CN111848798B - Nanometer antibody capable of combining BCMA and application thereof - Google Patents

Nanometer antibody capable of combining BCMA and application thereof Download PDF

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CN111848798B
CN111848798B CN202010732288.7A CN202010732288A CN111848798B CN 111848798 B CN111848798 B CN 111848798B CN 202010732288 A CN202010732288 A CN 202010732288A CN 111848798 B CN111848798 B CN 111848798B
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CN111848798A (en
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吴喜林
吴稚伟
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Y Clone Medical Science Co ltd
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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    • 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]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95

Abstract

The invention relates to a polypeptide capable of binding to human BCMA, which comprises 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NO. 1-104, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 105-207, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 208-311. BCMA is B cell mature antigen, the invention carries out nano antibody drug development and diagnostic reagent development aiming at B cell lymphoma, nano antibody VHH which is specifically combined with BCMA is screened by preparing BCMA protein, immune alpaca, utilizing a platform technology of phage library to display nano monoclonal antibody and the like, CDR sequence of the nano antibody VHH is identified, and humanized antibody BANB is constructed; and simultaneously, identifying the combination of the humanized antibody and the cell surface BCMA protein by using a flow cytometry detection method. The invention also provides a potential new nano-antibody medicament for clinical treatment of B cell lymphoma and a corresponding gene therapy method.

Description

Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
Technical Field
The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding human BCMA, and the application of said polypeptide in preparation of BCMA detection agent or B cell lymphoma therapeutic medicine.
Background
BCMA is a B cell maturation antigen, a transmembrane glycoprotein, also known as CD269 or TNFRSF17, and one of the TNF receptor superfamily members, whose ligands are B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA expression is restricted to the B cell lineage, occurring primarily on plasma cells and plasmablasts, and studies have shown that BCMA mRNA and protein is expressed much higher on malignant plasma cells than on normal plasma cells, but not on naive B cells and memory B cells and other normal tissue cells. BAFFF and APRIL promote the survival of malignant plasma cells through BCMA signaling. In addition, BCMA antibodies have been detected in multiple myeloma patients that remit after donor lymphocyte infusion and graft versus tumor reactions, and the results of these studies indicate that BCMA is a highly promising antigen for targeted therapy of B-cell lymphomas.
In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH2 and CH3), a hinge region and a heavy chain Variable region (VHH, i.e., antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of conventional antibodies, and has a molecular height and diameter at the nanometer level, which is the smallest functional antibody fragment currently available, and thus is also referred to as Nanobody (Nb). Because the nano monoclonal antibody has the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80 percent with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is widely applied to the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.
The development trend of therapeutic antibody drugs is from murine, human-murine chimeric, humanized to fully human, to recently focused nano-antibodies, and the development of nano-antibodies is rapidly progressing. The first nano-antibody drug was approved in 2018, 9 months. At present, a plurality of nano antibody medicines are in clinical research stage at home and abroad. We expect that by immunizing alpaca, high affinity therapeutic nanobodies targeting BCMA are obtained.
Disclosure of Invention
The invention obtains the alpaca source nano monoclonal antibody and VHH thereof by immunizing alpaca with antigen, and is used for diagnosing and treating B cell lymphoma patients. Based on these studies, the present invention provides a polypeptide that binds to BCMA, comprising 3 complementarity determining regions CDR1-3, CDR1 sequence is or comprises one of the sequences shown in SEQ ID NO:1-104, CDR2 sequence is or comprises one of the sequences shown in SEQ ID NO:105-207, CDR3 sequence is or comprises one of the sequences shown in SEQ ID NO: 208-311.
In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, wherein the FR1-4 is staggered from the CDR 1-3. For example, the FR1-4 sequence may be designed as shown in SEQ ID NO:312-315 (sources of alpaca), although the scope of the invention is not limited in this respect. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences have little influence and can be designed according to species, which is well known in the art. For example, FR region sequences of human, murine or alpaca origin can be designed to link the above CDRs, resulting in a polypeptide or domain that binds human BCMA. For example, the FR1-4 sequence can be designed as shown in SEQ ID NO:316-319 (humanized), and the humanized polypeptide has equivalent binding activity.
In a preferred embodiment, the polypeptide is a monoclonal antibody.
In a preferred embodiment, the polypeptide is VHH.
In a preferred embodiment, the polypeptide is a VHH of alpaca origin or a humanized VHH.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the CDR1 has the sequence GrieXTYV, and X at position 5 represents asparagine or serine;
II) the sequence of CDR2 is shown in one of SEQ ID NO 153-156 and
III) the sequence of CD3 is shown in one of SEQ ID NO 255, 257-260.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 50, the sequence of CDR2 is SEQ ID NO 153, and the sequence of CDR3 is SEQ ID NO 257; or
The sequence of CDR1 is SEQ ID NO:51, the sequence of CDR2 is SEQ ID NO:154, and the sequence of CDR3 is SEQ ID NO: 258; or
CDR1 has the sequence of SEQ ID NO 51, CDR2 has the sequence of SEQ ID NO 155, and CDR3 has the sequence of SEQ ID NO 259; or
The sequence of CDR1 is SEQ ID NO:51, the sequence of CDR2 is SEQ ID NO:156, and the sequence of CDR3 is SEQ ID NO: 255; or
The sequence of CDR1 is SEQ ID NO 51, the sequence of CDR2 is SEQ ID NO 153, and the sequence of CDR3 is SEQ ID NO 255; or
The sequence of CDR1 is SEQ ID NO:51, the sequence of CDR2 is SEQ ID NO:153, and the sequence of CDR3 is SEQ ID NO: 260.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CDR1 is GRTFSSYX, X at position 7 represents arginine, serine, tyrosine, or valine;
II) the sequence of CDR2 is shown in one of SEQ ID NO:167-170, and
III) the sequence of CD3 is shown in one of SEQ ID NO: 271-274.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 63, the sequence of CDR2 is SEQ ID NO 167, and the sequence of CDR3 is SEQ ID NO 271; or
The sequence of CDR1 is SEQ ID NO 64, the sequence of CDR2 is SEQ ID NO 168, and the sequence of CDR3 is SEQ ID NO 272; or
The sequence of CDR1 is SEQ ID NO 65, the sequence of CDR2 is SEQ ID NO 169 and the sequence of CDR3 is SEQ ID NO 273; or
The sequence of CDR1 is SEQ ID NO:66, the sequence of CDR2 is SEQ ID NO:170, and the sequence of CDR3 is SEQ ID NO: 274.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CDR1 is shown in 28 or 31;
II) the sequence of CDR2 is shown in SEQ ID NO:132, and
III) the sequence of CD3 is shown in SEQ ID NO:234 or 235.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 28, the sequence of CDR2 is SEQ ID NO 132, and the sequence of CDR3 is SEQ ID NO 234; or
The sequence of CDR1 is SEQ ID NO 28, the sequence of CDR2 is SEQ ID NO 132, and the sequence of CDR3 is SEQ ID NO 235; or
The sequence of CDR1 is SEQ ID NO. 31, the sequence of CDR2 is SEQ ID NO. 132, and the sequence of CDR3 is SEQ ID NO. 234.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO. 49, the sequence of CDR2 is SEQ ID NO. 152, and the sequence of CDR3 is SEQ ID NO. 256; or
The sequence of CDR1 is SEQ ID NO 52, the sequence of CDR2 is SEQ ID NO 152, and the sequence of CDR3 is SEQ ID NO 256.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO:62, the sequence of CDR2 is SEQ ID NO:155, and the sequence of CDR3 is SEQ ID NO: 270; or
The sequence of CDR1 is SEQ ID NO:74, the sequence of CDR2 is SEQ ID NO:177, and the sequence of CDR3 is SEQ ID NO: 270.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 68, the sequence of CDR2 is SEQ ID NO 172, and the sequence of CDR3 is SEQ ID NO 220; or
The sequence of CDR1 is SEQ ID NO 13, the sequence of CDR2 is SEQ ID NO 117, and the sequence of CDR3 is SEQ ID NO 220.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO:39, the sequence of CDR2 is SEQ ID NO:142, and the sequence of CDR3 is SEQ ID NO: 245; or
The sequence of CDR1 is SEQ ID NO:39, the sequence of CDR2 is SEQ ID NO:142, and the sequence of CDR3 is SEQ ID NO: 246.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 57, the sequence of CDR2 is SEQ ID NO 161, and the sequence of CDR3 is SEQ ID NO 265; or
The sequence of CDR1 is SEQ ID NO 57, the sequence of CDR2 is SEQ ID NO 162, and the sequence of CDR3 is SEQ ID NO 265.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CDR3 is HLYGAGGXLDA, wherein, X in 8 th represents isoleucine or arginine;
II) the sequence of CDR1 is shown in SEQ ID NO 98 or 100, and
III) the sequence of CD2 is shown in SEQ ID NO 201, 203 or 204.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 98, the sequence of CDR2 is SEQ ID NO 201, and the sequence of CDR3 is SEQ ID NO 304; or
The sequence of CDR1 is SEQ ID NO 100, the sequence of CDR2 is SEQ ID NO 203, and the sequence of CDR3 is SEQ ID NO 307; or
The sequence of CDR1 is SEQ ID NO 100, the sequence of CDR2 is SEQ ID NO 204, and the sequence of CDR3 is SEQ ID NO 307.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 99, the sequence of CDR2 is SEQ ID NO 202, and the sequence of CDR3 is SEQ ID NO 305; or
The sequence of CDR1 is SEQ ID NO 99, the sequence of CDR2 is SEQ ID NO 202, and the sequence of CDR3 is SEQ ID NO 306.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 10, the sequence of CDR2 is SEQ ID NO 114, and the sequence of CDR3 is SEQ ID NO 217; or
The sequence of CDR1 is SEQ ID NO. 27, the sequence of CDR2 is SEQ ID NO. 131, and the sequence of CDR3 is SEQ ID NO. 217.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 71, the sequence of CDR2 is SEQ ID NO 137, and the sequence of CDR3 is SEQ ID NO 278; or
The sequence of CDR1 is SEQ ID NO:34, the sequence of CDR2 is SEQ ID NO:137, and the sequence of CDR3 is SEQ ID NO: 240.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CDR2 is ITSDGXT, where X at position 6 represents serine, valine, or threonine;
II) the sequence of CDR1 is shown in SEQ ID NO 36, 70 or 94, and
III) the sequence of CD3 is shown in SEQ ID NO:242, 277 or 300.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO:36, the sequence of CDR2 is SEQ ID NO:139, and the sequence of CDR3 is SEQ ID NO: 242; or
The sequence of CDR1 is SEQ ID NO 70, the sequence of CDR2 is SEQ ID NO 174, and the sequence of CDR3 is SEQ ID NO 277; or
The sequence of CDR1 is SEQ ID NO:94, the sequence of CDR2 is SEQ ID NO:197, and the sequence of CDR3 is SEQ ID NO: 300.
