Single-domain antibody targeting cross binding of human and cynomolgus monkey CD46 and application of single-domain antibody

Abstract

The invention 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 of the single-domain antibody. The alpaca is utilized to generate the nanoscale antibody, the molecular weight of the nanoscale antibody is about 15 kDa, the nanoscale antibody can more efficiently penetrate solid tumor tissues or cross a blood brain barrier, and the binding efficiency of the antibody and a target spot is improved as much as possible. The 11 single-domain antibodies prepared by the invention have the specificity of resisting human and cynomolgus monkey CD46, the binding potency of the 11 single-domain antibodies is remarkably superior to that of an Anti-CD46 humanization antibody in the binding detection of CD46 target protein and CD46 overexpression cells, and the 11 single-domain antibodies can be used for treating tumors. Meanwhile, the single-domain antibody disclosed by the invention has cross-species cross binding capacity, can be directly used for preclinical pharmacodynamics and safety evaluation, does not need to develop a species-specific substitution antibody, and realizes'one target and double effects'.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and immunology, and specifically relates to a single-domain antibody targeting cross-binding of human and cynomolgus macaque CD46 and its application. Background Art

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

[0003] Despite the significant clinical value of CD46, existing targeted technologies face key bottlenecks. First, traditional monoclonal antibodies (such as the humanized antibody BNJ421) have a large molecular weight (approximately 150 kDa), making them difficult to penetrate solid tumors or cross the blood-brain barrier. They are easily cleared by the reticuloendothelial system, resulting in inefficient target binding and a high risk of immunogenicity (such as the HAMA effect). Second, non-humanized antibodies (such as the mouse antibody J4.48) are prone to eliciting anti-drug antibodies, leading to reduced efficacy or allergic reactions. Some antibodies also over-inhibit CD46's physiological functions, causing damage to normal tissues. Third, preclinical studies rely on non-human primate models (such as cynomolgus macaques). However, human CD46 and cynomolgus macaque CD46 (Cyno CD46) differ by 12% in a key structural domain (SCR2-3). This makes it impossible for existing antibodies (such as J4.48) to bind to both simultaneously, hindering preclinical pharmacodynamic and safety evaluations and increasing R&D costs and timelines.

[0004] Single-domain antibodies, also known as VHH antibodies or nanobodies, are antibody fragments composed solely of a single immunoglobulin variable domain (such as VH, VL, or VHH). They bind antigens independently of light chains and constant regions, with a molecular weight of approximately 12-15 kDa. They are the smallest known functional antigen-binding unit and offer advantages such as small molecular weight, strong penetrance, high stability, and ease of genetic engineering. They are considered the next generation of targeted therapeutic molecules. Currently, single-domain antibodies have achieved breakthrough applications in multiple fields, including disease treatment, diagnostics, scientific research tools, and industrial biotechnology. The diverse applications of single-domain antibodies are gradually rewriting the traditional development paradigm for antibody drugs, becoming a key tool in precision medicine and biotechnology innovation. However, single-domain antibodies that can cross-bind with human CD46 and cynomolgus macaque CD46 remain to be further developed. Summary of the Invention

[0005] The purpose of the present invention is to provide a single-domain antibody that cross-binds and targets human and cynomolgus macaque CD46 and its application. The single-domain antibody can specifically bind to the human and cynomolgus macaque CD46 target antigen, thereby exerting a cell-targeting effect and can be used for the diagnosis and / or treatment of tumors.

[0006] 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): 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.

[0007] 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.

[0008] The present invention also provides a nucleotide molecule, which is used to encode the single-domain antibody described in the above technical solution.

[0009] Preferably, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO: 12 to SEQ ID NO: 22.

[0010] The present invention also provides a biomaterial, which is an expression cassette, a recombinant vector or a recombinant cell line comprising the nucleotide molecule described in the above technical solution.

[0011] The present invention also provides the use of the single-domain antibody described in the above technical solution, the nucleotide molecule described in the above technical solution, or the biomaterial described in the above technical solution in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor clinical research and drug development.

[0012] Preferably, the tumor comprises a malignant tumor.

[0013] Preferably, the tumor includes a tumor whose cell surface is CD46 positive.

[0014] Preferably, the tumor comprises a colorectal tumor.

[0015] Preferably, the colorectal tumor includes a colorectal tumor that retains wild-type p53 and / or KRAS genes.

[0016] Beneficial effects: 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

[0017] 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.

[0018] Figure 1 This is a graph showing the purity of the CD46 recombinant antigen protein in Example 1; Figure 2 This is a flow separation diagram after two rounds of magnetic separation in Example 1; Figure 3 FIG2 is a diagram showing the binding of the candidate antibody to the CD46 target protein in Example 2; Figure 4 This is a diagram showing the binding of the candidate antibody in Example 3 to CD46-overexpressing human cells; 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

[0019] 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): 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); 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); 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); 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); 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); 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); 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); 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); 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); 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); 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).

