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3276 Flotetuzumab and Other Cellular Immunotherapies Upregulate MHC Class II Expression on Acute Myeloid Leukemia Cells in Vitro and In Vivo

Program: Oral and Poster Abstracts
Session: 704. Immunotherapies: Poster III
Hematology Disease Topics & Pathways:
Biological, Therapies, immunotherapy
Monday, December 7, 2020, 7:00 AM-3:30 PM

Joseph Rimando, MD1, Michael P. Rettig, PhD2, Matt Christopher, MD, PhD3, Julie K Ritchey4*, Miriam Y Kim, MD5, John Muth, MS6*, Jan Davidson6* and John F. DiPersio, MD3

1Department of Medicine, Washington University School of Medicine, St. Louis, MO
2Department of Internal Medicine, Division of Oncology, Washington Univ. School of Med., Saint Louis, MO
3Washington University School of Medicine, Saint Louis, MO
4Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
5Washington University in St Louis, Saint Louis, MO
6MacroGenics, Inc., Rockville, MD

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative therapy for patients with high-risk and refractory acute myeloid leukemia (AML). Unfortunately, up to 50 percent of patients relapse after allo-HCT. Recent research has shown that 30-50 percent of AML samples from patients relapsing after allo-HCT have downregulation of MHC class II (MHC-II) expression, which may promote immune effector evasion and disease relapse. These studies also report that interferon gamma (IFNγ) can restore MHC-II expression. IFNγ has never been systemically administered after allo-HCT and would likely cause significant and potentially life-threatening toxicities. Reinduction of MHC-II expression may lead to re-engagement of immune effectors, restoration of the graft-versus-malignancy effect, and disease control. We hypothesized that T cell immunotherapies targeting AML cells will lead to T cell activation, localized IFNγ release, and upregulation of MHC-II on AML cells.

Methods: For in vitro experiments, THP1 cells (THP1s), which have intermediate MHC-II expression, or primary human AML samples with low MHC-II expression from a patient relapsing after allo-HCT (AML-low cells) were co-cultured with or without T-cell immunotherapy and with or without human MHC-mismatched CD3+ T cells. The following T-cell immunotherapies were tested: flotetuzumab (FLZ), an investigational CD123 x CD3 bispecific DART® molecule; a CD33 x CD3 bispecific molecule (Creative Biolabs, Shirley, NY); and CD123-directed chimeric antigen receptor (CAR) T cells. THP1 IFNγ receptor-1 (IFNγR1) knockout cell lines were generated using CRISPR-Cas9. MHC-II expression was measured by flow cytometry and IFNγ concentrations via Luminex immunoflourescence assay. In order to rescue THP1s from FLZ-induced death and allow for longitudinal evaluation, a transwell plate system was used, placing THP1s, human CD3+ T cells, and FLZ in the top well and THP1s in the bottom well. This allowed for diffusion of IFNγ but not human T cells to the bottom wells, permitting MHC-II upregulation while limiting death. The upper and lower wells were coincubated together for 24 hours prior to harvesting of the THP1s in the lower well for longitudinal studies and mixed-lymphocyte reactions. For in vivo experiments, NOD-scid IL2Rgammanull mice expressing human IL-3, GM-CSF, and SCF (NSG-S) were irradiated with 250 rads and injected with 10e6 primary AML-low cells per mouse. After 5.5 weeks, mice were divided into the following groups: 1) untreated control; 2) FLZ only (2mg/kg); 3) human mismatched T cells only (10e7 T cells per mouse); 4) FLZ and T cells.

Results: In vitro co-culture of THP1 or AML-low cells with FLZ and T cells led to significantly increased MHC-II expression at 48 hours when compared with the control, FLZ only, and T cell only groups (Figure 1A-B). Co-culture of THP1s with the CD123 CAR-T cells led to similar results. Although co-incubation with a CD33 x CD3 bispecific led to a similar result, the MHC-II upregulation was not nearly as dramatic as that seen with CD123 targeting agents. Using a transwell system to rescue THP1s from FLZ-mediated toxicity, FLZ-induced MHC-II upregulation on THP1s peaked at 48-72 hours (similar kinetics to what is seen with IFNγ alone). These THP1s with upregulated MHC-II activated third-party donor mismatched human CD4+ T cells to a greater extent than untreated THP1s controls. Co-cultures of THP1s with CD4+ T cells and FLZ induced the secretion of very high concentrations of IFNγ, and blockade of IFNγ signaling through knockout of IFNγR1 led to abrogation of the effect (Figure 1C-D). Finally, in an in vivo model, NSG-S mice injected with AML-low samples and treated with FLZ and T cells showed significant upregulation of MHC-II expression on the AML cells. Single cell RNA-sequencing of AML cells purified from these mice is ongoing.

Conclusions: Use of FLZ and other T-cell immunotherapies targeting AML antigens led to both direct AML killing as well as significant upregulation of MHC-II expression on AML cells both in vitro and in vivo. The effect appears to be mediated primarily by IFNγ. T-cell immunotherapies represent a promising treatment approach for AML patients relapsing after allo-HCT and may lead to enhanced immune recognition in the 30-50% of patients who relapse after allo-HCT. Based on these results, a clinical trial treating patients relapsing after allo-HCT with FLZ is planned.

Disclosures: Christopher: Boulder Bioscience: Patents & Royalties: IP around the use of interferon gamma to treat stem cell transplant. Kim: Tmunity: Patents & Royalties: methods for gene editing in hematopoietic stem cells to enhance the therapeutic efficacy of antigen-specific immunotherapy (Licensed by University of Pennsylvania); Neoimmune Tech: Patents & Royalties: use of long-acting IL-7 analogs to enhance CAR T cells (licensed by Washington University). Muth: MacroGenics, Inc.: Current Employment, Current equity holder in publicly-traded company. Davidson: MacroGenics: Current Employment. DiPersio: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH