CN108218989A - Nano antibody PD-1/Nb18 of anti-PD-1 and preparation method and application - Google Patents
Nano antibody PD-1/Nb18 of anti-PD-1 and preparation method and application Download PDFInfo
- Publication number
- CN108218989A CN108218989A CN201711223594.2A CN201711223594A CN108218989A CN 108218989 A CN108218989 A CN 108218989A CN 201711223594 A CN201711223594 A CN 201711223594A CN 108218989 A CN108218989 A CN 108218989A
- Authority
- CN
- China
- Prior art keywords
- antibody
- nano
- nano antibody
- contain
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Nano antibody PD 1/Nb18 the invention discloses anti-PD 1 and preparation method and application.Nano antibody PD 1/Nb18 provided by the present invention, include antigenic determinant complementary region and skeleton area；The antigenic determinant complementary region of the nano antibody PD 1/Nb18 is made of CDR1, CDR2 and CDR3；The nano antibody PD 1/Nb18 nucleotide sequences and host cell announced by the present invention, nano antibody PD 1/Nb18 can in Escherichia coli high efficient expression, applied to the research and development of 1 Molecular Detection reagents of PD, prepare tumor inhibitor or inhibiting tumour cells agent and prepare the drug for inhibiting 1 activity of PD and promoting T cell proliferation.
Description
Technical field
Nano antibody PD-1/Nb18 the present invention relates to biomedical sector moderate resistance PD-1 and preparation method and application.
Background technology
Programmed death receptor 1 (programmed death 1, PD-1) is a kind of cross-film of activating T cell surface expression
Glycoprotein molecule belongs to CD28 superfamily members, negativity adjustment effect has been proliferated to T cell, important adjusting is played in immune response
Effect.PD-1 is mainly expressed in the T cell of activation and B cell, is a kind of surface receptor of activated form T cell, and PD-1 has two
A ligand, be respectively apoptosis-ligand 1 (PD-L1 also known as B7-H1) and apoptosis-ligand 1 (PD-L2, again
Name B7-DC).Tumor microenvironment in body can induce the T cell height of infiltration to express PD-1 molecules, and tumour cell can high expression
The ligand PD-L1 and PD-L2 of PD-1 causes after PD-1 accesses sustained activation PD-L1 couples with PD-1 in tumor microenvironment, and T is thin
Born of the same parents' function is suppressed, it is impossible to the signal of attack tumour is sent out to immune system.PD-1 inhibitor can block PD-1's and PD-L1
With reference to blocking negative regulation signal makes T cell activity recovery, so as to enhance immune response.High specific, hypoergia it is anti-
PD-1 monoclonal antibodies are affirmed in multinomial clinical test, such as the entitled Keytruda of MSD Corp.'s production
(Pembrolizumab) entitled Opdivo (Nivolumab) nivolumab's that drug and Shi Guibao companies produces
Drug.
But antibody drug is applied there are many problems, such as R＆D cycle are long, production cost is excessively high；It is difficult to extensive
Production；Stability difference is degradable, and storage is of high cost；It is easily contaminated, maintenance cost high cost；And there is immunogenicity etc.,
Limit its application range clinically.
Nano antibody technology, be biomedical science man on the basis of conventional antibodies, with Protocols in Molecular Biology knot
Close the antibody engineering revolution that the concept of nano-particle science carries out, the newest and minimum antibody molecule so as to develop.1993
The reports such as year Hamers, camel body is interior, and there is natural deletions light chain and the heavy chain antibody of heavy chain constant region 1 (CH1), Ke Longqi
Variable region obtains the single domain antibody being only made of a heavy chain variable region, referred to as VHH (variable domain of heavy
Chain of heavy-chain antibody), it is renamed as " nano antibody " (nanobody, Nb).Nanometer is anti-
Body has the minimum antigen-binding fragment of complete function, and crystal structure is oval, diameter 2.5nm, long 4nm.Nb has
Many unique properties are well suited for carrying out genetic modification, be presented in the Precise Diagnosis of disease and targeted therapy etc. wide
Application prospect.Nano antibody many simpler than antibody in chemical composition and in shape, without chemical drains, heat resistance
It is stronger with anti acid alkali performance, it is easier to be combined with each other or be combined with other compounds, energy coverlet gene code is easily closed with microorganism
Into.Nano antibody has good tolerance to environment, has the conformational stability of height, and molecular mass smaller, clinical
Therapeutic effect it is more preferable, while these little albumen molecules are easier to synthesize, and price is also lower.The unique property of nano antibody,
Its Precise Diagnosis in disease and immune targeted therapy etc. is made to present more wide application prospect.
Invention content
The technical problems to be solved by the invention are how to prepare the drug of effectively treatment tumour.
In order to solve the above technical problems, present invention firstly provides nano antibody PD-1/Nb18.
The first aspect of the present invention provides a kind of nano antibody, referred to as PD-1/Nb18, which includes antigen
Determinant complementary region CDR and skeleton area FR；The antigenic determinant complementary region CDR of the nano antibody is by CDR1, CDR2 and CDR3
Composition；The amino acid sequence of the CDR1 is the 23-32 amino acids of SEQ ID No.8 in sequence table；The ammonia of the CDR2
Base acid sequence is the 49-56 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the CDR3 is in sequence table
The 94-112 amino acids of SEQ ID No.8.
Preferably, the skeleton area FR of the nano antibody is made of FR1, FR2, FR3 and FR4；The amino acid of wherein described FR1
Sequence is the 1-22 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the FR2 is SEQ ID in sequence table
The 33-48 amino acids of No.8；The amino acid sequence of the FR3 is the 57-93 bit aminos of SEQ ID No.8 in sequence table
Acid；The amino acid sequence of the FR4 is the 113-123 amino acids of SEQ ID No.8 in sequence table.
Preferably, the amino acid sequence of the nano antibody PD-1/Nb18 is as shown in SEQ ID No.8 in sequence table.
The present invention also accordingly provides a kind of VHH chains of the nano antibody of PD-1, including skeleton area FR and complementary determining region
CDR, the amino acid sequence of the skeleton area FR FR selected from the group below：SEQ ID NO：FR1 shown in 1, SEQ ID NO：Shown in 2
FR2, SEQ ID NO：FR3 shown in 3, SEQ ID NO：FR4 shown in 4；The complementary determining region CDR is selected from the group below
The amino acid sequence of CDR：SEQ ID NO：CDR1 shown in 5, SEQ ID NO：CDR2 shown in 6, SEQ ID NO：Shown in 7
CDR3。
Preferably, the VHH chains of the nano antibody of the PD-1, it has SEQ ID NO：Amino acid sequence shown in 8.
In order to make the nano antibody PD-1/Nb18 convenient for purifying, can in sequence table SEQ ID No.8 1-123
The upper label as shown in Table 1 of amino terminal or carboxyl terminal connection of protein shown in amino acids.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
| HA | 9 | YPYDVPDYA |
Still further aspect of the present invention, the nano antibody PD-1/Nb18 can be artificial synthesized, also can first synthesize it and encode base
Cause, then carry out biological expression and obtain.The encoding gene of the nano antibody PD-1/Nb18 can be by by SEQ ID in sequence table
The codon of one or several amino acid residues is lacked in DNA sequence dna shown in No.9 and/or carries out one or several base-pairs
Missense mutation and/or the coded sequence of label shown in table 1 connected at its 5 ' end and/or 3 ' ends obtain.
In order to solve the above technical problems, the present invention also provides with the relevant biological materials of the nano antibody PD-1/Nb18
Material.
It is provided by the present invention with the relevant biomaterials of nano antibody PD-1/Nb18, be B1) to B12) in appoint
It is a kind of：
B1 the nucleic acid molecules of the nano antibody PD-1/Nb18) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B2) recombinant vector of the expression cassette；
B5) contain B1) recombinant microorganisms of the nucleic acid molecules；
B6) contain B2) recombinant microorganism of the expression cassette；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain B4) recombinant microorganism of the recombinant vector；
B9) contain B1) the transgenetic animal cell systems of the nucleic acid molecules；
B10) contain B2) the transgenetic animal cell system of the expression cassette；
B11) contain B3) the transgenetic animal cell system of the recombinant vector；
B12) contain B4) the transgenetic animal cell system of the recombinant vector.
In above-mentioned biomaterial, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The core
Acid molecule can also be RNA, such as mRNA or hnRNA.
In above-mentioned biomaterial, B2) described in the expression containing the nucleic acid molecules for encoding the nano antibody PD-1/Nb18
Box, also known as PD-1/Nb18 expression casettes are the DNA for referring to express nano antibody PD-1/Nb18 in host cell,
The DNA not only may include starting the promoter of nano antibody PD-1/Nb18 genetic transcriptions, may also include and terminates nano antibody PD-
The terminator of 1/Nb18 genetic transcriptions.Further, the expression cassette may also include enhancer sequence.
The recombinant vector of the nano antibody PD-1/Nb18 expression casettes can be contained with existing expression vector establishment.
In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.
In above-mentioned biomaterial, the recombinant vector can be by B1) nucleic acid molecules imported into pComb3 and obtain
Recombinant vector.In one embodiment of the invention, B3) recombinant vector is by the volume of the nano antibody PD-1/Nb18
Code gene (nucleotides sequence is classified as the 1-369 nucleotide of SEQ ID No.9 in sequence table) imports the weight obtained in pComb3
Nano antibody shown in group carrier pComb3-PD-1/Nb18, recombinant vector pComb3-PD-1/Nb18 expression SEQ ID No.8
PD-1/Nb18。
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi.
In above-mentioned biomaterial, the transgenetic animal cell system does not include propagating materials；The recombinant microorganism can be
By B1) nucleic acid molecules imported into the recombinant microorganism obtained in Escherichia coli WK6.
Those of ordinary skill in the art can easily adopt by known method, such as the side of orthogenesis and point mutation
Method, to the B1 of the present invention) nucleotide sequence of the nano antibody PD-1/Nb18 is mutated.Those by manually modified,
With the B1 with the present invention) nucleosides of more than 75% or 75% homogeneity of nucleotide sequence of the nano antibody PD-1/Nb18
Acid is derived from the present invention as long as encoding the nano antibody PD-1/Nb18 and with nano antibody PD-1/Nb18 activity
Nucleotide sequence and be equal to the present invention sequence.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " including with this hair
The nucleotide sequence of protein shown in bright coding SEQ ID No.8 has 75% or higher or 85% or higher or 90%
Higher or 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or computer software is evaluated.Make
With computer software, the homogeneity between two or more sequences can use percentage (%) to represent, can be used for evaluating phase
Close the homogeneity between sequence.
Above-mentioned 75% or more than 75% homogeneity can be 75%, 80%, 85%, 90% or more than 95% homogeneity.
In above-mentioned biomaterial, B1) nucleic acid molecules for it is following 1) or 2) or 3)：
1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.9 in sequence table；
2) nucleotide sequence with 1) limiting has 75% or more than 75% homogeneity, and encodes the nano antibody PD-
The cDNA molecules or genomic DNA molecule of 1/Nb18；
3) nucleotide sequence hybridization with 1) restriction, and encode the nano antibody PD-1/Nb18's under strict conditions
CDNA molecules or genomic DNA molecule.
In order to solve the above technical problems, the derivative antibody the present invention also provides the nano antibody PD-1/Nb18.
The derivative antibody of the nano antibody PD-1/Nb18 provided by the present invention, for it is following a) or b) or c) or d) or
e)：
A) single-chain antibody containing the nano antibody PD-1/Nb18；
B) contain the fusion antibody of a) single-chain antibody；
C) fusion antibody containing the nano antibody PD-1/Nb18；
D) Fab containing the nano antibody PD-1/Nb18；
E) complete antibody containing the nano antibody PD-1/Nb18.
In order to solve the above technical problems, the preparation method the present invention also provides the nano antibody PD-1/Nb18.
