CN110305200A - A kind of little albumen and its application - Google Patents
A kind of little albumen and its application Download PDFInfo
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- CN110305200A CN110305200A CN201810230338.4A CN201810230338A CN110305200A CN 110305200 A CN110305200 A CN 110305200A CN 201810230338 A CN201810230338 A CN 201810230338A CN 110305200 A CN110305200 A CN 110305200A
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Abstract
The invention discloses a kind of little albumen and its applications.The skelemin of the little albumen is the mutant of the IgG binding domain of streptococcal protein G, and the little albumen includes a α spiral and four β-pleated sheets, the α spiral runs through the region being enclosed by four β-pleated sheets, while being also distributed on the β-pleated sheet face formed by four β-pleated sheets can be with the protein bound key amino acid site PD-1.Little albumen and PD-L1 albumen of the invention has the structural similarity of height; it can inhibit PD-1/PD-L1 combination; it is potential PD-1/PD-L1 inhibitor; it is long to recycle the residence time with weak IgG binding ability in vivo for it, and has high structural stability; it can be used for inhibiting the interaction between PD-1/PD-L1; preparation is simple, can be used for large-scale production and application, so as to the immunization therapy of cancer.
Description
Technical field
The present invention relates to a kind of little albumens, and in particular to one kind is based on GB1 (streptococcal protein G (Streptococcal
Protein G) IgG binding domain (Immunoglobulin G-binding protein G), referred to as GB1) skeleton it is small
Albumen, and such little albumen has the application of the ability as well as PD-1/PD-L1 inhibitor in conjunction with IgG simultaneously, belongs to
PD-1/PD-L1 inhibitor technical field.
Background technique
In the confrontation with cancer, the effect of immune system is most important.Tumour cell due to inhereditary material change, carefully
Cellular surface expresses tumour specific antigen.Immunocyte can pass through the identification to these specific antigens, killing tumor cell.
However tumour cell can take Immune escaping mechanism that they are survived in antineoplastic immune.Therefore tumour cell is blocked
Escape be conducive to the killing to tumour cell.
PD-1 (programmed cell death 1, CD279) apoptosis albumen, is I type memebrane protein,
A member of CD28 family.There is expression in bone-marrow-derived lymphocyte, T lymphocyte, onthe surface of monocytes.The ligand PD-L1 of PD-1
(CD279, B7-H1) is distributed widely in lymph and non-lymphoid tissue, and it is upper that the knockout of PD-L1 gene causes T cell to respond
It adjusts.Block the antibody of PD-1/PD-L1 that antineoplastic immune activity can be improved.Another ligand PD-L2 (CD273, the B7- of PD-1
DC), distribution is extensive not as good as PD-L1, only has expression on the macrophage of activation and Dendritic Cells.Under normal physiological condition,
The T cell surface of activation can express PD-1, while generate the expression of PD-L1 in interferon-induced normal tissue.PD-1/PD-L1
Access can prevent the excessive activation of T cell, keep immune system stable state.However tumour cell (such as lymthoma, melanoma,
Lung cancer, liver cancer, glioblastoma, oophoroma etc.) on PD-L1 overexpression is often presented, the combination of PD-1/PD-L1 inhibits T cell
Proliferation, the release of cell factor and the enforcement of cytotoxicity function, eventually lead to the exhaustion of T cell, cause in tumour micro-loop
It is immunoreacted and is suppressed in border, tumour growth is uncontrolled.
As important immunologic test point, inhibitor is intended to release PD-1/PD-L1 and combines to T cell PD-1/PD-L1
Inhibiting effect, restore T cell mediate antineoplastic immune.
In recent years, the hope that the development of immunologic test point inhibitor has given treatment of cancer new, immunologic test point inhibitor are logical
Cross the function of blocking inhibition signal to restore T cell.2008 report murine PD-1/human PD-L1 and
The structure elucidation of murine PD-1/murine PD-L2 has established PD-1/PD-L1, the basis of-L2 interaction model；2015
The crystallographic structural analysis (PDB:4ZQK) for the human PD-1/human PD-L1 compound that year delivers, to study PD-1/PD-L1
The hotspot location of interaction provides important evidence.The faying face of the two is aboutIt is hydrophobic and relatively flat, not deep
Binding pocket, such faying face are unfavorable for designing micromolecular inhibitor.
