CN110144011B - Single domain antibodies against T lymphocyte immunoglobulin mucin3 - Google Patents

Single domain antibodies against T lymphocyte immunoglobulin mucin3 Download PDF

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CN110144011B
CN110144011B CN201810152107.6A CN201810152107A CN110144011B CN 110144011 B CN110144011 B CN 110144011B CN 201810152107 A CN201810152107 A CN 201810152107A CN 110144011 B CN110144011 B CN 110144011B
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CN110144011A (en
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万亚坤
朱敏
沈晓宁
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Shanghai Luoqi Biomedical 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®

Abstract

The invention relates to the technical field of antibody drugs, in particular to a nano antibody aiming at T lymphocyte immunoglobulin mucin 3(TIM3) and application thereof. The invention discloses a group of specific nanometer antibodies aiming at Tim3, discloses amino acid sequences of a framework region FR and a complementary determining region CDR of a VHH chain of the nanometer antibodies, and also discloses a gene sequence for encoding the single-domain heavy chain nanometer antibody. The anti-Tim 3 nano antibody provided by the invention can be effectively combined with Tim3 antigen, and has higher specificity and affinity. The gene sequence of the single-domain heavy chain nano antibody disclosed by the invention provides a new idea for research based on a Tim3 target spot, including gene detection and antibody drug development.

Description

Single domain antibodies against T lymphocyte immunoglobulin mucin3
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceutical, and discloses a single-domain antibody aiming at T lymphocyte immunoglobulin mucin 3(Tim3) and a coding sequence and application thereof.
Background
Recognition of tumor cells, presentation of tumor antigens to T cells, T cell activation, and specific killing of tumor cells. In this process, T cell activation requires costimulatory signals that require regulation of "immune checkpoints". T lymphocyte immunoglobulin mucin 3(T cell immunoglobulin and mucin3, TIM3) plays a crucial role in immune regulation in the tumor microenvironment as an important immune checkpoint in humans.
TIM3 is a member of the TIM family, encoding a type i membrane protein containing 281 amino acids. The common structure of TIM molecules includes the immunoglobulin N-terminal V region, mucin domain, transmembrane region and intracellular region. The extracellular part comprises an immunoglobulin-like region rich in cysteine and a mucin region rich in threonine, serine and proline, and the cytoplasmic region has 6 tyrosines, 1 of which belongs to a tyrosine phosphorylation motif part. A unique cleft, which is highly conserved in the immunoglobulin variable region consisting of 4 cysteines, distinct from other immunoglobulin superfamily members, can bind to Phosphatidylserine (PS). TIM3 is selectively expressed on IFN- γ secreting T helper cells (Th1 and Th17), T regulatory cells (tregs), Dendritic Cells (DCs), monocytes, mast cells, NK cells, Tumor Infiltrating Lymphocytes (TILs), and also on tumor cells, such as melanoma, gastric cancer, B cell lymphoma.
The TIM3 molecule is widely expressed on immune cells, can bind to a plurality of ligands such as galectin-9 (Gal-9), phosphatidylserine (PtdSer), high mobility group protein B-1 (HMGB-1) on the cell surface, is involved in regulating the differentiation of a variety of T cells including regulatory T cells (tregs), and plays an important role in immune homeostasis and clearance of apoptotic cells. TIM3 plays an important role in the development and maintenance of immune tolerance, and TIM3 can induce CD8+T cell 'exhaustion' can also inhibit growth of Th1 and Th17 cells by inducing apoptosis, reduce Th1 and Th17 type immune response, promote Treg differentiation and enhance Treg inhibition. In addition, Tim3 exerts an important regulatory role in autoimmune diseases by regulating macrophage activation and function, inhibiting tissue destructive immune responses. As a negative regulatory factor of tumor immune response, TIM3 has obvious tumor drug development potential and has been developed into a hot target molecule in the fields of immunotherapy and drug development. However, there is still a need to develop antibody drugs with good binding properties.
Disclosure of Invention
The invention provides a 29-strain specific nanometer antibody aiming at TIM3, and also provides a coding sequence and a preparation method of the nanometer antibody.
In a first aspect of the invention, the invention provides CDR regions of a nanobody specific for T lymphocyte immunoglobulin mucin 3(TIM 3).
In some embodiments, the TIM3 nanobody comprises one immunoglobulin single variable domain that specifically binds TIM 3. In some embodiments, the TIM3 nanobody comprises two or more immunoglobulin single variable domains that specifically bind TIM 3.
In some embodiments, the at least one immunoglobulin single variable domain comprises a CDRl, CDR2 and CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3 (corresponding to the CDR of antibody strain 1);
(2) CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6 (corresponding to the CDR of antibody strain 2);
(3) CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO.9 (corresponding to the CDR of antibody strain 3);
(4) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12 (corresponding to the CDR of antibody strain 4);
(5) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15 (corresponding to the CDR of antibody strain 5);
(6) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, CDR3 shown in SEQ ID NO:18 (corresponding to the CDR of antibody strain 6);
(7) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20, CDR3 shown in SEQ ID NO:21 (corresponding to the CDR of antibody strain 7);
(8) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23, CDR3 shown in SEQ ID NO. 24 (corresponding to the CDR of antibody strain 8);
(9) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 27 (corresponding to the CDR of antibody strain 9);
(10) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:29, CDR3 shown in SEQ ID NO:30 (corresponding to the CDR of antibody strain 10);
(11) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32, CDR3 shown in SEQ ID NO:33 (corresponding to the CDR of antibody strain 11);
(12) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35, CDR3 shown in SEQ ID NO:36 (corresponding to the CDR of antibody strain 12);
(13) CDR1 shown in SEQ ID NO:37, CDR2 shown in SEQ ID NO:38, CDR3 shown in SEQ ID NO:39 (corresponding to the CDR of antibody strain 13);
(14) CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:41, CDR3 shown in SEQ ID NO:42 (corresponding to the CDR of antibody strain 14);
(15) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, CDR3 shown in SEQ ID NO:45 (corresponding to the CDR of antibody strain 15);
(16) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, CDR3 shown in SEQ ID NO:48 (corresponding to the CDR of antibody strain 16);
(17) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50, CDR3 shown in SEQ ID NO:51 (corresponding to the CDR of antibody strain 17);
(18) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:53, CDR3 shown in SEQ ID NO:54 (corresponding to the CDR of antibody strain 18);
(19) CDR1 shown in SEQ ID NO:55, CDR2 shown in SEQ ID NO:56, CDR3 shown in SEQ ID NO:57 (corresponding to the CDR of antibody strain 19);
(20) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, CDR3 shown in SEQ ID NO:60 (corresponding to the CDR of antibody strain 20);
(21) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62, CDR3 shown in SEQ ID NO:63 (corresponding to the CDR of antibody strain 21);
(22) CDR1 shown in SEQ ID NO:64, CDR2 shown in SEQ ID NO:65, CDR3 shown in SEQ ID NO:66 (corresponding to the CDR of antibody strain 2);
(23) CDR20 shown in SEQ ID NO:67, CDR2 shown in SEQ ID NO:68, CDR3 shown in SEQ ID NO:69 (corresponding to the CDR of antibody strain 23);
(24) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, CDR3 shown in SEQ ID NO:72 (corresponding to the CDR of antibody strain 24);
(25) CDR1 shown in SEQ ID NO:73, CDR2 shown in SEQ ID NO:74, CDR3 shown in SEQ ID NO:75 (corresponding to the CDR of antibody strain 25);
(26) CDR1 shown in SEQ ID NO:76, CDR2 shown in SEQ ID NO:77, CDR3 shown in SEQ ID NO:78 (corresponding to the CDR of antibody strain 26);
(27) CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80, CDR3 shown in SEQ ID NO:81 (corresponding to the CDR of antibody strain 27);
(28) CDR1 shown in SEQ ID NO:82, CDR2 shown in SEQ ID NO:83, CDR3 shown in SEQ ID NO:84 (corresponding to the CDR of antibody strain 28);
(29) CDR1 shown in SEQ ID NO:85, CDR2 shown in SEQ ID NO:86, and CDR3 shown in SEQ ID NO:87 (corresponding to the CDR of antibody strain 29).
In another preferred embodiment, the CDRs 1, 2 and 3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
In a second aspect of the invention, there is provided a VHH chain of a TIM 3-specific nanobody, which is capable of specifically binding to TIM3, and which comprises one or more of the framework region FRs and the CDR regions of claim 1.
In another preferred embodiment, the VHH chain in said antibody is selected from one or more of SEQ ID NO 88-116.
In another preferred example, the nanobody is a nanobody combination, wherein the specific nanobody combination at least comprises the sequence shown in SEQ ID NO. 114.
In another preferred example, TIM 3-specific nanobodies of the present invention also encompass anti-TIM 3 antibody molecules capable of binding to the same epitope on TIM3 as the VHH consisting of the amino acid sequence of any one of SEQ ID NOs 88-116.
In a third aspect of the invention, there is provided an isolated polynucleotide sequence encoding a protein selected from the group consisting of: the CDR regions of the anti-TIM 3 nanobody of the first aspect of the invention, the VHH chain of the anti-TIM 3 nanobody of the second aspect of the invention, the VHH chain combination of the thirteenth aspect of the invention or the anti-TIM 3 nanobody of the invention.
In another preferred embodiment, the polynucleotide sequences are in combination, preferably the polynucleotide sequences comprise one or more of SEQ ID NO 117-145.
In another preferred embodiment, the present invention relates to nucleic acid molecules encoding the TIM3 nanobodies of the present invention. The nucleic acid of the present invention may be RNA, DNA or cDNA.
In a fourth aspect of the invention, there is provided an expression vector expressing the polynucleotide of the third aspect of the invention.
In a fifth aspect of the invention, there is provided a host cell comprising an expression vector according to the fifth aspect of the invention, or having a polynucleotide according to the fourth aspect of the invention integrated into its genome.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: escherichia coli, yeast cells.
In a sixth aspect of the present invention, there is provided a method for producing an anti-TIM 3 nanobody, comprising the steps of:
(a) culturing a host cell according to the fourth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture comprising said anti-TIM 3 nanobodies; and
(b) isolating or recovering said anti-TIM 3 nanobody from said culture; and optionally
(c) Purifying and/or modifying the TIM3 nanobody obtained in step (b).
In another preferred embodiment, the anti-TIM 3 nanobody has an amino acid sequence shown in SEQ ID NO 88-116.
In a seventh aspect of the present invention, there is provided a conjugate comprising:
(a) a VHH chain of an anti-TIM 3 nanobody according to the second aspect of the invention, or an anti-TIM 3 nanobody according to the second aspect of the invention; and operatively connected
(b) A modifying label selected from the group consisting of: chemical markers and biological markers.
In another preferred embodiment, the chemical label is an isotope, an immunotoxin and/or a chemical drug.
In another preferred embodiment, the biomarker is a biotin, avidin or enzyme label.
The present invention also provides a conjugate prepared by conjugating an anti-TIM 3 nanobody according to the second aspect of the present invention or a conjugate according to the seventh aspect of the present invention to a solid or semi-solid medium.
In another preferred embodiment, the conjugate consists of a conjugate of the seventh aspect of the invention with a coupling element selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.
In another preferred embodiment, the conjugate comprises: multivalent (e.g. bivalent) TIM3 nanobody according to the second aspect of the invention, VHH chain of TIM3 nanobody according to the third aspect of the invention.
In another preferred embodiment, said multivalent is the VHH chain of a TIM3 nanobody according to the second aspect of the invention, a TIM3 nanobody according to the third aspect of the invention, comprising multiple repeats in the amino acid sequence of said immunoconjugate.
In an eighth aspect of the present invention, there is provided use of a TIM3 nanobody of the second aspect of the present invention for the preparation of (a) a reagent for the detection of a TIM3 molecule; (b) a medicine for treating tumor.
In another preferred embodiment, the detection comprises flow detection and cell immunofluorescence detection.
