CN108084267B - Antigen binding fragment-dolastatin conjugate of antibody, preparation method and application thereof - Google Patents
Antigen binding fragment-dolastatin conjugate of antibody, preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C07K2319/00—Fusion polypeptide
Abstract
The invention discloses an antigen binding fragment-dolastatin conjugate of an antibody, a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) providing an antigen-binding fragment of anti-CD 20 monoclonal antibody with LPXTG sequence at C terminal, and dolastatin or dolastatin derivative with oligoglycine linker; (2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the antigen binding fragment is coupled with the dolastatin or dolastatin derivative with the oligoglycine linker; (3) after the reaction is finished, the antigen binding fragment-dolastatin conjugate is obtained by separation and purification. The antigen binding fragment-dolastatin coupling drug retains the specificity and affinity of a full-anti-dolastatin coupling drug and has a killing effect on CD20 positive tumor cells; and has a faster rate of tumor penetration and a higher amount of tumor site aggregation.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to an antigen binding fragment-dolastatin conjugate of an antibody, a preparation method and application thereof.
Background
Antibodies are a class of protein drugs with relatively large molecular weight (-150 kDa) and are difficult to penetrate through the intravascular cortex and intercellular spaces to reach tumor cells deep in solid tumors, so few Antibodies reach tumor sites, and Sedlacek et al studies show that only 0.01% of the injected dose of Antibodies reaches tumor sites (Sedlacek H-H, et al, Antibodies as receptors of cytotoxicity, contacts to Oncology, 1992; 43:1-145), which greatly limits the application of total Antibodies in solid tumors. Full antibodies show their disadvantages in terms of both economic cost and safety, and miniaturization modification of full antibodies has become a trend.
The main types of research in the early research of miniaturized antibodies were single-chain ScFv and Fab, which retain the affinity and specificity of the full antibody, but also show the disadvantage of short half-life, and thus the anti-tumor effect is not ideal. In order to increase the anti-tumor effect of such miniaturized antibodies, on the one hand, attempts have been made to improve the pharmacokinetic properties of fusion protein fragments (PEG, Fc, HSA, etc.) (Zhang H, et al, Therapeutic potential of an anti-HER2single chain antibody-DM 1conjugates for the Therapeutic of HER 2-reactive cancer, Signal Transduction and Targeted Therapy,17015 (2017)); on the other hand, the coupling of small molecule toxin drugs is tried, the toxin small molecules are transported to the tumor part in a targeted mode by utilizing the specificity of the antibody, and the tumor cells are killed and killed specifically by the functions of inhibiting cell DNA or protein synthesis, inhibiting cell silking and the like of the toxic small molecules.
The longer half-life of antibodies is mainly due to The interaction of The constant region Fc fragment with FcRn on The cell surface, and thus Fc fragments are often used to engineer The pharmacokinetic properties of miniaturised antibodies (Natalea, The comfort of The engineered multi-potent antibodies, Biotech Week,2015,20(5): 589-. Dimitrov et al, showed a stronger affinity for FcRn for the mutated CH3mut than the wild-type CH3, another option for improving the half-life of proteins, by amino acid mutation engineering of a fragment of the constant region CH3 of IgG-type antibodies (YING T L, et al, Engineered solid monomer IgG1CH3Domain, Journal of Biological Chemistry,2013,288(35): 25154-25164).
The DAR uniformity of the antibody coupled drug obtained by the traditional chemical coupling method is poor, the evaluation of the in vivo metabolic property is difficult, and the heterogeneity of the antibody causes the treatment window to be narrow and the treatment effect to be poor. The sortaseA enzyme is a membrane protein expressed in Staphylococcus aureus, and can specifically recognize That a molecule/polypeptide with glycine at the amino terminal and a protein are added to the protein terminal through nucleophilic addition by an LPXTG polypeptide sequence (Ton-eat H, Purification and characterization of a sortase, the transpeptidase from surfaces proteins of Staphylococcus aureus, Proc Natl Acad Sci.1999,96: 12424-9.). The antibody with LPXTG at the carboxyl terminal can be constructed by utilizing the property, and the toxic micromolecule with glycine at the amino terminal is quantitatively coupled to the carboxyl terminal of the antibody at a fixed point by utilizing sortaseA enzyme.
Disclosure of Invention
The invention provides an antigen binding fragment-dolastatin conjugate formed by coupling a miniaturized antibody (only an antigen binding fragment of the antibody is reserved) of the CD20 antibody and dolastatin (MMAE) and a preparation method and application thereof, aiming at the defects of the prior art, so that the problem that the existing antibody conjugate drug has too large molecular weight and is difficult to permeate through the intravascular cortex and the intercellular space to reach tumor cells at the deep part of a solid tumor is solved.
The antibody-dolastatin coupling drug has the specific affinity effect of the antibody on a target and the killing effect of MMAE on cells, and can directionally transport the MMAE to tumor cells under the synergistic effect of the antibody and the MMAE to play the specific anti-tumor effect.
A method for preparing an antigen-binding fragment of an antibody-dolastatin conjugate, comprising the steps of:
(1) providing an antigen-binding fragment of anti-CD 20 monoclonal antibody with LPXTG sequence at C terminal, and dolastatin or dolastatin derivative with oligoglycine linker;
(2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the antigen binding fragment is coupled with the dolastatin or dolastatin derivative with the oligoglycine linker;
(3) after the reaction is finished, the antigen binding fragment-dolastatin conjugate is obtained by separation and purification.
The C-terminus of the heavy chain of the antigen-binding fragment is linked to an LPXTG sequence.
The heavy chain LPXTG sequence of the antigen-binding fragment is also followed by a histidine purification tag. The histidine purification tag can be a 6 XHis tag, peptide bond between threonine (T) and glycine (G) in the LPXTG sequence tag under the catalytic action of Sortase A enzyme forms covalent thio intermediate, and simultaneously releases glycine and carboxyl C terminal peptide segment thereof, so the histidine purification tag is already cut off in the final antigen binding fragment-dolastatin conjugate, and the effect of the conjugate is not influenced.
The heavy chain amino acid sequence of the antigen binding fragment is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.
The heavy chain amino acid sequence of the antigen binding fragment is shown as SEQ ID No.5, and the light chain amino acid sequence is shown as SEQ ID No. 2.
The oligoglycine joint is a GGG sequence, and a valine-citrulline dipeptide joint is arranged between the oligoglycine joint and the dolastatin or the dolastatin derivative. The valine-citrulline dipeptide linker can be cleaved by the lysosomal enzyme cathepsin B.
The invention also provides the antigen binding fragment-dolastatin conjugate prepared by the preparation method.
The invention also provides the application of the antigen binding fragment-dolastatin conjugate in preparing antitumor drugs.
The invention also provides an anti-tumor drug which comprises the antigen binding fragment-dolastatin conjugate.
The tumor was a CD20 positive B cell lymphoma.
The invention carries out miniaturization test through the monoclonal antibody resisting CD20, obtains the antigen binding fragment of the antibody and couples with the aplysiatoxin, obtains the antigen binding fragment-aplysiatoxin coupling medicament with good DAR homogeneity, retains the specificity and affinity of the full-anti-aplysiatoxin coupling medicament, and has killing effect on CD20 positive tumor cells; and has a faster rate of tumor penetration and a higher amount of aggregation at the tumor site, is a very effective form of antibody conjugate for the treatment of solid tumors.
Drawings
FIG. 1 is a UV spectrum of Fab-vcMMAE enzymatic coupling reaction.
FIG. 2 is the UV spectrum of the Fab-CH3mut-vcMMAE enzymatic coupling reaction.
FIG. 3 is a graph showing the results of affinity assay.
