CN106188297B - Nano antibody Nb91 of anti-CTLA-4 and the preparation method and application thereof - Google Patents
Nano antibody Nb91 of anti-CTLA-4 and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses nano antibody Nb91 of anti-CTLA-4 and the preparation method and application thereof.Nano antibody provided by the present invention includes determinant complementary region and framework region；The determinant complementary region of the nano antibody is made of CDR1, CDR2 and CDR3；The amino acid sequence of the CDR1 is the 26-32 amino acids of SEQ ID No.7 in sequence table；The amino acid sequence of the CDR2 is the 48-55 amino acids of SEQ ID No.7 in sequence table；The amino acid sequence of the CDR3 is the 94-106 amino acids of SEQ ID No.7 in sequence table.Nano antibody Nb91 of the invention can be applied to the research and development of CTLA-4 Molecular Detection reagent in conjunction with T cell and CTLA-4, prepare tumor inhibitor or inhibiting tumour cells agent and preparation inhibits CTLA-4 activity and promotes the drug of T cell proliferation.
Description
Technical field
The present invention relates to nano antibody Nb91 of CTLA-4 anti-in field of biomedicine and the preparation method and application thereof.
Background technique
Cytotoxic t lymphocyte-associated antigen 4 (cytotoxic T lymphocytic associated
Antigen, CTLA-4/CTLA4) also known as CD152, it is a kind of leukocyte differentiation antigen, is one kind of activating T cell surface expression
Transmembrane glycoprotein molecule belongs to immunoglobulin superfamily member, negativity adjustment effect has been proliferated to T cell, in immune response
Play important regulative.CTLA-4Ig can in vivo and in vitro effectively, specifically inhibit cell and humoral immune reaction, to graft rejection
Reaction, tumour and various autoimmune diseases have significant therapeutic effect, and toxic side effect is extremely low, be presently considered to relatively have it is uncommon
The new immunosuppressive drug hoped.
Antibody drug is applied there are many problems, such as R&D cycle are long, and production cost is excessively high；It is difficult to be mass produced；
Stability difference is degradable, stores at high cost；It is easy to be contaminated, maintenance cost expense is high；And there is immunogenicity etc., it limits
Its application range clinically.
Nano antibody technology is biomedical science man on the basis of conventional antibodies, with Protocols in Molecular Biology knot
The antibody engineering revolution that the concept of nanoparticle science carries out is closed, thus the newest and the smallest antibody molecule developed.1993
The reports such as year Hamers, camel body is interior, and there is the heavy chain antibody of natural deletions light chain and 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 smallest antigen-binding fragment of complete function, and crystal structure is oval, diameter 2.5nm, long 4nm.Nb has
Many unique properties, be well suited for carry out genetic modification, disease Precise Diagnosis and in terms of present it is wide
Application prospect.Nano antibody many simpler than antibody in chemical composition and in shape does not have chemical drains, heat resistance
It is stronger with anti acid alkali performance, it is easier to be combined with each other or in conjunction with other compounds, can be encoded by single-gene, be easy to be closed with microorganism
At.Nano antibody has good tolerance to environment, has the conformational stability of height, and molecular mass is 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,
Make its disease Precise Diagnosis and it is immune in terms of present more extensive application prospect.
Summary of the invention
The technical problem to be solved by the present invention is to how treat tumour.
In order to solve the above technical problems, present invention firstly provides nano antibodies.
Nano antibody provided by the present invention, entitled Nb91 include determinant complementary region；
The determinant complementary region of the Nb91 is made of CDR1, CDR2 and CDR3；
The amino acid sequence of the CDR1 is the 26-32 amino acids of SEQ ID No.7 in sequence table；
The amino acid sequence of the CDR2 is the 48-55 amino acids of SEQ ID No.7 in sequence table；
The amino acid sequence of the CDR3 is the 94-106 amino acids of SEQ ID No.7 in sequence table.
In above-mentioned nano antibody, the Nb91 is made of the determinant complementary region and framework region.
In above-mentioned nano antibody, the Nb91 be it is following a) or b):
A) amino acid sequence is the nano antibody of the 1-117 amino acids of SEQ ID No.7 in sequence table；
B) amino acid sequence is the nano antibody of SEQ ID No.7 in sequence table.
