anti-CD 73-anti-PD-1 bispecific antibody and uses thereof

文档序号：127390 发布日期：2021-10-22 浏览：38次中文

阅读说明：本技术 抗cd73-抗pd-1双特异性抗体及其用途 (anti-CD 73-anti-PD-1 bispecific antibody and uses thereof ) 是由张鹏李百勇夏瑜王忠民于 2021-04-22 设计创作，主要内容包括：本发明涉及抗CD73-抗PD-1双特异性抗体、其药物组合物及用途。(The invention relates to an anti-CD 73-anti-PD-1 bispecific antibody, a pharmaceutical composition and application thereof.)

1. An anti-CD 73-anti-PD-1 bispecific antibody comprising:

a first protein functional region, said first protein functional region targeted to PD-1, and

a second protein functional region that targets CD73,

wherein

The first protein functional region comprises an amino acid sequence comprising the amino acid sequence of HCDR1, HCDR2 and HCDR3, preferably HCDR1, HCDR2 and HCDR3, respectively, of the heavy chain variable region shown in SEQ ID NO:44, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NOs:45-47, or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID NOs:45-47, and an amino acid sequence comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:44

The variable region of the light chain comprising the amino acid sequence shown as SEQ ID NO. 49 comprises the amino acid sequence of LCDR1, LCDR2 and LCDR3, preferably LCDR1, LCDR2 and LCDR3 shown as SEQ ID NOs:50-52 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NOs:50-52, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown as SEQ ID NOs: 50-52;

or the second protein functional region comprises an amino acid sequence comprising the amino acid sequence of HCDR1, HCDR2 and HCDR3, preferably HCDR1, HCDR2 and HCDR3, respectively, of the heavy chain variable region shown in SEQ ID NO:2, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NOs:3-5, or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID NOs:3-5, and a second protein functional region comprising an amino acid sequence comprising the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:2

The variable region of the light chain comprising the amino acid sequence shown as SEQ ID NO. 7 comprises LCDR1, LCDR2 and LCDR3, preferably LCDR1, LCDR2 and LCDR3, the amino acid sequences shown as SEQ ID NOs:8-10 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown as SEQ ID NOs:8-10, respectively, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown as SEQ ID NOs: 8-10.

2. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1, wherein:

the first protein functional region comprises

An amino acid sequence as set forth in SEQ ID NO:44 or SEQ ID NO:62 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:44 or 62, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to a sequence set forth in SEQ ID NOs:44 or 62; and comprises

Corresponding to an amino acid sequence selected from the group consisting of the sequence having an amino acid sequence as shown in SEQ ID NO:49 or SEQ ID NO:64 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NOs:49 or 64, respectively, or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID NOs:49 or 64,

and/or the presence of a gas in the gas,

the second protein functional region comprises an amino acid sequence selected from the group consisting of the sequence shown in SEQ ID NO. 2, SEQ ID NO. 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO. 2 or SEQ ID NO. 20, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID NO. 2 or SEQ ID NO. 20; and comprises

Corresponding to an amino acid sequence selected from SEQ ID NO 7, or SEQ ID NO 22 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO 7 or SEQ ID NO 22, or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID NO 7 or SEQ ID NO 22, respectively;

The second protein functional region comprises an amino acid sequence shown as SEQ ID NO:20 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID No. 20 or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID No. 20, and a polypeptide comprising a sequence as shown in SEQ ID No. 20 or a polypeptide comprising a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID No. 20 or a sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID No. 20

The sequence of amino acid sequence shown as SEQ ID NO. 24 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown as SEQ ID NO. 24 or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown as SEQ ID NO. 24.

3. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: the number of the first protein functional region and the number of the second protein functional region are independently 1 or more than 2.

4. The anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-3, wherein: the first protein functional region and the second protein functional region are directly connected or connected through a connecting fragment; preferably, the linker is (GGGGS) n, n being a positive integer, e.g. 1, 2, 3, 4, 5 or 6.

5. The anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-4, wherein: the first and second protein functional regions are independently an immunoglobulin or antigen binding fragment, e.g., a half antibody, Fab, F (ab')₂Or a single chain antibody, preferably, the first protein functional region is an immunoglobulin and the second protein functional region is an antigen binding fragment; alternatively, the first protein functional region is an antigen binding fragment and the second protein functional region is an immunoglobulin.

6. The anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-5, wherein: the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of CH1 of the immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of the light chain variable region CL of the immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of the light chain variable region CL of the immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of the heavy chain variable region CH1 of the immunoglobulin. Or

The C-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or via a linking fragment) linked to the N-terminus of the heavy chain of the immunoglobulin and the C-terminus of the light chain variable region of the antigen-binding fragment is directly (or via a linking fragment) linked to the N-terminus of the light chain of the immunoglobulin; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the N-terminus of the light chain of the immunoglobulin and the C-terminus of the light chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the N-terminus of the heavy chain of the immunoglobulin.

7. The anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-6, wherein: the antigen binding fragment is a single chain antibody, preferably, the first protein functional region is an immunoglobulin, and the second protein functional region is a single chain antibody; or, the first protein functional region is a single-chain antibody, and the second protein functional region is immunoglobulin.

8. The anti-CD 73-anti-PD-1 bispecific antibody of claim 7, wherein: the single-chain antibody is connected with the variable region (V) of the heavy chain of the antibody through a connecting fragment_H) And antibody light chain variable region (V)_L) A molecule of (a); preferably, the single chain antibody has the structure: NH2-V_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_L-COOH。

9. The anti-CD 73-anti-PD-1 bispecific antibody of claim 7 or 8, wherein: the single-chain antibody is connected to the C terminal (C) of the heavy chain of the immunoglobulin through a connecting fragment_H) (or the N-terminus of the heavy chain, the C-terminus of CH1 in the variable region of the heavy chain), the variable region of the antibody heavy chain of the single-chain antibody is first ligated (V)_H) Or, first, the antibody light chain variable region (V) of the single chain antibody is ligated_L) (ii) a Preferably, the single chain antibody may have the structure: connecting fragment-V_HConnecting fragment-V_L-COOH, or, connecting fragment-V_LConnecting fragment-V_H-COOH，

Preferably, the first and second electrodes are formed of a metal,

the heavy chain variable region of the immunoglobulin comprises CDRs with amino acid sequences of SEQ ID NO:3-5, and the light chain variable region of the immunoglobulin comprises CDRs with amino acid sequences of SEQ ID NO: 8-10;

the heavy chain variable region of the single-chain antibody comprises CDRs with amino acid sequences of SEQ ID NO:45-47, the light chain variable region of the single-chain antibody comprises CDRs with amino acid sequences of SEQ ID NO:50-52,

preferably, the single chain antibody (e.g., NH 2-V)_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_LCOOH) to the C-terminus of the heavy chain of an immunoglobulin by a linker fragment, the antibody heavy chain variable region (V) of the single chain antibody comprising CDRs having the amino acid sequences of SEQ ID NOS:45-47 may be first ligated_H) Or, the variable region of the antibody light chain comprising CDRs with amino acid sequences SEQ ID NOS:50-52 of the single-chain antibody is first linked (V)_L)，

Or, preferably, the number of bits in the bit stream,

the heavy chain variable region of the immunoglobulin comprises CDRs with amino acid sequences of SEQ ID NO:45-47, and the light chain variable region of the immunoglobulin comprises CDRs with amino acid sequences of SEQ ID NO: 50-52; and/or the presence of a gas in the gas,

the heavy chain variable region of the single-chain antibody comprises CDR with an amino acid sequence of SEQ ID NO. 3-5, the light chain variable region of the single-chain antibody comprises CDR with an amino acid sequence of SEQ ID NO. 8-10,

wherein the single chain antibody (e.g., NH 2-V)_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_LCOOH) to the C-terminus of the heavy chain of an immunoglobulin by a linker fragment, the antibody heavy chain variable region (V) of the single chain antibody comprising CDRs having the amino acid sequences of SEQ ID NOS:3-5 may be first ligated_H) Or, the variable region (V) of the antibody light chain comprising the CDRs of amino acid sequences SEQ ID NOS:8-10 of the single-chain antibody is first linked_L)，

Preferably, the first and second electrodes are formed of a metal,

two single chain antibody molecules are attached to one immunoglobulin molecule, more preferably, the two single chain antibody molecules are the same.

10. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein:

the immunoglobulin is IgG, IgA, IgD, IgE or IgM; preferably an IgG, such as IgG1, IgG2, IgG3 or IgG 4.

11. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: the single chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin, preferably one immunoglobulin molecule is linked to two single chain antibody molecules, more preferably, the two single chain antibody molecules are identical.

12. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: the heavy chain variable region of the immunoglobulin comprises CDRs with amino acid sequences of SEQ ID NO:45-47, and the light chain variable region of the immunoglobulin comprises CDRs with amino acid sequences of SEQ ID NO: 50-52;

and/or the presence of a gas in the gas,

preferably, when the single-chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker fragment, the variable region (V) of the heavy chain of the antibody comprising the CDRs having the amino acid sequences of SEQ ID NOS:3-5 of the single-chain antibody may be first linked_H) Or, the variable region (V) of the antibody light chain comprising the CDRs of amino acid sequences SEQ ID NOS:8-10 of the single-chain antibody is first linked_L)。

13. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein:

wherein, when the single-chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker fragment, the variable region (V) of the heavy chain of the antibody comprising the CDR having the amino acid sequence of SEQ ID NO:45-47 of the single-chain antibody may be first linked_H) Or, the variable region of the antibody light chain comprising CDRs with amino acid sequences SEQ ID NOS:50-52 of the single-chain antibody is first linked (V)_L)。

14. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:44 or SEQ ID NO: 62; the amino acid sequence of the variable region of the light chain of the immunoglobulin corresponds to the amino acid sequence selected from SEQ ID NO 49 or SEQ ID NO 64;

and/or the presence of a gas in the gas,

the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO:2 or SEQ ID NO: 20; the amino acid sequences of the light chain variable region of the single-chain antibody respectively correspond to the amino acid sequences selected from SEQ ID NO. 7and SEQ ID NO. 22; or the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO. 20, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO. 24;

wherein, when the single-chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker fragment, the variable region of the heavy chain (V) of the antibody of the single-chain antibody may be first linked_H) Or, first, the antibody light chain variable region (V) of the single chain antibody is ligated_L)。

15. The anti-CD 73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO. 2 or SEQ ID NO. 20; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO. 7 or SEQ ID NO. 22, or the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO. 20, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO. 24;

and/or the presence of a gas in the gas,

the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO. 44 or SEQ ID NO. 62; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO. 49 or SEQ ID NO. 64,

when the single-chain antibody is linked to the C-terminus of the heavy chain via a linker fragment, the variable region (V) of the heavy chain of the antibody of the single-chain antibody may be linked first_H) Or, first, an antibody light chain variable region (V) is ligated_L)。

16. The anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 5-14, wherein: the immunoglobulin comprises non-CDR regions from a species other than murine, such as from a human antibody, more preferably the immunoglobulin constant regions are humanized, e.g., the heavy chain constant regions are humanized with Ig gamma-1chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834, or

The heavy chain constant region of the immunoglobulin is mutated at any 2 or 3 of positions 234, 235 and 237 on the basis of Ig gamma-1chain C region, ACCESSION: P01857, and after mutation the bispecific antibody has a reduced affinity constant compared to that of Fc γ RIA, Fc γ RIIIa and/or C1q before mutation;

more preferably, the amino acid sequence has the following mutations at positions 234, 235 and/or 237 based on Ig gamma-1chain C region, ACCESSION: P01857 according to the EU numbering system:

L234A and L235A; or

L234A and G237A; or

L235A and G237A;

L234A、L235A、G237A，

Further preferably, the heavy chain constant region of the immunoglobulin further has one or more mutations selected from the group consisting of:

N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, a330R, C226S, C229S, E233P, P331P, S267P, L328P, a 330P, M252P, S254P, T256P, N297P, P238P, a 327P, P329P, K322P, T394P, G236P, L328P, a 330P, P331P, H268P, E318P and K320P, preferably, the anti-CD P-anti-PD-1 antibody structure is represented by a heavy chain-light chain-linking fragment 1-scFv, the scFv is selected from the group consisting of a C14H 1-C14H 1-C14, L1-C14H 1-C14H 14, the following specific linking fragment is selected from the group consisting of L12-L1-C14:

(1) NTPDV1, wherein the heavy chain amino acid sequence is shown as SEQ ID NO. 85, the light chain amino acid sequence is shown as SEQ ID NO. 28, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO. 79, the amino acid sequence of the 14C12H1V is shown as SEQ ID NO. 66, the amino acid sequence of the connecting fragment 2 is shown as SEQ ID NO. 81, the amino acid sequence of the 14C12L1V is shown as SEQ ID NO. 68,

(2) NTPDV2, wherein the heavy chain amino acid sequence is shown as SEQ ID NO. 85, the light chain amino acid sequence is shown as SEQ ID NO. 28, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO. 79, the amino acid sequence of the 14C12H1V is shown as SEQ ID NO. 66, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO. 79, the amino acid sequence of the 14C12L1V is shown as SEQ ID NO. 68,

(3) NTPDV3, wherein the heavy chain amino acid sequence is shown as SEQ ID NO. 85, the light chain amino acid sequence is shown as SEQ ID NO. 96, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO. 79, the amino acid sequence of the 14C12H1V is shown as SEQ ID NO. 66, the amino acid sequence of the connecting fragment 2 is shown as SEQ ID NO. 81, the amino acid sequence of the 14C12L1V is shown as SEQ ID NO. 68, and

(4) the amino acid sequence of the heavy chain of the NTPDV4 is shown as SEQ ID NO. 85, the amino acid sequence of the light chain is shown as SEQ ID NO. 96, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO. 79, the amino acid sequence of the 14C12H1V is shown as SEQ ID NO. 66, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO. 79, and the amino acid sequence of the 14C12L1V is shown as SEQ ID NO. 68.

17. The anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-16, wherein: the anti-CD 73-anti-PD-1 bispecific antibody is less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10K of M or less_DBinds to the CD73 protein and/or the PD-1 protein.

18. An isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain variable region of a bispecific antibody, wherein,

the heavy chain variable region of the antibody comprises:

CDR with amino acid sequence of SEQ ID NO. 3-5, CDR with amino acid sequence of SEQ ID NO. 45-47 and CDR with amino acid sequence of SEQ ID NO. 50-52;

CDR with amino acid sequence of SEQ ID NO. 45-47, CDR with amino acid sequence of SEQ ID NO. 3-5 and CDR with amino acid sequence of SEQ ID NO. 8-10;

and the bispecific antibody heavy chain variable region as part of the bispecific antibody specifically binds CD73 and PD-1 antigen, the bispecific antibody further comprising a light chain variable region comprising:

CDR with amino acid sequence of SEQ ID NO 8-10;

alternatively, the amino acid sequence is the CDR of SEQ ID NO 50-52.

Preferably, the CDRs of the light chain variable region are not identical to the CDRs comprised by the heavy chain variable region.

19. A vector comprising the isolated nucleic acid molecule of claim 18.

20. A host cell comprising the isolated nucleic acid molecule of claim 18, or the vector of claim 19.

21. A process for the preparation of an anti-CD 73-anti-PD-1 bispecific antibody according to any one of claims 1-17, comprising the steps of culturing the host cell of claim 20 under suitable conditions, and recovering the bispecific antibody from the cell culture.

22. A conjugate comprising the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 and a conjugate moiety, wherein the conjugate moiety is a detectable label; specifically, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

23. A kit comprising the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17, or the conjugate of claim 22; preferably the kit further comprises a second antibody that specifically recognizes the bispecific antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

24. Use of the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 in the preparation of a kit for detecting the presence or level of CD73 and/or PD-1 in a sample.

25. A pharmaceutical composition comprising the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 or the conjugate of claim 22; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

26. Use of the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 or the conjugate of claim 22 for the prevention and/or treatment of a tumor or anemia, or for the diagnosis of a tumor or anemia.

27. Use of the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 or the conjugate of claim 22 in the manufacture of a medicament for the prevention and/or treatment of a tumor or anemia, or in the manufacture of a medicament for the diagnosis of a tumor or anemia.

28. Use of the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 or the conjugate of claim 22 in the preparation of a medicament comprising:

(ii) a drug that detects the level of CD73 in the sample,

inhibiting the enzymatic activity of CD 73;

and/or

Drugs that block the binding of PD-1 to PDL1,

a drug that modulates (e.g., down-regulates) PD-1 activity or level,

a medicine for relieving the immunosuppression of PD-1 to the organism,

an agent for increasing IL-2 expression in T lymphocytes, or

A medicament for increasing IFN- γ expression in T lymphocytes.

29. An in vivo or in vitro method comprising the step of applying a cell or administering to a subject in need thereof an effective amount of the anti-CD 73-anti-PD-1 bispecific antibody of any one of claims 1-17 or the conjugate of claim 22,

30. a hybridoma cell line selected from the group consisting of:

hybridoma cell line LT014 (also called CD73-19F3) which is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 19 months in 2018 with the preservation number of CCTCC NO: C2018137; or

The hybridoma cell line LT003 (also called PD-1-14C12) is preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 16 days 2015 with the preservation number of CCTCC NO: C2015105.

31. The anti-CD 73 monoclonal antibody is 19F3H2L3(hG1TM), the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO:30, and the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO: shown at 28.

Technical Field

The invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-CD 73-anti-PD-1 bispecific antibody, a pharmaceutical composition thereof and application thereof.

Background

Extracellular-5 ' -Nucleotidase (Ecto-5 ' -Nucleotidase), CD73 protein, is a multifunctional glycoprotein encoded by NT5E gene and having a molecular weight of 70KD, which is anchored to the cell membrane by Glycosylphosphatidylinositol (GPI) (Zimmermann H.5 ' -Nucleotidase: molecular structure and functional assays. biochem J.1992; 285: 345-.

