Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Abstract

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells

“…The colocalization between GZ19-32 and CAIX was also demonstrated with another CAIX-specific monoclonal antibody M75 (Figure S4 A,B). In contrast, no yellow colocalization spots were seen when the antibody 1B10, specific for another membrane-associated isozyme CAXII 32 , was used (Figure S4 C,D), or when the primary antibody was omitted in the staining procedure (Figure S4 E,F).…”

Proanthocyanidins from Vaccinium vitis-idaea L. Leaves: Perspectives in Wound Healing and Designing for Topical Delivery

“…The PRF effect on human fibroblast migration was assessed using the ‘wound healing’ assay, as described elsewhere [ 66 ]. After trypsinization, HF cells were seeded in 24-well plates at a density of 4 × 10 4 cells/well and incubated for 48 h at 37 °C in a humidified atmosphere containing 5% CO 2 .…”

Carbonic anhydrase XII as biomarker and therapeutic target in ovarian carcinomas

“…Additionally, the enzyme contributes to the acidification of the tumor microenvironment, which in turn promotes tumor invasion and migration [10][11][12]. Consequently, CA XII is considered an attractive druggable target for specific therapies with either blocking antibodies [13][14][15][16][17][18] or small molecules [19][20][21][22][23].…”