Anti-CD39 Nanobodies and Uses Thereof

Abstract

The present invention relates to Nanobodies or antigen-binding fragments thereof capable of specifically binding to CD39, multispecific antibodies comprising Nanobodies or antigen-binding fragments thereof, nucleic acids encoding Nanobodies or antigen-binding fragments thereof and host cells comprising the nucleic acids, and related uses thereof. The present invention further relates to prophylactic, therapeutic, diagnostic and / or detection uses of the Nanobodies or antigen-binding fragments thereof or multispecific antibodies.

Description

[Technical Field]

[0001] The present invention relates to Nanobodies or antigen-binding fragments thereof capable of specifically binding to CD39, multispecific antibodies comprising same, nucleic acids encoding same and host cells comprising the nucleic acids, and related uses thereof. Furthermore, the present invention relates to prophylactic, therapeutic, diagnostic and / or detection uses of Nanobodies or antigen-binding fragments thereof or multispecific antibodies. [Background technology]

[0002] Human CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), is a member of the ectonucleoside hydrolases and a type II membrane protein with two transmembrane domains. It has a full-length extracellular region of 441 amino acids and exists in a truncated form, which can circulate in the form of soluble CD39 (sCD39). In the upstream adenosine pathway, CD39 hydrolyzes ATP to AMP, which is then hydrolyzed to adenosine by CD73. Adenosine acts on the adenosine receptor (A2AR) on immune cells, activating downstream protein kinase A (PKA) and CSK kinase, and inhibiting a series of signaling pathways related to immune activation, such as LCK, MAPK, and PKC (Mosenden et al., 2012), thereby resulting in immunosuppression. Studies have shown that CD39 is highly expressed in a variety of human tumors, including lymphomas, sarcomas, chronic lymphoblastic leukemia, lung cancer, pancreatic cancer, ovarian cancer, renal cancer, thyroid cancer, and testicular cancer. In some cases, tumor cells overexpress CD39 compared to normal cells, but the most consistent cell types that show high CD39 expression in the tumor microenvironment include vascular endothelial cells, fibroblasts, and several subsets of immune cells, including NK cells, CD4+CD25+ regulatory T (Treg) cells, macrophages, and tumor-specific effector T cells (Li, XY et al., 2019).

[0003] Blocking adenosine-mediated immunosuppression by targeting CD39 in the tumor microenvironment can inhibit tumor growth, which is mainly related to a two-part mechanism: on the one hand, blocking the ATPase activity of CD39 not only reduces adenosine production but also maintains ATP levels in the tumor microenvironment. ATP can activate dendritic cells (DC cells) and further promote T cell activation by DC cells; on the other hand, CD39 is highly expressed on regulatory T cells and exhausted T cells. Blocking the activity of CD39 can reduce the immunosuppressive function of regulatory T cells and reactivate exhausted T cells. However, in the tumor microenvironment, it has been found that CD39-targeted therapy can actually enhance antitumor immunity through various mechanisms, including reducing adenosine-mediated T cell immunosuppression, activating macrophage inflammasomes, affecting NK cell function, inhibiting the immunosuppressive function of Treg cells, increasing antigen-presenting cell (APC) maturation, etc. In general, these mechanisms mainly act by increasing eATP or reducing adenosine production.

[0004] Based on the above mechanism, several researchers have conducted exploratory studies on the anti-tumor effects of CD39. Preclinical mouse model results showed that CD39-targeting antibodies or CD39 gene knockout can effectively stop tumor growth and metastasis (Jackson et al., 2007). CD39, expressed by both immune and non-immune cells, can promote immune evasion, development, and metastasis of tumors. Additionally, preclinical mouse model results showed that CD39 inhibitors and PD-1 inhibitors can have a good synergistic effect on tumor inhibition. Summary of the Invention

[0005] The present inventors have conducted extensive research and have come up with a nanobody that exhibits high binding activity to human CD39 and cross-reactivity with cynomolgus monkey CD39. In particular, the nanobody of the present invention can effectively alleviate adenosine-mediated immunosuppression. Additionally, nanobodies are characterized by low molecular weight, excellent stability, and other features, which make them more advantageous than traditional antibodies in terms of drug research and development, for example, in terms of better tissue penetration, more flexible administration, and easier reconstitution of recombinant proteins. The present invention further provides anti-CD39 Nanobody-based multispecific antibodies, compositions comprising the Nanobody or antigen-binding fragment thereof or multispecific antibody, nucleic acids encoding the Nanobody or antigen-binding fragment thereof or multispecific antibody, and host cells comprising the nucleic acid, and related uses thereof.

[0006] Nanobodies or antigen-binding fragments thereof Thus, in a first aspect, the present invention provides a Nanobody or antigen-binding fragment thereof capable of specifically binding to CD39. The Nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 1 or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., one, two, or three amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 1; (b) a CDR2 having the sequence set forth in SEQ ID NO: 2 or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., one, two, or three amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 2; and (c) a CDR3 having the sequence set forth in SEQ ID NO: 3 or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., one, two, or three amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 3; Includes.

[0007] In some embodiments, the substitutions are conservative substitutions. In some embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO:1, a CDR2 set forth in SEQ ID NO:2, and a CDR3 set forth in SEQ ID NO:3. In some embodiments, the Nanobody or antigen-binding fragment thereof comprises the three CDRs of a VHH as set forth in any one of SEQ ID NOs: 4 to 8. In some embodiments, the three CDRs of the VHH are determined using the Kabat, Chothia, or IMGT numbering systems. In some embodiments, the nanobody or antigen-binding fragment thereof is (i) the sequence shown in SEQ ID NO: 4; (ii) a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 4; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4 The amino acid sequence is selected from the group consisting of:

[0008] In some embodiments, the substitutions are conservative substitutions. In some embodiments, the Nanobody or antigen-binding fragment thereof is humanized. In some embodiments, the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain embryoid body antibody gene), and the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to camelid residues.

[0009] In some embodiments, the nanobody or antigen-binding fragment thereof is (i) a sequence shown in any one of SEQ ID NOs: 5 to 8; (ii) a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in any one of SEQ ID NOs: 5 to 8; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 5 to 8. The amino acid sequence is selected from the group consisting of: In some embodiments, the substitutions are conservative substitutions.

[0010] In a second aspect, the present invention provides a Nanobody or antigen-binding fragment thereof capable of specifically binding to CD39. The Nanobody or antigen-binding fragment thereof comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 9 or 14, or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., one, two, or three amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 9 or 14; (b) a CDR2 having the sequence set forth in SEQ ID NO: 10 or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., one, two, or three amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 10; and (c) a CDR3 having the sequence set forth in SEQ ID NO: 11 or 15, or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., one, two, or three amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 11 or 15; Includes.

[0011] In some embodiments, the substitutions are conservative substitutions. In some embodiments, the Nanobody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 9 or 14, a CDR2 set forth in SEQ ID NO: 10, and a CDR3 set forth in SEQ ID NO: 11 or 15. In some embodiments, the nanobody or antigen-binding fragment thereof is (1) CDR1 shown in SEQ ID NO: 9, CDR2 shown in SEQ ID NO: 10, and CDR3 shown in SEQ ID NO: 11; (2) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 10, and CDR3 shown in SEQ ID NO: 15; or (3) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 10, and CDR3 shown in SEQ ID NO: 11 Includes.

[0012] In some embodiments, the Nanobody or antigen-binding fragment thereof comprises the three CDRs of a VHH as set forth in any one of SEQ ID NOs: 12, 13, and 16 to 18. In some embodiments, the three CDRs of the VHH are determined using the Kabat, Chothia, or IMGT numbering systems. In some embodiments, the nanobody or antigen-binding fragment thereof is (i) the sequence shown in SEQ ID NO: 12; (ii) a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO: 12; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12 The amino acid sequence is selected from the group consisting of:

[0013] In some embodiments, the substitutions are conservative substitutions. In some embodiments, the Nanobody or antigen-binding fragment thereof is humanized. In some embodiments, the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain embryoid body antibody gene), and the heavy chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to camelid residues. In some embodiments, the nanobody or antigen-binding fragment thereof is (i) a sequence shown in any one of SEQ ID NOs: 13 and 16 to 18; (ii) a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in any one of SEQ ID NOs: 13 and 16 to 18; or (iii) An amino acid sequence selected from sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 13, 16 to 18.

[0014] In some embodiments, the substitutions are conservative substitutions. In some embodiments, the CD39 as defined above in the first or second aspect is selected from human CD39 and / or cynomolgus CD39. In some embodiments, a Nanobody or antigen-binding fragment thereof as defined above in the first or second aspect is capable of blocking the enzymatic activity of CD39 to which it binds.

[0015] Peptide construction In a third aspect, the present invention further provides a polypeptide construct capable of specifically binding to CD39, comprising a Nanobody or an antigen-binding fragment thereof as described above in the first or second aspect, and an immunoglobulin Fc domain. As used herein, an Fc domain, also known as an Fc region, refers to the portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc domain comprises a hinge, CH2, and CH3. When an Fc domain comprises a hinge, the hinge mediates dimerization between two Fc-containing peptides. An Fc domain may be any isotype of antibody heavy chain constant region. In some embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0016] In some embodiments, the Fc domain contained in the polypeptide construct of the present invention is a native Fc region having the same amino acid sequence as that found in nature. For example, the Fc domain may have the same sequence as the native sequence of a human IgG1 Fc region, the native sequence of a human IgG2 Fc region, the native sequence of a human IgG3 Fc region, or the native sequence of a human IgG4 Fc region. The native Fc region may have an effector function. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); bacteriophage binding; cell surface receptor (e.g., B cell receptor) downregulation; and B cell activation. Functional alterations may result from the replacement of at least one amino acid residue in the native Fc region with a different residue or chemical modification. For example, effector function may be altered (e.g., reduced or enhanced) by changing the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q). Thus, in some embodiments, the Fc domain contained in the polypeptide construct of the invention may also be a mutant Fc region that comprises one or more (e.g., 1 to 10, e.g., 1 to 5) amino acid mutations or chemical modifications compared to a native Fc region to alter one or more of the following characteristics of the antibody of the invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0017] In some embodiments, the Fc domain contained in the polypeptide construct of the present invention has ADCC activity. In some embodiments, the Fc domain contained in the polypeptide construct of the present invention does not have ADCC activity. In some embodiments, the immunoglobulin Fc domain may be connected to the N-terminus and / or C-terminus (e.g., C-terminus) of the Nanobody or antigen-binding fragment thereof via a peptide linker. In some embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).

[0018] In some embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO:27, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:27, or a sequence having one or more amino acid substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or additions) compared to the sequence set forth in SEQ ID NO:27.

[0019] multispecific antibody In a fourth aspect, the present invention further provides a multispecific antibody comprising a Nanobody or antigen-binding fragment thereof or polypeptide construct as described in any of the preceding aspects. In some embodiments, the multispecific antibody is capable of specifically binding to CD39 and additionally is capable of specifically binding to one or more other targets. In some embodiments, the multispecific antibody further comprises at least one second antibody that has binding specificity for a second target. In some embodiments, the multispecific antibody comprises a Nanobody or antigen-binding fragment thereof described in the first aspect and at least one second antibody that has binding specificity for a second target. In some embodiments, the multispecific antibody comprises a Nanobody or antigen-binding fragment thereof described in the second aspect and at least one second antibody with binding specificity for a second target.

[0020] In a fifth aspect, the present application provides a multispecific antibody comprising a first antigen-binding domain specific for a first epitope on CD39 and a second antigen-binding domain specific for a second epitope on CD39, wherein the first antigen-binding domain comprises a nanobody or antigen-binding fragment thereof as described in the first aspect, and the second antigen-binding domain comprises a nanobody or antigen-binding fragment thereof as described in the second aspect. In a first exemplary embodiment of the fifth aspect, the first antigen-binding domain and the second antigen-binding domain are both VHHs, and the multispecific antibody comprises peptide chain II containing a monomeric Fc domain, the first antigen-binding domain and the second antigen-binding domain.

[0021] In some embodiments, the monomeric Fc domain comprises a CH2 and a CH3. In some embodiments, the multispecific antibody comprises two peptide chains II. In some embodiments, the monomeric Fc domains of the two peptide chains II form a dimer. In some embodiments, the two peptide chains II are identical. In some embodiments, the two peptide chains II are different. In some embodiments, individual domains may be connected through a linker (eg, a flexible peptide containing one or more glycines (G) and / or alanines (A)). It can be readily understood that the relative positions of the individual domains in peptide chain II are not limited, as long as activity can be maintained.

[0022] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are adjacent and connected between them, optionally via a linker (e.g., a flexible peptide containing one or more glycines (G) and / or alanines (A)). In some embodiments, the first antigen-binding domain is located at the N-terminus of the second antigen-binding domain. In some embodiments, the first antigen-binding domain is located at the C-terminus of the second antigen-binding domain. In some embodiments, peptide chain II comprises, in N-terminal to C-terminal order, adjacent first and second antigen-binding domains, or adjacent second and first antigen-binding domains, and further comprises a monomeric Fc domain. In some embodiments, peptide chain II comprises, in order from N-terminus to C-terminus, a monomeric Fc domain, a first antigen-binding domain, and a second antigen-binding domain.

[0023] In some embodiments, the first antigen-binding domain is connected to the C-terminus of the monomeric Fc domain through a linker (e.g., a flexible peptide comprising one or more glycines (G) and / or alanines (A)); and / or the second antigen-binding domain is connected to the C-terminus of the first antigen-binding domain through a linker (e.g., a flexible peptide comprising one or more glycines (G) and / or alanines (A)). In some embodiments, the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:7; and / or the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:16.

[0024] In some embodiments, peptide chain II comprises or consists of the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, peptide chain II comprises, in order from N-terminus to C-terminus, a second antigen-binding domain, a first antigen-binding domain, and a monomeric Fc domain. In some embodiments, a first antigen-binding domain is connected to the C-terminus of a second antigen-binding domain, optionally via a linker (e.g., a flexible peptide containing one or more glycines (G) and / or alanines (A)). In some embodiments, the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:7; and / or the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, peptide chain II comprises or consists of the amino acid sequence set forth in SEQ ID NO:22.

[0025] In a second exemplary embodiment of the fifth aspect, the first antigen-binding domain and the second antigen-binding domain are both VHHs and the multispecific antibody comprises: (i) a peptide chain IA comprising a first antigen-binding domain and a light chain constant region (CL); and (ii) a peptide chain IB comprising a second antigen-binding domain and a heavy chain constant region (CH) Includes. In some embodiments, the CL of peptide chain IA can dimerize with the CH1 domain of the heavy chain constant region of peptide chain IB.

[0026] In some embodiments, the multispecific antibody comprises two peptide chains IA and two peptide chains IB. In some embodiments, the heavy chain constant regions of the two peptide chains IB form a dimer. In some embodiments, the two peptide chains IA are identical. In some embodiments, the two peptide chains IA are different. In some embodiments, the two peptide chains IB are identical. In some embodiments, the two peptide chains IB are different. In some embodiments, (1) peptide chain IA comprises, in N-terminal to C-terminal order, a first antigen-binding domain and a light chain constant region (CL); and / or (2) Peptide chain IB comprises, in order from N-terminus to C-terminus, a second antigen-binding domain and a heavy chain constant region (CH).

[0027] In some embodiments, the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, peptide chain IA comprises or consists of the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, peptide chain IB comprises or consists of the amino acid sequence set forth in SEQ ID NO:19.

[0028] In a third exemplary embodiment of the fifth aspect, the multispecific antibody further comprises an antigen-binding domain specific for a target different from CD39. In some embodiments, the multispecific antibody further comprises one or more antigen-binding domains specific for other targets different from CD39. In some embodiments, the multispecific antibody further comprises a third antigen-binding domain specific for a target different from CD39. In a fourth exemplary embodiment of the fifth aspect, the multispecific antibody further comprises a third antigen-binding domain specific for PD-1. In some embodiments of the multispecific antibody, the first antigen-binding domain and the second antigen-binding domain are both VHHs, the third antigen-binding domain is a Fab, and the multispecific antibody comprises: (1) a peptide chain III-A comprising a light chain variable region and a light chain constant region (CL) of the third antigen-binding domain; and (2) Peptide chain III-B comprising a heavy chain variable region, a heavy chain constant region, a first antigen-binding domain, and a second antigen-binding domain of the third antigen-binding domain Includes.

[0029] In some embodiments, peptide chain III-B comprises, in N-terminal to C-terminal order, adjacent first and second antigen-binding domains, or adjacent second and first antigen-binding domains, and further comprises a heavy chain variable region and a heavy chain constant region of a third antigen-binding domain. In some embodiments, the CL of peptide chain III-A can form a dimer with the CH1 domain of the heavy chain constant region of peptide chain III-B. In some embodiments, the multispecific antibody comprises two peptide chains III-A and two peptide chains III-B. In some embodiments, the heavy chain constant regions of the two peptide chains III-B form a dimer. In some embodiments, the two peptide chains III-A are identical. In some embodiments, the two peptide chains III-A are different. In some embodiments, the two peptide chains III-B are identical. In some embodiments, the two peptide chains III-B are different.

[0030] In some embodiments, the individual domains are connected between them, and the connections may be through linkers (e.g., flexible peptides containing one or more glycines (G) and / or alanines (A)). In some embodiments of the multispecific antibody, (1) peptide chain III-A comprises, in N-terminal to C-terminal order, a light chain variable region and a light chain constant region (CL) of the third antigen-binding domain; and / or (2) Peptide chain III-B comprises, in order from the N-terminus to the C-terminus, a heavy chain variable region of the third antigen-binding domain, a heavy chain constant region, the first antigen-binding domain, and the second antigen-binding domain.

[0031] In some embodiments, the multispecific antibody has the following characteristics: (i) the first antigen-binding domain is connected to the C-terminus of the heavy chain constant region through a linker (e.g., a flexible peptide containing one or more glycines (G) and / or alanines (A)); and / or the second antigen-binding domain is connected to the C-terminus of the first antigen-binding domain through a linker (e.g., a flexible peptide containing one or more glycines (G) and / or alanines (A)); (ii) the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7; (iii) the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16; (iv) the heavy chain variable region of the third antigen-binding domain comprises VH CDRs 1 to 3 set forth in SEQ ID NOs: 36 to 38, respectively; (v) the light chain variable region of the third antigen-binding domain comprises VL CDRs 1 to 3 set forth in SEQ ID NOs: 39 to 41, respectively; (vi) the heavy chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; (vii) The light chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. It has one or more of the following.

[0032] In some embodiments, peptide chain III-A comprises or consists of the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, peptide chain III-B comprises or consists of the amino acid sequence set forth in SEQ ID NO:25. In a sixth aspect, the present invention provides a multispecific antibody capable of specifically binding to both CD39 and PD-1, comprising a Nanobody or antigen-binding fragment thereof or polypeptide construct as described in any of the preceding aspects, and an antigen-binding domain specific for PD-1. In some embodiments, the antigen-binding domain specific for PD-1 comprises VH CDRs 1-3 set forth in SEQ ID NOs: 36-38, respectively, and / or VL CDRs 1-3 set forth in SEQ ID NOs: 39-41, respectively. In some embodiments, the antigen-binding domain specific for PD-1 comprises a VH set forth in SEQ ID NO:23 and / or a VL set forth in SEQ ID NO:24.

[0033] In a seventh aspect, the present invention provides an isolated nucleic acid molecule capable of encoding a Nanobody or antigen-binding fragment thereof, polypeptide construct, or multispecific antibody according to any of the preceding aspects. In some embodiments, the isolated nucleic acid molecule is capable of encoding a Nanobody of the invention or an antigen-binding fragment thereof. In some embodiments, the isolated nucleic acid molecule is capable of encoding a polypeptide construct of the present invention. In some embodiments, the isolated nucleic acid molecule is capable of encoding a multispecific antibody of the invention.

[0034] It is readily apparent that the multispecific antibodies of the present invention can be composed of one or more polypeptide chains. The number of nucleic acid molecule chains in an isolated nucleic acid molecule encoding a multispecific antibody of the present invention is not limited. For example, in some embodiments, a multispecific antibody is composed of a first peptide chain and a second peptide chain, and the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first peptide chain and a second nucleotide sequence encoding the second peptide chain, with the first nucleotide sequence and the second nucleotide sequence being present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0035] In an eighth aspect, the present invention further provides a vector comprising the isolated nucleic acid molecule described above. In some embodiments, the vector is a cloning vector or an expression vector. It can be easily understood that the above-mentioned isolated nucleic acid molecule can be present in a vector in any form. For example, when the isolated nucleic acid molecule contains multiple nucleotide sequences encoding different peptide chains, the multiple nucleotide sequences may be located on the same vector or on different vectors. The orientation, relative position, and connection mode of the multiple nucleotide sequences on the vector are not limited. In some embodiments, a vector comprises a first nucleotide sequence encoding a first peptide chain of a multispecific antibody of the invention and a second nucleotide sequence encoding a second peptide chain of the multispecific antibody, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0036] In a ninth aspect, the present application provides a host cell comprising the above-described nucleic acid molecule or vector. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E. coli cells), eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells). In some embodiments, the host cell is a microorganism.

[0037] Nanobodies or antigen-binding fragments thereof, polypeptide constructs, or multispecific antibodies of the invention can be prepared by various methods known in the art, such as recombinant genetic engineering techniques. For example, DNA molecules encoding Nanobodies or antigen-binding fragments thereof, polypeptide constructs, or multispecific antibodies of the invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and transfected into host cells. The transfected host cells are then cultured under specific conditions to express the Nanobodies or antigen-binding fragments thereof, polypeptide constructs, or multispecific antibodies of the invention. In a tenth aspect, the present application provides a method for preparing a Nanobody or antigen-binding fragment thereof, polypeptide construct, or multispecific antibody as described in any of the preceding aspects, the method comprising culturing a host cell as described above under conditions that allow protein expression, and recovering the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody from the cultured host cell culture.

[0038] Preventive / therapeutic use In an eleventh aspect, the present invention provides a method for producing a composition comprising: (i) a Nanobody or antigen-binding fragment thereof, a polypeptide construct comprising a Nanobody or antigen-binding fragment thereof, a nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule or vector, as described in the first aspect; and (ii) a Nanobody or antigen-binding fragment thereof, a polypeptide construct comprising a Nanobody or antigen-binding fragment thereof, a nucleic acid molecule encoding a Nanobody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule or vector, as described in the second aspect. Further provided is a composition comprising: In a twelfth aspect, the present application further provides a pharmaceutical composition comprising a Nanobody or an antigen-binding fragment thereof, or a polypeptide construct, or a multispecific antibody, or an isolated nucleic acid molecule, or a vector, or a host cell, or a composition according to any of the preceding aspects, and a pharmaceutically acceptable carrier and / or excipient.

