A new anti-mesothelin antibody targets selectively the membrane-associated form

Abstract

Mesothelin is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that shows promise as a target for antibody-directed cancer therapy. High levels of soluble forms of the antigen represent a barrier to directing therapy to cellular targets. The ability to develop antibodies that can selectively discriminate between membrane-bound and soluble conformations of a specific protein, and thus target only the membrane-associated antigen, is a substantive issue. We show that use of a tolerance protocol provides a route to such discrimination. Mice were tolerized with soluble mesothelin and a second round of immunizations was performed using mesothelin transfected P815 cells. RNA extracted from splenocytes was used in phage display to obtain mesothelin-specific antigen-binding fragments (Fabs) that were subsequently screened by flow cytometry and ELISA. This approach generated 147 different Fabs in 34 VH-CDR3 families. Utilizing competition assays with soluble protein and mesothelin-containing serum obtained from metastatic cancer patients, 10 of these 34 VH-CDR3 families were found to bind exclusively to the membrane-associated form of mesothelin. Epitope mapping performed for the 1H7 clone showed that it does not recognize GPI anchor. VH-CDR3 sequence analysis of all Fabs showed significant differences between Fabs selective for the membrane-associated form of the antigen and those that recognize both membrane bound and soluble forms. This work demonstrates the potential to generate an antibody specific to the membrane-bound form of mesothelin. 1H7 offers potential for therapeutic application against mesothelin-bearing tumors, which would be largely unaffected by the presence of the soluble antigen.

Keywords: Competition assay; membrane-specific antibody; mesothelin; phage display; serum mesothelin; soluble mesothelin; therapeutic antibody; tolerance immunization.

Figure 1.

Generation of mouse anti-mesothelin Fabs. 5 mice were first tolerized with soluble form of mesothelin, then immunized with P815-Meso cells. After 4 cycles of immunization mice were killed and spleens were resected. Total RNA was extracted from splenocytes and cDNA was produced. Vh-Ch1 and Vk-Ck sequences were amplified, cutted and inserted to pCB3 phage vector. (A). 2 rounds of panning were performed on libraries with CHO-Meso (45%) vs CHO-WTcells (Round I) and P815-Meso (90%) vs P815 wt cells (Round II) respectively (B). Number of positive FABs screened on CHO-Meso and Hela cells for each mouse phage display library (C). Study pathway to identify non-competing Fabs which bind only membrane associated form of mesothelin (D).

Figure 2.

Competition tests with soluble recombinant mesothelin protein. Example of non-competing (3C2 and 1H7) and competing (3C1) Fabs with staining on Hela cells with or without presence of 0.8 µg soluble mesothelin protein. (A). ELISA assay to detect the binding capacity of Fabs (3C1, 3C2 and 1H7) to soluble recombinant mesothelin protein (B).

Figure 3.

Competition tests with patients' sera and concentrated Hela supernatant. Mesothelin concentration in patients' serum determined by ELISA assay (1 to 21). 2 safe donors (A and B) and PBS was used as a negative control for mesothelin expression and ELISA specificity, respectively (A). Mesothelin concentration in 10X concentrated Hela supernatant with ELISA assay. Hela cell lysate was used as a mesothelin containing control. (B). Examples of staining on Hela cells with non-competing Fabs (1H7 and 3C2) and competing FAB (3C1) in presence of 10X concentrated Hela supernatant and patients' sera high and low (C).

Figure 4.

Test on other cancer cell lines. FACS analysis of staining by 1H7-hFc on different cancer cells. CHO-Meso vs CHO-WT cells were used as a specificity control for 1H7-hFc.

Figure 5.

1h7-hFc does not bind to GPI. Mesothelin staining by 1H7-hFc on Hela cells after treatment with 1 and 10 U/ml of PtdIns-PLC for 1 hour (A). FACS analysis of PC3 and LN-Cap cells by CD59 and 1H7-hFc staining (B). RMFI on Hela cells by staining differents concentrations (25, 50 and 100 nM) of FLAER (C). FACS analysis of staining on Hela cells by 1H7-hFc with or without the presence of 50 nm of FLAER (D).

Figure 6.

VH-CDR3 sequence analysis of competing and non-competing Fabs. The frequency of individual amino acids at the specific 11 positions of competing (n = 10) (A) and non-competing binders'(n = 10) (B) VH CDR3 sequences. The color menu for amino acids is according to IMGT. CDR3 positions are shown according to the IMGT unique numbering. Only membrane associated form binders (non-competing Fabs) in panel A, soluble form binders (competing Fabs) in panel B.