CN111909267B - Low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and its application - Google Patents

Low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and its application Download PDF

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CN111909267B
CN111909267B CN202010350390.0A CN202010350390A CN111909267B CN 111909267 B CN111909267 B CN 111909267B CN 202010350390 A CN202010350390 A CN 202010350390A CN 111909267 B CN111909267 B CN 111909267B
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孙乐
任文林
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Abmax Biopharmaceuticals
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    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]

Abstract

The invention relates to the field of biotechnology, in particular to a low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and application thereof. The present invention provides an anti-TNF- α humanized monoclonal antibody TCX063 which is an anti-TNF- α humanized monoclonal antibody reduced in immunogenicity obtained by modifying the FR region of inflixb monoclonal antibody. The antibody provided by the invention has the same TNF-alpha antigen binding site as an inflixb monoclonal antibody, retains the antigen affinity and specificity of the inflixb monoclonal antibody, but has immunogenicity remarkably lower than that of the inflixb monoclonal antibody. The low-immunogenicity TNF-alpha antibody can reduce the risk of side effects of the drug caused by immune reaction caused by the immunogenicity of the antibody in a human body; reduces the antibody drug ADA; the in vivo half-life of the antibody is prolonged; meanwhile, the subcutaneous drug delivery mode becomes possible, and the application potential and the value are great.

Description

Low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and its application
Technical Field
The invention relates to the field of biotechnology, in particular to a low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and application thereof.
Background
Tumor necrosis factor alpha (TNF- α) is a cytokine secreted by immune cells that is naturally secreted in inflammatory and immune responses. Studies have shown that TNF- α levels tend to be up-regulated in patients with a variety of chronic diseases (e.g., crohn's disease, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, etc.) (e.g., elevated levels of TNF- α in synovial fluid of Rheumatoid Arthritis (RA) patients) and play an important role in pathological inflammation and joint destruction, among other processes. Human TNF-alpha has a molecular weight of 17kDa and can exist in vivo as a monomer or trimer, the active form of which is a trimer. TNF- α functions biologically by interacting with the cell surface receptors p55 and p 75. Currently, TNF-. alpha.in vivo neutralization is generally achieved using either monoclonal IgG antibodies or soluble TNF-. alpha.receptors.
Currently commercially available monoclonal antibodies targeting TNF- α mainly include etanercept, inflixib, adalimus, cetuximab, and the like, wherein etanercept is a fusion protein coupled to the Fc terminus by two human TNF- α receptors (p 75); inflixb is a humanized TNF- α murine monoclonal antibody and adalimumab is a humanized TNF- α antibody. Binding capacity assays using radiolabeled TNF- α indicated: inflixb is capable of binding soluble TNF- α in both monomeric (inactive) and trimeric (active) types; etanercept preferentially binds to active, multimeric forms of TNF-alpha and TNF-beta, whereas adalimumab binds only to TNF-alpha and not to TNF-beta.
Infliximab (Infliximab) is a chimeric antibody developed by the Yanssen Biotech (Janssen Biotech) (formerly Senno Tokor Corporation) with murine anti-TNF- α variable domain and human IgG constant domain that specifically binds to TNF- α and blocks the interaction of TNF- α with the cell surface TNF receptors p55 and p 75. The Infliximab monoclonal antibody has stronger immunogenicity in a host body, and the monoclonal antibody can generate immune reaction when being used by more than 35 percent of patients. The immune response may cause immune complex-mediated clearance of the antibody or fragment from circulation and result in repeated administrations that are not suitable for treatment, thereby reducing the patient's therapeutic benefit and limiting re-administration of the antibody. Meanwhile, due to the high immunogenicity, the inflixb monoclonal antibody needs to be administered by intravenous injection, and compared with the subcutaneous administration of adalimus and other monoclonal antibodies, the requirement on operators and administration environments is more strict, the administration mode is more complex, and the half-life period of the drug administered by intravenous injection is shorter. However, there are no reports of antibodies to TNF- α that are poorly immunogenic. Therefore, there is a need to develop anti-TNF- α antibodies with low immunogenicity while ensuring high affinity and specificity.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide an anti-TNF-alpha humanized monoclonal antibody with low immunogenicity, a preparation method and application thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows: according to the invention, the amino acid sequence of the inflixb monoclonal antibody is analyzed by utilizing the commercialized DNAstar TM, and the result shows that the immunogenicity coefficient of the inflixb monoclonal antibody is 16.4, and the inflixb monoclonal antibody has high immunogenicity. By performing sequence alignment analysis on Framework Regions (FR) of all light chains and heavy chains in a human antibody gene library, antibody FR segments having high sequence homology and relatively low immunogenicity with respect to inflixb monoclonal antibodies are screened. Replacing the corresponding section of the inflixb monoclonal antibody by the screened FR region; and 3D modeling is carried out on the amino acid sequence obtained after replacement, structural comparison is carried out on the amino acid sequence and the original inflixb monoclonal antibody, the amino acid sequence of the antibody which is relatively close to the conformation of the inflixb monoclonal antibody is selected as a screening target of the anti-TNF-alpha humanized monoclonal antibody with low immunogenicity, and the anti-TNF-alpha humanized monoclonal antibody with low immunogenicity while the affinity and the specificity of the inflixb monoclonal antibody are maintained is finally obtained through experimental screening such as analysis of antibody expression, antibody affinity, specificity and immunogenicity.
The first object of the present invention is to provide an anti-TNF- α humanized monoclonal antibody TCX063 which is an anti-TNF- α humanized monoclonal antibody with reduced immunogenicity obtained by modifying the FR region of inflixb monoclonal antibody, wherein the full-length amino acid sequence of the light chain of the inflixb monoclonal antibody is represented by SEQ ID No.1, and the full-length amino acid sequence of the heavy chain is represented by SEQ ID No. 2.
The amino acid sequence of the light chain FR1 region of the anti-TNF-alpha humanized monoclonal antibody TCX063 is shown as SEQ ID NO.3 or SEQ ID NO. 4;
the amino acid sequence of the light chain FR3 region of the anti-TNF-alpha humanized monoclonal antibody TCX063 is shown as SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7;
the amino acid sequence of the heavy chain FR1 region of the anti-TNF-alpha humanized monoclonal antibody TCX063 is shown as SEQ ID NO.8 or SEQ ID NO. 9;
the amino acid sequence of the heavy chain FR3 region of the anti-TNF-alpha humanized monoclonal antibody TCX063 is shown as SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO. 12.
The anti-TNF-alpha humanized monoclonal antibody TCX063 has the functions of binding human TNF-alpha and blocking the binding of human TNF-alpha and TNF receptor.
Preferably, the amino acid sequence of the light chain variable region of the anti-TNF-alpha humanized monoclonal antibody TCX063 is selected from any one of the amino acid sequences shown in SEQ ID No. 13-16, and the amino acid sequence of the heavy chain variable region is selected from any one of the amino acid sequences shown in SEQ ID No. 17-21. The anti-TNF-alpha humanized monoclonal antibody with the light chain and heavy chain variable region sequences has higher affinity and specificity and relatively lower immunogenicity.
In the antibody with the combination of the light chain variable region and the heavy chain variable region, 11 monoclonal antibodies with better expression level, affinity, specificity and immunogenicity performance are obtained by screening, wherein the 11 monoclonal antibodies all have the same antigen binding site as the inflixb monoclonal antibody, simultaneously maintain the expression level close to that of the inflixb monoclonal antibody and the affinity for binding with the human TNF-alpha, and can specifically block the binding of the TNF-alpha with cell surface TNF receptors p55 and p 75; meanwhile, the immunogenicity of the antibodies is effectively reduced by optimizing the FR regions of the antibodies.
In the present invention, the 11 humanized anti-TNF- α monoclonal antibodies with low immunogenicity are respectively named as H1L0, H1L1, H1L2, H2L0, H2L1, H2L2, H2L3, H2L4, H3L2, H3L4 and H5L 1.
The 11 low-immunogenicity humanized anti-TNF-alpha monoclonal antibodies have any one of the following variable region amino acid sequences:
(1) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.22, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17;
(2) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17;
(3) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17;
(4) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.22, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(5) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(6) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(7) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.15, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(8) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(9) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19;
(10) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19;
(11) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21.
In order to better ensure low immunogenicity, the anti-TNF-alpha humanized monoclonal antibody is a human IgG1 full antibody.
Preferably, the 11 anti-TNF- α humanized monoclonal antibodies have any one of the following amino acid sequences:
(1) the amino acid sequence of the light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 27;
(2) the amino acid sequence of the light chain is shown as SEQ ID NO.23, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 27;
(3) the amino acid sequence of the light chain is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 27;
(4) the amino acid sequence of the light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(5) the amino acid sequence of the light chain is shown as SEQ ID NO.23, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(6) the amino acid sequence of the light chain is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(7) the amino acid sequence of the light chain is shown as SEQ ID NO.25, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(8) the amino acid sequence of the light chain is shown as SEQ ID NO.26, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(9) the amino acid sequence of the light chain is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 29;
(10) the amino acid sequence of the light chain is shown as SEQ ID NO.26, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 29;
(11) the amino acid sequence of the light chain is shown as SEQ ID NO.23, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 31.
It is a second object of the present invention to provide a gene encoding the anti-TNF-. alpha.humanized monoclonal antibody TCX 063.
Preferably, the gene has any one of the following nucleotide sequences:
(1) the nucleotide sequence of the light chain is shown as SEQ ID NO.32, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 38;
(2) the nucleotide sequence of the light chain is shown as SEQ ID NO.34, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 38;
(3) the nucleotide sequence of the light chain is shown as SEQ ID NO.35, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 38;
(4) the nucleotide sequence of the light chain is shown as SEQ ID NO.32, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(5) the nucleotide sequence of the light chain is shown as SEQ ID NO.34, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(6) the nucleotide sequence of the light chain is shown as SEQ ID NO.35, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(7) the nucleotide sequence of the light chain is shown as SEQ ID NO.36, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(8) the nucleotide sequence of the light chain is shown as SEQ ID NO.37, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(9) the nucleotide sequence of the light chain is shown as SEQ ID NO.35, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 40;
(10) the nucleotide sequence of the light chain is shown as SEQ ID NO.37, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 40;
(11) the nucleotide sequence of the light chain is shown as SEQ ID NO.34, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 42.
It is a third object of the present invention to provide a biological material comprising the gene encoding the anti-TNF-alpha humanized monoclonal antibody TCX 063.
The biological material comprises an expression cassette, a vector, a host cell, an engineering bacterium or a cell line.
The fourth object of the present invention is to provide a method for preparing the anti-TNF- α humanized monoclonal antibody TCX063 by expressing the genes encoding the full length of the heavy chain and the full length of the light chain.
As one embodiment of the present invention, the preparation method of the anti-TNF-alpha humanized monoclonal antibody TCX063 comprises the following steps:
(1) synthesizing a heavy chain full-length gene and a light chain full-length gene of the anti-TNF-alpha humanized monoclonal antibody;
(2) connecting the synthetic gene to an expression vector;
(3) transforming the expression vector connected with the heavy chain full-length gene and the light chain full-length gene of the anti-TNF-alpha humanized monoclonal antibody into a host cell;
(4) screening to obtain a host cell stably expressing the anti-TNF-alpha humanized monoclonal antibody, culturing the host cell and expressing the anti-TNF-alpha humanized monoclonal antibody;
(5) extracting and purifying to obtain the anti-TNF-alpha humanized monoclonal antibody.
The fifth purpose of the invention is to provide the application of the anti-TNF-alpha humanized monoclonal antibody TCX063 or the gene coding the anti-TNF-alpha humanized monoclonal antibody TCX063 or the biological material containing the gene in the preparation of the drug taking human TNF-alpha as the target.
Preferably, the drug targeting human TNF- α is a drug for preventing or treating tumor, inflammation or autoimmune disease.
Such diseases include, but are not limited to, crohn's disease, rheumatoid arthritis, psoriatic arthritis, active ankylosing spondylitis, and the like.
The sixth purpose of the invention is to provide the application of the anti-TNF-alpha humanized monoclonal antibody TCX063 or the gene coding the anti-TNF-alpha humanized monoclonal antibody TCX063 or the biological material containing the gene in the preparation of human TNF-alpha detection reagents.
It is a seventh object of the present invention to provide a medicament or a detection reagent comprising the anti-TNF- α humanized monoclonal antibody TCX 063.
The medicine containing the anti-TNF-alpha humanized monoclonal antibody TCX063 can also contain other effective components or auxiliary materials allowed by the pharmaceutical field.
The invention has the beneficial effects that:
the humanized anti-TNF-alpha monoclonal antibody provided by the invention is obtained by manual modification on the basis of an inflixb monoclonal antibody, the humanized anti-TNF-alpha monoclonal antibody TCX063 and the inflixb monoclonal antibody have the same TNF-alpha antigen binding site, the antigen affinity and specificity of the inflixb monoclonal antibody are retained, but the immunogenicity of the humanized anti-TNF-alpha monoclonal antibody is obviously lower than that of the inflixb monoclonal antibody; lower immunogenicity will reduce the risk of drug side effects due to immune reactions caused by antibody immunogenicity in humans; on one hand, the reduction of immunogenicity avoids the elimination of immune compound mediated antibodies or fragments from circulation, prolongs the half-life period in vivo of the antibody drug prepared by the humanized anti-TNF-alpha monoclonal antibody TCX063, and avoids the reduction of drug effect caused by the antibody drug ADA to a greater extent; on one hand, the dosage of the antibody can be reduced, so that the treatment cost is reduced; on the other hand, the reduction of immunogenicity enables the subcutaneous administration mode to be possible besides the intravenous administration mode, so that the requirements of administration on operators and environment are reduced, and the administration is simpler and more convenient. The humanized anti-TNF-alpha monoclonal antibody TCX063 provided by the invention has great application potential and value, and is expected to become an ideal biological target therapeutic antibody.
Drawings
FIG. 1 shows the alignment of the amino acid sequences of the light chains of different humanized antibodies in example 1 of the present invention.
FIG. 2 shows the alignment of the amino acid sequences of the heavy chains of different humanized antibodies in example 1 of the present invention.
Fig. 3 is a diagram of the electrophoresis result of the double restriction enzyme identification of the gene encoding inflixb monoclonal antibody in example 2 of the present invention and the expression vector of the modified gene encoding monoclonal antibody provided by the present invention, wherein a is the double restriction enzyme result of Hind III and ecori of the expression plasmid of heavy chain H0, and the lanes are, from left to right: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; b is the HindIII and EcoRI double digestion results of the expression plasmid for heavy chain H1, lanes from left to right are: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; c is the HindIII and EcoRI double digestion results of the expression plasmid for heavy chain H2, lanes from left to right are: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; d is the HindIII and EcoRI double digestion results of the expression plasmid for heavy chain H3, lanes from left to right are: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; e is the HindIII and EcoRI double digestion results of the expression plasmid for heavy chain H4, lanes from left to right are: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; f is the HindIII and EcoRI double digestion results of the expression plasmid for heavy chain H5, lanes from left to right are: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; g Hind III and EcoRI double cleavage of the expression plasmid with light chain L0, lanes from left to right: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; h is the HindIII and EcoRI double digestion results of the expression plasmid of light chain L1, and the lanes are from left to right: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; the HindIII and EcoRI double digestion results of the expression plasmid with i as light chain L2 are shown in the left to right: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; j is the HindIII and EcoRI double digestion results of the expression plasmid of light chain L3, and the lanes are from left to right: plasmid before enzyme digestion, plasmid after enzyme digestion and DNA marker; the expression plasmid with k as light chain L4 has Hind III and EcoRI double digestion results, and the lanes from left to right are: plasmid before enzyme cutting, plasmid after enzyme cutting and DNA marker.
FIG. 4 is an SDS-PAGE electrophoresis chart of a part of the improved inflixb monoclonal antibody in example 4 of the present invention. Lane 1 is protein ladder marker; lane 2 is H0L 0; lane 3 is H1L 0; lane 4 is H1L 1; lane 5 is H2L 0; lane 6 is H2L 3; lane 7 is H2L 4; lane 8 is H3L 2; lane 9 is H3L 4; lane 10 is H5L 1.
FIG. 5 shows the improved infliximab affinity EC50 of example 5 of the present invention.
FIG. 6 shows the results of the mouse ADA evaluation experiment in example 6 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 sequence analysis and design of humanized anti-TNF-. alpha.monoclonal antibodies with reduced immunogenicity
The original sequence of inflixb monoclonal antibody is analyzed and evaluated by using commercial DNASTATM software, and the analysis result shows that the immunogenicity coefficient of the inflixb monoclonal antibody is 16.4 and the immunogenicity is high.
Searching all light chain and heavy chain FR sections in the NCBI human antibody gene library, performing immunogenicity analysis on FR region sequences of the humanized antibodies in the NCBI database by using DNASTATM software, screening out humanized FR region sequences with low immunogenicity, and constructing a humanized FR region database with low immunogenicity.
The non-antigen binding fragment (i.e., the FR region) in the variable region of inflixb antibody was evaluated for immunogenicity using dnastar software to find highly immunogenic fragments. BLAST is carried out on the high-immunogenicity fragment and the sequence in the low-immunogenicity human FR region database constructed above, and the human FR sequence with higher homology is selected to replace the corresponding FR region of the inflixb monoclonal antibody, so as to obtain the modified antibody sequence with low immunogenicity. The sequence of the human FR region with low immunogenicity and high homology with the Indonesia FR region is finally determined as follows:
light chain FR1 region: SEQ ID NO.3, SEQ ID NO. 4;
light chain FR3 region: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO. 7;
heavy chain FR1 region: SEQ ID NO.8, SEQ ID NO. 9;
heavy chain FR3 region: SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO. 12.
3D modeling is carried out on the obtained low-immunogenicity modified antibody sequence by utilizing Pymol protein molecular simulation software (www.pymol.org), a combination which has large influence on the space conformation of an antibody structure in the sequence replacement process is eliminated, and an antibody which is similar to the space conformation of the inflixb monoclonal antibody is reserved; the light chain is finally obtained with the following amino acid sequence: L1-L4 (the light chain variable region sequences are respectively shown as SEQ ID NO. 13-16, the full-length amino acid sequences of the light chain are respectively shown as SEQ ID NO. 23-26), and the light chain L0 of the inflixb monoclonal antibody (the variable region amino acid sequence is shown as SEQ ID NO.22, the full-length amino acid sequence of the light chain is shown as SEQ ID NO. 1), and a heavy chain variable region with the following amino acid sequences: H1-H5 (the sequences of the heavy chain variable regions are respectively shown in SEQ ID NO. 17-21, and the full-length amino acid sequences of the heavy chains are respectively shown in SEQ ID NO. 27-31) and H0 of the inflixb monoclonal antibody (the full-length amino acid sequences of the heavy chains are respectively shown in SEQ ID NO. 2). The above amino acid sequence alignment of the different light and heavy chains is shown in FIGS. 1 and 2, respectively. 30 combination modes can be obtained by the light chain and the heavy chain through random combination, and the antibody obtained by the combination modes keeps the original antigen binding site of the inflixb monoclonal antibody.
The coding gene sequences of 30 monoclonal antibodies obtained by arbitrarily combining the light chain and the heavy chain are subjected to whole-gene synthesis (synthesis by the Kingchi Authority company), the synthesized genes are sequenced (sequencing work by the Kingchi Authority company is entrusted), and correct coding gene sequences are selected according to sequencing comparison results for subsequent expression and antibody performance experimental verification. 30 combination of monoclonal antibody light chain and heavy chain full length coding gene nucleotide sequence as follows:
light chain L0: SEQ ID No. 32; heavy chain H0: SEQ ID No. 33; light chain L1: SEQ ID No. 34; light chain L2: SEQ ID No. 35; light chain L3: SEQ ID No. 36; light chain L4: SEQ ID No. 37; heavy chain H1: SEQ ID No. 38; heavy chain H2: SEQ ID No. 39; heavy chain H3: SEQ ID No. 40; heavy chain H4: SEQ ID No. 41; heavy chain H5: SEQ ID NO. 42.
Example 2 construction of an expression vector for improved infliximab TCX063
According to the nucleotide sequences of the full-length heavy chain and light chain of the anti-TNF-alpha humanized monoclonal antibody obtained in example 1, enzyme cutting sites on both sides of the light chain sequence are designed to be Hind III + EcoR I, enzyme cutting sites on both sides of the heavy chain sequence are designed to be Hind III + EcoR I, the full-length heavy chain and full-length light chain sequences carrying the enzyme cutting sites are delivered to a Jinzhi company to synthesize a whole gene sequence, and a connection vector used for synthesis is pUC 57. pEE12.4 (for heavy chain gene expression) and pEE6.4 (for light chain gene expression) are taken as expression vectors, the expression vectors and the synthesized gene sequences are respectively subjected to corresponding double enzyme digestion, the target gene and the expression vectors obtained by enzyme digestion are respectively subjected to agarose Gel electrophoresis separation, then target bands are cut off and recovered by a Qiagen Gel Extraction Kit, the target bands are connected overnight at 16 ℃ according to a T4 DNA enzyme connection system, then escherichia coli DH5 alpha is transformed, and the obtained expression vectors carrying the full-length genes of the heavy chains H0, H1, H2, H3, H4 and H5 and the full-length genes of the light chains L0, L1, L2, L3 and L4 are obtained through PCR identification, plasmid Extraction and sequence determination. The results of the double restriction enzyme identification electrophoresis of the expression vectors carrying the full-length genes of the above-mentioned various heavy and light chains, respectively, are shown in FIG. 3.
