CN1212967A - Parathormone related protein human source antibody and its preparation method - Google Patents

Abstract

The invention provides for the first time a humanized antibody specific to parathyroid hormone-related protein and a preparation method thereof. The antibody is mainly composed of heavy chain (VH+CH1) and light chain (L chain). Its preparation method is to use the method of genetic engineering. The antibody can be used as a medicine for diagnosis, treatment and prevention of tumors.

Description

Parathyroid hormone-related protein humanized antibody and preparation method thereof

The invention belongs to the field of humanized genetic engineering antibody

Parathyroid Hormone-Related Protein (PTHrP) has similar biological activity to Parathyroid Hormone PTH, and it was first discovered in Humoral Hypercalcemia of malignant tumors. In the blood of patients with Malignancy (HHM), squamous cell carcinoma, breast cancer, urinary system cancer, ovarian cancer and other tumor tissues can produce PTHrP, which is one of the factors that cause HHM. Clinically, HHM is characterized by loss of appetite, nausea, polyuria, mental abnormalities and even coma. It usually occurs in the late stage of cancer and seriously affects its prognosis. PTHrP can be detected in the blood of almost all tumor patients with HHM. Therefore, the research on PTHrP has attracted great attention from the medical community. One of the methods widely recognized by the medical community to reduce tumor-related hypercalcemia HHM is to use PTHrP neutralizing antibodies to immunoneutralize PTHrP, which not only reduces blood calcium, but also inhibits the growth of tumor cells that secrete PTHrP. growth for the purpose of treating tumors. So far, people have used polyclonal antiserum and mouse monoclonal antibody to immune neutralize PTHrP secreted by tumor cells, thereby reducing hypercalcemia and inhibiting the growth of tumor cells.

At present, relevant researchers have obtained the monoclonal antibody against PTHrP (patent No. US5217896) by using hybridoma technology, which can successfully detect, diagnose and treat hypercalcemia caused by malignant tumors. The method of obtaining monoclonal antibodies by establishing mouse-derived hybridoma cell lines is a great revolution in the history of antibody development. The obtained antibodies have strong specificity and have been widely used in clinical practice. However, the production cycle of monoclonal antibodies is long, the establishment of strains is unstable, and the reproducibility is poor. The most important thing is that the monoclonal antibody produced by the hybridoma is a mouse-derived antibody, which will cause an immune response after repeated application in the human body, thus limiting its popularization and application in clinic.

Since the first report in 1989 on the use of antibody library technology to prepare humanized monoclonal antibodies, the technology has made considerable progress (Ninnsim A, Hoogenboon HR, Tomlinson IM et al., Antibody fragments from a single pot phage display as immunochemical reagents. EMBO J, 1991; 13:692). At present, a variety of humanized antibodies or antibody fragments against haptens, proteins, virus particles, etc. can be screened from immunized or unimmunized natural antibody libraries.

In humans, a complete antibody consists of four chains, two light L chains (VL+CL) and two heavy H chains (VH+CH1+CH2+CH3). The modified and engineered humanized antibody fragments can be composed of VL+VH, or VL+CL and VH+CH1. If the antibody fragment is in the form of a single chain, the light chain and the heavy chain fragment can be connected by a linker (Linker). If the antibody fragment VL+CL and VH+CH1 are connected by a disulfide bond at the carboxyl end, the in vivo antibody fragment is formed. Fab. This humanized antibody or antibody fragment can not only achieve the effect similar to the monoclonal antibody produced by the hybridoma, but also has the incomparable advantages of the mouse monoclonal antibody.

But so far, there is no report on PTH-related protein humanized genetically engineered antibody and its preparation method.

The object of the present invention is to provide parathyroid hormone-related protein humanized genetic engineering antibody and preparation method thereof, and its technical route is as follows:

Lymphocytes were collected from peripheral blood of tumor patients, mRNA was extracted, reverse transcribed into cDNA, primers were designed, and VH+CH1 gene of heavy chain H and VL+CL gene of light chain L were amplified by PCR reaction. Primers were designed according to the reported conserved sequences of human antibody gene families, and the restriction sites designed at both ends of the primers were consistent with the corresponding cloning sites on the phage presentation vector. The VH+CH1 gene and VL+CL gene were amplified by polymerase chain reaction.

The heavy chain VH+CH1 gene and the light chain VL+CL gene are successively connected to the vector to transform Escherichia coli. With the help of the helper phage, the immunoglobulin molecular fragment Fab with binding function is expressed on the surface of the filamentous phage to form an antibody fragment gene combination library. There is a corresponding cloning site on the phage presentation vector pComb3, as shown in Figure 1.

The recombinant vector transforms Escherichia coli, and the heavy chain VH+CH1 and light chain VL+CL are assembled in the periplasmic cavity of Escherichia coli under the leadership of the guide peptide to form an immunoglobulin molecular fragment Fab with binding function, that is, the two form at the carboxyl terminal The disulfide bonds are held together, in the same way that antibodies are assembled in vivo. The Fab fragment and the phage outer membrane protein gIII protein (provided by the helper phage) are fused and expressed, and presented on the surface of the phage to form a combinatorial library of antibody fragment genes, referred to here as the library.

The library was amplified, panned and screened with PTHrP antigen to obtain positive strains. The library is amplified and added to the reaction wells coated with the antigen PTHrP for incubation. The Fab antibody presented on the surface of the phage is firmly bound to the antigen, and other non-specific phages are washed away, and the phages that bind to the antigen are amplified again. By repeating this process, the antibody fragment Fab against PTHrP can be screened, and the sequence analysis of the screened antibody gene heavy chain VH+CH1 and light chain VL+CL is compared with the human antibody gene sequence database to ensure its correctness.

The antibody gene fragments of positive strains were cloned into prokaryotic expression vectors to assemble high-efficiency expression plasmids. Transform Escherichia coli to obtain recombinant bacteria, and cultivate the recombinant bacteria under conditions that can express the antibody gene fragment. The expression vector pIG110 contains a bivalent polycistronic operon, SD sequence, transcription terminator and corresponding cloning restriction sites. After transforming Escherichia coli, the expression of its fusion protein was induced. SDS-PAGE electrophoresis showed that the fusion protein had a molecular weight of 52KD and existed in Escherichia coli in the form of inclusion body, accounting for about 30% of the bacterial protein.

Isolation of expressed antibodies. The inclusion bodies were collected, denatured and refolded, and purified antibody protein was obtained by passing through molecular sieves. Detect its biological activity at the cellular level. Fab antibody protein can neutralize the biological activity of PTHrP.

Humanized antibodies specific for PTH-related proteins were obtained by this method. Specifically, the present invention obtains a parathyroid hormone-related protein, and the present invention also obtains a gene encoding a parathyroid hormone-related protein. Amino acid sequence and nucleotide sequence are as follows: VH+CH1 chain: CCC CAG CTC ATA GTG GAA GTG TCT TTC AAC GGT ACA TTC GCT CAC AAA 48P Q L I V E E V S F N T G T G T G F GA GT A GC K GAT AAA TCG GTC TTC CCC AAG CTG CAC CCG GTC 96F V R C W D K -F P K L H P VAGC TGG GGG TAG TCG GAC TCG GAC TCT AAC D s n CTG AAC CAC TGG 240 S K Y y g k t t v f r l n h Wtct Gaa CCC ATG GCT GCT AAG GAG GAG AGG AGG AGG W RGAC ATA GGT CTG AAC CAC CAC TCT CAG CCG CAC CAC CAG AAG TTC ACA TAC 336D I G L N H S Q K F T YGGG ACA TGG TCA's GAC W f a r f s l d m e H Q K RCCC TTC GTC ATA TATA TTC AGA CTG GCT GGT GTC GGT GCT CAC 432P F V GTC GAG ATG ACA TAC AGA AGA GCT GCT GGT CTG 480W Q S W F K V E M T y R I A G LCCG GAC CAG TGG TCG GCC TAN P f y V T y PTTC TGG GGT GCT CAC ATG GAG GAG TAC CAC AAG CAG CAG TGC AGA TCT GTC 576F W G A H M K Q C R S vgct CCC TCT AGA CTG GTC CCC CCC CAG GAC CAG AAG 624A K W P S Q H S R L V p Q d Q KCTG GCTG AGA AGA AAC ACA CCC TGT TCT TC CCG AT 669L A Q W M R N T P S F P i NL Chain: TAC AAA TTC TCT AGA CTG GTC AAT AGA GCT CAG ATG ATG TCT 48y M K F S R L V N L v d a w p n GCC AAA TAT AAT TCT TGT TGT CTC AGT 192p N Q L P g T A K y I sgac Tac CCG GCC CCC CTC CCC CCC TCT CTT CAA AAG GCC 240D F Y P G A L F P P S L Q N K ATAC AGC TGC CAG GTC ACG Cat GAA ACC GCA TGG GAT GAT GAT GAT GAT GAT GAT AG 28y S C Q V T H E T V A W D D ACAG GCG AC's CTC GTC ACT 336Q A Q T M L N i C I F G S n AAC CAC TGG CAG AAG GGT TTC TTT AAG GCT CCC ATA TTC GGT 432y T N H W Q K F K A P GGAT GCG Act GCA ACG CAC GGG GGG TTC 480D S A T N S a T l h y t g s e FTCT CTG TGT CAG TGG AAG AAG ACC TCG ACG CCC AAC GTG 528S L C Q W K t s AAG ACC GCT GTC ACT TGC CTG 576A V F G L g q w f k k K LTTC CAG CAG AGG TCT AAC ATC GGC TCA CAG CTG L I P SCTG CTC TGT TAC ACT CTC CAG GTG TTC 651L L C Y T L Q V F