Currently, industry research staff is broadly divided into three kinds to the research of PD-1/PD-L1 inhibitor:
One, monoclonal antibody drug (mAbs): there are four the PD-1/PD-L1 inhibitor listed, Nivolumab
(Opdivo, Bristol Myers Squibb) pembrolizumab (Keytruda, Mo Shadong), Atezolizumab (Tecentriq, sieve
Family name), Durvalumab (Imfinzi, AstraZeneca), Avelumab (Merck/Pfizer), PDR001 (Novartis) in clinical test
Deng.Antibody is due to long half time, and affinity is high, and barrier effect is good, is that pharmaceuticals falls over each other the drug of exploitation and successfully listed.
But the high production cost of antibody, thereby increases and it is possible to generate the side effects such as T cell exhaustion, therefore be also required to the substitution of other inhibitor.
Two, micromolecular inhibitor: team, Harvard University delivers patent, sulphonamide derivatives within 2010
Sulfamonomethoxine and sulfamethizole can restore the function of T cell release IFN-γ；BMS company in 2015
Patents, in HTRF Binding experiment, IC50 is up to 0.1 μM or less；Team, Jilin University report is with resorcinol within 2016
For nuclear structure, amino connects phenyl dimethylcarbamates, HTRF the experiment effectively suppression of dimethylcarbamate
PD-1/PD-L1 processed is combined and effect is better than sulfamonomethoxine and sulfamethizole that front is reported, inhibiting rate
Up to 40% or more.Although however small molecule good penetrability, specificity it is not high, side effect is big.
Three, protein and peptide inhibitor: 2007, research team, Kyoto Univ Japan was the murine for reporting tetramerization
The affinity of PD-L1 and murine PD-1 significantly improves；2015, the team of Stanford University and Yale University utilized yeast
The mode of screening obtains the mutant HAC-PD-1 of PD-1 extracellular domain, has good tumor tissues permeability, in mouse tumor
It is more more effective than anti-PD-L1 antibody in model.With in 2015, the researcher of Tsinghua University and Zhejiang University uses mirror image bacteriophage
Screening technique obtained first can resistant to hydrolysis D type PD-1/PD-L1 antagonistDPPA-1, and in cell and bearing animals mould
It is proved in type effective.In addition, 2016, East China University of Science researcher by computer-aided from the beginning design polypeptide in the way of obtain
A series of PD-1 target polypeptides, it was demonstrated that can restore by the function of the HCT116 Jurkat T cell inhibited.But polypeptide presses down
The structural stability of preparation is poor, and affinity is not high enough, and circulation time in vivo is short to also limit its application.
Summary of the invention
The main purpose of the present invention is to provide a kind of little albumen and its applications, to overcome deficiency in the prior art.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
The embodiment of the invention provides a kind of little albumen, the skelemin (referred to as GB1) of the little albumen is streptococcus G
The IgG binding domain (Immunoglobulin G-binding protein G) of albumen (Streptococcal protein G)
Mutant, and the little albumen includes a α spiral and four β-pleated sheets, and the α spiral runs through to be enclosed by four β-pleated sheets
The region of formation, while being also distributed on the β-pleated sheet face formed by four β-pleated sheets can be protein bound with PD-1
Key amino acid site.
In some embodiments, the amino acid sequence of the little albumen is as shown in SEQ ID NO:12, and the amino
Acid sequence has following at least one amino acid replacement: it is replaced in the 5th Arg residue, the 7th Met residue displacement, the
9 Ser residue displacements, the 14th Ala residue displacement, the 15th Asp residue displacement, the 16th Tyr residue displacement,
17th Lys residue displacement, the 18th Arg residue displacement, the 45th Gln residue displacement, the 52nd Glu residue are set
It changes, the 54th Tyr residue displacement, the 56th Ile residue displacement.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:1.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:2.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:3.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:4.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:5.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:6.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:7.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:8.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:9.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:10.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:11.
Further, two β-pleated sheets close to α spiral side are distributed in be parallel to each other, and are distributed in the α spiral
Two β-pleated sheets of the other side are antiparallel.
