Hefazi, Mehrdad

Introduction: Fludarabine combined with melphalan 140 mg/m2 (FluMel140) is a commonly used reduced-intensity conditioning (RIC) regimen in patients undergoing matched related (MRD) and unrelated donor (MUD) allogeneic stem cell transplant (alloSCT). Ciurea et al Blood 2020, described outcomes of Flu 160 mg/m2 and Mel 100 mg/m2. In this study, we describe our experience of attenuated melphalan dosing 100-120 mg/m2 versus 140 mg/m2 with fludarabine 125 mg/m2 (attenuated FluMel versus regular dose FluMel). Methods: We retrospectively reviewed patients from November 2016 to April 2023 with AML, ALL or MDS undergoing alloSCT who received fludarabine and melphalan. Patients who received the lower dose of melphalan were considered to be the attenuated FluMel cohort, whereas patients receiving the melphalan 140 mg/m2 were considered to be the control cohort. Kaplan-Meier and log-rank tests were used to estimate OS. NRM and relapse incidence (RI) was calculated using competing risk analysis. R 4.2.0 (R Foundation for Statistical Computing) was used for statistical analyses. Results: A total of 134 patients [92 (68.7%) males] were evaluated. Thirty-seven (27.6%) patients received attenuated FluMel and 97 (72.4%) patients received regular dose FluMel (controls). Among the patients in the attenuated FluMel cohort, 5 (13.5%) patients also received fludarabine at doses less than 125 mg/m 2 [median 20 mg/m 2 (range 87.5-112.5 mg/m 2]. Median age at transplant was 65 years (range 30 - 76 years). The most common disease was AML [75 (56.0%) patients] ( Table 1). Patients in the attenuated FluMel group had comparable iothalamate clearance compared to control cohort (clearance <65 ml/min/1.73 m2: 18.9% vs. 12.4%, P = 0.52). Patients in the attenuated FluMel group had comparable rates of high/very high DRI to those in control cohort (51.4% vs. 43.3%, P = 0.91). Compared to the control cohort, patients in the attenuated FluMel group were less likely to have a matched related donor (21.6% vs. 45.4%, P = 0.02) and were more likely to receive PTCy for GVHD prophylaxis (37.8% vs. 16.5%, P = 0.02). Among the patients receiving attenuated FluMel, the median melphalan dose administered was 100 mg/m2. The median time to neutrophil engraftment was 19 days (IQR 16-21 days), and median time to platelet engraftment was 18 days (range 12-25 days). Eleven (29.7%) patients developed post-alloSCT mucositis, and 3 (8.1%) patients developed grade 3-4 mucositis. All the patients in the attenuated FluMel group achieved 100% CD33 donor chimerism at day +30 (n=35) and day +100 (n=28) after alloSCT. Similarly, 26 (74.3%, n = 35) patients achieved ≥ 95% CD3 donor chimerism at day +30 after alloSCT and 23 (82.1%, n = 28) patients achieved ≥ 95% CD3 donor chimerism at day +100 after alloSCT. Median follow-up of the entire cohort after alloSCT was 2.3 years (95% CI 2.1-2.5 years). Median follow-up was 1.5 years in the attenuated FluMel cohort and 2.5 years for the control cohort ( P<0.001) . Survival was similar among patients receiving attenuated FluMel vs. control cohort (2-year OS 74.7% vs. 65.0%, P = 0.28). At 2 years post-alloSCT, the attenuated FluMel and control cohort had similar non-relapse mortality (19.9% vs. 24.4%, P=0.58) and relapse (22% vs. 15.2%, P=0.51). The attenuated FluMel was not associated with an increased incidence of relapse (HR 1.36, 95% CI 0.50-3.73, P=0.55) or NRM (HR 0.74, 95% CI 0.28-1.95, P = 0.55). GVHD-free relapse free (GRFS) survival was also similar among the attenuated Flu/Mel and control cohort (1-year GRFS rate 41.1% vs. 45.6%, P = 0.8). Among the patients receiving PTCy for GVHD prophylaxis, the 2-year OS in patients receiving attenuated FluMel was higher than the control group, but it was not statistically significant (2-year OS rate: 90% vs. 55%, P=0.37, Figure 1). Compared to the control group, the attenuated FluMel group had similar rates of NRM at 100 days (10% vs. 12.5%), 1 year (NA vs. 25%) and at 2 years after alloSCT (NA vs. 25%, P=0.54). GRFS at 1 year was also similar between the attenuated FluMel and control group (68.6% vs. 43.8%, P = 0.69). Conclusion: The attenuated dosed FluMel conditioning is a safe and effective regimen for elderly patients with acute leukemia and MDS, and is non-inferior to regular dose FluMel. Among patients receiving PTCy for GVHD prophylaxis, the lower dose of melphalan was non-inferior to regular dose FluMel for survival outcomes. Larger studies are needed to confirm our findings.

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.

Abstract The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)–cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)–cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

Abstract Glioblastoma multiforme (GBM) is the most aggressive and common form of brain tumor, and it is characterized by an immunosuppressive tumor microenvironment (TME) that supports tumor growth and pathology. An integral part of the GBM stroma is the mesenchymal stem cells (MSC), which promote GBM growth. Receptor tyrosine kinase EphA3, a member of the Eph family, is overexpressed in GBM tumors and in MSCs within TME, making it an attractive target for Chimeric antigen receptor T-cell (CART) therapy. CART has proven incredible promise in certain hematological malignancies therapy but remained short in solid tumors, particularly GBM. We hypothesize that concurrent targeting of cancers and TME by EphA3-CART can be a potential therapeutic option in GBM. We first showed the expression of the EphA3 receptor by Immunohistochemistry and western blot on GBM patient-derived xenograft (PDX), as well as on the SNB-19 and U87 human GBM cell lines and adipose-derived MSCs using flow cytometry. We then generated EphA3-CAR construct derived from a clinically validated anti-EphA3 scFv cloned in a lentiviral vector containing CD28 and CD3ζ, and generated EphA3 CART cells with over 90% efficiency. EphA3 CART cells demonstrated potent antigen specific effector functions. To demonstrate the antitumor activity in vivo, we subcutaneously grafted 1 × 106 GBM cells per NOD-SCID-γ-/- mice and at ~150-300 mm3 tumor volume, mice were then treated with 5 × 106 EphA3-CART or un-transduced (UTD) T cells control. Tumors were monitored by bioluminescence imaging (BLI) and caliper measurements, and mice were followed for survival. Mice treated with EphA3 CART cells exhibited a significant reduction in tumor volume (p<0.005), and prolonged overall survival (p=0.0015). Finally, we tested the ability of EphA3-CART cells to overcome MSC induced CART cell suppression by co-culturing EphA3-CART cells with MSC and EphA3+ GBM target cells while using the CD19+ mantle cell lymphoma cell line JeKo-1 and CART19 as control. MSC conditioned coculture of GBM and CART showed significant suppression of CAR19 in the CART19 - JeKo-1 system (p=000.3), but no significant suppression of EphA3-CART cells. In summary, our results indicate that EphA3 CART cells exhibited potent and specific antitumor activity in vitro and in vivo and ameliorated MSC induced inhibition of CART cell functions, representing a potentially promising therapeutic option in GBM. Citation Format: Ekene J. Ogbodo, Michael W. Ruff, Reona L. Sakemura, Claudia ManriquezRoman, Truc Huynh, James H. Girsch, Olivia L. Sirpilla, Kun Yun, Carli M. Stewart, Ismail Can, Lionel K. Fonkoua, Mehrdad Hefazi, Elizabeth L. Siegler, Saad S. Kenderian. Simultaneous targeting of EphA3 on glioblastoma and tumor microenvironment to overcome resistance to CART cell therapy in brain cancer. [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 5017.

