Monoclonal antibodies generated in carbonic anhydrase IX-deficient mice recognize different domains of tumour-associated hypoxia-induced carbonic anhydrase IX.
Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for improving our knowledge of structure-function relationships in the CA IX molecule and a better understanding of the role of CA IX in cancer development. Moreover, the availability of the MAbs specific for distinct antigenic regions on two separate extracellular domains offers an opportunity to elaborate a sensitive assay that could be particularly important for CA IX detection in body fluids of cancer patients.
Zat'ovicová
Miriam
M
Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
Tarábková
Kvetoslava
K
Svastová
Eliska
E
Gibadulinová
Adriana
A
Mucha
Vojtech
V
Jakubícková
Lýdia
L
Biesová
Zuzana
Z
Rafajová
Monika
M
Ortova Gut
Marta
M
Parkkila
Seppo
S
Parkkila
Anna Kaisa
AK
Waheed
Abdul
A
Sly
Willam S
WS
Horak
Ivan
I
Pastorek
Jaromír
J
Pastoreková
Silvia
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D001667:Binding, Competitive; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions; D006801:Humans; D000860:Hypoxia / Q000201:enzymology*; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D009369:Neoplasms / Q000201:enzymology*; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C557569:VII-20 monoclonal antibody; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases