LT6331B - New recombinant antibody against human carbonic anhydrase xii - Google Patents

Abstract

A new recombinant antibody that targets transmembrane enzyme carbonic anhydrase XII was produced. The expression of the enzyme increases in certain types of cancer cells. Recombinant antibody produced is a new protein, which so far has not been described in scientific literature. First, cell line (hybridomas) producing monoclonal antibody to carbonic anhydrase XII was developed. Copy DNA sequences encoding an immunoglobulin variable regions were cloned from hybridoma cells, recombinant plasmids were created and recombinant antibody expression was attained. Well-known methods, such as hybridoma technology, genetic engineering, protein purification, immunochemical analysis were used for antibody development. Recombinant antibody produced may be applied in medical and biopharmaceutical branches due to capacity to inhibit growing of cancer cells containing carbonic anhydrase XII.

Description

Field of the Invention

The invention relates to the field of protein biotechnology; it describes the preparation of a recombinant antibody that recognizes and specifically binds to and neutralizes human carbonic anhydrase XII, and describes unique amino acid sequences of the immunoglobulin molecule required for antibody-antigen interaction.

BACKGROUND OF THE INVENTION

Carbonic anhydrase (CA) is a zinc metal enzyme found in almost every living organism, due to the importance of its catalyzed reaction in various physiological processes. 15 isoforms of the α-CA family have been detected in the human body and are found in cells of various organs and tissues (erythrocytes, intestines, adipose tissue, kidneys, lungs, gastrointestinal tract, brain, eyes, capillary endothelium, liver, saliva) [Gilmour KM Biochem Physiol Integr Physiol. 2010, 157, pp.193-197].

CA IX and CA XII are membrane enzymes with increased expression in cancerous lesions, particularly under conditions of hypoxia that are characteristic of solid tumors. For this reason, CA IX and CA XII expression is associated with cancer development. These enzymes are thought to have a direct effect on tumor progression by contributing to the acidification of the tumor environment, the regulation of intracellular pH - making cancer cells more susceptible to hypoxia and more likely to metastasize. [Kailio H. J Cancer Mol. 2010, 5, pp.73-78],

CA IX is currently recognized as a biomarker of light renal cell carcinoma. Determining the expression of this protein is important for the diagnosis of kidney cancer since CA IX is only synthesized in cancerous cells and not in normal cells. Studies are also being conducted to show the prognostic value of the enzyme, which will help predict the cancer's tendency to metastasize, its resistance to therapy and its likelihood of recovery [Tostain J. Eur J Cancer. 2010, 46, p. 3141-3148].

CA XII is an intensively studied enzyme to confirm its value as a marker for cancer cells. CA XII expression has been shown to be associated with more advanced oral carcinoma of the oral cavity, larger tumor size, and poorer patient survival [Chien M.H., Oncol. 2012, 48, p. 4172

423], more extensive research is needed to improve the diagnostics of cancers of various tissues, which encourages the development of novel diagnostic reagents and systems for studying CA XII expression in clinical samples.

Monoclonal Antibodies (MAks) are extremely popular diagnostic reagents used in clinical trials in a variety of fields: blood group, viral infections, cancer, vascular and heart disease diagnostics. Immunotherapy, another important area of antibody (Ak) application, is currently expanding rapidly. Mouse MAk are engineered to be engineered to retain their antigen recognition properties without triggering an immune response in the human body. Modified or recombinant antibodies may have various structures, such as the chimeric Ak composed of the murine MAk variable region (V), which is important for antigen recognition, and the human immunoglobulin constant domain (C) responsible for effector antibody functions; single-stranded Ak fragments (scFv) containing only the antigen-binding region and so on. Bacteria, yeast, plants, mammalian cells, or phage display systems are used to produce such recombinant antibodies [Frenzel A. Front Immunol. 2013, 4, p. 1-20],

Fig. 1 shows a schematic structure of recombinant antibodies (scFv and scFv-Fc) and their differences from full IgG class immunoglobulin [adapted from Frenzel A. Front Immunol. 2013, 4, p. 1-20].

Ak may activate the immune system by inducing Ak-induced cell-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or induce apoptosis, arrest proliferation, angiogenesis, or decrease cancer cell. spread of metastases. Often, the Ak effect is enhanced when used in combination with chemotherapy and substances that activate the immune system. The best results of AK treatment are obtained when used early in the disease. Rituximab, an Ak recognizing CD20 molecule, is the first registered chimeric MAk (1997) used to treat leukemia [Mellstedt H. Drugs Today (Bare). 2003, 39, p. 1-16].

RENCAREX® (Girentuximab) is a candidate for Phase 3 clinical trials for the treatment of non-metastatic light renal cell carcinomas. Girentuximab is a chimeric MAk that recognizes CA IX. It is the only Ak against carbonic anhydrase that has reached clinical trials.

Recombinant antibodies against CA XII are not described.

Drugs based on CA XII antibodies are also not described.

Lithuanian and foreign patents and publications describing the nearest analogs of the invention

Source Description PCT / US2006 / 046350 (name Carbonic anhydrase ix (g250) antibodies and methods of mustache paragraph ") The patent describes recombinant antibodies against human carbonic anhydrase IX, which also is a transmembrane enzyme and a marker for cancer cells but differs from carbonic anhydrase XII by amino acids sequence, structure and functions. Described antibodies do not react with human carbonic anhydrase XII. PCT / EP2011 / 056964 (Novel antibody to a carbonic anhydrase ") The patent describes mice monoclonal antibodies against carbonic anhydrase XII and provided by them variable region hyper variable sequences (CDRs): three heavy and three light sequences of immunoglobulin chains. The antibody sequences described do not match with an antibody of the present invention sequences. 1) Dekaminaviciute D, Lasickiene R, Parking S, Yoga V, Matuliene J, Matulis D, Sparrow A Development and characterization of new monoclonal antibodies against These publications describe other hybridomas we have created, producing monoclonal antibodies against carbonic anhydrase XII. Hybridoma 14D6 described in this application,

human recombinant CA XII. Biomed Res Int. 2014; 2014: 309307. doi:

10.1155 / 2014/309307.

2) Dekaminaviciute D, Kairys V, Zilnyte M, Petrikaite V, Yoga V, Matuliene J, Gudleviciene Z, Vullo D, Supuran CT, Žvirbliene A. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity. . J Enzyme Inhib Med Chem. January 9, 2014

not mentioned in our previous publications. In addition, the publications mention only monoclonal antibodies but not recombinant antibodies. Monoclonal antibodies differ from recombinant antibodies in that they are generated by other methods and have different structures.

The essence of the invention

The object of the present invention is to provide a novel recombinant antibody recognizing carbonic anhydrase XII. The present invention provides immunoglobulin heavy and light chain variable region (CDR) sequences obtained using genetic material from a 14D6 hybridoma cell line producing a native CAXII protein recognizing and neutralizing monoclonal antibody to CA XII.

Such an antibody has

(a) the amino acid sequences of SEQ ID NO. 1 (CDR 1), 2 (CDR 2), 3 (CDR 3), which define the heavy chain variable region (VH), and / or the amino acid sequence of SEQ ID NO. 4 (CDR 1), 5 (CDR 2), 4 (CDR 3), which define the light chain variable region (VL);

or

b) the amino acid sequences of part (a) wherein at least one amino acid is conservatively substituted with another amino acid of similar polarity in any one of SEQ ID NOs. 1-6.

SEQ ID NO: 1: GFTFSDYG. Length: 8 amino acids;

SEQ ID NO: 2: ISNLAYRI. Length: 8 amino acids;

SEQ ID NO: 3: VRATEYALDY. Length: 10 amino acids;

SEQ ID NO: 4: RASODINNYLN. Length: 11 amino acids;

SEQ ID NO: 5: YTSKLHS. Length: 7 amino acids;

SEQ ID NO: 6: OOGNTLPFTF. Length: 10 amino acids.

The present invention demonstrates that the 14D6 antibody recognizes the following amino acid sequence (CA-XII protein fragment from 27-valine to 130-glycine)

SEQ ID No: 7:

VNGSKWTYFGPDGENSWSKKYPSCGGLLQSPIDLHSDILQYDASLTPLEFQ

GYNLSANKQFLLTNNGHSVKLNLPSDMHIQGLQSRYSATQLHLHWGNPNDPHG or similar amino acid sequences with at least 95% identity [Basic Local Alignment Search Tool: http://blast.ncbi.nlm.nih.gov/Blast.cgn

The 14D6 antibody or fragment thereof according to the present invention is for use in inhibiting or neutralizing the biological activity of carbonic anhydrase XII. This antibody may be chemically or genetically conjugated to an enzyme, other protein or chemical compound and used as a diagnostic or therapeutic agent.

Brief description of the drawings

Fig. 1 shows a schematic structure of recombinant antibodies (scFv and scFv-Fc) and their differences from full IgG class immunoglobulin [adapted from Frenzel A. Front Immunol. 2013, 4, p. 1-20].

FIG. 2 shows an immunoblotting image obtained with recombinant CA XII synthesized in E. coli and HEK 293 cells and HEK 293 cell lysate.

FIG. 3 replicates the IFA results obtained using recombinant CA XII synthesized in E. coli (CA XII bacterium) or in the mammalian cell line HEK 293 (CA XII mammalian) as antigen.

FIG. 4 shows the interaction of the 14D6 antibody with the native protein present in the cell membrane.

FIG. 5 shows that the monoclonal antibody recognizes the amino acid moiety at the N-terminus of the protein and may have a linear epitope between the amino acid valine (position 27) and the amino acid glycine (position 130).

FIG. 6 shows the inhibitory effect of 14D6 on recombinant CA XII, its dissociation constant (~ 42nM) close to that of the classic carbonic anhydrase inhibitor acetazolamide Kd (-37.5 nM).

FIG. 7 shows the nucleotide and amino acid sequences of the variable regions of the immunoglobulin heavy (VH) and light (VL) chain variable regions (underlined).

FIG. 8 illustrates a schematic representation of the constructs VL- (G4S) 4-VH and VH- (G4S) 4-VL comprising the variable region fragments of antibody 14D6 with the human immunoglobulin Fc fragment (scFv-Fc-1 and scFv-Fc-2, respectively).

FIG. 9 shows an immunoblotting image obtained with a recombinant antibody synthesized in HEK 293 cells.

Detailed Description of the Invention

The present invention describes a heavy and light chain variable region (CDR) heavy chain and light chain antibody recombinant antibody consisting of a monoclonal antibody against carbonic anhydrase XII produced by a novel hybridoma line 14D $. Studies have shown that the resulting monoclonal antibody 14D6 reacts with both recombinant CA XII proteins synthesized in E. coli or the mammalian cell line HEK 293 and native CA XII synthesized in the cell membranes of various human cancer cell lines. The monoclonal antibody produced has also been shown to neutralize the biological activity of the recombinant CAXII protein in vitro by blocking the enzymatic reaction of carbon dioxide hydration.

The recombinant antibody of the present invention is novel and different from analogs from other sources and the monoclonal antibody against CA XII described above.

Recombinant CA XII synthesized in human embryonic kidney cells HEK 293 was used to create a new hybridoma line 14D6 producing a monoclonal antibody to CA XII and routine procedures known to those of ordinary skill in the art were performed, namely:

immunization - recombinant CA XII (antigen) was injected subcutaneously in BALB / c line mice. Prior to injection, the antigen solution was mixed in equal volume with two complete Freund's adjuvant;

re-immunization - after 4 weeks, mice were re-injected with the same dose of antigen without adjuvant; a small amount of blood is re-immunized from the tail vein of mice prior to the second immunization - again after 4 weeks, the same dose of antigen without adjuvant was injected again into the female. a small amount of blood is drawn from the tail vein of mice prior to the third immunization, and 4 weeks after the third immunization. By a known method, the enzyme-linked immunosorbent assay (IFA) was used to test for the presence of antibodies against the antigen in mouse serum.

mouse spleen hybridization - 5 weeks after the third immunization, the mouse with the highest amount of antigen antibody was injected with the same dose of antigen without adjuvant and 3 days later spleen cell hybridization was performed with mouse cancer cells - myeloma Sp2 / 0, fusion agent polyethylene glycol (PEG) [Sigma, USA] was used. This and other procedures were performed according to a known procedure used to obtain hybridomas [Kohler, Milstein, Nature 1975; 256, p. 495-497].

2 weeks after hybridization and receipt of hybrid cell clones (hybridomas), their specificity was tested, i. y. determining whether they produce antibodies against CA XII. the IFA method was used;

the resulting hybridima were characterized by various traits:

determining the isotype of the monoclonal antibody they produce;

testing antibody specificity by IFA and immunoblotting;

after identification of the hybridomas producing the monoclonal antibody to CAXII, they were cloned, propagated and frozen for further storage;

monoclonal antibodies produced by the hybridomas were characterized as follows:

Flow cytometry using IFA and immunoblotting using recombinant CA XII as antigen tests for the neutralizing effect of monoclonal antibodies using recombinant CAXII.

A new hybridoma line was deposited as described above and deposited at the Institute of Biotechnology, Vilnius University, Vilnius, Department of Immunology and Cell Biology:

Hybridoma 14D6, accession number CAXII-001, which produces an IgG1 subclass (subtype) antibody against CA XII.

Said novel hybridoma 14D6 produces a monoclonal antibody from which a recombinant antibody against CA XII, which is the subject of the present invention, was generated.

Characterization of hybridoma products

The properties of the novel hybridoma 14D6 and the monoclonal antibody it produces are illustrated in Figs. 2-6.

FIG. 2 shows an immunoblotting image obtained with recombinant CA XII synthesized in E. coli and HEK 293 cells and HEK 293 cell lysate.

FIG. 3 replicates the IFA results obtained using recombinant CA XII synthesized in E. coli (CA XII bacterium) or in the mammalian cell line HEK 293 (CA XII mammalian) as antigen. Cross-specificity with the recombinant HEK 293 recombinant protein of carbonic anhydrase IX was also tested.

FIG. 4 shows the interaction of the 14D6 antibody with the native protein present in the cell membrane. The interaction was investigated by flow cytometry using the A498 cell line (confirmed in the literature to have increased CA XII expression) and the HEK 293 cell line (CA XII expression was not detected in these cells by other methods).

FIG. 5 shows that the monoclonal antibody recognizes the amino acid moiety at the N-terminus of the protein and may have a linear epitope between the amino acid valine (position 27) and the amino acid glycine (position 130).

FIG. 6 shows the inhibitory effect of 14D6 on recombinant CA XII, with a dissociation constant (~ 42nM) close to the Kd (~ 37.5 nM) of the classical carbonic anhydrase inhibitor acetazolamide.

The method of obtaining the novel monoclonal antibodies and their properties are described in more detail in Examples 1-4 below and in the tables which illustrate but do not limit the scope of the invention.

example

Development of hybridomas producing monoclonal antibodies against carbonic anhydrase XII

The main steps in obtaining hybridomas and MAk are: immunization; culturing myeloma cells; cell fusion; blood serum collection; selection and cloning of hybrid cells; culturing hybridomas in vitro; freezing / thawing and storage of hybridomas.

Immunization

6- to 8-week-old BALB / c line mice bred at the Innovative Medical Center in the Department of Immunology were used for immunization. 3 mice were immunized with 50 pg antigen by recombinant CA XII protein synthesized in mammalian HEK293 cells. Subcutaneous injection. During the first immunization, the antigen was dissolved in phosphate buffered saline (PBS) and suspended in an equal volume with Freund's complete adjuvant (PFA). A second immunization was performed after 28 days using the same amount of protein dissolved in PBS. After 28 days mice were immunized a third time with the same amount of antigen in PBS. After each immunization, mouse blood was collected for antibody levels (8 pi blood mixed with 192 pi PBS). The mouse with the highest serum antibody levels was re-immunized with 50 pg of protein 5 weeks after the third immunization. Hybridization was performed 3 days after the last immunization.