The preparation method of the nano antibody PD-1/Nb18 provided by the present invention, including the nano antibody will be encoded
The nucleic acid molecules of PD-1/Nb18 import the transgenic cell that recipient cell obtains expressing the nano antibody PD-1/Nb18, culture
The transgenic cell obtains the nano antibody PD-1/Nb18.
In the preparation method of above-mentioned nano antibody PD-1/Nb18, the nucleic acid of the coding nano antibody PD-1/Nb18
The nucleotide sequence of molecule is as shown in SEQ ID No.9 in sequence table.
In the preparation method of above-mentioned nano antibody PD-1/Nb18, the recipient cell can be microbial cell, such as large intestine bar
Bacterium, concretely Escherichia coli WK6.
In order to solve the above technical problems, the present invention also provides any one of following A 1-A8 purposes：
The application of A1, the nano antibody PD-1/Nb18 in tumor inhibitor or inhibiting tumour cells agent is prepared；
The application of A2, the biomaterial in tumor inhibitor or inhibiting tumour cells agent is prepared；
Application of the derivative antibody in tumor inhibitor or inhibiting tumour cells agent is prepared of A3, the nano antibody；
A4, the nano antibody PD-1/Nb18 preparation method in tumor inhibitor or inhibiting tumour cells agent is prepared
Application；
A5, the nano antibody PD-1/Nb18 inhibit PD-1 activity in preparation or T cell are promoted to be proliferated answering in product
With；
A6, the biomaterial are preparing the application in inhibiting PD-1 activity or promoting T cell proliferation product；
A7, the derivative antibody are preparing the application in inhibiting PD-1 activity or promoting T cell proliferation product；
A8, the nano antibody PD-1/Nb18 preparation method prepare inhibit PD-1 activity or promote T cell proliferation production
Application in product.
The said goods can be drug.
The core of amino acid sequence or its any fragment amino acid sequence in amplification coding sequence table shown in SEQ ID No.8
The primer pair of acid molecule, also belongs to protection scope of the present invention.
The present invention provides a kind of nano antibody of anti-PD-1, the nucleotide sequence for encoding the nano antibody and host are thin
Born of the same parents, with and its preparation method and application.The nano antibody can in Escherichia coli high efficient expression, applied to PD-1 Molecular Detections
The research and development of reagent prepare tumor inhibitor or inhibiting tumour cells agent and prepare inhibition PD-1 activity and promote T cell proliferation
Drug.
Description of the drawings
Fig. 1 is the DNA electrophoretograms of nano antibody, and from left to right the DNA bands of gel pore are respectively：First is 2000bp
Molecular labeling, second is PCR product, and PCR product band is about 400bp；
Fig. 2 is the electricity of SDS-PAGEs of the PD-1 nano antibodies PD-1/Nb18 after nickel column resin gel affinitive layer purification
Swimming figure；Swimming lane M represents molecular weight of albumen Marker, unit KDa；
Fig. 3 A are the Binding experiment result of the 293T cells of nano antibody PD-1/Nb18 and untransfected PD-1；Fig. 3 B is receive
The Binding experiment result of meter Kang Ti PD-1/Nb18 and the 293T cells of transfection PD-1.
Specific embodiment
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
After Escherichia coli WK6 in following embodiments agrees to through the sub- female laboratory of life science institute of Southeast China University ten thousand,
The public can obtain the biomaterial from Guangxi Medical University, which only attaches most importance to used in the related experiment of duplicate invention, no
It can be used as other purposes.
The preparation of embodiment 1, nano antibody
The present invention provides a kind of nano antibody from camel, entitled PD-1/Nb18, nano antibody PD-1/
The amino acid sequence of Nb18 is as shown in SEQ ID No.8 in sequence table, by the nucleotide sequence coded of SEQ ID No.9.
The nucleotide electrophoretogram of nano antibody PD-1/Nb18 is as shown in Figure 1, be for wherein first the molecule mark of 2000bp
Note, the second duct are PCR product, and PCR product band is about 400bp.
