CN116023487B - Nanobody combined with various serum albumin and preparation method and application thereof - Google Patents

Nanobody combined with various serum albumin and preparation method and application thereof Download PDF

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CN116023487B
CN116023487B CN202210811567.1A CN202210811567A CN116023487B CN 116023487 B CN116023487 B CN 116023487B CN 202210811567 A CN202210811567 A CN 202210811567A CN 116023487 B CN116023487 B CN 116023487B
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serum albumin
nanobody
preparation
medicines
bsa
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CN116023487A (en
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张晓东
丁有斌
李志杰
徐小龙
赵善超
王继刚
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Third Affiliated Hospital Of Southern Medical University (academy Of Orthopaedics Guangdong Province)
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Abstract

The invention relates to the technical field of nanobodies, in particular to a nanobody combined with various serum albumin, and a preparation method and application thereof. The provided nano antibody Nb3 HAS higher binding force with human serum albumin (human serumalbumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA) at the same time, realizes slow release of the medicament, improves the solubility of the medicament, improves pharmacodynamics, and images or cooperatively treats diseases at the same time; the preparation method can increase aggregation of the medicine in target tissues, prolong half-life of various medicines, increase action time of the medicines, reduce dosage of the medicines, has a better broad-spectrum application range, and can be effectively applied to preparation of medicines for enhancing half-life of the medicines and preparation of tumor cell imaging reagents.

Description

Nanobody combined with various serum albumin and preparation method and application thereof
Technical Field
The invention relates to the technical field of nanobodies, in particular to a nanobody combined with various serum albumin, and a preparation method and application thereof.
Background
Whether a drug is effective depends on whether the drug can aggregate and act at the target site. The poor therapeutic effect and toxic and side effects of most of the tumor chemotherapeutic drugs in clinic at present are mainly due to the weak targeting property of the drugs and extremely short half-life period of the drugs, and the increase of half-life period and targeting property of the drugs are important points in the current drug development. Most of the current drugs are easily cleared by the kidney or liver during blood circulation due to their non-specific distribution and small molecular weight. Many scholars have used methods to extend the half-life of drugs, such as pegylation using polyethylene glycol (PEG) conjugation, and carriers such as liposomes, polymer nanoparticles, dendrimers, and solid lipid nanoparticles; however, these methods have not been able to avoid toxicity due to non-characteristic aggregation. Albumin is the most abundant plasma protein in plasma, with multiple binding sites and a circulatory half-life of about 19 days.
The solubility and in vivo stability of the drug can be improved by coupling with serum albumin or coupling with serum albumin binding protein, and the total molecular weight of the drug can be increased so as not to be filtered out quickly during blood circulation. Serum albumin is considered as a transport protein, which has a long half-life in vivo and is mainly related to a neonatal Fc receptor (neonatal Fc receptor, fcRn), which is a specific protein receptor expressed by various cells in vivo; fcRn is predominantly distributed in the endosome (Endosomes), and triggers specific binding of FcRn to intracellular serum albumin under conditions of endosomal acidification (endosomal acidification, < pH 6.5) and re-release of serum albumin to the outside of the cell under pH neutral conditions. Drug modification even with albumin antibodies has been used in large numbers by binding polypeptides, proteins with serum albumin, and by drug linking through the protein binding region of serum proteins. However, human serum albumin is difficult to obtain and has immunogenicity, so that the direct use of human serum albumin as a carrier for coupling drugs is limited.
The modification of the drug by the nanobody combined with serum albumin, especially the nanobody coupled drug, is a new technology. The tumor-targeted antibody is fused with the serum albumin-combined nano antibody, and then the cytotoxic drug is coupled, so that the half life period can be enhanced, and meanwhile, the accumulation of the drug on the target can be increased, thereby reducing the drug use amount and achieving a very good therapeutic effect.
Nanobody (Nb), also known as single domain antibody (single domain antibody, sdAb), is a new generation of miniantibodies derived from the single domain variable region (variable domain of the heavy chain antibody, VHH) of camel heavy chain antibodies; heavy chain antibodies (hcabs) lacking light chains naturally occur in blood of sharks and camels, and single domain antibodies (single domain antibody, sdabs) consisting of only one heavy chain variable region have been obtained by cloning their variable regions; such small molecule antibodies are the smallest unit currently available that has complete function and can bind antigen. Its relative molecular mass is about 15kd, its size is in the nanoscale range (length 4nm, diameter only 2.5 nm), and it is composed of about 120 amino acids, and is therefore called nanobody. Compared with the traditional antibody, the nano antibody has only 3 complementarity determining regions (complementarity determining region, CDR), but has better specific antigen binding capacity and high affinity, and is the minimum unit antibody with complete biological functions.
Nanobodies have many unique advantages not possessed by conventional antibodies: (1) Nanobodies are small in size and have the property of being prone to binding to concave epitopes, so that nanobodies have a higher tissue penetrating ability, and at the same time, nanobodies have a higher tissue penetrating ability, can rapidly penetrate tumor parenchyma and have a higher retention. The nano antibody can penetrate through the blood brain barrier, can overcome the defect that most medicaments cannot pass through the blood brain barrier, and can identify grooves, slits or hidden antigen epitopes on the target surface which are difficult to access by conventional antibodies; (2) VHH has a longer complementarity determining region (CDR 3) than conventional VH, and this long CDR3 structure is easy to form a convex loop structure, and can bind to a hidden epitope of an antigen molecule, thus compensating for the decrease in antigen binding ability of nanobodies due to light chain deletion; (3) The nanometer antibody is easy to carry out in vitro prokaryotic/eukaryotic cell expression and amplification in microbial systems such as bacteria, yeast, fungi and the like, so that mass production can be carried out at lower cost, (4) due to the filtration effect of kidneys, the half-life period of the nanometer antibody in blood is short, the nanometer antibody can be rapidly accumulated in tumors, and unbound parts can be rapidly cleared, so that the sensitivity and the specificity of tumor diagnosis are greatly improved; (5) Genes encoding camel-derived nanobodies have a high degree of homology with the human VH domain 3 (VH 3) and therefore are less immunogenic in humans; (6) Nanobodies comprise only one domain, which structure increases the stability of the nanobody, making it more suitable as a noninvasive molecular imaging agent and diagnostic tool; (7) The nano antibody has small molecular weight and good water solubility, can increase the solubility of the medicine, has better stability under the high temperature condition and better antigen binding activity, and is indicated to be easy to store and transport.
Because of the high homology of the serum protein genes of human, bovine and murine, the application of the serum albumin-bound nanobody to various animal models and human bodies can be expanded by screening any one of the serum albumin-bound nanobodies.
Therefore, there is an urgent need to develop new nanobodies that can bind to bovine serum albumin as well as both mouse and human serum albumin for enhancing half-life of various drugs.
Disclosure of Invention
The application aims to provide a nano antibody combined with various serum albumins, a preparation method and application thereof, and aims to solve the problem that the prior art lacks a half-life nano antibody which is combined with bovine serum albumin and simultaneously combined with mouse and human serum albumin and used for enhancing various medicines.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application discloses a nanobody that binds to a plurality of serum albumins, the nanobody that binds to a plurality of serum albumins comprises nanobody Nb3, and the amino acid sequence of nanobody Nb3 is shown as seq. Id No. 1.
In a second aspect, the application discloses the use of nanobodies bound to a plurality of serum albumins in the manufacture of a medicament for enhancing the half-life of the medicament.
In a third aspect, the application discloses the use of nanobodies bound to a plurality of serum albumin in the preparation of a reagent for tumor cell imaging.
The serum albumin binding nanobody provided by the first aspect of the application comprises a nanobody Nb3, the amino acid sequence of which is shown as seq.ID No.1, based on a phage natural nanobody library, three rounds of screening (Bovine serum albumin, BSA) proteins of bovine serum albumin are completed, ELISA verification is carried out, the nanobody Nb3 is obtained, and meanwhile, the binding force with human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA) is higher, so that the drug release is realized, the drug solubility is improved, the pharmacodynamics is improved, and meanwhile, imaging or synergistic treatment is carried out on diseases (including tumors); the preparation method can increase aggregation of the medicine in target tissues, prolong half-life of various medicines, increase action time of the medicines, reduce dosage of the medicines, has a better broad-spectrum application range, and can be effectively applied to preparation of medicines for enhancing half-life of the medicines and preparation of tumor cell imaging reagents.
The application of the serum albumin binding nanobody in preparing the medicament for enhancing the half-life of the medicament is characterized in that the obtained nanobody Nb3 not only HAS higher affinity to BSA, but also HAS better binding activity to human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA), so that the nanobody Nb3 is suitable for preparing the medicament for enhancing the half-life of the medicament.
The application of the serum albumin binding nanobody in preparing a reagent for tumor cell imaging is provided in the third aspect of the application, and the obtained nanobody Nb3 not only HAS higher affinity to BSA, but also HAS better binding activity to human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA), so that the nanobody Nb3 is suitable for preparing the reagent for tumor cell imaging.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Wherein:
fig. 1 is a screening analysis chart of serum albumin nanobodies provided in the examples of the present application.