[0020] The homology between the extracellular segment proteins of Human CD46 and Cyno CD46 is 89.25%, which is relatively high and provides the basis for screening cross-binding antibodies. Based on this, the present invention prepares CD46 recombinant antigen protein by targeting the cross-binding domains of human CD46 and cynomolgus macaque CD46; then uses the CD46 recombinant antigen protein to immunize alpacas to ensure that alpacas can produce antibodies that cross-bind to CD46; collects alpaca peripheral blood mononuclear cells to construct a single-domain antibody yeast display library, and selects candidate single-domain antibody sequences that recognize the target protein, ultimately obtaining 11 specifically binding single-domain antibody sequences, as shown in SEQ ID NO.1 to SEQ ID NO.11.

[0021] In one embodiment, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 11; the name and amino acid sequence of the single-domain antibody are as follows: 31-17-1-A12:QVQLVESGGGLVQPGGSLRLSCAAS GFTLDYN IGWFRQAPGKEREGVSC ISSDG ST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC ASSDSCGYYYTAGLNY WGQGTQVTVSS(SEQ IDNO:1); 31-17-1-C11:QLQLVESGGGLVQPGGSLRLSCAAS GFTLDYYD IGWFRQAPGKEREGVLC ISSDG ST YRADSVKGRFTISRDNAKNTVYLQMNSLKPEDIAVYC AASRYCGYYYTSHVVDY WGQGTQVTVSS (SEQ ID NO:2); 31-17-1-E03:AVQLVESGGGLVQPGGSLRLSCAA SGFTLDYNI GWFRQAPGKEREGVSC ITSDG ST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYC ASSVSCGYYYAAGLNY WGQGTQVTVSS (SEQ ID NO:3); 31-17-1-H06:QVQLVESGGGLVQPGESLRLSCAAS GFTLNSYA IGWFRQAPGKEREGVSC LSSSYG ST YYADSVKGRFTISRDNAKGTVYLQMNSLKPEDTAVYSC AAALGPDITSVETMCHVPLHIFGS WGQGTQVTVSS(SEQ ID NO:4); 34-35-3-A11:QLQLVESGGGLVQPGGSLRLSCVAS GFTLDYYA IGWFRQAPGKEREGVSC ISSDG ST YYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYC AIGNYCGYYSDYVPYDY WGQGTQVTVSS (SEQ ID NO:5); 34-35-3-A3:QLQLVESGGGLVQAGGSLRLSCAAS GFTFDYA IGWFRQAPGKEREGVSC ISSDGS T YYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYC AAASVCGYLLSSALDA WGQGTLVTVSS(SEQ ID NO:6); 34-35-3-A8:AVELVDSGGGLVQPGGSLRLSCAAS GFTLDYYA IGWFRQAPGKEREGVSC ISSDGS T YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYC ATDLTCGYYYPTAFGS WGQGTQVTVSS(SEQ ID NO:7); 34-35-3-C6:QLQLVESGGGLVQAGGSLRLSCAAS GFTFDYA IGWFRQAPGKEREGVSC ISSDGS T YYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC AADMYCGSYYPTRLGS WGQGTQVTVSS (SEQ ID NO:8); 34-35-3-D9:QVQLVESGGGLVQAGGSLRLSCAAS GFSFDDYA IGWFRQAPGKEREGVSC IRSDGT T YYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYC AIGNYCGYYSDYVPYDY WGQGTQVTVSS(SEQ IDNO:9); 34-35-3-E1:QVQLVESGGGLVQAGGSLRLSCAAS GFTDDDYA IGWFRQAPGKEREGVSC ISSDGS T FYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC ATGRTCGYYYTYVLDS WGQGTQVTVSS (SEQ ID NO:10); 34-35-3-E9:QLQLVESGGGLVQAGGSRLLSCLVS GFSFDEYA IGWFRQAPGKEREGVSC ISSDGS T YYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC AADMYCGSYYPTRLCS WGQGTQVTVFS (SEQ ID NO:11).

[0022] In SEQ ID NO: 1 to SEQ ID NO: 11, the bold italic underlined portions are the amino acid sequences of HCDR1 to HCDR3 of the single domain antibody.

