WO2018024162A1 - Glp-1 fusion protein comprising mutated immunoglobulin fc portion - Google Patents
Glp-1 fusion protein comprising mutated immunoglobulin fc portion Download PDFInfo
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- WO2018024162A1 WO2018024162A1 PCT/CN2017/094916 CN2017094916W WO2018024162A1 WO 2018024162 A1 WO2018024162 A1 WO 2018024162A1 CN 2017094916 W CN2017094916 W CN 2017094916W WO 2018024162 A1 WO2018024162 A1 WO 2018024162A1
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- amino acid
- fusion protein
- glp
- immunoglobulin
- seq
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Abstract
Provided a fusion protein comprising GLP-1 or an analog thereof, a peptide linker, and a mutated immunoglobulin Fc portion, which fusion protein has an increased half-life. Also provided a method for producing the fusion protein and the use of the fusion protein in the preparation of medicines.
Description
The present invention relates to a fusion protein of glucagon-like peptide-1 (GLP-1) , which comprises a mutated immunoglobulin Fc portion, and thus has an extended half-life in vivo. The fusion protein can be used for treating diabetes, obesity and other related diseases or conditions.
Background art
Glucagon-like peptide-1 (GLP-1) is initially isolated and extracted from intestinal mucosa, is a brain-gut peptide secreted by ileal endocrine cells, and is currently the main target for type 2 diabetes drugs. Since GLP-1 can inhibit gastric emptying and reduce intestinal peristalsis, it contributes to controlling ingestion and reducing body weight.
The GLP-1 initially generated by the intestinal tract is a 37-peptide. It is an inactive peptide chain and the N-terminal hexapeptide needs to be excised by enzymolysis, thus obtaining a bioactive GLP-1 (7-37) . The C-terminal glycine of the bioactive GLP-1 can be used as a substrate of an amidating enzyme, such that approximately 80％of the GLP-1 naturally generated in the intestinal tract is a GLP-1 (7-36) amide. The sequences of the GLP-1 (7-36) amides in currently researched mammals are all the same. C-terminal amidation increases the in vivo stability of GLP-1.
The amino acid sequence of a natural GLP-1 (7-37) is:
HisAlaGluGlyThrPheThrSerAspValSerSerTyrLeuGluGlyGlnAlaAlaLysGluPh eIleAlaTrpLeuValLysGlyArgGly
GLP-1 has an N-terminus and a C-terminus, wherein the N-terminus is associated with its physiological activity and the C-terminal is associated with its receptor binding. Dipeptidyl peptidase-IV (DPPIV) can catalyze the hydrolysis of the alanine at position 2 of the N-terminus in GLP-1. The formed GLP-1 (9-36)
NH2 is inactivated and acts as an in vivo natural antagonist for GLP-1R. The biological half-life of GLP-1 is relatively short and is 1-1.5 min. It is rapidly degraded by dipeptidyl peptidase-IV, therefore making its concentration in blood difficult to detect clinically. Therefore, it is the major subject during the development of such drugs for modifying the structure of GLP-1 to form a GLP-1 analog having the same pharmacological activity, as well as covering the DPP-IV binding site to extend the half-life thereof.
In the past few years, Eli Lilly and Company, Novo Nordisk and GSK etc. are competing to modify such proteins in order to obtain long-acting GLP-1 hypoglycemic drugs.
Exenatide is a bioactive peptide extracted from the salivary glands of lizards. Its amino acid sequence has 53％homology to GLP-1. Research has shown that its administration cycle can be extended to twice daily. The amino acid sequence of exenatide is shown as follows:
H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu -Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro -Pro-Pro-Ser-NH2
Since at position 2 of its N-terminus, Ala in GLP-1 is substituted with Gly, it is not degraded by DPP-IV and has a relatively long half-life and a relatively strong biological activity.
Liraglutide is a drug derived from GLP-1 protein via fatty acid chain modification. Its administration cycle is extended to once daily. Liraglutide is a GLP-1 (7-37) chain in which Lys at position 34 is substituted with Arg, and a hexadecanoic acid modified glutamine is connected to Lys at position 26. For GLP-1, its affinity with albumin is increased after fatty acid chain modification, thus reducing the hydrolysis rate by DPP-IV and reducing the renal clearance, and thereby extendingthe biological half-life.
Lixisenatide (trade name: Lyxumia) is co-developed by Sanofi-Aventis (France) and Zealand. The amino acid sequence of lixisenatide is shown as follows:
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-
Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH2
It can be seen from the structure that lixisenatide is exendin-4 in which Pro at position 38 is removed and 6-Lysines are connected to Ser at position 39. After being modified in structure, the half-life of lixisenatide is extended relative to that of exenatide, and lixisenatide can be subcutaneously injected once daily.
Albiglutide (trade name: Eperzan) is a long-acting GLP-1 analog which is subcutaneously injected once weekly and was developed by GlaxoSmithKline. It can be seen from the structure that Albiglutide is obtained by substituting Ala at position 8 in a GLP-1 (7-36) chain with Gly, and then fusing two modified GLP-1 peptide chains to a serum albumin comprising 585 residues in series, and this greatly extends the half-life.
Dulaglutide is a long-acting GLP-1 analog which is subcutaneously injected once weekly and was developed by Eli Lilly and Company. It can be seen from the structure that dulaglutide is obtained by substituting Ala at position 8 in a GLP-1 (7-37) chain with Gly, substituting Gly at position 22 with Glu, and substituting Arg at position 36 with Gly, and then fusing same to glutamic acid at position 216 of a recombinant IgG4 immunoalbumin (the Fc fragment comprising 227 amino acids) via a coupling bridge “GGGGSGGGGSGGGGSA” . Its average biological half-life is up to 90 hours.
Semeglutide is a long-acting GLP-1 analog which is subcutaneously injected once weekly and was developed by Nove Nordisk. It can be seen from the structure that semeglutide is obtained by substituting Ala at position 8 in a GLP-1 (7-37) chain with Aib, substituting Lys at position 34 with Arg, and connecting Lys at position 26 to the fatty acid chain of octadecanoic acid. Compared with liraglutide, the fatty acid chain of semeglutide is longer, and the hydrophobicity is increased, whereas after a short chain PEG modification, semeglutide will have a greatly enhanced hydrophilicity. Semeglutide can not only be closely bound to albumin after PEG modification to cover the hydrolysis site of the DPP-4 enzyme, but can also reduce the renal excretion, so its half-life can be extended, and the effect of long circulation is achieved.
Currently, the injection frequency of the longest-acting GLP-1 drug on the
market is once weekly. Developing long-acting GLP-1 drugs with a more optimized action time facilitates the reduction of frequent drug injections and improves patient compliance.
Summary of the invention
In one aspect of the present invention, provided is a fusion protein comprising or consisting of GLP-1 or an analog thereof, a peptide linker, and an immunoglobulin Fc portion, wherein the amino acid N at position 434 (numbered according to the EU numbering system) of the immunoglobulin Fc portion is substituted with a weakly hydrophobic amino acid selected from alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine, preferably alanine. Thus, an increased half-life in vivo in animals (preferably mammals, such as mice, and more preferably humans) is achieved.
In one embodiment of the fusion protein of the present invention, the C-terminus of the GLP-1 or an analog thereof is fused to the N-terminus of the Fc portion via the peptide linker, and/or wherein the immunoglobulin Fc portion is derived from human IgG1, IgG2, IgG3 or IgG4.
In another embodiment of the fusion protein of the present invention, the immunoglobulin Fc portion also comprises one or more (such as 1, 2, 3, 4, 5, 6, 7 or 8) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of S228P, F234A, L235A, M252Y, T256E, T307A, E380A and M428L, preferably comprises S228P, F234A, L235A and optionally one or more (such as 1, 2, 3, 4 or 5) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of M252Y, T256E, T307A, E380A and M428L, and more preferably comprises S228P, F234A, L235A and optionally one or more (such as 1, 2, 3, 4 or 5) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of T307A, E380A and M428L.
In another embodiment of the fusion protein of the present invention, the immunoglobulin Fc portion comprises a combination of amino acid substitutions (numbered according to the EU numbering system) , the combination is selected
from the group consisting of:
1) S228P+F234A+L235A+N434A；
2) S228P+F234A+L235A+M428L+N434A；
3) S228P+F234A+L235A+T307A+E380A+N434A； and
4) S228P+F234A+L235A+M252Y+T256E+N434A.
In one embodiment of the fusion protein of the present invention, the fusion protein has one or more features selected from the following:
1) the GLP-1 or an analog thereof has one or more (2 or 3) amino substitutions (numbered according to the EU numbering system) selected from A8G, G22E and R36G；
2) the GLP-1 or an analog thereof has (C12-24, such as C12, C14, C16 or C18 long chain) fatty acid modifications on 1 to 5 (such as 1, 2, 3, 4 or 5) amino acid residues； and
3) the amino acid at position 27 of the GLP-1 protein and the amino acid at position 216 of the immunoglobulin Fc (numbered according to the EU numbering system) of the fusion protein are negatively charged, and the amino acid at position 34 of the GLP-1 protein and the amino acid at position 218 of the immunoglobulin Fc (numbered according to the EU numbering system) are positively charged, and there is a continuous polar residue fragment (10 to 30 amino acid residues) between the GLP-1 protein and immunoglobulin Fc.
In another embodiment of the fusion protein of the present invention, the amino acid sequence of the fusion protein is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4； and/or wherein the amino acid sequence of the GLP-1 or an analog thereof is as shown by SEQ ID NO.6； and/or wherein the amino acid sequence of the peptide linker is as shown by SEQ ID NO. 7.
In another embodiment of the fusion protein of the present invention, the fusion protein exists in dimer structure form, preferably exists in homodimers form.
In another aspect of the present invention, provided is a polynucleotide which encodes the fusion protein of the present invention.
In another aspect of the present invention, also provided is a vector comprising
the polynucleotide according to the present invention.
In another aspect of the present invention, also provided is a host cell comprising the vector according to the present invention, and preferably, the host cell is a CHO cell.
In another aspect of the present invention, also provided is a method for producing the fusion protein according to the present invention, the method comprises the expression of the vector according to the present invention in a host cell.