In a ninth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) one or more of the complementarity determining regions CDRs of the anti-TIM 3 nanobody VHH chain of the first aspect of the invention, the VHH chain of the second aspect of the invention, the VHH combination of the thirteenth aspect of the invention, the anti-TIM 3 nanobody of the second aspect of the invention, or an antigen-binding fragment thereof; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymph cancer, adrenal gland tumor, or bladder tumor.
In a tenth aspect of the invention, there is provided a use of one or more of the anti-TIM 3 nanobodies of the second aspect of the invention:
(i) for detection of human TIM3 molecules;
(ii) for streaming detection;
(iii) for cellular immunofluorescence detection;
(iv) for the treatment of tumors;
(v) can be used for tumor diagnosis.
In another preferred embodiment, the use is non-diagnostic and non-therapeutic.
In an eleventh aspect of the present invention, there is provided a recombinant protein having:
(i) the sequence of a nanobody according to the second aspect of the invention or the sequence of a heavy chain variable region VHH according to the third aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification. In another preferred embodiment, the tag sequence comprises a 6His tag and an HA tag in another preferred embodiment, the recombinant protein specifically binds to TIM3 protein.
In a twelfth aspect of the invention, there is provided use of a nanobody according to the second aspect of the invention, a VHH chain according to the third aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention, for the preparation of a medicament, a reagent, a detection plate or a kit;
wherein the reagent, assay plate or kit is for: detecting TIM3 protein in a sample;
wherein the agent is for use in the treatment or prevention of a tumor that expresses TIM3 (i.e. TIM3 positive).
In a thirteenth aspect of the present invention, there is also provided a VHH chain combination of a TIM3 specific binding nanobody, wherein the VHH chain combination comprises at least a VHH chain having the following CDR regions:
CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80, and CDR3 shown in SEQ ID NO: 81.
In another preferred embodiment, the VHH chain combination further comprises VHH chains having one or more of the following CDR regions:
CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47, CDR3 shown in SEQ ID NO. 48;
CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 53, CDR3 shown in SEQ ID NO. 54
CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 59, CDR3 shown in SEQ ID NO. 60;
CDR1 shown in SEQ ID NO. 61, CDR2 shown in SEQ ID NO. 62, CDR3 shown in SEQ ID NO. 63;
CDR1 shown in SEQ ID NO. 64, CDR2 shown in SEQ ID NO. 65, CDR3 shown in SEQ ID NO. 66;
CDR1 shown in SEQ ID NO. 67, CDR2 shown in SEQ ID NO. 68, CDR3 shown in SEQ ID NO. 69;
CDR1 shown in SEQ ID NO. 70, CDR2 shown in SEQ ID NO. 71, CDR3 shown in SEQ ID NO. 72;
CDR1 shown in SEQ ID NO. 73, CDR2 shown in SEQ ID NO. 74, CDR3 shown in SEQ ID NO. 75;
CDR1 shown in SEQ ID NO. 76, CDR2 shown in SEQ ID NO. 77, and CDR3 shown in SEQ ID NO. 78.
In another preferred embodiment, the VHH chain combination further comprises VHH chains having one or more of the following CDR regions:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6;
CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9;
CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15;
CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17, CDR3 shown in SEQ ID NO. 18;
CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, CDR3 shown in SEQ ID NO. 21;
CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23, CDR3 shown in SEQ ID NO. 24;
CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 27;
CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 30;
CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 32, CDR3 shown in SEQ ID NO. 33;
CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35, CDR3 shown in SEQ ID NO. 36;
CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38, CDR3 shown in SEQ ID NO. 39;
CDR1 shown in SEQ ID NO. 40, CDR2 shown in SEQ ID NO. 41, CDR3 shown in SEQ ID NO. 42;
CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44, CDR3 shown in SEQ ID NO. 45;
CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 50, CDR3 shown in SEQ ID NO. 51;
CDR1 shown in SEQ ID NO. 82, CDR2 shown in SEQ ID NO. 83, CDR3 shown in SEQ ID NO. 84;
CDR1 shown in SEQ ID NO. 85, CDR2 shown in SEQ ID NO. 86 and CDR3 shown in SEQ ID NO. 87.
In another preferred embodiment, the combination comprises at least the amino acid sequence of Nb 27; preferably, the combination further comprises one or more sequences selected from Nb16, 18, 20, 21, 22, 23, 24, 25, or 26; more preferably, the combination further comprises one or more sequences selected from Nb1-15, Nb17, Nb19, Nb28, or Nb 29.
In a fourteenth aspect of the invention, there is provided a kit comprising an anti-TIM 3 nanobody of the second aspect of the invention, a conjugate of the seventh aspect of the invention, and/or a conjugate of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows that the constructed phage library was spread on a plate by gradient dilution and the library capacity was determined by counting the number of growing single clones. The figure shows that 1/5 is taken for gradient dilution 10410 times of510 times of6Doubling the number of clones, counting the number of single clones and determining the library size to be 9.0 × 108CFU。
FIG. 2 shows that 24 clones were randomly picked from the constructed library and tested by colony PCR to evaluate the insertion rate of the constructed library. The DNA bands of the gel wells from left to right in the figure are: the first path is a DNA molecular marker, the rest pore paths are PCR products for detecting the insert, and the PCR product band is about 500 bp; the insertion rate of the library reaches 91.7% through detection.
Figure 3 is a TIM3 nanobody screening enrichment process. No enrichment occurred after the first round of panning, 7.8-fold enrichment occurred in the second round of panning, and 408-fold enrichment occurred in the third round of panning.
FIG. 4 shows the purification of 29 strains of TIM3 nanobodies expressed by E.coli. The nanobody corresponding to SEQ ID NO 88-116 amino acid sequence is SDS-PAGE electrophoresis image of TIM3 nanobody after nickel column resin gel affinity chromatography purification. The result shows that the purity of the TIM3 nano antibody can reach more than 90% through the purification process.
FIG. 5 is a graph showing the detection of TIM-3 stable transfected cell line, wherein A is the protein purification result of TIM-3 positive antibody (TSR-022 at the stage of clinical phase I study), and B is the flow detection result of TIM-3 stable transfected cell line.
