WO2011083175A1 - Monoclonal antibody anti-tumor necrosis factor (tnf), which defines sequences, compositions comprising it and its applications - Google Patents

Abstract

The present disclosure relates to obtaining a monoclonal antibody against TNF or antibody fragments, the nucleotide sequences encoding both the antibody and the variable domains thereof. Methods for cloning and producing the antibody. The disclosure also considered formulations that include the products described above, as well as kits and/or means for the implementation of treatment or diagnosis. Additionally, the disclosure relates to treatment or diagnostic procedures related to the antibody, and the uses for which it is required.

Description

Monoclonal antibody anti-tumor necrosis factor (TNF), which defines sequences, compositions comprising St and its applications. The present invention is related to the field of immunology, particularly with the development of a monoclonal antibody anti-tumor human necrosis factor (TNF).

Description of state of the art. Antibodies (Ab) are the effector molecules of tumoral immune responses they correspond to glycoproteins produced by B cells, which bind to antigens and may trigger a range of effector functions that produce the activation and recruitment of molecules and cells that eliminate pathogens, foreign agents and, eventually, tumor cells, as well as neutralize and inhibit the action of some molecules such as toxins or cytokines.

The Ab have a common structure of four polypeptide chains, consisting of two heavy chains (Heavy-H~) and two identicai light chains (Light-L-) identical disulfide-bonded. They present constant and variable Globular domains, the latter characterized by its amino acid sequence variability, called variable regions (V) and located on the amino terminal heavy chain (VH) and light chain (VL). Constant domains (C), remaining portion of the H and L chain Ab, present a relatively constant stream sequence. In the V domains hypervariabie regions are found, called complementarity determining regions (CDR). The CDRs form the antigen binding site or paratope. Other amino acids of the V region are more conserved and are called Waste Framework-FR-, (scaffold waste). An antigen produces a polyclonal response, consisting of several clones of B lymphocytes by generating hybridoms, hybrid ceils composed of a B lymphocyte fused with a tumor cell that grows indefinitely in culture, secreting specific target antibody or Ab of interest, it is possible to obtain monoclonal antibodies (mAbs) that recognize a particular antigen epitope. The murine mAbs have been useful in research and in the medical field because of their exquisite specificity and high affinity. These features created expectation about which of them could be used as therapeutic agents in humans, by binding to target molecules and acting as blocking agents, neutralizing, cytolytic or killer. However, they have limited usefulness in this area, due to the immune response developed against murine mAbs by patients, known as HAMA (human anti-mouse antibodies). This response limits the efficacy of the mAbs due to the development of hypersensitivity responses and the reduction of the average life of the mAb administered. Another important disadvantage of murine mAbs as therapeutic agents, is that they are not efficient in developing effector functions in humans and have short half life in circulation (Chester and Hawkins, 1995).

In this sense, genetic engineering has been used to modify murine mAbs in order to use them as therapeutic agents in humans, with the goal of reducing the immunogenic sequences of the murine mAbs, partially or completely replacing murine sequences and human sequences Acc. Thus, chimeric mAbs have been generated in which the coding sequences of murine mAb variable domains are fused to sequences encoding the human Ig constant domains (Lazar et al., 2003; Boulianne, et al., 1984) . This type of modified mAbs presents about 30% of murine sequence and 70% of human sequence. This way a dramatic reduction in the human immune system response to an Ab is attained, because the C region contributes in approximately 90% of the immunogenicity of murine mAbs (Adair, JR, 1992).

Humanized mAbs have been generated, in which the coding sequences of the CDR regions of murine mAbs are exchanged in their respective coding sequences of human Ab and fully human Ab through human Ig transgenic mice and construction of expression library genes of human Ig V regions in filamentous phages (Humphrey and Glover, 2001 , Huls et al., 1999, Bruggemann and Taussig, 1997). Also new Ab formats have been developed based Ac fragments thereof (Brekke and Loset, 2003).

The modified mAbs are used primarily in the treatment of cancer and autoimmune diseases, treatment of cardiovascular disease, transplant rejection and infection, although its implementation may be more extensive. Several factors must be evaluated in the development of Ab's for therapeutic purposes according to the intended clinical application, such as affinity, stability, effector function, half- life, tissue penetration, distribution and production facility Ab, among others.

One of the applications of the antibodies is directed towards the human being molecules such as cytokines. TNF is a potent pro-inflammatory cytokine most important to the innate and acquired immunity and participates in inflammatory and autoimmune diseases. It is produced primarily by activated monocytes-macrophages, also B lymphocytes, T lymphocytes, endothelial cells and fibroblasts. TNF is expressed on the cell surface as a homotrimer and is subsequently serine cut metalloproteinase generating soluble TNF (Gearing et al., 1994).

Blocking TNF with modified mAbs has proven effective on diseases that overexpress TNF, mainly on rheumatoid arthritis (Elliott et al., 1994). Also, this therapy has demonstrated efficacy on other pathologies such as Crohn's disease and ulcerative colitis (Van Dullemen et al., 1995), ankilosing spondylitis (Braun and van der Heijde, 2003), Psoriasis (Chaudhari et al., 2001) and PsA Arthritis (Provenzano et al., 2003). Treatment with TNF blockers has become a routine clinical practice, more than 500,000 patients worldwide have been under this kind of treatment the past 6 years, without loss of efficacy and no significant adverse effects (Feldmann and Maini, 2003 ).

In the treatment of rheumatoid arthritis biological drugs are currently used: infliximab, chimeric lgG1 type mAb anti-TNF (Elliott et al., 1993, Knight et a!., 1993; Dentener et al. 2008; Nishida et al., 2008 ), Adalimumab and Golimumab, fully human antibodies anti-TNF, Etanercept, fusion protein of TNF receptor II (TNFRII) with human IgG Fc region (Smolen and Steiner, 2003). The CDP 571 antibody is formed by CDR murine monoclonal antibody anti human TNF and human lgG4 antibody (Sandborn et al. 2004). Monoclonal antibody Rituximab is a chimeric mouse/human that specifically binds to the CD20 antigen. Patients treated with these agents (Infliximab, Adalimumab, Etanercept) in combination with low dose Methotrexate (immunosuppressive drugs), showed a significant improvement in quality of life (Maini et al., 1998, Elliott et al., 1994) and additionally produces a significant inhibition of cartilage loss and articular bone erosion features of this disease (Weinblatt et al., 1999, Lipsky et al., 2000, Weinbiatt et al., 2003).

Similar efficacy has been demonstrated for the three biological agents used in patients with active rheumatoid arthritis (Hochberg et al., 2003). Additionally it has never been definitely concluded which one of the three drugs is the safest (Trotta et al., 2005).

The main problem in administering chimeric mAbs, which represents approximately 30% murine protein, could be the loss of efficiency due to the development of HACA response (human anti-chimeric antibodies), but it has been demonstrated in the treatment of Rheumatoid arthritis, which when administered in combination with low dose of Methotrexate, HACA response is reduced almost completely and no loss of efficiency occurs during the adminitration period (Maini et al., 1998).

The main mechanism by which the three drugs operate on biological therapies of TNF antagonists, is its binding to the TNF, thereby preventing interaction with its receptor. It was suggested that lysis of cells expressing TNF of the membrane (mainly macrophages), after the union of these drugs and complement activation and antibody-mediated cellular cytotoxicity (ADCC), constitute additional mechanisms of action (Scallon et al., 1995).

Description of the figures. Figure 1 , is a table which describes the primers used in this invention. The primers used were obtained from Cesate (Faculty of Medicine, University of Chile) and AlphaDNA (Montreal, Canada)

Figure 2, corresponds to the pcDNA3.1 (+/-) vector (!nvitrogen, USA) and pSecTag2B (Invitrogen, USA) used in cloning events of the present invention.

Figure 3, shows a schematic of the fusion of the cDNAs of murine V regions and human C immunoglobulin by overlap extension PCR. Figure 4, shows the nucleotides sequence and amino acids of the VH region of anti-TNF mAbs of this invention. The leader peptide sequence and the complementarity determining regions (CDRs) are indicated.

Figure 5, shows the nucleotide sequence and deduced amino acid of the VL region anti-TNF mAbs. The leader peptide sequence and the complementarity determining regions (CDRs) are indicated

Figure 6, shows a diagram of vectors pQH and PQL. A. The PQH vector containing cDNA of the murine VH region, including the coding sequence of the leader peptide (L) fused to the cDNA of the human CH region, between the sites Nhel (N) and BamHI (B). The vector contains the selectable marker for eukaryotic cells to neomycin resistance (Neo). B. PQL vector containing the cDNA of the murine VL region, including the coding sequence of the leader peptide (L) cDNA fused to human CL region, between the sites Nhei (N) and BamHI (B). This vector contains the selectable marker for eukaryotic cells of the resistance to Neomicin (Neo). PQL vector containing the cDNA of the murine VL region, including the coding sequence of the leader peptide (L) cDNA fused to human CL region, between the sites Nhei (N) and BamHI (B). This vector contains the selectable marker for eukaryotic cells resistance to Zeocin (Zeo). Both vectors have the cytomegalovirus (CMV) and bacterial selection marker for Ampicillin resistance (Amp) Figure 7 shows TNF-Reactivity of antibodies produced in culture supernatant of cells co-transfected with chimeric vectors, evaluated by ELISA. As antigen bound to the solid phase recombinant human TNF (2pg/ml) was used. Culture supernatant was added to different wells of cells co-transfected and Neomycin selected and used for detection of goat IgG anti-human gamma chain conjugated to peroxidase.

Figure 8, shows the H chain sequence pQH chimeric vector. The restriction sites used are pointed out at the beginning and at the end of the chimeric H chain. The VH region sequence is not shown, but is symbolized and is equivalent to, the one described in Figure 4.

Figure 9, shows the sequence of the chimeric L chain of the PQL vector. Indicates the restriction sites used. The VL region sequence is not shown, but it symbolized and is equivalent to the one described in Figure 5.

Figure 10, shows the alignment of the nucleotide sequence of the constant region of H chain (y1) of chimeric anti-TNF rnAbs of the present invention (identified as Query) with the code sequence gi | 49258105 | gb \ BC073773.1 | string H γ Homo sapiens immunoglobulin (labeled Subject). In position 1 1263 of the DNA sequence, there is a nucleotidic substitution of a thymidine instead of guanosine, however, it does not alter the corresponding amino acid, which is valine.

Figure 11 , shows the alignment of the nucleotide sequence of the constant region of L (K) of the chimeric anti-TNF mAbs of the present invention with the constant region of L (K) of Homo sapiens immunoglobulin (gene IGKC) code gi { 12054081 | emb I AJ294735.1 | HSA294735. This alignment shows 100% identity between sequences. Figure 12, shows the sequence of nucleotides and amino acid chain H (γ1 ) of chimeric anti-TNF mAbs of this invention.

Figure 13, shows the sequence of nucleotides and amino acids in the chain L (K) of the chimeric anti-TNF mAbs of this invention.

Description of the invention.

The present invention is related to obtaining a monoclonal antibody (mAb) against TNF, the nucleotide sequences encoding both the antibody and the variable domains thereof. It also includes cloning methods, production and evaluation of the antibody in vitro.

The invention also includes formulations and compositions comprising the products described above, as well as kits and / or means for the implementation of treatment or diagnosis.

Additionally, the invention relates to treatment or diagnosis procedures related to the antibody, and the uses or applications to which the Ab is required. Detailed description of the invention.

The present invention is an antibody against TNF, preferably human TNF. In one embodiment of the invention, it relates to a monoclonal antibody anti- human TNF, preferably a monoclonal antibody (mouse-human) and anti-TNF Fab and (Fab')₂ in addition to the formats called Fabs , TandAb, Diabody and Flexibody.

According to the above, the invention includes nucleotide sequences such as DNA, cDNA and RNA encoding the various domains and Ab chain of the invention, in addition to the amino acid sequences of the protein, an expert in the art may deduct such sequences from those indicated in the text. Preferably the invention comprises the nucleotide sequences of the antibody variable domains, preferably the VH and VL regions of the antibody. In a preferred form the invention comprises the CDR sequences of the variable regions of the antibody of the invention.

The Ab includes a sequence of nucleotides and amino acids for the heavy and light chains as described in Figures 8 and 9. In a preferred embodiment, the antibodies of the invention corresponds to Ab sequences comprising the variable regions shown in figures 4 and 5. Preferably, the antibodies of the invention includes nucleotide and amino acid sequences for the CDR1 , CDR2 and CDR3 as shown in Figures 4 and 5. The present invention includes allelic variants and sequences represented with addition, insertion, deletion and / or replacement of one or more nucleotides that encode for the variable domains of Ab and / or variable regions shown in figures 4 and 5, which encode for polypeptides that retain recognition function of TNF. Because the genetic code is degenerated, this invention also provides nucleic acid sequences that hybridize with the sequences described above, under standard stringency conditions, such as described by Sambrook et al 2001.

In one embodiment of the present invention the vectors include those vectors which include the nucleotide sequences of VH, VL, CH, CL In an embodiment the vectors of the invention includes vectors comprising pQH PQL and the sequences of the invention. In one embodiment, the invention comprises the cells and microorganisms including at least one of the sequences or vectors of the invention described above. In another embodiment the invention comprises formulations, compositions or preparations comprising the sequences, vectors, antibodies, fragments and formats as previously described. In a referred way, the formulations, compositions or preparations of the invention, comprise the antibodies, fragments thereof or formats with additional components required for the stability and efficiency of the same, where these formulations, compositions or preparations may be liquid, solid , lyophilized or other appropriate physical form in art. In addition, formulations, compositions or preparations are designed for the proper administration in a medically appropriate way, such as, and without being restricted to: oral, dermal, subcutaneous, intramuscular, intraperitoneal, intravenous, ocular, spinal, intracranial, among other.

