A single-domain antibody targeting human and cynomolgus monkey cd46 cross-binding and uses thereof

Abstract

The application belongs to the technical field of biological medicine and immunology, and particularly relates to a single-domain antibody targeting cross binding of human and cynomolgus monkey CD46 and application thereof. The application utilizes alpaca to generate nanoscale antibodies, the molecular weight of which is about 15 kDa, and the nanoscale antibodies can more efficiently penetrate solid tumor tissues or cross blood-brain barrier, and the binding efficiency of the antibodies and target points is as high as possible. The 11 single-domain antibodies prepared by the application have specificity of anti-human and anti-cynomolgus monkey CD46, and in the binding detection with CD46 target proteins and CD46 overexpression cells, the binding efficiency is significantly better than that of Anti-CD46 humanization antibody, and the single-domain antibodies can be used for tumor treatment. Meanwhile, the single-domain antibodies have cross binding ability of cross species, and can be directly used for preclinical pharmacodynamics and safety evaluation, without the need of developing species-specific alternative antibodies, so that the application achieves 'one target and double effect'.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine and immunology, and particularly relates to a single-domain antibody targeting cross-binding of human and cynomolgus monkey CD46 and application thereof. BACKGROUND

[0002] CD46 is also known as membrane cofactor protein, which is not only a key complement regulatory molecule, but also a multifunctional protein connecting innate immunity, adaptive immunity and reproductive physiology. Abnormal expression of CD46 has been proven to be closely related to the progression of various tumors, and has gradually become a popular target for treatment development in multiple fields.

[0003] Although the clinical value of CD46 is significant, there are key bottlenecks in existing targeting technologies. First, traditional monoclonal antibodies (such as humanized antibody BNJ421) have a large molecular weight (about 150 kDa), which is difficult to penetrate solid tumor tissues or cross the blood-brain barrier, and is easily cleared by the reticuloendothelial system, resulting in low target binding efficiency and easy triggering of immunogenicity reactions (such as HAMA effect). Second, non-humanized antibodies (such as murine antibody J4.48) can easily induce anti-drug antibodies, leading to decreased efficacy or allergic reactions, and some antibodies can excessively inhibit the physiological function of CD46, causing damage to normal tissues. Third, preclinical research relies on non-human primate models (such as cynomolgus monkeys), but there is a 12% difference in amino acids between human CD46 (Human CD46) and cynomolgus monkey CD46 (Cyno CD46) in the key structural domain (SCR2-3 region), which prevents existing antibodies (such as J4.48) from simultaneously binding to both, hindering preclinical pharmacodynamics and safety evaluation, and increasing research and development costs and cycle.

[0004] Single-domain antibodies, also known as VHH antibodies or nanobodies, refer to antibody fragments composed of only a single immunoglobulin variable domain (such as VH, VL or VHH), which can bind to antigens without relying on light chains and constant regions, with a molecular weight of about 12-15 kDa. It is the smallest known functional antigen-binding unit, has the advantages of small molecular weight, strong penetration, high stability, and easy genetic engineering modification, and is considered as the next generation of targeted therapy molecules. At present, single-domain antibodies have achieved breakthrough applications in multiple fields, including disease treatment, diagnosis, research tools, and industrial biotechnology. At the same time, the diversified application of single-domain antibodies is gradually rewriting the development paradigm of traditional antibody drugs, becoming an important tool in precision medicine and biotechnology innovation. However, single-domain antibodies that can target cross-binding of human CD46 and cynomolgus monkey CD46 still need to be further developed. SUMMARY

[0005] The present application aims to provide a single-domain antibody cross-bound to human and cynomolgus monkey CD46 and application thereof, which can specifically bind to human and cynomolgus monkey CD46 target antigen, thereby playing a role of targeting cells, and can be used for diagnosis and / or treatment of tumors.

[0006] The present application provides a single-domain antibody cross-bound to human and cynomolgus monkey CD46, which comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are shown in any one of 1) to 11):

[0007] 1) the amino acid sequence of HCDR1 is GFTLDYYN, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is ASSDSCGYYYTAGLNY;

[0008] 2) the amino acid sequence of HCDR1 is GFTLDYYD, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AASRYCGYYYTSHVVDY;

[0009] 3) the amino acid sequence of HCDR1 is SGFTLDYYNI, the amino acid sequence of HCDR2 is ITSSDGST, and the amino acid sequence of HCDR3 is ASSVSCGYYYAAGLNY;

[0010] 4) the amino acid sequence of HCDR1 is GFTLNSYA, the amino acid sequence of HCDR2 is LSSSYGST, and the amino acid sequence of HCDR3 is AAALGPDITSVETMCHVPLHIFGS;

[0011] 5) the amino acid sequence of HCDR1 is GFTLDYYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AIGNYCGYYSDYVPYDY;

[0012] 6) the amino acid sequence of HCDR1 is GFTFDDYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AAASVCGYYLLSALDA;

[0013] 7) the amino acid sequence of HCDR1 is GFTLDYYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is ATDLTCGYYYPTAFGS;

[0014] 8) the amino acid sequence of HCDR1 is GFTFDDYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AADMYCGSYYPTRLGS;

[0015] 9) the amino acid sequence of HCDR1 is GFTFDDYA, the amino acid sequence of HCDR2 is ISRSDGTT, and the amino acid sequence of HCDR3 is AIGNYCGYYSDYVPYDY;

[0016] 10) the amino acid sequence of HCDR1 is GFTDDDYA, the amino acid sequence of HCDR2 is ISSLDGST, and the amino acid sequence of HCDR3 is ATGRTCGYYYTYVLDS;

[0017] 11) the amino acid sequence of HCDR1 is GFSFDEYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AADMYCGSYYPTRLCS.

[0018] Preferably, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 11.

[0019] The application also provides a nucleotide molecule for encoding the single-domain antibody of the above technical solution.

[0020] Preferably, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO: 12 to SEQ ID NO: 22.

[0021] The application also provides a biological material, which is an expression cassette, a recombinant vector or a recombinant cell line containing the nucleotide molecule of the above technical solution.

[0022] The application also provides the use of the single-domain antibody of the above technical solution, or the nucleotide molecule of the above technical solution, or the biological material of the above technical solution in the preparation of one or more of tumor diagnosis, tumor treatment, tumor clinical research and drug development products.

[0023] Preferably, the tumor includes a malignant tumor.

[0024] Preferably, the tumor includes a tumor positive for cell surface CD46.

[0025] Preferably, the tumor includes a colorectal tumor.

[0026] Preferably, the colorectal tumor includes a colorectal tumor retaining wild-type p53 and / or KRAS gene.

