CD39 binding proteins

Abstract

The present disclosure provides antibodies, in particular heavy chain antibodies or nanoantibodies, capable of specifically binding to CD39, as well as nucleic acids, vectors or cells encoding the antibodies, and diagnostic or therapeutic uses of the antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to antigen binding proteins, heavy chain antibodies and nano antibodies, and uses thereof. Background Art

[0002] CD39, also known as extracellular ATP diphosphate hydrolase-1, is an extracellular nucleotide hydrolase encoded by the ENTPD1 gene. CD39 is a member of the ATP-adenosine pathway, which, together with CD73, is responsible for the cascade conversion of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and cyclic adenosine monophosphate (cAMP), ultimately leading to the release of an immunosuppressive form of extracellular adenosine (ADO) in the tumor microenvironment.

[0003] Various types of cells may release ATP into the microenvironment through designated channels or due to cell death or stress necrosis. High levels of extracellular ATP (eATP) provide a strong inflammatory signal through the involvement of P2 receptors (P2Y and P2X families), which are essential for the activation of innate and adaptive immune responses. Therefore, CD39 upregulation is an effective mechanism for tumors to evade anti-tumor strategies by depleting immunostimulatory eATP in the tumor microenvironment (TME). Due to its important role in the immunosuppressive response of the tumor microenvironment, CD39 has gradually attracted attention in recent years.

[0004] CD39 is overexpressed in human tumor cells such as lymphoma, breast cancer, bladder cancer, colon cancer, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cancer, melanoma, etc. CD39 is also expressed in vascular endothelial cells, cancer-associated fibroblasts (CAFs), and some immune cells (especially natural killer (NK) cells, tumor-associated macrophages (TAMs), and tumor-infiltrating lymphocytes (TILs), including Tregs and CD8+ T cells).

[0005] By blocking CD39, the conversion of ATP / AMP into extracellular adenosine can be limited, thereby achieving an inhibitory effect on extracellular adenosine (ADO)-mediated immunosuppression in the tumor microenvironment.

[0006] Monoclonal antibodies have been successfully applied in biological targeted therapy and cancer detection, and play an important role in biomedicine and immunotherapy. Compared with conventional monoclonal antibodies, nano antibodies have the advantages of small molecular weight, good solubility, strong stability, weak immunogenicity, strong penetration, high specificity, simple humanization, high expression, and easy production. Therefore, the development of a therapeutic nano antibody or a diagnostic reagent for detecting the expression of CD39 in cancer has broad applications. SUMMARY OF THE INVENTION

[0008] The present disclosure provides CD39 binding proteins that specifically bind to CD39, in particular, Nanobodies that specifically bind to CD39.

[0009] Therefore, in a first aspect of the present disclosure, a CD39 binding protein is provided.

[0010] The CD39 binding protein according to an embodiment of the present invention can specifically target and bind to CD39. The CD39 binding protein according to an embodiment of the present invention is a candidate for detecting CD39 or cells containing CD39.

[0011] In some embodiments, the CD39 binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NO: 88-116. In some embodiments, the CDR1, CDR2 and CDR3 are encoded according to Kabat, AbM, Chothia or IMGT.

[0012] In some embodiments of the above antibodies or antigen-binding fragments, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 are encoded according to IMGT, wherein:

[0013] (1) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 1, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 2, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 3;

[0014] (2) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:4, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:5, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:6;

[0015] (3) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:7, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:8, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:9;

[0016] (4) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 10, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 11, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 12;

[0017] (5) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 13, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 14, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 15;

[0018] (6) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 16, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 17, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 18;

[0019] (7) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 19, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 21;

[0020] (8) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:22, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:23, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:24;

[0021] (9) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:25, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:26, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:27;

[0022] (10) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:28, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:29, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:30;

[0023] (11) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:31, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:32, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:33;

[0024] (12) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:34, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:35, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:36;

[0025] (13) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:37, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:38, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:39;

[0026] (14) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:40, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:41, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:42;

[0027] (15) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:43, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:44, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:45;

[0028] (16) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:46, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:47, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:48;

[0029] (17) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:49, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:50, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:51;

[0030] (18) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:52, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:53, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:54;

[0031] (19) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:55, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:56, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:57;

[0032] (20) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:58, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:59, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:60;

[0033] (21) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:61, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:62, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:63;

[0034] (22) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:64, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:65, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:66;

[0035] (23) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:67, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:68, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:69;

[0036] (24) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:70, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:71, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:72;

[0037] (25) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:73, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:74, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:75;

[0038] (26) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:76, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:77, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:78;

[0039] (27) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:79, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:80, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:81;

[0040] (28) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:82, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:83, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:84; or

[0041] (29) The CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:85, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:86, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:87.

[0042] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs:88-116, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs:88-116.

[0043] In some embodiments of the present disclosure, the CD39 binding protein is monovalent, bivalent, or multivalent.

[0044] In some embodiments of the present disclosure, the CD39 binding protein is monospecific, bispecific, or multispecific.

[0045] In some embodiments of the present disclosure, the CD39 binding protein is a heavy chain antibody. In some embodiments, the heavy chain antibody further comprises human IgG1 Fc.

[0046] In some embodiments of the present disclosure, the CD39 binding protein is a nanobody. The nanobody according to the embodiments of the present invention can specifically target and bind to CD39. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in any one of SEQ ID NOs: 88-116. In some preferred embodiments, the nanobody comprises an amino acid sequence shown in any one of SEQ ID NOs: 88-116 or consists of an amino acid sequence shown in any one of SEQ ID NOs: 88-116.

[0047] The Nanobody with the amino acid sequence shown in SEQ ID NO: 88 corresponds to clone AHP18698 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 89 corresponds to clone AHP18706 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 90 corresponds to clone AHP18686 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 91 corresponds to clone AHP18697 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 92 corresponds to clone AHP18699 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 93 corresponds to clone AHP17815 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 94 corresponds to clone AHP17863 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 95 corresponds to clone AHP17849 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 96 corresponds to clone AHP17850 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 97 corresponds to the clone AHP17852 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 98 corresponds to the clone AHP17854 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 99 corresponds to the clone AHP17871 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 100 corresponds to the clone AHP18674 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 101 corresponds to the clone AHP18683 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 102 corresponds to the clone AHP18685 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 103 corresponds to the clone AHP18689 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 104 corresponds to the clone AHP18693 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 105 corresponds to the clone AHP18694 of the present disclosure; The Nanobody with the amino acid sequence shown in NO: 106 corresponds to the clone AHP18701 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 107 corresponds to the clone AHP18707 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 108 corresponds to the clone AHP24611 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 109 corresponds to the clone AHP24615 of the present disclosure;The Nanobody with the amino acid sequence shown in SEQ ID NO: 110 corresponds to clone AHP24616 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 111 corresponds to clone AHP24618 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 112 corresponds to clone AHP24620 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 113 corresponds to clone AHP24622 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 114 corresponds to clone AHP24626 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO: 115 corresponds to clone AHP24627 of the present disclosure; and the Nanobody with the amino acid sequence shown in SEQ ID NO: 116 corresponds to clone AHP24629 of the present disclosure;

[0048] In other embodiments, the nanobody comprises a heavy chain framework region, and at least a portion of the heavy chain framework region is from at least one of a mouse antibody, a human antibody, a primate antibody, and a mutant thereof. Preferably, when the heavy chain framework region is derived from a human antibody, the immunogenicity of the nanobody is low.

[0049] In a second aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the CD39 binding protein described in the first aspect of the present disclosure.

[0050] In some embodiments of the present disclosure, nucleic acid molecules can be introduced into host cells to express the CD39 binding protein described in the first aspect of the present disclosure.

[0051] In the third aspect of the present disclosure, an expression vector comprising the nucleic acid molecule described in the second aspect of the present disclosure is provided. As described above, the nucleic acid molecule encodes the CD39 binding protein described in the first aspect of the present disclosure. Therefore, the expression vector introduced into the host cell according to the embodiment of the present invention can express the CD39 binding protein described in the first aspect of the present disclosure under conditions suitable for protein expression.

[0052] In some embodiments of the present disclosure, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0053] In a fourth aspect of the present disclosure, a recombinant cell comprising the nucleic acid molecule described in the second aspect of the present disclosure or the expression vector described in the third aspect of the present disclosure is provided.

[0054] In some embodiments of the present disclosure, the recombinant cell is obtained by introducing the expression vector described in the third aspect of the present disclosure into a host cell.

[0055] In some embodiments of the present disclosure, the recombinant cell is a prokaryotic cell or a eukaryotic cell.

[0056] In some embodiments of the present disclosure, the recombinant cell is a mammalian cell, such as a HEK293 cell.

[0057] In a fifth aspect of the present disclosure, a conjugate is provided.

[0058] In some embodiments of the present disclosure, the conjugate comprises the CD39 binding protein of the first aspect of the present disclosure, and further comprises a therapeutic agent, a diagnostic agent or an imaging agent. In some embodiments, there is a linker between the CD39 binding protein of the first aspect of the present disclosure and the therapeutic agent, a diagnostic agent or an imaging agent.

[0059] In some embodiments of the present disclosure, the therapeutic agent is a small molecule cytotoxic drug.

[0060] The conjugate according to an embodiment of the present invention can be targeted and act on target cells containing CD39 under the guidance of the CD39 binding protein described in the first aspect of the present disclosure.

[0061] In the sixth aspect of the present disclosure, a composition is provided comprising the CD39 binding protein described in the first aspect of the present disclosure, the nucleic acid described in the second aspect of the present disclosure, the expression vector described in the third aspect of the present disclosure, the recombinant cell described in the fourth aspect of the present disclosure, and / or the conjugate described in the fifth aspect of the present disclosure.

[0062] In some embodiments of the present disclosure, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0063] In the seventh aspect of the present disclosure, provided is the use of the CD39 binding protein described in the first aspect of the present disclosure, the nucleic acid described in the second aspect of the present disclosure, the expression vector described in the third aspect of the present disclosure, the recombinant cell described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, and / or the composition described in the sixth aspect of the present disclosure in the preparation of a medicament for preventing, treating or alleviating CD39-related diseases.

[0064] Also provided is the use of the CD39 binding protein of the first aspect of the disclosure, the nucleic acid of the second aspect of the disclosure, the expression vector of the third aspect of the disclosure, the recombinant cell of the fourth aspect of the disclosure, the conjugate of the fifth aspect of the disclosure, and / or the composition of the sixth aspect of the disclosure in combination with another agent in the preparation of a drug for preventing, treating or alleviating a disease associated with CD39. In some embodiments, the other agent is an antibody or a chemotherapeutic agent.

[0065] In the eighth aspect of the present disclosure, provided is the CD39 binding protein described in the first aspect of the present disclosure, the nucleic acid described in the second aspect of the present disclosure, the expression vector described in the third aspect of the present disclosure, the recombinant cell described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, and / or the composition described in the sixth aspect of the present disclosure, which is used for preventing, treating or alleviating diseases associated with CD39.

[0066] In the ninth aspect of the present disclosure, a method for preventing, treating or alleviating a CD39-related disease in a subject is provided. In some embodiments, the method comprises administering to the subject the CD39 binding protein described in the first aspect of the present disclosure, the nucleic acid described in the second aspect of the present disclosure, the expression vector described in the third aspect of the present disclosure, the recombinant cell described in the fourth aspect of the present disclosure, the conjugate described in the fifth aspect of the present disclosure, and / or the composition described in the sixth aspect of the present disclosure.

[0067] In some embodiments of the seventh aspect to the ninth aspect of the present disclosure, the CD39-related disease or condition is a CD39-mediated disease or condition. In some embodiments, the CD39-mediated disease or condition is a tumor. In some embodiments, the CD39-mediated disease or condition is a solid tumor and / or a hematological tumor. In some specific embodiments, the CD39-mediated disease or condition is one or more tumors selected from the group consisting of: lymphoma, breast cancer, bladder cancer, colon cancer, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer and melanoma.

[0068] In the tenth aspect of the present disclosure, a kit for detecting CD39 or cells containing CD39 is provided. In some embodiments, the kit comprises the CD39 binding protein of the first aspect of the present disclosure, or the conjugate of the fifth aspect of the present disclosure.

[0069] In the eleventh aspect of the present disclosure, provided is use of the CD39 binding protein described in the first aspect of the present disclosure or the conjugate described in the fifth aspect of the present disclosure in preparing a kit for detecting CD39 or cells containing CD39.

[0070] In the twelfth aspect of the present disclosure, provided is the CD39 binding protein of the first aspect of the present disclosure or the conjugate of the fifth aspect of the present disclosure, which is used for detecting CD39 or cells containing CD39.

[0071] In the thirteenth aspect of the present disclosure, a method for detecting CD39 or cells containing CD39 is provided. In some embodiments of the present disclosure, the method comprises contacting the CD39 binding protein described in the first aspect of the present disclosure or the conjugate described in the fifth aspect of the present disclosure with a sample to be tested. A method for determining the presence and / or content of CD39 is also provided, comprising contacting the CD39 binding protein described in the first aspect of the present disclosure and / or the conjugate described in the fifth aspect of the present disclosure with a sample to be tested.

[0072] In the fourteenth aspect of the present disclosure, a chimeric antigen receptor is provided. In some embodiments of the present disclosure, the chimeric antigen receptor comprises an antigen recognition domain, a hinge region, a transmembrane binding domain, and an intracellular domain (including a co-stimulatory domain and a signal transduction domain), and the antigen recognition domain comprises the CD39 binding protein described in the first aspect of the present disclosure.

[0073] Further aspects and advantages will be described below, at least part of which will become apparent from the following description in conjunction with the accompanying drawings, and / or will be apparent to those of ordinary skill in the art from the embodiments described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The above features and advantages of the present invention and additional features and advantages thereof will be more clearly understood from the following detailed description of the embodiments in conjunction with the accompanying drawings.

[0075] Figure 1 The titer FACS test results (IgG antibody) of alpaca serum obtained after immunizing alpacas with human CD39 protein five times are shown. Black: CHO-K1(-); Red: CHO-K1 / CD39(+); Green: CHO-K1 / Cyno CD39(+).

[0076] Figure 2 The titer FACS test results of alpaca serum obtained after immunizing alpacas with human CD39 protein five times (VHH antibody) are shown. Black: CHO-K1(-); Red: CHO-K1 / CD39(+); Green: CHO-K1 / Cyno CD39(+).

[0077] Figure 3 The IMGT-CDR3 length frequency distribution plot of the library is shown.

[0078] Figure 4The FACS test results of 29 recombinantly expressed VHH-Fc (hIgG1) antibodies are shown. The black curve in each panel represents the FACS result of CHO-K1 (-) cells, the red curve represents the test result of CHO-K1 / CD39 (+) cells, and the green curve represents the test result of CHO-K1 / Cyno CD39 (+) cells. Human IgG represents an irrelevant IgG antibody, which is a negative control. Positive Ab represents Anti-human CD39 Antibody (Acro), which is a positive control. PBS represents phosphate-buffered saline without antibodies, which is a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0080] The above features and advantages of the present invention and additional features and advantages thereof will be more clearly understood from the following detailed description of the embodiments in conjunction with the accompanying drawings.

[0081] The embodiments described herein with reference to the accompanying drawings are explanatory, illustrative, and are used for a general understanding of the present invention. The embodiments should not be construed as limiting the scope of the present invention. The same or similar elements and elements with the same or similar functions are represented by the same reference numerals in the overall description.

[0082] Unless otherwise specified or defined, all terms used have the usual meaning in the art well known to the skilled person. For example, reference is made to standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology" (6th edition), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunology" (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), as well as the general background technology cited above.

[0083] Unless otherwise indicated, the term "immunoglobulin sequence" - whether it is used herein to refer to a heavy chain antibody or a conventional 4-chain antibody - is used as a general term to include full-length antibodies, single chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as V HH Domain or V H / V L In addition, the terms "sequence" (e.g., "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "V HH Terms such as "sequence" or "protein sequence" should generally be understood to include the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the relevant amino acid sequence, unless the context requires a more limited interpretation.

[0084] Unless otherwise indicated, as will be clear to the skilled person, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se. For example, reference is again made to the standard manuals and general background art mentioned herein and to the further references cited therein.

[0085] For comparison of two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at corresponding positions in the second sequence] by [the total number of nucleotides / amino acids in the first sequence] and multiplying by [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence is considered to be a difference of a single nucleotide (position) compared to the first nucleotide sequence.

[0086] Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment such as NCBI Blast v2.0 using standard settings.

[0087] Some other techniques, computer algorithms and setups for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357 768-A.

[0088] For comparison of two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions in the second amino acid sequence] by [the total number of nucleotides in the first amino acid sequence] multiplied by [100%], wherein the deletion, insertion, substitution or addition of each amino acid residue in the second amino acid sequence is considered to be a difference of a single amino acid residue (position) compared to the first amino acid sequence, i.e., an "amino acid difference" as defined herein.

[0089] Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms such as those described above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.

[0090] Typically, to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the most amino acid residues will be used as the "first" amino acid sequence and the other amino acid sequence will be used as the "second" amino acid sequence.

[0091] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04 / 037999, GB-A-2 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferably) the types and / or combinations of these substitutions can be selected according to the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and the further references cited therein.

[0092] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0093] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0094] Any amino acid substitutions described herein applied to polypeptides may also be based on an analysis of the frequency of amino acid variations between homologous proteins from different species as developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, an analysis of structure-forming potential as developed by Chou and Fasman, Biochemistry 13:211, 1974 and Adv. Enzymol., 47:45-149, 1978, and an analysis of protein hydrophobicity patterns as developed by Eisenberg et al., Proc. Nat. Acad Sci. USA 81:140-144, 1984; Kyte & Doolittle, J Mol. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986, the disclosures of which are incorporated herein by reference in their entirety.

[0095] Information on the primary, secondary and tertiary structures of Nanobodies is given in the description herein and in the general background art cited above. In addition, for this purpose, the crystal structure of the VHH domain from llama is given, for example, by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); Vol. 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). The conventional V H Some of the amino acid residues in the domains that form the VH / VL interface and further information on potential camelizing substitutions at these positions.

[0096] Amino acid sequences and nucleic acid sequences are said to be "identical" if they have 100% sequence identity (as defined herein) over their entire length.

[0097] A nucleic acid sequence or an amino acid sequence is considered to be "in substantially isolated form", e.g., when it has been separated from at least one other component with which it is normally associated in said source or medium, such as another nucleic acid, another protein / polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component, compared to its natural biological source and / or the reaction medium or culture medium from which it was obtained. In particular, a nucleic acid sequence or an amino acid sequence is considered to be "substantially isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. A nucleic acid sequence or an amino acid sequence "in substantially isolated form" is preferably substantially homogeneous, as determined using a suitable technique, such as a suitable chromatographic technique, such as polyacrylamide-gel electrophoresis.

[0098] The term "antigenic determinant" refers to an epitope on an antigen that is recognized by an antigen binding molecule (e.g., an antibody, heavy chain antibody, or nanobody of the invention), more specifically an epitope recognized by the antigen binding site of the molecule. The terms "antigenic determinant" and "epitope" may also be used interchangeably herein.

[0099] An amino acid sequence (such as a Nanobody, an antibody or a heavy chain antibody) that can bind, has affinity and / or has specificity for a specific antigenic determinant, epitope, antigen or protein (or at least a part, fragment or epitope thereof) is said to be "against" or "directed against" the antigenic determinant, epitope, antigen or protein.

[0100] The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen binding molecule or antigen binding protein (e.g., nanobodies or polypeptides of the present invention) molecule can bind to. The specificity of an antigen binding protein can be determined based on affinity and / or avidity. Affinity is represented by the equilibrium constant (KD) of the dissociation of the antigen and the antigen binding protein, and is a measure of the binding strength between an antigenic determinant and the antigen binding site of the antigen binding protein: the smaller the KD value, the stronger the binding strength between the antigenic determinant and the antigen binding molecule (or, affinity can also be expressed as an affinity constant (KA), which is 1 / KD). Avidity is a measure of the binding strength between an antigen binding molecule (e.g., nanobodies, antibodies, or heavy chain antibodies of the present invention) and the relevant antigen. Avidity relates to the affinity between an antigenic determinant and the antigen binding site of an antigen binding molecule and the number of relevant binding sites present on the antigen binding molecule. Typically, an antigen binding protein (e.g., nanobodies and / or heavy chain antibodies of the present invention) will be expressed as 10 -5 Up to 10 -12mol / L or less, preferably with a dissociation constant (KD) of 10 -7 Up to 10 -12 mol / L or less, more preferably 10 -8 Up to 10 -12 mol / L, and / or bind with a dissociation constant (KD) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , for example at least 10 12 M -1 It is generally considered that any binding affinity greater than 10 -4 The KD value in moles / liter represents non-specific binding. Preferably, the CD39 binding proteins of the invention, in particular nanobodies, will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 500 pM. The specific binding of the antigen-binding protein to the antigen or antigenic determinant can be determined by any suitable means known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays and different variants known per se in the art.

[0101] The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain of the present disclosure is a "domain antibody", such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae as defined below.

[0102] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e. "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VH domain") and the light chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VL domain"). The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.

[0103] In the context of the present disclosure, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody" and "nanobody" are used interchangeably.

[0104] As further described herein, the amino acid sequence and structure of Nanobodies can be considered - without being limited thereto - to comprise four framework regions or "FRs", referred to in the art and herein as "Framework Region 1" or "FR1"; "Framework Region 2" or "FR2"; "Framework Region 3" or "FR3"; and "Framework Region 4" or "FR4", respectively; these framework regions are interrupted by three complementarity determining regions or "CDRs", referred to in the art as "Complementarity Determining Region 1" or "CDR1"; "Complementarity Determining Region 2" or "CDR2"; and "Complementarity Determining Region 3" or "CDR3", respectively.

[0105] As also further described herein, the total number of amino acid residues in a Nanobody may be in the range of 120 - 150. It should however be noted that parts, fragments, analogs or derivatives of Nanobodies (as further described herein) have no particular limitation as to their length and / or size, as long as these parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.

[0106] The amino acid residues of the nanobody are based on the V given by Kabat et al. ("Sequence of protein of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). H The domains are numbered according to the common numbering used for the V domains of camelids, such as Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999). HH Domain (see, e.g., the above references Figure 2 ). In this regard, it should be noted that - as is known in the art for V H Domain and for V HH It is well known that the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues represented by the Kabat numbering. That is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering. This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0107] V H The alternative method of numbering the amino acid residues in the domains can also be applied in a similar manner to V from camelids. HH Domains and Nanobodies, which are the methods described by Chothia et al. (Nature 342, 877-883 (1989), the so-called "AbM definition" and the so-called "CONTACT definition". However, in the present specification, claims and drawings, unless otherwise stated, V will be followed. HH The domains are numbered according to IMGT.

[0108] Following the terminology used in the above references, the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “V HH domain" to combine it with the heavy chain variable domain present in a conventional 4-chain antibody (hereinafter referred to as "V H domain") and the light chain variable domain present in a conventional 4-chain antibody (hereinafter referred to as the "V L domains”).

[0109] As described in the prior art mentioned above, V HH The domains have many unique structural features and functional properties that make the isolated V HHDomain (and based on V HH The domains are similar to those of naturally occurring V HH domains share these structural features and functional properties) and contain isolated V HH Proteins containing V domains are very useful as functional antigen binding domains or proteins. In particular, but not limited to, V HH The V domain (which has essentially been "designed" to functionally bind to the antigen in the absence of and without any interaction with the light chain variable domain) and the Nanobody can act as a single, relatively small, functional antigen-binding structural unit, domain or protein. HH The domain structure is similar to that of a conventional 4-chain antibody. H and V L The latter are usually not suitable for practical application as single antigen-binding proteins or domains by themselves, but need to be combined in some form to provide a functional antigen-binding unit (e.g., in conventional antibody fragments such as Fab fragments; in L Domain covalently linked to V H domains).

[0110] Due to these unique characteristics, using V HH Domains and Nanobodies as single antigen binding proteins or antigen binding domains (i.e. as part of a larger protein or polypeptide) are comparable to those using conventional V H and V L Domains, ScFv or conventional antibody fragments (such as Fab or F(ab')2 fragments) offer a number of significant advantages: only a single domain is required to bind antigen with high affinity and selectivity, so there is no need for the presence of two separate domains, nor is there a need to ensure that the two domains are present in the correct spatial conformation and configuration (i.e., by using a specially designed linker as in ScFv).

[0111] V HH The domains and nanobodies can be expressed from a single gene, without the need for post-translational folding or modifications.

[0112] V HH Domains and nanobodies can be easily engineered into multivalent and multispecific formats.

[0113] V HH Domains and Nanobodies are highly soluble and have no tendency to aggregate (as described in "Mouse-derived antigen-binding domains" by Ward et al., Nature, Vol. 341, 1989, p. 544).

[0114] V HHDomains and Nanobodies are highly stable to heat, pH, proteases and other denaturing agents or conditions (see, e.g., Ewert et al., supra).

[0115] V HH Domains and nanobodies are easy to prepare relatively cheaply even on the scale expected for production. HH Domains, Nanobodies and proteins / polypeptides comprising them can be produced using microbial fermentation and do not require the use of mammalian expression systems as used, for example, for conventional antibody fragments.

[0116] Compared with traditional 4-chain antibodies and their antigen-binding fragments, V HH Domain and nanobodies are relatively small (approximately 15 kDa, or 10-fold smaller than traditional IgG) and therefore exhibit higher tissue (including but not limited to solid tumors and other dense tissue) penetration than such conventional 4-chain antibodies and antigen-binding fragments thereof.

[0117] V HH Domains and nanobodies can display so-called cavity binding properties (especially due to their affinity with conventional V H domains compared to the extended CDR3 loop), and thus can also access targets and epitopes that are inaccessible to conventional 4-chain antibodies and antigen-binding fragments. For example, it has been shown that V HH Domains and Nanobodies can inhibit enzymes (see for example WO 97 / 49805; Transue et al., (1998), supra; and Lauwereys et al., (1998), supra).

[0118] As described above, the present invention generally relates to Nanobodies against CD39, as well as polypeptides (eg antibodies or heavy chain antibodies or antigen-binding fragments thereof) comprising one or more such Nanobodies, which can be used for the prophylactic, therapeutic and / or diagnostic purposes described herein.

[0119] As also further described herein, the present invention also relates to nucleic acids encoding such Nanobodies, CD39 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, methods for preparing such Nanobodies, CD39 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, host cells expressing or capable of expressing such Nanobodies, CD39 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, compositions comprising such Nanobodies, CD39 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, nucleic acids or host cells, and uses of such Nanobodies, CD39 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, nucleic acids, host cells or compositions.

[0120] In general, it should be noted that the term Nanobody, CD39 binding protein or heavy chain antibody or antigen binding fragment thereof as used herein has its broadest meaning without being limited to a specific biological source or a specific method of preparation.

[0121] In a first preferred, but non-limiting aspect, the Nanobodies of the invention may have the structure

[0122] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0123] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively.

[0124] The humanized Nanobodies of the invention may be as defined herein, with the proviso that they are identical to naturally occurring V HH In some embodiments, a Nanobody has at least "one amino acid difference" (as defined herein) in at least one framework region compared to the corresponding framework region of a V domain of the present invention. More specifically, according to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it is identical to a naturally occurring V domain. HH A Nanobody has at least "one amino acid difference" (as defined herein) in at least one Hallmark residue compared to the corresponding framework regions of a V domain. Typically, a Nanobody differs from a naturally occurring V domain in at least one of FR2 and / or FR4, in particular at at least one Hallmark residue in FR2 and / or FR4. HH The domains have at least one such amino acid difference.

[0125] Heavy chain antibodies or HCAbs consist of only two heavy chains, each of which contains only the heavy chain variable region (VHH) as well as the hinge region, CH2 and CH3. The antigen-antibody binding region of heavy chain antibodies or HCAbs consists of three complementary determining regions, which also makes it have better antigen binding ability than ordinary antibodies. On the other hand, in addition to the lack of light chains, compared with ordinary antibodies, there is no CH1 region between its heavy chain variable region and hinge region, which is also a major difference of heavy chain antibodies.

[0126] Another embodiment of the invention is a nucleic acid capable of encoding a Nanobody, a CD39 binding protein or a heavy chain antibody as defined above.

[0127] Another embodiment of the invention is an antibody-drug conjugate comprising the nanobody, CD39 binding protein or heavy chain antibody or antigen binding fragment thereof, a linker and a small molecule cytotoxic drug.Antibody-drug conjugate (ADC) is a chemical connection connecting a biologically active small molecule drug to an antibody (e.g., a nanobody or antibody of the invention), which serves as a carrier to deliver the small molecule drug to target cells.

[0128] Another embodiment of the invention is a composition comprising a Nanobody, a CD39 binding protein, a heavy chain antibody, an antigen binding fragment, a nucleic acid, a cell and / or an antibody-drug composition as defined above. Another embodiment of the invention is a composition as defined above further comprising a pharmaceutically acceptable carrier.

[0129] Another embodiment of the invention is a CD39 binding protein, a heavy chain antibody, an antigen binding fragment or a Nanobody as defined above, or a nucleic acid as defined above, or a cell as defined above, or an antibody-drug conjugate as defined above, or a composition as defined above, for use as a medicament.

[0130] Another embodiment of the present invention is a CD39 binding protein, a heavy chain antibody, an antigen binding fragment or a Nanobody as defined above, or a nucleic acid as defined above, or a cell as defined above, or an antibody-drug conjugate as defined above, or a composition as defined above for use in treating, preventing and / or alleviating a CD39-related disease.

[0131] Another embodiment of the present invention is the use of a CD39 binding protein, a heavy chain antibody, an antigen binding fragment or a nanobody as defined above, or a nucleic acid as defined above, or a cell as defined above, or an antibody-drug conjugate as defined above, or a composition as defined above in the preparation of a medicament for treating, preventing and / or alleviating a CD39-related disease.

[0132] In some embodiments, the CD39-related disease or condition is a CD39-mediated disease or condition. In some embodiments, the CD39-mediated disease or condition is a tumor. In some embodiments, the CD39-mediated disease or condition is a solid tumor and / or a hematological tumor. In some specific embodiments, the CD39-mediated disease or condition is one or more tumors selected from the group consisting of: lymphoma, breast cancer, bladder cancer, colon cancer, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer and melanoma.

[0133] Another embodiment of the invention is the use of a Nanobody, a CD39 binding protein, a heavy chain antibody, a nucleic acid, a recombinant cell, a conjugate, or a composition as defined above, wherein the medicament is administered intravenously, subcutaneously, orally, sublingually, nasally or by inhalation.

[0134] Another embodiment of the invention is a method for the prophylactic or therapeutic treatment of a CD39-associated disease or disorder, comprising administering to the patient an effective dose of a Nanobody, antibody, heavy chain antibody, nucleic acid, recombinant cell, conjugate, or composition as defined above.

[0135] Another embodiment of the invention is a method of producing a Nanobody as defined above, comprising:

[0136] a) cultivating a host cell comprising a nucleic acid capable of encoding a polypeptide as defined above under conditions allowing expression of the polypeptide, and,

[0137] b) recovering the produced polypeptide from the culture.

[0138] Another embodiment of the invention is the method as defined above, wherein said host cell is a bacterial, yeast or mammalian cell.

[0139] Another embodiment of the present invention is a method for diagnosing a disease or condition mediated by CD39, comprising the steps of:

[0140] a) contacting the sample with a Nanobody, antibody, heavy chain antibody or antigen-binding fragment as defined above, and

[0141] b) detecting the binding of the Nanobody, antibody, heavy chain antibody or antigen-binding fragment to the sample, and

[0142] c) wherein a binding result above the cutoff value is indicative of a CD39-mediated disease or disorder in said sample.

[0143] In some embodiments, the sample is a biopsy sample. In some embodiments, the disease is a tumor. In some embodiments, the disease is a solid tumor and / or a hematological tumor. In some specific embodiments, the disease is one or more tumors selected from the group consisting of: lymphoma, breast cancer, bladder cancer, colon cancer, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer and melanoma.

[0144] Another embodiment of the invention is a kit for diagnosing a disease or disorder associated with CD39. In some embodiments, the reagent can be used in a method as defined above.

[0145] Another embodiment of the invention is a Nanobody, antibody, heavy chain antibody or antigen-binding fragment as defined above, further comprising one or more in vivo imaging agents.

[0146] One embodiment of the invention relates to a pharmaceutical composition comprising at least one Nanobody, antibody, heavy chain antibody or antigen-binding fragment of the invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0147] ELISA assays to measure the binding of anti-CD39 Nanobodies, antibodies, heavy chain antibodies or antigen binding fragments to CD39 are well known.

[0148] Conventional antibodies are unstable at room temperature and must be refrigerated for preparation and storage, which requires necessary refrigerated laboratory equipment, storage and transportation, thereby increasing time and cost. The anti-CD39 nanoantibodies of the present invention are stable at room temperature; therefore, they can be prepared, stored and / or transported without the use of refrigeration equipment, thereby saving cost, time and environment. In addition, conventional antibodies are not suitable for assays or kits performed at temperatures outside the biologically active temperature range (e.g., 37±20°C).

[0149] One aspect of the present invention is an anti-CD39 polypeptide comprising at least one anti-CD39 heavy chain antibody, particularly an anti-CD39 polypeptide derived from a nano antibody thereof. One aspect of the present invention is that such a polypeptide may comprise other components. Such other components may be a polypeptide sequence, such as one or more anti-CD39 nano antibodies or one or more anti-serum albumin nano antibodies. Other fusion proteins are also within the scope of the present invention and may include, for example, fusions with carrier polypeptides, signaling molecules, labels and enzymes. Other components may include, for example, radioactive labels, organic dyes, fluorescent compounds.

[0150] According to one aspect of the invention, the anti-CD39 polypeptide of the invention may comprise at least two identical or different anti-CD39 nanobody sequences. The anti-CD39 polypeptide may comprise at least two of the above sequences that do not have the same affinity for CD39, thereby forming an anti-CD39 polypeptide combining weak affinity and high affinity binding sequences.

[0151] Methods for constructing bivalent polypeptides are known in the art (eg US 2003 / 0088074) and are also described below.

[0152] It may be desirable to modify the CD39 binding proteins of the invention in terms of effector function to enhance their therapeutic efficacy. For example, nanobody fusions fused to certain Fc domains, especially to Fc domains of human origin, may be advantageous.

[0153] In sequential administration, the polypeptide may be administered once or any number of times before and / or after administration of the agent and in various doses. Sequential administration may be combined with simultaneous or sequential administration.

[0154] Another embodiment of the present invention is a CD39 binding protein as described herein, comprising one or more immunoglobulin single variable domains, wherein one or more of said immunoglobulin single variable domains are humanized.

[0155] Humanization refers to mutations that result in little or no potential immunogenicity when administered to human patients. According to the present invention, humanization of a polypeptide may include the following steps: replacing one or more non-human immunoglobulin amino acids with human counterparts present in a human consensus sequence or human germline gene sequence without causing the polypeptide to lose its typical characteristics, i.e., humanization will not significantly affect the antigen binding ability of the resulting polypeptide.

[0156] According to one aspect of the invention, humanized Nanobodies are defined as Nanobodies that have at least 50% homology (e.g. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) with human framework regions.

[0157] One embodiment of the invention relates to polypeptides comprising at least one Nanobody, in which one or more amino acid residues have been substituted without substantially altering the antigen binding capacity.

[0158] The skilled artisan will recognize that the CD39 binding proteins of the present invention can be modified, and such modifications are within the scope of the present invention. For example, the polypeptide can be used as a drug carrier, in which case it can be fused to a therapeutic activator, or its solubility characteristics can be changed by fusion with an ionic / bipolar group, or it can be used for imaging by fusion with a suitable imaging marker, or it can contain modified amino acids, etc. The polypeptide can also be prepared as a salt. Such modifications that substantially retain binding to CD39 are within the scope of the present invention.

[0159] It is clear from the disclosure herein that it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologues and orthologues of the Nanobodies of the invention as defined herein (collectively referred to herein as "analogues"), in particular analogues of the Nanobodies of SEQ ID NOs: 88 to 116. Thus, according to one embodiment of the invention, the term "Nanobody of the invention" in its broadest sense also encompasses such analogues.

[0160] Typically, in such analogs, one or more amino acid residues may have been replaced, deleted and / or added compared to the Nanobodies of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more framework regions and / or in one or more CDRs. When such substitutions, insertions or deletions are made in one or more framework regions, they may be made at one or more other positions in one or more Hallmark residues and / or framework residues, but substitutions, insertions or deletions of Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

[0161] Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobodies. Suitable labels and techniques for attaching, using and detecting Nanobodies will be clear to the skilled person and include, for example, but are not limited to fluorescent labels (e.g. fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and e.g. 152 Eu or other fluorescent metals of the lanthanide series), phosphorescent, chemiluminescent or bioluminescent labels (e.g., luminol, isoluminol, thermoacridinium esters, imidazoles, acridinium salts, oxalates, dioxetanes or GFP and its analogs), radioactive isotopes (e.g. 3 H. 125 I. 32 P. 35 S. 14 C. 51 Cr, 36 Cl, 57 Co. 58 Co. 59 Fe, and 75 Se), metals, metal chelates or metal cations (e.g. metal cations, e.g. 99m Tc, 123 I. 111 In, 131 I. 97 Such as 67 Cu, 67 Ga, and 68 Ga or other metals or metal cations particularly suitable for in vivo, in vitro or in situ diagnosis and imaging, such as 157 Gd, 55 Mn, 162 Dy, 52 Cr, and 56 Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels will be clear to the skilled person, for example including moieties that can be detected using NMR or ESR spectroscopy.

[0162] Depending on the choice of the specific label, such labeled Nanobodies and polypeptides of the invention can, for example, be used in in vitro, in vivo or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as for in vivo diagnostic and imaging purposes.

[0163] As will be clear to the skilled person, another modification may involve the introduction of a chelating group which, for example, chelates one of the above metals or metal cations. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0164] Another modification can include the introduction of a functional group that is part of a specific binding pair, such as a biotin-(streptavidin) avidin binding pair. Such a functional group can be used to connect the nanobody of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair (i.e., by forming a binding pair). For example, the nanobody of the invention can be conjugated with biotin and connected to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such conjugated nanobodies can be used as reporter genes, such as in a diagnostic system in which a detectable signal generator is conjugated to avidin or streptavidin. For example, such binding pairs can also be used to bind the nanobody of the invention to a carrier including a carrier suitable for pharmaceutical purposes. A non-limiting example is the case of liposome preparations described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs can also be used to connect therapeutic activators to the nanobodies of the invention.

[0165] Other potential chemical and enzymatic modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g., to study function-activity relationships). See, for example, Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

[0166] As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of at least one Nanobody of the invention. "Essentially consisting of" means that the amino acid sequence of the polypeptide of the invention is identical to the amino acid sequence of a Nanobody of the invention or corresponds to the amino acid sequence of a Nanobody of the invention with a limited number of amino acid residues, such as 1-20 amino acid residues, such as 1-10 amino acid residues, preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added to the amino-terminus, the carboxyl-terminus or both the amino-terminus and the carboxyl-terminus of the amino acid sequence of the Nanobody.

[0167] Said amino acid residues may or may not change, alter or otherwise affect the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody.

[0168] According to another embodiment, the polypeptide of the invention comprises a Nanobody of the invention, which is fused at its amino terminus, at its carboxyl terminus, or at its amino terminus and at its carboxyl terminus to at least one further amino acid sequence, i.e. to provide a fusion protein comprising said Nanobody of the invention and one or more further amino acid sequences. Such fusions will also be referred to herein as "Nanobody fusions".

[0169] The one or more additional amino acid sequences may be any suitable and / or desired amino acid sequences. The additional amino acid sequences may or may not change, alter or otherwise affect the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody or polypeptide of the invention. Preferably, the additional amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody or polypeptide of the invention.

[0170] The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA, preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention can be genomic DNA, cDNA or synthetic DNA (e.g., DNA with codon usage that has been specifically adapted for expression in the intended host cell or host organism).

[0171] According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated form as defined herein.

[0172] The nucleic acid of the invention may also be in the form of, present in and / or be part of a vector, such as a plasmid, cosmid or YAC, which may also be in essentially isolated form.

[0173] Nucleic acid of the present invention can also be the form of genetic construct, be present in genetic construct and / or as a part of genetic construct, which is clear to those skilled in the art.This type of genetic construct usually comprises at least one nucleic acid of the present invention, and this nucleic acid is optionally connected to one or more elements of genetic construct known per se, for example one or more suitable regulatory elements (for example suitable promoter, enhancer, terminator etc.) and other elements of genetic construct mentioned herein.This type of genetic construct comprising at least one nucleic acid of the present invention will also be referred to as " genetic construct of the present invention " in this article.

[0174] Genetic construct of the present invention can be DNA or RNA, and is preferably double-stranded DNA.Genetic construct of the present invention can also be the form suitable for transforming desired host cell or host organism, the form suitable for being integrated into the genomic DNA of desired host cell, or the form suitable for independently replicating, maintaining and hereditary in desired host organism.For example, genetic construct of the present invention can be the form of the carrier of for example plasmid, cosmid, YAC, viral vector or transposon.Especially, carrier can be expression vector, i.e. can provide the carrier of external and / or internal expression (for example in suitable host cell, host organism and / or expression system).

[0175] In a preferred, but non-limiting embodiment, the genetic construct of the invention comprises: a) at least one nucleic acid of the invention, operably linked to b) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally c) one or more other elements of a genetic construct known per se; wherein the terms "regulatory element", "promoter", "terminator" and "operably linked" have their usual meaning in the art (as further described herein); and wherein the "other elements" present in the genetic construct may be, for example, 3'-UTR or 5'-UTR sequences, leader sequences, selection markers, expression markers / reporter genes, and / or elements that may facilitate or increase transformation or integration (efficiency). These and other suitable elements for such genetic constructs are clear to the skilled person and may, for example, depend on the type of construct used, the desired host cell or host organism; the manner in which the nucleotide sequence of interest of the invention is expressed (e.g., by constitutive, transient or inducible expression); and / or the transformation technique to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

[0176] Preferably, in the genetic construct of the present invention, the at least one nucleic acid of the present invention and the regulatory element, and optionally the one or more other elements, are "operably connected" to each other, which generally means that there is a functional relationship between them. For example, if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of a coding sequence (wherein the coding sequence is understood to be "under the control of the promoter"), the promoter is considered to be "operably connected" to the coding sequence. Generally, when two nucleotide sequences are operably connected, they will be in the same orientation and generally in the same reading frame. It is also generally substantially continuous, although this may not be necessary.

[0177] Preferably, the regulatory elements and other elements of the genetic construct of the invention are such that it is able to provide its desired biological function in the desired host cell or host organism.

[0178] For example, a promoter, enhancer or terminator should be "operable" in the desired host cell or host organism, which means that (for example) the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence, such as a coding sequence, to which it is operably linked (as defined herein).

[0179] Some particularly preferred promoters include, but are not limited to, promoters known per se for expression in the host cells mentioned herein; and in particular promoters for expression in bacterial cells, such as those mentioned herein and / or used in the Examples.

[0180] Selectable markers should be those which allow - i.e. under appropriate selection conditions - host cells and / or host organisms which have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells / organisms which have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes which provide resistance to antibiotics (e.g. kanamycin or ampicillin), genes which provide temperature resistance, or genes which allow the host cell or host organism to be maintained in the absence of certain factors, compounds and / or (food) components in the culture medium which are essential for the survival of non-transformed cells or organisms.

[0181] The leader sequence should be one that allows the post-translational modification to be performed in the desired host cell or host organism and / or allows the mRNA to be directed to the desired part or organelle of the cell. The leader sequence can also allow the expression product to be secreted from the cell. Therefore, the leader sequence can be any prosequence, presequence or preprosequence that is operable in the host cell or host organism. Expression in bacterial cells may not require a leader sequence. For example, the leader sequence known per se for expressing and producing antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) can be used in a substantially similar manner.

[0182] The expression marker or reporter gene should be one that allows detection of the expression of the genetic construct (the gene or nucleotide sequence present on the genetic construct) in a host cell or host organism. The expression marker can also optionally allow localization of the expression product, such as in a specific part or organelle of a cell and / or in a specific cell, tissue, organ or part of a multicellular organism. Such reporter genes can also be expressed as proteins fused to the amino acid sequence of the present invention. Some preferred but non-limiting examples include fluorescent proteins such as GFP.

[0183] Some preferred but non-limiting examples of suitable promoters, terminators and other elements include those useful for expression in the host cells mentioned herein; and in particular those suitable for expression in bacterial cells, such as those mentioned herein and / or used in the examples below. Some (other) non-limiting examples of promoters, selection markers, leader sequences, expression markers and other elements that may be present / used in the genetic construct of the present invention, such as terminators, transcription and / or translation enhancers and / or integration factors, are referred to, for example, in the general handbooks of the people such as Sambrook and the people such as Ausubel mentioned above, and in WO 95 / 07463, WO 96 / 23810, WO 95 / 07463, WO 95 / 21191, WO 97 / 11094, WO 97 / 42320, WO 98 / 06737, WO 98 / 21355, US-A-6,207,410, US-A-5,693,492 and EP 1 085 089. Other examples will be clear to the technician. Reference is also made to the general background technology cited above and the further references cited herein.

[0184] The genetic constructs of the invention may generally be provided by appropriately ligating a nucleotide sequence of the invention to one or more of the other elements described above, for example using the techniques described in the general manuals of Sambrook et al. and Ausubel et al. mentioned above.

[0185] Typically, the genetic construct of the invention will be obtained by inserting the nucleotide sequence of the invention into a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the following examples and those mentioned herein.

[0186] The nucleic acids of the invention and / or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e. for expression and / or production of Nanobodies or polypeptides of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.

[0187] In general, for the prevention and / or treatment of the diseases and disorders mentioned herein, the Nanobodies and polypeptides of the invention are typically administered in an amount of 1 gram to 0.01 microgram per kg body weight per day, preferably 0.1 gram to 0.1 microgram per kg body weight per day, for example about 1, 10, 100 or 1000 micrograms per kg body weight per day, continuously (e.g. by infusion) as a single daily dose or as multiple divided doses throughout the day, depending on the specific disease or disorder to be treated, the potency of the specific Nanobodies and polypeptides of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used. Based on the factors mentioned herein, the clinician will generally be able to determine a suitable daily dose. It is also clear that in specific circumstances the clinician may choose to deviate from these amounts, for example based on the above factors and their professional judgment. In general, some guidance on the amount to be administered can be obtained from the usual amounts of comparable conventional antibodies or antibody fragments administered against the same target by essentially the same route, but differences in affinity / avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person need to be taken into account.

[0188] It should also be noted that when the Nanobodies of the invention contain one or more other CDR sequences that are different from the preferred CDR sequences described above, these CDR sequences can be obtained in any manner known per se, for example from Nanobodies (preferred), from V sequences from conventional antibodies, HThe present invention relates to a method for preparing an immunoglobulin sequence for an antibody or a polypeptide. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. The method comprises preparing an immunoglobulin sequence for an antibody or a polypeptide of the invention. Other techniques for producing immunoglobulins against specific targets include, for example, nanocloning technology (e.g. described in non-prepublished US provisional patent application 60 / 648,922), the so-called SLAM technology (e.g. described in European patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins or the well-known hybridoma technology (see, for example, Larrick et al., Biotechnology, Vol. 7, 1989, p. 934). All of these techniques can be used to produce immunoglobulins against A-beta, and the CDRs of such immunoglobulins can be used in the Nanobodies of the invention, i.e. as described above. For example, the sequences of such CDRs can be determined, synthesized and / or isolated, and inserted into the sequences of the Nanobodies of the invention (e.g., to replace the corresponding natural CDRs), all using techniques known per se such as those described herein, or Nanobodies of the invention comprising such CDRs (or nucleic acids encoding them) can be synthesized de novo, again using the techniques mentioned herein.

[0189] The invention will now be further described by way of the following non-limiting examples and figures. Example

[0190] Example 1 Alpaca Immunization and Detection

[0191] 1.1 Alpaca Immunity

[0192] Human CD39 protein (ACRO, CAT#CD9-H52H4) was added to Freund's complete adjuvant and Freund's incomplete adjuvant, and non-immunized Alpaca alpacas were immunized five times by subcutaneous multi-point injection.

[0193] 1.2 Potency detection

[0194] Serum titer was tested from the second immunization using ELISA and FACS, and data were collected from the third immunization.

[0195] 1) ELISA screening

[0196] 100 μL Human CD39 recombinant protein (ACRO, CAT#CD9-H52H4), Cyno CD39 recombinant protein (ACRO, CAT#CD9-C52H3), or Mouse CD39 recombinant protein (ACRO, CAT#CD9-M52H3) (final concentration 1 μg / mL) was coated on a 96-well ELISA plate at 4°C overnight; after washing three times with PBST (containing 0.05% Tween), blocking buffer (3% MPBS) was added and blocked at 37°C for 1 h, and the plate was washed three times with PBST; immune serum was added in a 2-fold serial dilution starting from 1:1000, incubated at 37°C for 1 h, washed three times with PBST, and 100 μL 0.1 μg / mL HRP-labeled MonoRab was added to each well. TM Rabbit Anti-Camelid VHH Cocktail (Genscript, CAT#A02014) or 0.1 μg / mL HRP-labeled Goat Anti-Llama IgG (Novus, CAT#NB7242) were added, incubated at 37°C for 0.5 h, washed 6 times with PBST, added 100 μL / well TMB colorimetric solution, incubated at room temperature in the dark for 10 min, added 50 μL stop solution to terminate the reaction, and read the OD450nm value in the well with a microplate reader.

[0197] Table 1. Serum ELISA test results of human CD39 as antigen

[0198]

[0199] Table 2. Serum ELISA test results of cynomolgus monkey CD39 as antigen

[0200]

[0201]

[0202] Table 3. Results of serum ELISA test using mouse CD39 as antigen

[0203]

[0204] 2) FACS screening

[0205] The Chinese hamster ovary cell line CHO-K1 (Probio), the Chinese hamster ovary cell line CHO-K1 / CD39(+) (Probio) expressing human CD39, and the Chinese hamster ovary cell line CHO-K1 / CynoCD39(+) (Probio) expressing cynomolgus monkey CD39 were divided into several portions, and the number of cells in each portion was 1×10 5 For each cell, 50 μL of immune serum diluted 1:100 and 1:1000 were used to incubate the target cells, mixed thoroughly, and incubated at room temperature for 1 h. The cells were washed 3 times with PBS and Alexa Fluor was added. 488AffiniPure Goat Anti-Alpaca IgG (H+L) (Jackson, CAT#128-545-003) and Alexa Fluor 647AffiniPure Goat Anti-Human IgG (H+L) (Jackson, CAT#109-605-088) were mixed thoroughly and incubated at room temperature in the dark for 30 min. The cells were washed 3 times with PBS and detected by flow cytometry. The results are shown in Figure 1 and Figure 2 The results showed that the serum of the alpaca after five immunizations had good FACS activity, indicating that good antibodies against CD39 protein were produced.

[0206] Example 2 Phage display library construction

[0207] 2.1 RNA extraction

[0208] Total RNA was extracted from isolated PBMCs according to the TRIzol Reagent instructions, and approximately 41 μg was extracted and used for reverse transcription into cDNA. The quantity and quality of total RNA were evaluated by gel electrophoresis and OD 260 / 280 ratio, respectively.

[0209] 2.2 VHH fragment amplification

[0210] Multiple forward primers and reverse primers were designed for amplification of the VHH fragment, in which two SfiI restriction enzyme sites were introduced. The products of different primer pairs were mixed and gel purified to obtain more than 3 μg of DNA encoding the VHH fragment.

[0211] 2.3 Library construction

[0212] The purified DNA encoding the VHH fragment was electroporated and inserted into the vector for library construction. 100 μL of the transformation solution was titrated and the library size was calculated based on the number of clones. 95 clones were randomly selected for sequencing to evaluate the quality of the library, and all the remaining bacterial solutions were amplified on a plate and finally frozen in the form of glycerol bacteria. The test summary results of the library are shown in Table 4. The constructed library capacity is 1.42×10 9 The frequency distribution of IMGT-CDR3 length in the library is shown in Figure 3 shown.

[0213] Table 4. Statistical results of library construction

[0214] Library capacity Insertion rate Correct translation rate Diversity Library capacity CD39 library <![CDATA[1.42×10 9 ]]> 100％(95 / 95) 95％(82 / 86) 98％(80 / 82)

[0215] Example 3 Phage Library Panning

[0216] After the alpaca immune phage library was prepared, solid phase panning, cell panning, and multiple rounds of liquid phase panning were used to select and enrich specific antibodies to achieve the purpose of screening targeted CD39 single domain antibodies.

[0217] 3.1 Solid phase panning

[0218] Human CD39 protein was diluted to 50 μg / mL with CBS coating solution and added to 8-well plates for solid phase screening at 125 μL / well. The plates were placed at 4°C overnight. The next day, the coated plates were washed twice with 0.05% PBST, 300 μL of 3% MPBS was added to each well, and the plates were blocked at 37°C for 1 hour. The phage library (1×10 12 pfu / pool) was diluted to 1mL with 3% MPBS and blocked for 1h at room temperature on a horizontal shaker. After blocking, discard the blocking solution on the solid phase strip, add the blocked phage library to the well plate, 125μL / well, and incubate at room temperature on a horizontal shaker for 30-45min. Discard the solution in the binding wells, add 300μL 0.05% PBST to each well and wash 8-10 times, then wash 3-5 times with 300μL PBS, and pat dry. Add 125μL 0.1M TEA to each well and slowly shake on a horizontal shaker to elute for 8-10min. Add 0.5mL 1M Tris-HCl (pH7.4) to a new 1.5mL protein low binding tube, and transfer the solution in the eluted microwell to the Tris-HCl solution after the elution time is reached, mix well, and neutralize for 10min. Take 1 mL of the eluted and neutralized phage product and add it to 10 mL of TG1 bacterial solution in the logarithmic growth phase. After mixing evenly, incubate at 37°C for 45 minutes for infection.

[0219] 3.2 Cell panning

[0220] Negative cells (CHO-K1) and positive cells expressing recombinant human CD39 (CHO-K1 / CD39) (Probio) were washed three times with pre-cooled, sterile PBS and placed on ice for later use. 7 cells) and phage library (1×10 12 pfu / pool), and fill with casein blocking solution to a final volume of 1mL, and block at room temperature on a horizontal shaker for 1h. After blocking, centrifuge the negative cells at low speed, discard the blocking solution, add the blocked phage library to the negative cells, resuspend, and incubate at room temperature on a horizontal shaker for 1h. Discard the negative cells, transfer the phage library supernatant to the positive cells, resuspend, and incubate at room temperature on a horizontal shaker for 1h. After incubation, wash 3 times with 1% BSA / PBS, then wash 3 times with 1×PBS, and finally resuspend the cells with 400μL of 1×PBS. Add 400μL 0.2M TEA to the cells, and shake slowly on a horizontal shaker for 8-10min to elute. Then add 400μL Tris-HCl solution, shake and neutralize on a horizontal shaker for 10min. Take 1mL of the eluted and neutralized phage product and add it to 10mL of TG1 bacterial solution in the logarithmic growth phase, mix well, and let it stand at 37℃ for 45min of infection.

[0221] 3.3 Liquid phase panning

[0222] 100 μL magnetic beads (Dynabeads M-280 Streptavidin) and phage library (2×10 12 pfu / pool), add 3% MPBS blocking solution to the final volume of 1mL, and incubate at room temperature on a horizontal shaker for 1h. Then take 5μg Biotin-humanCD39 protein and add it to the blocked phage library, and incubate at room temperature on a horizontal shaker for 30-45min. Discard the blocking solution of the magnetic beads, add the incubated antigen-phage library to the magnetic beads, resuspend, and incubate at room temperature on a horizontal shaker for 30-45min. Then wash the magnetic beads 8 times with 1mL 0.05% PBST, and then wash 3 times with PBS. Discard the washing solution, add 800μL 0.1M TEA to the magnetic beads, and slowly shake and elute on a horizontal shaker for 8-10min. When the elution is completed, discard the magnetic beads, transfer the elution solution to a low adsorption tube containing 400μL 1M Tris-HCl (pH7.4), mix well, and neutralize for 10min. Take 1 mL of the eluted and neutralized phage product and add it to 10 mL of TG1 bacterial solution in the logarithmic growth phase. After mixing evenly, incubate at 37°C for 45 minutes for infection.

[0223] Example 4 Monoclonal screening, detection and sequencing

[0224] After 1 / 2 rounds of panning, monoclones were selected for culture and expression, and phage supernatants were taken to detect the binding with Human CD39, Cyno CD39, and Mouse CD39 proteins by ELISA. The binding of the expressed monoclonal phage supernatants to the target cells (Chinese hamster ovary cell substrain CHO-K1, Chinese hamster ovary cells CHO-K1 / CD39(+) recombinantly expressing human CD39, and Chinese hamster ovary cells CHO-K1 / Cyno CD39(+) recombinantly expressing cynomolgus monkey CD39) was detected by FACS, and the results showed that most clones had binding activity with both CHO-K1 / CD39 and CHO-K1 / Cyno CD39 cells.

[0225] 1) ELISA screening

[0226] After adding M13KO7 helper phage to the monoclonal bacterial solution cultured in the 96-well plate, the plates were cultured overnight at 30°C. At the same time, 1 μg / mL Human, Cyno, and Mouse CD39 proteins were coated on the 96-well ELISA plate, 100 μL / well, and coated overnight at 4°C. After coating, 300 μL 3% MPBS was added to the ELISA plate, and the plates were blocked at 37°C for 1 hour. After washing, 50 μL of overnight culture supernatant and 50 μL 0.05% PBST were added to each well, and the plates were incubated and combined at room temperature for 2 hours. After washing, HRP-labeled Anti-M13 Monoclonal Antibody secondary antibody was added, and the plates were incubated at room temperature for 45 minutes. Finally, the plates were washed 6 times, 100 μL TMB color development solution was added to each well, and the color was developed at room temperature for 10 minutes, followed by 50 μL / well stop solution. After reading the results with an ELISA reader, positive clones were selected for FACS analysis based on the OD450nm value.

[0227] 2) FACS screening

[0228] Take 50 μL of supernatant sample and 50 μL of cells, incubate at 4°C for 40 min, then add 150 μL PBS, centrifuge at 300g for 3 min, and wash twice; add 100 μL Anti-fd Bacteriophage-Biotin antibody (Sigma, CAT#B2661), incubate at 4°C for 30 min, add 150 μL PBS, centrifuge at 300g for 3 min, wash twice; add 100 μL Streptavidin-Alexa Fluor 647 secondary antibody, incubate at 4°C for 30 min, add 150 μL PBS, centrifuge at 300g for 5 min, wash twice, and finally add 50-100 μL Flow Cytometry Staining Buffer to resuspend the cells, and use flow cytometer for detection and analysis.

[0229] The screened FACS positive clones were sequenced, and finally 29 unique VHH sequences were obtained. The sequences of CDR1, CDR2 and CDR3 of each VHH are shown in Table 5.

[0230] Table 5. Sequences of CDR1, CDR2 and CDR3 of positive clones VHH obtained by screening

[0231]

[0232]

[0233] Table 6 Amino acid sequences of positive clones VHH obtained by screening

[0234]

[0235]

[0236]

[0237] Example 5 Expression of recombinant antibodies

[0238] The coding sequences of the 29 obtained antibody sequences were respectively inserted into the eukaryotic expression vector pcDNA3.4 (Probio) containing the nucleic acid sequence encoding Fc, and the pcDNA3.4 inserted with the antibody coding sequence was transfected into Expi293F cells (Probio) to express the antibodies, and the expressed antibodies were purified.

[0239] Add Protein A Magnetic Beads to the sample to be purified and incubate in a shaking incubator at room temperature for 2 hours. Equilibrate Protein A Magnetic Beads with 10 column volumes of Binding Buffer. Add eluent to Protein A Magnetic Beads, collect the eluent, and add neutralizing solution (1 / 10 of the collected eluent volume) to adjust the pH to neutral. After elution and neutralization, transfer the eluted collected sample to a dialysis bag, dialyze with PBS at room temperature for 2 hours, then change the solution and continue dialysis at 2-8°C for 16 hours. Determine the concentration and purity of the antibody by OD280 and SDS-PAGE.

[0240] The SDS-PAGE results of the purified antibodies showed that the molecular weights of all antibodies were correct and the purity was >95%.

[0241] Example 6 Detection of purified antibodies

[0242] 1) ELISA screening

[0243] The antibody binding levels against Human CD39 protein, Cyno CD39 protein and Mouse CD39 protein were detected by ELISA EC50 experiment. 1μg / mL of Human CD39 protein, Cyno CD39 protein, or Mouse CD39 protein were added to the 96-well ELISA plate and coated overnight at 4°C. After washing the plate 3 times, 300μL of 3% MPBS was added to each well and blocked at 37°C for 1h. The antibody to be tested was diluted to a concentration of 100nM in the first well, followed by 3-fold gradient dilution, a total of 10 gradients, and the 12th well was a blank control. The diluted antibody was then added to the blocked ELISA plate, 100μL / well, incubated at 37°C, and bound for 1h. After washing the plate three times, add 100 μL of Mouse Anti-Human IgG Fc-HRP secondary antibody and incubate at 37°C for 30 min; finally wash the plate six times, then add 100 μL of TMB colorimetric solution to each well, develop at room temperature for 10 min, then add 50 μL / well of stop solution; read the OD450nm value using an enzyme reader.

[0244] The purified antibodies were tested by ELISA, and the results are shown in Tables 7 to 9. AHP18698 and AHP18706 all bind to human CD39 protein, monkey CD39 protein and mouse CD39 protein with strong binding ability. AHP18686, AHP18697 and AHP18699 also bind to human, monkey and mouse CD39 proteins with strong binding ability. AHP17815 and AHP18683 have strong cross-binding ability with human and monkey CD39.

[0245] Table 7 EC50 values ​​of each antibody binding to human CD39 detected by ELISA

[0246] Antibody ID EC50 / nM Antibody ID EC50 / nM Antibody ID EC50 / nM AHP17815 0.173 AHP18686 4.175 AHP24611 3.708 AHP17849 6.168 AHP18689 3.056 AHP24615 11.190 AHP17850 0.304 AHP18693 9.673 AHP24616 2.555 AHP17852 NA AHP18694 10.310 AHP24618 5.843 AHP17854 NA AHP18697 2.152 AHP24620 1.366 AHP17863 0.703 AHP18698 1.722 AHP24622 11.920 AHP17871 0.456 AHP18699 0.929 AHP24626 1.961 AHP18674 0.945 AHP18701 5.360 AHP24627 2.335 AHP18683 0.160 AHP18706 2.104 AHP24629 1.376 AHP18685 1.681 AHP18707 101.900 / /

[0247] Table 8 EC50 values ​​of each antibody binding to monkey CD39 detected by ELISA

[0248] Antibody ID EC50 / nM Antibody ID EC50 / nM Antibody ID EC50 / nM AHP17815 0.171 AHP18686 6.962 AHP24611 7.937 AHP17849 30.060 AHP18689 4.516 AHP24615 11.540 AHP17850 0.360 AHP18693 18.29 AHP24616 7.236 AHP17852 NA AHP18694 NA AHP24618 45.500 AHP17854 NA AHP18697 2.376 AHP24620 2.809 AHP17863 0.834 AHP18698 2.006 AHP24622 235.000 AHP17871 0.549 AHP18699 1.629 AHP24626 2.463 AHP18674 0.518 AHP18701 NA AHP24627 3.513 AHP18683 0.160 AHP18706 2.226 AHP24629 1.735 AHP18685 2.258 AHP18707 NA / /

[0249] Table 9 EC50 values ​​of each antibody binding to mouse CD39 detected by ELISA

[0250]

[0251]

[0252] 2) FACS screening

[0253] Take 50 μL of diluted antibody (10 μg / mL) and add it to the 96-well test plate. Then add 50 μL of test cells CHO-K1(-), CHO-K1 / CD39(+) or CHO-K1 / Cyno CD39(+) to each well. 5 / well, incubate at 4℃ for 40min; then add 150μL PBS, centrifuge at 300g for 3min, wash twice; add 100μL Alexa Fluor647AffiniPure Fab Fragment Goat Anti-Human IgG secondary antibody, incubate at 4℃ for 30min; then add 150μL PBS, centrifuge at 300g for 3min, wash twice; finally add 50μL PBS to resuspend the cells, and use flow cytometer for detection and analysis.

[0254] FACS results Figure 4 As shown, among the 29 antibodies, except for AHP17852 and AHP17854 antibodies which had weak FACS activity, the others had strong FACS activity.

Claims

1. A CD39 binding protein, wherein the binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NOs: 88-116.

2. The CD39 binding protein of claim 1, wherein the CDR1, CDR2 and CDR3 are encoded according to Kabat, AbM, Chothia or IMGT.

3. The CD39 binding protein of claim 2, wherein the CDR1, CDR2 and CDR3 are encoded according to IMGT, and: (1) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 1, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 2, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 3; (2) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:4, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:5, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:6; (3) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:7, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:8, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:9; (4) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 10, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 11, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 12; (5) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 13, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 14, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 15; (6) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 16, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 17, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 18; (7) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 19, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 20, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 21; (8) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:22, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:23, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:24; (9) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:25, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:26, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:27; (10) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:28, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:29, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:30; (11) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:31, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:32, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:33; (12) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:34, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:35, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:36; (13) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:37, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:38, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:39; (14) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:40, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:41, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:42; (15) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:43, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:44, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:45; (16) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:46, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:47, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:48; (17) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:49, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:50, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:51; (18) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:52, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:53, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:54; (19) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:55, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:56, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:57; (20) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:58, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:59, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:60; (21) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:61, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:62, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:63; (22) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:64, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:65, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:66; (23) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:67, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:68, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:69; (24) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:70, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:71, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:72; (25) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:73, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:74, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:75; (26) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:76, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:77, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:78; (27) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:79, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:80, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:81; (28) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:82, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:83, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:84; or (29) The CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:85, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:86, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:

87.

4. The CD39 binding protein of any one of claims 1-3, wherein the immunoglobulin single variable domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 88-116.

5. The CD39 binding protein of any one of claims 1 to 4, wherein the CD39 binding protein is monovalent, bivalent or multivalent.

6. The CD39 binding protein of any one of claims 1 to 5, wherein the CD39 binding protein is monospecific, bispecific or multispecific.

7. The CD39 binding protein of any one of claims 1 to 6, wherein the CD39 binding protein is a heavy chain antibody. The CD39 binding protein of claim 7 , wherein the heavy chain antibody further comprises human IgG1 Fc.

9. The CD39 binding protein of any one of claims 1 to 6, wherein the CD39 binding protein is a Nanobody.

10. The CD39 binding protein of claim 9, wherein the Nanobody comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 88-116, preferably, the Nanobody comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 88-116.

11. The CD39 binding protein of claim 9 or 10, wherein the Nanobody comprises a heavy chain framework region, and at least a portion of the heavy chain framework region is from at least one of a mouse antibody, a human antibody, a primate antibody, and a mutant thereof.

12. A nucleic acid molecule encoding the CD39 binding protein of any one of claims 1 to 11, optionally wherein the nucleic acid molecule is DNA.

13. An expression vector comprising the nucleic acid molecule of claim 12, optionally wherein the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

14. A cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13, optionally wherein the cell is a prokaryotic cell or a eukaryotic cell (eg, a mammalian cell).

15. A conjugate comprising the CD39 binding protein of any one of claims 1-11 conjugated to a therapeutic agent, a diagnostic agent or an imaging agent; optionally, wherein the therapeutic agent is a small molecule cytotoxic drug.

16. A composition comprising the CD39 binding protein of any one of claims 1-11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, or the conjugate of claim 15, optionally, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

17. Use of the CD39 binding protein of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, or the conjugate of claim 15 in the preparation of a medicament for preventing, treating or alleviating CD39-related diseases.

18. Use of the CD39 binding protein of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the expression vector of claim 13, the cell of claim 14, or the conjugate of claim 15 in combination with another agent in the preparation of a medicament for preventing, treating or alleviating a CD39-related disease, preferably, the other agent is an antibody or a chemotherapeutic agent.

19. A kit for detecting CD39 or cells containing CD39, comprising the CD39 binding protein according to any one of claims 1 to 11, or the conjugate according to claim 15.

20. Use of the CD39 binding protein according to any one of claims 1 to 11 or the conjugate according to claim 15 in preparing a kit for detecting CD39 or cells containing CD39.

21. A method for determining the presence and / or content of CD39, comprising contacting the CD39 binding protein according to any one of claims 1 to 11, and / or the conjugate according to claim 15 with a sample to be tested.

22. A chimeric antigen receptor comprising an antigen recognition domain, a hinge region, a transmembrane binding domain, and an intracellular domain (including a co-stimulatory domain and a signal transduction domain), wherein the antigen recognition domain comprises the CD39 binding protein according to any one of claims 1 to 11.

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