Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors

Abstract

Purpose: Given that heterogeneous expression and variants of antigens on solid tumors are responsible for relapse after chimeric antigen receptor (CAR)-T cell therapy, we hypothesized that combinatorial targeting two tumor-associated antigens would lessen this problem and enhance the antitumor activity of T cells. Methods: The co-expression level of CD70 and B7-H3 was analyzed in multiple tumor tissue samples. Further, two putative antigens were identified in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis database. Two CD70 targeted CARs with different antigen binding domain, truncated CD27 and CD70 specific single-chain antibody fragment (scFv), were designed to screen a more suitable target-antigen binding moiety. Accordingly, we designed a bivalent tandem CAR (TanCAR) and further assessed the anti-tumor efficacy of TanCAR-T cells in vitro and in vivo. Results: Our results indicated that co-expression of CD70 and B7-H3 was observed on multiple tumor types including kidney, breast, esophageal, liver, colon cancer, glioma as well as melanoma. The CD70 targeted CAR-T cells with binding moiety of CD70 specific scFv exhibit a higher affinity and antitumor effect against CD70⁺ tumor cells. TanCAR-T cells induced enhanced ability of cytolysis and cytokine release over unispecific CAR-T cells when encountering tumor cells expressing two target-antigens. Further, low doses of TanCAR-T cells could also effectively control the lung cancer and melanoma xenografts and improved overall survival of the treated animals. Conclusion: TanCAR-T cells targeting CD70 and B7-H3 exhibit enhanced antitumor functionality and improve the problem of antigenic heterogeneity and variant in the treatment against solid tumor and melanoma.

Keywords: B7-H3; CD70; Chimeric antigen receptor T cell; Immunotherapy; Solid tumor.

Figure 1

Expression of CD70 and B7-H3 on human tumor tissues. (A) Representative images of IHC staining of CD70 and B7-H3 on human tumor tissue microarrays were shown. (Scale bar, 20 μm) (B) IHC result of CD70 and B7-H3 staining in normal tissues including brain, esophagus, stomach, intestine, pancreas, appendix. The representative images were shown. (Scale bar, 50 μm) (C) Differential expression profile analysis of B7-H3 and CD70 in tumor and normal tissues based on the TCGA database.

Figure 2

Expression of CD70 and B7-H3 in human solid tumor cell lines. (A, B) Representative images showed the immunofluorescence staining of B7-H3 and CD70 together with DAPI in NCI-H460, A375, MDA-MB-435 and 786-O tumor cells. (Scale bar: 20 μm) (C, D) Flow cytometry result indicated high expression of CD70 and B7-H3 on the four solid tumor cell lines.

Figure 3

Construct of CAR (A) Schematic diagrams showing the composition of the four CARs used in this study: CD70 CAR₁, CD70 CAR₂, B7-H3 CAR and TanCAR. (B) The lentiviral backbone plasmid encodes the TanCAR. (C) Cartoon depicted of TanCAR targeting respective tumor antigens.

Figure 4

Generation of CAR-T cells. (A) Images of transduced CAR-T cells were captured using inverted fluorescent microscope. (Scale bar: 100 μm) (B) The transduction efficiency was measured by tdTomato positive cells using flow cytometric analysis. (C) Flow cytometry results illustrated the frequency of CD8⁺ T cells on 7 days post-transduction, compare with the non-transduced T cells.

Figure 5

Functional analysis of CD70 CAR₁ and CD70 CAR₂. (A) To assess the affinity of two CD70 binding fragments, immunofluorescence was performed using human trCD27.mFc and CD70 scFv.hFc chimeric protein as the primary antibody. Images showed the immunofluorescence staining of CD70 by NCI-H460 and A375 tumor cells. (Scale bar: 20 μm) (B) 4-hour ⁵¹Cr cytotoxicity assays indicated a higher tumor killing of CD70 CAR₂-T cells against target cells. (C) Microscopy images were captured 8 hours after A375 or H460 cells cocultured with CD70 CAR₁, CD70 CAR₂ and NT T cells at a ratio of 2 effector cell to 1 target cells. (Scale bar: 50 μm) (D) ELISA results showed the IFN-γ and IL-2 secretion levels by CD70 CAR₁, CD70 CAR2 and NT T cells encountering A375 or H460 cells.

Figure 6

Activity of TanCAR-T cells against tumor cells expressing CD70 and/or B7-H3. (A) Four-hour ⁵¹Cr cytotoxicity assays of TanCAR-T cells against tumor cells expressing CD70 and/or B7-H3, compared with unispecific CAR and NT T cells. (B) Analysis of IFNγ and IL2 secretion level from supernatants of co-cultures of TanCAR, B7-H3 CAR, CD70 CAR₂ and NT T cells with multiple tumor cells, as detected by ELISA. Shown are pooled data from 3 independent experiments done in triplicates.

Figure 7

Antitumor response of TanCAR-T cells against CD70 and B7-H3 positive tumors in vivo. (A) The treatment scheme showed the timing of subcutaneous injection of tumor cells, vein-tail injection of CAR-T cells T cells and in vivo optical imaging. (B) Antitumor response of TanCAR-T cells in human subcutaneous xenograft models. NCI-H460.ffLuc or A375.ffLuc tumor bearing (confirmed by imaging 6 days after tumor implantation, 8/group) mice were adoptively transferred through tail vein injection with NT, CD70 CAR₂, B7-H3 CAR or high/low doses (5×10⁶ or 1×10⁶/mouse) of TanCAR T cells on 7 days and 10 days post tumor inoculation. (C) Tumor growth was measured weekly by using Living Image software, and mean values per treated group were shown. (D) Kaplan-Meier survival curve were performed 75 days after T cells injection. Mice treated with TanCAR-T cells had a significantly longer survival probability in comparison with mice with NT, CD70 CAR₂ or B7-H3 CAR-T cells.