Example 3 transient expression and purification of modified Infinizumab TCX063
The E.coli DH 5. alpha. strains carrying different full-length heavy and light chain gene expression vectors obtained in example 2 were cultured, the culture was harvested, and the expression vectors carrying the full-length heavy and light chain genes were extracted and purified using Qiagen UltraPure plasmid DNA purification kit. The purified plasmid DNA was transfected into 293F cells using a kit for liposome method of Invitrogen, and the transfection method was as described in the kit.
293F cells were transfected with combinations of expression plasmids carrying different light and heavy chain genes, respectively, in a combined manner as shown in Table 1, for a total of 30 combinations of expression plasmids transient expression of 293F cells. After the transfected 293F cells were cultured for 7 days, the culture supernatant was taken and antibody expression level was measured, and the results are shown in Table 1, which indicates that the antibodies obtained in 30 combinations were all expressed.
TABLE 1 original and modified Inforti antibody expression level test results (mg/L)
Figure BDA0002471621770000051
(Note: the combinations in Table 1 are combinations of different light chains and heavy chains, H1L0 refers to the combination of original Infinigen light chain and modified Infinigen heavy chain H1, and so on; H0L0 refers to original Infinigen monoclonal antibody)
In order to screen antibodies with high binding affinity to TNF-alpha, the expressed antibodies are subjected to a pre-coating TNF-alpha experiment respectively, the 30 antibodies are added into a 96-well plate pre-coated with TNF-alpha respectively, and the activity of secreted antibodies for binding TNF-alpha is preliminarily evaluated by adopting an ELISA indirect method. The results of the 30 antibodies are shown in Table 2, where NC is a negative control with the addition of antibody dilutions. The results show that 30 antibodies can bind to the TNF-alpha, and the binding affinity of different antibodies to the TNF-alpha is different, wherein the expression amount and the binding affinity to the TNF-alpha are higher in H1L0, H1L1, H1L2, H2L0, H2L1, H2L2, H2L3, H2L4, H3L2, H3L4 and H5L 1.
Table 2 results of evaluation of antibody Activity of different combinations of heavy and light chains
Combination of Amount of expression (mg/L) OD value (1000 times dilution) Specific activity (OD 450/expression level) Specific activity (H0L0 normalization processing)
H0L0 2.86 0.604 0.21 1
H0L1 2.77 0.588 0.21 1
H0L2 5.2 0.85 0.16 0.77
H0L3 1.87 0.435 0.23 1.1
H0L4 3.66 0.762 0.21 0.99
H1L0 4.96 1.225 0.25 1.17
H1L1 8.09 1.475 0.18 0.86
H1L2 9.62 1.673 0.17 0.82
H1L3 5.68 1.195 0.21 1
H1L4 8.86 1.562 0.18 0.83
H2L0 1.98 0.529 0.27 1.27
H2L1 4.1 0.814 0.2 0.94
H2L2 10.06 1.254 0.12 0.59
H2L3 5.27 0.752 0.14 0.68
H2L4 7.37 1.101 0.15 0.71
H3L0 2.1 0.434 0.21 0.98
H3L1 4.48 0.785 0.18 0.83
H3L2 7.64 0.993 0.13 0.62
H3L3 3.71 0.74 0.2 0.94
H3L4 4.67 0.941 0.2 0.95
H4L0 0.84 0.246 0.29 1.38
H4L1 1.19 0.266 0.22 1.06
H4L2 2.21 0.316 0.14 0.68
H4L3 1.02 0.169 0.17 0.79
H4L4 1.99 0.351 0.18 0.84
H5L0 0.92 0.238 0.26 1.23
H5L1 2.47 0.652 0.26 1.25
H5L2 6.7 0.717 0.11 0.51
H5L3 0.82 0.173 0.21 1
H5L4 2.28 0.575 0.25 1.19
Example 4 expression and purification of modified Infinizumab TCX063
According to the detection result of the embodiment 3, expression plasmid combinations (H1L0, H1L1, H1L2, H2L0, H2L1, H2L2, H2L3, H2L4, H3L2, H3L4 and H5L1) with better quality detection results are selected for transient transfection and antibody purification.
293F cells were transfected by transient transfection, and cell culture supernatants were collected on the seventh day after transfection using 293 medium from Zhuhai happy Rui, Inc. and transfection reagents.
The culture supernatants of the 11 different combinations of heavy and light chains and 1 original combination (H0L0) were directly separated and purified using MabSelect Sure LX from GE to yield 11 modified human monoclonal antibodies (note: 11 of them were modified and 1 was original infliximab). The experimental process is detailed in the specification of GE company, the purified product is quantitatively detected by an ultraviolet spectrophotometer, and the calculation formula is as follows:
and (3) calculating the concentration: reading the collected elution peak to OD280Thereafter, the concentration was calculated. Antibody concentration ═ OD280/1.4。
SDS-PAGE electrophoresis verification is carried out after the antibody is purified, the result is shown in figure 4, and the electrophoresis result shows that the purity of the purified antibody exceeds more than 80%, the bands are relatively single, the concentration is equivalent, and later-stage experiments can be carried out.
Example 5 bioactivity assay of modified infliximab TCX063
1. Evaluation of affinity
This section evaluates antibody affinity using ELISA indirect assay for antibody EC 50.
The experimental method is as follows: TNF-alpha (from near shore technologies, Inc.) was diluted to 1. mu.g/ml with PBS; adding the diluted antigen into a 96-well plate according to 100 mu l/well, covering the plate, and standing overnight at 4 ℃; the liquid in the wells was spun off, washed three times with PBS, 200 ul/well, patted dry; sealing with 5% cow milk-PBS 200 μ l/hole for 1 hr, and tapping every 15 min; spin off the liquid in the well, wash with PBS once, 200 mul/well, pat dry; adding purified antibody (0-10 μ g/ml) in gradient, wherein the antibody name is shown in Table 3, diluting with 5% cow milk-PBS, 100 μ l/well, incubating for 1h, and tapping every 15 min; the liquid in the wells was spun off, washed three times with PBS, 200. mu.l/well, patted dry; adding secondary antibody, diluting with 5% cow milk-PBS, incubating for 1h, and tapping every 15 min; preheating a TMB substrate at room temperature, and simultaneously starting an enzyme-labeling instrument for preheating; washing with PBS 5 times, 250 μ l/well, the first three times for 5min, the second two times for 10min, and patting to dry; adding 50 mul/hole of TMB substrate A, B, developing for 20min at room temperature; the image was scanned and recorded using a scanner, 50. mu.l of stop buffer was added to each well, and OD450 was read using a microplate reader. The results are shown in FIG. 5, and the experimental data are shown in Table 3 below.
Table 3 EC50 results for the preferred modified light and heavy chain variable region combinations of Infinigen
Figure BDA0002471621770000061
The above results show that: the affinity of the improved inflixb monoclonal antibody is basically consistent with that of the original ground inflixb monoclonal antibody, and the EC50 of part of the improved inflixb monoclonal antibody is superior to that of the original ground inflixb monoclonal antibody.
