Chimeric antigen receptor preparation from hybridoma to T-cell expression

Abstract

The successful use of chimeric antigen receptor (CAR) for hematological cancer treatment has influenced the direction taken in translational research toward an increasing focus on personalized targeted immunotherapy. Thus, a growing number of labs worldwide are now interested in testing their old antibody collections in this format to broaden the spectrum of utility and improve safety and efficacy. We herein present a straightforward protocol for the identification of an antibody from a hybridoma and the design of the single chain fragment that will be placed on the extracellular part of the CAR construct. We further show how to test the expression and the activity of the construct in primary T cells. We illustrate our demonstration with two new CARs targeted against the B cell receptor, more precisely the light chains κ and λ, that represent potential alternatives to the CD19 CAR used in the treatment of B-cell malignancies.

Keywords: antibody; chimeric antigen receptor; hybridoma; single chain variable fragment.

© The Author(s) 2019. Published by Oxford University Press on behalf of Antibody Therapeutics.

Figures

Figure 1

The workflow of CAR development from a hybridoma. (A) The visual depiction of a second-generation CAR design. (B) A simplified roadmap of CAR development and assessment with the focus of the article indicated by the green part of the diagram. (C) Validation of target antigen expression in MAVER-1 (Igλ+, dark gray) and BL-41 (Igκ+, light gray) cell lines with the indicates antibodies. The upper panel was stained with commercial antibodies, and lower was done using the hybridoma supernatants in duplicates at the indicated dilutions. Representative data of two independent experiments are shown.

Figure 2

Investigating the hybridoma by 5′-RACE. (A) The depiction of 5′-RACE step by step protocol. (B) List of primers used in this study and references to already published ones. (C) Acquired gel pictures throughout the 5′-RACE protocol, each kappa and lambda hybridomas were divided into two and the protocol was followed in two replicates (e.g. named as K#1 and K#2). The top picture shows the result of the cDNA synthesis, the middle pictures the first PCR results of dC tailed samples and the bottom pictures the result of the second PCR performed with the first PCR products. The ladder information corresponding to 500 and 400 bp bands were specified as well as the primer dimer with an asterisk on each gel image.

Figure 3

The discovery of a scFv sequence and the assembly of the CAR. (A) A simplified depiction of the acquisition of the heavy and light chain’s sequences through IMGTV database and a general scFv structure. (B) A general description of a second-generation CAR design with the protein sequences of each domains.

Figure 4

In vitro validation of CAR constructs. (A) Jurkat cells were retrovirally transduced with either IGK (blue) or IGL CAR (red). The expression was assessed by flow cytometry detection of anti-Fab antibody. Non-transduced Jurkat cells (gray) were used as baseline control, representative of N > 2. (B) Activated primary T-cells were retrovirally transduced with either IGK (blue) or IGL CAR (red). The expression was assessed by flow cytometry, and mock transduced T cells (gray) were used as a baseline control, representative of N = 2. (C) CAR expressing T cells (IGK, blue; IGL CAR, red; and mock, black) specificity to their respective targets in functional assay. Inset: staining of the target cells with specific antibodies, MAVER-1 (Igλ+, black), BL-41 (Igκ+, light gray) and as a negative control Jurkat (Ig-, white). Cytokine secretion (TNF-α) was analyzed by flow cytometry and plotted. Data represent mean ± SD of triplicates, Student t-test where **P < 0.01, ****P < 0.0001, N = 2. (D) Bioluminescence-based killing assay of CAR expressing T cells (IGK, blue; IGL CAR, red; and mock, black) against the same target cells as in (C) and plotted as percentage of lysis over time. Data represent mean ± SD of quadruplicates, Student t-test where ***P < 0.001, ****P < 0.0001, N = 2.