Cancer Immunotherapy with T Cells Carrying Bispecific Receptors That Mimic Antibodies

Abstract

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo.

Figure 1.. Bi-EGFR.amR-T cells and conventional EGFR.CAR-T cells show comparable activity.

(A-C) Bio-layer Interferometry (BLI) was used to measure the affinity of the monomeric or heterodimeric single domain antibody mimics binding to recombinant Fc-EGFR on the FortéBio Octet system. 100 nM biotinylated antibody mimics were immobilized onto a streptavidin biosensor. Binding kinetics were measured against various concentrations of EGFR (0, 25, 50, 100 nM). The K_d was calculated based on kinetic fitting. (D) Representative expression of the Bi-EGFR.amR, EGFR.CAR or CD19.CAR in T cells as assessed by flow cytometry. Activated T cells were transduced with retroviral vectors encoding the Bi-EGFR.amR, EGFR.CAR or CD19.CAR. With the exception of the CD19.CAR, all constructs were detected using an anti-FLAG Ab. The CD19.CAR was detected using an anti-idiotype Ab. Shaded and unshaded histograms indicate non-transduced and specific mAb, respectively. (E) Summary of amR or CAR expression (n=4). (F) Expansion kinetics of Bi-EGFR.amR-T cells, EGFR.CAR-T cells or CD19.CAR-T cells NT (n=4); error bars denote SD (G) Phenotypic composition of Bi-EGFR.amR-T cells, EGFR.CAR-T cells or CD19.CAR-T cells 10 days post-transduction (n=4); error bars denote SD (H) Reducing immunoblots of Bi-EGFR.amR-T cell and EGFR.CAR-T cell lysates. Immunoblots were probed with anti-CD3ζ. Upper panel and lower panels represent detection of the receptors and endogenous ζ-chain, respectively. (I) EGFR.CAR-T cells and Bi-EGFR.amR-T cells were incubated at the indicated time points with the anti-FLAG Ab and cross-linked with a secondary Ab to induce the aggregation of the receptors. Cell lysates were immunoblotted to detect proximal (CD3ζ p-Y142) and distal (Akt p-S473 and ERK p-T202/204) phosphorylation events following receptor cross-linking. Total CAR.CD3ζ or amR.CD3ζ and endogenous CD3ζ were used as loading controls. Data are representative of 4 experiments.

Figure 2.. Bi-EGFR.amR-T cells show activity against EGFR-expressing tumor cells in vitro and in vivo.

(A) Control T cells (NTs or CD19.CAR-T cells), Bi-EGFR.amR-T cells and EGFR.CAR-T cells were co-cultured with MCF-7 cells (EGFR negative) or EGFR-expressing pancreatic adenocarcinoma cell lines (AsPC-1, BxPC-3, HPAF-II and Panc-1) at 1:5 E:T. Cells were collected and quantified by flow cytometry on day 5. The frequency of residual tumor cells was identified as CD4⁻CD8⁻ live cells (n=3–4), p<0.01 when Bi-EGFR.amR-T cells or EGFR.CAR-T cells are compared with control T cells; two-way ANOVA with Tukey correction (B) IFN-γ and (C) IL-2 released in the co-culture supernatant by Bi-EGFR.amR-T cells, EGFR.CAR-T cells and control T cells after 24 hours of co-culture with tumor cells as assessed by ELISA (n=3–4), p<0.01 when Bi-EGFR.amR-T cells or EGFR.CAR-T cells are compared with control T cells; two-way ANOVA with Tukey correction (D) Bi-EGFR.amR-T cells, EGFR.CAR-T cells or control T cells were labeled with CFSE and stimulated with irradiated EGFR-expressing AsPC-1 cells at 4:1 E:T. Representative CFSE dilution on day 5. (E) Proliferation index as assessed by CFSE dilution (n=4), p<0.01 when Bi-EGFR.amR-T cells or EGFR.CAR-T cells are compared with control T cells; two-way ANOVA with Tukey correction (F) Schematic representation of a metastatic pancreatic cancer model in NSG mice using the FFLuc-labeled human Panc-1 cell line. Representative images of tumor bioluminescence (BLI) (G) and kinetics (H) of tumor growth as assessed by BLI measurements. Data are representative of two independent experiments with 5 mice per group; p<0.01 when Bi-EGFR.amR-T cells or EGFR.CAR-T cells are compared with control T cells; two-way ANOVA with Tukey correction

Figure 3.. Bi-EGFR.amR-T cells are activated by recognition of two non-overlapping epitopes on EGFR.