FIG. 6 shows the result of affinity screening of the 29-strain nanobody. And (3) carrying out flow detection on the affinity of the 29-strain TIM-3 nano antibody to the TIM-3 antigen by taking PD-1, PD-L1 and CTLA-4 as negative controls.
Detailed Description
The inventor successfully obtains a group of anti-TIM 3 nano antibodies through extensive and intensive research and extensive screening, and experimental results show that the 29 strain TIM3(Nb1-29, respectively corresponding to SEQ ID NO. 88-116) nano antibodies obtained by the invention can be combined with TIM3 with high specificity and high affinity. The present invention has been completed based on this finding.
Specifically, the invention utilizes human TIM3 extracellular segment antigen protein to immunize camels to obtain a high-quality immune nano antibody gene library. Then, TIM3 protein molecules are coupled on an enzyme label plate to display the correct spatial structure of TIM3 protein, and the antigen in the form is used for screening an immune nano antibody gene library (camel heavy chain antibody phage display gene library) by using a phage display technology, so that the TIM3 specific nano antibody gene is obtained. Then the gene is transferred into escherichia coli, so that a nano antibody strain which can be efficiently expressed in the escherichia coli and has high specificity is obtained.
As used herein, the terms "nanobody of the invention", "anti-TIM 3 nanobody of the invention", "TIM 3 nanobody of the invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to TIM3 (including human TIM 3). Particularly preferred are nanobodies with the amino acid sequence of the VHH chain as shown in SEQ ID NO 88-116.
As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
As used herein, the terms "single domain antibody (sdAb, or VHH)", "nanobody" (nanobody) have the same meaning, referring to the cloning of the variable region of the antibody heavy chain, constructing a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Typically, single domain antibodies (VHHs) consisting of only one heavy chain variable region are constructed by first obtaining an antibody that naturally lacks the light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
The single domain antibody/nano antibody (Nanobody) is used as a novel small molecule antibody fragment and is obtained by cloning a camel natural heavy chain antibody heavy chain variable region (VHH). The Nanobody (Nb) has excellent biological characteristics, the molecular weight of 12-15kDa, which is one tenth of that of a complete antibody, good tissue penetration, high specificity and good water solubility. Due to the special structural properties, the antibody has the advantages of the traditional antibody and the micromolecule drug, almost perfectly overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody, gradually becomes a new force in the treatment of the new generation of antibody, and shows wide application prospect in immunodiagnosis and treatment.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which form the binding and specificity of each particular antibody for its particular antigen, however, the variability is not evenly distributed throughout the antibody variable region it is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions the more conserved portions of the variable regions are called Framework Regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, roughly in a β -fold configuration, connected by three CDRs forming a connecting loop, and in some cases may form a partial β fold structure.
As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention to form conjugates. The invention also includes cell surface markers or antigens that bind to the anti-TIM 3 protein antibodies or fragments thereof.
As used herein, the terms "heavy chain variable region" and "VH"may be used interchangeably.
As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and all refer to a polypeptide that specifically binds to the TIM3 protein, e.g., a protein or polypeptide having a heavy chain variable region. They may or may not contain the initial methionine.
The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of an antibody can be described by 3 specific regions located in the variable region of the heavy chain, called the variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction, the CDRs forming a loop structure, β folds formed by the FRs between them being spatially close to each other, the CDRs on the heavy chain and the corresponding CDRs on the light chain constituting the antigen binding site of the antibody.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the invention includes those molecules having an antibody heavy chain variable region with CDRs whose homology to the CDRs identified herein is greater than 90% (preferably greater than 95%, most preferably greater than 98%).
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
The antibody of the present invention refers to a polypeptide having TIM3 protein binding activity, including the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, fragments of the nanobodies of the invention are also encompassed by the present invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
TABLE 1
Initial residue(s) Representative substitutions Preferred substitutions
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 invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.
The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.
The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences, and the present invention particularly relates to polynucleotides which hybridize to the polynucleotides of the present invention under stringent conditions, where "stringent conditions" refer to (1) hybridization and elution at lower ionic strength and higher temperatures, e.g., 0.2 × SSC, 0.1% SDS, 60 ℃, or (2) hybridization with denaturing agents, e.g., 50% (v/v) formamide, 0.1% bovine serum/0.1% Ficoll, 42 ℃, etc., or (3) hybridization only if the identity between the two sequences is at least 90% or more, and more preferably 95% or more, and the polypeptides encoded by the hybridizable polynucleotides have the same biological function and activity as the mature polypeptides.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The sequences of the invention are shown in Table 2
TABLE 2
Figure BDA0001580153720000141
Figure BDA0001580153720000151
Figure BDA0001580153720000161
Figure BDA0001580153720000171
Figure BDA0001580153720000181
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. drug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. a therapeutic agent (e.g., cisplatin) or any form of nanoparticle, and the like.
VHH chain combinations
The invention also encompasses a combination of single domain antibody VHH chains. Wherein the combination comprises at least the following CDR regions:
CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80, and CDR3 shown in SEQ ID NO: 81.
Furthermore, the combination preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 CDR regions selected from the group or VHH chains selected from the group consisting of SEQ ID NO. 88-116.
Preferably, the first and second electrodes are formed of a metal,
the VHH chain combination also comprises a VHH chain with one or more CDR regions as follows:
CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47, CDR3 shown in SEQ ID NO. 48;
CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 53, CDR3 shown in SEQ ID NO. 54
CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 59, CDR3 shown in SEQ ID NO. 60;
CDR1 shown in SEQ ID NO. 61, CDR2 shown in SEQ ID NO. 62, CDR3 shown in SEQ ID NO. 63;
CDR1 shown in SEQ ID NO. 64, CDR2 shown in SEQ ID NO. 65, CDR3 shown in SEQ ID NO. 66;
CDR1 shown in SEQ ID NO. 67, CDR2 shown in SEQ ID NO. 68, CDR3 shown in SEQ ID NO. 69;
CDR1 shown in SEQ ID NO. 70, CDR2 shown in SEQ ID NO. 71, CDR3 shown in SEQ ID NO. 72;
CDR1 shown in SEQ ID NO. 73, CDR2 shown in SEQ ID NO. 74, CDR3 shown in SEQ ID NO. 75;
CDR1 shown in SEQ ID NO. 76, CDR2 shown in SEQ ID NO. 77, and CDR3 shown in SEQ ID NO. 78.
