Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology

Abstract

Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

Figure 1. Characterization of CAIX paramagnetic proteoliposomes (CAIX-PMPLs).

A (left), 3×10⁷ CAIX-PMPLs (lane 1) or M280 Dynal beads coated with 1D4 mAb only (lane 2) were treated with 2xSDS-buffer for 1 hr at 55°C followed by boiling for 5 min. The supernatant were used for SDS-PAGE analysis and proteins were visualized by Coomassie Blue staining. A (right), PMPLs were incubated with [³⁵S]-methionine/cysteine labeled 293T-CAIX-C9 (lane 1) or 293T (lane 2) cell lysates. Purity of the PMPLs was determined by SDS-PAGE followed by autoradiography analysis. B. Schema of the antibody panning and selection process. C. Summary of CAIX-PMPL panning results. D. Phylogenetic tree of the fourteen (14) unique anti-CA IX antibodies based on translational alignment of nucleotide sequence comparison using Geneious (http://www.geneious.com/) software with PHYML tree plugin (http://atgc.lirmm.fr/phyml). Jukes-Cantor genetic distance model were utilized. Consensus tree option was selected with 100 bootstraps. The unit of measure (the scale bar) represents the number of amino acid substitutions per site.

Figure 2. Characterization of selected anti-CAIX scFvFc antibodies.

A. Different anti-CAIX scFvFcs were expressed and purified from 293FT cells as discussed in Material and Methods. A total of 2 µg purified each anti-CAIX scFvFc were treated with 5XSDS-PAGE loading buffer (Pierce) in the presence (lower panel) and absence (upper panel) of reducing reagent DTT. The antibodies were separated by SDS- 10% Tris-Glycine PAGE gel electrophoresis and visualized by Coomassie Blue staining. B. Flow cytometric analysis of the anti-CAIX scFvFc binding activity. One microgram (1 µg) of each anti-CAIX scFvFc was incubated with 5×10⁵ sk-rc-52 (CAIX positive) or sk-rc-59 (CAIX negative) cells in 50 µl FACS buffer (1% BSA/PBS), followed by staining with PE-conjugated goat anti-human IgG. Binding activity of each antibody was examined by flow cytometric analysis and data is presented as overlapping histograms of the CAIX positive and negative cells as well as the secondary antibody only controls for each cell line. Similar findings were found in two independent experiments. C & D. Dosage-dependent binding of the anti-CAIX scFvFcs to 293T-CAIX cells. Anti-CAIX scFvFcs or a control of irrelevant anti-CXCR4 scFvFc (X33) at the concentrations indicated on the X-axis were incubated with 5×10⁵ 293T-CAIX cells followed by staining with FITC conjugated goat anti-human IgG. Flow cytometric analysis was performed and binding activity of each antibody is presented as both percentage of positive cells (C) and absolute geometric mean fluorescence intensity (GMFI) (D). A summary of the calculated EC₅₀ for half-maximal percent binding (C) and GMFI (D), along with maximal percent positive cells and GMFI reached, are presented in Table 1.

Figure 3. Binding specificity of the anti-CAIX antibodies as analyzed by domain mapping studies.

A. Amino acid sequence of CAIX with amino acid locations of the different segment/domains as indicated. B. Diagram of CAIX functional domains and fusion constructs. C. Coomassie Blue staining of 2 µg purified CAIX-ECD-Fc (lane 1 & 4), CAIX-CA-Fc (lane 2 & 5), and CAIX-PG-Fc (lane 3 & 6) separated by SDS-PAGE under non-reducing (lanes 1-3) or reducing (lanes 4-6) conditions. D. Binding of purified biotinylated CAIX-Fc constructs to the purified anti-CAIX scFvFcs analyzed by ELISA. E. Binding of purified anti-CAIX scFvFcs to the CAIX–GST fusion proteins analyzed by ELISA. For D and E, average OD450 of each duplicated sample is presented. Similar results were obtained in two independent experiments. Details on the ELISA assays performed are provided in Experimental Procedures.

Figure 4. Inhibition of carbonic anhydrase activity of CAIX by the anti-CAIX scFvFc antibodies.

The ability of the anti-CAIX scFvFc antibodies to inhibit the reaction CO₂+H₂O → H₂CO₃ catalyzed by CAIX (CAIX) was assessed using the electrometric assay as described in Experimental Procedures. Different molar ratio of antibody to CAIX enzyme was tested. The anti-CXCR4 scFvFc X33 and carbonic anhydrase inhibitor acetazolamide (AZT) were used as negative and positive control, respectively. Percent inhibition of CAIX enzymatic activity was calculated per formula discussed in the Experimental Procedures. A. Inhibition of CAIX enzymatic activity by anti-CAIX-scFvFc at molar ratio 25∶1. B. Inhibition of CAIX enzymatic activity by selected anti-CAIX-scFvFc at molar ratio 1∶1, 5∶1, and 25∶1. C. Inhibition of CAIX enzymatic activity by selected anti-CAIX-scFv antibodies at molar ratio 1∶1, 5∶1, and 25∶1. Values represent an average of three different experiments.

Figure 5. Induction of CAIX internalization from cell surface by anti-CAIX antibodies.

CypHer5E-labeled anti-CAIX scFvFc antibodies were tested for their abilities to induce CAIX internalization by flow cytometric analysis. The 293T cells (A) or 293T-CAIX cells (B) were incubated at 37°C with 2.5 µg/100 µl CypHer5E-labeled anti-CAIX scFvFc antibodies for 15, 30, 45, or 60 minutes. Two irrelevant scFvFc antibodies, 11A and X48, as well as CypHer5E dye were used as negative controls. C represents a similar experiment where CAIX internalization in sk-rc-52 (CAIX positive) cells were analyzed and compared at 37°C and 0°C. Fluorescence intensity of cells was analyzed using FACS Calibur and GMFI value (A & B) or percent of CypHer5E positive cells (C) were presented in corresponding panels.

Figure 6. Antibody-induced CAIX internalization as analyzed by ImageStream.

A. Single cells expressing EEA1 and Lamp1 were gated (R2), and the CypHer5E intensity was plotted for cells incubated with anti-CAIX G37, 119, or 36 (left, center, and right plots), with the relative percentage of CypHer5E+ events (R3) displayed in the upper right of each plot. Representative Brightfield, EEA1 (green), LAMP1 (orange), and CypHer5E (red) cell images from the three antibody treatment groups are shown below in the same order. B. The number of EEA1 or LAMP1 small punctate staining regions is plotted for CypHer5E+ (R3) or CypHer5E- cells from the indicated antibody treatment groups. The percentage of cells with greater than four spots (R4) is displayed in the upper right corner of each plot. Representative EEA1 images from the AbG36 sample of cells with zero (left), ten (center), or twenty EEA1 spots (right) are shown below the plots.