The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains

Zhenyu Dai, Wei Mu, Ya Zhao, Xiangyin Jia, Jianwei Liu, Qiaoe Wei, Taochao Tan, Jianfeng Zhou, Zhenyu Dai, Wei Mu, Ya Zhao, Xiangyin Jia, Jianwei Liu, Qiaoe Wei, Taochao Tan, Jianfeng Zhou

Abstract

T cell malignancies are a group of hematologic cancers with high recurrence and mortality rates. CD5 is highly expressed in ∼85% of T cell malignancies, although normal expression of CD5 is restricted to thymocytes, T cells, and B1 cells. However, CD5 expression on chimeric antigen receptor (CAR)-T cells leads to CAR-T cell fratricide. Once this limitation is overcome, CD5-targeting CAR-T therapy could be an attractive strategy to treat T cell malignancies. Here, we report the selection of novel CD5-targeting fully human heavy-chain variable (FHVH) domains for the development of a biepitopic CAR, termed FHVH3/VH1, containing FHVH1 and FHVH3, which were validated to bind different epitopes of the CD5 antigen. To prevent fratricide in CD5 CAR-T cells, we optimized the manufacturing procedures of a CRISPR-Cas9-based CD5 knockout (CD5KO) and lentiviral transduction of anti-CD5 CAR. In vitro and in vivo functional comparisons demonstrated that biepitopic CD5KO FHVH3/VH1 CAR-T cells exhibited enhanced and longer lasting efficacy; produced moderate levels of cytokine secretion; showed similar specificity profiles as either FHVH1, FHVH3, or the clinically tested H65; and is therefore suitable for further development.

Trial registration: ClinicalTrials.gov NCT03081910.

Keywords: CD5; T cell malignancies; biepitopic antibody; biepitopic chimeric antigen receptor; fully human antibody; heavy-chain-only antigen recognition domains; phage display antibody library; tumor escape.

Conflict of interest statement

Declaration of interests T.T., Y.Z., X.J., J.L., and Q.W. are employees of Nanjing IASO Biotherapeutics and held interests in the company. J.Z., T.T., Z.D., Y.Z., X.J., J.L., and Q.W. are among inventors of patent applications related to the fully human heavy-chain-only CD5 antibodies and CARs. J.Z. is a nonpaid member of Scientific and Medical Advisory Board of Nanjing IASO Biotherapeutics.

Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Phage display library screening and selection of fully human (FH) CD5-specific, heavy-chain variable domain (VH) (A) Schematic of the CD5-specific VH discovery process. (B) Binding activities of representative phage clones and KO7 (negative control phage) are shown. Binding to different antigens was detected after staining with mouse anti-M13 antibody and horseradish peroxidase (HRP)-goat anti-mouse IgG antibody before reading the optical density at 450 nm. Mouse anti-CD5 and HRP-goat anti-mouse IgG antibodies were used as the positive control. (C) Phage clones FHVH1–4 bind to Jurkat and CCRF-CEM (both CD5+) cells, but not to Raji and K562 (both CD5−) cells. Shown is analyzed using flow cytometry.
Figure 2
Figure 2
CD5 antigen binding assay and competitive binding analysis of VH and validation of anti-CD5 biepitopic antibodies (A) Schematic diagram of the anti-CD5 VH binding using a competitive fluorescence-activated cell sorting assay. (B) Representative flow cytometry analysis shows that H65, fully human (FH)VH1, and FHVH3 CAR-T cells all bind to CD5 antigen. FHVH3 recognizes the overlapped epitope of CD5 as H65, whereas FHVH1 binds different CD5 antigen epitopes with FHVH3 and H65. (C) Binding specificities of anti-CD5 biepitopic antibodies are shown. CD5+ cell lines (Jurkat and CCRF-CEM) and CD5− cell lines (CCRF-CD5KO and Raji) were stained with isotype control, FHVH1-hFc, FHVH3-hFc, FHVH1/VH3-hFc, and FHVH3/VH1-hFc antibody, respectively, followed by a secondary APC-labeled anti-human IgG, and analyzed using FACS. (D) Binding of FHVH3/VH1 to QT6 cells individually expressing 5,900 full-length human membrane proteins as determined by flow cytometry is shown.
Figure 3
Figure 3
Apoptosis analysis and phenotypic analysis of CD5 knockout anti-CD5 CARs (A) Schematic structures containing the anti-CD5 VH, anti-CD5 biepitopic VH domains, or murine-derived H65 scFv. (B) Gene editing efficiency in human primary T cells was determined 3 days after electroporation with Cas9 protein and CD5-specific gRNA-7 by FACS. The numbers indicated the frequency of CD5− cells. (C) Schematic outline of the optimized process for generating CD5KO anti-CD5 CAR-T cells is shown. (D) Proportion of CD4+ T cells and CD8+ T cells in CAR-T/T cells is shown. The data represent mean ± SD (n = 3). (E) The basal apoptosis of CAR-T/T cells is shown. The four CARs had similar background level of apoptosis as measured by annexin V and PI staining 10 days post-transduction. The data represent mean ± SD (n = 3). ns, not significant (one-way ANOVA). (F) CAR expression on CD5KO-T cells detected by anti-EGFR antibody and recombinant CD5-Fc staining on day 7 is shown. The data represent mean ± SD (n = 3). ns, not significant (two-way ANOVA). (G) Median fluorescence intensity ratios (CD5 antigen binding to EGFRt) of CD5KO-T cells expressing different CARs are shown. The data represent mean ± SD for three donors. ∗p < 0.05 (one-way ANOVA). (H) Representative flow cytometry analysis shows surface expression of CD5 and transduction efficiency of CD5KO anti-CD5 CAR-T/T and mock T cells on day 7. (I) Flow cytometry analysis of EGFRt expression on the surface of CD5 CAR-T cells at different time points is shown. The data represent mean ± SD for three donors. ns, not significant (two-way ANOVA). (J) Flow cytometry analysis of CD5 antigen expression on the surface of anti-CD5 CAR-T cells at different time points is shown. The data represent mean ± SD for three donors. ∗p < 0.05 (two-way ANOVA). (K) Frequency of effector and effector-memory (TEFF/TEM) (CCR7− CD45RA−), effector memory revertant (TEMRA) (CCR7− CD45RA+), naive (NAÏVE) (CCR7+ CD45RA+), and central memory (CM) (CCR7+ CD45RA−) in anti-CD5 CAR-T/T cells assessed using flow cytometry on days 7–10 post-transduction is shown. The data represent the average from three donors. (L) Representative dot plots show the phenotype of activated T cells in (K) after 10 days.
Figure 4
Figure 4
CD5KO anti-CD5 CAR-T cells were compared functionally (A) Surface expression of CD5 (red solid histograms) in T-ALL and T-lymphoma cell lines in comparison with isotype control (dotted line gray histograms) measured using flow cytometry. (B) Expression of the early T cell activation marker CD69 on anti-CD5 CAR-T cells following a 24-h co-incubation with CCRF-CEM or CCRF-CD5KO (gated on CD8+ EGFR+ cells) is shown. The data represent mean ± SD (n = 3). ∗∗∗∗p < 0.0001 (two-way ANOVA). (C) Expression of the T cell activation marker CD25 on anti-CD5 CAR-T cells following co-incubation as described in (B) is shown (gated on CD8+ EGFR+ cells). The data represent mean ± SD (n = 3). ∗∗∗∗p < 0.0001 (two-way ANOVA). (D) Median fluorescent intensity of FasL on anti-CD5 CAR-T cells following co-incubation as described in (B) is shown (gated on CD8+ EGFR+ cells). The data represent mean ± SD (n = 3). ∗∗∗∗p < 0.0001 (two-way ANOVA). (E) The degranulation assay of four CARs is shown. CAR-T cells were stimulated with target cells expressing different CD5 antigen densities. The CD5 antigen-specific increase in CD107a was assessed as a measure of degranulation. The data represent mean ± SD for three donors. ∗∗∗∗p < 0.0001 (two-way ANOVA). (F) Anti-CD5 CAR-T cells selectively kill CD5+ tumor cells. All CARs lyse CD5+ target cells in a dose-dependent manner. The killing ability of CAR-T/T cells for CD5+ cell lines was determined by luciferase-based cytotoxicity assay after 24 h incubated with target cells at different E:T ratios. The data indicate mean ± SD from three co-cultures. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001 (two-way ANOVA).
Figure 5
Figure 5
Cytokine secretion and repeated stimulation assay of CD5-targeting CAR-T cells in vitro (A) Quantification of cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-6, and IL-10) from the supernatant after CAR-T/T cells were co-cultured with CCRF-CEM and CCRF-CD5KO at an E:T ratio of 1:1 for 24 h. The results are displayed as mean ± SD (n = 3). ∗∗∗∗p 

