CN105801705B - Fused polypeptide and application thereof containing glucagon-like-peptide-1 and immunoglobulin heterozygosis Fc - Google Patents
Fused polypeptide and application thereof containing glucagon-like-peptide-1 and immunoglobulin heterozygosis Fc Download PDFInfo
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Abstract
The present invention relates to glucagon-like-peptide-1 and immunoglobulin heterozygosis Fc fused polypeptides, relate more specifically to the purposes of a kind of immunoglobulin heterozygosis Fc based on suitable glucagon-like-peptide-1 or its analog found and have the fused polypeptide for increasing half-life period and outstanding efficiency, the treating diabetes composition comprising above-mentioned fused polypeptide and the above-mentioned fused polypeptide for being used to prepare treating diabetes medicament compared to existing fused polypeptide.Compared to existing glucagon-like-peptide-1, fused polypeptide of the invention has increased half-life period, and there is outstanding effect of reducing blood sugar energy and the repellence to dipeptidyl peptidase-4, when treating diabetes, compared to existing medicament, outstanding drug efficiency is shown, pharmaceuticals can be effectively adapted to.
Description
Technical field
The present invention relates to glucagon-like-peptide-1 (GLP-1) and immunoglobulin heterozygosis Fc (Fc) fused polypeptides, more
Body it is related to the discovery of the immunoglobulin heterozygosis Fc based on suitable glucagon-like-peptide-1 or its analog a kind of and phase
Than there are the fused polypeptide, the diabetes comprising above-mentioned fused polypeptide that increase half-life period and outstanding efficiency in existing fused polypeptide
Therapeutic composition and be used to prepare treating diabetes medicament above-mentioned fused polypeptide purposes.Also, for above-mentioned
Polynucleotides, the expression vector comprising above-mentioned polynucleotides and the place comprising above-mentioned expression vector that fused polypeptide is encoded
Chief cell is also contained in the scope of the present invention.
Background technique
Diabetes be due to being influenced by environment and make insulin secretion go wrong or insulin function occur it is different
Often cause glucose in blood that can not become the energy source of cell, but with high specific gravity residual in blood show height
The metabolic disease of blood glucose disease (hyperglycemia) symptom, with various complication, becoming modern society will most weigh diabetes
Chronic disease.
Diabetes are roughly divided into a patients with type Ⅰ DM (Type 1diabetes) and type-II diabetes (Type 2diabetes).
One patients with type Ⅰ DM account for whole diabetics less than 10%, mainly fall ill in pediatric patient, report display, a patients with type Ⅰ DM
Pathogenic factor be that cannot achieve insulin synthesis and secretion since autoimmune disease etc. causes pancreatic beta cell to wreck
And cause to fall ill, need lifelong insulin injection.On the contrary, type-II diabetes account for 90% or more of whole diabetics, it is main
It to fall ill in adult patient, in particular, overweight and Obesity Adults more susceptible disease, report display, the pathogenic factor of type-II diabetes
It is the insulin secretion obstacle and insulin resistance of pancreas.
In order to treat this diabetes, according to previous mode, kinesiatrics or food therapy are used substantially, but by this
Mode is difficult in the case where adjusting blood glucose, will be implemented the independent of Remedies for diabetes or and is used method of administration.With reference to U.S.'s glycosuria
The guide of disease association (ADA), the medicament selected for the first time be melbine (metformin), second, third time drug be sulphonyl
Urea (sulfonylurea) class, Ge Lienai (glinide) class, thiazolidinedione (thiazolidinedione) class and dipeptides
Base peptase -4 (DPP-4) inhibitor etc., later using glucagon-like-peptide-1 (glucagon like peptide-1) excitement
The injections such as agent (agonist) or injection of insulin agent.
But in the case where the existing glycosuria therapeutic agent for oral use of current clinical use, in addition to Z can be continuously maintained blood glucose
Normalization this positive effect except, when taking for a long time, will lead to various side effects, such as induce hypoglycemia, diarrhea, body
Increase, cardiovascular disease, chronic hepatitis etc. again, and the β cell for eventually leading to pancreas wrecks, and finally needs to inject pancreas islet
Element.Also, insulin injection needs to implement daily 2~3 subcutaneous injections as last treatment method, not only very inconvenient,
But also it may cause and be induced as the hypoglycemia of maximum side effect.
In order to improve it is this there are problems, the important medicament as next-generation Remedies for diabetes is exactly the high blood of pancreas in recent years
Sugared element sample peptide -1.Glucagon-like-peptide-1 and the like and derivative are in the clinical test for type-II diabetes treatment
Good feasibility is shown, the secretion for the glucagon that stimulates insulin secretion, hinders hinders stomach empty stomachization, hinders stomach fortune
It moves property or intestinal motility and loses weight, induce many biology effects.Also, in the case where long-term use, also have
There is pancreas defencive function, risk of hypoglycemia is not present, reasonable blood glucose can be kept for a long time.
But in vivo, by the enzyme of referred to as dipeptidyl peptidase-4, above-mentioned glucagon-like-peptide-1 by cutting and
Cause to lose activity, there is very short biological Half-life in vivo as a result, is difficult to develop as therapeutic agent.In this regard, in order to both protect
Hold biological activity, while in order to extend the half-life period of glucagon-like-peptide-1, or in order to reduce the peptide of body
Removal rate implements various close to method.That is, currently energetically carrying out opening for various glucagon-like peptide-1 analogs
Hair activity, have also been attempted to immunoglobulin Fc site combine glucagon-like-peptide-1 close to method (United States Patent (USP) US7,
452,966B2 etc.).
But nonetheless, attempt so far limitation of the technology in half-life period of binding domain-immunoglobulin Fc, antibody according to
Cell mediated cytotoxicity (ADCC) induced-equation, dipeptidyl peptidase-4 Dependent Stability etc. is relied also to be not enough to commercialization
Change.
In this regard, the present inventor will exempt from as what is prepared in the Chinese patent CN 101687933 of the existing patent of the present inventor
Epidemic disease globulin heterozygosis Fc technology is combined with glucagon-like-peptide-1, and glucagon-like-peptide-1 is selected specifically to optimize
Immunoglobulin Fc, preparing not only has increased half-life period, but also with outstanding effect of reducing blood sugar energy and for dipeptidyl peptidase-
Thus the fused polypeptide of 4 repellence completes the present invention.
Summary of the invention
[technical problem]
It is an object of the present invention to provide fused polypeptide, above-mentioned fused polypeptide include: (a) glucagon-like-peptide-1 or
Its analog, and (b) the immunoglobulin Fc polypeptide of the IgD hinge area comprising limited quantity.
It is also another object of the present invention to provide the treating diabetes for including using above-mentioned fused polypeptide as effective component
Pharmaceutical composition.
It is a further object to provide the purposes for the above-mentioned fused polypeptide for being used to prepare treating diabetes medicament.
It is also an object of the present invention to provide for being encoded to above-mentioned fused polypeptide polynucleotides, comprising
State the expression vector of polynucleotides and the host cell comprising above-mentioned expression vector.
[means solved the problems, such as]
To achieve the goals above, as an embodiment of the invention, fused polypeptide, above-mentioned fused polypeptide packet are provided
It includes: (a) glucagon-like-peptide-1 or its analog；And (b) comprising limited quantity IgD hinge area immunoglobulin
Fc polypeptide.
Specifically, above-mentioned fused polypeptide can include: (a) glucagon-like-peptide-1 or its analog, and (b) be immunized
Immunoglobulin Fc polypeptide；Wherein, above-mentioned immunoglobulin Fc polypeptide may include (i) since the end C- of serial number 25 by 35 to 49
The isolated IgD hinge area that a continuous amino acid sequence is constituted；And (ii) is made of the amino acid sequence of serial number 29
The region CH2 and CH3.
Preferably, the end C- of above-mentioned glucagon-like-peptide-1 or its analog can be with the N- of immunoglobulin Fc polypeptide
End combines, and in immunoglobulin Fc polypeptide, the end C- of IgD hinge area can be with the end the N- phase in the region CH2 and CH3
In conjunction with.As a result, from the end N- along C- end direction can successively with glucagon-like-peptide-1 or its analog, IgD hinge area
Domain and the region CH2 and CH3 combine.In the present invention, " fused polypeptide " indicates the biologies such as above-mentioned glucagon-like-peptide-1
The form that bioactive molecule is combined with immunoglobulin Fc polypeptide is learned, it can be with the terms such as " Fc fused polypeptide ", " conjugated protein "
It is mixed.
Term " glucagon-like-peptide-1 (glucagon like peptide-1) " of the invention is by digestive organs point
A kind of protein of the long pancreotropic hormone for the hormone secreted relies on intake and secretes from the L cell in intestinal tube, can increase pancreas
Insulin secretion, the secretion of glucagon suppression, thus, it is possible to play the role of that postprandial blood sugar is inhibited to rise.In this way, at present
The treating diabetes purposes of glucagon-like-peptide-1 is well-known, may also participate in the Physiological effect of appetite, also has weight
Reduce effect.
On the one hand, above-mentioned natural glucagon sample peptide -1 is processed in vivo, so that preceding 6 amino acid is from molecule
It is middle to be cut.Therefore, the convention of affiliated industry, the amino terminal of glucagon-like-peptide-1 are appointed as No. 7, carboxylic according to the present invention
Base-end is appointed as No. 37.In the glucagon-like peptide-according to above-mentioned processing without the form of insulin secretion function
, can be processed in the form of glucagon-like-peptide-1 (7-37) in 1 (1-37), and become active form glucagon-like-peptide-1
(7-37) (amino acid sequence of serial number 1 and the nucleic acid sequence of serial number 33), carries out supplement deformation, so that the end C- in vivo
Glycine residue is removed, and is substituted with sulfophenyl, and then becomes glucagon-like-peptide-1 (7-36) amide (amino of serial number 13
The nucleic acid sequence of acid sequence and serial number 35) form.Therefore, glucagon-like-peptide-1 (7-37) hydroxyl (OH) and pancreas hyperglycemia
Plain sample peptide -1 (7-36) amide is equivalent to two kinds of natural type glucagon-like-peptide-1s.
But in vivo, it is cut at a very rapid rate by dipeptidyl peptidase-4, causes to lose activity
Deng, with drug development during exist many difficult, in order to increase biology intracorporal half-life period, persistently carried out various taste
Examination.In this regard, attempted to reduce the cutting by dipeptidyl peptidase-4 by making above-mentioned glucagon-like-peptide-1 mutate,
And then glucagon-like peptide-1 analogs are prepared, above-mentioned glucagon-like peptide-1 analogs are as a result, in order to realize above-mentioned mesh
, analog disclosed in industry belonging to the present invention can be used without limitation.
Glucagon-like-peptide-1 is lost specifically, the displacement of the amino acid of No. 8 positions can reduce dipeptidyl peptidase-4
A possibility that speed of deactivation, the displacement of the amino acid of No. 22 positions can reduce molecule agglutination and the efficiency for increasing molecule, 36
The displacement of the amino acid of number position can be reduced conjugated protein is into induction after human body repeatedly continuous injection and immune anti-
The risk answered.Therefore, though it is without being limited thereto, the form that the amino acid of above-mentioned No. 8, No. 22 and No. 36 positions can be used to be replaced
Analog, in addition to this it is possible to use No. 33, No. 34 and No. 37 positions as disclosed in United States Patent (USP) US 7,452,966B2 etc.
The analog of form that is replaced of amino acid.
As a more specific example, it is preferable that the analog of above-mentioned glucagon-like-peptide-1 can be by two peptidyls
Peptase -4 and the mutation that cleavage site occurs.Dipeptidyl peptidase-4 is in No. 8 of above-mentioned glucagon-like-peptide-1 and No. 9 amino acid
Between cut glucagon-like-peptide-1, be replaced as glycine (G) or figured silk fabrics ammonia as the alanine (A) of No. 8 amino acid as a result,
The glucagon-like peptide-1 analogs of sour (V) can reduce the cutting because of dipeptidyl peptidase-4.
Also, it can be replaced by glutamic acid (E) as the above-mentioned glycine (G) of No. 22 amino acid, as No. 36 amino acid
Arginine (R) can be replaced by glycine (G).
It is thus preferable that above-mentioned glucagon-like peptide-1 analogs can be from glucagon-like-peptide-1 (7-37)
In the ammonia with serial number 2,3,4,5,6,7,8,9,10,11 or 12 of form that is replaced of No. 8, No. 22 and/or No. 36 amino acid
The glucagon-like peptide-1 analogs of base acid sequence, can be from glucagon-like-peptide-1 (7-36) No. 8, No. 22 and/
Or the amino acid sequence with serial number 14,15,16,17,18,19,20,21,22,23 or 24 of form that No. 36 amino acid are replaced
The glucagon-like peptide-1 analogs of column.Also, it is highly preferred that can be as the cleavage site by dipeptidyl peptidase-4
The amino acid with serial number 2,3,6,7,8,9,11,12,14,15,18,19,20,21,23 or 24 that is mutated of No. 8 amino acid
The glucagon-like peptide-1 analogs of sequence, most preferably, can be replaced as the alanine (A) of No. 8 amino acid it is sweet
The glucagon-like peptide-1 analogs of the amino acid sequence with serial number 2 of propylhomoserin (G).Amino acid sequence with serial number 2
Above-mentioned glucagon-like peptide-1 analogs can be encoded by the nucleic acid sequence of serial number 34.In one embodiment of the invention
In, its efficiency is confirmed using the above-mentioned glucagon-like peptide-1 analogs of the amino acid sequence with serial number 2.The above-mentioned high blood of pancreas
The mutated site of sugared -1 analog of element sample peptide is as shown in the table.
[table 1]
Present invention recognize that glucagon-like-peptide-1 as described above or its analog are difficult to as medicine for treating diabetes
Object realizes that reasonable employment develops the immunoglobulin Fc polypeptide of suitable glucagon-like-peptide-1 in order to solve this problem,
And the fused polypeptide combined with this is prepared, here it is technical characteristic of the invention specifically, by dramatically increasing pancreas height
The biology intracorporal half-life period of blood glucose element sample peptide -1, so that glucagon-like-peptide-1 persistently plays drug effect in vivo
Energy.Addedly, above-mentioned fused polypeptide intraserous stability, dipeptidyl peptidase-4 repellence and in terms of
Also very outstanding.
For this purpose, in the present invention, first by will be prepared in the Chinese patent CN 101687933 as existing patent
The heterozygosis Fc5 according to immunoglobulin heterozygosis Fc technology combined with above-mentioned glucagon-like-peptide-1, come prepare fusion it is more
Peptide.The term " heterozygosis (hybrid) " being used in the present invention indicates more than two immunoglobulin Fcs in mutually different source
Fragment encoding se is present in single-chain immunoglobulins Fc segment.Above-mentioned heterozygosis Fc5 is the IgD hinge comprising 30 amino acid lengths
The heterozygosis Fc in chain region shows very big half-life period increase effect when being suitable for larger protein (Large protein)
Fruit, the small peptide short about relative length (Short peptide), such as when being suitable for the invention glucagon-like-peptide-1
When, compared to as described above be suitable for larger protein the case where, half-life period increase effect it is little, prepare as a result, effect into
The fused polypeptide that one step is improved.
The term " immunoglobulin Fc segments " being used in the present invention or " immunoglobulin Fc " indicate to include immune ball
The heavy chain constant region (CH) of albumen, the heavy chain of immunoglobulin and the Variable Area of light chain and chain constant region (CL) are indicated
It does not include protein.Above-mentioned Fc may also include hinge area, purpose according to the present invention, although including heavy chain constant region 2
(CH2) and heavy chain constant region 3 (CH3), but it may include or do not include heavy chain constant region (CH1).
Immunoglobulin Fc segments of the invention can include hinge area, CH2 structure along C- end direction from the end N-
Domain region and the domain region CH3.Specifically, immunoglobulin Fc segments of the invention can be hybrid immunoglobulin Fc
Segment, above-mentioned hinge area may include human Ig hinge region as a result, the above-mentioned domain region CH2 may include mankind IgD and
The amino acid residue part of IgG4CH2 structural domain, the above-mentioned domain region CH3 may include the amino of human IgG 4CH3 structural domain
Sour residue moiety.
The suitable immunoglobulin Fc polypeptide that can be combined with glucagon-like-peptide-1 or its analog of the invention,
It is characterized in that, the IgD hinge area comprising 35 to 49 amino acid lengths.The main function of above-mentioned hinge area be when and pancreas
When the biologically active molecules such as glucagon-like peptide -1 combine, by keeping its flexible (flexibility), to keep its knot
Structure.Specifically, in the amino acid sequence (being encoded with the nucleic acid sequence of serial number 36) of the serial number 25 as IgD hinge area
In, from the end C- along N- end direction, it may include the isolated IgD hinge area with 35 to 49 continuous amino acid sequences
Domain.And, it is preferable that in the amino acid sequence of serial number 25, can be from the end C- along N- end direction have 35 to
The IgD hinge area of 40 continuous amino acid sequences, it is highly preferred that can be with 35 or 40 continuous amino acid sequences
The IgD hinge area of column, it is highly preferred that can be the IgD hinge area with 40 continuous amino acid sequences.In serial number 25
Above-mentioned amino acid sequence in, serial number 26 is expressed as, by 40 by the IgD hinge area that 35 continuous amino acid sequences are constituted
The IgD that the IgD hinge area that continuous amino acid sequence is constituted is expressed as serial number 27, is made of 49 continuous amino acid sequences
The nucleic acid sequence that hinge area is expressed as serial number 28, is encoded to the amino acid sequence of serial number 26 is expressed as serial number 37, to sequence
The nucleic acid sequence that numbers 27 amino acid sequence is encoded is expressed as serial number 38, is encoded to the amino acid sequence of serial number 28
Nucleic acid sequence is expressed as serial number 39.
In the present invention, though the region immunoglobulin Fc CH2 and CH3 combined with IgD hinge area as described above
Do not change the pharmacology and drug efficiency of fused polypeptide of the invention, or unless causes Antibody -dependent cell cytotoxicity
The cytotoxicities such as (ADCC) and/or complement dependent cytotoxicity (CDC) are acted on, otherwise can be used without limitation, it is preferable that can make
The region heterozygosis Fc CH2 and CH3 of the IgD and IgG4 that are developed with the present inventor.Specifically, the amino by serial number 29 can be used
Acid sequence or the region CH2 and CH3 constituted with the amino acid sequence that the nucleic acid sequence of serial number 40 is encoded.
By glucagon-like-peptide-1 as described above or its analog and include IgD hinge area and the area CH2 and CH3
The immunoglobulin Fc polypeptide in domain combines and finally constitutes fused polypeptide of the invention.
Although being defined not to this, as an example, fused polypeptide of the invention can be by selected from by 30 to 32 group of serial number
At group in amino acid sequence constitute, more specifically, can be made of the amino acid sequence of serial number 30 or 31, or can be by serial number
31 amino acid sequence is constituted.The above-mentioned amino acid sequence of serial number 30 is the glucagon-like-peptide-1 class of serial number 2 of the invention
The form that is combined like the region CH2 and CH3 of object, the IgD hinge area of serial number 26 and serial number 29 and with " glucagon
Peptide -1- heterozygosis Fc8 " indicates, the amino acid sequence of serial number 31 be serial number 2 of the invention glucagon-like peptide-1 analogs,
Form that the IgD hinge area of serial number 27 and the region CH2 and CH3 of serial number 29 combine and with " glucagon-like-peptide-1-is miscellaneous
Closing Fc9 " indicates, the amino acid sequence of serial number 32 is the glucagon-like peptide-1 analogs of serial number 2 of the invention, serial number 28
Form that the region CH2 and CH3 of IgD hinge area and serial number 29 combines and with " glucagon-like-peptide-1-heterozygosis Fc11 "
It indicates.
