A single-domain antibody targeting human and cynomolgus pd-l1 cross-binding and uses thereof

Abstract

The present application belongs to the technical field of biological medicine and immunology, and particularly relates to a single-domain antibody targeting human and cynomolgus monkey PD-L1 cross-binding and application thereof. The single-domain antibody has specificity against human and cynomolgus monkey PD-L1, and shows strong binding efficiency in the binding detection with a PD-L1 target protein or a PD-L1 overexpression cell. In the detection of blocking PD-1 / PD-L1 function, part of the single-domain antibodies even show a blocking effect significantly better than Atezolizumab, which confirms that the single-domain antibody prepared by the present application can be used for the diagnosis, treatment and prognosis detection of PD-L1 positive tumors. Meanwhile, the single-domain antibody has cross-binding ability across species, and can be directly used for preclinical pharmacodynamics and safety evaluation, without the need to develop species-specific alternative antibodies, so as to achieve "one target with double effect".

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and immunology, and particularly relates to a single-domain antibody targeting human and cynomolgus monkey PD-L1 cross-binding and application thereof. BACKGROUND

[0002] PD-L1, also known as programmed death ligand-1, is a protein located on the cell surface, and is also a core target in the current field of tumor immunotherapy. PD-L1 is an important "talisman" used by cancer cells to escape the immune system, especially T cell attack. Inhibition of PD-L1 can block the binding of PD-L1 to PD-1 (a protein molecule located on the surface of immune cells, mainly T cells), thereby relieving immune suppression and awakening T cells to attack tumors again, resulting in a lasting therapeutic effect. Although the current PD-L1 targeting technology centered on antibody drugs has made significant progress in tumor treatment, its core defects and limitations are also very obvious, mainly in the aspects of drug properties, clinical efficacy, safety and the like. First, the currently marketed PD-L1 inhibitors are mostly large molecule drugs such as monoclonal antibodies, which have poor tissue penetration and are difficult to effectively penetrate the deep part of solid tumors, affecting the therapeutic effect. Second, in actual application, the single drug treatment of PD-L1 inhibitors has limited effectiveness (about 20 %), and is accompanied by secondary drug resistance and other problems. In addition, since the activation of the immune system is non-specific, PD-L1 inhibitors may "injure" normal tissues while attacking tumor cells, causing immune-related adverse reactions.

[0003] In view of the above defects, new strategies such as developing new molecular forms, innovative delivery systems and combination therapy are being actively explored. As a new star in the field of new molecular forms, single-domain antibody, also known as nanobody, is the smallest functional antigen-binding fragment known in nature (about 15 kDa), representing an important paradigm revolution in the field of antibody drugs. Single-domain antibody, with its unique advantages of small size, high stability, strong binding capacity, low production cost and easy engineering, provides a new idea for solving the challenges faced by traditional antibodies in targeting PD-L1 and other immune checkpoints.

[0004] It should be noted that the "human-macaque cross-binding" property should also be focused on during the development of antibodies. This is mainly due to the fact that before a drug enters human clinical trials, its safety and effectiveness must be fully evaluated in relevant animal models. Macaques are the closest relatives of humans, and their immune systems, physiological functions, and the development of many diseases are highly similar to those of humans, making them the most irreplaceable non-human primate models. If the species specificity is not fully considered during the development stage, it is likely that there will be no suitable animal model for preclinical safety evaluation, and the drug development process will be forced to stop, which highlights the strategic necessity of screening "cross-reactive" antibodies that can bind to both human and macaque target sites during the antibody development stage. However, single-domain antibodies that can target both human and macaque PD-L1 cross-binding still need to be further developed. SUMMARY

[0005] The present application aims to provide a single-domain antibody that targets both human and macaque PD-L1 cross-binding and its application. The single-domain antibody can specifically bind to human and macaque PD-L1 target antigens, thereby playing a role in targeting cells, and can be used for the diagnosis, treatment, and prognosis of tumors.

[0006] The present application provides a single-domain antibody that targets both human and macaque PD-L1 cross-binding 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 as shown in any one of 1) to 9):

[0007] 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;

[0008] 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;

[0009] 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;

[0010] 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;

[0011] 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;

[0012] 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;

[0013] 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;

[0014] 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;

[0015] 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.

[0016] Preferably, the single-domain antibody comprises framework regions FR1, FR2, FR3 and FR4, the amino acid sequences of which are as shown in any one of A) to I):

[0017] 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;

[0018] 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;

[0019] 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;

[0020] 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;

[0021] 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;

[0022] 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;

[0023] 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;

[0024] 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;

[0025] 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.

[0026] Preferably, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO:1~SEQ ID NO:9.

[0027] The present application also provides a nucleotide molecule for encoding the single-domain antibody of the above technical solution.

[0028] Preferably, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO:10~SEQ ID NO:18.

[0029] The present application also provides a biological material which is an expression cassette, a recombinant vector or a recombinant cell line containing the nucleotide molecule of the above technical solution.

[0030] The present application also provides the use of the single-domain antibody of the above technical solution or the nucleotide molecule of the above technical solution or the biological material of the above technical solution in the preparation of a PD-L1 inhibitor.

[0031] The present application also provides the use of the single-domain antibody of the above technical solution or the nucleotide molecule of the above technical solution or the biological material of the above technical solution in the preparation of one or more of tumor diagnosis, tumor treatment, tumor prognosis, tumor clinical research and drug development.

[0032] Preferably, the tumor includes a tumor positive for PD-L1 expression.

[0033] The present application also provides a PD-L1 inhibitor, the effective component of which includes the single-domain antibody of the above technical solution.