Culturing myeloma cells

The Sp2 / 0 myeloma cell line, which does not contain hypoxanthine-guanine-phosphoribosyltransferase, does not grow on selective hypoxanthine-aminopterin-thymidine (HAT) medium, and does not produce immunoglobulins or their chains, was used for hybridization. Prior to hybridization, the cells were grown in Dulbecco's modified Eagl's (DMEM) medium with 9% fetal calf serum (FBS) inoculated 3 times. Live cells in the logarithmic growth phase were used for fusion.

Cell fusion

The spleen of a mouse immunized with antigen was sterile removed and crushed to obtain a spleen cell suspension. Suspended myeloma and spleen cells were added to a single tube at a ratio of 1: 5, then slowly infused with 1 ml of cell fusion agent 50% polyethylene glycol (PEG-4000) solution. Over the next 3 minutes, the cells were incubated and 10 ml of serum-free medium was slowly added. After fusion, cells were suspended in already prepared selective HAT medium containing 15% FBS in which only hybrid cells grow. Macrophages harvested from the peritoneal cavity of the mouse during hybridization are required for better cell growth. Cells are seeded in sterile tissue culture 96-well plates at 150,000. into the well.

Non-fused myeloma cells begin to die a few days after hybridization due to the action of aminopterin. Macrophages begin to phagocyte their debris and the resulting clones become much more noticeable.

10-12 days after hybridization, growth media of hybrid clones were assayed by immunoenzyme assay (IFA) using the antigen used to immunize the mice. Hybrid clones producing the most potent antibodies (those with the highest optical density) were selected and cloned by limiting dilution. After cloning, the specificity and activity of the clones is checked again and then grown in larger wells for MAk propagation and cell freezing.

The hybridomas were cultured in DMEM growth medium containing 2 mM L-glutamine, 200 pg / ml gentamycin and 15% FBS in a 5% CO 2 incubator with 98% humidity at + 37 ° C.

Several hybridoma clones were obtained that produced monoclonal antibodies against carbonic anhydrase XII. Their specificity, neutralizing activity and antibody isotypes were investigated. The hybridoma producing neutralizing monoclonal antibodies against CA XII was named 14D6 and deposited in the collection of the Institute of Biotechnology (Vilnius, LT), Vilnius University.

example.

Monoclonal antibody specificity assay and isotype determination

FIG. 2 shows an immunoblotting image obtained with recombinant CA XII synthesized in E. coli and HEK 293 cells and HEK 293 cell lysate. Photograph of denaturing protein electrophoresis on the left and 14D6 monoclonal antibody immunoblotting (hybridoma growth medium, undiluted).

Lane M - stained protein molecular weight standard (kDa);

lane - recombinant purified CA XII synthesized in HEK 293 cells;

lane - recombinant purified CA XII synthesized in E. coli;

lane - HEK 293 cell lysate without CA XII;

FIG. 3 replicates the IFA results obtained using recombinant CA XII synthesized in E. coli (CA XII bacterium) or in the mammalian cell line HEK 293 (CA XII mammalian) as antigen. Cross-specificity with the recombinant HEK 293 recombinant protein of carbonic anhydrase IX was also tested. Hybridoma growth medium diluted 3 times was used.

FIG. 4 shows the interaction of the 14D6 antibody with the native protein present in the cell membrane. The interaction was investigated by flow cytometry using the A498 cell line (confirmed in the literature to have increased CA XII expression) and the HEK 293 cell line (CA XII expression was not detected in these cells by other methods). The primary antibodies used were: 1) black line - 14D6 antibody; 2) red line control IgG1 subclass antibody to porcine parvovirus, developed at the Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University (negative control). Secondary antibodies: Commercial goat antibodies against mouse immunoglobulins conjugated to FITC (BD Biosciences, USA). The signal intensity is increased by FITC glow and is recorded as a shift in the curve during the interaction of 14D6 with A498 cells (Figure left).

