CN116333143A - SCUBE2 neutralizing antibody and medical use thereof - Google Patents

SCUBE2 neutralizing antibody and medical use thereof Download PDF

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CN116333143A
CN116333143A CN202111582701.7A CN202111582701A CN116333143A CN 116333143 A CN116333143 A CN 116333143A CN 202111582701 A CN202111582701 A CN 202111582701A CN 116333143 A CN116333143 A CN 116333143A
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antibody
seq
scube2
protein
gly
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胡国宏
吴秋瑶
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Shanghai Institute of Nutrition and Health of CAS
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Shanghai Institute of Nutrition and Health of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Abstract

The invention provides a SCUBE2 neutralizing antibody and medical application thereof. In particular, the invention provides antibodies capable of competitively binding to a SCUBE2 protein. The antibody provided by the invention has high affinity, can bind to a CUB functional region of the SCUBE2 protein, competitively blocks the binding of a Hedgehog signal ligand (such as SHH protein) and the CUB structural region in the SCUBE2 protein, and thus inhibits the Hedgehog signal-mediated breast cancer bone metastasis process. The antibody has the prospect of treating breast cancer bone metastasis.

Description

SCUBE2 neutralizing antibody and medical use thereof
Technical Field
The invention relates to the field of biological medicine. In particular, the invention relates to a SCUBE2 neutralizing antibody and its medical use.
Background
Breast cancer is the most common female tumor worldwide and is the cancer source that poses a threat to female health. Remote metastasis, compared to in situ tumors, is a major cause of death in breast cancer patients. The metastatic organs of breast cancer are bone, liver, brain, lung, etc., with bone being the most common site of metastasis, and clinically more than 70% of patients with advanced breast cancer are diagnosed with bone metastasis, causing severe bone pain, bone fracture and other fatal complications. The current primary treatment for breast cancer bone metastases is the inhibition of osteoclast-induced osteolysis, such as bisphosphonates and diels, but these drugs only can alleviate the deterioration of the metastases and do not effectively increase the overall survival of the patient.
SCUBE2 is an evolutionarily highly conserved protein containing 9 classes of EGF repeats, three cysteine-rich repeats and one CUB region. SCUBE2 is an important protein in Hedgehog signaling and is responsible for the shear release of Hedgehog signaling ligands, such as SHH, anchored to the cell membrane, thereby allowing free SHH to bind to Hedgehog receptors and initiate downstream Hedgehog signaling. Studies have found that the CUB region of SCUBE2 plays a key role in cleavage release of SHH protein, and that truncated proteins lacking the CUB region cannot bind to SHH protein and cannot release it from the cell membrane. Earlier studies found that SCUBE2 plays an important role in breast cancer bone metastasis, and that SCUBE2 helps cancer cells to colonize the bone microenvironment by promoting the release of SHH by breast cancer cells, so targeting functional inhibition of SCUBE2 protein is a potential approach to treat breast cancer bone metastasis. At present, the inhibition molecules or drugs aiming at SCUBE2 are blank in the market.
There is therefore a need in the art to develop a novel therapeutic agent that targets SCUBE2 for breast cancer bone metastasis.
Disclosure of Invention
The invention aims to provide a novel therapeutic drug for targeting the breast cancer bone metastasis of SCUBE 2.
In a first aspect of the invention, there is provided an antibody that binds to a SCUBE2 protein, said antibody specifically binding to the CUB domain of a SCUBE2 protein and said antibody being capable of competitively blocking the binding of a Hedgehog signaling ligand to said SCUBE2 protein,
wherein the amino acid sequence of the CUB domain is shown as SEQ ID NO. 2.
In another preferred embodiment, the competitive blocking means that the binding rate of the Hedgehog signaling ligand to the SCUBE2 protein is reduced by 50%, preferably by 70%, more preferably by 90%.
In another preferred embodiment, the antibody comprises a heavy chain and a light chain,
wherein the variable region of the heavy chain has complementarity determining region CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO 9, 15 or 20,
VH-CDR2 shown in SEQ ID NO 10, 16 or 21, and
VH-CDR3 shown in SEQ ID No. 11, 17 or 22;
and, the variable region of the light chain has complementarity determining region CDRs selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO 12, 18 or 23,
VL-CDR2 shown in SEQ ID NO 13 or 24, and
VL-CDR3 as shown in SEQ ID NO 14, 19 or 25;
and, any one of the amino acid sequences described above further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid, and that allows a derivative antibody comprising the heavy and light chains of the derivative CDR sequence to retain SCUBE2 binding affinity.
In another preferred embodiment, the heavy chain variable region of the antibody has complementarity determining region CDRs selected from the group consisting of:
VH 1) a VH-CDR1 having an amino acid sequence shown in SEQ ID No. 9, a VH-CDR2 shown in SEQ ID No. 10, and a VH-CDR3 shown in SEQ ID No. 11;
VH 2) a VH-CDR1 having an amino acid sequence shown in SEQ ID No. 15, a VH-CDR2 shown in SEQ ID No. 16, and a VH-CDR3 shown in SEQ ID No. 17; or (b)
VH 3) a VH-CDR1 shown in SEQ ID NO. 20, a VH-CDR2 shown in SEQ ID NO. 21, and a VH-CDR3 shown in EQ ID NO. 22.
In another preferred embodiment, the light chain variable region of the antibody has complementarity determining region CDRs selected from the group consisting of:
VL 1) a VL-CDR1 having an amino acid sequence shown in SEQ ID NO. 12, a VL-CDR2 shown in SEQ ID NO. 13, and a VL-CDR3 shown in SEQ ID NO. 14;
VL 2) a VL-CDR1 having an amino acid sequence shown in SEQ ID NO. 18, a VL-CDR2 shown in SEQ ID NO. 13, and a VL-CDR3 shown in SEQ ID NO. 19; and
VL 3) the amino acid sequence is shown as VL-CDR1 of SEQ ID NO. 23, VL-CDR2 of SEQ ID NO. 24, and VL-CDR3 of SEQ ID NO. 25.
In another preferred embodiment, the heavy chain variable region comprises the following three complementarity determining region CDRs:
VH-CDR1 shown in SEQ ID No. 9, VH-CDR2 shown in SEQ ID No. 10, and VH-CDR3 shown in SEQ ID No. 11;
And, the light chain variable region includes the following three complementarity determining region CDRs:
VL-CDR1 shown in SEQ ID NO. 12, VL-CDR2 shown in SEQ ID NO. 13, and VL-CDR3 shown in SEQ ID NO. 14.
In another preferred embodiment, the heavy chain variable region comprises the following three complementarity determining region CDRs:
VH-CDR1 shown in SEQ ID No. 15, VH-CDR2 shown in SEQ ID No. 16, and VH-CDR3 shown in SEQ ID No. 17;
and, the light chain variable region includes the following three complementarity determining region CDRs:
VL-CDR1 shown in SEQ ID NO. 18, VL-CDR2 shown in SEQ ID NO. 13, and VL-CDR3 shown in SEQ ID NO. 19.
In another preferred embodiment, the heavy chain variable region comprises the following three complementarity determining region CDRs:
VH-CDR1 shown in SEQ ID NO. 20, VH-CDR2 shown in SEQ ID NO. 21, and VH-CDR3 shown in EQ ID NO. 22;
and, the light chain variable region includes the following three complementarity determining region CDRs:
VL-CDR1 shown in SEQ ID NO. 23, VL-CDR2 shown in SEQ ID NO. 24, and VL-CDR3 shown in SEQ ID NO. 25.
In another preferred embodiment, the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO. 3, 5 or 7.
In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human or murine origin.
In another preferred embodiment, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 4, 6 or 8.
In another preferred embodiment, the light chain further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human or murine origin.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 3, 5 or 7; and/or the light chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 4, 6 or 8.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence as set forth in SEQ ID NO 3, 5 or 7 of the sequence Listing.
In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence as set forth in SEQ ID NO. 4, 6 or 8 of the sequence Listing.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 3; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 4.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 5; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 6.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 7; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 8.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of murine origin, and/or the light chain variable region of the antibody further comprises a framework region of murine origin.
In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.
In another preferred embodiment, the antibody is a monoclonal antibody, or a polyclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen-binding fragment.
In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In another preferred embodiment, the antibody has one or more functions selected from the group consisting of:
(a) Inhibiting tumor cells from releasing Hedgehog signaling ligands;
(b) Inhibiting release of Hedgehog signaling ligands by peri-tumor endothelial cells;
(c) Inhibiting Hedgehog signaling ligand mediated tumor cell growth;
(d) Inhibit Hedgehog signaling ligand mediated tumor cell metastasis.
In another preferred embodiment, the Hedgehog signaling ligand is selected from the group consisting of: SHH protein, IHH protein, DHH protein.
In another preferred embodiment, the tumor is selected from the group consisting of: breast cancer, melanoma, lung cancer.
In another preferred embodiment, the tumor is breast cancer.
In another preferred embodiment, the transfer is selected from the group consisting of: bone metastasis.
In another preferred embodiment, the metastasis is bone metastasis.
In a second aspect of the present invention, there is provided a recombinant protein comprising:
(i) An antibody according to the first aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag, a GGGS sequence, a FLAG tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In another preferred embodiment, the recombinant protein specifically binds to a SCUBE2 protein.
In another preferred embodiment, the recombinant protein specifically binds to the CUB functional region of the SCUBE2 protein.
In another preferred embodiment, the recombinant protein is a fusion protein.
In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against a SCUBE2 protein), a bispecific antibody, or a multispecific antibody (e.g., a trispecific antibody).
In another preferred embodiment, the bispecific or multispecific antibody not only binds to an SCUBE2 protein, but also specifically binds to an additional target antigen (e.g., another tumor antigen, such as another antigen of breast cancer or another tumor antigen).
In a third aspect of the invention there is provided a Chimeric Antigen Receptor (CAR) construct, the scFv fragment of the antigen binding region of which specifically binds to a SCUBE2 protein and which scFv has a heavy chain variable region and a light chain variable region according to the first aspect of the invention.
In a fourth aspect of the invention, there is provided a recombinant immune cell expressing an exogenous CAR construct according to the third aspect of the invention.
In another preferred embodiment, the immune cells are selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In a fifth aspect of the invention, there is provided an antibody conjugate comprising:
(a) An antibody moiety selected from the group consisting of: an antibody according to the first aspect of the invention; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
In another preferred embodiment, the conjugate is 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 a detectable product, 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 proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In a sixth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the antibody according to the first aspect of the invention, the recombinant protein according to the second aspect of the invention, the antibody conjugate according to the fifth aspect of the invention, or a combination thereof, wherein the active ingredient is used for:
(a) Preparing a diagnostic reagent, a detection plate or a kit; and/or
(b) Preparing medicine for preventing and/or treating diseases related to SCUBE2 protein expression or dysfunction.
In another preferred embodiment, the reagent is used to detect a SCUBE2 protein.
In another preferred embodiment, the reagent, test plate or kit is used for detecting a disease associated with expression or dysfunction of a SCUBE2 protein.
In another preferred embodiment, the agent, test plate or kit is used to predict the risk of breast cancer bone metastasis.
In another preferred embodiment, the agent is for preventing and/or treating Hedgehog signaling ligand mediated tumor cell metastasis.
In another preferred embodiment, the agent is used for preventing and/or treating inhibiting breast cancer bone metastasis.
In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an immune cell according to the fourth aspect of the invention, an antibody conjugate according to the fifth aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody according to the first aspect of the invention, the recombinant protein according to the second aspect of the invention, the immune cell according to the fourth aspect of the invention, the antibody conjugate according to the fifth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, said percentages being mass percentages of the pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating Hedgehog signaling ligand mediated tumor cell metastasis.
In another preferred embodiment, the pharmaceutical composition is used for preventing and/or treating the inhibition of breast cancer bone metastasis.
In an eighth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) An antibody according to the first aspect of the invention;
(2) A recombinant protein according to the second aspect of the invention; or (b)
(3) A Chimeric Antigen Receptor (CAR) construct according to the third aspect of the invention.
In a ninth aspect of the invention there is provided a vector comprising a polynucleotide according to the eighth aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a tenth aspect of the invention there is provided an engineered host cell comprising a vector or genome according to the ninth aspect of the invention incorporating a polynucleotide according to the eighth aspect of the invention.
In an eleventh aspect of the invention, there is provided a method of detecting a SCUBE2 protein in a sample, the method comprising the steps of:
(1) Contacting the sample with an antibody according to the first aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of a SCUBE2 protein in the sample.
In another preferred embodiment, the detection is for non-therapeutic non-diagnostic purposes in vitro.
In a twelfth aspect of the invention, there is provided a composition for in vitro detection of a SCUBE2 protein in a sample comprising as an active ingredient an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an antibody conjugate according to the fifth aspect of the invention, an immune cell according to the fourth aspect of the invention, or a combination thereof.
In a thirteenth aspect of the present invention, there is provided a detection plate comprising: a substrate (support) and a test strip comprising an antibody according to the first aspect of the invention, a recombinant protein according to the second aspect of the invention, an antibody conjugate according to the fifth aspect of the invention, an immune cell according to the fourth aspect of the invention, or a combination thereof.
In a fourteenth aspect of the present invention, there is provided a kit comprising:
(1) A first container comprising an antibody according to the first aspect of the invention; and/or
(2) A second container comprising a second antibody against the antibody of the first aspect of the invention;
Or alternatively, the process may be performed,
the kit contains a detection plate according to the thirteenth aspect of the invention.
In a fifteenth aspect of the present invention, there is provided a method of producing a recombinant polypeptide, the method comprising:
(a) Culturing a host cell according to the tenth aspect of the invention under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.
In a sixteenth aspect of the present invention, there is provided a pharmaceutical combination comprising:
(i) A first active ingredient comprising an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an immune cell according to the fourth aspect of the invention, or an antibody conjugate according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, or a combination thereof;
(ii) A second active ingredient comprising a Hedgehog signaling inhibitor, an antiestrogen, or a chemotherapeutic agent.
In another preferred embodiment, the Hedgehog signaling inhibitor is selected from the group consisting of: SHH neutralizing antibodies or SMO inhibitors.
In another preferred embodiment, the antiestrogenic agent is selected from the group consisting of: tamoxifen, an ovarian function inhibitor, an aromatase inhibitor, or a combination thereof.
In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: anthracyclines (doxorubicin, epirubicin, daunorubicin, and aclarubicin), taxanes (paclitaxel, paclitaxel liposomes, albumin paclitaxel, and docetaxel), or combinations thereof.
In a seventeenth aspect of the invention there is provided the use of an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an antibody conjugate according to the fifth aspect of the invention, or an immune cell according to the fourth aspect of the invention, and/or a pharmaceutical composition according to the seventh aspect of the invention in combination with a Hedgehog signaling inhibitor or a chemotherapeutic agent, in the manufacture of a medicament for the treatment of a disease associated with abnormal expression or function of a SCUBE2 protein.
In another preferred embodiment, said abnormal expression of a SCUBE2 protein is referred to as overexpression of a SCUBE2 protein.
In another preferred embodiment, the overexpression is defined as the ratio of the expression level (F1) of the SCUBE2 protein to the expression level (F0) under physiological conditions (i.e., F1/F0) of 2.0 or more, preferably 3.0 or more, more preferably 5.0 or more.
In another preferred embodiment, the medicament is for the prevention and/or treatment of tumorigenesis, growth and/or metastasis.
In another preferred embodiment, the medicament is for preventing and/or treating breast cancer bone metastasis.
In another preferred embodiment, the Hedgehog signaling inhibitor is selected from the group consisting of: SHH neutralizing antibodies or SMO inhibitors.
In an eighteenth aspect of the invention there is provided a method of treating a disease associated with abnormal expression or function of a SCUBE2 protein, administering to a subject in need thereof an effective amount of an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an antibody conjugate according to the fifth aspect of the invention, or an immune cell according to the fourth aspect of the invention, or a pharmaceutical composition according to the sixteenth aspect of the invention, or a combination thereof.
In another preferred embodiment, the disease associated with expression or dysfunction of the SCUBE2 protein is cancer, tuberculosis of the central nervous system, arteriovenous malformations.
In another preferred embodiment, the treatment comprises preventing and/or treating tumor metastasis.
In another preferred embodiment, the tumor is selected from the group consisting of: breast cancer, melanoma, lung cancer.
In another preferred embodiment, the tumor metastasis is Hedgehog signal mediated tumor metastasis.