[0027] Beneficial effects:

[0028] The present invention provides a single-domain antibody that cross-binds human and cynomolgus macaque CD46. Using alpacas, the nanoscale antibodies, with a molecular weight of approximately 15 kDa, are produced. These antibodies are capable of more efficiently penetrating solid tumor tissue or crossing the blood-brain barrier, maximizing antibody-target binding efficiency. The 11 single-domain antibodies prepared in this invention exhibit specificity against both human and cynomolgus macaque CD46. In binding assays with both the CD46 target protein and CD46-overexpressing cells, their binding potency significantly outperformed the previously described Lenti-CMV-mCORI-hEF-mHVORI-puro [hEF-HTLV prom] (Anti-CD46 humanization) antibody, previously described in patent CN201811396656.4. These antibodies can be used to treat tumors, particularly those expressing CD46 on their cell surfaces. Furthermore, the cross-species cross-binding ability of the single-domain antibodies described in this invention allows for direct preclinical pharmacodynamic and safety evaluations, eliminating the need for developing species-specific surrogate antibodies and achieving a "single target, dual efficacy" approach. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0030] Figure 1 This is a graph showing the purity of the CD46 recombinant antigen protein in Example 1;

[0031] Figure 2 This is a flow separation diagram after two rounds of magnetic separation in Example 1;

[0032] Figure 3 This is a diagram showing the binding of the candidate antibody to the CD46 target protein in Example 2;

[0033] Figure 4 This is a diagram showing the binding of the candidate antibody in Example 3 to CD46-overexpressing human cells;

[0034] Figure 5 This is a graph showing the killing effect of the candidate antibodies in Example 5 on three CD46-positive tumor cells. DETAILED DESCRIPTION

[0035] The present invention provides a single-domain antibody that cross-binds and targets human and cynomolgus macaque CD46, wherein the single-domain antibody comprises heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as shown in any one of 1) to 11):

[0036] 1) the amino acid sequence of HCDR1 is GFTLDYYN (SEQ ID NO: 23), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is ASSDSCGYYYTAGLNY (SEQ ID NO: 25);

[0037] 2) the amino acid sequence of HCDR1 is GFTLDYYD (SEQ ID NO: 26), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is AASRYCGYYYTSHVVDY (SEQ ID NO: 27);

[0038] 3) the amino acid sequence of HCDR1 is SGFTLDYYNI (SEQ ID NO: 28), the amino acid sequence of HCDR2 is ITSSDGST (SEQ ID NO: 29), and the amino acid sequence of HCDR3 is ASSVSCGYYYAAGLNY (SEQ ID NO: 30);

[0039] 4) the amino acid sequence of HCDR1 is GFTLNSYA (SEQ ID NO: 31), the amino acid sequence of HCDR2 is LSSSYGST (SEQ ID NO: 32), and the amino acid sequence of HCDR3 is AAALGPDITSVETMCHVPLHIFGS (SEQ ID NO: 33);

[0040] 5) the amino acid sequence of HCDR1 is GFTLDYYA (SEQ ID NO: 34), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is AIGNYCGYYSDYVPYDY (SEQ ID NO: 35);

[0041] 6) the amino acid sequence of HCDR1 is GFTFDDYA (SEQ ID NO: 36), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is AAASVCGYYLLSALDA (SEQ ID NO: 37);

[0042] 7) the amino acid sequence of HCDR1 is GFTLDYYA (SEQ ID NO: 34), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is ATDLTCGYYYPTAFGS (SEQ ID NO: 38);

[0043] 8) the amino acid sequence of HCDR1 is GFTFDDYA (SEQ ID NO: 39), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is AADMYCGSYYPTRLGS (SEQ ID NO: 40);

[0044] 9) the amino acid sequence of HCDR1 is GFTFDDYA (SEQ ID NO: 36), the amino acid sequence of HCDR2 is ISRSDGTT (SEQ ID NO: 41), and the amino acid sequence of HCDR3 is AIGNYCGYYSDYVPYDY (SEQ ID NO: 42);

[0045] 10) the amino acid sequence of HCDR1 is GFTDDDYA (SEQ ID NO: 43), the amino acid sequence of HCDR2 is ISSLDGST (SEQ ID NO: 44), and the amino acid sequence of HCDR3 is ATGRTCGYYYTYVLDS (SEQ ID NO: 45);

[0046] 11) the amino acid sequence of HCDR1 is GFSFDEYA (SEQ ID NO: 46), the amino acid sequence of HCDR2 is ISSSDGST (SEQ ID NO: 24), and the amino acid sequence of HCDR3 is AADMYCGSYYPTRLCS (SEQ ID NO: 47).

[0047] The homology of the extracellular segment protein of human CD46 and Cyno CD46 is 89.25%, which is relatively high, and is the basis for screening the cross-binding antibody. Based on this, the application prepares a CD46 recombinant antigen protein by targeting the cross-binding domain of human CD46 and cynomolgus monkey CD46; then the CD46 recombinant antigen protein is used for immunizing a llama, so as to ensure that the llama can produce an antibody cross-binding CD46; the peripheral blood mononuclear cells of the llama are collected to construct a single-domain antibody yeast display library, and candidate single-domain antibody sequences recognizing the target protein are screened and selected, and finally 11 specific single-domain antibody sequences are obtained, as shown in SEQ ID NO. 1~SEQ ID NO. 11.

[0048] As an implementation manner, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO: 1~SEQ ID NO: 11; the name and amino acid sequence of the single-domain antibody are specifically as follows:

[0049] 31-17-1-A12: QVQLVESGGGLVQPGGSLRLSCAAS GFTLDYYN IGWFRQAPGKEREGVSC ISSSDGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYC ASSDSCGYYYTAGLNY WGQGTQVTVSS (SEQ ID NO:1);

[0050] 31-17-1-C11:QLQLVESGGGLVQPGGSLRLSCAAS GFTLDYYD IGWFRQAPGKEREGVLC ISSSDG ST YRADSVKGRFTISRDNAKNTVYLQMNSLKPEDIAVYC AASRYCGYYYTSHVVDY WGQGTQVTVSS (SEQ ID NO:2);

[0051] 31-17-1-E03:AVQLVESGGGLVQPGGSLRLSCAA SGFTLDYYNI GWFRQAPGKEREGVSC ITSSDG ST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYC ASSVSCGYYYAAGLNY WGQGTQVTVSS (SEQ ID NO:3);

[0052] 31-17-1-H06:QVQLVESGGGLVQPGESLRLSCAAS GFTLNSYA IGWFRQAPGKEREGVSC LSSSYG ST YYADSVKGRFTISRDNAKGTVYLQMNSLKPEDTAVYSC AAALGPDITSVETMCHVPLHIFGS WGQGTQVTVSS(SEQ ID NO:4);