FIG. 5 shows that the monoclonal antibody recognizes the amino acid moiety at the N-terminus of the protein and may have a linear epitope between the amino acid valine (position 27) and the amino acid glycine (position 130). Above is a comparison of the full length amino acid sequence of CA XII with the amino acid sequence of fragment # 1; bottom immunoblotting image obtained with CA XII fragments synthesized in E. coli. Photograph of denaturing protein electrophoresis on the left and immunoblotting with 14D6 monoclonal antibody on the right (5 times diluted hybridoma growth medium).

Lane M - stained protein molecular weight standard (kDa);

lane # 1 fragment, lysate of E. coli;

lane - fragment # 2, E. coli lysate;

lane # 3 fragment, lysate of E. coli;

lane - recombinant purified CA XII synthesized in HEK 293 cells.

example

CA XII Inhibition Assay

A recombinant CAXII protein produced in a mammalian cell line was used to measure the enzyme activity of carbonic anhydrase. CAXII hydration activity was measured by the stopped flow method [Khalifah. J Biol Chem 1971, 246, p. 2561-2673] using an Applied Photophysics SX.18MV-R spectrophotometer at 25 ° C. The indicator used was 30 μΜ phenol red, the absorbance of which was observed at 557 nm in the presence of 40 nM CAXII, 1717 mM CO2, 25 mM Hepes, 50 mM NaCl, pH 7.5 and different amounts (0-2 μΜ) of antibody or small molecule inhibitor. (acetazolamide). The rate of the catalyzed reaction was compared with that of the spontaneous CO2 reaction. The CAXII - antibody / suppressor complex was incubated for ~ 1 h before measurement. At 25 ° C. Dissociation constants were calculated using the Morrison equationFig. 6 shows the inhibitory effect of 14D6 on recombinant CA XII, its dissociation constant (~ 42nM) close to that of the classic carbonic anhydrase inhibitor acetazolamide Kd (-37.5 nM).

example.

Identification of antibody sequences and construction of recombinant antibody

Construction of the recombinant antibody began with the identification of the heavy chain and light chain variable region (CDR) sequences of the monoclonal antibody and the isolation of genetic material (RNA) from hybridoma 14D6. The following procedures were used:

1) Isolation of RNA from Hybridoma 14D6 - Total RNA was isolated from Hybridoma 14D6 cells using a single-step technique as described previously [Chomczynski and Sacchi, Anai. Biochem., 162, 1987, p. 156-159; Boom R., J. Clin. Microbiol. 1990, 28 pp. 495-503]. Cells are disrupted and homogenized in buffer with guanidine thiocyanate. The cell lysate is mixed with ethanol and applied to silica gel purification columns, where RNA is attached. The column is then rinsed and the pure RNA is unleashed with nuclease-free water.

2) Synthesis of the first copy DNA (cDNA) strand - The first cDNA strand is synthesized using RNA-dependent DNA polymerase (reverse transcriptase) in combination with poly (A) iRNA and random hexamer primers using the commercially available RevertAid

H Minus First Strand cDNA Synthesis Kit, [Thermo Fisher Scientific, Vilnius].

3) Final cDNA was amplified by polymerase chain reaction (PCR) using specific primers for murine immunoglobulin heavy and light chain variable domain (VH and VL, respectively):

(a) Mouse immunoglobulin heavy chain constant region primer 5'TTAATAGACAGATGGGGGTGTCGTTTTGGC;

b) Heavy degenerate primer 5-CATATGSARGTNMAGCTGSAGSAGTC (R - denotes A / G; S - C / G; N A / C / G / T) in the murine immunoglobulin heavy chain FR1 region;

c) Mouse immunoglobulin light kappa constant region primer 5-TTAGGATACAGTTGGTGCAGCATC;

d) Universal degenerate primer 5'CATATGGAYATTGTGMTSACMCARVVCTMCA (R- denotes A / G; S-C / G; W-A / T; M-14) in the murine immunoglobulin light kappa constant region constant region.