Fig. 2 is a graph of a BSA-binding nanobody positive clone analysis provided in the examples of the present application.
Fig. 3 is a purification analysis chart of BSA nanobody provided in the examples of the present application.
Fig. 4 is a purification and identification chart of BSA nanobodies provided in the examples of the present application.
Fig. 5 is an ELISA binding validation analysis chart of BSA Nb3 nanobodies provided in the examples of the present application.
Fig. 6 is an ELISA assay of BSA Nb3 nanobodies provided in the examples of the present application.
FIG. 7 shows the affinity measurement of Nb3 nanobodies and serum albumin from three sources according to the examples of the present application.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The first aspect of the embodiment of the application discloses a nanobody combined with a plurality of serum albumin, wherein the nanobody combined with the plurality of serum albumin comprises a nanobody Nb3, and the amino acid sequence of the nanobody Nb3 is shown as a seq. ID No. 1.
The serum albumin binding nanobody provided in the first aspect of the application comprises nanobody Nb3, the amino acid sequence of which is shown as seq.id No.1, three rounds of screening (Bovine serum albumin, BSA) proteins of bovine serum albumin are completed based on a phage natural nanobody library, and the nanobody Nb3 is obtained through ELISA verification, and meanwhile, the binding force with human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA) is higher, so that drug slow release is realized, drug solubility is improved, pharmacodynamics is improved, and imaging or synergistic treatment is performed on diseases (including tumors) at the same time; the preparation method can increase aggregation of the medicine in target tissues, prolong half-life of various medicines, increase action time of the medicines, reduce dosage of the medicines, has a better broad-spectrum application range, and can be effectively applied to preparation of medicines for enhancing half-life of the medicines and preparation of tumor cell imaging reagents.
In some embodiments, nanobodies that bind to a variety of serum albumins include nanobody Nb3, whose amino acid sequence is shown as seq.id No.1, seq.id No.1 is specifically as follows:
MAAVQLVESGGGLVQAGGFLRLSCAVSGRTFDSYTMGWFRQAPGKEREFVAAISRSGTYTRYADSVKARFTISRDNAKNTVYLQMSSLKPEDTAVYYCNAQRRWLGRSYDYWGQGTQVTVSS。
in some embodiments, the nanobody Nb3 comprises 4 framework regions FR1, FR2, FR3, FR4, and 3 complementarity determining regions CDR1, CDR2, CDR3.
Wherein, the amino acid sequence of FR1 is shown as SEQ ID NO.2, and SEQ ID NO.2 is specifically as follows:
MAAVQLVESGGGLVQAGGFLRLSCAVSGRTF;
the amino acid sequence of FR2 is shown as SEQ ID NO.3, and SEQ ID NO.3 is specifically as follows:
WFRQAPGKEREFVAAI;
the amino acid sequence of FR3 is shown as SEQ ID NO.4, and SEQ ID NO.4 is specifically as follows:
ADSVKARFTISRDNAKNTVYLQMSSLKPEDTAVYYCNA;
the amino acid sequence of FR4 is shown as SEQ ID NO.5, and SEQ ID NO.5 is specifically as follows:
WGQGTQVTVSS;
the amino acid sequence of CDR1 is shown as SEQ ID NO.6, and SEQ ID NO.6 is specifically as follows:
DSYTMG;
the amino acid sequence of CDR2 is shown as SEQ ID NO.7, SEQ ID NO.7 is specifically as follows:
SRSGTYTRY;
the amino acid sequence of CDR3 is shown as SEQ ID NO.8, SEQ ID NO.8 is specifically as follows:
QRRWLGRSYDY。
in some embodiments, the nanobody Nb3 has a base sequence as set forth in SEQ ID No. 9. SEQ ID NO.9 is specifically as follows:
ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTTTTTGAGACTCTCCTGTGCAGTCTCTGGACGCACCTTCGATAGCTATACCATGGGCTGGTTCCGTCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCGATTAGCCGGAGTGGTACTTACACACGCTATGCAGACTCCGTGAAGGCCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACGGCCGTCTATTATTGTAATGCGCAGAGGCGGTGGCTTGGGAGATCGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
correspondingly, the nano-antibody combined with the serum albumin is prepared by the following preparation method of the nano-antibody combined with the serum albumin, and comprises the following steps of:
screening of BSA nanobodies
Screening natural alpaca-derived phage display nanobody library by adopting immune tube method, wherein the selected phage display library capacity is 2X10 9 . The screening steps are as follows: a) Coating target protein on an immune tube according to the concentration of 25ug/ml, and carrying out 3 rounds of enrichment screening; b) Performing PCR amplification using a third round of phage eluate; d) Randomly selecting a monoclonal ELISA for detection, and then performing positive clone ELISA secondary verification on the result; e) After screening, 5 clones were selected for sequencing, 4 normal and 1 no signal were sequenced.
Expression purification of BSA nanobody
The nanobody gene sequence was cloned into pcoldi vector while fusion expressing hemagglutinin tag (hemagglutinin HA tag) for subsequent detection. The expression purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16 ℃ using IPTG at a concentration of 0.2 mM; b) Performing a large amount of induced expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition 1230Pa of the high-pressure bacteria breaker; c) Centrifuging at 12000rpm and 4 ℃ for 30min, taking supernatant and incubating at 4 ℃ with Ni filler for 1 hour; g) Performing molecular sieve separation after Ni column purification, setting AKATA parameters at a flow rate of 0.5 mL/min, and collecting once every 1 mL; the nano antibody combined with various serum albumin is obtained.
The second aspect of the embodiment of the application discloses application of nano-antibodies combined with various serum albumins in preparation of drugs for enhancing half-life of drugs.
The application of the serum albumin binding nanobody provided in the second aspect of the embodiment of the invention in preparing the medicament for enhancing the half-life of the medicament is applicable to preparing the medicament for enhancing the half-life of the medicament because the obtained nanobody Nb3 HAS higher affinity to BSA and HAS better binding activity to human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA).
The third aspect of the embodiment of the application discloses application of nanobodies combined with various serum albumin in preparation of tumor cell imaging reagents.
The application of the serum albumin binding nanobody provided by the third aspect of the embodiment of the application in preparing a reagent for tumor cell imaging, because the obtained nanobody Nb3 HAS higher affinity for BSA and HAS better binding activity for human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA), the nanobody Nb3 is suitable for preparing the reagent for tumor cell imaging.
The following description is made with reference to specific embodiments.
Example 1
Serum albumin binding nanobody Nb3 and preparation method thereof
The test steps are as follows:
(1) Screening of BSA nanobodies
Screening a natural alpaca-derived phage display nanobody library by adopting an immune tube method, wherein the selected phage display library capacity is 2X109. The screening steps are as follows: a) Coating target protein on an immune tube according to the concentration of 25ug/ml, and carrying out 3 rounds of enrichment screening; b) Performing PCR amplification using a third round of phage eluate; d) Randomly selecting a monoclonal ELISA for detection, and then performing positive clone ELISA secondary verification on the result; e) After screening, 5 clones were selected for sequencing, 4 normal and 1 no signal were sequenced.
(2) Expression purification of BSA nanobodies
The nanobody gene sequence was cloned into pcoldi vector while fusion expressing hemagglutinin tag (hemagglutinin HA tag) for subsequent detection. The expression purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16 ℃ using IPTG at a concentration of 0.2 mM; b) Performing a large amount of induced expression according to the pre-experiment induction conditions, and performing bacteria breaking under the working condition 1230Pa of the high-pressure bacteria breaker; c) Centrifuging at 12000rpm and 4 ℃ for 30min, taking supernatant and incubating at 4 ℃ with Ni filler for 1 hour; g) Performing molecular sieve separation after Ni column purification, setting AKATA parameters at a flow rate of 0.5 mL/min, and collecting once every 1 mL; the nano antibody combined with various serum albumin is obtained.
(3) ELISA assay for nanobodies
The HA tag is fused into a coding sequence of a nano antibody gene, the nano antibody with the HA tag is expressed, ELISA plates are respectively coated with bovine, murine and human serum albumin, the temperature is closed by skimmed milk powder for 2 hours at 4 DEG overnight, then the nano antibody with gradient concentration is added for incubation for 1 hour at the room temperature, PBST is rinsed for 3 times, after the anti-HA antibody is incubated for 1 hour at the room temperature, the signal of the anti-HA antibody marked by horseradish peroxidase is amplified, TMB color development is carried out, and meanwhile, the control of the irrelevant nano antibody and the blank control of the irrelevant protein antigen are carried out.
(4) Surface plasmon resonance experiments (surface plasmon resonance, SPR)
This experiment was used to verify the direct interaction of the in vitro purified nanobody expressed in vitro with the in vitro purified antigen protein and to calculate the equilibrium constants of the two. Purified antigen proteins are fixed on a chip, nano antibodies with different concentrations are sequentially added to analyze the affinity with the antigen proteins, reaction signals within 360 seconds are recorded, a kinetic curve is made, and relevant parameters are calculated.
Analysis of results:
purification of BSA/HSA/MSA Domain and selection of nanobodies
The molecular weight of the three BSA/MSA/HSA is about 65kDa (figure 1A), and after three rounds of screening of the natural alpaca nanobody library by taking BSA as a target, phage titer results show that the library is enriched by more than 200 times, indicating that nanobodies combined with BSA are amplified (figure 1B).
2. Positive clone ELISA validation
Randomly 192 phage clones were picked and ELISA verified to find 5 potential positive clones (OD 450 value of target antigen was 2.5 times greater than that of control OD450 value), these 5 clones were sequenced and 4 normal, 1 Nb4 no signal was sequenced. According to the nano antibody sequencing result, a nano antibody sequence is obtained, 4 sequences are translated into amino acids and then are sequenced and subjected to multi-sequence comparison, so that 4 different nano antibody sequences are obtained.
3. Purification and identification of serum albumin
Gene cloning and protein expression analysis were performed on 4 nanobody sequences, nb5 did not obtain protein, and the remaining three nanobodies Nb1,2, 3SDS-PAGE showed that the nanobody was about 15kDa (FIG. 3A), and purified Nb1,2,3 nanobodies were subjected to immunoblotting examination of His-tag (FIG. 3B) and HA-tag (FIG. 3C), and the results showed that the nanobodies were correctly expressed, and in vitro ELISA affinity verification showed that the nanobody No.3 Nb3 was strongly bound to BSA, HSA and MSA at the same time, while the control antibody did not show binding activity to BSA/HSA/MSA (FIGS. 4A-4C).
Identification of Nb3 nanobodies and ELISA further binding validation
Since the Nb3 nanobody showed binding activity to three serum albumins, we identified again the Nb3 nanobody by immunoblotting experiments with SDS-PAGE gel and antibodies to HA-tag and His-tag, SDS-PAGE showed that Nb3 nanobody was approximately 15kDa (fig. 5A), and purified No.3 nanobody was subjected to immunoblotting examination of His-tag (fig. 5B) and HA-tag (fig. 5C), confirming that nanobody expression was correct; again, the Nb3 nanobody was confirmed to have binding activity to HSA/BSA/MSA by ELISA assay, while neither control antibody showed binding activity to HSA/BSA/MSA (fig. 6A-6C).
Affinity assay of Nb3 nanobodies and serum Albumin from three sources
The affinity constant of the nanobody Nb3 with serum albumin from three sources was further determined by SPR, and the binding affinity constants of Nb3 with HSA/MSA/BSA were 7.32nM,5.55nM and 8.53nM, respectively (FIGS. 7A-7C).
In conclusion, the nanobody Nb3 provided by the application can combine with human serum albumin (human serum albumin, HAS), bovine serum albumin (Bovine serum albumin, BSA) and mouse serum albumin (Mouse serum albumin, MSA) at the same time, so that the drug release is realized, the drug solubility is improved, the pharmacodynamics is improved, and meanwhile, the imaging or the cooperative treatment of diseases (including tumors) is carried out; the preparation method can increase aggregation of the medicine in target tissues, prolong half-life of various medicines, increase action time of the medicines, reduce dosage of the medicines, has a better broad-spectrum application range, and can be effectively applied to preparation of medicines for enhancing half-life of the medicines and preparation of tumor cell imaging reagents.
The foregoing disclosure is illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

Claims (4)

1. A nanobody that binds to a plurality of serum albumins, wherein the nanobody that binds to a plurality of serum albumins comprises nanobody Nb3, and wherein the amino acid sequence of nanobody Nb3 is as shown in seq.id No. 1.
2. The nanobody for binding with various serum albumins according to claim 1, wherein the base sequence of the nanobody Nb3 is shown in SEQ ID No. 9.
3. Use of the nanobody according to any one of claims 1-2 in combination with a plurality of serum albumin for the preparation of a medicament for enhancing half-life of the medicament.
4. Use of the nanobody according to any one of claims 1-2 in combination with a plurality of serum albumin for the preparation of a reagent for tumor cell imaging.
CN202210811567.1A 2022-07-11 2022-07-11 Nanobody combined with various serum albumin and preparation method and application thereof Active CN116023487B (en)

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CN101528767A (en) * 2006-10-11 2009-09-09 埃博灵克斯股份有限公司 Amino acid sequences that bind to serum proteins in a manner that is essentially independent of the ph, compounds comprising the same, and use thereof
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