DNA fragmentation between PstI and NotI the identification sequence of carrier pComb3 (Biovector products) is replaced with into SEQ
DNA molecular shown in ID No.9, other sequences are constant, obtain recombinant vector pComb3-PD-1/Nb18, pComb3-PD-1/
The difference of Nb18 and pComb3 is only that replaces with SEQ ID by the DNA fragmentation between the PstI of pComb3 and NotI identification sequences
DNA molecular shown in No.9.Nano antibody PD-1/ shown in recombinant vector pComb3-PD-1/Nb18 expression SEQ ID No.8
Nb18.PComb3-PD-1/Nb18 is imported in Escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-PD-1/Nb18.
The specific preparation process of nano antibody is as follows：
(1) WK6-pComb3-PD-1/Nb18 is coated on to LB tablets (the LB tablets containing ampicillin and glucose
In, the concentration of ampicillin and glucose is respectively 100 μ g/mL and 20mg/mL) on, (10-14 is small for 25-37 DEG C of overnight incubation
When)；
(2) select single bacterium colony be seeded in LB culture solutions that 5mL contains ampicillin (in LB culture solutions, ammonia benzyl mould
A concentration of 100 μ g/mL of element) in, 25-37 DEG C of shaking table culture is overnight (10-14 hours)；
(3) culture solution of step (2) overnight incubation is seeded to according to 1 ﹕ 300-1 ﹕ 350 in fresh TB culture solutions, 25-37 DEG C
When shaking table culture to OD values reach 0.6-1.0, IPTG is added in, WK6-pComb3-PD-1/Nb18 culture solutions is obtained, makes WK6-
A concentration of 1mM of IPTG in pComb3-PD-1/Nb18 culture solutions, by WK6-pComb3-PD-1/Nb18 culture solutions at 20-30 DEG C
Under in overnight incubation (10-14 hours) on shaking table (rotating speed of shaking table be 220-250rpm), obtain WK6-pComb3-PD-1/
Nb18 induces liquid；
(4) WK6-pComb3-PD-1/Nb18 of step (3) induction liquid at 4 DEG C is centrifuged, collects thalline；
(5) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y is utilized:Combining magnetic
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
of influenza H3N2.Nanoscale Res Lett 2014,9:528.) osmosis in obtains antibody crude extract；
(6) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y is utilized:Combining magnetic
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
of influenza H3N2.Nanoscale Res Lett 2014,9:528.) the nickel column ion affinity chromatography method preparation in is received
Meter Kang Ti PD-1/Nb18.The SDA-PAGE electrophoretograms of nano antibody PD-1/Nb18 are respectively such as Fig. 2, nano antibody PD-1/Nb18
Size be about 15KDa.The results show that the purity of nano antibody PD-1/Nb18 that the above method obtains can reach 90% with
On.
The measure of embodiment 2, nano antibody PD-1/Nb18 and PD-1 Percentage bounds
Nano antibody PD-1/Nb18 measures (direct method) with PD-1 Percentage bounds
With the 293T cell detection nano antibody PD-1/Nb18 of PD-1 and PD-1 Percentage bounds is surely turned, by the nanometer of embodiment 1
Antibody PD-1/Nb18 (1 μ g) adds in 1-6 × 106Incubation 20-40min, PBS washing 2 times is protected from light in a above-mentioned 293T cells for 4 DEG C
Afterwards, 5 μ l PE anti-HA tag antibody (abcam, Clone are added in:20B12) 4 DEG C of incubation 20-40min, PBS washings 2
After secondary, by BACKMAN flow cytometers on sample, as a result as shown in Figure 3B, the 293T cells of untransfected PD-1 are used as control such as
Shown in Fig. 3 A.Fig. 3 A are blank control and PD-1 nano antibodies the PD-1/Nb18 knot with the 293T cells of untransfected PD-1 respectively
Close percentage；Fig. 3 B are blank control and PD-1 nano antibodies PD-1/Nb18 surely turn respectively PD-1 293T cells combination hundred
Divide rate；Horizontal axis is fluorescence intensity (PE) in Fig. 3 A and 3B, and the longitudinal axis is number percent (%of Max), and S2 represents blank control,
S1 represents PD-1 nano antibodies PD-1/Nb18.Graphical results can be seen that PD-1 nano antibodies PD-1/Nb18 can be good at
The steady 293T cell combinations for turning PD-1.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
The present invention is described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that：It still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent replacement to which part technical characteristic；
And these modification or replace, various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution spirit and
Range.