Additionally, the present invention includes devices, arrangements or kits that include strings, vectors, antibodies, fragments and formats as previously described. In a preferred embodiment, the invention comprises a kit of parts comprising the necessary means for in situ preparation of the formulations, preparations or compositions according to the invention required. In one embodiment the kit comprises the means ready for use with the products or formulations, preparations or compositions described above. Kit components and formulations, compositions or preparations of the invention can be used simultaneously or sequentially, together or separately.

The invention also includes uses for the products of this invention, their preparation, formulations or compositions, in one embodiment, the sequences of the invention are used in production processes or antibodies or fragments thereof formats described above.

In another embodiment of the invention, antibodies are used to treat, prevent, cure or diagnose, diseases related with TNF, also they can be used to control the TNF levels in conditions where the levels of these cytokines are modulators or promoters of the conditions and/ or symptoms of the disease. The main applications of the antibodies which are not limiting to, inflammatory bowel disease such as Crohn's disease and ulcerative colitis, rheumatic diseases, such as rheumatoid arthritis, sacroiliitis, ankylosing spondylitis and psoriatic arthritis, among other diseases, such as Sarcoidosis complicated system, proximal myopathy, Still's disease and Behcet syndrome; In addition to the treatment of psoriasis, systemic inflammatory response syndrome and multiple organ failure.

The present invention relates to methods and processes for the production of chimeric antibodies, in its preferred embodiment, a method characterized by the following steps: a) Purification or synthesis of DNA sequences described in Figures 4 and 5 (antibody variable regions), and the DNA sequences described in Figures 8 and 9 (antibody constant regions), or sequences that code for similar polypeptides such sequences, by general methodology found in the state of the art. b) Construction of chimeric vectors PQL and pQH binding fragments of variable and constant regions by overlap extension PCR, using methodology described in the prior art, in particular, using the region amplified with the primers VL and QLg9supR01 Iidkappag9, the Ck region with primers and IgKCas QLg9supF01 ; The VH region with the primers lid-nhe-g9-H and lgVH-G9supR and the region with primers CH~supF01 QHG9 and AGH, to generate H chain chimeric (fusion sequence murine VH region with the human Cy1) and L chain chimeric (fusion of the DNA sequence encoding the VL region of the murine with the human CK region) which are linked with the pcDNA3 vector and vector pSecTag2B respectively to generate the final vector pQH and PQL, respectively. c) Co-transfectiori of cells from hamster ovary K1 (Hamster Ovary Cells Chinese [CHO-K1]), ATCC CCL-61) or other expression cells of vectors and PQL pQH, using methods described in the state of the art. Example 1. Obtaining cDNA.

isolation of human PBMC.

Human mononuclear cells were isolated (PBMC) from peripheral blood by Ficoli gradient centrifugation, where 7 mL of heparinized blood was added 7 mL of saline buffer phosphate (PBS), mixed and added on 7 mL of Ficoli. The mixture was centrifuged at 1 ,200 rpm for 20 minutes and subsequently the PBMC layer was separated. This fraction was washed by adding PBS, centrifuged at 1 ,200 rpm for 5 minutes, the supernatant was removed. RNA isolation.

The RNA was purified with Trizol solution, according to the manufacturer's instructions. A pellet of 5-10x106 cells was lysed by adding 1 mL of Trizol. The homogenized sample was incubated for 5 minutes at room temperature and 0,2 mL of chloroform were added, mixed by shaking vigorously for 15 seconds. Then incubated for 2 to 3 minutes at room temperature and centrifuged at 12,000 xg for 15 minutes at 4°C. To precipitate the RNA the upper clear aqueous phase was recovered and 0,5 mL of isopropanol were added. Mixed and incubated for 10 minutes at room temperature and centrifuged at 12,000 xg for 15 minutes at 4 ° C. The RNA pellet was washed with 1 mL of 75% ethyl alcohol, mixed and centrifuged at 7,500 xg for 5 minutes at 4°C. The precipitate was allowed to dry for 5 to 10 minutes at room temperature, then the RNA was dissolved in 100 pL of water treated with diethylpyrocarbonate (DEPC). The extracted RNA was analyzed by agarose gel 1 % w/v, and its concentration was determined by absorbance at 260 nm.

RT-PCR reaction (cDNA synthesis).