The antibody obtained by the method of the present invention has a high affinity to PTHrP, and can neutralize the biological activity of PTHrP. When labeled with a biomarker (marker), such as enzyme, biotin, fluorescein, chromogenic chemical group or Radioactive isotopes can be used to detect and analyze the expression level of PTHrP in human normal tissues and tumor tissues, to detect blood calcium metabolism and regulation under normal and pathological conditions (such as osteoporosis and HHM), and also play a role To the purpose of diagnosing hypercalcemia caused by malignant tumors.

The antibody gene of the present invention can also be linked together with human interleukin-2, tumor necrosis factor and other tumor killing factors, and jointly expressed into a fusion protein. The antibody part not only plays the role of immune neutralization, but also plays the role of guiding in vivo, so as to kill tumor cells more accurately.

The antibody of the present invention can be administered alone or together with a pharmaceutically acceptable carrier.

The method of the present invention has the advantages of: (1) the process flow is simple, the cost is low, and a large amount of products can be obtained in a short period of time, which creates conditions for large-scale clinical application; (2) the specificity is high, which is unmatched by serum polyclonal antibodies (3) Eliminates the immunogenicity of mouse-derived monoclonal antibodies to the human body, ensuring the safety of the application to the human body; (4) The entire process has high technical content, high stability, and good repeatability, which is a genetic engineering Drugs can be applied to clinical basic conditions.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of the construction of the pComb3 vector. Figure 2 is a flow chart of the method of the present invention.

Example:

1. A set of primers was designed to clone a complete set of variable region genes of immunoglobulins. Construction of phage library.

According to the known human immunoglobulin variable region gene library data, it is known that the antibody gene has a family-conserved sequence. Based on this, six primers were designed, and a complete set of immunoglobulin variable region genes was cloned by polymerase chain reaction (PCR). , the six primers are: 5'(CG)AG GTG CAG CTC GAG (CG)AG TCT GGG 3'5'GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG 3'5'GA(AC) AT(CT) GAG CTC AC(CG) CAG TCT CCA 3'5'GCG CCG TCT AGA ACT AAC ACT CTC CCC 3'5'(GC)AG GTG CAG CTG CTC GAC TCT GGG 3'5'GAC ATC GAC CTG ACC CAG TCT CCA 3' The six primers were synthesized by a DNA synthesizer, purified and used for PCR. The template DNA was prepared from lymphocytes in the blood of tumor patients, and the total mRNA extracted from lymphocytes was reverse-transcribed into cDNA. The reverse transcription reaction system was provided by Promega. , the reaction system for the first cDNA strand is as follows:

4ul MgCl2, 25mM

2ul 5× reverse transcription buffer

2ul 10mM dNTP mix

0.5ul rRNasin ribonuclease inhibitor

1ul(15u) AMV Reverse Transcriptase

5ul RNA (2ug)

Add RNase-free water to 20ul

Mix well and react at 42°C for 30 minutes to obtain the first cDNA strand.

PCR amplification of H chain and L chain genes respectively:

5ul cDNA

1ul Taq polymerase (1-2u)

1ul 5' primer (50pmol)

1u1 3' primer (50pmol)

10ul 10×Taq enzyme buffer (containing magnesium chloride)

1.5ul dNTP

The PCR reaction conditions are: denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes. After 35 cycles, take 10ul of the reaction mixture and use 0.7% agarose gel electrophoresis to detect H chain bands of about 660bp. and the L chain band of about 660bp, which are similar to the variable region of the heavy chain and the variable region of the light chain of human immunoglobulin respectively.