SEQUENCE LISTING
<110>Guangxi Medical University
<120>Nano antibody PD-1/Nb18 of anti-PD-1 and preparation method and application
<130> GY17100593
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(22)
<223> FR1
<400> 1
Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg
1 5 10 15
Leu Thr Cys Ala Ala Ser
20
<210> 2
<211> 16
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(16)
<223> FR2
<400> 2
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile
1 5 10 15
<210> 3
<211> 37
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(37)
<223> FR3
<400> 3
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
1 5 10 15
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala
20 25 30
Met Tyr Tyr Cys Ala
35
<210> 4
<211> 11
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(11)
<223> FR4
<400> 4
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 5
<211> 10
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(10)
<223> CDR1
<400> 5
Gly Tyr Thr Tyr Ser Asn Tyr Tyr Val Gly
1 5 10
<210> 6
<211> 8
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(8)
<223> CDR2
<400> 6
Asp Thr Leu Gly Tyr Thr Arg Tyr
1 5
<210> 7
<211> 19
<212> PRT
<213>Artificial sequence
<220>
<221> CONFLICT
<222> (1)..(19)
<223> CDR3
<400> 7
Ala Pro Arg Ser Pro Tyr Tyr Arg Gly Gln Thr Phe Trp Glu Gly Ala
1 5 10 15
Tyr Asn Tyr
<210> 8
<211> 123
<212> PRT
<213>Artificial sequence
<400> 8
Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly Ser Leu Arg
1 5 10 15
Leu Thr Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Asn Tyr Tyr Val Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile
35 40 45
Asp Thr Leu Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Gln Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Pro Arg
85 90 95
Ser Pro Tyr Tyr Arg Gly Gln Thr Phe Trp Glu Gly Ala Tyr Asn Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 9
<211> 369
<212> DNA
<213>Artificial sequence
<400> 9
ctgcaggagt ctgggggagg ctcggtgcag tccggagggt ctctgagact cacctgtgca 60
gcctctggat acacctacag taactactac gtgggctggt tccgccaggc tccaggaaag 120
gagcgcgagg gggtcgcaag tattgatact cttggttata caagatacgc agactccgtg 180
aaggggcgat tcaccatctc ccgtgacaac gccaagaaca cgctgtatct gcaaatgaac 240
agcctgcaac cggaggacac ggccatgtat tactgtgcgg cccctcgttc tccttactac 300
cggggtcaga cgttctggga gggggcgtat aactactggg gccaggggac ccaggtcacc 360
gtctcctca 369
Claims (10)
1. nano antibody includes antigenic determinant complementary region；It is characterized in that：
The antigenic determinant complementary region of the nano antibody is made of CDR1, CDR2 and CDR3；
The amino acid sequence of the CDR1 is the 23-32 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR2 is the 49-56 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR3 is the 94-112 amino acids of SEQ ID No.8 in sequence table.
2. nano antibody according to claim 1, it is characterised in that：The nano antibody is by antigenic determinant complementation
Area and the skeleton district's groups into.
3. nano antibody according to claim 1 or 2, it is characterised in that：The amino acid sequence of the nano antibody such as sequence
In list shown in SEQ ID No.8.
Any one of 4. it is B1 with claims 1 or 2 or the 3 relevant biomaterials of nano antibody) to B12)：
B1 the nucleic acid molecules of claims 1 or 2 or 3 nano antibodies) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules；
B4) contain B2) recombinant vector of the expression cassette；
B5) contain B1) recombinant microorganisms of the nucleic acid molecules；
B6) contain B2) recombinant microorganism of the expression cassette；
B7) contain B3) recombinant microorganism of the recombinant vector；
B8) contain B4) recombinant microorganism of the recombinant vector；
B9) contain B1) the transgenetic animal cell systems of the nucleic acid molecules；
B10) contain B2) the transgenetic animal cell system of the expression cassette；
B11) contain B3) the transgenetic animal cell system of the recombinant vector；
B12) contain B4) the transgenetic animal cell system of the recombinant vector.