More preferably, the VHH chain combination further comprises a VHH chain having one or more of the following CDR regions:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6;
CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9;
CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15;
CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17, CDR3 shown in SEQ ID NO. 18;
CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, CDR3 shown in SEQ ID NO. 21;
CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23, CDR3 shown in SEQ ID NO. 24;
CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 27;
CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 30;
CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 32, CDR3 shown in SEQ ID NO. 33;
CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35, CDR3 shown in SEQ ID NO. 36;
CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38, CDR3 shown in SEQ ID NO. 39;
CDR1 shown in SEQ ID NO. 40, CDR2 shown in SEQ ID NO. 41, CDR3 shown in SEQ ID NO. 42;
CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44, CDR3 shown in SEQ ID NO. 45;
CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 50, CDR3 shown in SEQ ID NO. 51;
CDR1 shown in SEQ ID NO. 82, CDR2 shown in SEQ ID NO. 83, CDR3 shown in SEQ ID NO. 84;
CDR1 shown in SEQ ID NO. 85, CDR2 shown in SEQ ID NO. 86 and CDR3 shown in SEQ ID NO. 87.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the present invention can be used directly to bind TIM3 protein molecules and thus can be used to treat tumors. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the nanobody (or its conjugate) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Labeled nanobodies
In a preferred embodiment of the invention, the nanobody carries a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, coloured labels or fluorescent labels.
The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the nanobody against TIM3 is labeled with colloidal gold, resulting in a colloidal gold-labeled nanobody.
The anti-TIM 3 nano antibody has good specificity and high titer.
Detection method
The invention also relates to methods for detecting TIM3 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of TIM3 protein in the solubilized sample.
The sample used in the detection method of the present invention is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.
Reagent kit
The present invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
The invention also provides a detection kit for detecting the level of TIM3, which comprises an antibody for recognizing the TIM3 protein, a lysis medium for dissolving a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.
Applications of
As described above, the nanobody of the present invention has wide biological and clinical application values, and its applications relate to various fields such as diagnosis and treatment of diseases related to TIM3, research in basic medicine, and biological research. One preferred application is for clinical diagnosis and targeted therapy against TIM 3.
The main advantages of the invention include:
(a) the nano-antibody of the invention is highly specific for human TIM3 protein with correct spatial structure.
(b) The nano antibody has strong affinity.
(c) The production of the nano antibody is simple and convenient.
The nano antibody is suitable for prokaryotic expression and eukaryotic expression, and has the advantages of extremely high solubility, difficult aggregation, high temperature resistance, strong acid, strong alkali resistance and other denaturing conditions. Is suitable for laboratory and industrial development.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Example 1: expression purification of human TIM3 protein
(1) Synthesizing a nucleotide sequence of human TIM3(ECD) on a pFUSE-IgG1 vector, wherein a TEV enzyme cutting site is introduced at the C end of the TIM3(ECD) so as to prepare an Fc-tag-free TIM3(ECD) protein;
(2) extracting the constructed plasmid pF Mm SE-IgG1-TIM3(ECD) by using an Omega plasmid macroextraction kit;
(3) culturing HEK293F cells to OD 2.0 × 106Per mL;
(4) mixing the plasmid and the PEI 1:3 evenly, standing for 20min, adding into HEK293F cells at 37 ℃ and 6% CO2Culturing in a shaking incubator for 5-6 days;
(5) collecting cell supernatant, and combining with Protein A beads at room temperature for 1 h;
(6) after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M Glycine pH 3.0;
(7) ultrafiltering the eluted protein into PBS, sampling after determining yield, detecting the purity of the antigen by SDS-PAGE, and storing the rest proteins in a refrigerator at-80 ℃;
(8) selecting the antigen with the purity of more than 90 percent to carry out subsequent camel immunization.
Example 2: construction of TIM3 phage library
(1) Mixing 1mg of TIM3(ECD) -Fc antigen with Freund's adjuvant in equal volume, immunizing a Xinjiang bactrian camel once a week for 7 times, and stimulating B cells to express antigen-specific nano antibodies;
(2) after 7 times of immunization, extracting 100mL camel peripheral blood lymphocytes and extracting total RNA;
(3) synthesizing cDNA and amplifying VHH by using nested PCR;
(4) mu.g of pMECS phage display vector (supplied by Biovector) and 10. mu.g of VHH were digested with restriction enzymes Pst I and Not I and the two fragments were ligated;
(5) the ligation product is transformed into an electrotransformation competent cell TG1, a TIM3 nano antibody library is constructed, and the library capacity is determined, wherein the size of the library capacity is 9.0 × 108CFU (fig. 1);
meanwhile, 24 clones were randomly picked for colony PCR detection, and the insertion rate of the constructed library was 91.7% as shown in FIG. 2. The above results demonstrate that we obtained TIM3 nanobody phage display libraries that both qualified library capacity and insertion rate.
Example 3: screening and identification of TIM3 nano antibody
Antibody screening:
(1) the solution was dissolved in 100mM NaHCO 310 μ g of TIM3(ECD) -Fc antigen (10 μ g of Fc inNaHCO) at pH 8.23As a control) were coupled to NUNC enzyme plates and left overnight at 4 ℃;
(2) adding 100 μ L of 0.1% BSA the next day, blocking for 2h at room temperature;
(3) after 2h, 100. mu.L phage (2 × 10) was added11CFU immune camel nanometer antibody phage display gene library) and acting for 1h at room temperature;
(4) washing 5 times with 0.05% PBS + Tween-20 to wash away non-specific phage;
(5) phage specifically bound to TIM3 were dissociated with 100mM triethanolamine and infected with e.coli TG1 cells grown in log phase, incubated at 37 ℃ for 1h, phage generated and purified for the next round of screening, and the same screening procedure was repeated for 3 rounds with enrichment fold of no enrichment, 7.8 fold and 408 fold, respectively (fig. 3).