[0039] In some exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0040] In some embodiments, the pharmaceutical composition further comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD73 antibody, or a combination thereof. In some embodiments, the anti-PD-1 antibody comprises VH CDRs 1-3 set forth in SEQ ID NOs: 36-38, respectively, and / or VL CDRs 1-3 set forth in SEQ ID NOs: 39-41, respectively. In some embodiments, the anti-PD-1 antibody comprises a VH set forth in SEQ ID NO:23 and / or a VL set forth in SEQ ID NO:24. In some embodiments, the anti-CD73 antibody comprises a heavy chain set forth in SEQ ID NO:42 and / or a light chain set forth in SEQ ID NO:43.

[0041] In a thirteenth aspect, the present invention provides a method for producing a composition comprising: (1) To reduce the enzymatic activity of CD39 in vitro or in vivo (e.g., in humans); (2) to reduce adenosine-mediated immunosuppression in a subject (e.g., a human); (3) to prevent and / or treat tumors in a subject (e.g., a human); or (4) To prevent and / or treat infectious diseases in a subject (e.g., a human). The present invention further provides the use of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct, or a multispecific antibody, or an isolated nucleic acid molecule, or a vector, or a host cell, or a composition as described in any of the preceding aspects in the preparation of a medicament for treating a disease comprising administering to a subject a therapeutically effective amount of a compound according to the present invention. In some embodiments, the tumor is associated with CD39-positive tumor cells.

[0042] In some embodiments, the tumor is selected from a solid tumor or a hematological tumor (e.g., leukemia, lymphoma). In some embodiments, the tumor is selected from colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and melanoma. In some embodiments, the tumor is selected from breast cancer, ovarian cancer, testicular cancer, pancreatic cancer, kidney cancer, lung cancer, thyroid cancer, lymphoma, leukemia, myeloma, sarcoma, and melanoma.

[0043] In some embodiments, the infection is selected from a viral infection, a bacterial infection, a fungal infection, and a parasitic infection. In some embodiments, the subject is a mammal, such as a human or a monkey. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is used alone or in combination with other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is used in combination with other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered simultaneously with the other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered sequentially with the other pharmaceutically active agents.

[0044] In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is used in combination with an immune checkpoint inhibitor. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered simultaneously with the immune checkpoint inhibitor. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered sequentially with the immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD73 antibody, or any combination thereof.

[0045] In a fourteenth aspect, the present application provides a method for enhancing an immune response or preventing and / or treating a tumor or an infectious disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a Nanobody or antigen-binding fragment thereof, or a polypeptide construct, or a multispecific antibody, or an isolated nucleic acid molecule, or a vector, or a host cell, or a composition, or a pharmaceutical composition as described in any of the preceding aspects. In some embodiments, the tumor is associated with CD39-positive tumor cells. In some embodiments, the tumor is selected from a solid tumor or a hematological tumor (e.g., leukemia, lymphoma).

[0046] In some embodiments, the tumor is selected from colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and melanoma. In some embodiments, the tumor is selected from breast cancer, ovarian cancer, testicular cancer, pancreatic cancer, kidney cancer, lung cancer, thyroid cancer, lymphoma, leukemia, myeloma, sarcoma, and melanoma. In some embodiments, the infection is selected from a viral infection, a bacterial infection, a fungal infection, and a parasitic infection. In some embodiments, the subject is a mammal, such as a human or a monkey.

[0047] In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered alone or in combination with other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is used in combination with other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered simultaneously with the other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered sequentially with the other pharmaceutically active agents. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered in combination with an immune checkpoint inhibitor. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered simultaneously with the immune checkpoint inhibitor. In some embodiments, the Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or isolated nucleic acid molecule, or vector, or host cell, or composition is administered sequentially with the immune checkpoint inhibitor.

[0048] In some embodiments, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD73 antibody, or a combination thereof. The Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or pharmaceutical composition of the invention can be formulated into any dosage form known in the medical art, such as tablets, pills, suspensions, emulsions, liquids, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile injectable powders, and injectable concentrated solutions), inhalants, and sprays. The preferred dosage form depends on the expected mode of administration and therapeutic use. The Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or pharmaceutical composition of the invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection may be a sterile injectable solution. For example, sterile injectable solutions may be produced by mixing the required amount of a Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or pharmaceutical composition of the invention, optionally together with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof), in a suitable solvent, followed by filtration for sterilization. Additionally, sterile injectable solutions may be prepared into sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for storage and later use. Such sterile lyophilized powders may be dispersed in a suitable vehicle before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0049] A Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or pharmaceutical composition of the invention can be administered by any suitable means known in the art, including, but not limited to, oral, buccal, sublingual, periocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal administration. However, for many therapeutic purposes, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It should be understood that the route / mode of administration will vary with the intended purpose. In some embodiments, a Nanobody or antigen-binding fragment thereof, or polypeptide construct, or multispecific antibody, or pharmaceutical composition of the invention is administered intravenously or by bolus injection.

[0050] Test Purpose In a fifteenth aspect, the present application further provides a conjugate comprising a Nanobody or antigen-binding fragment thereof, or polypeptide construct as described in any of the preceding aspects, and a detectable marker linked to the Nanobody or antigen-binding fragment thereof, or polypeptide construct. In some embodiments, the detectable marker is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin. In a sixteenth aspect, the present application further provides a kit comprising a Nanobody or antigen-binding fragment thereof, or a polypeptide construct or conjugate according to any of the preceding aspects.

[0051] In some embodiments, the kit comprises a conjugate as described above. In some embodiments, the kit comprises a Nanobody or antigen-binding fragment thereof or polypeptide construct described in any of the preceding aspects, and a second antibody capable of specifically recognizing the Nanobody or antigen-binding fragment thereof, wherein the second antibody may further comprise a detectable marker, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0052] In a seventeenth aspect, the present application further provides a method for detecting the presence or level of CD39 in a sample, the method comprising use of a Nanobody or antigen-binding fragment thereof, or polypeptide construct or conjugate as defined in any one of the preceding aspects. In some embodiments, the method is an immunoassay, such as immunoblotting, enzyme immunoassay (eg, ELISA), chemiluminescence immunoassay, fluoroimmunoassay, or radioimmunoassay. In some embodiments, the methods include using a conjugate as described above.

[0053] In some embodiments, the method comprises using a Nanobody or antigen-binding fragment thereof, or polypeptide construct described in any one of the preceding aspects, and the method further comprises using a second antibody with a detectable marker (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect the Nanobody or antigen-binding fragment thereof, or polypeptide construct.

[0054] In some embodiments, the method comprises: (1) contacting a sample with a Nanobody or antigen-binding fragment thereof, polypeptide construct, or conjugate of the invention; and (2) detecting the formation of an antigen-antibody immune complex or determining the amount of the immune complex, wherein the formation of the immune complex is indicative of the presence of CD39 or a CD39-expressing cell. In an eighteenth aspect, the present application further provides the use of a Nanobody or antigen-binding fragment thereof, or polypeptide construct or conjugate of the invention, as described in any of the preceding aspects, in the preparation of a test reagent for detecting the presence or level of CD39 in a sample. In some embodiments, the test reagent detects the presence or level of CD39 in a sample by the methods described above for detecting the presence or level of CD39 in a sample. In some embodiments, the sample is a cell sample (eg, tumor cells) from a subject (eg, a mammal, preferably a human or monkey).

[0055] Definition of Terms In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Additionally, the laboratory procedures of virology, biochemistry and immunology used herein are all routine procedures commonly used in the corresponding fields. Nevertheless, for easy understanding of the present invention, definitions and explanations of relevant terms are provided below. When the terms "for example," "such as," "eg," "comprise," "include," or variations thereof are used herein, these terms should not be considered limiting and should be interpreted as indicating open-ended, i.e., "but not limited to," or "limited to." Unless otherwise specified herein or clearly contradicted by context, the terms "a / an," "one," and "the," and similar expressions, as used in the context of the present invention (particularly in the context of the appended claims), are to be construed as encompassing both singular and plural referents.

[0056] As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or shark antibody). Typically, a nanobody is composed of four framework regions and three complementarity determining regions in the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies may be truncated at the N- or C-terminus, and portions of FR1 and / or FR4 may be removed, or one or two of the framework regions may be deleted, so long as antigen-binding ability and specificity are substantially retained. Nanobodies are also called single domain antibodies (sdAbs), the two terms being used interchangeably herein.

[0057] As used herein, the term "antigen-binding fragment" of a Nanobody refers to a polypeptide that comprises a segment of a Nanobody and that retains the ability to specifically bind to the same antigen as the Nanobody and / or competes with the Nanobody for specific binding to an antigen; also referred to as an "antigen-binding portion." See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the Nanobodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, an "antigen-binding fragment" of a Nanobody may be truncated at the N-terminus or C-terminus compared to a full-length Nanobody, or portions of FR1 and / or FR4 may be removed, or one or two of their framework regions may be deleted, as long as antigen-binding ability and specificity are substantially retained. Antigen-binding fragments of a Nanobody can be obtained from a given Nanobody (e.g., a Nanobody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the resulting antigen-binding fragments may be screened for specificity in the same way as for intact Nanobodies. As used herein, unless the context clearly indicates otherwise, when the term "nanobody" is referenced it includes not only complete nanobodies but also antigen-binding fragments of nanobodies.

[0058] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificities for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen-binding domains that have binding specificities for different antigens (or epitopes), and is thus capable of binding to at least two different binding sites and / or target molecules. Each antigen-binding domain contained in a multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, an F(ab')2 fragment, or an scFv). In some cases, the antigen-binding domains are connected between them by a peptide linker.

[0059] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given Nanobody, one skilled in the art will readily identify its CDRs as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). Preferably, as used herein, the CDRs of Nanobodies are defined according to the Kabat numbering system.

[0060] As used herein, the term "framework region" or "FR" residues refers to amino acid residues other than the CDR residues defined above in antibody variable regions. As used herein, the term "Fab fragment" or "Fab" refers to an antibody fragment consisting of a light chain fragment comprising VL and CL and a heavy chain fragment comprising VH and CH1.

[0061] As used herein, the term "Fc domain" or "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. The Fc fragment of an antibody has several different functions but does not participate in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); bacteriophage; cell surface receptor (e.g., B cell receptor) downregulation; and B cell activation. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge regulates dimerization between two Fc-containing peptides. The Fc region may be any isotype of antibody heavy chain constant region, such as IgG1, IgG2, IgG3, or IgG4.

[0062] The Fc domain may be a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature. For example, native sequences of human Fc regions include the native sequences of the human IgG1 Fc region (both non-A and A isotypes); the native sequences of the human IgG2 Fc region; the native sequences of the human IgG3 Fc region; and the native sequences of the human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the native sequence of an Fc region as a result of at least one amino acid modification. In some embodiments, a variant Fc region may have an altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) that differs from that of a native Fc region.