[0034] Beneficial effects:

[0035] The present application provides a single-domain antibody targeting human and cynomolgus monkey PD-L1 cross-binding and its application, the single-domain antibody has specificity against human and cynomolgus monkey PD-L1, and exhibits 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 function, part of the above single-domain antibodies even exhibit a blocking effect significantly better than Atezolizumab, confirming that the single-domain antibody prepared by the present application can be used for the diagnosis, treatment and prognosis detection of PD-L1 positive tumors. At the same time, the single-domain antibody of the present application has cross-binding ability across species, and can be directly used for preclinical pharmacodynamics and safety evaluation, without the need to develop species-specific alternative antibodies, achieving "one target with double effect". BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0037] Figure 1 Figure for purity detection of PD-L1 recombinant antigen protein in Example 1;

[0038] Figure 2 Figure for flow cytometry results after one round of magnetic sorting in Example 1;

[0039] Figure 3 Figure for binding of candidate antibodies to PD-L1 target protein in Example 2;

[0040] Figure 4 Figure for binding of candidate antibodies to PD-L1 overexpressing cells in Example 3;

[0041] Figure 5 Figure for detection results of blocking PD-1 / PD-L1 function of candidate antibodies in Example 4. DETAILED DESCRIPTION

[0042] The present application provides a single-domain antibody targeting human and cynomolgus monkey PD-L1 cross-binding and application thereof, the single-domain antibody comprising complementarity determining regions CDR1, CDR2 and CDR3, the amino acid sequences of the CDR1, CDR2 and CDR3 are as shown in any one of 1)~9):

[0043] 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);

[0044] 2) the amino acid sequence of CDR1 is GTTISLIS (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);

[0045] 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);

[0046] 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);

[0047] 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);

[0048] 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);

[0049] 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);

[0050] 8) the amino acid sequence of CDR1 is GDTFRHYV (SEQ ID NO: 37), the amino acid sequence of CDR2 is ISWSGSST (SEQ ID NO: 38), and the amino acid sequence of CDR3 is AARRATTLGAVEAQSYDY (SEQ ID NO: 39);

[0051] 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).

[0052] As an embodiment, the single-domain antibody comprises framework regions FR1, FR2, FR3 and FR4, the amino acid sequences of which are as shown in any one of A) to I):

[0053] 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);

[0054] 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);

[0055] 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);

[0056] 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);

[0057] 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);

[0058] 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);

[0059] 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);

[0060] 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);

[0061] 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);

[0062] As an 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 specifically as follows:

[0063] 1-F07: QVKLEESGGGLVQAGGSLRLSCAAS GSSTSSIHV MGWYRQAPGKQRDWVAT LFTGGGNT IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC KAVEIGQAY WGQGTQVTVSS (SEQ ID NO: 1);

[0064] 2-E06: EVQLVESGGGLVQAGGSLILSCTAT GTTISLIS MGWYRQAPGKQRDWVAT LFTGGGNT IYADSVKGRFTISRDNAKNVLYLQMNRLKPEDTAVYYC KAVEIGQAY WGQGTQVTVSS (SEQ ID NO: 2);

[0065] 1-G1: QVQLVESGGGLVQPGGSLRLSCTAS GFTVDSSA TGWFRQAPGKQRELVAA ILSSGIT HYLDSVKGRFTISRDNAKKSVYLQMNSLKPEDTALYTC NIDDGVTAQDY WGQGTQVTVSS (SEQ ID NO: 3);

[0066] 1-H5: QVQLVESGGGLVQPGGSLTLSCTAS GFTFSASA INWVRQAPGKGREWVST IRSDGTT YYAASVKGRFTISRDNAGNTVNLQMNNLKPEDTALYYC ACGSK QGQGTQVTVSS (SEQ ID NO: 4);

[0067] 3-E7: QVQLVECGRGLAGAGGSLRLSCAPS ESSTSNIHE GGWYRRAPGKQREWVAT LFTGGGNT IYADSVKGRFTISRDNAKNVLYLLMNRLKPEDTAVYYC KAVNIREAY WGERTQVTVSS (SEQ ID NO: 5);

[0068] 14-D3: QVQLVESGGGLVQAGGFLRLSCAAS GSTFIINA IGWYRQAPGKQRELVAT ISSGGSA VYSPSVKGRFTISGDNAKNTVYLQMNSLKPEDTAVYIC NDWVRDY WGQGTQVTVSA (SEQ ID NO: 6);

[0069] 18-F1: QVQLVESGGGLVQPGGTLRLSCAAS GSTLSINA IGWYRQAPGKQREFVAT ISSTSRT IYADFVKGRFTISRDNAKNTVFLQMNNLKPEDTAVYYC NDWIRGY WGHGTQVTVSS (SEQ ID NO: 7);

[0070] 13-C1: QVQLVESGGGLVQAGGSLRLSCAAS GDTFRHYV MGWFRQAPGKEREFVSR ISWSGSST YYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYC AARRATTLGAVEAQSYDY WGQGNQVNVSS (SEQ ID NO: 8);

[0071] 14-F7: DVQLVESGGGLVQAGGSLRLSCAAS GSTFSNYD ITWYRQAPGKQREWVAL MTRFGHT NYAAPAKGRFTISRSNAKDTVYLQMNSLKPEDTAVYYC NTLELVSKGL WGQGTQVTVSS (SEQ ID NO: 9).

[0072] In SEQ ID NO: 1-SEQ ID NO: 9, the underlined part in bold italic is the amino acid sequence of CDR1-CDR3 of the single-domain antibody.