Figure 6

The in vivo antitumor activity…

Figure 6

The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model…

Figure 6
The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model established by CCRF-CEM (A) Average radiance quantification of each treatment group measured at the indicated time points. The results are displayed as mean ± SEM (n = 5). ∗p + EGFR+) in peripheral blood of mice on days 10 and 17 is shown, respectively (n = 3). ∗∗p < 0.01 (two-way ANOVA). (C) Overall Kaplan-Meier survival curve is shown. Survival curves were compared using the log rank test. Mice treated with CAR-T cells showed significantly increased survival (∗∗∗∗p < 0.0001) compared with those of CD5KO-T and PBS-treated groups. (D) Growth and staging of the tumor monitored by bioluminescence imaging are shown. (E) Body weight curve is shown. The results are displayed as mean ± SEM (n = 5). (F) Concentration of IFN-γ and IL-10 in the serum of indicated groups collected on day 17 is shown. The results are displayed as mean ± SD (n = 5). ∗p < 0.05 and ∗∗∗∗p < 0.0001 (one-way ANOVA). (G) Representative flow cytometry analysis shows the proportion of LAG-3+ and TIM-3+ cells in CAR-T cells in the peripheral blood of NCG mice collected on day 24. (H) Quantification and statistical analysis of the results in (G) are shown. The results are displayed as mean ± SD (n = 3). ∗p < 0.05 and ∗∗p < 0.01 (two-way ANOVA).

Figure 7

The in vivo antitumor activity…

Figure 7

The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model…

Figure 7
The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model established by SUP-T1 (A) Mouse tumor burden of each treatment group at the indicated time points. The results are displayed as mean ± SEM (n = 6). ∗p + EGFR+) in peripheral blood of mice on day 13, day 20, and day 26, respectively (n = 6). (D) Overall Kaplan-Meier survival curve is shown. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (log rank test).
All figures (8)
Figure 6
Figure 6
The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model established by CCRF-CEM (A) Average radiance quantification of each treatment group measured at the indicated time points. The results are displayed as mean ± SEM (n = 5). ∗p + EGFR+) in peripheral blood of mice on days 10 and 17 is shown, respectively (n = 3). ∗∗p < 0.01 (two-way ANOVA). (C) Overall Kaplan-Meier survival curve is shown. Survival curves were compared using the log rank test. Mice treated with CAR-T cells showed significantly increased survival (∗∗∗∗p < 0.0001) compared with those of CD5KO-T and PBS-treated groups. (D) Growth and staging of the tumor monitored by bioluminescence imaging are shown. (E) Body weight curve is shown. The results are displayed as mean ± SEM (n = 5). (F) Concentration of IFN-γ and IL-10 in the serum of indicated groups collected on day 17 is shown. The results are displayed as mean ± SD (n = 5). ∗p < 0.05 and ∗∗∗∗p < 0.0001 (one-way ANOVA). (G) Representative flow cytometry analysis shows the proportion of LAG-3+ and TIM-3+ cells in CAR-T cells in the peripheral blood of NCG mice collected on day 24. (H) Quantification and statistical analysis of the results in (G) are shown. The results are displayed as mean ± SD (n = 3). ∗p < 0.05 and ∗∗p < 0.01 (two-way ANOVA).
Figure 7
Figure 7
The in vivo antitumor activity of CD5-targeting CAR-T cells in the tumor model established by SUP-T1 (A) Mouse tumor burden of each treatment group at the indicated time points. The results are displayed as mean ± SEM (n = 6). ∗p + EGFR+) in peripheral blood of mice on day 13, day 20, and day 26, respectively (n = 6). (D) Overall Kaplan-Meier survival curve is shown. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (log rank test).

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