In one embodiment of this invention, existing comprising by 30 amino acid by will be developed by the present inventor first
The heterozygosis Fc5 of the IgD hinge area of Sequence composition is combined with glucagon-like-peptide-1 and is prepared glucagon-like peptide-
1- heterozygosis Fc5 (Fig. 1), half-life period and the independent peptide comparison result of existing glucagon-like-peptide-1, half-life period are realized
Increase.But it is suitable for the effect that is shown in the case where larger protein compared to by heterozygosis Fc5, when heterozygosis Fc5 is applicable in
When the glucagon-like-peptide-1 of the small peptide short as length, it is not high that the half-life period shown increases effect.
The present inventor attempts to find and can express higher than heterozygosis Fc5's when being suitable for glucagon-like-peptide-1 as a result,
The immunoglobulin Fc polypeptide of outstanding efficiency, as a result, it has been found that, for the short peptide of such as glucagon-like-peptide-1 equal length
The case where, when increasing the length of hinge area, can prepare more with outstanding half-life period and outstanding active fusion
Peptide.That is, in above-mentioned IgD hinge area there is be easy to by protease (protease) decompose and to above-mentioned enzyme reaction it is sensitive
Cleavage site, it is above-mentioned to cut in the case where the size of the physiologically active protein matter combined with immunoglobulin Fc polypeptide is big
It cuts site to be not exposed, to will not cause to go wrong, but in the feelings of the short peptide of glucagon-like-peptide-1 equal length
Under condition, cleavage site is exposed, and is combined by Fc polypeptide, does not occur expected half-life period increase effect.But this is analyzed
Out, when increasing the length of hinge area, have and solve the problems, such as a possibility that this.
In this regard, in one embodiment of this invention, preparing with the IgD hinge area being made of 40 amino acid sequences
Heterozygosis Fc9 (Fig. 3) prepare the heterozygosis with the IgD hinge area being made of 35 amino acid sequences and based on this
Fc8 and heterozygosis Fc11 with the IgD hinge area being made of 49 amino acid sequences, finally prepares glucagon
Peptide-1- heterozygosis Fc9, glucagon-like-peptide-1-heterozygosis Fc8 and glucagon-like-peptide-1-heterozygosis Fc11 fused polypeptide.And
And to the pharmacokinetic data (PK Profile) of prepared above-mentioned fused polypeptide of the invention be measured as a result,
Be higher than glucagon-like-peptide-1 independence peptide and glucagon-like-peptide-1-heterozygosis Fc5 half-life period (Fig. 4 and
Fig. 5), more effective drug efficiency is shown.
Also, as representative fused polypeptide in above-mentioned fused polypeptide, with glucagon-like-peptide-1-heterozygosis
Fc9 is that object and existing glucagon-like-peptide-1-heterozygosis Fc5 carry out various Effectiveness Comparisons as a result, showing in serum
Stability (Fig. 6), dipeptidyl peptidase-4 repellence (Fig. 7) and pharmacodynamic properties (PD Profile) (Fig. 8) it is all
Aspect shows apparent outstanding property.
Meanwhile in one more embodiment of the present invention, for above-mentioned glucagon-like-peptide-1-heterozygosis Fc9, compare pancreas height
What blood glucose element sample peptide -1- connector-IgG4-mut (United States Patent (USP) US 7,452,966B2) and inhibition antibody-dependant cell mediated
Cytotoxicity (ADCC) active ability as a result, glucagon-like-peptide-1-heterozygosis Fc9 inhibition antibody-dependant cell be situated between
Cytotoxicity (ADCC) the active ability led is outstanding (Fig. 9).Substantially, above-mentioned glucagon-like-peptide-1-connector-
IgG4-mut is in order to inhibit Antibody -dependent cell cytotoxicity to act on (ADCC), so that the part amino of immunoglobulin Fc
Sour site mutates, and there is glucagon-like-peptide-1 of the invention-heterozygosis Fc9 higher antibody-dependant cell to mediate
The rejection ability of cytotoxicity (ADCC).
Synthesis is this as a result, compared to existing glucagon-like-peptide-1, and fused polypeptide of the invention has increased
Half-life period has outstanding effect of reducing blood sugar energy and the repellence to dipeptidyl peptidase-4, is effectively used for treating diabetes.
In this regard, providing using above-mentioned fused polypeptide as effective component as yet further embodiment of the invention and including
Treating diabetes pharmaceutical composition.Also, as another embodiment, offer of the invention is used to prepare diabetes and controls
Treat the purposes of the above-mentioned fused polypeptide with medicament.At this point, above-mentioned diabetes can be type-II diabetes.
Fused polypeptide of the invention includes the existing glucagon-like-peptide-1 well-known as Remedies for diabetes
Or its analog, treating diabetes are effectively used for, compared to existing medicament, fused polypeptide of the invention has increased
Half-life period, outstanding effect of reducing blood sugar energy, the repellence to dipeptidyl peptidase-4 is also added, had compared to existing drug excellent
Elegant Profile, is applicable to pharmaceuticals.
Fused polypeptide of the invention can be widely used in the various diseases for the treatment of and symptom.Fused polypeptide of the invention is made first
Receptor for being referred to " glucagon-like peptide-1 receptor ", and play their biology effect.Therefore, of the invention to melt
Conjunction polypeptide, which can treat, makes the investment of glucagon-like peptide-1 receptor stimulation or glucagon-like-peptide-1 compound
The disease of close friend's reaction and/or the subject of symptom.This subject, which is referred to as, " needs glucagon-like-peptide-1 compound
Treatment " or " stimulation for needing glucagon-like peptide-1 receptor ".Including with adult-onset diabetes, insulin
Dependent diabetes mellitus, apoplexy (referring to WO 00/16797), myocardial infarction (referring to WO 98/08531), fat (reference WO 98/
19698), postoperative mutation (referring to U.S. Patent No. 6,006,753), functional dyspepsia FD and irritable bowel syndrome (ginseng
According to WO 99/64060) subject.The preventative-therapeutic subject of glucagon-like-peptide-1 compound is needed, such as also
Including the subject with adult-onset diabetes onset risk (referring to WO 00/07617).With impaired grape
Sugared patience or the subject of impaired glucose of going on a hunger strike, weight are more than the height figure of the subject in relation to the pact of normal type
25% subject, the subject for receiving local pancreatectomy operation, at least one in parent suffer from non-insulin-dependent
The subject of diabetes, the once subject with gestational diabetes mellitus and the subject for once suffering from acute or chronic pancreatitis
Deng with adult-onset diabetes onset risk.