Further, the little albumen has weak IgG binding ability.
Further, the little albumen and PD-L1 albumen have the structural similarity of height.
Further, the little albumen, which has, inhibits the protein bound ability of PD-1/PD-L1.
The embodiment of the invention also provides aforementioned little albumens to prepare the purposes in PD-1/PD-L1 inhibitor.
Further, the embodiment of the invention also provides a kind of PD-1/PD-L1 inhibitor, it includes little albumens above-mentioned.
Compared with prior art, beneficial effect of the present invention at least that:
1) little albumen provided by the invention and PD-L1 albumen have the structural similarity of height, can inhibit PD-1/PD-L1 knot
It closes, is potential PD-1/PD-L1 inhibitor, with weak IgG binding ability, it is long to recycle the residence time in vivo, and have
High structural stability, can be used for inhibiting the interaction between PD-1/PD-L1, and preparation is simple, can be used for large-scale production and
Using so as to the immunization therapy of cancer；
2) little albumen of stable structure provided by the invention, more can simulated albumin matter and albumen compared with small molecule compound
Interaction (PPIs) between matter, targeting and specificity are good.Compared with polypeptide, little albumen has more stable structure, is easy to obtain
The circulation time in vivo that must be suitable for.Compared with antibody, there is better penetration into tissue and the T cell mediated without Fc exhausts secondary make
With.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of small peptide backbone GB1 in a typical embodiments of the invention.
Fig. 2 is to move the key amino acid site in PD-L1 and PD-1 protein binding face in a typical embodiments of the invention
Plant the structural schematic diagram on the β-pleated sheet face corresponding position of little albumen.
Fig. 3 is the binding model schematic diagram of a kind of little albumen in a typical embodiments of the invention.
Fig. 4 is the polyacrylamide gel electrophoresis figure of mutant little albumen after purification in the embodiment of the present invention 1.
Fig. 5 is that small peptide backbone and PD-L1 protein conformation similarity compare schematic diagram in the embodiment of the present invention 2.
Fig. 6 is the surface plasma that mutant little albumen GBM8 inhibits PD-1/PD-L1 to combine in the embodiment of the present invention 3
Resonate testing result schematic diagram.
Fig. 7 is the table that mutant little albumen GBM7, GBM9, GBM11 inhibit PD-1/PD-L1 to combine in the embodiment of the present invention 3
Surface plasma resonance testing result schematic diagram.
Fig. 8 be in the embodiment of the present invention 4 circular dichroism detector characterization mutation after little albumen GBM1, GBM2, GBM3, GBM4 two
The comparing result schematic diagram of level structure and skelemin GB1.
Fig. 9 is the secondary structure and skeleton egg of little albumen GBM8 after the mutation of circular dichroism detector characterization in the embodiment of the present invention 4
The comparing result schematic diagram of white GB1.
Figure 10 is that the temperature of the secondary structure of little albumen GBM8 after the mutation of circular dichroism detector characterization in the embodiment of the present invention 4 is steady
Qualitative results schematic diagram.
Specific embodiment
As previously mentioned, inventor is studied for a long period of time and largely practiced in view of many defects of the prior art, mentioned
Technical solution of the present invention out mainly passes through the technologies such as molecular cloning, prokaryotic expression of protein, and PD-L1 and PD-1 is tied
The key amino acid site in conjunction face is transplanted on the β-pleated sheet face corresponding position of little albumen, and a kind of little albumen is obtained, its own has
Good stability has weak IgG binding ability, and the circulation residence time is long in vivo, can be used for inhibiting between PD-1/PD-L1
Interaction, so as to the immunization therapy of cancer.It as follows will be further to works such as the technical solution, its implementation process and principles
It illustrates.
A kind of little albumen that the one aspect of the embodiment of the present invention provides, the skelemin of the little albumen is (referred to as
It GB1 is) IgG binding domain (the Immunoglobulin G- of streptococcal protein G (Streptococcal protein G)
Binding protein G) mutant, and the little albumen includes a α spiral and four β-pleated sheets, and the α spiral passes through
The region being enclosed by four β-pleated sheets is worn, while energy is also distributed on the β-pleated sheet face formed by four β-pleated sheets
The enough and protein bound key amino acid site PD-1.
