, 41BB-CART19 showed enhanced antigen-specific proliferation upon stimulation with JeKo-1 (p = 0.008, 0.02, and 0.02 at 6 months, 12 months, and progression). Conclusion: Our results indicate significant improvement in T cell expansion and CART19 cell functions in patients with CLL following frontline ibrutinib-based therapy, and worsening T cell functions after FCR treatment. Improvement in T cell functions is independent of malignant B cell eradication and is more significant in 41BB-costimulated CART19. Citation Format: Truc Ngoc Huynh, Victoria Wang, Tait Shanafelt, Elizabeth L. Siegler, Carli M. Stewart, Kun Yun, Long K. Mai, Olivia Sirpilla, James H. Girsch, Claudia Manriquez-Roman, Jennifer M. Feigin, Makena Rodriguez, Brooke L. Kimball, Hong Xia, Reona L. Sakemura, Ismail Can, Omar L. Gutierrez-Ruiz, Ekene J. Ogbodo, Erin E. Tapper, Lionel A. Fonkoua, Mehrdad T. Hefazi, Michael W. Ruff, Sameer Parikh, Neil E. Kay, Saad J. Kenderian. Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912 [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 3849.