Screening of specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA):
(1) phage-containing cell culture after the 3-round selection600 single colonies were picked and inoculated in TB medium containing 100. mu.g/mL ampicillin (2.3 g KH in 1L TB medium)2PO4，12.52g K2HPO412g peptone, 24g yeast extract, 4mL glycerol), after growth to logarithmic phase, adding 1mM final concentration of IPTG and culturing overnight at 28 ℃;
(2) obtaining a crude antibody by using an osmosis method, transferring the antibody into an ELISA plate coated by an antigen, and standing for 1h at room temperature;
(3) washing away unbound antibody with PBST, adding mouse anti-HA antibody purchased from Beijing kang, century Biotechnology Co., Ltd.), and standing at room temperature for 1 h;
(4) washing away the unbound antibody by PBST, adding goat anti-mouse alkaline phosphatase labeled antibody, and standing at room temperature for 1 h;
(5) washing away the unbound antibody by PBST, adding an alkaline phosphatase developing solution, and reading an absorption value at a wavelength of 405nm of an ELISA instrument;
(6) when the OD value of the sample well is more than 3 times larger than that of the control well (Ratio +/- > 3), the sample well is judged to be a positive clone well. The result shows that 86 positive clones appear in 600 clones;
(7) the 86 positive clonal well of bacteria were shaken in LB liquid containing 2mL of ampicillin at a concentration of 100. mu.g/mL to extract plasmids and to sequence;
(8) comparing and analyzing 86 strain clone series, and finally obtaining 29 strain sequence difference nanobodies which are respectively numbered as Nb1-Nb29(SEQ ID NO. 88-116), wherein 16 types of nanometer antibodies with obviously different CDR3 regions are provided, 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 NO.:98-100) is family 3, Nb14, Nb15(SEQ ID NO.:101, 102) is family 4, Nb16-Nb29(SEQ ID NO.:103-116) is family 5-family 16.
Example 4: expression and purification of nano antibody in host escherichia coli
(1) Inoculating 29 strains of TIM3 specific clones obtained by sequencing analysis into a 5mL LA culture set for amplification culture;
(2) extracting corresponding plasmids, and performing electrotransformation on 1uL of the plasmids and 20uL of electrotransformation competence WK 6;
(3) plating the cells after the electric transfer on a plate containing ampicillin and glucose, and culturing overnight at 37 ℃;
(4) selecting single colony, inoculating in 5mL LB culture solution containing ampicillin, shaking at 37 deg.C
Culturing overnight; (ii) a
(5)3) inoculating 1mL of overnight strain into 330mL of TB culture solution, carrying out shake culture at 37 ℃, adding IPTG (isopropyl-beta-thiogalactoside) when the OD value reaches 0.6-1, and carrying out shake culture at 28 ℃ overnight;
(6) the thalli is collected by centrifugation, the thalli is subjected to hypertonic lysis to obtain antibody crude extract, then the antibody protein is purified by using a nickel ion affinity chromatography column, and the prokaryotic expression and purification result of the TIM3 nano antibody is shown in figure 4.
Example 5: TIM-3 Nanobody detection specificity analysis
5.1 construction of TIM-3 Stable transgenic cell line
(1) The TIM-3 gene sequence is amplified and then inserted into a pLVX-EF1 α -puro vector to construct a pLVX-EF1 α -puro-TIM-3 lentiviral plasmid.
(2) The TIM-3 stable cell line was established by passaging HEK293T cells in a 10cm diameter petri dish containing 5mL of DMEM complete medium, mixing 250uL Opti-MEM medium (Invitrogen) with 3ug of packaged plasmid (pLP 1: pLP 2: VSVG 1:1:1), 1ug of lentiviral plasmid, adding 20uL of Polyfect transfection reagent (QIAGEN), mixing thoroughly, standing at room temperature for 10min, adding to the petri dish containing HEK293T cells, culturing for 2 days in a 5% CO2 incubator at 37 deg.C, collecting the supernatant in a 50mL centrifuge tube, exchanging 10mL of fresh DMEM medium in the petri dish, culturing for 1 day, collecting the supernatant in a 50mL centrifuge tube for one day before, centrifuging at 4000rpm for 5min, filtering the supernatant with a 0.45um filter, adding 5. 5 × lentiviral concentrate, mixing thoroughly, placing in a 4 deg.C centrifuge tube, collecting supernatant after successful centrifugation at 4g every second day, selecting and re-inoculating the supernatant, and re-inoculating the My cell line, and re-culturing the strain with the My cell line, and re-cultured with 5mL DMEM medium, and re-separating the supernatant, and re-separating the strain, and re-cultured for 2mL of the strain.
5.2 flow cytometry for detection of TIM-3 Stable transgenic cell line
(1) Based on the sequence of TIM-3 antibody (TSR-022, U.S. Pat. No. 3, 20150218274, 1) in the clinical phase I study stage, its heavy and light chains were synthesized and constructed into pFUSE-hIgG1e4-Fc1 vectors, respectively. (2) Further using HEK293F eukaryotic expression system and protein A purification system to obtain TIM-3 positive antibody (BMK) for identification of TIM-3 stable transfer cell strain.
(2) In flow cytometry, 1.5 × 106The HEK293T TIM-3 stably transfected cells were divided into 3 centrifuge tubes, centrifuged at 3000rpm at 4 ℃ for 4min, the supernatant was discarded, and the pellet was resuspended in 100uL of precooled PBS, 5 × 10/105Cells were added with 6ug of antibody (TIM-3 positive antibody BMK or isotype control antibody AF70-Fc was added to the experimental group, no antibody was added to the blank group), mixed well and incubated at 4 ℃ for 20 min. 500uL PBS was washed once, the pellet was resuspended in 200uL PBS, 1uL of coat-anti-human (FITC) was added to each tube, mixed and incubated at 4 ℃ for 20 min. The supernatant was discarded after one wash with pre-cooled PBS, the pellet was resuspended in 200uL of pre-cooled PBS, and the proportion of TIM-3 positive cells was determined by flow cytometry (BD Co., USA). The results of the experiment are shown in FIG. 5. FIG. 5A shows the successful acquisition of high purity TIM-3BMK antibody, and FIG. 5B shows that the positive cell proportion of TIM-3 stable cell line reaches 98.1%.