In some embodiments, the amino acid sequence of the little albumen is as shown in SEQ ID NO:12, and the amino
Acid sequence has following at least one amino acid replacement: it is replaced in the 5th Arg residue, the 7th Met residue displacement, the
9 Ser residue displacements, the 14th Ala residue displacement, the 15th Asp residue displacement, the 16th Tyr residue displacement,
17th Lys residue displacement, the 18th Arg residue displacement, the 45th Gln residue displacement, the 52nd Glu residue are set
It changes, the 54th Tyr residue displacement, the 56th Ile residue displacement.
It further says, the key amino acid site that the PD-1/PD-L1 being transformed in the little albumen is combined is from N-terminal to C-terminal
It is followed successively by Arg (arginine), Met (methionine), Ser (serine), Ala (alanine), Asp (aspartic acid), Tyr (junket
Propylhomoserin), Lys (lysine), Arg (arginine), Gln (glutamine), Glu (glutamic acid), Tyr (tyrosine), Ile it is (different bright
Propylhomoserin).
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:1.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:2.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:3.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:4.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:5.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:6.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:7.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:8.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:9.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:10.
Further, the amino acid sequence of the little albumen can be as shown in SEQ ID NO:11.
Further, two β-pleated sheets close to α spiral side are distributed in be parallel to each other, and are distributed in the α spiral
Two β-pleated sheets of the other side are antiparallel.
Further, the skelemin includes naturally occurring or artificial synthesized α β little albumen.
Further, the little albumen has weak IgG binding ability.
Further, the little albumen and PD-L1 albumen have the structural similarity of height.
Further, the little albumen, which has, inhibits the protein bound ability of PD-1/PD-L1.
The embodiment of the invention also provides aforementioned little albumen in preparation for inhibiting to interact between PD-1/PD-L1
Purposes in inhibitor.
Further, the embodiment of the invention also provides a kind of PD-1/PD-L1 inhibitor, it includes little albumens above-mentioned.
In a typical embodiments of the invention, the present invention, will by technologies such as molecular cloning, prokaryotic expression of protein
The key amino acid site of PD-L1 and PD-1 faying face is transplanted on the β-pleated sheet face corresponding position of little albumen, and a kind of small egg is obtained
White (its typical structure sees Fig. 1-Fig. 3) makes it not only retain weaker IgG binding ability, but also has and inhibit PD-1/PD-L1
In conjunction with ability, play the effect of cancer immunotherapy.Wherein, the structural schematic diagram of little albumen skeleton GB1 is referring to Fig. 1, Fig. 2-
Fig. 3 shows the β-pleated sheet face corresponding position that the key amino acid site of PD-L1 and PD-1 protein binding face are transplanted to little albumen
On structural schematic diagram and binding model schematic diagram.
Below in conjunction with several preferred embodiments the technical solution of the present invention is further explained explanation, but reality therein
It tests condition and setup parameter is not construed as limitation to basic technical scheme of the present invention.And protection scope of the present invention is not limited to
Following embodiments.
More preferably little albumen sequence and wild type GB1 frame sequence compare embodiment 1
11 mutant little albumens being related in the present embodiment and skelemin GB1 are listed in table 1.Described is each prominent
Variant little albumen can be obtained by simple prokaryotic expression, obtain the prokaryotic expression carrier of little albumen through molecular cloning, then by
Pure mutant little albumen can be obtained in prokaryotic expression, affinity purification.Wherein, little albumen prokaryotic expression process are as follows: each mutant
The prokaryotic expression plasmid of little albumen can be obtained by molecular cloning.Enter BL21 (DE3) impression through chemical conversion after plasmid order-checking is correct
State Bacillus coli cells, cell is containing (such as pET28a, which uses to contain, blocks that with the consistent antibiotic LB culture medium of plasmid resistance gene
The LB culture medium of mycin) in overnight incubation, obtain stay overnight bacterium.When expressing albumen, 1/100 is added in antibiotic culture medium
Overnight bacterium (1ml stays overnight bacterium and is added in 100ml LB culture medium), when 37 DEG C of shaking table cultures are to OC600=0.5, IPTG is added
(final concentration 1mM), 37 DEG C of shaking tables continue culture 4 hours.Bacterium solution is collected, sonicated cells obtain after affinity purification pure
Mutant little albumen.It is illustrated in figure 4 the polyacrylamide gel electrophoresis figure of mutant little albumen after purification.
