Disclosure of Invention
The invention aims to solve the technical problems that the existing antibody aiming at B7H3 is mainly a mouse monoclonal antibody, has large molecular weight, poor tissue penetrability, difficult engineering transformation and the like.
The technical scheme for solving the technical problems is that a nano antibody for resisting B7H3 is provided. The anti-B7H 3 nanobody comprises a heavy chain variable region of CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR 1-3 is any one of the following in sequence:
As shown in SEQ ID No. 1-3, or as shown in SEQ ID No. 5-7, or as shown in SEQ ID No. 9-11, or as shown in SEQ ID No. 13-15, or as shown in SEQ ID No. 17-19, or as shown in SEQ ID No. 21-23, or as shown in SEQ ID No. 25-27, or as shown in SEQ ID No. 29-31, or as shown in SEQ ID No. 33-35, or as shown in SEQ ID No. 37-39, or as shown in SEQ ID No. 41-43, or as shown in SEQ ID No. 45-47, or as shown in SEQ ID No. 49-51, or as shown in SEQ ID No. 53-55, or as shown in SEQ ID No. 57-59.
The anti-B7H 3 nanobody further comprises a framework region, and the VHH chain of the nanobody has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
Further, in the above anti-B7H 3 nanobody, the amino acid sequence of the VHH chain of the nanobody is SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:28、SEQ ID NO:32、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:44、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:56 or any one of SEQ ID NO: 60.
Furthermore, the amino acid sequence of the CDR1 of the anti-B7H 3 nano antibody is shown as SEQ ID NO. 1, the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the CDR3 is shown as SEQ ID NO. 3, and the amino acid sequence of the VHH is shown as SEQ ID NO. 4; or the amino acid sequence of CDR1 is shown as SEQ ID NO. 5, the amino acid sequence of CDR2 is shown as SEQ ID NO. 6, the amino acid sequence of CDR3 is shown as SEQ ID NO. 7, and the amino acid sequence of VHH is shown as SEQ ID NO. 8; or the amino acid sequence of CDR1 is shown as SEQ ID NO. 9, the amino acid sequence of CDR2 is shown as SEQ ID NO. 10, the amino acid sequence of CDR3 is shown as SEQ ID NO. 11, the amino acid sequence of VHH is shown as SEQ ID NO. 12, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 13, the amino acid sequence of CDR2 is shown as SEQ ID NO. 14, the amino acid sequence of CDR3 is shown as SEQ ID NO. 15, the amino acid sequence of VHH is shown as SEQ ID NO. 16, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 17, the amino acid sequence of CDR2 is shown as SEQ ID NO. 18, the amino acid sequence of CDR3 is shown as SEQ ID NO. 19, the amino acid sequence of VHH is shown as SEQ ID NO. 20, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 21, the amino acid sequence of CDR2 is shown as SEQ ID NO. 22, the amino acid sequence of CDR3 is shown as SEQ ID NO. 23, the amino acid sequence of CDR1 is shown as SEQ ID NO. 24, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 25, the amino acid sequence of CDR1 is shown as SEQ ID NO. 16, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 26, or amino acid sequence of CDR3 is shown as SEQ ID NO. 2 is shown as SEQ ID NO. 31, amino acid sequence of CDR2 is shown as SEQ ID NO. 31, and amino acid sequence of CDR2 is shown as SEQ ID NO. 31 is shown as SEQ ID NO. 2 is shown as SEQ ID NO. 12 The amino acid sequence of (C) is shown as SEQ ID NO. 36; or the amino acid sequence of CDR1 is shown as SEQ ID NO. 37, the amino acid sequence of CDR2 is shown as SEQ ID NO. 38, the amino acid sequence of CDR3 is shown as SEQ ID NO. 39, the amino acid sequence of VHH is shown as SEQ ID NO. 40, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 41, the amino acid sequence of CDR2 is shown as SEQ ID NO. 42, the amino acid sequence of CDR3 is shown as SEQ ID NO. 43, the amino acid sequence of VHH is shown as SEQ ID NO. 44, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 45, the amino acid sequence of CDR2 is shown as SEQ ID NO. 46, the amino acid sequence of CDR3 is shown as SEQ ID NO. 47, the amino acid sequence of VHH is shown as SEQ ID NO. 48, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 49, the amino acid sequence of CDR2 is shown as SEQ ID NO. 50, the amino acid sequence of CDR3 is shown as SEQ ID NO. 51, the amino acid sequence of CDR3 is shown as SEQ ID NO. 52, or the amino acid sequence of CDR1 is shown as SEQ ID NO. 53, the amino acid sequence of CDR2 is shown as SEQ ID NO. 54, the amino acid sequence of CDR3 is shown as SEQ ID NO. 54, amino acid sequence of CDR3 is shown as SEQ ID NO. 58, the amino acid sequence of VHH is shown as SEQ ID NO. 58.
Wherein the above antibody sequences are shown in the following table 1:
TABLE 1 amino acid sequences of antibodies
Further, the anti-B7H 3 nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific nanobody, or a fusion nanobody.
Wherein the multispecific nano antibody is an anti-B7H 3/CD3 bispecific antibody.
Furthermore, the invention also provides an isolated polynucleotide for encoding the nano antibody.
Further, the nucleotide sequence of the isolated polynucleotide is shown in SEQ ID NO. 61-75.
The invention also provides a recombinant vector containing the isolated polynucleotide.
The invention also provides a host cell containing the recombinant vector.
Wherein the host cell is at least one of a prokaryotic host cell, a eukaryotic host cell or a phage. The prokaryotic host cell comprises escherichia coli, streptomyces, bacillus subtilis or mycobacterium, the eukaryotic host cell comprises animal cells, plant cells or fungi, the animal cells are selected from mammal cells, insect cells or caenorhabditis elegans, the mammal cells are selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, vero cells, BHK cells, CV1 cells, hela cells, MDCK cells, hep-2 cells and Per6 cells, and the fungi are selected from any one of Saccharomyces cerevisiae, pichia pastoris, hansen, candida, kluyveromyces lactis, aspergillus nidulans, schizosaccharomyces pombe and yarrowia lipolytica.
The invention also provides an immunoconjugate or pharmaceutical composition comprising the anti-B7H 3 nanobody.
Wherein the immunoconjugate further comprises a therapeutic agent comprising at least one of an immune checkpoint related agent, a toxin, a factor, a drug, a radionuclide, a kinase inhibitor, or a cytotoxic agent.
Wherein the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient, carrier or diluent.
Furthermore, the invention also provides application of the anti-B7H 3 nanobody, the isolated polynucleotide or the host cell in preparing medicaments for preventing, diagnosing or treating tumors.
Further, the tumor comprises at least one of head and neck tumor, craniopharyngeal tumor, prostatic cancer, glioma, skin squamous cell carcinoma, melanoma, intestinal adenocarcinoma, gastric adenocarcinoma, pancreatic cancer, renal clear cell carcinoma, breast cancer, liver cancer, bladder cancer, cervical cancer, skin cancer, neuroblastoma, medulloblastoma, ovarian cancer, lung adenocarcinoma or acute myeloid leukemia.
The invention has the following beneficial effects:
The invention takes the human B7H3 target as antigen, screens 15 anti-B7H 3 specific nano antibodies by phage display technology, can specifically bind to B7H3 antigen, has strong specificity and high affinity, and has the advantages of small molecular weight, high stability, easy transformation, strong solubility and the like. Meanwhile, the B7H3/CD3 bispecific antibody is prepared by connecting an anti-B7H 3 nano antibody and an anti-CD 3 antibody OKT3 in series, and in-vitro and in-vivo experiments show that the B7H3/CD3 bispecific antibody has good anti-tumor effect, and the B7H3/CD3 bispecific antibody can be applied to preparing anti-tumor medicines and has wide anti-tumor effect.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a B7H 3-resistant nanobody, which comprises a heavy chain variable region of a CDR1, a CDR2 and a CDR3, wherein the amino acid sequence of the CDR 1-3 is shown as SEQ ID NO 1-3, or as SEQ ID NO 5-7, or as SEQ ID NO 9-11, or as SEQ ID NO 13-15, or as SEQ ID NO 17-19, or as SEQ ID NO 21-23, or as SEQ ID NO 25-27, or as SEQ ID NO 29-31, or as SEQ ID NO 33-35, or as SEQ ID NO 37-39, or as SEQ ID NO 41-43, or as SEQ ID NO 45-47, or as SEQ ID NO 49-51, or as SEQ ID NO 53-55, or as SEQ ID NO 57-59.
The nanometer antibody for resisting B7H3 also comprises a framework region, and the structure of the heavy chain variable region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
Wherein the nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific nanobody, or a fusion nanobody.
The monovalent nanobody is an antigen-specific nanobody obtained by screening a nanobody library by using a specific antigen, and can keep a strict monomer structure due to a large number of hydrophilic residues on the surface of the monovalent nanobody, and can be combined with the antigen with high specificity and high affinity only in a monomer form.
The multivalent nanobody is a polymer of monovalent antibodies recognizing the same epitope, having a higher antigen affinity than the corresponding monovalent nanobody.
The multispecific antibody is a polymer of monovalent antibodies recognizing different epitopes, can bind to different targets or different epitopes of the same target, and has higher antigen recognition capability than the monovalent antibodies. The nanobody has a simple structure, and can be polymerized together through short connecting sequences, so that the nanobody can be converted into multivalent and multispecific forms.
Furthermore, the invention also provides an isolated polynucleotide for encoding the nano antibody.
Further, the nucleotide sequence of the isolated polynucleotide is shown in SEQ ID NO. 61-75.
SEQ ID NO. 61 nucleotide sequence encoding antibody Nb1
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGATACACCTACAGTAGCTACTGGATGGGCTGGTTCCGCCAGGCTCCAGGGA
AGGAGCGCGAGGGGGTCGCAGCTATTTATACTCGTGGTGGTACCACATACTATGCCGACTCCGT
GAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG
CCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATACGAAAGTGGGGGGTTGGGTACGGAATAACCCCGCCGGAATTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 62 encoding antibody Nb10
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGAATCACCTACGACAGCTATGCCATGGCCTGGTTCCGCCAGGCTCCAGGAA
AACAGCGCGAGGGCGTCGCAAGTCTTTACACTCGTGCTGGTACCACATACTATGCCGACTCCGT
AAAGGGCCGATTCACCATCTCCCACGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG
CCTGAAACCTGAGGACACCGCTATGTACTACTGTGCGACAGATCGAGTCTTCTGGGGTACTTCGTCCCTCCAGAGGACCCGCTATAACGTCTGGGGCCGTGGGACCCAGGTCACCGTCTCCTCA.
SEQ ID NO. 63 nucleotide sequence encoding antibody Nb12
CAGGTGCAGCTGCAGGAGTCTGGGGGAGACTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGAATCACCTACAACTGCTACTCCATGGCCTGGTTCCGCCAGGCTCCAGGAA
AGGAGCGCGAGGGCGTCGCAAGTCTTTATACTTGTGCTGGTACCACATACTATGCCGACTCCAT
AAAGGGCCGATTCACCATCTCCCACGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG
CCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGACAGATCGAGTCTTCTGGGGTACTTCGTCCCTCCAGAGGACCCGCTATAATTACTGGGGCCGTGGGACCCAGGTCACCGTCTCCTCA.
SEQ ID NO. 64 nucleotide sequence encoding antibody Nb15
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCGCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGTAGCCTCTGGATACGCCTACAGTAGAAACTGGGTGGGCTGGTTCCGCCAAACTCCAGGGA
AGGAGCGCGAGGCGGTCGCAGCTATTTATACTGGTGGTGGCAGCACATACTATGCCGACTCCGT
GAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG
CCTGATACCTGAGGACACTGCCATATACTACTGTGCGGCAGATCCGGCTGTCGGGGCTTGGGTTTCCGGGGACCCTTCTCGCCGCTTGAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 65 encoding antibody Nb25
CAGGTGCAGCTGCAGGAGTCTGGGGGAGACTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGAATCACCTACAGCAGCTACTCCATGGCCTGGTTCCGCCAGGCTCCAGGAA
AGGAGCGCGAGGGGGTCGCAAGTATTCATAGTCCTTCTGGTACCACATACTATGCCGACTCCAT
AAAGGGCCGATTCACCATCGCCCACGACAACGCCCTGAACACGGTGTATCTGGAAATGAACAG
CCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATCGAGTCTTCTGGGGTACTTCGTCCCTCCAGAGGACCCGCTATAAGTACTGGGGCCGTGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO 66 encoding antibody Nb52
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTACTTACACCTATAACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCG
AGGGGGTCGCAGCGTTTTATTCCAGAGGTACTAGGGTCTATGCCGACTCCGTGAAGGGCCGATT
CACCATCTCCCGTGACAACGCCAAGAACACGCTGTATCTAGACATTGACATGCTGAGACCTGAC
GACGCTGCCATGTACTACTGTGCGGCTGCCACGGAGTTGATTGGTACTGCTCCGTTAGATGCGAGGACGTATAAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 67 encoding antibody NbH59
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCCCTGGAAGCATCTATAGTAGGATGTGGATGGGCTGGTTCCGCCAGGCTCCAGGG
AAGGAGCGCGACGCGGTCGCAGCTATTTATACTGCTGCTGGTAGCACATACTATGCCGACTCCG
TGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAATACGGTGTATCTGCAAATGAACA
GCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATCCCGCCGTGGGATCATGGGTTGGCTCACGCCCCCTAGGGAGCGTGCGCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 68 encoding antibody Nb60
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCGCC
TGTGCAGCCTCTGGATACAGTCTAAGTAGCGATTTCGTGGCCTGGTTCCGCCAGGCTTCAGGGA
AGGATCGCGAAGACGTCGCAGGTATTTATCCTGGTGGTAGTATGGCACACTATGCCGACGCCGT
GAAGGGCCGATTCACCATCTCCCGAGACAACACCAAGAACATGGTGTATCTGCAAATGAACAG
CCTGAAACCTGAAGACACCGCCATGTACTACTGTGCATCACGGTTTCTTCCCAAGACCGGTCGAACGTGGGACCCGCTCAATTTTGCTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 69 encoding antibody Nb64
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGATACACCTTCAGTCGCCACTGGATGGGCTGGTTCCGCCAGGCTCCAGGGA
AGGAGCGCGAGGGGGTCGCAGCTATTTATACTAATGGTGGTAGCACATACTATGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCCAAGACAACGCCAAGAACACGGTATTTCTGCAAATGAATAG
CCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATTTAGCTGTTGGTAGTTGGCTG
AGACAGGGGGGCCCGTTGAGAATTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。
Nucleotide sequence of SEQ ID NO. 70 encoding antibody Nb70
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGCACTCTGAGACTCTCC
TGTGCAGCCTCTGGATACACCTTAAGTAGCAATTTCGTGGGCTGGTTCCGCCAGGCTTCAGGGA
AGGTCCGCGAAGAGGTCGCAGGTATCTATCCCGGTGATAGTCTTACGCACTATGCCGACGCCGT
GAAGGGCCGATTCACCATCTCCCGAGACAACGTCAAGAACACGGTGTATCTGCAAATGAACAG
CCTGAAACCTGAGGACACCGCCATGTACTACTGTGCGACACGGTTTCACCCCAAGACCAGTCGAACGTGGGACCCGCCCAACTTTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
SEQ ID NO. 71 nucleotide sequence encoding antibody Nb90
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCCCC
TGTGTAGCCTCTGGAATCACCCACATCGACTACTCCATGGCCTGGTTCCGCCAGGCTCCAGGAA
AGGAGCGCGAGGGGGTCGCAAGTATTCACGCTCGTAGCGGTACGACATACTATGCCAACTCCG
TACAGGGCCGATTCACCATCTCCCACGACAAGGCCAAGAACACGGTGTATCTGGAAATGAACA
GCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATCGAGTCTACTGGGGTACTTCGTCCCTCCAGAGGACCCGCTATAACTACTGGGGCCGTGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 72 encoding antibody NbH1
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTACAGCCTCTGGATTTACCGACAGTAGCTACTGGATGGGCTGGTTCCGCCAGGTTCCAGGAA
AGGAGCGCGAGGGGGTCGCAACTATTTATACTCAGCTTGGTACCACATACTATGCCGACTCCGT
GAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG
CCTTAAACCTGAGGACACTGCCATGTACTACTGTGCGGCAGATAGGAAAGTGATGTATTGGGTACAGAATAACCCCGCCGGGATTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
SEQ ID NO. 73 encoding the nucleotide sequence of antibody NbH51
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGAATCACCTACAACAGCTACTCCATGGCCTGGTTCCGCCAGGCTCCAGGAA
AGGAGCGCGAGGGCGTCGCAAGTCTTTACACTCTTGCTGGTACCACATACTATGCCGACTCCGT
AAAGGGCCGATTCACCATCTCCCACGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAG
CCTGAAACCTGAGGACACTGCTATGTACTACTGTGCGACAGATCGAGTCTTCTGGGGTACTTCGTCCCTCCAGAGGACCCGCTATAACGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
Nucleotide sequence of SEQ ID NO. 74 encoding antibody NbH68
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGATACATCGACAGTAGCAACTGGATGGGCTGGTTCCGCCAGGCTCCAGGG
AAGGAGCGCGAGGGGGTCGCAGCCATTTATGCTGATATTGGTACGACATACTATGCCGACTCC
GTGCAGGGCCGATTCACCATCTCGCAAGACACCGCCAAGAACACGGTATATCTGCAAATGAAC
GCACTGAAACCTGACGACACTGCCGCGTACTACTGTGCGATAGGGAGGAAGGTGGGCGCTTGG
TACAACTCCCGTTTCCGATCGTCAATTACTTACTGGGGCCGGGGAACCCAGGTCACCGTCTCCTCA。
SEQ ID NO. 75 nucleotide sequence encoding antibody NbH and 73
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGCAGACACCTACAGTCAGAACTTCATGACGTGGTTCCGCCAGGCTGCAGGGA
AGGAGCGGGAGGGGGTCGCAAGTGTTTATACTGGTAGTGGTGCCACAGTCTATGCCGACTCCG
TGAAGGGCCGATTCACCATCTCCCGAGACAACGCCGAGAACACGGTGTATCTACAAATGAACA
GCCTGAAACCTGAGGACACTGCCATGTACTATTGTGCGGCAAAACTTGTCAGTGGTCGCTGGTTGACGGGGACCTATACCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.