[0053] 34-35-3-A11:QLQLVESGGGLVQPGGSLRLSCVAS GFTLDYYA IGWFRQAPGKEREGVSC ISSSDG ST YYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYC AIGNYCGYYSDYVPYDY WGQGTQVTVSS (SEQ ID NO:5);

[0054] 34-35-3-A3:QLQLVESGGGLVQAGGSLRLSCAAS GFTFDDYA IGWFRQAPGKEREGVSC ISSSDGS TYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC AAASVCGYYLLSALDA WGQGTLVTVSS (SEQ ID NO: 6);

[0055] 34-35-3-A8: AVQLVESGGGLVQAGGSLRLSCAAS GFTLDYYA IGWFRQAPGKEREGVSC ISSSDGS T YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC ATDLTCGYYYPTAFGS WGQGTQVTVSS (SEQ ID NO: 7);

[0056] 34-35-3-C6: QVQLVESGGGLVQAGGSLRLSCAAS GFTFDDYA IGWFRQAPGKEREGVSC ISSSDGS T YYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC AADMYCGSYYPTRLGS WGQGTQVTVSS (SEQ ID NO: 8);

[0057] 34-35-3-D9: QVQLVESGGGLVQAGGSLRLSCAAS GFSFDDYA IGWFRQAPGKEREGVSC ISRSDGT T YYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYC AIGNYCGYYSDYVPYDY WGQGTQVTVSS (SEQ ID NO: 9);

[0058] 34-35-3-E1: QVQLVESGGGLVQAGGSLRLSCAAS GFTDDDYA IGWFRQAPGKEREGVSC ISSLDGS T FYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC ATGRTCGYYYTYVLDS WGQGTQVTVSS (SEQ ID NO: 10);

[0059] 34-35-3-E9: QVQLVESGGGLVQAGGSLRLSCLVS GFSFDEYA IGWFRQAPGKEREGVSC ISSSDGS TYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC AADMYCGSYYPTRLCS WGQGTQVTVFS (SEQ ID NO: 11).

[0060] In SEQ ID NO: 1-SEQ ID NO: 11, the underlined part in bold italic is the amino acid sequence of HCDR1-HCDR3 of the single-domain antibody.

[0061] The application also provides a nucleotide molecule for encoding the single-domain antibody described in the above technical solutions. As an implementation form, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO: 12-SEQ ID NO: 22, and specifically as follows:

[0062] 31-17-1-A12: caggtgcagctcgtggagtctggtggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattataacataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgagcagcgattcatgtggttactactacaccgccgggctcaactactggggccaggggacccaggtcaccgtctcctca (SEQ ID NO: 12);

[0063] 31-17-1-C11:cagttgcagctcgtggagtccgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattatgacataggctggttccgccaggccccagggaaggagcgtgagggggtcttatgtattagtagtagtgatggtagcacatacagagcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacatagccgtttattactgtgcagcaagcaggtattgcggttactactacacgtctcacgtggtggactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:13);

[0064] 31-17-1-E03:gcggtgcagctggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattataacataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattacaagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgagcagcgtttcatgtggttactactacgccgccgggctgaactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:14);

[0065] 31-17-1-H06: caggtacagctggtggagtctgggggaggcttggtgcagcctggggagtctctgagactctcctgtgcagcctctggattcactttaaattcttatgccataggctggttccgccaggccccaggaaaggagcgtgagggggtctcatgtcttagtagtagttatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagggcacggtgtatctgcaaatgaacagcctgaaaccggaggacacagccgtttatagctgtgcagccgccttagggcccgacattactagcgttgagactatgtgtcatgtaccccttcatatctttgggtcctggggccaggggacccaggtcaccgtctcctcg (SEQ ID NO: 15);

[0066] 34-35-3-A11: cagttgcagctcgtggagtctggggggggcttggtgcagcctgggggatctctgagactctcctgtgtagcctctggattcactttggattattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtctcatgtattagtagtagtgatggtagcacatactatccagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccatttattactgtgcgatcggaaattactgtggttactatagcgactatgtcccttatgactactggggccaggggacccaggtcaccgtctcctca (SEQ ID NO: 16);

[0067] 34-35-3-A3: cagttgcagctcgtggagtcaggcggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattcactttcgatgattatgctataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgcgcagcagctagcgtttgcggttactacctactctctgctttggacgcatggggccaggggaccctggtcactgtctcctca (SEQ ID NO: 17);

[0068] 34-35-3-A8: gccgtggagctggtggattctggaggaggattggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcgacagacctaacttgtggttactactacccgactgcttttggttcctggggccaggggacccaggtcaccgtctcctca (SEQ ID NO: 18);

[0069] 34-35-3-C6: cagttgcagctcgtggagtctggcggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattcactttcgatgattatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagctgacatgtattgcggtagttactacccgacgcgtttgggttcctggggccaggggacccaggtcaccgtctcctcg (SEQ ID NO: 19);

[0070] 34-35-3-D9: caggtgcagctcgtggagtcagggggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattctctttcgatgattatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtcgtagtgatggtaccacatactatccagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccatttattactgtgcgatcggaaattactgtggttactatagcgactatgtcccttatgactactggggccaggggacccaggtcaccgtctcctca (SEQ ID NO: 20);

[0071] 34-35-3-E1: caggtgcagctcgtggagtccggggggggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattcactgacgatgattatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtctagacggtagcacattctatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcgacagggaggacttgtggttactactacacctacgtacttgactcctggggccaggggacccaggtcaccgtctcctca (SEQ ID NO: 21);

[0072] 34-35-3-E9: cagttgcagctcgtggagtccgggggaggcttggtgcaggctggggggtctctgagactctcctgtttagtgtctggattcagtttcgatgagtatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagctgacatgtattgcggtagttactacccgacgcgtttgtgttcctggggccaggggacccaggtcaccgtcttctcg (SEQ ID NO: 22).

[0073] The present application also provides a biological material, which is an expression cassette, a recombinant vector or a recombinant cell line comprising the nucleotide molecule according to the above technical solution. As an embodiment, the initial vector in the recombinant vector of the present application is a plasmid vector or a lentivirus vector; as another embodiment, the initial vector in the recombinant vector is a eukaryotic expression vector. The present application does not have special limitations on the construction method of the biological material, and the conventional genetic engineering means in the art can be used.

[0074] The application detects the binding ability of the 11 single-domain antibodies to the target proteins (Human CD46-His and Cyno CD46-His) by enzyme-linked immunosorbent assay (ELISA), and detects the binding ability of the 11 antibodies to CD46 overexpressing cells (CHO-K1-CD46, CHO-S-Cyno CD46) by flow cytometry, thereby confirming that the single-domain antibodies have the specificity of anti-human and cynomolgus monkey CD46.