FIG. 4 is a graph showing the results of in vitro activity assays, wherein FIG. A, C, E uses cell Ramos, FIG. B, D, F uses cell Daudi, FIGS. A and B detect Fab and Fab-vcMAE, FIGS. C and D detect Fab-CH3mut and Fab-CH3 mut-vcMAE, and FIGS. E and F detect OFA and OFA-vcMAE.
FIG. 5 is a diagram of the result of apoptosis detection, in which diagram A is Fab, diagram B is Fab-CH3mut, diagram C is OFA, diagram D is Fab-vcMAE, diagram E is Fab-CH3 mut-vcMAE, diagram F is OFA-vcMAE, and diagram G is an indication diagram of diagrams A to F.
FIG. 6 is a graph showing the results of in vivo biodistribution imaging of Fab-cy5 and Fab-CH3mut-cy5 in nude mice.
FIG. 7 is a graph showing the results of in vivo anti-tumor experiments with Fab-vcMAE and Fab-CH3 mut-vcMAE.
Detailed Description
Example 1
The expression and purification of the SortaseA comprise:
(1) the gene sequence coding the sortaseA enzyme is obtained from the genome of staphylococcus aureus by PCR, and the His6 label is added at the N end of the gene sequence to be constructed in an expression vector PET28a and expressed by escherichia coli Rosetta strains 2(DE 3). The expression strain of sortaseA is inoculated into 1L LB culture medium with the final concentration of 1 percent and cultured in a shaker at 37 ℃ for about 6 hours at 180rpm, and when the OD value reaches about 0.6, 1mM IPTG is added for induced expression at 30 ℃ for about 12 hours.
(2) Centrifugally collecting bacterial liquid, dissolving the bacterial liquid in 150mM NaH2PO4300mM NaCl, wall broken with French press (Thermo), the supernatant collected by centrifugation, sterilized with a 0.45 μ M water membrane, purified with a HisTrap HP (5ml, GE) column, and the eluate concentrated by ultrafiltration with a 10kDa millipore ultrafiltration tube and replaced with 50mM tris-HCl, 150mM NaCl, pH7.4 buffer, and stored at-20 ℃.
Example 2
Fab or Fab-CH3mut monoclonal antibody.
The miniaturized anti-CD 20 antibody is modified on the basis of Ofatumumab (OFA) monoclonal antibody, and the OFA monoclonal antibody can specifically bind to CD20 antigen molecules, so that B lymphoma cells are killed specifically.
Fab represents an antigen binding fragment and comprises a heavy chain (H) and a light chain (L), wherein the amino acid sequence of the heavy chain is shown as SEQ ID No.1, the amino acid sequence of the light chain is shown as SEQ ID No.2, the coding gene sequence of the heavy chain is shown as SEQ ID No.3, and the coding gene sequence of the light chain is shown as SEQ ID No. 4. In the invention, Fab is further modified on the basis of an antigen binding fragment of the original OFA monoclonal antibody and is used for coupling with a drug, the modification mode is that a LPETGGHHHHHH polypeptide sequence is carried at the end of CH1 of a heavy chain, LPETG is a Sortase A enzyme recognition sequence, and a 6 × His tag is used for protein purification.
Fab-CH3mut is shown on the basis of antigen binding fragment of original OFA monoclonal antibody, CH3 fragment (named as CH3mut) with six amino acid mutations (P243C, L251S, T266R, L268H, P295K and A331C) is connected to the end of heavy chain CH1 through (GGGGS)3 linker, and then polypeptide sequence of LPETGGHHHHHH is connected to the end of CH3mut, heavy chain amino acid sequence of the modified Fab-CH3mut is shown as SEQ ID No.5, light chain amino acid sequence is shown as SEQ ID No.2, heavy chain coding gene sequence is shown as SEQ ID No.6, and light chain coding gene sequence is shown as SEQ ID No. 4.
