CN110144011A - For the single domain antibody of T lymphocyte immunoglobulin Mucin 3 - Google Patents

For the single domain antibody of T lymphocyte immunoglobulin Mucin 3 Download PDF

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Publication number
CN110144011A
CN110144011A CN201810152107.6A CN201810152107A CN110144011A CN 110144011 A CN110144011 A CN 110144011A CN 201810152107 A CN201810152107 A CN 201810152107A CN 110144011 A CN110144011 A CN 110144011A
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gly
ser
cdr3
cdr2
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CN110144011B (en
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万亚坤
朱敏
沈晓宁
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Shanghai Luoqi Bio Pharmaceutical Technology Co Ltd
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Shanghai Luoqi Bio Pharmaceutical Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

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  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to antibody drug technical fields, more particularly to nano antibody and its application for T lymphocyte immunoglobulin Mucin 3 (TIM3).The invention discloses the nano antibodies that a group-specific is directed to Tim3, disclose the amino acid sequence of the framework region FR and complementary determining region CDR of its VHH chain, while the gene order for encoding the single domain heavy chain nano antibody is also disclosed.Anti- Tim3 nano antibody provided by the invention can have effectively with Tim3 antigen binding compared with high specific and affinity.Single domain heavy chain nano antibody gene order announced through the invention includes that genetic test and antibody drug exploitation provide new approaches for the research based on Tim3 target spot.

Description

For the single domain antibody of T lymphocyte immunoglobulin Mucin 3
Technical field
The present invention relates to biomedical or biopharmaceutical technology, disclose glutinous for T lymphocyte immunoglobulin The single domain antibody and its coded sequence of albumen 3 (Tim3) and application.
Background technique
The identification of tumour cell, tumour antigen are presented to T cell, t cell activation and specific killing tumour cell.? During this, the activation of T cell needs costimulatory signal, and these signals need the regulation of " immunologic test point ".T lymph Cell immunoglobulin Mucin 3 (T cell immunoglobulin and mucin3, TIM3) is exempted from as human body is important Epidemic disease checkpoint plays a crucial role in the immunological regulation in tumor microenvironment.
TIM3 is TIM family member, encodes the I type memebrane protein containing 281 amino acid.The common structure packet of TIM molecule It includes: the area immunoglobulin N-terminal V, mucin domain, transmembrane region and intracellular region.Its extracellular portion includes one and is rich in half Guang The immunoglobulin-like region of propylhomoserin and a mucin area rich in threonine, serine and proline, cytoplasmic domain have 6 Tyrosine, wherein 1 belongs to tyrosine phosphorylation motif portion.It is made of in immune globulin variable region 4 cysteines Highly conserved unique crack different from other immunoglobulin superfamilies member: it can be tied with phosphatidylserine (PS) It closes.TIM3 selectively expresses t helper cell (Th1 and Th17), the T regulating cell (Treg), dendron shape in secretion of gamma-IFN Cell (DCs), monocyte, mast cell, NK cell, on tumor infiltrating lymphocyte (TILs), on tumour cell also There is expression, such as melanoma, gastric cancer, B cell lymphoma.
TIM3 molecule wide expression on immunocyte, can with the galactose agglutinin -9 of cell surface (galectin-9, Gal-9), phosphatidylserine (phosphatidylserine, PtdSer), high mobility group protein B-1 (high Mobility group box-1, HMGB-1) etc. multiple ligand bindings, participate in adjusting a variety of T cells including regulatory T cells The differentiation of (regulatory T cell, Treg), plays important work in the reset procedure of immunological homeostasis and apoptotic cell With.TIM3 plays an important role in the formation and maintenance of immune tolerance, and TIM3 can induce CD8+T cell " exhaustion ", can also pass through Apoptosis-induced mode inhibits Th1 and Th17 cell to grow, and lowers the immune response of Th1 and Th17 type, promotes Treg differentiation, enhancing Treg inhibiting effect.In addition to this, Tim3 inhibits tissue destructive is immune to answer by the activation and function of adjusting macrophage It answers, important adjustment effect is played in autoimmune disease.As the negative regulatory factor of tumor immune response, TIM3 has Apparent tumour medicine potentiality to be exploited has developed into the target molecule of immunization therapy Yu course of drug development hot topic.But still It so needs to develop the antibody drug with good combination performance.
Summary of the invention
The present invention provides 29 plants of specificity be directed to TIM3 nano antibody, while provide nano antibody coded sequence, Preparation method.
In the first aspect of the present invention, the present invention provides T lymphocyte immunoglobulin Mucin 3 (TIM3) specificity The CDR region of nano antibody.
In some embodiments, the TIM3 nano antibody includes the immunoglobulin list of a specific binding TIM3 One variable domains.In some embodiments, the TIM3 nano antibody includes two or more specific bindings TIM3 The single variable domains of immunoglobulin.
In some embodiments, the single variable domains of at least one immunoglobulin include selected from the following CDRl, CDR2 and CDR3:
(1) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 (CDR corresponding to antibody strain 1)；
(2) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 (CDR corresponding to antibody strain 2)；
(3) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 (CDR corresponding to antibody strain 3)；
(4) shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 CDR3 (CDR corresponding to antibody strain 4)；
(5) shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 CDR3 (CDR corresponding to antibody strain 5)；
(6) shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 CDR3 (CDR corresponding to antibody strain 6)；
(7) shown in CDR2 shown in CDR1 shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 CDR3 (CDR corresponding to antibody strain 7)；
(8) shown in CDR2 shown in CDR1 shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 CDR3 (CDR corresponding to antibody strain 8)；
(9) shown in CDR2 shown in CDR1 shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 CDR3 (CDR corresponding to antibody strain 9)；
(10) shown in CDR2 shown in CDR1 shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 CDR3 (CDR corresponding to antibody strain 10)；
(11) shown in CDR2 shown in CDR1 shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 CDR3 (CDR corresponding to antibody strain 11)；
(12) shown in CDR2 shown in CDR1 shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 CDR3 (CDR corresponding to antibody strain 12)；
(13) shown in CDR2 shown in CDR1 shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 CDR3 (CDR corresponding to antibody strain 13)；
(14) shown in CDR2 shown in CDR1 shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 CDR3 (CDR corresponding to antibody strain 14)；
(15) shown in CDR2 shown in CDR1 shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 CDR3 (CDR corresponding to antibody strain 15)；
(16) shown in CDR2 shown in CDR1 shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 CDR3 (CDR corresponding to antibody strain 16)；
(17) shown in CDR2 shown in CDR1 shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 CDR3 (CDR corresponding to antibody strain 17)；
(18) shown in CDR2 shown in CDR1 shown in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 CDR3 (CDR corresponding to antibody strain 18)；
(19) shown in CDR2 shown in CDR1 shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 CDR3 (CDR corresponding to antibody strain 19)；
(20) shown in CDR2 shown in CDR1 shown in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 CDR3 (CDR corresponding to antibody strain 20)；
(21) shown in CDR2 shown in CDR1 shown in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 CDR3 (CDR corresponding to antibody strain 21)；
(22) shown in CDR2 shown in CDR1 shown in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 CDR3 (CDR corresponding to antibody strain 2)；
(23) shown in CDR2 shown in CDR20 shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 CDR3 (CDR corresponding to antibody strain 23)；
(24) shown in CDR2 shown in CDR1 shown in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72 CDR3 (CDR corresponding to antibody strain 24)；
(25) shown in CDR2 shown in CDR1 shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75 CDR3 (CDR corresponding to antibody strain 25)；
(26) shown in CDR2 shown in CDR1 shown in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 CDR3 (CDR corresponding to antibody strain 26)；
(27) shown in CDR2 shown in CDR1 shown in SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 CDR3 (CDR corresponding to antibody strain 27)；
(28) shown in CDR2 shown in CDR1 shown in SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84 CDR3 (CDR corresponding to antibody strain 28)；
(29) shown in CDR2 shown in CDR1 shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87 CDR3 (CDR corresponding to antibody strain 29).
In another preferred example, described CDR1, CDR2 and CDR3 by VHH chain framework region FR1, FR2, FR3 and FR4 institute It separates.
Second aspect of the present invention provides a kind of VHH chain of TIM3 specific nano antibody, enough to specifically bind Tim3, and VHH chain includes one of framework region FR and CDR region described in claim 1 or a variety of.
In another preferred example, the VHH chain in the antibody is selected from one of SEQ ID NO:88-116 or a variety of.
In another preferred example, the nano antibody is nano antibody combination, wherein the specific nano antibody combination Including at least sequence shown in SEQ ID NO:114.