[0075] The application also evaluates the specific binding ability of the 11 antibodies to CD46 positive tumor cells (SW480, SW620, RKO) by flow cytometry, and detects the targeted killing ability of the 11 antibodies to CD46 positive tumor cells (SW480, SW620, RKO) by CCK-8 experiment, thereby confirming that the single-domain antibodies of the application can specifically recognize and kill tumor cells with positive CD46 on the surface. Therefore, the single-domain antibodies of the application can be applied to the immunotherapy of malignant tumors, especially CD46 positive tumors.

[0076] Based on the above advantages, the application also provides the use of the single-domain antibody of the above technical solution or the nucleotide molecule of the above technical solution or the biological material of the above technical solution in the preparation of one or more of tumor diagnosis, tumor treatment, tumor clinical research and drug development products. As an embodiment, the tumor includes a malignant tumor; as another embodiment, the malignant tumor is a tumor with positive CD46 on the cell surface. As an embodiment, the tumor is a colorectal tumor; as another embodiment, the colorectal tumor includes a colorectal tumor retaining a wild-type p53 and / or KRAS gene. As an embodiment, the drug development is tumor drug development; as another embodiment, the tumor drug development is pharmacodynamics and / or drug safety evaluation.

[0077] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0078] Example 1

[0079] The preparation of the single-domain antibody targeting the cross-binding of human and cynomolgus monkey CD46 is as follows:

[0080] The CD46 recombinant antigen protein is prepared by targeting the cross-binding domain of human CD46 and cynomolgus monkey CD46, and then the alpaca is immunized with the CD46 recombinant antigen protein, the peripheral blood mononuclear cells of the alpaca are collected to construct a single-domain antibody yeast display library, and the candidate single-domain antibody sequences recognizing the target protein are selected by panning, which are as follows:

[0081] 1. Preparation of recombinant antigen protein against human and cynomolgus monkey CD46 cross-binding domain CD46

[0082] According to the extracellular region amino acid sequence information of Human CD46 (Uniprot: P15529-3) and Cynomolgus Monkey CD46 (Uniprot: A0A2K5WCS2) proteins in the Uniprot database, a eukaryotic expression vector was constructed, and after transfection of mammalian cells using LVtransm transfection reagent (iCarEab, Cat# LVTran100), the culture medium supernatant was collected, and the target recombinant protein was prepared by affinity chromatography purification. The purity of the prepared recombinant protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein marker used was purchased from Solarbio, Cat# RP1930.

[0083] The amino acid sequence of the extracellular region of human CD46 protein is the amino acid at positions 35-313 of the amino acid with accession number Uniprot: P15529-3; the amino acid sequence of the extracellular region of Cynomolgus Monkey CD46 protein is the amino acid at positions 35-313 of the amino acid with accession number Uniprot: A0A2K5WCS2. The amino acid sequences of the CD46 recombinant proteins prepared by affinity chromatography purification are: >Human CD46 (Uniprot: P15529-3) 35-313AA: CEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVSTSSTTKSPASSASGPRPTYKPPVSNYGGGGSHHHHHH* (SEQ ID NO: 48); >Cynomolgus Monkey CD46 (Uniprot: A0A2K5WCS2) 35-313AA: CEEPPTFEAMELIGKPKPYYRVGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDEGCYREMCPHIRDPLNGEAILANGSYEFGAELHFICNEGYYLIGKDILYCELKDTVAIWSGKPPLCEKILCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESMIYCGNNSTWSHAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGYYLNGSDKIVCESNSTWDPPVPKCLKVSTSPTTKSPTSSASGPRPTYKPPVSNYGGGGSHHHHHH* (SEQ ID NO: 49), in the above-mentioned amino acid sequences of CD recombinant proteins, the bolded amino acids are His tags added at the C-terminus of the protein for subsequent protein purification.

[0084] The target protein was evaluated for binding activity by ELISA method using positive control antibody (PC) Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] (iCarEab). Among them, coating: pcDNA3.4-Cyno CD46-C35-Y328-His (iCarEab), 1 μg / mL, 4°C coating overnight; primary antibody (PC): Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] (disclosed in CN201811396656.4), 10 μg / mL, 3-fold dilution 7 points; secondary antibody: HRP-anti Human IgG (eBioscience, Cat#12-4998-82); the negative control (NC) group is the secondary antibody control hole, and the binding of Cyno CD46 protein and positive antibody is detected. The results are shown in Table 1 and Figure 1 Figure 1 Among them, M is a protein marker, lane 1 is Cyno CD46-C35-Y328-His, and lane 2 is Human CD46-His.

[0085] Among them, Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] is obtained in the preparation process of the previous patent CN201811396656.4, and is an antibody with anti-human CD46 gene activity, which is used as PC in the present patent.

[0086] Table 1 ELISA method for evaluating the binding activity of the target protein

[0087]

[0088] Note: The two values in Table 1 corresponding to each group are the detection results of two parallel duplicate wells.

[0089] From Figure 1 and Table 1, it can be seen that the purity of the prepared recombinant antigen protein is >95%, and the recombinant antigen protein has good binding activity with the positive antibody, and can be used for subsequent immunization and panning work.

[0090] 2, Camel immunization of CD46 recombinant antigen protein

[0091] ​Two alpacas (iCarEab) were immunized with the recombinant antigen protein described above, the immunization method was subcutaneous multi-point immunization, the immunization interval was 14 days, and Adjuvant immunoadjuvant (Gerbu, Cat#3030) was used during the interval. Starting from the second immunization, peripheral blood was collected from the alpacas one week after each immunization to monitor the immune serum titer. After the completion of immunization, 100 mL of peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated for single-domain antibody display library construction. The immunization schedule is shown in Table 2.

[0092] Table 2. Immunization schedule

[0093]

[0094] 3. Immune titer detection

[0095] 1) Collect 5 mL of peripheral blood from the alpaca, and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 h; then transfer the blood sample to 4°C overnight.

[0096] 2) Place the centrifuge tube containing the blood sample in a centrifuge, centrifuge at 5000 rpm for 20 min; separate the upper serum, and transfer the serum to a new sterile centrifuge tube to collect the immune serum.

[0097] 3) Dilute the target recombinant protein to a final concentration of 1 µg / mL using sterile carbonate buffer solution (CBS; Macklin, Cat#C885533).

[0098] 4) Take a new 96-well enzyme-labeled plate, add 1 µg / mL of the target recombinant protein, 100 μL / well, and coat overnight at 4°C.

[0099] 5) Remove the antigen coating solution, and wash 5 times with phosphate buffered saline containing 0.05% Tween 20 (PBST; Merck, Cat#P3563).