In another specific embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CDR2 is ITSGGXT, where X at position 6 represents serine, aspartic acid, or threonine;
II) the sequence of CDR1 is shown in SEQ ID NO 83, 87 or 104, and
III) the sequence of CD3 is shown in SEQ ID NO 289, 293 or 311.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 83, the sequence of CDR2 is SEQ ID NO 186, and the sequence of CDR3 is SEQ ID NO 289; or
The sequence of CDR1 is SEQ ID NO 87, the sequence of CDR2 is SEQ ID NO 190, and the sequence of CDR3 is SEQ ID NO 293; or
The sequence of CDR1 is SEQ ID NO 104, the sequence of CDR2 is SEQ ID NO 207, and the sequence of CDR3 is SEQ ID NO 311.
The invention also provides application of the polypeptide in preparing a B cell lymphoma treatment drug.
The present invention also provides the nucleic acid encoding sequence of the polypeptide.
In one embodiment, the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.
In a specific embodiment, the nucleic acid coding sequence is present in a gene expression cassette.
The invention also provides an expression vector containing the expression cassette of the nucleic acid coding sequence.
In a preferred embodiment, the expression vector is a viral vector.
In a preferred embodiment, the expression vector is an adeno-associated viral expression vector (AAV vector).
The invention also provides the application of the nucleic acid coding sequence and the expression vector in a B cell lymphoma treatment drug.
The invention also provides application of the polypeptide in preparing a BCMA detection agent. Can be used for detecting the specific expression condition of BCMA on the surface of cells.
In a specific embodiment, the detection agent is a cell flow detection agent.
In a preferred embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 17, the sequence of CDR2 is SEQ ID NO 121, and the sequence of CDR3 is SEQ ID NO 224; or
The sequence of CDR1 is SEQ ID NO 28, the sequence of CDR2 is SEQ ID NO 132, and the sequence of CDR3 is SEQ ID NO 234; or
The sequence of CDR1 is SEQ ID NO 28, the sequence of CDR2 is SEQ ID NO 132, and the sequence of CDR3 is SEQ ID NO 235; or
The sequence of CDR1 is SEQ ID NO. 31, the sequence of CDR2 is SEQ ID NO. 132, and the sequence of CDR3 is SEQ ID NO. 234; or
The sequence of CDR1 is SEQ ID NO 33, the sequence of CDR2 is SEQ ID NO 136, and the sequence of CDR3 is SEQ ID NO 239; or
The sequence of CDR1 is SEQ ID NO 42, the sequence of CDR2 is SEQ ID NO 145, and the sequence of CDR3 is SEQ ID NO 249; or
The sequence of CDR1 is SEQ ID NO 51, the sequence of CDR2 is SEQ ID NO 153, and the sequence of CDR3 is SEQ ID NO 255; or
The sequence of CDR1 is SEQ ID NO 51, the sequence of CDR2 is SEQ ID NO 153, and the sequence of CDR3 is SEQ ID NO 260; or
The sequence of CDR1 is SEQ ID NO 53, the sequence of CDR2 is SEQ ID NO 157, and the sequence of CDR3 is SEQ ID NO 261; or
The sequence of CDR1 is SEQ ID NO 57, the sequence of CDR2 is SEQ ID NO 162, and the sequence of CDR3 is SEQ ID NO 265; or
The sequence of CDR1 is SEQ ID NO 77, the sequence of CDR2 is SEQ ID NO 180, and the sequence of CDR3 is SEQ ID NO 283; or
The sequence of CDR1 is SEQ ID NO:78, the sequence of CDR2 is SEQ ID NO:181, and the sequence of CDR3 is SEQ ID NO: 284; or
The sequence of CDR1 is SEQ ID NO 96, the sequence of CDR2 is SEQ ID NO 199, and the sequence of CDR3 is SEQ ID NO 302.
The invention carries out nano antibody drug development and diagnostic reagent development aiming at B cell lymphoma therapeutic drugs, and selects the nano antibody VHH specifically combined with sBCMA by preparing sBCMA protein, immune alpaca, platform technology utilizing phage library to display nano monoclonal antibody and the like, identifies the CDR sequence thereof, and constructs humanized VHH-huFc1 (BANB); the binding of the humanized antibody to cell surface BCMA protein was also assessed using flow cytometry. The invention provides a potential nano-antibody new drug for clinical treatment of B cell lymphoma, and provides a corresponding detection reagent for diagnosis of patients with B cell lymphoma.
Drawings
FIG. 1 is a graph of the antiserum titer test one week after 3 rd and 4th immunizations of alpaca with sBCMA;
FIG. 2 is an electrophoretogram of PCR products amplified using a sBCMA-VHH phage antibody library as a template;
FIG. 3 is the panning identification of a sBCMA-VHH phage antibody library, wherein A is a statistical plot of ELISA detection after phage library panning against BCMA protein; b is the second wheel (2)nd) The third wheel (3)rd) And a fourth wheel (4)th) Respectively selecting 48, 46 and 47 clones from the panned phage antibody library to carry out phage ELISA detection statistical chart;
FIG. 4 is a statistical chart of ELISA detection of prokaryotically expressed VHH antibodies, each dot representing a clone, with the ordinate being OD450 for BCMA/OD 450 for blank, a positive being defined by a ratio greater than 2.5;
FIGS. 5 and 6 are graphs of the flow results of binding of eukaryotic expression supernatant of VHH-hfc1 to cell surface expressed membrane protein BCMA. The abscissa is the fluorescence signal value of anti BCMA VHH-hfc1 bound to membrane protein BCMA, and the ordinate is the fluorescence signal value of commercial BCMA antibody bound to membrane protein BCMA.
Detailed Description
1. Preparation of immunogens
According to the information of human BCMA protein sequence and gene sequence on NCBI website, polypeptide sBCMA capable of effectively inducing alpaca to generate specific antibody aiming at BCMA protein is analyzed and designed, and His-tag (sBCMA-His) or rabbit Fc (sBCMA-rFc) is connected to C terminal for subsequent purification and detection.
2. Preparation of alpaca immune and antiserum
Priming alpacas with an emulsified mixture of 250 mu g of sBCMA-rFc protein and 250 mu l of Freund's complete adjuvant, boosting the alpacas with the sBCMA-rFc protein and 250 mu l of Freund's incomplete adjuvant 3 times on 14 days, 28 days and 42 days, and collecting blood to detect the antiserum titer 1 week after the 3 rd and 4th immunization; after 1 week of 4th immunization, 100ml of blood was collected for phage antibody library construction.
The antiserum titer is detected by ELISA, BCMA protein with the concentration of 0.5 mug/ml is used for coating a detection plate, antiserum diluted in a gradient manner or purified antibody 100 mug (the contrast is alpaca serum before immunization) is added into each hole, the incubation is carried out for 1.5h at the temperature of 37 ℃, the washing is carried out for 2 times,add 1: 10000 diluted second antibody of horse radish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 mu L of TMB substrate is added, incubation is carried out for 10min at 37 ℃, and 50 mu L of 0.2M H is added2SO4The reaction was stopped and the OD450 nm was measured. ELISA assay serum titers were specified at the highest dilution of OD450 above 2.1-fold of blank and greater than 0.2.
As shown in FIG. 1, the antiserum titers of 3-and 4-immunization were 1.09X 10, respectively6And 3.28X 106. Therefore, the antigen can induce alpaca to generate high-titer antiserum specific to BCMA protein.
Construction and panning of VHH phage libraries
Collecting 100ml of immunized alpaca peripheral blood, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain alpaca PBMC, extracting RNA according to a TRIzol operation manual, inverting by using oligo (dT) into cDNA, cloning the VHH gene of the alpaca to phagemid plasmid through technologies such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. In order to further identify whether the sBCMA-VHH phage library is successfully constructed, the VHH target gene of immune sBCMA alpaca is amplified by PCR, and the target band is 500bp and the size is in line with the expectation (figure 2), which indicates that the sBCMA-VHH phage antibody library contains the VHH gene. 48 clones are selected for sequencing, and sequencing results show that the sequenced sequences do not have completely consistent repetitive sequences; the alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the library capacity of the constructed sBCMA-VHH phage antibody library is 1.6 multiplied by 109The positive rate was 100%, the sequence Diversity (Diversity) was 100%, and the effective insertion rate (In frame rate) was 85.4%.
The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The phage antibody library was enriched three times with 50. mu.g/ml coated sBCMA-His protein. And (3) carrying out elution, transformation, plate coating and monoclonal picking on the enriched phage, carrying out binding identification on the phage and sBCMA protein ELISA, sequencing the clone with the binding reading value of more than 1.0, cloning to an expression vector pVAX1, and transfecting 293F cells to express to produce the nano monoclonal antibody.
The panning library was tested for binding to BCMA protein. The phage ELISA results showed that the binding reading values of the sBCMA-VHH phage library and the sBCMA protein before enrichment were 0.50, and the reading values of the phage library after one, two and three rounds of enrichment were 1.17, 2.78 and 3.33, respectively (FIG. 3A). To further verify the positive phage rate of sBCMA-VHH protein binding in the enriched library, 48, 46, 47 clones were selected from the enriched libraries in round 2, round 3 and round 4, respectively, for single phage ELISA detection. The results showed that 6.25% of the individual phage clones were positive in the 2 nd round library, 13.04% of the phage clones were positive in the 3 rd round library, 34.04% of the phage clones were positive in the 4th round library, and the mean reading for binding was around 3.0 (fig. 3B), and the high binding sBCMA-VHH phage library was successfully enriched by sBCMA protein panning.
Construction of VHH prokaryotic expression library and VHH expression
Carrying out PCR amplification on the 4th-sBCMA-VHH phage antibody library after the four rounds of panning and enrichment; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; coating a plate on a prokaryotic expression antibody library, culturing overnight, randomly selecting 2016 monoclonal colonies the next day, inducing expression of an antibody supernatant by using IPTG, and carrying out ELISA binding detection on the antibody supernatant and sBCMA protein.
The results show that there was bacterial supernatant binding to sBCMA protein while not binding to the blank, with sBCMA binding/blank reading greater than 2.5 (figure 4 and table 1). Sequencing and aligning the sequences, and removing repeated sequences to finally obtain 114 VHH antibody sequences. Further experiments demonstrated that both these 114 VHH antibodies and CDRs derived from the VHH antibodies can specifically bind BCMA protein.
Table 1114 binding values of VHH antibodies to sBCMA protein and their CDR sequences
Figure BDA0002603752200000111
Figure BDA0002603752200000121
Figure BDA0002603752200000131
5. Detection of binding of VHH antibodies to tumor cells by flow cytometry
The antibody sequence is constructed into a humanized VHH-hfc1 antibody expression vector, and after the obtained vector is transfected into 293TT, antibody supernatant is collected to carry out the following flow-type experiment. Transfecting 293TT cells to express a membrane protein BCMA, mixing and incubating the BANB antibody and 293TT cells 293TT-BCMA expressing BCMA, and incubating at 100 mu l/sample at 4 ℃ for 1 h; after two washes with 0.5% PBSF, a fluorescent secondary antibody was added: AlexaFluor 488anti human FC, Alexa Fluor 647anti human BCMA, at 4 ℃ for 30 min; after washing twice with 0.5% PBSF, the machine is used for detection. Meanwhile, a negative cell control group, namely an untransfected 293TT cell group, is prepared. Flow-through results as shown in fig. 5 and 6, 14 VHH antibodies were able to bind to 293TT-BCMA cells but not 293TT cells, including BANB0017, BANB0047, BANB1081, BANB0108, BANB0117, BANB0162, BANB1912, BANB1981, BANB0199, BANB0205, BANB0312, BANB0309, BANB0333, BANB 0385. Positive binding was also seen in the flow assay of the above antibodies with RPMI-8226 cells expressing BCMA membrane protein. Therefore, the VHH antibody has the capability of targeting and binding membrane protein BCMA, and simultaneously can promote the phagocytosis of macrophages by targeting BCMA molecules on the surface of tumor cells so as to achieve the effect of treating or inhibiting the growth of tumors, so that the 14 VHH antibodies have the potential to become novel antibody medicines for treating tumors.
Since the 14 VHHs described above are able to recognize BCMA molecules on the cell surface, these VHH antibody sequences can also be applied in the treatment of tumors by CAR (Chimeric Antigen Receptor, consisting of VHH sequence fused to third or fourth generation CD28-4-1BB-CD3zeta molecule sequences) cells. In addition, because the VHH can recognize BCMA molecules on the surface of tumor cells, the VHH can be used for ADC (Antibody-drug conjugate) treatment by coupling drugs or for molecular image diagnosis depending on antibodies by coupling isotopes, and the like, so that a potential nano-new drug is provided for clinical treatment of tumors.
6. In vivo experiments using humanized VHH loaded AAV viral vectors
Adeno-associated virus (AAV) is derived from non-pathogenic wild adeno-associated virus, and is considered one of the most promising gene transfer vectors due to its high safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long time for expressing foreign genes in vivo, and is widely used in gene therapy and vaccine research worldwide.
AAV Helper-Free viral packaging system was purchased from Cell Biolabs, San Diego USA. Inserting the DNA coding sequence of the VHH into the pAAV-MCS plasmid by a molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids are used for co-transfecting AAV-293T cells by using a PEI transfection reagent according to the mass ratio of 1:1: 1. Supernatants were collected at 48, 72, 96 and 120 hours post transfection and concentrated with 5xPEG8000(sigma) and finally purified with 1.37g/ml cesium chloride. Purified AAV was dissolved in PBS, identified and stored at-80 ℃ after packaging.
Cg-prkdcsccill 2rgtm1Wjl/szj (ncg) mice, purchased from university of tokyo model animals, were deleted of the IL2 receptor gene on a SCID mouse basis, similar to NSG mice, resulting in the absence of mouse T cells, B cells, and very few NK cells in vivo. 1.0-15x 107PBMC were injected intraperitoneally into NCG mice for 4-6 weeks; three weeks later, human T cells were flow-tested by collecting blood and staining human CD45+、CD3+、CD4+And CD8+. The proportion of human CD45 positive cells reached 5% or more, and the mice were judged to be successfully humanized. Injecting RPMI-8226 cells 3 x10 into abdominal cavity of the mouse6One week later, mice received AAV-BANB (1X 10)11gc/100. mu.l) intramuscular injection, AAV-GFP as a control group. The result shows that AAV-BANB has a therapeutic effect on multiple myeloma.
From the above experimental results, it was found that both the VHH and the humanized VHH-hfc1 antibody specifically recognize and bind to sBCMA, and that they can effectively bind to BCMA membrane protein at the flow cell level and also have an inhibitory effect on the development of B-cell lymphoma in mice. The antibodies can recognize the cell surface membrane protein BCMA in the natural conformation, and can be coupled with therapeutic drugs by utilizing the targeting effect of the antibodies, so as to prepare Antibody-conjugated drug ADC (Antibody-drug conjugate) or conjugated isotopes for the molecular imaging diagnosis and the like depending on the antibodies. The antigen can also be applied to CAR therapy, and is used for treating B cell malignant tumors such as multiple myeloma, for example CAR-T or CAR-NK therapy, and T cells or natural killer cells (NK cells) are modified to express anti-BCMA CAR, so as to achieve the purpose of treatment. Furthermore, nucleic acids encoding the antibody VHH fragments described above can be loaded into AAV systems for the preparation of gene therapy agents for therapeutic purposes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Nanjing Anrui Biotechnology Ltd
<120> nanobody capable of binding BCMA and application thereof
<160> 319
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ala Phe Ser Leu Lys Tyr Val Gly
1 5
<210> 2
<211> 8
<212> PRT
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<400> 2
Ala Ser Leu Leu His Ile His Ser
1 5
<210> 3
<211> 8
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<400> 3
Glu Asp Phe Ser Asp Phe Tyr Ala
1 5
<210> 4
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<400> 4
Glu Pro Thr Phe Asn Ser Tyr Ala
1 5
<210> 5
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<400> 5
Glu Arg Ala Ala Ser Asn Tyr Ala
1 5
<210> 6
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<400> 6
Glu Thr Ile Ser Thr Ile Asn Ser
1 5
<210> 7
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Ala Ile Ser Ser Val Tyr Asp
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Ala Thr Ser Tyr His Lys Asp
1 5
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Asp Ser Ile Ser Ala Gly Leu Tyr Tyr
1 5 10
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Asp Ser Ile Thr Ala Gly Tyr Tyr Tyr
1 5 10
<210> 11
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gly Asp Thr Phe Ser Ser Tyr Ala
1 5
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gly Phe Ala Phe Asp Arg Tyr Ala
1 5
<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gly Phe Ile Phe Gly Ser His Val
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gly Phe Asn Phe Glu Asp Tyr Val
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Phe Pro Phe Gly Asp Tyr Ala
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gly Phe Arg Leu Gly Asp His Ala
1 5
<210> 17
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Gly Phe Ser Ile Arg Asp Tyr Gly
1 5
<210> 18
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gly Phe Thr Phe Ala Asp Ser Ala
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gly Phe Thr Phe Asp Asp Tyr Ala
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Gly Phe Thr Phe Asp Gly His Ala
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gly Phe Thr Phe Ser Thr Tyr Trp
1 5
<210> 22
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gly Phe Thr Ser Asp Ala Tyr Gly
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gly Phe Tyr Phe Ser Leu Tyr Arg
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Gly Gly Ala Trp Arg Phe Tyr Asp
1 5
<210> 25
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gly Gly Ser Ile Ser Thr Ser Tyr Phe His
1 5 10
<210> 26
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Gly Gly Ser Ile Thr Thr Asn Trp Asn Tyr
1 5 10
<210> 27
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Gly Gly Ser Ile Thr Thr Asn Tyr Phe Tyr
1 5 10
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Gly Gly Thr Phe Arg Ala Tyr Val
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Gly Gly Thr Phe Ser Arg Ser Thr
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Gly Gly Thr Leu Ser Thr Gly Ala
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Gly Gly Thr Ser Arg Ala Tyr Val
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Gly Ile Ile Asp Ser Ile Tyr Thr
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Gly Ile Ile Gly Ser Ile Lys Ser
1 5
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Gly Ile Ile Arg Arg Met Asn Thr
1 5
<210> 35
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Gly Ile Ser Val Asp Ser Ser Ala
1 5
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Gly Lys Ile Phe Ser Leu Ser Asp
1 5
<210> 37
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Gly Lys Ile Phe Ser Asn Met Ile
1 5
<210> 38
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Gly Leu Ser Phe Ser Thr Tyr Thr
1 5
<210> 39
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Gly Leu Thr Ser Asp Asp Tyr Gly
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Gly Asn Phe Tyr Asn Ile Asn Ser
1 5
<210> 41
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gly Asn Ile Phe Asn Ile Asn Asp
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Gly Asn Ile Phe Gln Val Lys Asp
1 5
<210> 43
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gly Asn Ile Phe Gln Val Tyr Asp
1 5
<210> 44
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Gly Asn Ser Arg Pro Ile Asn Ile
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Gly Pro Thr Ser Ser Tyr Arg Ala
1 5
<210> 46
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gly Gln Ile Ser Glu Thr Ser Thr
1 5
<210> 47
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gly Arg Ala Phe Ser Asn Tyr Ala
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gly Arg Ala Ser Ser Ile Tyr Arg
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Gly Arg Gly Phe Asp Thr Tyr Thr
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Gly Arg Ile Glu Asn Thr Tyr Val
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gly Arg Ile Glu Ser Thr Tyr Val
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Gly Arg Ile Phe Asp Thr Tyr Thr
1 5
<210> 53
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Gly Arg Ile Ile Arg Val Tyr Asp
1 5
<210> 54
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Gly Arg Ile Leu Ala Thr
1 5
<210> 55
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Gly Arg Ile Val Ser Leu Asp Asp
1 5
<210> 56
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gly Arg Ser Phe Ser Asn Tyr Lys
1 5
<210> 57
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Gly Arg Thr Asp Ser Ile Ile Thr
1 5
<210> 58
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Gly Arg Thr Phe Ala Ala Tyr Thr
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Gly Arg Thr Phe Gly Asn Tyr Lys
1 5
<210> 60
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Gly Arg Thr Phe Ile Thr Gly Ala
1 5
<210> 61
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Gly Arg Thr Phe Ser Asp Tyr Ala
1 5
<210> 62
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Gly Arg Thr Phe Ser Asn Tyr Ala
1 5
<210> 63
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Gly Arg Thr Phe Ser Ser Tyr Arg
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Gly Arg Thr Phe Ser Ser Tyr Ser
1 5
<210> 65
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Gly Arg Thr Phe Ser Ser Tyr Thr
1 5
<210> 66
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Gly Arg Thr Phe Ser Ser Tyr Val
1 5
<210> 67
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Gly Arg Thr Phe Thr Cys Ser Ala Cys Val
1 5 10
<210> 68
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Gly Arg Thr Asn Tyr Thr Tyr Thr
1 5
<210> 69
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Gly Arg Thr Ser Ala Ile Tyr Thr
1 5
<210> 70
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Gly Ser Phe Phe Ser Ile Asp Ser
1 5
<210> 71
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Gly Ser Phe Val Ser Ile Asp Ala
1 5
<210> 72
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Gly Ser Phe Val Ser Ile Asn Thr
1 5
<210> 73
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Gly Ser Gly Phe Leu Ile Asn Val
1 5
<210> 74
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Gly Ser Gly Phe Ser Ile Tyr Gly
1 5
<210> 75
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Gly Ser Ile Phe Asn Ile Asp Thr
1 5
<210> 76
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Gly Ser Ile Phe Arg Thr Asn Asp
