Mutated Flt3Lg Provides Reduced Flt3 Recycling Compared to Wild-Type Flt3Lg and Retains the Specificity of Flt3Lg-Based CAR T-Cell Targeting in AML Models

• Published in: International journal of molecular sciences Vol. 24; no. 8; p. 7626
• Main Authors: Maiorova, Varvara, Mollaev, Murad D, Vikhreva, Polina, Chudakov, Dmitriy M, Kibardin, Alexey, Maschan, Michael A, Larin, Sergey
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.04.2023; MDPI
• Subjects: acute myeloid leukemia; Animals; Antigens; Cancer; Chemotherapy; Comparative analysis; Cricetinae; Cricetulus; fms-like tyrosine kinase 3 (Flt3); fms-Like Tyrosine Kinase 3 - genetics; fms-like tyrosine kinase 3 ligand variant (Flt3Lg-L27P) bioactivity; Hematopoietic stem cells; Humans; Immunotherapy; Leukemia, Myeloid, Acute - genetics; Leukemia, Myeloid, Acute - therapy; ligand-based targeting; Ligands; natural-ligand chimeric antigen receptor (CAR) T; Receptors, Chimeric Antigen - genetics; Signal Transduction; T cells; Transplantation; Germany; Russia; natural-ligand chimeric antigen receptor (CAR) T; acute myeloid leukemia; fms-like tyrosine kinase 3 (Flt3); fms-like tyrosine kinase 3 ligand variant (Flt3Lg-L27P) bioactivity; ligand-based targeting
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24087626

The cells of acute myeloid leukemia are defined by clonal growth and heterogenous immunophenotypes. Chimeric antigen receptors (CARs) commonly recognize molecular targets by single-chain antibody fragments (scFvs) specific to a tumor-associated antigen. However, ScFvs may form aggregates, thus stimulating tonic CAR T-cell activation and reducing CAR T-cell functioning in vivo. Harnessing natural ligands as recognition parts of CARs, specific targeting of membrane receptors can be achieved. Previously, we presented ligand-based Flt3-CAR T-cells targeting the Flt3 receptor. The extracellular part of Flt3-CAR consisted of full-size Flt3Lg. Meanwhile, upon recognition, Flt3-CAR may potentially activate Flt3, triggering proliferative signaling in blast cells. Moreover, the long-lasting presence of Flt3Lg may lead to Flt3 downregulation. In this paper, we present mutated Flt3Lg-based Flt3m-CAR ('m'-for 'mutant') T-cells targeting Flt3. The extracellular part of Flt3m-CAR consists of full-length Flt3Lg-L27P. We have determined that ED for recombinant Flt3Lg-L27P produced in CHO cells is at least 10-fold higher than for the wild-type Flt3Lg. We show that the mutation in the recognizing domain of Flt3m-CAR did not affect the specificity of Flt3m-CAR T-cells when compared to Flt3-CAR T-cells. Flt3m-CAR T-cells combine the specificity of ligand-receptor recognition with reduced Flt3Lg-L27P bioactivity, leading to potentially safer immunotherapy.