[0100] 6) Add 200 μL / well of phosphate buffered solution containing 3% skim milk (MPBS; Beyotime, Cat#P0216; Gibco, Cat#14190-250) at 37°C for 2 h.

[0101] 7) After removing the blocking buffer, wash the plate 5 times with PBST, add 100 µL / well of gradient-diluted serum, and incubate at room temperature for 1 h. The control wells are phosphate buffered saline (PBS).

[0102] 8) Remove the liquid in the hole, and wash 5 times with PBST, add 100 μL HRP anti-Llama IgG (H+L) antibody (1:50000 dilution; Novus, Cat#NBP1-75095), incubate at room temperature for 1 h.

[0103] 9) After removing the liquid in the hole, wash the hole plate 5 times with PBST, add 100 μL / well TMB developing solution (Merck, Cat#T0440), incubate at room temperature for 10-15 min in the dark.

[0104] 10) Add 50 μL / well stop solution (Biosharp, Cat#BL1829B).

[0105] 11) Use the microplate reader to read the OD 450 value in the hole.

[0106] According to the ELISA experiment results, the llama negative serum titer is low, and can be immunized. After the second immunization, the immune serum can be combined with the target recombinant protein, and the OD value changes with the gradient dilution of the immune serum. Even at a serum dilution gradient of 1:64K, the second immunization group is about 5 times that of the negative serum group (≥ 3 times can be used), which confirms that the titer of the llama after the second immunization is significantly improved, and has reached the requirement of blood collection for library construction, and can be used for the construction of antibody display library. Therefore, after the third immunization, the immune titer detection is not repeated.

[0107] 4, Llama PBMC separation and VHH antibody fragment cloning

[0108] 1) Collect 100 mL of peripheral blood from each of the two llamas, and separate PBMC using lymphocyte separation medium (Biolab, Cat#JH0171). Since the immune titers of the two llamas are both high, the PBMC from the two llamas are mixed and used for subsequent experiments.

[0109] 2) Extract RNA by RNAiso Plus (TaKaRa, Cat#9109), and perform reverse transcription using PrimeScript™ II 1stStrand cDNA Synthesis Kit (TaKaRa, Cat#6210B) to prepare cDNA.

[0110] 3) Prepare the mixture Mix1 in a 200 μL PCR tube according to the PCR reaction system in Table 3, incubate at 65°C for 5 min, and then quickly cool on ice.

[0111] 4) The reaction solution was prepared in the PCR tube according to the reaction system in Table 4, and after mixing by blowing, 80 μL / tube was divided and placed in a PCR instrument for 1 h at 42°C, 15 min at 70°C for heat inactivation, and finally the cDNA sample was placed on ice or stored at -20°C for long-term preservation.

[0112] 5) The first round of PCR reaction system (50 μL / tube) was configured according to the reaction system in Table 5, and the upstream primer was combined with the signal peptide and the downstream primer was combined with the CH2 region. Among them, NuHi Power mix was purchased from Xinhai Biology, Cat# NH9303. After the PCR reaction system was configured, the PCR instrument was set according to the program in Table 6. The nucleotide sequences of the upstream primer and the downstream primer were 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO: 50); 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 51), respectively.

[0113] 6) The PCR product was analyzed by electrophoresis using 1% agarose (Merck, Cat# A6013), and PCR bands with molecular weights of about 750 bp and 1000 bp were obtained. The 750 bp PCR product was recovered as a second round PCR template using a gel recovery kit (Qiagen, Cat# 28706), and the concentration was measured using NanoDrop.

[0114] 7) The second round of PCR reaction system (50 μL / tube) was configured according to the reaction system in Table 7, and the upstream primer was combined with the antibody FR1 region and the downstream primer was combined with the anti-Hinge and FR4 region, and the enzyme cutting site was Sfil. After the PCR reaction system was configured, the PCR instrument was set according to the program in Table 6. The nucleotide sequences of the upstream primer and the downstream primer were 5'-AGKTGCAGCTCGTGGAGTCNGGNGG-3' (SEQ ID NO: 52); 5'-GATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 53), respectively.

[0115] 8) The second round of PCR product was analyzed by agarose electrophoresis, and the PCR product was analyzed by electrophoresis using 1% agarose. The VHH fragment with a molecular weight of about 500 bp was separated. The VHH PCR product was recovered using a gel recovery kit, and the concentration was measured using NanoDrop.

[0116] The recovered two-wheel PCR product was divided into 200 μL per 1.5 mL centrifuge tube, 1 / 10 volume (20 μL) of 3M sodium acetate (Sigma, Cat#126-96-5), 1 μg / μL glycogen (Glycogen; Beyotime, Cat#D0812) was added, and the mixture was mixed by blowing and sucking, 880 μL of anhydrous ethanol (Merck, Cat#459828) was added, and the mixture was mixed by inverting, and was stored at -80°C.

[0117] Table 3 Reverse transcription PCR mixture Mixl preparation system

[0118]

[0119] Table 4 Reverse transcription PCR reaction solution preparation system

[0120]

[0121] Table 5 First round PCR reaction system

[0122]

[0123] Table 6 PCR reaction program

[0124]

[0125] Table 7 Second round PCR reaction system

[0126]

[0127] 5. Construction and screening of single-domain antibody yeast display library

[0128] 1) Use Sfil (NEB, Cat#R0123L) to cut pYDisplay vector (iCarEab), and the linearization enzyme cutting system of yeast display vector pYDisplay is shown in Table 8, 100 μL / tube, 50°C enzyme cutting overnight.

[0129] Table 8 Linearization enzyme cutting system of yeast display vector pYDisplay

[0130]

[0131] 2) Use 1% agarose gel to separate pYDisplay vector fragments, cut 5000 bp of vector fragments for gel recovery, and use NanoDrop to determine the concentration.

[0132] 3) The recovered pYDisplay enzyme digestion product was aliquoted 200 μL per 1.5 mL centrifuge tube, 1 / 10 volume (20 μL) of 3 M sodium acetate, 1 μg / μL Glycogen was added, mixed by pipetting up and down, 880 μL of absolute ethanol was added, mixed by inverting, and stored at -80°C.

[0133] 4) The -80°C frozen competent yeast strain was streaked onto yeast extract peptone dextrose (YPD) solid medium plates and incubated at 30°C for 3-5 days.

[0134] 5) A single colony of competent yeast was inoculated into 50 mL of YPD medium and incubated at 250 rpm, 30°C for 1-2 days.

[0135] 6) After mixing the linearized vector fragment and PCR product, the mixture was added to an electroporation cuvette and electroporated. The electroporated competent yeast was transferred to a culture flask and incubated at 220 rpm, 30°C for 1 h to prepare the competent yeast strain.