To one pg of RNA was added 1 pL of oligo dT concentrated at 500 mg/mL and 12 pL DEPC water csp. Incubated at 70°C for 10 minutes, cooled rapidly in ice and centrifuged briefly. Subsequently 4 pL of RT-PCR 5x buffer, 2 pL of dithiothreitol (DTT) 0.1 M, 1 pL of 10 mM dNTP mix, were added to the tube, mixed, incubated for 2 minutes at 42°C and added 1 pL (200 U ) of Superscript II reverse transcriptase enzyme. Was incubated in the thermocycler at 42°C for 50 minutes and the enzyme was inactivated by incubating it at 70°C for 5 minutes. To verify the absence of genomic DNA contamination, the reaction was performed in the absence of reverse transcriptase enzyme. Subsequently PCR reaction was performed with 2 μΙ_. As a cDNA control PCR reaction was performed with specific primers for β-actin (Figure 1).

Example 2. Cloning of VH and VL variable regions of murine mAbs.

For the amplification of the VH and VL regions of murine mAbs 5'RACE system was used (Rapid Amplification of cDNA ends), following the manufacturer's instructions, RNA was isolated from hybridome cells producing the anti-TNF mAbs. CDNA was synthesized in the VH and VL region using the Superscript II reverse transcriptase and primers and MKCR3 MlgGCR (Figure 1), C region- specific gamma chain and the murine Ig kappa chain, respectively. RNA was digested by conditioning RNase enzyme and a poly-G 5'end was added of sing!e- stranded cDNAs. The V regions were amplified by PCR with Taq polymerase using the sense primer containing a sequence AAP poly-C and antisense primers MKCRN3 MlgGCR2 and complementary to the gamma chain C region and the murine Ig kappa chain, respectively. The PCR program was used consisting of 1 cycle at 94°C for 2 minutes, 32 cycles at 94°C for 40 seconds, 55°C for 40 seconds, 72°C for 60 seconds, followed by 1 cycle at 72°C for 5 minutes for the amplification of two V regions. The DNA obtained was cloned in a pGEMT Easy vector. E. coli DH5a bacteria were transformed and placed on LB plates /agar /ampiciilin /X-Gal /IPTG. The clones obtained were selected by their color (white colonies), because the multiple cloning region of vector, is inserted in the coding region of the peptide to the enzyme β-ga!actosidase, by inserting a DNA fragment does not produce peptide-a and recombinant clones do not have the blue color hydrolysis product of X-Gal for β-galactosidase. The selected clones were checked with colony PCR with the primers used in the amplification. Subsequently, the selected clones were stored at -80 ⁰ C in LB-Glycerol 5% from these piasmid DNA was prepared and sequenced using SP6 and 17 primers (Figure 1).

According to the above, aligning the sequences obtained from the murine Ig VH regions in database using ClustalW and BLAST-ig, The identified DNA sequence encoding the functional VH region anti-TNF Ab. This sequence, nucleotide and amino acid, is shown in Figure 4. Equivalentiy we proceeded to the VL region sequences, figure 5 shows the amino acid and nucleotide sequences of anti-TNF antibody of the present invention.

Example 3. Cloning the CH and CL regions of human immunoglobulins.

Total RNA was isolated from human PBMC, as described in Example 1 , and synthesized cDNA using oligo dT.

Primers were designed for the beginning and end of the gamma C region of the chain (SCGH and AGH) and kappa chain (IgKCsense and IgKCas) of human Ig, starting from sequence databases (GenBank, via NCBI). The primers designed were analyzed using BLAST and the program Oligo Explorer 1.2. Performing restriction analysis of sequences of regions C, in order to select the restriction site incorporated into the primers.

PCR was performed with Pfu enzyme using the primers designed. For the H chain the following PCR program was used: 1 cycle at 94°C for 20 seconds, 35 cycles at 94°C for 60 seconds, 60°C for 60 seconds, 72°C for 120 seconds, followed by 1 cycle at 72°C for 5 minutes. For the L chain was used: 7 cycles at 95°C for 30 seconds, 40°C for 40 seconds and 72°C for 40 seconds, 25 cycles of 95°C for 30 seconds, 60°C for 40 seconds, 72°C for 40 seconds, followed by 1 cycle at 72°C 5 minutes. The amplified products were digested with EcoRI and BamHI, respective sequences were incorporated by the primers, purified on agarose gel and bound into the vector pcDNA3.1 (-), previously digested with the same enzymes, dephosphorylated and purified on agarose gel. E. coli DH5a bacteria were transformed and the colonies obtained were checked for the presence of insert by colony PCR with the same primers used in the PCR reaction. The positive colonies were subjected to restriction analysis by digestion of plasmid DNA with EcoRI and BamHI. Cloning of each positive clone was selected at random, stored at -80°C in 15% LB-Glycerol from this plasmid DNA was prepared and sequenced using primers T7 and pcDNArev (Figure 1). The analysis of the sequence shown in Figure 8, shows a single nucleotide different from the C region of the human Ig g1 chain reported in the art, presenting 99% identity with the nucleotide sequence and 100% identity with the sequence of amino acids (Figure 10).

In the case of the C region of the L chain, the sequence obtained (Fig. 9) were compared with data bank sequences (BLAST) and confirmed a 100% identity with the nucleotide sequence of the C region of kappa chain of human Ig (Figure 11).

Example 4. Chimeric construction vectors and pQL pQH: binding fragments V and C regions by PCR extension superposition.

For the construction of the chimeric vectors pQH and PQL joining technique was used, of DNA fragments by overlap extension PCR (Higuchi et al., 1988). Briefly, DNA fragments encoding the murine V region and human C region, both H chain and the L, were amplified by PCR with Pfu DNA polymerase enzyme from the previously cioned cDNA sequences (PCR1 and PCR2, Figure 3). The VL region was amplified from previously cioned cDNA (Example 2) using the primers QLg9supR01 and Iidkappag9. Ck region was amplified from previously cloned cDNA (Example 3) with primers and IgKCas QLg9supF01.

The VH region was amplified from previously cloned cDNA (Example 2) using the primers lid-nhe-g9-H and fgVH~G9supR and CH region was amplified from previously cloned cDNA (Example 3) with primers QHG9- supFOI and AGH.

The internal primers (QLg9supR01 and QLg9supF01 , for the chain L and igVH-G9supR and QHG9-supF01 for H chain) are complementary and introduce mutations to eliminate the nucleotide changes and insertions introduced by the primers used in the cloning of these regions. The primers 5'-kappaG9 lid and lid- nhe-g9-H introduced Kozak sequence to the amplified DNA, which participates in the recognition by the ribosome in the process of translation in eukaryotic cells. The final sequences obtained are shown in Figures 12 and 13.

Subsequently, the amplified DNA obtained were purified on agarose gels and PCR was performed using equimolar amounts of fragments of DNA (3ng total DNA) in a total volume of 25 pL with Pfu enzyme (PCR3, Figure 3).

The DNA fragment obtained was digested with enzymes Nhel and BamHI, whose sequences were included in the 5'and 3' and was subsequently purified on agarose gel.

The chimeric H chain obtained from the fusion of the DNA sequence encoding the VH region of murine with human Νγ1 region, was iigated into the vector pcDNA3.1 (-) (Figure 2), previously digested with the same enzymes restricted, dephosphorylated and purified on agarose gel, obtaining the vector pQH (Fig. 6 A). The chimeric L chain, resulting from the merger of the DNA sequence encoding the VL region of the murine with the human Ck region, was bound into the vector pSecTag2B (Figure 2), PQL obtaining the vector (Fig. 6 B). Bacteria was obtained from transformed E. coli DH5a with the constructs obtained, clones were isolated and checked by colony PCR with specific primers for the 5'and 3' of each chimeric chain. The selected clones were stored at -80°C in 15% LB-Glycerol. A clone, plasmid DNA was prepared and sequenced.

Example 5. Co-transfection of cells from hamster ovary K1 (Hamster Ovary Cells Chinese [CHO-K1]), ATCC CCL-61) with vectors and PQL and pQH.

In order to verify that the constructed chimeric vectors produce a functional chimeric Ab, CHO-K1 cells were transfected with vectors linearized pQH and PQL for this plasmid DNA was a purified chimeric vector pQH and PQL, using QIAprep Spin Miniprep Kit (Qiagen, USA), which were subsequently linearized by digestion with Seal and Pvul enzymes, respectively. Subsequently were purified with QIAquick Gel Extraction Kit (Qiagen, USA) and were eluted in a TE buffer.

CHO-K1 cells were co-transfected with vectors, using SuperFect (Qiagen, USA), following the manufacturer's instructions. Briefly, cells were collected by centrifugation during 5 minutes at 1 ,100 rpm, the pellet was washed with PBS and 1x106 cells were seeded in 1 ,6 mL of RPMI supplemented. Mixed 3 pg of total DNA (1 ,5 pg and 1 ,5 pg pQH vector vector PQL) with un-supplemented RPMI qs 100 pL and added 10 pL of reagent SuperFect (Qiagen, USA). Mixed and incubated for 5 minutes at room temperature. Additional 600 pL of supplemented RPMI, mixed and added drop by drop to the ceils. Were incubated for 24 hours at 37°C, 5% CO₂.

Subsequently, to the transfected cells 11 pL were added of RPMI supplemented with neomycin 700 pg/mL and seeded in 100 pL/wells in a culture dish of 96 wells. They fed every 6 days by removing half the medium and adding the same voiume of medium with neomycin. Neomycin was used only for lines that stably expressed the Ab, because the H chain expression alone is toxic to cells.