The L chain was digested with SacI and XbaI, and the H chain was digested with XhoI and SpeI. Pcomb3 was also digested with the same enzyme and ligated with the above PCR products. The electroporation method was used to transfer into Escherichia coli XLl-Blue, and the H chain was correctly inserted. The plasmid with the L chain was named P24, and the positive clones were screened to confirm that the connection was correct. So far, a phage library of genetically engineered combined antibodies has been constructed.

2. Amplification panning and screening of libraries

The above-mentioned library was expanded and cultivated, and the helper phage M13 was added, and the H chain and L chain proteins of the antibody were fused with the M13 coat protein pIII and expressed on the surface of the filamentous phage. The amplification process was as follows:

1ml XL1-Blue (OD600≈1.0)+100ul library+LB to 10ml

Shake at 37°C for 1 hour.

Expand and add 100ml medium (LB), shake at 37°C for 1 hour

Add 1ml of helper phage M13 (titer > 10 ⁷ ), shake at 37°C for 2 hours

Add kanamycin to a final concentration of 70ug/ml and shake overnight at 37°C on a shaker. The next day, phages were extracted for panning and enrichment of the library.

Culture supernatant collected by centrifugation

Add PEG8000 to a final concentration of 4%, add NaCl to a final concentration of 3%

Ice bath for 30 minutes after fully dissolved

Centrifuge to collect the precipitate, fully dissolve the precipitate in 3ml phosphate buffer

Add to reaction wells coated with PTHrP antigen (2ug/well), incubate for 2 hours

Pour out the phage, fill with water and wash once

Wash with TBS/Tween 10-20 times, pipetting up and down several times during the process

TBS/Tween wash 10-20 times, blow up and down several times during this process

TBS/Tween buffer system: 50mM Tris-HCl, pH7.5

150mM NaCl

0.05% Tween 20

Finally, fill with water and wash once, remove TBS/Tween

Inject 50ul of eluent EB and leave it at room temperature for 10 minutes

EB eluent system: 0.4M glycine, 0.39M hydrochloric acid, 0.1% bovine serum albumin

Pipette up and down several times, suck out, add 3ul neutralizing solution for every 50ul eluent, fully neutralize to about pH7.0, neutralizing solution is 2M Tris.

Infect 2ml of fresh Escherichia coli XLl-Blue with each 50ul of the neutralized eluted product, and place it at room temperature for 15min

Add 10ml medium (SB) and shake on a shaker at 37°C for 1 hour

Pour 100ml medium (SB), expand culture at 37°C for 1 hour

Add helper phage M13 (10 ¹² PFU) and shake at 37°C for 2 hours

Add kanamycin to a final concentration of 70ug/ml and shake overnight at 37°C

On the third day, repeat the process of the first day and the second day, and so on three times.

The products obtained by elutriation and enrichment were spread on LB solid medium and grown overnight at 37°C.

Pick 100 positive clones and inoculate them in 2ml LB medium, and culture overnight at 37°C.

The collected phages were added to 100 phages coated with PTHrP antigen 0.5ug/well and incubated for 2 hours.

Screened by conventional ELISA method, strong positive clones were obtained.

3. Sequence Determination and Analysis of Parathyroid Hormone Related Protein Human Gene Engineering Antibody

The screened antibody was sequenced, and the United States Biochemical system provided by Amersham Life Science was selected for determination. The DNA sequence and amino acid sequence of the heavy chain VH+CH1 are shown in Figure 1, and the DNA sequence and amino acid sequence of the light chain VL+CL As shown in Figure 1.

4. Expression of Antibody Genes in Escherichia coli

Cut off the antibody gene H chain and L chain from the plasmid P24, and connect them to the same site of the prokaryotic expression plasmid pIG110, pIG110 contains bivalent polycistrons. After confirming that the H chain and L chain genes were correctly inserted, the plasmid was named pHLB1, and the engineering bacteria containing pHLB1 was named pHLB1.

Pick a single colony of PHLB1 into 3ml LB medium containing ampicillin, cultivate overnight at 30°C with shaking, transfer to 20ml LB medium according to 10% inoculum size, cultivate at 30°C until the OD600 value reaches 0.5, raise the temperature to 42°C, and induce 6 hours. Take 1ml of the culture solution and centrifuge to collect the cells and dissolve them in 100ul protein loading buffer.