[0073] The application also provides a nucleotide molecule for encoding the single-domain antibody described in the above technical solutions. As an implementation form, the nucleotide sequence of the nucleotide molecule is shown in any one of SEQ ID NO: 10-SEQ ID NO: 18, and specifically as follows:

[0074] 1-F07: CAGGTAAAGCTGGAGGAGTCTGGTGGAGGCTTGGTGCAGGCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCAGCACCTCTAGTATCCATGTGATGGGCTGGTACCGCCAGGCTCCAGGGAAACAGCGCGACTGGGTCGCAACTTTATTTACTGGTGGTGGTAACACAATCTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACGTGTTGTATCTGCAAATGAACCGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTAAAGCAGTGGAGATCGGGCAGGCCTACTGGGGCCATGGGACCCAGGTCACCGTCTCCTCA (SEQ ID NO: 10);

[0075] 2-E06: GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGATACTCTCCTGTACAGCCACCGGAACCACCATTAGTCTCATCTCCATGGGCTGGTATCGCCAGGCTCCAGGGAAGCAGCGCGACTGGGTCGCAACTTTATTTACTGGTGGTGGTAACACAATCTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACGTGTTGTATCTGCAAATGAACCGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTAAAGCAGTGGAGATCGGGCAGGCCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA (SEQ ID NO: 11);

[0076] 1-G1 : CAGGTTCAGCTGGTGGAGAGCGGTGGCGGTTTGGTCCAGCCAGGTGGCAGCCTGCGCCTGTCTTGCACAGCAAGCGGCTTTACAGTGGACAGCAGTGCCACTGGTTGGTTCAGGCAGGCACCTGGCAAACAGAGAGAACTGGTGGCAGCCATCTTGTCTAGCGGCATCACACACTATCTCGACAGCGTGAAAGGCAGGTTTACCATCTCCAGGGACAACGCCAAGAAGAGCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAAGACACAGCCTTGTACACCTGCAACATCGATGACGGTGTGACCGCACAGGACTATTGGGGACAGGGTACACAGGTGACTGTCAGCTCC (SEQ ID NO: 12);

[0077] 1-H5: CAGGTCCAGCTGGTCGAGTCCGGCGGCGGACTCGTGCAGCCTGGAGGCAGCCTCACACTGTCTTGTACAGCCAGCGGTTTCACCTTCTCCGCATCCGCCATCAACTGGGTGAGGCAGGCTCCAGGTAAGGGCAGAGAATGGGTTTCTACCATCAGATCCGACGGCACTACATACTACGCTGCTAGTGTGAAGGGCAGGTTTACCATCAGTCGGGACAATGCCGGCAACACCGTGAACCTGCAGATGAACAACCTCAAGCCTGAAGACACCGCTTTGTACTACTGTGCCTGCGGCAGCAAGCAAGGCCAGGGAACTCAGGTCACTGTTTCTAGC (SEQ ID NO: 13);

[0078] 3-E7: CAGGTGCAGCTGGTGGAGTGTGGCAGAGGCCTGGCAGGTGCTGGTGGAAGCCTGCGGCTGAGCTGCGCTCCAAGCGAAAGCAGCACCTCTAACATCCATGAGGGCGGCTGGTATAGACGCGCACCTGGCAAACAGAGAGAGTGGGTCGCCACCTTGTTCACCGGCGGCGGTAATACCATCTACGCTGACAGCGTCAAGGGCAGGTTCACCATCAGCCGGGATAACGCTAAGAACGTGCTGTACCTGCTCATGAACAGGCTTAAACCTGAAGATACAGCCGTCTATTACTGTAAGGCTGTCAACATTAGGGAGGCATACTGGGGTGAGCGGACACAGGTCACTGTGTCTTCC (SEQ ID NO: 14);

[0079] 14-D3: CAGGTGCAGCTGGTGGAAAGCGGTGGCGGTCTTGTCCAGGCTGGCGGATTCTTGCGACTGTCTTGTGCCGCAAGCGGCTCCACCTTCATCATCAACGCCATCGGTTGGTACAGACAGGCTCCAGGTAAGCAGAGAGAACTTGTCGCTACCATCTCTTCTGGAGGCTCTGCAGTGTATTCCCCTAGCGTCAAGGGCAGGTTCACCATCAGTGGCGATAACGCCAAGAACACAGTGTACCTGCAGATGAACAGCCTGAAGCCAGAAGACACCGCCGTGTACATCTGCAATGATTGGGTCAGAGACTACTGGGGCCAGGGCACACAGGTCACAGTCAGCGCT (SEQ ID NO: 15);

[0080] 18-F1 : CAGGTGCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCTGGCGGAACACTCCGCCTGAGCTGCGCTGCCTCTGGAAGCACACTCAGCATTAACGCCATCGGCTGGTACAGGCAGGCACCTGGTAAGCAGAGAGAGTTTGTGGCCACCATTAGCTCCACATCTCGCACAATCTACGCCGACTTTGTGAAAGGCCGGTTCACCATCTCACGCGACAATGCCAAGAACACCGTATTTCTGCAGATGAACAATCTGAAACCAGAAGATACAGCAGTGTACTATTGCAATGACTGGATTAGAGGCTACTGGGGTCACGGTACACAAGTGACCGTCTCTTCT (SEQ ID NO: 16);

[0081] 13-C1 : CAGGTGCAGCTGGTGGAGTCTGGTGGTGGTCTGGTGCAGGCTGGTGGCTCTCTGAGACTGAGCTGTGCTGCTTCAGGCGATACCTTCAGGCACTACGTCATGGGTTGGTTTCGGCAGGCACCAGGTAAAGAGAGAGAGTTCGTCAGCCGGATCAGCTGGAGCGGCTCATCCACCTATTATGCCGATTCCGTGAAGGGTAGATTCACCATGTCCAGGGATAACGCCAAGAACACAGTCTACCTCCAGATGAACAGCCTGAAGCCAGAAGACACTGCCGTCTACTACTGCGCAGCAAGAAGAGCCACAACCCTGGGTGCTGTGGAAGCACAGAGCTACGATTACTGGGGACAGGGTAATCAGGTGAACGTGTCTTCC (SEQ ID NO: 17);

