SCIENTIFIC ABSTRACT Acute myeloid leukemia (AML) comprises a genetically and clinically heterogeneous group of aggressive hematological malignancies. Despite advances in molecular characterization of AML, the majority of patients will relapse and die of their disease, indicating the urgent need for novel therapeutic approaches. Recently, antibody-based therapeutics have emerged as an effective tool in leukemia therapy but still in need of novel targets. Our proteomic studies using blast leukemia cells from different leukemia subtypes, have identified a putative therapeutic target HSPA1L, as being expressed on the cell surface of AML and other hematologic malignancies, but sparing normal tissues except for male reproductive system. We validated expression of this target by flow cytometry and confocal analyses. Our hypothesis is that immune therapy approaches targeting specific epitopes of the surface protein HSPA1L can selectively eliminate target-expressing AML while avoiding immune-related adverse effects. Importantly, we have already generated monoclonal antibodies (MAbs) against HSPA1L, selected positive clones by ELISA and validated cell surface target binding in AML cells. In this proposal, we will characterize expression patterns of HSPA1L in AML and AML stem/progenitor cells; and utilize first-time produced monoclonal antibodies for therapeutic applications, by testing antibodies alone, antibody-drug conjugates or as immune engaged targeted depletion effector function using in vitro and in vivo leukemia models. In Aim 1, we will characterize selected candidate antibodies exploring the feasibility of using them alone, to trigger complement depending cytotoxicity (CDC) or engage effector cells, such as NK and macrophages to trigger antibody dependent cell cytotoxicity or phagocytosis (ADCC or ADCP); or as antibody-drug conjugates (ADC) by conjugating MAbs to auristatin. The expression patterns of HSPA1L in AML, and AML stem/progenitor cells and in normal bone marrow cells and hematopoietic stem cells will be studied. In Aim 2, we will test the efficacy of the selected lead HSPA1L MAbs with or without conjugation as ADCs in vivo in bioluminescent-labeled AML cell line models using SCID mice, and in patient-derived xenograft (PDX) AML models in NSG mice. We have assembled a multi-faceted team of highly experienced investigators with deep background in MAbs generation and testing, proteomics and target discovery, pre-clinical assessment of novel agents in adult and pediatric leukemias in vitro and in vivo models, and clinical translation in adult and pediatric acute leukemias. If successful, this multi-investigator project will generate novel antibodies for future clinical development in target- expressing leukemias that will be tested in clinical trials, alone or in combination with standard chemotherapy.