[0136] 7) 20 μL of the resuspended solution was diluted 5000-fold with synthetic growth selection medium (SDCAA), 100 μL was pipetted and spread on an SDCAA plate, and incubated for 2-3 days. According to the calculation of the library capacity, the constructed yeast display library had a library capacity of 2.05 x 10 9 , which met the requirements and was arranged for diversity detection.

[0137] 8) Single clones were randomly selected for sequencing to analyze the diversity of the yeast display library. According to the sequencing results, all were antibody difference sequences, there were no empty loads and repeated sequences, and the library diversity was good.

[0138] 9) The remaining bacterial solution in step 6) was further incubated for 24 h and collected in a 50 mL centrifuge tube, centrifuged at 3000 x g for 5 min, the supernatant was discarded, 10 mL of SDCAA was added for resuspension, mixed with 50% glycerol at a ratio of 1:1, and stored at -80°C.

[0139] 10) The yeast cultured in SDCAA was added to a 250 mL shake flask containing 50 mL of galactose induction medium (SGCAA), and incubated at 30°C, 240 rpm for 16 h.

[0140] 11) After centrifugation, the supernatant was discarded, resuspended with 1 mL of 0.5% BSA-containing phosphate buffer (0.5% PBSA), added to a 1.5 mL centrifuge tube, centrifuged at 3000 x g for 5 min, the supernatant was discarded, and washed again with 0.5% PBSA.

[0141] 12) Wash the streptavidin magnetic beads incubated with Biotin antigen twice with 0.5% PBSA (incubating at 4°C with rotation for 5 minutes each time), place on a magnetic stand for 5 minutes, and discard the supernatant.

[0142] 13) Add the yeast solution to the antigen-bound magnetic beads, incubate with rotation at 4°C for 60 minutes, and place on a magnetic stand for 15 minutes.

[0143] 14) Discard the yeast solution and retain the magnetic beads. Wash three times with 0.5% PBSA (rotating and incubating at 4°C for 5 minutes each time).

[0144] 15) Resuspend the magnetic beads in 1 mL of SDCAA medium. Pipette 0.5-5 μL of the resuspension into 100 μL of SDCAA medium and spread on a plate. Divide the resuspension into two equal portions. Add 500 μL of 50% glycerol (stored at -80°C) to one portion; add the other portion to a shaker tube, add 2 mL of SDCAA medium, and incubate at 30°C, 240 rpm, for 16 h.

[0145] 16) Transfer the bacterial suspension from the shake tube to 50 mL of SDCAA medium (in a 250 mL shake flask) and culture overnight at 30°C and 240 rpm.

[0146] 17) Measure the OD of the bacterial solution 600 Value, according to OD 600 Take a portion of the bacterial solution, centrifuge it, resuspend it with SGCAA, and transfer it to 50 mL SGCAA medium to make the final OD 600 The culture was incubated at 30°C and 240 rpm overnight with a dilution of 1. The remaining bacterial solution was resuspended in SDCAA:50% glycerol at a ratio of 1:1 and stored at -80°C.

[0147] After magnetic separation using Biotin-Cyno CD46-His protein using flow cytometry, the positive rates for Human CD46 and Cyno CD46 in the display library were 16.464% and 14.071%, respectively. This library will be used for single clone detection and subsequent flow cytometry separation to further improve the positive rate of the display library. Biotin-Cyno CD46-His, Biotin-Human CD46-His, and V5 Tag Antibody [FITC], mAb (FITC-V5) were all purchased from iCarEab; Streptavidin APC (SA-APC) was purchased from Biolegend, Cat# 405207.

[0148] 79,263 cells from the P2 region were sorted using flow cytometry for subsequent analysis. Streptavidin-Phycoerythrin (SA-PE) was purchased from eBioscience, Cat# 12-4317-87.

[0149] 18) Spread the sorted yeast liquid on SDCAA plates, pick a single clone and culture it. Induce expression for 48 hours and then incubate it. After incubation, perform flow cytometry detection. Figure 2 As shown, in Figure 2 In the figure, A is NC; B is 1M+1F: primary antibody: Biotin-Human CD46-His, secondary antibody: FITC-V5+SA-APC; C is 1M+1F: primary antibody: Biotin-Cyno CD46-His, secondary antibody: FITC-V5+SA-APC; D is 1M+1F: primary antibody: Biotin-CD30-His (unrelated protein), secondary antibody: FITC-V5+SA-APC. Biotin-CD30-His (unrelated protein) was purchased from iCarEab.

[0150] Depend on Figure 2 It can be concluded that after flow cytometry sorting using Biotin-Cyno CD46-His protein, the positive proportions of HumanCD46 and Cyno CD46 in the display library were 34.556% and 52.077%, respectively. The library will be arranged for monoclonal picking and testing.

[0151] 6. Yeast monoclonal detection

[0152] Yeast clones that bound to the target antigen were lysed by incubation with 0.2% SDS (Beyotime, Cat#ST626) at 95°C for 10 min and centrifuged. 0.5 μL of the supernatant was used as a template for PCR amplification and sent to GenScript Biotech Co., Ltd. for sequencing. A total of 125 clones were sent for testing, and the remaining bacterial solution was stored at -20°C.

[0153] 7. Construction of antibody eukaryotic expression vector

[0154] 1) The positive yeast clones were subjected to PCR to obtain the antibody sequence, which was digested with SfiI and ligated into the eukaryotic expression vector pcDNA3.4-Fc (iCarEab). 18 new differential sequences were added for vector construction.

[0155] 2) The antibody eukaryotic expression vector was transiently transfected into 293F cells (ATCC cell bank) to obtain 14 antibody expression supernatants.

[0156] 3) Flow cytometry was used to detect the binding of 14 candidate antibodies and antigen proteins. Among them, the antigen protein used was a specific cell source, and three cell lines were CHO-S, CHO-K1-CD46 and CHO-S-Cyno CD46 (iCarEab Cell Library), 2 x 10 5 primary antibody: candidate antibody transfection supernatant, 100 μL / well; secondary antibody: PE-Goat anti-Human IgG Fc (1:1000 dilution; Invitrogen, Cat#12-4998-82); PC: Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom]; NC: secondary antibody control group, without target protein CD46 but with secondary antibody, which is used as the basis for circle gate in flow analysis.

[0157] Flow cytometry showed that, except for 34-35-3-C3 with weak binding, 31-17-5-H8 clone without binding, and 34-35-3-E8 clone with non-specific binding, the remaining 11 candidate antibodies all cross-bound with human and monkey cells, which was significantly better than the PC group and could be used for subsequent antibody purification and preparation work.

[0158] 8. Expression and purification of candidate single-domain antibodies

[0159] 1) According to the flow cytometry detection results of the candidate antibodies, positive clones were selected for antibody expression and preparation.