3. Experiment of cytotoxic Effect
The mouse fibroblast cell line L929 was subjected to apoptosis detection using a CCK-8 kit. The specific operation steps are as follows:
mu.l of modified infliximab (RPMI-1640 medium diluted with 10% FBS) diluted in a three-fold gradient was added to a 96-well plate (modified infliximab was added at a concentration of 3-5 gradients up and down, respectively, in accordance with the FDA reference to Inflate experiment IC 50), followed by 50. mu.l of rhTNF-. alpha.at a final concentration of 500pg/ml (RPMI-1640 medium diluted with 10% FBS), and incubated at room temperature for 30 min. 50 μ l L929 cells 5 × 10 were added to each well4Perwell, containing actinomycin-D at a final concentration of 1. mu.g/ml. Incubate overnight at 37 ℃ in an incubator (18-24 h). The control comprises a negative control and a positive control, wherein the negative control only adds RPMI-1640 and cells, the positive control only adds rhTNF alpha and cells, and the concentration of the rhTNF alpha is graded from 2ng/ml to 8.2 pg/ml. Add 20. mu.L of CCK8 solution to each well (taking care not to generate bubbles in the wells in order not to affect the OD reading). The plates were incubated for 1-4h at 37 ℃ in an incubator. Absorbance at 450nm was measured with a microplate reader. If OD is not to be measured temporarily, 10. mu.L of 0.1M HCl solution or 1% w/v SDS solution may be added to each well, and the plate may be covered and kept at room temperature under protection from light. The absorbance did not change when measured over 24 hours.
The cell killing rate and IC50 were calculated for the antibody at different concentrations.
Calculating the formula: modified Infinixib-treated cells TNF-. alpha.treated cells/TNF-. alpha.cells X100%.
The results are shown in Table 4.
TABLE 4 modified inflixb cytotoxicity IC50
H0L0 H1L0 H1L1 H1L2 H2L0 H2L1 H2L2 H2L3 H2L4 H3L2 H3L4
2.666E-10 5.39E-09 2.72E-09 5.68E-09 4.62E-09 8.44E-10 2.33E-09 7.36E-11 2.09E-09 3.42E-09 6.01E-11
Example 6 improved immunogenicity evaluation of Infinixib TCX063 mAb
An improved inflixb TCX063 monoclonal antibody immunization experiment is carried out in a mouse body, and the immunogenicity of the TCX063 monoclonal antibody is analyzed, wherein the immunogenicity is as follows:
1) basic immunity: the infliximab and the improved TCX063 antibody are mixed in equal volume with Freund's complete adjuvant and fully emulsified, and injected subcutaneously by several points, and the injection amount of each Balb/c mouse is 70 mug.
2) And (3) boosting immunity: the boosting immunity adopts infliximab and emulsion of improved TCX063 antibody medicine and Freund's incomplete adjuvant.
After the above experiment is completed, an ELISA detection test is performed, and the experimental method is as follows: diluting TCX063 improved medicine or Yinfuximab original grinding medicine to 1 μ g/ml with PBS; adding the diluted antigen into a 96-well plate according to 100 mu l/well, covering the plate, and standing overnight at 4 ℃; the liquid in the wells was spun off, washed three times with PBS, 200 ul/well, patted dry; sealing with 5% cow milk-PBS 200 μ l/hole for 1 hr, and tapping every 15 min; spin off the liquid in the well, wash with PBS once, 200 mul/well, pat dry; adding serum samples (1:500/1:1000/1:5000/1:10000/1:50000) with different dilution ratios in different days after immunization in a gradient manner, diluting with 5% cow milk-PBS, 100 μ l/hole, incubating for 1h, and tapping every 15 min; the liquid in the wells was spun off, washed three times with PBS, 200. mu.l/well, patted dry; adding secondary antibody, diluting with 5% cow milk-PBS, incubating for 1h, and tapping every 15 min; preheating a TMB substrate at room temperature, and simultaneously starting an enzyme-labeling instrument for preheating; washing with PBS 5 times, 250 μ l/well, the first three times for 5min, the second two times for 10min, and patting to dry; adding 50 mul/hole of TMB substrate A, B, developing for 20min at room temperature; the image was scanned and recorded using a scanner, 50. mu.l of stop buffer was added to each well, and OD450 was read using a microplate reader. The results are shown in fig. 6, and show that the immunogenicity of the improved infliximab is effectively reduced compared with the original infliximab.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing Tiancheng Xinmai Biotechnology Co., Ltd
<120> low-immunogenicity anti-TNF-alpha humanized monoclonal antibody TCX063 and its application
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Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser
115
<210> 20
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Gln Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser
115
<210> 21
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser
115
<210> 22
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys
100 105
<210> 23
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 24
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Met Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 25
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Ser Ala Ser Gln Phe Val Gly Ser Ser
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 26
<211> 214
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Ser Ala Ser Gln Phe Val Gly Ser Ser
20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Glu Ser Met Ser Gly Val Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 27
<211> 450
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Val Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 28
<211> 450
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Val Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Gln Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 29
<211> 450
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Val Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 30
<211> 450
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Gln Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 31
<211> 450
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Thr Asp Asp Ser Thr Ser Ala
65 70 75 80
Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr
85 90 95
Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 32
<211> 642
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gacattttac tgacacagag ccccgccatc ctcagcgtga gccccggtga gagagtgtcc 60