5. biomaterial according to claim 4, it is characterised in that：B1) nucleic acid molecules for it is following 1) or 2) or 3)：
1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.9 in sequence table；
2) nucleotide sequence with 1) limiting has 75% or more than 75% homogeneity, and encodes described in claims 1 or 2 or 3
The cDNA molecules or genomic DNA molecule of nano antibody；
3) nucleotide sequence hybridization with 1) restriction, and encode claims 1 or 2 or 3 nano antibodies under strict conditions
CDNA molecules or genomic DNA molecule.
6. the derivative antibody of claims 1 or 2 or 3 nano antibodies, for it is following a) or b) or c) or d) or e)：
A) single-chain antibody containing claims 1 or 2 or 3 nano antibodies；
B) contain the fusion antibody of a) single-chain antibody；
C) fusion antibody containing claims 1 or 2 or 3 nano antibodies；
D) Fab containing claims 1 or 2 or 3 nano antibodies；
E) complete antibody containing claims 1 or 2 or 3 nano antibodies.
7. the preparation method of claims 1 or 2 or 3 nano antibodies, including claims 1 or 2 or 3 nanometers will be encoded
The nucleic acid molecules of antibody import the transgenic cell that recipient cell obtains expressing the nano antibody, and it is thin to cultivate the transgenosis
Born of the same parents obtain the nano antibody.
8. according to the method described in claim 7, it is characterized in that：Described coding claims 1 or 2 or 3 nano antibodies
Nucleic acid molecules nucleotide sequence as shown in SEQ ID No.9 in sequence table.
9. according to the method described in claim 7, it is characterized in that：The recipient cell is microbial cell.
10. any one of following A 1-A8 purposes：
The application of A1, claims 1 or 2 or 3 nano antibodies in tumor inhibitor or inhibiting tumour cells agent is prepared；
Application of any biomaterial in tumor inhibitor or inhibiting tumour cells agent is prepared in A2, claim 4-5；
Application of the derivative antibody in tumor inhibitor or inhibiting tumour cells agent is prepared described in A3, claim 6；
The application of A4,7 or 8 the method for claim in tumor inhibitor or inhibiting tumour cells agent is prepared；
A5, claims 1 or 2 or 3 nano antibodies inhibit PD-1 activity in preparation or T cell are promoted to be proliferated answering in product
With；
Any biomaterial is in preparing inhibition PD-1 activity or promoting T cell proliferation product in A6, claim 4-5
Using；
Derivative antibody described in A7, claim 6 is preparing the application in inhibiting PD-1 activity or promoting T cell proliferation product；
A8, claim 7 or 8 or 9 the methods are preparing the application in inhibiting PD-1 activity or promoting T cell proliferation product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711223594.2A CN108218989B (en) | 2017-11-29 | 2017-11-29 | anti-PD-1 nano antibody PD-1/Nb18 and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711223594.2A CN108218989B (en) | 2017-11-29 | 2017-11-29 | anti-PD-1 nano antibody PD-1/Nb18 and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108218989A true CN108218989A (en) | 2018-06-29 |
| CN108218989B CN108218989B (en) | 2020-08-18 |
Family
ID=62652994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711223594.2A Active CN108218989B (en) | 2017-11-29 | 2017-11-29 | anti-PD-1 nano antibody PD-1/Nb18 and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108218989B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014022758A1 (en) * | 2012-08-03 | 2014-02-06 | Dana-Farber Cancer Institute, Inc. | Single agent anti-pd-l1 and pd-l2 dual binding antibodies and methods of use |
| CN105061597A (en) * | 2015-06-09 | 2015-11-18 | 北京东方百泰生物科技有限公司 | Anti-PD-1 monoclonal antibody and obtaining method thereof |
| WO2016168809A1 (en) * | 2015-04-17 | 2016-10-20 | Krishnan Nandabalan | Compositions and methods for preventing tumor growth and treating cancer by targeting lectin galactoside-binding soluble 3 binding protein |