[0023] The present invention also provides a nucleotide molecule for encoding the single-domain antibody described in the above technical solution. In one embodiment, the nucleotide sequence of the nucleotide molecule is as shown in any one of SEQ ID NO:12 to SEQ ID NO:22, specifically as follows: 31-17-1-A12:caggtgcagctcgtggagtctggtggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattataacataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgagcagcgattcatgtggttactactacaccgccgggctcaactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:12); 31-17-1-C11:cagttgcagctcgtggagtccgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattatgacataggctggttccgccaggccccagggaaggagcgtgagggggtcttatgtattagtagtagtgatggtagcacatacagagcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacatagccgtttattactgtgcagcaagcaggtattgcggttactactacacgtctcacgtggtggactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:13); 31-17-1-E03:gcggtgcagctggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattataacataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattacaagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacagccgtttattactgtgcgagcagcgtttcatgtggttactactacgccgccgggctgaactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:14); 31-17-1-H06:caggtacagctggtggagtctgggggaggcttggtgcagcctggggagtctctgagactctcctgtgcagcctctggattcactttaaattcttatgccataggctggttccgccaggccccaggaaaggagcgtgagggggtctcatgtcttagtagtagttatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagggcacggtgtatctgcaaatgaacagcctgaaaccggaggacacagccgtttatagctgtgcagccgccttagggcccgacattactagcgttgagactatgtgtcatgtaccccttcatatctttgggtcctggggccaggggacccaggtcaccgtctcctcg(SEQ ID NO:15); 34-35-3-A11:cagttgcagctcgtggagtctggggggggcttggtgcagcctgggggatctctgagactctcctgtgtagcctctggattcactttggattattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtctcatgtattagtagtagtgatggtagcacatactatccagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccatttattactgtgcgatcggaaattactgtggttactatagcgactatgtcccttatgactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:16); 34-35-3-A3:cagttgcagctcgtggagtcaggcggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattcactttcgatgattatgctataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgcgcagcagctagcgtttgcggttactacctactctctgctttggacgcatggggccaggggaccctggtcactgtctcctca(SEQ ID NO:17); 34-35-3-A8:gccgtggagctggtggattctggaggaggattggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcactttggattattatgccataggctggttccgccaggccccagggaaggagcgcgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcgacagacctaacttgtggttactactacccgactgcttttggttcctggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:18); 34-35-3-C6:cagttgcagctcgtggagtctggcggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattcactttcgatgattatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagctgacatgtattgcggtagttactacccgacgcgtttgggttcctggggccaggggacccaggtcaccgtctcctcg(SEQ ID NO:19); 34-35-3-D9:caggtgcagctcgtggagtcagggggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattctctttcgatgattatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtcgtagtgatggtaccacatactatccagactccgtgaagggccgattcaccatctccagagacaatgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccatttattactgtgcgatcggaaattactgtggttactatagcgactatgtcccttatgactactggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:20); 34-35-3-E1:caggtgcagctcgtggagtccggggggggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggattcactgacgatgattatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtctagacggtagcacattctatgcagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcgacagggaggacttgtggttactactacacctacgtacttgactcctggggccaggggacccaggtcaccgtctcctca(SEQ ID NO:21); 34-35-3-E9:cagttgcagctcgtggagtccgggggaggcttggtgcaggctggggggtctctgagactctcctgtttagtgtctggattcagtt tcgatgagtatgccataggctggttccgccaggccccagggaaggagcgtgagggggtctcatgtattagtagtagtgatggtagcacatactatg cagactccgtgaagggccgattcaccatctccagtgacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacggccgtttattactgtgcagctgacatgtattgcggtagttactacccgacgcgtttgtgttcctggggccaggggacccaggtcaccgtcttctcg (seq ID NO:22).

[0024] The present invention also provides a biomaterial comprising an expression cassette, recombinant vector, or recombinant cell line containing the nucleotide molecule described in the above technical solution. In one embodiment, the initial vector in the recombinant vector of the present invention is a plasmid vector or a lentiviral vector; in another embodiment, the initial vector in the recombinant vector is a eukaryotic expression vector. The present invention does not specifically limit the method for constructing the biomaterial; conventional genetic engineering techniques in the art can be employed.

[0025] The present invention tested the binding ability of the 11 single-domain antibodies to target proteins (HumanCD46-His and Cyno CD46-His) by enzyme-linked immunosorbent assay (ELISA), and combined with flow cytometry to detect the binding ability of the 11 antibodies to CD46-overexpressing cells (CHO-K1-CD46, CHO-S-Cyno CD46), confirming that the single-domain antibodies of the present invention have specificity against human and cynomolgus macaque CD46.

[0026] The present invention also evaluated the specific binding ability of the 11 aforementioned antibodies to CD46-positive tumor cells (SW480, SW620, and RKO) using flow cytometry. The CCK-8 assay also tested the targeted killing ability of the 11 aforementioned antibodies against CD46-positive tumor cells (SW480, SW620, and RKO), confirming that the single-domain antibodies of the present invention can specifically recognize and kill tumor cells expressing CD46 on their surfaces. Therefore, the single-domain antibodies of the present invention are applicable to the immunotherapy of malignant tumors, particularly CD46-positive tumors.

[0027] Based on the above advantages, the present invention also provides the use of the single-domain antibody described in the above technical solution, the nucleotide molecule described in the above technical solution, or the biomaterial described in the above technical solution in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor clinical research, and drug development. In one embodiment, the tumor includes a malignant tumor; in another embodiment, the malignant tumor is a tumor that is CD46-positive on the cell surface. In one embodiment, the tumor is a colorectal tumor; in another embodiment, the colorectal tumor includes a colorectal tumor that retains wild-type p53 and / or KRAS genes. In one embodiment, the drug development is tumor drug development; in another embodiment, the tumor drug development is pharmacodynamic and / or drug safety evaluation.