The invention also provides a recombinant vector containing the isolated polynucleotide.
The recombinant vector is an expression vector or a cloning vector, preferably an expression vector.
The embodiment of the invention also provides a host cell containing the recombinant vector according to any of the previous embodiments.
The embodiment of the invention also provides an immunoconjugate or pharmaceutical composition comprising the anti-B7H 3 nanobody of any of the embodiments described above or the antibody of any of the embodiments described above.
In some embodiments, the immunoconjugate further comprises a therapeutic agent.
In some embodiments, the therapeutic agent comprises at least one of an immune checkpoint related agent, a toxin, a factor, a drug, a radionuclide, a kinase inhibitor, and a cytotoxic agent.
In some embodiments, the pharmaceutical composition comprises at least one of a pharmaceutically acceptable excipient, carrier, and diluent.
In a preferred embodiment of the present invention, the carrier is a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes, but is not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talcum powder, and indigo.
The vehicle comprises at least one polar organic solvent and at least one thickener.
The diluent is for example selected from pharmaceutically acceptable water or salts.
Furthermore, the invention also provides application of the anti-B7H 3 nanobody, the isolated polynucleotide or the host cell in preparing medicaments for preventing, diagnosing or treating tumors.
Further, the tumor comprises at least one of head and neck tumor, craniopharyngeal tumor, prostatic cancer, glioma, skin squamous cell carcinoma, melanoma, intestinal adenocarcinoma, gastric adenocarcinoma, pancreatic cancer, renal clear cell carcinoma, breast cancer, liver cancer, bladder cancer, cervical cancer, skin cancer, neuroblastoma, medulloblastoma, ovarian cancer, lung adenocarcinoma or acute myeloid leukemia.
The term "treatment" as used herein includes curing, ameliorating, slowing the condition or pathological characteristics of a patient, or inhibiting the progression of a condition.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 construction of eukaryotic expression vector for 7H3-His protein, expression and purification thereof
1.1 Vector construction
The plasmid containing the full-length gene (gene number NM_ 001024736.2) of B7H3 is taken as a template, a primer is designed for amplification to obtain the extracellular domain (ECD) gene of B7H3, the gene is connected into a pVax-His vector subjected to double enzyme digestion by restriction enzymes PstI and XbaI in a homologous recombination mode, a coated Cana resistance plate is placed in DH5 alpha competence for culture overnight at 37 ℃, monoclonal sequencing is selected for identification, and the plasmid is extracted for the clone expansion culture which is successfully constructed.
1.2 Expression and purification of recombinant proteins
The recombinant plasmid pVax-B7H3 (ECD) -His containing the B7H3 extracellular region gene which is successfully constructed is transfected into HEK293T cells, the recombinant plasmid is changed into a fresh 293 fresh culture medium after 8 hours of transient transformation, the culture supernatant is collected after 5 days of culture, and the recombinant protein B7H3-His with high purity is purified by utilizing an NTA-Ni column through an affinity chromatography mode, and the result is shown in figure 1.
Example 2 screening and preparation of anti-B7H 3 protein nanobody
2.1 Protein emulsification and animal immunization
The purified 1mg of B7H3-His recombinant protein is emulsified with an equal volume of Freund's complete adjuvant and then subjected to subcutaneous immunization on an alashan Bactrian camel through the neck, and then 1mg of B7H3-His recombinant protein is emulsified with an incomplete Freund's complete adjuvant every 2 weeks and subjected to continuous immunization for 3 times, and peripheral blood is collected through the jugular vein. B7H3-His recombinant protein is placed in a 96-well ELISA plate according to 200 ng/Kong Baobei, serum of a camel before and after immunization is subjected to gradient dilution, and then the antibody titer of the B7H3 protein in the serum is detected through indirect ELISA, and the result is shown in a figure 2, wherein the antibody titer of the B7H3-His protein is 1:256,000, which indicates that the immune effect is good, and lays a foundation for the subsequent library construction.
2.2 Construction of the phage antibody library and panning
2.2.1 Isolation of peripheral blood lymphocytes
On day 7 after impact immunization, 200mL of peripheral anticoagulation is collected aseptically through jugular vein, diluted with equal volume of PBS, and 7.5X10-8 peripheral blood lymphocytes are obtained by separating through centrifugation with Ficoll-Paque Plus lymphocyte separation liquid (category 17144002, cytiva) and lymphocyte separation tube, and the obtained lymphocytes can be directly extracted for total RNA or frozen at-80 ℃ for later use.
2.2.2 VHH gene amplification
The total RNA (catalog 74134,RNeasy Plus Mini Kit,QIAGEN) of the obtained lymphocyte is firstly extracted according to the specification, then reverse transcription kit (catalog 18080051, superScript IIIfirst-STRAND SYNTHESIS SYSTEM, invitrogen) is used FOR carrying out reverse transcription by taking RNA as a template to obtain cDNA, the cDNA is used as a template, VHH genes are amplified by nested PCR, primers used in the first round of PCR are CALL001 (with a nucleotide sequence shown as SEQ ID NO: 76) and CALL002 (with a nucleotide sequence shown as SEQ ID NO: 77), about 700bp bands are separated and recovered by agarose gel electrophoresis, then the recovered 700bp product is used as a template FOR carrying out second round of PCR amplification, primers used in the second round of PCR are VHH-FOR (with a nucleotide sequence shown as SEQ ID NO: 78) and VHH-REV (with a nucleotide sequence shown as SEQ ID NO: 79), 400bp bands are separated and recovered by agarose gel electrophoresis, and the primer sequences are shown in the following table 2.
TABLE 2 VHH primer sequences required for Gene amplification
| Primer name | Sequence numbering | Primer sequence (5 '-3') |
| CALL001 | SEQ ID NO:76 | GTCCTGGCTGCTCTTCTACAAGG |
| CALL002 | SEQ ID NO:77 | GGTACGTGCTGTTGAACTGTTCC |
| VHH-FOR | SEQ ID NO:78 | CAGGTGCAGCTGCAGGAGTCTGGGGGAGR |
| VHH-REV | SEQ ID NO:79 | CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT |
2.2.3 Construction of VHH phage display vectors
Both the 400bp product recovered in 2.2.2 and phage display vector pMECS were digested with PstI and Not I and recovered, then ligated using T4 DNA ligase.
2.2.4 Ligation product electrotransformation TG1 competent cells and harvesting of phage antibody libraries
Adding the ligation product in 2.2.3 into competent cells of escherichia coli TG1, enabling the competent cells to enter the TG1 through electrotransformation, immediately adding an SOC culture medium after the electrotransformation is completed, culturing for 1h at 37 ℃ and 200rpm, coating the culture medium on an LB/AMP-GLU plate, culturing for 6-8 h at 37 ℃, collecting lawn, and adding 1/3 volume of 50% glycerol to obtain the phage library.
2.2.5 Determination of phage library diversity and storage Capacity
The electrotransformation product was diluted 10-fold, spread on LB/Amp-Glu plates, incubated at 37℃for 12h, and the number of transformants was counted, to finally obtain a phage library with a library capacity of 1.73X1010.
2.2.6 Screening of specific nanobodies against B7H3 protein
The phage library prepared was used as the source of antibodies and 3 rounds of screening were performed using phage display technology. Purified B7H3-His recombinant protein (2. Mu.g/mL) was first coated on a 96-well ELISA plate, the next day was blocked with 3% nonfat milk powder at 37℃for 1H, 1X 1010 nanobody-containing recombinant phages were added to each well, incubated at 37℃for 1H, washed 5 times with PBST, and the phages bound to B7H3-His were eluted with 0.1M glycine (pH 1.5), neutralized with 1M Tris-HCl (pH 8.0), the eluate was again infected with host strain TG1 and cultured in an expanded manner, and 3 rounds of screening were continued. From the third round of screening plates, 192 clones were randomly selected for expansion culture, nanobodies capable of specifically binding to B7H3 protein were identified by monoclonal ELISA, and the results showed that 128 of the 192 clones selected were positive clones (P/N >3.0, P represents the OD450 value of B7H3 well, and N represents the OD450 value of control well), and 15 specific nanobodies against B7H3 were obtained in total by comparing the sequencing results of the positive clones.
Example 3 preparation of specific nanobodies against B7H3 protein
The VHH gene is amplified by taking the plasmid containing the nanobody gene in the step 2.2.6 as a template, and is constructed into a eukaryotic expression vector pcDNA3.1-hFc in a homologous recombination mode, the plasmid is extracted after the sequencing is free, and is transfected into HEK293T cells, the supernatant is collected after the expression is carried out for 5 days, and the recombinant nanobody is obtained by purifying through an affinity chromatography mode by utilizing an NTA-Ni column.
Example 4 IFA detection of binding of anti-B7H 3 nanobodies to cellular level B7H3 antigen
The recombinant protein (Nbs-hFc) of the anti-B7H 3 nanobody prepared in example 3, the positive control antibody 8H9 (Ahmed M et al ,Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3.J Biol Chem.2015 Dec 11;290(50):30018-29) and isotype control antibody) of the anti-B7H 3 were incubated with Hela cells (B7H 3-Hela) over-expressing the B7H3 molecule at 37℃for 1H, washed 3 times with PBS, detected with Alexa FluorTM 594 Goat anti-human IgG (H+L) (ThermoFisher) secondary antibody (1:500), and observed, imaged and recorded with a fluorescence microscope, and the results show that the recombinant protein (Nbs-hFc) of the anti-B7H 3 nanobody of the invention has good binding activity with B7H3 at the cellular level.
Example 5 FACS detection of binding specificity of anti-B7H 3 nanobodies
The recombinant proteins (Nbs-hFc) of the anti-B7H 3 nanobody prepared in example 3, the positive control antibody 8H9 of the anti-B7H 3 and the isotype control antibody were incubated with B7H3-HeLa cells, wild-type A375 cells and A375 cells (A375-B7H 3-KO) knocked out of B7H3 molecules at 37 ℃ for 1H, washed 3 times with PBS, and then detected with Alexa FluorTM 647 Goat anti-human IgG (H+L) (ThermoFisher) secondary antibody (1:600), and analyzed with a flow cytometer, as shown in FIG. 4, the recombinant proteins (Nbs-hFc) of the anti-B7H 3 nanobody of the invention had good binding activities to B7H3-HeLa cells, wild-type HeLa cells and wild-type A375 cells, and were not bound to A-B7H 3-KO cells, indicating that the anti-B7H 3 nanobody prepared in the invention had good specific binding activities to B7H3 antigen.
Example 6 Biacore detection of affinity of nanobodies to B7H3 protein
The binding affinity of the recombinant nanobody against B7H3 with the B7H3-mFc antigen coated on CM5 chip (catalog 29104988, cytiva) was detected by using a Biacore 8k instrument, and the results are shown in Table 3, wherein the affinity of the recombinant nanobody against B7H3 with the B7H3 protein is 10-12M～10-9 M, and the antibodies are all high-affinity antibodies.
TABLE 3 in vitro binding affinity and kinetic analysis of anti-B7H 3 nanobodies to B7H3 protein
| Antibody numbering | Antibody name | Binding Rate ka (1/Ms) | Dissociation rate kd (1/s) | Affinity KD (M) |
| 1# | Nb1 | 2.52E+06 | 2.89E-03 | 1.15E-09 |
| 3# | Nb10 | 1.45E+06 | 9.31E-04 | 6.40E-10 |
| 4# | Nb12 | 4.17E+06 | 1.64E-03 | 3.94E-10 |
| 6# | Nb15 | 3.25E+06 | 6.39E-03 | 1.97E-09 |
| 8# | Nb25 | 3.65E+06 | 2.10E-03 | 5.76E-10 |
| 11# | Nb52 | 1.94E+05 | 2.75E-07 | 1.41E-12 |
| 12# | NbH59 | 6.00E+06 | 5.90E-03 | 9.84E-10 |
| 13# | Nb60 | 9.99E+05 | 2.73E-04 | 2.74E-10 |
| 14# | Nb64 | 3.07E+06 | 9.09E-03 | 2.97E-09 |
| 16# | Nb70 | 1.67E+06 | 8.89E-04 | 5.31E-10 |
| 17# | Nb90 | 1.13E+06 | 6.10E-04 | 5.37E-10 |
| 18# | NbH1 | 1.75E+06 | 7.77E-04 | 4.44E-10 |
| 19# | NbH51 | 3.16E+06 | 7.02E-04 | 2.22E-10 |
| 20# | NbH68 | 1.76E+06 | 8.06E-04 | 4.58E-10 |
| 21# | NbH73 | 6.12E+05 | 3.86E-04 | 6.30E-10 |
EXAMPLE 7RTCA detection of in vitro killing Activity of B7H3/CD3 bispecific antibodies against B7H3-Hela cell lines
The 15 nano antibodies provided by the invention are respectively constructed to eukaryotic expression vectors in series with single-chain antibodies OKT3 scFv (table 4, SEQ ID NO: 80) of anti-CD 3 molecules by using G4 S linker, transfected HEK293T cells for expression, and purified by using Ni columns.
TABLE 4 anti-CD 3 molecular antibody OKT3 scFv sequence information Table
B7H3-Hela cells are slowly added into a 96-well plate special for a non-labeled killing detector (RTCA) instrument according to 5000 cells per well and 100 mu L/well (compound well), the cells are placed into the instrument for culture until the cell index is between 1.0 and 2.0, T cells and B7H3/CD3 bispecific antibody (0.01 mu g/mL) are added according to the ratio of 5:1 of E to perform co-culture, and the instrument is started to continuously detect, so that the result is shown in figure 5, compared with a blank group and an irrelevant control group anti-human CD19-CD3 bispecific antibody (the anti-CD 19scFv antibody sequence is compared with approved marketed product Tisagenlecleucel), after co-culture is performed for 50 hours, the cell indexes of the 15B 7H3/CD3 bispecific antibody groups are all close to 0, and the anti-B7H 3/CD3 bispecific antibody has good killing activity on the B7H3-Hela cells.
Example 8 detection of anti-tumor Activity of B7H3/CD3 bispecific antibodies in AML models
The recombinant protein (Nbs-hFc) of the anti-B7H 3 nanobody prepared in example 3 and the positive control antibody 8H9 of the anti-B7H 3 were incubated with MV-4-11 Acute Myeloid Leukemia (AML) cells at 37℃for 1H, and after washing 3 times with PBS, the recombinant protein (Nbs-hFc) of the anti-B7H 3 nanobody of the invention was detected by using Alexa FluorTM 647 Goat anti-human IgG (H+L) (ThermoFisher) secondary antibody (1:600), and the binding between the recombinant protein and MV-4-11 cells was positive (FIG. 6A).
MV-4-11-luciferase cells are inoculated into NCG mice of 6-8 weeks old through tail veins according to each 2X 106 cells, tumor growth is detected in an in-vivo imager, and the mice are randomly divided into 17 groups after tumor formation, and 3 mice are used in each group. A blank control group (PBS) was set, experimental group (B7H 3/CD3 bispecific antibody) and an irrelevant control group (CD 19-CD3 bispecific antibody) were given by intraperitoneal administration 1 time every 2 days, 7 times continuously, at a dose of 2.5mg/kg, and the same volume of PBS was given to the blank control group (PBS), wherein the experimental group (B7H 3/CD3 bispecific antibody) and the irrelevant control group (CD 19-CD3 bispecific antibody) mice were each vaccinated with 1×107 T cells via the tail vein at the first administration, and the blank control group (PBS) mice were each injected with the same volume of PBS via the tail vein. The survival status of each mouse was observed for the statistical test group (B7H 3/CD 3), the unrelated control group (CD 19-CD 3) and the blank control group (PBS), and the statistical time period was observed to be 40 days. The survival curves of mice are drawn by Kaplan-Meier method, and the results are shown in FIG. 6B, wherein the blank control group (PBS) and the irrelevant control group (CD 19-CD3 bispecific antibody) all die at 16 days after tumor grafting, and the survival rate of the 15B 7H3/CD3 bispecific antibody groups prepared by the invention is 100% at 40 days after tumor grafting, which indicates that the 15B 7H3/CD3 bispecific antibodies prepared by the invention have good anti-tumor activity in the mouse xenograft model of AML.
The experimental results show that the 15 anti-B7H 3 nano antibodies obtained by the invention have good antigen binding activity and antigen binding specificity, and belong to high-affinity antibodies. The B7H3/CD3 bispecific antibody formed by combining the anti-B7H 3 nanobody and the anti-CD 3 molecular antibody OKT3 scFv has good killing activity on tumor cells in vivo and in vitro, and can be used for preparing medicaments for preventing or treating tumors.