ttcagctgca gagccagcca gttcgtgggc agcagcatcc actggtacca gcagaggacc 120
aacggctccc ctcgtctgct gatcaagtac gccagcgaga gcatgtccgg aatcccctct 180
cgttttagcg gcagcggaag cggcaccgac ttcactttaa gcatcaacac agtggagtcc 240
gaggacatcg ccgactacta ctgccagcag agccacagct ggcctttcac attcggcagc 300
ggcaccaatc tggaggtgaa aaggaccgtg gccgccccct ccgtctttat cttccccccc 360
agcgacgagc agctgaagtc cggaaccgcc agcgtggtgt gtttactgaa caacttctac 420
cctcgtgagg ccaaggtgca gtggaaggtg gacaacgctt tacagtccgg caacagccaa 480
gaaagcgtga ccgagcaaga tagcaaggac agcacctact ctttaagcag cacactgact 540
ttaagcaagg ccgattacga gaagcacaag gtgtacgctt gtgaggtgac acaccaaggt 600
ttaagctccc ccgtgaccaa gagcttcaat cgtggagagt gc 642
<210> 33
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gaagtgaagc tcgaggaaag cggaggaggt ttagtgcagc ccggtggctc catgaagctc 60
tcttgtgtgg cctccggctt tatcttcagc aaccactgga tgaattgggt tcgtcagagc 120
cccgaaaaag gcctcgagtg ggtcgccgaa attcgtagca agtccatcaa cagcgccaca 180
cactatgccg agagcgtcaa gggtcgtttt accatctcca gggacgatag caagagcgcc 240
gtgtatttac agatgacaga tctgagaacc gaggacaccg gcgtgtacta ctgctctcgt 300
aactactacg gatccaccta cgactactgg ggccaaggca ccactttaac cgtgagctcc 360
gccagcacca agggacccag cgtgttcccc ctcgccccca gcagcaagtc cacaagcgga 420
ggaacagctg ccctcggctg tttagtgaag gactacttcc ccgaacccgt gaccgtgtct 480
tggaacagcg gagccctcac ctccggcgtc cataccttcc ccgctgtgct ccagagctcc 540
ggtttatact ctttaagctc cgtggtgaca gtgcccagca gctctttagg cacccagacc 600
tatatctgta atgtgaacca caagccctcc aataccaagg tggataagaa ggtcgagccc 660
aagagctgcg acaagacaca cacttgtcct ccttgtcccg ctcccgaact gctgggcggc 720
cctagcgttt ttttatttcc tcccaagccc aaggacactt taatgatcag cagaaccccc 780
gaagtcactt gtgtggtcgt ggacgtctcc cacgaggacc ccgaggtgaa gtttaactgg 840
tacgtcgacg gcgtggaagt ccacaatgcc aagaccaagc ctagggaaga gcagtacaac 900
agcacctatc gtgtcgtctc cgtgctgaca gtgctgcatc aagattggct gaacggcaaa 960
gagtacaaat gcaaggtgag caataaggct ttacccgccc ctatcgagaa gaccatcagc 1020
aaggccaagg gccagcctcg tgaacctcaa gtttatacac tgcccccctc tcgtgaggag 1080
atgaccaaga accaagttag cctcacatgt ttagtgaaag gcttctaccc tagcgacatt 1140
gccgtggaat gggagagcaa cggccagccc gaaaacaatt acaagaccac ccctcccgtt 1200
ctggacagcg acggctcctt ctttctgtac agcaagctga cagtggacaa gagcagatgg 1260
cagcaaggta acgtgttcag ctgcagcgtg atgcacgagg ctctgcacaa ccactacaca 1320
cagaagtctt tatctttatc ccccggtaag 1350
<210> 34
<211> 642
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gatattttac tgacccagtc ccccgccatt ctgtccgtga gccccggaga gagagtgtcc 60
ttcagctgtc gtgccagcca gttcgtgggc agctccatcc actggtatca gcagaggacc 120
aacggcagcc ctcgtctgct gatcaagtac gcctccgaaa gcatgagcgg catcccctct 180
cgttttagcg gcagcggaag cggaaccgac ttcactttaa gcattagctc tttagagccc 240
gaggacttcg ccgtgtacta ctgccagcag agccacagct ggcccttcac atttggctcc 300
ggcaccaatt tagaagtgaa gaggaccgtg gccgccccca gcgtgttcat cttccctccc 360
agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gtttactgaa caacttttac 420
cctcgtgagg ccaaggtgca gtggaaggtg gacaatgctt tacagtccgg caacagccaa 480
gaaagcgtga ccgagcaaga tagcaaggac agcacctact ctttaagcag cactttaact 540
ttatccaagg ccgactacga gaagcacaag gtgtacgctt gtgaggtgac acaccaaggt 600
ttatccagcc ccgtgaccaa gagcttcaat agaggcgagt gc 642
<210> 35
<211> 642
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gacattctgc tgacccagag ccccgctatt ttaagcgtga gccccggcga gagagtgagc 60
tttagctgta gggccagcca gttcgtgggc agcagcattc actggtacca gcagaggacc 120
aacggctccc ctcgtctgct catcaagtac gccagcgaaa gcatgagcgg cgtgcccgct 180
cgttttagcg gaagcggctc cggcaccgac ttcactttaa ccatcagctc tttagagccc 240
gaggacttcg ccgtgtacta ctgccagcag tcccacagct ggcccttcac cttcggcagc 300
ggcaccaatt tagaggtgaa gagaaccgtg gccgccccta gcgtcttcat cttccctccc 360
agcgacgagc agctgaagag cggcaccgct agcgtggtgt gtttactgaa caacttctac 420
cctcgtgagg ccaaggtgca gtggaaggtg gacaacgctt tacagagcgg caactcccaa 480
gagtccgtga cagaacaaga tagcaaggac agcacctact ctttaagctc cacactgact 540
ttaagcaagg ccgactacga gaagcataag gtgtacgctt gtgaggtgac acatcaaggt 600
ttatccagcc ccgtgaccaa gagcttcaat cgtggcgagt gc 642
<210> 36
<211> 642
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gatattttac tgacccagtc ccccgccatt ctgtccgtga gccccggaga gagagtgtcc 60
ttcagctgta gcgccagcca gttcgtgggc agctccatcc actggtatca gcagaggacc 120
aacggcagcc ctcgtctgct gatcaagtac gcctccgaaa gcatgagcgg catcccctct 180
cgttttagcg gcagcggaag cggaaccgac ttcactttaa gcattagctc tttagagccc 240
gaggacttcg ccgtgtacta ctgccagcag agccacagct ggcccttcac atttggctcc 300
ggcaccaatt tagaagtgaa gaggaccgtg gccgccccca gcgtgttcat cttccctccc 360
agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gtttactgaa caacttttac 420
cctcgtgagg ccaaggtgca gtggaaggtg gacaatgctt tacagtccgg caacagccaa 480
gaaagcgtga ccgagcaaga tagcaaggac agcacctact ctttaagcag cactttaact 540
ttatccaagg ccgactacga gaagcacaag gtgtacgctt gtgaggtgac acaccaaggt 600
ttatccagcc ccgtgaccaa gagcttcaat agaggcgagt gc 642
<210> 37
<211> 642
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gacattctgc tgacccagag ccccgctatt ttaagcgtga gccccggcga gagagtgagc 60
tttagctgta gcgccagcca gttcgtgggc agcagcattc actggtacca gcagaggacc 120
aacggctccc ctcgtctgct catcaagtac gccagcgaaa gcatgagcgg cgtgcccgct 180
cgttttagcg gaagcggctc cggcaccgac ttcactttaa ccatcagctc tttagagccc 240
gaggacttcg ccgtgtacta ctgccagcag tcccacagct ggcccttcac cttcggcagc 300
ggcaccaatt tagaggtgaa gagaaccgtg gccgccccta gcgtcttcat cttccctccc 360
agcgacgagc agctgaagag cggcaccgct agcgtggtgt gtttactgaa caacttctac 420
cctcgtgagg ccaaggtgca gtggaaggtg gacaacgctt tacagagcgg caactcccaa 480
gagtccgtga cagaacaaga tagcaaggac agcacctact ctttaagctc cacactgact 540
ttaagcaagg ccgactacga gaagcataag gtgtacgctt gtgaggtgac acatcaaggt 600
ttatccagcc ccgtgaccaa gagcttcaat cgtggcgagt gc 642
<210> 38
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gaagtccagc tgctcgaaag cggaggcggt ttagtgcagc ccggtggcag catgaagctg 60
tcttgtgctg tcagcggctt catcttcagc aaccactgga tgaactgggt gagacagagc 120
cccgaaaagg gtttagagtg ggtcgccgag attcgtagca agtccatcaa ctccgccaca 180
cactacgccg agtccgtgaa aggtcgtttc accatctcca gggacgactc caagtccgcc 240
gtgtatttac agatgaccga tttaagaaca gaggacaccg gcgtgtacta ctgttctcgt 300
aactactacg gctccaccta tgactactgg ggacaaggta ccactttaac cgtgagcagc 360
gcctccacaa aaggccctag cgtgttccct ctggccccca gctccaagtc cacaagcggc 420
ggaacagctg ctttaggctg tctcgtgaag gactatttcc ccgaacccgt gacagtgagc 480
tggaacagcg gcgctttaac aagcggagtg cacaccttcc ccgctgtgct gcagagcagc 540