[0028] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 Preparation of a single-domain antibody targeting human and cynomolgus macaque CD46 cross-linking, the steps are as follows: By preparing CD46 recombinant antigen protein by targeting the cross-binding domains of human CD46 and cynomolgus macaque CD46, alpacas were immunized with the CD46 recombinant antigen protein, and alpaca peripheral blood mononuclear cells were collected to construct a single-domain antibody yeast display library, and candidate single-domain antibody sequences that recognize the target protein were selected, as follows: 1. Preparation of recombinant CD46 antigen protein targeting the cross-binding domains of human and cynomolgus macaque CD46 Based on the extracellular domain amino acid sequences of human CD46 (Uniprot: P15529-3) and Cynomolgus monkey CD46 (Uniprot: A0A2K5WCS2) proteins from the Uniprot database, eukaryotic expression vectors were constructed. Mammalian cells were transiently transfected using LVtransm transfection reagent (iCarEab, Cat# LVTran100). The culture supernatant was collected and purified by affinity chromatography to produce the target recombinant protein. The purity of the recombinant protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein marker used was purchased from Solarbio, Cat# RP1930.

[0030] The extracellular amino acid sequence of the human CD46 protein is the amino acids at positions 35-313 of the accession number Uniprot: 15529-3; the extracellular amino acid sequence of the Cynomolgus Monkey CD46 protein is the amino acids at positions 35-313 of the accession number Uniprot: A0A2K5WCS2. The amino acid sequences of the CD46 recombinant proteins prepared by affinity chromatography purification are as follows: >Human CD46 (Uniprot: P15529-3) 35-313AA: CEEPPTFEAMELIGKPKPYYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFGYQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVSTSSTTKSPASSASGPRPTYKPPVSNYGGGGSHHHHHH* (SEQ ID NO: 48); >Cynomolgus MonkeyCD46 (Uniprot: A0A2K5WCS2) 35-313AA: CEAPPTFEAMELIGKPKPYYRVGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDEGCYREMCPHIRDPLNGEAILANGSYEFGAELHFICNEGYYLIGKDILYCELKDTVAIWSGKPPLCEKILCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAPGPDPFSLIGESMIYCGNNSTWSHAAPECKVVKCRFPVVENGKQISGFGKKFYYKATVMFECDKGYYLNGSDKIVCESNSTWDPPVPKCLKVSTSPTTKSPTSSASGPRPTYKPPVSNYGGGGSHHHHHH* (SEQ ID NO:49). In the above CD recombinant protein amino acid sequence, the bold amino acids represent the His tag added to the C-terminus of the protein to facilitate subsequent protein purification.

[0031] The binding activity of the target protein was evaluated by ELISA using the positive control antibody (PC) Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] (iCarEab). The coating was pcDNA3.4-Cyno CD46-C35-Y328-His (iCarEab), 1 μg / mL, coated overnight at 4°C; the primary antibody (PC) was Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] (disclosed in CN201811396656.4), 10 μg / mL, 3-fold dilution at 7 points; the secondary antibody was HRP-anti Human IgG (eBioscience, Cat#12-4998-82); the negative control (NC) group was the secondary antibody control well, detecting the binding of Cyno CD46 protein to the positive antibody. The results are shown in Tables 1 and Figure 1 As shown, in Figure 1 In the figure, M is a protein marker, lane 1 is Cyno CD46-C35-Y328-His, and lane 2 is Human CD46-His.

[0032] Among them, Lenti-CMV-mCORI-hEF-mHVORI-puro[hEF-HTLV prom] was obtained in the early preparation process of patent CN201811396656.4. It is an antibody with anti-human CD46 gene activity and is used as PC in this patent.

[0033] Table 1 Results of ELISA binding activity evaluation of target proteins

[0034] Note: The two values ​​corresponding to each group in Table 1 are the test results of two parallel replicate wells.

[0035] Depend on Figure 1 As can be seen from Table 1, 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.

[0036] 2. Alpaca immunization with CD46 recombinant antigen protein Two alpacas (iCarEab) were immunized with the recombinant antigen protein described above. Multiple subcutaneous immunizations were administered 14 days apart, using Adjuvant (Gerbu, Cat#3030) as an adjuvant. Starting with the second immunization, peripheral blood was collected from the alpacas one week after each immunization to monitor immune serum titers. After immunization, 100 mL of peripheral blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated for the construction of a single-domain antibody display library. The immunization schedule is shown in Table 2.

[0037] Table 2 Immunization process schedule

[0038] 3. Immune titer detection 1) Collect 5 mL of peripheral blood from alpacas and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight.

[0039] 2) Place the centrifuge tube containing the blood sample in a centrifuge and 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.

[0040] 3) Dilute the target recombinant protein to a final concentration of 1 µg / mL in sterile carbonate-buffered saline (CBS; Macklin, Cat# C885533).

[0041] 4) Take a new 96-well microtiter plate, add 1 µg / mL target recombinant protein, 100 µL / well, and coat overnight at 4°C.

[0042] 5) Remove the antigen coating solution and wash five times with phosphate-buffered saline containing 0.05% Tween 20 (PBST; Merck, Cat# P3563).

[0043] 6) Add 200 μL / well of phosphate-buffered saline (MPBS; Beyotime, Cat# P0216; Gibco, Cat# 14190-250) containing 3% skim milk powder and block at 37°C for 2 h.