CD73 is widely distributed on the cell surface of human tissue, and it has been found that CD73 is highly expressed in various solid tumors, such as cancer cells, dendritic cells, regulatory T cells (tregs), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), etc. in tumor microenvironment. The expression of CD73 is regulated by molecules such as TGF-beta, EGFR, AKT, beta-catenin, etc., and particularly HIF-1, which functions as a transcription factor, is the most critical. An important feature of the tumor microenvironment is hypoxia, the upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1), which in turn leads to the widespread expression of CD73 in the tumor microenvironment (Synnestdt K, et al, Ecto-5' -nucleotidase (CD73) regulation by hypoxia-inhibitor factor-1 peptides metabolism in intracellular expression. J Clin invest.2002; 110: 993-1002.). Analysis of clinical tumor samples shows that the high expression of CD73 is a potential biomarker and is closely related to the poor prognosis of various tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer and the like.

CD73 has hydrolase and non-hydrolase activity. The enzymes of CD73 are present in tumor-associated processes concurrently with non-enzymatic functions and promote each other, maintaining the progression of the tumor. CD73 has been found to be a key regulatory molecule of tumor cell proliferation, metastasis and invasion in vitro, tumor angiogenesis and tumor immune escape mechanism in vivo, wherein an important immunosuppressive mechanism is mediated by CD 73-Adenosine (Adenosine) metabolic signaling pathway, CD39 upstream of CD73 can catalyze ATP to generate Adenosine Monophosphate (AMP), the generated AMP is converted into Adenosine by CD73, and Adenosine can bind to a downstream Adenosine receptor (A2AR), A2AR inhibits a series of signal pathways related to immune activation such as LCK, MAPK, PKC and the like by activating Protein Kinase A (PKA) and Csk kinase, so as to inhibit immune killing of T cells, thereby playing an immunosuppressive role and achieving immune escape of tumors (Antonioli L, Immunity, flexibility and migration: a vacant role for Adenosine Reccer.2013; 13: 842). Preclinical animal model studies show that CD73 expressed by immune cells and non-immune cells can promote immune escape, development and metastasis of tumors, and the inhibition of CTL (cytotoxic T cells) and NK cell functions by Treg cell-related CD 73-adenosine signals is most obvious.

For the treatment of solid Tumor, one important aspect to overcome drug resistance and improve curative effect is to relieve the inhibition of Tumor Microenvironment (TME) on immune effector cells. TME is a very complex system, consisting of various cells, matrix, enzymes, cytokines, surrogate products, etc., has the remarkable characteristics of low hydrogen, low pH and high pressure, and is very different from normal tissues. Hypoxia or ATP enrichment caused by killing tumor cells by radiotherapy and chemotherapy can promote the cascade reaction of CD39-CD73 adenosine signals, is beneficial to the proliferation and functions of various cancer-promoting cells and is not beneficial to inhibiting the cancer cells. (Regateiro, F.S., Cobbold, S.P. & Waldmann, H.CD73 and adenosine generation in the creation of relationship microorganisms. Clin.Exp.Immunol.2013; 171: 1-7).

The use of antibodies targeting CD73 or gene knockout CD73 in animal models can effectively block tumor growth and metastasis. Recently, CD73 monoclonal antibody, small interfering RNA technology, specific inhibitor APCP and the like are utilized to obtain remarkable curative effect in the anti-tumor treatment of animal experiments, and a new approach is provided for the anti-tumor treatment. Evidence from in vivo studies shows that targeted blockade of CD73 would be an effective therapeutic approach for tumor patients.

The link between CD73 overexpression and tumor subtype, prognosis and patient drug response has shown that CD73 can be an important marker for future tumor treatment and detection in individuals. Therefore, the study of the CD73 target is indispensable.

The transmembrane receptor PD-1 (programmed cell death-1) is one of the members of the CD28 family and is expressed on activated T cells, B cells, and myeloid lineage cells. Both receptors PDL1 and PDL2 of PD-1 belong to the B7 superfamily, where PDL1 is expressed by a wide variety of cells, including T cells, B cells, and endothelial and epithelial cells, and PDL2 is only expressed by antigen presenting cells such as dendritic cells and macrophages.

PD-1 plays a very important role in the activation process of the negative regulation T cells, PD-1 mediated negative regulation on the T cells is one of important mechanisms for tumor immunity escape, PDL-1 expressed on the surface of the tumor can be combined with PD-1 on the surface of immune cells, and then the killing of the tumor tissues by the immune cells is inhibited through a PD-1/PDL-1 signal pathway, and tumors with high expression of PD-L1 are accompanied by cancers which are difficult to detect (Hamanishi et al, Proc. Natl.Acad.Sci.2007; 104: 3360-5). An effective method of antagonizing PD-1, and thereby inhibiting the PD-1/PDL-1 signaling pathway, is the in vivo injection of anti-PD-1 antibodies.

PD-1 antibodies have broad anti-tumor prospects and surprising potency, and antibodies directed against the PD-1 pathway will bring breakthrough advances in the treatment of a variety of tumors: for the treatment of non-small cell lung Cancer, renal cell carcinoma, ovarian Cancer, melanoma (Homet M.B., Parisi G., et al, Anti-PD-1 Therapy in Melanoma. Semin Oncol. 2015Jun; 42 (3): 466-.

Bifunctional antibodies, also known as Bispecific antibodies (Bispecific antibodies), are specific Antibody drugs that target two different antigens simultaneously, and can be produced by immunoselection purification or can be obtained by genetic engineering. Genetic engineering has corresponding flexibility in the aspects of binding site optimization, consideration of synthetic forms, yield and the like, and therefore has certain advantages. Currently, over 45 forms of bispecific antibodies exist (Muller D, Kontermann RE. Bispecific antibodies for cancer immunology: Current Perspecific. BioDrugs 2010; 24: 89-98). The IgG-ScFv form, i.e., the Morrison model (Coloma MJ, Morrison SL. design and description of novel biological antibodies. Nature Biotechnology, 1997; 15: 159. 163) has proven to be an ideal form of bifunctional antibodies due to its advantages in antibody engineering, expression and purification (Miller, Demarest SJ, et al., Stability engineering of the recombinant and biological antibodies. protein Eng variants Sel 2010; 23: 549-57; Fitzgerald J, Lugovsky A. ratio engineering of biological antibodies. 2011. polypeptide of biological antibodies; 2011. 3: 299. multiple. 3) because of its similarity to naturally occurring IgG forms.

ADCC (antibody-dependent cell-mediated cytotoxicity) is an antibody-dependent cell-mediated cytotoxicity, and refers to binding of a Fab fragment of an antibody to an epitope of a virus-infected cell or a tumor cell, and binding of an Fc fragment thereof to an Fc Receptor (FcR) on the surface of a killer cell (NK cell, macrophage, etc.) to mediate direct killing of the killer cell to a target cell.

CDC (complementary dependent cytotoxicity) refers to complement dependent cytotoxicity. The CDC effect is caused by the first binding of the antibody to the corresponding antigen on the surface of the cell membrane and further binding to complement C1q, and then C2-C9 is activated to form a membrane attack complex to exert a lytic effect on the target cell.

The IgG family comprises four members, IgG1, IgG2, IgG3, and IgG4, which differ in their affinity for Fc γ Rs due to amino acid differences in the fragment crystallizable (Fc) region of the heavy chain constant region. Wild-type IgG1 was able to bind various Fc γ Rs, and was able to elicit ADCC as well as CDC effects. Studies of Zhang et al (Zhang T et al Cancer immunol. 2018; 67 (7): 1079. 1090.) and Dahan et al (Dahan R et al Cancer cell. 2015; 28 (3): 285-95.) indicate that binding of the Fc fragment of an antibody targeting an immune checkpoint such as PD-1 to the Fc receptor negatively affects anti-antibody mediated anti-Cancer activity, probably due to Fc-dependent effector function-induced immune cell damage, including antibody-dependent cell-mediated cytotoxicity being an important mechanism leading to immune cell damage.

Interleukin-8 (IL-8) is a Chemotactic cytokine (Chemotactic cytokines) and is secreted mainly by monocytes and the like. IL-8 plays an important role in the proliferation of normal cells and tumor cells, and especially plays an important role in promoting the occurrence and development of tumors. Research shows that IL-8 can promote the generation of tumors; tumor cells themselves also secrete IL-8, promoting tumor growth and metastasis (Lo MC et al cancer letters, 2013, 335 (1): 81-92.). Therefore, IL-8 has become an important inflammatory factor indispensable in the tumor microenvironment.

IL-8 is closely related to the occurrence and development of tumors as a proinflammatory factor. During the process of inducing malignant transformation of non-kidney cancer cells by methylarsinic acid (methylarsonate), the expression of IL-8 gene is increased, IL-8 gene silencing can obviously inhibit the growth of transplanted tumor in mice, and in addition, the reduction of IL-8 level can inhibit the expression of Matrix metalloproteinase-9 (Matrix metalloproteinase-9), Cyclin D1(Cyclin D1), apoptosis-promoting protein Bcl-2 and Vascular Endothelial Growth Factor (VEGF) related to the growth and metastasis of tumor (accu-sources C et al. toxicity and applied pharmacology, 2012, 258 (1): 10-18.). Inoue et al found that IL-8 induced malignant and invasive growth of non-neoplastic bladder cell lines (233JP), while the incidence of malignant transformation of 233JP cells was significantly reduced in IL-8 knockdown mice (Inoue K et al cancer Res, 2000, 60 (8): 2290-2299.). Furthermore, in prostate cancer, IL-8 can promote the development of castration-resistant prostate cancer (CRPC) in patients (Chen K et al. cancer research, 2015, 75 (10): 1992-; gene silencing of IL-8 or its receptor induces cell cycle arrest in tumor cells and inhibits tumor proliferation (Singh RK, Lokeshwar BL. molecul Cancer, 2009, 8: 57.). The above studies indicate that the level of IL-8 is closely related to the development and development of tumors. Further studies (Mian BM et al. Clin Cancer Res, 2003, 9 (8): 3167-3175.) have shown that IL-8 can be a novel target for tumor therapy. In a tumor model of bladder cancer, the use of anti-IL-8 antibody can significantly inhibit tumor growth.

IL-6 is rapidly produced primarily by macrophages, responds to pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP), and cures damaged tissue by removing infectious agents, inducing acute phases and an immune response, and serves as a protective effect. Although IL-6 plays an important role in the resistance and repair of infection and tissue injury, high levels of IL-6 can activate the coagulation pathway and vascular endothelial cells, thereby inhibiting myocardial function, and can even cause "cytokine storm" to produce severe acute systemic inflammatory responses. Cytokine storms are a lethal complication and adverse reaction in viral infections, tumor immunotherapy, and the like.

Immune-related adverse reactions are a common and dangerous adverse reaction in Immune Checkpoint Inhibitor (ICI) antitumor therapy (Spain L et al cancer Treat rev.2016; 44: 51-60.). In recent years, immune checkpoint inhibitors have had great success in tumor immunotherapy, but also have resulted in a completely new toxicity profile due to off-target effects. In which severe immune-related adverse events (irAE), especially of major organs including heart, lung and brain, may be life threatening (Bergqvist V, et al. cancer Immunol immunol.2017; 66 (5): 581-. Data have shown that ICI may induce off-target effects by 4 mechanisms, including direct binding to immune checkpoint molecules expressed on the surface of normal cells, activating complement hypersensitivity; the normal tissue and the tumor cell have homologous antigen/epitope; generating autoantibodies; increasing The levels of pro-inflammatory cytokines, such as IL-6 and The like (Martins F et al, The Lancet Oncology, 20(1), e54-e 64.).

Currently, anti-IL-6 therapy, such as Tolizumab, a recombinant humanized anti-IL-6R monoclonal antibody, has been used to treat acute stage severe iraE, severe or refractory arthritis, macrovasculitis, uveitis, myocarditis, pneumonia, myasthenia gravis, etc. (Martins F et al, The Lancet Oncology, 20(1), e54-e 64.).

The Fc section of the antibody is mutated by inducing the Fc section of the macrophage to secrete IL-8 and IL-6(Kinder M et al, mAbs.2015), so that the Fc section is eliminated from being combined with the Fc gamma RIA, and the secretion of the IL-8 can be effectively inhibited, thereby increasing the safety and effectiveness of the antibody.

Disclosure of Invention

The inventor utilizes a mammalian cell expression system to respectively express recombinant CD73 and PD-1 as antigens to immunize mice, and obtains hybridoma cells by fusing mouse spleen cells and myeloma cells. The inventors obtained the following hybridoma cell lines by screening a large number of samples:

the hybridoma cell line LT014 (also called CD73-19F3) is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 19 months in 2018, with the preservation number of CCTCC NO: c2018137; and

hybridoma cell line LT003 (also called PD-1-14C12), which is preserved in China Center for Type Culture Collection (CCTCC) at 6 month and 16 days 2015 with the preservation number of CCTCC NO: C2015105.

the inventors have surprisingly found that:

the hybridoma cell line LT014 can secrete and generate a specific monoclonal antibody (named as 19F3) specifically combined with human CD73, and the monoclonal antibody can effectively inhibit the enzyme activity reaction of CD73 in a non-substrate competition mode, reduce the generation of adenosine, and promote the activity of T cells and the tumor inhibition effect.

Hybridoma cell line LT003 was able to secrete a specific monoclonal antibody (designated 14C12) that specifically binds to PD-1 and was able to block very effectively the binding of PD-1 to PDL 1.

Further, the present inventors have creatively produced humanized antibodies against CD73 (designated as 19F3H2L2, 19F3H2L3, 19F3H2L3(hG1M), 19F3H2L3(hG1TM), respectively) and humanized antibodies against PD-1 (designated as 14C12H1L1 and 14C12H1L1(hG1 TM)).

Further, the inventors creatively fuse two types of humanized antibodies into a new antibody through protein recombination, and obtain humanized bifunctional antibodies (respectively named as P1D7V01, P1D7V03, NTPDV1, NTPDV2, NTPDV3 and NTPDV4 (also written as NTPDV1(hG1TM), NTPDV2(hG1TM), NTPDV3(hG1TM) and NTPDV4(hG1TM) in the Chinese patent with the application number of 202110270671X) which can bind to CD73 and PD-1, inhibit the activity of CD73 and block the binding of PD-1 and PDL1, and have the potential for preparing drugs for preventing and treating solid tumors and blood tumors.

The following invention is thus provided:

one aspect of the present invention relates to an anti-CD 73-anti-PD-1 bispecific antibody comprising:

a first protein functional region, said first protein functional region targeted to PD-1, and

a second protein functional region, said second protein functional region targeted to CD 73.

In one embodiment of the invention, in said bispecific antibody

The first protein functional region comprises an amino acid sequence shown as SEQ ID NO:44, and the amino acid sequences of HCDR1, HCDR2 and HCDR3, preferably HCDR1, HCDR2 and HCDR3, contained in the heavy chain variable region are set forth in SEQ ID NOs:45-47 or a sequence substantially identical to SEQ ID NOs:45-47, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence set forth in SEQ ID NOs:45-47, and amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence set forth in

The amino acid sequence is shown as SEQ ID NO:49 and preferably LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, are set forth in SEQ ID NOs:50-52 or a sequence substantially identical to SEQ ID NOs:50-52, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence set forth in SEQ ID NOs:50-52 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions);

the second protein functional region comprises an amino acid sequence shown as SEQ ID NO:2, and the amino acid sequences of HCDR1, HCDR2 and HCDR3, preferably HCDR1, HCDR2 and HCDR3, contained in the heavy chain variable region shown in SEQ ID NOs:3-5 or a sequence corresponding to SEQ ID NOs:3-5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:3-5 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, and comprises

The amino acid sequence is shown as SEQ ID NO:7, the amino acid sequences of LCDR1, LCDR2 and LCDR3, preferably LCDR1, LCDR2 and LCDR3, contained in the light chain variable region shown in SEQ ID NOs:8-10 or a sequence corresponding to SEQ ID NOs:8-10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:8-10, with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In one embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody:

the first protein functional region comprises

The amino acid sequence is shown as SEQ ID NO:44 or SEQ ID NO:62 or a sequence corresponding to SEQ ID NOs:44 or 62, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:44 or 62 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and comprises

Respectively corresponding to amino acid sequences selected from SEQ ID NO:49 or SEQ ID NO:64 or a sequence substantially identical to SEQ ID NOs:49 or 64, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:49 or 64 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions);

and/or the presence of a gas in the gas,

the second protein functional region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2. SEQ ID NO:20 or a sequence corresponding to SEQ ID NO: 2. SEQ ID NO:20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 2. SEQ ID NO:20 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and comprises

Respectively correspond to a sequence selected from SEQ ID NO:7, or SEQ ID NO:22 or a sequence identical to SEQ ID NO:7 or SEQ ID NO:22, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NO:7 or SEQ ID NO:22, having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence set forth in seq id no;

The second protein functional region comprises a sequence with an amino acid sequence shown as SEQ ID NO20 or a sequence similar to SEQ ID NO:20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, and a sequence comprising one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in seq id no

The amino acid sequence is shown as SEQ ID NO:24 or a sequence corresponding to SEQ ID NO:24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NO:24 compared to an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions).

One aspect of the present invention relates to an anti-CD 73-anti-PD-1 bispecific antibody comprising:

a first protein functional region, said first protein functional region targeting CD73, and

a second protein functional region, said second protein functional region targeted to PD-1.