[0160] 2) Take LVTransm transfection reagent and pcDNA3.4-Fc antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly with a pipette gun. Take PBS buffer and warm it to room temperature. Take 2 mL PBS into one well of a 6-well plate, add 20 μg of antibody expression vector, mix thoroughly with a pipette gun, then add 60 μL of LVTransm, immediately mix with a pipette, and incubate at room temperature for 10 min.

[0161] 3) Add the above DNA / LVTransm complex to 20 mL of 293F cells, mix gently and thoroughly. Incubate the cells in a 37℃, 5% CO2, 130 rpm incubator.

[0162] 4) After continuous culture for 5-7 days, centrifuge to collect the culture supernatant, filter with a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube.

[0163] 5) Since the pcDNA3.4-Fc antibody expression vector can specifically bind to Protein A, the antibody was purified using a Protein A column (Suzhou Bioengineering Technology Co., Ltd., Cat# BG18-0010-02).

[0164] The 11 candidate antibody sequences obtained by the above method are shown in SEQ ID NO: 1~SEQ ID NO: 11, and the molecular weight is about 15 kDa. The heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 are marked in the amino acid sequences shown in SEQ ID NO: 1~SEQ ID NO: 11. The RNA sequence corresponding to the candidate antibody can be deduced from the triplet codon, as shown in SEQ ID NO: 12~SEQ ID NO: 22.

[0165] Example 2

[0166] ELISA was used to detect the binding of the candidate antibody to the CD46 target protein. The steps are as follows:

[0167] 1) Dilute the CD46 recombinant protein to a final concentration of 2 µg / mL using sterile CBS. Take a new 96-well enzyme plate, add 100 μL to each well, and coat overnight at 4°C.

[0168] 2) Remove the antigen coating solution, wash 5 times with PBST, and add 200 μL / well of 3 % MPBS. Incubate at 37°C for 2 h.

[0169] 3) After removing the blocking buffer, wash the plate 5 times with PBST. Add the expressed recombinant antibody, transfection supernatant 100 μL / well, or positive antibody (starting concentration 10 µg / mL, 5-fold gradient dilution 7 points, 100 μL / well). PC is Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom]. Incubate at room temperature for 1 h. The control wells are PBS.

[0170] 4) Remove the liquid in the wells, wash 5 times with PBST, and add 100 μL / well of HRP-Goat anti-Human IgG Fc antibody (1:30000 dilution; Abeam, Cat# ab97225). Incubate at room temperature for 1 h.

[0171] 5) After removing the liquid in the wells, wash the plate 5 times with PBST, add 100 μL / well of TMB developing solution, and incubate at room temperature in the dark for 10~15 min.

[0172] 6) Add 50 μL / well of stop solution.

[0173] 7) Read OD in the wells using a microplate reader 450 Values.

[0174] The results are shown in Figure 3 , where A is the binding of the first batch of candidate antibodies to Human CD46-His; B is the binding of the second batch of candidate antibodies to Human CD46-His; C is the binding of the first batch of candidate antibodies to Cyno CD46-His; and D is the binding of the second batch of candidate antibodies to Cyno CD46-His.

[0175] It can be concluded that after coating with Human CD46-His antigen (1 μg / mL) and Cyno CD46-His antigen (1 μg / mL), 11 candidate antibodies bind to Human CD46-His and Cyno CD46-His with strong binding, which is significantly better than the PC group, confirming that the antibodies can specifically bind to the CD46 target protein. Figure 3 Example 3

[0176] The binding of the candidate antibodies to CD46-overexpressing cells was detected as follows:

[0177] 1) Resuscitate CHO-S, CHO-K1-CD46 and CHO-S-Cyno CD46 cells from liquid nitrogen, and adjust the cell state to the logarithmic growth phase.

[0178] 5 2) Divide the cells into several portions, with 2×10

[0179] 3) Incubate the candidate antibodies (10 μg / mL, 100 μL / well) with the target cells CHO-S and CHO-S-Cyno CD46; in addition, adjust the candidate antibodies to an initial concentration of 20 μg / mL, 100 μL / well, and perform 8-point dilution, and incubate with the target cells CHO-K1-CD46. After thorough mixing, incubate at room temperature for 1 h, where the PC group is Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] and the NC group is the secondary antibody control group.

[0180] Among them, the gradient dilution of the candidate antibodies when incubated with the target cells CHO-K1-CD46 is because the candidate antibodies need to be applied to human tumor cells for evaluation later, so the specific binding potency of the candidate antibodies to CHO-K1-CD46 cells at multiple concentrations is detected first.

[0181] Among them, the gradient dilution of the candidate antibodies when incubated with the target cells CHO-K1-CD46 is because the candidate antibodies need to be applied to human tumor cells for evaluation later, so the specific binding potency of the candidate antibodies to CHO-K1-CD46 cells at multiple concentrations is detected first.

[0182] ​4) 800 x g centrifugation at room temperature for 3 min, remove the supernatant containing the antibody, and wash the cells with PBS for 3 times.

[0183] 5) Add secondary antibody PE-Goat anti-Human IgG Fc (1:1000 dilution), mix well, and incubate at room temperature for 30 min.

[0184] 6) 800 x g centrifugation at room temperature for 3 min, remove the supernatant containing the secondary antibody, and wash the cells with PBS for 3 times.

[0185] 7) Resuspend the cells with 500 μL PBS for flow cytometry analysis.

[0186] The results are shown in Tables 9-10 and Figure 4 , wherein A is the flow cytometry detection result of the binding of the first batch of candidate antibodies to CD46-overexpressing human cells; and B is the flow cytometry detection result of the binding of the second batch of candidate antibodies to CD46-overexpressing human cells. Figure 4 Table 9: Flow cytometry detection result of the binding of the first batch of candidate antibodies to CD46-overexpressing cynomolgus monkey cells

[0187]

[0188] Table 10: Flow cytometry detection result of the binding of the second batch of candidate antibodies to CD46-overexpressing cynomolgus monkey cells

[0189]

[0190] From Tables 9-10 (target cells are CHO-S and CHO-S-Cyno CD46 cells) and

[0191] (target cells are CHO-K1-CD46), it can be concluded that the 11 candidate antibodies can specifically bind to CD46-overexpressing human and cynomolgus monkey cells, and the binding effect is significantly better than that of the PC group. It is confirmed that the antibodies can specifically bind to CD46-overexpressing cells. Figure 4 Example 4

[0192] The binding detection of the candidate antibodies to CD46-positive tumor cells is as follows:

[0193] 1) Take CD46-positive tumor cells in the logarithmic growth phase: SW480, SW620, and RKO (ATCC cell bank), and adjust the cell state to the logarithmic growth phase.