[0082] 14-F7: GACGTGCAGCTGGTGGAGTCTGGTGGTGGTCTGGTGCAGGCCGGTGGTAGCCTCAGGTTGAGCTGCGCTGCCAGCGGCTCTACCTTCAGCAACTACGATATTACCTGGTATAGGCAGGCTCCAGGTAAACAGCGGGAATGGGTGGCACTGATGACTAGGTTCGGCCACACCAACTACGCCGCACCAGCCAAGGGCCGCTTCACTATCAGCAGGTCCAACGCCAAAGACACCGTGTACCTGCAGATGAACTCTCTGAAACCAGAAGATACCGCCGTCTACTACTGCAACACCTTGGAGCTTGTCTCTAAGGGCCTGTGGGGACAGGGAACACAGGTGACAGTCAGCTCC (SEQ ID NO: 18).

[0083] The present application also provides a biological material, which is an expression cassette, a recombinant vector or a recombinant cell line comprising the nucleotide molecule according to the above technical solution. As an embodiment, the initial vector in the recombinant vector according to the present application is a plasmid vector or a lentivirus vector; as another embodiment, the initial vector in the recombinant vector is a eukaryotic expression vector. The present application does not have special limitations on the construction method of the biological material, and the conventional genetic engineering means in the art can be used.

[0084] For the 18 single-domain antibodies obtained by panning identification, the present application screens 9 single-domain antibodies capable of cross-binding with human / cynomolgus monkey PD-L1 stably overexpressed cells through single-point binding verification of candidate antibodies; after expression and purification of the above antibodies, the binding ability of the antibodies to target proteins (Human PD-L1-His and Cyno PD-L1-His) is detected by ELISA, and the binding ability of the antibodies to PD-L1 overexpressed cells (CHO-S-PD-L1) is detected by FACS, which confirms that the 9 single-domain antibodies have the specificity of anti-human and cynomolgus monkey PD-L1.

[0085] In addition, the present application also evaluates the blocking PD-1 / PD-L1 efficiency of the above antibodies, and finds that some of the above antibodies even show a blocking effect significantly better than that of Atezolizumab antibody, which confirms that the 9 single-domain antibodies of the present application can specifically recognize and kill tumor cells positive for surface PD-L1, and can be applied in the treatment of tumors, especially PD-L1 positive tumors.

[0086] Based on the above advantages, the application also provides the use of the single-domain antibody, the nucleotide molecule or the biological material in the preparation of a PD-L1 inhibitor, and in the preparation of a product for one or more of tumor diagnosis, tumor treatment, tumor prognosis, tumor clinical research and drug development. As an embodiment, the tumor can be a PD-L1 expression positive tumor.

[0087] The application also provides a PD-L1 inhibitor, and the effective component includes the single-domain antibody. The auxiliary materials in the PD-L1 inhibitor are not particularly limited in the application, and can be selected according to the needs.

[0088] In order to further illustrate the application, the technical effects provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0089] Example 1

[0090] Preparation of a single-domain antibody targeting human and cynomolgus monkey PD-L1 cross-binding

[0091] The PD-L1 target recombinant antigen protein is prepared by targeting the human PD-L1 and cynomolgus monkey PD-L1 cross-binding domain, and then the above-mentioned recombinant antigen protein is used to immunize a llama, and the immune serum titer is monitored to ensure that the llama can produce a recombinant antibody that cross-binds PD-L1. Then, the peripheral blood mononuclear cells (PBMC) of the llama are collected to construct a single-domain antibody yeast display library, and candidate single-domain antibody sequences that recognize the target protein are selected by panning. The specific steps are as follows:

[0092] 1. Preparation of a PD-L1 target recombinant antigen protein targeting human and cynomolgus monkey PD-L1 cross-binding domain

[0093] According to the amino acid sequence information of Human PD-L1 (Uniprot: Q9NZQ7) and Cynomolgus Monkey PD-L1 (NCBI: G7PSE7-1) protein, a eukaryotic expression vector is constructed, and after transfection of mammalian cells by using LVtransm transfection reagent (iCarEab, Cat# LVTran100), the culture medium supernatant is collected, and the target recombinant protein is prepared by affinity chromatography purification, and the purity of the prepared recombinant protein is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Protein marker used is purchased from Solarbio, Cat# RP1930.

[0094] The amino acid sequence of human PD-L1 protein is the amino acid at positions Phe 19-Arg 238 of the amino acid with accession number Uniprot: Q9NZQ7; the amino acid sequence of Cynomolgus Monkey PD-L1 protein is the amino acid at positions Phe 19-Arg 238 of the amino acid with accession number NCBI: G7PSE7-1. The amino acid sequences of the PD-L1 recombinant proteins prepared by affinity chromatography purification are respectively:

[0095] >Human PD-L1:

[0096] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO: 73);

[0097] >Cynomolgus Monkey PD-L1:

[0098] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNER (SEQ ID NO: 74).

[0099] In the above-mentioned amino acid sequences of recombinant proteins, the bolded amino acids are the added linker (GGGGS) and His tag label (HHHHHH) at the C-terminal of the protein, so as to facilitate subsequent protein purification.