[0044] 7) After removing the blocking buffer, wash the plate five times with PBST, add 100 µL / well of serially diluted serum, and incubate at room temperature for 1 hour. The control wells contain phosphate-buffered saline (PBS).

[0045] 8) Remove the liquid from the wells and wash five times with PBST. Add 100 µL of HRP anti-Llama IgG (H+L) antibody (1:50,000 dilution; Novus, Cat# NBP1-75095) and incubate at room temperature for 1 hour.

[0046] 9) After removing the liquid from the wells, wash the plate five times with PBST, add 100 μL / well of TMB colorimetric solution (Merck, Cat# T0440), and incubate at room temperature in the dark for 10-15 minutes.

[0047] 10) Add 50 μL / well of stop solution (Biosharp, Cat#BL1829B).

[0048] 11) Use a microplate reader to read the OD in the wells 450 value.

[0049] ELISA results indicated that the titer of the alpaca negative serum was low, indicating that it was suitable for immunization. After the second immunization, the immune serum was able to bind to the target recombinant protein, and the OD value showed a gradient change with the serial dilution of the immune serum. Even at a serum dilution gradient of 1:64K, the titer of the second-immunization group was still approximately 5 times that of the negative serum group (≥ 3 times is acceptable), confirming that the titer of the alpaca increased significantly after the second immunization, meeting the requirements for blood collection and library construction, and suitable for use in the construction of an antibody display library. Therefore, no repeated immune titer testing was performed after the third immunization.

[0050] 4. Alpaca PBMC Isolation and VHH Antibody Fragment Cloning 1) 100 mL of peripheral blood was collected from each alpaca and PBMCs were isolated using Lymphocyte Separation Medium (BioLab, Cat# JH0171). Because both alpacas had high immune titers, PBMCs from the two alpacas were pooled for subsequent experiments.

[0051] 2) RNA was extracted using RNAiso Plus (TaKaRa, Cat#9109) and reverse transcribed using the PrimeScript™ II 1stStrand cDNA Synthesis Kit (TaKaRa, Cat#6210B) to prepare cDNA.

[0052] 3) Prepare Mix 1 in a 200 μL PCR tube according to the PCR reaction system in Table 3. Incubate at 65°C for 5 minutes and then quickly cool on ice.

[0053] 4) Prepare the reaction solution in the PCR tubes described in Table 4. Mix thoroughly by pipetting. Aliquot 80 μL into each tube. Incubate in a PCR instrument at 42°C for 1 hour, then heat-inactivate at 70°C for 15 minutes. Finally, store the cDNA samples on ice or at -20°C for long-term storage.

[0054] 5) Prepare the first-round PCR reaction system (50 μL / tube) according to the reaction system in Table 5, with the upstream primer binding to the signal peptide and the downstream primer binding to the CH2 region. NuHi Power mix was purchased from New Sea Biotechnology, Cat# NH9303. After preparing the PCR reaction system, set up the PCR instrument according to the protocol in Table 6. The nucleotide sequences of the upstream and downstream primers are 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO: 50) and 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 51), respectively.

[0055] 6) PCR products were analyzed by electrophoresis using 1% agarose gel (Merck, Cat# A6013). PCR bands of approximately 750 bp and 1000 bp were obtained. The 750 bp PCR product was recovered using a gel extraction kit (Qiagen, Cat# 28706) and used as template for the second round of PCR. The concentration was determined using a NanoDrop flow cytometer.

[0056] 7) Prepare a second-round PCR reaction system (50 μL / tube) according to the reaction system in Table 7. The upstream primer binds to the antibody FR1 region, the downstream primer binds to the anti-hinge and FR4 regions, and the restriction enzyme site is SfiI. After preparing the PCR reaction system, set up the PCR instrument according to the program in Table 6. The nucleotide sequences of the upstream primer and downstream primer are 5'-AGKTGCAGCTCGTGGAGTCNGGNGG-3' (SEQ ID NO: 52) and 5'-GATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO: 53), respectively.

[0057] 8) Perform agarose gel electrophoresis on 1% agarose gel to analyze the PCR products from the second round. Isolate a VHH fragment with a molecular weight of approximately 500 bp. Recover the VHH PCR product using a gel recovery kit and determine its concentration using a NanoDrop.

[0058] Aliquot 200 μL of the recovered second-round PCR product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate (Sigma, Cat#126-96-5) and 1 μg / μL glycogen (Glycogen; Beyotime, Cat#D0812), mix by pipetting, add 880 μL of anhydrous ethanol (Merck, Cat#459828), mix by inversion, and freeze at -80°C.

[0059] Table 3 Reverse transcription PCR mixture Mix 1 preparation system

[0060] Table 4 Reverse transcription PCR reaction solution preparation system

[0061] Table 5 First round PCR reaction system

[0062] Table 6 PCR reaction program

[0063] Table 7 Second round PCR reaction system

[0064] 5. Construction and panning of single domain antibody yeast display library 1) Use SfiI (NEB, Cat#R0123L) to digest the pYDisplay vector (iCarEab). The linearization enzyme digestion system for the yeast display vector pYDisplay is shown in Table 8. Aliquot 100 μL / tube and digest at 50°C overnight.

