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Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

ABSTRACT: The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pH_i), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

SUBMITTER: Lenferink AEG

PROVIDER: S-EPMC8632296 | biostudies-literature | 2021 Jan-Dec

REPOSITORIES: biostudies-literature

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Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

Lenferink Anne E G AEG McDonald Paul C PC Cantin Christiane C Grothé Suzanne S Gosselin Mylene M Baardsnes Jason J Banville Myriam M Lachance Paul P Robert Alma A Cepero-Donates Yuneivy Y Radinovic Stevo S Salois Patrick P Parat Marie M Oamari Hafida H Dulude Annie A Patel Mehul M Lafrance Martin M Acel Andrea A Bousquet-Gagnon Nathalie N L'Abbé Denis D Pelletier Alex A Malenfant Félix F Jaramillo Maria M O'Connor-Mccourt Maureen M Wu Cunle C Durocher Yves Y Duchesne Mélanie M Gadoury Christine C Marcil Anne A Fortin Yves Y Paul-Roc Beatrice B Acchione Maurizio M Chafe Shawn C SC Nemirovsky Oksana O Lau Joseph J Bénard Francois F Dedhar Shoukat S

mAbs 20210101 1

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these p ...[more]

PMID: 34806527

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Project description:BackgroundHypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first- or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy.MethodsParental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma, we obtained 25 clones. Each clone was tested for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system.ResultsBased on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity.ConclusionCA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.

Project description:BackgroundCarbonic anhydrase IX (CA IX) is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1) was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes.Methodology/principal findingsVarious fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy) was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues.ConclusionsOur data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of the ligand in clinical approaches.

Dataset Information

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

Publications

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

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