In one embodiment of the invention, in the bispecific antibody,

the first protein functional region comprises an amino acid sequence shown as SEQ ID NO:2, and preferably HCDR1, HCDR2 and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3, preferably HCDR1, HCDR2 and HCDR3, respectively, are set forth in SEQ ID NOs:3-5 or a sequence corresponding to SEQ ID NOs:3-5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence set forth in SEQ ID NOs:3-5 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, and comprises

The amino acid sequence is shown as SEQ ID NO:7, the amino acid sequences of LCDR1, LCDR2 and LCDR3, preferably LCDR1, LCDR2 and LCDR3, contained in the light chain variable region shown in SEQ ID NOs:8-10 or a sequence corresponding to SEQ ID NOs:8-10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:8-10 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions);

the second protein functional region comprises an amino acid sequence shown as SEQ ID NO:44, and the amino acid sequences of HCDR1, HCDR2 and HCDR3, preferably HCDR1, HCDR2 and HCDR3, contained in the heavy chain variable region are set forth in SEQ ID NOs:45-47 or a sequence substantially identical to SEQ ID NOs:45-47, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:45-47, and amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence set forth in

In one embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody:

the first protein functional region comprises

The amino acid sequence is shown as SEQ ID NO: 2. SEQ ID NO:20 or a sequence corresponding to SEQ ID NOs: 2 or 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs: 2 or 20 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and comprises

Respectively corresponding to amino acid sequences selected from SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO:24 or a sequence corresponding to SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO:24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs: 7, 22 or 24, having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence;

and/or the presence of a gas in the gas,

the second protein functional region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:44 or SEQ ID NO:62 or a sequence corresponding to SEQ ID NO:44 or SEQ ID NO:62, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:44 or SEQ ID NO:62 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and comprises

Corresponding to a sequence selected from SEQ ID NOs:49 or 64 or a variant of SEQ ID NOs:49 or 64, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in SEQ ID NOs:49 or 64 with one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions).

In one embodiment of the present invention, in the anti-CD 73-anti-PD-1 bispecific antibody, the first protein functional region and the second protein functional region are linked directly or via a linking fragment; preferably, the linker is (GGGGS) n, n being a positive integer, e.g. 1, 2, 3, 4, 5 or 6.

In one embodiment of the invention, the first protein functional region and the second protein functional region in the anti-CD 73-anti-PD-1 bispecific antibody are independently an immunoglobulin or an antigen-binding fragment, e.g., a half-antibody, Fab, F (ab')₂Or a single chain antibody, preferably, the first protein functional region is an immunoglobulin and the second protein functional region is an antigen binding fragment; alternatively, the first protein functional region is an antigen binding fragment and the second protein functional region is an immunoglobulin.

In one embodiment of the present invention, the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of CH1 of the immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of the light chain variable region CL of the immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of the light chain variable region CL of the immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the C-terminus of the heavy chain variable region CH1 of the immunoglobulin.

In one embodiment of the present invention, the C-terminus of the heavy chain variable region of the antigen-binding fragment is directly linked (or via a linking fragment) to the N-terminus of the heavy chain of the immunoglobulin and the C-terminus of the light chain variable region of the antigen-binding fragment is directly linked (or via a linking fragment) to the N-terminus of the light chain of the immunoglobulin; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the N-terminus of the light chain of the immunoglobulin and the C-terminus of the light chain variable region of the antigen-binding fragment is directly (or through a linking fragment) linked to the N-terminus of the heavy chain of the immunoglobulin.

In one embodiment of the present invention, the antigen-binding fragment is a single chain antibody, preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain antibody; or, the first protein functional region is a single-chain antibody, and the second protein functional region is immunoglobulin.

In one embodiment of the present invention, the bispecific antibody is one wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2.

In one embodiment of the present invention, in the anti-CD 73-anti-PD-1 bispecific antibody, the single chain antibody is a heavy chain variable region (V) of the antibody linked by a Linker_H) And antibody light chain variable region (V)_L) A molecule of (a); preferably, it may have the general structure: NH2-V_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_L-COOH。

In one embodiment of the present invention, in the anti-CD 73-anti-PD-1 bispecific antibody, the single chain antibody is linked to the C-terminus (C) of the heavy chain of an immunoglobulin via a linking fragment_H) (or the N-terminus of the heavy chain, the C-terminus of CH1 in the variable region of the heavy chain), the variable region of the antibody heavy chain of the single-chain antibody may be first ligated (V)_H) Or, first, the antibody light chain variable region (V) of the single chain antibody is ligated_L) (ii) a Preferably, the single chain antibody may have the general structure: c_H-connectingfragment-V_HConnecting fragment-V_L-COOH, or, C_HConnecting fragment-V_LConnecting fragment-V_H-COOH，

Preferably, the first and second electrodes are formed of a metal,

the heavy chain variable region of the immunoglobulin comprises an amino acid sequence of SEQ ID NO:3-5, wherein the light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NO:8-10 CDR;

the heavy chain variable region of the single-chain antibody comprises an amino acid sequence of SEQ ID NO:45-47, wherein the light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NO: the CDRs of 50-52 are,

preferably, the single chain antibody (e.g., NH 2-V)_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_L-COOH) is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker fragment, the C-terminus of the heavy chain of the immunoglobulin comprising the amino acid sequence of SEQ ID NO: antibody heavy chain variable region (V) of CDR of 45-47_H) Or, first linking a single chain antibody comprising the amino acid sequence of SEQ ID NO: antibody light chain variable region (V) of CDR 50-52_L)，

Or, preferably, the number of bits in the bit stream,

the heavy chain variable region of the immunoglobulin comprises an amino acid sequence of SEQ ID NO:45-47, wherein the light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NO: 50-52; and/or the presence of a gas in the gas,

the heavy chain variable region of the single-chain antibody comprises an amino acid sequence of SEQ ID NO:3-5, wherein the light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NO: the CDRs of 8-10 are described,

wherein the single chain antibody (e.g., NH 2-V)_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_L-COOH) is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker fragment, the C-terminus of the heavy chain of the immunoglobulin comprising the amino acid sequence of SEQ ID NO:3-5 CDR antibody heavy chain variable region (V)_H) Or, first linking a single chain antibody comprising the amino acid sequence of SEQ ID NO: antibody light chain variable region (V) of CDR of 8-10_L)，

Preferably, the first and second electrodes are formed of a metal,

two single chain antibody molecules are attached to one immunoglobulin molecule, more preferably, the two single chain antibody molecules are the same.

In one embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody, the immunoglobulin is IgG, IgA, IgD, IgE, or IgM; preferably an IgG, such as IgG1, IgG2, IgG3 or IgG 4.

In one embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody, the single chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin. Since an immunoglobulin consists of two heavy chains, two single-chain antibody molecules are linked to one immunoglobulin molecule. Preferably, the two single chain antibody molecules are identical.

In one embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody:

and/or the presence of a gas in the gas,

In another embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody:

In one embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody:

the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO:2 or SEQ ID NO: 20; the amino acid sequences of the variable regions of the light chains of the immunoglobulins correspond to the amino acid sequences selected from SEQ ID NOs: 7 or SEQ ID NO:22, or the heavy chain variable region of said immunoglobulin has the amino acid sequence of SEQ ID NO: 20; the amino acid sequence of the variable region of the light chain of the immunoglobulin is SEQ ID NO: 24;

and/or the presence of a gas in the gas,

the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO:44 or SEQ ID NO:62, a first step of mixing; the amino acid sequences of the light chain variable regions of the single-chain antibody respectively correspond to the amino acid sequences selected from SEQ ID NO:49 or SEQ ID NO: 64;

wherein, when the single-chain antibody is connected to the C-terminal of the heavy chain of the immunoglobulin through the Linker, the antibody heavy chain variable region (V) of the single-chain antibody can be firstly connected_H) Or, first, the antibody light chain variable region (V) of the single chain antibody is ligated_L)。

In another embodiment of the invention, in the anti-CD 73-anti-PD-1 bispecific antibody:

the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO:44 or SEQ ID NO:62, a first step of mixing; the amino acid sequence of the variable region of the light chain of the immunoglobulin corresponds to the amino acid sequence selected from the group consisting of SEQ ID NO:49 or SEQ ID NO:64, or the heavy chain variable region of said single chain antibody is selected from the group consisting of SEQ ID NOs: 2 or SEQ ID NO:20, the variable region of the light chain of the single chain antibody has an amino acid sequence corresponding to a sequence selected from SEQ ID NO:7 or SEQ ID NO:22 or the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO:20, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO:24, or a sequence shown in fig. 24.

Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain variable region of a bispecific antibody, wherein,

the heavy chain variable region of the antibody comprises:

the amino acid sequence is SEQ ID NO:3-5 has the CDR and amino acid sequence shown in SEQ ID NO:45-47 are the CDR and amino acid sequences of SEQ ID NO: 50-52;

the amino acid sequence is SEQ ID NO:8-10 CDR;

preferably, the CDRs of the light chain variable region are not identical to the CDRs comprised by the heavy chain variable region.

In one embodiment of the invention, in the bispecific antibody,

the immunoglobulin includes a non-CDR region, and the non-CDR region is from a species other than murine, such as from a human antibody.

In one embodiment of the invention, the constant region of the immunoglobulin is humanized, e.g., the heavy chain constant region is replaced with Ig gamma-1chain C region, ACCESSION: p01857; the light chain constant region was obtained using Ig kappa chain C region, ACCESSION: p01834; wherein the heavy chain constant region of the immunoglobulin is mutated at any 2 or 3 of positions 234, 235 and 237 according to the EU numbering system (EU numbering system), and the bispecific antibody has a reduced pre-mutation affinity constant compared to the affinity constant of Fc γ RIa, Fc γ RIIIa and/or C1q after the mutation; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.

In one or more embodiments of the invention, the bispecific antibody wherein the immunoglobulin heavy chain constant region has the following mutations at position 234, 235 and/or 237 according to the EU numbering system:

L234A and L235A; or

L234A and G237A; or

L235A and G237A;

L234A、L235A、G237A。

In the present invention, letters before the site indicate amino acids before mutation, and letters after the site indicate amino acids after mutation, if not otherwise specified.

In one or more embodiments of the invention, the bispecific antibody further comprises one or more mutations selected from the group consisting of:

N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, a330R, C226S, C229S, E233P, P331S, S267E, L328F, a330L, M252Y, S254T, T256E, N297Q, P238S, P238A, a327Q, a327G, P329A, K322A, T394D, G236R, G236A, L328R, a330S, P331S, H268A, E318A, and K320A.

In a specific embodiment, the anti-CD 73-anti-PD-1 bispecific antibody is structurally represented by a heavy chain-light chain-linking fragment 1-scFv selected from the group consisting of 14C12H 1V-linking fragment 2-14C12L1V, 14C12H 1V-linking fragment 1-14C12L1V, 14C12H 1V-linking fragment 2-14C12L1V and 14C12H 1V-linking fragment 1-14C12L1V, specifically selected from the group consisting of:

(1) NTPDV1, the heavy chain amino acid sequence of which is set forth in SEQ ID NO:85, and the light chain amino acid sequence is shown as SEQ ID NO:28, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO:79, the amino acid sequence of 14C12H1V is shown as SEQ ID NO:66, the amino acid sequence of the connecting fragment 2 is shown as SEQ ID NO:81, and the amino acid sequence of 14C12L1V is shown as SEQ ID NO: as shown at 68, the flow of gas is,

(2) NTPDV2, the heavy chain amino acid sequence of which is set forth in SEQ ID NO:85, and the light chain amino acid sequence is shown as SEQ ID NO:28, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO:79, the amino acid sequence of 14C12H1V is shown as SEQ ID NO:66, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO:79, the amino acid sequence of 14C12L1V is shown as SEQ ID NO: as shown at 68, the flow of gas is,

(3) NTPDV3, the heavy chain amino acid sequence of which is set forth in SEQ ID NO:85, and the light chain amino acid sequence is shown as SEQ ID NO:96, and the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO:79, the amino acid sequence of 14C12H1V is shown as SEQ ID NO:66, the amino acid sequence of the connecting fragment 2 is shown as SEQ ID NO:81, and the amino acid sequence of 14C12L1V is shown as SEQ ID NO:68, and

(4) NTPDV4, the heavy chain amino acid sequence of which is set forth in SEQ ID NO:85, and the light chain amino acid sequence is shown as SEQ ID NO:96, and the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO:79, the amino acid sequence of 14C12H1V is shown as SEQ ID NO:66, the amino acid sequence of the connecting fragment 1 is shown as SEQ ID NO:79, the amino acid sequence of 14C12L1V is shown as SEQ ID NO: as shown at 68.

In one embodiment of the invention, the bispecific antibody is administered in an amount of less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10K of M or less_DBinds to the CD73 protein and/or the PD-1 protein.

Yet another aspect of the invention relates to a vector comprising an isolated nucleic acid molecule of the invention.

Yet another aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention.

Yet another aspect of the invention relates to a method of making a bispecific antibody of the invention comprising the steps of culturing a host cell of the invention under suitable conditions, and recovering the bispecific antibody from the cell culture.

Yet another aspect of the invention relates to a conjugate comprising a bispecific antibody and a conjugate moiety, wherein the bispecific antibody is a bispecific antibody of the invention and the conjugate moiety is a detectable label; specifically, the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

Yet another aspect of the invention relates to a kit comprising a bispecific antibody of the invention, or comprising a conjugate of the invention; preferably the kit further comprises a second antibody that specifically recognizes the bispecific antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

Yet another aspect of the invention relates to the use of a bispecific antibody of the invention in the preparation of a kit for detecting the presence or level of CD73 and/or PD-1 in a sample.

Yet another aspect of the invention relates to a pharmaceutical composition comprising a bispecific antibody of the invention or a conjugate of the invention; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.

Yet another aspect of the invention relates to the use of a bispecific antibody of the invention or a conjugate of the invention for the prevention and/or treatment of a tumor or anemia, or for the diagnosis of a tumor or anemia.

Yet another aspect of the invention relates to the use of a bispecific antibody of the invention or a conjugate of the invention for the preparation of a medicament for the prevention and/or treatment of a tumor or anemia, or for the preparation of a medicament for the diagnosis of a tumor or anemia.

A further aspect of the invention relates to the use of a bispecific antibody of the invention or a conjugate of the invention for the preparation of a medicament:

(ii) a drug that detects the level of CD73 in the sample,

inhibiting the enzymatic activity of CD 73;

and/or

Drugs that block the binding of PD-1 to PDL1,

a drug that modulates (e.g., down-regulates) PD-1 activity or level,

a medicine for relieving the immunosuppression of PD-1 to the organism,

an agent for increasing IL-2 expression in T lymphocytes, or

A medicament for increasing IFN- γ expression in T lymphocytes.

A further aspect of the invention relates to an in vivo or in vitro method comprising the step of applying a cell or administering to a subject in need thereof an effective amount of a bispecific antibody of the invention or a conjugate of the invention,

the anti-CD 73-anti-PD-1 bispecific antibody can inhibit the activity of CD73 enzyme on the surface of a cell membrane, can induce the secretion of IFN gamma and IL-2, and can activate immune response.

The variable regions of the light and heavy chains determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called Complementarity Determining Regions (CDRs) (CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3 and CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR 3; named by Kabat et al, see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3: 91-3242).

Preferably, the CDRs are also defined by the IMGT numbering system, see Ehremann F, Kaas Q, Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibiotics, T cell receptors, MHC, IgSF and MhcSF [ J ]. Nucleic acids research, 2009; 38(suppl _ 1): D301-D307.

The amino acid sequences of the CDRs of the monoclonal antibody sequences in items (1) to (11) below were analyzed by technical means well known to those skilled in the art, for example, according to the IMGT definition, and the results are as follows:

(1)19F3

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 7.

The amino acid sequences of the 3 CDRs of the heavy chain variable region are as follows:

HCDR1：GYSFTGYT(SEQ ID NO：3)，

HCDR2：INPYNAGT(SEQ ID NO：4)，

HCDR3：ARSEYRYGGDYFDY(SEQ ID NO：5)；

the amino acid sequences of the 3 CDRs of the light chain variable region are as follows:

LCDR1：QSLLNSSNQKNY(SEQ ID NO：8)，

LCDR2：FAS(SEQ ID NO：9)，

LCDR3：QQHYDTPYT(SEQ ID NO：10)。

(2)19F3H2L2

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:20, the amino acid sequence of the light chain variable region is shown as SEQ ID NO:22, respectively.

The amino acid sequence of 3 CDRs of its heavy chain variable region is identical to 19F 3.

The amino acid sequence of 3 CDRs of its light chain variable region is identical to 19F 3.

(3)19F3H2L3

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:20, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: as shown at 24.

The amino acid sequence of 3 CDRs of its heavy chain variable region is identical to 19F 3.

The amino acid sequence of 3 CDRs of its light chain variable region is identical to 19F 3.

(4)14C12

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:44, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 49.

The amino acid sequences of the 3 CDRs of the heavy chain variable region are as follows:

HCDR1：GFAFSSYD(SEQ ID NO：45)

HCDR2：ISGGGRYT(SEQ ID NO：46)

HCDR3：ANRYGEAWFAY(SEQ ID NO：47)

the amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:

LCDR1：QDINTY(SEQ ID NO：50)

LCDR2：RAN(SEQ ID NO：51)

LCDR3：LQYDEFPLT(SEQ ID NO：52)

(5)14C12H1L1

the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:62, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: as shown at 64.

The amino acid sequence of 3 CDRs in its heavy chain variable region is identical to 14C 12.

The amino acid sequence of 3 CDRs in its light chain variable region is identical to 14C 12.

(6)NTPDV1、NTPDV2、NTPDV3、NTPDV4The amino acid sequences of the 9 CDRs contained in the heavy chain are consistent with the CDR amino acid sequences of the heavy chain region 13F9, the heavy chain region 14C12 and the light chain region 14C12 respectively according to the sequence from N end to C end, and the sequences are arranged as follows:

HCDR1：GYSFTGYT(SEQ ID NO：3)

HCDR2：INPYNAGT(SEQ ID NO：4)

HCDR3：ARSEYRYGGDYFDY(SEQ ID NO：5)

HCDR4：GFAFSSYD(SEQ ID NO：45)

HCDR5：ISGGGRYT(SEQ ID NO：46)

HCDR6：ANRYGEAWFAY(SEQ ID NO：47)

HCDR7：QDINTY(SEQ ID NO：50)

HCDR8：RAN(SEQ ID NO：51)

HCDR9：LQYDEFPLT(SEQ ID NO：52)

the amino acid sequence of the 3 CDRs of the light chain is identical to the amino acid sequence of the three CDRs of the 19F3 light chain, and the sequence is as follows:

LCDR1：QSLLNSSNQKNY(SEQ ID NO：8)

LCDR2：FAS(SEQ ID NO：9)

LCDR3：QQHYDTPYT(SEQ ID NO：10)。

still another aspect of the present invention relates to hybridoma cell line LT014, which is deposited in the chinese typical culture collection center (CCTCC) with a collection number of CCTCC NO: C2018137.

still another aspect of the present invention relates to hybridoma cell line LT003, which is deposited in the chinese typical culture collection center (CCTCC) with a collection number of CCTCC NO: C2015105.

in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term EC₅₀Refers to the concentration of the half maximal effect (concentration for 50% of the maximum effect), and refers to the concentration that causes 50% of the maximal effect.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987and 1991)), or Chothia & Lesk J.mol.biol.1987; 196: 901-; chothia et al Nature 1989; 342: 878-: a database and a tool for immunoglobulins or antibiotics, T cell receptors, MHC, IgSF and MhcSF [ J ]. Nucleic acids research, 2009; 38(suppl _ 1): definitions of D301-D307.