[0100] The binding activity of the target protein was evaluated by ELISA experiment using positive control (PC) antibody Atezolizumab (iCarEab). Among them, coating: Human PD-L1-His (iCarEab), Cyno PD-L1-His (iCarEab), 2 μg / mL, 100 μL / well, 4°C overnight; primary antibody (PC): Atezolizumab, 10 μg / mL, 5-fold dilution of 7 points, 100 μL / well; secondary antibody: HRP-Goat anti-human IgG Fc (Abcam, Cat# ab97225), 1:50000 dilution, 100 μL / well; the negative control (NC) group is the secondary antibody control hole, and the binding of the candidate antibodies Human PD-L1 and Cyno PD-L1 to the target protein is detected. The results are shown in Table 1 and Figure 1 Figure 1 , wherein M is a protein marker, lane 1 is Human PD-L1-His, and lane 2 is Cyno PD-L1-His.

[0101] Table 1. Results of binding activity evaluation of target protein by ELISA method

[0102]

[0103] Note: The two values in Table 1 corresponding to each group are the detection results of two parallel duplicate wells.

[0104] From Figure 1 and Table 1, it can be seen that the purity of the prepared recombinant antigen protein is >95 %, the target recombinant protein has good binding activity with the positive antibody, and can be used for subsequent immunization experiments and panning work.

[0105] 2. Llama immunization of PD-L1 recombinant antigen protein

[0106] Two llamas (iCarEab) were immunized with the above-mentioned recombinant antigen protein, and the immunization method was subcutaneous multi-point immunization, with an immunization interval of 14 days, and Adjuvant immunoadjuvant (Gerbu, Cat#3030) was used during the interval. Starting from the second immunization, peripheral blood was collected from the llama 7 days after each immunization for immune serum titer monitoring. After immunization was completed, 100 mL of peripheral blood was collected, and peripheral blood PBMC was separated for single-domain antibody display library construction. The immunization schedule is shown in Table 2.

[0107] Table 2. Immunization schedule

[0108]

[0109] 3. Immune titer detection ​

[0110] 1) Collect 5 mL of peripheral blood from a llama, and place the centrifuge tube containing the blood sample in a 37 °C incubator for 1 h; then transfer the blood sample to 4 °C overnight.

[0111] 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.

[0112] 3) Dilute the target recombinant protein to a final concentration of 1 µg / mL using sterile carbonate buffer solution (CBS; Macklin, Cat# C885533).

[0113] 4) Take a new 96-well enzyme-labeled plate, add 1 µg / mL of the target recombinant antigen protein, 100 μL / well, and coat at 4 °C overnight, wherein Human PD-L1-His and Cyno PD-L1-His are both purchased from iCarEab.

[0114] 5) Remove the antigen coating solution, and wash 5 times using phosphate buffer solution containing 0.05 % Tween 20 (PBST; Merck, Cat# P3563).

[0115] 6) Add 200 μL / well of phosphate buffer solution containing 3 % skimmed milk (MPBS; Beyotime, Cat# P0216; Gibco, Cat# 14190-250), and block at 37 °C for 2 h.

[0116] 7) After removing the blocking buffer, wash the plate 5 times using PBST, add 100 µL / well of gradient-diluted serum, and incubate at room temperature for 1 h; the control wells are phosphate buffer solution (PBS; Gibco, Cat# 14190-250).

[0117] 8) Remove the liquid in the wells, and wash 5 times using PBST.

[0118] 9) Add 100 µL of anti-Llama IgG (H+L) Secondary Antibody [HRP] antibody (1:50000 dilution; Novus, Cat# NBP1-75095), and incubate at room temperature for 1 h.

[0119] 10) Remove the liquid in the wells, and wash the plate 5 times using PBST.

[0120] 11) Add 100 μL / well of TMB developing solution (Merck, Cat# T0440), and incubate at room temperature in the dark for 10-15 min.

[0121] 12) Add 50 μL / well of stop solution (Biosharp, Cat#BL1829B).

[0122] 13) Read the OD value in the wells using a microplate reader. 450

[0123] According to the results of the ELISA experiment, the llama immune serum can bind to the target recombinant protein, and the OD value changes in gradient with the gradient dilution of the immune serum. The titer of the llama is significantly improved after immunization, reaching the requirement of blood sampling for library construction, and can be used for the construction of antibody display library.

[0124] 4. Llama PBMC separation and VHH antibody fragment cloning

[0125] 1) Collect 100 mL of peripheral blood from each of the two llamas, and separate PBMC using lymphocyte separation medium (Biolab, Cat#JH0171). Since the titer of the two llamas is high, the PBMC from the two llamas is mixed and used for subsequent experiments.

[0126] 2) Extract RNA using RNAiso Plus (TaKaRa, Cat#9109), and perform reverse transcription using PrimeScript™ II 1stStrand cDNA Synthesis Kit (TaKaRa, Cat#6210B) to prepare cDNA.

[0127] 3) Prepare Mix1 in a 200 μL PCR tube according to the PCR reaction system in Table 3, incubate at 65°C for 5 min, and then cool rapidly on ice.

[0128] 4) Prepare the reaction solution in the above PCR tube according to the reaction system in Table 4, mix well by blowing, aliquot 80 μL / tube, and place in a PCR instrument for 42°C reaction for 1 h, then 70°C heat inactivation for 15 min. Finally, store the cDNA sample on ice or at -20°C for long-term storage.

[0129] 5) Prepare the first round of 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. The NuHi Power mix used is purchased from Xinhai Biology, Cat#NH9303. After preparing the PCR reaction system, set the PCR instrument according to the program in Table 6. The nucleotide sequences of the upstream primer and the downstream primer are 5'-AGKTGCAGCTCGTGGAGTCNGGNGG-3' (SEQ ID NO:75); 5'-GATCACTAGTGGGGTCTTCGCTGTGGTGCG-3' (SEQ ID NO:76)​

[0130] 6) Agarose gel electrophoresis was used to analyze the PCR products with 1 % agarose (Merck, Cat#A6013). The PCR bands with molecular weight of 750 bp and 1000 bp were obtained. The band with molecular weight of 750 bp was separated and the PCR product was recovered using a gel recovery kit (Qiagen, Cat#28706). The concentration was determined using a NanoDrop. The recovered PCR product was used as the template for the second round of PCR.