The amino acid sequence of 11 mutant little albumens and skelemin GB1 being related in 1 embodiment 1 of table
2 little albumen skeleton of embodiment is compared with PD-L1 protein conformation similarity
Using the pair fitting function of PyMOL software Wiazrd menu, 12 for choosing PD-L1 and PD-1 faying face
The C of key amino acidαWith little albumen skeleton β-pleated sheet face opposite position CαIt is compared, it is inclined according to the calculated root mean square of software
(root-mean-square deviation, RMSD, unit are difference) evaluation little albumen face journey similar to PD-L1 structure
Degree, the smaller space structure that represents of numerical value is more close, whenWhen, representative structure is completely coincident.Choose several small eggs
The RMSD comparison value of bones of the dead frame and PD-L1 existLeft and right, the RMSD value of GB1 skeleton and PD-L1 that the present invention selects are only As shown in Fig. 5 and table 2, table 2 lists small peptide backbone in the present embodiment and compares with PD-L1 protein conformation similarity
Gained RMSD value, structural similarity are very high.Therefore GB1 has features designed to the potentiality of PD-1/PD-L1 inhibitor.
Small peptide backbone and PD-L1 protein conformation similarity compare gained RMSD value in 2 the present embodiment of table
Embodiment 3
1) surface plasma body resonant vibration (the Surface Plasmon that mutant little albumen 8 inhibits PD-1/PD-L1 to combine
Resonance, SPR) detection
SPR detection be by the change of detection chip refractive index, the variation of reaction chip surface conjugate weight, when point
Ligand binding of the logistics through being coupled on chip surface, with chip is analysed, causes the change of chip refractive index, changes under instrument record
Response (Response Unit, RU).RU value embodies the variation of chip surface conjugate weight, response analysis object and ligand
Combination or dissociation.As shown in fig. 6, being coupled PD-1 albumen on chip, and the GB1 of various concentration is mixed in analyte PD-L1
Mutant GBM8, with the increase of GB1 mutant GBM8 concentration, the combination of PD-L1 and PD-1 are reduced, and embody GB1 mutant
The inhibiting effect that GBM8 combines PD-1/PD-L1.
2) surface that mutant little albumen 7, mutant little albumen 9, mutant little albumen 11 inhibit PD-1/PD-L1 to combine
Plasma resonance (Surface Plasmon Resonance, SPR) detection
SPR detection be by the change of detection chip refractive index, the variation of reaction chip surface conjugate weight, when point
Ligand binding of the logistics through being coupled on chip surface, with chip is analysed, causes the change of chip refractive index, changes under instrument record
Response (Response Unit, RU).RU value embodies the variation of chip surface conjugate weight, response analysis object and ligand
Combination or dissociation.As shown in fig. 7, being coupled PD-1 albumen on chip, and the GB1 of various concentration is mixed in analyte PD-L1
Mutant GBM7, GBM9, GBM11, with the increase of GB1 mutant GBM7, GBM9, GBM11 concentration, the knot of PD-L1 and PD-1
It closes and reduces, embody the inhibiting effect that GB1 mutant GBM7, GBM9, GBM11 combines PD-1/PD-L1.
In addition, remaining mutant little albumen such as mutant little albumen 1 (GBM1), mutant little albumen 2 in the present invention
(GBM2), mutant little albumen 3 (GBM3), mutant little albumen 4 (GBM4), mutant little albumen 5 (GBM5), the small egg of mutant
The result for the surface plasma body resonant vibration detection that white 6 (GBM6), mutant little albumen 10 (GBM10) inhibit PD-1/PD-L1 to combine
It is similar to Fig. 6 and Fig. 7.