A / C; Y - C / T).

4) The PCR products were cloned into a vector plasmid and the nucleotide sequence was sequenced. Sequences were analyzed using BLASTN, pDRAW32, and Chrome Lite programs, and GenBank and IgBLAST databases. Immunoglobulin sequences of SEQ ID: no. 1 (CDR 1), 2 (CDR 2), 3 (CDR 3), which define the heavy chain variable region (VH), and the amino acid sequence of SEQ ID NO. 4 (CDR 1), 5 (CDR 2), 4 (CDR 3), which define the light chain variable region (VL) (Fig. 7).

FIG. 7 shows the nucleotide and amino acid sequences of the variable regions of the immunoglobulin heavy (VH) and light (VL) chain variable regions (underlined).

By cloning these sequences from hybridoma 14D6, a single-stranded antibody fragment (scFv) was constructed by introducing a linker (linker) sequence (G4S) 4 between VL (SEQ ID NO: 9) and VH (SEQ ID NO: 8). The final construct encoding VL- (G4S) 4-VH or VH- (G4S) 4-VL was cloned into the expression vector pCEP4dS [FreeStyle Max 293 expression system, Invitrogen, Life Technologies, Carlsbad, California, USA]. The construct comprises the human immunoglobulin Fc domain. The N-terminus of the protein contains the secretion signal (SS) sequence. The final plasmid was transfected with human embryonic kidney HEK 293 line cells. Transient transfection and protein induction were performed according to the manufacturer's recommendations.

FIG. 8 illustrates a schematic representation of the constructs VL- (G4S) 4-VH and VH- (G4S) 4-VL comprising the variable region fragments of antibody 14D6 with the human immunoglobulin Fc fragment (scFv-Fc-1 and scFv-Fc-2, respectively). VH immunoglobulin heavy chain variable region hyper reading region (SEQ ID NO: 8); VL-immunoglobulin light chain variable region hyper reading region (SEQ ID NO: 9); (G4S) 4 - Linker (linker) between VL and VH consisting of 4 glycines and 1 serine sequence repeating 4 times; Fc is the C-terminal portion of the human immunoglobulin heavy chain.

FIG. 9 shows an immunoblotting image obtained with a recombinant antibody synthesized in HEK 293 cells. On the left, photo of denaturing protein electrophoresis, on the right, immunoblotting with polyclonal rabbit antibodies recognizing human immunoglobulin Fc chain conjugated to horseradish peroxidase, diluted 1000-fold [Dako, Agilent Technologies, Denmark, Glostrup].

Lane M - stained protein molecular weight standard (kDa);

lane Lysate of HEK 293 cells producing recombinant antibody VL- (G4S) 4VH-Fc;

lane Lysate of HEK 293 cells producing recombinant antibody VH- (G4S) 4VL-Fc;

lane Lysate of HEK 293 cells producing the recombinant human immunoglobulin Fc fragment;

lane HEK 293 cell lysate;

lane - Human immunoglobulins IgG from serum [Sigma-Aldrich Co., USA, Missouri, St. Louis].

Immunoblotting shows that HEK 293 is transfected into cells after transfection with recombinant expression of antibodies consisting of the constructs VL- (G4S) 4-VH and VH- (G4S) 4-VL, comprising antibody 14D6 variable region fragments with the human immunoglobulin Fc fragment.

