Sakemura, Reona

Brexucabtagene autoleucel (brexu-cel), a CD28-costimulated CD19-targeted chimeric antigen receptor T cell (CART19) product, was FDA-approved in 2020 after demonstrating remarkable efficacy in the pivotal ZUMA-2 trial of patients with mantle cell lymphoma (MCL). However, despite the impressive initial activity of CART19 cell therapy in MCL, most patients relapse within the first 3 years following treatment. Myeloid cells, such as monocytes and macrophages, have been demonstrated to be major inhibitors of other immune cells, including T cells. Growing evidence shows that monocytes and macrophages contribute to CART cell failure, although the exact mechanisms are not fully understood. To better understand the mechanisms contributing to CART treatment resistance in MCL, we first utilized brexu-cel products from patients with MCL on the ZUMA-2 trial to uncover their transcriptomic features by single cell RNA sequencing (scRNAseq) analysis. To understand if myeloid cells had negative impacts on clinical outcomes, we then analyzed autologous baseline peripheral blood mononuclear cell (PBMC) samples by scRNAseq. Our analyses of the brexu-cel samples revealed reduced response to IL-1β and its downstream signaling pathway as well as impaired T cell functions in non-responders. Transcriptomic analyses of the autologous baseline monocyte populations showed downregulation of IL-1β production and its associated pathways as well as upregulation of M2 phenotype-associated genes and IL-1 receptor antagonist (IL-1ra) in non-responder monocytes, implying a potential role of myeloid cells in CART resistance through impairment of IL-1 signaling in brexu-cel products. To validate our findings from the ZUMA-2 samples, we further studied the interactions between macrophages, MCL cells, and CART19 cells in preclinical models. First, CD28-costimulated CART19 cells generated from healthy donors were cocultured with JeKo-1, a CD19+ MCL cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression by M2-like macrophages was contact-independent. Cytokine profile analysis of coculture supernatants showed significant elevation of IL-1ra in M2-like macrophage cocultures (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292). This led us to hypothesize that M2-like macrophages inhibit CART19 by secreting IL-1ra to block IL-1 signaling in CART19. To test this hypothesis, we interrogated the role of IL-1ra in CART19. CART19 + JeKo-1 cocultures were supplemented with IL-1β + IL-1ra or IL-1β + IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 proliferation was improved by IL-1β (vehicle control vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra, p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Next, we assessed the impact of IL-1ra on CART functions in mouse MCL xenografts in the presence of M2-like macrophages. NOD-SCID-γ-/- (NSG) mice were subcutaneously injected with 0.5 million of human macrophages and 1 million of luciferase+ JeKo-1. Mice were then randomized once tumor engraftment was confirmed by bioluminescence imaging (BLI), followed by treatment with 2 million CART19 with 10 mg/kg IL-1ra neuAb or control IgG for 3 weeks. Combination of CART19 cells and IL-1ra neuAb led to improved antitumor activity based on BLI measurements compared to control IgG (IL-1ra neuAb vs control IgG, p=0.0058). Additionally, expression of IL-1RI, receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which might potentially desensitize CART response to IL-1β, resulting in further suppression in CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Overall, our study elucidates a potential mechanism of IL-1 pathway modulation mediated by myeloid cells in contributing to CART19 failure in MCL.

CD19-targeted chimeric antigen receptor T (CART19) cell therapy has shown outstanding clinical success. However, patients can develop CART-induced toxicities, including cytokine release syndrome (CRS) and neurotoxicity. Studies have found that activation of myeloid cells by granulocyte monocyte-colony stimulating factor (GM-CSF) can contribute to toxicity development. GM-CSF depletion leads to reduced myeloid activation, prevention of CART-induced toxicities, and improved CART efficacy. CART cells upregulate cytokine receptors upon activation, and our results indicate that CART19 cells significantly upregulate GMCSFR upon antigen-specific stimulation (<10% vs >30%, p<0.01). This led us to hypothesize that GMCSFR signaling contributes to CART cell activity. To understand the role of GM-CSFR upregulation on CART cell functions, we compared the impacts of neutralizing GM-CSF or blocking GM-CSF receptor alpha (GM-CSFRα) on CART19. First, 4-1BB-costimulated CART19 cells generated from healthy donors were stimulated with CD3/CD28 beads at a 1:3 ratio for 6 days. CART cells were maintained at 1 x 106/mL throughout the culture and supplemented with IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade at 10 μg/mL, 20 μg/mL, or 100 μg/mL. On day 6, cells were debeaded and rested for 2 days. Then, CART cells were cocultured with NALM6, a CD19+ acute lymphoblastic leukemia cell line, at a ratio of 1:1 for 3 days. The cocultures were supplemented with the same doses of antibodies listed above. Antigen-specific proliferation of CART cells was measured by flow cytometry. Our data suggested that CART19 proliferated is not impacted by GM-CSF neutralization but is inhibited by GM-CSFRα blockade (IgG isotype vs 10 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 20 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 100 μg/mL GM-CSFRα blockade, p=0.009). CART cells were assessed in a cytotoxicity assay with NALM6 luciferase+ cells as well, which did not show significant impacts from GM-CSF neutralization or GM-CSFRα blockade. To evaluate the impacts of GM-CSF neutralization and GM-CSFRα blockade in a mouse model, we engrafted NSG mice with 1 million NALM6 luciferase+ cells intravenously. Mice were then treated with 1.4 million CART19 plus 10 mg/kg IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade intraperitoneally. GM-CSFRα blockade drastically suppressed antitumor activities of CART (CART19+GM-CSFRα blockade vs CART19+IgG isotype control, p<0.0001; CART19+GM-CSFRα blockade vs CART19+GM-CSF neutralizing antibody, p<0.0001; CART19+ GM-CSFRα blockade vs untransduced T cell, n.s.). Serum cytokines were analyzed 2 days after CART treatment, showing that mice with GM-CSFRα blockade had significantly lower levels of human IFNγ (p=0.0189) and IL-2 (p=0.0121) compared to IgG isotype control group, while GM-CSF neutralization did not show significant impacts. Mice treated with CART plus IgG isotype control or GM-CSF neutralizing antibody showed prolonged survival compared to mice treated with GM-CSFRα blockade or untransduced T cells (p=0.0285). To unravel the mechanisms behind functional differences between GM-CSFRα blockade and GM-CSF neutralization, CART cells (n = 3 biological replicates) were cocultured with NALM6 at a 1:1 ratio supplemented with 10 μg/mL GM-CSF neutralizing antibody, GM-CSFRα blockade, or IgG isotype control overnight, followed by T cell isolation, bulk RNA isolation, and RNA sequencing (RNAseq) analysis. Our RNAseq data indicated significant upregulation (Log2FC>0.5, padj. <0.05) of T cell exhaustion- and dysfunction-associated genes (RGS16, PDCD1, CTLA4, NR4A3, CD101, LAG3) as well as negative regulators of T cell functions (G0S2, PIK3IP1, LGALS3) in the GM-CSFRα blockade group compared to GM-CSF neutralizing antibody or IgG isotype control groups. T cell activation-associated pathways were enriched in the GM-CSFRα blockade group. Overall, our data show that GM-CSFRα blockade impacts CART functions differently from GM-CSF neutralization. Specifically, GM-CSFRα blockade inhibits CART proliferation in vitro and antitumor activity in vivo. Interestingly, GM-CSFRα blockade leads to enrichment of T cell exhaustion- and dysfunction-associated genes in CART19, which is worth further investigation. These findings may be applicable to other cytokine receptors upregulated on activated CART cells.

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 CD19-directed chimeric antigen receptor T (CART19) cell therapy has shown remarkable outcomes in B cell malignancies and was FDA approved in multiple indications, but durable remissions are limited to ~40%. Inhibitory myeloid cells in the tumor microenvironment have been found to suppress T cell expansion and contribute to CART19 failure. Here, we studied interactions between monocytes, CART19, and tumor cells to understand the impact of monocytes on CART19 effector functions. First, CD28-costimulated CART19 (CART19-28ζ) generated in the lab from healthy donors were cocultured with JeKo-1, a CD19+ mantle cell lymphoma cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression from M2-like macrophages was not contact-dependent. IL-1 receptor antagonist (IL-1ra) was significantly elevated in M2-like macrophage coculture supernatant (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292) and intracellularly in M2-like macrophages after coculture (90%+). We therefore hypothesized that M2-like macrophages inhibit CART19 by secreting IL-1ra, which blocks IL-1 signaling in CART19. We interrogated the role of IL-1ra in CART19. JeKo-1 was cocultured with CART19, supplemented with rhIL-1β only, IL-1β + IL-1ra, or IL-1β +IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 antigen-specific proliferation was improved by IL-1β (PBS vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Additionally, expression of IL-1RI, the receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which is a potential mechanism of blocking CART response to IL-1β, resulting in further suppression of CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Next, we assessed the impact of IL-1ra on CART cell functions in the presence of M2-like macrophage in a mantle cell lymphoma xenograft model. NOD-SCID-γ−/- (NSG) mice were subcutaneously injected with human macrophages and luciferase+ JeKo-1 cells. Upon tumor engraftment by bioluminescent imaging, mice were randomized to treatment with CART19 + IL-1ra neuAb or control IgG for 3 weeks. CART19 + IL-1ra neuAb led to improved antitumor activity (IL-1ra neuAb vs control IgG, p=0.0033). Overall, our study revealed a role of IL-1ra in macrophage-induced CART inhibition mediated by blocking IL-1β signaling through IL-1RI. Citation Format: Kun Yun, Reona Sakemura, Truc Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli Stewart, James Girsch, Ekene Ogbodo, Ismail Can, Jennifer Feigin, Long Mai, Hong Xia, Brooke Kimball, Makena Rodriguez, Lionel Kankeu Fonkoua, Mehrdad Hefazi, Michael Ruff, Elizabeth Siegler, Saad Kenderian. Immunosuppressive monocytes suppress CART19 functions through modulation of the IL-1 pathway [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 6813.

