Development of a monoclonal antibody for the detection of anti-canine CD20 chimeric antigen receptor expression on canine CD20 chimeric antigen receptor-transduced T cells

Abstract

Chimeric antigen receptor (CAR) CAR-T cell therapy targeting CD20 can be a novel adoptive cell therapy for canine patients with B-cell malignancy. After injection of the CAR-T cells in vivo, monitoring circulating CAR-T cells is essential to prove in vivo persistence of CAR-T cells. In this study, we developed a novel monoclonal antibody against canine CD20 CAR, whose single-chain variable fragment was derived from the our previously reported anti-canine CD20 therapeutic antibody. Furthermore, we proved that this monoclonal antibody can detect therapeutic anti-canine CD20 chimeric antibody in the serum from healthy beagle dogs injected with the therapeutic antibody for safety study. This monoclonal antibody is a useful tool for monitoring both canine CD20-CAR-T cells and anti-canine CD20 therapeutic antibody for canine lymphoma.

Keywords: CD20; adoptive immunotherapy; canine; chimeric antigen receptor T cell; monoclonal antibody.

Fig. 1.

Generation of anti-CD20-chimeric antigen receptor (CAR) monoclonal antibody. (A) Schematic diagram of the canine CD20-CAR-expressing construct and ritCAR-expressing construct used as a control. (B) Establishment of CD20 CAR-expressing murine cells for immunization. NIH3T3 cells were retrovirally transduced with the CAR-expressing vector (NIH3T3/CAR). Wild-type NIH3T3 (red) and NIH3T3/CAR (blue) cells were assessed by Venus expression. (C) CD20 CAR detection using the established antibody, 1F6-11D-1F. NIH3T3/CAR and NIH3T3/ritCAR cells were stained with 1F6-11D-1F and anti-canine CD8α antibodies. Both cells expressed canine CD8α, but CD20 -CAR was expressed only in NIH3T3/CAR, indicating 1F6-11D-1F is specific to CD20-CAR. (D) CAR-T cells were stained with 1F6-11D-1F. Peripheral Blood Mononuclear Cells were isolated from a healthy beagle, and stimulated by Iinterleukin-2 and phytohemagglutinin, followed by the transduction of CD20 CAR, as described in the text. CD20 CAR-T cells were stained with the established anti-CD20-CAR antibody (1F6-11D-1F). The dot plot shows Venus expression versus 1F6-11D-1F staining.

Fig. 2.

The measurement of chimeric anti-canine CD20 antibody. (A) Plates were coated with 1F6-11D-1F (1 μg/ml), followed by incubation with serially diluted chimeric anti-canine CD20 antibody (4E1-7-B). Antibody binding was detected by HRP-conjugated anti-dog IgG antibody at indicated dilutions (1:500, 1:1,000 and 1:2,500). Substrate solution was added and absorbance at 405 nm was measured. (B) Pharmacokinetic profile of chimeric anti-canine CD20 antibody in the serum from healthy beagle dogs. Fifteen healthy beagles (of three dogs in each group) were injected with either of rat-dog chimeric anti-canine CD20 antibody (4E1-7-B) or the defucosylated antibody (4E1-7-B_f) at the indicated dose. The concentration of anti-canine CD20 antibody in dog sera was measured by ELISA using plate-coated 1F6-11D-1F. Error bars indicate standard deviation.