The effective quantity for the fused polypeptide recorded in the present invention is directed to need the stimulation of glucagon-like peptide-1 receptor
Subject can be realized when putting into fused polypeptide neither induce side effect can reach again goal treatment and/or preventive effect perhaps
It can measure." objective response " includes following at least one.1) mitigate disease or symptom in relation to each sign；2) postpone disease
It is sick or the related sign of symptom to start to occur；3) compared to not treating the case where, service life increase；And 4) compared to not treating
Situation, quality of life improve.For example, " effective quantity " of the fused polypeptide of the invention for treating diabetes, compared to not controlling
The case where treatment, can preferably control blood sugar concentration, can postpone the diabetic complications such as retinopathy, neurological disorder or kidney diaseases
Morbidity.For preventing " effective quantity " of the fused polypeptide of diabetes, compared to untreated situation, can postpone to need with sulphonyl
The blood glucose level for the rising that the hypoglycemias drug such as ureas, thiazolidinediones, insulin and (or) biguanides is treated
Morbidity.
In one embodiment of this invention, it is authenticated and is illustrated except the experiment of pharmacokinetic data really by half-life period,
Confirmed, fused polypeptide of the invention also has outstanding Remedies for diabetes efficiency.Specifically, (in vivo
Vivo) experiment in, to glucagon-like-peptide-1-heterozygosis Fc9 fused polypeptide pharmacodynamic properties (PD profile) into
Go confirming as a result, there is obvious outstanding effect of reducing blood sugar energy (Fig. 8) compared to glucagon-like-peptide-1-heterozygosis Fc5,
In vitro in (in vitro) experiment, the obvious outstanding repellence to dipeptidyl peptidase-4 is also shown, it was demonstrated that in two peptidyls
Peptase -4 is in relation to also having outstanding efficiency (Fig. 7) in terms of stability.In treating diabetes, glucose content in blood
Keep critically important, it is contemplated that dipeptidyl peptidase-4 inhibitors are typically used as diabetes refrigerant, and fused polypeptide of the invention is being controlled
It may be used as outstanding drug during treating diabetes.
In the present invention, term " treatment " refers to by injecting fused polypeptide of the invention or comprising the drug of fused polypeptide
Composition, so that all behaviors that the symptom of diabetes improves or it is made to take a favorable turn.
It can include to melt with various weight % if pharmaceutical composition of the invention can express the therapeutic effect of diabetes
Polypeptide is closed as effective component.
Also, pharmaceutical composition of the invention can also include according to the carrier appropriate of usual way, excipient or
Diluent.It include but is not limited to lactose, grape as may include in the carrier, excipient and diluent of composition of the invention
Sugar, sucrose, sorbierite, mannitol, xylitol, erythrite, maltitol, starch, acacia gum, alginates, gelatin, phosphoric acid
Calcium, calcium silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, to hydroxyl
Yl benzoic acid propyl ester, talcum, magnesium stearate and mineral oil.
Pharmaceutical composition of the invention can be according to usual way with powder, granule, pastille, capsule, suspension, cream
The oral types such as shape liquid, slurries, aerosol dosage form, external preparation, suppository or sterilizing injecting solution form dosage form after use.Specifically
For, in the case where dosage form, common filler, diluent, bonding agent, wetting agent, disintegrating agent, surface-active can be used
It is prepared by the diluents such as agent or excipient.There are pastille, pill, powder, granule, capsule etc. for oral solid pharmaceutical preparation,
In this solid pharmaceutical preparation, above compound can heterozygosis at least one excipient prepare, for example, starch, calcium carbonate, sucrose,
Lactose, gelatin etc..Also, other than simple excipient, the lubricants such as magnesium stearate, talcum can also be used.For mouth
The liquid preparation of clothes has suspending agent, interior liquor, emulsion, slurry agent etc., in addition to the usually used water as simple diluent, liquid
It can also include various excipient, such as wetting agent, sweetener, aromatic, antistaling agent etc. except body paraffin.Non-oral formulation
Aqueous solution, non-aqueous solvent, suspending agent, emulsion, lyophilized preparation and suppository including sterilizing.As non-aqueous solvent, suspending agent,
The ethyl ester etc. of the injectables such as plant oils, the ethyl oleates such as propylene glycol, polyethylene glycol, olive oil can be used.System as suppository
Acrawax, polyethylene glycol, Tween61, cocoa butter, laurine, glycerin gelatine etc. can be used in agent.
Composition of the invention is with pharmaceutically effectively amount medication.Above-mentioned effective quantity pharmaceutically refers to be applicable to cure
Reasonable benefit/the risk ratio for learning treatment is enough to treat disease, and does not cause the amount of side effect, and effective dose level can be according to packet
Include the health status of patient, the type of disease, severe degree, the activity of drug, the sensibility to drug, administrated method, medication
It time, medication path and excludes generally to recognize in ratio, treatment time limit, the element of cooperation or concomitant medicament and other medical domains
The element known and determine.
Also, pharmaceutical composition of the invention can be used alone or with the other drugs that show treating diabetes effect
It is used cooperatively.Pharmaceutical compositions of the invention can be by various paths to the mammals medication such as rat, mouse, domestic animal, mankind.
Above-mentioned medication indicates to provide defined substance, as long as destination organization can be reached, the present invention to patient in all suitable methods
Composition medication path it is unrestricted.E.g., including but be not limited to intra-articular injection, intraperitoneal injection, intravenous injection,
Intramuscular injection, subcutaneous injection, intracutaneous injection, oral, locally injecting, nasal injection, pulmonary injection, intrarectal injection.
Preferably, fused polypeptide of the invention is primary with surrounding, biweekly or weekly period medication.According to institute
The disease for the treatment of, fused polypeptide can 2 to 3 more frequent period medications weekly.
As yet another embodiment, the present invention is provided to the polynucleotides that above-mentioned fused polypeptide is encoded,
The host cell of expression vector comprising above-mentioned polynucleotides and the above-mentioned expression vector of protection.
The present invention is characterized in that above-mentioned fused polypeptide, the polynucleotides encoded to above-mentioned fused polypeptide include upper
The host cell of the expression vector and the above-mentioned expression vector of protection of stating polynucleotides is also contained in the scope of the present invention, as long as
It can express above-mentioned fused polypeptide, type is unrestricted.
Above-mentioned polynucleotides are unrestricted, such as can be by being encoded to " glucagon-like-peptide-1-heterozygosis Fc8 "
The nucleic acid sequence of serial number 41, the nucleic acid sequence or right for the serial number 42 that " glucagon-like-peptide-1-heterozygosis Fc9 " is encoded
The nucleic acid sequence for the serial number 43 that " glucagon-like-peptide-1-heterozygosis Fc11 " is encoded is constituted, or can also be by serial number 42
Nucleic acid sequence constitute.
