CN106046165A - Anti-CTLA-4 nanobody Nb30 as well as preparation method and application thereof - Google Patents
Anti-CTLA-4 nanobody Nb30 as well as preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The invention discloses an anti-CTLA-4 nanobody Nb30 as well as a preparation method and application thereof. The nanobody provided by the invention comprises a complementarity-determining region and a framework region; the complementarity-determining region of the nanobody consists of CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is that of a 26th-35th amino acid of SEQ ID No. 3 in a sequence table; the amino acid sequence of CDR2 is that of a 51st-58th amino acid of SEQ ID No. 3 in the sequence table; the amino acid sequence of CDR3 is that of a 97th-115th amino acid of SEQ ID No. 3 in the sequence table. The nanobody Nb30 can be combined to T cells and CTLA-4, can be used for research and development of a CTLA-4 molecular detection reagent, and can be used for preparing a tumor inhibitor or a tumor cell inhibitor and preparing drugs for inhibiting CTLA-4 activity and promoting proliferation of the T cells.
Description
Technical field
Nano antibody Nb30 that the present invention relates to anti-CTLA-4 in biomedical sector and preparation method and application.
Background technology
Cytotoxic t lymphocyte-associated antigen 4 (cytotoxic T lymphocytic associated
Antigen, CTLA-4/CTLA4) have another name called CD152, it is a kind of leukocyte differentiation antigen, is the one of activating T cell surface expression
Transmembrane glycoprotein molecule, belongs to immunoglobulin superfamily member, T cell has been bred negativity regulation effect, in immunne response
Play important regulative.CTLA-4Ig can suppress cell and humoral immune reaction, the most effectively, specifically to transplant rejection
Reaction, tumor and various autoimmune disease have significant therapeutic effect, and toxic and side effects is extremely low, be presently considered to relatively to have uncommon
The new immunosuppressive drug hoped.
It is long to there is a lot of problem, such as R&D cycle in antibody drug application, and production cost is too high；It is difficult to large-scale production；
Poor stability is degradable, and storage cost is high；The most contaminated, maintenance cost is costly；And there is immunogenicity etc., limit
Its range of application clinically.
Nano antibody technology, be Biomedical Science man on the basis of conventional antibodies, use Protocols in Molecular Biology knot
The antibody engineering revolution that the concept of conjunction nanoparticle science is carried out, thus the up-to-date and minimum antibody molecule developed.1993
The reports such as year Hamers, also exist natural deletions light chain and the heavy chain antibody of CH1 (CH1), Ke Longqi in camel body
Variable region obtains the single domain antibody being only made up of, referred to as VHH (variable domain of heavy a variable region of heavy chain
Chain of heavy-chain antibody), renamed as " nano antibody " (nanobody, Nb).Nanometer resists
Body has the minimum Fab of complete function, its crystal structure ovalize, diameter 2.5nm, long 4nm.Nb has
Many unique character, are well suited for carrying out genetic modification, present wide at aspects such as the Precise Diagnosis of disease and targeted therapies
Application prospect.Nano antibody, in chemical composition and simpler many than antibody in shape, does not have chemical drains, its heat resistance
Higher with anti acid alkali performance, it is easier to be combined with each other or be combined with other compounds, energy coverlet gene code, easily closes with microorganism
Become.Nano antibody has good toleration to environment, possesses the conformational stability of height, and molecular mass is less, clinical
Therapeutic effect more preferable, these little protein moleculars are easier to synthesis simultaneously, and price is the lowest.Unique character of nano antibody,
It is made to present the most wide application prospect at aspects such as the Precise Diagnosis of disease and immunity targeted therapies.
Summary of the invention
The technical problem to be solved is how to treat tumor.
For solving above-mentioned technical problem, present invention firstly provides nano antibody.
Nano antibody provided by the present invention, its entitled Nb30, comprise determinant complementary region；
The determinant complementary region of described Nb30 is made up of CDR1, CDR2 and CDR3；
The aminoacid sequence of described CDR1 is the 26-35 amino acids of SEQ ID No.3 in sequence table；
The aminoacid sequence of described CDR2 is the 51-58 amino acids of SEQ ID No.3 in sequence table；
The aminoacid sequence of described CDR3 is the 97-115 amino acids of SEQ ID No.3 in sequence table.