1 5
<210> 77
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Gly Ser Ile Phe Arg Val His Asp
1 5
<210> 78
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Gly Ser Ile Phe Ser Ala Tyr Asp
1 5
<210> 79
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Gly Ser Ile Phe Ser Ile Asn Ala
1 5
<210> 80
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Gly Ser Ile Phe Ser Thr Tyr Trp
1 5
<210> 81
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Gly Ser Ile Phe Ser Tyr Asn Pro
1 5
<210> 82
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Gly Ser Ile Phe Tyr Ser Asp Val
1 5
<210> 83
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Gly Ser Ile Leu Arg Val Asp Asp
1 5
<210> 84
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 84
Gly Ser Ile Arg Asp Ile Asn Val
1 5
<210> 85
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Gly Ser Ile Arg Ser Ile Asp Thr
1 5
<210> 86
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 86
Gly Ser Ile Ser Gly Phe Asn Val
1 5
<210> 87
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Gly Ser Ile Ser Ser Ile Asn Ala
1 5
<210> 88
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 88
Gly Ser Ile Val Ser Phe Asn Val
1 5
<210> 89
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Gly Ser Ile Val Ser Ile Asn Ser
1 5
<210> 90
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Gly Ser Ile Tyr Gly Ile Asn Thr
1 5
<210> 91
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Gly Ser Thr Leu Arg Arg Thr Val
1 5
<210> 92
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 92
Gly Ser Val Ser Ile Ser Pro Arg Ile Asn
1 5 10
<210> 93
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Gly Thr Ile Phe Met Ile Asp Thr
1 5
<210> 94
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 94
Gly Thr Ile Phe Ser Ile Asn Gly
1 5
<210> 95
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Gly Thr Ile Phe Ser Arg Phe Pro
1 5
<210> 96
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 96
Gly Thr Asn Val Ser Leu Asn Ile
1 5
<210> 97
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Gly Trp Tyr Val Ser Ser Asn Ala
1 5
<210> 98
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 98
Lys Val Pro Tyr Ser Phe Asp Arg
1 5
<210> 99
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Arg Ser Ile Ser Glu Ile Asn Thr
1 5
<210> 100
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 100
Arg Val Ala Tyr Ser Phe Asp Arg
1 5
<210> 101
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Ser Ser Ile Phe Met Leu Ser Thr
1 5
<210> 102
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 102
Ser Thr Asp Ile Met Gly Asp
1 5
<210> 103
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 103
Thr Gly Ser Phe Ser Ser Tyr Ala
1 5
<210> 104
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 104
Thr Thr Phe Phe Ser Ile Arg Thr
1 5
<210> 105
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 105
Ile Ser Thr Leu Asp Asp Ser Ser
1 5
<210> 106
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 106
Ile Leu Ser Asn Tyr Glu Ala
1 5
<210> 107
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 107
Ile Asn Lys Leu Pro Gly Ile Ile
1 5
<210> 108
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 108
Ile Ser Trp Asn Gly Gly Gly Thr
1 5
<210> 109
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 109
Leu Ser Trp Lys Gly Pro Thr Pro
1 5
<210> 110
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 110
Ile Thr Gly Thr Gly Thr Ala
1 5
<210> 111
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 111
Ile Thr Gly Ser Gly Arg Thr
1 5
<210> 112
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 112
Ile Thr Ser Ala Gly Ser Thr
1 5
<210> 113
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 113
Gln Tyr Ser Val Gly Gly Thr
1 5
<210> 114
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 114
Val Thr Tyr Ser Asp Ser Thr
1 5
<210> 115
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 115
Ile Ser Trp Ser Gly Gly Ser Thr
1 5
<210> 116
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 116
Ile Thr Asn Asn Asp Gly Asp Thr
1 5
<210> 117
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 117
Met Thr Arg Asn Gly Asp Gly Thr
1 5
<210> 118
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 118
Ile Ser Val Thr Asp Gly Ser Asp Gly Ser Thr
1 5 10
<210> 119
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 119
Ile Ser Gln Arg Ser Thr Thr Thr
1 5
<210> 120
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 120
Ile Ser Val His Glu Arg Ser Thr
1 5
<210> 121
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 121
Val Thr Leu Arg Gly Glu Thr
1 5
<210> 122
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 122
Ile Asn Trp Asn Gly Gly Ser Thr
1 5
<210> 123
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 123
Ile Ser Ser Ser Gly Ser Asp Thr Ser Thr
1 5 10
<210> 124
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 124
Ile Met Trp Asn Gly Ala Ser Ala
1 5
<210> 125
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 125
Ile Asn Ser Gly Gly Ser Ser Ile
1 5
<210> 126
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 126
Ile Ser Asp Val Ala Gly Asn Ser
1 5
<210> 127
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 127
Ile Arg Arg Asp Gly Gly Ala Thr
1 5
<210> 128
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 128
Ile Thr Val Asp Gly Gln Thr
1 5
<210> 129
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 129
Ile Gly Tyr Ala Gly Ser Thr
1 5
<210> 130
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 130
Ile Ala Tyr Ser Gly Ser Thr
1 5
<210> 131
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 131
Ile Thr Tyr Ser Gly Asn Thr
1 5
<210> 132
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 132
Ile Gln Trp Ser Asp Gly Thr Thr
1 5
<210> 133
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 133
Glu Ser Trp Ser Gly Arg Thr
1 5
<210> 134
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 134
Ile Ser Ala Ser Gly Gly Asn Thr
1 5
<210> 135
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 135
Val Thr Ser Ser Ser Asp Lys
1 5
<210> 136
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 136
Ile Ser Asp Gly Asp Ile Thr
1 5
<210> 137
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 137
Ile Thr Ser Gly Gly Val Thr
1 5
<210> 138
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 138
Ile Ser Gly Ser Gly Gly Thr Thr
1 5
<210> 139
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 139
Ile Thr Ser Asp Gly Val Thr
1 5
<210> 140
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 140
Ile Thr Ser Asp Thr Val Thr
1 5
<210> 141
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 141
Ile Leu Trp Ala Gly Gly Ser Thr
1 5
<210> 142
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 142
Phe Ser Thr Gly Asp Ala Ser Thr
1 5
<210> 143
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 143
Ile Thr Arg Gly Gly Glu Thr
1 5
<210> 144
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 144
Ile Thr Arg Gly Gly Arg Thr
1 5
<210> 145
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 145
Ile Gly Phe Arg Gly Glu Pro
1 5
<210> 146
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 146
Ile Gly Phe Arg Gly Asp Thr
1 5
<210> 147
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 147
Val Ser Lys Asn Gly Glu Thr
1 5
<210> 148
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 148
Val Ser Trp Asn Gly Glu Gly Thr
1 5
<210> 149
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 149
Ile Ala Asn Gly Gly Ser Thr
1 5
<210> 150
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 150
Ile Asp Trp Thr Ser Gly Val Ser
1 5
<210> 151
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 151
Ile Gln Tyr Arg Gly Phe Thr Thr
1 5
<210> 152
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 152
Ile Leu Trp Ser Gly Gly Ser Thr
1 5
<210> 153
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 153
Ile Met Trp Ser Asp Ser Ile Thr
1 5
<210> 154
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 154
Leu Ser Ile Ser Gly Arg Ser
1 5
<210> 155
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 155
Ile Ser Trp Asp Val Ser Asp Thr
1 5
<210> 156
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 156
Ile Met Trp Ser Asp Ser Ile Ala
1 5
<210> 157
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 157
Ile Ser Arg His Asn Asp Thr
1 5
<210> 158
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 158
Ile Ser Trp Ser Gly Gly Asn Thr
1 5
<210> 159
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 159
Ile Arg Ser Gly Gly Leu Val
1 5
<210> 160
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 160
Ile Ser Arg Ser Gly Asp Arg Thr
1 5
<210> 161
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 161
Ile Ala Trp Ser Thr Asn Lys Thr
1 5
<210> 162
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 162
Ile Val Trp Ser Thr Asn Lys Thr
1 5
<210> 163
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 163
Ile Leu Trp Thr Arg Ser Thr
1 5
<210> 164
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 164
Ile Arg Trp Ser Gly Gly Gly Arg
1 5
<210> 165
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 165
Ile Asn Trp Asn Gly Gly Ser Asn
1 5
<210> 166
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 166
Ile Thr Trp Asn Gly Gly Thr Thr
1 5
<210> 167
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 167
Ile Ile Trp Asn Gly Gly Asn Thr
1 5
<210> 168
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 168
Ile Thr Trp Ser Ala Gly Asn Thr
1 5
<210> 169
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 169
Ile Asn Trp Ser Gly Gly Ser Thr
1 5
<210> 170
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 170
Ile Ser Arg Phe Gly Gly Thr Thr
1 5
<210> 171
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 171
Ile Ile Trp Ser Asp Gly Ser Thr
1 5
<210> 172
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 172
Leu Lys Trp Asp Asn Ser Pro
1 5
<210> 173
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 173
Leu Val Trp Pro Gly Thr Ser Thr
1 5
<210> 174
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 174
Ile Thr Ser Asp Gly Thr Thr
1 5
<210> 175
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 175
Ile Arg Thr Gly Gly Ser Pro
1 5
<210> 176
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 176
Ile Asn Tyr Ser Gly Arg Thr
1 5
<210> 177
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 177
Ile Thr Arg Asp Gly Ile Thr
1 5
<210> 178
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 178
Ile Ser Ser Gly Gly Ser Thr
1 5
<210> 179
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 179
Ile Gly Lys Gly Gly Thr Thr
1 5
<210> 180
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 180
Ile Arg Arg Asn Gly Glu Thr
1 5
<210> 181
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 181
Ile Asn Asn Ser Asp Gly Gly Thr
1 5
<210> 182
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 182
Ile Thr Thr Ala Gly Gly Thr
1 5
<210> 183
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 183
Ile Ser Thr Gly Asp Leu Thr Thr
1 5
<210> 184
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 184
Ile Ser Asn Ser Gly Met Thr
1 5
<210> 185
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 185
Ile Gly Arg Asp Gly Arg Thr
1 5
<210> 186
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 186
Ile Thr Ser Gly Gly Ser Thr
1 5
<210> 187
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 187
Ile Thr Glu Gly Gly Ser Thr
1 5
<210> 188
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 188
Ile Ser Arg Gly Gly Ser Thr
1 5
<210> 189
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 189
Phe Thr Ser Ala Gly Arg Thr
1 5
<210> 190
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 190
Ile Thr Ser Gly Gly Thr Thr
1 5
<210> 191
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 191
Ile Thr Ser Thr Gly Ser Thr
1 5
<210> 192
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 192
Val Thr Ser Gly Gly Ser Thr
1 5
<210> 193
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 193
Ile Thr Arg Arg Ser Ser Thr
1 5
<210> 194
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 194
Ile Ser Arg Gly Gly Gly Tyr Thr
1 5
<210> 195
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 195
Ile Asp Gly Gly Gly Thr Pro
1 5
<210> 196
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 196
Ile Thr Arg Ser His Lys Ala
1 5
<210> 197
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 197
Ile Thr Ser Asp Gly Ser Thr
1 5
<210> 198
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 198
Val Ser Arg His Gly Asp Thr
1 5
<210> 199
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 199
Ile Ser Ser Tyr Gly Val Arg
1 5
<210> 200
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 200
Ile Ala Lys Ser Gly Gly Ser Thr
1 5
<210> 201
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 201
Ile Ile Asn Gly Gly Ser Ile
1 5
<210> 202
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 202
Leu Thr Ser Asn Asp Asp Ala
1 5
<210> 203
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 203
Ile Ser Asn Gly Gly Thr Ile
1 5
<210> 204
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 204
Ile Ser Asn Gly Gly Thr Val
1 5
<210> 205
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 205
Ile Ser Arg Gly Arg Ser Ala
1 5
<210> 206
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 206
Ile Ser Trp Asp Ala Lys Asn Lys
1 5
<210> 207
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 207
Ile Thr Ser Gly Gly Asp Thr
1 5
<210> 208
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 208
Ala Ile Gly Thr Arg Tyr Thr Gly Ser Tyr Tyr Val Gly Asp Cys Pro
1 5 10 15
Asn Glu Phe Asp Tyr
20
<210> 209
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 209
Asn Leu Glu Trp Ser Val Asn Pro Gly Val Gly Val Leu Arg Asp Phe
1 5 10 15
<210> 210
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 210
Ala Val Arg Ser Phe Tyr Ser Gly Ala Asn Phe Pro Glu Trp Asn Ser
1 5 10 15
Asp Tyr Glu Tyr Asp Tyr
20
<210> 211
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 211
Ala Ile Asp Met Leu Pro Arg Arg Ile Met Ala Thr Pro Glu Leu Pro
1 5 10 15
Pro Asn Ser Ser Gly Ala Phe Glu Tyr
20 25
<210> 212
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 212
Ala Gly Gly Pro Tyr Tyr Thr Glu Ile Glu Arg Asn Tyr Ile His
1 5 10 15
<210> 213
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 213
Thr Ala Asp Leu Val Tyr Met Asn Asp Phe Gly Asp Arg Tyr Arg Pro
1 5 10 15
Leu Asp Leu
<210> 214
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 214
Asn Ile Arg Leu Asp Gly Ala Phe Trp Asn Thr Asn Asn Tyr
1 5 10
<210> 215
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 215
Asn Leu Glu Thr His Asp Leu Leu Ala Gly Ser Leu Asp Arg Tyr Arg
1 5 10 15
Val Val
<210> 216
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 216
Thr Arg Gly Asp Trp Leu Ile Asn Thr Ile Gly Arg Leu Asp Asp Phe
1 5 10 15
Asp Tyr
<210> 217
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 217
Thr Arg Gly Asp Trp Leu Ile Asn Lys Val Asn Arg Leu Asp Asp Phe
1 5 10 15
Asp Ser
<210> 218
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 218
Ala Ser Thr Phe Ser Val Ser Trp Arg Ser Gly Val Arg Tyr Asp Ser
1 5 10 15
<210> 219
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 219
Ala Ala Ala Pro Leu Thr Asp Trp Gly Leu Ser Cys Ser Asp His Thr
1 5 10 15
Leu His Phe Asp Tyr Glu Tyr Leu Tyr
20 25
<210> 220
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 220
Ala Ala Arg Arg Ala Phe Lys Ile Thr Ser Val Arg Ser Thr Asp Tyr
1 5 10 15
Asp Tyr
<210> 221
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 221
Ala Thr Asp Ser Gly Thr Pro Pro Tyr Asp Ser Ser Trp Cys Ser Arg
1 5 10 15
Asn Tyr Gly Met Asp Tyr
20
<210> 222
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 222
Ala Ala Asp Gln Tyr Phe Gly Cys Ser Asp Tyr Gly Phe Gly Ser Tyr
1 5 10 15
Asp Tyr
<210> 223
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 223
Ala Met Glu Arg Tyr Phe Tyr Cys Ser Lys Trp Asp Gly Pro Arg Asp
1 5 10 15
Gly
<210> 224
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 224
Asn Arg Asn Pro Ser Ser Phe Asn Tyr
1 5
<210> 225
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 225
Ala Lys Asp Arg Ser Arg Leu Tyr Ser Gly Ser Tyr Tyr His Tyr Glu
1 5 10 15
Ala Asp Tyr
<210> 226
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 226
Ala Ala Glu Leu Leu Asp Tyr Cys Ile Asn Tyr Glu Asp His Asp Tyr
1 5 10 15
Met Asp Tyr
<210> 227
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 227
Ala Lys Ala Gly Pro Tyr Arg Asp Tyr Thr Glu Glu Tyr Asp Gly
1 5 10 15
<210> 228
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 228
Ala Arg Asp Ile Val Ala Pro Pro Gly Arg Leu Val Leu Ile Thr Thr
1 5 10 15
Gln Leu Arg Gln Arg
20
<210> 229
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 229
Gly Ala Arg Ser Leu Tyr Tyr Cys Ser Asp Trp Leu His Gln Asn Gln
1 5 10 15
Met His His
<210> 230
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 230
Ala Lys Pro Lys Met Asp Thr Ala Tyr Pro Asp Ala Phe Ser Ser Asp
1 5 10 15