[0065] Table 8 Yeast display vector pYDisplay linearization enzyme digestion system

[0066] 2) Separate the pYDisplay vector fragment using a 1% agarose gel, excise a 5000 bp fragment, recover it from the gel, and determine its concentration using a NanoDrop.

[0067] 3) Aliquot 200 μL of the recovered pYDisplay digestion product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3 M sodium acetate and 1 μg / μL Glycogen, pipette to mix thoroughly, add 880 μL of anhydrous ethanol, invert to mix, and store at -80°C.

[0068] 4) Streak the competent yeast strain frozen at -80°C onto a Yeast Extract Peptone Dextrose (YPD) solid medium plate and activate at 30°C for 3-5 days.

[0069] 5) Inoculate a single competent yeast colony into 50 mL of YPD medium and shake at 250 rpm and 30°C for 1–2 days.

[0070] 6) Mix the linearized vector fragment and PCR product, add them to an electroporation cuvette, and electroporate. After electroporation, shake the culture in a yeast competent transfection flask at 220 rpm and 30°C for 1 hour to prepare a yeast competent strain.

[0071] 7) Take 20 μL of the resuspension, dilute it 5000 times with growth selection synthetic medium (SDCAA), aspirate 100 μL, apply it to SDCAA plate, and culture it for 2-3 days. According to the library capacity calculation, the constructed yeast display library capacity is 2.05×10 9 , meet the requirements and arrange diversity testing.

[0072] 8) Randomly select single clones for sequencing to analyze the diversity of the yeast display library. Comparison of the sequencing results shows that all of them are antibody differential sequences, with no empty or repeated sequences, indicating good library diversity.

[0073] 9) Continue culturing the remaining bacterial suspension from step 6) for 24 h, collect it into a 50 mL centrifuge tube, centrifuge at 3000 × g for 5 min, discard the supernatant, add 10 mL of SDCAA to resuspend, and mix with 50% glycerol: resuspension solution in a 1:1 ratio. Store at -80°C.

[0074] 10) Add the yeast cultured in SDCAA to a 250 mL shake flask containing 50 mL of galactose induction medium (SGCAA) and culture on a shaker at 30°C and 240 rpm for 16 h.

[0075] 11) After centrifugation, discard the supernatant, resuspend the tube in 1 mL of phosphate buffered saline (PBSA) containing 0.5% BSA (0.5% PBSA), add the tube to a 1.5 mL centrifuge tube, centrifuge at 3000 × g for 5 min, discard the supernatant, and wash again with 0.5% PBSA.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 2In 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.

[0085] 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.

[0086] 6. Yeast monoclonal detection 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.

[0087] 7. Construction of antibody eukaryotic expression vector 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.

[0088] 2) The antibody eukaryotic expression vector was transiently transfected into 293F cells (ATCC cell bank) to obtain 14 antibody expression supernatants.

[0089] 3) Flow cytometry was used to detect the binding of 14 candidate antibodies to antigen proteins. The antigen proteins used were derived from specific cell lines, namely CHO-S, CHO-K1-CD46, and CHO-S-Cyno CD46 (iCarEab cell bank), 2×10 5100 μL / well. Primary antibody: candidate antibody transfection supernatant, 100 μL / well; secondary antibody: PE-Goat anti-Human IgGFc (1:1000 dilution; Invitrogen, Cat#12-4998-82); PC: Lenti-CMV-mCORI-hEF-mHVORI-puro [hEF-HTLV prom]; NC: secondary antibody control, without the target protein CD46 but with the secondary antibody, used as the basis for gating during flow cytometry analysis.

[0090] Flow cytometric analysis revealed that, except for 34-35-3-C3, which had weak binding, 31-17-5-H8 clone, which had no binding, and 34-35-3-E8 clone, which had nonspecific binding, the remaining 11 candidate antibodies all cross-bound to human and monkey cells, with significantly better effects than the PC group, and could be used for subsequent antibody purification and preparation.

[0091] 8. Expression and purification of candidate single domain antibodies 1) Based on the flow cytometry test results of the candidate antibodies, positive clones are selected and the antibody expression and preparation is performed.

[0092] 2) Remove LVTransm transfection reagent and pcDNA3.4-Fc antibody expression vector from the refrigerator. Thaw at room temperature and mix thoroughly by pipetting up and down. Remove PBS buffer and warm to room temperature. Dispense 2 mL of PBS into each well of a 6-well plate and add 20 μg of antibody expression vector to each well. Mix thoroughly by pipetting up and down. Then, add 60 μL of LVTransm and immediately mix by pipetting up and down. Let stand at room temperature for 10 minutes.

[0093] 3) Add the DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Continue culturing the cells in a 37°C, 5% CO2, 130 rpm incubator.

[0094] 4) After 5-7 days of continuous culture, collect the supernatant by centrifugation, filter through a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube.

[0095] 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 Bojin Biotechnology Co., Ltd., Cat# BG18-0010-02).