Abstract Despite the remarkable efficacy of chimeric antigen receptor T -cell therapy (CART) for the treatment of B-cell malignancies, durable remission remains limited often due to tumor relapse. We have previously demonstrated that tumor-derived extracellular vesicles (EVs) induced CART cell failure in preclinical models. To characterize the impact of tumor EV induced CART cell dysfunction, we assessed microRNA (miRNA) profiles contained within tumor EVs from two independent patient cohorts and studied how they contributed to CART dysfunction. Our first cohort included patients with chronic lymphocytic leukemia (n=50). We previously showed that CLL patient derived EVs induce a state of CART cell dysfunction. To understand this dysfunction, we interrogated the miRNA cargo significantly enriched in CLL EVs and how their coculture impacts CART signaling by RNAseq. Here, we found a significant enrichment in miR-125a within CLL EVs as compared to healthy donor controls (p=.0001). CART cocultured with CLL EVs showed alterations in T cell activation pathways, where miR-125a targets TNFAIP3 and NFkB pathways were activated. These findings implicate miR-125a from CLL EVs in CART dysfunction. Next, we aimed to validate the impact of miRNA on CART cell outcomes in the clinic. We interrogated miRNA profiles from patients with diffuse large B cell lymphoma (DLBCL) treated with CART cell therapy (CART19-28ζ, axicabtagene ciloleucel). EV miRNA cargo was compared between responders (complete remission at 6 months, n=10) and non-responders (no response 1 month post CART19, n=10) to identify differentially enriched miRNAs across groups. miR-125a was again significantly upregulated in EVs of non-responders as compared to responders to CART19 treatment (fold change=3.28, padj=.018). To study the direct effect of miR-125a on CART cell functions, we generated and studied the effector functions of CART19-28z following transfection with miR-125a or negative miR control. CART19 cells transfected with miR-125a showed a dose dependent decrease in killing of lymphoma cells as compared to miR-control (p=.0001). miR-125a also significantly increased the rate of apoptosis in CART19 cells (fold change=1.95, p=.024) and decreased the level of CART proliferation (fold change=.71, p=.013) as compared to controls. Notably, miR-125a did not impact tumor cell growth, further verifying a direct CART effect. In summary, our studies demonstrate the impact of tumor derived miRNAs on CART cell outcomes. They identify that miR-125a is 1) associated with CLL EV induced CART dysfunction, 2) significantly elevated in non-responders to CART therapy in the clinic, and 3) able to decrease CART19 efficacy through reduced CART killing/proliferation and increased apoptosis. These results highlight a novel mechanism of CART19 failure induced through miRNA cargo delivery by tumor-derived EVs and the role of miR-125a in inducing CART19 dysfunction. Citation Format: Olivia Sirpilla, R. Leo Sakemura, Ismail Can, Truc N. Huynh, James H. GIrsch, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ekene J. Ogbodo, Long Mai, Omar Gutierrez Ruiz, Makena Rodriguez, Brooke Kimball, Hong Xia, Jose C. Villasboas, Stephen Ansell, Nora Bennani, Patrick B. Johnston, Jonas Paludo, Januario Castro, Mohamed Kharfandabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth Siegler, Saad Kenderian. Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies [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 3841.

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.