50mmol/L Tris-HCl pH6.8

100mmol/L DTT

2% SDS

0.1% bromophenol blue

10% glycerin

Take 10ul for 10% SDS-PAGE electrophoresis, identify the expression product, select the strain with the highest expression, and store it in a glycerol tube as a strain.

5. Fermentation of engineering bacteria and purification after fermentation

LKB-Bromma fermenter: PHLB1 single colony was cultured overnight at 30°C in 6ul LB-Amp culture medium, and used as the primary seed solution for fermentation.

According to the ratio of 1:50, inoculate the primary seed solution into 300ml M9CA-Amp culture medium, shake at 30°C for 16 hours, and use it as the secondary seed solution for fermentation.

Sterilize the fermenter with the highest pressure steam (15P, 30 minutes), place it on the operating table after cooling, set the temperature at 30°C, pH 7.0, dissolved oxygen at 85%, and stirring speed at 450 times/min. After the parameters are stable, inoculate the secondary seed liquid at 1:10, keep the above parameters unchanged, raise the temperature to 42°C after 5 hours and continue to induce for 10 hours, automatically adjust the pH to about 7.0, and the dissolved oxygen is greater than 100% at this time .

Bacteria were collected, ultrasonically broken, and inclusion bodies were collected. After repeated washing, denaturing solution containing 8M urea was added.

8M urea

50mmol/L Tris-HCl (pH8.5)

1ul/ml β-ME

Dissolve the precipitate, pass the dissolved inclusion body through a Sephrose CL600 molecular sieve column, adjust the concentration of the loaded inclusion body to 10mg/ml, collect the peak sample, detect it with 12% SDS-PAGE, combine the solutions of each tube containing the target protein, add 1M urea refolding solution.

50mmol/L Tris-HCl (pH9.0)

1mol/L urea

Finally, after dialysis with a urea-free refolding solution, the sample was recovered, and the sample was passed through a molecular sieve column again under the same elution and equilibrium conditions, and each elution peak was collected, and the target protein was identified and combined together for ultrafiltration and concentration.

The purified finished product was tested for its biological activity on the Solo cell line, which is a cell line that secretes PTHrP, provided by the University of Melbourne, Australia. After testing, the product can inhibit the growth of Solo cells. Please refer to US Patent No. 5,127,896 for the detection method.

According to the content disclosed herein, those of ordinary skill in the art can obtain the humanized antibody specific for parathyroid hormone-related protein described herein. As we all know, the variable regions of antibodies are highly variable and unpredictable, so the antibodies obtained by repeating this experiment will not be completely identical in amino acid primary structure. Thus disclosed in the present invention, and also obtainable according to the method of the present invention, is a class of humanized antibodies whose common feature is specificity for parathyroid hormone-related protein.

Claims (10)