Degeneracy (degeneracy) by codon or the biological institute in view of attempting to show above-mentioned fused polypeptide
The codon of preference, above-mentioned polynucleotides can be realized respectively in the range of not changing the amino acid sequence of showed fused polypeptide
Kind deformation.
[The effect of invention]
Compared to existing glucagon-like-peptide-1, fused polypeptide of the invention has increased half-life period, and has
Outstanding effect of reducing blood sugar energy and the repellence to dipeptidyl peptidase-4, during treating diabetes, compared to existing medicament,
Outstanding drug efficiency is shown, pharmaceuticals can be effectively served as.
Detailed description of the invention
Fig. 1 is to prepare related figure with glucagon-like-peptide-1-heterozygosis Fc5, indicates that glucagon-like-peptide-1-is miscellaneous
Close the synoptic diagram and glucagon-like-peptide-1-heterozygosis Fc5 sequence of Fc5.
Fig. 2 is to prove glucagon-like-peptide-1 peptide and glucagon-like-peptide-1-heterozygosis Fc5 pharmacokinetics
The figure of data.
Fig. 3 is to prepare related figure with glucagon-like-peptide-1-heterozygosis Fc9, indicates that glucagon-like-peptide-1-is miscellaneous
Close Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 sequence and synoptic diagram.
Fig. 4 be and glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 pharmacokinetics
The related figure of data indicates area (AUC) value under albumen quality and drug concentration profile remaining in the blood of each period.
Fig. 5 be and glucagon-like-peptide-1-heterozygosis Fc5, glucagon-like-peptide-1-heterozygosis Fc8 and glucagon
The related figure of pharmacokinetic data of sample peptide -1- heterozygosis Fc9, indicates albumen quality remaining in the blood of each period.
Fig. 6 is to prove in glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 serum surely
Qualitatively figure indicates the albumen quality of the remaining of each reaction period and area under the curve (AUC) value of time plot.
Fig. 7 is to prove glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 dipeptidyl peptidase
The figure of the repellence of enzyme -4 indicates the albumen quality of the remaining of each reaction period and the area under the curve of time plot
(AUC) value.
Fig. 8 be and glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 pharmacodynamics
The related figure of characteristic finds out area under the curve value, and by measurement the concentration of glucose in blood variation to compare negative control group
Area under the curve value content % indicate.
Fig. 9 is to compare glucagon-like-peptide-1-heterozygosis Fc9 and glucagon-like-peptide-1-connector-IgG4-mut
The figure of Antibody -dependent cell cytotoxicity effect (ADCC) rejection ability, it was demonstrated that the binding ability of Fc γ receptor.
Specific embodiment
In the following, the present invention will be described in more details by following embodiments.These embodiments are used for the purpose of
The present invention is illustrated, the scope of the present invention is not restricted by the embodiments.
Embodiment 1: glucagon-like-peptide-1-heterozygosis Fc5 preparation and its pharmacokinetic data confirmation
Glucagon-like-peptide-1 (Glucagon like peptide-1) is effective protein in treating diabetes,
In vivo, glucagon-like-peptide-1 is cut at a very rapid rate by dipeptidyl peptidase-4, and half-life period is about 3
To 5 minutes, the time was very short, therefore, if existing many difficult as drug development.In this regard, implementing many how in life
Increase the trial of half-life period in object.
In this regard, being based on glucagon-like-peptide-1 (7- first of all for the cutting reduced by above-mentioned dipeptidyl peptidase-4
37) it, in alanine, at glycine, and then will be made as the cleavage site by dipeptidyl peptidase-4 No. 8 amino acid replacements
Standby glucagon-like peptide-1 analogs (serial number 2).It then, will be in the Chinese patent CN of the existing patent as the present inventor
Heterozygosis Fc5 (hybrid Fc 5) polypeptide and the above-mentioned glucagon-like peptide-1 analogs peptide phase prepared in 101687933
In conjunction with preparing glucagon-like-peptide-1-heterozygosis Fc5 fused polypeptide (Fig. 1).Above-mentioned glucagon-like-peptide-1-heterozygosis Fc5
The full sequence of fused polypeptide is as shown in Figure 1.
In order to confirm prepared above-mentioned glucagon-like-peptide-1-heterozygosis Fc5 fused polypeptide pharmacokinetic properties
(pharmacokinetic profile, PK profile) as a control group by the glucagon-like-peptide-1 of synthesis is executed
Following experiment.
Albumen is injected by vein (IV) injection path respectively for every group of four male Sprague Dawley Rats
Matter (glucagon-like-peptide-1 and glucagon-like-peptide-1-heterozygosis Fc5).Before the injection with after injection 0.08,0.16,
0.5, blood is taken respectively within 1,2,4,6,12,24,48,72 and 96 hour.In order to realize condensation, blood sample takes care of 30 in room temperature
Minute.After the centrifuge separation of 3000rpm implementation 10 minutes, the serum of each sample is extracted, ultra-low temperature cold frozen storehouse is stored in
(deep freezer).Sample is diluted using glucagon-like-peptide-1 kit (ALPCO, cat#43-GP1HU-E01)
With quantitatively, make it possible to be analyzed on the straight line of standard curve.
As a result, as shown in Fig. 2, the glucagon-like-peptide-1 independent peptide of unbonded heterozygosis Fc polypeptide the case where
Under, half-life period is 4 minutes, but in contrast, in the glucagon-like peptide-for combining glucagon-like-peptide-1 and heterozygosis Fc5
In the case where 1- heterozygosis Fc5 polypeptide, half-life period is 8 hours or more, increases about 116 times than the former.
Embodiment 2: glucagon-like-peptide-1-heterozygosis Fc9, glucagon-like-peptide-1-heterozygosis Fc8 and glucagon
The preparation of sample peptide -1- heterozygosis Fc11
Attempt to prepare half-life period greater than glucagon-like-peptide-1-heterozygosis Fc5 half-life period and in terms of other activity
Also outstanding fused polypeptide.
Specifically, being prepared by carrying out various adjustings to IgD hinge area in heterozygosis Fc5 with more outstanding
Half-life period and more outstanding active fused polypeptide, it is demonstrated experimentally that increase IgD hinge area amino acid the case where
Under, it is possible to meet this condition.