In particular, the heavy chain may also comprise more than 3 CDRs, for example 6, 9, or 12. For example, in a bispecific antibody of the invention, the heavy chain may be an IgG antibody with an ScFv linked to the C-terminus of the heavy chain, in which case the heavy chain contains 9 CDRs.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained by the hybridoma technique first reported by Kohler et al (G，Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J]Nature, 1975; 256(5517): 495) but can also be obtained using recombinant DNA techniques (see, e.g., U.S. patent 4,816,567).

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some amino acid residues of the Framework Region (FR) of the acceptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., Jones et al, Nature 1986; 321: 522525, respectively; reichmann et al, Nature, 1988; 332: 323329, respectively; presta, curr.op.struct.biol.1992; 2: 593-; and Clark, immunol. today 2000; 21: 397402. in some cases, the antigen-binding fragment of an antibody is a diabody (Diabodies), wherein V_HAnd V_LDomains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between two domains of the same chain, thereby forcing the domains to pair with complementary domains of another chain and generating two antigen binding sites (see, e.g., Holliger P.et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-.

As used herein, the term "single chain fragment variable (ScFv)" refers to a variable region comprising heavy chains of antibodies (V) linked by a linker_H) And antibody light chain variable region (V)_L) The molecule of (1). Wherein V_LAnd V_HThe domains form monovalent molecules by pairing linkers that enable them to be generated as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242: 423-. Such scFv molecules may haveGeneral structure: NH2-V_LConnecting fragment-V_H-COOH or NH2-V_HConnecting fragment-V_L-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-. Other linkers useful in the present invention are prepared by Alfthan et al, Protein Eng.1995; 8: 725-731, Choi et al, Eur.J.Immunol.2001; 31: 94-106, Hu et al, Cancer Res.1996; 56: 3055-3061, Kipriyanov et al, J.Mol.biol.1999; 293: 41-56 and Rovers et al, Cancer Immunology, immunotherpy, 2001, 50 (1): 51-59.

As used herein, the term "isolated" or "isolated" refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less affinity (K)_D) Binding the antigen.

As used herein, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^- ⁹M or 10^-10Dissociation equilibrium constant (K) of M or less_D) Binding antigen (e.g., PD-1 protein). K can be determined using methods known to those skilled in the art_DE.g. using a Fortebio molecular interaction device。

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR, 19th ed. pennsylvania: mach Publishing Company, 1995), and include, but are not limited to: pH regulator, surfactant, adjuvant, and ionic strength enhancer. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a prophylactically effective amount (e.g., tumor) refers to an amount sufficient to prevent, or delay the onset of a disease (e.g., tumor); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

Advantageous effects of the invention

The monoclonal antibody (such as 13F9H2L3) disclosed by the invention can be well specifically combined with CD73, and can effectively inhibit the enzyme activity reaction of CD73 in a non-substrate competition manner, reduce the generation of adenosine and promote the activity of T cells and the tumor inhibition effect.

The bispecific antibody related to the invention, such as NTPDV1, NTPDV2, NTPDV3 and NTPDV4, can be well and specifically combined with PD-1 and CD73, can effectively block the combination of PD-1 and PDL1, specifically relieve the immune suppression of PD-1 and inhibit the catalytic activity of CD73, relieve the suppression of adenosine on immune cells, activate T lymphocytes, do not cause the release of cytokines IL-8 and IL-6, and effectively increase the safety and effectiveness.

The bifunctional antibody has the potential of being applied to preparing anti-tumor medicines.

Drawings

FIG. 1 ELISA detects the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, Nivolumab to PD-1-mFc.

FIG. 2 ELISA detects binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3, MEDI9447 to human NT 5E-Biotin.

FIG. 3, the results of activity assays of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab in competition with human PD-L1-mFc for binding to human PD-1-mFc-Biotin.

FIG. 4.P1D7V01 determination of affinity constant for PD-1-mFc.

FIG. 5.14C 12H1L1 determination of affinity constant for PD-1-mFc

FIG. 6. determination of affinity constant of Nivolumab for PD-1-mFc.

FIG. 7.P1D7V01 determination of affinity constant for human NT5E (1-552) -his.

FIG. 8 measurement of affinity constants of MEDI9447 with human NT5E (1-552) -his.

FIG. 9 FACS assays of the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on the cell surface of 293T-PD 1.

FIG. 10 FACS detection of binding Activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the cell surface of MDA-MB-231

FIG. 11 inhibitory Effect of anti-CD 73-anti-PD-1 bispecific antibody on the enzymatic activity of CD73 on the membrane surface of MDA-MB-231.

FIG. 12 inhibition of the enzyme activity of U87-MG membrane surface CD73 by anti-CD 73-anti-PD-1 bispecific antibody.

FIG. 13 detection of the bioactivity of anti-CD 73-anti-PD-1 bispecific antibody in promoting IFN-gamma secretion in Raji-PDL1 mixed lymphocyte reaction system.

FIG. 14 detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody in promoting IL-2 secretion in Raji-PDL1 mixed lymphocyte reaction system.

FIG. 15 detection of the bioactivity of anti-CD 73-anti-PD-1 bispecific antibody in promoting IFN-gamma secretion in DC mixed lymph reaction system.

FIG. 16 shows the detection of the bioactivity of anti-CD 73-anti-PD-1 bispecific antibody in promoting IL-2 secretion in DC mixed lymph reaction system.

FIG. 17.14C 12H1L1(hG1TM) was determined with the PD-1-mFc affinity constant.

FIG. 18 measurement of affinity constant of Nivolumab for PD-1-mFc

FIG. 19 determination of affinity constants of NTPDV1 with PD-1-mFc.

FIG. 20 determination of affinity constants of NTPDV2 with PD-1-mFc.

FIG. 21. determination of affinity constants of NTPDV3 with PD-1-mFc.

FIG. 22. determination of affinity constants of NTPDV4 with PD-1-mFc.

FIG. 23.19F 3H2L3(hG1M) was determined as compared to human NT5E (1-552) -his affinity constants.

FIG. 24. determination of affinity constants of NTPDV1 with human NT5E (1-552) -his.

FIG. 25 NTPDV2 determination of affinity constant for human NT5E (1-552) -his.

FIG. 26. determination of affinity constants of NTPDV3 with human NT5E (1-552) -his.

FIG. 27 NTPDV4 determination of affinity constant for human NT5E (1-552) -his.

FIG. 28 detection of the inhibition of the enzyme activity of CD73 on the cell membrane surface of U87-MG by anti-CD 73-anti-PD-1 bispecific antibody.

FIG. 29 Mixed lymphocyte reaction MLR assays for the biological activity of anti-CD 73-anti-PD-1 bispecific antibodies in promoting IFN-. gamma.and IL-2 secretion.

FIG. 30 Effect of isotype control, 19F3H2L3(hG1M), different doses of NTPDV2 on mouse tumor volume.

FIG. 31 Effect of isotype control, 19F3H2L3(hG1M), varying doses of NTPDV2 on mouse body weight.

FIG. 32 mutation of the amino acids in the Fc region was effective in abolishing IL-8 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody in the CHO-K1-PD1 cell and human macrophage co-culture system.

FIG. 33, in CHO-K1-PD1 cell and human macrophage co-culture system, Fc segment amino acid mutation effectively abolished IL-6 secretion by human macrophages mediated by PD-1/CD73 bispecific antibody.

FIG. 34 mutation of the amino acid in the Fc region effectively abolished IL-8 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody in a co-culture system of U87-MG cells and human macrophages.

FIG. 35 in a co-culture system of U87-MG cells and human macrophages, the mutation of the Fc region amino acid effectively abolished IL-6 secretion by human macrophages mediated by the PD-1/CD73 bispecific antibody.

Biological material retention information:

the hybridoma cell line LT014 (also called CD73-19F3) is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 21 months in 2018, with the preservation number of CCTCC NO: c2018137, the preservation address is China, Wuhan university, postcode: 430072.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, according to the techniques or conditions described in the literature of the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, of J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, but are conventional products available on the market. For example, MDA-MB-231 cells and U87-MG cells can be purchased from ATCC.

In the following examples of the present invention, BALB/c mice used were purchased from the center for medical laboratory animals in Guangdong province.

In the following examples of the invention, the positive control antibody MEDI9447 (generic name: Oleclumab), produced by Zhongshan Kangfang biomedical Co., Ltd, was used, the sequence of which is described in Medmmune Limited, publication No.: the antibody SEQ ID NOs: 21-24 are identical.

In the following examples of the invention, the co-targeted marketed pharmaceutical antibody Nivolumab, under the name Opdivo, was used and purchased from Bethes-Mich.

In the following examples of the present invention, 293T-PD1 cell line used was constructed by Zhongshan kang biomedical corporation. The 293T-PD1 cell line was prepared from HEK293T cells by virus infection using 3rd Generation leviral Systems, see, e.g., A Third Generation levirus Vector with a Conditional Packaging System, Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol, 1998.72 (11): 8463-8471, wherein the lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (wherein PD1, Genebank ID: NM-005018; vector pCDH-CMV-Puro, purchased from Youbao, product number: VT 1480).

In the following examples of the present invention, the Raji-PDL1 cell line used was constructed by Zhongshan kang biomedical corporation. Raji-PDL1 cell line was prepared by infecting Raji cells with viruses prepared using 3rd Generation viral Systems, see, e.g., A Third Generation viral Vector with a Conditional Packaging System, Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72 (11): 8463-8471, wherein the lentiviral expression vector used was plenti6.3-PDL1 (wherein PDL1, Genebank ID: NP-054862.1; vector plenti6.3, available from Invitrogen, cat # K5315-20).

In the following examples of the present invention, the CHO-K1-PD1 cell line used was constructed by Zhongshan Kangfang biomedical Co., Ltd. CHO-K1-PD1 cell lines were prepared from CHO-K1 cells by infection with viruses prepared using 3rd Generation leviral Systems, see, e.g., A Third Generation levirus Vector with a conditioned Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J virol.1998.72 (11): 8463-8471, wherein the lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (wherein PD1, Genebank ID: NM-005018; vector pCDH-CMV-Puro, purchased from Youbao, product number: VT 1480).

In the experimental examples, Nivolumab (trade name Opdivo) which is an IgG4 subtype anti-PD-1 antibody carrying the mutation S228P was used as a control antibody and was purchased from Bethes Baikh, McSt.

In the following experimental examples of the present invention, the isotype control antibodies used, i.e., hIgG1, were antibodies targeted to human anti-Hen Egg Lysosome (HEL), and the variable region sequences of the antibodies were derived from Affinity matching of the domains of anti-protein antibodies published by Acierno et al (Acierno et al J Mol biol. 2007; 374 (1): 130-46.), and the constant region fragments of hIgG1 were obtained from Ig gamma-1chain C region, ACCESSION: p01857 as the heavy chain constant region, Ig kappa chain C region, access: p01834 is a light chain constant region; the hIgG1 was made in the laboratory of zhongshan kang biomedical corporation, ltd.

Example 1: preparation of anti-CD 73 antibody 19F3

1. Preparation of hybridoma cell line LT014

The antigen used to prepare the anti-CD 73 antibody was human NT5E-his (NT5E is GenbankID: NP-002517.1, position: 1-552). Spleen cells of immunized mice are taken and fused with myeloma cells of mice to prepare hybridoma cells, the hybridoma cells are screened by an indirect ELISA method by taking human NT5E (NT5E is GenbankID: NP-002517.1, position: 1-552) -Biotin as antigen, and the hybridoma cells capable of secreting antibodies specifically combined with CD73 are obtained. And (3) carrying out limited dilution on the hybridoma obtained by screening with an ELISA method to obtain a stable hybridoma cell strain. The hybridoma cell line was designated as hybridoma cell line LT014, and the secreted monoclonal antibody was designated as 19F 3.

The hybridoma cell line LT014 (also called CD73-19F3) is preserved in China Center for Type Culture Collection (CCTCC) in 6 and 21 months in 2018, with the preservation number of CCTCC NO: c2018137, the preservation address is China, Wuhan university, postcode: 430072.

2. preparation of anti-CD 73 antibody 19F3

The LT014 cell line prepared above was cultured in CD Medium (CD Medium containing 1% streptomycin in 5% CO)₂Cultured in a cell culture chamber at 37 ℃) and after 7 days, the cell culture supernatant was collected and purified by high-speed centrifugation, microfiltration membrane vacuum filtration and HiTrap protein A HP column to obtain antibody 19F 3.

Example 2: sequence analysis of anti-CD 73 antibody 19F3

mRNA was extracted from each of the LT014 cell lines cultured in example 1 according to the method of the cultured cell bacterial total RNA extraction kit (Tiangen, cat # DP 430).

According to InvitrogenIII First-Strand Synthesis System for RT-PCR kit instructions Synthesis of cDNA, and PCR amplification.

The PCR amplification product was directly subjected to TA Cloning, and the specific operation was carried out with reference to the pEASY-T1 Cloning Kit (Transgen CT101) Kit instruction.

The products of the TA clones were sequenced directly, with the following results:

the nucleic acid sequence (363bp) of the 19F3 heavy chain variable region is shown in SEQ ID NO: 1, and the coded amino acid sequence (121aa) is shown as SEQ ID NO:2, respectively.

According to the IMGT numbering system, wherein the sequence of the heavy chain CDR1 is as set forth in SEQ ID NO:3, the sequence of heavy chain CDR2 is shown in SEQ ID NO:4, the sequence of heavy chain CDR3 is shown in SEQ ID NO:5, respectively.

The nucleic acid sequence (339bp) of the 19F3 light chain variable region is shown as SEQ ID NO:6, and the coded amino acid sequence (113aa) is shown as SEQ ID NO: shown at 7.

According to the IMGT numbering system, wherein the light chain CDR1 has the sequence shown in SEQ ID NO:8, the light chain CDR2 has the sequence shown in SEQ ID NO:9, the light chain CDR3 has the sequence shown in SEQ ID NO: shown at 10.

The amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of the 19F3 heavy chain are respectively shown in SEQ ID NO: 11 to SEQ ID NO: 14 is shown in the figure; 19F3 light chain 4 Framework regions (Framework regions) (FR-H1 to FR-H4) amino acid sequences are respectively shown in SEQ ID NO: 15 to SEQ ID NO: 18, respectively.

Example 3: design, preparation and detection of humanized antibody against human CD73

1. Design of light and heavy chain sequences for humanized antibodies 19F3H2L3 and 19F3H2L2

Based on the three-dimensional crystal structure of the human CD73 protein (Hage T, Reinier P, Sebald W. Crystals of a 1: 1complex between human interferon-4 and the extracellular domain of its receptor alpha chain. Eur. J biochem. 1998; 258 (2): 831-6) and the sequence of the antibody murine antibody 19F3 obtained in example 2, the variable region sequences of antibodies 19F3H1L1, 19F3H2L3 and 19F3H2L3 were obtained by computer modeling and designing mutations based on the model, the light chain variable region sequences of which are shown in SEQ ID NO: 93 and SEQ ID NO: 97, and the light chain variable region sequences of which are shown in SEQ ID NO: 1, 19F3L2, 19F3H2 (amino acid sequences are shown in SEQ ID NO: 93 and SEQ ID NO: 3699, respectively) and the amino acid sequences of which are shown in SEQ ID NO: 19F3L1, 19F3L2, 19F3L 19L 567 (amino acid sequences of which are shown in SEQ ID NO: 3695, and SEQ ID NO: 99, and the constant region sequences of heavy chain variable region of antibodies using the antibody C-1, SEQ ID NO: 98, ACCESSION: p01857; the light chain constant region is Ig kappa chain C region, ACCESSION: p01834. Among them, 19F3H2L3 is also written as 19F3H2L3(hG1WT) in chinese patent with application number 202110270671.X, and the light and heavy chain variable regions of 19F3H1L1, 19F3H2L2 and 19F3H2L3 can be also marked as 19F3H1V (or 19F3H 1)_V) 19F3H2V (or 19F3H 2)_V) 19F3L1V (or 19F3L 1)_V) 19F3L2V (or 19F3L 2)_V) 19F3L3V (or 19F3L3)_V)。

(1) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 19F3H1L1

The nucleic acid sequence (363bp) of the heavy chain variable region is shown in SEQ ID NO: 92, and the coded amino acid sequence (121aa) is shown as SEQ ID NO: 93, respectively.

The variable region in the light chain has the nucleic acid sequence (339bp) as shown in SEQ ID NO: 94, and the coded amino acid sequence (113aa) is shown as SEQ ID NO: 95 is shown.

(2) The sequences of the heavy chain variable region and the light chain variable region of the humanized monoclonal antibody 19F3H2L2 are as follows:

the nucleic acid sequence (363bp) of heavy chain variable region 19F3H2 is set forth in SEQ ID NO: 19, and the coded amino acid sequence (121aa) is shown as SEQ ID NO: shown at 20.

The variable region in light chain 19F3L3 has the nucleic acid sequence (339bp) as shown in SEQ ID NO: 21, and the coded amino acid sequence (113aa) is shown as SEQ ID NO:22, respectively.

(3) The sequences of the heavy chain variable region and the light chain variable region of the humanized monoclonal antibody 19F3H2L3 are as follows:

the nucleic acid sequence (363bp) of heavy chain variable region 19F3H2 is set forth in SEQ ID NO: 19, and the coded amino acid sequence (121aa) is shown as SEQ ID NO: shown at 20.

