T Cell Receptor-Directed Bispecific T Cell Engager Targeting MHC-Linked NY-ESO-1 for Tumor Immunotherapy

Abstract

Antibody-based bispecific T cell engagers (TCEs) that redirect T cells to kill tumor cells have shown a promising therapeutic effect on hematologic malignancies. However, tumor-specific targeting is still a challenge for TCEs, impeding the development of TCEs for solid tumor therapy. The major histocompatibility complex (MHC) presents almost all intracellular peptides (including tumor-specific peptides) on the cell surface to be scanned by the TCR on T cells. With the premise of choosing optimal peptides, the final complex peptide-MHC could be the tumor-specific target for TCEs. Here, a novel TCR-directed format of a TCE targeting peptide-MHC was designed named IgG-T-TCE, which was modified from the IgG backbone and prepared in a mammalian cell expression system. The recombinant IgG-T-TCE-NY targeting NY-ESO-1_157-165/HLA-A*02:01 could be generated in HEK293 cells with a glycosylated TCR and showed potency in T cell activation and redirecting T cells to specifically kill target tumor cells. We also found that the in vitro activity of IgG-T-TCE-NY could be leveraged by various anti-CD3 antibodies and Fc silencing. The IgG-T-TCE-NY efficiently inhibited tumor growth in a tumor-PBMC co-engrafted mouse model without any obvious toxicities.

Keywords: NY-ESO-1; T cell receptor; bispecific T cell engager; immunotherapy; pMHC; soluble expression.

Figure 1

Structure and characterization of IgG-T-TCE. (a) Schematic diagram of IgG-T-TCE-NY structure. Red arrows indicate the simultaneous binding of IgG-T-TCE-NY to NY-ESO-1_157–165/HLA-A*02:01 on tumor cell and human CD3 on T cell. The MW of each chain was estimated using ExPASy. (b) Flow diagram of IgG-T-TCE preparation. (c) Western blot analysis of IgG-T-TCE-NY. (d) Reducing SDS-PAGE analysis of IgG-T-TCE-NY with/without deglycosylation operation. The position of each band matched the estimated size of each chain of IgG-T-TCE-NY after deglycosylation. (e) ELISA assay for testing the affinity of IgG-T-TCE-NY to NY-ESO-1_157–165/HLA-A*02:01. The blank group was incubated with PBS instead of IgG-T-TCE-NY. The t-test of these two groups was performed using GraphPad. p value was lower than 0.0001. (f) ELISA assay for comparing the affinity of IgG-T-TCE-NY to NY-ESO-1_157–165/HLA-A*02:01 with/without the deglycosylation operation. PNGase F alone and a blank group incubated with PBS instead of IgG-T-TCE-NY were set as controls. (g) Binding ability of IgG-T-TCE-NY to Jurkat cells measured with FACS. (h) The simultaneous binding of IgG-T-TCE-NY to NY-ESO-1_157–165/HLA-A*02:01 and human CD3 was identified with FACS using the sandwich method. The pMHC multimer tagged with PE could bind IgG-T-TCE-NY caught by the hCD3 on Jurkat cells. The blank group was incubated with PBS instead of IgG-T-TCE-NY. Data are means ± SD, n = 3.

Figure 2

IgG-T-TCE induced T cell activation. (a,b) Expression of T cell activation markers CD69 and CD25 after 72 h co-culture of PBMCs and positive tumor cells (U266) (E:T, 4:1) in the presence of IgG-T-TCE-NY, measured with FACS. Both CD69 and CD25 expression on T cells increased with the increased concentration of IgG-T-TCE-NY. The colors in the density map represent the cell density at the same location. The cell density increases from blue to red. CD3, FITC; CD69, pacific blue; CD25, APC. (c) Analysis of IL-2 secretion after 72 h co-culture (positive tumor cell line: A375). (d) Analysis of T cell proliferation after 96 h co-culture (positive tumor cell: A375-NY-GFP). CFSE was used to label PBMC before co-culture. The proportion of proliferated T cells to T cells significantly increased with the increase in IgG-T-TCE-NY concentration. CD3, APC. (e) Analysis of granzyme B⁺ CD8⁺ T cells after 48 h co-culture (positive tumor cell line: A375). The proportion of granzyme B⁺ T cells to CD8⁺ T cells significantly increased with the increase in IgG-T-TCE-NY concentration. Granzyme B, PE; CD8, APC. (f) Analysis of granzyme B⁺ tumor cells after 48 h co-culture (positive tumor cell line: A375-NY-GFP). The proportion of granzyme B⁺ tumor cells significantly increased with the increase in IgG-T-TCE-NY concentration. Granzyme B, PE. Data are means ± SD, n = 3.

Figure 3

In vitro cytotoxicity assay of IgG-T-TCE. (a) Percentage of tumor cell lysis (A375) measured with the LDH assay (left) after 48 h co-culture and tumor cell apoptosis (A375-NY-GFP) detected with FACS after 48 h co-culture (right). (b) Representative photomicrographs (40× magnifications) of co-culture cells (positive tumor cell line: A375) in the presence of 0 pM and 10,000 pM IgG-T-TCE-NY after 48 h. (c) Relative NY-ESO-1_157–165/HLA-A*02:01 expression on constructed K562-based cell lines (left) and corresponding tumor cell apoptosis detected with FACS after 48 h co-culture (right). Red line, tumor cells stained with APC-labeled 1G4-113 TCR; black line, blank control. (d) Percentage of tumor cell lysis (A375) after 48 h co-culture in the presence of IgG-T-TCE-NY or Fc-silenced IgG-T-TCE-NY measured with the LDH assay. (e) Percentage of tumor cell lysis (A375) after 48 h co-culture in the presence of modified IgG-T-TCE-NY with different affinities to human CD3 measured with the LDH assay. Rank order of CD3 affinity: UCHT1 > hOKT3 > hOKT3-LT. Data are means ± SD, n = 3.

Figure 4

In vivo antitumor activity of IgG-T-TCE. (a) Schematic depiction of animal model and experimental design. Female NOD-SCID mice engrafted with A375 melanoma cells (1.5 × 10⁶) and PBMCs (3.75 × 10⁶) were treated with IgG-T-TCE at 0.1 mg/kg according to the schedule depicted by the purple triangle and yellow inverted triangles. As controls, A375 cells were engrafted without or with PBMCs, and mice were dosed with the vehicle (PBS). (b,c) Average weight and tumor volume change for mice in the three groups. (d) Tumor volume change for each mouse in the three groups. (e) Digital image of the stripped tumors. Data are means ± SD, n = 5.