In another preferred example, TIM3 specific nano antibody of the invention also cover can with by SEQ ID NO:88- The anti-TIM3 antibody molecule of same epitope in 116 on the VHH combination TIM3 of any amino acid sequence composition.
Third aspect present invention, provides a kind of isolated polynucleotide sequence, and the polynucleotide encoding is selected from the group Protein: anti-TIM3 described in the CDR region of anti-TIM3 nano antibody, second aspect of the present invention described in first aspect present invention The VHH chain combination of the VHH chain of nano antibody, the 13rd aspect of the present invention or the anti-TIM3 nano antibody of the present invention.
In another preferred example, the polynucleotide sequence is combining form, it is preferable that the polynucleotide sequence includes One of SEQ ID NO:117-145 or a variety of.
In another preferred example, the present invention relates to the nucleic acid molecules for encoding TIM3 nano antibody of the invention.Of the invention Nucleic acid can be RNA, DNA or cDNA.
Fourth aspect present invention, provides a kind of expression vector, and the expression vector expresses the more of third aspect present invention Nucleotide.
Fifth aspect present invention, provides a kind of host cell, and the host cell contains described in fifth aspect present invention Expression vector or its genome in be integrated with polynucleotides described in fourth aspect present invention.
In another preferred example, the host cell includes prokaryotic cell or eukaryocyte.
In another preferred example, the host cell is selected from the group: Escherichia coli, yeast cells.
Sixth aspect present invention provides a kind of method for generating anti-TIM3 nano antibody, comprising steps of
(a) under conditions of being suitble to generate nano antibody, host cell described in fourth aspect present invention is cultivated, to obtain The culture of the anti-TIM3 nano antibody must be contained；And
(b) the anti-TIM3 nano antibody is separated or recycled from the culture；And optional
(c) purifying and/or modification step (b) TIM3 nano antibody obtained.
In another preferred example, the anti-TIM3 nano antibody has the amino acid as shown in SEQ ID NO:88-116 Sequence.
Seventh aspect present invention provides a kind of conjugate, and the conjugate contains:
(a) the VHH chain of anti-TIM3 nano antibody as described in respect of the second aspect of the invention or as described in respect of the second aspect of the invention Anti- TIM3 nano antibody；It is connected with operability
(b) modification label selected from the group below: chemical labeling and biomarker.
In another preferred example, the chemical labeling is isotope, immunotoxin and/or chemicals.
In another preferred example, the biomarker is biotin, Avidin or enzyme label.
The present invention also provides a kind of conjugate, the conjugate anti-TIM3 nano antibody as described in second aspect of the present invention Or seventh aspect present invention conjugate is made with solid dielectric or semi-solid medium coupling.
In another preferred example, the conjugate is by seventh aspect present invention conjugate and coupling element structure selected from the following At: fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (Electronic Computer Tomography Technology) contrast agent or can generate the enzyme of detectable product, radionuclide, biotoxin, cell factor (such as IL-2), Antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/nanometer rods, virion, liposome, magnetic nanosphere, prodrug Kinase (for example, DT- diaphorase (DTD) or biphenyl base hydrolase-sample protein (BPHL)), chemotherapeutics (for example, cis-platinum) Or any type of nano particle etc..
In another preferred example, the conjugate contains: multivalence (such as divalent) as described in respect of the second aspect of the invention The VHH chain of TIM3 nano antibody, TIM3 nano antibody as described in the third aspect of the present invention.
In another preferred example, the multivalence refers to, comprising multiple heavy in the amino acid sequence of the immune conjugate Multiple TIM3 nano antibody as described in respect of the second aspect of the invention, TIM3 nano antibody as described in the third aspect of the present invention VHH chain.
Eighth aspect present invention provides the purposes of TIM3 nano antibody described in second aspect of the present invention, is used to prepare (a) for detecting the reagent of TIM3 molecule；(b) for treating the drug of tumour.
In another preferred example, the detection includes flow cytometer detection, cellular immunofluorescence detection.
Ninth aspect present invention provides a kind of pharmaceutical composition, contains:
(i) the complementary determining region CDR of the anti-TIM3 nano antibody VHH chain of first aspect present invention, second aspect of the present invention institute The VHH chain stated, the 13rd aspect VHH of present invention combination, anti-TIM3 nano antibody or its antigen described in second aspect of the present invention One of binding fragment is a variety of；And
(ii) pharmaceutically acceptable carrier.
In another preferred example, the pharmaceutical composition is injection type.
In another preferred example, the pharmaceutical composition is used to prepare the drug for the treatment of tumour, and the tumour is selected from The following group: gastric cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, large intestine Cancer, prostate cancer, cervical carcinoma, lymph cancer, adrenal tumor or tumor of bladder.
Tenth aspect present invention provides one or more use of anti-TIM3 nano antibody described in second aspect of the present invention On the way:
(i) for detecting people's TIM3 molecule；
(ii) it is used for flow cytometer detection；
(iii) it is detected for cellular immunofluorescence；
(iv) for treating tumour；
(v) it is used for diagnosing tumor.
In another preferred example, the purposes is non-diagnostic and non-treatment.
On the one hand the present invention the tenth, provides a kind of recombinant protein, the recombinant protein includes
(i) sequence of nano antibody as described in respect of the second aspect of the invention or heavy chain as described in the third aspect of the present invention can Become the sequence of area VHH；And
(ii) sequence label of optional assistance expression and/or purifying.In another preferred example, the sequence label packet Include 6His label and HA label in another preferred example, the recombinant protein specifically binds TIM3 albumen.
The twelfth aspect of the present invention provides nano antibody as described in respect of the second aspect of the invention, third party such as of the present invention The purposes of immune conjugate described in VHH chain or seventh aspect present invention described in face, they be used to prepare medicament, reagent, Detection plate or kit；
Wherein, the reagent, detection plate or kit are used for: TIM3 albumen in test sample；
Wherein, the medicament is used to treat or prevent the tumour of expression TIM3 (i.e. TIM3 is positive).
The 13rd aspect of the present invention, additionally provides a kind of specific binding TIM3 nano antibody VHH chain combination, and feature exists In at least containing the VHH chain with following CDR region in the VHH chain combination:
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81.
In another preferred example, also containing the VHH chain with one or more of CDR region in VHH chain combination:
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78.
In another preferred example, also containing the VHH chain with one or more of CDR region in VHH chain combination:
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87.
In another preferred example, the combination is at least containing the amino acid sequence of Nb27；Preferably, the combination also contains Have one or more selected from Nb16,18,20,21,22,23,24,25 or 26 sequence；More preferably, the combination also contains one Kind or a variety of sequences selected from Nb1-15, Nb17, Nb19, Nb28 or Nb29.
Fourteenth aspect of the present invention, provides a kind of kit, and the kit contains the anti-of second aspect of the present invention The conjugate and/or conjugate of the present invention of TIM3 nano antibody, seventh aspect present invention.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1, which shows, is coated on the phage library of building on plate by gradient dilution, and by calculating growth Monoclonal number carries out storage capacity measurement.Display takes 1/5 gradient dilution 10 in figure4Again, 105Again, 106Colony counts again calculate single It clones number and determines that library size is 9.0 × 108CFU。
Fig. 2 is that 24 clones of random picking carry out bacterium colony PCR detection from the library of building, to the insertion rate in building library It is evaluated.The DNA band of attached drawing from left to right gel pore is respectively: first is DNA molecular marker, remaining duct is detection The PCR product of Insert Fragment, PCR product band are about 500bp；Through detecting, the insertion rate in the library reaches 91.7%.
Fig. 3 is TIM3 nano antibody screening enrichment process.It is not enriched with after first round elutriation, the second wheel elutriation occurs There are 408 times of enrichments in 7.8 times of enrichments, third round elutriation.
Fig. 4 is the purifying figure of 29 plants of TIM3 nano antibodies of Bacillus coli expression.Corresponding SEQ ID NO:88-116 amino acid The nano antibody of sequence is the electrophoretogram of the SDS-PAGE of TIM3 nano antibody after nickel column resin gel affinitive layer purification. The results show that TIM3 nano antibody passes through the purification process, purity can reach 90% or more.
Fig. 5 is TIM-3 stable cell strain detection figure, and A figure is TIM-3 positive antibody (in clinicalⅰstage conceptual phase TSR-022 protein purification) is as a result, B figure is the flow cytometer detection result of TIM-3 stable cell strain.
Fig. 6 is the affinity the selection result of 29 plants of nano antibodies.With PD-1, PD-L1 and CTLA-4 as negative control, stream Formula detects 29 plants of TIM-3 nano antibodies to the affinity of TIM-3 antigen.