Asp Tyr Asp Ser
20
<210> 231
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 231
Gly Ala Arg Gly Tyr
1 5
<210> 232
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 232
Ala Arg Gly Leu Thr Val Gln Gly Gly Gly Pro Ala His Asp Tyr
1 5 10 15
<210> 233
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 233
Ala Gln Asn Gly Ala Met Pro Tyr Asp Phe Gly Ser
1 5 10
<210> 234
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 234
Gly Ala Ala Arg Ser Arg Phe Ser Glu Tyr Glu Tyr
1 5 10
<210> 235
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 235
Val Val Arg Pro Val Val Gly Phe Arg Ser Met Asn Ile
1 5 10
<210> 236
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 236
Val Ala Asp Arg Glu Ser Leu Arg Val Ala Pro Arg Pro Ala His Asn
1 5 10 15
Tyr Glu Phe
<210> 237
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 237
Ala Ala Ala Lys Trp Gly Gly Thr Arg Trp Ser Ile Arg Asp Ser Ser
1 5 10 15
Arg Val Asp Tyr
20
<210> 238
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 238
Asn Val Arg Gly Ser Phe Gly Gln Tyr
1 5
<210> 239
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 239
Arg Val Glu Arg Gly Ile Arg Pro Arg Val Ile Ser
1 5 10
<210> 240
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 240
Thr Tyr Ala Pro Leu Tyr Gln Pro Ser Ser Ala Ser
1 5 10
<210> 241
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 241
Ala Val Gly Phe Phe Ser Ala Arg Ala Gly Cys Gly Leu Ile Asn Arg
1 5 10 15
Pro Asp Leu Phe Arg Ser
20
<210> 242
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 242
Asn Val Gln Gly Arg Asn Trp Trp Arg Ala Ala Phe Asp Ser
1 5 10
<210> 243
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 243
Asn Ala Arg Tyr Thr Phe Tyr Asn Phe
1 5
<210> 244
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 244
Ala Ala Asn Pro Tyr Gly Gly Val Thr Glu Lys Glu Asp Gly Tyr Glu
1 5 10 15
Tyr
<210> 245
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 245
Ala Leu Arg Gly Phe Tyr His Cys Ser Ile Tyr Gly Pro Gly Asp Met
1 5 10 15
Asp Tyr
<210> 246
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 246
Ala Leu Arg His Tyr Tyr His Cys Ser Ile Trp Gly Pro Gly Asp Met
1 5 10 15
Asp Tyr
<210> 247
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 247
His Ala Ile Trp Val Asp Pro Lys Gly Tyr Asn Ile Asp Tyr
1 5 10
<210> 248
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 248
Lys Met Gln Ile Val Arg Ile Gln Arg Asp Ser Phe Phe Ser Thr Asp
1 5 10 15
Tyr
<210> 249
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 249
Tyr Ala Asp Val Gln Thr Phe Arg Ser Asp Ile Leu Asp Glu Tyr Thr
1 5 10 15
Tyr
<210> 250
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 250
Ser Ala Asp Val His Thr Phe Arg Ala Tyr Ile Leu Asp Glu Tyr Thr
1 5 10 15
Tyr
<210> 251
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 251
Asn Val Lys Pro Glu Asn Tyr
1 5
<210> 252
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 252
Ala Ala Gly Ala Leu Tyr Ala Arg Ala Glu Asn Leu Arg Gln Lys Ala
1 5 10 15
Asp Tyr Asn Asn
20
<210> 253
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 253
Tyr Gly Asn Phe Arg Arg Leu Gly Trp Thr Lys Phe Leu Asp Tyr
1 5 10 15
<210> 254
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 254
Ala Ala Arg Asp Gly Tyr Arg Pro Lys Asn Glu Tyr Asp Tyr
1 5 10
<210> 255
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 255
Ala Ala Ala Arg Ser Arg Tyr Ser Glu Tyr Val Tyr
1 5 10
<210> 256
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 256
Ala Ala Asp Gln Trp Gly Ser Val Val Ala Thr Ala Pro Glu Tyr Asp
1 5 10 15
Tyr
<210> 257
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 257
Ala Ala Ala Arg Ile Arg Tyr Ser Glu Tyr Gly Tyr
1 5 10
<210> 258
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 258
Tyr Ala Arg Phe Leu Thr Asp Glu Phe
1 5
<210> 259
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 259
Asn Leu His Ser Thr Trp Pro Ser Tyr Asn Leu
1 5 10
<210> 260
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 260
Ala Ala Ala Arg Ser Arg Tyr Phe Glu Tyr Val Tyr
1 5 10
<210> 261
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 261
Thr Ala Glu Ala Gln Thr Pro Gly Arg Phe Ala Leu Val Asn Ala
1 5 10 15
<210> 262
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 262
Ala Ala Asp Glu Ser Ala Tyr Gly Gly Gly Arg Pro Asn Glu Tyr Asp
1 5 10 15
Tyr
<210> 263
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 263
Asn Ala His Asn Ala Val Asp Phe Arg Asp Tyr
1 5 10
<210> 264
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 264
Asn Ala Val Ala Lys Arg Thr Ile Val Thr Thr Pro Glu Pro Tyr
1 5 10 15
<210> 265
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 265
Ala Ser Ala Ala Phe Asp Ala Gly Ser Trp Pro Gly Gly Tyr Asn Tyr
1 5 10 15
<210> 266
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 266
Asn Ala Asn Thr Ser Pro Tyr Gly Arg Ala Asn Tyr
1 5 10
<210> 267
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 267
Ala Val Asp Val Asp Ser Leu Gly Phe Pro Thr Thr Ala Gln Tyr Tyr
1 5 10 15
Glu Tyr
<210> 268
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 268
Ala Ala Asp Arg Ser Arg Leu Leu Ser Ile Arg Arg Gly Ile Ser Asp
1 5 10 15
Tyr Asp Tyr
<210> 269
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 269
Ala Gln Ser Lys Gly Gly Trp Tyr Thr Val Ser Thr Ser Thr Tyr Thr
1 5 10 15
Phe
<210> 270
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 270
Ala Ala Lys Lys Phe Val Leu Thr Ala Thr Pro His Gln Tyr Asp Tyr
1 5 10 15
<210> 271
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 271
Ala Ala Ala Leu Lys Ile Pro Phe Thr Asn Leu Phe Arg Ser Tyr Asp
1 5 10 15
Tyr
<210> 272
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 272
Ala Thr Asp Pro Leu Asp Gln Leu Arg Val Gly Leu Asp Phe Arg Ser
1 5 10 15
<210> 273
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 273
Ala Ala Asp His Ser Ser Asp Tyr Thr Arg Gly Val Tyr Tyr Gly Met
1 5 10 15
Asp Tyr
<210> 274
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 274
Ala Ala Ser Thr Glu Val Thr Glu Val Phe Ser Gly Leu Leu His Pro
1 5 10 15
Glu Ser Tyr Lys Tyr
20
<210> 275
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 275
Ala Tyr Ala Pro Ser Arg Tyr Glu Glu Tyr His Tyr
1 5 10
<210> 276
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 276
Ala Gly Glu Thr Gln Tyr Arg Lys Thr Trp Tyr Ala Tyr Thr Ile Pro
1 5 10 15
Glu Lys Tyr Asp Arg
20
<210> 277
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 277
Asn Val Glu Gly Arg Arg Phe Pro Trp Glu Ser Arg Arg Ala Tyr
1 5 10 15
<210> 278
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 278
Asn Ala Lys Trp Arg Asp Gly Ile Thr Thr Ile Glu Gly Tyr Glu Tyr
1 5 10 15
Asp Tyr
<210> 279
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 279
Asn Gln Arg Val Thr Arg Trp Asn Asn Val Phe Glu Tyr
1 5 10
<210> 280
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 280
His Tyr Gly Thr Ser Thr
1 5
<210> 281
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 281
Thr Ala Lys Gly Tyr Gly Ser Ile Leu Asp Ala Leu Asn Lys Val Leu
1 5 10 15
Gln Thr Tyr
<210> 282
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 282
Asn Val Ile Phe Ala Thr Tyr Leu Pro
1 5
<210> 283
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 283
Ser Ala Arg Ile Ile Arg Met Gly Ser Ser Gln Glu Tyr Thr Trp
1 5 10 15
<210> 284
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 284
Asn Ala Lys Asn Arg Ile Phe Arg Asn Gln Gly Thr Gly Tyr
1 5 10
<210> 285
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 285
Asn Ala Glu Leu Glu Gly Trp Gly Ser Pro Val Ser
1 5 10
<210> 286
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 286
Ala Arg Ala Arg Gly Ala Pro Ser Phe Thr Gly Arg Arg Phe Asp Tyr
1 5 10 15
Asp Tyr Arg
<210> 287
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 287
Asn Thr Lys Ser Leu Ser Arg Asp Tyr
1 5
<210> 288
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 288
Asn Leu Glu Arg Gly Trp Thr Asp Tyr
1 5
<210> 289
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 289
Asn Ala Lys Ile Val Pro Arg Ala Ile Met Trp Arg Leu Thr Asp Tyr
1 5 10 15
<210> 290
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 290
Asn Ala Tyr Arg Thr Ser Trp Ser Gly Arg His Glu Tyr
1 5 10
<210> 291
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 291
Asn Ala Asp Leu Tyr Gly Trp Gly Gln Leu Thr Leu Gln Asn Tyr
1 5 10 15
<210> 292
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 292
Lys Ile Thr Val Phe Phe Pro Ser Glu Thr Thr Tyr
1 5 10
<210> 293
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 293