In another aspect of the present invention, also provided is use of the fusion protein according to the present invention in the manufacture of medicaments, and preferably, the medicaments are used for treating diabetes or obesity.
In another aspect of the present invention, also provided is a method of treating diabetes or obesity in a patient comprising administrating the fusion protein of the present invention to the patient.
In another aspect of the present invention, also provided is the fusion protein of the present invention for use in treating diabetes or obesity in a patient.
Figure 1. The schematic diagram of the GLP-1 fusion protein comprising a mutated immunoglobulin Fc portion of the present invention (from left to right: N-terminus to C-terminus) ；
Figure 2. The amino acid sequence of the fusion protein (Na) comprising Fc mutant 1 (SEQ ID NO. 1) ；
Figure 3. The amino acid sequence of the fusion protein (MNa) comprising Fc mutant 2 (SEQ ID NO. 2) ；
Figure 4. The amino acid sequence of the fusion protein (TENa) comprising Fc mutant 3 (SEQ ID NO. 3) ；
Figure 5. The amino acid sequence of the fusion protein (MTNa) comprising Fc mutant 4 (SEQ ID NO. 4) ；
Figure 6. The amino acid sequence of dulaglutide (SEQ ID NO. 5) ；
Figure 7. The amino acid sequence of YES (S228P, F234A, L235A, M252Y,
T256E and N434S) (SEQ ID NO. 8) ；
Figure 8. The amino acid sequence of YTE (S228P, F234A, L235A, M252Y, S254T and T256E) (SEQ ID NO. 9) ；
Figure 9. The amino acid sequence of YTELS (S228P, F234A, L235A, M252Y, S254T, T256E, M428L and N434S) (SEQ ID NO. 10) ；
Figure 10. The amino acid sequence of YE (228P, F234A, L235A, M252Y and T256E) (SEQ ID NO. 11) ；
Figure 11. In vitro activity assay of the fusion protein of the present invention； and
Figure 12. The design schematic diagram of a homodimeric fusion protein in the embodiments of the present invention, wherein the Fc fusion protein comprises an Fc region of an antibody and a unique drug fusion fragment (GLP-1 or an analog thereof) , the unique drug fusion fragment comprises a 5 nm flexible region, both position E27 of the GLP-1 protein and position E216 of the Fc protein (numbered according to the EU numbering system) are negatively charged, whereas both position K34 of the GLP-1 protein and position K218 of the Fc protein (numbered according to the EU numbering system) are positively charged. This structure makes there be a potential interaction between the 5 nm length of the drug fusion fragment and the charged residues and polar residues on the Fc fragment, which further affects the spatial state of the drug fragment.
Detailed description of embodiments
Fc fusion protein drugs are a kind of new functional recombinant protein obtained by fusing a functional protein and an immunoglobulin Fc fragment via technologies such as genetic engineering. Fc fusion proteins and antibodies are different types of proteins. Their essential difference is: the antibodies comprise two heavy chains and two light chains, and the Fc fragment is in the constant regions of the heavy chains, whereas the Fc fusion protein comprises a functional protein and an Fc fragment. This feature of Fc fusion proteins makes them retain the biological activity of functional proteins, and further embody the nature of antibodies, such as having a long-acting half-life.
The present research obtains a long-acting drug having an obvious advantage in terms of pharmacokinetic half-life by means of a structural modification on an Fc region of such Fc fusion proteins.
More particularly, provided in the present invention is a GLP-1 Fc fusion protein having a long-acting hypoglycemic ability. The protein is obtained from a GLP-1 analog and an Fc mutant connect through a peptide, wherein the Fc mutant has at least one mutation: amino acid N434 is substituted with A434 (numbered under the EU index) .
According to one particular embodiment of the present invention, provided is a fusion protein comprising or consisting of GLP-1 or an analog thereof, a peptide linker, and an immunoglobulin Fc portion, wherein the amino acid N at position 434 (numbered according to the EU numbering system) of the immunoglobulin Fc portion is substituted with a weakly hydrophobic amino acid, and preferably, the immunoglobulin Fc portion comprises amino acid substitution N434A (numbered according to the EU numbering system) (note: in this context, the amino acid substitution of the immunoglobulin Fc portion is named as follows: initial amino acid, position (numbered according to the EU numbering system) , and substitution amino acid； and multiple mutations are separated by a plus sign (+)) .
Natural GLP-1 is processed in vivo, wherein the first six amino acids are excised, and therefore, the amino terminus (N-terminus) of GLP-1 is generally specified as being position 7, and its carboxyl terminus (C-terminus) is position 37 in the art.
Other GLP-1 analogs retaining the natural biological activity of GLP-1 are well known to a person skilled in the art or can be determined by conventional experiments.
Preferably, the GLP-1 analog of the present invention comprises 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions (such as conservative amino acid substitutions) , deletions or insertions in the natural GLP-1 sequence, and the half-life in vivo of GLP-1 is thus extended, and at the same time, the natural biological activity of GLP-1 is retained. Preferably, for example, the GLP-1 analogs disclosed in CN 1802386 A, in particular, SEQ ID NOs. 1-6 disclosed in CN
1802386 A.
Preferably, the GLP-1 analog of the present invention comprises (C12-24, such as C12, C14, C16 or C18 long chain) fatty acid modifications on 1-5 (such as 1, 2, 3, 4 or 5) amino acid residues in a natural GLP-1 sequence, and thus the half-life in vivo is extended.