Specific embodiment
The present inventor is successfully obtained one group anti-TIM3 nanometers and resisted by extensive and in-depth research by largely screening Body, the experimental results showed that, 29 plants of TIM3 (Nb1-29 respectively corresponds SEQ ID NO.:88-116) nanometer that the present invention obtains is anti- Body can Gao Teyi, high affine in conjunction with TIM3.The present invention is completed on this basis.
Specifically, camel is immunized using the TIM3 extracellular fragment antigen protein of source of people in the present invention, obtains the immune of high quality and receives Rice library of antibody genes.Then TIM3 protein molecular is coupled on ELISA Plate, shows the correct space structure of TIM3 albumen, with The antigen of this form screens immune nano Antibody geometric mean titer (camel heavy chain antibody phage display base using display technique of bacteriophage Yin Ku), to obtain the nano antibody gene of TIM3 specificity.This gene is gone in Escherichia coli again, to obtain It can be in E. coli and specific high nano antibody strain.
As used herein, term " nano antibody of the present invention ", " anti-TIM3 nano antibody of the invention ", " TIM3 of the present invention Nano antibody " is used interchangeably, and refers both to specific recognition and the nano antibody for being incorporated into TIM3 (including people TIM3).Particularly preferably Be VHH chain amino acid sequence nano antibody as shown in SEQ ID NO:88-116.
As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical structure feature Different four glycan albumen is made of two identical light chains (L) and two identical heavy chains (H).Every light chain is total by one Valence disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Each heavy chain and The intrachain disulfide bond at light chain also regular interval.One end of each heavy chain has variable region (VH), is followed by multiple constant regions.Every One end of light chain has variable region (VL), and the other end has constant region；The constant region of light chain is opposite with first constant region of heavy chain, gently The variable region of chain and the variable region of heavy chain are opposite.Special amino acid residue forms boundary between the variable regions of the light chain and the heavy chain Face.
As used herein, term " single domain antibody (single domain antibody, sdAb or VHH) ", " nanometer is anti- Body " (nanobody) has the same meaning, and refers to the variable region of monoclonal antibody heavy chain, and building is only made of a heavy chain variable region Single domain antibody, it is the smallest antigen-binding fragment with complete function.Usually first obtain natural deletions light chain and heavy chain After the antibody of constant region 1 (CH1), then the variable region of monoclonal antibody heavy chain, construct the single domain being only made of a heavy chain variable region Antibody (VHH).
Single domain antibody/nano antibody (Nanobody) small antibody fragments novel as one kind, natural by hunchbacked class Heavy chain antibody heavy chain variable region (VHH) clone obtains.Nanobody (Nb) has excellent biological characteristics, molecular weight 12- 15kDa is 1/10th of complete antibody, has good penetration into tissue, specificity is high, good water solubility.Because its is special Structural property has had both the advantage of conventional antibodies and small-molecule drug, and the almost ideal development cycle for overcoming conventional antibodies is long, The defects of stability is lower, and preservation condition is harsh, the newly emerging force being increasingly becoming in Antybody therapy of new generation, in immunodiagnosis and Wide application prospect is shown in treating.
As used herein, " variable " the certain parts for indicating variable region in antibody of term are different in sequence, its shape Combination and specificity at various specific antibodies to its specific antigen.However, changeability and being unevenly distributed over entire anti- In body variable region.It concentrates in light chain and heavy chain variable region three segments being known as in complementary determining region (CDR) or hypervariable region In.More conservative part is known as framework region (FR) in variable region.Four FR are respectively contained in the variable region of native heavy and light chain Area, they are generally in beta sheet configuration, are connected by three CDR of formation connection ring, can form part β folding in some cases Stack structure.CDR in every chain is by the area FR firmly against the antigen for together forming antibody together and with the CDR of another chain Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody Cellular toxicity.
As it is known by the man skilled in the art, immune conjugate and fusion expressed product include: drug, toxin, cell factor (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule are formed in conjunction with antibody of the invention or its segment Conjugate.The invention also includes the cell surface marker object in conjunction with the anti-TIM3 protein antibodies or its segment or resist It is former.
As used herein, term " heavy chain variable region " and " VH" be used interchangeably.
As used herein, term " variable region " and " complementary determining region (complementarity determining Region, CDR) " it is used interchangeably.
In a preferred embodiment of the invention, the heavy chain variable region of the antibody includes three complementary determining regions CDR1, CDR2 and CDR3.
In a preferred embodiment of the invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain Constant region.
In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all Refer to the polypeptide of specific binding TIM3 albumen, such as albumen or polypeptide with heavy chain variable region.They can with or without rise Beginning methionine.
The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention Including with the heavy chain containing variable region any protein or protein conjugate and fusion expressed product (i.e. immune conjugate and Fusion expressed product), as long as the variable region is identical as the heavy chain variable region of antibody of the present invention or at least 90% homology, preferably At least 95% homology.
Generally, the antigenic binding property of antibody can be described by being located at 3 specific regions of heavy chain variable region, referred to as variable The section is partitioned into 4 frame areas (FR) by region (CDR), and the amino acid sequence of 4 FR is relatively conservative, does not join directly With association reaction.These CDR form cyclic structure, and the β-pleated sheet formed by FR therebetween is close to each other on space structure, weight The CDR on CDR and corresponding light chain on chain constitutes the antigen binding site of antibody.It can be by comparing the antibody of same type Amino acid sequence determines be which Amino acid profile FR or CDR region domain.
The variable region of the heavy chain of antibody of the present invention is particularly interesting, because being at least partly related to combining in them anti- It is former.Therefore, the present invention includes those molecules with antibody heavy chain variable region with CDR, as long as its CDR and identify herein CDR has the homology of 90% or more (preferably 95% or more, most preferably 98% or more).
The present invention not only includes complete antibody, further includes the segment or antibody and other sequences with immunocompetent antibody Arrange the fusion protein formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.
As used herein, term " segment ", " derivative " refer to that be kept substantially antibody of the present invention identical with " analog " Biological function or active polypeptide.Polypeptide fragment of the invention, derivative or the like, which can be (i), one or more Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino Sour residue, which can be, may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second Glycol) fusion is formed by polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (as before Lead sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or merge egg with what 6His label was formed It is white).According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.
Antibody of the present invention refers to polypeptide with TIM3 protein binding activity, including above-mentioned CDR region.The term further includes tool There is the variant form of polypeptide with antibody identical function of the present invention, comprising above-mentioned CDR region.These variant forms include (but simultaneously It is not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid Missing, insertion and/or substitution, and it is one or several (usually within 20, preferably in C-terminal and/or N-terminal addition Ground is within 10, is more preferably within 5) amino acid.For example, in the art, with amino acid similar in performance When being replaced, the function of protein is not usually changed.For another example, one or several ammonia are added in C-terminal and/or N-terminal Base acid will not generally also change the function of protein.The term further includes the active fragment and reactive derivative of antibody of the present invention.
The variant form of the polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induction Mutant, the encoded albumen of DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency, with And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.
The present invention also provides other polypeptides, such as the fusion protein comprising nano antibody or its segment.In addition to almost overall length Polypeptide outside, the invention also includes the segments of nano antibody of the present invention.In general, the segment has antibody of the present invention at least about 50 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably extremely Few about 100 continuous amino acids.
In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention, There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and generate.
Table 1
Initial residue Representative substitution It is preferred to replace
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The present invention also provides encoding such antibodies or the polynucleotide molecules of its segment or its fusion protein.Of the invention Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA can To be single-stranded or double-strand.DNA can be coding strand or noncoding strand.
The polynucleotides for encoding mature polypeptide of the invention include: the coded sequence of an encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume Code sequence.
The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include The polynucleotides of additional code and/or non-coding sequence.
The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90% with On, more preferably 95% or more when, just hybridizes.Also, the polypeptide of interfertile polynucleotide encoding has identical with mature polypeptide Biological function and activity.
The nucleotide full length sequence of antibody of the invention or its segment can usually use PCR amplification method, recombination method or artificial Synthetic method obtains.A kind of feasible method is that manually synthetic method synthesizes related sequence, especially fragment length When shorter.In general, being then attached the very long segment of available sequence again by first synthesizing multiple small fragments.In addition, may be used also The coded sequence of heavy chain and expression label (such as 6His) are fused together, fusion protein is formed.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method. Biomolecule (nucleic acid, albumen etc.) according to the present invention includes existing biomolecule in a separate form.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
Sequence of the present invention is as shown in table 2
Table 2
The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This A little carriers can be used for converting host cell appropriate, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell；Fungal cell's such as yeast；The insect cell of drosophila S2 or Sf9；CHO, COS7, zooblast of 293 cells etc..
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute With the step of it is generally well-known in the art.Another method is using MgCl2.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
Antibody of the invention can be used alone, can also be with detectable marker (for diagnostic purpose), therapeutic agent, PK (egg White kinases) combination of modified part or any the above substance combines or coupling.
Detectable marker includes but is not limited to for diagnostic purposes: fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.
Can in conjunction with antibody of the present invention or coupling therapeutic agent include but is not limited to: 1. radionuclides；2. biological poison；3. Cell factor such as IL-2 etc.；4. gold nano grain/nanometer rods；5. virion；6. liposome；7. magnetic nanosphere；8. medicine activates Enzyme (for example, DT- diaphorase (DTD) or biphenyl base hydrolase-sample protein (BPHL))；9. treating agent (for example, cis-platinum) or appointing The nano particle etc. of what form.
The combination of VHH chain
The present invention further comprises a kind of combination of single domain antibody VHH chain.Wherein, the combination at least contains following CDR region:
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81.
In addition, the combination further comprises preferably, the combination at least contains 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 Kind, 9 kinds, 10 kinds, 11 kinds, 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds, 22 kinds, 23 kinds, 24 kinds, 25 kinds, 26 kinds, 27 kinds, 28 kinds or 29 kinds CDR regions selected from the group below are selected from VHH shown in SEQ ID NO.:88-116 Chain.
Preferably,
Also containing the VHH chain with one or more of CDR region in the VHH chain combination:
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78.
It is highly preferred that also containing the VHH chain with one or more of CDR region in the VHH chain combination:
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84；
CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87.
Pharmaceutical composition
The present invention also provides a kind of compositions.Preferably, the composition is pharmaceutical composition, it contains above-mentioned Antibody or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, nothing can be formulated in these substances In poison, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is usually about 5-8, and preferably pH is about 6-8, Although pH value can be varied with the property and illness to be treated for being formulated substance.Prepared pharmaceutical composition can be with It is administered by conventional route, including (but being not limited to): tumor is interior, peritonaeum is interior, intravenous or local administration.
Pharmaceutical composition of the invention can be directly used for combining TIM3 protein molecular, thus can be used for treating tumour.In addition, It also can be used simultaneously other therapeutic agents.
Pharmaceutical composition of the invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more Good ground 0.1-80wt%) the above-mentioned nano antibody (or its conjugate) and pharmaceutically acceptable carrier or figuration of the present invention Agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Pharmaceutical preparation It should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain The aqueous solution of glucose and other adjuvants is prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably in sterile item It is manufactured under part.The dosage of active constituent is therapeutically effective amount, such as about 50 mg/kg of about 10 micrograms/kg body weight-daily Weight.In addition, polypeptide of the invention can be also used together with other therapeutic agents.
It is that the immune conjugate of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety Effective quantity typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg weight, compared with Good ground dosage is about 10 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration way The factors such as diameter, patient health situation, within the scope of these are all skilled practitioners technical ability.
The nano antibody of label
In a preference of the invention, the nano antibody has detectable marker.More preferably, the label Object is selected from the group: isotope, colloid gold label object, colored labels or fluorescent marker.
Method known to those skilled in the art progress can be used in colloid gold label.In a preferred scheme of the invention In, the nano antibody colloid gold label of anti-TIM3 obtains the nano antibody of colloid gold label.
Anti- TIM3 nano antibody of the invention has specificity well, very high potency.
Detection method
The invention further relates to the methods of detection TIM3 albumen.This method step approximately as: obtain cell and/or tissue sample This；In the medium by sample dissolution；Detect the level of the TIM3 albumen in the sample of the dissolution.
In detection method of the invention, used sample is not particularly limited, and representative example is to be present in carefully Born of the same parents save the celliferous sample in liquid.
Kit
The present invention also provides a kind of kits containing antibody (or its segment) or detection plate of the invention, in the present invention A preference in, the kit further includes container, operation instructions, buffer etc..
The present invention also provides the detection kit for detecting TIM3 level, which includes identification TIM3 albumen Antibody detects required common reagent and buffer for dissolving the cracking medium of sample, such as various buffers, detection label, Detection substrate etc..The detection kit can be in-vitro diagnosis device.
Using
As described above, nano antibody of the invention has, extensive biologic applications are worth and clinical value, application are related to The multiple fields such as diagnosing and treating, basic medical research, biological study to disease relevant to TIM3.One is preferably answered Be for for TIM3 clinical diagnosis and targeted therapy.
Main advantages of the present invention include:
(a) nano antibody high specific of the present invention is directed to the TIM3 albumen with correct space structure of people.
(b) affinity of nano antibody of the present invention is strong.
(c) production of nano antibody of the present invention is easy.
Nano antibody of the present invention is suitable for prokaryotic expression and eukaryotic expression, has solubility high, is not easy to assemble, and is resistant to high Temperature, strong acid, highly basic etc. cause the advantages of Denaturing.Suitable for laboratory and industrial development.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.
Embodiment 1: the expression and purification of people's TIM3 albumen
(1) nucleotide sequence of people TIM3 (ECD) is synthesized on pFUSE-IgG1 carrier, wherein in the C of TIM3 (ECD) End introduces a TEV restriction enzyme site, convenient for preparing TIM3 (ECD) albumen without Fc label；
(2) pF Μ SE-IgG1-TIM3 (ECD) plasmid of building is extracted with the big extraction reagent kit of Omega plasmid；
(3) culture HEK293F cell to OD be 2.0 × 106A/mL；
(4) plasmid and transfection reagent PEI 1:3 are stood into 20min after mixing, are then added to HEK293F cell In, 37 DEG C, 6%CO2It is cultivated 5-6 days in shaking table culture case；
(5) cell conditioned medium is collected, is combined 1h at room temperature with Protein A pearl；
(6) with phosphate buffer pH 7.0 wash pearl after, then with 0.1M pH 3.0Glycine elution albumen；
(7) by the albumen ultrafiltration of elution into PBS, sampling progress SDS-PAGE examines antigen purity after measuring yield It surveys, remaining albumen is stored in -80 DEG C of refrigerators；
(8) it chooses antigen of the purity greater than 90% and carries out the immune of subsequent camel.
The building of embodiment 2:TIM3 phage library
(1) 1mg TIM3 (ECD)-Fc antigen is mixed in equal volume with Freund's adjuvant, an Xinjiang two-humped camel is immunized, weekly Once, it is immunized 7 times altogether, stimulation B cell expresses the nano antibody of antigentic specificity；
After (2) 7 times immune, extract 100mL camel peripheral blood lymphocytes and extract total serum IgE；
(3) it synthesizes cDNA and expands VHH using sleeve type PCR；
(4) 20 μ g pMECS Vector for Phage Display (Biovector of restriction enzyme Pst I and Not I digestion is utilized Supply) and two segments of 10 μ g VHH and connection；
(5) connection product is converted to electricity and is turned in competent cell TG1, construct TIM3 nano antibody library and measure library Hold, storage capacity size is 9.0 × 108CFU (Fig. 1)；
At the same time, 24 clones of random picking carry out bacterium colony PCR detection, by Fig. 2 the result shows that the insertion in built library Rate is up to 91.7%.The above results illustrate the TIM3 nano antibody phage display qualified we obtain storage capacity and insertion rate Library.
The screening and identification of embodiment 3:TIM3 nano antibody
Antibody screening:
(1) it is dissolved in 100mM NaHCO3, 10 μ g TIM3 (ECD)-Fc antigen (10 μ g Fc in pH 8.2 NaHCO3As control) it is coupled on NUNC ELISA Plate, 4 DEG C stand overnight；
(2) second days 100 μ L 0.1%BSA of addition, room temperature close 2h；
(3) after 2h, 100 μ L bacteriophages (2 × 10 are added11Camel nano antibody phage display gene pool is immunized in CFU), room temperature Act on 1h；
(4) it is washed 5 times with 0.05%PBS+Tween-20, to wash off non-specific bacteriophage；
(5) it under being dissociated the bacteriophage specifically bound with TIM3 with 100mM triethanolamine, and infects raw in logarithmic phase Long e. coli tg1 cell, 37 DEG C of culture 1h generate the screening that simultaneously purified phage is used for next round, identical screening process 3 wheels are repeated, enrichment times are respectively 7.8 times and 408 times (Fig. 3) without enrichment.
The single positive colony of specificity is screened with the enzyme-linked immunoassay method (ELISA) of bacteriophage:
(1) after above-mentioned 3 wheel screening in the Tissue Culture Dish containing bacteriophage, 600 single bacterium colonies is selected and are inoculated in The TB culture medium of ampicillin containing 100 μ g/mL (contains 2.3g KH in 1L TB culture medium2PO4, 12.52g K2HPO4, 12g peptone, 24g yeast extract, 4mL glycerol) in, after growing to logarithmic phase, add the IPTG of final concentration 1mM, 28 DEG C of cultures Overnight；
(2) antibody slightly is mentioned using osmosis acquisition, and antibody is transferred in the elisa plate through antigen coat, room temperature is quiet Set 1h；
(3) unbonded antibody is washed away with PBST, the anti-HA antibody of mouse is added, and is that century biotechnology is limited purchased from Beijing health Company), it is placed at room temperature for 1h；
(4) unbonded antibody is washed away with PBST, and goat-anti-mouse alkaline phosphatase enzyme mark antibody is added, is placed at room temperature for 1h；
(5) unbonded antibody is washed away with PBST, alkaline phosphatase developing solution is added, and is read at ELISA instrument 405nm wavelength Take absorption value；
(6) when sample well OD value is greater than 3 times of control wells OD value or more (Ratio+/- > 3), it is judged to positive colony hole.Knot Fruit shows that 86 positive colonies occur altogether in 600 clones；
(7) bacterium in 86 positive colony holes is turned to shake the LB liquid being 100 μ g/mL containing 2mL ampicillin concentration In to extract plasmid and to be sequenced；
(8) 86 strain clone of comparative analysis series, we finally obtain 29 plants of sequence difference nano antibodies, and number is respectively Nb1-Nb29 (SEQ ID NO.:88-116), wherein having the 16 kinds of area notable difference CDR3 nano antibodies, wherein Nb1-Nb5 (SEQ ID NO.:88-92) is family 1；Nb6-Nb10 (SEQ ID NO.:93-97) is family 2, Nb11-Nb13 (SEQ ID It NO.:98-100) is family 3, Nb14, Nb15 (SEQ ID NO.:101,102) is family 4, Nb16-Nb29 (SEQ ID NO.: It 103-116) is family 5- family 16.
Embodiment 4: expression and purification of the nano antibody in host e. coli
(1) sequencing analysis 29 plants of TIM3 specificity clone obtained is seeded to 5mL LA culture concentration and carries out expansion training It supports；
(2) corresponding plasmid is extracted, and takes 1uL plasmid, 20uL electricity turns competence WK6, and electricity turns；
(3) on the plate containing ampicillin and glucose, 37 DEG C are incubated overnight plating cells after turning electricity；
(4) it selects single bacterium colony to be seeded in the LB culture solution that 5mL contains ampicillin, 37 DEG C of shaking tables
Overnight incubation；；
(5) 3) the overnight strain of 1mL is inoculated with into 330mL TB culture solution, and 37 DEG C of shaking table cultures, culture reaches to OD value When 0.6-1, IPTG is added, 28 DEG C of shaking table cultures are stayed overnight；
(6) thalline were collected by centrifugation, and then the hypertonic cracking of thallus is utilized the affine layer of nickel ion to obtain antibody crude extract Column purification antibody protein is analysed, TIM3 nano antibody Prokaryotic expression, purification result is as shown in Figure 4.
The detection specificity analysis of embodiment 5:TIM-3 nano antibody
The building of 5.1TIM-3 stable cell strain
(1) it will be inserted into pLVX-EF1 α-puro carrier after the amplification of TIM-3 gene order, constructs pLVX-EF1 α-puro- TIM-3 slow virus plasmid.
(2) method for building up of TIM-3 stable cell strain is as follows: HEK 293T cell being passed on and is trained completely in DMEM containing 5mL In the 10cm diameter Petri dishes for supporting base, by 250uL Opti-MEM culture medium (Invitrogen company) and 3ug packaging plasmid (pLP1:pLP2:VSVG=1:1:1), 1ug slow virus plasmid mixes, and 20uL Polyfect transfection reagent is then added (QIAGEN company), mixes well, is placed at room temperature for 10min, is added in the culture dish of the cell containing HEK293T, and 37 DEG C, 5%CO2 It is cultivated 2 days in incubator.Collection supernatant changes 10mL fresh DMEM medium into 50mL centrifuge tube, in culture dish and continues culture 1 It.Supernatant is collected into proxima luce (prox. luc) 50mL centrifuge tube, 4000rpm is centrifuged 5min, supernatant is filtered with the filter of 0.45um to new Centrifuge tube in.5 × slow virus concentrate is added, mixes well, is placed in 4 DEG C overnight.Second day 4000g, 4 DEG C of centrifugation 25min, Supernatant is removed, slow virus is resuspended with 1mL DMEM culture medium.The TIM-3 lentiviral particle of harvest and 9mL DMEM culture medium are mixed It is even, it is added into the HEK 293T cell in new biography generation.Again vitellophag every three days, with the puromycin of 2.0ug/mL into Row screening.After screening 3-4 wheel, whether success is constructed using Flow Cytometry detection stable cell strain.
5.2 Flow Cytometries detect TIM-3 stable cell strain
(1) according to the TIM-3 antibody (TSR-022, the patent No.: US20150218274A1) for being in clinicalⅰstage conceptual phase Sequence, its heavy chain and light chain are synthesized, and are built into pFUSE-hIgG1e4-Fc1 carrier respectively.(2) HEK is further utilized 293F eukaryotic expression system and protein A protein purification system obtain TIM-3 positive antibody (bench marker, BMK), With the identification for TIM-3 stable cell strain.
(2) in FCM analysis, by 1.5 × 106A HEK 293T TIM-3 surely turns cell point to 3 centrifuge tubes In, 3000rpm, is centrifuged 4min by 4 DEG C, abandons supernatant, and the PBS that precipitating 100uL is pre-chilled is resuspended, and every 5 × 105It is anti-that 6ug is added in cell Body (TIM-3 positive antibody BMK is added in experimental group or antibody is not added in isotype control Ab AF70-Fc, blank group), 4 after mixing DEG C be incubated for 20min.500uL PBS is washed one time, and precipitating is resuspended with 200uL PBS, and 1uL goat-anti-human is added in every pipe (FITC), 4 DEG C of incubation 20min are mixed.Pre-cooling PBS abandons supernatant after washing one time, precipitating is resuspended with 200uL pre-cooling PBS, utilizes streaming The ratio of cell instrument (U.S. company BD) detection TIM-3 positive cell.Experimental result is shown in Fig. 5.Fig. 5 A show successfully obtain it is high-purity The TIM-3BMK antibody of degree, Fig. 5 B show that the positive cell ratio of TIM-3 stable cell strain reaches 98.1%.
5.3 screenings have the TIM-3 nano antibody of high specific, high-affinity
(1) by 1.65 × 107A HEK293T TIM-3 surely turns cell (HEK 293T is compareed as negative cells) and is resuspended in It in ice-cold PBS, and is transferred in 33 holes of U-shaped 96 orifice plate, every hole 100uL cell sample.
(2) every hole be added 6ug antibody (experimental group is 29 plants of TIM-3 nano antibodies, Isotype control PD-1, PD-L1 or Antibody is not added in CTLA-4 nano antibody, blank group), 4 DEG C of incubation 20min after mixing.
(3) it after PBS washes one time, is diluted with the antibody of 100uL mouse-anti-His containing secondary antibody (Alexa Fluor 488) Liquid is resuspended, 4 DEG C of incubation 20min after mixing.
(4) it is resuspended after PBS washes one time with 200uL PBS, turns cell table with steady using flow cytomery nano antibody The ratio for the positive cell that face TIM-3 is combined.Experimental result is shown in Fig. 6.It is compareed using HEK293T as negative cells, result of study Show that 29 kinds of TIM-3 nano antibodies and HEK293T without (not shown) is combined, there are 12 kinds to have with TIM-3 stable cell strain In conjunction with, wherein 10 kinds of TIM-3 nano antibodies and surely to turn the TIM-3 binding force on strain surface be more than 80%, and homotype negative control resists Body (PD-1, PD-L1 and CTLA-4 nano antibody), without combination, tests with the TIM-3 for surely turning strain surface and shows elutriation obtains 29 There are 10 kinds to show that high specificity and affinity, nano antibody number are followed successively by TIM-3 antigen in kind nano antibody Nb16, Nb18, Nb20, Nb21SEQ ID NO.:108, Nb22SEQ ID NO.:109, Nb23, Nb24, Nb25, Nb26, Nb27. correspond to sequence and be followed successively by SEQ ID NO.:103, SEQ ID NO.:105, SEQ ID NO.:107, SEQ ID NO.: 110, SEQ ID NO.:111, SEQ ID NO.:112, SEQ ID NO.:113, SEQ ID NO.:114), the knot of remaining sequence It is higher to close affinity.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
SEQUENCE LISTING
<110>Shanghai Luo Qi biological medicine technology Co., Ltd
<120>it is directed to the single domain antibody of T lymphocyte immunoglobulin Mucin 3
<130> P2018-0335
<160> 145
<170> PatentIn version 3.5
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Gly Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Thr
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Gly Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Thr
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Gly Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Thr
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Gly Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Thr
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Gly Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Ile
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Gly Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Ile
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Gly Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Ile
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Gly Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg Asp Asp Gly Ser Thr
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Ala Ala Asp Ile Thr Cys Arg His Ala Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg Asp Gly Gly Ser Thr
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Ile Arg Asp Gly Gly Ser Thr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg Asp Asp Gly Ser Thr
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Ala Ala Asp Ile Thr Cys Arg His Ala Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Ile Arg His Asp Gly Ser Ile
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Gly Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr
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Gly Tyr Thr Tyr Ser Arg Ala Cys Met Gly
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Val Arg His Asp Gly Ser Ile
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Gly Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr
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Val Trp Ile Phe Ser Asn Cys Ala Met Ala
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Ile Gly Ser Phe Arg Asp Thr
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Lys Ile Gln Cys Gly Thr Gln Val Asn
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Val Trp Ile Phe Ser Asn Cys Ala Met Ala
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Ile Gly Ser Phe Arg Asp Thr
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Lys Ile Gln Cys Gly Thr Gln Val Asn
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Arg Ser Thr Tyr Cys Met Gly
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Ile Ser Ser Asp Gly Arg Thr
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Ala Thr Arg Pro Gly Asn Ser Cys Gly Thr Gly Ile Asp Met Pro Tyr
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Thr Thr Leu Tyr Ile Ala Ser Leu Gly
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Val Asp Arg Asp Gly Asn Leu
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Ser Ala Ala Leu Ser Tyr Val Pro Ala Gly Arg Arg Leu Gln Pro Asp
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Ala Tyr Asn Tyr
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Gly Phe Thr Phe Ser Val Ala Asp Met Ser
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Ile Asn Ser Asp Gly Val Ser Thr
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Ala Ile Gly His Thr Pro Cys Thr Ala Gly Ser Cys Arg
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Thr Tyr Thr Val Val Arg Asn Cys Phe Gly
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Ile Asp Arg Asp Gly Ser Thr Arg
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Ala Ala Asp Trp Asn Asn Asp Arg Ser Cys Pro Leu Trp Ala Asp Gly
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Phe Gly
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Gly Phe Thr Phe Ser Val Ala Asp Met Thr
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Val Asn Ser Asp Gly Gly Ser Thr
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Ala Ile Gly Arg Thr Pro Cys Thr Gly Gly Phe Cys His
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Lys Ile Thr Tyr Val Ser Ser Cys Met Gly
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Ile Asp Arg Asp Gly Ser Thr Thr
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Ala Ala Asp Trp Gly Arg Trp Cys Ser Leu Glu Lys Ala Val Asp Phe
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Val Tyr
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Gly Tyr Thr Tyr Ser Leu Tyr Cys Met Gly
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Ile Ser Ala Gly Gly Gly Thr Thr
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Ala Arg Ser Gly Gly Tyr Cys Gly Leu Leu Glu Tyr Pro Phe Thr Ser
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Gly Phe Thr Phe Ser Ser Ala Asp Met Ser
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Ile Asn Ser Gly Gly Gly Trp Thr
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Thr Ile Gly Asn Thr Tyr Cys Ser Arg Gly Ala Cys Leu
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Gly Phe Thr Phe Ser Val Ala Asp Met Ser
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Ile Asn Ser Gly Gly Gly Arg Thr
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Thr Ile Gly His Thr Tyr Cys Ser Gly Gly Ala Cys Leu
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Val Asp Ile Tyr Arg Thr Tyr Cys Met Gly
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Ile Ser Thr Asp Gly Arg Ile
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Ala Ala Asp Ser Ala Arg Cys Gly Leu Trp Leu Gly Gly Gly Tyr Pro
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Asn Tyr
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Gly Ser Thr Phe Ser Thr Ala Leu Met Gly
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Ile Ser Ala Gly Gly Gly Ser Thr
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Ala Ser Ser Asn Ser Leu Trp Thr Arg Glu Ser Arg Tyr Phe Gly Tyr
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Gly Phe Thr Phe Ser Ser Ser Glu Met Thr
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Ser Ser Thr Ser Ala Phe Thr
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Ala Lys Asp Val Tyr Cys Gly Gly Gln Tyr Cys Pro Pro
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Lys Ala Leu Val Ile Ser Cys Leu Ala
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Ile Gly Arg Asp Gly Ser Thr
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Ala Ala Ala Thr Lys Gln Asp Gly Ile Pro Leu Asn Pro Ala Asp Tyr
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Asp Ile
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Gly Thr Thr Ser Arg Asn Tyr Cys Met Gly
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Ile Asp Lys Tyr Gly Thr Ser
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Ala Ile Ser Ser Gln Tyr Gly Leu Cys Leu Ala Gln Thr Gly Asp Tyr
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Ala Tyr
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr Trp Gly Gln Gly
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Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Asp Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gln Ser Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Ile Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Ile Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 95
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 95
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Ile Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 96
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 96
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg Asp Asp Gly Ser Thr Ala Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Ile Thr Cys Arg His Ala Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 97
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 97
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg Asp Gly Gly Ser Thr Ala Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Ile Thr Cys Arg His Ala Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 98
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 98
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg Asp Asp Gly Ser Thr Ala Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Ile Thr Cys Arg His Ala Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 99
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 99
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Ile Arg His Asp Gly Ser Ile Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 100
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 100
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Arg Ala
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Trp Val
35 40 45
Ser Gly Val Arg His Asp Gly Ser Ile Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Gly
85 90 95
Ala Asp Ile Asp Cys Arg Gly Thr Arg Pro Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 101
<211> 115
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 101
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Thr Ala Ser Val Trp Ile Phe Ser Asn Cys
20 25 30
Ala Met Ala Trp Tyr Arg Gln Ala Pro Arg Lys Glu Arg Glu Phe Val
35 40 45
Ser Ala Ile Gly Ser Phe Arg Asp Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys Lys
85 90 95
Ile Gln Cys Gly Thr Gln Val Asn Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 102
<211> 115
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 102
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Thr Ala Ser Val Trp Ile Phe Ser Asn Cys
20 25 30
Ala Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ser Ala Ile Gly Ser Phe Arg Asp Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys Lys
85 90 95
Ile Gln Cys Gly Thr Gln Val Asn Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 103
<211> 119
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 103
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Arg Ser Thr Tyr Cys Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ile Ile
35 40 45
Ser Ser Asp Gly Arg Thr Asn Tyr Ala Asp Pro Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Met Phe Leu Gln Met Asn
65 70 75 80
Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Arg Pro
85 90 95
Gly Asn Ser Cys Gly Thr Gly Ile Asp Met Pro Tyr Trp Gly Lys Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 104
<211> 125
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 104
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Thr Leu Tyr Ile Ala Ser
20 25 30
Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
35 40 45
Ala Val Asp Arg Asp Gly Asn Leu Asp Tyr Ala Asp Ser Val Lys Gly
50 55 60
Arg Phe Thr Phe Ser Arg Asp His Ala Lys Asn Thr Leu Tyr Leu Gln
65 70 75 80
Met Asn Ser Leu Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ser Ala
85 90 95
Ala Leu Ser Tyr Val Pro Ala Gly Arg Arg Leu Gln Pro Asp Ala Tyr
100 105 110
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 105
<211> 120
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 105
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Ala
20 25 30
Asp Met Ser Trp Val Arg Gln Gly Pro Gly Lys Gly Phe Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Asp Gly Val Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Tyr
65 70 75 80
Leu Arg Ala Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Gly His Thr Pro Cys Thr Ala Gly Ser Cys Arg Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 106
<211> 125
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 106
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Tyr Thr Val Val Arg Asn
20 25 30
Cys Phe Gly Trp Phe Arg Gln Ala Pro Gly Lys Lys Arg Glu Gly Val
35 40 45
Ala Val Ile Asp Arg Asp Gly Ser Thr Arg Tyr Ala Ala Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Ala Leu Tyr Leu
65 70 75 80
Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Trp Asn Asn Asp Arg Ser Cys Pro Leu Trp Ala Asp Gly Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 107
<211> 120
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 107
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Ala
20 25 30
Asp Met Thr Trp Val Arg Gln Gly Leu Gly Lys Gly Phe Glu Trp Val
35 40 45
Ala Ser Val Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Arg Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ile Gly Arg Thr Pro Cys Thr Gly Gly Phe Cys His Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 108
<211> 124
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 108
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Tyr Cys Ala Ala Ser Lys Ile Thr Tyr Val Ser Ser
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ser Ile Asp Arg Asp Gly Ser Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Trp Gly Arg Trp Cys Ser Leu Glu Lys Ala Val Asp Phe Val
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 109
<211> 123
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 109
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Arg Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Leu Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Ser Ala Gly Gly Gly Thr Thr Tyr Tyr Ser Asp Ala Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Cys Gly Leu Leu Glu Tyr Pro Phe Thr Ser
100 105 110
Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 110
<211> 120
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 110
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Ala
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Gly Gly Gly Trp Thr Asp Tyr Ala Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Gly Asn Thr Tyr Cys Ser Arg Gly Ala Cys Leu Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 111
<211> 120
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 111
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Ala
20 25 30
Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asn Ser Gly Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Met
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Gly His Thr Tyr Cys Ser Gly Gly Ala Cys Leu Arg Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 112
<211> 124
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 112
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Val Asp Ile Tyr Arg Thr Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val
35 40 45
Ala Ala Ile Ser Thr Asp Gly Arg Ile Arg Tyr Ala Asn Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Asn Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Phe Cys Ala
85 90 95
Ala Asp Ser Ala Arg Cys Gly Leu Trp Leu Gly Gly Gly Tyr Pro Asn
100 105 110
Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 113
<211> 123
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 113
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Thr Phe Ser Thr Ala
20 25 30
Leu Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Val Ile Ser Ala Gly Gly Gly Ser Thr Trp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Phe
65 70 75 80
Leu Gln Met Ile Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ser Ser Asn Ser Leu Trp Thr Arg Glu Ser Arg Tyr Phe Gly Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 114
<211> 120
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 114
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ala Gly Phe Thr Phe Ser Ser Ser
20 25 30
Glu Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Ser Ser Thr Ser Ala Phe Thr Gln Tyr Ala Gly Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Lys Asp Val Tyr Cys Gly Gly Gln Tyr Cys Pro Pro Val Gly Pro
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 115
<211> 123
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 115
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Thr Lys Ala Leu Val Ile Ser Cys
20 25 30
Leu Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala
35 40 45
Leu Ile Gly Arg Asp Gly Ser Thr Ser Tyr Pro Asp Ser Val Lys Gly
50 55 60
Arg Phe Thr Phe Ser Lys Asn Thr Ala Lys Asn Thr Leu Glu Leu Gln
65 70 75 80
Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Ala Tyr Tyr Cys Ala Ala
85 90 95
Ala Thr Lys Gln Asp Gly Ile Pro Leu Asn Pro Ala Asp Tyr Asp Ile
100 105 110
Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 116
<211> 124
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 116
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Ser Arg Asn Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val
35 40 45
Ala His Ile Asp Lys Tyr Gly Thr Ser Asp Tyr Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ile Ser Ser Gln Tyr Gly Leu Cys Leu Ala Gln Thr Gly Asp Tyr Ala
100 105 110
Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 117
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 117
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcac aagctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtcagt cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 118
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 118
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ttggaggatc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcac aagctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaaca ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtcagt cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 119
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 119
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ttggaggatc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgactg ggtctcaggt attagacatg atggtagcac aagctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtcagt cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 120
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 120
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcac aagctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtcagt cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 121
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 121
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ttggaggatc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcac aagctatgca 180
gactccgtga agggccgatc caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtcagt cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 122
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 122
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ttggagggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcat cacctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtggta cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 123
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 123
caggtgcagc tgcaggagtc tgggggagga tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcat cacctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtggta cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 124
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 124
caggtgcagc tgcaggagtc tggaggagga tcggtgcagg ccggaggctc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcat cacctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca acctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtggta cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 125
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 125
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ttggagggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagagatg atggtagcac cgcctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc cgacataact 300
tgtcgtcatg cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 126
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 126
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagagatg gtggtagcac cgcctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc cgacataact 300
tgtcgtcatg cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 127
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 127
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagagatg atggtagcac cgcctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc cgacataact 300
tgtcgtcatg cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 128
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 128
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagc ctggggggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt attagacatg atggtagcat cacctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtggta cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 129
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 129
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ttggagggtc tctgagactc 60
tcctgtacag cctctggata cacctacagc cgcgcctgca tgggttggtt ccgccaggct 120
ccagggaagc agcgcgagtg ggtctcaggt gttagacatg atggtagcat cacctatgca 180
gactccgtga agggccgatt caccatctcc cgggacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtggggc agacatagat 300
tgtcgtggta cccgaccgcc ctactggggc caggggaccc aggtcaccgt ctcctca 357
<210> 130
<211> 345
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 130
caggtgcagc tgcaggagtc tggaggaggc tcggtgcaga ctggagggtc tctgaaactc 60
tcctgtacag cctctgtctg gatcttcagt aactgcgcga tggcctggta ccgccaggct 120
ccaaggaagg agcgcgagtt cgtctcagct attggctctt ttcgtgacac aaactacgcc 180
gactccgtga agggccgatt caccatttcc cgagacaacg ccaagaacac ggggtatcta 240
caaatgaaca gcctgaaacc tgaggactcg gccatgtatt attgtaaaat ccaatgcgga 300
acgcaagtaa actggggcca ggggacccag gtcaccgtct cctca 345
<210> 131
<211> 345
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 131
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgaaactc 60
tcctgtacag cctctgtctg gatcttcagt aactgcgcga tggcctggta ccgccaggct 120
ccagggaagg agcgcgagtt cgtctcagct attggctctt ttcgtgacac aaactacgcc 180
gactccgtga agggccgatt caccatttcc cgagacaacg ccaagaacac ggggtatcta 240
caaatgaaca gcctgaaacc tgaggactcg gccatgtatt attgtaaaat ccaatgcgga 300
acgcaagtaa actggggcca ggggacccag gtcaccgtct cctca 345
<210> 132
<211> 357
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 132
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcgtgtacag cctctagatc cacatattgc atgggctggt tccgccaggc tccagggaag 120
gagcgcgagg gggtcgcaat aatttccagt gatgggcgca caaactacgc agaccccgtg 180