6) The amino acid sequence of the antibody, which differs from the antibody sequence described in PCT / EP2011 / 05696, has been determined as follows:

14D6 antibody 6A10 antibody (analogue described in PCT / EP2011 / 056964) SEQ ID NO: 1: GFTFSDYG. Length: 8 amino acids; 1: GFSLTTYSVS

SEQ ID NO: 2: ISNLAYRI. Length: 8 Amino acids; 2: RMVVYDGDTV SEQ ID NO: 3: VRATEYALDY. Length: 10 amino acids; 3: DFGYFDGSSPFDY SEQ ID NO: 4: RASODINNYLN. Length: 11 amino acids: 4: RASOGISTSHIRE SEQ ID NO: 5: YTSKLHS. Length: 7 Amino acids; 5: FASOSIS SEQ ID NO: 6: OOGNTLPFTF. Length: 10 amino acids. 6: OOTYSLPYTF

References

Gilmour K. M. Perspectives on Carbonic Anhydrase, Comp Biochem Physiol A Mol Integr Physiol. 2010; 157: 193-197.

Kailio H., Martinez A.R., Hilvo M., Hyrskyluoto A., Parkkila S. CancerAssociated Carbonic Anhydrases IX and XII: Effect of Growth Factors on Gene Expression in Human Cancer Cell Lines, J. Cancer Mol. 5: 73-78, 2010

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Chien M.H., Ying T.H., Hsieh Y.H., Lin C.H., Shih C.H., Wei L.H., Yang S.F. Tumor-associated carbonic anhydrase XII is linked to the growth of primary weather squamous cell carcinoma and its poor prognosis. Weather in Oncol. 2012 May; 48 (5): 417-23.

Frenzel A., Hust M., Schirrmann T. Expression of Recombinant Antibodies Front Immunol. 2013; 4: 217.

Mellstedt H. Monoclonal antibodies in human cancer. Drugs Today (Bare). 2003; 39 Suppl C: 1-16.

Chomczynski, P. Sacchi. N. Single-step method for RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anna. Biochem. 1987,162: 156159.

Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar; 28 (3): 495-503.

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Khalifah RG. Carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on native human isoenzymes B and C. J Biol Chem (1971) 246 (8): 2561-73.

Claims (14)

Definition of the Invention An isolated antibody or antibody fragment that specifically binds carbonic anhydrase XII comprising: (a) a heavy chain variable region CDR1 comprising SEQ ID NO. 1, a heavy chain variable region CDR2 comprising SEQ ID NO. 2 and a heavy chain variable region CDR3 comprising SEQ ID NO: 2. 3, and / or b) a light chain variable region CDR1 comprising SEQ ID NO. 4, a light chain variable region CDR2 comprising SEQ ID NO. 5 and a light chain variable region CDR3 comprising SEQ ID NO. 6th An isolated antibody or antibody fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 9th The antibody of claim 1 or 2, which is a monoclonal antibody. The antibody fragment of claim 1 or 2, which is a single-chain antibody fragment (scFv). The antibody or antibody fragment of claim 1 or 2, which recognizes carbonic anhydrase XII in human cells. The antibody or antibody fragment of claim 1 or 2 which neutralizes the enzymatic activity of carbonic anhydrase XII. The antibody or antibody fragment of claim 1 or 2, wherein at least one amino acid is conservatively substituted with another amino acid of similar polarity in any one of the amino acid sequences of SEQ ID NO. 1-6 or SEQ ID NO. 8 and 9th The antibody or antibody fragment of claim 7, which recognizes the amino acid sequence of carbonic anhydrase XII from 27-valine to 130-glycine (SEQ ID NO: 7) or a similar amino acid sequence that is at least 95% identical to SEQ ID NO: 7. The conjugate of an antibody or antibody fragment according to any one of the preceding claims. An isolated nucleic acid molecule encoding the amino acid sequence of any of the antibodies and antibody fragments of any one of the preceding claims. An expression vector comprising the nucleic acid molecule of claim 10. A recombinant host cell comprising the expression vector of claim 11. A composition comprising an antibody or antibody fragment according to any one of claims 1 to 9, a nucleic acid molecule encoding an antibody or antibody fragment, an expression vector comprising a nucleic acid molecule and an expression vector comprising a host cell for use in the diagnosis of cancer. The antibody or antibody fragment of any one of claims 1 to 9 for use in inhibiting the growth of cancer cells.