CN101891818B - Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof - Google Patents

Abstract

The anti-human prostate-specific membrane antigen (PSMA) extracellular domain single-chain antibody of the present invention is a large-capacity single-chain antibody library constructed from peripheral blood mononuclear cells of prostate cancer patients using phage display technology. obtained after rigorous screening. The antibody can be soluble expressed in Escherichia coli, and the affinity with the extracellular region of the prostate specific membrane antigen reaches Kd=0.2nM. The antibody can not only bind to the extracellular region of recombinant PSMA, but also bind to NEK293 cells expressing PSMA on the surface and prostate cancer cell line LNCaP. This lays the foundation for the imaging diagnosis and guided treatment of prostate cancer using the antibody.

Description

Anti-human prostate-specific membrane antigen extracellular region single-chain antibody and its application

technical field

The invention belongs to genetic engineering antibodies, and relates to a humanized single-chain antibody against the extracellular region of human prostate-specific membrane antigen (PSMA) and its application in the development of prostate cancer diagnosis and treatment drugs.

Background technique

Prostate cancer is one of the malignant tumors that seriously endanger human health today. Its early diagnosis and treatment, especially the early diagnosis and treatment of metastasis and recurrence are difficult medical problems for people to overcome. With the continuous development of tumor immunology and molecular biology, especially the continuous improvement of phage surface display technology, new opportunities have been brought for the early diagnosis and cure of prostate cancer. Currently, anti-prostate cancer antibodies are undergoing clinical trials abroad, and these antibodies are basically humanized murine antibodies. Although humanization can reduce the immunogenicity of murine antibodies to a large extent, it is impossible to completely remove its immunogenicity, so it will inevitably lead to the appearance of neutralizing antibodies in clinical treatment and reduce its therapeutic value . Direct screening of high-affinity single-chain antibodies from human-derived single-chain antibody libraries constructed from prostate cancer patients will undoubtedly become an important means for the early diagnosis of prostate cancer and the research of guiding therapeutic drugs.

Contents of the invention

The purpose of the present invention is to screen a human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody using phage antibody library technology.

Another object of the present invention is the application of the above-mentioned single-chain antibody in the early diagnosis and guided treatment of prostate cancer. Using this specific human single-chain antibody can specifically detect the expression level of human prostate-specific membrane antigen (PSMA), as a standard for diagnosing prostate cancer; at the same time, using this human prostate-specific membrane antigen (PSMA) extracellular The region-specific human single-chain antibody is used as a biological carrier, combined with optimized anti-tumor drugs, targeted delivery to prostate cancer cells, targeted to kill cancer cells, and plays an important role in the treatment of prostate cancer.

The present invention uses phage surface display technology to extract mRNA from peripheral blood mononuclear cells of prostate cancer patients and amplify a full set of human antibody variable region genes, then assemble into single-chain antibody gene fragments and clone them into phagemid vectors and transform large intestine Bacillus constructs a large-capacity human single-chain antibody library. Then, the extracellular region of recombinant human prostate-specific membrane antigen (PSMA) was used to screen to obtain its specific high-affinity antibody. The antibody can be soluble expressed in Escherichia coli, and the amino acid sequence of the antibody is as follows:

L P V L T Q S P S A S A S L G A S V K L T C T L S S W H S S N A I

A W H Q L R P E K G L R Y L M K V N S D G S H N W G D G I P D R F

S G S S S G A E R Y L I I S S L Q S E D E A D Y Y C Q T W G T G I H

V V F G G G T K L T V L G G G G S G G G G S G G G G S E V Q L L Q

S G G G V V R P G R S L R L S C A A S G F T F S N Y A M H W V R

Q A P G K G L E W V A V M S Y D G N N K Y Y A D S V K G R F T I S

R D N S K N T L Y L Q M S S L R A G D T A V Y Y C A R D L D I A A

R F H Y C A M D V W G Q G T A V T V S S

The amino acid sequence is designated as SEQ ID NO: 1.

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody of the present invention has a heavy chain variable region gene encoding 124 amino acids, and the sequence is as follows:

E V Q L L Q S G G G V V R P G R S L R L S C A A S G F T F S N Y A

M H W V R Q A P G K G L E W V A V M S Y D G N N K Y Y A D S V K

G R F T I S R D N S K N T L Y L Q M S S L R A G D T A V Y Y C A R

D L D I A A R F H Y C A M D V W G Q G T A V T V S S

The amino acid sequence is designated as SEQ ID NO: 2.

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody of the present invention has a light chain variable region gene encoding 112 amino acids, the sequence of which is as follows:

L P V L T Q S P S A S A S L G A S V K L T C T L S S W H S S N A I

A W H Q L R P E K G L R Y L M K V N S D G S H N W G D G I P D R F

S G S S S G A E R Y L I I S S L Q S E D E A D Y Y C Q T W G T G I H

V V F G G G T K L T V L

The amino acid sequence is designated as SEQ ID NO: 3.

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody of the present invention consists of ×753 nucleotides, and its sequence is as follows:

CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG

CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT

GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA

ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT

TACCTCATCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC

TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG

CGGTGGCGGATCTGGCGGAGGTGGCTCCGGAGGTGGCGGTTCTGAGGTGCAGCTGC

TGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCA

GCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC

AAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGC

AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT

ATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGA

GATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGG

GACTGCGGTCACCGTCTCCTCA

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody heavy chain variable region of the present invention consists of 372 nucleotides, and its sequence is as follows:

GAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCG

CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATA

ATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC

AAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATA

TTACTGTGCGAGA GATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGT

CTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody heavy chain variable region of the present invention has a single open reading frame formed by rearrangement of GHV3-30, IGHD6-6 and IGHJ6 genes Functional variable region genes. The nucleotide sequence of CDR3 is GAT CTG GAT ATA GCA GCT CGT TTC, and the amino acid sequence is D L D I A A RF , which consists of IGHD6-6 and the unique sequence of the antibody.

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody light chain variable region of the present invention consists of 339 nucleotides, and its sequence is as follows:

CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG

CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT

GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA

ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT

TACCTCATCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC

TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG

C

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody light chain variable region of the present invention is formed by rearrangement of IGLV4-69 and IGLJ3 gene fragments and has a single open reading frame. variable region gene. The nucleotide sequence of CDR3 is CAG ACC TGG GGC ACT GGC ATT, and the amino acid sequence is: Q TW G T G I.

The humanized single-chain antibody against the extracellular region of human prostate-specific membrane antigen (PSMA) can be used in the development of prostate cancer diagnosis and therapeutic drugs.

The phage antibody library technology can directly obtain specific high-affinity antibody molecules without going through the hybridoma technology or even the immunization process. Single-chain antibodies have the advantages of small molecular weight, strong penetrability, and short half-life. They are ideal diagnostic reagents for tumor recurrence and distant metastasis, and can also provide guidance for guided drug therapy. However, after the screened single-chain antibody is transformed into a whole antibody, it will have a long half-life and can directly mediate the effect of complement and cellular immunity (ADCC) on killing tumors. Therefore, the human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody of the present invention has important value in the early diagnosis and guided treatment of prostate cancer.

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody of the present invention can be displayed and expressed on the surface of phage, and can also be soluble expressed in Escherichia coli. It was confirmed by protein electrophoresis and Western blot that the molecular weight of the soluble single-chain antibody expressed after IPTG induction was about 30kD. Analysis by ELISA and flow cytometry showed that the affinity of the antibody to the recombinant human prostate-specific membrane antigen (PSMA) extracellular region protein was Kd=0.2nM; the antibody could not only bind to the recombinant PSMA extracellular region, but also bind NEK293 cells expressing PSMA on their surface and the prostate cancer cell line LNCaP. This lays the foundation for the imaging diagnosis and guided treatment of prostate cancer using the antibody.

Description of drawings

Fig. 1 is the purification of human prostate-specific membrane antigen (PSMA) extracellular region-specific human single chain antibody of the present invention;

Lane 1: Marker; Lane 2: flow-through; Lane 3: first wash; lane 4: second wash; lane 5: purified single-chain antibody.

Fig. 2 is the determination of the affinity between the extracellular region-specific human single-chain antibody of human prostate-specific membrane antigen (PSMA) of the present invention and the extracellular region of recombinant human PSMA.

Fig. 3 is the identification of the natural PSMA binding activity of human prostate-specific membrane antigen (PSMA) extracellular region specific human single chain antibody of the present invention and cell surface expression;

A: NEK293 cells were transfected with anti-V5-APC only; B: NEK293 cells were transfected with the single-chain antibody of the present invention and anti-V5-APC; C: NEK293 cells transfected with PSMA were only stained with anti-V5-APC; D: NEK293 cells were transfected with PSMA The inventive single-chain antibody and anti-V5-APC were used to stain PSMA-transfected NEK293 cells; E: only anti-V5-APC was used to stain LNCaP cells; F: the inventive single-chain antibody and anti-V5-APC were used to stain LNCaP cells.

Detailed ways

The following is a detailed description of the embodiments of the human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody of the present invention.

Example

(1) Isolation of lymphocytes

A total of 500ml of peripheral blood was collected from 50 prostate cancer patients, added an equal volume of lymphocyte separation medium, and centrifuged to separate layers at 2500g for 10min. Carefully absorb the middle lymphocytes and centrifuge at 2500g for 10min. Discard the supernatant, and use 1ml Trizol/5ml peripheral blood to lyse the lymphocytes until the solution is completely transparent. Store at -80°C.

(2) Extraction of RNA

Thaw the lymphocyte lysate stored at -70°C to room temperature, add 0.2ml chloroform/ml Trizol, shake with Votex for 15s, let it stand at room temperature for 3min, 12000g, 5min. Aspirate the clear water phase of the upper layer, and be careful not to mix into the middle layer of protein and DNA. Add 0.5ml isopropanol/ml Trizol, mix upside down, precipitate at -20°C for 30-60min, and centrifuge at 12000g for 10min. Keep the precipitate, wash it once with 70% ethanol, air dry, and dissolve the precipitated RNA in DEPC water.

(3) Purification of mRNA

Purification steps are common techniques in the art, and are carried out according to the instructions of the kit (OMEGA, USA).

(4) Reverse transcription

Take 13 μg of mRNA and incubate at 70°C for 10 minutes to open the secondary structure, then quickly place it on ice, add each reagent in turn according to the instructions, and perform reverse transcription of random 6-mers. Leave at room temperature for 10 minutes to extend the primers, then carry out reverse transcription at 42°C for 60 minutes, and finally inactivate the reverse transcriptase at 95°C for 5 minutes, store in ice bath for 5 minutes, and store at -20°C.

(5) Full set of variable region gene amplification

The reverse transcription product was divided into three parts as templates for the first round of PCR, and the 3' end primers of VH, VL, and VK were mixed separately and then amplified with the 5' end primers respectively. The reaction conditions were: hot start at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 1 minute, a total of 35 cycles. Finally a further extension of 7min. The reaction volume is 100 μl. The product VH of the first round of PCR was mixed, VL and VK were mixed at a ratio of 1:2, purified and recovered by electrophoresis, and diluted 100 times as a template for the second round of PCR reaction for the second round of PCR reaction to introduce a longer protection sequence and Overlap extension sequence, the conditions are: hot start at 94°C for 5min, denaturation at 94°C for 1min, annealing at 55°C for 1min, extension at 72°C for 1min, a total of 25 cycles. The reaction volume is 100 μl.

(6) Random splicing and amplification of scFv (single-chain antibody) fragments

The VH, VL and VK gene fragments were separated and recovered by 2% agarose gel electrophoresis, and then mixed according to the ratio of VH:VK:VL:Llinker=3:2:1:3 for overlapping extension to assemble scFv fragments, The conditions were denaturation at 94°C for 1min, annealing at 55°C for 1min, and extension at 72°C for 1min, a total of 7 cycles. The overlapping extension product was diluted 1:100, and used as a template to amplify the scFv fragment using the protection sequences at the VL 5' end and VH 3' end as primers. After 10 cycles of annealing at ℃ for 1 min, and extension at 72 °C for 1 min, the annealing temperature was lowered to 58 °C for 15 cycles. The PCR product is the single-chain antibody gene fragment, which is recovered with 1.5% agarose gel.

(7) Ligation of scFv to phagemid vector and preparation of single-chain antibody library

1. Preparation of competent cells for electroporation

Pick the TG1 monoclonal clone from the M9 plate and shake it overnight, transfer it to 500ml LB at 1:100, shake for 2.5h, the _OD600 value is between 0.5-0.6, centrifuge to collect the bacteria, 3000rpm, 10min. Wash twice with an equal volume of ice-cold 1mM HEPES, 3000rpm, 10min, wash twice with 1/10 volume of ice-cold 10% glycerol, 4000rpm, 10min, resuspend the pellet with an equal volume of ice-cold 10% glycerol, each tube 50 μl was stored at -80°C. Note: Competent preparations were carried out at 0-4°C.

2. Preparation of single-chain antibody library

The above-mentioned purified single-chain antibody gene fragment was digested with Sfi I/Not I, and connected with the phagemid vector PCANTAB5E cut by the same enzyme. The ligation product was incubated at 70°C for 10 min to inactivate T4 DNA ligase, and then electrotransformed into Escherichia coli TG1. Spread the transformed bacteria on a 2YT plate containing 1% glucose and 100 mg/L ampicillin, culture overnight at 30°C, and scrape off the clones the next day with 2YT containing 15% glycerol and store them at -80°C.

3. Phage Surface Display of Antibody Libraries

Inoculate 3×10 ¹⁰ bacterial antibody library in 1500ml 2YTGA (2YT+1% glucose+amp), shake at 37°C until OD600=0.5. Take 150ml (the bacterial concentration of OD600=0.1 is 8×10 ⁷ ) to infect with helper phage M13K07 at a ratio of 20:1 at 37° C. in a water bath for 30 minutes. Then centrifuge at 3300g for 10min at room temperature, discard the supernatant, and transfer the bacterial pellets to 1500ml of 2YTGAK (2YT+1% glucose+amp+kan) preheated at 30°C, shake overnight at 30°C (should reach 12 hours) . The next day, centrifuge for 10 minutes (4°C), and collect the supernatant. Add 1/5 volume of PEG/NaCl (20% PEG6000, 2.5M NaCl) to the supernatant, mix well, and let stand at 4° C. for more than 1 hour to precipitate phage. Then centrifuge at 10000rpm at 4°C for 15min, collect the phage pellet and resuspend in 5ml PBS. Centrifuge again at 10,000 rpm for 15 minutes, take the supernatant (precipitated as residual bacterial fragments and PEG), and store at 4°C for screening. At the same time, 1 ul ratio dilution was taken for titer analysis.

(8) Determination of phage antibody library titer

Pick TG1 monoclonal clones from M9 medium in 5ml 2YT and shake overnight. Inoculate 0.5ml overnight bacteria into 50ml 2YT (100ml Erlenmeyer flask), shake at 37°C until OD600=0.2. The phage antibody library was diluted tenfold, and 10 μl from each dilution was added to 200 μl TG1 with OD600=0.2, mixed evenly, and bathed in 37°C water for 30 minutes. Spread the 200 μl mixture of bacteria and phage on the 2TY agar plate containing ampicillin. Place it upside down at 37°C, and calculate the titer of the phage library based on the number of clones the next day.

(9) Preparation of helper phage M13K07

Pick TG1 monoclonal clones from M9 medium in 5ml 2YT and shake overnight. Take 0.5ml overnight bacteria and inoculate into 50ml 2YT (100ml Erlenmeyer flask), shake at 37°C until OD600=0.2. Dilute M13K07 ten-fold, and add 10 μl from each dilution to 200 μl TG1 with OD600=0.2, mix well, and bathe in water at 37°C for 30 minutes. Add the mixture of 200 μl of bacteria and phage to 3ml of the upper layer of agar gel that is not hot to the touch, shake it a little, and immediately pour it on the agar plate prepared with 2TY preheated at 37°C. Place it upside down at 37°C, and plaques will appear the next day. Pick a single plaque and inoculate it in 3-4ml TG1 with OD600=0.5 (the preparation method is the same as before, that is, pick the clone from M9 the night before, shake the bacteria overnight, and transfer it to 50ml 2YT at 1:100 the next day, shake Shake for about 2-2.5 hours), shake at 37°C for 2 hours. Inoculate 3-4ml of TG1 infected with M13K07 into 500ml of 2YT and shake at 37°C for 1 hour. Add kana (50-70 μg/ml), continue to shake at 37°C for 8-16 hours, centrifuge at 10000rpm for 15min, and collect the supernatant. Add 1/5 volume of PEG/NaCl (20% PEG6000, 2.5M NaCl) to the supernatant, mix well, let stand at 4°C for more than 1 hour, centrifuge at 10,000 rpm for 15 min. Resuspend the phage with 20 ml of PBS containing 15% glycerol, centrifuge at 10,000 rpm for 15-20 min, and collect the supernatant (precipitated as bacterial debris and PEG). The phage solution was filtered and sterilized by a 0.45 μm filter membrane, then subpackaged, frozen at -80°C, and its titer was measured by plaque method.

(10) Screening of specific antibodies

Coat the 25cm culture flask with 5ml of 50ug/ml antigen (the first round), overnight at 4°C (the coating solution is 200mM sodium bicarbonate buffer, pH9.6), after the second round, the concentration of the coated antigen is reduced to 5ml 10ug/ml. After washing with PBST, add 5ml of 4% MPBS containing 1×10 ¹⁴ phage, shake lightly on a decolorizing shaker for 30 minutes, and then place horizontally at room temperature for more than 90 minutes for antigen-antibody binding. Then wash with PBS and PBST 20 times each, and finally add 10ml of TG1 that has grown to the logarithmic growth phase into the culture bottle, and let the phages bound to the antigen infect TG1 in a water bath at 30°C for 30 minutes. Infected TG1 was spread on a 15cm 2YTGA plate after centrifugation, overnight at 30°C. Phages were then prepared from the infected and amplified TG1 by superinfection with helper phages, and the second round of screening was performed. The screening process of "adsorption-elution-infection-propagation" was repeated for 4 rounds.

(11) Identification of Antigen Binding Activity of Monoclonal Phage Antibody

1. Preparation of monoclonal phage antibody

Select a single clone from the last round of screening plate in a 96-well culture plate equipped with 2YTGA, shake overnight at 37°C, transfer to new 2YTGA at a ratio of 1:100 the next day, shake at 37°C for 2.5 hours, and use M13K07 Perform superinfection at a ratio of 20:1, centrifuge at 3500 rpm for 10 min, resuspend the bacterial pellet with 200ul 2YTGAK, and shake overnight at 30°C. The next day, centrifuge at 11,000 rpm for 15 min, and take the supernatant for phage ELISA identification.

2. Phage ELISA identification of antigen-antibody binding activity

Coat the ELISA plate with PSMA at 4°C overnight, 50ul per well, the concentration is 1ug/ml. The next day, fill the wells with PBS containing 5% skimmed milk powder and block at room temperature for 2 hours, wash 5 times with PBST, dilute the antibody with PBS containing 2% skimmed milk powder, and place the diluted antibody at room temperature for 30 minutes. Wash the plate 6 times with PBST, then add 200ul of antibody, incubate at room temperature for 2 hours, wash the plate with PBST, 6 times. Add 200ul of HRP-labeled anti-M13 phage antibody, and the dilution ratio is 1:5000. Incubate at room temperature for 1 hour, wash the plate 8 times with PBST, and add TMB for color development.

(12) Expression of soluble single-chain antibody

Positive phages need to infect HB2151 for soluble expression. The phages of the positive clones were diluted 10 or 100 times and passed through a 0.45 filter membrane (the filter membrane step is to remove TG1 Escherichia coli mixed in the phage. TG1 has a growth advantage over HB2151, so it should be removed), and the diluted A good 10 μl was used to infect 200 μl of HB2151 in logarithmic growth phase. The method is as follows: the HB2151 monoclonal grown on the M9 medium was placed in 5ml 2YTG, transferred to 50ml 2YTG at 1:100 the next day, and shaken at 37°C until OD600=0.5. Add 10 μl of filtered and doubly diluted phage, mix well, and bathe in water at 37°C for 30 minutes. Spread 2YTG agar plates coated with netidone acid and ampicillin, overnight at 37°C. Pick a single clone in 5ml 2YTGA (containing 1% glucose), shake overnight at 37°C. The next day, transfer to 50ml 2YTGA containing 0.1% glucose at a ratio of 1:100, shake at 37°C until OD600=0.9 (about 3 hours and 40 minutes), add IPTG to a final concentration of 1 mM, shake overnight at 30°C, and take the supernatant The expression was analyzed by polyacrylamide gel electrophoresis, and the antigen-binding activity of the soluble expressed single-chain antibody was identified by ELISA.

(14) Purification of soluble single-chain antibody

Since the single-chain antibody is fused with 6 histidine tags, the soluble expression supernatant can be affinity purified with a nickel column. The specific steps are: equilibrate the affinity column with PBS, put the sample on the column, and wash with 50mM imidazole. To remove non-specific binding proteins, elute the single-chain antibody bound to the nickel column with 500 mM imidazole, remove imidazole with molecular sieves, and replace the buffer with PBS. (see picture 1)

(15) Affinity identification of soluble single chain antibody

Coat the ELISA plate with PSMA at two different concentrations of 1ug/ml and 0.5ug/ml, then add purified single-chain antibody diluted tenfold from 1ug/ml to 1pg/ml, and then add anti-V5-HRP Antibodies (scFv expressed as fusion with V5 tag), finally displayed with TMB. Draw the affinity curve according to the ELISA readings, and then calculate the antibody concentrations corresponding to 1ug/ml and 0.5ug/ml PSMA at OD50, which are respectively recorded as [Ab] (corresponding to 1ug/ml PSMA) and [Ab]' (corresponding to 0.5ug/ml PSMA) /ml PSMA), and then use the formula Kd=2[Ab]'-[Ab] to calculate the affinity of the single-chain antibody. (See Figure 2)

(16) Identification of binding activity of soluble antibody to natural PSMA expressed on cell surface

Since the conformation of recombinant PSMA may be different from the native conformation of PSMA expressed on the surface of prostate cancer cells, scFv with binding activity to recombinant PSMA may not be able to bind PSMA expressed on the cell surface. Therefore, in order to identify the binding activity of the single-chain antibody we screened to PSMA naturally expressed on the cell surface, we used flow cytometry to identify the binding activity of the antibody to NEK293 cells, NEK293 cells transfected with PSMA, and the prostate cancer cell line LNCaP A comparison was made. The method is to incubate the antibody with the above three kinds of cells for 1 hour, and the control group does not add the antibody, then stain with anti-V5-APC antibody, and finally compare the intensity of APC signal by flow cytometry. (See Figure 3A-F)

It should be noted that Figure 1 provided by the present invention is a picture collected under a gel imager, and Figures 3A-F are pictures automatically generated after detection by a flow cytometer, which are the clearest pictures for medical research.

The parts that are not described in detail in the above embodiments belong to the known technologies of this industry or related industries, and the equipment used is industry conventional equipment.

sequence listing

1. SEQ ID: 1.

sequence:

L P V L T Q S P S A S A S L G A S V K L T C T L S S W H S S N A I

A W H Q L R P E K G L R Y L M K V N S D G S H N W G D G I P D R F

S G S S S G A E R Y L I I S S L Q S E D E A D Y Y C Q T W G T G I H

V V F G G G T K L T V L G G G G S G G G G S G G G G S E V Q L L Q

S G G G V V R P G R S L R L S C A A S G F T F S N Y A M H W V R

Q A P G K G L E W V A V M S Y D G N N K Y Y A D S V K G R F T I S

R D N S K N T L Y L Q M S S L R A G D T A V Y Y C A R D L D I A A

R F H Y C A M D V W G Q G T A V T V S S

Composed of 753 nucleotides, its sequence is as follows:

CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG

CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT

GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA

ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT

TACCTCATCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC

TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG

CGGTGGCGGATCTGGCGGAGGTGGCTCCGGAGGTGGCGGTTCTGAGGTGCAGCTGC

TGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCA

GCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC

AAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGC

AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT

ATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGA

GATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGG

GACTGCGGTCACCGTCTCCTCA

2. SEQ ID NO: 2.

sequence:

E V Q L L Q S G G G V V R P G R S L R L S C A A S G F T F S N Y A

M H W V R Q A P G K G L E W V A V M S Y D G N N K Y Y A D S V K

G R F T I S R DN S K N T L Y L Q M S S L R A G D T A V Y Y C A R

D L D I A A R F H Y C A M D V W G Q G T A V T V S S

Composed of 372 nucleotides, its sequence is as follows:

GAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCG

CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATA

ATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC

AAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATA

TTACTGTGCGAGAGATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGT

CTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody heavy chain variable region of the present invention has a single open reading frame formed by rearrangement of GHV3-30, IGHD6-6 and IGHJ6 genes Functional variable region genes. The nucleotide sequence of CDR3 is GAT CTG GAT ATA GCA GCT CGT TTC, and the amino acid sequence is D L D I A A RF , which consists of IGHD6-6 and the unique sequence of the antibody.

3. SEQ ID NO: 3

sequence:

L P V L T Q S P S A S A S L G A S V K L T C T L S S W H S S N A I

A W H Q L R P E K G L R Y L M K V N S D G S H N W G D G I P D R F

S G S S S G A E R Y L I I S S L Q S E D E A D Y Y C Q T W G T G I H

V V F G G G T K L T V L

Composed of 339 nucleotides, its sequence is as follows:

CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG

CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT

GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA

ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT

TACCTCATCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC

TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG

C

The human prostate-specific membrane antigen (PSMA) extracellular region-specific human single-chain antibody light chain variable region of the present invention is formed by rearrangement of IGLV4-69 and IGLJ3 gene fragments and has a single open reading frame. variable region gene. The nucleotide sequence of CDR3 is CAG ACC TGG GGC ACT GGC ATT, and the amino acid sequence is: Q TW G T G I.

Claims (5)

1. A humanized single-chain antibody against the extracellular region of human prostate-specific membrane antigen, characterized in that: the amino acid sequence of the single-chain antibody is SEQ ID NO: 1 The amino acid sequence of the antibody is as follows: L P V L T Q S P S A S A S L G A S V K L T C T L S S W H S S N A I A W H Q L R P E K G L R Y L M K V N S D G S H N W G D G I P D R F S G S S S S G A E R Y L I I S S L Q S E D E A D Y Y C Q T W G T G I H V V V F G G G T K L T V L G G G G G S G G G G S G G G G G S E V Q L L Q S G G G G V V R P G R S L R L S C A A S G F T F S N Y A M H W V R Q A P G K G L E W V A V M S Y D G N N K Y Y A D S V K G R F T I S R D N S K N T L Y L Q M S S L R A G D T A V Y Y C A R D L D I A A A R F H Y C A M D V W G Q Q G T A V T V S S. 2. The humanized single-chain antibody against the extracellular region of human prostate specific membrane antigen according to claim 1, characterized in that: the humanized single-chain antibody consists of 753 nucleotides, and its sequence is as follows : CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACTGCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACAACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGTTACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGCGGTGGCGGATCTGGCGGAGGTGGCTCCGGAGGTGGCGGTTCTGAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGAGATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA。 3. The humanized single-chain antibody against the extracellular region of human prostate-specific membrane antigen according to claim 1, characterized in that: the humanized single-chain antibody specific for the extracellular region of human prostate-specific membrane antigen The heavy chain variable region consists of 372 nucleotides, and its sequence is as follows: GAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGAGATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA。 4. The humanized single-chain antibody against the extracellular region of human prostate-specific membrane antigen according to claim 1, characterized in that: the humanized single-chain antibody specific for the extracellular region of human prostate-specific membrane antigen The light chain variable region consists of 339 nucleotides, and its sequence is as follows: CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACTGCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACAACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGTTACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGC。 5. Use of the humanized single-chain antibody against the extracellular region of human prostate specific membrane antigen according to claim 1 in the preparation of a medicament for treating prostate cancer.

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