The variable region in light chain 19F3L3 has the nucleic acid sequence (339bp) as shown in SEQ ID NO: 23, and the coded amino acid sequence (113aa) is shown as SEQ ID NO: as shown at 24.

2. Preparation of humanized antibodies 19F3H1L1, 19F3H2L2, and 19F3H2L3

The heavy chain constant region was determined using Ig gamma-1chain C region, ACCESSION: p01857; the light chain constant region was obtained using Ig kappa chain C region, ACCESSION: p01834.

Heavy chain cDNA and light chain cDNA of 19F3H1L1, 19F3H2L2 and 19F3H2L3, respectively, were cloned into pUC57simple (supplied by Kinsley) vectors to obtain pUC57simple-19F3H1, pUC57simple-19F3L1, pUC57simple-19F3H2, pUC57simple-19F3L2 and pUC57simple-19F3L3, respectively. Referring to the standard technique introduced in molecular cloning instructions (second edition), EcoRI & HindIII was used to cleave the synthesized full-length heavy and light chains, and subcloned into expression vector pcDNA3.1 by restriction of restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-19F3H1, pcDNA3.1-19F3L1, pcDNA3.1-19F3H2, pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3, and further to sequence and analyze the heavy and light chain genes of the recombinant expression plasmids. Then co-transfecting 293F cells with corresponding light and heavy chain recombinant plasmid design gene combinations (pcDNA3.1-19F3H1/pcDNA3.1-19F3L1, pcDNA3.1-19F3H2/pcDNA3.1-19F3L2, pcDNA3.1-19F3H2/pcDNA3.1-19F3L3), and collecting and purifying culture solution. And (3) after sequencing verification is correct, preparing an expression plasmid with endotoxin removed level, transiently transfecting HEK293 cells with the plasmid to express the antibody, culturing for 7 days, collecting cell culture solution, and performing affinity purification by adopting a Protein A column to obtain the humanized antibody.

3. Design of light and heavy chain sequences of humanized antibodies 19F3H2L3(hG1M) and 19F3H2L3(hG1TM)

Based on 19F3H2L3 obtained in example 31, the present inventors introduced a point mutation from leucine to alanine at position 234 (L234A) and introduced a point mutation from leucine to alanine at position 235 (L235A) of the heavy chain, to obtain 19F3H2L3(hG1M), and the nucleotide and amino acid sequences of the heavy chain of 19F3H2L3(hG1M) are shown in SEQ ID NOs: 25 and SEQ ID NO: 26 is shown; the 19F3H2L3(hG1M) light chain constant region is Ig kappa chain C region, ACCESSION: the nucleotide and the amino acid sequences of the light chain of P01834 and 19F3H2L3(hG1M) are respectively shown as SEQ ID NO: 27 and SEQ ID NO: shown at 28.

The present inventors obtained 19F3H2L3 obtained in example 31, and introduced a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A), to obtain 19F3H2L3(hG1TM), whose heavy chain amino acid and amino acid sequences are shown in SEQ ID NOs: 29 and SEQ ID NO:30 is shown in the figure; the light chain was identical to 19F3H2L3(hG 1M).

The amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of 19F3H2 are respectively shown in SEQ ID NO: 31 to SEQ ID NO: 34;

the amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of the 19F3L2 light chain are respectively shown as SEQ ID NO: 35 to SEQ ID NO: 38;

the amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of the 19F3L3 light chain are respectively shown as SEQ ID NO: 39 to SEQ ID NO: shown at 42.

4. Preparation of humanized antibody 19F3H2L3(hG1M)

Heavy chain cDNA and light chain cDNA of 19F3H2L3(hG1M) were cloned into pUC57simple (supplied by Kinsyru Co.) vectors to obtain pUC57simple-19F3H2(hG1M) and pUC57simple-19F3L3, respectively. Referring to the standard technique introduced in molecular cloning instructions (second edition), EcoRI & HindIII was used to cleave the synthesized full-length heavy and light chains, and subcloned into expression vector pcDNA3.1 by restriction of restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-19F3H2(hG1M) and pcDNA3.1-19F3L3, and further to sequence and analyze the heavy and light chain genes of the recombinant expression plasmids. Then co-transfecting 293F cells with the corresponding light and heavy chain recombinant plasmid design gene combination pcDNA3.1-19F3H2(hG1M)/pcDNA3.1-19F3L3, and collecting the culture solution for purification. After the sequencing verification is correct, an expression plasmid with endotoxin removed level is prepared and transiently transfected into HEK293 cells for antibody expression, and after 7 days of culture, cell culture fluid is collected and affinity purification is carried out by adopting a Protein A column to obtain a humanized antibody 19F3H2L3(hG 1M).

Example 4: preparation of anti-PD-1 antibody 14C12

1. Preparation of hybridoma cell line LT003

Spleen cells of immunized BALB/c mice (purchased from Guangdong center for medical laboratory animals) were fused with mouse myeloma cells to hybridoma cells using PD-1-mFc fusion protein (PD-1 GenBank: NM-005018, mFc SEQ ID NO: 89) as an antigen, in accordance with the currently established Methods (e.g., Stewart, S.J., "Monoclonal Antibody Production", in Basic Methods in Antibody Production and Characterization, eds.G.C.Howard and D.R.Bell, Boca Raton: CRC Press, 2000).

PD-1-hFc (PD-1 Genbank ID: NM-005018, hFc is a human IgG Fc purification label, specifically Ig gamma-1chain C region, Genbank ID: P01857 position 114-330) is used as an antigen coated enzyme label plate to carry out indirect ELISA screening to obtain hybridoma secreting new antibody specifically combined with PD-1.

And (2) screening a hybridoma cell line capable of secreting a monoclonal antibody which competes with a ligand PD-L1-hFc (PD-L1 Genbank ID: NP-054862.1) for binding with PD-1 by competition ELISA, obtaining a stable hybridoma cell line by a limiting dilution method, obtaining an LT003 stable cell line (PD-1-14C12) by the limiting dilution method, and naming the secreted monoclonal antibody as 14C 12.

Hybridoma cell line LT003 (also called PD-1-14C12), which is preserved in China Center for Type Culture Collection (CCTCC) at 6 month and 16 days 2015 with the preservation number of CCTCC NO: c2015105, wherein the preservation address is China, Wuhan university, postcode: 430072.

2. preparation of anti-PD-1 antibody 14C12

The LT003 cell line prepared above was cultured in IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium containing 1% streptomycin in 5% CO)₂And cultured in a cell culture box at 37 ℃), and after 7 days, cell culture supernatant was collected and purified to obtain antibody 14C 12.

Example 5: sequence analysis of anti-PD-1 antibody 14C12

mRNA was extracted from hybridoma cell line LT003 obtained in example 1 by the method of the cultured cell bacterial total RNA extraction kit (Tiangen, cat # DP 430).

According to InvitrogenIII First-Strand Synthesis System for RT-PCR kit instructions Synthesis of cDNA, and PCR amplification.

The PCR amplification product was directly subjected to TA Cloning, and the specific operation was carried out with reference to the pEASY-T1 Cloning Kit (Transgen CT101) Kit instruction.

The products of the TA clones were sequenced directly, with the following results:

the nucleic acid sequence (354bp) of the heavy chain variable region is as shown in SEQ ID NO: 43, and the coded amino acid sequence (118aa) is shown as SEQ ID NO: as shown at 44.

According to the IMGT numbering system, wherein the sequence of the heavy chain CDR1 is as set forth in SEQ ID NO:45, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 46, the sequence of heavy chain CDR3 is shown in SEQ ID NO: shown at 47.

The nucleic acid sequence (321bp) of the light chain variable region is shown as SEQ ID NO: 48, and the coded amino acid sequence (107aa) is shown as SEQ ID NO: shown at 49.

According to the IMGT numbering system, wherein the light chain CDR1 has the sequence shown in SEQ ID NO:50, the light chain CDR2 has the sequence shown in SEQ ID NO: 51, the light chain CDR3 has the sequence shown in SEQ ID NO: shown at 52.

The amino acid sequences of 4 Framework regions (Framework regions) of the 14C12 heavy chain (FR-H1 to FR-H4) are respectively shown in SEQ ID NO: 53 to SEQ ID NO: 56 is shown; the amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of the 14C12 light chain are respectively shown in SEQ ID NO: 57 to SEQ ID NO: shown at 60.

Example 6: design and preparation of humanized antibodies 14C12H1L1 and 14C12H1L1(hG1TM) against PD-1

1. Design of humanized antibody 14C12H1L1 against PD-1

The light and heavy chain sequences of humanized antibody 14C12H1L1 were designed based on the three-dimensional crystal Structure of PD-1 protein (Shinohara T, et al, Structure and chromosomal localization of the human PD-1 gene (PDCD1), Genomics 1995, 23 (3): 704-6) and the sequence of antibody 14C12 obtained in example 5, by computer modeling the antibody model and designing mutations based on the model to obtain the variable region sequences of antibody 14C12H1L 1.

The variable region sequences were designed as follows:

the variable region of the heavy chain of the humanized monoclonal antibody 14C12H1L1, the nucleic acid sequence of 14C12H1 (354bp) is shown in SEQ ID NO: 61, and the encoded amino acid sequence (118aa) is shown as SEQ ID NO: shown at 62.

The variable region of the light chain of the humanized monoclonal antibody 14C12H1L1, the nucleic acid sequence of 14C12L1 (321bp) is shown in SEQ ID NO: 63, and the coded amino acid sequence (107aa) is shown as SEQ ID NO: as shown at 64.

Antibody constant region sequence of antibody 14C12H1L1 from NCBI database, heavy chain constant region was determined using Ig gamma-1chain C region, access: p01857; the light chain constant region is Ig kappa chain C region, ACCESSION: p01834), the nucleotide sequence and the amino acid sequence of the 14C12H1L1 heavy chain are respectively shown as SEQ ID NO:65 and 66, the nucleotide sequence and amino acid sequence of the 14C12H1L1 light chain are set forth in SEQ ID NOs: 67 and 68. Among them, 14C12H1L1 is also written as 14C12H1L1(hG1WT) in this text and in chinese patent application No. 202110270671.X, and among them, the heavy chain variable region and light chain variable region of 14C12H1L1 are also written as 14C12H1V (or 14C12H 1) in chinese patent application No. 202110270671.X_V) And 14C12L1_V(or 14C12L1_V)。

2. Design of light and heavy chain sequences of humanized antibody 14C12H1L1(hG1TM)

The present inventors obtained 14C12H1L1 obtained in example 6, and obtained 14C12H1L1(hG1TM) by introducing a leucine to alanine point mutation at position 234 (L234A), a leucine to alanine point mutation at position 235 (L235A), and a glycine to alanine point mutation at position 237 (G237A). The nucleotide and amino acid sequences of the heavy chain of 14C12H1L1(hG1TM) are set forth in SEQ ID NO: 69 and SEQ ID NO: 70 is shown; the variable light chain region amino acid sequence thereof is identical to the variable light chain region of antibody 14C12H1L 1.

The amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of 14C12H1 are respectively shown in SEQ ID NO: 71 to SEQ ID NO: 74 is shown;

the amino acid sequences of 4 Framework regions (Framework regions) (FR-H1 to FR-H4) of the 14C12L1 light chain are respectively shown as SEQ ID NO: 75 to SEQ ID NO: 78, respectively;

3. preparation of humanized antibodies 14C12H1L1, 14C12H1L1(hG1TM)

The heavy chain cDNA and light chain cDNA of 14C12H1L1(hG1TM) and 14C12H1L1, respectively, were cloned into pUC57simple (supplied by Kinsley) vectors to obtain pUC57simple-14C12H1, pUC57simple-14C12L1 and pUC57simple-14C12H1(hG1TM), respectively. Referring to the standard technique introduced in molecular cloning instructions (second edition), EcoRI & HindIII was digested to synthesize the full-length heavy and light chains, which were subcloned into expression vector pcDNA3.1 by restriction of restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-14C12H1, pcDNA3.1-14C12L1, and pcDNA3.1-14C12H1(hG1TM), and further the heavy and light chain genes of the recombinant expression plasmids were subjected to sequencing analysis. Then co-transfecting 293F cells with the corresponding light and heavy chain recombinant plasmid design gene combinations (pcDNA3.1-14C12H1(hG1TM)/pcDNA3.1-14C12L1, and pcDNA3.1-14C12H1/pcDNA3.1-14C12L1) respectively, and collecting and purifying culture solution. And (3) after sequencing verification is correct, preparing an expression plasmid with endotoxin removed level, transiently transfecting HEK293 cells with the plasmid to express the antibody, culturing for 7 days, collecting cell culture solution, and performing affinity purification by adopting a Protein A column to obtain the humanized antibody.

Example 7: sequence design and expression of anti-PD-1/CD 73 bifunctional antibody

1. Sequence design

The structural pattern of the bifunctional antibody of the present invention belongs to the Morrison pattern (IgG-scFv), i.e., scFv fragments of one IgG antibody, in which both heavy chains are connected at their C-termini to another antibody, and the main compositions of their heavy and light chains are designed as shown in Table 1 below. Table 1: compositional design of the heavy and light chains of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, P1D7V07, P1D7V08

In table 1 above:

(1) the lower right hand corner is labeled "V" and refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. The corresponding heavy or light chain, not labeled "V", is the full length comprising the constant region. The variable region or the full-length amino acid sequence and the nucleic acid sequence encoding the same are referred to the corresponding sequences described in the above examples.

(2) The amino acid sequence of Linker1 was (GGGGS)4 ((nucleotide sequence SEQ ID NO: 80, amino acid sequence SEQ ID NO: 79), and the amino acid sequence of Linker2 was (GGGGS)3 ((nucleotide sequence SEQ ID NO: 82, amino acid sequence SEQ ID NO: 81).

2. Expression and purification of antibodies

The heavy chain cDNA sequence and the light chain cDNA sequence of P1D7V01 were cloned into pUC57simple (supplied by Kinsley) vectors to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.

The plasmids pUC57simple-VP101H and pUC57simple VP101L were digested with enzymes (HindIII & EcoRI), the heavy chain and light chain recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmid was extracted and transfected into 293F cell. After 7 days of cell culture, the culture fluid was subjected to high speed centrifugation, supernatant was concentrated, and then applied to a HiTrap MabSelect Sure column, proteins were eluted in one step using an Elution Buffer, and the target sample was recovered and changed to PBS.

Purified antibodies P1D7V02R, P1D7V03, P1D7V04R, P1D7V07, P1D7V08 were obtained according to the expression and purification method of P1D7V01 described above.

Example 8: ELISA method for determining the binding Activity of anti-CD 73-anti-PD-1 bispecific antibody to antigen

ELISA method for determining the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R to the antigen PD-1-mFc, respectively. The specific method comprises the following steps:

the PD-1-mFc, 0.5 mug/ml coated ELISA plate is placed at 4 ℃ for incubation overnight, then the antigen coated ELISA plate is washed by PBST for 1 time, and then the ELISA plate is blocked for 2 hours at 37 ℃ by using 1% BSA PBS solution as blocking solution. After the enzyme label plate is sealed, washing the plate 3 times by using PBST, adding the antibody diluted by the PBST solution in a gradient manner (the antibody dilution gradient is detailed in table 2), incubating the enzyme label plate added with the antibody to be detected for 30 minutes at 37 ℃, and washing the plate 3 times by using PBST after the incubation is finished. Adding 1: a5000-ratio dilution of the HR-labeled goat anti-human IgG (H + L) (Jackson, cat # 109. 035. sup. 088) secondary antibody working solution was incubated at 37 ℃ for 30 minutes. After the incubation, the plate was washed 4 times with PBST, then TMB (Neogen, 308177) was added and developed for 5min in the dark, and a stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software.

The results are shown in table 2 and fig. 1. As can be seen from the figure, P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R can effectively bind to the antigen PD-1-mFc, the binding efficiency is in a dose-dependent relationship, the absorbance intensity of each dose is shown in Table 2, the binding efficiency EC50 of the antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab (as a control) is respectively 0.078nM, 0.075nM and 0.089nM, 0.033nM and 0.051nM by performing absorbance quantitative analysis on the bound antibodies and curve simulation calculation.

The above experimental results show that under the same experimental conditions, the activity of binding PD-1-mFc of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R is equivalent to that of the homotarget positive drugs 14C12H1L1 and Nivolumab, and suggest that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have the activity of effectively binding PD-1-mFc.

Table 2: ELISA detected P1D7V01, P1D7V02R, P1D7V03, P1D7V 04R. Binding of 14C12H1L1, Nivolumab to PD-1-mFc

ELISA method for determining the binding activity of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to human antigen NT5E-Biotin

Streptavidin, 2. mu.g/ml coated microplate was incubated overnight at 4 ℃. After incubation, the streptavidin-coated ELISA plate was washed 1 time with PBST, and 1% BSA in PBS was used as the ELISA plate blocking solution to block the ELISA plate for 2 hours at 37 ℃. After the end of the enzyme plate blocking, the plate was washed 3 times with PBST. Then 0.5. mu.g/ml of antigen human NT5E-Biotin is added and incubated at 37 ℃ for 30 minutes, and then the plate is washed 3 times with PBST. Adding antibody diluted by PBST solution in a gradient manner (the antibody dilution gradient is detailed in table 3) into the wells of the ELISA plate, incubating the ELISA plate added with the antibody to be detected for 30 minutes at 37 ℃, and washing the plate 3 times by using PBST after the incubation is finished. After washing the plate, a working solution of HR-labeled goat anti-human IgG (H + L) (Jackson, cat # 109. sup. 035. sup. 088) secondary antibody diluted 1: 5000 was added and incubated at 37 ℃ for 30 minutes. After the incubation, the plate was washed 4 times with PBST, then TMB (Neogen, 308177) was added and developed for 5min in the dark, and a stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software.

The results of the measurements are shown in Table 3 and FIG. 2. As can be seen from the figure, P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R can effectively bind to the antigen human NT5E-Biotin, and the binding efficiency is dose-dependent (the absorbance intensity of each dose is shown in Table 3). The binding efficiencies EC50 of the antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3 and MEDI9447 (as control antibodies) were 0.063nM, 0.230nM, 0.068nM and 0.439nM, 0.045nM, 0.042nM, respectively, as calculated by curve simulation by absorbance quantification of bound antibodies.