[0063] As used herein, the term "humanization" refers to the modification of a non-human antibody by genetic engineering, such that the amino acid sequence is altered to improve homology with that of a human antibody. Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (receptor antibody). In some embodiments, a humanized antibody has its CDR regions derived from a non-human antibody (donor antibody) and all or part of its non-CDR regions (e.g., variable region FRs and / or constant regions) derived from a human immunoglobulin (receptor antibody). A humanized antibody typically retains the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. As used herein, the donor antibody can be a camelid antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To prepare humanized antibodies, CDR regions from immunized animals are inserted into human framework sequences using methods known in the art. In the context of nanobodies, humanized antibodies may also refer to humanized VHHs, i.e., VHHs in which one or more framework regions have been replaced with corresponding human framework regions. In some cases, some framework regions (FRs) of a human immunoglobulin are replaced with corresponding non-human residues. Additionally, humanized VHHs may contain residues that are not found in either the initial VHH sequence or the human framework sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing peptide.

[0064] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or two nucleic acids. In determining the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the two molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences have the same length. Determining the percentage identity between two sequences can also be accomplished using a mathematical algorithm. Non-limiting examples of mathematical algorithms for comparing two sequences include the algorithm of Karlin and Altschul (1990, Proc Natl. Acad. Sci. USA 87: 2264-2268), and its modification by Karlin and Altschul (1993, Proc Natl. Acad. Sci. USA 90: 5873-5877) by incorporating the algorithm into the NBLAST and XBLAST programs of Altschul et al. (1990, J. Mol. Biol. 215: 403).

[0065] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D As used herein, the term "K D" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and describes the binding affinity between an antibody and an antigen. A smaller equilibrium dissociation constant indicates tighter antibody-antigen binding and a higher affinity between the antibody and the antigen.

[0066] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the formation and dissociation rates of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentrations and the actual association and dissociation rates (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of k / k is the dissociation constant, K D (See Davies et al., Annual Rev Biochem, 1990, 59: 439-473). K D The k, k and k values ​​may be measured by any practical method. In some embodiments, surface plasmon resonance (SPR) may be used in a Biacore to determine the dissociation constant. Additionally, bioluminescence interferometry or Kinexa may also be used to determine the dissociation constant.

[0067] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide is inserted. When a vector expresses a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector may be transferred into a host cell through transformation, transduction, or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including, but not limited to, plasmids; bacteriophages; Cox plasmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or artificial chromosomes from P1 sources (PACs); phages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (e.g., SV40). The vector may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may further contain a replication origin.

[0068] As used herein, the term "host cell" refers to a cell into which a vector is introduced, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as an S2 Drosophila cell or an Sf9, or an animal cell such as a fibroblast; a CHO cell; a COS cell; an NSO cell; a HeLa cell; a BHK cell; a HEK 293 cell; or a human cell.

[0069] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or adversely alter the expected properties of a protein / peptide containing the amino acid sequence. For example, conservative substitutions may be introduced through standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing an amino acid residue with another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Lists of groups of amino acid residues with similar side chains have been defined in the art, including amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, corresponding amino acid residues are preferably replaced with other amino acid residues from the same group. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12 (10): 879-884 (1999); and Burks et al., Proc Natl Acad. Set USA 94: 412-417 (1997), all of which are incorporated herein by reference).

[0070] With respect to the 20 conventional amino acids in the present specification, they are written according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Green, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Furthermore, as used herein, amino acids are usually represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0071] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, as is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, agents for maintaining osmolality, agents for delaying absorption, and preservatives. 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 enhancing agents include, but are not limited to, sodium chloride. Agents for maintaining osmolality include, but are not limited to, sugars, NaCl, and analogs thereof. Absorption delaying agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerin). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, p-hydroxybenzoate, tert-butyl trichloride, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art, i.e., they are capable of stabilizing the expected activity of the pharmaceutical active ingredient, and include, but are not limited to, sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, glucan, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysates), and the like. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution).In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solutions), Ringer's solution, and any combination thereof.

[0072] Beneficial Effects of the Invention The present invention provides Nanobodies that exhibit high binding activity to human CD39 and cross-reactivity with cynomolgus monkey CD39. In particular, the Nanobodies of the present invention can effectively alleviate adenosine-mediated immunosuppression. Additionally, Nanobodies are characterized by low molecular weight, excellent stability, and other features, which make them more advantageous than traditional antibodies in terms of drug research and development, for example, in terms of better tissue penetration, more flexible administration, and easier reconstitution of recombinant proteins.

[0073] Additionally, the present invention further provides anti-CD39 nanobody-based multispecific antibodies that significantly inhibit tumor growth when administered in animal models and therefore have significant clinical value.

[0074]

[0023] The embodiments of the present invention are described in detail below in combination with drawings and examples. However, it should be understood that the following drawings and examples are illustrative only and not limiting. Based on the drawings and the following detailed description of the preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. [Brief explanation of the drawings]

[0075] [Figure 1] 1 is a graph showing the results of testing the binding activity of anti-CD39 nanobodies to CHO-hCD39 cells. [Figure 2] 1 is a graph showing the results of testing the binding activity of anti-CD39 nanobodies to CHO-cyCD39 cells. [Figure 3] Graph showing the results of testing anti-CD39 nanobodies binding to epitope bins. [Figure 4] 1 is a graph showing the results of testing anti-CD39 nanobodies for blocking CD39 enzymatic activity in human CD39-overexpressing cells. [Figure 5] 1 is a graph showing the results of testing anti-CD39 nanobodies for blocking CD39 enzymatic activity in a PBMC system. [Figure 6] 1 is a graph showing the results of testing anti-CD39 nanobodies for blocking the enzymatic activity of soluble CD39. [Figure 7] Graph showing the results of affinity testing of humanized nanobodies for CHO-hCD39 cells. [Figure 8] Graph showing the results of affinity testing of humanized nanobodies for CHO-cyCD39 cells. [Figure 9] Figure 1 shows the results of testing humanized nanobodies for blocking CD39 enzymatic activity in human CD39-overexpressing cells. [Figure 10] Figure 1 shows the results of testing humanized nanobodies for blocking CD39 enzymatic activity in a PBMC system. [Figure 11] Figure 1 shows the results of testing humanized nanobodies for blocking the enzymatic activity of soluble CD39. [Figure 12] Figure 1 shows the results of testing humanized nanobodies for blocking CD39 enzymatic activity in human CD39-overexpressing cells. [Figure 13] Figure 1 shows the results of testing humanized nanobodies for blocking CD39 enzymatic activity in a PBMC system. [Figure 14] Figure 1 shows the results of testing humanized nanobodies for blocking the enzymatic activity of soluble CD39. [Figure 15] Graph showing results of testing humanized nanobodies to reverse CD4+ T cell proliferation inhibition. [Figure 16]Graph showing the results of testing humanized nanobodies to reverse CD8+ T cell proliferation inhibition. [Figure 17] FIG. 1 shows the structural design of two-epitope antibodies (Bi307 / 308 and Fc-37-46). [Figure 18] 1 is a graph showing the results of testing two epitope antibodies (Bi307 / 308 and Fc-37-46) for blocking CD39 enzymatic activity in human CD39-overexpressing cells. [Figure 19] 1 is a graph showing the results of testing two epitope antibodies (Bi307 / 308 and Fc-37-46) for blocking the enzymatic activity of soluble CD39. [Figure 20] 1 is a graph showing the results of testing two epitope antibodies (Bi307 / 308 and Fc-37-46) for blocking CD39 enzymatic activity in a PBMC system. [Figure 21] 1 is a graph showing the results of a PK study of two epitope antibodies (Bi307 / 308 and Fc-37-46) in mice. [Figure 22] 1 is a graph showing the results of a pharmacodynamic study of two epitope antibodies (Bi307 / 308 and Fc-37-46) in tumor-bearing mice inoculated with MDA-MB-231 cells overexpressing hCD39. [Figure 23] 1 is a graph showing the results of a pharmacodynamics test of two epitope antibodies (Bi307 / 308 and Fc-37-46) in Molp-8 tumor model mice. [Figure 24] FIG. 1 shows the structural design of a two-epitope antibody (46-37-Fc) having an N-terminal base structure. [Figure 25] 1 is a graph showing the results of testing a bi-epitope antibody for blocking enzymatic activity in MOLP-8 tumor cells. [Figure 26] 1 is a graph showing the results of testing bi-epitope antibodies for blocking the enzymatic activity of soluble CD39. [Figure 27] 1 is a graph showing the results of testing bi-epitope antibodies for blocking CD39 enzymatic activity in a PBMC system. [Figure 28]1 is a graph showing the results of testing a bi-epitope antibody to reverse T cell proliferation inhibition. [Figure 29] 1 is a graph showing the results of a PK test of a two-epitope antibody (Fc-37-46) in mice. [Figure 30] 1 is a graph showing the results of a pharmacodynamic study of a two-epitope antibody in tumor-bearing mice inoculated with A375 cells overexpressing hCD39. [Figure 31] FIG. 1 shows the structural design of anti-PD1xCD39 antibodies. [Figure 32] 1 is a graph showing the results of testing the binding activity of anti-PD1xCD39 antibodies to CHO-hCD39 cells. [Figure 33] 1 is a graph showing the results of a test of the binding activity of anti-PD1xCD39 antibodies to CHO-cynoCD39 cells. [Figure 34] 1 is a graph showing the test results of the binding activity of anti-PD1xCD39 antibodies to CHO-hPD1 cells. [Figure 35] 1 is a graph showing the results of testing the binding activity of anti-PD1xCD39 antibodies to CHO-cynoPD1 cells. [Figure 36] 1 is a graph showing the results of testing anti-PD1xCD39 antibodies for blocking enzyme activity in MOLP-8 tumor cells. [Figure 37] 1 is a graph showing the results of testing anti-PD1xCD39 antibodies for blocking CD39 enzyme activity in PBMCs. [Figure 38] Figure 10 is a graph showing the results of testing the blocking activity of anti-PD1xCD39 antibodies in blocking PD1 / PD-L1 binding. [Figure 39] 10 is a graph showing the results of a pharmacodynamic study of anti-PD1xCD39 antibodies in tumor-bearing mice inoculated with A375 cells overexpressing hCD39. DETAILED DESCRIPTION OF THE INVENTION

[0076] Sequence information The sequences involved in the present invention are set out in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Example]

[0077] The present invention will now be described with reference to the following embodiments, which are intended to be illustrative of the invention, rather than limiting thereof. Unless otherwise specified, the molecular biological experimental methods and immunoassay methods used herein basically refer to Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989, and F M. Ausubel et al., Short protocols in molecular biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases are used according to the conditions recommended by the manufacturer. It should be understood that the following examples are provided as illustrations and are not intended to limit the scope of protection of the present invention.

[0078] Example 1 Immunization and screening of anti-CD39 nanobodies Alpacas (llamas) were immunized with human CD39 antigen (commercially available from Sinobiological, product number 16020-H08B). Total RNA was extracted from alpaca peripheral lymphocytes and subjected to reverse transcription to obtain cDNA. The cDNA PCR products were ligated with a yeast display vector and then electrotransformed into Saccharomyces cerevisiae cells (commercially available from ATCC, product number 208289) to construct an anti-CD39 nanobody library.