[0131] 7) The reaction system for the second round of PCR was configured according to Table 7 (50 μL / tube). The upstream primer was combined with the FR1 region of the antibody and the downstream primer was combined with the Hinge and FR4 regions. The enzyme cutting site was Sfil. After the PCR reaction system was configured, the PCR instrument was set according to the program in Table 6. The primer sequences in this process were confidential and could not be provided.

[0132] 8) Agarose gel electrophoresis was used to analyze the PCR products of the second round. The PCR products were analyzed by electrophoresis using 1 % agarose. The VHH fragment with a molecular weight of about 500 bp was obtained. The VHH PCR product was recovered using a gel recovery kit and the concentration was determined using a NanoDrop.

[0133] 9) The recovered second round of PCR product was divided into 200 μL per 1.5 mL centrifuge tube. 1 / 10 volume (20 μL) of 3M sodium acetate (Sigma, Cat#126-96-5), 1 μg / μL glycogen (Glycogen; Beyotime, Cat#D0812) were added, mixed by blowing and sucking, 880 μL of anhydrous ethanol (Merck, Cat#459828) was added, and after mixing by inversion, it was stored at -80°C.

[0134] Table 3 Preparation system of reverse transcription PCR mixture Mix1

[0135]

[0136] Table 4 Preparation system of reverse transcription PCR reaction solution

[0137]

[0138] Table 5 First round of PCR reaction system

[0139]

[0140] Table 6 PCR reaction program

[0141]

[0142] Table 7 Second round of PCR reaction system

[0143]

[0144] 5. Construction and panning of single domain antibody yeast display library

[0145] 1) Linearization of yeast display vector pYDisplay (iCarEab) with Sfil (NEB, Cat#R0123L) enzyme, enzyme linearization system as shown in Table 8, 100 μL / tube, 50°C enzyme digestion overnight.

[0146] Table 8. Yeast display vector pYDisplay linearization enzyme digestion system

[0147]

[0148] 2) Use 1% agarose gel to separate pYDisplay vector fragments, cut 5000 bp of vector fragments for gel recovery, and use NanoDrop to determine the concentration.

[0149] 3) Divide the recovered pYDisplay enzyme digestion product 200 μL per 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3 M sodium acetate, 1 μg / μL Glycogen, mix well by blowing and sucking, add 880 μL of anhydrous ethanol, mix well by inverting, and store at -80°C.

[0150] 4) The -80°C frozen yeast competent strain is streaked onto yeast extract powder peptone glucose medium (YPD) solid medium plates, and incubated at 30°C for 3-5 days.

[0151] 5) Inoculate single colony yeast competent into 50 mL YPD medium, 250 rpm, 30°C shake culture for 1-2 days.

[0152] 6) After mixing the linearized vector fragments and PCR products, add them to the electroporation cup, and then perform electroporation; the electroporated yeast competent strain is transferred to a culture bottle, 220 rpm, 30°C shake culture for 1 h, to prepare the yeast competent strain.

[0153] 7) Take 20 μL of the resuspension, dilute 5000-fold with growth selection synthetic medium (SDCAA), take 100 μL, and spread on SDCAA plates, and incubate for 2-3 days. According to the calculation of the library capacity, the constructed yeast display library has a library capacity of 2.83 x 10 9 , which meets the requirements and arranges the diversity detection.

[0154] 8) Randomly select single clones for sequencing to analyze the diversity of the yeast display library. According to the sequencing results, all are antibody difference sequences, there are no empty loads and repeated sequences, and the library diversity is good.

[0155] 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℃.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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).

[0161] 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.

[0162] 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.

[0163] 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℃.

[0164] Using flow cytometry, after Biotin-Cyno PD-L1-His (iCarEab) protein magnetic beads sorting, the results are shown in Figure 2 Figure 2 In the above table, A is NC; B is 1 MACS: primary antibody: Human PD-L1-Fc (iCarEab), secondary antibody: PE-anti-Human IgG (Invitrogen, Cat#12-4998-82); C is 1 MACS: 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 is 1 MACS: primary antibody: Human PD-L1-Fc, secondary antibody: Biotin-Human PD-1-His (iCarEab), tertiary antibody: SA-APC + FC-PE.

[0165] From the above table, it can be concluded that after using Biotin-Cyno PD-L1-His protein flow sorting, the positive proportion of the display library capable of binding Human PD-L1 and blocking its binding with PD-1 is 12.382 %, and monoclonal picking plate and detection will be arranged. Figure 2

[0166] 6、Yeast monoclonal detection

[0167] Yeast clones that bind to target antigens are subjected to binding detection and blocking detection.

[0168] 1) Dilute Human PD-L1-Fc to a final concentration of 2 μg / mL using sterile CBS, take a new 96-well enzyme plate, add 100 μL per well, and coat overnight at 4°C.

[0169] 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.

[0170] 3) After removing the blocking buffer, wash the plate 5 times with PBST.

[0171] 4) Add the expressed recombinant antibody, transfection supernatant 100 μL / well, or purified antibody (starting concentration 10 μg / mL, 5-fold gradient dilution 7 points, 100 μL / well), wherein PC is Atezolizumab. Incubate at room temperature for 1 h, and the control well is PBS.

[0172] ​​5) Add 50 μL of Biotin-Human PD-1-His at a final concentration of 0.2 μg / mL and 50 μL of candidate clone expression supernatant, incubate at room temperature for 1 h, the negative control well is 50 μL of PBS and 50 μL of Biotin-Human PD-1-His at a final concentration of 0.2 μg / mL, the positive control well is 50 μL of Atelizumab positive control antibody at a final concentration of 25 μg / mL and 50 μL of Biotin-Human PD-1-His at a final concentration of 0.2 μg / mL.

