CN114621354A - Fab-albumin binding peptide fusion protein and preparation method and application thereof - Google Patents

Fab-albumin binding peptide fusion protein and preparation method and application thereof Download PDF

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CN114621354A
CN114621354A CN202111511772.8A CN202111511772A CN114621354A CN 114621354 A CN114621354 A CN 114621354A CN 202111511772 A CN202111511772 A CN 202111511772A CN 114621354 A CN114621354 A CN 114621354A
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fab
ser
binding peptide
albumin binding
ala
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冯军
张庆彬
张喜全
吴勇
赵文杰
徐宏江
黄宗庆
于敏
杜萱
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Shanghai Duomirui Biological Technology Co ltd
Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Shanghai Duomirui Biological Technology Co ltd
Shanghai Institute of Pharmaceutical Industry
Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Abstract

The invention relates to a Fab-albumin binding peptide fusion protein, a preparation method and application thereof, wherein the Fab-albumin binding peptide fusion protein comprises a Fab fragment binding TNF-alpha, an albumin binding peptide (ABD) and a connecting peptide segment. Wherein the albumin binding peptide is linked to the C-terminus of the Fab fragment by a linker peptide segment. The Fab-albumin binding peptide fusion protein not only retains the antigen specific binding capacity of a Fab fragment, but also has the capacity of binding albumin, and can be specifically bound with plasma albumin in vivo to obviously prolong the self half-life. The Fab-albumin binding peptide fusion protein can be efficiently expressed in prokaryotic cells, and the purification process is simple and efficient.

Description

Fab-albumin binding peptide fusion protein and preparation method and application thereof
Technical Field
The invention relates to the technical field of molecular biology, in particular to the field of development of long-acting biological drugs, and specifically relates to a Fab-albumin binding peptide fusion protein and a preparation method and application thereof.
Background
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine with wide biological activity and generated by multinucleated giant cells, and plays an important role in immune response regulation, T cell-mediated tissue injury, occurrence and development of chronic inflammation and the like. Normal levels of TNF- α can enhance the body's ability to fight viruses, bacteria and parasites and mediate apoptosis of tumor cells. However, it has been shown that TNF- α secreted in the body in excess can cause immune dysfunction, promote the production of various inflammatory factors, and finally cause various pathological injuries to the body through inflammatory cascade, such AS Rheumatoid Arthritis (RA), Crohn's Disease (CD), and Ankylosing Spondylitis (AS).
Research has found that TNF-alpha is a proinflammatory cytokine which is the central position in the pathogenesis of inflammatory diseases such as RA and CD, and the like, so the chronic inflammatory diseases can be effectively treated by blocking a signal transduction pathway of the TNF-alpha and inhibiting the biological activity of the TNF-alpha. A variety of TNF-alpha inhibitors, including golimumab, have been successfully marketed and have been shown to have good clinical therapeutic efficacy
Figure BDA0003395590210000011
adalimumab
Figure BDA0003395590210000012
infliximab
Figure BDA0003395590210000013
certolizumab pegol
Figure BDA0003395590210000014
And etanercept
Figure BDA0003395590210000015
Wherein the golimumab, adalimumab and infliximab are full-length monoclonal antibodies, certolizumab pegol is a PEG-modified antibody Fab fragment, and etanercept is a fusion protein of TNFR2 (tumor necrosis factor receptor 2) and hIgG1 Fc fragment.
The full-length monoclonal antibody has large molecular weight, needs mammalian cells for expression, has long fermentation period and high purification and quality control difficulty, so the production cost is high, the price is high, and heavy economic burden is brought to the treatment of patients. The miniaturized antibody prepared by the genetic engineering method, such as an antibody Fab fragment, becomes a new research and development hotspot in the field of monoclonal antibody medicines due to the small molecular weight, structural function integrity and easy expression. The molecular weight of Fab is one third of that of full-length antibody, the smaller molecular weight makes it suitable for prokaryotic expression system, the cost of fermentation expression is greatly reduced, and it contains no Fc segment and does not produce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In addition, the Fab fragment has strong in-vivo penetrability and is easy to permeate into inflammatory tissues to play a role, so that the Fab fragment medicine targeting TNF-alpha has a great development prospect in the field of treatment of inflammatory diseases such as RA.
However, the Fab fragment does not contain an Fc segment, cannot participate in an in vivo circulating transport pathway mediated by FcRn (neonatal Fc receptor), has a small molecular weight, is easily cleared by glomerulus filtration, and has a fast in vivo clearing rate, a half-life period in a human body is only 16-20 h, and the short half-life period greatly limits the clinical application of the Fab fragment. In order to apply Fab fragment to the field of inflammatory disease treatment, UCB pharmaceutical company and Nektar company in Belgium collaboratively developed, PEG-MAL (40 KDa) is site-specific modified to free hinge region of heavy chain of Fab fragment of human anti-TNF-alphaCysteine, thereby producing a PEG-modified Fab fragment (Certolizumab PEG), not only reducing immunogenicity, but also increasing its in vivo half-life to 14 d (Bourn T, Fossati G, Nesbit A.A PEGylated Fab' fragment obtained from the patient tumor tissue for the treatment of Crohn disease: expanding a new mechanism of action [ J].Biodrugs Clinical Immunotherapeutics Biopharmaceuticals&Gene Therapy,2008,22(5): 331-. Another report on Adalilimumab Fab fragments (mutSS Fab)SH) The carboxyl Terminal of the heavy chain is modified by site-directed PEG-MAL (20 KDa), and the half-life of the PEG-modified Fab product in rats is prolonged by 28 times compared with that of the unmodified Fab fragment (Nakamura H, Antaku M, Odaueda N, et al, C-Terminal Cysteine PEGylation of Adalamumab Fab with an Engineered Interchain SS Bond [ J].Biological&Pharmaceutical Bulletin,2020,43(3): 418-423). However, the long-acting strategy based on the PEG modification technology has the problems of complex modification process, difficult separation of modified products, reduced activity of modified proteins and the like.
Disclosure of Invention
The invention provides a Fab-albumin binding peptide fusion protein, a preparation method and application thereof.
Therefore, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a Fab-albumin binding peptide fusion protein comprising an albumin binding peptide and a Fab fragment that binds TNF- α, the albumin binding peptide being linked to the C-terminus of the Fab fragment.
In the Fab-albumin binding peptide fusion protein, the N-terminus or C-terminus of the albumin binding peptide can be linked to the C-terminus of the Fab fragment, wherein the linkage can be direct or via a linker peptide segment. In some embodiments, the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment and/or the light chain of the Fab fragment, either directly or through a linking peptide segment.
Further, in some embodiments, the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment, either directly or via a linker peptide segment. Preferably, in some embodiments, the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment via the linker peptide segment.
In other embodiments, the N-terminus of the albumin binding peptide is linked to the C-terminus of the light chain of the Fab fragment, either directly or through a linker peptide segment. In other embodiments, the N-terminus of the albumin binding peptide is linked to the C-terminus of the light chain of the Fab fragment and the C-terminus of the heavy chain of the Fab fragment, either directly or through a linking peptide segment.
The Fab fragment may be murine, chimeric, humanized or fully human.
In some embodiments, the Fab fragment comprises CDRs selected from the group consisting of:
a) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of infliximab;
b) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of adalimumab;
c) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of golimumab; or
d) The amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of trastuzumab.
In a preferred embodiment, the Fab fragment comprises the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of adalimumab.
In some embodiments, the Fab fragment comprises the amino acid sequence of the light chain variable region and the heavy chain variable region of infliximab, the amino acid sequence of the light chain variable region and the heavy chain variable region of adalimumab, the amino acid sequence of the light chain variable region and the heavy chain variable region of golimumab, or the amino acid sequence of the light chain variable region and the heavy chain variable region of certolizumab ozogamicin. In a preferred embodiment, the Fab fragment comprises the amino acid sequence of the light chain variable region and the heavy chain variable region of adalimumab.
In some embodiments, the Fab fragment is a Fab fragment of infliximab, a Fab fragment of adalimumab, a Fab fragment of golimumab, or a Fab fragment of certolizumab, preferably a Fab fragment of adalimumab.
Infliximab (infliximab), trade name
Figure BDA0003395590210000021
The amino acid sequences of the light chain CDR1-3 are respectively shown as SEQ ID NO:24-26 in the patent application WO2017102835, and the amino acid sequences of the heavy chain CDR1-3 are respectively shown as SEQ ID NO:27-29 in the patent application WO 2017102835; the amino acid sequences of the light chain variable region and the heavy chain variable region are shown as SEQ ID NO 40 and 41 in WO2017102835, respectively.
Golimumab (golimumab), trade name
Figure BDA0003395590210000022
The amino acid sequences of the light chain CDR1-3 are respectively shown as SEQ ID NO:30-32 in the patent application WO2017102835, and the amino acid sequences of the heavy chain CDR1-3 are respectively shown as SEQ ID NO:33-35 in the patent application WO 2017102835; the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NO:42 and 43 in WO2017102835, respectively.
Cetuzumab pegol (certolizumab pegol), trade name
Figure BDA0003395590210000023
The amino acid sequences of the light chain CDR1-3 are respectively shown as SEQ ID NO:18-20 in the patent application WO2017102835, and the amino acid sequences of the heavy chain CDR1-3 are respectively shown as SEQ ID NO:21-23 in the patent application WO 2017102835; the amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NO:38 and 39, respectively, in WO 2017102835.
Adalimumab (adalimumab), trade name
Figure BDA0003395590210000024
The amino acid sequences of the light chain CDR1-3 are respectively shown in SEQ ID NO. 13-15, the amino acid sequences of the heavy chain CDR1-3 are respectively shown in SEQ ID NO. 16-18, and the amino acid sequences of the light chain variable region and the heavy chain variable region are respectively shown in SEQ ID NO. 19 and 20; the light chain amino acid sequence of the Fab fragment is shown as SEQ ID NO. 1, and the heavy chain amino acid sequence of the Fab fragment is shown as SEQ ID NO. 2.
The albumin binding peptides (ABDs) are capable of specifically binding human serum albumin, including domains derived from screening of microbial native protein sequences and various engineered derivatives thereof. In some embodiments, the albumin binding peptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences selected from the group consisting of those set forth as SEQ ID NOs 3-6. In some embodiments, the albumin binding peptide comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOS 3-6. In some specific embodiments, the amino acid sequence of the albumin binding peptide is as set forth in SEQ ID No.3, 4, 5 or 6. Experiments prove that the fusion protein of the amino acid sequence shown in one of SEQ ID NO 3-6 and the Fab fragment of anti-TNF-alpha not only retains the specific antigen binding capacity of the Fab fragment, but also has the albumin binding capacity of the ABD sequence, and can be specifically bound with albumin in plasma in vivo to prolong the half life.
In some embodiments, the linker is (G)4S)n、(ED)n、(PA)n、(GSP)nWherein n is an integer of 1-12. In some embodiments, the linker is (G)4S)nWherein n is an integer of 1 to 12, preferably an integer of 1 to 6, more preferably an integer of 2 to 4. In some embodiments, the linker is (G)4S)3。
In some embodiments, the Fab-albumin binding peptide fusion protein comprises two polypeptide chains, wherein one polypeptide chain has an amino acid sequence as set forth in SEQ ID No. 1 and the other polypeptide chain has an amino acid sequence selected from any one of the amino acid sequences set forth in SEQ ID nos. 7-10.
In a specific example, the Fab-albumin binding peptide fusion protein is Fab-ABD094, which is composed of two polypeptide chains, wherein one polypeptide chain has the amino acid sequence shown in SEQ ID NO. 1, and the other polypeptide chain has the amino acid sequence shown in SEQ ID NO. 7. In another specific example, the Fab-albumin binding peptide fusion protein is Fab-ABD035, consisting of two polypeptide chains, one of which has the amino acid sequence shown in SEQ ID NO. 1 and the other of which has the amino acid sequence shown in SEQ ID NO. 8. In another specific example, the Fab-albumin binding peptide fusion protein is Fab-ABDwt, and consists of two polypeptide chains, wherein the amino acid sequence of one polypeptide chain is shown as SEQ ID NO. 1, and the amino acid sequence of the other polypeptide chain is shown as SEQ ID NO. 9. In another specific example, the Fab-albumin binding peptide fusion protein is Fab-ABDcon, which is composed of two polypeptide chains, wherein one polypeptide chain has the amino acid sequence shown in SEQ ID NO. 1, and the other polypeptide chain has the amino acid sequence shown in SEQ ID NO. 10.
In a second aspect of the invention, there is provided an isolated nucleic acid encoding a Fab-albumin binding peptide fusion protein according to any one of the first aspect of the invention. In some embodiments, the Fab-albumin binding peptide fusion proteins, non-limiting examples such as Fab-ABDwt, Fab-ABDcon, Fab-ABD035, and Fab-ABD 094.
In a third aspect of the invention, there is provided a recombinant expression vector comprising at least one copy of a nucleic acid according to the second aspect of the invention. The origin vector of the recombinant expression vector may alternatively be any expression vector capable of expressing the Fab-albumin binding peptide fusion proteins described herein, in particular the origin vector is pET28 a.
In a fourth aspect of the invention, there is provided a recombinant host cell comprising a recombinant expression vector according to the third aspect of the invention or an isolated nucleic acid according to the second aspect. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is escherichia coli BL21(DE 3).
The Fab-albumin binding peptide fusion proteins can be derived from recombinant methods. Although the genetic recombination process in the preparation of proteins varies, the recombination process typically involves constructing a nucleic acid encoding the desired polypeptide or protein, cloning the nucleic acid into an expression vector, transforming a host cell, and expressing the nucleic acid to produce the desired polypeptide or protein. Methods for producing and expressing recombinant proteins in vitro and in host cells are known to those of ordinary skill in the art.
The host cell may be a eukaryotic cell or a prokaryotic cell, and includes, but is not limited to, bacteria, such as escherichia coli (e.coli), mammals, yeasts, such as pichia pastoris (p.pastoris), and plant cells, insect cells, and the like. Expression may be by exogenous expression or by endogenous expression. In some embodiments, the Fab-albumin binding peptide fusion protein is expressed in escherichia coli (e.
After the Fab-albumin binding peptide fusion protein is expressed in cells, the protein concentration can be determined by enzyme-linked immunosorbent assay (ELISA), High Performance Liquid Chromatography (HPLC), polyacrylamide gel electrophoresis (SDS-PAGE) or other methods.
In a fifth aspect of the invention, there is provided a method of preparing a Fab-albumin binding peptide fusion protein according to any one of the first aspect of the invention, comprising the steps of: (a) constructing a nucleic acid encoding the Fab-albumin binding peptide fusion protein according to any one of the first aspect of the invention, cloning the nucleic acid into an expression vector, and constructing to obtain a recombinant expression vector; (b) transforming the recombinant expression vector into a host cell to obtain a recombinant host cell, then fermenting and culturing the recombinant host cell, and inducing the Fab-albumin binding peptide fusion protein to express; and, (c) isolating and purifying the Fab-albumin binding peptide fusion protein. The nucleic acid can be obtained by various methods known in the art, such as gene splicing and chemical synthesis.
In some embodiments, the original vector of the recombinant expression vector includes, but is not limited to, pET28 a. In some embodiments the host cell includes, but is not limited to, BL21(DE 3). In some embodiments, the original vector of the recombinant expression vector is pET28a and the host cell is BL21(DE 3).
In some embodiments, the purification is one or a combination of ion exchange chromatography, hydrophobic chromatography, affinity chromatography and molecular exclusion chromatography; preferably, affinity chromatography is used, and more preferably, a combination of affinity chromatography using HSA as a ligand and affinity chromatography using Protein L as a ligand is used, for example, affinity chromatography using HSA as a ligand is performed first, and then affinity chromatography using Protein L as a ligand is performed.
The purity of the Fab-albumin binding peptide fusion protein can be determined by any of a variety of known analytical methods, including gel electrophoresis (SDS-PAGE), High Performance Liquid Chromatography (HPLC), and the like, and the molecular weight verified by mass spectrometry.
In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a Fab-albumin binding peptide fusion protein according to any one of the first aspect of the invention, and a pharmaceutically acceptable carrier.
In a seventh aspect of the invention, there is provided a Fab-albumin binding peptide fusion protein according to any one of the first aspect of the invention or a pharmaceutical composition according to the sixth aspect of the invention for use in the preparation of a medicament for the prevention and/or treatment of a disease associated with TNF- α. In some embodiments, there is provided a method of preventing and/or treating a TNF- α related disease in a subject in need thereof, wherein a prophylactically and/or therapeutically effective amount of a Fab-albumin binding peptide fusion protein of any one of the first aspect of the invention or a pharmaceutical composition of the sixth aspect of the invention is administered to the subject.
The TNF-alpha related diseases are diseases caused by the increase of human TNF-alpha; in some embodiments, the TNF- α -related disorder is selected from osteoarthritis, pouchitis, behcet's disease, lumbar spondylitis, hidradenitis suppurativa, rheumatoid arthritis, autoimmune uveitis, crohn's disease, psoriatic psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, or juvenile idiopathic arthritis. The dosage of the anti-TNF-alpha Fab-albumin binding peptide fusion protein or the pharmaceutical composition of the invention for treating the TNF-alpha related diseases can be compared with the conventional dosage of the existing TNF-alpha antibody.
The Fab-albumin binding peptide fusion protein provided by the invention fuses the Fab fragment of an antibody with albumin binding peptide, has the capacity of binding albumin, and obviously prolongs the half life in vivo.
The anti-TNF-alpha Fab-albumin binding peptide fusion protein maintains the antigen binding effect of the specificity of the Fab fragment. In vitro experiments show that the compound can effectively inhibit the killing effect of TNF-alpha on L929 cells.
The Fab-albumin combined peptide fusion protein can be efficiently expressed in a prokaryotic system, has simple and efficient purification process, and can be industrially produced in a large scale.
Drawings
FIG. 1 is a schematic diagram of the structure of Fab-ABD.
FIG. 2 is a schematic diagram of a recombinant expression vector containing an expressed Fab-ABD094 gene.
FIG. 3 is an electropherogram of Fab-ABD094 prepared by affinity chromatography purification: lane 1: a periplasmic protein extract; lane 2: flowing through an HSA affinity chromatography column; lane 3: eluting HSA affinity chromatographic column; lane 4: eluting the Protein L affinity chromatography column; lane 5: protein L affinity chromatography column eluate (dithiothreitol (DTT) reduction); lane M: marker.
FIG. 4 is a diagram of purified Fab-ABD electrophoretograms: lane 1: Ada-Fab; lane 2: Fab-ABDwt; lane 3: Fab-ABD 035; lane 4: Fab-ABDcon; lane 5: Fab-ABD 094; lane M: marker; lane 6: Fab-ABD094(DTT reduction); lane 7: Fab-ABDcon (DTT reduction); lane 8: Fab-ABD035(DTT reduction); lane 9: Fab-ABDwt (DTT reduction); lane 10: Ada-Fab (DTT reduction).
FIG. 5 is a graph of the binding and dissociation signals during the determination of the affinity of Fab-ABDwt for TNF- α by the biofilm interference technique (BLI).
FIG. 6 is a graph of the binding and dissociation signals during determination of the affinity of Fab-ABDwt for HSA by the biofilm interference technique (BLI) at pH 7.4.
FIG. 7 is a graph of the binding and dissociation signals during determination of the affinity of Fab-ABD035 for MSA by biofilm interference technique (BLI) at pH 6.0.
FIG. 8 is a signal map of Fab-ABDcon binding to TNF- α and HSA as determined by biofilm interference technique (BLI).
FIG. 9 is a signal map for determining the binding of Fab-ABDcon, HSA and FcRn to each other by the biofilm interference technique (BLI).
Detailed Description
Detailed description of the invention
The present invention provides the following embodiments.
Embodiment [1 ]: a Fab-albumin binding peptide fusion protein, wherein the Fab-albumin binding peptide fusion protein comprises an albumin binding peptide and a Fab fragment that binds TNF-a, the albumin binding peptide being linked to the C-terminus of the Fab fragment.
Embodiment [2 ]: the Fab-albumin binding peptide fusion protein of embodiment [1], wherein the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment and/or the light chain of the Fab fragment either directly or through a linker peptide segment; preferably, the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment via the linker peptide segment.
Embodiment [3 ]: the Fab-albumin binding peptide fusion protein of embodiment [1] or [2], wherein the Fab fragment comprises a CDR selected from the group consisting of:
a) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of infliximab;
b) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of adalimumab;
c) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of golimumab; or
d) The amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of trastuzumab.
Embodiment [4 ]: the Fab-albumin binding peptide fusion protein of embodiment [3], wherein the Fab fragment comprises the amino acid sequences of the light chain variable region and the heavy chain variable region of infliximab, the amino acid sequences of the light chain variable region and the heavy chain variable region of adalimumab, the amino acid sequences of the light chain variable region and the heavy chain variable region of golimumab, or the amino acid sequences of the light chain variable region and the heavy chain variable region of certolizumab pegol.
Embodiment [5 ]: the Fab-albumin binding peptide fusion protein according to embodiment [4], wherein the Fab fragment is a Fab fragment of infliximab, a Fab fragment of adalimumab, a Fab fragment of golimumab or a Fab fragment of certolizumab ozogamicin, preferably a Fab fragment of adalimumab.
Embodiment [6 ]: the Fab-albumin binding peptide fusion protein of any one of embodiments [1] to [5], wherein the albumin binding peptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one sequence selected from the group consisting of SEQ ID NOs 3-6.
Embodiment [7]: according to embodiment [2]-[6]The Fab-albumin binding peptide fusion protein of any one of, wherein the linker is (G)4S)n、(ED)n、(PA)n、(GSP)nWherein n is an integer of 1 to 12.
Embodiment [8]: according to embodiment [7]The Fab-albumin binding peptide fusion protein, wherein the connecting peptide segment is (G)4S)n(ii) a Wherein n is an integer from 1 to 12, preferably an integer from 1 to 6, more preferably an integer from 2 to 4.
Embodiment [9 ]: the Fab-albumin binding peptide fusion protein of embodiment [1], wherein the Fab-albumin binding peptide fusion protein comprises two polypeptide chains, wherein the amino acid sequence of one polypeptide chain is set forth in SEQ ID NO. 1 and the amino acid sequence of the other polypeptide chain is selected from any one of the amino acid sequences set forth in SEQ ID NO. 7-10.
Embodiment [10 ]: an isolated nucleic acid encoding a Fab-albumin binding peptide fusion protein according to any one of embodiments [1] to [9 ].
Embodiment [11 ]: a recombinant expression vector comprising at least one copy of the nucleic acid of embodiment [10 ].
Embodiment [12 ]: a recombinant host cell comprising the recombinant expression vector according to embodiment [11 ].
Embodiment [13 ]: a method of making a Fab-albumin binding peptide fusion protein of any one of embodiments [1] to [9], comprising:
a) constructing a nucleic acid encoding the Fab-albumin binding peptide fusion protein according to any one of the embodiments [1] to [9], cloning the nucleic acid into an expression vector, and constructing to obtain a recombinant expression vector;
b) transforming the recombinant expression vector into a host cell to obtain a recombinant host cell, then fermenting and culturing the recombinant host cell, and inducing the Fab-albumin binding peptide fusion protein to express; and (c) and (d),
c) and (3) separating and purifying the Fab-albumin binding peptide fusion protein.
Embodiment [14 ]: the method according to embodiment [13], wherein the original vector of the recombinant expression vector is pET28a and the host cell is BL21(DE 3).
Embodiment [15 ]: the method according to embodiment [13] or [14], wherein the purification is one or a combination of ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and size exclusion chromatography; preferably, the combination of affinity chromatography using HSA as a ligand and affinity chromatography using Protein L as a ligand is used.
Embodiment [16 ]: a pharmaceutical composition, wherein said pharmaceutical composition comprises a Fab-albumin binding peptide fusion protein of any one of embodiments [1] to [9], and a pharmaceutically acceptable carrier.
Embodiment [17 ]: use of a Fab-albumin binding peptide fusion protein according to any one of embodiments [1] to [9] for the preparation of a medicament for the prevention and/or treatment of a disease associated with TNF- α.
Embodiment [18 ]: use of the pharmaceutical composition according to embodiment [16] for the preparation of a medicament for the prevention and/or treatment of diseases associated with TNF- α.
Embodiment [19 ]: the use according to embodiment [17] or [18], wherein the disease associated with TNF- α is osteoarthritis, pouchitis, behcet's disease, lumbar spondylitis, hidradenitis suppurativa, rheumatoid arthritis, autoimmune uveitis, crohn's disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, or juvenile idiopathic arthritis.
Definition of
The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art.
The term "Fab fragment" refers to the heavy chain V derived from an immunoglobulinHAnd C H1 Domain (the heavy chain of the "Fab fragment") and the light chain VLAnd CLAntibody fragments consisting of the domains ("light chains of the Fab fragments").
The term "CDR" (complementarity determining region), also known as "hypervariable region (HVR)", generally refers to each region of an antibody variable region that is hypervariable in sequence and/or forms structurally defined loops. Natural four-chain antibodies or Fab fragments typically comprise six CDRs, three in the heavy chain variable region (heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3), and three in the light chain variable region (light chain CDR1, light chain CDR2, and light chain CDR 3).
"ABD", "albumin binding domain" and "albumin binding peptide" are used interchangeably herein.
As used herein, a "Fab-albumin binding peptide fusion protein" refers to a fusion protein formed by the attachment of a Fab fragment to an albumin binding peptide. "Fab-albumin binding peptide fusion protein" and "Fab-ABD" are used interchangeably herein and include Fab-ABDwt, Fab-ABDcon, Fab-ABD035, and Fab-ABD094, as shown by the following non-limiting examples.
The term "EC50"refers to the effective concentration, binding protein (such as antibody, antibody fragment or Fab-albumin binding peptide fusion protein) 50% of the maximum response. The term "IC50By "is meant the inhibitory concentration, 50% of the maximal response of the binding protein. EC (EC)50And IC50Can be measured by ELISA or FACS analysis or any other method known in the art.
The term "Ka"or" binding rate constant "refers to the binding rate constant of a binding protein (e.g., an antibody, antibody fragment, or Fab-albumin binding peptide fusion protein) bound to a ligand (e.g., an antigen or HSA) to form a binding protein/ligand complex.
The term "Kd"or" off rate constant "refers to the off rate constant for dissociation of a binding protein (e.g., an antibody, antibody fragment, or Fab-albumin binding peptide fusion protein) from a binding protein/ligand complex.
The term "KD"or" equilibrium dissociation constant "refers to the dissociation rate constant (K) at equilibrium, or by the addition ofd) Divided by the binding rate constant (K)a) The obtained value. The term "K" will also be usedDThe term "or" equilibrium dissociation constant "is referred to as the" affinity constant, "and is used interchangeably herein. Using Ka、KdAnd KDIndicating the binding affinity between the binding protein and the ligand. Determination of KaAnd KdMethods of (a) are well known in the art.
The term "TNF- α" refers to human tumor necrosis factor α, a human cytokine present in a secreted form of 17kD and a membrane bound form of 26 kD. The biologically active form consists of a trimer of non-covalently linked 17kD molecules. Their structures are further described, for example, in Pennica, D.et al (1984) Nature 312: 724-729; davis, J.M. et al (1987) Biochemistry 26: 1322-1326; and Jones, E.Y. et al (1989) Nature 338: 225-.
The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer encoded information to an expression system (e.g., a host cell or an in vitro expression system). One type of vector is a "plasmid," which refers to a circular double-stranded DNA (dsdna) molecule into which additional DNA fragments can be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
The term "expression vector" refers to a vector that can be used to direct the translation of a polypeptide encoded by a polynucleotide sequence present in an expression vector in a biological or reconstituted biological system.
The term "recombinant host cell" refers to a cell into which a recombinant expression vector has been introduced, such as a recombinant Escherichia coli. It is understood that such terms refer not only to the particular subject cell, but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "recombinant host cell" as used herein.
The term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compounds described herein. Such a substance is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorants, dyes, and the like, and combinations thereof, as are well known to those skilled in the art (Remington's Pharmaceutical Sciences,18th ed. mac Printing Company,1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, use thereof is contemplated in the therapeutic or pharmaceutical compositions.
The term "treatment" refers to an attempt to alter the natural course of disease in a treated individual, and may be a clinical intervention performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis.
The term "therapeutically effective amount" refers to the amount of Fab-ABD or composition or other administration necessary to provide a therapeutic and/or prophylactic benefit to a subject.
The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. Preferably, the subject according to the invention is a human. The terms "patient" or "subject" are used interchangeably unless indicated otherwise.
The term "specific binding" or "specific binding" means that the binding is selective for the antigen or albumin and can be distinguished from interactions that are not required or are non-specific. The ability of a Fab-ABD to bind to an antigen or to HSA can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.
The term "isolated" refers to a compound of interest, such as a nucleic acid, that has been isolated from its natural environment.
The term sequence "identity", also called identity. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity ═ number of identical positions/total number of positions x 100), where the number of gaps required to be introduced and the length of each gap to produce an optimal alignment of the two sequences are taken into account. As shown in the following non-limiting examples, comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. The percent identity between two amino acid sequences can be determined using the algorithm of e.meyers and w.miller (comput.appl.biosci.,4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 residue weight table with a gap length penalty of 12 and a gap penalty of 4. In addition, percent identity of two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (j.mol.biol.484453 (1970)) which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, GAP weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.
Amino acid residues in proteins are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; glycine is Gly or G.
The present invention is further described below with reference to specific examples, which, however, are only illustrative and not intended to limit the scope of the present invention. Likewise, the present invention is not limited to any particular preferred embodiment described herein. It will be appreciated by those skilled in the art that equivalent substitutions for the features of the invention, or corresponding modifications, may be made without departing from the scope of the invention. The reagents used in the following examples are commercially available products, and the solutions can be prepared by techniques conventional in the art, except where otherwise specified.
Unless otherwise specified, the procedures of the present invention will be performed using conventional techniques of organic synthesis, biochemistry, protein purification, etc., within the skill of the art, or according to product specifications, which are well explained in the literature. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.
Example 1: construction of recombinant Escherichia coli expressing Fab-ABD094
The light and heavy chains of the Fab fragment form heterodimeric structures via disulfide bonds, retaining the antigen binding activity of the full-length antibody. The light and heavy chains of the Fab fragment are secreted into the periplasmic space of Escherichia coli under the guidance of a signal peptide, and the light and heavy chains can complete folding in the periplasmic space and form correct intra-chain and inter-chain disulfide bonds to become a biologically active Fab protein. Based on the above theoretical basis, the present embodiment is realized.
Fab-ABD094 is derived from the albumin binding peptide ABD094 (amino acid sequence shown in SEQ ID NO: 3) attached to the C-terminus of the heavy chain of adalimumab Fab fragment, which has two polypeptide chains with amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:7, respectively.
The amino acid sequences of the light chain and the heavy chain of the adalimumab Fab fragment were converted into DNA sequences (the nucleotide sequences are shown in SEQ ID NO:22 and 23, respectively) according to the codon preference of Escherichia coli BL21(DE 3). The gene encoding the STII signal peptide (amino acid sequence shown in SEQ ID NO: 11) was added to the 5 'end of the gene encoding the light chain of the adalimumab Fab fragment and the gene encoding the heavy chain of the Fab fragment, respectively, the signal peptide sequence directed secretion and expression of the target polypeptide into the periplasmic space of Escherichia coli, the intergenic sequence shown in SEQ ID NO:21 was added between the gene encoding the light chain of the Fab fragment containing the STII signal peptide sequence and the gene encoding the heavy chain of the Fab fragment containing the STII signal peptide sequence, and the gene encoding ABD094 (nucleotide sequence shown in SEQ ID NO: 24) was added to the 3' end of the gene encoding the heavy chain of the Fab fragment, thereby constructing the target gene sequence STII-Ada-ABD094, which had restriction sites NcoI (C ↓. CATGG) and BamHI (G ↓. GATCC) added to the 5 'and 3' ends, respectively. The gene encoding the light chain of the Fab fragment and the gene encoding the heavy chain of the Fab fragment are expressed under the control of the same promoter.
The gene STII-Ada-ABD094 was synthesized and cloned into the NcoI and BamHI cleavage sites of the expression vector pET28a to construct a recombinant expression vector pET28a-STII-Ada-ABD094, as shown in FIG. 2. The recombinant expression vector is transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3)/STII-Ada-ABD094, which is named as DMR 466.
Example 2: fermentation of DMR466 and preparation of periplasmic protein extract
A high density fermentation process for DMR466 is described in [ Newton J M, Vlahopoulou J, Zhou Y. investing and modifying the effects of cell physiology on the microbiological properties of fermentation broth [ J].Biochemical Engineering Journal,2017,121:38-48]. A single colony of DMR466 was inoculated in a liquid LB medium and cultured at 37 ℃ and 220rpm for about 7 hours to prepare a seed solution. Then, the seed solution was inoculated into 3L of SM6Gc fermentation medium in an amount of 10% and kanamycin was added thereto to a final concentration of 25. mu.g/mL. The initial culture temperature of fermentation is 30 deg.C, air flux is 3L/min, and controlThe pH was about 7.0 and the rotation speed was 300 rpm. In the fermentation process, the dissolved oxygen is kept to be not less than 30 percent by adjusting the rotating speed and the tank pressure, the pH is adjusted by 2mol/L sulfuric acid solution and 50 percent (v/v) ammonia water solution, and defoaming agent is added for defoaming. When fermentation broth OD600When the value reaches 40.0, the fermentation culture temperature is reduced to 25 ℃, and 2.1g of magnesium sulfate is added into the fermentation tank in time. When the dissolved oxygen and the pH value in the fermentation liquor are obviously increased, the fermentation liquor needs to be fed. At this time, 80% (w/w) of glycerol solution was added at a constant flow rate of 12mL/h, and IPTG was added to the fermentor at a final concentration of 0.1mmol/L for induction expression, the total fermentation time being about 80 h.
After the high-density fermentation is finished, taking the fermentation liquor, centrifuging at 6000rpm for 10min at room temperature, and pouring off the supernatant. The cells obtained by precipitation were suspended in 20mM Tris-HCl (pH 8.0) at a mass-to-volume ratio (w: v) of 1:10, washed, and the cell suspension was centrifuged at 6000rpm at 4 ℃ for 10min, and the supernatant was decanted. The washing step was repeated twice.
The cells were suspended in a buffer (pH 4.0) containing 60mM citric acid and 50mM magnesium sulfate at a mass/volume ratio (w: v) of 1: 5. The suspension of the above-mentioned strain was filtered with a fine gauze to remove impurities, and then homogenized for three times at 4 ℃ under 550bar pressure in a cell disruptor (Guangzhou energy-gathering nanotechnology GmbH Co., Ltd.). Then centrifuging the obtained homogenate at 4 ℃ and 10000rpm for 10min, collecting supernatant, and adjusting pH to 7.2 to obtain the periplasmic protein extracting solution.
Example 3: affinity chromatography (with HSA as ligand) purification and identification of Fab-ABD094
In the research process of the invention, the Fab-ABD is found to be degraded to a certain degree inevitably in the secretion and expression process of the Fab-ABD in the periplasm space of the thallus, and degradation sites are all in the ABD sequence, so that firstly, the ABD sequence is used as a purification label, HSA is coupled to a solid phase carrier, and the Fab-ABD094 with correct structure is specifically enriched and purified from the periplasm extract by utilizing the specific affinity action of the ABD and the HSA (human serum albumin).
(1) Immobilization of HSA
HSA (Sigma) was dissolved in 200mM NaHCO3500mM NaCl, pH 8.3In solution, HSA was then immobilized on NHS-activated SepharoseTM4Fast Flow packing (GE company), HSA affinity chromatography column was finally prepared.
(2) Affinity chromatography purification of Fab-ABD094 with HSA as ligand
The periplasmic protein extract of example 2 was filtered through a 0.22 μm water phase filter to obtain a sample for use. The HSA affinity column was first equilibrated with equilibration buffer (PBS, pH 7.4) and the sample loaded after baseline, pH and conductance were stabilized. After the sample loading is finished, the chromatographic column is washed by using the equilibrium buffer solution, and the hybrid protein which is not specifically bound on the chromatographic column is eluted. Then, the column was washed with an elution buffer (0.1M Gly-HCl solution, pH 2.7) to collect the eluate in which the target protein was present.
(3) Identification of Fab-ABD094
The purified samples were analyzed by SDS-PAGE and the results are shown in FIG. 3.
Example 4: affinity chromatography (Protein L as ligand) purification and identification of Fab-ABD094
Considering that albumin is a natural carrier molecule having several interaction sites for different proteins, fatty acids, sterols, ions, etc., non-specific adsorption of HSA during purification contaminates Fab-ABD094, Fab-ABD094 purified in example 3 is further subjected to affinity chromatography with Protein L as a ligand, enriching Fab-ABD094 and further improving its purity.
The Fab-ABD094 solution prepared by affinity chromatography using HSA as the ligand was adjusted to pH 7.2 and then subjected to affinity chromatography using Protein L as the ligand. The HiTrap Protein L (5mL, GE Healthcare) affinity column was first equilibrated with equilibration buffer (PBS, pH 7.4) and sample loading was started after baseline, pH and conductance had stabilized. After the loading is finished, the affinity column is washed by the balance buffer solution, and impurities which are not specifically bound on the affinity column are eluted. The column was then washed with elution buffer (0.1M Gly-HCl solution, pH 2.7) and the collection of the eluate was started, with the desired protein present in the eluate. The purified samples were analyzed by SDS-PAGE and the results are shown in FIG. 3. Affinity chromatography by using HSA as ligandAnd affinity chromatography with Protein L as ligand, and the final Fab-ABD094 has purity over 95%. Molecular weight of Fab-ABD094 was confirmed by ESI-MS, and M/z was 1608.1467[ M +33H ]]33+This corresponds to the theoretical molecular weight (53036.21 Da).
By using
Figure BDA0003395590210000091
The purified Fab-ABD094 was replaced in PBS (pH 7.4) by desaling (GE healthcare) desalting column. The protein concentration was determined by BCA assay and frozen at-70 ℃.
Example 5: preparation of Fab-ABD035
Fab-ABD035 is obtained by connecting albumin binding peptide ABD035 (amino acid sequence shown in SEQ ID NO:4, nucleotide sequence shown in SEQ ID NO: 25) to the C-terminal of the heavy chain of adalimumab Fab fragment, which has two polypeptide chains, and the amino acid sequences are shown in SEQ ID NO:1 and SEQ ID NO:8, respectively.
Construction of recombinant Escherichia coli expressing Fab-ABD035, fermentation of the strain, preparation of periplasmic extract, and purification and characterization of Fab-ABD035 the procedures of examples 1-4 were followed, and the characterization results are shown in FIG. 4. Molecular weight of Fab-ABD035 was confirmed by ESI-MS, M/z is 1614.2236[ M +33H ]]33+In agreement with the theoretical molecular weight (53237.68 Da).
Example 6: preparation of Fab-ABDwt
Fab-ABDwt is obtained by connecting albumin binding peptide ABDwt (amino acid sequence is shown as SEQ ID NO:5, nucleotide sequence is shown as SEQ ID NO: 26) to the end of heavy chain C of adalimumab Fab fragment, and has two polypeptide chains, and the amino acid sequences are respectively shown as SEQ ID NO:1 and SEQ ID NO: 9.
Construction of recombinant Escherichia coli expressing Fab-ABDwt, fermentation of the strain, preparation of periplasmic extract, and purification and identification of Fab-ABDwt the methods of examples 1 to 4 were referred to, and the results are shown in FIG. 4. Molecular weight of Fab-ABDwt is confirmed by ESI-MS, and M/z is 1564.9583[ M +34H ]]34+In agreement with the theoretical molecular weight (53175.47 Da).
Example 7: preparation of Fab-ABDcon
The Fab-ABDcon is obtained by connecting albumin binding peptide ABDcon (the amino acid sequence is shown as SEQ ID NO:6, and the nucleotide sequence is shown as SEQ ID NO: 27) to the end of heavy chain C of adalimumab Fab fragment, and has two polypeptide chains, and the amino acid sequences are shown as SEQ ID NO:1 and SEQ ID NO:10 respectively.
Construction of recombinant Escherichia coli expressing Fab-ABDcon, fermentation of the strain, preparation of periplasmic extract, and purification and identification of Fab-ABDcon are carried out by the methods described in examples 1 to 4, and the results are shown in FIG. 4. Molecular weight confirmation of Fab-ABDcon by ESI-MS, M/z is 1526.0492[ M +35H [ ]]35+In agreement with the theoretical molecular weight (53377.75 Da).
Example 8: preparation of Ada-Fab
Ada-Fab is the histidine purification tag His6By (G4S)3The LPETGG is connected to the C end of the heavy chain of the adalimumab Fab fragment and is used as a negative control of an experiment, the light chain amino acid sequence of the LPETGG is shown as SEQ ID NO. 1, and the heavy chain amino acid sequence of the LPETGG is shown as SEQ ID NO. 12. Construction of recombinant Escherichia coli expressing Ada-Fab protein, fermentation of the strain, preparation of periplasmic extract, and purification and identification of Ada-Fab protein the methods of examples 1, 2 and 4 were referenced, and the results are shown in FIG. 4. Molecular weight of Ada-Fab is confirmed by ESI-MS, and M/z is 1457.5347[ M +34H ]]34+In agreement with the theoretical molecular weight (49523.12 Da).
Example 9: indirect ELISA method for determining binding activity of Fab-ABD and TNF-alpha
The antigen for detection TNF-. alpha. (GenScript, Cat. No.Z01001) was diluted to 1. mu.g/mL with coating buffer (carbonate buffer, pH 9.6), added to a MaxiSorp plate (Immuno) (100. mu.L/well), and incubated at 37 ℃ for 1 h. After coating was completed, the plates were washed 4 times in succession with a wash solution (PBST containing 0.05% (v/v) Tween-20, pH 7.4), 200. mu.L/well blocking solution (PBS containing 5% (w/v) skim milk powder, pH 7.4) was added, and incubated at 37 ℃ for 1 h. After blocking, the plates were washed 4 times with washing solution and 100. mu.L/well of 2-fold serial dilutions of the blocking solution (concentration range 150nM to 0.018nM) were added while positive and negative controls were set up, respectively, and incubated for 1h at 37 ℃. Then, the plate was washed 4 times with a washing solution, and an enzyme-labeled goat anti-human Fab color-developing antibody (Sigma, catalog No. A0293) was added theretoThe solution was incubated at 100. mu.L/well for 0.5h at 37 ℃. Then, the plate was washed 4 times with a washing solution, 100. mu.L/well of TMB color developing solution (Thermo Fisher Scientific Inc., catalog No.34021) was added, and after incubation at 37 ℃ for 15min, stop solution (2M H) was added2SO4Solution) 50. mu.L/well, measuring absorbance at a detection wavelength of 450nm and calculating EC50The value is obtained.
The detection results are shown in Table 1, and the prepared four Fab-ABDs retain the TNF-alpha specific binding capacity of the adalimumab Fab fragment, have the activity equivalent to that of Ada-Fab, and have good biological activity.
Table 1: binding Activity of Fab-ABD to TNF-alpha
Sample (I) EC50(nM)
Adalimumab((Fab)2-Fc) 0.515
Ada-Fab 1.366
Fab-ABD094 1.517
Fab-ABD035 1.977
Fab-ABDwt 1.494
Fab-ABDcon 1.696
Example 10: indirect ELISA method for determining binding activity of Fab-ABD and HSA
HSA was diluted to 1. mu.g/mL with a coating buffer (carbonate buffer, pH 9.6), added to a high adsorption microplate (Biotechnology engineering (Shanghai) Co., Ltd.) (100. mu.L/well), and incubated at 37 ℃ for 1 h. After coating was completed, the plates were washed 4 times in succession with a wash solution (PBST containing 0.05% (v/v) Tween-20, pH 7.4), 200. mu.L/well blocking solution (PBS containing 5% (w/v) skim milk powder, pH 7.4) was added, and incubated at 37 ℃ for 1 h. After blocking, the plate was washed 4 times with washing solution, 100. mu.L/well of a sample diluted 2-fold in blocking solution of pH 6.0 and pH 7.4, respectively (concentration ranging from 62.5nM to 0.0076nM) was added, while positive and negative controls were set, respectively, and incubated at 37 ℃ for 1 h. The plate was then washed 4 times with washing solution, 100. mu.L/well of enzyme-labeled goat anti-human Fab chromogenic antibody (Sigma, catalog No. A0293) was added, and incubation was carried out at 37 ℃ for 0.5 h. Then, the plate was washed 4 times with a washing solution, 100. mu.L/well of TMB color developing solution (Thermo Fisher Scientific Inc., catalog No.34021) was added, and after incubation at 37 ℃ for 10min, stop solution (2M H) was added2SO4Solution) 50. mu.L/well, measuring absorbance at a detection wavelength of 450nm and calculating EC50The value is obtained.
The detection results are shown in Table 2, and Adalilimumab and Ada-Fab have no HSA binding capacity and are consistent with theoretical results; the four Fab-ABDs have good HSA binding activity under neutral (pH 7.4) and weakly acidic (pH 6.0) conditions.
Table 2: binding Activity of Fab-ABD with HSA
Figure BDA0003395590210000101
Example 11: determination of binding kinetics parameters of Fab-ABD and TNF-alpha by biofilm interference (BLI) technique
Respectively and uniformly mixing Fab-ABD, Ada-Fab and Adalilimumab with NHS-LC-biotin (Bomeida) in a molar ratio of 1:3, reacting at room temperature for 50min, removing residual NHS-LC-biotin through a desalting column (biotinylation kit from Jiangsu Bomeida Life sciences, Inc.) to obtain biotinylated Fab-ABD, Ada-Fab and Adalilimumab samples for later use.
The binding kinetic parameters of Fab-ABD, Ada-Fab and Adalilimumab with TNF-alpha were determined using the BLItz system (Fortebio) Kinetics of the biofilm interference (BLI) technique, and the experimental procedure setup was performed according to the instrument instructions. 100nM biotinylated Fab-ABD, Ada-Fab and Adalilimumab, respectively, were bound to a streptavidin-rich biosensor (streptavidin biosensor) via a Loading buffer (PBST containing 0.02% (v/v) Tween-20, pH 7.4). TNF-. alpha.was diluted to different concentrations in PBST (containing 0.02% (v/v) Tween-20, pH 7.4) at 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.563 nM. Then the following steps are carried out:
(1) and (5) balancing. The biosensor combined with biotinylated Fab-ABD, Ada-Fab or Adalilimumab was immersed in an equilibration buffer (PBST containing 0.02% (v/v) Tween-20, pH 7.4) for 30-60 s, and the signal was detected and collected.
(2) And (3) a combination stage. And taking out the biosensor from the equilibrium buffer solution, immersing the biosensor in a sample solution containing TNF-alpha with a specific concentration for 5-15 min, and detecting and collecting signals.
(3) And (4) a dissociation phase. The biosensor is taken out from a sample solution containing TNF-alpha, immersed in an equilibrium buffer (PBST containing 0.02% (v/v) Tween-20, pH 7.4) for 5-15 min, and a collected signal is detected.
(4) And (4) a regeneration stage. The biosensor was removed from the equilibration buffer, immersed in regeneration buffer (10mM Gly-HCl solution, pH 3.0) for 10s, and this step was repeated several times.
Samples of TNF-. alpha.at various concentrations were assayed by the equilibration-association-dissociation-regeneration procedure described above, with PBST (containing 0.02% (v/v) Tween-20, pH 7.4) as a blank.
The association rate constant (K) was calculated by fitting the association and dissociation signal profiles (as shown in FIG. 5) using the 1:1 association model using the BLItz system softwarea) Dissociation rate constant (K)d) And affinity constant (K)D)。
The results are shown in Table 3, and the Fab-ABD, Ada-Fab and Adalilimumab have comparable affinity to TNF-alpha, and the binding of Fab-ABD to TNF-alpha is not interfered by the binding of ABD sequence.
Table 3: kinetic parameters of Fab-ABD binding to TNF-alpha
Sample(s) Ka(1/Ms) Kd(1/s) KD(M)
Adalimumab 3.11E+05 1.01E-04 3.25E-10
Ada-Fab 5.12E+05 2.69E-04 5.25E-10
Fab-ABD094 5.46E+05 3.34E-04 6.12E-10
Fab-ABD035 5.95E+05 1.20E-04 2.02E-10
Fab-ABDwt 6.86E+05 1.55E-04 2.25E-10
Fab-ABDcon 3.83E+05 1.31E-04 3.40E-10
Example 12: determination of binding kinetics parameters of Fab-ABD and albumin by BLI technology
100nM biotinylated Fab-ABD, Ada-Fab or Adalilimumab was bound to a streptavidin-rich biosensor (streptavidin biosensor) via a Loading buffer (PBST containing 0.02% (v/v) Tween-20, pH 7.4). Albumin (HSA or MSA) was serially diluted 2-fold into different concentrations with PBST (containing 0.02% (v/v) Tween-20, pH 6.0) and PBST (containing 0.02% (v/v) Tween-20, pH 7.4), where the concentration of HSA ranged from 250nM to 7.813nM and the concentration of MSA ranged from 312.5nM to 9.766 nM. Referring to the method of example 11, albumin samples at different concentrations under the corresponding pH conditions were assayed according to the equilibration-association-dissociation-regeneration procedure at pH 6.0 or pH 7.4, and PBST (containing 0.02% (v/v) Tween-20) at the corresponding pH was used as a blank.
The association rate constant (K) was calculated by fitting the association and dissociation signal profiles (as shown in FIGS. 6 and 7) using the 1:1 association model by the BLItz system softwarea) Dissociation rate constant (K)d) And affinity constant (K)D)。
The results of the calculations are shown in tables 4 and 5, and Adalilimumab and Ada-Fab have no binding activity to HSA or Mouse Serum Albumin (MSA); the four Fab-ABDs have different degrees of affinity for HSA and different degrees of affinity for MSA; for the same albumin, the difference in affinity between the four Fab-ABDs arises from the difference in ABD sequences to which the Fab fragments are attached. Fab-ABDcon and Fab-ABD035 have better affinity for HSA. When the solution pH was lowered to 6.0, the affinity of the same Fab-ABD to the same albumin remained essentially unchanged.
Table 4: kinetic parameters of binding of Fab-ABD to HSA under different pH conditions
Figure BDA0003395590210000111
Table 5: kinetic parameters of binding of Fab-ABD to MSA at different pH conditions
Figure BDA0003395590210000112
Example 13: BLI technology for detecting simultaneous binding of Fab-ABD and TNF-alpha and HSA
The previous experiments showed that Fab-ABD has not only TNF- α binding activity but also albumin binding activity. In this example, Fab-ABDcon was used as an example and its simultaneous binding to TNF-. alpha.and HSA was examined by BLI technique.
100nM biotinylated Fab-ABDcon was bound to a surface streptavidin-rich biosensor via a Loading buffer (PBST containing 0.02% (v/v) Tween-20, pH 7.4). TNF-. alpha.and HSA were diluted in PBST (containing 0.02% (v/v) Tween-20, pH 7.4) to prepare sample 1 containing 12.5nM TNF-. alpha., sample 2 containing 62.5nM HSA, and sample 3 containing both 12.5nM TNF-. alpha.and 62.5nM HSA.
Referring to the method of example 11, the sample solutions 1, 2 and 3 were measured by the equilibration-binding-regeneration procedure, and binding signals were detected, and the signal profile is shown in FIG. 8. As shown in Table 6, the signal value generated by the sample liquid 3 was substantially equal to the sum of the signal values generated by the sample liquids 1 and 2. Therefore, the Fab-ABDcon not only has good affinity with TNF-alpha and HSA, but also can be simultaneously combined with TNF-alpha and HSA, and the combination of HSA and Fab-ABDcon does not interfere the antigen combination capability of Fab-ABDcon.
Table 6: binding Signal values for Fab-ABDcon with HSA and TNF-alpha
Δλ(nm)
Assay sample 1(12.5nM TNF-. alpha.) 0.27
Assay sample 2(62.5nM HSA) 0.37
Assay sample 3(12.5nM TNF-. alpha. +62.5nM HSA) 0.65
Example 14: effect function of Fab-ABD: l929 cytoprotective assay
Incubation of TNF- α with mouse L929 cells resulted in apoptosis, and actinomycin D enhanced the cytotoxic effects of TNF- α. The anti-TNF-alpha antibody has an inhibiting effect on cell killing caused by TNF-alpha by specifically binding to the TNF-alpha. Therefore, the biological activity of the Fab-ABD can be detected by inhibiting the killing of TNF-alpha on a target cell L929 cell strain. The experiment adopts L929 cells as target cells killed by TNF-alpha, and the cell survival rate is detected by a CCK-8(Dojindo) reagent, so that the biological activity of a sample is finally reflected. The experimental procedure was as follows:
l929 cells were cultured to logarithmic phase, and cells were washed with PBS (pH 7.4) and trypsinized cell layer added. Cells were diluted to 2X 10 with MEM medium (containing 10% FBS)5one/mL of the cells was added to a 96-well cell culture plate (Corning) at 100. mu.L/well. And (3) placing the cell culture plate in a 5% carbon dioxide incubator at 37 ℃ for culture for 23-25 h. Using MEM medium (containing 2% FBS) containing actinomycin D (2. mu.g/mL) or simultaneouslyA MEM medium (containing 2% FBS) containing actinomycin D (2. mu.g/mL) and HSA (250. mu.g/mL) was used to serially dilute the Fab-ABD2 fold to different concentrations (ranging from 256nM to 0.25nM concentration). A96-well cell culture plate is taken, supernatant is discarded, and 50 mu L/well of the diluted Fab-ABD solution and 50 mu L/well of the TNF-alpha solution with the concentration of 20ng/mL are added. Positive and negative control wells were also set. And (3) placing the 96-hole cell culture plate in a 5% carbon dioxide incubator at 37 ℃ for culturing for 16-18 h. Taking out 96-well cell culture plate, adding CCK-8 solution for staining for 2h, measuring light absorption value by enzyme labeling instrument at detection wavelength of 450nm and calculating IC50The value is obtained.
The calculation result is shown in the table 7, and the Fab-ABD can inhibit the killing effect of TNF-alpha on L929 cells; in addition, HSA can be specifically combined with Fab-ABD in the presence of HSA to form a Fab-ABD/HSA complex, but the formation of the complex does not influence the combination of the Fab-ABD and the TNF-alpha and can still exert the biological activity of inhibiting the L929 cell killing by the TNF-alpha.
Table 7: Fab-ABD inhibition of killing effect of TNF-alpha on L929 cells
Figure BDA0003395590210000121
Example 15: BLI technology to examine the Effect of Fab-ABD on pH-dependent binding of HSA to FcRn
The invention connects the ABD sequence with Fab fragment to construct Fab-ABD, which is combined with HSA in vivo specifically, and realizes long-acting of Fab fragment by utilizing the albumin endogenous FcRn mediated in vivo circulation transport pathway. In this example, Fab-ABDcon was used as an example, and whether the binding of Fab-ABD to HSA would interfere with the pH-dependent binding of HSA to FcRn was investigated by BLI technology.
Sample solution 1 was prepared by diluting HSA to 125nM with PBST (containing 0.02% (v/v) Tween-20, pH 6.0); sample solution 2 was prepared by diluting recombinant human FcRn (rhFcRn, beijing baiolaibo) to 1000nM with PBST (containing 0.02% (v/v) Tween-20, pH 6.0); sample 3 was prepared by diluting rhFcRn to 1000nM with PBST (containing 0.02% (v/v) Tween-20, pH 7.4). 100nM biotinylated Fab-ABDcon were bound to a surface streptavidin-rich biosensor (streptavidin biosensor) by a Loading buffer (PBST containing 0.02% (v/v) Tween-20, pH 7.4) and the following assays were performed, respectively, with reference to the method of example 11:
test 1: immersing the biosensor in a PBST (containing 0.02% (v/v) Tween-20 and pH 6.0) solution for 60s, and detecting and collecting signals; taking out the biosensor, immersing the biosensor in the sample solution 1 for 4min, and detecting and collecting signals; the biosensor was taken out, immersed in the sample solution 2 for 4min, and the signal was detected and collected. The signal map is shown in fig. 9 "run 1".
Test 2: immersing the regenerated biosensor in a PBST (containing 0.02% (v/v) Tween-20 and pH 6.0) solution for 60s, and detecting and collecting signals; taking out the biosensor, immersing the biosensor in the sample solution 1 for 4min, and detecting and collecting signals; and taking out the biosensor, immersing the biosensor in the sample liquid 3 for 4min, and detecting and collecting signals. The signal map is shown in fig. 9 "run 2".
Test 3: immersing the regenerated biosensor in a PBST (containing 0.02% (v/v) Tween-20 and pH 6.0) solution for 60s, and detecting and collecting signals; taking out the biosensor, immersing the biosensor in the sample liquid 2 for 4min, and detecting and collecting signals; and taking out the biosensor, immersing the biosensor in the sample solution 1 for 4min, and detecting and collecting signals. The signal map is shown in fig. 9 "run 3".
The results are shown in fig. 9, where HSA bound to Fab-ABDcon, and bound to rhFcRn under acidic conditions (pH 6.0) and not under neutral conditions (pH 7.4). Binding of Fab-ABDcon to HSA did not interfere with pH-dependent binding of HSA to FcRn.
Example 16: in vivo pharmacokinetic experiment of mice for detecting circulating half-life of Fab-ABD
In this example, Fab-ABDcon is taken as an example, and is subjected to in vivo pharmacokinetic study in mice to detect whether Fab-ABD can achieve the purpose of prolonging half-life by specifically binding plasma albumin.
Approximately 20g of female BALB/c mice were divided into two groups A and B, 10 per group. Two groups of mice were injected intravenously with 5mg/kg of Fab-ABDcon or Ada-Fab, respectively. Collecting blood samples from mouse orbit at 10min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h and 72h after administration, placing in EDTA-K2-containing containerAnd centrifuging in an anticoagulation tube to obtain a plasma sample. ELISA method is adopted to detect the concentration of Fab-ABDcon or Ada-Fab in plasma samples at different time points, and the elimination half-life (t) of pharmacokinetic parameter is calculated1/2) Apparent volume of distribution (V)d) Clearance (CL) and area under the concentration-time curve (AUC) presumed to be infinite(0-∞))。
The results of the calculation are shown in Table 8, and Fab-ABDcon shows 21.5-fold prolonged plasma half-life (t) compared to Ada-Fab1/2) And its Clearance (CL) in mice slowed by 63.5-fold, AUC(0-∞)The value increased 62.3 times. Thus the specific binding of Fab-ABD to plasma albumin can significantly improve its pharmacokinetic properties.
Table 8: pharmacokinetic parameters of Fab-ABDcon and Ada-Fab in BALB/c mice
Fab-ABDcon Ada-Fab
t1/2(h) 28.20±6.33 1.31±0.38
Vd(mL·kg-1) 56.5±18.4 59.2±16.5
CL(mL·h-1·kg-1) 2.8±0.2 177.7±24.1
AUC(0-∞)(mg·L-1·h) 1782.31±236.92 28.59±3.62
。
Sequence listing
<110> Ningda Ningqing pharmaceutical industry group, Inc
Shanghai Pharmaceutical Industry Research Institute
SHANGHAI DUOMIRUI BIOTECHNOLOGY Co.,Ltd.
<120> Fab-albumin binding peptide fusion protein, preparation method and application thereof
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20
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Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu
225 230 235 240
Pro Glu Thr Gly Gly His His His His His His
245 250
<210> 13
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala
1 5 10
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Ala Ala Ser Thr Leu Gln Ser
1 5
<210> 15
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gln Arg Tyr Asn Arg Ala Pro Tyr Thr
1 5
<210> 16
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Asp Tyr Ala Met His
1 5
<210> 17
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu
1 5 10 15
Gly
<210> 18
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr
1 5 10
<210> 19
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 20
<211> 121
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
taagaaggag atatacat 18
<210> 22
<211> 642
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gacatccaga tgacccagtc tccgtcttct ctgtctgcat ctgttggtga ccgtgttacc 60
atcacctgcc gtgcttctca gggtatccgt aactacctgg cttggtacca gcagaaaccg 120
ggtaaagctc cgaaactgct gatctacgct gcatctaccc tgcagtctgg tgttccgtct 180
cgtttctctg gttcgggttc tggtaccgac ttcacgctga ccatctcgtc tctgcagccg 240
gaagatgtgg ctacctacta ctgccagcgt tacaaccgtg ctccgtatac ctttggtcag 300
ggtaccaaag tggaaatcaa acgtaccgtt gctgctccgt ctgtgttcat ctttccgccg 360
tccgatgaac agctgaaatc tggtaccgct tctgttgttt gcctgctgaa caacttctac 420
ccgcgtgaag cgaaagttca gtggaaagtg gataacgctc tgcagtctgg taactctcag 480
gaatctgtta ccgaacagga ttcgaaagat tccacctatt ccctgtcttc taccctgacc 540
ctgtctaaag ctgattacga aaaacacaaa gtgtatgctt gcgaagttac ccatcagggt 600
ctgtcttctc cggttaccaa atcgttcaac cgtggtgaat gc 642
<210> 23
<211> 672
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gaagttcagc tggtggaatc tggtggtggt ctggttcagc cgggtcgttc tctgcgtctg 60
tcttgcgctg cttctggttt caccttcgac gactacgcta tgcattgggt tcgtcaggct 120
ccgggtaaag gtctggaatg ggtttctgct atcacctgga actctggtca catcgactac 180
gctgattctg tggaaggtcg tttcaccatc tctcgtgaca acgcgaaaaa ctctctgtat 240
ctgcagatga actctctgcg tgctgaagat accgctgtgt actattgcgc taaagtgtcc 300
tatctgtcta ccgcttcttc tctggattac tggggtcagg gtaccctggt taccgtttct 360
tctgcttcta ccaaaggtcc gtctgtgttt ccgctggctc cgtcttccaa atctacctct 420
ggtggtaccg ctgctctggg ttgcctggtg aaagactact tcccggaacc ggttaccgtt 480
tcttggaact ctggtgctct gacctctggt gttcatacct ttccggctgt tctgcagtct 540
tctggtctgt attctctgtc ttctgtggtt accgttccgt cttcctctct gggtacccag 600
acctacatct gcaacgtgaa ccacaaaccg tctaacacca aagtggacaa aaaagtggaa 660
ccgaaatctt gc 672
<210> 24
<211> 138
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ctggcagaag cgaaagaagc ggctaacgcc gaactggata gctatggcgt tagcgacttc 60
tacaaacgtc tgatcgacaa agcgaaaacc gttgaaggtg tggaagctct gaaagatgcg 120
atcctggcag ctctgccg 138
<210> 25
<211> 138
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ctggctgaag ctaaagtgct ggctaaccgt gaactggaca aatacggtgt ttctgacttc 60
tacaaacgtc tgatcaacaa agccaaaacc gtggaaggtg tggaagctct gaaactgcac 120
atcctggctg cactgccg 138
<210> 26
<211> 138
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ctggctgaag ctaaagtgct ggctaaccgt gaactggaca aatacggtgt ttctgactac 60
tacaaaaacc tgatcaacaa cgctaaaacc gtggaaggtg tgaaagctct gatcgacgaa 120
atcctggctg cactgccg 138
<210> 27
<211> 141
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctgaaagaag ctaaagagaa agccatcgaa gaactgaaga aagcaggtat cacctctgac 60
tactacttcg acctgatcaa caaagccaaa accgtggaag gtgtgaacgc tctgaaagac 120
gaaatcctga aagcactgcc g 141

Claims (10)

1. A Fab-albumin binding peptide fusion protein comprising an albumin binding peptide and a Fab fragment that binds TNF-a, the albumin binding peptide being linked to the C-terminus of the Fab fragment.
2. The Fab-albumin binding peptide fusion protein of claim 1, wherein the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment and/or the light chain of the Fab fragment, either directly or via a linker peptide segment; preferably, the N-terminus of the albumin binding peptide is linked to the C-terminus of the heavy chain of the Fab fragment via the linker peptide segment.
3. The Fab-albumin binding peptide fusion protein of claim 1 or 2, wherein the Fab fragment comprises CDRs selected from the group consisting of:
a) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of infliximab;
b) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of adalimumab;
c) the amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of golimumab; or
d) The amino acid sequence of the light chain CDR1-3 and the amino acid sequence of the heavy chain CDR1-3 of trastuzumab.
4. The Fab-albumin binding peptide fusion protein of any one of claims 1 to 3, wherein the albumin binding peptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences selected from the group consisting of SEQ ID NOS 3-6.
5. The Fab-albumin binding peptide fusion protein of any one of claims 2 to 4, wherein the linker is (G)4S)n、(ED)n、(PA)n、(GSP)nWherein n is an integer of 1 to 12.
6. The Fab-albumin binding peptide fusion protein of claim 1, wherein the Fab-albumin binding peptide fusion protein comprises two polypeptide chains, wherein one polypeptide chain has an amino acid sequence as set forth in SEQ ID NO. 1 and the other polypeptide chain has an amino acid sequence selected from any one of the amino acid sequences set forth in SEQ ID NO. 7-10.
7. A pharmaceutical composition comprising a Fab-albumin binding peptide fusion protein of any one of claims 1-6, and a pharmaceutically acceptable carrier.
8. Use of a Fab-albumin binding peptide fusion protein of any one of claims 1-6 in the manufacture of a medicament for the prevention and/or treatment of a disease associated with TNF-a.
9. Use of the pharmaceutical composition of claim 7 for the preparation of a medicament for the prevention and/or treatment of TNF- α related diseases.
10. Use according to claim 8 or 9, characterized in that the TNF-alpha related disease is osteoarthritis, pouchitis, behcet's disease, lumbar spondylitis, hidradenitis suppurativa, rheumatoid arthritis, autoimmune uveitis, crohn's disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis or juvenile idiopathic arthritis.
CN202111511772.8A 2020-12-11 2021-12-07 Fab-albumin binding peptide fusion protein and preparation method and application thereof Pending CN114621354A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011460273 2020-12-11
CN2020114602736 2020-12-11

Publications (1)

Publication Number Publication Date
CN114621354A true CN114621354A (en) 2022-06-14

Family

ID=81898651

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (1)

Country Link
CN (1) CN114621354A (en)

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