CN110218254A - Nano antibody CD3/Nb25 of AntiCD3 McAb and the preparation method and application thereof - Google Patents
Nano antibody CD3/Nb25 of AntiCD3 McAb and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses nano antibody CD3/Nb25 of AntiCD3 McAb 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；Nano antibody nucleotide sequence and host cell announced through the invention, the nano antibody can in Escherichia coli high efficient expression, applied to the research and development of CD3 Molecular Detection reagent, prepares tumor inhibitor or inhibiting tumour cells agent and preparation inhibits CD3 activity and promotes the drug of T cell proliferation.
Description
Technical field
The present invention relates to nano antibody CD3/Nb25 of AntiCD3 McAb in field of biomedicine and the preparation method and application thereof.
Background technique
CD3 molecule is the important differentiation antigen on T cell film, is the characteristic mark of mature T cells.It and T cell table
Face receptor TCR forms compound, plays an important role during signal transduction in the cell.CD3 antibody can specifically identify T
Cell surface CD3 molecule causes the crosslinking of T cell TCR-CD3 complex, directly generation activation signals, leads to T cell activation simultaneously
Proliferation.Current study show that CD3 antibody has important role in oncotherapy.
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, stores at high cost；It is easy to be contaminated, maintenance cost expense is high；And there is immunogenicity etc.,
Limit 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
How the technical problem to be solved by the present invention is to prepare the drug of effectively treatment tumour.
In order to solve the above technical problems, present invention firstly provides nano antibodies.
The first aspect of the present invention provides a kind of nano antibody, referred to as CD3/Nb25, which includes determinant
Complementary region CDR and framework region FR；The determinant complementary region CDR of the nano antibody is made of CDR1, CDR2 and CDR3；It is described
The amino acid sequence of CDR1 is the 23-32 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the CDR2 is
The 49-57 amino acids of SEQ ID No.8 in sequence table；The amino acid sequence of the CDR3 is SEQ ID in sequence table
The 95-112 amino acids of No.8.
Preferably, the framework region FR of the nano antibody is made of FR1, FR2, FR3 and FR4；The wherein amino acid of the 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 58-94 bit amino 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 is as shown in SEQ ID No.8 in sequence table.
The present invention also accordingly provides a kind of VHH chain of the nano antibody of CD3, including framework region FR and complementary determining region
CDR, the amino acid sequence of the framework region FR FR selected from the group below: shown in FR1 shown in SEQ ID NO:1, SEQ ID NO:2
FR2, FR4 shown in FR3 shown in SEQ ID NO:3, SEQ ID NO:4；The complementary determining region CDR is selected from the group below
The amino acid sequence of CDR: shown in CDR2 shown in CDR1 shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7
CDR3。
Preferably, the VHH chain of the nano antibody of the CD3, it has amino acid sequence shown in SEQ ID NO:8.
It, can be 1-123 of SEQ ID No.8 in sequence table in order to make the nano antibody CD3/Nb25 convenient for purifying
The amino terminal or carboxyl terminal of protein shown in amino acid 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 |
Still further aspect of the present invention, the nano antibody CD3/Nb25 can be artificial synthesized, can also first synthesize its encoding gene,
Biological expression is carried out again to obtain.The encoding gene of the nano antibody CD3/Nb25 can be by by SEQ ID No.9 in sequence table
Shown in the codon of one or several amino acid residues is lacked in DNA sequence dna, and/or carry out the mistake of one or several base-pairs
Justice mutation, and/or obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.
In order to solve the above technical problems, the present invention also provides biological materials relevant to the nano antibody CD3/Nb25
Material.
Biomaterial relevant to the nano antibody CD3/Nb25 provided by the present invention is B1) appointing into B12)
It is a kind of:
B1 the nucleic acid molecules of the nano antibody CD3/Nb25) 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 the expression containing the nucleic acid molecules for encoding the nano antibody CD3/Nb25
Box, also known as CD3/Nb25 expression casette are the DNA for referring to express nano antibody CD3/Nb25 in host cell, should
DNA not only may include the promoter for starting nano antibody CD3/Nb25 genetic transcription, may also include and terminates nano antibody CD3/
The terminator of Nb25 genetic transcription.Further, the expression cassette may also include enhancer sequence.