aagggccgat tcaccatctc caaagacaac gccaagaata ccatgtttct gcaaatgaac 240
agcctgaaac ctgaggacac tgccatgtac tactgtgcga cccgcccggg taattcctgc 300
ggcaccggta tagatatgcc atattggggc aaaggaaccc aggtcaccgt ctcctca 357
<210> 133
<211> 375
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 133
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctacaac tctctacatc gcctcattgg gctggttccg ccaggctcca 120
gggaaggagc gcgagggggt cgcggctgtt gatcgtgatg gtaatttaga ctacgcagac 180
tccgtgaagg gccgattcac cttctccaga gaccacgcta agaataccct gtatctgcaa 240
atgaacagcc tgaaacctga ggacgctgcc atgtactact gttcggcagc actatcttac 300
gttccggcag gacggagact acaaccagat gcatataact actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 134
<211> 360
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 134
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcttgtgcag cctctggatt cacattcagt gtggccgata tgagctgggt ccgccagggt 120
ccagggaagg ggttcgagtg ggtctcatct attaatagtg atggtgttag tacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccgagaa cacgctgtat 240
ctgcgagcga acagcctgaa aactgaggac actgccgtgt attactgcgc cataggacat 300
acgccttgta ctgctggtag ctgtcgccga ggccagggga cccaggtcac cgtctcctca 360
<210> 135
<211> 375
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 135
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctccacata caccgtggtt cgcaattgtt tcggctggtt ccgccaggct 120
ccagggaaga agcgcgaggg ggtcgcagtt attgacaggg atggtagtac aaggtacgca 180
gcctccgtga agggccgatt caccatctcc aaagacaacg ccaagaatgc cctgtatctg 240
caaatgagca gcctggaacc cgaggacact gccgtttact actgtgcggc tgattggaat 300
aacgatcgta gctgtcctct atgggccgac ggctttggtt actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 136
<211> 360
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 136
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctccggatt cacattcagt gtagccgaca tgacctgggt ccgccagggt 120
ctagggaagg ggttcgagtg ggtagcatcc gttaatagtg atggtggttc cacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcgattga acagcctgaa aactgaggac actgccgtgt attactgcgc cataggacga 300
acgccttgta ctggtggttt ctgtcaccga ggccagggga cccaggtcac cgtctcctca 360
<210> 137
<211> 372
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 137
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tactgtgcag cctctaaaat cacctacgtt agctcctgca tgggctggtt ccgccaggct 120
ccagggaagg agcgcgaggg ggtcgcaagt attgatcgtg atggtagcac aacgtacgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaaggacag tctgtatcta 240
caaatgaata gcctgaaacc tgaggacact gccatgtact actgtgcggc agattggggc 300
cggtggtgta gcctagagaa ggcggtggac tttgtttact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 138
<211> 369
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 138
caggtgcagc tgcaggagtc tgggggaggc tcggtgcggg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cacctacagt ctctactgca tgggctggtt ccgccaggct 120
ccagggaagg agcgcgaggg ggtcgctgct atttcagctg gtggaggtac cacatactat 180
agcgacgccg tgaagggccg attcaccatc tcccgagaca acgccaagaa aacgctctat 240
ctgcaaatga acagcctgaa tcctgaggac actgccatgt actactgtgc gaggagtggt 300
ggttactgcg gccttcttga atatccgttt acttcctggg gcccggggac ccaggtcacc 360
gtctcctca 369
<210> 139
<211> 360
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 139
caggtgcagc tgcaggagtc tggaggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgtacag cctctggatt cacattcagt agcgccgaca tgagctgggt ccgccaggct 120
ccagggaagg ggctcgagtg ggtctcatct atcaatagtg gtggtggttg gacagactac 180
gcagactccg tgcagggccg attcaccatc tccagagaca acggcaagaa cacgctgtat 240
ctgcaaatga acagcctgaa aactgaagac actgccgtgt attactgcac tataggtaat 300
acatattgta gtcgtggcgc ctgcttacga ggccagggga cccaggtcac cgtctcctca 360
<210> 140
<211> 360
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 140
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt cacattcagt gtcgccgaca tgagctgggt ccgccaggct 120
ccagggaagg ggctcgagtg ggtctcatct atcaatagtg gtggtggtag gacatactat 180
gcagactcca tgaagggccg attcaccatc tccagagaca acggcaagaa cacgctgtat 240
ctgcaaatga acagcctgaa aactgaggac actgccgtgt attactgcac cataggacat 300
acatattgta gtggtggcgc ctgcttacga ggccagggga cccaggtcac cgtctcctca 360
<210> 141
<211> 372
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 141
caggtgcagc tgcaggagtc tgggggaggc tcggtacagg ctggagggtc tctgagactc 60
tcctgtgcag cctctgtaga catctatagg acctactgca tgggctggtt ccgccaggct 120
ccaggaaagc agcgcgaggg ggtcgcagcc attagtactg atggtaggat acgttacgcg 180
aattccgtgg agggccgatt caccatctcc aaagacaacg ccaacaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact tctgtgcggc agattcggcc 300
agatgtggtc tatggcttgg cggcggttac cctaactact ggggccgggg gacccaggtc 360
accgtctcct ca 372
<210> 142
<211> 369
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 142
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtacag cctctggatc tacctttagt accgcattaa tgggctggtt tcggcaggct 120
ccagggaagg agcgcgaggg ggtcgcagtt atttcggctg gtggtggaag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tcccgagaca acaccaagaa cacggtgttt 240
ctgcaaatga tcagcctgaa acctgaggac actgccatgt actactgtgc gtctagtaac 300
agcttgtgga cccgagagag tcgttatttt ggttactggg gccaggggac ccaggtcacc 360
gtctcctca 369
<210> 143
<211> 360
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 143
caggtgcagc tgcaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag ccgctggatt caccttcagt tcgtctgaga tgacgtgggt ccgccaggct 120
ccaggaaagg gactcgagtg ggtcgccagg attagtagta ctagtgcttt cacgcagtat 180
gccggctccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgctgtat 240
ctgcaattga acagcctgaa aactgaggac acggccatgt attactgtgc aaaagatgtt 300
tattgtggtg gtcaatactg cccccccgta ggcccgggga cccaggtcac cgtctcctca 360
<210> 144
<211> 369
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 144
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag ccactaaagc actcgttatc agctgcttgg cctggttccg ccaggctcca 120
gggaaggagc gcgagtgggt cgcacttatt ggtcgtgatg gtagcacaag ctacccagac 180
tccgtgaagg gccgattcac tttctccaaa aacaccgcca agaacactct agaactgcaa 240
atgaacaatc tgaaacctga agacaccggc gcgtactact gtgcggcggc cacaaagcag 300
gatggtatac cgttaaatcc cgccgattat gacatctggg gccgggggac ccaggtcacc 360
gtctcctca 369
<210> 145
<211> 372
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 145
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ttggagggtc tctgagactc 60
tcctgtgcag cctctggaac cacctctcgc aactactgta tgggttggtt tcgccaggct 120
ccagggaagg agcgcgaatg ggtcgcacat attgataaat atggtacatc tgactacact 180
gactccgtga agggccgatt caccatctcc aaagacaacg ccaagaacac gctatatcta 240
caaatgaaca gcctgaaacc cgaggacact gccatgtact actgcgcgat ctcgtcccaa 300
tatgggttgt gtctagccca aaccggcgac tatgcctact taggccaggg gacccaggtc 360
accgtctcct ca 372

Claims (10)

  1. The CDR region of 1.T lymphocyte immune globulin Mucin 3 (TIM3) specific nano antibody, which is characterized in that it can Tim3 is specifically bound, and the CDR region is selected from the group below one or more:
    (1) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3；
    (2) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6；
    (3) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9；
    (4) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12；
    (5) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15；
    (6) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18；
    (7) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21；
    (8) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24；
    (9) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27；
    (10) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30；
    (11) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33；
    (12) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36；
    (13) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39；
    (14) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42；
    (15) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45；
    (16) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48；
    (17) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51；
    (18) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54；
    (19) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57；
    (20) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60；
    (21) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63；
    (22) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66；
    (23) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69；
    (24) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72；
    (25) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75；
    (26) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78；
    (27) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81；
    (28) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84；
    (29) CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87.
  2. 2. a kind of VHH chain of TIM3 specific nano antibody, which is characterized in that VHH chain includes framework region FR and claim 1 institute One of CDR region stated is a variety of.
  3. 3. a kind of specific binding TIM3 nano antibody VHH chain combination, which is characterized in that at least contain in the VHH chain combination VHH chain with following CDR region:
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81.
  4. 4. combination as claimed in claim 3, which is characterized in that in the VHH chain combination also containing have it is following a kind of or The VHH chain of a variety of CDR regions:
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78.
  5. 5. combination as claimed in claim 4, which is characterized in that in the VHH chain combination also containing have it is following a kind of or The VHH chain of a variety of CDR regions:
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84；
    CDR3 shown in CDR2 shown in CDR1 shown in SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87.
  6. 6. a kind of TIM3 nano antibody, which is characterized in that it enough specifically binds Tim3, and the VHH chain in the antibody is selected from One of SEQ ID NO:88-116 or a variety of.
  7. 7. TIM3 nano antibody as claimed in claim 6, which is characterized in that wherein the specific nano antibody includes at least SEQ Sequence shown in ID NO:114.
  8. 8. a kind of isolated polynucleotide sequence, which is characterized in that the polynucleotide encoding protein selected from the group below: right It is required that the CDR region of anti-TIM3 nano antibody described in 1, the VHH chain of anti-TIM3 nano antibody as claimed in claim 2, right are wanted Ask the 4 VHH chain combinations or anti-TIM3 nano antibody as claimed in claim 3.
  9. 9. polynucleotide sequence as claimed in claim 8, the polynucleotide sequence includes in SEQ ID NO:117-145 It is one or more.
  10. 10. a kind of expression vector, which is characterized in that the expression vector contains any polynucleotides of claim 8-9.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112094348A (en) * 2020-09-22 2020-12-18 首都医科大学附属北京胸科医院 Anti-human Tim3 antibody or functional fragment thereof and application thereof
CN113214396A (en) * 2020-07-31 2021-08-06 北京市神经外科研究所 Single-chain antibody of anti-TIM 3 and application thereof in preparing medicine for treating tumor
CN113831411A (en) * 2021-09-18 2021-12-24 南京融捷康生物科技有限公司 Single-domain antibody aiming at L1CAM and derivative protein and application thereof
CN114478778A (en) * 2022-04-01 2022-05-13 中国人民解放军军事科学院军事医学研究院 anti-Tim-3 nano antibody and application thereof
WO2023142309A1 (en) 2021-02-05 2023-08-03 上海洛启生物医药技术有限公司 Anti-tslp nanobody and use thereof

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