Asn Leu Arg Arg Ala Arg Phe Leu Val Tyr
1 5 10
<210> 294
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 294
Asn Ile Ala Leu His Asn Pro Trp Gly Thr Phe Asp Thr Tyr
1 5 10
<210> 295
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 295
Asn Ala Lys Gly Arg Asn Pro Pro Val Trp Gly Leu Asp Tyr Asn Ser
1 5 10 15
<210> 296
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 296
Asn Ala Asp Ile Ala Ala Met Pro Asp Trp Tyr Ser Ala Tyr
1 5 10
<210> 297
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 297
Ala Ala Asp Ser Gly Ser Glu Pro Ser Pro Leu Tyr Tyr Ser Tyr Glu
1 5 10 15
Tyr Asp Tyr
<210> 298
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 298
Thr Ala Leu Asn Tyr
1 5
<210> 299
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 299
Lys Ala Glu Ala Tyr Arg Ser Phe Ser Tyr Tyr Val Ser Arg Arg Leu
1 5 10 15
Asp Asp Tyr
<210> 300
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 300
Gln Met Thr Ile Arg Glu Arg Phe Ile Pro Phe Arg Thr Ser Asp Tyr
1 5 10 15
<210> 301
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 301
Asn Ala Gly Arg Thr Ser Thr Leu His Phe Gly Asp Ile Trp Pro Gly
1 5 10 15
Ile Asp Tyr
<210> 302
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 302
His Gly Arg Asn Ser Glu Ile Phe Pro Pro Arg Glu Tyr
1 5 10
<210> 303
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 303
Ala Ala Ser Glu Tyr Pro Asn Thr Trp Arg Asp Pro Gly Arg Tyr Ala
1 5 10 15
His
<210> 304
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 304
His Leu Tyr Gly Ala Gly Gly Ile Leu Asp Ala
1 5 10
<210> 305
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 305
Asn Leu Val Trp Ser Tyr His Thr Pro Gly His Asn Tyr
1 5 10
<210> 306
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 306
Asn Leu Val Trp Ser Tyr His Thr Pro Gly Gln Asn Tyr
1 5 10
<210> 307
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 307
His Leu Tyr Gly Ala Gly Gly Arg Leu Asp Ala
1 5 10
<210> 308
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 308
Arg Ala Asp Arg Ile Val Ser Gly Ala Tyr
1 5 10
<210> 309
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 309
Phe Ala Gln Ala Phe Asp Gln Gln His Met Tyr
1 5 10
<210> 310
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 310
Thr Ala Gly Phe Asp Pro Asp Arg Asp Trp Ser Arg Tyr Asp Leu
1 5 10 15
<210> 311
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 311
Lys Leu Gly Val Thr Arg Ser Tyr
1 5
<210> 312
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 312
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 313
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 313
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala
1 5 10 15
Gly
<210> 314
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 314
Ser Tyr Thr Asp Ser Ala Lys Asp Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Thr Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Leu Cys
35
<210> 315
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 315
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 316
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 316
Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu
1 5 10 15
Thr Leu Arg Leu Ser Cys Thr Ala Ser
20 25
<210> 317
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 317
Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala
1 5 10 15
<210> 318
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 318
Ala Asp Ser Thr Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys
1 5 10 15
His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly
20 25 30
Val Tyr Tyr Cys
35
<210> 319
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 319
Gly Gln Gly Thr Arg Val Thr Val Ser Ser
1 5 10

Claims (7)

1. A nanobody capable of binding BCMA, comprising 3 CDRs 1-3 of Complementarity Determining Region (CDR) sequence as follows:
the sequence of CDR1 is shown in SEQ ID NO. 51, the sequence of CDR2 is shown in SEQ ID NO. 153, and the sequence of CDR3 is shown in SEQ ID NO. 255; or
The sequence of CDR1 is shown in SEQ ID NO. 51, the sequence of CDR2 is shown in SEQ ID NO. 153, and the sequence of CDR3 is shown in SEQ ID NO. 260.
2. The nanobody of claim 1, further comprising 4 framework regions FR1-4, wherein the FR1-4 is sequentially staggered from the CDR 1-3.
3. Use of the nanobody of claim 1 or 2 for the preparation of a BCMA detection agent or a B-cell lymphoma therapeutic drug.
4. A nucleic acid encoding the nanobody of claim 1 or 2.
5. Use of the nucleic acid of claim 4 for the preparation of a medicament for the treatment of B-cell lymphoma.
6. A BCMA detection agent, wherein the detection antibody and the coating antibody are each one of the nanobodies of claim 1 or 2.
7. The BCMA assay reagent of claim 6, wherein the BCMA assay reagent is a flow assay reagent.
CN202010732288.7A 2020-07-27 2020-07-27 Nanometer antibody capable of combining BCMA and application thereof Active CN111848798B (en)

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Publication number Priority date Publication date Assignee Title
CN112592405B (en) * 2020-12-08 2022-05-03 博奥信生物技术（南京）有限公司 Anti-human BCMA nano antibody and preparation method and application thereof
CN112538115B (en) * 2020-12-08 2022-05-03 博奥信生物技术（南京）有限公司 Anti-human BCMA nano antibody and preparation method and application thereof
CN113265001B (en) * 2021-02-08 2022-07-29 华道(上海)生物医药有限公司 Nanometer antibody for resisting B cell mature antigen and application thereof
CN112812186B (en) * 2021-02-08 2022-07-29 华道(上海)生物医药有限公司 Nanometer antibody for resisting B cell mature antigen and application thereof
CN115109156B (en) * 2021-03-22 2024-03-08 浙江纳米抗体技术中心有限公司 BCMA-targeted nano antibody and application thereof
CN114276452A (en) * 2021-12-29 2022-04-05 源道隆（苏州）医学科技有限公司 Nano antibody capable of being combined with BCMA (brain cell activating antigen) and application thereof
CN117430709B (en) * 2023-12-21 2024-03-26 康维众和(中山)生物药业有限公司 Nanometer antibody and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019000223A1 (en) * 2017-06-27 2019-01-03 Nanjing Legend Biotech Co., Ltd. Chimeric antibody immune effctor cell engagers and methods of use thereof
CN109134665A (en) * 2018-08-24 2019-01-04 深圳普瑞金生物药业有限公司 A kind of BCMA Chimeric antigen receptor and application based on single domain antibody
CN109311999A (en) * 2015-08-11 2019-02-05 南京传奇生物科技有限公司 Chimeric antigen receptor and its application method based on single domain antibody
CN109942708A (en) * 2019-03-28 2019-06-28 上海科棋药业科技有限公司 The single domain antibody of anti-BCMA a kind of and its application
CN109942709A (en) * 2019-04-22 2019-06-28 广州百暨基因科技有限公司 The single domain antibody of anti-BCMA a kind of and its application
TW202016136A (en) * 2018-06-01 2020-05-01 瑞士商諾華公司 Binding molecules against bcma and uses thereof
CN111333729A (en) * 2020-03-17 2020-06-26 深圳市南科生物工程有限公司 Nano antibody for resisting B cell mature antigen and application

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109311999A (en) * 2015-08-11 2019-02-05 南京传奇生物科技有限公司 Chimeric antigen receptor and its application method based on single domain antibody
WO2019000223A1 (en) * 2017-06-27 2019-01-03 Nanjing Legend Biotech Co., Ltd. Chimeric antibody immune effctor cell engagers and methods of use thereof
TW202016136A (en) * 2018-06-01 2020-05-01 瑞士商諾華公司 Binding molecules against bcma and uses thereof
CN109134665A (en) * 2018-08-24 2019-01-04 深圳普瑞金生物药业有限公司 A kind of BCMA Chimeric antigen receptor and application based on single domain antibody
CN109942708A (en) * 2019-03-28 2019-06-28 上海科棋药业科技有限公司 The single domain antibody of anti-BCMA a kind of and its application
CN109942709A (en) * 2019-04-22 2019-06-28 广州百暨基因科技有限公司 The single domain antibody of anti-BCMA a kind of and its application
CN111333729A (en) * 2020-03-17 2020-06-26 深圳市南科生物工程有限公司 Nano antibody for resisting B cell mature antigen and application

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Anti-BCMA antibodies in the future management of multiple myeloma;Maria Gavriatopoulou等;《Expert Review of Anticancer Therapy》;20190318;第19卷(第4期);第319-326页 *
B 细胞成熟抗原的抗肿瘤免疫治疗研究进展;张航等;《药物生物技术》;20181231;第25卷(第1期);第1-6页 *
Development and Evaluation of CART Targeting Bcma with Humanized Alpaca-Derived Single Domain Antibody As Antigen Recognition Domain;Lu Han等;《BLOOD》;20181129;第132卷;第1-3页 *
T cells expressing an anti–B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma;Syed Abbas Ali等;《BLOOD》;20160929;第128卷(第13期);第1688-1700页 *

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