ggactgtact ctttaagctc cgtggtgaca gtgccctcct cctctttagg aacccagacc 600
tacatttgta atgtcaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 660
aagtcttgtg acaagaccca cacttgtccc ccttgtcccg cccccgaact gctgggagga 720
ccctccgtgt ttttattccc tcccaagcct aaggacactt taatgatctc tcgtacaccc 780
gaggtgactt gtgtggtcgt cgacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 840
tacgtggacg gcgtcgaggt ccataacgcc aaaacaaagc ctcgtgagga gcagtacaac 900
agcacctata gggtggtcag cgtgctgacc gtgctccacc aagattggct gaatggcaaa 960
gagtacaaat gcaaggtcag caataaggcc ctccccgccc ccattgagaa gacaatcagc 1020
aaggccaagg gccaacccag agagccccaa gtttacactt tacctccctc tcgtgaagag 1080
atgaccaaga atcaagtttc tttaacttgt ctcgtgaagg gcttttatcc cagcgacatc 1140
gctgtggaat gggagagcaa cggacagccc gagaacaatt acaagaccac cccccccgtt 1200
ctggactccg atggcagctt ctttttatac agcaagctga cagtggataa gtctcgttgg 1260
caacaaggta acgtcttcag ctgctccgtg atgcacgagg ctttacacaa tcactacacc 1320
cagaagtctt tatctttatc ccccggtaaa 1350
<210> 39
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gaagtccagc tgctcgaaag cggaggcggt ttagtgcagc ccggtggcag catgaagctg 60
tcttgtgctg tcagcggctt catcttcagc aaccactgga tgaactgggt gagacagagc 120
cccgaaaagg gtttagagtg ggtcgccgag attcgtagca agtccatcaa ctccgccaca 180
cactacgccg agtccgtgaa aggtcgtttc accatctcca cagacgactc cacctccgcc 240
gtgtatttac agatgaccga tttacagaca gaggacaccg gcgtgtacta ctgttctcgt 300
aactactacg gctccaccta tgactactgg ggacaaggta ccactttaac cgtgagcagc 360
gcctccacaa aaggccctag cgtgttccct ctggccccca gctccaagtc cacaagcggc 420
ggaacagctg ctttaggctg tctcgtgaag gactatttcc ccgaacccgt gacagtgagc 480
tggaacagcg gcgctttaac aagcggagtg cacaccttcc ccgctgtgct gcagagcagc 540
ggactgtact ctttaagctc cgtggtgaca gtgccctcct cctctttagg aacccagacc 600
tacatttgta atgtcaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 660
aagtcttgtg acaagaccca cacttgtccc ccttgtcccg cccccgaact gctgggagga 720
ccctccgtgt ttttattccc tcccaagcct aaggacactt taatgatctc tcgtacaccc 780
gaggtgactt gtgtggtcgt cgacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 840
tacgtggacg gcgtcgaggt ccataacgcc aaaacaaagc ctcgtgagga gcagtacaac 900
agcacctata gggtggtcag cgtgctgacc gtgctccacc aagattggct gaatggcaaa 960
gagtacaaat gcaaggtcag caataaggcc ctccccgccc ccattgagaa gacaatcagc 1020
aaggccaagg gccaacccag agagccccaa gtttacactt tacctccctc tcgtgaagag 1080
atgaccaaga atcaagtttc tttaacttgt ctcgtgaagg gcttttatcc cagcgacatc 1140
gctgtggaat gggagagcaa cggacagccc gagaacaatt acaagaccac cccccccgtt 1200
ctggactccg atggcagctt ctttttatac agcaagctga cagtggataa gtctcgttgg 1260
caacaaggta acgtcttcag ctgctccgtg atgcacgagg ctttacacaa tcactacacc 1320
cagaagtctt tatctttatc ccccggtaaa 1350
<210> 40
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gaagtccagc tgctcgaaag cggaggcggt ttagtgcagc ccggtggcag catgaagctg 60
tcttgtgctg tcagcggctt catcttcagc aaccactgga tgaactgggt gagacagagc 120
cccgaaaagg gtttagagtg ggtcgccgag attcgtagca agtccatcaa ctccgccaca 180
cactacgccg agtccgtgaa aggtcgtttc accatctcca cagacgactc cacctccgcc 240
gtgtatttac agatgaccga tttaagaaca gaggacaccg gcgtgtacta ctgttctcgt 300
aactactacg gctccaccta tgactactgg ggacaaggta ccactttaac cgtgagcagc 360
gcctccacaa aaggccctag cgtgttccct ctggccccca gctccaagtc cacaagcggc 420
ggaacagctg ctttaggctg tctcgtgaag gactatttcc ccgaacccgt gacagtgagc 480
tggaacagcg gcgctttaac aagcggagtg cacaccttcc ccgctgtgct gcagagcagc 540
ggactgtact ctttaagctc cgtggtgaca gtgccctcct cctctttagg aacccagacc 600
tacatttgta atgtcaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 660
aagtcttgtg acaagaccca cacttgtccc ccttgtcccg cccccgaact gctgggagga 720
ccctccgtgt ttttattccc tcccaagcct aaggacactt taatgatctc tcgtacaccc 780
gaggtgactt gtgtggtcgt cgacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 840
tacgtggacg gcgtcgaggt ccataacgcc aaaacaaagc ctcgtgagga gcagtacaac 900
agcacctata gggtggtcag cgtgctgacc gtgctccacc aagattggct gaatggcaaa 960
gagtacaaat gcaaggtcag caataaggcc ctccccgccc ccattgagaa gacaatcagc 1020
aaggccaagg gccaacccag agagccccaa gtttacactt tacctccctc tcgtgaagag 1080
atgaccaaga atcaagtttc tttaacttgt ctcgtgaagg gcttttatcc cagcgacatc 1140
gctgtggaat gggagagcaa cggacagccc gagaacaatt acaagaccac cccccccgtt 1200
ctggactccg atggcagctt ctttttatac agcaagctga cagtggataa gtctcgttgg 1260
caacaaggta acgtcttcag ctgctccgtg atgcacgagg ctttacacaa tcactacacc 1320
cagaagtctt tatctttatc ccccggtaaa 1350
<210> 41
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gaagtgaagc tcgaggaaag cggaggaggt ttagtgcagc ccggtggctc catgaagctc 60
tcttgtgtgg cctccggctt tatcttcagc aaccactgga tgaattgggt tcgtcagagc 120
cccgaaaaag gcctcgagtg ggtcgccgaa attcgtagca agtccatcaa cagcgccaca 180
cactatgccg agagcgtcaa gggtcgtttt accatctcca ccgacgatag caccagcgcc 240
gtgtatttac agatgacaga tctgcagacc gaggacaccg gcgtgtacta ctgctctcgt 300
aactactacg gatccaccta cgactactgg ggccaaggca ccactttaac cgtgagctcc 360
gccagcacca agggacccag cgtgttcccc ctcgccccca gcagcaagtc cacaagcgga 420
ggaacagctg ccctcggctg tttagtgaag gactacttcc ccgaacccgt gaccgtgtct 480
tggaacagcg gagccctcac ctccggcgtc cataccttcc ccgctgtgct ccagagctcc 540
ggtttatact ctttaagctc cgtggtgaca gtgcccagca gctctttagg cacccagacc 600
tatatctgta atgtgaacca caagccctcc aataccaagg tggataagaa ggtcgagccc 660
aagagctgcg acaagacaca cacttgtcct ccttgtcccg ctcccgaact gctgggcggc 720
cctagcgttt ttttatttcc tcccaagccc aaggacactt taatgatcag cagaaccccc 780
gaagtcactt gtgtggtcgt ggacgtctcc cacgaggacc ccgaggtgaa gtttaactgg 840
tacgtcgacg gcgtggaagt ccacaatgcc aagaccaagc ctagggaaga gcagtacaac 900
agcacctatc gtgtcgtctc cgtgctgaca gtgctgcatc aagattggct gaacggcaaa 960
gagtacaaat gcaaggtgag caataaggct ttacccgccc ctatcgagaa gaccatcagc 1020
aaggccaagg gccagcctcg tgaacctcaa gtttatacac tgcccccctc tcgtgaggag 1080
atgaccaaga accaagttag cctcacatgt ttagtgaaag gcttctaccc tagcgacatt 1140
gccgtggaat gggagagcaa cggccagccc gaaaacaatt acaagaccac ccctcccgtt 1200
ctggacagcg acggctcctt ctttctgtac agcaagctga cagtggacaa gagcagatgg 1260
cagcaaggta acgtgttcag ctgcagcgtg atgcacgagg ctctgcacaa ccactacaca 1320
cagaagtctt tatctttatc ccccggtaag 1350
<210> 42
<211> 1350
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gaagtgaagc tcgaggaaag cggaggaggt ttagtgcagc ccggtggctc catgaagctc 60
tcttgtgtgg cctccggctt tatcttcagc aaccactgga tgaattgggt tcgtcagagc 120
cccgaaaaag gcctcgagtg ggtcgccgaa attcgtagca agtccatcaa cagcgccaca 180
cactatgccg agagcgtcaa gggtcgtttt accatctcca ccgacgatag caccagcgcc 240