In one preferred embodiment of the present invention, the GLP-1 analog of the present invention is selected from the group consisting of exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide and semeglutide. In the embodiment, the albiglutide and dulaglutide refer to the GLP-1 portion in their structures.
One representative sequence in the GLP-1 analog of the present invention (from left to right: N-terminus to C-terminus) is shown as follows:
HisGly8GluGlyThrPheThrSerAspValSerSerTyrLeuGluGlu22GlnAlaAlaLysGlu PheIleAlaTrpLeuValLysGlyGly36Gly (SEQ ID NO: 6)
Compared with natural active GLP-1 (7-37) in the human body, three amino acids are substituted: A8G, G22E, and R36G, wherein the purpose is to reduce the degradation of the analog by an endogenous enzyme, reduce the potential for molecular aggregation and/or reduce the immunogenicity. In this sequence, amino acid E27 at position 27 is negatively charged, and amino acid K34 at position 34 is positively charged. The distribution of these residues demonstrates the specific charge distribution characteristics of GLP-1 active protein residues.
The GLP-1 fusion protein of the present invention has the following features: the amino acid at position 27 (such as E27) of the GLP-1 protein and the amino acid at position 216 of the immunoglobulin Fc (numbered according to the EU numbering system, such as E216) are negatively charged, and the amino acid at position 34 (such as K34) of the GLP-1 protein and the amino acid at position 218 of the immunoglobulin Fc (numbered according to the EU numbering system, such as K218) are positively charged. Furthermore, there is a continuous polar residue fragment (such as GGGGSGGGGSGGGG) between the GLP-1 protein and immunoglobulin Fc.
The peptide linker in the fusion protein of the present invention can be selected from peptide linkers which are well known in the art (such as the peptide linkers
disclosed in CN 1802386 A, in particular, SEQ ID NOs. 8, 19 and 21 disclosed thereby) , as long as it does not adversely affect the activity of GLP-1 in the fusion protein and/or the stability of the fusion protein. The amino acid sequence of a representative preferred peptide linker used in the fusion protein of the present invention consists of a repeated sequence of GGGGS plus A, and the representative sequence is:
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla (SEQ ID NO: 7)
According to one particular embodiment of the present invention, the C-terminus of the GLP-1 or an analog thereof is fused to the N-terminus of the Fc portion via the peptide linker.
The Fc portion of the present invention can be derived from human IgG1, IgG2, IgG3 or IgG4.
Preferably, the Fc portion of the present invention is mutated, such that the effector function is minimized (such as L235A and F234A, which are numbered according to the EU numbering system, see Kabat, E. A. et al., (1991) , Sequences of Proteins of Immunological Interest, fifth edition, published by the U.S. Dept. of Health and Human Services, Bethesda, MD, NIH, 91-3242) . In addition, preferably, the Fc portion of the present invention is mutated (such as S228P) , such that a stable dimer structure can be formed.
The inventors have further found that the mutation at position N434A can significantly increase the pharmacokinetic half-life of GLP-1/Fc in blood, and is expected to develop into a more long-acting GLP-1 hypoglycemic drug by systematic modification research on residues in the Fc segment of the fusion protein.
According to one particular embodiment of the present invention, in addition to N434A substitution, the immunoglobulin Fc portion in the fusion protein of the present invention also comprises one or more (such as 1, 2, 3, 4, 5, 6, 7 or 8) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of S228P, F234A, L235A, M252Y, T256E, T307A, E380A and M428L, preferably comprises S228P, F234A, L235A and optionally one or more (such as 1, 2, 3, 4 or 5) additional amino acid substitutions (numbered according to
the EU numbering system) selected from the group consisting of M252Y, T256E, T307A, E380A and M428L, and more preferably comprises S228P, F234A, L235A and optionally one or more (such as 1, 2, 3, 4 or 5) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of T307A, E380A and M428L.
In one preferred embodiment of the present invention, the immunoglobulin Fc portion comprises a combination of amino acid substitutions (numbered according to the EU numbering system) , the combination is selected from the group consisting of:
1) S228P+F234A+L235A+N434A；
2) S228P+F234A+L235A+M428L+N434A；
3) S228P+F234A+L235A+T307A+E380A+N434A； and
4) S228P+F234A+L235A+M252Y+T256E+N434A.
In one preferred embodiment of the present invention, the fusion protein has one or more features selected from the following:
1) the GLP-1 or an analog thereof has one or more (2 or 3) amino substitutions (numbered according to the EU numbering system) selected from A8G, G22E and R36G；
2) the GLP-1 or an analog thereof has (C12-24, such as C12, C14, C16 or C18 long chain) fatty acid modifications on 1 to 5 (such as 1, 2, 3, 4 or 5) amino acid residues； and
3) the amino acid at position 27 of the GLP-1 protein and the amino acid at position 216 of the immunoglobulin Fc (numbered according to the EU numbering system) of the fusion protein are negatively charged, and the amino acid at position 34 of the GLP-1 protein and the amino acid at position 218 of the immunoglobulin Fc (numbered according to the EU numbering system) are positively charged, and there is a continuous polar residue fragment (10 to 30 amino acid residues) between the GLP-1 protein and immunoglobulin Fc.