[0079] Human CD39 protein was labeled with a biotin labeling kit (commercially available from Thermo, product number 90407) according to the manufacturer's instructions. After amplification, the anti-CD39 nanobody yeast library was labeled with biotin-labeled CD39 protein and enriched for positively labeled yeast cells with magnetic beads. After amplification, the enriched yeast cells were added with anti-c-Myc antibody (commercially available from Thermo, product number MA1-980) diluted 1:200 and an appropriate amount of biotin-labeled CD39 for staining. After washing with PBS, the yeast cells were added with goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (commercially available from Invitrogen, product number A32723TR) and streptavidin-APC conjugated fluorescent antibody (commercially available from Invitrogen, product number SA1005) diluted 1:500, followed by a 15-minute incubation. The cells were resuspended in PBS and sorted on a BD FACSAria II instrument to obtain yeast cells with high binding capacity to human CD39.

[0080] The resulting yeast cell liquid with high binding ability to human CD39, obtained through magnetic bead enrichment and flow cytometry sorting, was cultured overnight in amplification medium at 30°C and 225 rpm, and then subjected to yeast plasmid extraction using a yeast plasmid extraction kit (commercially available from Tiangen, product number: DP112) according to the manufacturer's instructions. The plasmid was electrotransformed into Top10 recipient cells (commercially available from Tiangen, product number: CB104-02), plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were selected for sequencing to obtain the VHH (variable region) gene sequences.

[0081] Example 2 Vector construction, protein expression and purification of anti-CD39 nanobodies The VHH-encoding sequence of the anti-CD39 nanobody obtained from the screening was subjected to homologous recombination with a human IgG1 Fc segment-encoding sequence (see SEQ ID NO: 27 for the amino acid sequence) to construct a fusion protein expression sequence. Using the ExpiCHO™ Expression System kit (commercially available from Thermo, product number: A2910001), the fusion protein expression plasmid from the media preparation was transformed into Expi-CHO cells (commercially available from Thermo, product number: A2910002) according to the product instructions. After 5 days of incubation, the supernatant was collected and purified for the target protein using a selection procedure using Protein A magnetic beads (commercially available from Genscript Biotech Corporation, product number: L00723). The magnetic beads were suspended in an appropriate volume (1-4 times the volume of the magnetic beads) of binding buffer (PBS + 0.1% Tween 20, pH 7.4), added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking during the period. The sample was placed on a magnetic shelf (commercially available from Beaver Biosciences Inc.). After discarding the supernatant, the magnetic beads were washed three times with binding buffer, and an appropriate volume (3-5 times the volume of the magnetic beads) of elution buffer (0.1 M sodium citrate, pH 3.2) was added, followed by shaking at room temperature for 5-10 minutes. After placing the sample on the magnetic shelf again, the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1 M Tris, pH 8.54) and mixed until homogenous. Thus, the preparation procedure was completed, yielding purified anti-CD39 nanobodies, R-Ye-19(1)-037 and R-Ye-19(1)-046, whose CDR and variable region amino acid sequences are listed in Table 1.

[0082] Example 3 Affinity testing of anti-CD39 nanobodies at the protein level The ForteBio affinity test was performed according to a conventional method (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and affinity binding. MAbs, 2013.5 (2): pp. 270-8). Briefly, the sensor was equilibrated offline in the analysis buffer for 30 minutes, and then tested online for 60 seconds to establish a baseline. The purified antibody obtained above was then loaded online onto the AHQ sensor, and placed in 100 nM human CD39 (SEQ ID NO: 28) for 5 minutes. The sensor was then transferred to PBS for 5 minutes for dissociation. A 1:1 binding model was used for kinetic analysis. The test was performed using a control antibody I394 BMK (the antibody I394 BMK was derived from the humanized monoclonal antibody I-394, developed by Innate Pharma and disclosed in WO2019068907, which was regarded as the most potent CD39 inhibitor antibody in the landscape), and the light and heavy chain amino acid sequences of the control antibody I394 BMK are shown in SEQ ID NOs: 34 and 35, respectively. The test results are shown in Table 2.

[0083] [Table 2]

[0084] Example 4 Affinity testing of anti-CD39 nanobodies at the cellular level CHO cells overexpressing human CD39 (CHO-hCD39 cells) were produced by transfection with the pCHO1.0 vector (commercially available from Invitrogen, product number HG-VPI0983) carrying CD39 cDNA. 2 × 10 expanded CHO-CD39 cells were cultured at 4°C for 12 h. 6The cells were adjusted to a cell density of 100 cells / ml and added to a 96-well flow plate at 100 μL / well, followed by centrifugation for future use. Purified anti-CD39 antibodies prepared according to Example 2 were diluted in PBS in 3-fold dilutions starting from 400 nM for a total of 12 concentration values. 100 μL / well of the diluted samples were added to each of the 96-well flow plates containing the cells. After incubation at 4°C for 30 minutes, the plate was washed twice with PBS, and then 100 μL / well of goat F(ab')2 anti-human IgG-Fc(PE) (commercially available from Abcam, ab98596), diluted 100-fold in PBS, was added. After incubation at 4°C for 30 minutes, the plate was washed twice with PBS, and then 100 μL / well of PBS was added to resuspend the cells. The cells were then examined using a CytoFlex (Beckman) flow cytometer, and the corresponding MFI values ​​were calculated.

[0085] The test results determined by the above method are shown in FIG. 1 and Table 3, and the results indicated that all purified anti-CD39 antibody samples of the present invention had binding activity to CHO-hCD39 cells. [Table 3] To identify the affinity of antibodies to cynomolgus monkey CD39 (cyCD39) at the cellular level, a CHO cell line overexpressing cynomolgus monkey CD39 (CHO-cyCD39 cells) was constructed according to the method described above, and the affinity of purified antibodies to CHO-cyCD39 at the cellular level was tested. The test results are shown in Figure 2 and Table 4.

[0086] [Table 4]

[0087] Example 5 Determination of epitope bins of anti-CD39 nanobodies The anti-human IgG Fc sensor was first loaded with 100 nM anti-CD39 antibody, equilibrated for 30 seconds, quenched with rituximab for 10 minutes, then loaded with 100 nM human CD39 recombinant protein for 30 seconds, equilibrated for 120 seconds, and then another antibody to be tested was added. If there was a further response, it was an antibody in a different bin; if there was no response, it was an antibody in the same bin. This cycle was repeated until all antibodies were classified by bin. The results are shown in Figure 3 and indicated that R-Ye-19(1)-037 and I394 BMK were from the same bin, but R-Ye-19(1)-046 was from a different bin.

[0088] Example 6 Testing anti-CD39 nanobodies for blocking CD39 enzymatic activity HuCD39-overexpressing cells were added to wells of a 96-well plate at 100,000 cells / well. The plate was centrifuged to remove the supernatant, and purified anti-CD39 antibody prepared according to Example 2 (for dose dependency: starting at 200 nM, 3-fold dilutions, 12 concentration values) was added at 100 μL / well and incubated at 4°C for 30 minutes. 20 μM ATP was added to the plate at 100 μL / well, incubated at 37°C for 30 minutes, and centrifuged. The supernatant was added to a white 96-well plate at 50 μL / well, and then CellTiter-Glo (CTG) was added to the white 96-well plate at 50 μL / well, and then chemiluminescence was read under a Spectra i3x. The results are shown in Figure 4 and Table 5 and indicated that both of the two candidate anti-CD39 nanobodies were capable of inhibiting CD39 enzymatic activity on the surface of CD39-overexpressing cells.

[0089] [Table 5] By referring to the above experimental method, the activity of inhibiting human CD39 enzyme activity was tested in a PBMC system. The results are shown in Figure 5 and Table 6, and indicate that both of the two candidate anti-CD39 nanobodies have the ability to inhibit CD39 enzyme activity in PBMC.

[0090] [Table 6] The inhibitory activity of soluble human CD39 on the CD39 enzymatic activity was tested by referring to the above experimental method. The results are shown in Figure 6 and Table 7. The results indicated that both of the two candidate anti-CD39 nanobodies were capable of inhibiting the CD39 enzymatic activity of soluble CD39. [Table 7]

[0091] Example 7 Affinity testing of humanized anti-CD39 nanobodies The sequences of two antibodies obtained from the screening, namely R-Ye-19(1)-037 and R-Ye-19(1)-046, were humanized. The humanized sequences were subjected to humanized antibody vector construction, expression, and purification according to the methods described in Example 2. Finally, four humanized antibodies, encoded by HZ-R-Ye-19(1)-037-1, HZ-R-Ye-19(1)-037-2, HZ-R-Ye-19(1)-037-3, and HZ-R-Ye-19(1)-037-4, respectively, were obtained from R-Ye-19(1)-037; four humanized modified antibodies, encoded by HZ-R-Ye-19(1)-046-1, HZ-R-Ye-19(1)-046-2, HZ-R-Ye-19(1)-046-3, and HZ-R-Ye-19(1)-046-4, respectively, were obtained from R-Ye-19(1)-046. The CDR and variable region amino acid sequences of each of these humanized antibodies are shown in Table 1. The purified humanized antibodies were subjected to affinity testing at the protein level according to the method described in Example 3. The results are shown in Table 8.

[0092] [Table 8]

[0093] Example 8 Affinity testing of humanized anti-CD39 nanobodies to CHO-hCD39 cells and their species cross-reactivity The purified humanized antibodies were subjected to affinity testing for CHO-hCD39 at the cellular level according to the method described in Example 4. The results are shown in Figure 7 and Table 9 and indicated that the humanized antibodies had cell binding activity equivalent to that of the non-humanized antibodies.

[0094] [Table 9]

[0095] To identify the affinity of the humanized antibodies for cynomolgus monkey CD39 (cyCD39) at the cellular level, a cell line (CHO-cyCD39) was constructed according to the method described in Example 4, and the purified humanized antibodies were subjected to affinity testing for CHO-cyCD39 at the cellular level. The results are shown in Figure 8 and Table 10.

[0096] [Table 10]

[0097] Example 9 Testing humanized anti-CD39 nanobodies for blocking CD39 enzymatic activity The humanized anti-CD39 Nanobodies were tested for their activity in blocking and inhibiting CD39 enzymatic activity on the surface of human CD39-overexpressing cells by reference to the methods described in Example 6. The results are shown in Figure 9 and Table 11 and indicated that the blocking activity of the humanized Nanobodies was slightly better than that of the parent antibody.

[0098] [Table 11]

[0099] Similarly, the humanized anti-CD39 Nanobodies were further tested for their activity in blocking and inhibiting human CD39 enzymatic activity in a PBMC system by reference to the methods described in Example 6. The results are shown in Figure 10 and Table 12 and indicated that some humanized Nanobodies were slightly better than the parental antibodies.

[0100] [Table 12] Similarly, the humanized anti-CD39 Nanobodies were further tested for their ability to block the enzymatic activity of soluble human CD39 by reference to the methods described in Example 6. The results are shown in Figure 11 and Table 13 and indicated that the humanized Nanobodies were slightly better than the parental antibody.

