Single-domain antibody targeting PD-L1 cross binding of human and cynomolgus monkeys 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 PD-L1 cross binding of human and cynomolgus monkeys and application of the single-domain antibody. The single-domain antibody disclosed by the invention has the specificity of resisting PD-L1 of human and cynomolgus monkeys, and shows relatively strong binding efficacy in the binding detection of PD-L1 target protein or PD-L1 overexpression cells; in the detection of blocking PD-1 / PD-L1 functions, a part of the single-domain antibodies even show a blocking effect which is obviously superior to that of Atezolizumab, so that the single-domain antibodies prepared by the invention can be used for diagnosis, treatment and prognosis detection of PD-L1 positive 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] This invention belongs to the fields of biomedicine and immunology, specifically relating to a single-domain antibody that targets the cross-binding of PD-L1 in humans and cynomolgus monkeys and its applications. Background Technology

[0002] PD-L1, also known as programmed death-ligand-1, is a protein located on the cell surface and a core target in current tumor immunotherapy. PD-L1 is a crucial "shield" used by cancer cells to evade the immune system, especially T cells. Inhibiting PD-L1 blocks its binding to PD-1 (a protein molecule located on the surface of immune cells, primarily T cells), thereby relieving immunosuppression, awakening T cells to attack tumors again, and producing a durable therapeutic effect. While current PD-L1 targeting technology, primarily based on antibody drugs, has made significant progress in tumor treatment, its core defects and limitations are also very obvious, mainly reflected in the characteristics of the drugs themselves, clinical efficacy, and safety. First, most currently marketed PD-L1 inhibitors are large molecule drugs such as monoclonal antibodies, which have poor tissue penetration and are difficult to effectively penetrate deep into solid tumors, affecting efficacy. Second, in practical applications, the efficacy rate of PD-L1 inhibitor monotherapy is limited (approximately 20%), and problems such as drug resistance may arise. In addition, because the activation of the immune system is non-specific, PD-L1 inhibitors may also "accidentally" damage normal tissues while attacking tumor cells, triggering immune-related adverse reactions.

[0003] To address the aforementioned shortcomings, new strategies such as developing novel molecular forms, innovative delivery systems, and combination therapies are being actively explored. As a rising star among novel molecular forms, single-domain antibodies, also known as nanobodies, are the smallest known functional antigen-binding fragments in nature (approximately 15 kDa), representing a significant paradigm shift in the field of antibody drugs. Single-domain antibodies, with their unique advantages such as small size, high stability, strong binding capacity, low production cost, and ease of engineering, offer new insights into solving the challenges faced by traditional antibodies in targeting immune checkpoints such as PD-L1.

[0004] It is important to note that the "human-cynomolgus monkey cross-binding" characteristic must be emphasized during antibody development. This is primarily because before a drug can enter human clinical trials, its safety and efficacy must be thoroughly evaluated in relevant animal models. Cynomolgus monkeys, due to their close relationship to humans, share highly similar immune systems, physiological functions, and the development of many diseases, making them the most irreplaceable non-human primate model. Failure to fully consider species specificity during the development phase could lead to difficulties in finding suitable animal models for preclinical safety evaluations, forcing the termination of drug development. This highlights the strategic necessity of screening for "cross-reactive" antibodies that can simultaneously bind to both human and cynomolgus monkey targets during the antibody development stage. However, single-domain antibodies capable of cross-binding to PD-L1 in both humans and cynomolgus monkeys still require further development. Summary of the Invention

[0005] The purpose of this invention is to provide a single-domain antibody that targets and cross-binds with human and cynomolgus monkey PD-L1 and its application. The single-domain antibody can specifically bind to the human and cynomolgus monkey PD-L1 target antigen, thereby playing a cell-targeting role and can be used for tumor diagnosis, treatment and prognosis.

[0006] This invention provides a single-domain antibody targeting the cross-binding of PD-L1 in humans and cynomolgus monkeys, and its application. The single-domain antibody includes complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in any one of 1) to 9). 1) The amino acid sequence of CDR1 is GSSTSSIHV, the amino acid sequence of CDR2 is LFTGGGNT, and the amino acid sequence of CDR3 is KAVEIGQAY. 2) The amino acid sequence of CDR1 is GTTISLIS, the amino acid sequence of CDR2 is LFTGGGNT, and the amino acid sequence of CDR3 is KAVEIGQAY. 3) The amino acid sequence of CDR1 is GFTVDSSA, the amino acid sequence of CDR2 is ILSSGIT, and the amino acid sequence of CDR3 is NIDDGVTAQDY. 4) The amino acid sequence of CDR1 is GFTFSASA, the amino acid sequence of CDR2 is IRSDGTT, and the amino acid sequence of CDR3 is ACGSK. 5) The amino acid sequence of CDR1 is ESSTSNIHE, the amino acid sequence of CDR2 is LFTGGGNT, and the amino acid sequence of CDR3 is KAVNIREAY. 6) The amino acid sequence of CDR1 is GSTFIINA, the amino acid sequence of CDR2 is ISSGGSA, and the amino acid sequence of CDR3 is NDWVRDY. 7) The amino acid sequence of CDR1 is GSTLSINA, the amino acid sequence of CDR2 is ISSTSRT, and the amino acid sequence of CDR3 is NDWIRGY. 8) The amino acid sequence of CDR1 is GDTFRHYV, the amino acid sequence of CDR2 is ISWSGSST, and the amino acid sequence of CDR3 is AARRATTLGAVEAQSYDY. 9) The amino acid sequence of CDR1 is GSTFSNYD, the amino acid sequence of CDR2 is MTRFGHT, and the amino acid sequence of CDR3 is NTLELVSKGL.

[0007] Preferably, the single-domain antibody comprises frame regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in any one of A) to I). A) The amino acid sequence of FR1 is QVKLEESGGGLVQAGGSLRLSCAAS, the amino acid sequence of FR2 is MGWYRQAPGKQRDWVAT, the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC, and the amino acid sequence of FR4 is WGHGTQVTVSS. B) The amino acid sequence of FR1 is EVQLVESGGGLVQAGGSLILSCTAT, the amino acid sequence of FR2 is MGWYRQAPGKQRDWVAT, the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS. C) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCTAS, the amino acid sequence of FR2 is TGWFRQAPGKQRELVAA, the amino acid sequence of FR3 is HYLDSVKGRFTISRDNAKKSVYLQMNSLKPEDTALYTC, and the amino acid sequence of FR4 is WGQGTQVTVSS. D) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCTAS, the amino acid sequence of FR2 is INWVRQAPGKGREWVST, the amino acid sequence of FR3 is YYAASVKGRFTISRDNAGNTVNLQMNNLKPEDTALYYC, and the amino acid sequence of FR4 is QGQGTQVTVSS. E) The amino acid sequence of FR1 is QVQLVECGRGLAGAGGSLRLSCAPS, the amino acid sequence of FR2 is GGWYRRAPGKQREWVAT, the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLLMNRLKPEDTAVYYC, and the amino acid sequence of FR4 is WGERTQVTVSS. F) The amino acid sequence of FR1 is QVQLVESGGGLVQAGGFLRLSCAAS, the amino acid sequence of FR2 is IGWYRQAPGKQRELVAT, the amino acid sequence of FR3 is VYSPSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYIC, and the amino acid sequence of FR4 is WGQGTQVTVSA. G) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGTLRLSCAAS, the amino acid sequence of FR2 is IGWYRQAPGKQREFVAT, the amino acid sequence of FR3 is IYADFVKGRFTISRDNAKNTVFLQMNNLKPEDTAVYYC, and the amino acid sequence of FR4 is WGHGTQVTVSS. H) The amino acid sequence of FR1 is QVQLVESGGGLVQAGGSLRLSCAAS, the amino acid sequence of FR2 is MGWFRQAPGKEREFVSR, the amino acid sequence of FR3 is YYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGNQVNVSS. I) The amino acid sequence of FR1 is DVQLVESGGGLVQAGGSLRLSCAAS, the amino acid sequence of FR2 is ITWYRQAPGKQREWVAL, the amino acid sequence of FR3 is NYAAPAKGRFTISRSNAKDTVYLQMNSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS.

[0008] Preferably, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO:1 to SEQ ID NO:9.

[0009] The present invention also provides a nucleotide molecule for encoding the single-domain antibody described in the above technical solution.

[0010] Preferably, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO:10 to SEQ ID NO:18.

[0011] The present invention also provides a biomaterial, which is an expression cassette, recombinant vector or recombinant cell line containing the nucleotide molecules described in the above technical solutions.

[0012] The present invention also provides the application of the single-domain antibody, the nucleotide molecule, or the biomaterial described in the above-described technical solutions in the preparation of PD-L1 inhibitors.

[0013] This invention also provides the application of the single-domain antibody, nucleotide molecule, or biomaterial described in the above-described technical solutions in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor prognosis, tumor clinical research, and drug development.

[0014] Preferably, the tumor includes tumors that are positive for PD-L1 expression.

[0015] The present invention also provides a PD-L1 inhibitor, the active ingredient of which includes the single-domain antibody described in the above technical solution.

[0016] Beneficial effects: This invention provides a single-domain antibody that cross-binds to PD-L1 in humans and cynomolgus monkeys, and its applications. The single-domain antibody exhibits specificity against PD-L1 in both humans and cynomolgus monkeys, demonstrating strong binding efficacy in binding assays with PD-L1 target proteins or PD-L1-overexpressing cells. In PD-1 / PD-L1 function blocking assays, some of these single-domain antibodies even showed significantly superior blocking effects compared to atezolizumab, confirming that the single-domain antibody prepared by this invention can be used for the diagnosis, treatment, and prognostic detection of PD-L1-positive tumors. Furthermore, the single-domain antibody of this invention possesses cross-species binding capability, allowing it to be directly used for preclinical pharmacodynamic and safety assessments without the need to develop species-specific alternative antibodies, achieving a "one-target, two-effect" approach. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a purity detection graph of the PD-L1 recombinant antigen protein in Example 1; Figure 2 This is a flow cytometry result diagram after one round of magnetic separation in Example 1; Figure 3 This is a diagram showing the binding of the candidate antibody to the PD-L1 target protein in Example 2; Figure 4 This is a diagram showing the binding of the candidate antibody to PD-L1 overexpressing cells in Example 3; Figure 5 This is a graph showing the results of detecting the PD-1 / PD-L1 function of the candidate antibody in Example 4. Detailed Implementation

[0019] This invention provides a single-domain antibody targeting the cross-binding of PD-L1 in humans and cynomolgus monkeys, and its application. The single-domain antibody includes complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in any one of 1) to 9). 1) The amino acid sequence of CDR1 is GSSTSSIHV (SEQ ID NO:19), the amino acid sequence of CDR2 is LFTGGGNT (SEQ ID NO:20), and the amino acid sequence of CDR3 is KAVEIGQAY (SEQ ID NO:21). 2) The amino acid sequence of CDR1 is GTTIS (SEQ ID NO:22), the amino acid sequence of CDR2 is LFTGGGNT (SEQ ID NO:20), and the amino acid sequence of CDR3 is KAVEIGQAY (SEQ ID NO:21). 3) The amino acid sequence of CDR1 is GFTVDSSA (SEQ ID NO:23), the amino acid sequence of CDR2 is ILSSGIT (SEQ ID NO:24), and the amino acid sequence of CDR3 is NIDDGVTAQDY (SEQ ID NO:25). 4) The amino acid sequence of CDR1 is GFTFSASA (SEQ ID NO:26), the amino acid sequence of CDR2 is IRSDGTT (SEQ ID NO:27), and the amino acid sequence of CDR3 is ACGSK (SEQ ID NO:28). 5) The amino acid sequence of CDR1 is ESSTSNIHE (SEQ ID NO:29), the amino acid sequence of CDR2 is LFTGGGNT (SEQ ID NO:20), and the amino acid sequence of CDR3 is KAVNIREAY (SEQ ID NO:30). 6) The amino acid sequence of CDR1 is GSTFIINA (SEQ ID NO:31), the amino acid sequence of CDR2 is ISSGGSA (SEQ ID NO:32), and the amino acid sequence of CDR3 is NDWVRDY (SEQ ID NO:33). 7) The amino acid sequence of CDR1 is GSTLSINA (SEQ ID NO:34), the amino acid sequence of CDR2 is ISSTSRT (SEQ ID NO:35), and the amino acid sequence of CDR3 is NDWIRGY (SEQ ID NO:36). 8) The amino acid sequence of CDR1 is GDTFRHYV (SEQ ID NO:37), the amino acid sequence of CDR2 is ISWGSST (SEQ ID NO:38), and the amino acid sequence of CDR3 is AARRATTLGAVEAQSYDY (SEQ ID NO:39). 9) The amino acid sequence of CDR1 is GSTFSNYD (SEQ ID NO:40), the amino acid sequence of CDR2 is MTRFGHT (SEQ ID NO:41), and the amino acid sequence of CDR3 is NTLELVSKGL (SEQ ID NO:42).

[0020] In one embodiment, the single-domain antibody includes frame regions FR1, FR2, FR3, and FR4, the amino acid sequences of which are shown in any one of A) to I). A) The amino acid sequence of FR1 is QVKLEESGGGLVQAGGSLRLSCAAS (SEQ ID NO:43), the amino acid sequence of FR2 is MGWYRQAPGKQRDWVAT (SEQ ID NO:44), the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC (SEQ ID NO:45), and the amino acid sequence of FR4 is WGHGTQVTVSS (SEQ ID NO:46). B) The amino acid sequence of FR1 is EVQLVESGGGLVQAGGSLILSCTAT (SEQ ID NO:47), the amino acid sequence of FR2 is MGWYRQAPGKQRDWVAT (SEQ ID NO:44), the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC (SEQ ID NO:45), and the amino acid sequence of FR4 is WGQGTQVTVSS (SEQ ID NO:48). C) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCTAS (SEQ ID NO:49), the amino acid sequence of FR2 is TGWFRQAPGKQRELVAA (SEQ ID NO:50), the amino acid sequence of FR3 is HYLDSVKGRFTISRDNAKKSVYLQMNSLKPEDTALYTC (SEQ ID NO:51), and the amino acid sequence of FR4 is WGQGTQVTVSS (SEQ ID NO:48). D) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCTAS (SEQ ID NO:49), the amino acid sequence of FR2 is INWVRQAPGKGREWVST (SEQ ID NO:52), the amino acid sequence of FR3 is YYAASVKGRFTISRDNAGNTVNLQMNNLKPEDTALYYC (SEQ ID NO:53), and the amino acid sequence of FR4 is QGQGTQVTVSS (SEQ ID NO:54). E) The amino acid sequence of FR1 is QVQLVECGRGLAGAGGSLRLSCAPS (SEQ ID NO:55), the amino acid sequence of FR2 is GGWYRRAPGKQREWVAT (SEQ ID NO:56), the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLLMNRLKPEDTAVYYC (SEQ ID NO:57), and the amino acid sequence of FR4 is WGERTQVTVSS (SEQ ID NO:58). F) The amino acid sequence of FR1 is QVQLVESGGGLVQAGGFLRLSCAAS (SEQ ID NO:59), the amino acid sequence of FR2 is IGWYRQAPGKQRELVAT (SEQ ID NO:60), the amino acid sequence of FR3 is VYSPSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYIC (SEQ ID NO:61), and the amino acid sequence of FR4 is WGQGTQVTVSA (SEQ ID NO:62). G) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGTLRLSCAAS (SEQ ID NO:63), the amino acid sequence of FR2 is IGWYRQAPGKQREFVAT (SEQ ID NO:64), the amino acid sequence of FR3 is IYADFVKGRFTISRDNAKNTVFLQMNNLKPEDTAVYYC (SEQ ID NO:65), and the amino acid sequence of FR4 is WGHGTQVTVSS (SEQ ID NO:46). H) The amino acid sequence of FR1 is QVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO:66), the amino acid sequence of FR2 is MGWFRQAPGKEREFVSR (SEQ ID NO:67), the amino acid sequence of FR3 is YYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO:68), and the amino acid sequence of FR4 is WGQGNQVNVSS (SEQ ID NO:69). I) The amino acid sequence of FR1 is DVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO:70), the amino acid sequence of FR2 is ITWYRQAPGKQREWVAL (SEQ ID NO:71), the amino acid sequence of FR3 is NYAAPAKGRFTISRSNAKDTVYLQMNSLKPEDTAVYYC (SEQ ID NO:72), and the amino acid sequence of FR4 is WGQGTQVTVSS (SEQ ID NO:48).