In above-mentioned nano antibody, described Nb30 is made up of described determinant complementary region and framework region.
In above-mentioned nano antibody, described Nb30 be following a) or b):
A) nano antibody of the 1-126 amino acids of SEQ ID No.3 during aminoacid sequence is sequence table；
B) nano antibody of SEQ ID No.3 during aminoacid sequence is sequence table.
In order to make described Nb30 be easy to purification, can be in sequence table shown in the 1-126 amino acids of SEQ ID No.3
The amino terminal of protein or carboxyl terminal connect upper label as shown in table 1.
Table 1, the sequence of 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 |
Described Nb30 can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain.Described Nb30's
Encoding gene can be by by the 1-378 position nucleotide of SEQ ID No.4 in sequence table or the DNA sequence shown in SEQ ID No.4
Row lack the codon of one or several amino acid residue, and/or carries out the missense mutation of one or several base pair, and/
Or hold the coded sequence connecting the label shown in table 1 to obtain at its 5 ' end and/or 3 '.
Wherein, the receiving of the 1-126 amino acids of the 1-378 position nucleotide coding SEQ ID No.3 of SEQ ID No.4
The nanometer of the 127-145 amino acids of the 379-435 position nucleotide coding SEQ ID No.3 of meter Kang Ti, SEQ ID No.4
Antibody, the nano antibody shown in DNA encoding SEQ ID No.3 shown in SEQ ID No.4.
For solving above-mentioned technical problem, present invention also offers the biomaterial relevant to described Nb30.
Biomaterial relevant for Nb30 provided by the present invention and described, for B1) to B12) in any one:
B1) nucleic acid molecules of described Nb30 is encoded；
B2) containing B1) expression cassette of described nucleic acid molecules；
B3) containing B1) recombinant vector of described nucleic acid molecules；
B4) containing B2) recombinant vector of described expression cassette；
B5) containing B1) recombinant microorganism of described nucleic acid molecules；
B6) containing B2) recombinant microorganism of described expression cassette；
B7) containing B3) recombinant microorganism of described recombinant vector；
B8) containing B4) recombinant microorganism of described recombinant vector；
B9) containing B1) the transgenetic animal cell system of described nucleic acid molecules；
B10) containing B2) the transgenetic animal cell system of described expression cassette；
B11) containing B3) the transgenetic animal cell system of described recombinant vector；
B12) containing B4) the transgenetic animal cell system of described recombinant vector.
In above-mentioned biomaterial, described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described core
Acid molecule can also be RNA, such as mRNA or hnRNA etc..
In above-mentioned biomaterial, B2) described in expression cassette (the Nb30 gene table containing the nucleic acid molecules encoding described Nb30
Reach box), refer to express the DNA of Nb30, this DNA in host cell and not only can include starting the startup of Nb30 genetic transcription
Son, may also include the terminator terminating Nb30 genetic transcription.Further, described expression cassette may also include enhancer sequence.
Available existing expression vector establishment contains the recombinant vector of described Nb30 expression casette.
In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, phage or viral vector.
In above-mentioned biomaterial, described recombinant vector can be by B1) described nucleic acid molecules imports to obtain in pComb3
Recombinant vector.In one embodiment of the invention, B3) described recombinant vector is by the encoding gene (nucleotide of described Nb30
Sequence is the 1-378 position nucleotide of SEQ ID No.4 in sequence table) import the recombinant vector pComb3-obtained in pComb3
Nb30, recombinant vector pComb3-Nb30 express the nano antibody Nb30 shown in SEQ ID No.3.
In above-mentioned biomaterial, described microorganism can be yeast, antibacterial, algae or fungus.
In above-mentioned biomaterial, described transgenetic animal cell system does not include propagating materials；Described recombinant microorganism can be
By B1) described nucleic acid molecules imports to the recombinant microorganism that obtains in escherichia coli WK6.
Those of ordinary skill in the art can use the side of known method, such as orthogenesis and point mutation easily
Method, the B1 to the present invention) nucleotide sequence of described Nb30 suddenlys change.Those have and the present invention through manually modified
B1) nucleotide of more than 75% or 75% homogeneity of nucleotide sequence of described Nb30, as long as encoding described Nb30 and having
Nb30 activity, is all derived from the nucleotide sequence of the present invention and is equal to the sequence of the present invention.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this
Bright coding SEQ ID No.3 or the nucleotide sequence of the protein shown in 1-126 amino acids of SEQ ID No.3 have
75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.Homogeneity can
With with the naked eye or computer software is evaluated.Using computer software, the homogeneity between two or more sequences can be used
Percentage ratio (%) represents, it can be used to the homogeneity evaluating between correlated series.
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 75%, 80%, 85%, 90% or more than 95%.
In above-mentioned biomaterial, the coded sequence of the CDR1 of described Nb30 is the 76-of SEQ ID No.4 in sequence table
105 nucleotide；
The 151-174 position nucleotide of SEQ ID No.4 in the coded sequence of the CDR2 of described Nb30 such as sequence table；
The coded sequence of the CDR3 of described Nb30 is the 289-345 position nucleotide of SEQ ID No.4 in sequence table.
In above-mentioned biomaterial, B1) described nucleic acid molecules is following 1) or 2) or 3) or 4):
1) the cDNA molecule of SEQ ID No.4 or DNA molecular during nucleotide sequence is sequence table；
2) during nucleotide sequence is sequence table, cDNA molecule or the DNA of the 1-378 position nucleotide of SEQ ID No.4 divide
Son；
3) with 1) or 2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and encode described Nb30's
CDNA molecule or genomic DNA molecule；
4) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and encode described Nb30 cDNA molecule or
Genomic DNA molecule.
For solving above-mentioned technical problem, present invention also offers the derivative antibody of described Nb30.
The derivative antibody of described Nb30 provided by the present invention, for following a) or b) or c) or d) or e):
A) single-chain antibody containing described Nb30；
B) fusion antibody containing a) described single-chain antibody；
C) fusion antibody containing described Nb30；
D) Fab containing described Nb30；
E) complete antibody containing described Nb30.
For solving above-mentioned technical problem, present invention also offers the preparation method of described Nb30.
The preparation method of described Nb30 provided by the present invention, imports receptor including by the nucleic acid molecules encoding described Nb30
Cell obtains expressing the transgenic cell of described Nb30, cultivates described transgenic cell, obtains described Nb30.
In the preparation method of above-mentioned Nb30, in the nucleotide sequence such as sequence table of the nucleic acid molecules of the described Nb30 of described coding
Shown in the 1-378 position nucleotide of SEQ ID No.4 or SEQ ID No.4.
In the preparation method of above-mentioned Nb30, described recipient cell can be microbial cell, such as escherichia coli, the most greatly
Enterobacteria WK6.
For solving above-mentioned technical problem, present invention also offers any one purposes in following A 1-A12:
A1, described Nb30 application in preparing tumor inhibitor or inhibiting tumour cells agent；
A2, the application in preparing tumor inhibitor or inhibiting tumour cells agent of the described biomaterial；
A3, the derivative antibody application in preparing tumor inhibitor or inhibiting tumour cells agent of described nano antibody；
A4, the preparation method application in preparing tumor inhibitor or inhibiting tumour cells agent of described Nb30；
A5, described Nb30 are in preparation suppression CTLA-4 activity or promote the application in T cell propagation product；
A6, described biomaterial are in preparation suppression CTLA-4 activity or promote the application in T cell propagation product；
A7, described derivative antibody are in preparation suppression CTLA-4 activity or promote the application in T cell propagation product；
A8, the preparation method of described Nb30 in preparation suppression CTLA-4 activity or promote the application in T cell propagation product；
A9, the described Nb30 application in preparation with CTLA-4 bonded products；
The application in preparation with CTLA-4 bonded products of A10, described biomaterial；
The application in preparation with CTLA-4 bonded products of A11, described derivative antibody；
A12, the preparation method of described Nb30 are being prepared and the application in CTLA-4 bonded products.
The said goods can be medicine.
Aminoacid sequence shown in SEQ ID No.3 or the core of its arbitrary fragment amino acid sequence in amplification coding sequence table
The primer pair of acid molecule, falls within protection scope of the present invention.
The invention discloses a kind of nano antibody (Nb30) being directed to CTLA-4 peptide molecule epitope, simultaneously the most public
Cloth encodes the gene order of this nano antibody and can express the host cell of this nano antibody.Announced by the present invention
Nano antibody gene order and host cell, this nano antibody (Nb30) can in escherichia coli high efficient expression, can be with T
Cell and CTLA-4 combine, and can be applicable to the research and development of CTLA-4 Molecular Detection reagent, prepare tumor inhibitor or tumor cell presses down
Preparation and the active medicine with promotion T cell propagation of preparation suppression CTLA-4.
Accompanying drawing explanation
Fig. 1 is the SDA-PAGE electrophoretogram of nano antibody Nb16.Wherein, swimming lane M represents molecular weight of albumen Marker.
Fig. 2 is the SDA-PAGE electrophoretogram of nano antibody Nb30.Wherein, swimming lane M represents molecular weight of albumen Marker.
Fig. 3 is the SDA-PAGE electrophoretogram of nano antibody Nb36.Wherein, swimming lane M represents molecular weight of albumen Marker.
Fig. 4 is the SDA-PAGE electrophoretogram of nano antibody Nb91.Wherein, swimming lane M represents 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 Fig. 5-Fig. 8, CTLA-4mAb represents PE mouse anti-human CD152.
Detailed description of the invention
Being further described in detail the present invention below in conjunction with detailed description of the invention, the embodiment be given is only for explaining
The bright present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Escherichia coli WK6 (Yan et al., Characterization and appl in following embodiment
ications of Nanobodies against human procalcitonin selected from a novelNanobody phage display l ibrary, Journal of Nanobiotechnology (2015) 13:
33) after the sub-female laboratory of life science institute of Southeast China University ten thousand is agreed to, the public can obtain this biology from Guangxi Medical University
Material, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes.
Embodiment 1, the preparation of nano antibody
The invention provides the four kinds of nano antibodies deriving from camel, its title be respectively Nb16, Nb30, Nb36 and
Nb91.Wherein, the aminoacid 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 aminoacid sequence of Nb30 as shown in SEQ ID No.3 in sequence table, nucleotide sequence coded by SEQ ID No.4；
The aminoacid sequence of Nb36 as shown in SEQ ID No.5 in sequence table, nucleotide sequence coded by SEQ ID No.6；Nb91
Aminoacid sequence as shown in SEQ ID No.7 in sequence table, nucleotide sequence coded by SEQ ID No.8.
DNA fragmentation between PstI and the NotI recognition sequence of carrier pComb3 (Biovector product) is replaced with SEQ
DNA molecular shown in ID No.2, other sequences are the most constant, obtain recombinant vector pComb3-Nb16, pComb3-Nb16 with
DNA fragmentation between the difference of pComb3 is only that PstI and the NotI recognition sequence of pComb3 replaces with SEQ ID No.2 institute
The DNA molecular shown.Recombinant vector pComb3-Nb16 expresses the nano antibody Nb16 shown in SEQ ID No.1.By pComb3-
Nb16 imports in escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb16.
DNA fragmentation between PstI and the NotI recognition sequence of carrier pComb3 (Biovector product) is replaced with SEQ
DNA molecular shown in ID No.4, other sequences are the most constant, obtain recombinant vector pComb3-Nb30, pComb3-Nb30 with
DNA fragmentation between the difference of pComb3 is only that PstI and the NotI recognition sequence of pComb3 replaces with SEQ ID No.4 institute
The DNA molecular shown.Recombinant vector pComb3-Nb16 expresses the nano antibody Nb30 shown in SEQ ID No.3.By pComb3-
Nb30 imports in escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb30.
DNA fragmentation between PstI and the NotI recognition sequence of carrier pComb3 (Biovector product) is replaced with SEQ
DNA molecular shown in ID No.6, other sequences are the most constant, obtain recombinant vector pComb3-Nb36, pComb3-Nb36 with
DNA fragmentation between the difference of pComb3 is only that PstI and the NotI recognition sequence of pComb3 replaces with SEQ ID No.6 institute
The DNA molecular shown.Recombinant vector pComb3-Nb36 expresses the nano antibody Nb36 shown in SEQ ID No.5.By pComb3-
Nb36 imports in escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb36.
DNA fragmentation between PstI and the NotI recognition sequence of carrier pComb3 (Biovector product) is replaced with SEQ
DNA molecular shown in ID No.8, other sequences are the most constant, obtain recombinant vector pComb3-Nb91, pComb3-Nb91 with
DNA fragmentation between the difference of pComb3 is only that PstI and the NotI recognition sequence of pComb3 replaces with SEQ ID No.8 institute
The DNA molecular shown.Recombinant vector pComb3-Nb91 expresses the nano antibody Nb91 shown in SEQ ID No.7.By pComb3-
Nb91 imports in escherichia coli WK6, obtains recombinant bacterium WK6-pComb3-Nb91.
The concrete preparation process of nano antibody is as follows:
(1) WK6-pComb3-Nb16 is coated on LB flat board containing ampicillin and glucose (in LB flat board, ammonia
The concentration of benzylpcnicillin and glucose is respectively 100 μ g/mL and 20mg/mL) on, 37 DEG C of overnight incubation (12 hours)；
(2) select LB culture fluid that single colony inoculation contains ampicillin at 5mL (in LB culture fluid, ammonia benzyl penicillium sp
The concentration of element is 100 μ g/mL) in, 37 DEG C of shaking table overnight incubation (12 hours)；
(3) culture fluid taking 1mL step (2) overnight incubation is seeded in 330mL TB culture fluid, and 37 DEG C of shaking tables are cultivated extremely
When OD value reaches 0.6-1, add IPTG, obtain WK6-pComb3-Nb16 culture fluid, make in WK6-pComb3-Nb16 culture fluid
The concentration of IPTG is 1mM, is above trained in shaking table (rotating speed of shaking table is 220rpm) by WK6-pComb3-Nb16 culture fluid at 28 DEG C
Support overnight (12 hours), obtain WK6-pComb3-Nb16 and induce liquid；
(4) WK6-pComb3-Nb16 of step (3) induces liquid centrifugal at 4 DEG C, collect thalline；
(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, it is thus achieved that 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 post ion affinity chromatography method preparation receive
Meter Kang Ti Nb16.
According to the method for above-mentioned steps (1)-(6), respectively WK6-pComb3-Nb16 is replaced with WK6-pComb3-Nb30,
WK6-pComb3-Nb36 and WK6-pComb3-Nb91, other steps are the most constant, respectively obtain nano antibody Nb30, Nb36 and
Nb91。
The SDA-PAGE electrophoretogram of nano antibody Nb16, Nb30, Nb36 and Nb91 is distinguished the most as shown in Figure 1, Figure 2, Fig. 3 and Fig. 4 institute
Show.Result shows, the purity of nano antibody Nb16, Nb30, Nb36 and Nb91 that said method obtains all reaches more than 90%.
Embodiment 2, nano antibody and the mensuration of CTLA-4 combination rate
One, nano antibody measures (direct method) with CTLA-4 combination rate
From healthy volunteer human peripheral blood cell, separate T cell, be incubated in 96 orifice plates, by 10 μ g/mL PHA
After (sigma, L9017) addition T cell co-cultures 72h, the Nb16 (1 μ g) of embodiment 1 is added 1 × 106Individual above-mentioned T cell
In 4 DEG C of lucifuges hatch after 30min, PBS wash three times, add 1 μ g PE anti-HA tag antibody (abcam, Clone:
16B12) hatching 30min, PBS and wash after three times for 4 DEG C, by BACKMAN flow cytometer on sample, result is as shown in A in Fig. 5.
According to the method described above, Nb16 replacing with Nb30, Nb36 and Nb91 respectively, other steps are the most constant, obtain Nb30,
The combination result of Nb36 and Nb91 and CTLA-4, respectively as shown in A in A, Fig. 8 in A, Fig. 7 in Fig. 6.
Two, nano antibody measures (competition law) with CTLA-4 combination rate
1, from healthy volunteer human peripheral blood cell, separate T cell, be incubated in 96 orifice plates, by 10 μ g/mL PHA
(sigma, L9017) addition T cell co-cultures 72h, obtains the T cell that PHA stimulates.
2, by PE mouse anti-human CD152 (BD Clone:BNI3) (20 μ L) and 1 × 106Individual above-mentioned T cell
4 DEG C of lucifuges are hatched after 30min, PBS wash three times, by BACKMAN flow cytometer on sample.
3, by the Nb16 (1 μ g) of embodiment 1 and PE mouse anti-human CD152 (BD Clone:BNI3) (20 μ
L) 4 DEG C of lucifuges hatch 30min, then by this mixture and the 1 × 10 of step 164 DEG C of lucifuges of T cell that individual PHA stimulates are hatched
After 30min, PBS wash three times, by BACKMAN flow cytometer on sample.Result is as shown in B in Fig. 5.
According to the method described above, Nb16 replacing with Nb30, Nb36 and Nb91 respectively, other steps are the most constant, obtain Nb30,
The combination result of Nb36 and Nb91 and CTLA-4, respectively as shown in B in B, Fig. 8 in B, Fig. 7 in Fig. 6.
Result shows, nano antibody Nb16, Nb30, Nb36 and Nb91 all can be combined with T cell and CTLA-4.
Claims (10)
1. nano antibody, comprises determinant complementary region；
The determinant complementary region of described nano antibody is made up of CDR1, CDR2 and CDR3；
The aminoacid sequence of described CDR1 is the 26-35 amino acids of SEQ ID No.3 in sequence table；
The aminoacid sequence of described CDR2 is the 51-58 amino acids of SEQ ID No.3 in sequence table；
The aminoacid sequence of described CDR3 is the 97-115 amino acids of SEQ ID No.3 in sequence table.
Nano antibody the most according to claim 1, it is characterised in that: described nano antibody by described determinant complementary region and
Framework region forms.
Nano antibody the most according to claim 1 and 2, it is characterised in that: described nano antibody be following a) or b):
A) nano antibody of the 1-126 amino acids of SEQ ID No.3 during aminoacid sequence is sequence table；
B) nano antibody of SEQ ID No.3 during aminoacid sequence is sequence table.
4. the biomaterial relevant to nano antibody described in claim 1 or 2 or 3, for B1) to B12) in any one:
B1) nucleic acid molecules of nano antibody described in coding claim 1 or 2 or 3；
B2) containing B1) expression cassette of described nucleic acid molecules；
B3) containing B1) recombinant vector of described nucleic acid molecules；
B4) containing B2) recombinant vector of described expression cassette；
B5) containing B1) recombinant microorganism of described nucleic acid molecules；
B6) containing B2) recombinant microorganism of described expression cassette；
B7) containing B3) recombinant microorganism of described recombinant vector；
B8) containing B4) recombinant microorganism of described recombinant vector；
B9) containing B1) the transgenetic animal cell system of described nucleic acid molecules；
B10) containing B2) the transgenetic animal cell system of described expression cassette；
B11) containing B3) the transgenetic animal cell system of described recombinant vector；
B12) containing B4) the transgenetic animal cell system of described recombinant vector.
Biomaterial the most according to claim 4, it is characterised in that:
The coded sequence of the CDR1 of described nano antibody is the 76-105 position nucleotide of SEQ ID No.4 in sequence table；
The 151-174 position nucleotide of SEQ ID No.4 in the coded sequence of the CDR2 of described nano antibody such as sequence table；
The coded sequence of the CDR3 of described nano antibody is the 289-345 position nucleotide of SEQ ID No.4 in sequence table.
6. according to the biomaterial described in claim 4 or 5, it is characterised in that: B1) described nucleic acid molecules is following 1) or 2) or
3) or 4):
1) the cDNA molecule of SEQ ID No.4 or DNA molecular during nucleotide sequence is sequence table；
2) the cDNA molecule of the 1-378 position nucleotide of SEQ ID No.4 or DNA molecular during nucleotide sequence is sequence table；
3) with 1) or 2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and coding claim 1 or 2 or 3
The cDNA molecule of described nano antibody or genomic DNA molecule；
4) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and nanometer described in coding claim 1 or 2 or 3
The cDNA molecule of antibody or genomic DNA molecule.
7. the derivative antibody of nano antibody described in claim 1 or 2 or 3, for following a) or b) or c) or d) or e):
A) single-chain antibody containing nano antibody described in claim 1 or 2 or 3；
B) fusion antibody containing a) described single-chain antibody；
C) fusion antibody containing nano antibody described in claim 1 or 2 or 3；
D) Fab containing nano antibody described in claim 1 or 2 or 3；
E) complete antibody containing nano antibody described in claim 1 or 2 or 3.
