Project 6: Bi-specific antibody-based therapies for AML after allogeneic hematopoietic stem cell transplantation

John F. DiPersio, MD, PhD, and Peter Westervelt, MD, PhD

The long-term goal of this project is to develop a bispecific antibody-based therapeutic to treat acute myelogenous leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (alloHCT). Roughly 40% of AML patients will relapse after alloHCT and face a dismal prognosis with a 2-year survival of approximately 20%. Relapse occurs for one of two reasons: (1) because the residual leukemic cells that were not eliminated by the transplant conditioning regimen evade the immune response provided by the donor immune cells or (2) because the immune response cannot be sustained. Donor lymphocyte infusion (DLI) represents a non-specific form of adoptive cell therapy which involves infusion of a pool of allogeneic immune cells, including T cells, NK cells and antigen presenting cells. The DLI is believed to work in part through the graft-versus-leukemia (GvL) effect of alloHCT, in which donor immune cells control leukemic blasts through both direct cellular cytotoxicity and secretion of inflammatory cytokines such as interferon gamma (IFN-g). However, response rates following DLI alone for relapsed AML post-HCT are 15-40%, with only 15-20% survival at 2 years. Flotetuzumab is a CD123 x CD3 bispecific antibody designed to target CD123-positive cells for recognition and elimination by CD3-expressing T lymphocytes. CD123 is expressed by leukemic blasts and leukemia stem cells but not by normal hematopoietic stem cells (HSCs). The potential of T cell engaging therapies, such as flotetuzumab, to reestablish and retarget GvL in the post-HCT relapse setting may be an effective approach to treat AML relapse after alloHCT. We hypothesize that flotetuzumab for relapsed AML following alloHCT will be safe, tolerable and facilitate preferential T cell killing of leukemic cells resulting in improved patient outcomes. Furthermore, administration of a donor lymphocyte infusion (DLI) in combination with flotetuzumab will be safe, tolerable and may provide additional therapeutic efficacy. We will investigate these hypotheses via the following specific aims:

Specific Aim 1: To conduct a Phase II clinical trial of flotetuzumab, a CD123 x CD3 dual affinity retargeting agent in patients with relapsed or refractory (r/r) AML after allogeneic stem cell transplant ( Id: NCT04582864). This is an open-label, single-arm, single-center Phase 2 study to evaluate for efficacy, safety and tolerability of flotetuzumab administered as a single agent and/or in combination with DLI in patients with relapsed AML post-alloHCT. Patients will receive flotetuzumab for the first 28 days of the study (cycle 1). Based on day 28 response assessment, patients with CR/CRi will continue on flotetuzumab for another 28 days (cycle 2); patients with PR/SD will receive donor lymphocyte infusion (DLI) plus flotetuzumab (cycle 2); and patients with PD will come off study.

Specific Aim 2: To determine the immunophenotype and transcriptional profiles of r/rAML and T cells before and after treatment with of flotetuzumab. A. To characterize the effect of flotetuzumab on the induction of cytokine production by immune effectors. B. To determine the effect of flotetuzumab on the absolute number and phenotype of leukemic blasts and normal hematopoietic cell subsets in the peripheral blood and bone marrow of AML patients over time using flow cytometry, single cell RNA sequencing (CITE-seq), single cell proteomics (Isoplexis Isocode single cell polyfunctional strength analyses) and gene expression profiling (NanoString PanCancer IO360 panel evaluating 770 genes involved in the immune response against tumors).

Specific Aim 3: To identify novel approaches to mitigate flotetuzumab-associated cytokine release syndrome (CRS) while maintaining anti-leukemia activity using in vitro and in vivo preclinical models. A. To develop novel approaches to mitigate CRS induced by T cell-based therapeutics. B. To test the efficacy of our novel approaches to mitigate flotetuzumab-associated CRS in immunodeficient NSGS mice engrafted with human hematopoietic stem cells and in immunocompetent human CD3e knock-in mice.