[0101] [Table 13]

[0102] Example 10 Testing the combination of two humanized anti-CD39 nanobodies with different epitopes for blocking CD39 enzymatic activity By referring to the method described in Example 6, a combination of two humanized anti-CD39 nanobodies with different epitopes (i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2, with a molar concentration ratio of HZ-R-Ye-19(1)-037-3 to HZ-R-Ye-19(1)-046-2 of 1:1) was tested for their activity in blocking and inhibiting human CD39 enzymatic activity on the surface of overexpressing cells. The results are shown in Figure 12 and Table 14, and indicated that there was a strong synergistic effect between the two antibodies with different epitopes, i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2, and the combination produced better results than the control antibody.

[0103] [Table 14]

[0104] Similarly, by referring to the method described in Example 6, a combination of two humanized anti-CD39 nanobodies with different epitopes (i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2, with a molar concentration ratio of HZ-R-Ye-19(1)-037-3 to HZ-R-Ye-19(1)-046-2 of 1:1) was tested for their activity in blocking and inhibiting human CD39 enzymatic activity in a PBMC system. The results are shown in Figure 13 and Table 15, and indicated a strong synergistic effect between the two antibodies with different epitopes, i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2.

[0105] [Table 15] Similarly, by referring to the method described in Example 6, a combination of two humanized anti-CD39 nanobodies with different epitopes (i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2, with a molar concentration ratio of HZ-R-Ye-19(1)-037-3 to HZ-R-Ye-19(1)-046-2 of 1:1) was tested for their activity in blocking the enzymatic activity of soluble human CD39. The results are shown in Figure 14 and Table 16, and indicated that there was a strong synergistic effect between the two antibodies with different epitopes, i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2, and the combination produced better results than the control antibody.

[0106] [Table 16]

[0107] Example 11 Testing the combination of two humanized anti-CD39 nanobodies with different epitopes for reversal of T cell proliferation inhibition The experiment was carried out as follows: cryopreserved PBMC cells (commercially available from Sailybio, product number: XFB-HP100B) were thawed, resuspended in X-VIVO15 (commercially available from Lonza, product number: 04-418Q), added with a small amount of DNase, and transferred into a T75 square flask. The flask was placed in a 37°C incubator and incubated for 2 hours to allow adhesion to the flask wall. The suspended cells were pipetted from the culture bottle and centrifuged at 400 × g for 5 minutes, and 10 cells were collected. 8 CTV (commercially available from Invitrogen, product number C34557) diluted 1000 times with PBS was added to each well and incubated at 37°C for 10 minutes. After washing twice with PBS, the cell density was adjusted to 6.0 × 10 6 The cells were conditioned with X-VIVO15 culture medium at 100 μL / well. Simultaneously, a 96-well flat-bottom plate was coated with 1 μg / mL anti-human CD3 OKT-3 (commercially available from Biogene, product number 317348) diluted in PBS at 100 μL / well, incubated in a 37°C incubator for 2 hours, rinsed twice with 100 μL / well of PBS, and the supernatant was discarded. The resulting CTV-labeled cells were added to the 96-well flat-bottom plate at 50 μL / well along with 50 μL / well of gradient-diluted anti-CD39 antibody samples and incubated in a 37°C incubator for 1 hour.

[0108] ATP was diluted to a working concentration of 1500 μM in X-VIVO15 medium, and purified anti-human CD28 (commercially available from BioLegend, product number 302902) was added to a working concentration of 3 μg / mL. The resulting liquid mixture was added at 50 μL / well to a 96-well flat-bottom plate coated with 1 μg / mL anti-human CD3 and incubated in a 37°C incubator for 3-5 days. Wells without ATP were used as positive controls. Flow cytometry was used to determine the proliferation ratio of CTV-labeled T cells. The results are shown in Figures 15 and 16 and indicate that there was a strong synergistic effect between the two antibodies with different epitopes, i.e., HZ-R-Ye-19(1)-037-3 and HZ-R-Ye-19(1)-046-2, and the combination maximally reversed T cell proliferation inhibition, resulting in better results than the control antibody.

[0109] Example 12 Construction and affinity testing of a two-epitope anti-CD39 antibody Two types of biepitope antibodies were designed, and their structures are shown in Figure 17. One had an IgG-like structure (designated Bi307 / 308), and the other had an Fc-C-terminal structure (designated Fc-37-46). Bi307 / 308 consisted of two peptide chains IA and two peptide chains IB. Peptide chain IA contained, from N- to C-terminus, the HZ-R-Ye-19(1)-037-3 VHH (SEQ ID NO: 7)-light chain constant region CL (SEQ ID NO: 32). Peptide chain IB contained, from N- to C-terminus, the HZ-R-Ye-19(1)-046-2 VHH (SEQ ID NO: 16)-heavy chain constant region CH (SEQ ID NO: 33). Fc-37-46 is composed of two peptide chains II, each of which contains, from N- to C-terminus, the monomeric Fc domain (SEQ ID NO: 27)-HZ-R-Ye-19(1)-037-3 VHH (SEQ ID NO: 7)-HZ-R-Ye-19(1)-046-2 VHH (SEQ ID NO: 16). Affinity testing at the protein level was carried out according to the method described in Example 3, and the results are shown in Table 17.

[0110] [Table 17]

[0111] Example 13 Testing a biepitope anti-CD39 antibody that blocks CD39 enzymatic activity By referring to the method described in Example 6, bi-epitope anti-CD39 antibodies were tested for blocking human CD39 enzyme activity in different systems. The results are shown in Figures 18 to 20, respectively, and indicate that among the bi-epitope antibodies, Bi307 / 308, which resembles an IgG structure, exhibited activity comparable to or slightly superior to that of the combination group, and Fc-37-46, which has an Fc-C-terminal structure, exhibited activity comparable to that of the combination group, both of which were superior to the positive control antibody I394 BMK.

[0112] Example 14 PK study of a dual epitope anti-CD39 antibody in mice Half male and half female Balb / c mice were maintained on a 12 / 12 light / dark cycle at 24 ± 2°C, humidity 40-70%, and with food and water ad libitum. On the day of the experiment, Balb / c mice received a single injection of the specific antibody molecule at a dose of 10 mg / kg via the tail vein. Blood samples were collected from the mouse orbit at the following time points post-dose: 5 min, 0.5 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, 336 h, and 504 h. The whole blood samples were left at 2-8°C for 30 min and then centrifuged at 12,000 rpm for 5 min to collect serum. The resulting serum was then centrifuged at 12,000 rpm for 5 min at 2-8°C and stored at -80°C for further determination of the serum concentration of anti-CD39 antibodies by ELISA. The results are shown in Figure 21 and indicate that the half-lives of Bi307 / 308 and Fc-37-46 in mice were approximately 69 h and 141 h, respectively, indicating that the blood concentration of Bi307 / 308 in mice rapidly declined.

[0113] Example 15 Pharmacodynamic testing of a biepitope anti-CD39 antibody for antitumor efficacy in tumor-bearing mice inoculated with hCD39-overexpressing MDA-MB-231 cells In this experiment, anti-CD39 antibodies were tested for anti-tumor efficacy using MDA-MB-231 cells overexpressing hCD39 in B2M KO NDG mice. First, a tumor-bearing mouse model was established by subcutaneous inoculation of MDA-MB-231 cells overexpressing hCD39. Tumors were approximately 50-60 mm. 3 When tumors reached tumor size, mice were intraperitoneally injected with different doses of different antibodies. Mice in each group were monitored for tumor volume and body weight changes at 3-4 day intervals for 6-7 weeks. The dosages and administration modes are shown in Table 18. The results are shown in Figure 22 and indicated that Fc-37-46 produced better in vivo antitumor effects than Bi307 / 308 in this tumor model. [Table 18]

[0114] Example 16 Pharmacodynamic testing of a biepitope anti-CD39 antibody for antitumor efficacy in tumor-bearing mice inoculated with Molp-8 tumor cells In this experiment, anti-CD39 antibodies were tested for anti-tumor efficacy using Molp-8 cells in CB-17 SCID mice. First, mice were subcutaneously inoculated with Molp-8 tumor cells to establish a mouse model bearing the tumors, and then treated with different doses of different antibodies via intraperitoneal injection. Mice in each group were monitored for tumor volume and body weight changes at 3-4 day intervals for 6-7 weeks. The dosages and administration modes are shown in Table 19. The results are shown in Figure 23 and indicate that Fc-37-46 produced better in vivo anti-tumor effects than Bi307 / 308 in this tumor model, while Bi307 / 308 produced little effect. Pharmacodynamic results from two tumor animal models indicated that Bi307 / 308 had weak antitumor effects. Based on its PK data in mice, it was preliminarily determined that Bi307 / 308 had low blood concentrations in mice, resulting in unsustainable pharmacodynamic effects.

[0115] [Table 19]

[0116] Example 17 Construction and affinity testing of a biepitope anti-CD39 antibody with an N-terminal base structure As indicated by the results of previous experiments, Bi307 / 308, which resembles an IgG structure, had unclear antitumor effects due to its poor PK properties in mice. Therefore, we redesigned a biepitope antibody (designated 46-37-Fc) with an N-terminal base structure, which has the structure shown in Figure 24, composed of two peptide chains II, in the order from N-terminus to C-terminus, HZ-R-Ye-19(1)-046-2 VHH (SEQ ID NO: 16)-HZ-R-Ye-19(1)-037-3 VHH (SEQ ID NO: 7)-monomer Fc domain (SEQ ID NO: 27). The two-epitope antibody with an N-terminal base structure, namely, 46-37-Fc, was subjected to affinity testing at the protein level according to the method described in Example 3, and the results are shown in Table 20. [Table 20]

[0117] Example 18 Testing a biepitope anti-CD39 antibody with an N-terminal base structure for blocking CD39 enzymatic activity The biepitope anti-CD39 antibodies were tested for blocking human CD39 enzyme activity in different systems using the method described in Example 6. The results are shown in Figures 25 to 27, respectively, and indicate that among the biepitope antibodies, the biepitope antibody 46-37-Fc with an N-terminal base structure exhibited superior activity compared to the combination group and the positive control antibody I394 BMK, as well as the C-terminal base biepitope antibody Fc-37-46.

[0118] Example 19 Testing a biepitope anti-CD39 antibody with an N-terminal base structure for reversal of T cell proliferation inhibition By referring to the method described in Example 11, bi-epitope anti-CD39 antibodies with N-terminal base structures were tested for reversal of T cell proliferation inhibition, and the results are shown in FIG.

[0119] Example 20 PK study of a biepitope anti-CD39 antibody with an N-terminal base structure in mice Half male and half female Balb / c mice were maintained on a 12 / 12 light / dark cycle at 24 ± 2°C, humidity 40-70%, and with food and water ad libitum. On the day of the experiment, Balb / c mice received a single injection of the specific antibody molecule at a dose of 10 mg / kg via the tail vein. Blood samples were collected from the mouse orbit at the following time points after administration: 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, and 504 hours. The whole blood samples were left at 2-8°C for 30 minutes and then centrifuged at 12,000 rpm for 5 minutes to collect serum. The resulting serum was then centrifuged at 12,000 rpm for 5 minutes at 2-8°C and stored at -80°C for further determination of the serum concentration of anti-CD39 antibody in the serum by ELISA. The results are shown in Figure 29 and indicate that the half-life of 46-37-Fc in mice was 161 hours, and that the serum concentration of 46-37-Fc in mice declined slowly.

