Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen

Abstract

Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.

Fig. 1

Characterization of MCSP-specific BiTE antibodies. a Structures and binding affinities of MCSP-specific BiTE antibodies. All four BiTE antibodies share the same CD3-specific single-chain antibody L2K (open boxes), but differ in their MCSP-specific single-chain antibody (filled boxes). All had a C-terminal hexahistidine sequence for detection and affinity purification; N N-terminus; C C-terminus. b MCSP-derived constructs as expressed on transfected CHO cell lines. D1, D2, and D3 refer to the three domains of the dumbbell-shaped MCSP molecule. MCSP domain 1 corresponds to aa 30–668, D2 to aa 669–1,537, and D3 to aa 1,538–2,221 of human MCSP. All single and double domain constructs contain the transmembrane and cytoplasmic domain of human MCSP corresponding to aa 2,222–2,322; PM plasma membrane. c Surface expression of FLAG-tagged MCSP constructs on stably transfected CHO cells. d Number of binding sites for various anti-MCSP BiTE antibodies on stably transfected CHO lines. Binding sites were determined by the QIFIKIT kit. e Model of MCSP showing binding sites for four BiTE antibodies

Fig. 2

Redirected lysis of human MCSP-expressing CHO line and human melanoma cells by MCSP domain-specific BiTE antibodies. Dose–response analysis for redirected lysis of a human MCSP-expressing CHO line and b human melanoma line 888 by three BiTE antibodies. ⁵¹Cr-labeled target cells were co-cultured for 18 h with stimulated human CD8⁺ T cells at an E:T ratio of 10:1 with the indicated BiTE concentrations. c Values for half maximal lysis (EC₅₀) and overall lysis during the 18-h assay period are shown. Standard deviations of the mean are shown from three independent experiments for MCSP-CHO and two for Mel888

Fig. 3

Redirected lysis of CHO lines expressing MCSP subdomains by MCSP-specific BiTE antibodies. Dose–response analyses of redirected lysis were performed for a, b domain D1-specific BiTE antibody MCSP128, c, d domain D2-specific BiTE antibody MCSP113, and e, f for domain D3-specific BiTE antibody MCSP120 by a ⁵¹Cr release assay. Standard deviations of the mean are shown from three independent experiments

Fig. 4

Characterization of CHO cell lines stably expressing fusions between EpCAM and MCSP domains. a Schematic depiction of fusion proteins between EpCAM and MCSP. All fusion proteins use the transmembrane and cytoplasmic domain of human MCSP; PM plasma membrane. b Structure of EpCAM/CD3-bispecific BiTE antibody MT110. The overall structure is similar to that of MCSP-specific BiTE antibodies (see Fig. 1a). For purification and detection MT110 also contains a hexahistidine tag. c Expression of EpCAM/MCSP fusion proteins on the surface of stably transfected CHO cell lines. Cells were stained for FACS analysis with the parental anti-EpCAM antibody of MT110 and a PE-conjugated monoclonal anti-mouse-IgG antibody. d Quantitation of EpCAM-specific binding sites on the surface of transfected CHO cell lines

Fig. 5

Redirected lysis of CHO lines expressing EpCAM/MCSP fusion proteins by BiTE antibody MT110. a MT110 dose–response analysis of redirected lysis of CHO lines stably expressing fusion proteins between EpCAM and MCSP. b Quantitation of assay results for maximal lysis and half maximum lysis (EC₅₀). Standard deviations of the mean are shown from three independent experiments

Fig. 6

The effect of antigen size on redirected lysis of transfected CHO cells by domain D3-specific BiTE antibody MCSP120. a CHO cell lines expressing EpCAM/MCSP fusion proteins or EpCAM alone were used as targets. b Dose–response analysis of redirected lysis for D3-specific BiTE antibody MCSP120 or EpCAM-specific BiTE antibody MT110 of CHO lines stably expressing fusion EpCAM/MCSP proteins or EpCAM. c Quantitation of assay results for maximal lysis and half maximum lysis (EC₅₀)