5.3 screening of TIM-3 Nanobodies with high specificity and high affinity
(1) 1.65 × 107Individual HEK293T TIM-3 stable cells (HEK 293T as negative cell control) were resuspended in ice-cold PBS and transferred to 33 wells of U-shaped 96-well plates, 100uL cell samples per well.
(2) 6ug of antibody (29 strain TIM-3 nanometer antibody in experimental group, PD-1, PD-L1 or CTLA-4 nanometer antibody in isotype control, no antibody in blank group) was added into each well, mixed, and incubated at 4 deg.C for 20 min.
(3) After one PBS wash, the suspension was resuspended in 100uL of antibody dilution containing the second antibody mouse-anti-His (Alexa Fluor 488), mixed and incubated at 4 ℃ for 20 min.
(4) After washing once with PBS, the suspension was resuspended in 200uL PBS, and the ratio of positive cells bound by the nanobody to the TIM-3 on the surface of the stabilized cells was determined by flow cytometry. The results of the experiment are shown in FIG. 6. The results of the study using HEK293T as negative cell control showed that 29 TIM-3 nanobodies did not bind HEK293T (not shown), and 12 did bind to TIM-3 stable transformant, wherein 10 TIM-3 nanobodies bound more than 80% to TIM-3 on the surface of stable transformant, while isotype negative control antibodies (PD-1, PD-L1 and CTLA-4 nanobodies) did not bind to TIM-3 on the surface of stable transformant, and the experiments showed that 10 of the 29 nanobodies obtained by panning showed very high specificity and affinity for TIM-3 antigen, and their nanobodies were numbered Nb16, Nb18, Nb20, Nb21SEQ ID NO. 108, Nb22SEQ ID NO. 109, Nb23, Nb24, Nb25, Nb26, Nb27. corresponding to the sequence of SEQ ID NO. 103, SEQ ID NO. 105, SEQ ID NO. 107, SEQ ID NO. 110, 111, 112, 113, 114) with higher binding affinity of the remaining sequences.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
SEQUENCE LISTING
<110> Shanghai Luoqi biomedical technology, Inc
<120> Single Domain antibody against T lymphocyte immunoglobulin mucin3
<130>P2018-0335
<160>145
<170>PatentIn version 3.5
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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 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 AlaCys 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
1 5
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Lys Ile Gln Cys Gly Thr Gln Val Asn
1 5
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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
1 5
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Lys Ile Gln Cys Gly Thr Gln Val Asn
1 5
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Arg Ser Thr Tyr Cys Met Gly
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Ile Ser Ser Asp Gly Arg Thr
1 5
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Ala Thr Arg Pro Gly Asn Ser Cys Gly Thr Gly Ile Asp Met Pro Tyr
1 5 10 15
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Thr Thr Leu Tyr Ile Ala Ser Leu Gly
1 5
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Val Asp Arg Asp Gly Asn Leu
1 5
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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
20
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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
1 5
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Ala Ala Asp Trp Asn Asn Asp Arg Ser Cys Pro Leu Trp Ala Asp Gly
1 5 10 15
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
1 5
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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
1 5
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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
1 5
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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
1 5
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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
1 5
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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
1 5
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Ala Ala Asp Ser Ala Arg Cys Gly Leu Trp Leu Gly Gly Gly Tyr Pro
1 5 10 15
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
1 5
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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
1 5
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Ala Lys Asp Val Tyr Cys Gly Gly Gln Tyr Cys Pro Pro
1 5 10
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Lys Ala Leu Val Ile Ser Cys Leu Ala
1 5
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Ile Gly Arg Asp Gly Ser Thr
1 5
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Ala Ala Ala Thr Lys Gln Asp Gly Ile Pro Leu Asn Pro Ala Asp Tyr
1 5 10 15
Asp Ile
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Gly Thr Thr Ser Arg Asn Tyr Cys Met Gly
1 5 10
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Ile Asp Lys Tyr Gly Thr Ser
1 5
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Ala Ile Ser Ser Gln Tyr Gly Leu Cys Leu Ala Gln Thr Gly Asp Tyr
1 5 10 15
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
115
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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 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
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 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 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 7580
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
115
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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 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
115
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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 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
115
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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 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>94
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<400>94
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 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
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<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
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<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
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<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
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<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 2530
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
3540 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 TyrTrp 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 LeuArg 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 AlaAsp 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 7580
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 (32)

1. A T lymphocyte immunoglobulin mucin 3(TIM3) -specific nanobody, which is capable of specifically binding to TIM3, and in which 3 CDR regions are respectively as follows:
CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80, and CDR3 shown in SEQ ID NO: 81.
2. The nanobody of claim 1, wherein the VHH chain of the nanobody further comprises a framework region FR.
3. A nanobody combination specifically binding to TIM3, comprising at least 3 CDR regions of nanobodies respectively as follows:
CDR1 shown in SEQ ID NO:79, CDR2 shown in SEQ ID NO:80, and CDR3 shown in SEQ ID NO: 81.
4. The combination of claim 3, wherein the nanobody combination further comprises one or more nanobodies having 3 CDR regions as follows:
CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47, CDR3 shown in SEQ ID NO. 48;
CDR1 shown in SEQ ID NO. 52, CDR2 shown in SEQ ID NO. 53, CDR3 shown in SEQ ID NO. 54
CDR1 shown in SEQ ID NO. 58, CDR2 shown in SEQ ID NO. 59, CDR3 shown in SEQ ID NO. 60;
CDR1 shown in SEQ ID NO. 61, CDR2 shown in SEQ ID NO. 62, CDR3 shown in SEQ ID NO. 63;
CDR1 shown in SEQ ID NO. 64, CDR2 shown in SEQ ID NO. 65, CDR3 shown in SEQ ID NO. 66;
CDR1 shown in SEQ ID NO. 67, CDR2 shown in SEQ ID NO. 68, CDR3 shown in SEQ ID NO. 69;
CDR1 shown in SEQ ID NO. 70, CDR2 shown in SEQ ID NO. 71, CDR3 shown in SEQ ID NO. 72;
CDR1 shown in SEQ ID NO. 73, CDR2 shown in SEQ ID NO. 74, CDR3 shown in SEQ ID NO. 75;
CDR1 shown in SEQ ID NO. 76, CDR2 shown in SEQ ID NO. 77, and CDR3 shown in SEQ ID NO. 78.