In one preferred embodiment of the present invention, the fusion protein exists in dimer structure form； In one particularly preferred embodiment of the present invention, the fusion protein exists in homodimers form.
In one particularly preferred embodiment of the present invention, the amino acid sequence of the immunoglobulin Fc portion is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4； and/or wherein the amino acid sequence of the GLP-1 or an analog thereof is as shown by SEQ ID NO. 6； and/or wherein the amino acid sequence of the peptide linker is as shown by SEQ ID NO. 7.
The mutant of the immunoglobulin Fc portion of the present invention can be prepared by using any mutation method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis and shuffling, and so on.
Site-directed mutagenesis is technology in which one or more (several) mutations are made at one or more definitive sites in the parent encoding polynucleotide.
Site-directed mutagenesis can be performed in vitro by PCR, wherein the PCR is related to the use of oligonucleotide primers comprising an expected mutagenesis. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, wherein the cassette mutagenesis relates to: the lysis at one site in a plasmid comprising the parent encoding polynucleotide by means of a restriction enzyme and the subsequent ligation of same to the oligonucleotide comprising a mutation in the polynucleotide. Generally, the restriction enzymes for digesting the plasmid and the oligonucleotide are the same, such that the sticky end of the plasmid and an inserting sequence are ligated to each other. Site-directed mutagenesis can also be performed in vivo by a method known in the art.
Any site-directed mutagenesis program can be used in the present invention. Many available commercial kits can be used for preparing a variant.
Mutagenesis/shuffling methods can be in combination with a high throughput automatic screening method to detect the activity of the cloned-and mutagenesis-treated polypeptide which is expressed by a host cell. The mutagenic DNA molecule which encodes an active polypeptide can be recovered from the host cell and rapidly sequenced by using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid
residues in the polypeptide.
Semi-synthetic gene construction is performed by using synthetic gene construction and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling in combination. Typically, semi-synthetic gene construction uses the combination of a method for synthezing a polynucleotide fragment and PCR technology. Therefore, a definitive gene region can be synthesized de novo, whereas the other regions can be amplified by using site-specific mutagenesis primers, or the other regions can be amplified by error prone PCR or non-error prone PCR. Then, the polynucleotide subsequence can be shuffled.
Also provided in the present invention are a polynucleotide which encodes the fusion protein of the present invention, a vector (in particular, an expression vector) comprising the polynucleotide, and a host cell (preferably, a CHO cell) which comprises the vector. The terms “polynucleotide” , “vector” and “host cell” have meanings which are well known in the art, unless otherwise indicated.
Also provided in the present invention is a method for producing the fusion protein of the present invention, and the method comprises the expression of the vector comprising the polynucleotide which encodes the fusion protein of the present invention in a host cell. The method can be performed according to a method for expressing a recombinant protein which is well known to a person skilled in the art.
In addition, also provided in the present invention is the use of the fusion protein of the present invention in the preparation of medicines, and preferably, the medicines are used for treating diabetes or obesity.
The present invention also relates to a pharmaceutical composition comprising the fusion protein of the present invention, and optionally at least one pharmaceutical carrier, diluent or excipient.
Also provided in the present invention is a method for treating diabetes or obesity, and the method comprises administrating a therapeutically effective amount of the fusion protein of the present invention to a subject in need thereof (for example, mammals, preferably any primate, and in particular, humans) .
The present invention is further described by means of the following
non-limiting experimental section and drawings hereafter.
Summary of technical route:
1. Designing mutation sites of the immunoglobulin Fc portion and fusing a GLP-1 analog and an IgG-Fc mutant by means of a peptide linker, wherein the mutant fusion protein comprises the three domains as shown by figure 1, and wherein GLP-1 is a GLP-1 active protein sequence, and the representative sequence is:
HisGly8GluGlyThrPheThrSerAspValSerSerTyrLeuGluGlu22GlnAlaAlaLysGlu PheIleAlaTrpLeuValLysGlyGly36Gly
Compared with natural active GLP-1 in the human body, three amino acids are substituted: A8G, G22E, and R36G, wherein the peptide linker consists of a repeated sequence of GGGGS plus A, and the representative sequence is:
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerAla
Fc represents the immunoglobulin Fc mutant.