Control (NTs), AFF.EGFR.amR-T cells, FN3.EGFR.amR-T cells and Bi-EGFR.amR-T cells were seeded in tissue culture plates coated with either recombinant human EGFR WT protein (rEGFR) or the truncated mutant EGFRvIII recombinant protein (rEGFRvIII). (A) Representative expression of CD69 on NTs, AFF.EGFR.amR-T cells, FN3.EGFR.amR-T cells and Bi-EGFR.amR-T cells 6 hours post-stimulation with plate-bound rEGFR or rEGFRvIII protein as assessed by flow cytometry. Shaded and dashed histograms indicate media and PMA/Iono controls, respectively. Solid and dashed lines indicate rEGFR and rEGFRvIII protein stimulation, respectively. (B) Summary of CD69 expression on total live T cells (n=4), p<0.01 when comparing FN3.EGFR.amR-T cells seeded in rEGFR and rEGFRvIII-coated wells; two-way ANOVA with Tukey correction. (C) IFN-γ released in the supernatant by AFF.EGFR.amR-T cells, FN3.EGFR.amR-T cells and Bi-EGFR.amR-T cells as assessed by ELISA (n=3), p<0.01 when comparing FN3.EGFR.amR-T cells seeded in rEGFR and rEGFRvIII-coated wells; two-way ANOVA with Tukey correction. (D) mAFF.EGFR.amR-T cells or mBi-EGFR.amR-T cells were co-cultured with BV173-WT or BV173-EGFR cells at 1:5 E:T for 3 days. CD19.CAR-T cells were used as a positive control. Cells were collected and T cells (CD3⁺) and tumor cells (CD19⁺) were quantified by flow cytometry. Representative flow plots are illustrated. (E) Quantification of BV173-WT or BV173-EGFR cells remaining after 3 days of co-culture. (F) IFN-γ released in the supernatant by NT, CD19.CAR-T, mAFF.EGFR.amR-T and mBi-EGFR.amR-T cells as assessed by ELISA (n=2–4), p<0.01 when comparing the percentage of residual BV173-WT and BV173-EGFR cells remaining in the mBi-EGFR.amR-T wells; two-way ANOVA with Tukey correction

Figure 4.. Bi-HER2.amR-T cells have anti-tumor activity against HER2-expressing primary cells in vitro and in vivo.

(A-C) Bio-layer Interferometry (BLI) was used to measure the affinity of the monomeric or heterodimeric single domain antibody mimics binding to recombinant Fc-HER2 using the FortéBio Octet system. 100 nM biotinylated antibody mimics were immobilized onto a streptavidin biosensor. Binding kinetics was measured against various concentrations of HER2 (0, 25, 50, 100 nM). The K_d was calculated based on kinetic fitting. (D) Control T (NT), Bi-HER2.amR-T cells and EGFR.CAR-T cells were co-cultured with Panc-1 cells (HER negative) or HER2-expressing pancreatic adenocarcinoma cell lines (AsPC-1 and HPAF-II) at 1:5 E:T. Cells were collected and quantified by flow cytometry on day 5. The frequency of residual tumor cells was identified as CD4⁻ CD8⁻ live cells (n=3–6), p<0.01 when comparing Bi-HER2.amR-T cells or EGFR.CAR-T cells with NTs; two-way ANOVA with Tukey correction (E) IFN-γ and (F) IL-2 released in the co-culture supernatant by NTs, Bi-HER2.amR-T cells or EGFR.CAR-T cells after 24 hours of co-culture with tumor cells as assessed by ELISA (n=3–6), p<0.01 when comparing Bi-HER2.amR-T cells or EGFR.CAR-T cells with NTs; two-way ANOVA with Tukey correction (G) Schematic representation of a metastatic pancreatic cancer model in NSG mice using the using the FFLuc-labeled human HPAF-II cell line. Representative images of tumor bioluminescence (BLI) (H) and kinetics (I) of tumor growth as assessed by BLI measurements. Data are representative of two independent experiments with 5 mice per group; p<0.01 when Bi-HER2.amR-T cells or EGFR.CAR-T cells are compared with NTs; two-way ANOVA with Tukey correction

Figure 5.. EGFR-HER2.amR-T cells show dual specificity.

Co-culture experiment in which T cells were plated with its respective BV173 target cells, and 3 days later T cells were collected and re-plated with the same BV173 tumor cell line or BV173 cells expressing the non-specific target. (A) Schema of the repetitive co-culture experiments with control (CD19.CAR-T cells), monospecific FN3.EGFR.amR-T cells, DARPin.HER2.amR-T cells and bispecific EGFR-HER2.amR-T cells all plated at 1:2 E:T. (B) Representative flow plots of the repetitive co-culture experiments on the second round. Cells were collected and quantified by flow cytometry on day 3. The frequency of residual tumor cells was identified as CD3⁻CD19⁺ live cells. (C) Quantification of tumor cells remaining after the second co-culture (n=2–3), p<0.01 when comparing FN3.EGFR.amR-T cells and DARPin.HER2.amR-T cells and their respective targets against WT tumor cells; two-way ANOVA with Tukey correction (D) IFN-γ released in the co-culture supernatant by CD19.CAR-T cells, FN3.EGFR.amR-T cells, DARPin.HER2-amR-T cells or EGFR-HER2.CAR-T cells after 24 hours of co-culture with tumor cells as assessed by ELISA (n=2–3), p<0.01 when comparing FN3.EGFR.amR-T cells and DARPin.HER2.amR-T cells and their respective targets against WT cells; two-way ANOVA with Tukey correction (E) Schematic representation of the re-challenge tumor model in NSG mice using the EGFR⁺HER2⁻ human Panc-1 cell line labeled with the FF-Luc and the EGFR⁻HER2 human BV173-HER2 cell line. Representative images of the Pan-1 tumor BLI (F) and BLI kinetics (G) of tumor growth of one experiment 3 – 7 mice per group. (H) Kaplan-Meier survival curve of NSG mice after re-challenge with the BV173-HER2 cell line; p<0.01 when mice treated with EGFR.amR-T cells are compared with mice treated with EGFR-HER2.amR-T cells.