Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies

Abstract

MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.

Figure 1. Isolation and characterization of TARP/HLA-A2 specific TCR-like antibodies

1. ELISA reactivity assay of Fab antibody clones with recombinant purified TARP/HLA-A2 and control complexes. Biotinylated scHLA-A2-peptide complexes were immobilized through BSA-streptavidin. Detection of Fab binding was with Peroxidase-labeld anti-human Fab.

2. Expression and purification of TCR-like D2 Fab. SDS-PAGE analysis of purified D2 Fab. Fab clone was expressed in E.coli and purified by metal affinity chromatography as described. M: Molecular weight markers, NR: Non-reduced SDS-PAGE.

3. ELISA assay for D2 Fab reactivity with TRAP/HLA-A2 and control recombinant purified complexes. Detection was with Peroxidase-labeled anti-human Fab.

4. Binding of D2 Fab by flow cytometry to peptide-loaded APCs. JY EBV-transformed HLA-A2 positive B cells were loaded with the TARP29-37 and control HLA-A2-restricted peptides and the reactivity with purified D2 Fab (10-50μg/ml) was tested. Detection of binding was with PE-labeled anti-human Fab. Control peptides were: MART1, hTERT (540), gp 100-derived peptides G9-209, G9-280, Human T cell lymphotropic virus type 1(HTLV-1) derived peptide TAX and Cytomegalovirus (CMV)- derived peptide. Data are representative of 5 experiments.

Figure 2. Sensitivity of D2 Fab in the detection of TARP/HLA-A2 complexes

Titration of D2 Fab (A) and TARP29-37 peptide (C) by flow cytometry on JY cells pulsed with the TARP29-37 peptide. Controls are JY cells pulsed with the melanoma differentiation-derived HLA-A2-restricted peptide 207-217 derived from gp100. To quantify the minimal number of TARP-HLA-A2 complexes recognized by the D2 Fab, The level of fluorescence intensity on stained cells in A was compared with the fluorescence intensities of calibration beads with known numbers of Phycoerythrin (PE) molecules per bead (QuantiBRITE PE beads; BD Biosciences), thus providing a mean of quantifying PE-stained cells using a flow cytometry. The calculated numbers of complexes/cell at various concentrations of peptide are indicated.

Figure 3. Fine specificity of D2 Fab to TARP enhanced epitopes

1. ELISA binding assay of D2 Fab to recombinant purified HLA-A2 complexes generated with wild-type and epitope enhanced TARP peptides. scHLA-A2/peptide complexes were generated as described in materials and methods. The stability of the wild-type and modified complexes was monitored by reactivity with MAb W6/32 which recognizes properly folded complexes that contain peptide. Reactivity was monitored with Peroxidase-labeled anti-human Fab.

2. Stabilization of class I MHC carrying wild type and epitope enhanced TARP peptides on the cell surface. Flow cytometric analysis of RMAS-HHD cells which are TAP-mutant APCs stably transfected with HLA-A2. Cells were loaded with wild type TARP29-37, TARP29-37-9V, TARP29-37-3A and control Tax peptides. The stabilization of complexes on the cell surface was monitored by the reactivity of BB7.2, a MAb to HLA-A2. Detection was with FITC-labeled anti-mouse IgG.

3. Reactivity of D2 Fab with wild-type and epitope enhanced TARP peptides on the surface of cells. Flow cytometry analysis of RMAS-HHD cells that were loaded with TARP29-37, TARP29-37-9V, TARP29-37-3A and control Tax peptides and the binding of D2 Fab was monitored with PE-labeled anti-human Fab.

Figure 4. Binding of D2 Fab to TARP/HLA-A2 complexes generated by active intracellular processing

JY HLA-A2+ or APD HLA-A2− APCs were transfected with pCDNA containing the intact full length TARP gene (pCTARP), full length melanoma differentiation antigen MART1 (pCMART) or with empty pCDNA control vector. 24 (A) or 48 (B) hrs after transfection cells were stained by flow cytometry using the HLA-A2/TARP-specific D2 Fab or a control CLA12 TCR-like Fab specific for melanoma differentiation antigen MART1 epitope. In C, control APD cells which are HLA-A2−/HLA-A1+ were transfected with pCTARP and pCMART and reacted with the D2 Fab. The efficiency of TARP gene transduction into JY cells was monitored by transfection of the pCDNA vector carrying the GFP gene (D).

Figure 5. Binding of D2 Fab to tumor cells

Flow cytometry analysis of the binding of D2 Fab to tumor cells. TARP and HLA-A2 positive MCF7 and LNCaP cells or TARP and HLA-A2 negative cells were stained with D2 Fab. HLA-A2 expression was monitored by MAb BB7.2. Detection was with FITC-labeled anti-human Fab (for D2 Fab) or FITC-labeled anti-mouse IgG (for BB7.2).

Figure 6. Binding of D2 Fab-PE38 fusion molecule to tumor cells

1. A. Flow cytometry binding analysis of D2 Fab-PE38 fusion to JY APCs. JY cells were loaded with TARP and control Tax and G9-209 peptides and the reactivity of the D2 Fab-PE38 fusion molecule was determined by using rabbit anti-PE38 antibodies. Detection was with FITC-anti rabbit IgG.

2. B-J. Flow cytometry binding analysis of D2 Fb-PE38 fusion to tumor cells. Binding of D2 Fab-PE38 to TARP positive and negative tumor cells was monitored by rabbit anti-PE38 antibodies. In B,E, and H binding to tumor cells was tested. In C,F, and I binding was to tumor cells pulsed with the TARP peptide. Monitoring of HLA-A2 expression was with BB7.2 (D,G,and J). Binding of PE38 fusion was detected with FITC-labeled anti rabbit IgG. Binding of BB7.2 was with FITC-labeled anti-mouse IgG.

Figure 7. Cytotoxic activity of D2 Fab-PE38 fusion

Inhibition of protein synthesis in tumor cells by D2 Fab-PE38 fusions. TARP positive and control tumor cells were incubated for 20 hrs with increasing concentrations of D2 Fab-PE38 (A) or D2 Fab-PE38KDEL (B). Protein synthesis was determined by incorporation of ³H-Leucine into cellular proteins for 4 hrs.

Figure 8. D2 Fab-PE38 prevents tumor growth in athymic nude mice

Athymic mice were injected subcutaneously with 8 × 10⁶ MCF-7 cells diluted in 0.2 ml PBS containing 1μg D2 Fab-PE38. Control athymic mice were injected subcutaneously with 8 × 10⁶ MCF-7 cells diluted in 0.2 ml PBS only or with 1μg TA2 Fab-PE38 which targets Tyrosinase/HLA-A2 pMHC complexes. Tumor growth was observed in mice treated with PBS (◇) or control immunotoxin TA2 Fab-PE38 (▲) at wk 4, the tumor volume had increased >8-fold. In contrast, no tumor growth was observed in mice treated with the D2 Fab-PE38 (■). Tumors were monitored, and final scoring was evaluated at 33 days after implant.