The above experimental results show that under the same experimental conditions, the activity of bispecific antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R for binding human NT5E-Biotin is equivalent to that of targeted positive drugs 19F3H2L3 and MEDI9447, suggesting that P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have the activity of effectively binding human NT 5E-Biotin.

Table 3: ELISA for detecting the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3, MEDI9447 and human NT5E-Biotin

Example 9: competitive ELISA method for respectively determining activity of antibody anti-CD 73-anti-PD-1 bispecific antibody and human PD-L1-mFc for competing binding with human PD-1-mFc-Biotin

Human PD-L1-mFc (PD-L1 Genbank ID: NP-054862.1, mFc SEQ ID NO: 143) was coated on an enzyme plate at 2. mu.g/mL and incubated overnight at 4 ℃. After the incubation, the plate was blocked with 1% BSA in PBS for 2 hours at 37 deg.C, and after the blocking, the plate was washed three times and blotted dry. At 10 μ g/mL as starting concentration on dilution plates according to 1: 3 to 7 concentration, setting blank control, adding equal volume of 0.3 mug/mL human PD-1-mFc-Biotin solution, mixing evenly, and incubating for 20 minutes at room temperature. Then the mixed solution after reaction is added to the coated enzyme label plate and incubated for 30 minutes at 37 ℃. After the incubation, the plates were washed three times with PBST and patted dry, SA-HRP (KPL, 14-30-00) working solution was added, and incubated at 37 ℃ for 30 minutes. After incubation, the plate was washed dry for four times, then TMB (Neogen, 308177) was added and developed for 5min in the dark, and stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, selecting the wavelength of light with 450nm to read the OD value of each hole of the ELISA plate, and analyzing and processing the data by using SoftMax Pro 6.2.1 software.

The results of the detection are shown in FIG. 3. The OD values for each dose are shown in Table 4. Bound EC50 was obtained by performing absorbance intensity quantification of bound antibody and the curve simulates the binding efficiency of the antibody (table 4).

The results show that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab (as controls) can effectively block the binding of antigen human PD-1-mFc-Biotin and its receptor human PD-L1-mFc, and the blocking efficiency presents a dose-dependent relationship, and that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab block the binding of human PD-1-mFc-Biotin and its ligand human PD-L1-mFc with EC50 of 1.115nM, 1.329nM, 1.154nM, 1.339nM, 1.459nM and 1.698nM, respectively.

Table 4: activity detection results of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab which compete with human PD-L1-mFc to bind to human PD-1-mFc-Biotin

Example 10: determination of kinetic parameters of the binding of anti-CD 73-anti-PD-1 bispecific antibody to the antigen human PD-1-mFc Using Fortebio molecular interactor

The sample dilution buffer was PBST, 0.1% BSA, pH 7.4. The antibody was immobilized on the AHC sensor at a concentration of 5. mu.g/mL, with an immobilization height of about 0.4nM, the sensor was equilibrated in buffer for 60s, the antibody immobilized on the sensor bound to the antigen PD-1-mFc at an antigen concentration of 0.6-50nM (three-fold dilution) for 120s, and the protein dissociated in buffer for 180 s. The detection temperature was 37 degrees, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000 rpm. The data were analyzed by fitting a 1: 1 model to obtain affinity constants.

The results of the determination of affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and Nivolumab (as a control antibody) and human PD-1-mFc are shown in Table 5, and the results of the determination are shown in FIG. 4, FIG. 5 and FIG. 6. The affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and Nivolumab to human PD-1-mFc are 1.76E-10M, 1.64E-10M and 2.32E-10M in this order. The above experimental results show that: the binding capacity of P1D7V01 to 14C12H1L1 and Nivolumab is equivalent, and the fact that the humanized antibody P1D7V01 has stronger binding capacity to human PD-1-mFc is suggested.

Table 5: determination of P1D7V01, 14C12H1L1, Nivolumab, affinity constant for PD-1-mFc

Antibodies to be tested

KD(M)

kon(1/Ms)

SE(kon)

kdis(1/s)

S E(kdis)

Rmax(nm)

P1D7V01

1.76E-10

4.19E+05

1.52E+04

7.37E-05

3.12E-05

0.13-0.17

14C12H1L1

1.64E-10

4.55E+05

1.61E+04

7.47E-05

2.98E-05

0.24-0.28

Nivolumab

2.32E-10

5.85E+05

2.03E+04

1.36E-04

3.47E-05

0.02-0.14

K_DIs the affinity constant; k_D＝kdis/kon

Example 11: kinetic parameters of the binding of the anti-CD 73-anti-PD-1 bispecific antibody to the antigen human NT5E (1-552) -his were determined using a Fortebio molecular interactor.

The sample dilution buffer was PBST, pH 7.4. The antibody was immobilized on the Protein A sensor at a concentration of 5. mu.g/mL for 15s, the sensor was equilibrated in buffer for 120s, the antibody immobilized on the sensor bound to the antigen human NT5E (1-552) -his at an antigen concentration of 3.125-200nM (two-fold dilution) for 120s, and the Protein was dissociated in buffer for 600 s. The sensor was regenerated using 10mM Gly, pH1.5 solution. The detection temperature was 37 degrees, the detection frequency was 0.6Hz, and the sample plate vibration rate was 1000 rpm. The data are fit analyzed by a 1: 1 model to obtain an affinity constant

The results of the determination of affinity constants of humanized antibodies P1D7V01 and MEDI9447 (as a control antibody) and human NT5E (1-552) -his are shown in Table 6, and the results are shown in FIGS. 7and 8. The affinity constants of humanized antibodies P1D7V01 and MEDI9447 for human NT5E (1-552) -his were, in order, 2.29E-10M, 1.04E-10M.

The above experimental results show that: the binding capacity of P1D7V01 is equivalent to that of MEDI9447, suggesting that the humanized antibody P1D7V01 has stronger binding capacity with human NT5E (1-552) -his.

Table 6: determination of affinity constants of P1D7V01, MEDI9447 and human NT5E (1-552) -his

Antibodies to be tested

KD(M)

kon(1/Ms)

S E(kon)

kdis(1/s)

S E(kdis)

Rmax(nm)

P1D7V01

2.29E-10

1.81E+05

2.30E+03

4.13E-05

4.54E-06

0.47-0.57

MEDI 9447

1.04E-10

2.34E+05

3.20E+03

2.44E-05

5.02E-06

0.59-0.82

K_DIs the affinity constant; k_D＝kdis/kon

Example 12: FACS detection of the binding Activity of an anti-CD 73-anti-PD-1 bispecific antibody

FACS detection of the binding Activity of an anti-CD 73-anti-PD-1 bispecific antibody to PD-1 on the surface of 293T-PD1 membranes

293T-PD1 cells in logarithmic growth phase were collected at 3X10⁵Transferring the cells to a 1.5ml centrifuge tube per cell/tube, adding 500 μ L PBSA, centrifuging at 5600rpm for 5min, and removing the supernatant; adding 100 μ L of antibody diluted with PBSA (final concentrations of 100, 33.33, 11.11, 3.7, 1.23, 0.41, 0.14, 0.05nM), mixing gently, and incubating on ice for 1 h; adding 500 μ L PBSA, centrifuging at 5600rpm for 5min, and removing supernatant; adding a 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, cat # 109-; adding 500 μ LPBSA, centrifuging at 5600rpm for 5min, and removing supernatant; the cell pellet was resuspended by adding 200. mu.L of LPBSA and transferred to a flow tube for FACSCalibur detection.

The experimental results are shown in table 7and fig. 9, and P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R all bind specifically to the membrane surface PD-1 of 293T-PD1 and are dose-dependent, and all are stronger than 14C12H1L1 compared with PD1 single-target control antibody 14C12H1L 1.

Under the same experimental conditions, EC50 bound by P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 293T-PD1 are 1.000nM, 1.075nM, 1.377nM, 1.57nM, and EC50 bound by 14C12H1L1 and 293T-PD1 is 2.111nM, respectively.

The above experimental results show that the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 293T-PD1 is better than that of PD1 single-target control antibody 14C12H1L1 under the same experimental conditions, and indicate that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have the activity of effectively binding to PD-1 on the membrane surface of 293T-PD 1.

Table 7: FACS detection of the binding Activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on the cell surface of 293T-PD1

Concentration (nM)	0.05	0.14	0.41	1.23	3.70	11.11	33.33	100.00	EC50
										14C12H1L1	35.69	57.70	129.42	437.62	974.97	1384.91	1174.75	1500.20	2.111
P1D7V01	26.65	58.65	150.57	307.19	530.77	577.76	479.34	531.15	1.000
										P1D7V02R	23.44	42.98	108.44	217.56	404.33	426.06	405.29	301.40	1.075
P1D7V03	24.05	49.21	118.03	289.08	384.62	459.40	808.38	343.48	1.377
										P1D7V04R	24.79	52.43	112.56	288.79	466.72	1284.73	400.33	360.79	1.57.0

FACS detection of the binding Activity of an anti-CD 73-anti-PD-1 bispecific antibody to the CD73 on the surface of the MDA-MB-231 Membrane

Conventional trypsinization of log phase MDA-MB-231 cells at 3X10⁵Transferring the cells to a 1.5ml centrifuge tube per cell/tube, adding 500 μ L PBSA, centrifuging at 5600rpm for 5min, and removing the supernatant; adding 100 μ L of antibody diluted with PBSA (final concentrations of 100, 33.33, 11.11, 3.7, 1.23, 0.41, 0.14, 0.05nM), mixing gently, and incubating on ice for 1 h; adding 500 μ LPBSA, centrifuging at 5600rpm for 5min, and removing supernatant; adding a 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, cat # 109-; adding 500 μ LPBSA, centrifuging at 5600rpm for 5min, and removing supernatant; the cell pellet was resuspended by adding 200. mu.L of LPBSA and transferred to a flow tube for FACSCalibur detection.

The experimental results are shown in Table 8 and FIG. 10, and the binding activity with CD73 on the membrane surface of MDA-MB-231, P1D7V01 and P1D7V03 are superior to 19F3H2L3, wherein P1D7V01 is superior to that of the co-target positive drug MEDI 9447. Under the same experimental conditions, the EC50 of P1D7V01 and P1D7V03 for binding with MDA-MB-231 membrane surface CD73 is 1.384nM and 2.009nM, respectively, and the EC50 of MEDI9447 and 19F3H2L3 for binding with MDA-MB-231 membrane surface CD73 is 1.589nM and 2.773nM, respectively.

The above experimental results show that P1D7V01, P1D7V03, 19F3H2L3 and the co-target positive drug MEDI9447 can be in dose-dependent specific binding with MDA-MB-231 membrane surface CD73, the binding activity of P1D7V01 and P1D7V03 is better than that of 19F3H2L3, and the binding activity of P1D7V01 is better than that of the co-target positive drug MEDI 9447. The P1D7V01 and P1D7V03 are suggested to have the activity of effectively binding CD73 on the surface of the MDA-MB-231 membrane.

Table 8: FACS measures the binding activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the cell surface of MDA-MB-231.

Concentration (nM)	0.05	0.14	0.41	1.23	3.70	11.11	33.33	100.00	EC50
										MEDI9447	32.59	64.38	157.75	388.51	697.80	829.29	930.26	878.04	1.589
19F3H2L3	25.95	45.13	102.56	189.07	466.87	658.72	843.09	639.57	2.773
										P1D7V01	23.37	38.02	76.84	271.72	571.56	560.81	525.91	705.50	1.384
P1D7V03	16.96	44.05	106.39	233.32	457.61	525.37	576.19	677.66	2.009

Example 13: detection of anti-CD 73-anti-PD-1 bispecific antibody for inhibiting activity of CD73 enzyme on cell membrane surface

1. Detection of inhibition of anti-CD 73-anti-PD-1 bispecific antibody on activity of CD73 enzyme on surface of MDA-MB-231 cell membrane

The experimental procedure was as follows: taking the good-state MDA-MB-231 cells in the logarithmic phase, and re-suspending and counting the cells by using a serum-free RPMI-1640 culture solution; MDA-MB-231 cells were seeded into 96-well plates, 2X10⁴Individual cells/100 μ L/well; carrying out gradient dilution on the antibody by serum-free RPMI-1640 culture solution according to 2.5 times; adding 50. mu.L of the antibody to a 96-well plate, incubating at 37 ℃ for 1 hour, and after 1 hour, adding 50. mu.L of 1200. mu.M RPMI-1640 diluted AMP (TCL, cat # A0157) to each well; after 3 hours, 25. mu.L of the cell culture supernatant was transferred to a new 96-well plate, and 25. mu.L of 100. mu.M ATP (TCL, cat # A0158) was added to each well; mu.L of CTG (CellTiterGlo, Promega, cat # G8641) developing solution was added to each well for development, and relative fluorescence intensity RLU was read in a multi-labeled microplate detector (PerkinElmer 2140-.

As shown in fig. 11, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R were comparable to the AMP content of the positive control MEDI9447 and were concentration-dependent.

The above experimental results show that the added AMP is catalyzed by the enzyme activity of CD73 on the cell surface of MDA-MB-231 to be converted into adenosine A in the absence of antibody, and after the antibody is added, the enzyme activity is reduced by the combination of the antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R with CD73, so that the AMP is not converted into adenosine A. The antibody is prompted to effectively inhibit the enzyme activity reaction in a non-substrate competition mode, and the production of adenosine is reduced.

2. Detection of inhibition of anti-CD 73-anti-PD-1 bispecific antibody on enzymatic activity of U87-MG cell membrane surface CD73

Taking well-conditioned log-phase U87-MG cells, and resuspending and counting the cells by using serum-free RPMI-1640 culture solution; seeding U87-MG cells into 96-well plates, 2X10⁴Individual cells/100 μ L/well; diluting the antibody with serum-free RPMI-1640 culture solution in a gradient manner by 2.5 times, adding the antibody into a 96-well plate, incubating at 37 ℃ for 1 hour, wherein each well contains 50 mu L of antibody; after 1 hour, 50. mu.L of 1200uM RPMI-1640 diluted AMP was added per well; after 3 hours, 25. mu.L of cell culture supernatant was taken and transferred to a new 96-well plate, and 25. mu.L of 100. mu.M ATP was added to each well; 50 μ l of CTG (CellTiterGlo) developing solution was added to each well, and after development, relative fluorescence intensity RLU was read in a multi-labeled microplate detector (PerkinElmer 2140-.

As shown in fig. 12, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R were comparable to the AMP content of the positive control MEDI9447 and were concentration-dependent.

The above experimental results show that the added AMP is converted into adenosine A by the enzyme activity of CD73 on the surface of U87-MG cells in the absence of an antibody, and the enzyme catalytic function is reduced by the binding of the antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to CD73 after the addition of the antibody, so that the AMP is not converted into adenosine A. The antibody is prompted to effectively inhibit the enzyme activity reaction in a non-substrate competition mode, and the production of adenosine is reduced.

Example 14: mixed lymphocyte reaction MLR detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting IFN-gamma and IL-2 secretion

1. Detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting Raji-PDL1 mixed lymph reaction system to secrete IFN-gamma

Normal subculturing of Raji-PDL1 cells; resuscitate PBMC, incubate with 10mL 1640 complete medium, 0.5. mu.g/mL SEB (Denocatek, cat # S010201) for two days; Raji-PDL1 cells were treated with 25. mu.g/mL of MMC (Sigma, cat # M4287) and placed in an incubator at 37 ℃ for 1 hour; collecting PBMC and Raji-PDL1 cells treated by MMC for 1 hour 2 days after SEB stimulation, washing with PBS twice, performing complete culture basis suspension counting, and adding to a U-shaped 96-well plate, 100000 cells/well respectively; adding the antibody according to the experimental design, and culturing in an incubator for 3 days; after 3 days, cell culture supernatants were collected and subjected to IFN-. gamma.detection by ELISA.

As shown in FIG. 13, human PBMC and Raji-PDL1 cells have a significant promoting effect on secretion of IFN-gamma from PBMC after mixed culture, and the antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have activity equivalent to that of the master PD-1 single-target antibody 14C12H1L1 in terms of the level of activity of promoting secretion of IFN-gamma, which can be significantly induced by adding antibodies into a mixed culture system.

2. Detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting IL-2 secretion of Raji-PDL1 mixed lymph reaction system

Normal subculturing of Raji-PDL1 cells; resuscitate PBMC, incubate with 10mL 1640 complete medium, SEB (0.5. mu.g/mL) stimulate for two days; Raji-PDL1 cells were treated with 25. mu.g/mL MMC and placed in an incubator at 37 ℃ for 1 hour; collecting PBMC after 2 days of SEB stimulation and Raji-PDL1 cells treated by MMC for 1 hour, washing twice with PBS, performing complete culture basis suspension counting, and adding to a U-shaped 96-well plate with 100000 cells/well; adding antibody according to experimental design, and culturing for 3 days; cell culture supernatants were collected and tested for IL-2 by ELISA.

As shown in fig. 14, human PBMC and raini-PDL 1 cells have certain promotion effect on the secretion of IL-2 from PBMC after mixed culture, the antibody added in the mixed culture system can significantly induce PBMC to further secrete IL-2, and has significant dose-dependent relationship, and the level of IL-2 secretion activity is slightly lower than that of the parent PD-1 single-target antibody 14C12H1L1 in terms of bifunctional antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R at low concentration, while the high concentration is equivalent to the high concentration, and compared with the PD1 target positive control drug Nivolumab, P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have better IL-2 secretion potential at three different antibody concentration levels.

3. Detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting IFN-gamma secretion of DC mixed lymph reaction system

Separating normal human peripheral blood PBMC, re-suspending the complete culture medium, inoculating the PBMC to a culture dish, placing the culture dish in an incubator for overnight culture, collecting and removing the suspended PBMC, washing the adherent cells at the bottom of the dish by PBS buffer solution, and then carrying out DC maturation induction: adding 10mL of RPMI1640 complete culture medium containing GM-CSF and IL-4 into each dish, wherein the concentrations of GM-CSF and IL-4 are both 1000U/mL, and placing the dishes in a 5% carbon dioxide incubator at 37 ℃ for culturing for three days; changing the liquid by half, supplementing 1000U/mL of GM-CSF and IL-4 respectively, placing in a 5% carbon dioxide incubator at 37 ℃, and continuously culturing for three days; after three days, half amount of the solution is changed again, GM-CSF and IL-4 are added, 1000U/mL and 100U/mL of TNF-alpha are added respectively, and the culture is continued for two days; PBMCs from additional donors were freshly isolated, plated into 96-well plates, 100000 cells/well after cell counting; collecting induced mature DC cells, washing once with a complete culture medium, counting the cells, and inoculating to a 96-well plate containing PBMC, 10000 cells/well; adding the antibody according to the experimental design, uniformly mixing, and then putting into a 5% carbon dioxide incubator for co-culture for 5 days at 37 ℃; after 5 days, cell culture supernatants were collected and IFN-. gamma.was quantitatively determined by ELISA.

Results as shown in fig. 15, mixed culture of DC and PBMC significantly promoted secretion of IFN- γ compared to DC alone or PBMC culture; compared with an isotype control, the IFN-gamma secretion level is further obviously improved by adding the anti-CD 73-anti-PD-1 bispecific antibody on the basis of mixed culture of the DC and the PBMC.

Separating normal human peripheral blood PBMC (peripheral blood mononuclear cell) by detecting the bioactivity of an anti-CD 73-anti-PD-1 bispecific antibody promoting DC mixed lymph reaction system to secrete IL-2, resuspending a complete culture medium, inoculating the complete culture medium in a culture dish, placing the culture dish in an incubator for overnight culture, collecting and removing suspended PBMC, washing adherent cells at the bottom of the dish by using a PBS (phosphate buffer solution) and then carrying out DC maturation induction; adding 10mL of RPMI1640 complete culture medium containing GM-CSF and IL-4 into each dish, wherein the concentrations of GM-CSF and IL-4 are both 2000U/mL, and placing the dishes in a 5% carbon dioxide incubator at 37 ℃ for culturing for three days; half the amount of the solution was changed, 50ng/mL IFN-. gamma.and 100ng/mL LPS were added, and the culture was continued for two days; resuscitating PBMCs from additional donors, plating into 96-well plates, 100000 cells/well after cell counting; collecting induced mature DC cells, washing once with a complete culture medium, counting the cells, and inoculating to a 96-well plate containing PBMC, 10000 cells/well; adding the antibody according to the experimental design, uniformly mixing, and then putting into a 5% carbon dioxide incubator for co-culture for 5 days at 37 ℃; after 5 days, cell culture supernatants were collected and assayed for IL-2 by ELISA. Results as shown in fig. 16, mixed culture of DC and PBMC significantly promoted secretion of IL-2 compared to DC or PBMC culture alone; compared with an isotype control, the IL-2 secretion level is further obviously improved by adding the anti-CD 73-anti-PD-1 bispecific antibody on the basis of mixed culture of DC and PBMC.

Example 15: preparation of anti-PD-1/CD 73 bispecific antibody NTPDV1, NTPDV2, NTPDV3, NTPDV4

The structural pattern of bispecific antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 belongs to the Morrison pattern (IgG-scFv), i.e. the scFv fragments of one IgG antibody are linked at the C-terminus of both heavy chains of the other antibody by a linking fragment, the design composition of their heavy and light chains is as in table 9 below.

NTPDV1, NTPDV2, NTPDV3, NTPDV4 are also written herein and in chinese patent application No. 202110270671.X as NTPDV1(hG1TM), NTPDV2(hG1TM), NTPDV3(hG1TM), NTPDV4(hG1TM), because the constant region of the immunoglobulin part thereof introduces amino acid mutations to eliminate its binding activity with Fc γ R.

Table 9: sequence design of NTPDV1, NTPDV2, NTPDV3 and NTPDV4

In table 9 above:

the lower right hand corner is labeled "V" and refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. The corresponding heavy or light chain, not labeled "V", is the full length comprising the constant region. The variable region or the full-length amino acid sequence and the nucleic acid sequence encoding the same are referred to the corresponding sequences described in the above examples.

The amino acid sequence of Linker1 is (GGGGS) structure repeated 4 times, namely (GGGGS)4 or (G4S)4 (nucleotide sequence SEQ ID NO: 80, amino acid sequence SEQ ID NO: 79);

the amino acid sequence of Linker2 is (GGGGS) structure repeated 3 times, i.e., (GGGGS)4 or (G4S)3 (nucleotide sequence SEQ ID NO: 82, amino acid sequence SEQ ID NO: 81).

The 3 CDR sequences of light chain 19F3L2 and 19F3L3 in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3, NTPDV4 were identical to the light chain CDR sequence in 19F 3.

The 3 CDR sequences of 19F3H2(hG1TM) in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the heavy chain CDR sequences in 19F 3.

The CDR sequences of 14C12H1V-Linker2-14C12L1V and 14C12H1V-Linker1-14C12L1V of the scFv portion in NTPDV1, NTPDV2, NTPDV3, NTPDV4 are identical to the heavy and light chain CDRs in 14C 12.

The amino acid sequences of the heavy chains of NTPDV2 and NTPDV4 are identical and are marked as NTPDH2/4(SEQ ID NO: 83), and the nucleotide sequences of the heavy chains of NTPDV2 and NTPDV4 are SEQ ID NO: 84.

the amino acid sequences of the heavy chains of NTPDV1 and NTPDV3 are identical and are marked as NTPDH1/3(SEQ ID NO: 85), and the nucleotide sequences of the heavy chains of NTPDV1 and NTPDV3 are SEQ ID NO: 86.

the 3 CDR sequences of 19F3L3 and 19F3L2 in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3, NTPDV4 were identical to the three CDRs of the light chain of antibody 19F 3.

The amino acid sequence of the light chain 19F3L3 of the immunoglobulin fraction in NTPDV1, NTPDV2 was identical to the light chain sequence of antibody 19F3H2L3(G1M) (SEQ ID NO: 28), and the nucleotide sequence of the light chain 19F3L3 of the immunoglobulin fraction in NTPDV1, NTPDV2 was SEQ ID NO: 27. the amino acid sequence of light chain 19F3L2 of the immunoglobulin part in NTPDV3, NTPDV4 is SEQ ID NO: 96) and the nucleotide sequence of the light chain 19F3L2 of the immunoglobulin part in NTPDV3 and NTPDV4 is SEQ ID NO: 100.

Expression and purification of antibodies

Heavy chain cDNA of NTPDV1 and NTPDV3, heavy chain cDNA sequences of NTPDV2 and NTPDV4, respectively, and cDNA sequences of light chains thereof were cloned into pUC57simple (supplied by Kinsrui Co.) vectors to obtain pUC57simple-NTPDH2/4, pUC57simple-NTPDH1/3, and pUC57simple-19F3L3 plasmids and pUC57simple-19F3L2 plasmids, respectively.

Plasmids pUC57simple-NTPDH2/4 and pUC57simple-19F3L3, pUC57simple-NTPDH2/4 and pUC57simple-19F3L2, pUC57simple-NTPDH1/3 and pUC57simple-19F3L3, pUC57simple-NTPDH1/3 and pUC57simple-19F3L2 were digested respectively, and the heavy chain and light chain recovered by electrophoresis were subcloned into pcDNA3.1 to obtain pcDNA3.1-NTPDH2/4 and pcDNA3.1-19F3L3, pcDNA3.1-NTPDH2/4 and pcDNA3.1-19F3L2, pcDNA3.1-NTPDH 1/4 and pcDNA3.84-19F 3L3, pcDNA3.1-NTDNA3/19 and pUC 57-19F 3L2, and recombinant plasmids pCDNA8945-893.7 were transfected to obtain pcDNA3.8, respectively. After 7 days of cell culture, the culture fluid was subjected to high speed centrifugation, supernatant was concentrated, and then applied to a HiTrap MabSelect Sure column, proteins were eluted in one step using an Elution Buffer, and the target sample was recovered and changed to PBS.

Purified antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 were obtained according to the expression and purification methods mentioned in the previous preparation examples.

Example 16: ELISA method for determining the binding Activity of anti-CD 73-anti-PD-1 bispecific antibody to antigen

The binding activity of NTPDV1, NTPDV2, NTPDV3, NTPDV4 to the antigen PD-1-mFc was determined by ELISA method, respectively.

The ELISA plate coated with PD-1-mFc (0.5. mu.g/ml) was incubated overnight at 4 ℃, then the antigen-coated ELISA plate was washed 1 time with PBST, and then blocked for 2 hours at 37 ℃ using 1% BSA in PBS as a blocking solution. After the enzyme label plate is sealed, washing the plate 3 times by using PBST, adding the antibody diluted by the PBST solution in a gradient manner (the antibody dilution gradient is detailed in table 2), incubating the enzyme label plate added with the antibody to be detected for 30 minutes at 37 ℃, and washing the plate 3 times by using PBST after the incubation is finished. Adding 1: a5000-ratio dilution of the HR-labeled goat anti-human IgG Fc (Jackson, cat # 109-035-098) secondary antibody working solution was incubated at 37 ℃ for 30 minutes. After the incubation, the plate was washed 4 times with PBST, then TMB (Neogen, 308177) was added and developed for 8min in the dark, and a stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software.

The results are shown in Table 10. It is known that NTPDV1, NTPDV2, NTPDV3, NTPDV4 can bind to antigen PD-1-mFc effectively, and the binding efficiency is dose-dependent, the absorbance intensity of each dose is shown in table 10, and the binding efficiency EC50 of antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1(hG1TM) (as a control) obtained by quantitative analysis of the absorbance of the bound antibodies by curve simulation is 0.101nM, 0.119nM, and 0.110nM, 0.123nM, 0.031nM, respectively.

The above experimental results show that under the same experimental conditions, the binding activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to PD-1-mFc is equivalent to that of the same-target positive drug 14C12H1L1(hG1TM), suggesting that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have the effective binding activity to PD-1-mFc.

Table 10: ELISA detection of binding of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1(hG1TM) to PD-1-mFc

ELISA method for determining the binding activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to human antigen NT5E-Biotin

Streptavidin SA (2. mu.g/ml) was coated on the microplate and incubated overnight at 4 ℃. After incubation, the streptavidin-coated ELISA plate was washed 1 time with PBST, and 1% BSA in PBS was used as the ELISA plate blocking solution to block the ELISA plate for 2 hours at 37 ℃. After the end of the enzyme plate blocking, the plate was washed 3 times with PBST. Then 0.5. mu.g/ml of antigen human NT5E-Biotin is added and incubated at 37 ℃ for 30 minutes, and then the plate is washed 3 times with PBST. The antibody diluted by the PBST solution in a gradient manner is added into the hole of the ELISA plate (the antibody dilution gradient is detailed in Table 11), the ELISA plate added with the antibody to be detected is placed at 37 ℃ for incubation for 30 minutes, and the plate is washed by the PBST for 3 times after the incubation is finished. After washing the plate, a working solution of HR-labeled goat anti-human IgG Fc (Jackson, cat # 109-. After the incubation, the plate was washed 4 times with PBST, then TMB (Neogen, 308177) was added and developed for 7min in the dark, and a stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software.

The results are shown in Table 11. It is known that NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3(hG1M) can effectively bind to human NT5E-Biotin, and the binding efficiency is dose-dependent (the absorbance intensity of each dose is shown in table 11). The binding efficiencies EC50 of the antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3(hG1M) (as control antibodies) were calculated by curve simulation by absorbance quantification of the bound antibodies as 0.079nM, 0.082nM and 0.084nM, 0.077nM, 0.029nM, respectively.

The above experimental results show that under the same experimental conditions, the activity of bispecific antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 for binding human NT5E-Biotin is equivalent to that of the same-target positive drug 19F3H2L3(hG1M), suggesting that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have the activity of effectively binding human NT 5E-Biotin.

Table 11: ELISA detection of the binding of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3(hG1M) to human NT5E-Biotin

Example 17: competitive ELISA method for respectively determining activity of anti-CD 73-anti-PD-1 bispecific antibody and human PD-L1-mFc for competing binding to human PD-1-mFc-Biotin

Human PD-L1-mFc (PD-L1 Genbank ID: NP-054862.1, mFc SEQ ID NO: 89) was coated on an ELISA plate at 2. mu.g/mL and incubated overnight at 4 ℃. After the incubation, the enzyme-labeled plate is blocked by 1% BSA PBS solution at 37 ℃ for 2 hours, and after the blocking is finished, the plate is washed once and is dried by beating. At 10 μ g/mL as starting concentration on dilution plates according to 1: 3 to 7 concentration, setting blank control, adding equal volume of 0.3 mug/mL human PD-1-mFc-Biotin solution, mixing evenly, and incubating for 10 minutes at room temperature. Then the mixed solution after reaction is added to the coated enzyme label plate and incubated for 30 minutes at 37 ℃. After the incubation, the plates were washed three times with PBST and patted dry, SA-HRP (KPL, 14-30-00) working solution was added, and incubated at 37 ℃ for 30 minutes. After incubation, the plate was washed dry for four times, then TMB (Neogen, 308177) was added and developed for 5min in the dark, and stop solution was added to terminate the color development reaction. Immediately putting the ELISA plate into an ELISA reader, selecting the wavelength of light with 450nm to read the OD value of each hole of the ELISA plate, and analyzing and processing the data by using SoftMax Pro 6.2.1 software.

The OD values for each dose are shown in Table 12. Bound EC50 was obtained by performing absorbance intensity quantification of bound antibody and the curve simulates the binding efficiency of the antibody (table 12).

The results show that NTPDV1, NTPDV2, NTPDV3, NTPDV 4and 14C12H1L1(hG1TM) (as a control) can effectively block the binding of antigen human PD-1-mFc-Biotin and receptor human PD-L1-mFc thereof, and the blocking efficiency shows a dose-dependent relationship, and the EC50 of the combination of NTPDV1, NTPDV2, NTPDV3, NTPDV 4and 14C12H1L1(hG1TM) blocking the binding of human PD-1-mFc-Biotin and ligand human PD-L1-mFc thereof is 2.249nM, 2.253nM, 2.332nM, 2.398nM, 2.216nM and 2.231nM respectively.

Table 12: activity detection results of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1(hG1TM) and human PD-L1-mFc competitive binding human PD-1-mFc-Biotin

Example 18: determination of kinetic parameters of the binding of anti-CD 73-anti-PD-1 bispecific antibody to the antigen human PD-1-mFc Using Fortebio molecular interactor

The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. PD1-mFc was immobilized on the AMC sensor at a concentration of 5. mu.g/mL at a height of about 0.1nM (time 60s), the sensor was equilibrated in buffer for 60s, and PD1-mFc immobilized on the sensor was bound to the antibody at a concentration of 0.62-50nM (triple dilution) for 120s, and the protein was dissociated in buffer for 300 s. The detection temperature was 30 degrees, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000 rpm. The data were analyzed by fitting a 1: 1 model to obtain affinity constants.

The results of determining the affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV 4and Nivolumab (as a control antibody) and human PD-1-mFc are shown in table 13, and the results of detection are shown in fig. 19, fig. 20, fig. 21, fig. 22 and fig. 18. The affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV 4and Nivolumab with human PD-1-mFc are 1.40E-10M, 7.39E-11M, 1.25E-10M, 1.13E-11M and 2.26E-10M in sequence. The above experimental results show that: the binding capacity of NTPDV1, NTPDV3 and Nivolumama is equivalent, the binding capacity of NTPDV2 and NTPDV4 is superior to that of Nivolumama, and humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have stronger binding capacity with human PD-1-mFc.

Table 13: determination of affinity constants of 14C12H1L1(hG1TM), NTPDV1, NTPDV2, NTPDV3, NTPDV4, Nivolumab and PD-1-mFc

KD is the affinity constant; KD ═ kdis/kon

Example 19: kinetic parameters of the binding of the anti-CD 73-anti-PD-1 bispecific antibody to the antigen human NT5E (1-552) -his were determined using a Fortebio molecular interactor.

The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. HNT5E (1-552) -His was immobilized on the HIS1K sensor at a concentration of 5. mu.g/mL, at a height of about 0.4nM (time 50s), the sensor was equilibrated in buffer for 60s, and HNT5E (1-552) -His immobilized on the sensor was bound to the antibody at a concentration of 0.31-25nM (three-fold dilution) for 100s, and the protein was dissociated in buffer for 180 s. The detection temperature was 30 degrees, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000 rpm. The data were analyzed by fitting a 1: 1 model to obtain affinity constants.

The results of affinity constant measurement of humanized antibody 19F3H2L3(hG1M) (as a control antibody), NTPDV1, NTPDV2, NTPDV3, NTPDV 4and human NT5E (1-552) -his are shown in Table 14, and the results of detection are shown in FIGS. 24-27. The humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have affinity constants of 3.29E-11M, 2.88E-11M, 7.92E-11M and 5.77E-11M for human NT5E (1-552) -his in this order.

The above experimental results show that: the binding capacity of 19F3H2L3(hG1M), NTPDV1, NTPDV2, NTPDV3 and NTPDV4 is equivalent, and the humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have stronger binding capacity with human NT5E (1-552) -his.

Table 14: determination of affinity constants for 19F3H2L3(hG1M), NTPDV1, NTPDV2, NTPDV3, NTPDV 4and human NT5E (1-552) -his

K_DIs the affinity constant; k_D＝kdis/kon

Example 20: detection of anti-CD 73-anti-PD-1 bispecific antibody for inhibiting activity of U87-MG cell membrane surface CD73 enzyme

Taking good log phase U87-MG cells, resuspending and counting the cells by using serum-free RPMI-1640 culture solution; U87-MG cells were seeded into 96-well plates, 2.5X10⁴Individual cells/60 μ L/well; carrying out gradient dilution on the antibody by using serum-free RPMI-1640 culture solution according to the experimental design concentration, adding the antibody into a 96-well plate, incubating for 1 hour at 37 ℃ and 60 mu L per well; after 1 hour, 60 μ L of 600uM RPMI-1640 diluted AMP was added per well; taking 100 mu L of cell culture supernatant after 3 hours, transferring the cell culture supernatant to a new 96-well plate, adding 40 mu L of CTG (CellTiterGlo) color development liquid into each well, and placing the cell culture supernatant for 5min at room temperature in a dark place; after 5min, 10. mu.L of 300. mu.M ATP was added to each well, and after color development, relative fluorescence intensity RLU was read in a multi-labeled microplate detector (PerkinElmer 2140-.