[0120] Example 21 Pharmacodynamic testing of a biepitope anti-CD39 antibody for antitumor efficacy in tumor-bearing mice inoculated with hCD39-overexpressing A375 cells In this experiment, anti-CD39 antibodies were tested for anti-tumor efficacy using hCD39-overexpressing A375 cells in B2M KO NDG mice. First, a tumor-bearing mouse model was established by subcutaneously inoculating mice with hCD39-overexpressing A375 cells mixed with a specific ratio of PBMC cells. Tumors were approximately 50-60 mm. 3When the tumors reached tumor size, mice were intraperitoneally injected with different doses of different antibodies. Mice in each group were monitored for tumor volume and body weight changes at 2-3 day intervals for 2 weeks. The dosages and administration modes are shown in Table 21. The results are shown in Figure 30 and indicate that both biepitope anti-CD39 antibodies had anti-tumor efficacy in this tumor model, with the in vivo anti-tumor effect of 46-37-Fc being superior to that of Fc-37-46; and the combination of Fc-37-46 and anti-CD73 antibody (the amino acid sequences of its light and heavy chains are shown in Table 1) produced even better in vivo anti-tumor effects.

[0121] [Table 21]

[0122] Example 22 Construction and affinity testing of anti-PD1xCD39 antibodies Many publications (e.g., Simoni et al., 2018; Paulino et al., 2021) have reported that co-expression of PD1 and CD39 on tumor-infiltrating CD8+ lymphocytes is of great significance for anti-tumor immunity. Based on the anti-PD1 antibody molecule (ADI-54872, the amino acid sequences of its light and heavy chain variable regions are shown in Table 1) (fully independent intellectual property owned by BIOTHEUS INC.), an anti-PD1xCD39 antibody will be designed and its in vitro and in vivo activity will be investigated. As indicated by the structure shown in Figure 31, the anti-PD1xCD39 antibody is composed of two peptide chains III-A and two peptide chains III-B. Peptide chain III-A comprises, from N-terminus to C-terminus, the anti-PD-1 antibody VL (SEQ ID NO: 24)-light chain constant region CL (SEQ ID NO: 32). Peptide chain III-B comprises, from N-terminus to C-terminus, the anti-PD-1 antibody VH (SEQ ID NO: 23)-heavy chain constant region CH (SEQ ID NO: 33)-HZ-R-Ye-19(1)-037-3 VHH (SEQ ID NO: 7)-HZ-R-Ye-19(1)-046-2 VHH (SEQ ID NO: 16).

[0123] Example 23 Cell binding activity of anti-PD1xCD39 antibodies Referring to the experimental method of Example 4, the binding activity of anti-PD1xCD39 antibodies to cells overexpressing human CD39, cynomolgus monkey CD39, and PD1 was tested (the amino acid sequences of the human CD39 and cynomolgus monkey CD39 used are set forth in SEQ ID NOs: 28 and 29, respectively, and the amino acid sequences of the human PD-1 and cynomolgus monkey PD-1 used are set forth in SEQ ID NOs: 30 and 31, respectively). The results are shown in Figures 32 to 35, and indicate that the binding activity of anti-PD1xCD39 antibodies to cells overexpressing CD39 or PD1 was consistent with that of the original antibodies.

[0124] Example 24 Functional activity of anti-PD1xCD39 antibodies The activity of anti-PD1xCD39 antibodies to block human CD39 enzymatic activity was first tested by referring to the experimental method in Example 6. The results are shown in Figures 36 and 37, and indicate that the activity of anti-PD1xCD39 antibodies to block CD39 enzymatic activity is comparable to that of the original anti-CD39 antibody molecule. Additionally, to investigate the activity of anti-PD1xCD39 antibodies to block PD1, anti-PD1xCD39 antibodies were tested for blocking PD-1 / PD-L1 binding according to the experimental method provided by the PD-1 / PD-L1 Blocking Bioassay (commercially available from Promega, product number: J1250). The activity data are shown in Figure 38, and the data indicated that the blocking activity of anti-PD1xCD39 antibodies was consistent with that of anti-PD-1 monoclonal antibody molecules.

[0125] Example 25 Pharmacodynamic testing of anti-PD1xCD39 antibodies for anti-tumor efficacy in tumor-bearing mice inoculated with hCD39-overexpressing A375 cells In this experiment, we tested the antitumor efficacy of hCD39-overexpressing A375 cells in B2M KO NDG mice. First, we established a tumor-bearing mouse model by subcutaneously inoculating mice with hCD39-overexpressing A375 cells mixed with a specific ratio of PBMC cells. When tumors reached approximately 150-200 mm 3 When tumors reached tumor size, mice were intraperitoneally injected with different doses of different antibodies. Mice in each group were monitored for tumor volume and body weight changes every 2-3 days for 2 weeks. The dosages and administration modes are shown in Table 22. The results are shown in Figure 39 and indicate that in this tumor model, the anti-tumor effect of the anti-PD1xCD39 antibody was significantly better than that of the monoclonal antibody and was comparable to or slightly better than that of the combination group.

[0126] [Table 22]

[0127] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on the above disclosure, and all such modifications are within the scope of protection of the present invention. The full scope of protection of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. A nanobody or antigen-binding fragment thereof that has the ability to specifically bind to CD39, (a) CDR1 having the sequence shown in SEQ ID NO: 1 or 44, or a sequence having one or more amino acid substitutions, deletions and / or additions (e.g., one, two or three amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 1 or 44; (b) CDR2 having the sequence shown in SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions and / or additions (e.g., one, two or three amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 2; and (c) CDR3 having the sequence shown in SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions and / or additions (for example, one, two or three amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 3 Including; Preferably, the substitution is a conservative substitution; Preferably, the nanobody or its antigen-binding fragment comprises CDR1 as shown in SEQ ID NO: 1 or 44, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; Preferably, the nanobody or antigen-binding fragment thereof comprises three CDRs of VHH as shown in any one of SEQ ID NOs: 4 to 8; preferably, the nanobody or antigen-binding fragment thereof comprises three CDRs of VHH as determined using the Kabat, Chothia, or IMGT numbering system.

2. (i) The sequence shown in Sequence ID No. 4; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4 or 5 amino acids) compared to the sequence shown in Sequence ID No. 4; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in Sequence ID No.

4. It includes an amino acid sequence selected from; Preferably, the nanobody or antigen-binding fragment thereof according to claim 1, wherein the substitution is a conservative substitution.

3. The nanobody or its antigen-binding fragment is humanized; Preferably, the nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain embryoid antibody gene), wherein the heavy chain framework region may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reverse mutations from human residues to camel residues, according to claim 1.

4. Nanobodies or their antigen-binding fragments, (i) The sequence shown in any one of sequence numbers 5 to 8; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, or 5 amino acids) compared to any one of the sequences shown in Sequence ID No. 5 to 8; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in any one of sequence numbers 5 to 8. It includes an amino acid sequence selected from; Preferably, the nanobody or antigen-binding fragment thereof according to claim 3, wherein the substitution is a conservative substitution.

5. A nanobody or antigen-binding fragment thereof that has the ability to specifically bind to CD39, (a) CDR1 having the sequence shown in SEQ ID NO: 9 or 14, or a sequence having one or more amino acid substitutions, deletions and / or additions (e.g., one, two or three amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 9 or 14; (b) CDR2 having the sequence shown in SEQ ID NO: 10, or a sequence having one or more amino acid substitutions, deletions and / or additions (e.g., one, two or three amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 10; and (c) CDR3 having the sequence shown in SEQ ID NO: 11 or 15, or a sequence having one or more amino acid substitutions, deletions and / or additions (for example, one, two or three amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 11 or 15 Including; Preferably, the substitution is a conservative substitution; Preferably, the nanobody or its antigen-binding fragment comprises CDR1 shown in SEQ ID NO: 9 or 14, CDR2 shown in SEQ ID NO: 10, and CDR3 shown in SEQ ID NO: 11 or 15; preferably, the nanobody or its antigen-binding fragment comprises (1) CDR1 shown in Sequence ID 9, CDR2 shown in Sequence ID 10, and CDR3 shown in Sequence ID 11; (2) CDR1 shown in Sequence ID No. 14, CDR2 shown in Sequence ID No. 10, CDR3 shown in Sequence ID No. 15; or (3) CDR1 shown in Sequence ID No. 14, CDR2 shown in Sequence ID No. 10, and CDR3 shown in Sequence ID No. 11 Including; Preferably, the nanobody or antigen-binding fragment thereof comprises three CDRs of VHH as shown in any one of SEQ ID NOs: 12, 13 and 16-18; preferably, the nanobody or antigen-binding fragment thereof comprises three CDRs of VHH as determined using the Kabat, Chothia or IMGT numbering system.

6. (i) The sequence shown in sequence number 12; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in Sequence ID No. 12; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in Sequence ID No.

12. It includes an amino acid sequence selected from; Preferably, the nanobody or antigen-binding fragment thereof according to claim 5, wherein the substitution is a conservative substitution.

7. The nanobody or its antigen-binding fragment is humanized; Preferably, the nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain embryoid antibody gene), wherein the heavy chain framework region may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reverse mutations from human residues to camel residues, according to claim 5.

8. Nanobodies or their antigen-binding fragments, (i) The sequence shown in any one of sequence numbers 13 and 16-18; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, or 5 amino acids) compared to any one of the sequences shown in Sequence ID No. 13 and 16-18; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in any one of sequence numbers 13 and 16-18. It includes an amino acid sequence selected from; Preferably, the nanobody or antigen-binding fragment thereof according to claim 7, wherein the substitution is a conservative substitution.

9. CD39 is selected from human CD39 and / or cynomolgus monkey CD39; Preferably, the nanobody or antigen-binding fragment thereof can block the enzymatic activity of CD39 to which it binds, according to any one of claims 1 to 8.

10. A polypeptide construct having the ability to specifically bind to CD39, comprising a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 8, and an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain may be connected to the N-terminus and / or C-terminus (e.g., the C-terminus) of a nanobody or its antigen-binding fragment via a peptide linker; Preferably, the immunoglobulin Fc domain is the Fc domain of IgG (for example, the Fc domain of IgG1); Preferably, a polypeptide construct having an immunoglobulin Fc domain that includes the sequence shown in SEQ ID NO: 27, or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 27, or has a sequence that has one or more amino acid substitutions, deletions and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO:

27.

11. A polyspecific antibody comprising a nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 8; Preferably, the polyspecific antibody has the ability to specifically bind to CD39, and additionally, has the ability to specifically bind to one or more other targets; Preferably, the polyspecific antibody further comprises at least one second antibody having binding specificity for a second target.

12. A polyspecific antibody comprising a first antigen-binding domain specific to a first epitope of CD39 and a second antigen-binding domain specific to a second epitope of CD39, wherein the first antigen-binding domain comprises a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 4, and the second antigen-binding domain comprises a nanobody or antigen-binding fragment thereof as described in any one of claims 5 to 8.

13. The first antigen-binding domain and the second antigen-binding domain are VHH, and the polyspecific antibody comprises a peptide chain II containing a monomeric Fc domain, the first antigen-binding domain, and the second antigen-binding domain; Preferably, the monomeric Fc domain comprises CH2 and CH3; Preferably, the polyspecific antibody comprises two peptide chains II; preferably, the two monomeric Fc domains of the two peptide chains II form a dimer; Preferably, the individual domains may be linked by linkers (e.g., flexible peptides containing one or more glycine (G) and / or alanine (A)); Preferably, the multispecific antibody according to claim 12, wherein the peptide chain II comprises, in the order from the N-terminus to the C-terminus, an adjacent first antigen-binding domain and a second antigen-binding domain or an adjacent second antigen-binding domain and a first antigen-binding domain, and further comprises a monomeric Fc domain.