8. the preparation method of nano antibody described in claim 1 or 2 or 3, including by nanometer described in coding claim 1 or 2 or 3
The nucleic acid molecules of antibody imports recipient cell and obtains expressing the transgenic cell of described nano antibody, cultivates described transgenic thin
Born of the same parents, obtain described nano antibody.
Method the most according to claim 8, it is characterised in that: nano antibody described in described coding claim 1 or 2 or 3
Nucleic acid molecules nucleotide sequence such as sequence table in the 1-378 position nucleotide of SEQ ID No.4 or SEQ ID No.4 institute
Show；
And/or,
Described recipient cell is microbial cell.
10. any one purposes in following A 1-A12:
Nano antibody application in preparing tumor inhibitor or inhibiting tumour cells agent described in A1, claim 1 or 2 or 3；
Arbitrary described biomaterial application in preparing tumor inhibitor or inhibiting tumour cells agent in A2, claim 4-6；
Derivative antibody application in preparing tumor inhibitor or inhibiting tumour cells agent described in A3, claim 7；
Method application in preparing tumor inhibitor or inhibiting tumour cells agent described in A4, claim 8 or 9；
Nano antibody described in A5, claim 1 or 2 or 3 is in preparation suppression CTLA-4 activity or promotes in T cell propagation product
Application；
In A6, claim 4-6, arbitrary described biomaterial in preparation suppression CTLA-4 activity or promotes in T cell propagation product
Application；
Derivative antibody described in A7, claim 7 is in preparation suppression CTLA-4 activity or promotes the application in T cell propagation product；
Method described in A8, claim 8 or 9 is in preparation suppression CTLA-4 activity or promotes the application in T cell propagation product；
The application in preparation with CTLA-4 bonded products of the nano antibody described in A9, claim 1 or 2 or 3；
Arbitrary described biomaterial application in preparation with CTLA-4 bonded products in A10, claim 4-6；
Derivative antibody application in preparation with CTLA-4 bonded products described in A11, claim 7；
The application in preparation with CTLA-4 bonded products of the method described in A12, claim 8 or 9.
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| CN2015104277183 | 2015-07-20 | ||
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| CN107474135A (en) * | 2017-02-17 | 2017-12-15 | 广西医科大学 | Anti- PD 1 nano antibody PD 1/Nb20 and preparation method and application |
| WO2019120232A1 (en) * | 2017-12-20 | 2019-06-27 | Harbour Biomed (Shanghai) Co., Ltd | Antibodies binding ctla-4 and uses thereof |
| CN114560942A (en) * | 2022-01-24 | 2022-05-31 | 宁夏医科大学 | anti-CTLA-4 nano antibody, encoding gene, recombinant nano antibody, recombinant vector, recombinant bacterium and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017198212A1 (en) * | 2016-05-19 | 2017-11-23 | 苏州康宁杰瑞生物科技有限公司 | Single domain antibody for ctla4 and derived protein thereof |
| US11091549B2 (en) | 2016-05-19 | 2021-08-17 | Suzhou Alphamab Co., Ltd. | Single domain antibody and derivative proteins thereof against CTLA4 |
| US11912768B2 (en) | 2016-05-19 | 2024-02-27 | Suzhou Alphamab Co., Ltd. | Single domain antibody and derivative proteins thereof against CTLA4 |
| CN107474135A (en) * | 2017-02-17 | 2017-12-15 | 广西医科大学 | Anti- PD 1 nano antibody PD 1/Nb20 and preparation method and application |
| 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 |
| WO2019120232A1 (en) * | 2017-12-20 | 2019-06-27 | Harbour Biomed (Shanghai) Co., Ltd | Antibodies binding ctla-4 and uses thereof |
| US11098122B2 (en) | 2017-12-20 | 2021-08-24 | Harbour Biomed (Shanghai) Co., Ltd. | Antibodies binding CTLA-4 and uses thereof |
| CN114560942A (en) * | 2022-01-24 | 2022-05-31 | 宁夏医科大学 | anti-CTLA-4 nano antibody, encoding gene, recombinant nano antibody, recombinant vector, recombinant bacterium and application thereof |
| CN114560942B (en) * | 2022-01-24 | 2023-05-19 | 宁夏医科大学 | anti-CTLA-4 nanobody, coding gene, recombinant nanobody, recombinant vector, recombinant bacterium and application thereof |
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