The stability of the measurement of mutant little albumen secondary structure and little albumen secondary structure after embodiment 4 is transformed
Circular dichroism spectra can characterize Secondary structure feature.As shown in Figure 8 and Figure 9, through 10 amino acid mutations
Little albumen afterwards is still able to maintain secondary structure similar with wild type little albumen skeleton, and has good temperature stability.Such as
Shown in Figure 10, measurement is caused the variation of GBM8 structure by 10 DEG C to 90 DEG C temperature, and display GBM8 has preferable temperature stability, and
From 90 DEG C be cooled to 20 DEG C after, the recovery rate of structure is up to 86%.
In addition, remaining mutant little albumen such as mutant little albumen 1 (GBM1), mutant little albumen 2 in the present invention
(GBM2), mutant little albumen 3 (GBM3), mutant little albumen 4 (GBM4), mutant little albumen 5 (GBM5), the small egg of mutant
White 6 (GBM6), mutant little albumen 7 (GBM7), mutant little albumen 9 (GBM9), mutant little albumen 10 (GBM10), mutation
The secondary structure measurement result of body little albumen 11 (GBM11) is similar to Fig. 8 and Fig. 9, the stability test result of secondary structure
It is similar to FIG. 10.
By above-mentioned technical proposal, the present invention is by technologies such as molecular cloning, prokaryotic expression of protein, by PD-L1 and PD-
The key amino acid site of 1 faying face is transplanted on the β-pleated sheet face corresponding position of little albumen, and a kind of little albumen or its mutation are obtained
Body has the structural similarity of height with PD-L1 albumen, can inhibit PD-1/PD-L1 combination, is potential PD-1/PD-L1 suppression
Preparation it is long to recycle the residence time, and have high structural stability in vivo, can be used for pressing down with weak IgG binding ability
Interaction between PD-1/PD-L1 processed, preparation is simple, can be used for large-scale production and application, immune so as to cancer is controlled
It treats.
Finally, it is to be noted that, the terms "include", "comprise" or its any other variant be intended to it is non-exclusive
Property include so that include a series of elements process, method, article or equipment not only include those elements, but also
Further include other elements that are not explicitly listed, or further include for this process, method, article or equipment it is intrinsic
Element.
It will be appreciated by those skilled in the art that the above described specific embodiments of the present invention, are not constituted to the present invention
The restriction of protection scope.Any any other various changes and modifications in accordance with the technical idea of the present invention, should all
Comprising within the scope of the invention as claimed.
Sequence table
<110>Shanghai University
Suzhou Institute of Nano-tech. and Nano-bionics, Chinese Academy of Sciences
<120>a kind of little albumen and its application
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Ala Thr Leu Gly Pro Thr Val Ile Gly
50 55
<210> 8
<211> 57
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 8
Gly Ala Thr Thr Ala Leu Met Leu Ser Gly Leu Thr Leu Ala Ala Thr
1 5 10 15
Leu Ala Thr Gly Ala Val Ala Ala Ala Thr Ala Gly Leu Val Pro Leu
20 25 30
Gly Thr Ala Ala Ala Ala Gly Val Ala Gly Gly Thr Gly Thr Ala Ala
35 40 45
Ala Thr Leu Thr Pro Thr Val Ile Gly
50 55
<210> 9
<211> 55
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 9
Ala Thr Thr Ala Leu Met Leu Ser Gly Thr Leu Ala Ala Thr Leu Ala
1 5 10 15
Thr Gly Ala Val Ala Ala Ala Thr Ala Gly Leu Val Pro Leu Gly Thr
20 25 30
Ala Ala Ala Ala Gly Val Ala Gly Gly Thr Gly Thr Ala Ala Ala Thr
35 40 45
Leu Thr Pro Thr Val Ile Gly
50 55
<210> 10
<211> 56
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 10
Gly Ala Thr Thr Ala Leu Met Leu Ser Gly Leu Leu Ala Ala Thr Leu
1 5 10 15
Ala Thr Gly Ala Val Ala Ala Ala Thr Ala Gly Leu Val Pro Leu Gly
20 25 30
Thr Ala Ala Ala Ala Gly Val Ala Gly Gly Thr Gly Thr Ala Ala Ala
35 40 45
Thr Leu Thr Pro Thr Val Ile Gly
50 55
<210> 11
<211> 56
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 11