[0021] As 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:9. The name and amino acid sequence of the single-domain antibody are as follows: 1-F07:QVKLEESGGGLVQAGGSLRLSCAAS GSSTSSIHV MGWYRQAPGKQRDWVAT LFTGGGNT IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC KAVEIGQAY WGHGTQVTVSS(SEQ ID NO:1); 2-E06:EVQLVESGGGLVQAGGSLILSCTAT GTTISLIS MGWYRQAPGKQRDWVAT LFTGGGNT IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC KAVEIGQAY WGQGTQVTVSS(SEQ ID NO:2); 1-G1:QVQLVESGGGLVQPGGSLRLSCTAS GFTVDSSA TGWFRQAPGKQRELVAA ILSSGIT HYLDSVKGRFTISRDNAKKSVYLQMNSLKPEDTALYTC NIDDGVTAQDY WGQGTQVTVSS(SEQ ID NO:3); 1-H5:QVQLVESGGGLVQPGGSLTLSCTAS GFTFSASA INWVRQAPGKGREWVST IRSDGTT YYAASVKGRFTISRDNAGNTVNLQMNNLKPEDTALYYC ACGSK QGQGTQVTVSS(SEQ ID NO:4); 3-E7:QVQLVECGRGLAGAGGSLRLSCAPS ESSTSNIHE GGWYRRAPGKQREWVAT LFTGGGNT IYADSVKGRFTISRDNAKNVLYLLMNRLKPEDTAVYYC KAVNIREAY WGERTQVTVSS(SEQ ID NO:5); 14-D3:QVQLVESGGGLVQAGGFLRLSCAAS GSTFIINA IGWYRQAPGKQRELVAT ISSGGSA VYSPSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYIC NDWVRDY WGQGTQVTVSA(SEQ ID NO:6); 18-F1:QVQLVESGGGLVQPGGTLRLSCAAS GSTLSINA IGWYRQAPGKQREFVAT ISSTSRT IYADFVKGRFTISRDNAKNTVFLQMNNLKPEDTAVYYC NDWIRGY WGHGTQVTVSS(SEQ ID NO:7); 13-C1:QVQLVESGGGLVQAGGSLRLSCAAS GDTFRHYV MGWFRQAPGKEREFVSR ISWSGSST YYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYC AARRATTLGAVEAQSYDY WGQGNQVNVSS(SEQ ID NO:8); 14-F7:DVQLVESGGGLVQAGGSLLRLSCAAS GSTFSNYD ITWYRQAPGKQREWVAL MTRFGHT NYAAPAKGRFTISRSNAKDTVYLQMNSLKPEDTAVYYC NTLELVSKGL WGQGTQVTVSS (SEQ ID NO:9).

[0022] In SEQ ID NO:1~SEQ ID NO:9, the bold italicized and underlined portions are the amino acid sequences of CDR1~CDR3 of the single-domain antibody.

[0023] The present invention also provides a nucleotide molecule for encoding the single-domain antibody described in the above-described technical solution. As one embodiment, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO:10 to SEQ ID NO:18, specifically as follows: 1-F07: CAGGTAAAGCTGGAGGAGTCTGGTGGAGGCTTGGTGCAGGCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCAGCACCTCTAGTATCCATGTGATGGGCTGGTACCGCCAGGCTCCAGGGAAACAGCGCGACTGGGTCGCAACTTTATTTACTGGTGGTGGTAAC ACAATCTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACGTGTTGTATCTGCAAATGAACCGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTAAAGCAGTGGAGATCGGGCAGGCCTACTGGGGCCATGGGACCCAGGTCACCGTCTCCTCA (SEQ ID NO:10); 2-E06:GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGATACTCTCCTGTACAGCCACCGGAACCACCATTAGTCTCATCTCCATGGGCTGGTATCGCCAGGCTCCAGGGAAGCAGCGCGACTGGGTCGCAACTTTATTTACTGGTGGTGGTAACACAATCTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACGTGTTGTATCTGCAAATGAACCGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTAAAGCAGTGGAGATCGGGCAGGCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO:11); 1-G1:CAGGTTCAGCTGGTGGAGAGCGGTGGCGGTTTGGTCCAGCCAGGTGGCAGCCTGCGCCTGTCTTGCACAGCAAGCGGCTTTACAGTGGACAGCAGTGCCACTGGTTGGTTCAGGCAGGCACCTGGCAAACAGAGAGAACTGGTGGCAGCCATCTTGTCTAGCGGCATCACACACTATCTCGACAGCGTGAAAGGCAGGTTTACCATCTCCAGGGACAACGCCAAGAAGAGCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAAGACACAGCCTTGTACACCTGCAACATCGATGACGGTGTGACCGCACAGGACTATTGGGGACAGGGTACACAGGTGACTGTCAGCTCC(SEQ ID NO:12); 1-H5:CAGGTCCAGCTGGTCGAGTCCGGCGGCGGACTCGTGCAGCCTGGAGGCAGCCTCACACTGTCTTGTACAGCCAGCGGTTTCACCTTCTCCGCATCCGCCATCAACTGGGTGAGGCAGGCTCCAGGTAAGGGCAGAGAATGGGTTTCTACCATCAGATCCGACGGCACTACATACTACGCTGCTAGTGTGAAGGGCAGGTTTACCATCAGTCGGGACAATGCCGGCAACACCGTGAACCTGCAGATGAACAACCTCAAGCCTGAAGACACCGCTTTGTACTACTGTGCCTGCGGCAGCAAGCAAGGCCAGGGAACTCAGGTCACTGTTTCTAGC(SEQ ID NO:13); 3-E7:CAGGTGCAGCTGGTGGAGTGTGGCAGAGGCCTGGCAGGTGCTGGTGGAAGCCTGCGGCTGAGCTGCGCTCCAAGCGAAAGCAGCACCTCTAACATCCATGAGGGCGGCTGGTATAGACGCGCACCTGGCAAACAGAGAGAGTGGGTCGCCACCTTGTTCACCGGCGGCGGTAATACCATCTACGCTGACAGCGTCAAGGGCAGGTTCACCATCAGCCGGGATAACGCTAAGAACGTGCTGTACCTGCTCATGAACAGGCTTAAACCTGAAGATACAGCCGTCTATTACTGTAAGGCTGTCAACATTAGGGAGGCATACTGGGGTGAGCGGACACAGGTCACTGTGTCTTCC(SEQ ID NO:14); 14-D3:CAGGTGCAGCTGGTGGAAAGCGGTGGCGGTCTTGTCCAGGCTGGCGGATTCTTGCGACTGTCTTGTGCCGCAAGCGGCTCCACCTTCATCATCAACGCCATCGGTTGGTACAGACAGGCTCCAGGTAAGCAGAGAGAACTTGTCGCTACCATCTCTTCTGGAGGCTCTGCAGTGTATTCCCCTAGCGTCAAGGGCAGGTTCACCATCAGTGGCGATAACGCCAAGAACACAGTGTACCTGCAGATGAACAGCCTGAAGCCAGAAGACACCGCCGTGTACATCTGCAATGATTGGGTCAGAGACTACTGGGGCCAGGGCACACAGGTCACAGTCAGCGCT(SEQ ID NO:15); 18-F1:CAGGTGCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCTGGCGGAACACTCCGCCTGAGCTGCGCTGCCTCTGGAAGCACACTCAGCATTAACGCCATCGGCTGGTACAGGCAGGCACCTGGTAAGCAGAGAGAGTTTGTGGCCACCATTAGCTCCACATCTCGCACAATCTACGCCGACTTTGTGAAAGGCCGGTTCACCATCTCACGCGACAATGCCAAGAACACCGTATTTCTGCAGATGAACAATCTGAAACCAGAAGATACAGCAGTGTACTATTGCAATGACTGGATTAGAGGCTACTGGGGTCACGGTACACAAGTGACCGTCTCTTCT(SEQ ID NO:16); 13-C1: CAGGTGCAGCTGGTGGAGTCTGGTGGTGGTCTGGTGCAGGCTGGTGGCTCTCTGAGACTGAGCTGTGCTGCTTCAGGCGATAACCTTCAGGCACTACGTCATGGGTTGGTTTCGGCAGGCACCAGGTAAAGAGAGAGTTCGTCAGCCGGATCAGCTGGAGCGGCTCATCCACCTATTATGCCGAT TCCGTGAAGGGTAGATTCACCATGTCCAGGGATAACGCCAAGAACACAGTCTACCTCCAGATGAACAGCCTGAAGCCAGAAGACACTGCCGTCTACTACTGCGCAGCAAGAAGAGCCACAACCCTGGGTGCTGTGGAAGCACAGAGCTACGATTACTGGGGACAGGGTAATCAGGTGAACGTGTCTTCC (SEQ ID NO:17); 14-F7: GACGTGCAGCTGGTGGAGTCTGGTGGTGGTCTGGTGCAGGCCGGTGGTAGCCTCAGGTTGAGCTGCGCTGCCAGCGGCTCTACCTTCAGCAACTACGATATTACCTGGTATAGGCAGGCTCCAGGTAAACAGCGGGAATGGGTGGCACTGATGACTAGGTTCGGCCACACCAA CTACGCCGCACCAGCCAAGGGCCGCTTCACTATCAGCAGGTCCAACGCCAAAGACACCGTGTACCTGCAGATGAACTCTCTGAAACCAGAAGATACCGCCGTCTACTACTGCAACACCTTGGAGCTTGTCTCTAAGGGCCTGTGGGGACAGGGAACACAGGTGACAGTCAGCTCC (SEQ ID NO:18).

[0024] This invention also provides a biological material, which is an expression cassette, recombinant vector, or recombinant cell line containing the nucleotide molecules described in the above-described technical solutions. As one embodiment, the initial vector in the recombinant vector of this invention is a plasmid vector or a lentiviral vector; as another embodiment, the initial vector in the recombinant vector is a eukaryotic expression vector. This invention does not impose any particular limitation on the construction method of the biological material; conventional genetic engineering techniques in the art can be used.

[0025] Of the 18 single-domain antibodies identified through screening, this invention screened out 9 single-domain antibodies that could cross-bind with human / cynomolgus monkey PD-L1 stably overexpressing cells by single-point binding verification. After expressing and purifying the above antibodies, their binding ability to target proteins (Human PD-L1-His and Cyno PD-L1-His) was detected by ELISA, and their binding ability to PD-L1 overexpressing cells (CHO-S-PD-L1) was detected by FACS, confirming that these 9 single-domain antibodies have specificity against human and cynomolgus monkey PD-L1.

[0026] Furthermore, by evaluating the PD-1 / PD-L1 blocking efficiency of the above-mentioned antibodies, this invention found that some of the antibodies even showed significantly better blocking effects than Atezolizumab antibody, confirming that the nine single-domain antibodies of this invention can specifically recognize and kill PD-L1-positive tumor cells on the surface, and can be applied to the treatment of tumors, especially PD-L1-positive tumors.

[0027] Based on the above advantages, this invention also provides the application of the single-domain antibody, nucleotide molecule, or biomaterial described in the above technical solutions in the preparation of PD-L1 inhibitors, and their application in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor prognosis, tumor clinical research, and drug development. As one embodiment, the tumor can be a PD-L1-positive tumor.

[0028] This invention also provides a PD-L1 inhibitor, the active ingredient of which includes the single-domain antibody described in the above-described technical solution. This invention does not impose any special limitations on the excipients in the PD-L1 inhibitor; conventional selection is acceptable as needed.

[0029] To further illustrate the present invention, the technical effects provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Preparation of single-domain antibodies targeting human and cynomolgus monkey PD-L1 cross-binding A recombinant PD-L1 target antigen protein was prepared targeting the cross-binding domains of human PD-L1 and cynomolgus monkey PD-L1. This recombinant antigen protein was then used to immunize alpacas, and the serum titer was monitored to ensure that the alpacas produced recombinant antibodies that cross-bind with PD-L1. Subsequently, peripheral blood mononuclear cells (PBMCs) were collected from alpacas to construct a yeast display library of single-domain antibodies. Candidate single-domain antibody sequences recognizing the target protein were screened. The specific steps are as follows: 1. Preparation of recombinant antigen protein targeting the PD-L1 cross-binding domain of human and cynomolgus monkey Based on the amino acid sequences of Human PD-L1 (Uniprot: Q9NZQ7) and Cynomolgus Monkey PD-L1 (NCBI: G7PSE7-1) proteins, a eukaryotic expression vector was constructed. After transient transfection of mammalian cells using LVtransm transfection reagent (iCarEab, Cat#LVTran100), the culture supernatant was collected, and the target recombinant protein was purified by affinity chromatography. The purity of the prepared recombinant protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein marker used was purchased from Solarbio, Cat#RP1930.

[0031] The amino acid sequence of the human PD-L1 protein is the amino acid at position Phe19-Arg 238 of the amino acid sequence in Uniprot:Q9NZQ7; the amino acid sequence of the Cynomolgus Monkey PD-L1 protein is the amino acid at position Phe19-Arg 238 of the amino acid sequence in NCBI:G7PSE7-1. The amino acid sequences of the recombinant PD-L1 protein prepared by affinity chromatography are as follows: Human PD-L1: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO:73); Cynomolgus Monkey PD-L1: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNER (SEQ ID NO:74).

[0032] In the above recombinant protein amino acid sequence, the bolded amino acids are the linker (GGGGS) and His tag (HHHHHH) added to the C-terminus of the protein for subsequent protein purification.

[0033] The binding activity of the target protein was assessed using an ELISA assay with the positive control (PC) antibody Atezolizumab (iCarEab). The coating consisted of Human PD-L1-His (iCarEab) and Cyno PD-L1-His (iCarEab), 2 μg / mL, 100 μL / well, incubated overnight at 4°C. The primary antibody (PC) was Atezolizumab, starting at 10 μg / mL, diluted 5-fold across 7 spots, 100 μL / well. The secondary antibody was HRP-Goat anti-human IgG Fc (Abcam, Cat# ab97225), diluted 1:50000, 100 μL / well. The negative control (NC) group served as the secondary antibody control wells, detecting the binding of candidate antibodies Huaman PD-L1 and Cyno PD-L1 to the target protein. The results are shown in Table 1 and [Table data missing]. Figure 1 As shown, in Figure 1 In the diagram, M represents the protein label, lane 1 is Human PD-L1-His, and lane 2 is Cyno PD-L1-His.

[0034] Table 1. Results of ELISA assessment of the binding activity of the target protein.

[0035] Note: The two values ​​corresponding to each group in Table 1 are the test results of two parallel duplicate holes.

[0036] Depend on Figure 1 As shown in Table 1, the purity of the prepared recombinant antigen protein is >95%, and the target recombinant protein has good binding activity with the positive antibody, which can be used for subsequent immunoassays and panning.

[0037] 2. Alpaca Immunization with PD-L1 Recombinant Antigen Protein Two alpacas (iCarEab) were immunized using the recombinant antigen protein described above. The immunization was performed subcutaneously at multiple sites, with immunization intervals of 14 days, during which Adjuvant (Gerbu, Cat#3030) was used. Starting from the second immunization, peripheral blood was collected from the alpacas 7 days after each immunization for monitoring serum titers. After immunization, 100 mL of peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated for constructing a single-domain antibody display library. The immunization schedule is shown in Table 2.

[0038] Table 2 Immune Progress Schedule

[0039] 3. Immunotiter testing 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.

[0040] 2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum layer and transfer the serum to a new sterile centrifuge tube to collect the immune serum.

[0041] 3) Dilute the target recombinant protein to a final concentration of 1 µg / mL using sterile carbonate buffer (CBS; Macklin, Cat#C885533).

[0042] 4) Take a new 96-well microplate, add 1 µg / mL of the target recombinant antigen protein, 100 μL / well, and coat overnight at 4°C. Human PD-L1-His and Cyno PD-L1-His were both purchased from iCarEab.

[0043] 5) Remove the antigen coating solution and wash 5 times with phosphate buffer (PBST; Merck, Cat#P3563) containing 0.05% Tween 20.

[0044] 6) Add 200 μL / well of phosphate buffer solution containing 3% skim milk powder (MPBS; Beyotime, Cat#P0216; Gibco, Cat#14190-250), and block at 37°C for 2 h.

[0045] 7) After removing the blocking buffer, wash the plate 5 times with PBST, add 100 µL of serially diluted serum per well, and incubate at room temperature for 1 h. The control wells contain phosphate buffer (PBS; Gbico, Cat#14190-250).

[0046] 8) Remove the liquid from the well and wash 5 times with PBST.

[0047] 9) Add 100 µL of anti-Llama IgG (H+L) Secondary Antibody [HRP] (1:50000 dilution; Novus, Cat#NBP1-75095) and incubate at room temperature for 1 h.

[0048] 10) Remove the liquid from the wells and wash the plate 5 times with PBST.

[0049] 11) Add 100 μL / well TMB colorimetric solution (Merck, Cat#T0440) and incubate at room temperature in the dark for 10-15 min.

[0050] 12) Add 50 μL / well stop solution (Biosharp, Cat#BL1829B).

[0051] 13) Use a microplate reader to read the OD in the wells. 450 value.

[0052] According to the ELISA results, alpaca immune serum can bind to the target recombinant protein, and the OD value changes with gradient dilution of the immune serum. The titer of alpaca serum is significantly higher than that of serum after four immunizations, meeting the requirements for blood collection and library construction, and can be used for the construction of antibody display libraries.

[0053] 4. Isolation of alpaca PBMCs and cloning of VHH antibody fragments 1) 100 mL of peripheral blood was collected from each of the two alpacas, and PBMCs were isolated using lymphocyte separation medium (Bio-Lapis, Cat#JH0171). Since the immunogenicity of both alpacas was high, the PBMCs from the two alpacas were mixed before subsequent experiments.

[0054] 2) RNA was extracted using RNAiso Plus (TaKaRa, Cat#9109), and cDNA was prepared by reverse transcription using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Cat#6210B).

[0055] 3) Prepare Mix 1 in a 200 μL PCR tube according to the PCR reaction system in Table 3, incubate at 65℃ for 5 min, and then cool rapidly on ice.

[0056] 4) Prepare the reaction solution in the PCR tubes according to the reaction system in Table 4. Mix well by pipetting and dispensing, dispense 80 μL / tube, place in a PCR instrument at 42℃ for 1 h, then inactivate at 70℃ for 15 min. Finally, store the cDNA sample on ice or at -20℃ for long-term storage.

[0057] 5) Prepare the first-round PCR reaction system (50 μL / tube) according to the reaction system in Table 5. The upstream primer binds to the signal peptide, and the downstream primer binds to the CH2 region. The NuHi Power mix used was purchased from Xinhai Biotechnology, Cat#NH9303. After preparing the PCR reaction system, set up the PCR instrument according to the procedure in Table 6. The nucleotide sequences of the upstream and downstream primers are 5'-AGKTGCAGCTCGTGGAGTCNGGNGG-3' (SEQ ID NO:75) and 5'-GATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO:76), respectively. 6) Electrophoresis analysis of PCR products was performed using 1% agarose (Merck, Cat#A6013) to obtain PCR bands with molecular weights of approximately 750 bp and 1000 bp. The band with a molecular weight of approximately 750 bp was separated, and the PCR product was recovered using a gel extraction kit (Qiagen, Cat#28706). The concentration was determined using NanoDrop and used as template for the second round of PCR.

[0058] 7) Prepare the 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, and the downstream primer binds to the anti-Hinge and FR4 regions. The restriction enzyme site is SfiI. After preparing the PCR reaction system, set up the PCR instrument according to the procedure in Table 6. The primer sequences used in this process are confidential and cannot be provided at this time.

[0059] 8) The second round of PCR products were analyzed by agarose gel electrophoresis using 1% agarose. A VHH fragment with a molecular weight of approximately 500 bp was isolated. The VHH PCR product was recovered using a gel extraction kit, and its concentration was determined using NanoDrop.

[0060] 9) Aliquot 200 μL of the recovered second-round PCR products 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 store at -80℃.

[0061] Table 3. Preparation system of Mix1 for reverse transcription PCR

[0062] Table 4. Preparation system of reverse transcription PCR reaction solution

[0063] Table 5 First-round PCR reaction system

[0064] Table 6 PCR reaction procedure

[0065] Table 7 Second round PCR reaction system

[0066] 5. Construction and screening of single-domain antibody yeast display library 1) The pYDisplay vector (iCarEab) was digested with SfiI (NEB, Cat#R0123L). The linearized digestion system of the yeast display vector pYDisplay is shown in Table 8. 100 μL / tube was dispensed and digested overnight at 50℃.

[0067] Table 8 Linearized Enzymatic Digestion System for Yeast Display Vector pYDisplay

[0068] 2) The pYDisplay vector fragment was separated using a 1% agarose gel, a 5000 bp fragment was excised and recovered from the gel, and the concentration was determined using NanoDrop.

[0069] 3) Aliquot 200 μL of the recovered pYDisplay enzyme 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, mix by pipetting and aspiration, add 880 μL of anhydrous ethanol, mix by inversion, and store at -80℃.

[0070] 4) Streak the competent yeast strains frozen at -80℃ onto yeast extract peptone glucose (YPD) solid medium plates and activate them at 30℃ for 3-5 days.

[0071] 5) Inoculate single colony yeast competent cells into 50 mL of YPD medium and incubate at 250 rpm and 30℃ for 1-2 days.

[0072] 6) After mixing the linearized vector fragment and PCR product, add it to an electroporation cuvette and electroporate; then transfect the electroporated yeast competent cells into a culture flask and incubate at 220 rpm and 30℃ for 1 h to prepare yeast competent strains.

[0073] 7) Take 20 μL of the resuspension, dilute it 5000 times with growth-selective synthetic medium (SDCAA), take 100 μL, spread it on an SDCAA plate, and incubate for 2-3 days. Based on the calculated library capacity, the constructed yeast display library has a capacity of 2.83 × 10⁻⁶. 9 The requirements are met, and diverse testing is arranged.

[0074] 8) Randomly selected single clones were sequenced to analyze the diversity of the yeast display library. Sequencing results showed that all sequences were antibody-differential sequences, with no empty vectors or repetitive sequences, indicating good library diversity.

[0075] 9) After culturing the remaining bacterial culture from step 6) for 24 h, collect it into a 50 mL centrifuge tube, centrifuge at 3000 rcf for 5 min, discard the supernatant, add 10 mL SDCAA to resuspend, mix with 50% glycerol: resuspension solution = 1:1, and store at -80℃.

[0076] 10) Add the yeast cultured in SDCAA to a 250 mL shake flask containing 50 mL of galactose-inducing medium (SGCAA) and culture at 30℃ and 240 rpm for 16 h on a shaker.

[0077] 11) After centrifugation, discard the supernatant, resuspend in 1 mL of phosphate buffer (0.5% PBSA) containing 0.5% BSA, add to a 1.5 mL centrifuge tube, centrifuge at 3000 rcf for 5 min, discard the supernatant, and wash again with 0.5% PBSA.

[0078] 12) Wash the streptavidin magnetic beads incubated with biotin antigen twice with 0.5% PBSA (incubate at 4°C for 5 min each time), place them on a magnetic rack for 5 min, and discard the supernatant.

[0079] 13) Add the yeast culture to the magnetic beads that have been bound to the antigen, incubate at 4°C for 60 min by rotation, and then place on a magnetic rack for 15 min.

[0080] 14) Discard the yeast culture and keep the magnetic beads. Wash three times with 0.5% PBSA (incubate at 4°C for 5 min each time).

[0081] 15) Resuspend the magnetic beads in 1 mL of SDCAA medium, and transfer 0.5-5 μL of the resuspension into 100 μL of SDCAA medium to a plate. Divide the resuspension into two portions. Add 500 μL of 50% glycerol to one portion (store at -80℃); add the other portion to a shaker tube, add 2 mL of SDCAA medium, and incubate at 30℃ and 240 rpm for 16 h.

[0082] 16) Transfer the bacterial culture from the shake tube to 50 mL of SDCAA medium (250 mL shake flask) and incubate overnight at 30°C and 240 rpm.

[0083] 17) Measure the OD of the bacterial culture 600 Value, based on OD 600 Centrifuge a portion of the bacterial culture, resuspend it in SGCAA, and transfer it to 50 mL of SGCAA medium to allow the final OD to reach the target value. 600 The value was 1, and the culture was carried out overnight at 30℃ and 240 rpm. The remaining bacterial culture was resuspended in SDCAA: 50% glycerol = 1:1 and stored at -80℃.

[0084] After sorting using Biotin-Cyno PD-L1-His (iCarEab) protein magnetic beads by flow cytometry, the results are as follows: Figure 2 As shown. In Figure 2 In the table, A represents NC; B represents 1MACS: primary antibody: Human PD-L1-Fc (iCarEab), secondary antibody: PE-anti-Human IgG (Invitrogen, Cat#12-4998-82); C represents 1MACS: primary antibody: Biotin-Cyno PD-L1-His, secondary antibody: Streptavidin APC (SA-APC; Biolegend, Cat#405207) + V5 Tag Antibody [FITC], mAb (V5-FITC; iCarEab); D represents 1MACS: primary antibody: Human PD-L1-Fc, secondary antibody: Biotin-Human PD-1-His (iCarEab), tertiary antibody: SA-APC + FC-PE.

[0085] Depend on Figure 2 It can be concluded that after using Biotin-Cyno PD-L1-His protein flow cytometry sorting, the positive percentage of the display library that can bind to Human PD-L1 and block its binding to PD-1 is 12.382%, and monoclonal picking and detection will be arranged.

[0086] 6. Yeast monoclonal detection The yeast clones that bind to the target antigen are subjected to binding detection and blocking detection.

[0087] 1) Dilute Human PD-L1-Fc to a final concentration of 2 µg / mL using sterile CBS. Take a new 96-well microplate, add 100 μL to each well, and coat overnight at 4°C.

[0088] 2) Remove the antigen coating solution, wash 5 times with PBST (containing 0.05% Tween 20), add 200 μL / well of 3% MPBS, and block at 37°C for 2 h.

[0089] 3) After removing the blocking buffer, wash the plate 5 times with PBST.

[0090] 4) Add the expressed recombinant antibody and transfect 100 μL of supernatant per well, or 100 μL of purified antibody (starting concentration 10 µg / mL, serially diluted 5-fold for 7 spots, 100 μL / well). PC is Atezolizumab. Incubate at room temperature for 1 h. Control wells contain PBS.

[0091] 5) Add 50 μL of Biotin-Human PD-1-His to a final concentration of 0.2 μg / mL and 50 μL of candidate clone expression supernatant, and incubate at room temperature for 1 h. The negative control well contains 50 μL of PBS and 50 μL of Biotin-Human PD-1-His to a final concentration of 0.2 μg / mL, and the positive control well contains 50 μL of Atelizumab positive control antibody to a final concentration of 25 μg / mL and 50 μL of Biotin-Human PD-1-His to a final concentration of 0.2 μg / mL.

[0092] 6) Remove the liquid from the wells, wash 5 times with PBST, add 100 μL / well HRP-Streptavidin (Boster, Cat#BA1088), dilute 1:50000, and incubate at room temperature for 1 h.

[0093] 7) Remove the liquid from the wells, wash the plate 5 times with PBST, add 100 μL / well TMB colorimetric solution, and incubate at room temperature in the dark for 10-15 min; 8) Add 50 μL / well stop solution.

[0094] 9) Use a microplate reader to read the OD in the wells. 450 value.

[0095] Based on the test results, candidate clones with blocking activity were selected and sent to GenScript Biotech for sequencing. The remaining bacterial culture was stored at -20°C.

[0096] 7. Construction of antibody eukaryotic expression vector 1) The positive yeast clones were subjected to PCR to obtain the antibody sequence, which was then digested with SfiI (NEB, Cat#R0123L) and ligated into the eukaryotic expression vector pcDNA3.4-Fc (iCarEab) to construct the antibody expression vector.

[0097] 2) The antibody eukaryotic expression vector was transiently transfected into 293F cells (ATCC cell bank), and 18 antibody expression supernatants were finally obtained.

[0098] 3) The binding of 18 candidate antibodies to antigen proteins was detected by FACS. The antigen proteins used were of specific cell origin; three cell lines were from the iCarEab cell bank: CHO-S, CHO-S-PD-L1 (a cell line stably overexpressing human PD-L1), and CHO-S-Cyno-PD-L1 (a cell line stably overexpressing monkey PD-L1), 2 × 10⁻⁶ cells. 5 100 μL / well for candidate antibody transfection supernatant; 100 μL / well for secondary antibody: HRP-Goat anti-human IgG Fc (Abcam, Cat#ab97225); 100 μL / well for PC: Atezolizumab (iCarEab); 100 μL / well for NC: secondary antibody control group, without target protein PD-L1 but with secondary antibody, used as the basis for gating during flow cytometry analysis.

[0099] FACS results showed that among the 18 candidate antibodies, 9 were able to cross-bind with human / monkey PD-L1 overexpressing cells, and could be used for subsequent antibody purification and preparation.

[0100] 8. Expression and purification of candidate single-domain antibodies 1) Remove the LVTransm transfection reagent and pcDNA3.4-Fc antibody expression vector from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Transfer 2 mL of PBS to one well of a 6-well plate, add 20 μg of antibody expression vector, mix thoroughly by pipetting, then add 60 μL of LVTransm, immediately mix by pipetting, and let stand at room temperature for 10 min.

[0101] 2) Add the DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C, 5% CO2, 130 rpm.

[0102] 3) After continuous culture for 5 to 7 days, centrifuge to collect the supernatant of the culture medium, filter it through a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube.

[0103] 4) Since the pcDNA3.4-Fc antibody expression vector can specifically bind to Protein A, Protein A column was used to purify the antibody (Suzhou Bojin Biotechnology Co., Ltd., Cat#BG18-0010-02).

[0104] The nine candidate antibody sequences obtained by the above method are shown in SEQ ID NO:1 to SEQ ID NO:9, and their molecular weights are all around 15 kDa. After adding a linker and a His tag to their C-terminus, the overall molecular weight of the prepared single-domain antibodies is around 80 kDa. CDR1, CDR2, and CDR3, which have been added to their heavy chain complementarity-determining regions, are respectively labeled in the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:9. The RNA sequences corresponding to the candidate antibodies can be derived from triplet codons, as shown in SEQ ID NO:10 to SEQ ID NO:18.

[0105] Example 2 The steps for detecting the binding of candidate antibodies to the PD-L1 target protein using ELISA are as follows: 1) Dilute the recombinant proteins (Human PD-L1-His, Cyno PD-L1-His) to a final concentration of 2µg / mL using sterile CBS. Take a new 96-well microplate, add 100 μL to each well, and coat overnight at 4°C.

[0106] 2) Remove the antigen coating solution, wash 5 times with PBST, add 200 μL / well of 3% MPBS, and block at 37°C for 2 h.

[0107] 3) After removing the blocking buffer, wash the plate 5 times with PBST, add the expressed recombinant antibody, and transfect 100 μL of supernatant per well or purified antibody (starting concentration 10 µg / mL, serially diluted 5-fold to 7 spots, 100 μL / well), where PC is Atezolizumab. Incubate at room temperature for 1 h, with PBS as the control well.

[0108] 4) Remove the liquid from the wells, wash 5 times with PBST, add 100 μL / well HRP-Streptavidin (1:50000 dilution), and incubate at room temperature for 1 h.

[0109] 5) Remove the liquid from the wells, wash the plate 5 times with PBST, add 100 μL / well TMB colorimetric solution, and incubate at room temperature in the dark for 10-15 min.

[0110] 6) Add 50 μL / well stop solution.

[0111] 7) Use a microplate reader to read the OD in the wells. 450 value.

[0112] The results are as follows Figure 3As shown, A represents the binding of the first batch of candidate antibodies with Human PD-L1-His; B represents the binding of the second batch of candidate antibodies with Human PD-L1-His; C represents the binding of the first batch of candidate antibodies with Cyno PD-L1-His; and D represents the binding of the second batch of candidate antibodies with Cyno PD-L1-His.

[0113] Depend on Figure 3 It can be concluded that after coating with Human PD-L1-His antigen and Cyno PD-L1-His antigen, the nine candidate antibodies can specifically bind to the PD-L1 target protein.

[0114] Example 3 The steps for detecting the binding of candidate antibodies to PD-L1 overexpressing cells are as follows: 1) Resuscitate CHO-S and CHO-S-PD-L1 cells in liquid nitrogen and adjust the cell state to the logarithmic growth phase.

[0115] 2) Divide the cells into several portions, with each portion containing 3 × 10 cells. 5 Each cell.

[0116] 3) Incubate the candidate antibody (starting concentration of 10 µg / mL, 100 μL / well) or 100 μL of transfection supernatant with the target cells, mix thoroughly, and incubate at room temperature for 1 h. The PC group was Atezolizumab.

[0117] 4) Centrifuge at 800 rcf at room temperature for 3 min, remove the supernatant containing antibodies, and wash the cells 3 times with PBS.

[0118] 5) Add secondary antibody PE-anti-Human IgG (1:1000 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes.

[0119] 6) Centrifuge at 800 rcf at room temperature for 3 min, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS.

[0120] 7) Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0121] The results are as follows Figure 4 As shown, A is the flow cytometry result of the binding of the first batch of candidate antibodies to human cells stably overexpressing PD-L1; B is the flow cytometry result of the binding of the second batch of candidate antibodies to human cells stably overexpressing PD-L1.

[0122] Depend on Figure 4It can be concluded that, except for 3-E7, the other 8 single-domain antibodies among the 9 candidate antibodies can specifically bind to PD-L1 overexpressing cells, and their binding efficiency is significantly higher than that of the positive antibody Atezolizumab. This confirms that the antibodies can bind specifically to PD-L1 overexpressing cells effectively.

[0123] Example 4 The steps for detecting the PD-1 / PD-L1 function of candidate antibodies are as follows: 1) After 48-72 h of cell resuscitation, observe the cells to grow to the logarithmic growth phase. If the cells are in normal condition and the viability reaches more than 90%, subsequent experiments can be carried out.

[0124] 2) Antibody preparation: The positive control Atezolizumab and the test antibody were diluted to 200 μg / mL with RPMI 1640 (Gibco, Cat#31870082) + 10% fetal bovine serum (FBS; Gibco, Cat#A5256701) medium, and serially diluted 5-fold nine times. 100 μL of each solution was added to each well of a 96-well plate. Control wells (RLU) were also included. Control Add 100 μL of culture medium to the corresponding well; background wells (RLU) Blank Add 50 μL of culture medium to the corresponding well.

[0125] 3) Cell preparation: Jurkat-PD-1 effector cells (iCarEab) and CHO-S-PD-L1 target cells were centrifuged at 500 rcf for 5 min at room temperature, washed once with PBS, and then resuspended in RPMI 1640 medium containing 10% FBS to a density of 4 × 10⁻⁶ cells / mL. 6 / mL. After thoroughly mixing the two cell types at a 1:1 ratio, aspirate 100 μL of the cell suspension and add it to a 96-well plate (excluding background wells). Add 50 μL of Jurkat-PD-1 cells to the background wells. Transfer the 96-well cell culture plate to a cell culture incubator and incubate at 37°C, 5% CO2 for 18 h.

[0126] 4) After the culture is complete, remove the 96-well plate and add 100 μL of the self-made luciferase detection reagent to each well. Mix well by pipetting and transfer to the 96-well white plate, avoiding air bubbles during transfer. Protect from light and incubate at room temperature for 5 min. Use a Tecan M1000 multi-functional microplate reader to read the luciferase fluorescence value.

[0127] 5) Data Processing: Calculate the Fold of Induction for each hole using the following formula: Fold of Induction = RLU (Sample-Blank) / RLU (Control-Blank) The results are shown in Table 9 and Figure 5 As shown.

[0128] Table 9 Results of PD-1 / PD-L1 blocking function of candidate antibodies

[0129] From Table 9 and Figure 5 It can be concluded that 7 out of the 9 candidate antibodies can effectively block the binding of PD-1 / PD-L1, and the blocking effect of some single-domain antibodies (such as 13-C1 and 18-F1) is even significantly better than that of Atezolizumab in the PC group, confirming that the antibodies can specifically target and kill PD-L1 positive tumor cells.

[0130] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A single-domain antibody targeting the cross-binding of PD-L1 in humans and cynomolgus monkeys and its application, characterized in that, The single-domain antibody includes complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are shown in any of 1) to 9). 1) The amino acid sequence of CDR1 is GSSTSSIHV, the amino acid sequence of CDR2 is LFTGGGNT, and the amino acid sequence of CDR3 is KAVEIGQAY. 2) The amino acid sequence of CDR1 is GTTISLIS, the amino acid sequence of CDR2 is LFTGGGNT, and the amino acid sequence of CDR3 is KAVEIGQAY. 3) The amino acid sequence of CDR1 is GFTVDSSA, the amino acid sequence of CDR2 is ILSSGIT, and the amino acid sequence of CDR3 is NIDDGVTAQDY. 4) The amino acid sequence of CDR1 is GFTFSASA, the amino acid sequence of CDR2 is IRSDGTT, and the amino acid sequence of CDR3 is ACGSK. 5) The amino acid sequence of CDR1 is ESSTSNIHE, the amino acid sequence of CDR2 is LFTGGGNT, and the amino acid sequence of CDR3 is KAVNIREAY. 6) The amino acid sequence of CDR1 is GSTFIINA, the amino acid sequence of CDR2 is ISSGGSA, and the amino acid sequence of CDR3 is NDWVRDY. 7) The amino acid sequence of CDR1 is GSTLSINA, the amino acid sequence of CDR2 is ISSTSRT, and the amino acid sequence of CDR3 is NDWIRGY. 8) The amino acid sequence of CDR1 is GDTFRHYV, the amino acid sequence of CDR2 is ISWSGSST, and the amino acid sequence of CDR3 is AARRATTLGAVEAQSYDY. 9) The amino acid sequence of CDR1 is GSTFSNYD, the amino acid sequence of CDR2 is MTRFGHT, and the amino acid sequence of CDR3 is NTLELVSKGL.

2. The single-domain antibody according to claim 1, characterized in that, The single-domain antibody includes frame regions FR1, FR2, FR3, and FR4, and the amino acid sequences of FR1, FR2, FR3, and FR4 are shown in any of A) to I). A) The amino acid sequence of FR1 is QVKLEESGGGLVQAGGSLRLSCAAS, the amino acid sequence of FR2 is MGWYRQAPGKQRDWVAT, the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC, and the amino acid sequence of FR4 is WGHGTQVTVSS. B) The amino acid sequence of FR1 is EVQLVESGGGLVQAGGSLILSCTAT, the amino acid sequence of FR2 is MGWYRQAPGKQRDWVAT, the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS. C) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCTAS, the amino acid sequence of FR2 is TGWFRQAPGKQRELVAA, the amino acid sequence of FR3 is HYLDSVKGRFTISRDNAKKSVYLQMNSLKPEDTALYTC, and the amino acid sequence of FR4 is WGQGTQVTVSS. D) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGSLRLSCTAS, the amino acid sequence of FR2 is INWVRQAPGKGREWVST, the amino acid sequence of FR3 is YYAASVKGRFTISRDNAGNTVNLQMNNLKPEDTALYYC, and the amino acid sequence of FR4 is QGQGTQVTVSS. E) The amino acid sequence of FR1 is QVQLVECGRGLAGAGGSLRLSCAPS, the amino acid sequence of FR2 is GGWYRRAPGKQREWVAT, the amino acid sequence of FR3 is IYADSVKGRFTISRDNAKNVLYLLMNRLKPEDTAVYYC, and the amino acid sequence of FR4 is WGERTQVTVSS. F) The amino acid sequence of FR1 is QVQLVESGGGLVQAGGFLRLSCAAS, the amino acid sequence of FR2 is IGWYRQAPGKQRELVAT, the amino acid sequence of FR3 is VYSPSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYIC, and the amino acid sequence of FR4 is WGQGTQVTVSA. G) The amino acid sequence of FR1 is QVQLVESGGGLVQPGGTLRLSCAAS, the amino acid sequence of FR2 is IGWYRQAPGKQREFVAT, the amino acid sequence of FR3 is IYADFVKGRFTISRDNAKNTVFLQMNNLKPEDTAVYYC, and the amino acid sequence of FR4 is WGHGTQVTVSS. H) The amino acid sequence of FR1 is QVQLVESGGGLVQAGGSLRLSCAAS, the amino acid sequence of FR2 is MGWFRQAPGKEREFVSR, the amino acid sequence of FR3 is YYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGNQVNVSS. I) The amino acid sequence of FR1 is DVQLVESGGGLVQAGGSLRLSCAAS, the amino acid sequence of FR2 is ITWYRQAPGKQREWVAL, the amino acid sequence of FR3 is NYAAPAKGRFTISRSNAKDTVYLQMNSLKPEDTAVYYC, and the amino acid sequence of FR4 is WGQGTQVTVSS.

3. The single-domain antibody according to claim 1 or 2, 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:

9.

4. A nucleotide molecule, characterized in that, The nucleotide molecule is used to encode the single-domain antibody according to any one of claims 1 to 3.

5. The nucleotide molecule according to claim 4, characterized in that, The nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO:10 to SEQ ID NO:

18.

6. A biomaterial, characterized in that, The biomaterial is an expression cassette, recombinant vector, or recombinant cell line containing the nucleotide molecules described in claim 4 or 5.

7. The use of the single-domain antibody according to any one of claims 1 to 3, the nucleotide molecule according to claim 4 or 5, or the biomaterial according to claim 6 in the preparation of PD-L1 inhibitors.

8. The use of the single-domain antibody according to any one of claims 1 to 3, the nucleotide molecule according to claim 4 or 5, or the biomaterial according to claim 6 in the preparation of one or more products for tumor diagnosis, tumor treatment, tumor prognosis, tumor clinical research, and drug development.

9. The application according to claim 8, characterized in that, The tumors mentioned include those that are positive for PD-L1 expression.

10. A PD-L1 inhibitor, characterized in that, The active ingredient includes the single-domain antibody as described in any one of claims 1 to 3.

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