CN119874908A

CN119874908A - CD39 binding proteins

Info

Publication number: CN119874908A
Application number: CN202311393681.8A
Authority: CN
Inventors: 何景昌; 李维; 张敏; 张琪; 胡炳旭; 于红亮
Original assignee: Beijing Tide Pharmaceutical Co Ltd
Current assignee: Beijing Tide Pharmaceutical Co Ltd
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2025-04-25

Abstract

本公开提供了能够特异性结合CD39的抗体，特别是重链抗体或纳米抗体，以及编码所述抗体的核酸、载体或细胞，以及所述抗体的诊断或治疗用途。The present disclosure provides antibodies capable of specifically binding to CD39, in particular heavy chain antibodies or nanobodies, as well as nucleic acids, vectors or cells encoding the antibodies, and diagnostic or therapeutic uses of the antibodies.

Description

CD39 binding proteins

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an antigen binding protein, a heavy chain antibody, a nano antibody and application thereof.

Background

CD39, also known as apyrase-1, is an extracellular nucleotide hydrolase encoded by the ENTPD gene. CD39 is a member of the ATP-adenosine pathway, which together with CD73 is responsible for converting the Adenosine Triphosphate (ATP) cascade into Adenosine Diphosphate (ADP) and adenosine cyclophosphate (cAMP), ultimately leading to release of immunosuppressive forms of extracellular Adenosine (ADO) in the tumor microenvironment.

Various types of cells may release ATP into the microenvironment through designated channels, or due to death or stress cell necrosis. High levels of extracellular ATP (ATP) provide a powerful inflammatory signal through the involvement of P2 receptors (P2Y and P2X families), P2 receptors being critical for the activation of innate and adaptive immune responses. Thus, CD39 upregulation is an effective mechanism for tumors to evade anti-tumor strategies by depleting immunostimulatory etap in the Tumor Microenvironment (TME). Since CD39 plays an important role in tumor microenvironment immunosuppressive responses, it has been receiving attention in recent years.

CD39 is overexpressed in human tumor cells such as lymphomas, breast cancers, bladder cancers, colon cancers, sarcomas, lung cancers, pancreatic cancers, ovarian cancers, renal cancers, and melanomas. CD39 is also expressed in vascular endothelial cells, cancer-associated fibroblasts (CAF) and some immune cells (in particular Natural Killer (NK) cells, tumor-associated macrophages (TAM) and tumor-infiltrating lymphocytes (TIL), including Tregs and cd8+ T cells).

By blocking CD39, ATP/AMP can be restricted from being converted into extracellular adenosine, thereby realizing the inhibition effect of extracellular Adenosine (ADO) mediated tumor microenvironment immunosuppression.

Monoclonal antibodies find successful application in biological targeted therapy and detection of cancer, playing an important role in biomedical and immunotherapeutic. Compared with the conventional monoclonal antibody, the nano antibody has the advantages of small molecular weight, good solubility, strong stability, weak immunogenicity, strong penetrability, high specificity, simplicity in humanization, high expression, easiness in production and the like. Therefore, development of a therapeutic nanobody or a diagnostic reagent for detecting the expression level of CD39 in cancer has a wide range of applications.

Summary of The Invention

The present disclosure provides CD39 binding proteins that specifically bind CD39, particularly nanobodies that specifically bind CD 39.

Thus, in a first aspect of the present disclosure, a CD39 binding protein is provided.

CD39 binding proteins according to embodiments of the invention may specifically target and bind CD39. The CD39 binding proteins according to embodiments of the invention are candidates for detecting CD39 or cells comprising CD39.

In some embodiments, the CD39 binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 comprised in a VHH as set forth in any one of SEQ ID NOS 88-116. In some embodiments, the CDR1, CDR2, and CDR3 are encoded according to Kabat, abM, chothia or IMGT.

In some embodiments of the antibodies or antigen binding fragments described above, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3, the CDR1, CDR2, and CDR3 being encoded according to IMGT, wherein:

(1) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 1, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 2, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 3;

(2) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 4, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 5, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 6;

(3) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 7, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 8, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 9;

(4) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 10, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 11, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 12;

(5) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 13, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 14, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 15;

(6) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 16, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 17, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 18;

(7) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 19, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 20, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 21;

(8) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 22, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 23, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 24;

(9) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 25, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 26, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 27;

(10) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 28, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 29, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in 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 set forth in 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 set forth in 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 set forth in SEQ ID No. 33;

(12) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 34, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 35, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 36;

(13) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 37, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 38, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 39;

(14) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 40, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 41, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 42;

(15) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 43, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 44, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 45;

(16) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 46, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 47, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 48;

(17) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 49, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 50, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 51;

(18) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 52, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID No. 53, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in 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 set forth in SEQ ID No. 81;

(28) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 82, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 83, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 84, or

(29) The CDR1 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 85, the CDR2 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 86, and the CDR3 comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO. 87.

In some embodiments, the immunoglobulin single variable domain comprises 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 set forth in any one of SEQ ID NOS: 88-116.

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

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

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

In some embodiments of the disclosure, the CD39 binding protein is a nanobody. Nanobodies according to embodiments of the invention may specifically target and bind 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 to the amino acid sequence set forth in any one of SEQ ID NOS 88-116. In some preferred embodiments, the nanobody comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS 88-116.

The nanobody with the amino acid sequence shown in SEQ ID NO. 88 corresponds to clone AHP18698 of the present disclosure; the nanobody having the amino acid sequence shown in SEQ ID NO. 89 corresponds to clone AHP18706 of the present disclosure; the nanobody having the amino acid sequence shown in SEQ ID NO. 90 corresponds to clone AHP18686 of the present disclosure; the nanobody having the amino acid sequence shown in SEQ ID NO. 91 corresponds to clone AHP18697 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 92 corresponds to clone AHP18699 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 93 corresponds to clone AHP17815 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 94 corresponds to clone AHP17863 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 95 corresponds to clone AHP17849 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 96 corresponds to clone AHP17850 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 97 corresponds to clone AHP17854 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 99 corresponds to clone AHP17871 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 94 corresponds to clone AHP 18103 of the present disclosure, the nanobody having the amino acid sequence shown in SEQ ID NO. 96 corresponds to clone AHP 68103 of the amino acid sequence shown in SEQ ID NO. 98 corresponds to clone AHP17854 of the present disclosure, and the nanobody having the amino acid sequence shown in SEQ ID NO. 9 corresponds to clone AHP 68102 of the present disclosure The nanobody corresponds to clone AHP18693 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 105 corresponds to clone AHP18694 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 106 corresponds to clone AHP18701 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 107 corresponds to clone AHP18707 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 108 corresponds to clone AHP24611 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 109 corresponds to 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 AHP24620 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 113 corresponds to clone AHP 622 of the present disclosure, the nanobody with the amino acid sequence shown in SEQ ID NO. 113 corresponds to clone AHP24626 of the present disclosure, and the nanobody with the amino acid sequence shown in SEQ ID NO. 116 corresponds to clone AHP 24116 of the present disclosure;

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, nanobodies are less immunogenic when the heavy chain framework region is derived from a human antibody.

In a second aspect of the present disclosure, there is provided a nucleic acid molecule encoding a CD39 binding protein according to the first aspect of the present disclosure.

In some embodiments of the present disclosure, a nucleic acid molecule may be introduced into a host cell to express a CD39 binding protein of the first aspect of the present disclosure.

In a third aspect of the present disclosure, there is provided an expression vector comprising a nucleic acid molecule as set forth in the second aspect of the present disclosure. As described above, the nucleic acid molecule encodes a CD39 binding protein according to the first aspect of the disclosure. Thus, an expression vector introduced into a host cell according to an embodiment of the invention may express a CD39 binding protein according to the first aspect of the present disclosure under conditions suitable for expression of the protein.

In some embodiments of the disclosure, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

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

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

In some embodiments of the disclosure, the recombinant cell is a prokaryotic cell or a eukaryotic cell.

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

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

In some embodiments of the present disclosure, the conjugate comprises a CD39 binding protein of the first aspect of the present disclosure, further comprising a therapeutic, diagnostic, or imaging agent. In some embodiments, the CD39 binding protein of the first aspect of the present disclosure has a linker between the CD39 binding protein and the therapeutic, diagnostic or imaging agent.

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

Conjugates according to embodiments of the present invention may target and act on target cells comprising CD39 under the guidance of a CD39 binding protein according to the first aspect of the present disclosure.

In a sixth aspect of the present disclosure, there is provided a composition comprising a CD39 binding protein according to the first aspect of the present disclosure, a nucleic acid according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a recombinant cell according to the fourth aspect of the present disclosure, and/or a conjugate according to the fifth aspect of the present disclosure.

In some embodiments of the present disclosure, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

In a seventh aspect of the present disclosure there is provided the use of a CD39 binding protein according to the first aspect of the present disclosure, a nucleic acid according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a recombinant cell according to the fourth aspect of the present disclosure, a conjugate according to the fifth aspect of the present disclosure, and/or a composition according to the sixth aspect of the present disclosure in the manufacture of a medicament for the prevention, treatment or alleviation of a CD39 related disease.

Also provided is the use of a CD39 binding protein according to the first aspect of the present disclosure, a nucleic acid according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a recombinant cell according to the fourth aspect of the present disclosure, a conjugate according to the fifth aspect of the present disclosure, and/or a composition according to the sixth aspect of the present disclosure in combination with another agent for the manufacture of a medicament for preventing, treating or alleviating a CD 39-related disease. In some embodiments, the other agent is an antibody or a chemotherapeutic agent.

In an eighth aspect of the present disclosure, there is provided a CD39 binding protein according to the first aspect of the present disclosure, a nucleic acid according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a recombinant cell according to the fourth aspect of the present disclosure, a conjugate according to the fifth aspect of the present disclosure, and/or a composition according to the sixth aspect of the present disclosure for use in preventing, treating or alleviating a CD 39-related disease.

In a ninth aspect of the present disclosure, there is provided a method of preventing, treating or alleviating a CD 39-related disorder in a subject. In some embodiments, the method comprises administering to the subject a CD39 binding protein of the first aspect of the disclosure, a nucleic acid of the second aspect of the disclosure, an expression vector of the third aspect of the disclosure, a recombinant cell of the fourth aspect of the disclosure, a conjugate of the fifth aspect of the disclosure, and/or a composition of the sixth aspect of the disclosure.

In some embodiments of the seventh to ninth aspects of the disclosure, the CD 39-related disease or disorder is a CD 39-mediated disease or disorder. In some embodiments, the CD39 mediated disease or disorder is a tumor. In some embodiments, the CD39 mediated disease or disorder is a solid tumor and/or hematological tumor. In some specific embodiments, the CD39 mediated disease or disorder 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.

In a tenth aspect of the present disclosure, a kit for detecting CD39 or a cell comprising CD39 is provided. In some embodiments, the kit comprises a CD39 binding protein of the first aspect of the disclosure, or a conjugate of the fifth aspect of the disclosure.

In an eleventh aspect of the present disclosure there is provided the use of a CD39 binding protein according to the first aspect of the present disclosure or a conjugate according to the fifth aspect of the present disclosure in the manufacture of a kit for detecting CD39 or a cell comprising CD 39.

In a twelfth aspect of the present disclosure, there is provided a CD39 binding protein according to the first aspect of the present disclosure, or a conjugate according to the fifth aspect of the present disclosure, for detecting CD39 or a cell comprising CD 39.

In a thirteenth aspect of the present disclosure, methods of detecting CD39 or a cell comprising CD39 are provided. In some embodiments of the present disclosure, the method comprises contacting the CD39 binding protein of the first aspect of the present disclosure, or the conjugate of the fifth aspect of the present disclosure, with a sample to be tested. Also provided are methods of determining the presence and/or amount of CD39 comprising contacting a CD39 binding protein according to the first aspect of the present disclosure, and/or a conjugate according to the fifth aspect of the present disclosure, with a test sample.

In a 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 comprising a CD39 binding protein of the first aspect of the present disclosure, a hinge region, a transmembrane binding domain, and an intracellular domain (including a co-stimulatory domain and a signaling domain).

Further aspects and advantages will be described below, at least some of which will become apparent to those of ordinary skill in the art from the following description taken in conjunction with the accompanying drawings and/or from the embodiments described below.

Drawings

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

FIG. 1 shows the results of FACS detection of the titers of alpaca serum (IgG antibodies) obtained after 5 immunizations of alpaca with human CD39 protein. Black for CHO-K1 (-), red for CHO-K1/CD39 (+), and green for CHO-K1/Cyno CD39 (+).

FIG. 2 shows the results of FACS detection of the titers of alpaca serum (VHH antibodies) obtained after 5 alpaca immunizations with human CD39 protein. Black for CHO-K1 (-), red for CHO-K1/CD39 (+), and green for CHO-K1/Cyno CD39 (+).

FIG. 3 shows the frequency distribution of the length of the IMGT-CDR3 of the library.

FIG. 4 shows the FACS detection results of 29 recombinantly expressed VHH-Fc (hIgG 1) antibodies. The black curve in each panel shows the FACS results of CHO-K1 (-) cells, the red curve shows the detection results of CHO-K1/CD39 (+) cells, and the green curve shows the detection results of CHO-K1/Cyno CD39 (+) cells. Human IgG represents an irrelevant IgG antibody, which is a negative control. The Positive Ab represents Anti-human CD39 Antibody (Acro), which is a Positive control. PBS represents phosphate buffered saline without antibody, and is a negative control.

Detailed Description

The embodiments described herein with reference to the drawings are illustrative, explanatory and are intended to be generally understood. The embodiments should not be construed as limiting the scope of the invention. The same or similar elements and elements having the same or similar functions are denoted by the same reference numerals throughout the description.

Unless otherwise indicated or defined, all terms used have the ordinary meaning known to the skilled artisan. For example, reference is made to standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2 nd edition), vols.1-3,Cold Spring Harbor Laboratory Press (1989); F.Ausubel et al, "Current protocols in molecular biology", green Publishing and WILEY INTERSCIENCE, new York (1987); roitt et al, "Immunology" (6 th edition), mosby/Elsevier, edinburgh (2001); and Janeway et al, "Immunology" (6 th edition), GARLAND SCIENCE Publishing/Churchill Livingstone, new York (2005), and the general background art cited above.

Unless otherwise indicated, the term "immunoglobulin sequence", whether used herein to refer to a heavy chain antibody or a conventional 4 chain antibody, is used as a general term, including full length antibodies, single chains thereof, and all portions, domains, or fragments thereof (including, but not limited to, antigen binding domains or fragments, such as the V _HH domain or V _H/V_L domain, respectively). Furthermore, the term "sequence" (e.g., "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "V _HH sequence" or "protein sequence" and like terms) as used herein is generally 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.

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

To compare two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be determined by dividing [ the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions in the second sequence ] by [ the total number of nucleotides/amino acids in the first sequence ] and multiplying by [100% ], wherein each nucleotide in the second nucleotide sequence is deleted, inserted, substituted or added, as compared to the first nucleotide sequence, as a single nucleotide (position) difference.

Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using standard settings using known computer algorithms for sequence alignment, such as NCBI Blast v 2.0.

Some other techniques, computer algorithms and settings 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 2 357 768-A.

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 considered as the difference in single amino acid residues (positions) as compared to the first amino acid sequence, i.e. "amino acid difference" as defined herein, by dividing [ the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues in the corresponding position in the second amino acid sequence ] by [ the total number of nucleotides in the first amino acid sequence ] times [100% ].

Or the degree of sequence identity between two amino acid sequences can be calculated using standard settings as well, using for example those known computer algorithms described above for determining the degree of sequence identity of nucleotide sequences.

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 referred to as the "first" amino acid sequence, and the other amino acid sequence will be referred to as the "second" amino acid sequence.

Furthermore, in determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g. WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300, and (preferably) the type and/or combination of such substitutions may be selected in accordance with the relevant teachings from WO 04/037999 and WO 98/49185, and further references cited therein.

Such conservative substitutions are preferably substitutions of one amino acid in groups (a) to (e) with another amino acid residue in the same group (a) small aliphatic, nonpolar 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, nonpolar residues Met, leu, he, val and Cys, and (e) aromatic residues Phe, tyr and Trp.

Particularly preferred conservative substitutions are 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.

Any amino acid substitutions described herein as being useful for polypeptides can also be based on analysis of the frequency of amino acid variation between homologous proteins of different species developed by Schulz et al, PRINCIPLES OF PROTEIN STRUCTURE, springer-Verlag,1978, based on analysis of the structure forming potential developed by Chou and Fasman, biochemistry 13:211,1974 and adv. Enzymol, 47:45-149,1978, and based on analysis of the protein hydrophobicity pattern developed by Eisenberg et al, proc. Nat. Acad Sci. USA81:140-144,1984;Kyte&Doolittle,J Mol.Biol.157:105-132,1981, and Goldman et al, ann. Rev. Biophys. Chem.15:321-353,1986, which are incorporated herein by reference in their entirety.

Information on the primary, secondary and tertiary structures of nanobodies is given in the description herein and in the general background art cited above. Furthermore, for this purpose, the crystal Structure of the VHH domain from llama (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). Further information is provided for some amino acid residues forming the VH/VL interface in the conventional V _H domain and potential camelized substitutions at these positions.

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.

A nucleic acid sequence or amino acid sequence is considered to be "(in) substantially isolated (form)", e.g., when it has been separated from at least one other component typically associated with it 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, as compared to its natural biological source and/or the reaction medium or culture medium in which it is obtained. In particular, a nucleic acid sequence or amino acid sequence is considered "substantially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold and up to 1000-fold or more. The nucleic acid sequence or amino acid sequence "in a substantially isolated form" is preferably substantially homogeneous, as determined using a suitable technique, such as a suitable chromatographic technique, e.g., polyacrylamide-gel electrophoresis.

The term "epitope" 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 by the antigen binding site of the molecule. The terms "epitope" and "epitope" are also used interchangeably herein.

Amino acid sequences (e.g., nanobodies, antibodies or heavy chain antibodies) that can bind, have affinity and/or have specificity for a particular epitope, antigen or protein (or at least a portion, fragment or epitope thereof) are referred to as being "directed against" or "directly against" the epitope, antigen or protein.

The term "specific" refers to the number of different types of antigens or antigenic determinants that a particular antigen binding molecule or antigen binding protein (e.g., nanobody or polypeptide of the invention) molecule can bind. The specificity of an antigen binding protein may be determined based on affinity and/or avidity. Affinity is expressed by the equilibrium constant (KD) for the dissociation of an antigen from an antigen binding protein and is a measure of the strength of binding between an epitope and the antigen binding site of an antigen binding protein, the smaller the KD value, the stronger the strength of binding between the epitope and the antigen binding molecule (or affinity can also be expressed as affinity constant (KA), which is 1/KD). avidity is a measure of the strength of binding between an antigen binding molecule (e.g., nanobody, antibody or heavy chain antibody of the invention) and the associated antigen. Avidity relates to the affinity between an epitope and the antigen binding site of an antigen binding molecule and the number of relevant binding sites present on the antigen binding molecule. Typically, antigen binding proteins (e.g., nanobodies and/or heavy chain antibodies of the invention) will bind with a dissociation constant (KD) of 10 ^-5 to 10 ^-12 moles/liter or less, preferably 10 ^-7 to 10 ^-12 moles/liter or less, more preferably 10 ^-8 to 10 ^-12 moles/liter, and/or with a binding affinity of at least 10 ⁷M^-1, preferably at least 10 ⁸M^-1, more preferably at least 10 ⁹M^-1, e.g., at least 10 ¹²M^-1. Any KD value greater than 10 ^-4 moles/liter is generally considered to represent non-specific binding. Preferably, the CD39 binding proteins of the invention, particularly nanobodies, will bind the desired antigen with an affinity of less than 500nM, preferably less than 200nM, more preferably less than 10nM, for example less than 500 pM. Specific binding of the antigen binding protein to the antigen or antigenic determinant may 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.

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

The term "VHH domain", also known as heavy chain single domain antibodies, VHH antibody fragments, and VHH antibodies, is used by the term "VHH domain" for the variable domain (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)). of an antigen-binding immunoglobulin called "heavy chain antibody" (i.e., an "antibody lacking a light chain") to distinguish the variable domain from the heavy chain variable domain (which is referred to herein as a "VH domain") present in a conventional 4-chain antibody, and the light chain variable domain (which is referred to herein as a "VL domain") present in a conventional 4-chain antibody. The VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain.

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

As further described herein, the amino acid sequence and structure of a nanobody may be considered to include, but is not limited to, four framework regions or "FR", referred to in the art and herein as "framework region 1" or "FR1", respectively, "framework region 2" or "FR2", respectively, "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, which are interrupted by three complementarity determining regions or "CDRs", referred to in the art as "complementarity determining region 1" or "CDR1", respectively, "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively.

As also further described herein, the total number of amino acid residues in the nanobody may be in the range of 120-150. It should be noted, however, that portions, fragments, analogs, or derivatives of nanobodies (as further described herein) are not particularly limited in their length and/or size, so long as such portions, fragments, analogs, or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein.

The amino acid residues of nanobodies are numbered according to the general numbering of the V _H domain given by Kabat et al ("Sequence of protein of immunological Interest",US Public Health Services,NIH Bethesda,MD,Publication No.91), as applied to the V _HH domain of camelids in Riechmann and Muyldermans, j.immunol methods 231,25-38 (1999) (see e.g. figure 2 of the above references). In this regard, it should be noted that the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues represented by Kabat numbering, as is well known in the art for the V _H domain and for the V _HH domain. That is, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows. This means that in general, numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

Alternative methods of numbering the amino acid residues of the V _H domain can also be applied in a similar manner to the V _HH domain from camelids and nanobodies, which are methods described by Chothia et al (Nature 342,877-883 (1989), so-called "AbM definition" and so-called "CONTACT definition". However, in this specification, claims and figures, unless otherwise indicated, numbering according to IMGT of the V _HH domain will be followed.

According to the terminology used in the above references, the variable domain present in a naturally occurring heavy chain antibody will also be referred to as the "V _HH domain" to distinguish it from the heavy chain variable domain present in a conventional 4 chain antibody (which will be referred to as the "V _H domain" hereinafter) and the light chain variable domain present in a conventional 4 chain antibody (which will be referred to as the "V _L domain" hereinafter).

As described in the prior art mentioned above, V _HH domains have a number of unique structural and functional characteristics that make isolated V _HH domains (and nanobodies based on V _HH domains sharing these structural and functional characteristics with naturally occurring V _HH domains) and proteins containing isolated V _HH domains very advantageous for use as functional antigen binding domains or proteins. In particular, but not limited thereto, the V _HH domain (which has been "designed" in nature to functionally bind to an antigen in the absence of and without any interaction with the light chain variable domain) and nanobody may be as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the V _HH domain from the V _H and V _L domains of conventional 4-chain antibodies, which are generally not suitable per se for practical use as a single antigen binding protein or domain, but rather 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 ScFv fragments consisting of the V _H domain covalently linked to the V _L domain).

Because of these unique properties, the use of V _HH domains and nanobodies as single antigen-binding proteins or antigen-binding domains (i.e., as part of a larger protein or polypeptide) provides a number of significant advantages over the use of conventional V _H and V _L domains, scFv, or conventional antibody fragments (e.g., fab or F (ab') 2 fragments) in that only a single domain is required to bind antigen with high affinity and selectivity, and thus there is no need for the presence of two separate domains, nor for ensuring that the two domains exist in the correct spatial conformation and configuration (i.e., by using specifically designed linkers as in ScFv).

The V _HH domain and nanobody can be expressed from a single gene without post-translational folding or modification.

V _HH domains and nanobodies can be easily engineered into multivalent and multispecific forms.

The V _HH domains and nanobodies are highly soluble and have no tendency to aggregate (e.g. "mouse-derived antigen binding domains" as described in Ward et al, nature, vol.341,1989, p.544).

V _HH domains and nanobodies are highly stable to heat, pH, proteases and other denaturing agents or conditions (see, e.g., ewert et al, supra).

V _HH domains and nanobodies are easy to prepare relatively cheaply, even on the scale expected for production. For example, V _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.

The V _HH domains and nanobodies are relatively small compared to conventional 4-chain antibodies and antigen-binding fragments thereof (about 15kDa, or 10-fold smaller than conventional IgG), and thus exhibit higher tissue permeability than such conventional 4-chain antibodies and antigen-binding fragments thereof, including but not limited to solid tumors and other dense tissues.

V _HH domains and nanobodies may exhibit so-called cavity binding properties (especially due to their extended CDR3 loops compared to conventional V _H domains) and thus may also gain access to targets and epitopes that conventional 4-chain antibodies and antigen binding fragments cannot gain access to. For example, V _HH domains and nanobodies have been shown to inhibit enzymes (see, e.g., WO 97/49805; transue et al, (1998) supra; and Lauwereys et al, (1998) supra).

As noted above, the present invention relates generally to nanobodies against CD39, and polypeptides (e.g., antibodies or heavy chain antibodies or antigen-binding fragments thereof) comprising one or more such nanobodies, which are useful for the prophylactic, therapeutic and/or diagnostic purposes described herein.

As further described herein, the invention also relates to nucleic acids encoding such nanobodies, CD39 binding proteins or heavy chain antibodies or antigen binding fragments thereof, methods of making 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.

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 and is not limited to a particular biological source or a particular method of preparation.

In a first preferred but non-limiting aspect, nanobodies of the invention can have the structure

FRl-CDRl-FR2-CDR2-FR3-CDR3-FR4

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.

The humanized nanobody of the invention may be as defined herein, provided that it has at least one "amino acid difference" in at least one framework region (as defined herein) compared to the corresponding framework region of the naturally occurring V _HH domain. More specifically, according to one non-limiting aspect of the invention, nanobodies may be as defined herein, provided that they have at least "one amino acid difference" (as defined herein) at least one tag residue compared to the corresponding framework region of the naturally occurring V _HH domain. Typically, nanobodies have at least one such amino acid difference from the naturally occurring V _HH domain in at least one of FR2 and/or FR4, particularly at least one marker residue in FR2 and/or FR 4.

Heavy chain antibodies or hcabs consist of only two heavy chains, each of which comprises only a heavy chain variable region (VHH) and hinge, CH2 and CH3. The antigen-antibody binding region of a heavy chain antibody or HCAb consists of three complementarity determining regions, which also makes it more antigen-binding than a typical antibody. On the other hand, in addition to the lack of a light chain, there is no CH1 region between the heavy chain variable region and the hinge region, which is also a great difference in heavy chain antibodies, compared to conventional antibodies.

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.

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. An antibody-drug conjugate (ADC) is a chemical linkage linking a biologically active small molecule drug to an antibody (e.g., a nanobody or antibody of the invention) that serves as a carrier to deliver the small molecule drug to a target cell.

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-pharmaceutical composition as defined above. Another embodiment of the invention is a composition as defined above further comprising a pharmaceutically acceptable carrier.

Another embodiment of the invention is a CD39 binding protein, heavy chain antibody, antigen binding fragment or 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.

Another embodiment of the invention is a CD39 binding protein, heavy chain antibody, antigen binding fragment or 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 the treatment, prevention and/or alleviation of a CD39 related disease.

Another embodiment of the invention is the use of a CD39 binding protein, heavy chain antibody, antigen binding fragment or 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 manufacture of a medicament for the treatment, prevention and/or alleviation of a CD39 related disease.

In some embodiments, the CD 39-related disease or disorder is a CD 39-mediated disease or disorder. In some embodiments, the CD39 mediated disease or disorder is a tumor. In some embodiments, the CD39 mediated disease or disorder is a solid tumor and/or hematological tumor. In some specific embodiments, the CD39 mediated disease or disorder 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.

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

Another embodiment of the invention is a method of prophylactic or therapeutic treatment of a CD 39-related disease or disorder, comprising administering to a patient an effective dose of a nanobody, antibody, heavy chain antibody, nucleic acid, recombinant cell, conjugate, or composition as defined above.

Another embodiment of the invention is a method of producing a nanobody as defined above comprising:

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

B) Recovering the produced polypeptide from the culture.

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

Another embodiment of the invention is a method of diagnosing a disease or condition mediated by CD39 comprising the steps of:

a) Contacting the sample with a nanobody, antibody, heavy chain antibody or antigen binding fragment as defined above, and

B) Detecting binding of the nanobody, antibody, heavy chain antibody or antigen binding fragment to the sample, and

C) Wherein a binding result above the cutoff value indicates that the sample is a CD39 mediated disease or disorder.

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 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, renal cancer, and melanoma.

Another embodiment of the invention is a kit for diagnosing a disease or disorder associated with CD 39. In some embodiments, the agent may be used in a method as defined above.

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.

One embodiment of the present 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.

ELISA assays for measuring the binding of anti-CD 39 nanobodies, antibodies, heavy chain antibodies or antigen binding fragments to CD39 are well known.

Conventional antibodies are unstable at room temperature and must be refrigerated for preparation and storage, thereby requiring the necessary refrigerated laboratory equipment, storage and transportation, thereby increasing time and expense. The anti-CD 39 nanobody of the present invention is stable at room temperature, and thus, it can be prepared, stored and/or transported without using a refrigerating apparatus, thereby saving costs, time and environment. Furthermore, conventional antibodies are not suitable for use in assays or kits that are performed at temperatures outside the bioactive temperature range (e.g., 37±20 ℃).

One aspect of the invention is an anti-CD 39 polypeptide comprising at least one anti-CD 39 heavy chain antibody, in particular nanobodies derived therefrom. One aspect of the invention is that such polypeptides may comprise other components. Such other components may be polypeptide sequences, such as one or more anti-CD 39 nanobodies or one or more anti-serum albumin nanobodies. Other fusion proteins are also within the scope of the invention and may include, for example, fusions with carrier polypeptides, signal molecules, tags, and enzymes. Other components may include, for example, radiolabels, organic dyes, fluorescent compounds.

According to one aspect of the invention, an anti-CD 39 polypeptide of the invention may comprise at least two identical or non-identical anti-CD 39 nanobody sequences. The anti-CD 39 polypeptide may comprise at least two of the above sequences that do not have the same affinity for CD39, thus forming an anti-CD 39 polypeptide that combines a weak affinity and a high affinity binding sequence.

Methods of constructing bivalent polypeptides are known in the art (e.g. US 2003/0088074) and are also described below.

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 with certain Fc domains, particularly with Fc domains of human origin, may be advantageous.

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

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

Humanization refers to mutation such that potential immunogenicity is little or absent upon administration in a human patient. According to the present invention, humanizing a polypeptide may include the step of replacing one or more non-human immunoglobulin amino acids with human counterparts present in a human consensus sequence or human germline gene sequence without losing the polypeptide's typical characteristics, i.e., humanizing does not significantly affect the antigen binding ability of the resulting polypeptide.

According to one aspect of the invention, a humanized nanobody is defined as a nanobody having at least 50% homology (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) with a human framework region.

One embodiment of the invention relates to polypeptides comprising at least one nanobody, wherein one or more amino acid residues have been substituted without substantially altering the antigen binding capacity.

The skilled artisan will recognize that the CD39 binding proteins of the invention may be modified and that such modifications are within the scope of the invention. For example, the polypeptide may be used as a pharmaceutical carrier, in which case it may be fused to a therapeutic activator, or its solubility characteristics may be altered by fusion to an ionic/bipolar group, or it may be used for imaging by fusion to a suitable imaging marker, or it may comprise modified amino acids or the like. The polypeptides may also be prepared as salts. Such modifications that substantially retain binding to CD39 are within the scope of the invention.

It is clear from the disclosure herein that analogs of the nanobody of the invention, particularly SEQ ID NOS 88-116, using natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (collectively referred to herein as "analogs") of the nanobody of the invention as defined herein are also within the scope of the invention. Thus, according to one embodiment of the present invention, the term "nanobody of the invention" also encompasses such analogs in its broadest sense.

In general, one or more amino acid residues may have been replaced, deleted and/or added in such analogs as 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 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 tag residues and/or one or more other positions in the framework residues, but substitution, insertion, or deletion of tag residues is generally less preferred (unless these are suitable humanized substitutions as described herein).

Yet another modification may include the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labeled nanobody. Suitable labels and techniques for attaching, using and detecting nanobodies are apparent to the skilled artisan and include, for example, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as ¹⁵² Eu or other lanthanide metals), phosphorescent labels, chemiluminescent or bioluminescent labels (e.g., luminol, isoluminol, thermal acridinium ester, imidazole, acridinium salt, oxalate, dioxetane or GFP and analogs thereof), radioisotopes (e.g., ³H、¹²⁵I、³²P、³⁵S、¹⁴C、⁵¹Cr、³⁶Cl、⁵⁷Co、⁵⁸Co、⁵⁹Fe、 and ⁷⁵ Se), metals, metal chelates or metal cations (e.g., metal cations such as ^99mTc、¹²³I、¹¹¹In、¹³¹I、⁹⁷Ru、⁶⁷Cu、⁶⁷ Ga, and ⁶⁸ Ga or other metals or metal cations particularly suitable for in vivo, in vitro or in situ diagnosis and imaging, e.g., ¹⁵⁷Gd、⁵⁵Mn、¹⁶²Dy、⁵² Cr, and ⁵⁶ Fe), and chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, phospho-triose, bio-peroxidase, asparaginase, glucose oxidase, and the like). Other suitable tags will be apparent to the skilled person, including for example parts that can be detected using NMR or ESR spectroscopy.

Such labeled nanobodies and polypeptides of the invention can be used, for example, 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, depending on the choice of the particular label.

As will be clear to a person skilled in the art, another modification may involve the introduction of chelating groups, for example chelating one of the metals or metal cations described above. Suitable chelating groups include, for example, but are not limited to, diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA).

Yet another modification may include the introduction of a functional group as part of a specific binding pair, such as a biotin- (streptavidin) binding pair. Such functional groups may be used to attach the nanobody of the invention to another protein, polypeptide or chemical compound that binds to the other half of the binding pair (i.e., by forming the binding pair). For example, the nanobody of the invention may be conjugated to biotin and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such conjugated nanobodies may be used as reporter genes, for example in diagnostic systems where a detectable signal generating agent is conjugated to avidin or streptavidin. For example, such binding pairs may also be used to bind nanobodies of the invention to a carrier comprising a carrier suitable for pharmaceutical purposes. One non-limiting example is the case of the liposome formulation described in Cao and sursh, journal of Drug Targeting,8,4,257 (2000). Such binding pairs may also be used to attach therapeutic activators to nanobodies of the invention.

Other potential chemical and enzymatic modifications will be apparent to the skilled artisan. Such modifications may also be introduced for research purposes (e.g., to study functional-activity relationships). See, e.g., lundblad and Bradshaw, biotechnol. Appl. Biochem.,26,143-151 (1997).

As noted above, the present invention also relates to proteins or polypeptides consisting essentially of at least one nanobody of the invention. By "consisting essentially of" is meant that the amino acid sequence of the polypeptide of the invention is identical to or corresponds to the amino acid sequence of the nanobody of the invention having a limited number of amino acid residues, e.g. 1-20 amino acid residues, e.g. 1-10 amino acid residues, preferably 1-6 amino acid residues, e.g. 1, 2, 3, 4, 5 or 6 amino acid residues, added to the amino terminus, carboxy terminus or amino terminus and carboxy terminus of the amino acid sequence of the nanobody.

The amino acid residues may or may not alter, alter or otherwise affect the (biological) properties of the nanobody, and may or may not add further functionality to the nanobody.

According to another embodiment, the polypeptide of the invention comprises a nanobody of the invention fused at its amino-terminus, at its carboxy-terminus, or at its amino-terminus and at its carboxy-terminus with at least one additional amino acid sequence, i.e. to provide a fusion protein comprising said nanobody of the invention and one or more additional amino acid sequences. Such fusion will also be referred to herein as "nanobody fusion".

The one or more additional amino acid sequences may be any suitable and/or desired amino acid sequence. The additional amino acid sequence may or may not alter, 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 imparts one or more desired properties or functionalities to the nanobody or polypeptide of the invention.

The nucleic acid of the invention may 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 invention may be genomic DNA, cDNA or synthetic DNA (e.g., DNA having a codon usage that has been specifically adapted for expression in a desired host cell or host organism).

According to one embodiment of the invention, the nucleic acid of the invention is in a substantially isolated form as defined herein.

The nucleic acids of the invention may also be in the form of, in and/or as part of a vector, such as a plasmid, cosmid or YAC, which may also be in a substantially isolated form.

The nucleic acids of the invention may also be in the form of, present in and/or be part of a genetic construct, as will be clear to a person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention, optionally linked to one or more elements of the genetic constructs known per se, for example one or more suitable regulatory elements (e.g. suitable promoters, enhancers, terminators, etc.) and other elements of the genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention".

The genetic construct of the invention may be DNA or RNA, and is preferably double stranded DNA. The genetic construct of the invention may also be in a form suitable for transformation of a desired host cell or host organism, for integration into the genomic DNA of a desired host cell, or for independent replication, maintenance and inheritance in a desired host organism. For example, the genetic construct of the invention may be in the form of a vector such as a plasmid, cosmid, YAC, viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that may provide for in vitro and/or in vivo expression (e.g. in a suitable host cell, host organism and/or expression system).

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 the 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 said "other elements" present in the genetic construct may be, for example, 3'-UTR or 5' -UTR sequences, leader sequences, selectable markers, expression markers/reporter genes, and/or elements that may promote or increase transformation or integration (efficiency). These and other suitable elements for such genetic constructs will be apparent to the skilled person and may depend, for example, on the type of construct used, the desired host cell or host organism, the manner in which the nucleotide sequences of interest of the present invention are 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 expression and production of antibodies and antibody fragments, including but not limited to (single) domain antibodies and ScFv fragments, may be used in a substantially similar manner.

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

Preferably, the regulatory and other elements of the genetic construct of the invention enable it to provide its desired biological function in a desired host cell or host organism.

For example, a promoter, enhancer or terminator should be "operable" in a desired host cell or host organism, which means that, for example, the promoter should be capable of initiating or otherwise controlling/regulating transcription and/or expression of a nucleotide sequence, such as a coding sequence, to which it is operably linked (as defined herein).

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 those used in the examples.

The 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 sequences 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 that provide resistance to antibiotics (e.g. kanamycin or ampicillin), genes that provide temperature resistance, or genes that allow a host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) ingredients in the medium critical to the survival of non-transformed cells or organisms.

The leader sequences should be those that allow for the desired post-translational modification in the desired host cell or host organism and/or such that they direct the transcribed mRNA to the desired portion of the cell or organelle. The leader sequence may also allow secretion of the expression product from the cell. Thus, the leader sequence may be any pro-sequence, prepro-sequence, or prepro-sequence operable in the host cell or host organism. Leader sequences may not be required for expression in bacterial cells. For example, leader sequences known per se for expression and production of antibodies and antibody fragments (including, but not limited to, single domain antibodies and ScFv fragments) may be used in a substantially similar manner.

An expression marker or reporter gene should be one that allows for the detection of the expression of a genetic construct (a gene or nucleotide sequence present on the genetic construct) in a host cell or host organism. Expression markers may also optionally allow for localization of the expression product, for example 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 may also be expressed as proteins fused to the amino acid sequences of the invention. Some preferred but non-limiting examples include fluorescent proteins such as GFP.

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 particularly those suitable for expression in bacterial cells, such as those mentioned herein and/or those used in the examples below. For some (other) non-limiting examples of promoters, selectable markers, leaders, expression markers and other elements that may be present/used in the genetic constructs of the invention, e.g., terminators, transcriptional and/or translational enhancers and/or integration factors, reference is made to the general handbooks of Sambrook et al and Ausubel et al, as mentioned above, and examples given 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 apparent to the skilled person. Reference is also made to the general background art cited above and to the further references cited herein.

The genetic constructs of the invention may generally be provided by appropriately ligating the nucleotide sequences of the invention with one or more of the other elements described above, for example using techniques as described in the general handbooks of Sambrook et al and Ausubel et al, as mentioned above.

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 examples below and those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e., for expression and/or production of the nanobody or polypeptide of the invention. Suitable hosts or host cells will be apparent to the skilled person and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism.

In general, for the prevention and/or treatment of the diseases and conditions mentioned herein, the nanobodies and polypeptides of the invention are typically administered as a single daily dose or as multiple divided doses throughout the day, in an amount of from 1 gram to 0.01 microgram per kg body weight per day, preferably from 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, depending on the particular disease or condition to be treated, the potency of the particular nanobodies and polypeptides of the invention to be used, the particular route of administration, and the particular pharmaceutical formulation or composition used. Depending on the factors mentioned herein, a clinician is generally able to determine an appropriate daily dose. It is also clear that in certain situations, the clinician may choose to deviate from these amounts, for example, based on the factors described above and their professional judgment. In general, some guidance regarding the amount administered may be obtained from the usual amount of a comparable conventional antibody or antibody fragment administered by essentially the same route against the same target, but taking into account differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

It should also be noted that when the nanobody of the invention contains one or more other CDR sequences different from the preferred CDR sequences described above, these CDR sequences may be obtained in any manner known per se, for example from the nanobody (preferred), the V _H domain from a conventional antibody (in particular from a human antibody), the heavy chain antibody, a conventional 4 chain antibody (for example a conventional human 4 chain antibody) or other immunoglobulin sequences directed against a- β. Such immunoglobulin sequences directed against a-beta may be generated in any manner known per se, i.e. by immunization with a-beta or by screening a library of suitable immunoglobulin sequences with a-beta or any suitable combination, as will be clear to a person skilled in the art. Optionally, techniques such as random or site-directed mutagenesis and/or other affinity maturation techniques known per se may follow. Suitable techniques for producing such immunoglobulin sequences are apparent to the skilled artisan and include, for example, screening techniques reviewed by Hoogenboom, nature Biotechnology,23,9,1105-1116 (2005). Other techniques for producing immunoglobulins against a specific target include, for example, nanocloning techniques (e.g., as described in U.S. provisional patent application 60/648,922, which is not previously published), so-called SLAM techniques (e.g., as described in european patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins, or well-known hybridoma techniques (see, e.g., larrick et al, biotechnology, vol.7,1989, p.934). All of these techniques can be used to generate immunoglobulins directed against a- β, and CDRs of such immunoglobulins can be used in 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 nanobody of the invention (e.g., to replace the corresponding natural CDRs), all of which can be synthesized de novo using techniques known per se such as those described herein, or the nanobody of the invention comprising such CDRs (or nucleic acids encoding the same) can be synthesized de novo using the techniques mentioned herein.

The invention will now be further described by the following non-limiting examples and figures.

Examples

Example 1 alpaca immunization and detection

1.1 Alpaca immunization

The non-immunized Alpaca alpaca was immunized with human CD39 protein (ACRO, cat#cd9-H52H 4), with complete freund's adjuvant and incomplete freund's adjuvant, using subcutaneous multiple injections.

1.2 Potency detection

Serum titers were detected from the second immunization by ELISA and FACS. Data were counted starting from the third immunization.

1) ELISA screening

A96-well ELISA plate was coated with 100. Mu.L of Human CD39 recombinant protein (ACRO, CAT#CD9-H52H 4), cyno CD39 recombinant protein (ACRO, CAT#CD9-C52H 3), or Mouse CD39 recombinant protein (ACRO, CAT#CD9-M52H 3) (final concentration 1. Mu.g/mL) overnight at 4 ℃,. Mu.C, 3 times of washing with PBST (containing 0.05% Tween), 37℃blocking buffer (3% MPBS), 3 times of washing with PBST, 2-fold serial dilutions of immune serum from 1:1000 were added, 37℃were incubated for 1H, 3 times of PBST washing, 100. Mu.L of 0.1. Mu.g/mL of HRP-8624 Rabbit Anti-CAMELID VHH Cocktail (Gensc, CAT#A02014) or 0.1. Mu.g/mL of labeled Goat Anti-Llama (HRP, CAT#A42), 3 times of light-blocking buffer (6. Mu.5 nm) was added, each well was stopped, 50. Mu.L of the PCR reaction was stopped, and the 50. Mu.L of the PCR plate was stopped by a light-resistant device.

TABLE 1 serum ELISA assay results with human CD39 as antigen

TABLE 2 serum ELISA assay results with cynomolgus monkey CD39 as antigen

TABLE 3 serum ELISA assay results with mouse CD39 as antigen

2) FACS screening

Chinese hamster ovary cell sub-strain CHO-K1 (Probio), chinese hamster ovary cell CHO-K1/CD39 (+) (Probio) recombinantly expressing human CD39 and chinese hamster ovary cell CHO-K1/Cyno CD39 (+) (Probio) recombinantly expressing cynomolgus monkey CD39 were divided into several parts, each cell number was 1×10 ⁵ cells, target cells were incubated with 50 μl of immune serum diluted 1:100 and 1:1000 fold, respectively, and after thoroughly mixing, incubated at room temperature for 1h. Cells were washed 3 times with PBS and Alexa was added488AffiniPure 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), after thoroughly mixing, incubating for 30min at room temperature in the absence of light, washing the cells with PBS 3 times, and detecting the cells with a flow cytometer, the results of which are shown in FIGS. 1 and 2. The results showed that the serum from 5 immunized alpaca had better FACS activity, indicating that very good antibodies to CD39 protein were produced.

EXAMPLE 2 phage display library construction

2.1RNA extraction

Total RNA was extracted from isolated PBMC according to TRIzol Reagent instructions, and a total of about 41. Mu.g was extracted and used for reverse transcription into cDNA. The amount and quality of total RNA were assessed by gel electrophoresis and OD 260/280 ratio, respectively.

2.2VHH fragment amplification

A plurality of forward and reverse primers were designed for the amplification of VHH fragments, respectively, into which two SfiI restriction sites were introduced. The products of the different primer pairs were mixed and gel purified to obtain more than 3. Mu.g of DNA encoding VHH fragments.

2.3 Library construction

The purified DNA encoding VHH fragments was electroinserted into a vector for library construction. 100. Mu.L of the transformation was titrated to count the library size. 95 clones were randomly selected for sequencing to assess library quality and all remaining bacterial fluids were subjected to plate amplification and finally cryopreserved as glycerol bacteria. The results of library detection and summary are shown in Table 4, the constructed library capacity is 1.42×10 ⁹, and the frequency distribution of the length and the frequency of the IMGT-CDR3 of the library is shown in FIG. 3.

TABLE 4 statistical results of library construction

Library capacity	Insertion rate	Correct translation rate	Diversity of	Library capacity
					CD39 library	1.42×10⁹	100%(95/95)	95%(82/86)	98%(80/82)

EXAMPLE 3 phage library panning

After the alpaca immune phage library is prepared, solid phase panning, cell panning and multi-round liquid phase panning are respectively adopted to perform panning and enrichment of specific antibodies, so that the aim of screening targeted CD39 single domain antibodies is fulfilled.

3.1 Solid phase panning

The Human CD39 protein was diluted to 50. Mu.g/mL with CBS coating solution, added to the 8-well plate strips of the solid phase screen, 125. Mu.L/well, and left overnight at 4 ℃. The next day the coated strips were washed twice with 0.05% PBST, 300. Mu.L 3% MPBS was added to each well and blocked at 37℃for 1h. Phage library (1X 10 ¹² pfu/pool) was simultaneously taken and diluted to 1mL with 3% MPBS and blocked for 1h on a horizontal shaker at room temperature. After the end of the blocking, the blocking solution on the solid-phase lath is discarded, the blocked phage library is added into the pore plate, 125 mu L/pore is added, and the mixture is incubated for 30-45min at room temperature by a horizontal shaking table. The solution in the binding wells was discarded, washed 8-10 times with 300. Mu.L of 0.05% PBST per well, and 3-5 times with 300. Mu.L PBS, and the wells were dried by pipetting. 125. Mu.L of 0.1M TEA was added to each well and eluted with slow shaking on a horizontal shaker for 8-10min. To a new 1.5mL protein low binding tube, 0.5mL 1M Tris-HCl (pH 7.4) was added, and after the elution time had arrived, the solution in the eluted microwells was transferred to Tris-HCl solution for mixing and neutralization for 10min. 1mL of phage product after elution and neutralization is added into 10mL of TG1 bacterial liquid in logarithmic growth phase, and the mixture is mixed uniformly and then kept stand at 37 ℃ for infection for 45min.

3.2 Cell panning

The negative cells (CHO-K1) and the positive cells (CHO-K1/CD 39) (Probio) recombinantly expressing human CD39 were each washed 3 times with pre-chilled, sterile PBS and placed on ice for use. Negative cells (1X 10 ⁷ cells) and phage library (1X 10 ¹² pfu/pool) were taken and each made up to a final volume of 1mL with casein blocking solution and blocked for 1h on a horizontal shaker at room temperature. After the end of the blocking, the negative cells are centrifuged at a low speed, blocking solution is discarded, the blocked phage library is added into the negative cells, and after the phage library is resuspended, the phage library is incubated for 1h at room temperature and in a horizontal shaker. Negative cells were discarded, phage library supernatant was transferred to positive cells, and after resuspension, incubated for 1h at room temperature on a horizontal shaker. After the incubation, the cells were resuspended in 400. Mu.L of 1 XPBS after 3 washes with 1% BSA/PBS and 3 washes with 1 XPBS. 400. Mu.L of 0.2M TEA was added to the cells and eluted with slow shaking on a horizontal shaker for 8-10min. Then 400. Mu.L of Tris-HCl solution was added thereto, and the mixture was shaken and neutralized on a horizontal shaker for 10 minutes. 1mL of phage product after elution and neutralization is added into 10mL of TG1 bacterial liquid in logarithmic growth phase, and the mixture is mixed uniformly and then kept stand at 37 ℃ for infection for 45min.

3.3 Liquid phase panning

Mu.L of magnetic beads (Dynabeads M-280 strepitavidins) and phage library (2X 10 ¹² pfu/pool) were added with 3% MPBS blocking solution to a final volume of 1mL and incubated for 1h at room temperature on a horizontal shaker. Then 5. Mu.g of Biotin-human CD39 protein was added to the blocked phage library and incubated at room temperature for 30-45min on a horizontal shaker. Removing the sealing liquid of the magnetic beads, adding the well-incubated antigen-phage library into the magnetic beads, and incubating for 30-45min at room temperature in a horizontal shaking table after re-suspending. The beads were then washed 8 times with 1mL of 0.05% PBST and 3 times with PBS. The wash was discarded, 800. Mu.L of 0.1M TEA was added to the beads and eluted by slow shaking on a horizontal shaker for 8-10min. After the elution was completed, the magnetic beads were discarded, and the eluted solution was transferred to a low adsorption tube containing 400. Mu.L of 1M Tris-HCl (pH 7.4), mixed well, and neutralized for 10min. 1mL of phage product after elution and neutralization is added into 10mL of TG1 bacterial liquid in logarithmic growth phase, and the mixture is mixed uniformly and then kept stand at 37 ℃ for infection for 45min.

Example 4 monoclonal screening assay and sequencing

After 1 round/2 round of panning, monoclonal culture and expression are respectively selected, phage supernatant is taken, and ELISA is used for detecting the binding condition of Human CD39, cyno CD39 and Mouse CD39 proteins. The binding of the expressed monoclonal phage supernatant to target cells (chinese hamster ovary cell sub-strain 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 CD 39) was examined by FACS and showed that most clones had binding activity to both CHO-K1/CD39 and CHO-K1/Cyno CD39 cells.

1) ELISA screening

The monoclonal bacterial solutions cultured in 96-well plates were shake-cultured overnight at 30℃after adding M13KO7 helper phage, respectively. Meanwhile, 96-well ELISA plates were coated with 1. Mu.g/mL of Human, cyno, mouse CD39 protein, 100. Mu.L/well, respectively, and coated overnight at 4 ℃. After the coating, 300. Mu.L of 3% MPBS was added to each of the ELISA plates, and the plates were blocked at 37℃for 1 hour. After washing the plates, 50. Mu.L of overnight culture broth supernatant and 50. Mu.L of 0.05% PBST were added to each well, and incubated and bound for 2 hours at room temperature. After washing the plates, HRP-labeled Anti-M13 Monoclonal Antibody secondary antibody was added and incubated for 45min at room temperature. Finally, the plate was washed 6 times, 100. Mu.L of TMB developing solution was added to each well, the color was developed at room temperature for 10min, and then 50. Mu.L/well of stop solution was added. After reading by an ELISA reader, positive clones were selected for FACS detection based on OD450 nm.

2) FACS screening

Taking 50 mu L of supernatant samples and 50 mu L of cells, incubating for 40min at 4 ℃, then adding 150 mu L of PBS, centrifuging for 3min at 300g, washing for 2 times, adding 100 mu L of Anti-fd bacterial-Biotin Anti-body (Sigma, CAT#B2661), incubating for 30min at 4 ℃, adding 150 mu L of PBS, centrifuging for 3min at 300g, washing for 2 times, adding 100 mu L STREPTAVIDIN-Alexa Fluor 647 secondary antibody, incubating for 30min at 4 ℃, adding 150 mu L of PBS, centrifuging for 5min at 300g, washing for 2 times, finally adding 50-100 mu L of Flow Cytometry Staining Buffer resuspended cells, and detecting and analyzing by using a flow cytometer.

The screened FACS positive clones were sequenced, and a total of 29 unique VHH sequences were finally obtained. The sequences of CDR1, CDR2 and CDR3 for each VHH are shown in table 5.

TABLE 5 sequences of CDR1, CDR2 and CDR3 of the positive clones VHH obtained by screening

TABLE 6 amino acid sequence of the Positive clone VHH obtained by screening

EXAMPLE 5 expression of recombinant antibodies

The coding sequences of the 29 antibody sequences obtained were inserted into eukaryotic expression vectors pcdna3.4 (Probio) containing nucleic acid sequences encoding Fc, respectively, and the pcdna3.4 into which the antibody coding sequences were inserted was transfected into an Expi293F cell (Probio), thereby performing antibody expression, and purifying the expressed antibodies.

Protein A MAGNETIC Beads were added to the sample to be purified and incubated for 2h in a room temperature shaking incubator. Protein A MAGNETIC reads were equilibrated with 10 column volumes of Binding Buffer. Eluent was added to Protein A MAGNETIC heads, the eluent was collected, and at the same time, neutralization solution (1/10 of the volume of eluent collected) was added to adjust the pH to neutral. After elution and neutralization, the eluted and collected sample is transferred to a dialysis bag, dialyzed with PBS at room temperature for 2h, and then the solution is changed, and dialysis is continued for 16h at 2-8 ℃. The concentration and purity of the antibodies were determined by OD280 and SDS-PAGE.

SDS-PAGE detection of purified antibodies showed that all antibodies were of the correct molecular weight and purity >95%.

EXAMPLE 6 detection of purified antibodies

1) ELISA screening

Antibody binding levels to the Human CD39 protein, the Cyno CD39 protein, and the Mouse CD39 protein were detected by ELISA EC50 experiments, respectively. The 96-well ELISA plate was coated overnight at 4℃with 1. Mu.g/mL of Human CD39 protein, cyno CD39 protein, or Mouse CD39 protein, respectively. After 3 plate washes, 300 μl of 3% mpbs was added to each well and blocked at 37 ℃ for 1h. The antibodies to be tested were diluted to a concentration of 100nM in the first well, followed by a 3-fold gradient dilution for 10 gradients, with the 12 th well being the blank. The diluted antibodies were then added to the blocked elisa plate, 100 μl/well, incubated at 37 ℃ and bound for 1h. After washing the plate 3 times, 100. Mu.L of Mouse Anti-Human IgG Fc-HRP secondary antibody is added, incubation is carried out at 37 ℃ for 30min, finally the plate is washed 6 times, then 100 mu LTMB color development liquid is added into each hole, color development is carried out at room temperature for 10min, then 50 mu L/hole termination liquid is added, and the OD450nm value is read through an enzyme label instrument.

The results of ELISA detection of the purified antibodies are shown in tables 7 to 9, and the results show that AHP18698 and AHP18706 are combined with human CD39 protein, monkey CD39 protein and mouse CD39 protein and have strong combination ability. AHP18686, AHP18697 and AHP18699 bind to human, monkey and mouse CD39 protein and have strong binding capacity. AHP17815 and AHP18683 cross-bind to human and monkey CD 39.

Table 7ELISA detection of EC50 values for binding of each antibody to human CD39

Antibody numbering	EC50/nM	Antibody numbering	EC50/nM	Antibody numbering	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	/	/

Table 8ELISA detection of EC50 values for binding of each antibody to monkey CD39

Antibody numbering	EC50/nM	Antibody numbering	EC50/nM	Antibody numbering	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	/	/

Table 9ELISA detection of EC50 values for binding of each antibody to murine CD39

2) FACS screening

50. Mu.L of diluted antibody (10. Mu.g/mL) was added to a 96-well assay plate, then 50. Mu.L of assay cells CHO-K1 (-), CHO-K1/CD39 (+) or CHO-K1/Cyno CD39 (+), 1X 10 ⁵ cells/well were added per well, incubated at 4℃for 40min, then 150. Mu.L of PBS was added, centrifuged at 300g for 3min, washed 2 times, 100. Mu.L of Alexa Fluor 647AffiniPure Fab Fragment Goat Anti-Human IgG secondary antibody was added, incubated at 4℃for 30min, then 150. Mu.L of PBS was added, centrifuged at 300g for 3min, washed 2 times, and finally 50. Mu.L of resuspended cells were added and detected using a flow cytometer.

As shown in FIG. 4, the 29 antibodies had stronger FACS activity except for the weaker FACS activity of the AHP17852 and AHP17854 antibodies.

Claims

1. A CD39 binding protein, wherein said binding protein comprises an immunoglobulin single variable domain, wherein said immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 comprised in a VHH set forth 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:

4. A CD39 binding protein according to any one of claims 1-3, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs 88-116.

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

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

7. The CD39 binding protein of any one of claims 1-6, wherein the CD39 binding protein is a heavy chain antibody.

8. The CD39 binding protein of claim 7, wherein the heavy chain antibody further comprises a human IgG1 Fc.

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

10. The CD39 binding protein of claim 9, said nanobody comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in any one of SEQ ID NOs 88 to 116, preferably said nanobody comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs 88 to 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 1-11, 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 (e.g., a mammalian cell).

15. A conjugate comprising the CD39 binding protein of any one of claims 1-11 conjugated to a therapeutic, diagnostic or 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 a CD39 binding protein according to any one of claims 1-11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, or a cell according to claim 14, or a conjugate according to claim 15, in the manufacture of a medicament for the prevention, treatment or alleviation of a CD39 related disease.

18. Use of a CD39 binding protein according to any one of claims 1-11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, or a cell according to claim 14, or a conjugate according to claim 15 in combination with another agent, preferably an antibody or a chemotherapeutic agent, for the manufacture of a medicament for the prevention, treatment or alleviation of a CD39 related disease.

19. A kit for detecting CD39 or a cell comprising CD39, comprising the CD39 binding protein of any one of claims 1-11, or the conjugate of claim 15.

20. Use of a CD39 binding protein according to any one of claims 1-11 or a conjugate according to claim 15 in the manufacture of a kit for detecting CD39 or a cell comprising CD 39.

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

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