In order to evaluate the efficiency of transfection, culture supernatant was collected after 3 weeks post-transfection and was essayed with ELISA and its reactivity essayed with the TNF.

As indicated above, the plates of polyvinyl chloride (PVC) were sensibilized with 100 pl/well of TNF (2 mg/ml), diluted in 0,1 M with a carbonate buffer, to a pH 9,3, incubated at 4°C overnight. Subsequently washed three times with PBS- Tween 0.05% and blocked with 200 pi PBS-Tween-BSA 1 %, at 37°C for 2 hours in a humid environment. Subsequently washed and added in triplicate 100 pi culture supernatant transfected cells or Acm murine anti-TNF (control positive.) Incubated at 37°C for 2 hours in a humid environment. After washing, Ig anti-human IgG (gamma chain specific) conjugated was added to peroxidase for detection of mAbs or Ig chimeric anti-murine IgG conjugated to peroxidase for detection of murine anti-TNF mAbs. Reaction was revealed by adding, to each well, 100 ul of peroxidase substrate solution, 2,2'azino bis 3-ethyl benziltiazonil-6-suif0nico/H₂O2. The optical density reading was performed at a wavelength of 405 nm in a standard EL!SA reader.

As a negative control, culture supernatant was added, which was obtained from the same cells without being transfected and wells or vials which were coated only with blocking protein, culture supernatant of the transfectomas cells without transfect was added. Shown in Figure 7 that the antibodies Ab of the invention, display reactivity against TNF in an ELISA assay, thus the vectors encode a functional Ab, whose chains are expressed, assembled, and secreted molecule binds to TNF. This is important because it shows that these vectors can be used to make eucharyotic cell lines which express the Ab in a stable way and can be used in the production of recombinant Ab.

Example 6. Determination of kinetic constants of the designed chimeric antibody

The determination of evolved chimeric antibody affinity was performed on a BIAcore 2000 (BIAcore Tokyo, Japan) equipment. Used as antigen recombinant human TNF, immobilized on a chip CM5 sensor to 10 pg / mL at pH 5,5. The antibody was passed over the chip in a HBS-EP buffer (0,01 M HEPES pH 7,4, 10 mM NaCI, 3 mM EDTA and P-20 surfactant) at various concentrations (0,6, 1 ,2 and 2,4 nM) and the interaction was monitored for 3 minutes. The sensor surface was washed with an HBS-EP buffer so as to detect dissociation. Regeneration of the chip was performed with glycine-HCI (pH 1 ,5) at the end of each determination test. The association rate constant (Ka), the dissociation constant (Kd)rate and the dissociation constant (KD) were calculated by the BlAevalution software (Version 3.1 , BIAcore). The values obtained for the different constants were: k = 17,8 M- s- 1 , kd = 1 x 0~5 s-1 and KD = 5,6 x 10-7 M.

Claims

SET OF CLAIMS

1. A monoclonal antibody that recognizes as an antigen the tumor necrosis factor (TNF), CHARACTERIZED in that it recognizes as an antigen the human

TNF.

2. A monoclonal antibody according to the preceding claim CHARACTERIZED in that the monoclonal antibody is chimeric, murine-human.

3. Nucleotide sequence (DNA, RNA, cDNA) encoding monoclonal antibodies or fragments thereof that recognize antigen to the TNF, CHARACTERIZED in that:

a) the complete sequence or fragments is described in Figure 4,

b) a nucleotide sequence, where the genetic code is degenerate, which encodes a polypeptide having the same amino acid sequence encoded by the nucleotide sequence in a),

c) A nucleotides sequence which hybridises the complement of the nucleotide sequence of a) and b) under standard stringency conditions.

4. Nucleotide sequence (DNA, RNA, cDNA) encoding monoclonal antibodies or fragments thereof that recognize antigen to TNF, CHARACTERIZED in that:

a) the complete sequence or fragments of it are described in Figure 5, b) a nucleotide sequence, because the genetic code is degenerated, encodes a polypeptide having the same amino acid sequence encoded by the nucleotide sequence a),

c) A nucleotide sequence hybridizing the complement of the nucleotide sequence of a) and b) under standard stringency conditions.

5. Nucleotide sequence, such as DNA, RNA, cDNA encoding monoclonal antibodies or fragments thereof that recognize antigen to TNF, CHARACTERIZED in that,

a) comprises at least one sequence CDR1 , CDR2 and CDR3 described in Figures 4 and 5

b) a nucleotide sequence, because the genetic code is degenerated, encodes a polypeptide having the same amino acid sequence encoded by the nucleotide sequence a), c) A nucleotide sequence hybridizing the complement of the nucleotide sequence of a) and b) under standard stringency conditions.