It, can be in sequence table shown in the 1-117 amino acids of SEQ ID No.7 in order to make the Nb91 convenient for purifying
The amino terminal or carboxyl terminal of protein connect upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
| HA | 9 | YPYDVPDYA |
The Nb91 can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.The Nb91's
Encoding gene can be by by DNA sequence shown in the 1-351 nucleotide or SEQ ID No.8 of SEQ ID No.8 in sequence table
The codon of one or several amino acid residues is lacked in column, and/or carries out the missense mutation of one or several base-pairs, and/
Or it is obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.
Wherein, the 1-117 amino acids of the 1-351 nucleotide coding SEQ ID No.7 of SEQ ID No.8 are received
The nanometer of the 118-136 amino acids of the 352-408 nucleotide coding SEQ ID No.7 of meter Kang Ti, SEQ ID No.8
Antibody, nano antibody shown in DNA encoding SEQ ID No.7 shown in SEQ ID No.8.
In order to solve the above technical problems, the present invention also provides biomaterials relevant to the Nb91.
Biomaterial relevant to the Nb91 provided by the present invention is B1) any one of to B12):
B1 the nucleic acid molecules of the Nb91) 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 is also possible to RNA, such as mRNA or hnRNA.
In above-mentioned biomaterial, B2) described in expression cassette (the Nb91 gene table containing the nucleic acid molecules for encoding the Nb91
Up to box), it is the DNA for referring to express Nb91 in host cell, which not only may include the starting for starting Nb91 genetic transcription
Son may also include the terminator for terminating Nb91 genetic transcription.Further, the expression cassette may also include enhancer sequence.
The recombinant vector of the Nb91 expression casette 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 that the B1) nucleic acid molecules are imported into obtained in pComb3
Recombinant vector.In one embodiment of the invention, B3) recombinant vector is by the encoding gene (nucleotide of the Nb91
Sequence is the 1-351 nucleotide of SEQ ID No.8 in sequence table) import recombinant vector pComb3- obtained in pComb3
Nb91, recombinant vector pComb3-Nb91 express nano antibody Nb91 shown in SEQ ID No.7.
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 propagation material；The recombinant microorganism can be
B1) the nucleic acid molecules are imported into 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 directed evolution and point mutation
Method, to B1 of the invention) nucleotide sequence of the Nb91 is mutated.Those have and the present invention by manually modified
B1) Nb91 75% or 75% or more identity of nucleotide sequence nucleotide, as long as encoding the Nb91 and having
Nb91 activity is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
The nucleotide sequence of protein shown in the 1-117 amino acids of bright coding SEQ ID No.7 or SEQ ID No.7 has
The nucleotide sequence of 75% or higher or 85% or higher or 90% or higher or 95% or higher identity.Identity can
With with the naked eye or computer software is evaluated.Using computer software, the identity between two or more sequences can be used
Percentage (%) indicates, can be used to evaluate the identity between correlated series.
Above-mentioned 75% or 75% or more identity can be 75%, 80%, 85%, 90% or 95% or more identity.
In above-mentioned biomaterial, the coded sequence of the CDR1 of the Nb91 is the 75-96 of SEQ ID No.8 in sequence table
Position nucleotide；
The 142-165 nucleotide of SEQ ID No.8 in the coded sequence of the CDR2 of the Nb91 such as sequence table；
The coded sequence of the CDR3 of the Nb91 is the 280-318 nucleotide of SEQ ID No.8 in sequence table.
In above-mentioned biomaterial, B1) nucleic acid molecules be it is following 1) or 2) or 3) or 4):
1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.8 in sequence table；
2) nucleotide sequence is that the cDNA molecule of the 1-351 nucleotide of SEQ ID No.8 or DNA divide in sequence table
Son；
1) or 2) 3) there is 75% or 75% or more identity with the nucleotide sequence limited, and encodes the Nb91's
CDNA molecule or genomic DNA molecule；
4) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and encode the Nb91 cDNA molecule or
Genomic DNA molecule.