5. The combination of claim 4, wherein the nanobody combination further comprises one or more nanobodies having 3 CDR regions as follows:
CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5, CDR3 shown in SEQ ID NO. 6;
CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8, CDR3 shown in SEQ ID NO. 9;
CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 15;
CDR1 shown in SEQ ID NO. 16, CDR2 shown in SEQ ID NO. 17, CDR3 shown in SEQ ID NO. 18;
CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, CDR3 shown in SEQ ID NO. 21;
CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 23, CDR3 shown in SEQ ID NO. 24;
CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 27;
CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 30;
CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 32, CDR3 shown in SEQ ID NO. 33;
CDR1 shown in SEQ ID NO. 34, CDR2 shown in SEQ ID NO. 35, CDR3 shown in SEQ ID NO. 36;
CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38, CDR3 shown in SEQ ID NO. 39;
CDR1 shown in SEQ ID NO. 40, CDR2 shown in SEQ ID NO. 41, CDR3 shown in SEQ ID NO. 42;
CDR1 shown in SEQ ID NO. 43, CDR2 shown in SEQ ID NO. 44, CDR3 shown in SEQ ID NO. 45;
CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 50, CDR3 shown in SEQ ID NO. 51;
CDR1 shown in SEQ ID NO. 82, CDR2 shown in SEQ ID NO. 83, CDR3 shown in SEQ ID NO. 84;
CDR1 shown in SEQ ID NO. 85, CDR2 shown in SEQ ID NO. 86 and CDR3 shown in SEQ ID NO. 87.
6. The combination of claim 3, wherein the combination comprises at least the nanobody having an amino acid sequence set forth in SEQ ID NO. 114.
7. The combination of claim 6, further comprising one or more nanobodies having the amino acid sequence set forth in SEQ ID NOS 103, 105, 107, 108, 109, 110, 111, 112 and 113.
8. The combination of claim 6, further comprising one or more nanobodies having the amino acid sequence set forth in SEQ ID Nos. 88-102, 104, 106, 115 and 116.
9. An isolated polynucleotide encoding a protein selected from the group consisting of: the nanobody of claim 1 or the combination of claim 3.
10. The polynucleotide according to claim 9, wherein the sequence of the polynucleotide comprises one or more of SEQ ID NO 117 and 145.
11. An expression vector comprising the polynucleotide of any one of claims 9-10.
12. A host cell comprising the expression vector of claim 11, or having the polynucleotide of claim 9 integrated into its genome.
13. The host cell of claim 12, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell.
14. The host cell of claim 12, wherein the host cell is selected from the group consisting of: escherichia coli, yeast cells.
15. A method of producing an anti-TIM 3 nanobody, comprising the steps of:
(a) culturing the host cell of claim 12 under conditions suitable for nanobody production, thereby obtaining a culture comprising the anti-TIM 3 nanobody; and
(b) isolating or recovering the anti-TIM 3 nanobody from the culture.
16. The method of claim 15, further comprising the step of:
(c) purifying and/or modifying the TIM3 nanobody obtained in step (b).
17. A conjugate, comprising:
(a) the nanobody of claim 1; and operatively connected
(b) A modifying label selected from the group consisting of: chemical markers and biological markers.
18. The conjugate of claim 17, wherein the chemical label is an isotope, an immunotoxin, and/or a chemical drug.
19. The conjugate of claim 17, wherein the biomarker is a biotin, avidin, or enzyme label.
20. A conjugate prepared by conjugating the nanobody of claim 1 or the conjugate of claim 17 to a solid or semi-solid medium.
21. The conjugate of claim 20, wherein the conjugate is comprised of the conjugate of claim 17 and a coupling element selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI or CT contrast agents, gold nanoparticles/nanorods, liposomes, nanomagnets or chemotherapeutic agents.
22. The conjugate of claim 21, wherein the radioactive label is a radionuclide.
23. The conjugate of claim 21, wherein the chemotherapeutic agent is cisplatin.
24. The conjugate of claim 20, wherein the conjugate comprises: a multivalent nanobody according to claim 1.
25. The conjugate of claim 24, wherein the multivalent is divalent.
26. The conjugate of claim 24, wherein multivalent is the TIM3 nanobody of claim 1 comprising multiple repeats in the amino acid sequence of the immunoconjugate.
27. A pharmaceutical composition comprising:
(i) the nanobody of claim 1, or the combination of claim 3; and
(ii) a pharmaceutically acceptable carrier.
28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is in an injectable dosage form.
29. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is for use in the preparation of a medicament for treating a tumor selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, lymph cancer, adrenal gland tumor, or bladder tumor.
30. Use of a nanobody according to claim 1, or a conjugate according to claim 20, for the preparation of a medicament, a reagent, a detection plate or a kit;
wherein the reagent, assay plate or kit is for: detecting TIM3 protein in a sample;
wherein the agent is for use in the treatment or prevention of a TIM 3-expressing tumor;
wherein the TIM 3-expressing tumor is selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, lymph cancer, adrenal tumor, bladder tumor, or a combination thereof.
31. The use of claim 30, wherein said detection comprises flow detection, cellular immunofluorescence detection.
32. A kit comprising a nanobody according to claim 1, a conjugate according to claim 17 and/or a conjugate according to claim 20.
CN201810152107.6A 2018-02-14 2018-02-14 Single domain antibodies against T lymphocyte immunoglobulin mucin3 Active CN110144011B (en)

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CN112094348B (en) * 2020-09-22 2021-11-30 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
CN114853888B (en) 2021-02-05 2023-11-03 上海洛启生物医药技术有限公司 anti-TSLP nanobody and application thereof
CN113831411B (en) * 2021-09-18 2023-05-02 南京融捷康生物科技有限公司 Single-domain antibody for L1CAM and derived protein and application thereof
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