gtgtatttac agatgacaga tctgagaacc gaggacaccg gcgtgtacta ctgctctcgt 300
aactactacg gatccaccta cgactactgg ggccaaggca ccactttaac cgtgagctcc 360
gccagcacca agggacccag cgtgttcccc ctcgccccca gcagcaagtc cacaagcgga 420
ggaacagctg ccctcggctg tttagtgaag gactacttcc ccgaacccgt gaccgtgtct 480
tggaacagcg gagccctcac ctccggcgtc cataccttcc ccgctgtgct ccagagctcc 540
ggtttatact ctttaagctc cgtggtgaca gtgcccagca gctctttagg cacccagacc 600
tatatctgta atgtgaacca caagccctcc aataccaagg tggataagaa ggtcgagccc 660
aagagctgcg acaagacaca cacttgtcct ccttgtcccg ctcccgaact gctgggcggc 720
cctagcgttt ttttatttcc tcccaagccc aaggacactt taatgatcag cagaaccccc 780
gaagtcactt gtgtggtcgt ggacgtctcc cacgaggacc ccgaggtgaa gtttaactgg 840
tacgtcgacg gcgtggaagt ccacaatgcc aagaccaagc ctagggaaga gcagtacaac 900
agcacctatc gtgtcgtctc cgtgctgaca gtgctgcatc aagattggct gaacggcaaa 960
gagtacaaat gcaaggtgag caataaggct ttacccgccc ctatcgagaa gaccatcagc 1020
aaggccaagg gccagcctcg tgaacctcaa gtttatacac tgcccccctc tcgtgaggag 1080
atgaccaaga accaagttag cctcacatgt ttagtgaaag gcttctaccc tagcgacatt 1140
gccgtggaat gggagagcaa cggccagccc gaaaacaatt acaagaccac ccctcccgtt 1200
ctggacagcg acggctcctt ctttctgtac agcaagctga cagtggacaa gagcagatgg 1260
cagcaaggta acgtgttcag ctgcagcgtg atgcacgagg ctctgcacaa ccactacaca 1320
cagaagtctt tatctttatc ccccggtaag 1350

Claims (9)

1. An anti-TNF- α humanized monoclonal antibody TCX063, wherein the anti-TNF- α humanized monoclonal antibody TCX063 has an amino acid sequence of any one of:
(1) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.22, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17;
(2) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17;
(3) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17;
(4) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.22, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(5) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(6) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(7) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.15, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(8) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 18;
(9) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19;
(10) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19;
(11) the amino acid sequence of the light chain variable region is shown as SEQ ID NO.13, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21.
2. The anti-TNF- α humanized monoclonal antibody TCX063 of claim 1 wherein the monoclonal antibody TCX063 is a human IgG1 whole antibody.
3. The anti-TNF- α humanized monoclonal antibody TCX063 of claim 2 wherein the anti-TNF- α humanized monoclonal antibody TCX063 has the amino acid sequence of any of:
(1) the amino acid sequence of the light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 27;
(2) the amino acid sequence of the light chain is shown as SEQ ID NO.23, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 27;
(3) the amino acid sequence of the light chain is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 27;
(4) the amino acid sequence of the light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(5) the amino acid sequence of the light chain is shown as SEQ ID NO.23, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(6) the amino acid sequence of the light chain is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(7) the amino acid sequence of the light chain is shown as SEQ ID NO.25, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(8) the amino acid sequence of the light chain is shown as SEQ ID NO.26, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 28;
(9) the amino acid sequence of the light chain is shown as SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 29;
(10) the amino acid sequence of the light chain is shown as SEQ ID NO.26, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 29;
(11) the amino acid sequence of the light chain is shown as SEQ ID NO.23, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 31.
4. A gene encoding the anti-TNF-alpha humanized monoclonal antibody TCX063 according to any one of claims 1 to 3.
5. The gene according to claim 4, wherein the gene has any one of the following nucleotide sequences:
(1) the nucleotide sequence of the light chain is shown as SEQ ID NO.32, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 38;
(2) the nucleotide sequence of the light chain is shown as SEQ ID NO.34, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 38;
(3) the nucleotide sequence of the light chain is shown as SEQ ID NO.35, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 38;
(4) the nucleotide sequence of the light chain is shown as SEQ ID NO.32, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(5) the nucleotide sequence of the light chain is shown as SEQ ID NO.34, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(6) the nucleotide sequence of the light chain is shown as SEQ ID NO.35, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(7) the nucleotide sequence of the light chain is shown as SEQ ID NO.36, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(8) the nucleotide sequence of the light chain is shown as SEQ ID NO.37, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 39;
(9) the nucleotide sequence of the light chain is shown as SEQ ID NO.35, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 40;
(10) the nucleotide sequence of the light chain is shown as SEQ ID NO.37, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 40;
(11) the nucleotide sequence of the light chain is shown as SEQ ID NO.34, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 42.
6. Biomaterial comprising the genes of claim 4 or 5, characterized in that it comprises an expression cassette, a vector, a host cell, an engineered bacterium or a cell line.
7. Use of the monoclonal antibody TCX063 of any one of claims 1 to 3 or the gene of claim 4 or 5 or the biomaterial of claim 6 in the manufacture of a medicament for the prevention or treatment of inflammatory or autoimmune diseases.
8. Use of the monoclonal antibody TCX063 of any one of claims 1 to 3 or the gene of claim 4 or 5 or the biomaterial of claim 6 in the preparation of a human TNF- α detection reagent.
9. A medicament or a detection reagent comprising the monoclonal antibody TCX063 according to anyone of claims 1 to 3.
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