Comparison of methods generating antibody-epitope conjugates for targeting cancer with virus-specific T cells

Abstract

Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.

Keywords: antibody-epitope conjugates (AECs); conjugation strategies; immunotherapy; redirecting virus-specific T-cells; targeted therapy.

Figure 1

Three strategies to generate AECs: chemical, enzymatical and genetic. (A) Schematic representation of the reaction and/or the end-product of the 3 conjugation strategies: maleimide conjugation, Sortase A conjugation, and genetic modification. For the maleimide conjugation an example with 1 epitope is given after reduction of the disulfide bridges. (B) Analysis of the FLAG-tag epitope conjugated to CTX by Western blot with in red the molecular weight marker and FLAG-tag signal, and in green the human IgG signal. For CTX-SrtA-FLAG, the parental antibody with sortase recognition domain (S-His6) and the reaction mixture (RXM) are shown. (C) FACs analysis to visualize the binding of the CTX conjugates with FLAG-tag, to HeLa cells expressing EGFR. As a control a single anti-human-IgG-PE or anti-FLAG-PE staining was taken along.

Figure 2

An EBV T-cell epitope preceded by a protease cleavage sequence can be conjugated chemically, enzymatically, or genetically to tumour-specific antibodies. (A) The amino acid sequences of the peptides that are used in the reaction for maleimide- and sortase A conjugation and the peptide sequence that is attached genetically to CTX. The three conjugation strategies analysed by SDS-PAGE visualized with an Instant Blue staining (B) under reducing and (C) non-reducing conditions. With the conjugation possibilities for CTX-MAL depicted next to the gel. The conjugation possibilities were determined by looking at the most abundant bands. (D) To determine whether the antibody conjugates still bind their target, titrations were performed with CTX antibody conjugates on HeLa-A2 cells and with TRS antibody conjugates on HeLa-A2 Her2 cells and binding potential was analysed by flow cytometry. Bound antibody conjugates were visualized with anti-human IgG-PE and representative data of two independent experiments are shown.

Figure 3

EBV-AECs can sensitize tumour cell lines to be recognized and killed by EBV-specific T cells, irrespective of the strategy used for conjugation. CTX-MAL, -SrtA and -GEN AECs (A) and TRS-MAL, -SrtA and -GEN AECs (B) were titrated on HeLa cells transduced with HLA-A2 (HeLa-A2) for CTX-AECs and on HeLa-A2 tHer2 for TRS-AECs and incubated overnight with EBV-BRFL1/A2-specific T-cells. Supernatant was harvested and IFN-y production as measure for T-cell activation was analysed by IFN-y ELISA. In (C) the EC₅₀s of the different AECs were calculated and plotted. For the statistical analysis, a RM one-way ANOVA with Tukey`s multiple comparisons was performed. (D) To test whether proteolytic activity is necessary CTX-MAL and CTX-MAL_D-AA were taken along in a coculture assay and T-cell activation was analysed by IFN-y ELISA. (E) To check for target cell killing, an AlamarBlue assay was performed. (A, B, D, E) Plotted values are means of duplicates (SEM) and graphs shown are representative figures of an n>3.

Figure 4

Recognition and activation of the T-cells is target specific and shows differences at high concentrations. (A) For both targets KO cells were generated by using CRISPR/Cas9 technology and analysed with FACS for EGFR and HLA-A2 expression (B–D) The three CTX conjugates were also titrated on the HeLa-A2 KO EGFR alongside the HeLa-A2 cells with wildtype (wt) EGFR expression for the three highest concentrations and T-cell activation was measured with an IFN-y ELISA. Plotted values are the means of duplicates within one experiment of at least three independently performed experiments. For the statistical analysis, a repeated measurements one-way ANOVA with Šidák multiple comparisons was performed.

Figure 5

The CMV epitope NLV/A2 epitope was conjugated with sortase A with three different cleavage sites. (A) These conjugates were titrated on HeLa-A2 (B) or HeLa-A2 EGFR KO cells and incubated with CMV NLV/A2-specific T-cells. T-cell activation was measured with an IFN-y ELISA. Plotted values are the means of duplicates (+/- SEM) and graphs shown are representative figures of an n=3.