[0194] Among them, the SW480 cells are derived from in situ rectal adenocarcinoma, p53 and KRAS gene mutations;

[0195]

[0196] ​SW620 cells are derived from lymph node metastasis, p53 and KRAS gene mutation;

[0197] RKO cells are derived from poorly differentiated colorectal cancer, retaining wild-type p53 and KRAS gene.

[0198] 2) Divide the cells into several portions, and the number of cells in each portion is 2x10 5 cells.

[0199] 3) Incubate the candidate antibody (10 μg / mL, 100 μL / well) or positive antibody with the target cells, mix well, and incubate at room temperature for 1 h. PC is Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom], and NC is still the secondary antibody control group.

[0200] 4) Centrifuge at 800xg at room temperature for 3 min, remove the supernatant containing the antibody, and wash the cells with PBS 3 times.

[0201] 5) Add secondary antibody PE-Goat anti-Human IgG Fc (1:1000 dilution), mix well, and incubate at room temperature for 30 min in the dark.

[0202] 6) Centrifuge at 800xg at room temperature for 3 min, remove the supernatant containing the secondary antibody, and wash the cells with PBS 3 times.

[0203] 7) Resuspend the cells with 500 μL PBS for flow analysis.

[0204] The results are shown in Table 11.

[0205] Table 11 Binding detection results of candidate antibodies on three CD46-positive tumor cells

[0206]

[0207] As can be seen from Table 11, the binding of the 11 candidate antibodies to the three CD46-positive tumor cells is strong, which confirms that the antibodies can specifically bind to CD46-positive tumor cells.

[0208] Example 5

[0209] The steps of the killing detection of the candidate antibodies on CD46-positive tumor cells are as follows:

[0210] 1) Take CD46-positive tumor cells in the logarithmic growth phase: SW480, SW620, and RKO, resuspend in DMEM (Gibco, Cat#41965-062) + 10 % FBS (Merck, Cat#F0193) after digestion to 3x10 46 x 10 4 7 x 10 4

[0211] 2) After the incubation, the secondary antibody Anti-Fc-MMAE (iCarEab) and the candidate antibody were configured at 2 μg / mL (0.5 μg / mL final concentration) using DMEM + 10% FBS, and mixed with the candidate antibody at 1:1 (molar ratio of candidate antibody: secondary antibody = 2:1), 100 μL of the mixture was added to the SW480 and SW620 wells; the secondary antibody and the candidate antibody were configured at 0.4 μg / mL (0.1 μg / mL final concentration), 100 μL of the mixture was added to the RKO wells; the secondary antibody control wells were set: 50 μL + 50 μL medium + 100 μL cells, and the cell control wells were set: 100 μL medium + 100 μL cells. Two parallel wells were set for each group. Incubation was performed in a 37°C incubator with a 5% CO2concentration.

[0212] 3) The cell state was observed every day, and after 4 days of incubation, 100 μL of the cell viability detection reagent was added to each well, and then mixed evenly, 100 μL of the liquid was transferred from each well to a white opaque 96-well plate, and the luminescence value was read using a Tecan M1000Pro. The results are shown in Table 12 and Figure 5

[0213] Table 12. Results of the detection of the killing of CD46-positive tumor cells by the candidate antibodies

[0214]

[0215] Note: The two values in Table 12 corresponding to each group are the detection results of two parallel wells.

[0216] As can be seen from Table 12 and Figure 5 the 11 candidate antibodies have strong killing ability on the three CD46-positive tumor cells, and the effect is significantly better than that of the PC group, which confirms that the antibodies can specifically target and kill CD46-positive tumor cells. Moreover, since the antibodies have the strongest killing ability on RKO cells among the three cells, it is confirmed that the antibodies have the best killing effect on CD46-positive tumor cells that retain wild-type p53 and / or KRAS genes.

[0217] ​​Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained on the basis of the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A single domain antibody that targets human and cynomolgus CD46 cross-binding, characterized in that, The single-domain antibody comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are as shown in any one of 1)~11): 1) the amino acid sequence of HCDR1 is GFTLDYYN, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is ASSDSCGYYYTAGLNY; 2) the amino acid sequence of HCDR1 is GFTLDYYD, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AASRYCGYYYTSHVVDY; 3) the amino acid sequence of HCDR1 is SGFTLDYYNI, the amino acid sequence of HCDR2 is ITSSDGST, and the amino acid sequence of HCDR3 is ASSVSCGYYYAAGLNY; 4) the amino acid sequence of HCDR1 is GFTLNSYA, the amino acid sequence of HCDR2 is LSSSYGST, and the amino acid sequence of HCDR3 is AAALGPDITSVETMCHVPLHIFGS; 5) the amino acid sequence of HCDR1 is GFTLDYYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AIGNYCGYYSDYVPYDY; 6) the amino acid sequence of HCDR1 is GFTFDDYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AAASVCGYYLLSALDA; 7) the amino acid sequence of HCDR1 is GFTLDYYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is ATDLTCGYYYPTAFGS; 8) the amino acid sequence of HCDR1 is GFTFDDYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AADMYCGSYYPTRLGS; 9) the amino acid sequence of HCDR1 is GFTFDDYA, the amino acid sequence of HCDR2 is ISRSDGTT, and the amino acid sequence of HCDR3 is AIGNYCGYYSDYVPYDY; 10) the amino acid sequence of HCDR1 is GFTDDDYA, the amino acid sequence of HCDR2 is ISSLDGST, and the amino acid sequence of HCDR3 is ATGRTCGYYYTYVLDS; 11) the amino acid sequence of HCDR1 is GFSFDEYA, the amino acid sequence of HCDR2 is ISSSDGST, and the amino acid sequence of HCDR3 is AADMYCGSYYPTRLCS.

2. The single-domain antibody of claim 1, wherein, The amino acid sequence of the single-domain antibody is as shown in any one of SEQ ID NO:1~SEQ ID NO:

11.

3. A nucleotide molecule, characterized in that, The nucleotide molecule is used for encoding the single-domain antibody of claim 1 or 2.

4. The nucleotide molecule of claim 3, wherein, The nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO: 12~SEQ ID NO:

22.

5. A biomaterial, characterized by, The biological material is an expression cassette, a recombinant vector or a recombinant cell line comprising the nucleotide molecule of claim 3 or 4.

6. Use of the single-domain antibody of claim 1 or 2 or the nucleotide molecule of claim 3 or 4 or the biological material of claim 5 in the preparation of one or more of a tumor diagnosis, a tumor treatment, a tumor clinical study and a drug development product; the tumor is a colorectal tumor, and the colorectal tumor is a colorectal tumor retaining a wild-type p53 and / or KRAS gene.

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