6. Amino acid sequence CHARACTERIZED because amino acid sequence is encoded by at least one of the sequences described in claims 3 to 5.

7. Amino acid sequence according to claim 6, CHARACTERIZED in that it has a 24% sequence homology with that claim, maintaining the same functionality.

8. An anti-TNF monoclonal antibody according to claims 1 and 2 CHARACTERIZED in that it comprises at least one of the amino acid sequences described in claim 6 or 7,

9. An anti-TNF monoclonal antibody according to claim 8,

CHARACTERIZED in that it comprises at least one of the amino acid sequences for heavy and light chains described in Figures 8 and 9.

10. Fab, (Fab ') 2, Fabs, TandAb, Diabody, Flexibody or other antibody fragment CHARACTERIZED in that it comprises at least one of the amino acid sequences claimed in claims 6 or 7.

11. A vector used to transform cells or microorganisms CHRARACTERIZED in that it comprises at least one of the sequences described in claims 3 to 5.

12. A vector according to claim 11 , CHARACTERIZED in that it comprises the vector ADO and PQH PQL.

13. Transformed cells or microorganisms, CHARACTERiZED in that it comprises at least one of the sequences or vectors of the invention described above.

14. Formulations, compositions or preparations CHARACTERIZED in that it comprises the sequences, vectors, antibodies, fragments and formats as described in previous claims.

15. Formulations, compositions or preparations according to the previous claim CHARACTERIZED in that it comprises the additional components required for the stability and efficiency of them.

16. Formulations, compositions or preparations according to claim 15,

CHARACTERIZED in that they may be liquid, solid, lyophilized or other appropriate physical form in art.

17. Formulations, compositions or preparations according to claim 16, CHARACTERIZED in that they are designed for the proper administration by the medically appropriate way, as can be without being restricted to oral, dermal, subcutaneous, intramuscular, intraperitoneal, intravenous, ocular, spinal, intracranial, among others.

18. Devices, or kits arrangements CHARACTERIZED in that it comprises the sequences, vectors, antibodies, their fragments and formats described in the previous claims.

19. Kit of parts CHARACTERIZED in that it comprises the means necessary for site preparation of the formulations, preparations or compositions described in claims 14 to 7.

20. Kit CHARACTERIZED in that it comprises parts of the kit components and formulations, compositions or preparations described in the claims 14 to 17, which can be used simultaneously or sequences, together or separately.

21. Use of monoclonal antibodies or fragments thereof, described in claims 1 , 2 and 8 to 10, CHARACTERIZED in that they serve to treat, prevent, cure or diagnose diseases related to TNF, also serve to control the levels of TNF in diseases where levels of these cytokines are modulators or promoters of the conditions and / or symptoms of the disease, among the main applications of the antibodies are not limiting to, inflammatory bowel disease such as Crohn's disease and ulcerative colitis, rheumatic diseases eg rheumatoid arthritis, sacroi!iitis, ankylosing spondylitis and psoriatic arthritis, among other diseases such as Sarcoidosis complicated system, proximal myopathy, Still's disease and Behcet syndrome; addition to the treatment of Psoriasis, Systemic Inflammatory Response Syndrome and Multiple Organ Failure .

22. Method of production of chimeric monoclonal antibody according to claim 9, CHARACTERIZED in that it comprises the following steps:

Purification and synthesis of DNA sequences described in Figures 4 and 5 (antibody variable regions), and the DNA sequences described in Figures 8 and 9 (antibody constant regions), or sequences that code for similar polypeptides such sequences, by general methodology found in the state of the art

Construction of chimeric vectors and PQL pQH joining fragments of variable and constant regions by overlap extension PCR, using methodology described in the prior art, in particular, using the region amplified with the primers VL and QLg9supR01 Iidkappag9, the Ck region with primers and IgKCas QLg9supF01 ; The VH region with the primers Hd-nhe-g9-H and lgVH-G9supR and the region with primers CH-supF01 QHG9 and AGH, to generate H chain chimeric (fusion sequence murine VH region with the human Ng1) and L chain chimeric {fusion of the DNA sequence encoding the VL region of the murine with the human Ck region) which are linked with the pcDNA3 vector and vector pSecTag2B respectively to generate the final vector pQH and PQL, respectively; c) Co-transfeccion de ceiulas de ovario de hamster K1 (Chinese Hamster Ovary Cells [CHO-K1]), ATCC CCL-61) u otras ceiulas de expresion compatibles con vectores pQH y pQL, utilizando metodos descritos en el estado del arte. References

Sambroock, J., Russell, D, W. 2001. Molecular cloning. A laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.