The experimental results are shown in table 15 and fig. 28, where NTPDV2 is comparable to the AMP content of the positive control MEDI 9447.

The above experimental results show that the added AMP can be catalyzed by the enzyme activity of CD73 on the surface of U87-MG cells and converted into adenosine A without antibody, and after the antibody is added, the enzyme-catalyzed function of the AMP is reduced due to the combination of the NTPDV2 antibody and CD73, so that the AMP is not converted into adenosine A. The antibody is prompted to effectively inhibit the enzyme activity reaction in a non-substrate competition mode, and the production of adenosine is reduced.

TABLE 15 detection of the inhibition of the enzyme activity of U87-MG cell membrane surface CD73 by the anti-CD 73-anti-PD-1 bispecific antibody

Name of antibody	MEDI9447	19F3H2L3(hG1M)	NTPDV2
				IC50(nM)	0.1189	0.3647	0.5551

Example 21: mixed lymphocyte reaction MLR detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting IFN-gamma and IL-2 secretion

1. Detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting Raji-PDL1 mixed lymph reaction system to secrete IFN-gamma

Normal subculturing of Raji-PDL1 cells; resuscitate PBMC, incubate with 10mL 1640 complete medium, 0.5. mu.g/mL SEB (Denocatek, cat # S010201) for two days; Raji-PDL1 cells were treated with 25. mu.g/mL of MMC (mitomycin C, Stressmarkq, Cat: SIH-246, lot number: SM286474) and placed in an incubator at 37 ℃ for 1 hour; PBMC and MMC-treated Raji-PDL1 cells for 1 hour 2 days after SEB stimulation were collected, washed twice with PBS, counted in full culture medium by weight suspension, added to a U-shaped 96-well plate, 1X10, respectively⁵Individual cells/well; adding the antibody according to the experimental design, and culturing in an incubator for 3 days; after 3 days, cell culture supernatants were collected and subjected to IFN-. gamma.detection by ELISA.

As shown in FIG. 29, human PBMC and Raji-PDL1 cells have a significant effect on promoting IFN-gamma secretion of PBMC after mixed culture, and the NTPDV2 antibody has activity equivalent to that of the master PD-1 single-target antibody 14C12H1L1 in terms of the level of IFN-gamma secretion activity significantly induced by the PBMC when the antibody is added into a mixed culture system.

2. Detection of bioactivity of anti-CD 73-anti-PD-1 bispecific antibody for promoting IL-2 secretion of Raji-PDL1 mixed lymph reaction system

Normal subculturing of Raji-PDL1 cells; resuscitate PBMC, incubate with 10mL 1640 complete medium, SEB (0.5. mu.g/mL) stimulate for two days; Raji-PDL1 cells were treated with 25. mu.g/mL MMC and placed in an incubator at 37 ℃ for 1 hour; PBMC after 2 days of SEB stimulation and Raji-PDL1 cells treated for 1 hour with MMC were collected, washed twice with PBS, counted in a complete media resuspension and added to a U-shaped 96-well plate, 1X10, respectively⁵Individual cells/well; adding antibody according to experimental design, and culturing for 3 days(ii) a Cell culture supernatants were collected and tested for IL-2 by ELISA.

As shown in fig. 29, human PBMC and Raji-PDL1 cells have certain promotion effect on the secretion of IL-2 from PBMC after mixed culture, and the antibody added into the mixed culture system can significantly induce PBMC to further secrete IL-2, which has significant dose-dependent relationship, and the level of the activity of promoting IL-2 secretion is that bifunctional antibody NTPDV2 has activity at low concentration equivalent to that of its master PD-1 single-target antibody 14C12H1L1, while the middle-high concentration is slightly lower than that of master PD-1 single-target antibody 14C12H1L 1.

Example 22: anti-CD 73-anti-PD-1 bispecific antibody in vivo tumor growth inhibition experiment

To test the in vivo anti-tumor activity of the anti-CD 73-anti-PD-1 bispecific antibody, MC38-hPDL1/hCD73 cells (from Jiangsu Jiejicakang Biotech Co., Ltd.) were first subcutaneously inoculated into 5-7 week old female C57BL6-hPDlhPDL1hCD73 transgenic mice (from Jiangsu Jiejicakang Biotech Co., Ltd.) and the model and specific administration method are shown in Table 16. The length and width of each group of tumors were measured after administration, and the tumor volume was calculated.

Table 16: and (4) experimental design.

The results of the experiment are shown in fig. 30 and 31, and show that: compared with the isotype control antibody hIgG, 19F3H2L3(hG1M), the NTPDV2 with different doses can effectively inhibit the growth of the tumor of the mouse, and the inhibition of the tumor by the high-dose NTPDV2 is better than that by the low-dose NTPDV 2. In addition, NTPDV2 had no effect on tumor-bearing mouse body weight.

Experimental example 23: fc segment mutation can effectively eliminate secretion of IL-8 and IL-6 mediated by bispecific immune checkpoint inhibitor PD-1/CD73 bispecific antibody

HPMM was induced from PBMC. PBMCs used in this study were all prepared separately in zhongshan kang biomedical limited and with the informed consent of the providers.

Ficoll-Paque PLUS lymphocyte isolate (GE, cat # 17-1440-03); RPMI1640 (Gibco, cat # 22400-105); CHO-K1-PD1 cells (constructed by Zhongshan Kangfang biomedicine Co., Ltd.); U87-MG cells (cells from ATCC, original technology of China limited, Beijing); FBS (Fetal bone Serum, Excell bio, cat # FSP 500); human IFN-gamma protein (sinobio, cat # 11725-HNAS-100); LPS (Lipopolysaccharides), lipopolysaccharide (sigma, cat # L4391); 96-well cell culture plates (corning).

Healthy human PBMC were isolated and resuspended in 1640 medium containing 2% FBS according to the isolate Ficoll-Paque (TM) Plus reagent instructions and placed in a 5% CO2 cell culture box at 37 ℃. After 2h the supernatant was removed, adherent cells were washed 2 times with PBS and induced for 7 days by addition of 1640 complete medium (containing 10% FBS) and 100ng/mL human M-CSF. Fluid was changed and supplemented with M-CSF to induce HPMM on days 3 and 5. After completion of HPMM induction on day 7, the cells were collected, adjusted to a concentration of 10 ten thousand/mL with the use of the whole medium, dispensed into 96-well plates, and recombinant human IFN-. gamma.was added (50ng/mL), and left in an incubator for 24 hours. CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 were collected at log phase after 24h, and the concentration was adjusted to 30 ten thousand/mL with complete medium after resuspension. The antibody was diluted with complete medium to working concentrations of 25nM, 2.5nM, 0.25 nM. And isotype control antibody and blank control were designed simultaneously. The supernatant in the 96-well plate is removed, and CHO-K1-PD1 or U87-MG cell suspension and antibody (final volume 200. mu.L) are added and mixed evenly, and the mixture is placed in an incubator and incubated for 24 h. Centrifuging at 500Xg for 5min, collecting supernatant, and detecting IL-8 and IL-6 secretion amount with Dake kit. LPS was used as a positive control drug and was adjusted to a concentration of 100ng/mL from complete medium in the experiment.

In this example, the co-culture of CHO-K1-PD1 and U87-MG cells as target cells with HPMM induces the activation of HPMM, and after the activated HPMM is linked to the target cells by antibody Fab, the Fc fragment of the antibody acts on Fc γ R on HPMM to cause the secretion of cytokines by HPMM.

3. Results of the experiment

As shown in fig. 32-35.

The results show that the anti-PD-1/CD 73 bispecific antibody carrying an Fc fragment mutation can effectively eliminate IL-6 and/or IL-8 secretion from immune cells compared to the PD-1 antibody or CD73 antibody of wild-type IgG1 subtype.

While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that the present invention is not limited to the details of the embodiments shown and described, but is capable of numerous equivalents and substitutions without departing from the spirit of the invention and its scope is defined by the claims appended hereto.

Sequence listing

The heavy chain variable region nucleotide sequence of 19F 3:

(SEQ ID NO: 1, CDR sequences underlined)

19F3 heavy chain variable region amino acid sequence:

(SEQ ID NO:2, CDR sequences underlined)

HCDR1 of 19F 3: GYSFTGYT

(SEQ ID NO：3)

HCDR2 of 19F 3: INPYNAGT

(SEQ ID NO：4)

HCDR3 of 19F 3: ARSEYRYGGDYFDY

(SEQ ID NO：5)

Nucleotide sequence of the 19F3 light chain variable region:

(SEQ ID NO:6, CDR sequences underlined)

19F3 light chain variable amino acid sequence:

(SEQ ID NO:7, CDR sequences underlined)

LCDR1 amino acid sequence of 19F 3: QSLLNSSNQKNY

(SEQ ID NO：8)

LCDR2 amino acid sequence of 19F 3: FAS

(SEQ ID NO：9)

LCDR3 amino acid sequence of 19F 3: QQHYDTPYT

(SEQ ID NO：10)

Framework amino acid sequence of 19F3 heavy chain:

FR-H1：EVQLQQSGPELVKPGASMRMSCKAS(SEQ ID NO：11)

FR-H2：MNWVKQSHGKNLEWIGL(SEQ ID NO：12)

FR-H3：SYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC(SEQ ID NO：13)

FR-H4：WGQGTTLTVSS(SEQ ID NO：14)

framework amino acid sequence of 19F3 light chain:

FR-L1：DIVMTQSPSSLAMSVGQKVTMSCKSS(SEQ ID NO：15)

FR-L2：LAWYQQKPGQSPKLLVY(SEQ ID NO：16)

FR-L3：TRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFC(SEQ ID NO：17)

FR-L4：FGGGTKLEIK(SEQ ID NO：18)

nucleotide sequence of 19F3H 2: (SEQ ID NO: 19, CDR sequences underlined)

Amino acid sequence of 19F3H 2: (SEQ ID NO:20, CDR sequences underlined)

Nucleotide sequence of 19F3L 2: (SEQ ID NO: 21, CDR sequences underlined)

Amino acid sequence of 19F3L 2: (SEQ ID NO:22, CDR sequences underlined)

Nucleotide sequence of 19F3L 3: (SEQ ID NO: 23, CDR sequences underlined)

Amino acid sequence of 19F3L 3: (SEQ ID NO:24, CDR sequences underlined)

19F3H2L3(G1M) heavy chain nucleotides (SEQ ID NO: 25, non-variable region sequence underlined region)

19F3H2L3(G1M) heavy chain amino acids (SEQ ID NO: 26, underlined region non-variable region sequence)

Light chain nucleotides of 19F3H2L3(G1M) (SEQ ID NO: 27, non-variable region sequence underlined region)

Light chain amino acid of 19F3H2L3(G1M) (SEQ ID NO:28, underlined region non-variable region sequence)

19F3H2L3(hG1TM) heavy chain nucleotides (SEQ ID NO: 29, non-variable region sequence underlined region)

Heavy chain amino acid sequence of 19F3H2L3(hG1 TM): (SEQ ID NO: 30)

Framework region amino acid sequence of 19F3H 2:

FR-H1：QVQLVQSGAEVVKPGASVKVSCKAS(SEQ ID NO：31)

FR-H2：MNWVRQAPGQNLEWIGL(SEQ ID NO：32)

FR-H3：SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC(SEQ ID NO：33)

FR-H4：WGQGTTLTVSS(SEQ ID NO：34)

framework region amino acid sequence of 19F3L 2:

FR-L1：DIVMTQSPSSLAVSVGERVTISCKSS(SEQ ID NO：35)

FR-L2：LAWYQQKPGQAPKLLIY(SEQ ID NO：36)

FR-L3：TRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYC(SEQ ID NO：37)

FR-L4：FGGGTKLEIK(SEQ ID NO：38)

framework region amino acid sequence of 19F3L 3:

FR-L1：DIVMTQSPSSLAVSVGERVTISCKSS(SEQ ID NO：39)

FR-L2：LAWYQQKPGQAPKLLIY(SEQ ID NO：40)

FR-L3：TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC(SEQ ID NO：41)

FR-L4：FGGGTKLEIK(SEO ID NO：42)

14C12 heavy chain variable region nucleotide sequence: (SEQ ID NO: 43)

14C12 heavy chain variable region amino acid sequence: (SEQ ID NO: 44)

HCDR1 amino acid sequence of 14C 12: GFAFSSYD

(SEQ ID N0：45)

HCDR2 amino acid sequence of 14C 12: ISGGGRYT

(SEQ ID NO：46

HCDR3 amino acid sequence of 14C 12: ANRYGEAWFAY

(SEQ ID NO：47)

14C12 light chain variable region nucleotide sequence: (SEQ ID NO: 48)

14C12 light chain variable region amino acid sequence: (SEQ ID NO: 49)

LCDR1 amino acid sequence of 14C 12: QDINTY (SEQ ID NO: 50)

LCDR2 amino acid sequence of 14C 12: RAN (SEQ ID NO: 51)

LCDR3 amino acid sequence of 14C 12: LQYDEFPLT (SEQ ID NO: 52)

14C12 heavy chain framework region amino acid sequence:

FR-H1：EVKLVESGGGLVKPGGSLKLSCAAS(SEQ ID NO：53)

FR-H2：MSWVRQTPEKRLEWVAT(SEQ ID NO：54)

FR-H3：YYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYC(SEQ ID NO：55)

FR-H4：WGQGTLVTVSA(SEQ ID NO：56)

14C12 light chain framework region amino acid sequence:

HR-L1：DIKMTQSPSSMYASLGERVTFTCKAS(SEQ ID NO：57)

HR-L2：LSWFQQKPGKSPKTLIY(SEQ ID NO：58)

HR-L3：RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC(SEQ ID NO：59)

HR-L4：FGAGTKLELK(SEQ ID NO：60)

nucleotide sequence of 14C12H 1: (SEQ ID NO: 61)

Amino acid sequence of 14C12H 1: (SEQ ID NO: 62)

Nucleotide sequence of 14C12L 1: (SEQ ID NO: 63)

Amino acid sequence of 14C12L 1: (SEQ ID NO: 64)

14C12H1L1 heavy chain nucleotide (SEQ ID NO: 65)

Heavy chain amino acid sequence of 14C12H1L 1: (SEQ ID NO: 66)

Light chain nucleotide sequence of 14C12H1L 1: (SEQ ID NO: 67)

Light chain amino acid sequence of 14C12H1L 1: (SEQ ID NO: 68)

Heavy chain nucleotide sequence of 14C12H1L1(G1TM) (SEQ ID NO: 69)

Heavy chain amino acid of 14C12H1L1(G1TM) (SEQ ID NO: 70)

Heavy chain framework region amino acid sequence of 14C12H 1:

FR-H1：EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO：71)

FR-H2：MSWVRQAPGKGLDWVAT(SEQ ID NO：72)

FR-H3：YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC(SEQ ID NO：73)

FR-H4：WGQGTLVTVSS(SEQ ID NO：74)

14C12L1 light chain framework region amino acid sequence:

FR-L1：DIQMTQSPSSMSASVGDRVTFTCRAS(SEQ ID NO：75)

FR-L2：LSWFQQKPGKSPKTLIY(SEQ ID NO：76)

FR-L3：RLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYC(SEQ ID NO：77)

FR-L4：FGAGTKLELK(SEQ ID NO：78)

amino acid sequence of Linker 1: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79)

The nucleotide sequence of Linker 1: (SEQ ID NO: 80)

Amino acid sequence of Linker 2: GGGGSGGGGSGGGGS (SEQ ID NO: 81)

The nucleotide sequence of Linker 2: (SEQ ID NO: 82)

Amino acid sequences of the heavy chains of NTPDV2 and NTPDV 4(SEQ ID NO: 83): wherein the CDR region of 19F3H2(hG1TM) in the immunoglobulin moiety is usedUnderlined bold identificationCDR regions of 14C12H1V-Linker1-14C12L1V in the scFv moieties were used respectivelyUnderlined and boldedIdentification, heavy chain region mutant amino acids are marked in italics and linker regions are marked in bold:

nucleotide sequences of the heavy chains of NTPDV2 and NTPDV 4: (SEQ ID NO: 84)

Amino acid sequences of the heavy chains of NTPDV1 and NTPDV 3(SEQ ID NO: 85): wherein the CDR region of 19F3H2(hG1TM) in the immunoglobulin moiety is usedUnderlined bold identificationCDR regions of 14C12H1V-Linker2-14C12L1V in the scFv moieties were used respectivelyUnderlined and boldedIdentification, heavy chain region mutant amino acids are marked in italics and linker regions are marked in bold:

nucleotide sequences of the heavy chains of NTPDV1 and NTPDV 3: (SEQ ID NO: 86)

NT5E (1-552) -his amino acid sequence (SEQ ID NO: 87)

NT5E (1-552) -his nucleotide sequence (SEQ ID NO: 88)

Amino acid sequence of mFc: (SEQ ID NO: 89)

19F3H1V-Linker-19F3L 2V: (SEQ ID NO: 90)

19F3H1V-Linker2-19F3L2V nucleotide sequence: (SEQ ID NO: 91)

Nucleotide sequence of 19F3H 1: (SEQ ID NO: 92)

Amino acid sequence of 19F3H 1: (SEQ ID NO: 93)

Nucleotide sequence of 19F3L 1: (SEQ ID NO: 94)

Amino acid sequence of 19F3L 1: (SEQ ID NO: 95)

19F3L2 light chain amino acid (full-length) (SEQ ID NO:96, underlined region non-variable region sequence)

19F3H2 variable region, the CDR sequences are underlined: (SEQ ID NO: 97)

19F3L2 variable region, the CDR sequences are underlined: (SEQ ID NO: 98)

19F3L3 variable region, the CDR sequences are underlined: (SEQ ID NO: 99)

Light chain nucleotide of 19F3L2 (full length) (SEQ ID NO: 100)

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