[0173] 6) Remove the liquid in the well, wash 5 times with PBST, add 100 μL / well of HRP-Streptavidin (Boster, Cat#BA1088) diluted 1:50000, incubate at room temperature for 1 h.

[0174] 7) Remove the liquid in the well, wash the well plate 5 times with PBST, add 100 μL / well of TMB developing solution, incubate at room temperature in the dark for 10-15 min;

[0175] 8) Add 50 μL / well of stop solution.

[0176] 9) Use a microplate reader to read the OD 450 value in the well.

[0177] According to the detection results, select the candidate clones with blocking activity to send to Kings River Biotech Co., Ltd. for sequencing, and store the remaining bacterial liquid at -20°C.

[0178] 7. Construction of antibody eukaryotic expression vector

[0179] 1) Perform PCR on the positive yeast clone to obtain the antibody sequence, then perform Sfil enzyme digestion (NEB, Cat#R0123L) and connect with the eukaryotic expression vector pcDNA3.4-Fc (iCarEab) to construct the antibody expression vector.

[0180] 2) Transiently transfect 293F cells (ATCC Cell Bank) with the antibody eukaryotic expression vector to finally obtain 18 antibody expression supernatants.

[0181] 3) Detect the binding of 18 candidate antibodies and antigen proteins by FACS. Among them, the antigen protein used is a specific cell source, and three cell lines are from the iCarEab cell bank, namely CHO-S, CHO-S-PD-L1 (a cell strain stably overexpressing human PD-L1) and CHO-S-Cyno-PD-L1 (a cell strain stably overexpressing monkey PD-L1), 2x10 5One antibody: candidate antibody transfection supernatant, 100 μL / well; secondary antibody: HRP-Goat anti-human IgG Fc (Abeam, Cat# ab97225); PC: atezolizumab (iCarEab); NC: secondary antibody control group, without adding target protein PD-L1 but adding secondary antibody, which is used as the basis for circle gate when flow analysis.

[0182] FACS results show that among the 18 candidate antibodies, 9 candidate antibodies can cross-bind with human / monkey PD-L1 overexpression cells, which can be used for subsequent antibody purification and preparation work.

[0183] 8. Expression and purification of candidate single domain antibodies

[0184] 1) Take LVTransm transfection reagent and pcDNA3.4-Fc antibody expression vector from the refrigerator, and mix thoroughly after thawing at room temperature. Take PBS buffer and warm it to room temperature. Take 2 mL PBS into one well of a 6-well plate, add 20 μg of antibody expression vector, mix thoroughly with a pipette, then add 60 μL of LVTransm, mix immediately with a pipette, and incubate at room temperature for 10 min.

[0185] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells and mix thoroughly. Continue to culture the cells in a 37°C, 5% CO2, 130 rpm incubator.

[0186] 3) After continuous culture for 5-7 days, centrifuge to collect the culture supernatant, filter with a 0.45 μm filter, and transfer the filtrate to a sterile centrifuge tube.

[0187] 4) Since the pcDNA3.4-Fc antibody expression vector can specifically bind to Protein A, Protein A column is used to purify the antibody (Suzhou Bio-Gen Biotech Co., Ltd., Cat# BG18-0010-02).

[0188] The nine candidate antibody sequences obtained by the above method are shown as SEQ ID NO: 1~SEQ ID NO: 9, and the molecular weight of each is about 15 kDa. After adding a linker and a His tag label to the C-terminus, the overall molecular weight of the prepared single-domain antibody is about 80 kDa. The CDR1, CDR2 and CDR3 of the heavy chain complementarity determining region are marked in the amino acid sequences shown in SEQ ID NO: 1~SEQ ID NO: 9. The RNA sequence corresponding to the candidate antibody can be derived from the triplet codon, as shown in SEQ ID NO: 10~SEQ ID NO: 18.

[0189] Example 2

[0190] The steps of the ELISA detection of the binding of the candidate antibody to the PD-L1 target protein are as follows:

[0191] 1) Dilute the recombinant protein (Human PD-L1-His, Cyno PD-L1-His) to a final concentration of 2 µg / mL using sterile CBS, take a new 96-well enzyme plate, add 100 μL to each well, and coat overnight at 4°C.

[0192] 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.

[0193] 3) After removing the blocking buffer, wash the plate 5 times with PBST, add the expressed recombinant antibody, transfection supernatant 100 μL / well, or purified antibody (starting concentration 10 µg / mL, 5-fold gradient dilution 7 points, 100 μL / well), and PC is Atezolizumab. Incubate at room temperature for 1 h, and the control wells are PBS.

[0194] 4) Remove the liquid in the wells, wash 5 times with PBST, add 100 μL / well of HRP-Streptavidin (1:50000 dilution), and incubate at room temperature for 1 h.

[0195] 5) Remove the liquid in the wells, wash the plate 5 times with PBST, add 100 μL / well of TMB developing solution, and incubate at room temperature in the dark for 10~15 min.

[0196] 6) Add 50 μL / well of stop solution.

[0197] 7) Read the OD 450 value in the wells using an enzyme labeler.

[0198] The results are as follows: Figure 3Figure 2 shows the binding of the candidate antibodies to Human PD-L1-His and Cyno PD-L1-His. A is a graph showing the binding of the first batch of candidate antibodies to Human PD-L1-His; B is a graph showing the binding of the second batch of candidate antibodies to Human PD-L1-His; C is a graph showing the binding of the first batch of candidate antibodies to Cyno PD-L1-His; D is a graph showing the binding of the second batch of candidate antibodies to Cyno PD-L1-His.