The representative sequences of the mutant fusion protein are SEQ ID NOs. 1-4, wherein:
the EU number corresponding to the Fc portion in SEQ ID NO. 1 is E216-G446, which comprises the following amino acid substitutions: S228P, F234A, L235A and N434A；
the EU number corresponding to the Fc portion in SEQ ID NO. 2 is E216-G446, which comprises the following amino acid substitutions: S228P, F234A, L235A, M428L and N434A；
the EU number corresponding to the Fc portion in SEQ ID NO. 3 is E216-G446, which comprises the following amino acid substitutions: S228P, F234A, L235A, T307A, E380A and N434A； and
the EU number corresponding to the Fc portion in SEQ ID NO. 4 is E216-G446, which comprises the following amino acid substitutions: S228P, F234A, L235A, M252Y, T256E and N434A；
2. performing the gene design on the designed mutant fusion protein, and performing whole gene synthesis (entrusted to GenScript Co., Ltd. ) ；
3. by using the molecular cloning method, constructing the whole synthetic gene into a eukaryotic expression vector (pcDNA3.3, Invitrogen) to obtain an expression construct (Plasmid-X) ；
4. transfecting a host cell (aCHO cell) with the expression construct (Plasmid-X) through an electric transfection method, and performing a pressure screening with 800 μg/mL of G418 (Geneticin) ；
5. expressing the Fc mutant fusion protein in the mixed clone strains；
6. performing affinity chromatography purification on the Fc mutant fusion protein using Protein A packing；
7. performing mass spectrum identification on the collected protein to confirm the correctness of the product, and at the same time, performing an SDS-PAGE research to determine the purity of the purified protein； and
8. diluting the purified fusion protein to a concentration of 0.1 mg/ml using 10 mM of PBS, and injecting approximately 0.03-0.04 mg protein into each rat according to an injection dose of 0.1 mg/kg； furthermore, sampling at 0 h, 2 h, 5 h, 8 h, 24 h, 48 h, 72 h, 120 h and 168 h, and performing a detection using a GLP-1 kit, and finally calculating the pharmacokinetic half-life.
Based on the above-mentioned technical route, various expression vectors of the present invention hereinbelow (from the N-terminus to the C-terminus) are constructed using a molecular cloning method:
The GLP-1 analog (the amino acid sequence is as shown by SEQ ID NO. 6) +peptide linker (the amino acid sequence is as shown by SEQ ID NO. 7) + mutated immunoglobulin Fc portion.
The amino acid sequences of the constructed fusion proteins are as shown by SEQ ID NOs. 1-4 and 8-11 respectively.
As a control, a vector encoding the following (from the N-terminus to the C-terminus) is constructed:
Dulaglutide (WT) , and the amino acid sequence thereof is as shown by SEQ ID NO. 5.
The mutated immunoglobulin Fc portions of the present invention are summarized as follows:
Table 1
The nucleotide sequences which encode the WT fusion protein (dulaglutide) and IgG4-Fc mutant fusion protein are obtained by means of chemical synthesis based on the amino acid sequences encoded thereby through entrusting them to
GenScript Co., Ltd. (Nanjing, China) . After double enzyme digestion, the obtained synthetic sequence is inserted into a eukaryotic expression vector, between the sites which are digested by the same double enzymes, so as to construct a series of vectors of Plasmid-GLP-1-Fc and the mutant thereof. Then, a series of correct expression vectors through verification are extracted by an Invitrogen plasmid extraction kit, and are linearized by a restriction enzyme, followed by purification and recovery, and stored at -20℃.
After a resuscitation culture using a CHO culture medium, and when the cell density is approximately 8 x 105 cells/mL, the CHO host cells are collected for transfection. The transfected cells are approximately 1 x 107 cells, the plasmid is approximately 40 μg, and transfection is performed by using an electric shock method. The cells are cultured in 20 mL of a CHO culture medium after the electric shock. On the second day of the culture, the cells are centrifuged and collected, and then re-suspended and cultured in 20 mL of a CHO culture medium supplemented with G418 (Geneticin) to a final concentration of 800 μg/mL. When the cell density is approximately 0.6 x 106 cells/mL, the obtained mixed clone strains are passaged using a CHO culture medium and the density of the passaged cells is approximately 0.2 x 106 cells/mL. When the cell survival rate is approximately 90％, the cell culture liquid is collected.
The series of fusion proteins in embodiment 1 are detected at the translational level. A small amount ofcell culture liquid is enriched using Protein A packing, and fusion proteins are collected. The obtained fusion proteins are homodimers which are formed by disulfide bonds and various non-covalent interactions. The molecular weight is approximately 62 KD through mass spectrometric detection, which is identical to the theoretical molecular weight. The collected fusion proteins are purified using a Protein A chromatographic column. After reduction, the collected sample is detected by 10％SDS-PAGE electrophoresis. The electrophoresis pattern
shows a single band at approximately 36 KD. The purified sample is dialyzed overnight at 4℃, using 10 mM of a PBS buffer at pH 7.2.
The series of purified fusion proteins are diluted to a concentration of 0.1mg/mL with 10 mM of PBS. Screened SD rats with a body weight of 0.3-0.4 kg are selected randomly, and are subjected to calculated administration by injecting 0.1 mg/kg of the fusion protein into each SD rat, wherein each fusion protein is subcutaneously injected into 3 rats.
Before the administration, 200 μl of blood is taken from the jugular vein of each rat, then subjected to an anti-coagulation treatment using EDTA-K2 and DPP-4 inhibitors, and stored at -20℃. 2 h, 5 h, 8 h, 24 h, 48 h, 72 h, 120 h and 168 h after the administration, blood is taken from each animal, and then likewise subjected to an anti-coagulation treatment and stored at -20℃.
For each blood sample, drug residues are detected using a GLP-1 ELISA detection kit (Millipore) , and the pharmacokinetic data is calculated, with the results being shown in the following table:
Table 2
The purified fusion proteins are quantified using the BCA method, and are then subjected to a three-fold gradient dilution with the Assay buffer (DMEM 20 ml,
FBS 200 μl, and IBMX 20 μl) . The cAMP content in cells is determined using a cAMP detection kit (manufacturer: Cisbio) after GLP-1R/HEK293 cells are stimulated by the fusion proteins. That is to say, 5 μl of diluted sample solution is added to a 384 shallow well plate, then 5 μl of cell suspension (the cell density being 100/μl) is added, and the plate is incubated in a carbon dioxide incubator for 30 min. Then, the fluorescence values at 665 nm and 620 nm are detected in a multifunctional microplate reader after a reaction reagent is added thereto and reacted. A standard curve is drawn according to the ratio of the concentration of the cAMP standard and its corresponding fluorescence values. The cAMP amounts generated by the GLP-1R/HEK293 cells stimulated by the test samples at different concentrations are calculated. The curve is drawn using the logarithm values of the concentrations of test samples as the horizontal axis, and the nM values of cAMP as the vertical axis. The results show that the content curve of cAMP in the cells stimulated by the test samples is a typical S curve, and the EC50 values are calculated according to these curves. The results are as shown in the following table and figure 11:
Table 3
The crystal structures of the Fc region of the antibody and the receptor thereof (PDB: 1FRT, 4N0U) show that the Fc region recognizes FcRn by its flanked amino acids comprising M252, S254, T256, M428, N434, etc. (figure 12) . The site-directed mutagenesis of these flanked amino acids can change the binding ability of the Fc to the receptor thereof. For example, by mutating M252, S254 and T256 to Y252, T254 and E256, a research group has found that the binding ability of the mutant to the receptor can be increased, and the mutations introduce a negatively charged residue E256 and a polar amino acid T254. There are also research groups which mutate N434 to S434, etc., and introduce a polar amino
acid so as to increase the binding of the mutant to the receptor.
The Fc fusion proteins designed in the present invention comprise an Fc region of an antibody and a unique drug fusion fragment (GLP-1 or an analog thereof) (figure 12) . In the Fc fusion proteins, the unique drug fusion fragment comprises an about 5 nm flexible region. In the Fc fusion proteins, both position E27 of the GLP-1 protein and position E216 of the Fc protein (numbered according to the EU numbering system) are negatively charged, whereas both position K34 of the GLP-1 protein and position K218 of the Fc protein (numbered according to the EU numbering system) are positively charged. Furthermore, there is a continuous polar residue fragment, i.e. the peptide linker (G38GGGSGGGGSGGGGS52) between the GLP-1 protein and the immunoglobulin. This unique structural feature makes there be a potential interaction between the 5 nm length of the drug fusion fragment and the new introduced charged residues and polar residues on the Fc fragment, which further affects the spatial state of the drug fragment.
The mutation and pharmacokinetic experiments of the present invention also demonstrate this view (the experimental methods and steps are the same as embodiments above) . In addition to the above-mentioned fusion proteins of the present invention, the inventors also introduced a negatively charged residue E256 into the Fc region in the corresponding fusion protein drugs, and constructed four candidate drugs, YES (the amino acid sequence being as shown by SEQ ID NO. 8, and the Fc region comprising a mutation S228P+F234A+L235A+M252Y+T256E+N434S) , YTE (the amino acid sequence being as shown by SEQ ID NO. 9, and the Fc region comprising a mutation S228P+F234A+L235A+M252Y+S254T+T256E) , YTELS (the amino acid sequence being as shown by SEQ ID NO. 10, and the Fc region comprising a mutation S228P+F234A+L235A+M252Y+S254T+T256E+M428L+N434S) and YE (the amino acid sequence being as shown by SEQ ID NO. 11, and the Fc region comprising a mutation S228P+F234A+L235A+M252Y+T256E) , wherein the Cmax values are reduced and the t1/2 is shortened, and thus the four candidate drugs do not have a druggability of potential long-acting drugs (for comparison results with the fusion proteins of the present invention, please make reference to table 2) . It can be seen therefrom that the introduction of a polar residue S434 leads to a shortened t1/2
and an absence of druggability of potential long-acting drugs. In the process of the pharmacokinetic experiment, through screening, we have found that introducing a weakly hydrophobic residue A434 into the Fc region can significantly extend t1/2, and its indexes such as Cmax also demonstrates a pharmacokinetic advantage. Therefore, the inventors surprisingly found that the introduction of a weakly hydrophobic residue (such as A434) (rather than a polar or charged residue) into the drug Fc fragment can reduce the structural self-inhibition between the drug fusion fragment and the Fc fragment, thereby increase the pharmacokinetic effect, and completed the present invention.
Although for clear understanding, the above-mentioned invention has been described in some detail by means of drawings and embodiments, the description and embodiments should not be considered as limiting the scope of the present invention. The disclosures of all the patents and scientific documents cited in the context are completely and clearly incorporated herein by reference.