The recombinant vector of the nano antibody CD3/Nb25 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 coding of the nano antibody CD3/Nb25
Gene (the 1-369 nucleotide that nucleotides sequence is classified as SEQ ID No.9 in sequence table) is imported and is recombinated obtained in pComb3
Carrier pComb3-CD3/Nb25, recombinant vector pComb3-CD3/Nb25 express nano antibody CD3/ shown in SEQ ID No.8
Nb25。
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 nano antibody CD3/Nb25 is mutated.Those by manually modified,
Nucleosides with 75% or 75% or more the identity of nucleotide sequence with B1 of the invention) the nano antibody CD3/Nb25
Acid is derived from of the invention as long as encoding the nano antibody CD3/Nb25 and having nano antibody CD3/Nb25 activity
Nucleotide sequence and it is 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 bright coding SEQ ID No.8 has 75% or higher or 85% or higher or 90%
Or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software is evaluated.Make
With computer software, the identity between two or more sequences can be indicated with percentage (%), can be used to evaluate phase
Close the identity between sequence.
Above-mentioned 75% or 75% or more identity can be 75%, 80%, 85%, 90% or 95% or more identity.
In above-mentioned biomaterial, B1) nucleic acid molecules be 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) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes the nano antibody CD3/
The cDNA molecule or genomic DNA molecule of Nb25；
3) and encode the nano antibody CD3/Nb25's with the nucleotide sequence hybridization that 1) limits under strict conditions
CDNA molecule or genomic DNA molecule.
In order to solve the above technical problems, the present invention also provides the derivative antibody of the nano antibody CD3/Nb25.
The derivative antibody of the nano antibody CD3/Nb25 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 nano antibody CD3/Nb25；
B) contain the fusion antibody of a) single-chain antibody；
C) contain the fusion antibody of the nano antibody CD3/Nb25；
D) contain the Fab of the nano antibody CD3/Nb25；
E) contain the complete antibody of the nano antibody CD3/Nb25.
In order to solve the above technical problems, the present invention also provides the preparation methods of the nano antibody CD3/Nb25.
The preparation method of the nano antibody CD3/Nb25 provided by the present invention, including the nano antibody will be encoded
The nucleic acid molecules of CD3/Nb25 import the transgenic cell that recipient cell obtains expressing the nano antibody CD3/Nb25, cultivate institute
Transgenic cell is stated, the nano antibody CD3/Nb25 is obtained.
In the preparation method of above-mentioned nano antibody CD3/Nb25, the nucleic acid point of the coding nano antibody CD3/Nb25
The nucleotide sequence of son is as shown in SEQ ID No.9 in sequence table.
In the preparation method of above-mentioned nano antibody CD3/Nb25, 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 purposes in following A 1-A8:
A1, the nano antibody CD3/Nb25 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 nano antibody CD3/Nb25 preparation method in preparing tumor inhibitor or inhibiting tumour cells agent
Using；
A5, the nano antibody CD3/Nb25 inhibit CD3 activity in preparation or promote the application in T cell proliferation product；
A6, the biomaterial inhibit CD3 activity in preparation or promote the application in T cell proliferation product；
A7, the derivative antibody inhibit CD3 activity in preparation or promote the application in T cell proliferation product；
A8, the nano antibody CD3/Nb25 preparation method preparation inhibit CD3 activity or promote T cell be proliferated product
In application.
The said goods can be drug.
The core of amino acid sequence shown in SEQ ID No.8 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 present invention provides a kind of nano antibody of AntiCD3 McAb, the nucleotide sequence and host cell of the nano antibody are encoded,
With and its preparation method and application.The nano antibody can in Escherichia coli high efficient expression, be applied to CD3 Molecular Detection reagent
Research and development, prepare tumor inhibitor or inhibiting tumour cells agent and preparation inhibit CD3 activity and promote T cell proliferation medicine
Object.
Detailed description of the invention
Fig. 1 is the DNA electrophoretogram of nano antibody, and from left to right the DNA band of gel pore is respectively: first is 2000bp
Molecular labeling, the second duct be CD3 nano antibody DNA electrophoretic band, PCR product band is about 450bp.
Fig. 2 is the electrophoresis of SDS-PAGE of the CD3 nano antibody CD3/Nb25 after nickel column resin gel affinitive layer purification
Figure；Swimming lane M indicates molecular weight of albumen Marker.
Fig. 3 A is the Binding experiment result of nano antibody CD3/Nb25 and B cell；Fig. 3 B is nano antibody CD3/Nb25 and T
The Binding experiment result of cell.
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 in following embodiments after the Southeast China University laboratory life science institute Wan Yakun is agreed to,
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 to use.
The preparation of embodiment 1, nano antibody
The present invention provides a kind of nano antibody for deriving from camel, entitled CD3/Nb25, nano antibody CD3/
The amino acid sequence of Nb25 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 CD3/Nb25 as shown in Figure 1, wherein first be 2000bp molecular labeling,
Second duct is CD3 nano antibody DNA electrophoretic band, which is about 450bp.
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.9, other sequences are constant, obtain recombinant vector pComb3-CD3/Nb25, pComb3-CD3/
The difference of Nb25 and pComb3, which is only that, replaces with SEQ ID for the DNA fragmentation between the PstI of pComb3 and NotI identification sequence
DNA molecular shown in No.9.Recombinant vector pComb3-CD3/Nb25 expresses nano antibody CD3/ shown in SEQ ID No.8
Nb25.PComb3-CD3/Nb25 is imported in Escherichia coli WK6, recombinant bacterium WK6-pComb3-CD3/Nb25 is obtained.
Specific preparation process is as follows for nano antibody:
(1) WK6-pComb3-CD3/Nb25 is coated on to LB plate (the LB plate containing ampicillin and glucose
In, the concentration of ampicillin 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-CD3/Nb25 culture solution, makes WK6-pComb3-CD3/Nb25
The concentration of IPTG is 1mM in culture solution, by WK6-pComb3-CD3/Nb25 culture solution in the shaking table (revolving speed of shaking table at 28 DEG C
For overnight incubation on 220rpm) (12 hours), WK6-pComb3-CD3/Nb25 induction liquid is obtained；
(4) the WK6-pComb3-CD3/Nb25 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；Tool
Body step are as follows: the bacterium solution of collection step (4) containing thallus is centrifuged 20min in 6000rpm, discards supernatant after bacterial sediment, in bacterium
4-5ml lysate (20 milliliters of cell cracking formula of liquid: 40 μ L 0.5M EDTA (PH8.0), 4ml 10% are added in body precipitating
SDS, 1ml 1M Tris-cl (PH6.8), 14.96ml distilled water), 250rpm, 4 DEG C of cracking 2h obtain cracking mixed liquor.Then
The lysate for being 4:1 with the volume ratio of cracking mixed liquor is added, in 4 DEG C of cracking 2h.It is centrifuged 30min in 4000rpm again, is collected
Supernatant lysate.
(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 CD3/Nb25.Specifically: step (5) is split to the supernatant lysate being collected into after bacterium, it is mixed that 600 μ L of magnesium chloride is added in every pipe
After even, after the mixing of 1.5ml nickel magnetic bead is added, 4 DEG C of concussions combine 1h.Chromatographic column is poured into, is first cleaned with PBS to nickel bead color and is become
Then indigo plant washes 2 column volumes with the imidazoles of 20mM, if nickel column is dirty, can increase to 3 column volumes.With the miaow of 2ml 50mM
Azoles is washed once, is washed once with the imidazoles of 1ml 100mM, and the imidazoles of 1ml 500mM is added, and places 10min, collects albumen；Again plus
Enter the imidazoles of 2ml 500mM, place 10min, collects albumen；The imidazoles of 1ml 500mM is added, 10min is placed, collects egg
It is white.It is protein crude extract the albumen mixing collected three times.Protein crude extract is put into ultra-filtration centrifuge tube, is washed repeatedly with PBS
Protein crude extract, until imidazole concentration is 1mM.Albumen is exactly nano antibody CD3/Nb25 obtained on super filter tube.
The SDA-PAGE electrophoretogram of nano antibody CD3/Nb25 is respectively such as Fig. 2.The results show that nanometer obtained by the above method
The purity of antibody CD3/Nb25 reaches 90% or more.
The measurement of embodiment 2, nano antibody and CD3 Percentage bound
Nano antibody CD3/Nb25 and CD3 Percentage bound measurement (direct method)
T cell and B cell are separated from healthy volunteer human peripheral blood cell, by the nano antibody CD3/Nb25 of example 1
(1 μ g) is separately added into 1 × 106Incubation 30min is protected from light for 4 DEG C in a above-mentioned T cell and B cell, and after PBS is washed 2 times, 5 μ l are added
4 DEG C of incubation 30min of PE anti-HA tag antibody (abcam, Clone:20B12) will be on sample after PBS is washed 2 times
BACKMAN flow cytometer, as a result such as Fig. 3 A, shown in 3B.Fig. 3 A be blank control and CD3 nano antibody CD3/Nb25 respectively with
The combination percentage of B cell；Fig. 3 B be blank control and CD3 nano antibody CD3/Nb25 respectively and the combination percentage of T cell；
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, and S1 is represented
CD3 nano antibody CD3/Nb25.Graphical results can be seen that CD3 nano antibody CD3/Nb25 can be good in conjunction with T cell,
Percentage bound is up to 35.8%.
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
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
SEQUENCE LISTING
<110>Guangxi Medical University
<120>the nano antibody CD3/Nb25 and the preparation method and application thereof of AntiCD3 McAb
<130> GY18100128
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> PRT
<213>artificial sequence
<220>
<221> CONFLICT
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<223> FR1
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Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg
1 5 10 15
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20
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<212> PRT
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<220>
<221> CONFLICT
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1 5 10 15
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<212> PRT
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<221> CONFLICT
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<223> FR3
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Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
20 25 30
Met Tyr Tyr Cys Ser
35
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<221> CONFLICT
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<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 Asp Thr Tyr Ser Arg Tyr Tyr Met Gly
1 5 10
<210> 6
<211> 9
<212> PRT
<213>artificial sequence
<220>
<221> CONFLICT
<222> (1)..(9)
<223> CDR2
<400> 6
Ser Pro Asp Gly Gly Ser Thr Asn Tyr
1 5
<210> 7
<211> 18
<212> PRT
<213>artificial sequence
<220>
<221> CONFLICT
<222> (1)..(18)
<223> CDR3
<400> 7
Ala Asp Leu Asn Gly Thr Trp Tyr Arg Arg Leu Ser Gly Arg Ala His
1 5 10 15
Asn Tyr
<210> 8
<211> 123
<212> PRT
<213>artificial sequence
<400> 8
Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg
1 5 10 15
Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr Ser Arg Tyr Tyr Met Gly
20 25 30
Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile
35 40 45
Ser Pro Asp Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg
50 55 60
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ser Ala Asp
85 90 95
Leu Asn Gly Thr Trp Tyr Arg Arg Leu Ser Gly Arg Ala His 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 gctggagggt ctctgagact ctcctgtgca 60
gcctctggag acacctacag taggtactac atgggctggt tccgccaggt tccagggaag 120
gagcgtgagg gggtcgcaag tatttcacct gatggtggaa gcacaaacta tgccgactcc 180
gtgaagggcc gattcaccat ctcccgagac aacgccaaga acacgctgta tctacaaatg 240
aacagcctga aacctgagga cactgccatg tactactgtt cggcagatct caacggtact 300
tggtaccgac gcctgtccgg gcgggcgcat aactactggg gccaggggac ccaggtcacc 360
gtctcctca 369
Claims (10)
1. nano antibody includes determinant complementary region；It is characterized by:
The 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 49-57 amino acids of SEQ ID No.8 in sequence table；
The amino acid sequence of the CDR3 is the 95-112 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 by the determinant complementary region and
The framework region composition.
