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Integrated scFv identification and CAR T cell generation for AML targeting in vivo
Yi Liu1,2, Annika Lauk1, David Sedloev1
1Medical Faculty Heidelberg and Department of Internal Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany.
Abstract:
Cancer immunotherapy has witnessed remarkable advancements, particularly in the development of chimeric antigen receptor (CAR) T cell therapy. Here, we integrated single chain variable fragment (scFv) development with CAR T cell generation based on a newly developed scFv phagemid library. High-throughput long-read PacBio sequencing identified 4.5 × 107 unique full-length scFv proteins within the generated library. As a proof of principle, we screened for scFvs targeting C-type lectin-like molecule-1 (CLL1) with subsequent cloning into a third generation retroviral CAR backbone. Functional assays revealed the specificity and potency of these CAR T cells in targeting CLL1-positive AML cells in vitro. In vivo studies reduced tumor burden and improved survival rates compared to controls. Taken together, screening for tumor-specific scFvs against CLL1 can rapidly generate AML-specific CAR T cells with effective tumor killing in vivo.
Insights
This study developed a novel method for creating chimeric antigen receptor (CAR) T cells by screening a large library of single chain variable fragments (scFvs). The resulting CAR T cells effectively targeted and killed acute myeloid leukemia (AML) cells expressing C-type lectin-like molecule-1 (CLL1) in preclinical models.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) T cell therapy represents a significant advancement in cancer immunotherapy.
- Development of novel CAR T cells requires efficient methods for identifying and generating specific targeting moieties.
Purpose of the Study:
- To establish a high-throughput method for single chain variable fragment (scFv) discovery and CAR T cell generation.
- To develop CAR T cells targeting C-type lectin-like molecule-1 (CLL1) for acute myeloid leukemia (AML) treatment.
Main Methods:
- Construction and high-throughput sequencing (PacBio) of a large scFv phagemid library (4.5 × 10^7 unique scFvs).
- Screening for CLL1-specific scFvs and their subsequent cloning into a third-generation retroviral CAR backbone.
- In vitro functional assays and in vivo preclinical studies using AML models.
Main Results:
- Identification of a vast number of unique full-length scFv proteins.
- Demonstration of CAR T cell specificity and potency against CLL1-positive AML cells in vitro.
- Significant reduction in tumor burden and improved survival rates in vivo.
Conclusions:
- The integrated scFv library screening and CAR T cell generation platform enables rapid development of targeted immunotherapies.
- CLL1-specific CAR T cells show promising therapeutic potential for AML treatment.