In order to solve the above technical problems, the present invention also provides the derivative antibody of the Nb91.
The derivative antibody of the Nb91 provided by the present invention, for it is following a) or b) or c) or d) or e):
A) contain the single-chain antibody of the Nb91；
B) contain the fusion antibody of a) single-chain antibody；
C) contain the fusion antibody of the Nb91；
D) contain the Fab of the Nb91；
E) contain the complete antibody of the Nb91.
In order to solve the above technical problems, the present invention also provides the preparation methods of the Nb91.
The preparation method of the Nb91 provided by the present invention, the nucleic acid molecules including that will encode the Nb91 import receptor
Cell obtains expressing the transgenic cell of the Nb91, cultivates the transgenic cell, obtains the Nb91.
In the preparation method of above-mentioned Nb91, in the nucleotide sequence of the nucleic acid molecules of the coding Nb91 such as sequence table
Shown in the 1-351 nucleotide or SEQ ID No.8 of SEQ ID No.8.
In the preparation method of above-mentioned Nb91, the recipient cell can be microbial cell, such as Escherichia coli, concretely greatly
Enterobacteria WK6.
In order to solve the above technical problems, the present invention also provides any purposes in following A 1-A12:
A1, the Nb91 are preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A2, the biomaterial are preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A3, the nano antibody derivative antibody preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A4, the Nb91 preparation method preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A5, the Nb91 inhibit CTLA-4 activity in preparation or promote the application in T cell proliferation product；
A6, the biomaterial inhibit CTLA-4 activity in preparation or promote the application in T cell proliferation product；
A7, the derivative antibody inhibit CTLA-4 activity in preparation or promote the application in T cell proliferation product；
A8, the Nb91 preparation method preparation inhibit CTLA-4 activity or promote T cell proliferation product in application；
A9, the Nb91 are in preparation and the application in CTLA-4 combination product；
A10, the biomaterial are in preparation and the application in CTLA-4 combination product；
A11, the derivative antibody are in preparation and the application in CTLA-4 combination product；
A12, the Nb91 preparation method preparation with CTLA-4 combination product in application.
The said goods can be drug.
The core of amino acid sequence shown in SEQ ID No.7 or its any fragment amino acid sequence in amplification coding sequence table
The primer pair of acid molecule, also belongs to protection scope of the present invention.
The invention discloses a kind of nano antibodies (Nb91) for being directed to CTLA-4 peptide molecule epitope, while also public affairs
Cloth encodes the gene order of the nano antibody and can express the host cell of the nano antibody.It is announced through the invention
Nano antibody gene order and host cell, the nano antibody (Nb91) can in Escherichia coli high efficient expression, can be with T
Cell and CTLA-4 are combined, and can be applied to the research and development of CTLA-4 Molecular Detection reagent, prepare tumor inhibitor or tumour cell suppression
Preparation and preparation inhibit CTLA-4 activity and promote the drug of T cell proliferation.
Detailed description of the invention
Fig. 1 is the SDA-PAGE electrophoretogram of nano antibody Nb16.Wherein, swimming lane M indicates molecular weight of albumen Marker.
Fig. 2 is the SDA-PAGE electrophoretogram of nano antibody Nb30.Wherein, swimming lane M indicates molecular weight of albumen Marker.
Fig. 3 is the SDA-PAGE electrophoretogram of nano antibody Nb36.Wherein, swimming lane M indicates molecular weight of albumen Marker.
Fig. 4 is the SDA-PAGE electrophoretogram of nano antibody Nb91.Wherein, swimming lane M indicates molecular weight of albumen Marker.
Fig. 5 is the Binding experiment result of nano antibody Nb16 and antigen.
Fig. 6 is the Binding experiment result of nano antibody Nb30 and antigen.
Fig. 7 is the Binding experiment result of nano antibody Nb36 and antigen.
Fig. 8 is the Binding experiment result of nano antibody Nb91 and antigen.
In figure 5-8, CTLA-4mAb indicates PE mouse anti-human CD152.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and 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 unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Escherichia coli WK6 (Yan et al., Characterization and appl in following embodiments
ications of Nanobodies against human procalcitonin selected from a novelNanobody phage display l ibrary, Journal of Nanobiotechnology (2015) 13:
33) after the Southeast China University laboratory life science institute Wan Yakun is agreed to, the public can obtain the biology from Guangxi Medical University
Material, the biomaterial are only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and are used.
The preparation of embodiment 1, nano antibody
The present invention provides derive from camel four kinds of nano antibodies, title be respectively Nb16, Nb30, Nb36 and
Nb91.Wherein, the amino acid sequence of Nb16 is as shown in SEQ ID No.1 in sequence table, by the nucleotide sequence of SEQ ID No.2
Coding；The amino acid sequence of Nb30 is as shown in SEQ ID No.3 in sequence table, by the nucleotide sequence coded of SEQ ID No.4；
The amino acid sequence of Nb36 is as shown in SEQ ID No.5 in sequence table, by the nucleotide sequence coded of SEQ ID No.6；Nb91
Amino acid sequence as shown in SEQ ID No.7 in sequence table, by the nucleotide sequence coded of SEQ ID No.8.
The PstI and NotI of carrier pComb3 (Biovector product) are identified that the DNA fragmentation between sequence replaces with SEQ
DNA molecular shown in ID No.2, other sequences are constant, obtain recombinant vector pComb3-Nb16, pComb3-Nb16 with
The difference of pComb3, which is only that, replaces with SEQ ID No.2 institute for the DNA fragmentation between the PstI of pComb3 and NotI identification sequence
The DNA molecular shown.Recombinant vector pComb3-Nb16 expresses nano antibody Nb16 shown in SEQ ID No.1.By pComb3-
Nb16 is imported in Escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb16.
The PstI and NotI of carrier pComb3 (Biovector product) are identified that the DNA fragmentation between sequence replaces with SEQ
DNA molecular shown in ID No.4, other sequences are constant, obtain recombinant vector pComb3-Nb30, pComb3-Nb30 with
The difference of pComb3, which is only that, replaces with SEQ ID No.4 institute for the DNA fragmentation between the PstI of pComb3 and NotI identification sequence
The DNA molecular shown.Recombinant vector pComb3-Nb16 expresses nano antibody Nb30 shown in SEQ ID No.3.By pComb3-
Nb30 is imported in Escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb30.
The PstI and NotI of carrier pComb3 (Biovector product) are identified that the DNA fragmentation between sequence replaces with SEQ
DNA molecular shown in ID No.6, other sequences are constant, obtain recombinant vector pComb3-Nb36, pComb3-Nb36 with
The difference of pComb3, which is only that, replaces with SEQ ID No.6 institute for the DNA fragmentation between the PstI of pComb3 and NotI identification sequence
The DNA molecular shown.Recombinant vector pComb3-Nb36 expresses nano antibody Nb36 shown in SEQ ID No.5.By pComb3-
Nb36 is imported in Escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb36.
The PstI and NotI of carrier pComb3 (Biovector product) are identified that the DNA fragmentation between sequence replaces with SEQ
DNA molecular shown in ID No.8, other sequences are constant, obtain recombinant vector pComb3-Nb91, pComb3-Nb91 with
The difference of pComb3, which is only that, replaces with SEQ ID No.8 institute for the DNA fragmentation between the PstI of pComb3 and NotI identification sequence
The DNA molecular shown.Recombinant vector pComb3-Nb91 expresses nano antibody Nb91 shown in SEQ ID No.7.By pComb3-
Nb91 is imported in Escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb91.
Specific preparation process is as follows for nano antibody:
(1) by WK6-pComb3-Nb16 be coated on containing ampicillin and glucose LB plate (in LB plate, ammonia
The concentration of parasiticin and glucose is respectively 100 μ g/mL and 20mg/mL) on, 37 DEG C of overnight incubations (12 hours)；
(2) select single bacterium colony be seeded in LB culture solution that 5mL contains ampicillin (in LB culture solution, ammonia benzyl mould
The concentration of element is 100 μ g/mL) in, 37 DEG C of shaking table cultures are overnight (12 hours)；
(3) culture solution of 1mL step (2) overnight incubation is taken to be seeded in 330mL TB culture solution, 37 DEG C of shaking table cultures are extremely
When OD value reaches 0.6-1, IPTG is added, obtains WK6-pComb3-Nb16 culture solution, makes in WK6-pComb3-Nb16 culture solution
The concentration of IPTG is 1mM, and WK6-pComb3-Nb16 culture solution is trained at 28 DEG C on shaking table (revolving speed of shaking table is 220rpm)
It supports overnight (12 hours), obtains WK6-pComb3-Nb16 induction liquid；
(4) the WK6-pComb3-Nb16 induction liquid of step (3) is centrifuged at 4 DEG C, collects thallus；
(5) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y:Combining magnetic is utilized
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
Of influenza H3N2.Nanoscale Res Lett 2014,9:528.) in osmosis, obtain antibody crude extract；
(6) document (Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S, Wan Y:Combining magnetic is utilized
nanoparticle with biotinylated nanobodies for rapid and sensitive detection
Of influenza H3N2.Nanoscale Res Lett 2014,9:528.) in nickel column ion affinity chromatography method preparation receive
Meter Kang Ti Nb16.
According to above-mentioned steps (1)-(6) method, respectively by WK6-pComb3-Nb16 replace with WK6-pComb3-Nb30,
WK6-pComb3-Nb36 and WK6-pComb3-Nb91, other steps are constant, respectively obtain nano antibody Nb30, Nb36 and
Nb91。
The SDA-PAGE electrophoretogram of nano antibody Nb16, Nb30, Nb36 and Nb91 distinguish as shown in Figure 1, Figure 2, Fig. 3 and Fig. 4 institute
Show.The results show that the purity of nano antibody Nb16, Nb30, Nb36 and Nb91 obtained by the above method reach 90% or more.
The measurement of embodiment 2, nano antibody and CTLA-4 Percentage bound
One, nano antibody and CTLA-4 Percentage bound measurement (direct method)
T cell is separated from healthy volunteer human peripheral blood cell, is incubated in 96 orifice plates, by 10 μ g/mL PHA
(sigma, L9017) is added in T cell after co-cultivation 72h, and the Nb16 (1 μ g) of embodiment 1 is added 1 × 106A above-mentioned T cell
In 4 DEG C be protected from light be incubated for 30min, PBS washing three times after, be added 1 μ g PE anti-HA tag antibody (abcam, Clone:
16B12) after 4 DEG C of incubation 30min, PBS washings three times, by BACKMAN flow cytometer on sample, as a result as shown in A in Fig. 5.
According to the method described above, Nb16 is replaced with into Nb30, Nb36 and Nb91 respectively, other steps are constant, obtain Nb30,
The combination of Nb36 and Nb91 and CTLA-4 is as a result, respectively as shown in A in A, Fig. 8 in A, Fig. 7 in Fig. 6.
Two, nano antibody and CTLA-4 Percentage bound measurement (competition law)
1, T cell is separated from healthy volunteer human peripheral blood cell, be incubated in 96 orifice plates, by 10 μ g/ml PHA
(sigma, L9017) is added in T cell and co-cultures 72h, obtains the T cell of PHA stimulation.
2, by PE mouse anti-human CD152 (BD Clone:BNI3) (20 μ l) and 1 × 106A above-mentioned T cell
After 4 DEG C are protected from light incubation 30min, PBS washing three times, by BACKMAN flow cytometer on sample.
3, by the Nb16 of embodiment 1 (1 μ g) and PE mouse anti-human CD152 (BD Clone:BNI3) (20 μ
L) incubation 30min is protected from light for 4 DEG C, then by the mixture and the 1 × 10 of step 164 DEG C of T cell of a PHA stimulation are protected from light incubation
After 30min, PBS washing three times, by BACKMAN flow cytometer on sample.As a result as shown in B in Fig. 5.
According to the method described above, Nb16 is replaced with into Nb30, Nb36 and Nb91 respectively, other steps are constant, obtain Nb30,
The combination of Nb36 and Nb91 and CTLA-4 is as a result, respectively as shown in B in B, Fig. 8 in B, Fig. 7 in Fig. 6.