Gly Ala Thr Thr Ala Leu Met Leu Ser Gly Gly Leu Ala Ala Thr Leu
1 5 10 15
Ala Thr Gly Ala Val Ala Ala Ala Thr Ala Gly Leu Val Pro Leu Gly
20 25 30
Thr Ala Ala Ala Ala Gly Val Ala Gly Gly Thr Gly Thr Ala Ala Ala
35 40 45
Thr Leu Thr Pro Thr Val Ile Gly
50 55
<210> 12
<211> 57
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 12
Gly Ala Thr Thr Leu Leu Ile Leu Ala Gly Leu Thr Leu Leu Gly Gly
1 5 10 15
Thr Thr Thr Gly Ala Val Ala Ala Ala Thr Ala Gly Leu Val Pro Leu
20 25 30
Gly Thr Ala Ala Ala Ala Gly Val Ala Gly Gly Thr Thr Thr Ala Ala
35 40 45
Ala Thr Leu Thr Pro Thr Val Thr Gly
50 55
Claims (16)
1. a kind of little albumen, which is characterized in that the skelemin of the little albumen is the prominent of the IgG binding domain of streptococcal protein G
Variant, and the little albumen includes a α spiral and four β-pleated sheets, and the α spiral runs through to be enclosed by four β-pleated sheets
Region, while being also distributed on the β-pleated sheet face formed by four β-pleated sheets can be with the protein bound crucial ammonia of PD-1
Base acid site.
2. little albumen according to claim 1, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 12, and the amino acid sequence has following at least one amino acid replacement: it is replaced in the 5th Arg residue, the
7 Met residue displacements, the 9th Ser residue displacement, the 14th Ala residue displacement, the 15th Asp residue displacement,
16th Tyr residue displacement, the 17th Lys residue displacement, the 18th Arg residue displacement, the 45th Gln residue are set
It changes, the 52nd Glu residue displacement, the 54th Tyr residue displacement, the 56th Ile residue displacement.
3. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 1.
4. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 2.
5. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 3.
6. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 4.
7. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 5.
8. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 6.
9. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID NO:
Shown in 7.
10. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID
Shown in NO:8.
11. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID
Shown in NO:9.
12. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID
Shown in NO:10.
13. little albumen according to claim 2, it is characterised in that: the amino acid sequence of the little albumen such as SEQ ID
Shown in NO:11.
14. little albumen according to claim 1, it is characterised in that: be distributed in two β close to α spiral side and roll over
It is folded to be parallel to each other, and two β-pleated sheets for being distributed in the α spiral other side are antiparallel.
15. little albumen described in any one of claim 1-14 is preparing the purposes in PD-1/PD-L1 inhibitor.
16. a kind of PD-1/PD-L1 inhibitor, it is characterised in that include little albumen described in any one of claim 1-14.
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| CN201810230338.4A CN110305200B (en) | 2018-03-20 | 2018-03-20 | Small protein and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104159911A (en) * | 2012-03-07 | 2014-11-19 | 奥瑞基尼探索技术有限公司 | Peptidomimetic compounds as immunomodulators |
| WO2015036927A1 (en) * | 2013-09-10 | 2015-03-19 | Aurigene Discovery Technologies Limited | Immunomodulating peptidomimetic derivatives |
| CN108727470A (en) * | 2017-04-17 | 2018-11-02 | 上海大学 | A kind of polypeptide and its application |
-
2018
- 2018-03-20 CN CN201810230338.4A patent/CN110305200B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104159911A (en) * | 2012-03-07 | 2014-11-19 | 奥瑞基尼探索技术有限公司 | Peptidomimetic compounds as immunomodulators |
| WO2015036927A1 (en) * | 2013-09-10 | 2015-03-19 | Aurigene Discovery Technologies Limited | Immunomodulating peptidomimetic derivatives |
| CN108727470A (en) * | 2017-04-17 | 2018-11-02 | 上海大学 | A kind of polypeptide and its application |
Non-Patent Citations (1)
| Title |
|---|
| SAMAN MALEKI VAREKI等: "Biomarkers of response to PD-1/PD-L1 inhibition", 《CRITICAL REVIEWS IN ONCOLOGY/HEMATOLOGY》 * |
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