Our group has reported that transient CD19 antigen masking of leukemic cells with the CD19 monoclonal antibody (mAb), tafasitamab, resulted in reduction of CD19 chimeric antigen receptor T cell (CART19) apoptosis, enhanced CAR T proliferation, improved anti-tumor activity, diminished tumor pyroptosis, and reduced severity of cytokine release syndrome (CRS) in preclinical models. In this report, we aimed to study the mAb characteristics responsible for these observations. Specifically, we hypothesized that binding affinity of CD19 mAbs might affect the subsequent activation and apoptosis of CART19 as well as tumor cell pyroptosis in preclinical models. To test our hypothesis, we first determined the affinities of the CD19-targeting antibodies tafasitamab, HD37 and FMC63 to recombinant human CD19 protein. Tafasitamab demonstrated the strongest binding affinity among the three tested antibodies [tafasitamab ( K D: 1.0±0.2 nM), HD37 ( K D: 4.1±0.3 nM), FMC63 ( K D: 3.4±1.2 nM)]. Next, we studied CART19 apoptosis in the presence of these CD19 mAbs with different binding affinities. Here, we co-cultured CART19, CD19 + mantle cell lymphoma JeKo, and 400 μg/mL of CD19 mAb for 1 hour and measured CD3 + 7-AAD - Annexin V + T cells with flow cytometry. The number of apoptotic CART19 cells was significantly reduced by tafasitamab co-treatment compared to HD37, FMC63, or control IgG [control IgG (mean = 19.1, standard deviation (SD) = 0.8), tafasitamab (mean = 13.1, SD = 2.9), HD37 (mean = 17.8, SD = 0.8), FMC63 (mean = 18.4, SD = 1.0)]. We also performed an in vitro high mobility group box 1 (HMGB1) assay to assess if different affinities of the CD19 mAbs affect tumor cell pyroptosis triggered by CART19. Here, we co-cultured CART19, CD19 + JeKo, and 400 μg/mL of CD19 mAb for 24 hours and assessed HMGB1 on JeKo. In analogy to the apoptosis data, tafasitamab was the only mAb that prevented tumor cell pyroptosis, indicated by reduced HMGB1 expression (Fig 1A). Given these results, we aimed to study the impact of CD19 occupancy with different CD19 mAbs prior to CART19 infusion on tumor pyroptosis in vivo. We utilized a mouse model that mimics CAR T toxicities. Immunocompromised NSG mice were inoculated via tail vein with 5x10 6 leukemic cells derived from patients with relapsed/refractory acute lymphoblastic leukemia. Two weeks later (day -7), tumor burden was assessed by peripheral blood sampling. Leukemic cells were defined as murine CD45 -, human CD45 + CD19 + CD20 + via flow cytometry, and volumetric absolute number was calculated. Mice were then randomized according to tumor burden to receive IgG control [Group 1 (G1)], tafasitamab (G2), HD37 (G3), or FMC63 (G4) (5 mg/kg, ip) at day -7. CRS was determined by weight loss, development of motor weakness, poor body posture, and elevated serum cytokine levels. At day 0, all mice received 3x10 6 luciferase + CART19 via tail vein. Mice were monitored with daily bioluminescent imaging (BLI) to track and quantify CART19 expansion and physical assessment to detect toxicity. BLI on day 4 revealed that CART19 expansion in the tafasitamab-treated group (G2, mean = 1x10 7, SD = 2.3x10 7) was significantly lower than in the control group (mean = 3.5x10 7, SD = 9.3x10 7), the HD37-treated group (G3, mean = 4x10 7, SD = 6.6x10 7), and the FMC63-treated group (G4, mean = 2x10 7, SD = 3.59x10 7), [G1 vs G2: 95% confidence interval (CI) 3.5x10 7 to 1.1x10 8, p <0.0001, G1 vs G3: 95% CI -7.5x10 7 to -1.1x10 8, p=0.005, G1 vs G4: 95% CI -4.5x10 7 to 1.9x10 7, p=0.71] indicating that tafasitamab pretreatment significantly reduced early activation of CART19 compared to IgG control and HD37 in this model. Mice were bled on day 4 to analyze plasma cytokine. Tafasitamab-treated mice (G2) secreted significantly less IFNγ (Fig 1B), IL10 (Mean G1: 78, G2: 2.52, G3: 12.2, G4: 13.9 pg/mL), MDC (Mean G1: 4072, G2: 445, G3: 921, G4: 1364 pg/mL), and MIP1β (Mean G1: 283, G2: 59.1, G3: 148, G4: 123 pg/mL) compared to G1, G3, and G4. Satellite mice were euthanized on day 1 and spleens were harvested. Western blot analysis of splenocytes revealed that G1, 3, and 4 showed significantly lower expression of apoptosis-inducing protein gasdermin E on leukemic cells compared to G2, indicating reduced tumor pyroptosis in G2. In summary, application of the high affinity mAb tafasitamab prior to CART19 resulted in reduced CAR T apoptosis, diminished tumor cell pyroptosis, and reduction of CRS compared to lower affinity CD19 mAbs in preclinical models.

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

Abstract In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.