To target heterogeneous AML, Zoine et al. designed bispecific CARs with a GRP78 peptide- binding domain and an anti-CD123 scFv, linked by molecules of varying length and rigidity. In tumor coculture, bispecific CAR T cells formed an immune synapse, secreted cytokines, and lysed target cells, dependent on linker selection, which was consistent with structures predicted using AlphaFold2. In vivo, bispecific CAR T cells treated AML tumor lines and patient-derived xenografts, comparable or superior to monospecific CAR T cells. Adjusting the CAR scFv produced functional bispecific CAR T cells against alternative targets; higher linker flexibility ((G4S)3) improved antigen detection.
Contributed by Alex Najibi
ABSTRACT: The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.
Author Info: (1) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (2) Department of Bone Marrow Transplantation and
Author Info: (1) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (2) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Department of Structural Biology and Center of Excellence for Data Driven Discovery, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (3) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (4) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (5) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (6) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (7) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (8) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (9) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (10) Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (11) Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (12) Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (13) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (14) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (15) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (16) Department of Structural Biology and Center of Excellence for Data Driven Discovery, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. (17) Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address: paulina.velasquez@stjude.org.