As a result, increase the amino acid of the hinge area of the heterozygosis Fc5 with the hinge by 30 Amino acid profiles, first
Prepare with by 40 Amino acid profiles hinge (serial number 27) heterozygosis Fc9 (Fig. 3), then, prepare respectively have by
The heterozygosis Fc8 of the hinge (serial number 26) of 35 Amino acid profiles and and with the hinge (serial number 28) by 49 Amino acid profiles
Heterozygosis Fc11.Meanwhile by above-mentioned heterozygosis Fc9, heterozygosis Fc8 and heterozygosis Fc11 respectively with glucagon-like peptide-1 analogs contracting ammonia
Acid combines, and prepares glucagon-like-peptide-1-heterozygosis Fc9 (serial number 31), glucagon-like-peptide-1-heterozygosis Fc8 respectively
(serial number 30) and glucagon-like-peptide-1-heterozygosis Fc11 (serial number 32) fused polypeptide.
Embodiment 3: glucagon-like-peptide-1-heterozygosis Fc9, glucagon-like-peptide-1-heterozygosis Fc8 and glucagon
Sample peptide -1- heterozygosis Fc11, pharmacokinetic data confirmation
3-1: glucagon-like-peptide-1-heterozygosis Fc9 pharmacokinetic data confirmation
Firstly, partly declining by comparing the glucagon-like-peptide-1-heterozygosis Fc9 fused polypeptide prepared in example 2
Phase and the glucagon-like-peptide-1-heterozygosis Fc5 fused polypeptide half-life period prepared in embodiment 1, it is intended to confirm that medicine generation is dynamic
Mechanical Data.
Albumen is injected by subcutaneous (SC) injection path respectively for every group of four male Sprague Dawley Rats
Matter.Blood is taken respectively with 2,6,12,26,48,72,96,120,144 and 168 hours after injection before the injection.In order to realize
Condensation, blood sample are taken care of 30 minutes in room temperature.After the centrifuge separation of 3000rpm implementation 10 minutes, each sample is extracted
Serum is stored in ultra-low temperature cold frozen storehouse.Sample is carried out dilute using glucagon-like-peptide-1 kit (IBL, Cat#27784A)
It releases and quantifies, make it possible to be analyzed on the straight line of standard curve.
Its result is with area (AUC, Area under albumen quality and drug concentration profile remaining in the blood of each period
Under the Curve) value expression.As shown in figure 4, compared to glucagon-like-peptide-1-heterozygosis Fc5, in glucagon
About the former 12 times of area under the curve value is shown in sample peptide -1- heterozygosis Fc9.Based on this result it is found that compared to pancreas height
Blood glucose element sample peptide-1- heterozygosis Fc5, glucagon-like-peptide-1-heterozygosis Fc9 half-life period further increase, show more added with
The drug efficiency of effect.
3-2: glucagon-like-peptide-1-heterozygosis Fc8 pharmacokinetic data confirmation
Then, glucagon-like-peptide-1-heterozygosis Fc8 fused polypeptide half-life period and glucagon-like-peptide-1-is miscellaneous
It closes Fc9 fused polypeptide to be compared, it is intended to confirm glucagon-like-peptide-1-heterozygosis Fc8 pharmacokinetic data.With pancreas height
Blood glucose element sample peptide-1- heterozygosis Fc5, glucagon-like-peptide-1-heterozygosis Fc9 and glucagon-like-peptide-1-heterozygosis Fc8 are pair
As executing experiment identical with above-described embodiment 3-1.
As a result, as shown in figure 5, glucagon-like-peptide-1-heterozygosis Fc9 and glucagon-like-peptide-1-heterozygosis Fc8 is aobvious
The pharmacokinetic data of similar level is shown, glucagon-like-peptide-1-heterozygosis Fc8 performance level is slightly higher.Compared to conduct
The glucagon-like-peptide-1 of control group-heterozygosis Fc5, glucagon-like-peptide-1-heterozygosis Fc8 show significant outstanding water
It is flat.Based on this result it is found that glucagon-like-peptide-1-heterozygosis Fc8 half-life period equally further increases, show more
Effective drug efficiency.
Embodiment 4: glucagon-like-peptide-1-heterozygosis Fc9 complementarity efficiency confirmation
Hereinafter, by be shown compared to glucagon-like-peptide-1 as a control group-heterozygosis Fc5 in above-described embodiment 3
Showing glucagon-like-peptide-1-heterozygosis Fc9 in the fused polypeptide of outstanding pharmacokinetic data is object, it is intended to be confirmed
The various efficiency of complementarity.As a control group, using glucagon-like-peptide-1-heterozygosis Fc5.
4-1: glucagon-like-peptide-1-heterozygosis Fc9 serum internal stability confirmation
In order to be directed to glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9, confirm in serum
Factoring Dependent Stability executes the test of rat blood serum internal stability.
Firstly, diluting two kinds of substances using rat blood serum, each sample of preparation is put into 37 DEG C of thermostats, is made anti-
After answering 0,6,10,24,48 hour, each substance is quantified using enzyme-linked immunosorbent assay (ELISA) method.
Its result with the area under the curve of albumen quality and time plot remaining in the blood of each period (AUC,
Area Under the Curve) value expression.As shown in fig. 6, compared to glucagon-like-peptide-1-heterozygosis Fc5, in the high blood of pancreas
About the former 1.2 times of area under the curve value is shown in sugared element sample peptide -1- heterozygosis Fc9.Based on this result it is found that compared to
Glucagon-like-peptide-1-heterozygosis Fc5, glucagon-like-peptide-1-heterozygosis Fc9 serum internal stability are more outstanding.
4-2: glucagon-like-peptide-1-heterozygosis Fc9 dipeptidyl peptidase-4 repellence confirmation
In order to confirm glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 to as main
The repellence and Dependent Stability of the dipeptidyl peptidase-4 (Sigma, #D4943-1VL) of metabolic enzyme, perform dipeptidyl peptidase
The test of -4 repellence of enzyme.
Two kinds of substances and dipeptidyl peptidase-4 are put into 37 by reaction time (0,2,8,24,48 hour) according to schedule
DEG C thermostat, respectively quantitative material.
Its result is with area under the curve (AUC) value of albumen quality and time plot remaining in the blood of each period
It indicates.As shown in fig. 7, compared to glucagon-like-peptide-1-heterozygosis Fc5, the table in glucagon-like-peptide-1-heterozygosis Fc9
Reveal about the former 7 times or more of dipeptidyl peptidase-4 repellence.Based on this result it is found that compared to glucagon
Peptide -1- heterozygosis Fc5, the dipeptidyl peptidase-4 effect stability aspect of cleavable glucagon-like-peptide-1, glucagon
Peptide -1- heterozygosis Fc9 is also significant outstanding.
4-3: glucagon-like-peptide-1-heterozygosis Fc9 PD Profile confirmation
In order to confirm prepared glucagon-like-peptide-1-heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9
Pharmacodynamic properties execute following experiment.
For every group of ten CD-1 mouse, by subcutaneous (SC) injection path, after injecting protein respectively, every 0,1,2,
4 and 8 days, glucose is put into, measures the variation of glucose in blood, confirmation blood glucose reduces ability.
Glucose in blood is measured from during 1 minute to 180 minutes after investment glucose in each measurement day
Concentration variation, finds out the area under the curve value of each test day, and compares negative control group (vehicle), indicates glucagon
Sample peptide-1- heterozygosis Fc5 and glucagon-like-peptide-1-heterozygosis Fc9 area under the curve content %.
As a result, as shown in figure 8, in the case where glucagon-like-peptide-1-heterozygosis Fc5, since second day, blood glucose
Reduction ability disappears and restores normalization (normalization), on the contrary, in glucagon-like-peptide-1-heterozygosis Fc9 feelings
Under condition, the low state of Glucose in Blood by Cyclic was also maintained until the 8th day.This result means using pancreas hyperglycemia
In the case where plain sample peptide -1- heterozygosis Fc9, it was also able to maintain blood glucose until the 8th day and reduces ability.
In summary, as shown in the table, compared to glucagon-like-peptide-1-heterozygosis Fc5, glucagon-like-peptide-1-
Heterozygosis Fc9 shows more excellent efficiency at all aspects for completing test.
[table 2]
Embodiment 5: with glucagon-like-peptide-1-connector-IgG4-mut Antibody -dependent cell cytotoxicity
Effect (ADCC) rejection ability compares
Then, for showing the glucagon-like-peptide-1-of outstanding efficiency in all respects through the foregoing embodiment
Heterozygosis Fc9 fused polypeptide passes through Antibody -dependent cell cytotoxicity effect related with existing fused polypeptide carry out
(ADCC) comparison of rejection ability, it is intended to prove its outstanding property.
Using glucagon-like-peptide-1-connector-IgG4-mut disclosed in United States Patent (USP) US 7,452,966B2 as pair
According to group, it is to be mutated at a kind of site IgG4 3 and Antibody -dependent cell cytotoxicity is hindered to act on (ADCC, Antibody
Dependent Cell-mediated Cytotoxicity) polypeptide.
Above-mentioned glucagon-like-peptide-1-connector-IgG4-mut and glucagon-like-peptide-1-heterozygosis of the invention
Fc9 includes the position CH2-CH3 of IgG4 in structure, and complement dependent cytotoxicity (Complement is not present
Dependent Cytotoxicity) related security issues, but there are Antibody -dependent cell cytotoxicity effect is related
Therefore safety issue in order to be confirmed to this, is measured in the process for inducing Antibody -dependent cell cytotoxicity effect
In the Fc γ receptor correlation binding ability that plays an important role, utilize surface plasma resonant vibration (SPR, Bio- for this purpose, performing
Rad, #Proteon XPR36) combining power test.
Firstly, being reacted by NHS/EDC, using acetate buffer, to Bole's fragment (bio-rad of amine pairing
Chip Fc γ receptor is dripped in each channel), makes ligand immobilisation.As the concept of negative control group, drippage is added with polysorbas20
Phosphate buffered saline (PBS) (Phosphate Buffered Saline).It is dripped on the fragment for being combined with each Fc γ receptor each
Substances measure binding force.
As a result, as shown in figure 9, glucagon-like-peptide-1-connector-IgG4-mut is in order to eliminate immunoglobulin Fc
The residual effect function at position deforms multiple amino acid sites, is inducing Antibody -dependent cell cytotoxicity effect
It shows to be higher than glucagon-like-peptide-1-heterozygosis Fc9 binding ability on main Fc γ receptor, it follows that having potential
A possibility that causing cytotoxicity.On the contrary, compared to glucagon-like-peptide-1-connector-IgG4-mut, the high blood of pancreas
Sugared element sample peptide -1- heterozygosis Fc9 is low with the binding ability of all Fc γ receptors, it follows that in long-term administration, it can be achieved that into one
The safety of step.
Claims (11)
1. a kind of fused polypeptide, which is characterized in that the fused polypeptide is made of the amino acid sequence of serial number 30 or 31.
2. fused polypeptide according to claim 1, which is characterized in that the fused polypeptide by serial number 31 amino acid sequence
It constitutes.
3. a kind for the treatment of diabetes pharmaceutical composition, which is characterized in that make comprising fused polypeptide of any of claims 1 or 2
For effective component.
4. treating diabetes pharmaceutical composition according to claim 3, which is characterized in that the diabetes are two types sugar
Urine disease.
5. treating diabetes pharmaceutical composition according to claim 3, which is characterized in that the composition has drop blood
Sugared efficiency increases the repellence to dipeptidyl peptidase-4.
6. fused polypeptide of any of claims 1 or 2 is preparing the purposes in the drug for treating diabetes.
7. a kind of polynucleotides encode fused polypeptide of any of claims 1 or 2.
8. polynucleotides according to claim 7, which is characterized in that the polynucleotides by serial number 41 or 42 nucleic acid sequence
Column are constituted.
9. polynucleotides according to claim 7, which is characterized in that the polynucleotides by serial number 42 nucleic acid sequence structure
At.
10. a kind of expression vector, which is characterized in that the polynucleotides including claim 7.
11. a kind of host cell, which is characterized in that the expression vector including claim 10.
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| CN106432511A (en) * | 2016-09-29 | 2017-02-22 | 上海凯茂生物医药有限公司 | GLP-1 analogue-Fc fusion protein and preparation method and application thereof |
| CN109021093B (en) * | 2018-08-29 | 2021-09-07 | 上海生物制品研究所有限责任公司 | Polyethylene glycol modified GLP-1 derivatives and medicinal salts thereof |
| CN111217915B (en) * | 2019-01-08 | 2021-05-14 | 东莞太力生物工程有限公司 | GLP-1 analogue Fc fusion polypeptide and application thereof |
| CN113573739A (en) * | 2019-03-08 | 2021-10-29 | 格纳西尼有限公司 | GLP-1 fusion proteins and uses thereof |
| CN110256580B (en) * | 2019-06-24 | 2022-07-29 | 王跃驹 | Application of plant produced oral dolabrin and nattokinase hypoglycemic capsule |
| CN117510646A (en) * | 2022-08-03 | 2024-02-06 | 无锡市华盛康肽生物技术有限公司 | Dual-function fusion protein and application thereof |
| WO2024141054A1 (en) * | 2022-12-30 | 2024-07-04 | 苏州康宁杰瑞生物科技有限公司 | Pharmaceutical composition comprising fusion protein and use thereof |
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