14. Peptide chain II comprises, in order from the N-terminus to the C-terminus, a monomeric Fc domain, a first antigen-binding domain, and a second antigen-binding domain; Preferably, a first antigen-binding domain is connected to the C-terminus of the monomeric Fc domain via a linker (e.g., a flexible peptide containing one or more glycine (G) and / or alanine (A)); and / or a second antigen-binding domain is connected to the C-terminus of the first antigen-binding domain via a linker (e.g., a flexible peptide containing one or more glycine (G) and / or alanine (A)); Preferably, the first antigen-binding domain includes or consists of the amino acid sequence shown in SEQ ID NO: 7; and / or the second antigen-binding domain includes or consists of the amino acid sequence shown in SEQ ID NO: 16; Preferably, peptide chain II contains or consists of the amino acid sequence shown in SEQ ID NO: 21; Preferably, the multispecific antibody according to claim 13, wherein peptide chain II comprises a monomeric Fc domain including, in order from the N-terminus to the C-terminus, the sequence shown in SEQ ID NO: 27, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions and / or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions and / or additions) compared to the sequence shown in SEQ ID NO: 27; a first antigen-binding domain including or consisting of the amino acid sequence shown in SEQ ID NO: 7; and a second antigen-binding domain including or consisting of the amino acid sequence shown in SEQ ID NO:

16.

15. Peptide chain II comprises, in order from the N-terminus to the C-terminus, a second antigen-binding domain, a first antigen-binding domain, and a monomeric Fc domain; Preferably, the first antigen-binding domain may be connected to the C-terminus of the second antigen-binding domain via a linker (e.g., a flexible peptide containing one or more glycine (G) and / or alanine (A)); Preferably, the first antigen-binding domain includes or consists of the amino acid sequence shown in SEQ ID NO: 7; and / or the second antigen-binding domain includes or consists of the amino acid sequence shown in SEQ ID NO: 16; Preferably, the multispecific antibody according to claim 13, wherein peptide chain II contains or consists of the amino acid sequence shown in SEQ ID NO:

22.

16. The first antigen-binding domain and the second antigen-binding domain are VHH, and the polyspecific antibody is, (i) Peptide chain I-A comprising a first antigen-binding domain and a light chain constant region (CL); and, (ii) Peptide chain I-B including a second antigen-binding domain and a heavy chain constant region (CH) Including; Preferably, the CL of peptide chain I-A can form a dimer with the CH1 domain of the heavy chain constant region of peptide chain I-B; Preferably, the polyspecific antibody comprises two peptide chains I-A and two peptide chains I-B; preferably, the heavy chain constant regions of the two peptide chains I-B form a dimer, according to claim 12.

17. (1) Peptide chain I-A comprises a first antigen-binding domain and a light chain constant region (CL) in the order from the N-terminus to the C-terminus; and / or (2) The polyspecific antibody according to claim 16, wherein peptide chain I-B comprises a second antigen-binding domain and a heavy chain constant region (CH) in the order from the N-terminus to the C-terminus.

18. (i) The first antigen-binding domain contains or consists of the amino acid sequence shown in Sequence ID No. 7; Preferably, peptide chain I-A contains or consists of the amino acid sequence shown in SEQ ID NO: 20; and / or, (ii) The second antigen-binding domain contains or consists of the amino acid sequence shown in Sequence ID No. 16; Preferably, the multispecific antibody according to claim 16, wherein peptide chain I-B contains or consists of the amino acid sequence shown in SEQ ID NO:

19.

19. The polyspecific antibody according to claim 12, further comprising an antigen-binding domain specific to a target different from CD39.

20. The polyspecific antibody according to claim 12, further comprising a third antigen-binding domain specific to PD-1.

21. The first and second antigen-binding domains are VHH; the third antigen-binding domain is Fab, and the polyspecific antibody is (1) Peptide chain III-A comprising a light chain variable region and a light chain constant region (CL) of a third antigen-binding domain; and, (2) comprising peptide chain III-B including a heavy chain variable region, a heavy chain constant region, a first antigen-binding domain and a second antigen-binding domain of a third antigen-binding domain; preferably, peptide chain III-B includes adjacent first antigen-binding domains and second antigen-binding domains or adjacent second antigen-binding domains and first antigen-binding domains in the order from N-terminus to C-terminus, and further comprising a heavy chain variable region and a heavy chain constant region of a third antigen-binding domain; Preferably, the CL of peptide chain III-A can form a dimer with the CH1 domain of the heavy chain constant region of peptide chain III-B; Preferably, the polyspecific antibody comprises two peptide chains III-A and two peptide chains III-B; preferably, the heavy chain constant regions of the two peptide chains III-B form a dimer; Preferably, the individual domains may be linked to each other through a linker (e.g., a flexible peptide comprising one or more glycine (G) and / or alanine (A)) as described in claim 20, the polyspecific antibody.

22. (1) Peptide chain III-A comprises, in order from the N-terminus to the C-terminus, a light chain variable region and a light chain constant region (CL) of the third antigen-binding domain; and / or, (2) The polyspecific antibody according to claim 21, wherein the peptide chain III-B comprises, in the order from the N-terminus to the C-terminus, a heavy chain variable region of a third antigen-binding domain, a heavy chain constant region, a first antigen-binding domain, and a second antigen-binding domain.

23. The following features: (i) The first antigen-binding domain is connected to the C-terminus of the heavy chain constant region via a linker (e.g., a flexible peptide containing one or more glycine (G) and / or alanine (A)); and / or the second antigen-binding domain is connected to the C-terminus of the first antigen-binding domain via a linker (e.g., a flexible peptide containing one or more glycine (G) and / or alanine (A)); (ii) The first antigen-binding domain includes or consists of the amino acid sequence shown in Sequence ID No. 7; (iii) The second antigen-binding domain includes or consists of the amino acid sequence shown in Sequence ID No. 16; (iv) The heavy chain variable region of the third antigen-binding domain contains VH CDR 1-3 as described in SEQ ID NOs. 36-38; (v) The light chain variable region of the third antigen-binding domain contains VL CDRs 1-3 as described in SEQ ID NOs: 39-41; (vi) The heavy chain variable region of the third antigen-binding domain includes or consists of the amino acid sequence shown in SEQ ID NO: 23; (vii) The light chain variable region of the third antigen-binding domain includes or consists of the amino acid sequence shown in SEQ ID NO:

24. A polyspecific antibody according to claim 22, having one or more of the above.

24. A polyspecific antibody having the ability to specifically bind to both CD39 and PD-1, comprising a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 8, and an antigen-binding domain specific to PD-1; Preferably, a polyspecific antibody comprising PD-1-specific antigen-binding domains, each containing VH CDR 1-3 as described in SEQ ID NOs. 36-38 and / or VL CDR 1-3 as described in SEQ ID NOs. 39-41.

25. An isolated nucleic acid molecule encoding a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

26. A vector comprising an isolated nucleic acid molecule as described in claim 25, preferably a cloning vector or an expression vector.

27. A host cell comprising the nucleic acid molecule described in claim 25.

28. A method for preparing a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 8, comprising culturing a host cell containing a nucleic acid molecule encoding a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 8 under conditions that enable protein expression, and recovering the nanobody or an antigen-binding fragment thereof from the cultured host cell culture.

29. (i) a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, a polypeptide construct comprising the nanobody or antigen-binding fragment thereof, a nucleic acid molecule encoding the nanobody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule or vector; and (ii) A nanobody or antigen-binding fragment thereof according to any one of claims 5 to 8, a polypeptide construct comprising the nanobody or antigen-binding fragment thereof, a nucleic acid molecule encoding the nanobody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule or vector A composition containing the following:

30. A pharmaceutical composition comprising a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 8, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor is selected from anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD73 antibody, or a combination thereof; Preferably, the anti-PD-1 antibody comprises VH CDR 1-3 as described in SEQ ID NOs. 36-38 and / or VL CDR 1-3 as described in SEQ ID NOs. 39-41; Preferably, the pharmaceutical composition comprises an anti-PD-1 antibody VH as shown in SEQ ID NO: 23 and / or VL as shown in SEQ ID NO:

24.

31. (1) To reduce the enzymatic activity of CD39 in vitro or in vivo (for example, in humans); (2) To reduce adenosine-mediated immunosuppression in the target population (e.g., humans); (3) For the prevention and / or treatment of tumors in subjects (e.g., humans); or (4) To prevent and / or treat infectious diseases in subjects (e.g., humans) The use of a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a pharmaceutical; Preferably, the tumor is accompanied by CD39-positive tumor cells; Preferably, the tumor is selected from solid tumors or hematological malignancies (e.g., leukemia, lymphoma); Preferably, the tumor is selected from colorectal cancer, colon cancer, bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell carcinoma, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and melanoma; Preferably, the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections; Preferably, the subject is a mammal, such as a human or a monkey; Preferably, the nanobody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents; Preferably, the nanobody or its antigen-binding fragment is used in combination with an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor used is selected from anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD73 antibody, or a combination thereof.

32. A conjugate comprising a nanobody or an antigen-binding fragment thereof according to any one of claims 1 to 8, and a detectable marker connected to the nanobody or the antigen-binding fragment; Preferably, the detectable marker is a conjugate selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine ester compounds, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins), radionuclides, or biotin.

33. A kit comprising a nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 8; Preferably, the kit comprises a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 8, and a second antibody having the ability to specifically recognize the nanobody or antigen-binding fragment thereof; the second antibody may further comprise a detectable marker, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

34. A method for detecting the presence or level of CD39 in a sample, comprising the use of a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 8; Preferably, the method is an immunoassay, such as an immunoblotting assay, an enzyme immunoassay (e.g., ELISA), a chemiluminescent immunoassay, a fluoroimmunoassay, or a radioimmunoassay; Preferably, the method comprises using a nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 8, and further comprising using a second antibody having a detectable marker (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridine ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin) to detect the nanobody or antigen-binding fragment.

35. (1) Contacting a sample with a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 8; (2) The method according to claim 34, comprising detecting the formation of an antigen-antibody immune complex or determining the amount of an immune complex, wherein the formation of the immune complex indicates the presence of CD39 or CD39-expressing cells.

36. The use of a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a test reagent for detecting the presence or level of CD39 in a sample; Preferably, the sample is a cell sample (e.g., tumor cells) derived from the subject (e.g., a mammal, preferably a human or monkey).

37. The polyspecific antibody according to claim 22, wherein peptide chain III-A contains or consists of the amino acid sequence shown in SEQ ID NO: 26 and / or peptide chain III-B contains or consists of the amino acid sequence shown in SEQ ID NO:

25.

38. The polyspecific antibody according to claim 24, wherein the antigen-binding domain specific to PD-1 comprises VH as shown in SEQ ID NO: 23 and / or VL as shown in SEQ ID NO:

24.

39. The polyspecific antibody according to claim 24, comprising or consisting of a light chain of the amino acid sequence shown in SEQ ID NO: 26 and / or a heavy chain of the amino acid sequence shown in SEQ ID NO: 25.

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