[0096] The 11 candidate antibody sequences obtained by the above method are shown in SEQ ID NOs: 1 to 11, all with molecular weights of approximately 15 kDa. Their heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) are annotated in the amino acid sequences shown in SEQ ID NOs: 1 to 11. The RNA sequences corresponding to the candidate antibodies can be deduced from the codon triplets, as shown in SEQ ID NOs: 12 to 22.

[0097] Example 2 ELISA test for binding of candidate antibodies to CD46 target protein is performed as follows: 1) Dilute CD46 recombinant protein in sterile CBS to a final concentration of 2 µg / mL. Add 100 µL to each well of a new 96-well microtiter plate and coat overnight at 4°C.

[0098] 2) Remove the antigen coating solution, wash with PBST five times, add 200 μL / well of 3% MPBS, and block at 37°C for 2 h.

[0099] 3) After removing the blocking buffer, wash the plate five times with PBST. Add the expressed recombinant antibody (100 μL / well of transfection supernatant) or the positive antibody (starting at 10 μg / mL, diluted 5-fold over 7 points, 100 μL / well). For PC, use Lenti-CMV-mCORI-hEF-mHVORI-puro [hEF-HTLV prom]. Incubate at room temperature for 1 hour. Control wells should contain PBS.

[0100] 4) Remove the liquid from the wells and wash five times with PBST. Add 100 μL / well of HRP-Goat anti-Human IgG Fc antibody (1:30,000 dilution; Abcam, Cat#ab97225) and incubate at room temperature for 1 h.

[0101] 5) After removing the liquid from the wells, wash the plate five times with PBST, add 100 μL / well of TMB colorimetric solution, and incubate at room temperature in the dark for 10-15 minutes. 6) Add 50 μL / well of stop solution.

[0102] 7) Use a microplate reader to read the OD in the well 450 value.

[0103] The results are as follows Figure 3As shown, A is a diagram showing the binding of the first batch of candidate antibodies to Human CD46-His; B is a diagram showing the binding of the second batch of candidate antibodies to Human CD46-His; C is a diagram showing the binding of the first batch of candidate antibodies to Cyno CD46-His; and D is a diagram showing the binding of the second batch of candidate antibodies to Cyno CD46-His.

[0104] Depend on Figure 3 It can be concluded that after coating with Human CD46-His antigen (1 μg / mL) and Cyno CD46-His antigen (1 μg / mL), the 11 candidate antibodies bound strongly to Human CD46-His and Cyno CD46-His, and the effect was significantly better than that of the PC group, confirming that the antibodies can specifically bind to the CD46 target protein.

[0105] Example 3 Binding of candidate antibodies to CD46-overexpressing cells is tested as follows: 1) Resuscitate CHO-S, CHO-K1-CD46, and CHO-S-Cyno CD46 cells from liquid nitrogen and adjust the cells to the logarithmic growth phase.

[0106] 2) Divide the cells into several portions, with the number of cells in each portion being 2×10 5 cells.

[0107] 3) Incubate the candidate antibody (10 µg / mL, 100 µL / well) with target cells CHO-S and CHO-S-Cyno CD46. Additionally, adjust the candidate antibody to a starting concentration of 20 µg / mL, 100 µL / well, and incubate with target cells CHO-K1-CD46 at eight 5-fold dilutions. Mix thoroughly and incubate at room temperature for 1 hour. The PC group was Lenti-CMV-mCORI-hEF-mHVORI-puro [hEF-HTLV prom], and the NC group was the secondary antibody control group.

[0108] Among them, the candidate antibody was gradiently diluted when incubated with the target cells CHO-K1-CD46. This is because the candidate antibody needs to be applied to human tumor cells for evaluation in the later stage. Therefore, the specific binding efficacy of the candidate antibody to CHO-K1-CD46 cells at multiple concentrations was first tested here.

[0109] 4) Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the antibody, and wash the cells three times with PBS.

[0110] 5) Add secondary antibody PE-Goat anti-Human IgG Fc (1:1000 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 min.

[0111] 6) Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS.

[0112] 7) Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0113] The results are shown in Tables 9-10 and Figure 4 As shown, in Figure 4 In the figure, A is a flow cytometry result diagram of the binding of the first batch of candidate antibodies to CD46-overexpressing human cells; B is a flow cytometry result diagram of the binding of the second batch of candidate antibodies to CD46-overexpressing human cells.

[0114] Table 9 Binding test results of the first batch of candidate antibodies and CD46-overexpressing cynomolgus monkey-derived cells

[0115] Table 10 Binding test results of the second batch of candidate antibodies and CD46-overexpressing cynomolgus monkey-derived cells

[0116] From Tables 9 to 10 (target cells are CHO-S and CHO-S-Cyno CD46 cells) Figure 4 (Target cells: CHO-K1-CD46) It can be concluded that all 11 candidate antibodies were able to specifically bind to CD46-overexpressing human and cynomolgus macaque cells, with significantly better binding than the PC group. This confirms that the antibodies can specifically bind to CD46-overexpressing cells.

[0117] Example 4 Binding of candidate antibodies to CD46-positive tumor cells is tested as follows: 1) Obtain CD46-positive tumor cells in the logarithmic growth phase: SW480, SW620, RKO (ATCC cell bank) and adjust the cells to the logarithmic growth phase.

[0118] Among them, SW480 cells are derived from in situ rectal adenocarcinoma and have p53 and KRAS gene mutations; The origin of SW620 cells is lymph node metastasis, p53 and KRAS gene mutations; RKO cells are derived from poorly differentiated colorectal cancer that retains wild-type p53 and KRAS genes.

[0119] 2) Divide the cells into several portions, with the number of cells in each portion being 2×10 5 cells.

[0120] 3) Incubate target cells with candidate antibodies (10 µg / mL, 100 µL / well) or positive antibodies, mix thoroughly, and incubate at room temperature for 1 hour. PC represents Lenti-CMV-mCORI-hEF-mHVORI-puro [hEF-HTLV prom], and NC represents the secondary antibody control.

[0121] 4) Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the antibody, and wash the cells three times with PBS.

[0122] 5) Add secondary antibody PE-Goat anti-Human IgG Fc (1:1000 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 min.

[0123] 6) Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS.

[0124] 7) Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0125] The results are shown in Table 11.

[0126] Table 11 Binding test results of candidate antibodies to three CD46-positive tumor cells

[0127] It can be seen from Table 11 that the 11 candidate antibodies all strongly bound to the three CD46-positive tumor cells, confirming that the antibodies can specifically bind to CD46-positive tumor cells.

[0128] Example 5 The steps for testing the killing of CD46-positive tumor cells by candidate antibodies are as follows; 1) CD46-positive tumor cells in the logarithmic growth phase were obtained: SW480, SW620, and RKO. After digestion, they were resuspended in DMEM (Gibco, Cat#41965-062) + 10% FBS (Merck, Cat#F0193) to a volume of 3×10 4 / mL, 6×10 4 / mL and 7×10 4 100 μL of the solution was added to each well of a 96-well plate. The wells around the 96-well plate were not used, and 200 μL of PBS buffer was added to prevent marginal effects. The plate was placed in a CO2 concentration of 5% and incubated at 37°C for 24 h.

[0129] 2) After incubation, the secondary antibody, Anti-Fc-MMAE (iCarEab), and the candidate antibody were diluted to 2 μg / mL (final concentration 0.5 μg / mL) in DMEM + 10% FBS and mixed with the candidate antibody at a 1:1 ratio (candidate antibody:secondary antibody molar ratio = 2:1). 100 μL of this mixture was added to the SW480 and SW620 wells. The secondary antibody and candidate antibody were diluted to 0.4 μg / mL (final concentration 0.1 μg / mL) and 100 μL of this mixture was added to the RKO wells. Control wells for the secondary antibody were prepared with 50 μL of secondary antibody plus 50 μL of culture medium plus 100 μL of cells, and control wells for the cells were prepared with 100 μL of culture medium plus 100 μL of cells. Two replicate wells were set up for each group. Incubate the wells in a 37°C incubator with 5% CO2.

[0130] 3) Observe the cell status every day. After 4 days of culture, add 100 μL of cell viability detection reagent to each cell well. After pipetting evenly, transfer 100 μL of liquid from each well to an opaque 96-well plate with a white bottom. Use Tecan M1000Pro to read the luminescence value. The results are shown in Table 12 and Figure 5 shown.

[0131] Table 12 Results of the killing test of candidate antibodies on CD46-positive tumor cells

[0132] Note: The two values ​​corresponding to each group in Table 12 are the test results of two parallel replicate wells.

[0133] From Table 12 and Figure 5 It can be concluded that all 11 candidate antibodies showed strong killing abilities against the three CD46-positive tumor cell lines, with significantly better results than the PC group, confirming that the antibodies can specifically target and kill CD46-positive tumor cells. Furthermore, since the antibodies showed the strongest killing ability against RKO cells among the three cell lines, they were confirmed to be most effective against CD46-positive tumor cells that retain wild-type p53 and / or KRAS genes.

[0134] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A single domain antibody targeting human and cynomolgus macaque CD46 cross-binding, characterized in that The single-domain antibody comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as shown in any one of 1) to 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 according to claim 1, characterized in that The amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO: 1 to SEQ ID NO:

11.

3. A nucleotide molecule, characterized in that The nucleotide molecule is used to encode the single domain antibody according to claim 1 or 2.

4. The nucleotide molecule according to claim 3, characterized in that The nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO: 12 to SEQ ID NO:

22.

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

6. Use of the single domain antibody according to claim 1 or 2, the nucleotide molecule according to claim 3 or 4, or the biomaterial according to claim 5 in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor clinical research, and drug development.

7. The use according to claim 6, characterized in that The tumor includes a malignant tumor.

8. The use according to claim 6 or 7, characterized in that The tumor includes a tumor whose cell surface is CD46 positive.

9. The use according to claim 8, characterized in that The tumors include colorectal tumors.

10. The use according to claim 9, characterized in that The colorectal tumors include colorectal tumors that retain wild-type p53 and / or KRAS genes.

Images