1. A class of humanized antibodies specific to parathyroid hormone-related proteins. 2. The humanized antibody according to claim 1, characterized in that it is mainly composed of VH+CH1 and L chains. 3. The humanized antibody according to claim 2, characterized in that the VH+CH1 and L chains are linked by a disulfide bond at the carboxyl terminal. 4. The humanized antibody according to claim 1, characterized in that it has the following amino acid sequences of VH+CH1 and L chains: VH+CH1 chain: P Q L I V E V S F N G T F A H KF V r C w d k s v f p k l h p v y e s s d r d s n f w gn m d s p h q r s n t Y y d g k t v f r l n h ws e i q p m v a f a m k v e w rd I g l n S l d m e h q k RP f v V t Y M K f s r l v n G t F g s n v tl f k p h y y y, m r r f v iy t n h w q k f k a p i f gd s a t l h y t g s e Fs l c q k t L Q V F 5. A method for preparing a humanized antibody to parathyroid hormone-related protein, characterized in that it comprises the following steps: (1) Collect lymphocytes from the peripheral blood of tumor patients, extract mRNA, reverse transcribe into cDNA, amplify the VH+CH1 gene of heavy chain H and the gene of light chain L by PCR reaction, (2) The heavy chain VH+CH1 gene and the light chain L gene are successively connected to the carrier to transform Escherichia coli, and with the help of the helper phage, the immunoglobulin molecule fragment Fab with binding function is expressed on the surface of the filamentous phage, Construct antibody fragment gene combinatorial library. (3) Amplify, wash and screen the library for PTHrP antibodies with PTHrP antigens to obtain positive strains, (4) Cloning the antibody gene fragment of the positive strain into a prokaryotic expression vector to assemble a high-efficiency expression plasmid, transforming Escherichia coli to obtain a recombinant bacterium, and cultivating the engineered bacterium under conditions that allow the expression of the antibody gene fragment. (5) Isolating the expressed antibody. 6. A gene encoding the humanized antibody of any one of claims 1-4. 7. The humanized antibody gene according to claim 6, characterized in that it has the following VH+CH1 and L chain nucleotide sequences: VH+CH1 chain: CCC CAG CTC ATA GTG GAA GTG TCT TTC AAC GGT ACA TTC GCT CAC AAA  48TTC GTG AGA TGC TGG GAT AAA TCG GTC TTC CCC AAG CTG CAC CCG GTC  96AGC TGG GTT GGG TAT GAG TCT TCG GAT AGA GAC TCT AAC TTT TGG GGC 144AAT ATG GAC TCT CCC CAC CAG AGA TCG AAC ACA GAC GAT AAG ATG TCG 192TCT AAG TAC TAT GAC GGT AAA ACC ACT GTG TTC AGA CTG AAC CAC TGG 240TCT GAA ATA CAG CCC ATG GTC GCT TTC GCT ATG AAG GTC GAG TGG AGA 288GAC ATA GGT CTG AAC CAC TCT CAG CCG ATG CAC CAG AAG TTC ACA TAC 336GGG ACA TGG TTC GCT AGA TTT TCT CTG GAC ATG GAG CAC CAG AAG AGA 384CCC TTC GTC ATA TAT ACT TTC AGA CTG GCT GGT TCT GTC GGT GCT CAC 432TGG CAG TCT TGG TTC AAG GTC GAG ATG ACA TAC AGA ATA GCT GGT CTG 480CCG GAC AAT ATC CAG TGG TCG GCC TAT CCC TTC TAT GTC ACA TAC CCC 528TTC TGG GGT GCT CAC ATG GAG TAC CAC ATG AAG CAG TTC AGA TCT GTC 576GCT AAA TGG CCC TCT CAG CAC TCT AGA CTG GTC CCC CAG GAC CAG AAG 624CTG GCT CAG TGG ATG AGA AAC ACA CCC TCG TCG TCT TTC CCG ATC AAT 669 light chain LTAC AAA TTC TCT AGA CTG GTC AGA GCT CAG ATGG AT CCG CCC TCC TCT GAG GAG GCA TGG GAT GAT AGC CTG AGT GGC 144CCT AAT CAG GTC CCA GGA ACG GCC AAA TAT AAT TCT TGT CTC ATA AGT 192GAC TTC TAC CCG GGA GCC CTG TTC CCG CCC TCT CTT CAA AAC AAG GCC 240TAC AGC TGC CAG GTC ACG CAT GAA ACC GTG GCA TGG GAT GAT AGC TGG 288CAG GCG CAG ACT ATG CTC AAC ATA TGT ATA TTC GGA TCT AAT GTC ACT 336CTG TTC AAA CCC CAC TAT TGG GAG TTC GCT ATG AGA AGA TTC GTC ATA 384TAT ACA AAC CAC TGG CAG AAG GGT TTC TTT AAG GCT CCC ATA TTC GGT 432GAT TCT GCG ACT AAT TGT GCA ACT CTG CAC TAC ACG GGG AGC GAG TTC 480TCT CTG TGT CAG TGG AAG ACT TCC TAC TCG ACG CCC AAC GTG ACA GTG 528GCC GTG TTC GGA CTG GGA CAG TGG TTC AAG ACC GCT GTC ACT TCA CTG 576TTC CAG CTG ACC AGG TCT AAC ATC GGC TCA CAG TGG CTG ATC CCT TCC 624CTG CTC TTC TAC ACT CTC CAG GTG TTC 5 1 8. The humanized antibody according to any one of claims 1 to 4, characterized in that it can be used as a drug for diagnosis, treatment or prevention of tumors. 9. The humanized antibody according to any one of claims 1 to 4, characterized in that it is used in medicine for diagnosis, treatment or prevention of tumors. 10. The humanized antibody gene according to any one of claims 6 or 7, characterized in that it is used in the preparation of fusion proteins composed of human interleukin-2, tumor necrosis factor and other tumor killing factors .

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