[0199] Figure 3 shows the binding of the candidate antibodies to Human PD-L1-His and Cyno PD-L1-His. A is a graph showing the binding of the first batch of candidate antibodies to Human PD-L1-His; B is a graph showing the binding of the second batch of candidate antibodies to Human PD-L1-His; C is a graph showing the binding of the first batch of candidate antibodies to Cyno PD-L1-His; D is a graph showing the binding of the second batch of candidate antibodies to Cyno PD-L1-His. Figure 3 It can be concluded that after coating with Human PD-L1-His antigen and Cyno PD-L1-His antigen, 9 candidate antibodies can specifically bind to PD-L1 target protein.

[0200] Example 3

[0201] The binding of the candidate antibodies to PD-L1 overexpressing cells was detected by the following steps:

[0202] 1) The CHO-S and CHO-S-PD-L1 cells were recovered from liquid nitrogen, and the cell state was adjusted to the logarithmic growth phase.

[0203] 2) The cells were divided into several portions, and the number of cells in each portion was 3 x 10 5

[0204] 3) The candidate antibodies (initial concentration of 10 μg / mL, 100 μL / well) or 100 μL of transfection supernatant were incubated with the target cells, and after mixing well, the cells were incubated at room temperature for 1 h, and the PC group was Atezolizumab.

[0205] 4) Centrifugation at 800 rcf for 3 min at room temperature, remove the supernatant containing the antibody, and wash the cells with PBS for 3 times.

[0206] 5) Add secondary antibody PE-anti-Human IgG (1:1000 dilution), mix well, and incubate at room temperature for 30 min in the dark.

[0207] 6) Centrifugation at 800 rcf for 3 min at room temperature, remove the supernatant containing the secondary antibody, and wash the cells with PBS for 3 times.

[0208] 7) Resuspend the cells with 500 μL of PBS for flow cytometry analysis.

[0209] The results are shown in Figure 4, A is a graph showing the binding of the first batch of candidate antibodies to stable PD-L1 overexpressing human cells; B is a graph showing the binding of the second batch of candidate antibodies to stable PD-L1 overexpressing human cells. Figure 4 Figure 5 shows the binding of the candidate antibodies to stable PD-L1 overexpressing human cells. A is a graph showing the binding of the first batch of candidate antibodies to stable PD-L1 overexpressing human cells; B is a graph showing the binding of the second batch of candidate antibodies to stable PD-L1 overexpressing human cells.

[0210] Figure 4 ​​It can be concluded that 8 single domain antibodies except 3-E7 can specifically bind to PD-L1 overexpressed cells, and the binding efficiency is significantly higher than that of positive antibody Atezolizumab. It is proved that the antibody can specifically bind to PD-L1 overexpressed cells.

[0211] Example 4

[0212] The steps of the detection of the blocking of PD-1 / PD-L1 function by the candidate antibody are as follows:

[0213] 1) After 48~72 h of cell recovery, the cell growth is observed to be in the logarithmic growth phase, the cell state is normal, and the survival rate is more than 90%, so that the subsequent experiment can be carried out.

[0214] 2) Antibody preparation: positive control Atezolizumab and the antibody to be tested are diluted to 200 μg / mL with RPMI 1640 (Gibco, Cat#31870082) + 10 % fetal bovine serum (FBS; Gibco, Cat#A5256701) medium, 9 gradients of 5-fold dilution are prepared, 100 μL per well is added to the 96-well plate, and the control wells (RLU Control ) are set, 100 μL of medium is added to the corresponding wells; background wells (RLU Blank ) are set, and 50 μL of medium is added to the corresponding wells.

[0215] 3) Cell preparation: Jurkat-PD-1 effector cells (iCarEab) and CHO-S-PD-L1 target cells are centrifuged at 500rcf for 5 min at room temperature, washed once with PBS, and resuspended in RPMI 1640 medium containing 10 % FBS to 4×10 6 / mL. After mixing the two kinds of cells 1:1, 100 μL of cell suspension is taken and added to the 96-well plate (excluding background wells). 50 μL of Jurkat-PD-1 cells are added to the background wells. The 96-well cell plate is transferred to a cell incubator, 37℃, 5 % CO2, and continues to be cultured for 18 h.

[0216] 4) After the culture is completed, the 96-well plate is taken out, 100 μL of self-made luciferase detection reagent is added to each well. After mixing, it is transferred to a 96-well white plate, avoiding bubbles during transfer, avoiding light, and placing at room temperature for 5 min. The Tecan M1000 multifunctional enzyme labeler is used to read the Luciferase fluorescence value.

[0217] 5) Data processing: the Fold of Induction of each well is calculated according to the following formula:

[0218] Fold of Induction =RLU (Sample-Blank) / RLU (Control-Blank)

[0219] Results are shown in Table 9 and Figure 5 Table 10.

[0220] Table 9. Results of detection of PD-1 / PD-L1 blocking function of candidate antibodies

[0221]

[0222] From Table 9 and Figure 5 Table 10, it can be concluded that 7 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.

[0223] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A single-domain antibody that targets the cross-binding of PD-L1 in humans and cynomolgus monkeys, characterized in that, The single-domain antibody includes complementarity-determining regions CDR1, CDR2, and CDR3, with the amino acid sequence of CDR1 being GSSTSSIHV, the amino acid sequence of CDR2 being LFTGGGNT, and the amino acid sequence of CDR3 being KAVEIGQAY.

2. The single-domain antibody according to claim 1, characterized in that, The single-domain antibody includes frame regions FR1, FR2, FR3 and FR4. 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.

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 SEQ ID NO:

1.

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 SEQ ID NO:

10.

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; The tumor is a PD-L1 positive tumor.

9. 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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