Claims (14)
- A fusion protein comprising or consisting of GLP-1 or an analog thereof, a peptide linker, and an immunoglobulin Fc portion, wherein the amino acid (such as amino acid N) at position 434 (numbered according to the EU numbering system) of the immunoglobulin Fc portion is substituted with a hydrophobic amino acid selected from alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine, preferably alanine, more preferably the immunoglobulin Fc portion comprises amino substitution N434A.
- The fusion protein according to claim 1, wherein the C-terminus of the GLP-1 or an analog thereof is fused to the N-terminus of the Fc portion via the peptide linker, and/or wherein the immunoglobulin Fc portion is derived from human IgG1, IgG2, IgG3 or IgG4.
- The fusion protein according to claim 1 or 2, wherein the immunoglobulin Fc portion also comprises one or more (such as 1, 2, 3, 4, 5, 6, 7 or 8) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of S228P, F234A, L235A, M252Y, T256E, T307A, E380A and M428L, preferably comprises S228P, F234A, L235A and optionally one or more (such as 1, 2, 3, 4 or 5) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of M252Y, T256E, T307A, E380A and M428L, and more preferably comprises S228P, F234A, L235A and optionally one or more (such as 1, 2, 3, 4 or 5) amino acid substitutions (numbered according to the EU numbering system) selected from the group consisting of T307A, E380A and M428L.
- The fusion protein according to claim 3, wherein the immunoglobulin Fc portion comprises a combination of amino acid substitutions (numbered according to the EU numbering system) , the combination is selected from the group consisting of:1) S228P+F234A+L235A+N434A；2) S228P+F234A+L235A+M428L+N434A；3) S228P+F234A+L235A+T307A+E380A+N434A； and4) S228P+F234A+L235A+M252Y+T256E+N434A.
- The fusion protein according to claim 1, wherein the fusion protein has one or more features selected from the following:1) the GLP-1 or an analog thereof has one or more (2 or 3) amino substitutions (numbered according to the EU numbering system) selected from A8G, G22E and R36G；2) the GLP-1 or an analog thereof has (C12-24, such as C12, C14, C16 or C18 long chain) fatty acid modifications on 1 to 5 (such as 1, 2, 3, 4 or 5) amino acid residues； and3) the amino acid at position 27 of the GLP-1 protein and the amino acid at position 216 of the immunoglobulin Fc (numbered according to the EU numbering system) of the fusion protein are negatively charged, and the amino acid at position 34 of the GLP-1 protein and the amino acid at position 218 of the immunoglobulin Fc (numbered according to the EU numbering system) are positively charged, and there is a continuous polar residue fragment (10 to 30 amino acid residues) between the GLP-1 protein and immunoglobulin Fc.
- The fusion protein according to claim 4 or 5, wherein the amino acid sequence of the fusion protein is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4； and/or wherein the amino acid sequence of the GLP-1 or an analog thereof is as shown by SEQ ID NO. 6； and/or wherein the amino acid sequence of the peptide linker is as shown by SEQ ID NO. 7.
- The fusion protein according to claim 1， which existing in a dimer structure form, preferably existing in a homodimers form.
- A polynucleotide which encodes the fusion protein according to any one of claims 1-7.
- A vector comprising the polynucleotide according to claim 8.
- A host cell comprising the vector according to claim 9, and preferably, the host cell being a CHO cell.
- A method for producing the fusion protein according to any one of claims 1-7, wherein the method comprises the expression of the vector according to claim 9 in a host cell.
- Use of the fusion protein according to any one of claims 1-7 in the manufacture of medicaments, and preferably, the medicaments being used for treating diabetes or obesity.
- A method of treating diabetes or obesity in a patient comprising administrating the fusion protein according to any one of claims 1-7 to the patient.
- The fusion protein according to any one of claims 1-7 for use in treating diabetes or obesity in a patient.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020063628A1 (en) * | 2018-09-26 | 2020-04-02 | 北京辅仁瑞辉生物医药研究院有限公司 | Glp1-fc fusion protein and conjugate thereof |
| WO2020219922A1 (en) * | 2019-04-24 | 2020-10-29 | The Trustees Of The University Of Pennsylvania | Bi-functional humanized anti-c5 antibodies and factor h fusion proteins and uses thereof |
| CN114245807A (en) * | 2019-06-25 | 2022-03-25 | 吉利德科学公司 | FLT3L-FC fusion proteins and methods of use |
| EP3838925A4 (en) * | 2018-09-06 | 2022-05-18 | Zhejiang Palo Alto Pharmaceuticals, Inc. | Long-acting recombinant glp1-fc-cd47 protein, preparation method and use thereof |
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| MX2020012518A (en) * | 2018-06-18 | 2021-02-16 | Denali Therapeutics Inc | Fusion proteins comprising progranulin. |
| CN117143242B (en) * | 2023-10-30 | 2024-03-29 | 南京佰抗生物科技有限公司 | Monoclonal antibody composition for resisting Galectin-3 protein and application thereof |
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Also Published As
| Publication number | Publication date |
|---|---|
| CN107698684B (en) | 2021-09-28 |
| CN107698684A (en) | 2018-02-16 |
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