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.
4. biomaterial relevant to claims 1 or 2 or 3 nano antibodies, is B1) any one of 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 be 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) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes described in claims 1 or 2 or 3
The cDNA molecule or genomic DNA molecule of nano antibody；
3) nucleotide sequence hybridization with 1) restriction, and coding claims 1 or 2 or 3 nano antibodies under strict conditions
CDNA molecule 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 by: 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 by: the recipient cell is microbial cell.
10. any purposes in following A 1-A8:
A1, claims 1 or 2 or 3 nano antibodies are preparing the application in tumor inhibitor or inhibiting tumour cells agent；
Any biomaterial is preparing the application in tumor inhibitor or inhibiting tumour cells agent in A2, claim 4-5；
Derivative antibody described in A3, claim 6 is preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A4, claim 7 or 8 the methods are preparing the application in tumor inhibitor or inhibiting tumour cells agent；
A5, claims 1 or 2 or 3 nano antibodies inhibit CD3 activity in preparation or promote answering in T cell proliferation product
With；
Any biomaterial inhibits CD3 activity in preparation or promotes in T cell proliferation product in A6, claim 4-5
Using；
Derivative antibody described in A7, claim 6 inhibits CD3 activity in preparation or promotes the application in T cell proliferation product；
A8, claim 7 or 8 or 9 the methods inhibit CD3 activity in preparation or promote the application in T cell proliferation product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810175293.5A CN110218254B (en) | 2018-03-02 | 2018-03-02 | CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810175293.5A CN110218254B (en) | 2018-03-02 | 2018-03-02 | CD 3-resistant nano antibody CD3/Nb25 and preparation method and application thereof |
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| Publication Number | Publication Date |
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| CN110218254B CN110218254B (en) | 2020-12-04 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100303777A1 (en) * | 2006-12-14 | 2010-12-02 | Actogenix N.V. | Delivery of binding molecules to induce immunomodulation |
| WO2016179003A1 (en) * | 2015-05-01 | 2016-11-10 | Genentech, Inc. | Masked anti-cd3 antibodies and methods of use |
| CN106117359A (en) * | 2016-06-17 | 2016-11-16 | 中山大学 | AntiCD3 McAb single domain antibody |
| CN106939048A (en) * | 2017-03-25 | 2017-07-11 | 康众（北京）生物科技有限公司 | A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof |
-
2018
- 2018-03-02 CN CN201810175293.5A patent/CN110218254B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100303777A1 (en) * | 2006-12-14 | 2010-12-02 | Actogenix N.V. | Delivery of binding molecules to induce immunomodulation |
| WO2016179003A1 (en) * | 2015-05-01 | 2016-11-10 | Genentech, Inc. | Masked anti-cd3 antibodies and methods of use |
| CN106117359A (en) * | 2016-06-17 | 2016-11-16 | 中山大学 | AntiCD3 McAb single domain antibody |
| CN106939048A (en) * | 2017-03-25 | 2017-07-11 | 康众（北京）生物科技有限公司 | A kind of CD3 ε × CD19 bispecific nano antibodies and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| PETER BANNAS等: "Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics", 《FRONTIERS IN IMMUNOLOGY》 * |
| SOPHIE STEELAND: "Nanobodies as therapeutics: big opportunities for small antibodies", 《DRUG DISCOVERY TODAY 》 * |
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| CN110218254B (en) | 2020-12-04 |
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