ALBERT

Description: Background: Outcomes for adults and children with acute myeloid leukemia (AML) are dismal with 20-40% and 60% 5-year event-free survival, respectively. Alternative therapeutic strategies for AML are thus needed to improve outcomes. Chimeric antigen receptor (CAR) T cell immunotherapy has induced remarkable clinical responses in multiple phase 1 clinical trials for patients with relapsed or chemorefractory B cell leukemias, encouraging great interest in developing similar approaches for AML. Prior studies have demonstrated efficacy of CD33 or CD123-redirected CAR T cells in AML models, although the genetic heterogeneity of AML will likely require identification of additional therapeutic targets. In the current studies, we report preliminary in vitro and in vivo efficacy of new CAR T cells targeting the FMS-like tyrosine kinase 3 (FLT3) in human AML. FLT3 mutations via internal tandem duplication or kinase domain point mutations occur in approximately 25% of AML and result in FLT3 surface protein overexpression, suggesting potential efficacy of FLT3-targeting therapies. Both types of FLT3 alterations induce ligand-independent activation of FLT3 signaling, further demonstrating a critical role of FLT3 in AML pathogenesis. Hypothesis: FLT3 is a promising target for CAR T cell immunotherapy based treatment of AML. Results: Quantitative flow cytometric analysis of human AML cell lines demonstrated FLT3 surface expression ranging from 1338 (MOLM-13), 2594 (MOLM-14), and 2710 (MV4;11) receptors/cell versus 623 receptors/cell on negative control U937 cells. We first generated FLT3-redirected CAR construct consisting of a single chain variable fragment (scFv) derived from a well-characterized anti-human FLT3 antibody coupled to T cell 4-1BB (CD137) costimulatory and CD3-zeta activation domains. CD33 CAR T cells based on Gemtuzumab created by identical methodologies were also used as AML CAR T cell controls. In vitro studies verified that human T cells transduced with the FLT3 CAR construct induced interferon-gamma and interleukin-2 production after co-culture with AML cell lines MOLM-13, MOLM-14, and MV4;11. One dose of FLT3 CAR T cells inhibited leukemia proliferation in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with FLT3-mutant MOLM-13 or MOLM14 cell lines. These first data demonstrate potent preclinical activity of FLT3 CAR T cells and warrant further study in additional AML models. However, on target/off tumor toxicities can occur with AML antigen-targeted immunotherapies, as previously reported in studies of CD33 and CD123 CAR T cells. Normal expression of FLT3 has been mainly described on CD34+ hematopoietic progenitor stem cell populations, and FLT3-targeted therapies have potential to induce aplastic anemia. To address this question of hematologic toxicity of FLT3 CAR T cells, we created normal human hematopoiesis xenograft models in NOD scid gamma Il3-GM-SF (NSGS) mice engrafted with CD34+ cord blood cells for treatment with anti-AML CAR T cells. No difference in human granulocyte numbers was observed in marrows of engrafted mice treated with FLT3 CAR T cells, CD33 CAR T cells, or non-transduced T cells. A significant reduction in monocytes was observed in FLT3 CAR T cell-treated animals, however (p

Description: Background: Cytokine release syndrome (CRS) in the setting of CAR T cell therapy manifests as a wide constellation of symptoms with multi-organ involvement. CRS can vary from a mild, self-limited course to a life-threatening systemic inflammatory response which, in severe cases, may be associated with manifestations similar to those seen in hemophagocytic lymphohistiocytosis (HLH). Tocilizumab, an anti-human IL-6 receptor antibody, has become a widely accepted pharmacologic intervention of first choice in severe CRS based on the observation of elevated levels of inflammatory cytokines, most notably interleukin (IL)-6 and interferon (IFN)-γ. Indeed, following administration of tocilizumab, most patients show rapid signs of improvement. However, in rare circumstances, CRS may be refractory to tocilizumab, and repeat administration or institution of additional immunosuppression is needed. Here we summarize cytokine profiles and CRS seen in patients treated with anti-CD22-CAR T cells and propose tocilizumab-refractory CRS as a potentially distinct pathophysiological entity from typical CRS that may merit alternative immunosuppressive interventions other than tocilizumab. Methods: Children and young adults with relapsed/refractory CD22+ ALL were treated with anti-CD22 CAR T cells. Serial samples for serum cytokine levels (IFN-γ, IL-6, IL-2, IL-10, IL-12p70, IL-1β, IL-15, IL-13, IL-4, IL-8, TNF-α, GM-CSF, MIP1-α) were obtained at pre-specified time points (0, 12, 24, 48, 72 hours, then daily on days 4 - 14 and 28 following CAR T cell infusion). Transduced CAR T cell dosage ranged from 3x10e5 cells/kg (dose level [DL] 1), 1x10e6 cells/kg (DL2), and 3x10e6 cells/kg (DL3). CRS severity was determined according to recently proposed grading system (Lee DW et al., Blood. 2014). Disease burden was assessed using standard morphology and flow cytometry analysis of bone marrow and peripheral blood samples. Results: Cytokine profiles are available on 10 patients treated: first 9 patients enrolled in our phase I trial (NCT02315612), and the tenth patient was treated off-protocol on an emergency investigational new drug protocol given lack of alternative treatment option for rapidly progressing disease. All subjects, median age 20 years (range, 6-22 years), had a diagnosis of multiply relapsed ALL. Seven of 10 subjects developed CRS. Five subjects with CRS were complete responders to CAR therapy (Table). The median time to the onset of CRS was 9 days (range, 7-12 days) post-infusion and resolved within 1 week with supportive care alone except in one patient who received pharmacologic intervention for grade 4 CRS. Rise in C-reactive protein (CRP) tended to correlate with clinical severity of CRS. Chronological changes in the level of IFN-γ, IL-6, IL-1β, IL-8, TNF-α, and MIP1-α generally mirrored the CRP trend, typically preceding CRP change by 1-2 days. In contrast to the CRS (maximum grade 2) seen in the first 9 patients, the 10th patient treated at DL3 developed grade 4 CRS with manifestations characteristic of HLH unresponsive to tocilizumab. Cytokine profile for this patient, compared to those of other CRS patients, was notable for a substantially higher serum IL-2 (35 pg/mL vs median 6.1 (range, 1.2-13.5)) and GM-CSF level (28 pg/mL vs median 1.0 (range, 0-6.1)) at 12 hours post infusion. Subsequent CRP elevation was not initially accompanied by a rise in IL-6 as in other patients, which may have explained the lack of response to tocilizumab (Figure). Evaluation for a genetic cause of HLH did not reveal any mutations (PRF1, MUNC13-4, RAB27A, STX11, STXBP2). Although this patient was a complete responder to therapy, the clinical course was complicated by pre-existing respiratory compromise and bacteremia, which may have contributed to increased CAR toxicity with variability in the cytokine profile. Conclusion: Based on our early experience, we postulate that patients with an early increase in GM-CSF and IL-2 may potentially experience more atypical and severe CRS, which without a concomitant rise in IL-6, may not respond to tocilizumab and thus early intervention with other immunosuppression may be indicated. Analysis of larger numbers of patients is required to better delineate clinical confounders and to develop rational pharmacological approach to CAR-mediated inflammatory responses. Ongoing efforts are underway to further analyze clinical samples for biomarkers. Disclosures Mackall: NCI: Patents & Royalties: B7H3 CAR.

Description: The myelodysplastic syndromes (MDS) are clonal hematopoietic malignancies characterized by dysplasia, ineffective hematopoiesis and a propensity for progression to acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for the majority of patients. However, overall survival (OS) of patients with MDS following allogeneic HSCT is only about 40%, due to both relapse and non-relapse mortality (NRM) including graft versus host disease (GVHD). Available data suggests that long-term survival following HSCT for MDS is due both to myeloablative therapy and a graft versus tumor (GVT) effect. Mice that express a NUP98-HOXD13 (NHD13) transgene develop MDS with virtually 100% penetrance. In order to develop a pre-clinical model for the study of MDS HSCT, we transplanted NHD13 mice, which are bred on a C57 Bl6 background, with bone marrow harvested from syngeneic C57Bl6 donors. Sub lethally irradiated (650 rad) recipient NHD13 mice transplanted with syngeneic donor cells relapsed early, with no therapeutic benefit in terms of hematologic parameters in peripheral blood or survival. However, lethally irradiated (1000 rad) recipient mice that were transplanted with syngeneic donor bone marrow (BM) showed complete normalization of peripheral blood counts significantly enhanced survival (median survival of 15 months) compared with non-transplanted NHD13 mice (median survival 10 months). Although there were no detectable MDS cells for up to 38 weeks post-transplant, all mice eventually relapsed and died. In order to determine if a GVT effect could enhance survival, we performed 3 types of allogeneic HSCT with donor BM that was mismatched at minor histocompatibility antigen loci (C3H.SW x C57Bl6 donors); donor BM only, donor BM with donor splenocytes (6 x 10E06 CD3+ T cells), and donor BM with donor regulatory T cells (Treg). None of these forms of allogeneic HSCT let to enhanced survival compared to that achieved with syngeneic HSCT. The early relapse rate for allogeneic HSCT with donor BM only was decreased compared to the syngeneic HSCT group (8.3% vs 28% at post-transplantation week 6 and 17% vs 43% at post-transplantation week 16); however, the relapse rate at 38 weeks was similar between the two groups (83.3% vs 85.7%). Adding donor splenocytes, containing reactive T-cells, dramatically decreased the relapse rate, such that the relapse rate was only 20% at post-transplantation week 38, suggesting a GVT effect. This GVT effect was accompanied by a severe GVH effect, and OS was not different between the allogeneic BM + splenocyte and the syngeneic HSCT groups. In an attempt to induce a GVT effect without a severe GVHD, we transplanted allogeneic Treg cells along with allogeneic BM, however, survival and relapse rates were similar to those with allogeneic BM only. Taken together, these findings suggest that a lethal dose of ionizing radiation (1000 rads) is insufficient to eradicate the MDS initiating cell, and that transplantation of donor CD3+ splenocytes leads to decreased relapse rates, but at the cost of severe GVHD. We suggest that the NHD13 mice are a feasible pre-clinical model for the study of HSCT for MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Background: With advances in immunotherapeutic approaches and the recognition of antigen modulation as a mechanism of relapse, it is imperative to understand the impact of sequential targeting strategies and the role it may have on outcomes of future therapies to optimize timing of therapeutic interventions. We previously reported on the safety, feasibility, and efficacy on our phase I dose escalation anti-CD22 CAR protocol (clinicaltrials.gov/NCT02315612).1 Based on our initial experience, we identified CD22 loss or diminution of CD22 as a risk factor for relapse following CD22 directed CAR therapy. With development of other CD22 directed therapies, we retrospectively analyzed impact of prior CD22 targeted therapy on response to CD22 CAR in our ongoing clinical trial. Design: Children and young adults with relapsed/refractory CD22+ hematologic malignancies eligible for our phase I dose escalation anti-CD22 CAR protocol were enrolled on study (Clinicaltrials.gov NCT02315612). All had bone marrow evaluations at baseline, prior to lympho-depleting chemotherapy (Fludarabine 25 mg/m2 x 3 days and Cyclophosphamide 900 mg/m2 x 1 day) and again at day 28 (+/- 4 days) post-CAR infusion. We retrospectively analyzed the impact of prior CD22 directed therapy on outcomes following CD22 CAR and specifically looked at the variables of CD22 antigen expression prior to CAR infusion (% positive and antigen density) and compared responses to CD22 CAR for those who did and did not receive prior CD22 targeted therapy. Results: From December 2014 to July 2018, 43 subjects with ALL were treated. All had active bone marrow involvement at baseline, the majority with an M2 marrow (〉5% blasts) or higher disease burden. 34 had a prior transplant and 26 were previously treated with CD19 CAR. Fourteen subjects had received prior CD22 directed therapy, including CD22 CAR elsewhere (n=2) or inotuzumab ozagamicin (Ino) (n=13). Subjects received a median of 3 doses of Ino (3-6 doses) and the median time from last Ino exposure was 2 months (range 1-20 months). Median CD22 antigen expression on bone marrow leukemic blasts prior to planned lymphodepletion for those who had received prior CD22 therapy compared to those who did not was 2527 (882-9079) vs 3929 (846-13452), respectively (one-tailed p=0.05, Figure 1). (Figure 1). Complete remission (CR) rates following CAR-T infusion for those who had prior CD22 directed therapy compared to those who did not was 57% and 71%, respectively with MRD negativity by flow cytometry achieved in only 5/8 (62.5%) patients versus 18/20 (90%) respectively and residual disease in those not achieving MRD negativity was CD22 dim. Both subjects who had received prior CD22 CAR elsewhere were non-responders to our construct with a first-infusion, however one subject converted to a CR with an intensified lymphodepletion and a second infusion. Two subjects who had received prior Ino were noted to have partial CD22 expression on at least one time point (69-89% positivity) prior to enrollment. One of these patients with pre-existing CD22 partial positivity (69% positivity) had evidence for CAR-T cell expansion but had residual low CD22 expressing disease at restaging. Notably, another subject who received 6 doses of Ino prior to receiving CD22 CAR T-cells and had uniformly CD22+ disease at enrollment, emerged with CD22 negative disease following CD22 CAR. Durability of remission also significantly differed amongst the two groups. Median time to relapse in patients who received prior CD22 directed therapy was 2 months (range 2-5 months) versus 6 months (range 2-13 months) for those who did not receive prior CD22 targeted therapy, with the majority relapsing with CD22 negative disease. Conclusion: Sequential targeting of CD19 has anecdotally increased the possibility of CD19 negative relapses, and our data provide evidence for a similar phenomenon with sequential targeting of CD22. Most notably, CD22 expression in patients who had received prior CD22 targeted therapies was lower compared to those who did not. This may have ultimately contributed to both of the observed findings of decreased response rates and decreased durability of remission, the majority of whom relapsed with CD22 negative disease following sequential targeting. This observation contributes to the increasing fund of knowledge regarding optimization of targeted therapies. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Acute graft versus host disease (GVHD) remains as the major complication after allogeneic bone marrow transplant (BMT) resulting in organ toxicity and immune dysfunction. Indeed, we have previously demonstrated that GVHD impairs responses to dendritic cell vaccines. The pathophysiology of GVHD involves preparative regimen-induced inflammation of target organs, release of inflammatory mediators such as gamma interferon (IFNg), and subsequent activation of alloreactive T cells. Given that IFNg can both contribute to GVHD and provide beneficial immune responses, we explored the potential role of IFNg on GVHD and post-transplant immunocompetence. METHODS: We utilized a minor histocompatibility antigen mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) as a means of controlling the induction of acute GVHD and to provide a source of immunocompetence in a thymectomized mouse. To study the role of IFNg on GVHD, we chose either IFNg receptor 1 (IFNgR1) −/− marrow or DLI to permit the normal production of IFNg in GVHD while influencing which cells that can respond to the cytokine. Normal C57BL/6 (B6) or IFNgR1 −/− B6 mice were used as bone marrow donors on day 0 into lethally irradiated, thymectomized B6 × C3H.SW (F1) mice. Normal B6 or IFNgR1 −/− DLIs given with or without a dendritic cell vaccine were introduced at days 14 and 28 post-BMT both to control the induction of GVHD and to provide a population of vaccine-responding cells. F1 recipients were observed for signs of GVHD. ELISPOT of the number of antigen-reactive IFNg-producing splenocytes were also performed to measure functional response to vaccine. RESULTS: The absence of IFNgR1 in the DLI abrogates GVHD as shown by % change in weight (B6 DLI = −6.3 +/− 4.7 vs. IFNgR1−/− DLI = 6.6 +/− 6.1, p=0.001) and allows for greater doses of DLI to be tolerated by the host, however, there is also decreased vaccine responses by ELISPOT (B6 DLI = 1631 vs. IFNgR1−/− DLI = 72, p=0.03). Surprisingly, using IFNgR1−/− bone marrow also abrogates GVHD as shown by % change in weight (B6 marrow = −6.3 +/− 4.7 vs. IFNgR1−/− marrow = 4.4 +/− 4.2, p less than 0.05) and splenocyte count (B6 marrow = 31.48 +/− 12.54 vs. IFNgR1−/− marrow = 63.54 +/− 15.92, p=0.008), but vaccine responses by ELISPOT can be restored to levels that are equivalent of syngeneic control mice, even in the presence of a normal B6 DLI (B6 marrow = 1631 vs. IFNgR1−/− marrow = 9283, p=0.0002). The abrogation of GVHD by IFNgR1−/− marrow does not appear to be a dominant effect since mixtures of IFNgR1 −/− and normal B6 bone marrow still cause GVHD. CONCLUSIONS: Recipients of allogeneic bone marrow and T cells developed GVHD and had decreased vaccine responses by ELISPOT. Loss of IFNgR1 on allogeneic donor lymphocytes abrogates their ability to cause GVHD, but also diminished their ability to respond to vaccine. Surprisingly, loss of IFNgR1 on a donor bone marrow-derived, non T cell results in equivalent abrogation of GVHD, while restoring immunocompetence through favorable responses to a vaccine. Further studies will attempt to identify the phenotype of the responsible bone marrow-derived cell. These results demonstrate a strategy of providing higher doses of DLIs to enhance anti-tumor activity without exacerbating GVHD, and thus, have implications for immune modulation post-allogeneic BMT.

Description: Despite recent progress in our understanding of the biology of T-cell homeostasis, clinically available therapies to substantially improve immune reconstitution in patients sustaining T-cell depletion are lacking. T cells are regenerated via a dynamic interplay between thymopoiesis and thymic-independent homeostatic peripheral expansion (HPE). Using athymic mice subjected to T-cell depletion, we observed that HPE is critically dependent on dendritic cells (DCs) for presentation of antigen, raising the possibility that the availability of DCs might be limiting in vivo for HPE to occur efficiently. Indeed, flt3 ligand (flt3L) treatment of athymic mice subjected to T-cell depletion (without DC depletion) substantially enhanced HPE and improved immune competence. Following bone marrow transplantation (BMT) in athymic hosts, both dendritic cells and T cells were profoundly depleted and flt3L therapy restored DC numbers and enhanced HPE. In addition, thymus-bearing BMT recipients treated with flt3L regenerated increased numbers of thymic-dependent progeny with increased numbers of T-cell receptor excision circle (TREC)-positive T cells, indicating increased thymopoiesis. Therefore, flt3L is a potent immunorestorative agent that enhances both thymic-dependent and thymic-independent pathways of T-cell regeneration. (Blood. 2004;104:2794-2800)