BACKGROUND: Pulmonary function test (PFT) impairments are common after allogeneic hematopoietic stem cell transplantation. The prognostic significance of these declines on outcomes is not well understood. The objectives were to determine the frequency of declines in pulmonary function (FVC, FEV 1 , and diffusing capacity for carbon monoxide [D LCO ]) in the early post-transplantation period; and to determine the prognostic significance of these declines on mortality or development of bronchiolitis obliterans syndrome. METHODS: This was a retrospective cohort study conducted at Mayo Clinic, Rochester, Minnesota. PFTs were obtained at baseline and at day +100. Competing risk survival models were developed, which accounted for pre-transplantation pulmonary function and relapse status. RESULTS: Between January 1, 2005, and December 31, 2020, 1,145 subjects underwent allogeneic hematopoietic stem cell transplantation and had a pre-transplantation PFT performed. Of these, 900 (78.6%) survived to day 100 and had post-transplantation PFTs performed (median [interquartile range] 97 [94–103] d). A decline of ≥10% in FEV 1, FVC, or D LCO was seen in 401 of 900 subjects (44.5%). Declines of ≥20% in FEV 1 (hazard ratio 1.65, 95% CI 1.07–2.56; P = .02), FVC (hazard ratio 1.72, 95% CI [1.11–2.67]; P = .02), and D LCO (hazard ratio 1.46, 95% CI 1.04–2.07; P = .028) were all associated with reduced survival when compared with those with < 10% decline in PFT measures. These findings were independent of pre-transplantation pulmonary function or relapse status. Bronchiolitis obliterans syndrome was diagnosed in 118 subjects (10.3%), and there was no relationship between early PFT decline and a subsequent diagnosis of bronchiolitis obliterans syndrome. The subjects who received myeloablative conditioning with cyclophosphamide plus total body irradiation or cyclophosphamide plus fludarabine plus total body irradiation were more likely to have lower spirometry values after hematopoietic stem cell transplantation. The subjects who received reduced intensity conditioning or nonmyeloablative conditioning with fludarabine plus total body irradiation were more likely to have higher post–hematopoietic stem cell transplantation FEV 1 , FVC, and D LCO . CONCLUSIONS: An absolute decline of ≥20% in FEV 1 , FVC, or D LCO were associated with reduced survival independent of pre-transplantation pulmonary function or relapse status. In contrast to previous work, early declines in PFT measures were not associated with future development of bronchiolitis obliterans syndrome.

Abstract CD19 directed chimeric antigen receptor T (CART19) cell therapy has resulted in remarkable outcomes in B cell malignancies and was FDA approved in multiple indications. However, durable remissions are limited to 40% of treated patients. Inhibitory myeloid cells in tumor microenvironment have been found to suppress T cell expansion and contribute to failure of CART19 cell therapy. In this study, we aimed to unravel the interactions between monocytes, CART19 cells and tumor cells to understand how monocytes-CART19 cell interactions impact CART19 cell effector functions and clinical outcomes. Two sets of experiments were conducted, 1) use of healthy CART19 cells, CD19+ tumor cells, and healthy monocytes; 2) use of brexu-cel products from ZUMA-2 clinical trial treating mantle cell lymphoma (MCL), patient-matched monocytes and circulating MCL tumor cells (n = 11; 6 durable responders, 2 relapsed after initial response and 3 non-responders). CD28 costimulated CART19 (CART19-28ζ) cells generated in the lab from healthy donors were co-cultured with donor freshly isolated monocytes in the presence of Jeko-1 cells (a CD19+ MCL cell line). CART19 antigen specific proliferation was not inhibited by freshly isolated monocytes. When monocytes were co-cultured with CART19 and tumor cells, higher levels of eotaxin, GRO, MCP-3 and IL-7 were detected. When CART19 cells were co-cultured with the CD19+ JeKo-1 cells in the presence of ex vivo M2 polarized macrophages, CART19 antigen specific proliferation was inhibited (p=0.0045). Transwell experiments demonstrate that M2-induced CART19 inhibition is not contact dependent. Cytokine profile analysis indicated increased level of IL-1ra, IP-10 and MCP-1 and decreased level of IL-17A, sCD40L, IL-9 and MIP-1α when M2 macrophages were co-cultured with CART19 and tumor cells compared to co-cultures of tumor cells and CART19. Then we conducted ex vivo co-cultures of brexu-cel products, autologous monocytes and circulating MCL tumor cells from MCL patients (ZUMA-2) collected prior to CART19 cell infusion. Here we observed trends of elevation of IL-13 and IL-5 and reduction of GRO, MCP-3, MIP-1β and IL-8 in non-responders, compared to responders (durable responses or relapsed patients). Our results support that monocyte- and macrophage-dependent cytokine release could modulate CART19 effector and trafficking functions, and thus CART19 clinical outcomes. This warrants further investigation around strategies to improve durable responses to CART cell therapy. Citation Format: Kun Yun, Reona Leo Sakemura, Michelle J. Cox, Truc Huynh, Claudia Manriquez-Roman, Olivia Sirpilla, Carli M. Stewart, James H. Girsch, Ekene J. Ogbodo, Ismail Can, Brooke Kimball, Lionel A. Kankeu Fonkoua, Mehrdad Hefazi, Michael W. Ruff, Elizabeth L. Siegler, Mike Mattie, Sao-Mai Nguyen-Mau, Simone Filosto, Saad S. Kenderian. Impact of immunosuppressive monocytes on CART19 cell effector functions and outcomes. [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 4082.