The result shows that nano antibody Nb16, Nb30, Nb36 and Nb91 can be in conjunction with T cells and CTLA-4.
Claims (7)
1. nano antibody, it is characterised in that: the nano antibody is as shown in SEQ ID No.7 in sequence table.
2. it is B1 biomaterial relevant to nano antibody described in claim 1) any one of to B12):
B1 the nucleic acid molecules of nano antibody described in claim 1) 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.
3. biomaterial according to claim 2, it is characterised in that: B1) nucleic acid molecules are SEQ ID in sequence table
DNA molecular shown in No.8.
4. the derivative antibody of nano antibody described in claim 1, for it is following a) or b) or c) or d) or e):
A) single-chain antibody containing nano antibody described in claim 1；
B) contain the fusion antibody of a) single-chain antibody；
C) fusion antibody containing nano antibody described in claim 1；
D) Fab containing nano antibody described in claim 1；
E) complete antibody containing nano antibody described in claim 1.
5. the preparation method of nano antibody described in claim 1, the nucleic acid point including nano antibody described in claim 1 will be encoded
Son imports the transgenic cell that recipient cell obtains expressing the nano antibody, cultivates the transgenic cell, obtains described receive
Meter Kang Ti.
6. according to the method described in claim 5, it is characterized by: the nucleic acid of nano antibody described in the coding claim 1
The nucleotide sequence of molecule is as shown in SEQ ID No.8 in sequence table；
And/or
The recipient cell is microbial cell.
7. any purposes in following A 1-A12:
Nano antibody described in A1, claim 1 inhibits CTLA-4 activity in preparation or promotes the application in T cell proliferation product；
Biomaterial described in A2, Claims 2 or 3 inhibits CTLA-4 activity in preparation or promotes answering in T cell proliferation product
With；
Derivative antibody described in A3, claim 4 inhibits CTLA-4 activity in preparation or promotes the application in T cell proliferation product；
A4, claim 5 or 6 the methods inhibit CTLA-4 activity in preparation or promote the application in T cell proliferation product；
Nano antibody described in A5, claim 1 is in preparation and the application in CTLA-4 combination product；
Biomaterial described in A6, Claims 2 or 3 is in preparation and the application in CTLA-4 combination product；
Derivative antibody is in preparation and the application in CTLA-4 combination product described in A7, claim 4；
A8, claim 4 or 5 the methods are in preparation and the application in CTLA-4 combination product.
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| CN107400166A (en) | 2016-05-19 | 2017-11-28 | 苏州康宁杰瑞生物科技有限公司 | for CTLA4 single domain antibody and its derived protein |
| CN107474135B (en) * | 2017-02-17 | 2020-08-18 | 广西医科大学 | anti-PD-1 nano antibody PD-1/Nb20 and preparation method and application thereof |
| EP4295915A3 (en) * | 2017-12-20 | 2024-05-22 | Harbour Biomed (Shanghai) Co., Ltd | Antibodies binding ctla-4 and uses thereof |
| EP3768721A4 (en) * | 2018-03-19 | 2021-12-29 | Wuxi Biologics Ireland Limited. | Novel anti-ctla-4 antibody polypeptide |
| CN109021099B (en) * | 2018-08-20 | 2020-05-05 | 山东农业大学 | Nano antibody for specifically recognizing type 1 duck hepatitis A virus |
| US20220106406A1 (en) * | 2020-10-01 | 2022-04-07 | University Of Kentucky Research Foundation | Polypeptide directed against protein tyrosine phosphatase 4a proteins, and compositions and methods for use thereof |
| WO2024088424A1 (en) * | 2022-10-28 | 2024-05-02 | 嘉和生物药业有限公司 | Novel anti-ctla4 antibody |
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2016
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| CTLA-4抗体抑制小鼠Lewis肺癌生长的实验研究;崔倪 等;《中国实验诊断学》;20150430;第19卷(第4期);全文 * |
| Nanobody, New Agent for Combating Against Breast Cancer Cells;Fatemeh Rahbarizadeh et al.;《Breast Cancer – Current and Alternative Therapeutic Modalities》;20111231;全文 * |
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