The light chain and heavy chain coding gene sequences of the antigen binding fragment of the OFA monoclonal antibody are synthesized by the generation of the biological engineering (Shanghai) GmbH (Shanghai) corporation (synthesized by the inventor in the early stage experiment, see the Chinese invention patent with the application number of ZL201310046396.9 for details), and then the transformation is completed by a PCR method. Then, the pMH3 expression vectors were ligated to each other and transiently transferred into human embryonic kidney cells (293F), and the antibody was purified using His Trap HP affinity column, and Fab or Fab-CH3mut monoclonal antibody was obtained.
Example 3
Preparation of Fab-vcMMAE and Fab-CH3mut-vcMMAE conjugates. The coupling reaction is carried out on the principle that sortaseA enzyme attacks the LPETG fragment at the tail end of an antibody, specifically cuts off amino acid residues T and G, GGG-vcMMAE is subjected to affinity addition to the tail end of the LPET to form a peptide bond through dehydration condensation, and finally the Fab-vcMMAE and Fab-CH3mut-vcMMAE conjugates are formed.
The preparation method of the Fab-vcMAE and Fab-CH3 mut-vcMAE conjugate comprises the following steps:
(1) mu.M Fab or Fab-CH3mut monoclonal antibody (dissolved in 50mM Tris-HCl, 150mM NaCl, pH7.4), 300. mu.M GGG-vcMMAE (synthesized by Nanjing Binning Biotechnology Ltd., GGG-vcMMAE in which GGG represents three glycine residues, vc represents a dipeptide linker (valine-citrulline) cleavable by the lysosomal enzyme cathepsin B, MMAE is a highly toxic small molecule drug, 50 μ M sortaseA, 5mM CaCl, were added to a 10ml reaction system2And reacting in water bath at 37 ℃ for 12 h.
(2) The reaction solution was purified using protein L (1ml, GE) column, and the eluate was eluted to give an antibody conjugate, which was adjusted to pH 7.0 with 1M Tris, concentrated by ultrafiltration using a 10kDa millipore ultrafiltration tube and replaced with PBS buffer, and stored at-20 ℃ for further use.
(3) The Fab-vcMMAE coupling efficiency was analyzed by HPLC, as shown in FIG. 1 for the light (L) and heavy (H) chains of Fab, and in addition for the peaks of sortaseA (Srt) and GGG-vcMMAE (M). The delay in time of peak appearance after heavy chain conjugation to GGG-vcMMAE was labeled H1 (indicating that a MMAE was attached to the heavy chain), and the efficiency of enzymatic conjugation could reach around 85% from peak area analysis. The same FIG. 2 shows the ultraviolet spectrum of the coupling efficiency of Fab-CH3mut-vcMMAE, and the analysis result shows that the coupling efficiency can reach about 85%.
Example 4
Flow cytometry was used to detect the affinity of Fab-vcMAE and Fab-CH3 mut-vcMAE conjugates.
(1) Collecting 1X 106Each Daudi cell (positive for CD 20) was incubated with 10. mu.g/ml of Fab-vcMAE, Fab-CH3 mut-vcMAE and OFA-vcMAE at 4 ℃ for 30 min.
(2) After three PBS washes, 200. mu.l of anti-human murine mAb Fab (1: 1500 dilution) was added to each tube and incubated at 4 ℃ for 30 min.
(3) After three PBS washes, 200. mu.l of FITC-labeled goat anti-mouse IgG (H + L) (1: 250 dilution) was added to each tube, and after incubation at 4 ℃ for 30min, the tubes were washed 3 times with PBS and examined by flow cytometry.
(4) The affinity of the antibody conjugate to CD20 positive cells as detected by flow cytometry is mainly indicated by the mean fluorescence intensity of FITC after secondary antibody labeling.
(5) As shown in FIG. 3, Fab-vcMAE and Fab-CH3 mut-vcMAE showed a slight decrease in affinity compared to the all-anti-OFA-vcMAE but substantially retained the affinity of the all-anti.
Example 5
In vitro activity assays for Fab-vcMMAE and Fab-CH3mut-vcMMAE conjugates.
(1) The CD20 positive cells Ramos and Daudi were collected, counted and plated with RPMI-1640 (10% FBS) medium at 3000 cells/well and 100 μ Ι/well, respectively;
(2) adding 100 μ l/well of Fab, Fab-CH3mut, Fab-vcMAE and Fab-CH3 mut-vcMAE dilutions in triplicate at 37 deg.C and 5% CO2Culturing in an incubator for 4 days;
(3) 50 μ l of 25% CCK (diluted in blank RPMI-1640 medium), 37 ℃ 5% CO was added to each well2Culturing for 2-4 h in an incubator, and detecting the OD value of 450nm by using an enzyme-labeling instrument. Calculating IC50 values for the antibody conjugates using Graphpadprism software;
(4) the results in FIG. 4 show that Fab-vcMMAE and Fab-CH3mut-vcMMAE have significantly improved killing ability against CD20 positive cells compared with Fab and Fab-CH3mut, wherein IC50 of Fab-vcMMAE and OFA-vcMMAE are not much different, and IC50 of Fab-CH3mut-vcMMAE is about 5-10 times lower than that of Fab-vcMMAE and OFA-vcMMAE.
Example 6
Apoptosis assay.
Ramos cells were collected at 1X 105cell/ml amount was plated in six well plates, incubated 20nM Fab, Fab-CH3mut, OFA, Fab-vcMAE, Fab-CH3 mut-vcMAE and OFA-vcMAE for a total of 48h, centrifuged to remove medium, washed with PBS, resuspended with 100. mu.l of binding solution containing 5. mu.l of annexin V and 5. mu.l of PI, incubated at room temperature for 15min, supplemented with 400. mu.l of binding solution, and the percent apoptosis was detected by flow cytometry.
From the results of FIG. 5, it can be seen that Fab, Fab-CH3mut and OFA have weak killing effect on the cells, and the apoptosis rates of the Fab, Fab-CH3mut and OFA are substantially consistent with those of the blank group, but the phenomena of inducing apoptosis by Fab-vcMAE and Fab-CH3 mut-vcMAE are very obvious, and the early regulation/late regulation is 24.2%/23.7%, 15.4%/20.9% and 21.8%/30.4% respectively, which are not different from each other.
Example 7
In vivo biodistribution assay.
(1) Fab/Fab-CH3mut/OFA Small molecule GGG- (PEG)3-N3 (GPN for short, synthesized by Nanjing Linning Biotechnology Co., Ltd.) was coupled according to the reaction system in example 3; protein L purification conjugate Fab-GPN/Fab-CH3 mut-GNP/OFA-GNP;
(2) Fab-GPN/Fab-CH3mut-GPN/OFA-GPN is reacted with equal molar DBCO-Cy5(lumiprobe, USA) in PBS buffer solution at room temperature overnight, ultrafiltered and concentrated by 10kDa Millipore ultrafilter tube in dark place, and replaced by PBS buffer solution to obtain Fab-Cy5/Fab-CH3mut-Cy5/OFA-Cy5, which is stored at-20 ℃ for later use.
(3) Culturing in vitro CD20 positive cells Ramos, centrifuging, collecting, washing twice with PBS, counting, resuspending with a certain volume of PBS to 7 × 107cell/ml cell suspension;
(4) the cell suspension was prepared in 7X 10 portions6The amount of the cell is inoculated on the left axillary skin of a nude mouse (Balb/Cnud) until the average tumor size in the nude mouse is 1000mm33 nude mice were administered 7nM Fab-cy5, Fab-CH3mut-cy5, and OFA-cy5 tail vein respectively, and the distribution of antibody drug in nude mice was observed at 1.5h, 4h, 8h, 12h, 24h, and 48h with maestro in vivo imaging system instrument;
(5) as shown in FIG. 6, Fab-cy5 showed significant aggregation at the tumor site at 4h, whereas Fab-CH3mut-cy5 showed significant aggregation at about 8h, OFA-cy5 showed significant aggregation at 24h, indicating that the rate of entry into the tumor site was Fab > Fab-CH3mut > OFA; from the aspect of tumor aggregation, Fab-CH3-cy5 has obvious aggregation at 8h but slightly reduces the aggregation with the time, Fab-cy5 and OFA-cy5 respectively reach the maximum aggregation at 12h and 24h, but the aggregation is not reduced with the time, so that the Fab has the advantages of both OFA and Fab-CH3mut, has a faster tumor penetrating rate and can keep higher aggregation for a longer time.
Example 8
In vivo antitumor activity test.
(1) Culturing in vitro CD20 positive cells Ramos, centrifuging, collecting, washing twice with PBS, counting, resuspending with a certain volume of PBS to 7 × 107cell/ml cell suspension;
(2) the cell suspension was prepared in 7X 10 portions6The amount of the cell is inoculated on the left axillary skin of a nude mouse (Balb/C nud) until the average tumor size in the nude mouse is 100mm3Then, 54 nude mice were divided into 9 groups of which two control groups, two PBS groups, HER2-Fab-vcMMAE (110 nM/kg); seven groups of experimental groups: fab group (220 nM)/kg), Fab-CH3mut (220nM/kg), Fab-vcMAE (220nM/kg high, 110nM/kg medium, 55nM/kg low) Fab-CH3 mut-vcMAE (110nM/kg), OFA-vcMAE (220nM/kg), 6 per group. The tail vein was administered once every three days for a total of four times. The nude mice were observed and measured for body weight and tumor volume after drug withdrawal and data were recorded.
(3) As a result, as shown in FIG. 7, tumors of 6 nude mice were completely disappeared after four times of administration of OFA-vcMAE of the positive control group. 6 out of 4 in the Fab-vcMAE high dose group the dosing was complete (-100 mm)3) The postero-tumor mass disappears, with 2 of the initial tumors being larger (-150 mm)3) The tumor was still growing slowly after the end of the dosing. The tumor inhibition effect of the Fab-vcMAE group and the Fab-CH3 mut-vcMAE group with medium dosage is poor, compared with the PBS and HER 2-vcMAE of the negative control group, the tumor is continuously increased, but the increase speed is obviously lower than that of the negative group, wherein the tumor inhibition effect of the Fab-vcMAE 110nM/kg is better than that of the Fab-CH3 mut-vcMAE 110nM/kg, and the p-0.0488 is analyzed by t-test<0.05 had a significant difference. The Fab-vcMAE is proved to be superior to the Fab-CH3 mut-vcMAE in the tumor treatment, and the Fab-vcMAE retains the high-efficiency anti-tumor activity of the full anti-OFA-vcMAE.
Sequence listing
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35 40 45
Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
225 230 235 240
Gly Gln Cys Arg Glu Pro Gln Val Tyr Thr Ser Pro Pro Ser Arg Glu
245 250 255
Glu Met Thr Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe
260 265 270
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
275 280 285
Asn Asn Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe
290 295 300
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
305 310 315 320
Asn Val Phe Ser Cys Ser Val Met His Glu Cys Leu His Asn His Tyr
325 330 335
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Ser Leu Pro
340 345 350
Glu Thr Gly Leu Glu His His His His His His
355 360
<210> 6
<211> 1092
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gaagtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttaat gattatgcca tgcactgggt ccggcaagct 120
ccagggaagg gcctggagtg ggtctcaact attagttgga atagtggttc cataggctat 180
gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa gtccctgtat 240
ctgcaaatga acagtctgag agctgaggac acggccttgt attactgtgc aaaagatata 300
cagtacggca actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360
tcctcagcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420
tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480
gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540
tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660
gagcccaaat cttgtggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcg 720
gggcagtgcc gagaaccaca ggtgtacacc tcgcccccat cccgggagga gatgaccaag 780
aaccaggtca gcctgcgctg ccatgtcaaa ggcttctatc ccagcgacat cgccgtggag 840
tgggagagca atgggcagcc ggagaacaac tacaagacca cgaagcccgt gctggactcc 900
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960
aacgtcttct catgctccgt gatgcatgag tgtctgcaca accactacac gcagaagagc 1020
ctctccctgt ctccgggtaa aggtgggtct ctgccggaga ctggtctgga acatcaccac 1080
catcaccatt aa 1092
<210> 7
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Leu Pro Glu Thr Gly
1 5
<210> 8
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Leu Pro Glu Thr Gly Gly His His His His His His
1 5 10
<210> 9
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 10
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Gly Gly
1
Claims (5)
1. A method for preparing an antigen-binding fragment of an antibody-dolastatin conjugate, comprising the steps of:
(1) providing antigen-binding fragments of anti-CD 20 monoclonal antibody with LPXTG sequence at C terminal, and dolastatin with oligoglycine linker;
(2) under the catalysis of Sortase A enzyme, the LPXTG sequence and the oligoglycine linker generate transpeptidation reaction, so that the antigen binding fragment is coupled with the dolastatin with the oligoglycine linker;
(3) after the reaction is finished, separating and purifying to obtain the antigen binding fragment-dolastatin conjugate,
the C end of the heavy chain of the antigen binding fragment is connected with LPXTG sequence,
the heavy chain LPXTG sequence of the antigen binding fragment is connected with a histidine purification tag,
the heavy chain amino acid sequence of the antigen binding fragment is shown as SEQ ID No.1, the light chain amino acid sequence is shown as SEQ ID No.2, the antigen binding fragment is assembled into an Fab structure by a heavy chain and a light chain,
the oligoglycine joint is a GGG sequence, and a valine-citrulline dipeptide joint is arranged between the oligoglycine joint and the dolastatin.
2. An antigen-binding fragment-dolastatin conjugate prepared according to the method of claim 1.
3. Use of the antigen-binding fragment-dolastatin conjugate of claim 2 in the preparation of an anti-tumor medicament.
4. An anti-tumor drug comprising the antigen-binding fragment-dolastatin conjugate of claim 2.
5. The anti-neoplastic drug of claim 4, wherein said tumor is a CD20 positive B cell lymphoma.
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| CN109836502B (en) * | 2018-11-12 | 2021-09-07 | 浙江大学 | Bispecific antibody and application thereof |
| CN110317275B (en) * | 2019-05-27 | 2021-03-30 | 浙江大学 | Recombinant antibody-like T cell antigen receptor, T cell antigen receptor coupled drug, bispecific molecule and application |
| CN114555117A (en) * | 2019-10-12 | 2022-05-27 | 百奥泰生物制药股份有限公司 | anti-CD 20 antibody preparation and application of anti-CD 20 antibody in treatment of CD20 positive diseases |
| CN112675126A (en) * | 2019-10-18 | 2021-04-20 | 百奥泰生物制药股份有限公司 | anti-CD 20 antibody preparation and application thereof |
| CN111848816B (en) * | 2020-07-24 | 2021-06-08 | 斯普迈(北京)生物科技有限公司 | Fusion proteins and uses thereof |
| CN113398242B (en) * | 2021-06-04 | 2022-04-29 | 浙江大学 | Antibody coupling medicine and application thereof |
| WO2023143319A1 (en) * | 2022-01-28 | 2023-08-03 | 启德医药科技（苏州）有限公司 | Antibody-drug conjugate, and pharmaceutical composition and use thereof |
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| CN106237341B (en) * | 2016-07-12 | 2019-07-30 | 浙江大学 | A kind of antibody coupling drug and its preparation method and application |
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