In another preferred embodiment, the tumor metastasis is bone metastasis.
In another preferred embodiment, the method further comprises: a safe and effective amount of a second active ingredient is administered to the subject before, during and/or after administration of the first active ingredient.
In another preferred embodiment, the second active ingredient comprises a Hedgehog signaling inhibitor, an antiestrogen, or a chemotherapeutic agent.
In another preferred embodiment, the Hedgehog signaling inhibitor is selected from the group consisting of: SHH neutralizing antibodies or SMO inhibitors.
In another preferred embodiment, the antiestrogenic agent is selected from the group consisting of: tamoxifen, an ovarian function inhibitor, an aromatase inhibitor, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 shows the purification and identification of SCUBE2 monoclonal antibodies. (FIG. 1A) Coomassie brilliant blue staining pattern of purified antibodies in ascites fluid in mice after hybridoma cell injection. (FIG. 1B) ELISA kit identified SCUBE2 monoclonal antibody subtype. (FIG. 1C) SCUBE2 expression was detected using monoclonal antibodies in the human breast cancer cell line MDA-MB-231 that overexpressed SCUBE 2.
FIG. 2 shows that a SCUBE2 monoclonal antibody inhibits the function of a SCUBE2 protein in vitro. (FIG. 2A) after treatment with IgG or SCUBE2 monoclonal antibodies, MCF7 cell supernatants induced GLI reporting system activation. (FIG. 2B) after treatment with IgG or SCUBE2 monoclonal antibody, MCF7 cell supernatant induced osteoblast differentiation. (FIG. 2C) expression of SHH protein in MCF7 cell supernatants after treatment with different concentrations of IgG or SCUBE2 monoclonal antibody.
FIG. 3 shows the in vivo therapeutic effect of SCUBE2 monoclonal antibodies. (FIGS. 3A-B) control IgG or SCUBE2 monoclonal antibody treated mice in the left ventricle injected with human breast cancer cell lines were light signals for systemic bone metastasis and schematic representation (FIG. 3A) and light signals for in vitro bone tissue metastasis (FIG. 3B). (fig. 3C-D) mice in the control group or SCUBE2 monoclonal antibody treated group after left ventricular injection of the mouse breast cancer cell line were light signals for total bone metastasis and schematic (fig. 3C) and light signals for bone tissue metastasis in vitro (fig. 3D).
Detailed Description
The present inventors have made extensive and intensive studies to obtain a neutralizing monoclonal antibody against SCUBE2 for the first time. The neutralizing antibodies of the invention are capable of competitively binding to SCUBE2 protein and have high affinity. Unexpectedly, the antibody of the invention can inhibit not only the capability of scission release of SHH of the SCUBE2 protein in vitro, but also the release of SHH mediated by breast cancer cells and the bone metastasis process induced thereby in vivo, so that the antibody of the invention has the prospect of treating breast cancer bone metastasis. On this basis, the present invention has been completed.
Experiments show that the CUB functional region of the SCUBE2 neutralizing antibody can bind to the CUB functional region of the SCUBE2 protein, competitively block the binding of a Hedgehog signaling ligand (such as SHH protein) to the CUB structural domain in the SCUBE2 protein, and therefore prevent the Hedgehog signaling mediated breast cancer bone metastasis process.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).
As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent comprising an antibody of the invention directed against a respiratory syncytial virus fusion protein (preferably a pre-fusion F protein) and compositions thereof to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that there may be, but need not be, 1, 2, or 3 antibody heavy chain variable regions of a particular sequence.
"sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,100%.
SCUBE2
SCUBE2 is an evolutionarily highly conserved protein containing 9 classes of EGF repeats, three cysteine-rich repeats and one CUB region. SCUBE2 is an important protein in Hedgehog signaling and is responsible for the shear release of Hedgehog signaling ligands, such as SHH, anchored to the cell membrane, thereby allowing free SHH to bind to Hedgehog receptors and initiate downstream Hedgehog signaling. Studies have found that the CUB region of SCUBE2 plays a key role in cleavage release of SHH protein, and that truncated proteins lacking the CUB region cannot bind to SHH protein and cannot release it from the cell membrane. Earlier studies found that SCUBE2 plays an important role in bone metastasis of breast cancer, and that SCUBE2 helps the cancer cells colonize the bone microenvironment by promoting SHH release by the breast cancer cells.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a 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 a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; 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. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in 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 fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly 5 classes of immunoglobulins: igA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called 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. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge over the longer chain region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
The invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments such as Fab or (Fab') 2 Fragments; antibody heavy chain; an antibody light chain.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. Epitopes may be discrete on an antigen, three-dimensional spatial sites recognized by an antibody or antigen binding fragment of the invention.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 -7 M, e.g. less than about 10 -8 M、10 -9 M or l0 -10 Affinity (KD) binding of M or less.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies. Chimeric antibodies are a molecule in which different portions are derived from different animal species, e.g., chimeric antibodies having variable regions from murine monoclonal antibodies, and constant regions from human immunoglobulins (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, incorporated herein by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH".
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface.
In a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region described above and a light chain constant region, which may be murine or human in origin.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.
Table A
Initial residues Representative substitution Preferred substitution
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
SCUBE2 antibodies
As used herein, the terms "antibody of the invention", "SCUBE 2 antibody of the invention" and "SCUBE 2 neutralizing antibody of the invention" are used interchangeably and refer to antibodies that bind to a SCUBE2 protein as described in the first aspect of the invention.
The function of the antibodies of the invention is determined by the antibody light and heavy chain variable region gene-specific gene sequences. The antibody of the invention can competitively bind with high affinity to the SCUBE2 protein, competitively block the binding of the Hedgehog signaling ligand (such as SHH protein) to the CUB domain in the SCUBE2 protein, thereby further inhibiting the Hedgehog signaling mediated breast cancer bone metastasis process. Using the variable region genes or Complementarity Determining Region (CDR) genes of the antibodies of the invention, genetically engineered antibodies of different forms can be engineered and produced in any expression system that utilizes prokaryotic and eukaryotic cells.
In a first aspect of the invention, there is provided an antibody that binds to a SCUBE2 protein, said antibody specifically binding to a CUB domain of a SCUBE2 protein, and said antibody being capable of competitively blocking the binding of a Hedgehog signaling ligand to said SCUBE2 protein, wherein the amino acid sequence of said CUB domain is shown in SEQ ID No. 2.
In a preferred embodiment of the invention, the antibody comprises a heavy chain and a light chain,
wherein the variable region of the heavy chain has complementarity determining region CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO 9, 15 or 20,
VH-CDR2 shown in SEQ ID NO 10, 16 or 21, and
VH-CDR3 shown in SEQ ID No. 11, 17 or 22;
and, the variable region of the light chain has complementarity determining region CDRs selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO 12, 18 or 23,
VL-CDR2 shown in SEQ ID NO 13 or 24, and
VL-CDR3 as shown in SEQ ID NO 14, 19 or 25;
and, any one of the amino acid sequences described above further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid, and that allows a derivative antibody comprising the heavy and light chains of the derivative CDR sequence to retain SCUBE2 binding affinity.
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 3; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 4.
In another preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 5; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 6.
In another preferred embodiment of the invention, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 7; and the light chain variable region of the antibody contains an amino acid sequence shown in SEQ ID NO. 8.
And, the amino acid sequence further includes a sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence, preferably an amino acid sequence having homology or sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
Methods of determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer molecular biology (Computational Molecular Biology), lesk, a.m. editions, oxford university press, new york, 1988; biological calculation: informatics and genome project (Biocomputing: informatics and Genome Projects), smith, d.w. editions, academic press, new york, 1993; computer analysis of sequence data (Computer Analysis of Sequence Data), first part, griffin, a.m. and Griffin, h.g. editions, humana Press, new jersey, 1994; sequence analysis in molecular biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and sequence analysis primer (Sequence Analysis Primer), gribskov, m. and deveverux, j. Code M Stockton Press, new york, 1991 and carllo, h. and Lipman, d., SIAM j.applied math.,48:1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: GCG package (Devereux, J. Et al, 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. Et al, 1990). BLASTX programs are available to the public from NCBI and other sources (BLAST handbook, altschul, S. Et al, NCBI NLM NIH Bethesda, md.20894; altschul, S. Et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
Preferably, the antibodies described herein are one or more of full length antibodies, antigen-antibody binding domain protein fragments, bispecific antibodies, multispecific antibodies, single chain antibodies (single chain antibody fragment, scFv), single domain antibodies (single domain antibody, sdAb) and single domain antibodies (sign-domain antibodies), and monoclonal or polyclonal antibodies made from the above antibodies. The monoclonal antibodies can be developed by a variety of routes and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the main stream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
The antibody full-length protein is a conventional antibody full-length protein in the art, and comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region form the full-length protein of the fully human antibody. Preferably, the antibody full-length protein is IgG1, igG2, igG3, or IgG4.
The antibody of the present invention may be a double-or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.
The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as IgA, igD, igE, igG and any one or more of IgM antibodies or antibodies of other subtypes.
The single chain antibody is a conventional single chain antibody in the field, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
Wherein the animal is preferably a mammal, such as a mouse.
The antibodies of the invention can be chimeric, humanized, CDR grafted and/or modified antibodies that target SCUBE2 (e.g., human SCUBE 2).
In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.
In the above aspect of the present invention, more preferably, the number of the added, deleted, modified and/or substituted amino acids may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, still more preferably 1 to 2.
The SCUBE2 antibody can cut off the source of SHH released by tumor cells, and simultaneously, a Hedgehog signal inhibitor, such as a SHH neutralizing antibody or a SMO inhibitor, can be combined to inhibit Hedgehog signals and further inhibit tumor progression.
Recombinant proteins
The invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR 1), heavy chain CDR2 (VH-CDR 2) and heavy chain CDR3 (VH-CDR 3) of the antibody of the invention, and/or one or more of the light chain CDR1 (VL-CDR 1), light chain CDR2 (VL-CDR 2) and light chain CDR3 (VL-CDR 3) of the SCUBE2 of the invention.
Preferably, the recombinant protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, wherein the antibody heavy chain constant region is conventional in the art, preferably a rat or human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.
In another preferred embodiment, the recombinant protein comprises an antibody of the invention.
The recombinant protein is a protein conventional in the art, preferably, it is one or more of an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody fragment, scFv), a single domain antibody (single domain antibody, sdAb) and a single domain antibody (signaling-domain antibody), and a monoclonal antibody or polyclonal antibody produced by the above antibodies. The monoclonal antibodies can be developed by a variety of routes and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the main stream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
The antibody full-length protein is a conventional antibody full-length protein in the art, and comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region form the full-length protein of the fully human antibody. Preferably, the antibody full-length protein is IgG1, igG2, igG3, or IgG4.
The single chain antibody is a conventional single chain antibody in the field, and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, which comprises the Fd segment of the light chain variable region, the light chain constant region and the heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F (ab').
The single domain antibody is a conventional single domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region.
The single region antibody is a conventional single region antibody in the art, which comprises only the heavy chain variable region.
Wherein, the preparation method of the recombinant protein is a preparation method conventional in the field. The preparation method preferably comprises the following steps: isolated from expression transformants recombinantly expressing the protein or obtained by artificially synthesizing the protein sequence. The isolation from the expression transformant recombinantly expressing the protein preferably comprises the following steps: cloning the nucleic acid molecule which codes for the protein and has point mutation into a recombinant vector, transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant, and culturing the obtained recombinant expression transformant to obtain the recombinant protein by separation and purification.
Nucleic acid
The invention also provides a nucleic acid encoding an antibody or recombinant protein of the invention or a Chimeric Antigen Receptor (CAR) construct of an antibody of the invention as described above.
The preparation method of the nucleic acid is a preparation method conventional in the art, and preferably comprises the following steps: the nucleic acid molecules encoding the above proteins are obtained by gene cloning techniques or by artificial total sequence synthesis.
It is known to those skilled in the art that a nucleotide sequence encoding the amino acid sequence of the above protein may be appropriately introduced into a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide. Homologs of the polynucleotides of the invention may be obtained by substitution, deletion or addition of one or more bases of the gene encoding the protein sequence within a range that retains antibody activity.
Carrier body
The invention also provides a recombinant expression vector comprising the nucleic acid.
Wherein said recombinant expression vector is obtainable by methods conventional in the art, namely: the nucleic acid molecule is constructed by connecting the nucleic acid molecule to various expression vectors. The expression vector is a variety of vectors conventional in the art, as long as it can accommodate the aforementioned nucleic acid molecule. The carrier preferably comprises: various plasmids, cosmids, phage or viral vectors, and the like.
The invention also provides a recombinant expression transformant containing the recombinant expression vector.
Wherein, the preparation method of the recombinant expression transformant is a preparation method conventional in the field, preferably: the recombinant expression vector is transformed into a host cell. The host cell is a variety of host cells conventional in the art, so long as the recombinant expression vector can stably replicate itself and the nucleic acid carried thereby can be expressed efficiently. Preferably, the host cell is an E.coli TG1 or E.coli BL21 cell (expressing a single chain antibody or Fab antibody), or HEK293 or CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell, so that the preferred recombinant expression transformant of the invention can be obtained. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.
Preparation of antibodies
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is already possible to obtain the DNA sequences encoding the antibodies of the invention (or fragments or derivatives thereof) described, entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of 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. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. Biochem.,107:220 (1980).
The antibodies of the invention may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such 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 (salting-out method), centrifugation, osmotic sterilization, 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 of these methods.
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
The antibody of the invention may be coupled to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).
Certain residues on antibodies (e.g., cys or Lys, etc.) are useful in connection with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly attached to the antibody, or indirectly attached through a linker.
Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.
Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimides, halogenated amides (e.g., iodine, bromine, or chlorine); halogenated esters (e.g., iodine, bromine, or chlorinated); halomethyl ketone (e.g., iodine, bromine, or chlorine), benzyl halide (e.g., iodine, bromine, or chlorine); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-di- (mercuromethyl) dioxane, while the counterion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.
The drug may be any cytotoxic, cytostatic or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody.
Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/duocarmycin (duocarmycins), etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolobenzodiazepines (oxazolodiazenes)) and vinca alkaloids (vinca alkaloids).
In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkyne group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.
The invention also provides a method of making an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
In certain embodiments, the methods of the invention comprise: the antibody is bound to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is conjugated to the drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody through the linker.
Application of
The invention also provides the use of an antibody, antibody conjugate ADC, recombinant protein, chimeric Antigen Receptor (CAR) construct and/or immune cell of the invention, e.g. for the preparation of a diagnostic formulation or for the preparation of a medicament.
Preferably, the medicament is a medicament for preventing and/or treating diseases associated with abnormal expression or function of SCUBE 2.
In the present invention, the disease associated with expression or dysfunction of SCUBE2 is a disease associated with expression or dysfunction of SCUBE2, which is conventional in the art. Preferably, the diseases associated with abnormal expression or function of SCUBE2 are cancer, central nervous system tuberculosis and cerebral arteriovenous malformations.
In the present invention, the disease associated with abnormal expression or function of SCUBE2 further includes cancer metastasis, preferably, the cancer metastasis is bone metastasis, for example, breast cancer bone metastasis.
In the present invention, the cancer is a cancer conventional in the art, preferably breast cancer, melanoma, lung cancer.
The antibody of the present invention can effectively prevent and/or reduce the excessive release of SHH caused by the expression or function abnormality of SCUBE2, thereby preventing and/or treating cancer bone metastasis. In cancers where excessive release of SHH has been observed, the antibodies of the invention may be used in combination with SHH neutralizing antibodies, SMO inhibitors, or any other therapeutic agent with Hedgehog signaling inhibition to enhance inhibition of cancer bone metastasis.
Detection application and kit
The antibodies of the invention, or ADCs thereof, may be used in detection applications, for example, for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include, for example, resected samples of tumors, tissue samples prepared by endoscopic methods or puncture of organs or needle biopsies.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may 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. Typically, the route of administration of the pharmaceutical compositions of the present invention is preferably injection or oral. The injection administration preferably comprises intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical compositions are in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be in the form of aqueous solutions, non-aqueous solutions or suspensions, more preferably tablets, capsules, granules, injections or infusions, etc.
The antibodies of the invention may also be used for cellular therapy where the nucleotide sequence is expressed intracellularly, e.g., for chimeric antigen receptor T cell immunotherapy (CAR-T), etc.
The pharmaceutical composition is used for preventing and/or treating diseases related to the expression or the dysfunction of the SCUBE 2.
The pharmaceutical composition of the invention can be directly used for combining a SCUBE2 protein molecule, blocking the combination of SCUBE2 and SHH, and inhibiting the release of SHH, thus being used for preventing and treating diseases such as tumor, tumor metastasis and the like.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the 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 formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The 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 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In a preferred embodiment of the invention, the polypeptides of the invention may be used in combination with therapeutic agents such as Hedgehog signaling inhibitors, antiestrogens or chemotherapeutic agents for the treatment and/or prevention of cancer and/or cancer metastasis. Wherein the Hedgehog signaling inhibitor can be a SHH neutralizing antibody or a SMO inhibitor; the antiestrogens may be tamoxifen, an ovarian function inhibitor, an aromatase inhibitor, or a combination thereof. The chemotherapeutic agent may be an anthracycline (doxorubicin, epirubicin, daunorubicin, and aclarubicin), a taxane (paclitaxel, paclitaxel liposomes, albumin paclitaxel, and docetaxel), or a combination thereof.
In the present invention, the pharmaceutical composition of the present invention preferably further comprises one or more pharmaceutically acceptable carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the protein and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentages are mass percentages of the pharmaceutical composition.
In the present invention, the pharmaceutical composition is preferably administered in an amount effective to reduce or delay the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the symptoms to be treated and the consideration of the results sought. The skilled artisan can determine the effective amount by using the factors described above on an individual basis and the like and using no more than routine experimentation.
When a pharmaceutical composition is used, 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 per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The invention provides application of the pharmaceutical composition in preparing medicines for preventing and/or treating diseases related to expression or dysfunction of SCUBE 2. Preferably, the diseases associated with abnormal expression or function of SCUBE2 are cancer, central nervous system tuberculosis and cerebral arteriovenous malformations. More preferably, the disease associated with abnormal expression or function of SCUBE2 is breast cancer or breast cancer bone metastasis.
Methods and compositions for detecting SCUBE2 protein in a sample
The invention also provides a method of detecting a SCUBE2 protein in a sample (e.g., detecting a cell that overexpresses SCUBE 2), comprising the steps of: the antibody is contacted with a sample to be detected in vitro, and whether the antibody is combined with the sample to be detected to form an antigen-antibody complex is detected.
The meaning of overexpression is conventional in the art and refers to overexpression of the SCUBE2 protein in the sample to be examined (due to increased transcription, post-transcriptional processing, translation, post-translational processing and altered protein degradation), as well as to local overexpression and increased functional activity due to altered protein transport patterns (increased nuclear localization), as in the case of increased enzymatic hydrolysis of the substrate.
In the present invention, the above-mentioned detection mode of whether or not an antigen-antibody complex is formed by binding is a conventional detection mode in the art, preferably a flow cytometry (FACS) detection.
The present invention provides a composition for detecting a SCUBE2 protein in a sample, comprising the above-described antibody, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient. Preferably, it further comprises a compound composed of the functional fragment of the above antibody as an active ingredient.
The main advantages of the invention include:
1) The antibody can competitively bind with the SCUBE2 protein, prevent the combination of the SCUBE2 protein and SHH, inhibit the capability of shearing and releasing the SHH of the SCUBE2 protein in vitro, obviously inhibit the release of the SHH mediated by breast cancer cells and the bone metastasis process induced by the release of the SHH in vivo, and has the prospect of treating breast cancer bone metastasis.
2) The SCUBE2 neutralizing antibody provided by the invention has therapeutic potential for other tumors which highly express SCUBE2 and start downstream Hedgehog signals besides breast cancer bone metastasis, and has a corresponding application prospect.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
EXAMPLE 1 screening of SCUBE2 monoclonal antibodies
In this example, a murine monoclonal neutralizing antibody against the human SCUBE2 protein was screened.
In this example, the full-length sequence of SCUBE2 (SEQ ID NO: 1) was cloned from human breast cancer cell MCF7, and the CUB functional region (809-918aa SEQ ID NO:2) in the full-length sequence of SCUBE2 annotated in NCBI was selected and constructed onto pGEX4T-AB1 vector, and the prokaryotic system expressed the purified protein.
Full length sequence of SCUBE 2:
MGVAGRNRPGAAWAVLLLLLLLPPLLLLAGAVPPGRGRAAGPQEDVDECAQGLDDCHADALCQNTPTSYKCSCKPGYQGEGRQCEDIDECGNELNGGCVHDCLNIPGNYRCTCFDGFMLAHDGHNCLDVDECLENNGGCQHTCVNVMGSYECCCKEGFFLSDNQHTCIHRSEEGLSCMNKDHGCSHICKEAPRGSVACECRPGFELAKNQRDCILTCNHGNGGCQHSCDDTADGPECSCHPQYKMHTDGRSCLEREDTVLEVTESNTTSVVDGDKRVKRRLLMETCAVNNGGCDRTCKDTSTGVHCSCPVGFTLQLDGKTCKDIDECQTRNGGCDHFCKNIVGSFDCGCKKGFKLLTDEKSCQDVDECSLDRTCDHSCINHPGTFACACNRGYTLYGFTHCGDTNECSINNGGCQQVCVNTVGSYECQCHPGYKLHWNKKDCVEVKGLLPTSVSPRVSLHCGKSGGGDGCFLRCHSGIHLSSDVTTIRTSVTFKLNEGKCSLKNAELFPEGLRPALPEKHSSVKESFRYVNLTCSSGKQVPGAPGRPSTPKEMFITVEFELETNQKEVTASCDLSCIVKRTEKRLRKAIRTLRKAVHREQFHLQLSGMNLDVAKKPPRTSERQAESCGVGQGHAENQCVSCRAGTYYDGARERCILCPNGTFQNEEGQMTCEPCPRPGNSGALKTPEAWNMSECGGLCQPGEYSADGFAPCQLCALGTFQPEAGRTSCFPCGGGLATKHQGATSFQDCETRVQCSPGHFYNTTTHRCIRCPVGTYQPEFGKNNCVSCPGNTTTDFDGSTNITQCKNRRCGGELGDFTGYIESPNYPGNYPANTECTWTINPPPKRRILIVVPEIFLPIEDDCGDYLVMRKTSSSNSVTTYETCQTYERPIAFTSRSKKLWIQFKSNEGNSARGFQVPYVTYDEDYQELIEDIVRDGRLYASENHQEILKDKKLIKALFDVLAHPQNYFKYTAQESREMFPRSFIRLLRSKVSRFLRPYK(SEQ ID NO:1)
CUB domain (809-918 aa of SEQ ID NO: 1)
CGGELGDFTGYIESPNYPGNYPANTECTWTINPPPKRRILIVVPEIFLPIEDDCGDYLVMRKTSSSNSVTTYETCQTYERPIAFTSRSKKLWIQFKSNEGNSARGFQVPY(SEQ ID NO:2)
Balb/C mice were immunized using the purified recombinant CUB domain protein as antigen. After 3 immunizations, ELISA was performed on the mouse serum to detect the binding capacity to the antigen, and mice with anti-SCUBE 2 immunoglobulin titers were obtained. Meanwhile, human recombinant SHH protein is used as competitive binding protein for SCUBE2 function detection, serum competition ELISA detection is carried out together with antigen protein, and 2 mice with the best detection effect are selected to be killed, and spleen and SP2/0 myeloma cells are taken out for fusion.
Antibody screening is carried out on the fused hybridoma cells, and meanwhile, competition ELISA detection is carried out on hybridoma cell supernatants by using human recombinant SHH proteins. And selecting positive monoclonal antibodies to perform subsequent subcloning twice, and finally screening to obtain 3 positive mouse monoclonal antibodies which are respectively named as LTMA1F1, LTMA16D5 and LTMA24C10.
EXAMPLE 2 determination of the binding Capacity of the SCUBE2 monoclonal antibody to the CUB Domain
In this example, the SCUBE2 monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 screened in example 1 were tested for their ability to bind to the CUB domain (SCUBE 2 proteins 809-918 aa).
First, a 96-well ELISA plate is coated with CUB domain antigen protein, and after hybridoma cell supernatant is added, the mixture is incubated for 2 hours at room temperature. After 3 washes, goat secondary antibody conjugated with mouse antibody was added and incubated for 1 hour at room temperature. After 3 washes the reaction substrate was added, the reaction was terminated after 5-10 minutes and immediately the absorbance signal at 450nM was detected using an microplate reader.
Results: as shown in Table 1, compared with the negative control and the blank control, 3 strains of the SCUBE2 murine monoclonal antibodies screened by the invention all have stronger binding capacity with the CUB domain in the SCUBE2 protein.
Table 1: binding ability of SCUBE2 monoclonal antibodies to CUB Domain
SCUBE2 murine monoclonal antibodies Absorption value of 450nM
LTMA1F1 1.268
LTMA16D5 1.279
LTMA24C10 0.695
Negative control 0.067
Blank control 0.061
EXAMPLE 3 murine monoclonal antibody to SCUBE2 blocks protein function of SCUBE2
In this example, it was examined whether 3 strains of the murine monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 selected in example 1 blocked the protein function of SCUBE 2.
Since the CUB domain of the SCUBE2 protein binds to the SHH protein, it functions to cleave and release SHH. The antibodies screened in example 1, if capable of competitively blocking binding of the CUB domain to SHH protein, inhibit the ability of the SCUBE2 protein to cleave and release SHH.
In this example, competition ELISA experiments were first used for verification. The 96-well ELISA plate is coated with the SCUBE2 antigen protein, hybridoma cell supernatants and different concentrations of competitive protein SHH are added, and incubated for 2 hours at room temperature, and negative control is the competitive binding result of adding commercial SCUBE2 antibody (the antibody recognition site is a non-CUB region). After 3 washes, goat secondary antibody conjugated with mouse antibody was added and incubated for 1 hour at room temperature. After 3 washes the reaction substrate was added, the reaction was terminated after 5-10 minutes and immediately the absorbance signal at 450nM was detected using an microplate reader.
Results: the case of different antibodies competing with SHH protein for binding to SCUBE2 protein is shown in Table 2.
Table 2: SCUBE2 monoclonal antibodies and SHH proteins competitively bind to SCUBE2 proteins
Figure BDA0003427493640000251
The SHH protein binds specifically to the CUB region of the SCUBE2 protein. Table 2 shows that as the concentration of the competitor SHH added to the system increases, the binding of the negative control antibody with the non-CUB region at the antibody recognition site to the SCUBE2 was not significantly changed, while the binding of the three screened SCUBE2 murine monoclonal antibodies of the present invention to the CUB domain was gradually decreased.
This suggests that the binding sites of these three mouse monoclonal antibodies to SCUBE2 are all binding sites for SHH protein to SCUBE2 protein, and thus have the ability to competitively block binding of SHH protein to SCUBE2 protein.
EXAMPLE 4 SCUBE2 monoclonal antibodies
In this example, the positive hybridoma cells producing the three antibodies described above were subjected to second-generation sequencing, and the corresponding amino acid sequences of the antibody variable region sequences were determined as shown in Table 3.
TABLE 3 variable region sequences of antibodies of the invention
Figure BDA0003427493640000261
Note that: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence is italic FR sequence, and the bottom line is CDR sequence.
The amino acid sequences corresponding to the antibody heavy and light chain CDR regions are shown in Table 4.
TABLE 4 CDR region sequences for each heavy and light chain
Figure BDA0003427493640000271
EXAMPLE 5 purification of SCUBE2 monoclonal antibody and subtype detection
In this example, the antibody was collected and purified. Firstly, 200 ten thousand hybridoma cells are injected into the abdominal cavity of a Balb/C mouse sensitized in advance, after 7-14 days of observation, the mouse is sacrificed to collect ascites, and a Protein G antibody affinity column is used for purification to obtain a high-concentration SCUBE2 monoclonal antibody. The purity of the resulting antibodies was then detected using coomassie brilliant blue staining. A difference in the size of the heavy and light chain band positions of the different monoclonal antibodies was observed in the Coomassie brilliant blue staining results, and antibody subtypes were detected using an antibody subtype identification ELISA kit.
Results: clear heavy and light chain bands of the antibodies appeared in the protein lanes after purification, and no impurity bands, indicating that the purity of the obtained antibodies was high (fig. 1A). Antibodies LTMA1F1 and LTMA16D5 were detected as belonging to the IgG1 class, and antibody LTMA24C10 was detected as belonging to the IgM subtype (FIG. 1B).
EXAMPLE 6 detection of binding specificity of SCUBE2 monoclonal antibody
In order to detect the binding specificity of the monoclonal antibodies obtained by the screening of the present invention, in this example, the recognition of the full-length SCUBE2 protein in breast cancer cells by three SCUBE2 murine monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 of the present invention was detected by immunoblotting (western blot).
Results: in the protein lanes over-expressing SCUBE2, the SCUBE2 monoclonal antibodies all exhibited accurate and specific recognition effects (fig. 1C). The SCUBE2 monoclonal antibody obtained by the invention has higher recognition specificity to the human full-length SCUBE2 protein.
Example 7 in vitro neutralizing Effect of SCUBE2 monoclonal antibodies
In this example, the in vitro neutralizing effect of a SCUBE2 monoclonal antibody on the CUB region was examined.
GLI reporter system activation following SCUBE2 mab treatment to inhibit MCF7 cell supernatant induction
The SCUBE2 neutralizing antibody blocks the ability of SCUBE2 to cleave and release SHH by competitively binding to the functional domain CUB region of SCUBE2, thereby blocking the binding of SHH to SCUBE 2. GLI reporting systems are commonly used in the art to detect the release levels of cellular SHH. Thus, in this example, an in vitro GLI reporter system initiation experiment was used to detect the effect of SCUBE2 antibodies on tumor cell SHH release.
After 24 hours of co-incubation of the SCUBE2 monoclonal antibody with breast cancer cells MCF7, a supernatant suspension of cancer cells was collected for treatment of the target cells MC3T3 transferred into the GLI reporter system.
Results: the supernatant of breast cancer in the control IgG treated group significantly started the GLI reporting system compared to the blank DMEM, while the ability of MCF7 cell supernatant to induce GLI initiation was significantly inhibited after SCUBE2 monoclonal antibody addition (fig. 2A). It was demonstrated that SCUBE2 monoclonal antibodies inhibited the release of SHH extracellular by breast cancer cells.
Inhibition of MCF7 cell supernatant induced osteoblast differentiation following SCUBE2 mab treatment
SHH significantly promotes osteoblast differentiation, manifesting as an upregulation of ALP enzyme activity. In this example the effect of SCUBE2 monoclonal antibodies on the release of SHH from tumor cells was examined by assaying ALP enzyme activity. MCF7 cell supernatants after control IgG and SCUBE2 antibody treatment were collected, respectively, and osteoblast differentiation experiments were induced in vitro.
Results: the control IgG treated group MCF7 supernatant significantly promoted the level of ALP enzyme activity in osteoblasts, indicating that breast cancer cells released high levels of SHH, which could initiate osteoblast differentiation. While the SCUBE2 monoclonal antibody treatment groups all inhibit the degree of the breast cancer cells inducing the osteoblast differentiation (figure 2B), which shows that the three neutralizing antibodies in the invention can inhibit the release of the breast cancer cells SHH, thereby inhibiting the osteogenic differentiation induced by tumor cells.
Expression of SHH in breast cancer cell supernatants after treatment with different concentrations of SCUBE2 mab
This example also examined SHH expression in breast cancer cell supernatants after treatment with varying SCUBE2 monoclonal antibody concentrations.
Results: it was shown that with increasing concentration of SCUBE2 monoclonal antibody, the lower the content of SHH released extracellular by breast cancer cells (fig. 2C).
The results of this example demonstrate that three SCUBE2 monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 of the invention each inhibit the cleavage release of SHH from SCUBE2 protein in vitro, and act to neutralize the function of SCUBE2 protein.
EXAMPLE 8 inhibition of breast cancer bone metastasis by SCUBE2 monoclonal antibodies
In this example, it was examined whether or not a SCUBE2 monoclonal antibody has an effect of inhibiting bone metastasis of breast cancer in vivo.
After injecting MCF7 human breast cancer cells with luciferase label into immunodeficient mice, luciferase substrate was injected and light signals corresponding to the metastatic load were observed. The results showed that tail vein injection of SCUBE2 monoclonal antibody can significantly inhibit bone metastasis of breast cancer in mice (fig. 3A, B).
The left ventricle of 4T1.2 mouse breast cancer cells with luciferase label was injected into Balb/C mice. The results showed that intraperitoneal injection of SCUBE2 monoclonal antibody also significantly inhibited bone metastasis signals in immunized normal mice (fig. 3C, D).
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Shanghai nutrition and health institute of China academy of sciences
<120> SCUBE2 neutralizing antibody and medical use thereof
<130> P2021-2822
<160> 25
<170> PatentIn version 3.5
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<211> 999
<212> PRT
<213> Homo sapiens (Homo sapiens)
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Met Gly Val Ala Gly Arg Asn Arg Pro Gly Ala Ala Trp Ala Val Leu
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Leu Leu Leu Leu Leu Leu Pro Pro Leu Leu Leu Leu Ala Gly Ala Val
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Pro Pro Gly Arg Gly Arg Ala Ala Gly Pro Gln Glu Asp Val Asp Glu
35 40 45
Cys Ala Gln Gly Leu Asp Asp Cys His Ala Asp Ala Leu Cys Gln Asn
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Thr Pro Thr Ser Tyr Lys Cys Ser Cys Lys Pro Gly Tyr Gln Gly Glu
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Gly Arg Gln Cys Glu Asp Ile Asp Glu Cys Gly Asn Glu Leu Asn Gly
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Gly Cys Val His Asp Cys Leu Asn Ile Pro Gly Asn Tyr Arg Cys Thr
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Cys Phe Asp Gly Phe Met Leu Ala His Asp Gly His Asn Cys Leu Asp
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Val Asp Glu Cys Leu Glu Asn Asn Gly Gly Cys Gln His Thr Cys Val
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Asn Val Met Gly Ser Tyr Glu Cys Cys Cys Lys Glu Gly Phe Phe Leu
145 150 155 160
Ser Asp Asn Gln His Thr Cys Ile His Arg Ser Glu Glu Gly Leu Ser
165 170 175
Cys Met Asn Lys Asp His Gly Cys Ser His Ile Cys Lys Glu Ala Pro
180 185 190
Arg Gly Ser Val Ala Cys Glu Cys Arg Pro Gly Phe Glu Leu Ala Lys
195 200 205
Asn Gln Arg Asp Cys Ile Leu Thr Cys Asn His Gly Asn Gly Gly Cys
210 215 220
Gln His Ser Cys Asp Asp Thr Ala Asp Gly Pro Glu Cys Ser Cys His
225 230 235 240
Pro Gln Tyr Lys Met His Thr Asp Gly Arg Ser Cys Leu Glu Arg Glu
245 250 255
Asp Thr Val Leu Glu Val Thr Glu Ser Asn Thr Thr Ser Val Val Asp
260 265 270
Gly Asp Lys Arg Val Lys Arg Arg Leu Leu Met Glu Thr Cys Ala Val
275 280 285
Asn Asn Gly Gly Cys Asp Arg Thr Cys Lys Asp Thr Ser Thr Gly Val
290 295 300
His Cys Ser Cys Pro Val Gly Phe Thr Leu Gln Leu Asp Gly Lys Thr
305 310 315 320
Cys Lys Asp Ile Asp Glu Cys Gln Thr Arg Asn Gly Gly Cys Asp His
325 330 335
Phe Cys Lys Asn Ile Val Gly Ser Phe Asp Cys Gly Cys Lys Lys Gly
340 345 350
Phe Lys Leu Leu Thr Asp Glu Lys Ser Cys Gln Asp Val Asp Glu Cys
355 360 365
Ser Leu Asp Arg Thr Cys Asp His Ser Cys Ile Asn His Pro Gly Thr
370 375 380
Phe Ala Cys Ala Cys Asn Arg Gly Tyr Thr Leu Tyr Gly Phe Thr His
385 390 395 400
Cys Gly Asp Thr Asn Glu Cys Ser Ile Asn Asn Gly Gly Cys Gln Gln
405 410 415
Val Cys Val Asn Thr Val Gly Ser Tyr Glu Cys Gln Cys His Pro Gly
420 425 430
Tyr Lys Leu His Trp Asn Lys Lys Asp Cys Val Glu Val Lys Gly Leu
435 440 445
Leu Pro Thr Ser Val Ser Pro Arg Val Ser Leu His Cys Gly Lys Ser
450 455 460
Gly Gly Gly Asp Gly Cys Phe Leu Arg Cys His Ser Gly Ile His Leu
465 470 475 480
Ser Ser Asp Val Thr Thr Ile Arg Thr Ser Val Thr Phe Lys Leu Asn
485 490 495
Glu Gly Lys Cys Ser Leu Lys Asn Ala Glu Leu Phe Pro Glu Gly Leu
500 505 510
Arg Pro Ala Leu Pro Glu Lys His Ser Ser Val Lys Glu Ser Phe Arg
515 520 525
Tyr Val Asn Leu Thr Cys Ser Ser Gly Lys Gln Val Pro Gly Ala Pro
530 535 540
Gly Arg Pro Ser Thr Pro Lys Glu Met Phe Ile Thr Val Glu Phe Glu
545 550 555 560
Leu Glu Thr Asn Gln Lys Glu Val Thr Ala Ser Cys Asp Leu Ser Cys
565 570 575
Ile Val Lys Arg Thr Glu Lys Arg Leu Arg Lys Ala Ile Arg Thr Leu
580 585 590
Arg Lys Ala Val His Arg Glu Gln Phe His Leu Gln Leu Ser Gly Met
595 600 605
Asn Leu Asp Val Ala Lys Lys Pro Pro Arg Thr Ser Glu Arg Gln Ala
610 615 620
Glu Ser Cys Gly Val Gly Gln Gly His Ala Glu Asn Gln Cys Val Ser
625 630 635 640
Cys Arg Ala Gly Thr Tyr Tyr Asp Gly Ala Arg Glu Arg Cys Ile Leu
645 650 655
Cys Pro Asn Gly Thr Phe Gln Asn Glu Glu Gly Gln Met Thr Cys Glu
660 665 670
Pro Cys Pro Arg Pro Gly Asn Ser Gly Ala Leu Lys Thr Pro Glu Ala
675 680 685
Trp Asn Met Ser Glu Cys Gly Gly Leu Cys Gln Pro Gly Glu Tyr Ser
690 695 700
Ala Asp Gly Phe Ala Pro Cys Gln Leu Cys Ala Leu Gly Thr Phe Gln
705 710 715 720
Pro Glu Ala Gly Arg Thr Ser Cys Phe Pro Cys Gly Gly Gly Leu Ala
725 730 735
Thr Lys His Gln Gly Ala Thr Ser Phe Gln Asp Cys Glu Thr Arg Val
740 745 750
Gln Cys Ser Pro Gly His Phe Tyr Asn Thr Thr Thr His Arg Cys Ile
755 760 765
Arg Cys Pro Val Gly Thr Tyr Gln Pro Glu Phe Gly Lys Asn Asn Cys
770 775 780
Val Ser Cys Pro Gly Asn Thr Thr Thr Asp Phe Asp Gly Ser Thr Asn
785 790 795 800
Ile Thr Gln Cys Lys Asn Arg Arg Cys Gly Gly Glu Leu Gly Asp Phe
805 810 815
Thr Gly Tyr Ile Glu Ser Pro Asn Tyr Pro Gly Asn Tyr Pro Ala Asn
820 825 830
Thr Glu Cys Thr Trp Thr Ile Asn Pro Pro Pro Lys Arg Arg Ile Leu
835 840 845
Ile Val Val Pro Glu Ile Phe Leu Pro Ile Glu Asp Asp Cys Gly Asp
850 855 860
Tyr Leu Val Met Arg Lys Thr Ser Ser Ser Asn Ser Val Thr Thr Tyr
865 870 875 880
Glu Thr Cys Gln Thr Tyr Glu Arg Pro Ile Ala Phe Thr Ser Arg Ser
885 890 895
Lys Lys Leu Trp Ile Gln Phe Lys Ser Asn Glu Gly Asn Ser Ala Arg
900 905 910
Gly Phe Gln Val Pro Tyr Val Thr Tyr Asp Glu Asp Tyr Gln Glu Leu
915 920 925
Ile Glu Asp Ile Val Arg Asp Gly Arg Leu Tyr Ala Ser Glu Asn His
930 935 940
Gln Glu Ile Leu Lys Asp Lys Lys Leu Ile Lys Ala Leu Phe Asp Val
945 950 955 960
Leu Ala His Pro Gln Asn Tyr Phe Lys Tyr Thr Ala Gln Glu Ser Arg
965 970 975
Glu Met Phe Pro Arg Ser Phe Ile Arg Leu Leu Arg Ser Lys Val Ser
980 985 990
Arg Phe Leu Arg Pro Tyr Lys
995
<210> 2
<211> 110
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 2
Cys Gly Gly Glu Leu Gly Asp Phe Thr Gly Tyr Ile Glu Ser Pro Asn
1 5 10 15
Tyr Pro Gly Asn Tyr Pro Ala Asn Thr Glu Cys Thr Trp Thr Ile Asn
20 25 30
Pro Pro Pro Lys Arg Arg Ile Leu Ile Val Val Pro Glu Ile Phe Leu
35 40 45
Pro Ile Glu Asp Asp Cys Gly Asp Tyr Leu Val Met Arg Lys Thr Ser
50 55 60
Ser Ser Asn Ser Val Thr Thr Tyr Glu Thr Cys Gln Thr Tyr Glu Arg
65 70 75 80
Pro Ile Ala Phe Thr Ser Arg Ser Lys Lys Leu Trp Ile Gln Phe Lys
85 90 95
Ser Asn Glu Gly Asn Ser Ala Arg Gly Phe Gln Val Pro Tyr
100 105 110
<210> 3
<211> 116
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ile Tyr
20 25 30
Asn Ile His Trp Val Lys Leu Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ser Ile Tyr Pro Gly Asp Gly His Thr Ser Tyr Asn Pro Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Phe Cys
85 90 95
Ala Arg Leu Glu Lys Tyr Thr Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 4
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly
85 90 95
Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 5
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Ile Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Gly Arg Phe Thr Pro Val Val Glu Asp Tyr Trp Gly Gln Gly Ser Thr
100 105 110
Leu Thr Val Ser Ser
115
<210> 6
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Ala
85 90 95
Thr His Phe Pro His Thr Phe Gly Ser Gly Thr Glu Leu Glu Ile Lys
100 105 110
<210> 7
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45
Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Asp Tyr Tyr Gly Ser Ser Ser Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 8
<211> 111
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser
20 25 30
Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 9
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Gly Tyr Thr Phe Thr Ile Tyr Asn
1 5
<210> 10
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Ile Tyr Pro Gly Asp Gly His Thr
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Ala Arg Leu Glu Lys Tyr Thr Asp Tyr
1 5
<210> 12
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Gln Ser Leu Leu Ala Ser Asp Gly Lys Thr Tyr
1 5 10
<210> 13
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Leu Val Ser
1
<210> 14
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Trp Gln Gly Thr His Phe Pro Phe Thr
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Gly Tyr Thr Phe Thr Asp Tyr Ser
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Ile Asn Thr Glu Thr Gly Glu Pro
1 5
<210> 17
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Gly Arg Phe Thr Pro Val Val Glu Asp Tyr
1 5 10
<210> 18
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Gln Ser Leu Leu Tyr Ser Asn Gly Lys Thr Tyr
1 5 10
<210> 19
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Val Gln Ala Thr His Phe Pro His Thr
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Gly Tyr Ser Phe Thr Gly Tyr Thr
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Ile Asn Pro Tyr Asn Gly Gly Thr
1 5
<210> 22
<211> 13
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Ala Arg Arg Asp Tyr Tyr Gly Ser Ser Ser Phe Asp Tyr
1 5 10
<210> 23
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr
1 5 10
<210> 24
<211> 3
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Tyr Ala Ser
1
<210> 25
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Gln His Ser Trp Glu Ile Pro Trp Thr
1 5

Claims (10)

1. An antibody that binds to a SCUBE2 protein, wherein the antibody specifically binds to the CUB domain of a SCUBE2 protein and is capable of competitively blocking the binding of a Hedgehog signaling ligand to the SCUBE2 protein,
wherein the amino acid sequence of the CUB domain is shown as SEQ ID NO. 2.
2. The antibody of claim 1, wherein the antibody comprises a heavy chain and a light chain,
wherein the variable region of the heavy chain has complementarity determining region CDRs selected from the group consisting of:
the amino acid sequence is shown as VH-CDR1 shown as SEQ ID NO 9, 15 or 20,
VH-CDR2 having the amino acid sequence shown in SEQ ID No. 10, 16 or 21, and
VH-CDR3 with amino acid sequence as shown in SEQ ID No. 11, 17 or 22;
And, the variable region of the light chain has complementarity determining region CDRs selected from the group consisting of:
VL-CDR1 with the amino acid sequence shown as SEQ ID NO 12, 18 or 23,
VL-CDR2 having an amino acid sequence shown in SEQ ID NO 13 or 24, and
the amino acid sequence is shown as VL-CDR3 of SEQ ID NO 14, 19 or 25.
3. The antibody of claim 1, wherein the heavy chain variable region of the antibody has complementarity determining region CDRs selected from the group consisting of:
VH 1) a VH-CDR1 having an amino acid sequence shown in SEQ ID No. 9, a VH-CDR2 shown in SEQ ID No. 10, and a VH-CDR3 shown in SEQ ID No. 11;
VH 2) a VH-CDR1 having an amino acid sequence shown in SEQ ID No. 15, a VH-CDR2 shown in SEQ ID No. 16, and a VH-CDR3 shown in SEQ ID No. 17; or (b)
VH 3) a VH-CDR1 shown in SEQ ID NO. 20, a VH-CDR2 shown in SEQ ID NO. 21, and a VH-CDR3 shown in EQ ID NO. 22.
4. The antibody of claim 1, wherein the light chain variable region of the antibody has complementarity determining region CDRs selected from the group consisting of:
VL 1) a VL-CDR1 having an amino acid sequence shown in SEQ ID NO. 12, a VL-CDR2 shown in SEQ ID NO. 13, and a VL-CDR3 shown in SEQ ID NO. 14;
VL 2) a VL-CDR1 having an amino acid sequence shown in SEQ ID NO. 18, a VL-CDR2 shown in SEQ ID NO. 13, and a VL-CDR3 shown in SEQ ID NO. 19; and
VL 3) the amino acid sequence is shown as VL-CDR1 of SEQ ID NO. 23, VL-CDR2 of SEQ ID NO. 24, and VL-CDR3 of SEQ ID NO. 25.
5. The antibody of claim 1, wherein the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID No. 3, 5 or 7 and/or the light chain variable region of the antibody has the amino acid sequence set forth in SEQ ID No. 4, 6 or 8.
6. A recombinant protein, wherein said recombinant protein comprises:
(i) The antibody of claim 1; and
(ii) Optionally a tag sequence to assist expression and/or purification.
7. A Chimeric Antigen Receptor (CAR) construct, wherein the scFv fragment of the antigen binding region of the chimeric antigen receptor construct specifically binds to a SCUBE2 protein and the scFv has the heavy chain variable region and the light chain variable region of the antibody of claim 1.
8. A recombinant immune cell expressing the CAR construct of claim 7 exogenously.
9. An antibody conjugate, comprising:
(a) An antibody moiety selected from the group consisting of: the antibody of claim 1; and
(b) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof.
10. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 6, the antibody conjugate of claim 9, or a combination thereof, wherein the active ingredient is used to:
(a) Preparing a diagnostic reagent, a detection plate or a kit; and/or
(b) Preparing medicine for preventing and/or treating diseases related to SCUBE2 protein expression or dysfunction.
CN202111582701.7A 2021-12-22 2021-12-22 SCUBE2 neutralizing antibody and medical use thereof Pending CN116333143A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111582701.7A CN116333143A (en) 2021-12-22 2021-12-22 SCUBE2 neutralizing antibody and medical use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111582701.7A CN116333143A (en) 2021-12-22 2021-12-22 SCUBE2 neutralizing antibody and medical use thereof

Publications (1)

Publication Number Publication Date
CN116333143A true CN116333143A (en) 2023-06-27

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111582701.7A Pending CN116333143A (en) 2021-12-22 2021-12-22 SCUBE2 neutralizing antibody and medical use thereof

Country Status (1)

Country Link
CN (1) CN116333143A (en)

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