| CN106432494A (en) * | 2015-08-11 | 2017-02-22 | 哈尔滨誉衡药业股份有限公司 | Novel anti-PD-1 antibody |
-
2017
- 2017-11-29 CN CN201711223594.2A patent/CN108218989B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014022758A1 (en) * | 2012-08-03 | 2014-02-06 | Dana-Farber Cancer Institute, Inc. | Single agent anti-pd-l1 and pd-l2 dual binding antibodies and methods of use |
| WO2016168809A1 (en) * | 2015-04-17 | 2016-10-20 | Krishnan Nandabalan | Compositions and methods for preventing tumor growth and treating cancer by targeting lectin galactoside-binding soluble 3 binding protein |
| CN105061597A (en) * | 2015-06-09 | 2015-11-18 | 北京东方百泰生物科技有限公司 | Anti-PD-1 monoclonal antibody and obtaining method thereof |
| CN106432494A (en) * | 2015-08-11 | 2017-02-22 | 哈尔滨誉衡药业股份有限公司 | Novel anti-PD-1 antibody |
Non-Patent Citations (1)
| Title |
|---|
| FEI ZHANG等: "Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade", 《CITATION: CELL DISCOVERY》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108218989B (en) | 2020-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108218988B (en) | Nano antibody PD-1/Nb52 of anti-PD-1 and the preparation method and application thereof | |
| CN107474135A (en) | Anti- PD 1 nano antibody PD 1/Nb20 and preparation method and application | |
| CN106188297B (en) | Nano antibody Nb91 of anti-CTLA-4 and the preparation method and application thereof | |
| CN106220732B (en) | Nano antibody Nb16 of anti-CTLA-4 and the preparation method and application thereof | |
| CN106046164B (en) | Nano antibody Nb36 of anti-CTLA-4 and the preparation method and application thereof | |
| Giessen et al. | Converting a natural protein compartment into a nanofactory for the size-constrained synthesis of antimicrobial silver nanoparticles | |
| Ku et al. | Nucleic acid aptamers: An emerging tool for biotechnology and biomedical sensing | |
| CN106046165B (en) | Nano antibody Nb30 of anti-CTLA-4 and the preparation method and application thereof | |
| Bognár et al. | Aptamers against immunoglobulins: Design, selection and bioanalytical applications | |
| WO2018184267A1 (en) | Mrna-encoded nanobody and application thereof | |
| Kalousková et al. | Tumor marker B7-H6 bound to the coiled coil peptide-polymer conjugate enables targeted therapy by activating human natural killer cells | |
| Cho et al. | Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins | |
| CN108218989A (en) | Nano antibody PD-1/Nb18 of anti-PD-1 and preparation method and application | |
| CN110218255A (en) | Nano antibody CD3/Nb47 of AntiCD3 McAb and the preparation method and application thereof | |
| CN110218256A (en) | Nano antibody CD3/Nb29 of AntiCD3 McAb and the preparation method and application thereof | |
| KR101755266B1 (en) | Composition for detection or diagnosis of diseases containing transcription activator-like effector | |
| KR20100001091A (en) | Fusion peptides introducing antibody into cells, and cellular imaging and targeted drug delivery system using the same | |
| CN110218254B (en) | CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof | |
| CN114349859B (en) | anti-EGFRvIII nanobody EGFRvIII/Nb1-11 and preparation method and application thereof | |
| US20220411472A1 (en) | Self-assembling circular tandem repeat proteins with increased stability | |
| CN110218253A (en) | Nano antibody CD3/Nb14 of AntiCD3 McAb and the preparation method and application thereof | |
| CN114539412B (en) | Single domain antibody of anti-HLA-A 2/WT1 complex, preparation method and application thereof | |
| Choi et al. | Cyclic Peptide-Decorated Self-Assembled Nanohybrids for Selective Recognition and Detection of Multivalent RNAs | |
| Legaz et al. | A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly (lactic acid) nanoparticle surface is transcriptionally active in cellulo | |
| US20220259268A1 (en) | Adhesive protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |