WO2024109585A1 - 抗cd39抗体及其应用 - Google Patents

抗cd39抗体及其应用 Download PDF

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Publication number
WO2024109585A1
WO2024109585A1 PCT/CN2023/131347 CN2023131347W WO2024109585A1 WO 2024109585 A1 WO2024109585 A1 WO 2024109585A1 CN 2023131347 W CN2023131347 W CN 2023131347W WO 2024109585 A1 WO2024109585 A1 WO 2024109585A1
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seq
amino acid
acid sequence
antibody
antigen
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PCT/CN2023/131347
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English (en)
French (fr)
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王媛媛
周新新
张轶博
肖亮
欧颖烨
张喆
霍永庭
芦迪
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广东菲鹏制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present disclosure relates to the field of biotechnology, and in particular, to an anti-CD39 antibody and an application thereof.
  • TAE tumor microenvironment
  • ATP-adenosine pathway plays a key role.
  • CD39 or ectonucleoside triphosphate diphosphohydrolase 1 (ENTPDase1), is a member of the ATP-adenosine pathway and plays an important role in the immunosuppressive response of the tumor microenvironment.
  • CD39 is a single-channel type II transmembrane protein that converts extracellular eATP or ADP into AMP, and then CD73 converts AMP into adenosine, which is a metabolite that exerts an immunosuppressive effect in the tumor microenvironment.
  • adenosine When the produced adenosine binds to adenosine receptors on the surface of CD4T, CD8T cells, and natural killer (NK) cells, it can inhibit T cell and NK cell responses, thereby inhibiting the immune system and promoting tumor growth.
  • adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, thereby inhibiting phagocytosis and antigen presentation, and increasing the secretion of tumor-promoting factors (such as VEGF, TGF ⁇ , and IL6).
  • Adenosine promotes the immunosuppressive activity of Treg.
  • the adenosine pathway refers to the conversion of eATP to adenosine by adenylate hydrolases and the signaling of adenosine through A2A/A2B adenosine receptors on immune cells.
  • CD39 maintains a balance between the extracellular levels of immunosuppressive adenosine and immunostimulatory ATP.
  • ATP is barely detectable in the extracellular environment because ATP is rapidly broken down by CD39 to generate ADP or AMP, which is then converted to adenosine by CD73.
  • eATP levels increase significantly, but are subsequently hydrolyzed by CD39 and CD73 and converted to adenosine, which hinders the immune system's recognition of tumors and their killing.
  • the efficacy of anti-CD39 antibodies in blocking CD39 hydrolysis of ATP varies greatly, and screening for anti-CD39 antibodies with high blocking activity is difficult.
  • One of the purposes of the present disclosure is to provide an antibody that can specifically bind to CD39 and its application.
  • the present disclosure provides an anti-CD39 antibody or an antigen-binding fragment thereof.
  • the antibody comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the complementarity determining region of the light chain variable region comprises LCDR1, LCDR2 and LCDR3;
  • the above HCDR1 comprises an amino acid sequence as shown in SEQ ID NO.2 (GX 2-2 TFX 2-5 NX 2-7 G) or SEQ ID NO.1 (X 1-1 X 1-2 SX 1-4 TX 1-6 YX 1-8 ), wherein X 2-2 is Y or F; X 2-5 is T or S; X 2-7 is Y or F; X 1-1 is G or D; X 1-2 is Y, F or L; X 1-4 is F or L; X 1-6 is D or N; X 1-8 is N or A;
  • the HCDR2 comprises an amino acid sequence as shown in SEQ ID NO.6 (INTNTGEP), SEQ ID NO.3 (IDPYNGX 3-7 T), SEQ ID NO.4 (IWRRGST) or SEQ ID NO.5 (ISSGSNTI), wherein X 3-7 is A or V;
  • the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO.7 (ARX 7-3 X 7-4 IYYDYVWFFDV), SEQ ID NO.8 (AX 8-2 YGYDX 8-7 X 8-8 X 8-9 X 8-10 YFDY) or SEQ ID NO.9 (AKGGTATSWFAY), wherein X 7-3 is R or S; X 7-4 is A or G; X 8-2 is I or V; X 8-7 is G or E; X 8-8 is G or V; X 8-9 is A or P; X 8-10 is Y or N;
  • the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO.10 (QDINX 10-5 Y), SEQ ID NO.12 (SSVNY), SEQ ID NO.11 (QNVDTX 11-6 ) or SEQ ID NO.13 (KSLLHSNGNTY), wherein X 10-5 is N or K; X 11-6 is N or T;
  • the LCDR2 comprises an amino acid sequence as shown by amino acid residues YT, GT, SA or RM;
  • the LCDR3 comprises an amino acid sequence as shown in SEQ ID NO.16 (LQYHDLIT), SEQ ID NO.14 ( X14-1 QX14-3 X14-4 X14-5 X14-6 X14-7 X14-8 T) or SEQ ID NO.15 (MQHLEYPFT), wherein X14-1 is Q or L; X14-3 is R or Y; X14-4 is S, D or N; X14-5 is S or N; X14-6 is Y or L; X14-7 is P or missing; X14-8 is F or L.
  • SEQ ID NO.16 LQYHDLIT
  • SEQ ID NO.14 X14-1 QX14-3 X14-4 X14-5 X14-6 X14-7 X14-8 T
  • SEQ ID NO.15 MQHLEYPFT
  • the present disclosure provides an anti-CD39 antibody or an antigen-binding fragment thereof, the antibody comprising:
  • HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 are determined according to the Kabat definition, Chothia definition, AbM definition and Contact definition.
  • the present disclosure provides an anti-CD39 antibody or an antigen-binding fragment thereof, which competitively binds to CD39 with the aforementioned anti-CD39 antibody, or the epitope thereof that binds to the CD39 antigen is the same as the epitope that the aforementioned anti-CD39 antibody binds to the CD39 antigen.
  • the present disclosure provides a multispecific antibody comprising the aforementioned anti-CD39 antibody or an antigen-binding fragment thereof.
  • the present disclosure provides a biomaterial, characterized in that the biomaterial is selected from any one of (i) to (v):
  • a chimeric antigen receptor (i) a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular region, wherein the extracellular region comprises an antigen binding domain; and wherein the antigen binding domain comprises the anti-CD39 antibody or antigen binding fragment thereof according to any of the preceding aspects;
  • an engineered immune cell wherein the immune cell expresses the chimeric antigen receptor described in (i) or contains a nucleic acid encoding the chimeric antigen receptor described in (i);
  • a recombinant cell wherein the recombinant cell carries the nucleic acid molecule described in (iii), or contains the vector described in (iv), or expresses the antibody or antigen-binding fragment thereof described in any one of the preceding aspects.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned anti-CD39 antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, and the aforementioned biological material;
  • the above-mentioned pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the present disclosure provides the use of the aforementioned anti-CD39 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned biomaterial or the aforementioned pharmaceutical composition in the preparation of a drug for preventing, treating, or diagnosing tumor diseases associated with CD39.
  • the present disclosure provides a method for producing the aforementioned anti-CD39 antibody or antigen-binding fragment thereof, comprising the following steps:
  • step (b) isolating and purifying the anti-CD39 antibody or antigen-binding fragment thereof obtained in step (a).
  • the present disclosure provides a method for treating, preventing or alleviating a disease, disorder or condition associated with CD39, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition to a subject.
  • the present disclosure also provides an anti-CD39 antibody or antigen-binding fragment as described in any of the aforementioned aspects for use as a medicament.
  • the present disclosure provides a kit for detecting CD39, comprising the aforementioned anti-CD39 antibody or antigen-binding fragment thereof.
  • the present disclosure provides use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned biomaterial or the aforementioned pharmaceutical composition in preparing a kit for detecting CD39 or diagnosing a disease associated with CD39.
  • FIG1 is a flow cytometric peak graph of CHO-K1 cells transfected and stably expressing cynoCD39 or mCD39 after binding to the corresponding antibodies.
  • Figure 2 shows the binding activity of CD39 monoclonal antibodies to Mino cells measured by flow cytometry fluorescence sorting technology.
  • A shows the binding activity of antibodies #2, #66, #75, and #95 to Mino cells;
  • B shows the binding activity of antibodies #103 and #110 to Mino cells.
  • Figure 3 shows the binding activity of CD39 monoclonal antibodies to MOLP-8 cells measured by flow cytometry fluorescence sorting technology.
  • A shows the binding activity of antibodies #2, #66, #75, and #95 to MOLP-8 cells;
  • B shows the binding activity of antibodies #103 and #110 to MOLP-8 cells.
  • Figure 4 shows the binding activity of CD39 monoclonal antibodies to CHO-K1-hCD39 cells measured by flow cytometry fluorescence sorting technology.
  • A shows the binding activity of antibodies #2, #66, #75, and #95 to CHO-K1-hCD39 cells;
  • B shows the binding activity of antibodies #103 and #110 to CHO-K1-hCD39 cells.
  • FIG5 shows the binding activity of CD39 monoclonal antibody to CHO-K1-cynoCD39 cells measured by flow cytometric fluorescence sorting technology.
  • FIG6 shows the binding activity of CD39 monoclonal antibody to CHO-K1-mCD39 cells measured by flow cytometric fluorescence sorting technology.
  • Figure 7 shows the inhibitory effect of CD39 monoclonal antibodies on the ATPase activity of CD39 on Mino cells.
  • A shows the inhibitory effect of antibodies #2, #66, #75, and #95 on the ATPase activity of CD39 on Mino cells;
  • B shows the inhibitory effect of antibodies #103 and #110 on the ATPase activity of CD39 on Mino cells.
  • Figure 8 shows the inhibitory effect of CD39 monoclonal antibodies on the ATPase activity of CD39 on MOLP-8 cells.
  • A shows the inhibitory effect of antibodies #66, #75, and #95 on the ATPase activity of CD39 on MOLP-8 cells;
  • B shows the inhibitory effect of antibodies #103 and #110 on the ATPase activity of CD39 on MOLP-8 cells.
  • FIG. 9 shows the binding activity of CD39 antibodies of the mIgG1 subtype to the extracellular ECD region of the CD39 protein measured by ELISA.
  • Figure 10 shows the binding activity of CD39 antibodies of the mIgG1 subtype to Mino cells measured by flow cytometry fluorescence sorting technology.
  • A shows the binding activity of antibodies R1445, R1446, and R1476 to Mino cells;
  • B shows the binding activity of antibodies R1478 and R1448 to Mino cells.
  • Figure 11 shows the binding activity of CD39 antibodies of the mIgG1 subtype to MOLP-8 cells measured by flow cytometry fluorescence sorting technology.
  • A shows the binding activity of antibodies R1445, R1446, and R1476 to MOLP-8 cells;
  • B shows the binding activity of antibodies R1478 and R1448 to MOLP-8 cells.
  • Figure 12 shows the inhibitory effect of CD39 antibodies of the mIgG1 subtype on the ATPase activity of CD39 on Mino cells.
  • A shows the inhibitory effect of antibodies R1445, R1446, and R1476 on the ATPase activity of CD39 on Mino cells;
  • B shows the inhibitory effect of antibodies R1478 and R1448 on the ATPase activity of CD39 on Mino cells.
  • FIG. 13 shows the inhibitory effect of CD39 antibody of the mIgG1 subtype on the ATPase activity of CD39 on MOLP-8 cells.
  • Figure 14 shows the blocking effect of CD39 monoclonal antibodies of the mIgG1 subtype on ATP-mediated inhibition of CD3T or CD8T cell proliferation.
  • A shows the blocking effect of antibodies R1445, R1446, and R1476 on ATP-mediated inhibition of CD3T cell proliferation;
  • B shows the blocking effect of antibodies R1478 and R1448 on ATP-mediated inhibition of CD3 T cell proliferation;
  • C shows the blocking effect of antibodies R1445, R1446 and R1476 on ATP-mediated inhibition of CD8 T cell proliferation;
  • D shows the blocking effect of antibodies R1478 and R1448 on ATP-mediated inhibition of CD8 T cell proliferation.
  • FIG. 15 shows the binding properties of CD39 antibodies to human CD39 protein and its homologous family proteins determined by ELISA.
  • Figure 16 shows the effect of CD39 antibody of mIgG1 subtype on tumor and body weight in MOLP-8 xenograft mice.
  • A shows the inhibition of tumor growth in MOLP-8 xenograft mice by CD39 antibody of mIgG1 subtype
  • B shows the effect of CD39 antibody of mIgG1 subtype on body weight in MOLP-8 xenograft mice.
  • kits and reagents obtained from commercial sources are generally performed according to the protocols and/or parameters defined by the manufacturer.
  • an antibody refers to one antibody or more than one antibody.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
  • a feature defined as “first” or “second” may explicitly or implicitly include at least one of the features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • CD39 is also referred to as ENTPD1 or ENTPD enzyme 1, which is a membrane protein that can convert ATP into AMP.
  • CD39 contains two transmembrane domains, a smaller cytoplasmic domain, and a larger extracellular hydrophobic domain.
  • the CD39 is human CD39.
  • the CD39 used in the present disclosure can also be from other animal species, such as mice and cynomolgus monkeys.
  • An exemplary sequence of human CD39 protein is disclosed in NCBI Ref Seq No. NP_001767.3.
  • An exemplary sequence of mouse (Mus musculus) CD39 protein is disclosed in NCBI Ref Seq No. NP_033978.1.
  • An exemplary sequence of cynomolgus (Cynomolgus) CD39 protein is disclosed in NCBI Ref Seq No. XP_015311945.1.
  • the ENTPD enzyme family also includes several other members, including ENTPD enzymes 2, 3, 4, 5, 6, 7 and 8 (also referred to as ENTPD2, 3, 4, 5, 6, 7 and 8, which are used interchangeably in the present disclosure).
  • ENTPD enzymes 2, 3, 4, 5, 6, 7 and 8 also referred to as ENTPD2, 3, 4, 5, 6, 7 and 8, which are used interchangeably in the present disclosure.
  • the antibodies or antigen-binding fragments thereof provided by the present disclosure specifically bind to CD39 (i.e., ENTPD enzyme 1), but do not bind to other family members, such as ENTPD enzymes 2, 3, 5 or 6.
  • CD39 and CD39 antigen and CD39 protein are used interchangeably in the present disclosure. Unless otherwise indicated, the term includes human CD39, subtypes and species homologs expressed by cells naturally expressed or transfected with CD39 genes.
  • the anti-CD39 antibodies or antigen-binding fragments thereof provided by the present disclosure are capable of specifically binding to CD39.
  • the present disclosure provides an anti-CD39 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the complementarity determining region of the light chain variable region comprises LCDR1, LCDR2 and LCDR3;
  • the above HCDR1 comprises an amino acid sequence as shown in SEQ ID NO.2 (GX 2-2 TFX 2-5 NX 2-7 G) or SEQ ID NO.1 (X 1-1 X 1-2 SX 1-4 TX 1-6 YX 1-8 ), wherein X 2-2 is Y or F; X 2-5 is T or S; X 2-7 is Y or F; X 1-1 is G or D; X 1-2 is Y, F or L; X 1-4 is F or L; X 1-6 is D or N; X 1-8 is N or A; and
  • the HCDR2 comprises an amino acid sequence as shown in SEQ ID NO.6 (INTNTGEP), SEQ ID NO.3 (IDPYNGX 3-7 T), SEQ ID NO.4 (IWRRGST) or SEQ ID NO.5 (ISSGSNTI), wherein X 3-7 is A or V;
  • the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO.7 (ARX 7-3 X 7-4 IYYDYVWFFDV), SEQ ID NO.8 (AX 8-2 YGYDX 8-7 X 8-8 X 8-9 X 8-10 YFDY) or SEQ ID NO.9 (AKGGTATSWFAY), wherein X 7-3 is R or S; X 7-4 is A or G; X 8-2 is I or V; X 8-7 is G or E; X 8-8 is G or V; X 8-9 is A or P; X 8-10 is Y or N;
  • the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO.10 (QDINX 10-5 Y), SEQ ID NO.12 (SSVNY), SEQ ID NO.11 (QNVDTX 11-6 ) or SEQ ID NO.13 (KSLLHSNGNTY), wherein X 10-5 is N or K; X 11-6 is N or T;
  • the LCDR2 comprises an amino acid sequence as shown by amino acid residues YT, GT, SA or RM;
  • the LCDR3 comprises an amino acid sequence as shown in SEQ ID NO.16 (LQYHDLIT), SEQ ID NO.14 ( X14-1 QX14-3 X14-4 X14-5 X14-6 X14-7 X14-8 T) or SEQ ID NO.15 (MQHLEYPFT), wherein X14-1 is Q or L; X14-3 is R or Y; X14-4 is S, D or N; X14-5 is S or N; X14-6 is Y or L; X14-7 is P or missing; X14-8 is F or L.
  • SEQ ID NO.16 LQYHDLIT
  • SEQ ID NO.14 X14-1 QX14-3 X14-4 X14-5 X14-6 X14-7 X14-8 T
  • SEQ ID NO.15 MQHLEYPFT
  • the term “antibody” includes any immunoglobulin that can bind to a specific antigen.
  • the term “antibody” is used in the broadest sense, covering various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, full-length antibodies, antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.
  • a natural complete antibody contains two heavy (H) chains and two light (L) chains.
  • H heavy
  • L light
  • antibodies can be divided into five major classifications or isotypes: IgA, IgD, IgE, IgG and IgM.
  • Each heavy chain consists of a variable region (VH) and the first, second, third and fourth (optionally) constant regions (CH1, CH2, CH3, CH4, respectively).
  • the light chains of mammals can be divided into ⁇ or ⁇ , and each light chain consists of a variable region (VL) and a constant region (CL).
  • variable regions of the light chain and heavy chain determine the binding of the antigen.
  • the variable region of each chain usually contains three hypervariable regions, called “complementarity determining regions (CDR)", among which the light chain CDR contains LCDR1, LCDR2, LCDR3, and the heavy chain CDR contains HCDR1, HCDR2, HCDR3.
  • anti-CD39 antibody refers to an antibody capable of specifically binding to CD39 (eg, human or monkey CD39).
  • amino acid refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids include amino acids encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
  • Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., alpha carbon bound to hydrogen, carboxyl, amino, and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.
  • the HCDR1 includes an amino acid sequence as shown in SEQ ID NO.22, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.21
  • the HCDR2 includes an amino acid sequence as shown in SEQ ID NO.6, SEQ ID NO.23, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.24
  • the HCDR3 includes an amino acid sequence as shown in SEQ ID NO.28, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36 or SEQ ID NO.37.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO.32, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.29, SEQ ID NO.30 or SEQ ID NO.31
  • the LCDR2 includes the amino acid sequence shown in amino acid residues YT, SA, GT or RM
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO.16, SEQ ID NO.33, SEQ ID NO.15, SEQ ID NO.34, SEQ ID NO.35 or SEQ ID NO.36.
  • the complementarity determining region of the above antibody is selected from any one of the following (a)-(f):
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO.22
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO.6
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO.28
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO.32
  • the amino acid sequence shown LCDR2 includes the amino acid sequence shown by amino acid residue YT
  • LCDR3 includes the amino acid sequence shown by SEQ ID NO.16;
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO.17
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO.23
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO.25
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO.12
  • LCDR2 includes the amino acid sequence shown in amino acid residue GT
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO.33;
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO.21
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO.4
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO.9
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO.31
  • LCDR2 includes the amino acid sequence shown in amino acid residue SA
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO.36;
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO.18
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO.24
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO.26
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO.13
  • LCDR2 includes the amino acid sequence shown in amino acid residue RM
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO.15;
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO.19
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO.5
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO.27
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO.29
  • LCDR2 includes the amino acid sequence shown in amino acid residue YT
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO.34;
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO.20
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO.4
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO.9
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO.30
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO.35
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO.35;
  • the above CDR is determined according to the IMGT definition.
  • the present disclosure provides an anti-CD39 antibody or an antigen-binding fragment thereof, comprising:
  • a heavy chain variable region wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO.85, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83 and SEQ ID NO.84; and,
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of SEQ ID NO.91, SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.88, SEQ ID NO.89 and SEQ ID NO.90;
  • HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 are determined according to the Kabat definition, the Chothia definition, the AbM definition or the Contact definition.
  • CDR boundaries of the antibodies or antigen-binding fragments thereof disclosed in the present disclosure are defined or identified according to the IMGT, Kabat or Chothia definitions. CDRs defined in other ways, such as AbM, Contact, etc., also fall within the scope of protection of the present disclosure (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005);
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the above-mentioned anti-CD39 antibody or antigen-binding fragment thereof can be selected from Table 1.
  • Table 1 shows exemplary antibodies #2 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.80, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.86), #66 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.81 The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.84, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.90), #103 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.84, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.90), and #110 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.85, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.91) are CDR amino acid sequences defined according to different definition methods.
  • the above-mentioned antibody contains a heavy chain framework region and/or a light chain framework region, and the above-mentioned heavy chain framework region and/or light chain framework region are derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.
  • the three CDRs of the anti-CD39 antibody are separated by flanking portions called framework regions (FRs, light chain FRs comprising LFR1, LFR2, LFR3 and LFR4, heavy chain FRs comprising HFR1, HFR2, HFR3 and HFR4).
  • FRs framework regions
  • light chain FRs comprising LFR1, LFR2, LFR3 and LFR4
  • heavy chain FRs comprising HFR1, HFR2, HFR3 and HFR4
  • the framework region of the heavy chain variable region of the anti-CD39 antibody comprises HFR1, HFR2, HFR3 and HFR4, and the framework region of the light chain variable region comprises LFR1, LFR2, LFR3 and LFR4, wherein
  • the HFR1 comprises an amino acid sequence as shown in any one of SEQ ID NO.41, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40, or comprises a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.41, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, and SEQ ID NO.40;
  • the HFR2 comprises an amino acid sequence as shown in any one of SEQ ID NO.46, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, or comprises a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.46, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45;
  • the HFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO.52, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51, or a sequence having at least 65% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.52, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, and SEQ ID NO.51;
  • the HFR4 comprises an amino acid sequence as shown in any one of SEQ ID NO.55, SEQ ID NO.53, SEQ ID NO.54, and SEQ ID NO.56, or a sequence having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.55, SEQ ID NO.53, SEQ ID NO.54, and SEQ ID NO.56;
  • the LFR1 comprises an amino acid sequence as shown in any one of SEQ ID NO.62, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, and SEQ ID NO.61, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.62, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, and SEQ ID NO.61;
  • the LFR2 comprises an amino acid sequence as shown in any one of SEQ ID NO.67, SEQ ID NO.63, SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66, or a sequence having at least 80% (e.g., 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.67, SEQ ID NO.63, SEQ ID NO.64, SEQ ID NO.65, and SEQ ID NO.66;
  • the LFR3 comprises an amino acid sequence as shown in any one of SEQ ID NO.73, SEQ ID NO.68, SEQ ID NO.69, SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence shown in any one of SEQ ID NO.73, SEQ ID NO.68, SEQ ID NO.69, SEQ ID NO.70, SEQ ID NO.71, and SEQ ID NO.72;
  • the LFR4 includes an amino acid sequence as shown in any one of SEQ ID NO.79, SEQ ID NO.74, SEQ ID NO.75, SEQ ID NO.76, SEQ ID NO.77, and SEQ ID NO.78, or a sequence having at least 92% (e.g., 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.79, SEQ ID NO.74, SEQ ID NO.75, SEQ ID NO.76, SEQ ID NO.77, and SEQ ID NO.78.
  • the HFR1 of the above-mentioned anti-CD39 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO.41, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.41;
  • HFR2 comprises the amino acid sequence shown in SEQ ID NO.46, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.46.
  • HFR3 comprises the amino acid sequence as shown in SEQ ID NO.52, or a sequence having at least 65% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO.52;
  • HFR4 comprises the amino acid sequence as shown in SEQ ID NO.55, or a sequence having at least 65% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO.52;
  • HFR4 comprises the amino acid sequence as shown in SEQ ID NO.55, or a sequence having at least 65% (e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9
  • LFR1 includes the amino acid sequence as shown in SEQ ID NO.62, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.62;
  • LFR2 includes the amino acid sequence as shown in SEQ ID NO.67, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence as shown in SEQ ID NO.
  • LFR3 includes the amino acid sequence as shown in SEQ ID NO.73, or a sequence having at least 75% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.73; and LFR4 includes a sequence having at least 92% (e.g., 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in SEQ ID NO.79.
  • the above FR can be determined according to the IMGT definition method.
  • percent identity refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned.
  • the comparison of amino acid sequence identity percentages can be performed in various ways known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA software known in the art.
  • One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.
  • the antibodies disclosed herein comprise appropriate framework region (FR) sequences, as long as the antibodies can specifically bind to CD39.
  • FR framework region
  • the CDR sequences shown in Table 1 above are obtained from mouse antibodies, but can be grafted to any suitable FR sequences of any suitable species (e.g., mouse, human, rat, rabbit, and others) using suitable methods known in the art (e.g., recombinant technology).
  • the HFR1, the HFR2, the HFR3, the HFR4, the LFR1, the LFR2, the LFR3 and the LFR4 of the anti-CD39 antibody are selected from Table 2.
  • FR amino acid sequences of exemplary antibodies #2, #66, #75, #95, #103 and #110 are shown below in Table 2.
  • FR boundaries are defined or identified according to the IMGT definition, and FRs defined in other ways, such as Kabat, Chothia, AbM, Contact or Lesk definitions, are also within the scope of the present disclosure.
  • the above-mentioned antibody has a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO.85, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, and SEQ ID NO.84, or having at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.85, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, and SEQ ID NO.84;
  • the above-mentioned antibody has a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO.91, SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.88, SEQ ID NO.89, and SEQ ID NO.90, or having at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO.91, SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.88, SEQ ID NO.89, and SEQ ID NO.90;
  • amino acid sequences of the heavy chain variable regions and light chain variable regions of exemplary antibodies #2, #66, #75, #95, #103 and #110 are shown in Table 3 below.
  • the above-mentioned anti-CD39 antibody contains a heavy chain framework region and/or a light chain framework region, and the heavy chain framework region and/or the light chain framework region of the above-mentioned antibody is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.
  • the heavy chain constant region of the above antibody is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region of the above antibody is ⁇ or ⁇ chain.
  • the above-mentioned antibody is a full-length antibody or an antigen-binding fragment thereof, and the antigen-binding fragment is any one or more of F(ab')2, Fab', Fab, scFv, Fv, and dsFV.
  • antigen-binding fragment refers to an antibody fragment formed by a part of an antibody having antigen-binding activity and containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not have a complete native antibody structure.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies).
  • the above antigen-binding fragments are capable of binding to the same antigen as the parent antibody.
  • Fab of an antibody refers to a portion of an antibody composed of a single light chain (including variable and constant regions) and the variable region and first constant region of a single heavy chain bound together by disulfide bonds.
  • Fab' fragment refers to the Fab fragment that includes part of the hinge region.
  • F(ab')2 refers to a dimer of Fab'.
  • An "Fv fragment” consists of the variable region of a single light chain combined with or without the variable region of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an antibody fragment composed of a light chain variable region and a heavy chain variable region directly linked to each other or connected by a peptide linker sequence.
  • the anti-CD39 antibody or antigen-binding fragment thereof has one or more of the following properties (a)-(l):
  • the determination method can be carried out with reference to the step described in item 4 of embodiment 2;
  • the determination method can be carried out with reference to the step described in item 12 of embodiment 2;
  • the determination method can be carried out with reference to the step described in item 13 of embodiment 2;
  • not more than 10 ⁇ 9 M for example, not more than 5x10 ⁇ 10 M, not more than 4x10 ⁇ 10 M, not more than 3x10 ⁇ 10 M, not more than 2x10 ⁇ 10 M, not more than 1x10 ⁇ 10 M, not more than 9x10 ⁇ 11 M, not more than 8x10 ⁇ 11 M, not more than 7x10 ⁇ 11 M, not more than 6x10 ⁇ 11 M, not more than 5x10 ⁇ 11 M, not more than 4x10 ⁇ 11 M, not more than 3x10 ⁇ 11 M, not more than 2x10 ⁇ 11 M, not more than 1x10 ⁇ 11 M, not more than 9x10 ⁇ 12 M, not more than 8x10 ⁇ 12 M, not more than 7x10 ⁇ 12 M, not more than 6x10 ⁇ 12 M, not more than 5x10 ⁇ 12 M, not more than 4x10 ⁇ 12 M, not more than 3x10 ⁇ 12 M, or not more than 2x10 -12
  • IC50 inhibiting ATPase activity in Mino cells expressing CD39 with an IC50 of no more than 7 nM (e.g., no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM, no more than 1 nM, no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM or no more than 0.09 nM), wherein the IC50 is determined by an ATPase activity assay.
  • the determination method can refer to the description of item 14 in embodiment 2;
  • the assay method may refer to that described in Item 15 of Embodiment 2;
  • IC50 blocks ATP and inhibits CD8T cell proliferation, and the above IC50 is determined by flow cytometry fluorescence sorting analysis. The determination method can be carried out with reference to the step 16 in embodiment 2;
  • the determination method can be carried out with reference to the step 16 of Embodiment 2;
  • (j) specifically binds to human CD39, but has no cross-binding to CD39 homologous family proteins (e.g. ENTPD2, 3, 5, and 6);
  • (k) capable of inhibiting tumor growth in a mammal (e.g., capable of inhibiting tumor growth in MOLP-8 xenografted mice);
  • the CD39 in the anti-CD39 antibody or antigen-binding fragment thereof is human CD39 or monkey CD39.
  • the term “specific binding” or “specifically binds” refers to a non-random binding reaction between two molecules, for example, a reaction between an antibody and an antigen.
  • the antibodies or antigen-binding fragments thereof provided by the present disclosure specifically bind to human CD39, but not to mouse CD39.
  • the antibodies or antigen-binding fragments thereof provided by the present disclosure specifically bind to cynomolgus monkey CD39.
  • affinity refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.
  • the strength or affinity of an immunological binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction, wherein a smaller KD value indicates a higher affinity.
  • KD can be determined using any conventional method known in the art, including but not limited to Biacore assays, Octet methods, microthermophoresis, HPLC-MS methods, and flow cytometry fluorescence sorting techniques.
  • the binding of the antibodies or antigen-binding fragments thereof provided by the present disclosure to human CD39 can also be expressed as "half-maximal effective concentration (EC50)", which refers to the concentration of the drug or antibody that can achieve 50% of the maximum biological effect after a specific exposure time.
  • EC50 half-maximal effective concentration
  • the EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technology, and other binding assays).
  • IC50 half-maximal inhibitory concentration
  • the antibody or its Fab provided by the present disclosure inhibits the ATPase activity in the Mino cells expressing CD39 with an IC50 of no more than 5nM, and the IC50 is determined by ATPase activity analysis.
  • the ATPase activity in the MOLP-8 cells expressing CD39 is inhibited with an IC50 of no more than 5nM, and the IC50 is determined by ATPase activity analysis.
  • Methods known in the art can be used to determine the ATPase activity assay, such as by colorimetric detection as a result of ATPase activity The phosphate released.
  • the ATPase activity is determined by those methods described in the 7th item in the embodiment 2 of the present disclosure.
  • IC50 can also be used to represent the inhibitory activity of antibodies on cell proliferation.
  • the antibodies or antigen-binding fragments thereof provided by the present disclosure can block ATP with an IC50 of no more than 10 nM, inhibiting CD8 T cell proliferation, and the IC50 is determined by flow cytometry analysis. Blocking ATP with an IC50 of no more than 10 nM inhibits CD3 T cell proliferation, and the IC50 is determined by flow cytometry analysis.
  • the proliferation of T cells can be determined using methods known in the art (such as those described in Item 8 or Item 16 of Embodiment 2 of the present disclosure).
  • Mino cell is a human non-Hodgkin's lymphoma cell line that naturally expresses human CD39 protein on its cell surface.
  • MOLP-8 cells refers to a human multiple myeloma cell line that naturally expresses human CD39 protein on its cell surface.
  • the multiple myeloma model established by inoculating the cells into NOD-SCID mice can be used to detect the in vivo anti-tumor effect of anti-CD39 antibodies.
  • the present disclosure provides an anti-CD39 antibody or an antigen-binding fragment thereof, which competitively binds to CD39 with the anti-CD39 antibody described in the first or second aspect, or the epitope thereof that binds to the CD39 antigen is the same as the epitope of the anti-CD39 antibody described in the first or second aspect that binds to the CD39 antigen.
  • epitope refers to any antigenic determinant on an antigen that is bound by the paratope of an antibody.
  • Antigenic determinants are typically special chemical groups with a certain composition and structure.
  • An epitope can be linear (i.e., continuous) or conformational (i.e., comprising spaced amino acid residues, non-continuous).
  • An epitope defines the minimum binding site of an antibody and is therefore a specific target of an antibody or its antigen-binding fragment.
  • An epitope can be determined by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy).
  • the present disclosure provides a multispecific antibody comprising the anti-CD39 antibody or antigen-binding fragment thereof as shown in any one of the aforementioned items.
  • multispecific antibody refers to an antibody molecule that can bind to multiple (two or more) different antigenic epitopes of the same antigen or multiple (two or more) different antigens.
  • the present disclosure provides a biomaterial, characterized in that the biomaterial is selected from any one of (i) to (v):
  • a chimeric antigen receptor (i) a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular region, wherein the extracellular region comprises an antigen binding domain; and wherein the antigen binding domain comprises the anti-CD39 antibody or antigen binding fragment thereof as described in any of the preceding aspects;
  • an engineered immune cell wherein the immune cell expresses the chimeric antigen receptor described in (i) or contains a nucleic acid encoding the chimeric antigen receptor described in (i);
  • a recombinant cell wherein the recombinant cell carries the nucleic acid molecule described in (iii), or contains the vector described in (iv), or expresses the antibody or antigen-binding fragment thereof described in any one of the preceding aspects.
  • the "chimeric antigen receptor” in the present disclosure is an artificial receptor that simulates the function of TCR. It is composed of an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence.
  • the antigen (receptor) on the surface of the tumor cell binds to the antibody (ligand) of the chimeric antigen receptor, the signal can be transmitted to the cell through the hinge region and the transmembrane region.
  • the intracellular domain then converts the signal into an activation signal to activate effector cells.
  • the effector cells kill tumor cells by secreting perforin or producing cytokines. At the same time, the effector cells themselves also proliferate, further expanding the immune killing effect.
  • the extracellular domain is generally composed of a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to the antigen and a hinge region (Hinge) that acts as a connector.
  • the single-chain variable fragment is the antigen binding domain of CAR, which determines the specificity and function of CAR-immune cells.
  • the hinge region is the extracellular structural region of CAR that connects the single-chain variable fragment and the transmembrane domain. It usually maintains the stability required for robust CAR expression and activity in effector cells.
  • the hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28. The type and length of the hinge region have an important influence on the functional activity of CAR.
  • the transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain.
  • Commonly used transmembrane domains are derived from CD4, CD8, CD28 and CD3 ⁇ or their derivatives. The choice of transmembrane domain affects the degree of activation of the CAR structure in cell function.
  • the intracellular domain consists of a costimulatory domain and a signal transduction domain.
  • the costimulatory domain is usually from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27).
  • the signal transduction domain is usually a T cell receptor TCR/CD3 ⁇ chain or an immunoglobulin Fc receptor Fc ⁇ RI ⁇ chain.
  • the CAR of the present disclosure (including functional parts and functional variants thereof) can be obtained by methods known in the art, for example, can be prepared by any suitable method for preparing a polypeptide or protein.
  • engineered immune cells refers to immune cells expressing CAR or immune cells modified by CAR, wherein the immune cells include but are not limited to T cells, natural killer cells (NK cells), macrophages (M cells), and Treg cells.
  • nucleic acid molecule is generally RNA or DNA, which can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid molecule is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • DNA is used when it is linked into a vector.
  • the term "vector” refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element.
  • the vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell.
  • vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc.
  • the vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells.
  • the vector may be an expression vector or a cloning vector.
  • the vectors e.g., expression vectors
  • the vectors contain a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in the present disclosure, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
  • the term "recombinant cell” refers to a cell into which an exogenous polynucleotide and/or vector can be or has been introduced.
  • the recombinant cell contains the vector, which can be introduced into a mammalian cell to construct a recombinant cell, and then the recombinant cell is used to express the antibody or antigen-binding fragment provided by the present disclosure.
  • the recombinant cell is cultured to obtain the corresponding antibody.
  • the mammalian cell that can be used can be a CHO cell, etc.
  • the present disclosure provides a pharmaceutical composition comprising the aforementioned anti-CD39 antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, and the aforementioned biological material.
  • the term "pharmaceutical composition” is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.
  • the antibody contained in the above-mentioned pharmaceutical composition or the expressed antibody can specifically target and bind to CD39, blocking CD39 from hydrolyzing ATP.
  • the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the term "pharmaceutically acceptable carrier” may include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible and serve to prolong the shelf life or effectiveness of the antibody.
  • the present disclosure provides the use of the aforementioned anti-CD39 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned biomaterial or the aforementioned pharmaceutical composition in the preparation of a drug for preventing, treating, or diagnosing and treating tumor diseases associated with CD39.
  • the present disclosure provides a method for producing the aforementioned anti-CD39 antibody or antigen-binding fragment thereof, comprising the following steps:
  • step (b) isolating and purifying the anti-CD39 antibody or antigen-binding fragment thereof obtained in step (a).
  • the present disclosure provides a method for treating, preventing or alleviating a disease, disorder or condition associated with CD39, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-CD39 antibody or its antigen-binding fragment and/or the aforementioned pharmaceutical composition.
  • the present disclosure also provides an anti-CD39 antibody or antigen-binding fragment as described in any of the aforementioned aspects for use as a medicament.
  • the term "subject” or “patient” refers to a mammalian subject or patient.
  • exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats, and sheep.
  • the subject is a human.
  • the subject is a human suspected of having cancer, an autoimmune disease or condition, and/or an infection.
  • diagnosis refers to the identification of a pathological state, disease or condition, such as the identification of a CD39-related disease, or the identification of a subject with a CD39-related disease who may benefit from a particular treatment regimen.
  • diagnosis comprises identification of abnormal levels or activity of CD39.
  • diagnosis refers to identification of cancer or an autoimmune disease in a subject.
  • a "CD39-associated" disease, disorder or condition refers to any disease or condition caused by, aggravated by, or associated with an increase or decrease in the expression or activity of CD39.
  • a CD39-associated disease, disorder or condition is a disorder associated with excessive cell proliferation, such as cancer.
  • a CD39-associated disease or condition is characterized by expression or overexpression of CD39 and or CD39-associated genes, such as ENTPD1, 2, 3, 4, 5, 6, 7 or 8 genes.
  • CD39-expressing cancer refers to a cancer in which CD39 is expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells, and the level of CD39 expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells is significantly higher than the expected level in normal cells.
  • CD39-associated diseases, disorders or conditions include: cancer, autoimmune diseases and infections.
  • the cancer is selected from the group consisting of anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer
  • the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer.
  • the term "effective amount” refers to a therapeutic amount, which is sufficient to reduce or improve the severity and/or duration of a condition or one or more symptoms thereof; prevent the progression of a disease; cause disease regression; prevent the recurrence, development or progression of one or more symptoms associated with a disease; detect a disease; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent).
  • the therapeutically effective dose of the antibody or antigen-binding fragment thereof described in the present disclosure depends on a variety of factors known in the art, such as body weight, age, past medical history, current treatment, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity and side effects, as well as the route of administration and the degree of tumor development.
  • a person skilled in the art e.g., a physician or veterinarian may proportionally reduce or increase the dose according to these or other conditions or requirements.
  • antibodies or antigen-binding fragments as described herein can be administered at a therapeutically effective dose of about 0.001 mg/kg to about 1000 mg/kg.
  • the dosage can vary with the course of treatment.
  • the initial dosage can be higher than the subsequent dosage.
  • the dosage is adjusted during the course of treatment based on the response of the subject being administered.
  • the antibodies or antigen-binding fragments thereof disclosed in the present disclosure can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent.
  • the antibodies or antigen-binding fragments thereof disclosed in the present disclosure can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer agent, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine).
  • a second therapeutic agent e.g., a chemotherapeutic agent, an anticancer agent, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an
  • the antibodies or antigen-binding fragments thereof disclosed in the present disclosure when used in combination with one or more additional therapeutic agents, they can be administered simultaneously with the one or more additional therapeutic agents. In some such embodiments, the antibodies or antigen-binding fragments thereof and the additional therapeutic agents can be administered simultaneously as part of the same pharmaceutical composition. However, antibodies or antigen-binding fragments thereof "used in combination” with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agent.
  • the meaning of "used in combination” in the present disclosure also includes that antibodies or antigen-binding fragments thereof administered before or after another therapeutic agent are also considered to be "used in combination" with the therapeutic agent, that is, the antibodies or antigen-binding fragments thereof and the second substance are administered by different administration methods.
  • the present disclosure provides a kit for detecting CD39, comprising the aforementioned anti-CD39 antibody or antigen-binding fragment thereof.
  • the present disclosure provides use of the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned recombinant cell or the aforementioned pharmaceutical composition in the preparation of a kit, wherein the aforementioned kit is used to detect CD39 or diagnose CD39-related diseases.
  • the aforementioned anti-CD39 antibody or its antigen-binding fragment can specifically target and bind to CD39.
  • the kit according to the embodiment of the present disclosure can achieve specific detection of CD39.
  • a fluorescent detection device can be used to achieve the localization or real-time detection of CD39.
  • it can be used for immunoblotting, immunoprecipitation, and other kits involving the use of the specific binding properties of CD39 antigens and antibodies for detection.
  • kits may contain any one or more of the following: antagonists, anti-CD39 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc.
  • the kit can also be used to detect diseases, disorders or conditions associated with CD39.
  • the anti-CD39 antibodies provided by the present disclosure can specifically bind to CD39 protein, block CD39 from hydrolyzing ATP, reduce adenosine in the tumor microenvironment, exert anti-tumor effects, and provide new possibilities for the treatment and/or prevention of cancer.
  • Embodiment 1 Generation of mouse monoclonal antibodies against CD39
  • human CD39 extracellular domain fusion protein hCD39-his (Acro, Cat.CD9-H52H4) carrying a his tag was used as an immunogen to produce anti-human CD39 antibodies.
  • the commonly used mouse hybridoma system was used to produce anti-human CD39 mouse monoclonal antibody. The process is as follows:
  • the recombinant human CD39 protein powder was prepared into a solution with a concentration of 0.5 mg/mL, and then mixed with titermax adjuvant (sigma, Cat. T2684) in equal volumes to obtain an oily emulsion.
  • titermax adjuvant Sigma, Cat. T2684
  • a dose of 0.2 mL was subcutaneously administered to the back site of 6-week-old female BALB/c mice (Guangdong Medical Experimental Animal Center).
  • the recombinant human CD39 protein and alum After mixing equal volumes of adjuvant (Thermo, Cat.77161), intraperitoneal immunization was performed. After 4-5 injections, tail blood was collected for titer detection.
  • mice The spleen cells of immunized BALb/c mice were fused with mouse myeloma cells, and then the obtained hybridoma cells were screened for antigen-specific antibodies.
  • the spleen cells of mice were isolated based on the standard protocol, and the immune spleen cells were mixed with mouse myeloma SP2/0 cells at a cell number ratio of 1:1, transferred to a 50mL centrifuge tube, and washed once with DMEM basal medium. The supernatant was discarded, and the cells were mixed with 40mL of electrofusion solution (BTX, Cat.47-0001), added to the electrofusion tank, and electrofusion was performed according to the established parameters.
  • electrofusion solution BTX, Cat.47-0001
  • DMEM complete medium containing HAT was added and gently suspended, and divided equally into 96-well plates, a total of 40 pieces, 50 ⁇ L/well, and cultured statically in a cell culture incubator at 37°C and 5% CO2 . After culture for the sixth day, the DMEM complete medium containing HT was replaced once.
  • HRP-labeled goat anti-mouse IgG (sigma, Cat.A0168-1ML) at 100 ⁇ L/well, incubate at 37°C for 30min, wash 3 times with 1 ⁇ PBS, and then perform a color reaction.
  • CHO-K1-hCD39 cells (humanized CHO-K1 Chinese hamster ovarian cancer cell line expressing human CD19 protein) were used to screen the binding activity of anti-human CD39 antibodies.
  • 2E5 cells/well were added to a 96-well flow cytometry plate at 100 ⁇ L/well, and cell supernatant was added at 100 ⁇ L/well and incubated at 4°C for 1 hour.
  • fluorescently labeled goat anti-mouse IgG antibody was added at 100 ⁇ L/well, incubated at 4°C for 1 hour, washed 3 times with 2% BSA, and resuspended with 100 ⁇ L/well PBS before testing on the machine.
  • Hybridoma cells were inoculated into a dialysis-based bioreactor at 2 ⁇ 10 6 cells/mL, and the supernatant containing the antibody was harvested once a week.
  • Mouse monoclonal antibodies were purified using Protein A (GE-MabSelectSuRe LX, Cat#17-5438-03) by FPLC.
  • Antibody concentration was determined by BCA kit or A280 absorbance, antibody purity was determined by SEC (size exclusion chromatography), and purity was checked by SDS (sodium dodecyl sulfate) gel electrophoresis and Coomassie brilliant blue staining.
  • Embodiment 2 Binding characteristics of monoclonal antibodies against CD39
  • cynomolgus CD39 an exemplary sequence of cynomolgus CD39 protein is disclosed in NCBI Ref Seq No. XP_015311944.1, and its amino acid sequence is shown in SEQ ID NO. 12
  • mouse CD39 an exemplary sequence of mouse (Mus musculus) CD39 protein is disclosed in NCBI Ref Seq No. NP_033978.1, and its amino acid sequence is shown in S CHO-K1 cells were transfected with a plasmid containing SEQ ID NO.
  • CHO cells and CHO-K1-mCD39 cells stably expressing mouse CD39 were collected separately, centrifuged at 350g for 5 minutes, resuspended with PBS, plated on a V-shaped 96-well plate at 2E5/well, added with PE anti-mCD39, and incubated at 4°C for 30 minutes. After centrifugation, the cells were washed once with PBS, resuspended, and the differences in CD39 expression in the cell lines were evaluated using a Cytoflex flow cytometer (Beckman Countler).
  • CHO-K1-mCD39 cell lines that highly express mouse CD39 and CHO-K1-cynoCD39 cell lines that highly express cynomolgus monkey CD39 were selected for subsequent expansion.
  • Mino cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated on a V-shaped 96-well plate at 2E5/well.
  • CD39 monoclonal antibody and positive control antibody R1048-mIgG1 (D265A) were prepared at 40 ⁇ g/mL in culture medium, and then 3-fold gradient dilution was performed, with a total of 10 concentration points.
  • the iso group (mIgG1, Biolegend, Cat#401411) was only prepared at a concentration of 40 ⁇ g/mL. 50 ⁇ L of the antibody was added to each well of the plate, and the antibody was incubated at 4°C for 30 minutes.
  • the binding curves are shown in FIG2 , wherein antibodies #75, #2, #103, and #110 bind to Mino cells with EC50 of 0.2492 nM, 0.9503 nM, 0.6152 nM, and 0.4378 nM, respectively, and have relatively good affinity.
  • MOLP-8 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated on a V-shaped 96-well plate at 2E5/well.
  • CD39 monoclonal antibody and positive antibody R1048 were prepared at 40 ⁇ /mL in culture medium, and then 3-fold gradient dilution was performed, with a total of 10 concentration points. The iso group was only configured with a concentration of 40 ⁇ g/mL. The antibody was added to the plate at 50 ⁇ L per well and incubated at 4°C for 30 minutes.
  • the binding curves are shown in FIG3 , wherein antibodies #75, #95, #110, and #103 bind to MOLP-8 cells with EC50 of 0.4398 nM, 0.5699 nM, 1.067 nM, and 0.891 nM, respectively, and have relatively good affinity.
  • CHO-K1-hCD39 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated on a V-shaped 96-well plate at 2E5/well.
  • CD39 monoclonal antibody and positive antibody R1048 were prepared at 40 ⁇ g/mL in culture medium, and then 3-fold gradient dilution was performed, with a total of 10 concentration points. The iso group was only configured with a concentration of 40 ⁇ g/mL. The antibody was added to the plate at 50 ⁇ L per well and incubated at 4°C for 30 minutes.
  • the binding activity is shown in FIG4 , where antibodies #2, #66, and #95 bind to CHO-K1-hCD39 cells with EC50 of 2.048 nM, 3.769 nM, and 3.606 nM, respectively.
  • CHO-K1-cynoCD39 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated on a V-shaped 96-well plate at 2E5/well.
  • CD39 monoclonal antibody and positive antibody R1048 were prepared at 40 ⁇ g/mL in culture medium, and then 3-fold gradient dilution was performed, with a total of 10 concentration points. The iso group was only configured with a concentration of 40 ⁇ g/mL. The antibody was added to the plate at 50 ⁇ L per well and incubated at 4°C for 30 minutes.
  • CHO-K1-mCD39 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated on a V-shaped 96-well plate at 2E5/well.
  • CD39 monoclonal antibody and positive antibody R1048 were prepared at 40 ⁇ g/mL in culture medium, and then 3-fold gradient dilution was performed, with a total of 10 concentration points. The iso group was only configured with a concentration of 40 ⁇ g/mL. The antibody was added to the plate at 50 ⁇ L per well and incubated at 4°C for 30 minutes.
  • the binding status is shown in FIG6 . All CD39 antibodies had no binding activity with CHO-K1-mCD39 cells, indicating that these CD39 antibodies had no binding activity with mouse CD39 protein.
  • the antibody medium is configured to start at 20 ⁇ g/mL. 3-fold dilution, a total of 10 concentrations, add 100 ⁇ L of antibody to the cells per well, mix well, and incubate at 37°C for 60 minutes; ATP is configured to 200 ⁇ M with medium, added to the cells at 50 ⁇ L/well, mix well, and incubate at 37°C for 45 minutes; the cells are centrifuged at 350G for 5 minutes, 80 ⁇ L of the supernatant is taken, transferred to a white plate, and an equal volume of Cell Titer-Glo is added, incubated at room temperature in the dark for 10 minutes, and tested on a machine.
  • the blocking activity curve of the antibody is shown in Figure 7, among which antibody #75 blocked the ATPase hydrolysis of CD39 on Mino cells with an IC50 of 0.2102nM, which was better than the positive control antibody R1048; antibodies #95, #110, and #103 blocked the ATPase hydrolysis of CD39 on Mino cells with IC50 of 0.3216nM, 0.4047nM, and 0.3696nM, respectively; the platform values of #2, #75, and #66 were higher than the positive control antibody R1048.
  • Antibodies #75, #95, #103, and #110 blocked ATPase hydrolysis of CD39 on MOLP-8 cells with IC50s of 0.506 nM, 0.6498 nM, 0.2488 nM, and 0.3138 nM, respectively.
  • the amino acid sequence of the heavy chain variable region of antibody #95 is shown in SEQ ID NO.83, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.89.
  • RNA was isolated from the hybridoma cells corresponding to the six monoclonal antibodies, and reverse transcribed using a commercial kit to obtain their respective cDNAs. Then, using cDNA as a template, the heavy chain variable region and light chain variable region were amplified using primers from Mouse Ig-Primer Set (Novagen). The PCR products with the correct molecular size were gel-recovered and then connected to a suitable vector. The connected products were transformed into DH5 ⁇ competent cells. After screening cloning and DNA sequencing analysis, the heavy chain variable region sequences and light chain variable region sequences of the six antibodies were obtained; then, the respective variable region sequences were connected to the constant region of the mouse IgG1 subtype to produce antibodies of the mIgG1 subtype. The corresponding numbering relationship and variable region sequences are shown in Table 6.
  • Table 6 shows the mouse monoclonal antibodies after replacing the mIgG1 subtype.
  • the control antibody R1048 is numbered R1444 after replacing the mIgG1 subtype. These antibodies will be further evaluated in functional experiments including human CD39 binding activity, blocking experiments, and in vitro immune cell activation experiments.
  • Biacore was used to characterize the binding affinity of CD39 antibodies and reference antibodies to human CD39.
  • a column of Octet HIS1K Biosensor (SARTORIUS) was immersed in the solidification solution to capture an appropriate amount of human CD39 ECD antigen (ACRO, Cat#CD9-H52H4) with a His tag, and the probe with the solidified antigen was immersed in the buffer solution as a baseline, and then immersed in different concentrations of the test antibody solution for binding for 180 seconds, and then immersed in the buffer solution for dissociation for 240 seconds (kinetic mode).
  • the binding and dissociation curves were fitted with a 1:1 binding model, and the Kon/Kd/KD values of each antibody were calculated. The results are shown in Table 7.
  • the CD39 protein was added to 50mM CB coating solution at a concentration of 1 ⁇ g/mL, and added to the ELISA plate at a volume of 100 ⁇ L per well, and coated at 4°C overnight; after washing twice with PBST, 200 ⁇ L of 1% BSA was added to each well, and the sample was diluted with 1% BSA, and the initial concentration of the test antibody was set to 10 ⁇ g/mL.
  • test antibody was diluted ten points, and the diluted sample was added to the ELISA plate at a volume of 100 ⁇ L per well, and the sample was coated at 37°C for 1h; the secondary antibody was added, and the secondary antibody enzyme was diluted with 1% BSA at a dilution ratio of 1:15K, and the ELISA plate was washed 5 times with PBST; the diluted secondary antibody was added to the ELISA plate at a volume of 100 ⁇ L per well, and the sample was coated at 37°C for 0.5h.
  • the ELISA plate was washed 5 times with PBST, and 50 ⁇ L of colorimetric solution A and solution B were added, and the sample was coated at 37°C for 10min, and then 50 ⁇ L of stop solution was added, and the ELISA reader read the data.
  • the binding curves are shown in FIG9 .
  • R1448, R1476, and R1478 bind to CD39 protein with EC50 of 0.4509 nM, 0.1654 nM, and 0.07216 nM, respectively, and have better affinity.
  • Mino cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, and plated on a V-shaped 96-well plate at 2E5/well.
  • CD39 monoclonal antibody and control antibody were added respectively, and incubated at 4°C for 30 minutes.
  • 200 ⁇ L of PBS was added to each well to wash the cells, and then 100 ⁇ L/well of PE Goat anti mo ⁇ se IgG Fc (1:500 dilution) was added, and the cells were resuspended and incubated at 4°C for 30 minutes.
  • 200 ⁇ L of PBS was added to each well to wash the cells, and then 100 ⁇ L of PBS was added to each well. After the cells were resuspended, the binding difference of CD39 antibody to Mino cells was evaluated using a Cytoflex flow cytometer (Beckman Countler).
  • the binding curve is shown in FIG10A , wherein R1476 binds to Mino cells with an EC50 of 0.3307 nM, having a relatively good affinity; as shown in FIG10B , the platform value of R1448 is higher than that of the positive control R1444.
  • MOLP-8 cells were collected, centrifuged at 350g for 5 minutes, resuspended in PBS, plated on a V-shaped 96-well plate at 2E5/well, and CD39 monoclonal antibody and control antibody were added respectively, and incubated at 4°C for 30 minutes. After centrifugation, 200 ⁇ L of PBS was added to each well to wash the cells, and then 100 ⁇ L/well of PE Goat anti mouse IgG Fc (1:500 dilution) was added, and the cells were resuspended and incubated at 4°C for 30 minutes. After centrifugation, 200 ⁇ L of PBS was added to each well to wash the cells, and then 100 ⁇ L of PBS was added to each well. After resuspending the cells, the binding difference of CD39 antibody to Molp cells was evaluated using a Cytoflex flow cytometer (Beckman Countler).
  • the binding curve is shown in FIG11A , where R1476 binds to MOLP-8 cells with an EC50 of 0.3064 nM, having a better affinity; as shown in FIG11B , the platform value of R1448 is higher than that of the positive control R1444.
  • the blocking curves are shown in FIG12 , wherein R1448, R1478, R1446, and R1476 blocked ATPase hydrolysis of CD39 on Mino cells with IC50 of 0.404 nM, 0.187 nM, 0.8614 nM, and 0.08273 nM, respectively, and had better blocking activity.
  • the blocking activity curve of the antibody is shown in Figure 13, where R1476 and R1446 blocked ATPase hydrolysis of CD39 on MOLP-8 cells with IC50 of 0.1473nM and 0.2404nM, respectively, which was better than the positive control antibody R1444; the platform values of R1448, R1445, R1446, and R1476 were higher than the positive control antibody R1444.
  • the T cell density was adjusted to 5E6/mL, and DPBS was used as buffer to label T cells with CFSE (10mM stock solution) to a final concentration of 0.5 ⁇ M, and the labeling was carried out in a 37°C water bath in the dark for 20 minutes; 12mL of complete culture medium (10% FBS, pre-cooled at 4°C) was added to terminate the CFSE reaction; the antibody was diluted with cell culture medium to a concentration of 400nM, 10-fold gradient dilution, and 5 points were set; the labeled cells were centrifuged at 300g for 5 minutes, the supernatant was removed, and the complete culture medium was added to resuspend and the cells were counted , adjust the density to 2E6 (total cells are counted here, not viable cells), add 50 ⁇ L/well to a 96-well U-shaped plate, i.e., the final cell volume is 1E5/well; add the prepared antibody to the cells at 50 ⁇ L/well; dilute the beads according
  • CD39 and its family proteins were added to 50mM CB coating solution at a concentration of 1 ⁇ g/mL, and added to the ELISA plate at a volume of 100 ⁇ L per well, and coated at 4°C overnight; after washing twice with PBST, 200 ⁇ L of 1% BSA was added to each well and coated at 37°C for 60min; the sample was diluted with 1% BSA, and the initial concentration of the test antibody was set to 10 ⁇ g/mL, and each test antibody was diluted ten times; the diluted sample was added to the ELISA plate at a volume of 100 ⁇ L per well, and coated at 37°C for 1h; the secondary antibody was added at a ratio of 1:15K Dilute the secondary antibody enzyme with 1% BSA at a dilution ratio; wash the ELISA plate with PBST 5 times, add 100 ⁇ L of the diluted secondary antibody to each well of the ELISA plate, and incubate at 37°C for 0.5 h; wash the ELISA plate with PBST 5 times,
  • CD39 monoclonal antibodies specifically bind to human CD39 (ENTPD1), but do not bind to the homologous family proteins of human CD39 (ENTPD2, ENTPD3, ENTPD5, ENTPD6).
  • mice Female NOD SCID mice aged 6-8 weeks were subcutaneously injected with 5x106 MOLP-8 cells on day 0, and CD39 antibody or control antibody R0859 (mIgG1, whose heavy chain amino acid sequence is shown in SEQ ID NO.131, and whose light chain amino acid sequence is shown in SEQ ID NO.132) was administered at a dose of 30 mg/kg on days 1, 5, 8 and 12, respectively.
  • CD39 antibody or control antibody R0859 mIgG1, whose heavy chain amino acid sequence is shown in SEQ ID NO.131, and whose light chain amino acid sequence is shown in SEQ ID NO.132
  • Figure 16A The results are shown in Figure 16A, where the curve depicts the average tumor volume and the error bar represents the SEM.
  • the triangle on the X-axis marks the time point of administration, the X-axis represents the number of days after implantation, and the Y-axis represents the volume of the tumor.
  • Figure 16B shows the weight of mice, and it can be seen that there is no significant difference in the weight of mice between groups during the administration process.
  • the present disclosure provides an anti-CD39 antibody and its application, which can specifically bind to CD39 protein, block CD39 from hydrolyzing ATP, reduce adenosine in the tumor microenvironment, exert anti-tumor effects, and has excellent industrial practicability.

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Abstract

提供了一种抗CD39抗体及其应用，涉及生物技术领域。该抗体能够特异性结合CD39蛋白，阻断CD39水解ATP，减少肿瘤微环境中的腺苷，发挥抗肿瘤作用，为癌症的治疗和/或预防提供新的可能。

Description

抗CD39抗体及其应用
相关申请的交叉引用
本申请要求于2022年11月23日提交中国国家知识产权局的申请号为202211471910.9、名称为“抗CD39抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物技术领域，具体而言，涉及一种抗CD39抗体及其应用。
背景技术
免疫疗法是当下肿瘤主要治疗手段之一，但其效果会受多种因素影响，肿瘤微环境(TME)就是重要的影响因素之一。研究发现，肿瘤微环境中的免疫细胞组成常常会影响其对某种特定疗法的响应，从而决定癌症免疫疗法的成败。而在肿瘤微环境的先天性和适应性免疫中，ATP-腺苷通路起着关键作用。
CD39即外核苷三磷酸二磷酸水解酶1(ENTPDase1)是ATP-腺苷通路中的一员，在肿瘤微环境免疫抑制反应中扮演的重要角色。CD39是一种单通道的II型跨膜蛋白，可将胞外的eATP或ADP转化为AMP，然后CD73将AMP转化为腺苷，腺苷是肿瘤微环境中发挥免疫抑制作用的代谢产物。当产生的腺苷与CD4T、CD8T细胞和自然杀伤(NK)细胞表面的腺苷受体结合时，能够抑制T细胞和NK细胞应答，从而抑制免疫系统并促进肿瘤的生长。此外，腺苷还与巨噬细胞和树突细胞上的A2A或A2B受体结合，从而抑制吞噬作用和抗原呈递，并增加促癌因子(例如VEGF、TGFβ和IL6)的分泌。而腺苷对于Treg则会促进Treg的免疫抑制活性。在TME内，腺苷途径是指eATP在腺苷酸水解酶的作用下向腺苷转化，以及腺苷通过免疫细胞上的A2A/A2B腺苷受体的信号传导。
在正常条件下，CD39维持免疫抑制性腺苷与免疫刺激性ATP的细胞外水平的平衡。在健康组织中，ATP在细胞外环境中几乎检测不到，因为ATP被CD39快速分解生成ADP或AMP，然后AMP被CD73转化为腺苷。在包括癌症在内的细胞应激条件下，eATP水平显著升高，但由于被CD39和CD73相继水解而转化为腺苷，从而导致免疫系统对肿瘤的识别以及对肿瘤的杀伤产生阻碍。抗CD39抗体对阻断CD39水解ATP的功效差异很大，筛选高阻断活性的抗CD39抗体难度较高。
发明内容
本公开的目的之一在于提供一种可以特异性结合CD39的抗体及其应用。
本公开是这样实现的：
在本公开的第一方面，本公开提供了一种抗CD39抗体或其抗原结合片段。在一些具体的实施方式中，上述抗体含有重链可变区和轻链可变区，其中，上述重链可变区的互补决定区包括HCDR1、HCDR2和HCDR3，上述轻链可变区的互补决定区包括LCDR1、LCDR2和LCDR3；
其中，上述HCDR1包括如SEQ ID NO.2(GX2-2TFX2-5NX2-7G)或SEQ ID NO.1(X1-1X1-2SX1-4TX1-6YX1-8)所示的氨基酸序列，其中X2-2是Y或F；X2-5是T或S；X2-7是Y或F；X1-1是G或D；X1-2是Y、F或L；X1-4是F或L；X1-6是D或N；X1-8是N或A；
上述HCDR2包括如SEQ ID NO.6(INTNTGEP)、SEQ ID NO.3(IDPYNGX3-7T)、SEQ ID NO.4(IWRRGST)或SEQ ID NO.5(ISSGSNTI)所示的氨基酸序列，其中X3-7是A或V；
上述HCDR3包括如SEQ ID NO.7(ARX7-3X7-4IYYDYVWFFDV)、SEQ ID NO.8(AX8-2YGYDX8-7X8-8X8-9X8-10YFDY)或SEQ ID NO.9(AKGGTATSWFAY)所示的氨基酸序列，其中X7-3是R或S；X7-4是A或G；X8-2是I或V；X8-7是G或E；X8-8是G或V；X8-9是A或P；X8-10是Y或N；
上述LCDR1包括如SEQ ID NO.10(QDINX10-5Y)、SEQ ID NO.12(SSVNY)、SEQ ID NO.11(QNVDTX11-6)或SEQ ID NO.13(KSLLHSNGNTY)所示的氨基酸序列，其中X10-5是N或K；X11-6 是N或T；
上述LCDR2包括如氨基酸残基YT、GT、SA或RM所示的氨基酸序列；
上述LCDR3包括如SEQ ID NO.16(LQYHDLIT)、SEQ ID NO.14(X14-1QX14-3X14-4X14-5X14-6X14-7X14-8T)或SEQ ID NO.15(MQHLEYPFT)所示的氨基酸序列，其中X14-1是Q或L；X14-3是R或Y；X14-4是S、D或N；X14-5是S或N；X14-6是Y或L；X14-7是P或缺失；X14-8是F或L。
在本公开的第二方面，本公开提供了一种抗CD39抗体或其抗原结合片段，所述抗体包含：
(a)重链可变区，其包含SEQ ID NO.85、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、和SEQ ID NO.84中任一项所示重链可变区的HCDR1,HCDR2和HCDR3；以及，
(b)轻链可变区，其包含SEQ ID NO.91、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、和SEQ ID NO.90中任一项所示轻链可变区的LCDR1,LCDR2和LCDR3；
上述HCDR1,HCDR2和HCDR3及LCDR1,LCDR2和LCDR3按照Kabat定义方式、Chothia定义方式、AbM定义方式、Contact定义方式确定。
在本公开的第三方面，本公开提供了一种抗CD39抗体或其抗原结合片段，其与前述的抗CD39抗体竞争性结合CD39，或者其结合CD39抗原的表位与前述的抗CD39抗体结合CD39抗原的表位相同。
在本公开的第四方面，本公开提供了一种多特异性抗体，其含有前述抗CD39抗体或其抗原结合片段。
在本公开的第五方面，本公开提供了一种生物材料，其特征在于，所述生物材料选自(ⅰ)～(v)中任一项：
(i)嵌合抗原受体(CAR)，所述嵌合抗原受体包括胞外区，所述胞外区包括抗原结合结构域；并且，所述抗原结合结构域含有前述任一方面所述的抗CD39抗体或其抗原结合片段；
(ii)工程化的免疫细胞，所述免疫细胞表达(i)所述的嵌合抗原受体或含有编码(i)所述的嵌合抗原受体的核酸；
(iii)分离的核酸分子，包括编码前述任一方面所述的抗体或其抗原结合片段的核苷酸序列；
(iv)载体，所述载体携带前述(iii)核酸分子；
(v)重组细胞，所述重组细胞携带(iii)中核酸分子，或含有(iv)中载体，或表达前述任一方面所述的抗体或其抗原结合片段。
在本公开的第六方面，本公开提供了一种药物组合物，其含有前述的抗CD39抗体或其抗原结合片段、前述的多特异性抗体、前述的生物材料；
在一些具体的实施例中，上述述药物组合物还包括药学上可接受的载体。
在本公开的第七方面，本公开提供了前述的抗CD39抗体或其抗原结合片段、前述的多特异性抗体、前述的生物材料或前述的药物组合物在制备用于预防、治疗、或诊断与CD39相关的肿瘤疾病的药物中的应用。
在本公开的第八方面，本公开提供了一种用于产生前述抗CD39抗体或其抗原结合片段的方法，其包括以下步骤：
(a)在表达抗CD39抗体或其抗原结合片段的表达条件下培养前述的重组细胞；
(b)分离和纯化步骤(a)中获得的抗CD39抗体或其抗原结合片段。
在本公开的第九方面，本公开提供了一种治疗、预防或减轻与CD39相关的疾病、病症或状况的方法，其包括向受试者施用治疗有效量的前述的药物组合物。本公开还提供了一种用作药物的前述任一方面所述的抗CD39抗体或抗原结合片段。
在本公开的第十方面，本公开提供了一种检测CD39的试剂盒，其包含前述的抗CD39抗体或其抗原结合片段。
在本公开的第十一方面，本公开提供了前述的抗体或其抗原结合片段、前述的生物材料或前述的药物组合物在制备试剂盒中的用途，上述试剂盒用于检测CD39或者诊断与CD39相关的疾病。
本公开的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本公开的实践了解到。
附图说明
为了更清楚地说明本公开实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施方式，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为转染并稳定表达cynoCD39或mCD39的CHO-K1细胞在结合相应的抗体后的流式峰图。
图2显示了通过流式细胞荧光分选技术测定的CD39单克隆抗体与Mino细胞的结合活性。A显示了抗体#2、#66、#75、#95与Mino细胞的结合活性；B显示了抗体#103、#110与Mino细胞的结合活性。
图3显示了通过流式细胞荧光分选技术测定的CD39单克隆抗体与MOLP-8细胞的结合活性。A显示了抗体#2、#66、#75、#95与MOLP-8细胞的结合活性；B显示了抗体#103、#110与MOLP-8细胞的结合活性。
图4显示了通过流式细胞荧光分选技术测定的CD39单克隆抗体与CHO-K1-hCD39细胞的结合活性。A显示了抗体#2、#66、#75、#95与CHO-K1-hCD39细胞细胞的结合活性；B显示了抗体#103、#110与CHO-K1-hCD39细胞细胞的结合活性。
图5显示了通过流式细胞荧光分选技术测定的CD39单克隆抗体与CHO-K1-cynoCD39细胞的结合活性。
图6显示了通过流式细胞荧光分选技术测定的CD39单克隆抗体与CHO-K1-mCD39细胞的结合活性。
图7显示了CD39单克隆抗体对Mino细胞上CD39的ATP酶活性的抑制作用。A显示了抗体#2、#66、#75、#95对Mino细胞上CD39的ATP酶活性的抑制作用；B显示了抗体#103、#110对Mino细胞上CD39的ATP酶活性的抑制作用。
图8显示了CD39单克隆抗体对MOLP-8细胞上CD39的ATP酶活性的抑制作用。A显示了抗体#66、#75、#95对MOLP-8细胞上CD39的ATP酶活性的抑制作用；B显示了抗体#103、#110对MOLP-8细胞上CD39的ATP酶活性的抑制作用。
图9显示了通过ELISA测定的mIgG1亚型的CD39抗体与CD39蛋白的胞外ECD区域的结合活性。
图10显示了通过流式细胞荧光分选技术测定的mIgG1亚型的CD39抗体与Mino细胞的结合活性。A显示了抗体R1445、R1446、R1476与Mino细胞的结合活性；B显示了抗体R1478、R1448与Mino细胞的结合活性。
图11显示了通过流式细胞荧光分选技术测定的mIgG1亚型的CD39抗体与MOLP-8细胞的结合活性。A显示了抗体R1445、R1446、R1476与MOLP-8细胞的结合活性；B显示了抗体R1478、R1448与MOLP-8细胞的结合活性。
图12显示了mIgG1亚型的CD39抗体对Mino细胞上CD39的ATP酶活性的抑制作用。A显示了抗体R1445、R1446、R1476对Mino细胞上CD39的ATP酶活性的抑制作用；B显示了抗体R1478、R1448对Mino细胞上CD39的ATP酶活性的抑制作用。
图13显示了mIgG1亚型的CD39抗体对MOLP-8细胞上CD39的ATP酶活性的抑制作用。
图14显示了mIgG1亚型的CD39单克隆抗体对ATP介导的CD3T或CD8T细胞增殖抑制的阻断。A显示了抗体R1445、R1446、R1476对ATP介导的CD3T细胞增殖抑制的阻断；B 显示了抗体R1478、R1448对ATP介导的CD3T细胞增殖抑制的阻断；C显示了抗体R1445、R1446、R1476对ATP介导的CD8T细胞增殖抑制的阻断；D显示了抗体R1478、R1448对ATP介导的CD8T细胞增殖抑制的阻断。
图15显示了通过ELISA测定的CD39抗体与人CD39蛋白以及其同家族蛋白的结合特性。
图16显示了mIgG1亚型的CD39抗体对MOLP-8异种移植小鼠中的肿瘤和体重的影响。A显示了mIgG1亚型的CD39抗体对MOLP-8异种移植小鼠中的肿瘤生长抑制，B显示了mIgG1亚型的CD39抗体对MOLP-8异种移植小鼠中的体重的影响。
具体实施方式
为了更容易理解本公开，以下具体定义了某些技术和科学在本公开中，术语。除非本公开明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。除非另有说明，本公开中使用自商业途径获得的试剂盒及试剂的方法通常根据厂商定义的方案和/或参数进行。
本公开中的冠词“一种”(a/an)、“一个”(a/an)和“所述”包括复数指代，除非上下文清楚表明其并非如此。举例来说，“一种抗体”指一种抗体或多于一种抗体。
在本公开中，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本公开的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本公开中，术语“CD39”也被称为ENTPD1或ENTPD酶1，是一种可将ATP转化为AMP的膜蛋白。CD39含有两个跨膜结构域、一个较小的胞质域和一个较大的胞外疏水结构域。在一些实施方式中，所述CD39是人类CD39。本公开所用的CD39也可以来自其他动物物种，例如小鼠和食蟹猴等。人类CD39蛋白的示例性序列公开于NCBI Ref Seq No.NP_001767.3中。小鼠(Mus musculus)CD39蛋白的示例性序列公开于NCBI Ref SeqNo.NP_033978.1中。食蟹猴(Cynomolgus)CD39蛋白的示例性序列公开于NCBI Ref SeqNo.XP_015311945.1中。
除了CD39之外，ENTPD酶家族还包含其他几个成员，包括ENTPD酶2、3、4、5、6、7和8(也称为ENTPD2、3、4、5、6、7和8，在本公开中可互换使用)。在一些具体的实施方式中，本公开提供的抗体或其抗原结合片段特异性结合CD39(即ENTPD酶1)，但不与其他家族成员结合，例如ENTPD酶2、3、5或6。
“CD39”和“CD39抗原(CD39antigen)”以及“CD39蛋白”在本公开中可互换使用。除非另行说明，所述术语包括由细胞天然表达或经CD39基因转染的细胞表达的人CD39、亚型及物种同源物。本公开提供的抗CD39抗体或其抗原结合片段能够与CD39特异性结合。
在本公开的第一方面，本公开提供了一种抗CD39抗体或其抗原结合片段，其含有重链可变区和轻链可变区，其中，上述重链可变区的互补决定区包括HCDR1、HCDR2和HCDR3，上述轻链可变区的互补决定区包括LCDR1、LCDR2和LCDR3；
其中，上述HCDR1包括如SEQ ID NO.2(GX2-2TFX2-5NX2-7G)或SEQ ID NO.1(X1-1X1-2SX1-4TX1-6YX1-8)所示的氨基酸序列，其中X2-2是Y或F；X2-5是T或S；X2-7是Y或F；X1-1是G或D；X1-2是Y、F或L；X1-4是F或L；X1-6是D或N；X1-8是N或A；并且
上述HCDR2包括如SEQ ID NO.6(INTNTGEP)、SEQ ID NO.3(IDPYNGX3-7T)、SEQ ID NO.4(IWRRGST)或SEQ ID NO.5(ISSGSNTI)所示的氨基酸序列，其中X3-7是A或V；
上述HCDR3包括如SEQ ID NO.7(ARX7-3X7-4IYYDYVWFFDV)、SEQ ID NO.8(AX8-2YGYDX8-7X8-8X8-9X8-10YFDY)或SEQ ID NO.9(AKGGTATSWFAY)所示的氨基酸序列，其中X7-3是R或S；X7-4是A或G；X8-2是I或V；X8-7是G或E；X8-8是G或V；X8-9是A或P；X8-10是Y或N；
上述LCDR1包括如SEQ ID NO.10(QDINX10-5Y)、SEQ ID NO.12(SSVNY)、SEQ ID NO.11(QNVDTX11-6)或SEQ ID NO.13(KSLLHSNGNTY)所示的氨基酸序列，其中X10-5是N或K；X11-6 是N或T；
上述LCDR2包括如氨基酸残基YT、GT、SA或RM所示的氨基酸序列；
上述LCDR3包括如SEQ ID NO.16(LQYHDLIT)、SEQ ID NO.14(X14-1QX14-3X14-4X14-5X14-6X14-7X14-8T)或SEQ ID NO.15(MQHLEYPFT)所示的氨基酸序列，其中X14-1是Q或L；X14-3是R或Y；X14-4是S、D或N；X14-5是S或N；X14-6是Y或L；X14-7是P或缺失；X14-8是F或L。
在本公开中，术语“抗体”包括任何可结合某特定抗原的免疫球蛋白。所述术语“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体\多克隆抗体，单特异性抗体\多特异性抗体，全长抗体\抗原结合片段，只要它们展现出期望的抗原结合活性。通常，一个天然的完整抗体包含两条重(H)链和两条轻(L)链。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或同种型：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。每条重链由一个可变区(VH)以及第一、第二、第三和第四(可选地)恒定区(分别为CH1、CH2、CH3、CH4)组成。哺乳动物的轻链可分为λ或κ，而每条轻链由一个可变区(VL)以及一个恒定区(CL)组成。通常，天然的完整抗体呈“Y”型，“Y”型结构的茎部由通过二硫键结合的两条重链的第二和第三恒定区组成。“Y”型结构的每条臂包括重链的VH和CH1(VH-CH1)以及轻链(VL-CL)。轻链和重链的可变区决定抗原的结合。每条链的可变区通常含有三个高变区，称为“互补决定区(CDR)”，其中，轻链CDR包含LCDR1、LCDR2、LCDR3，重链CDR包含HCDR1、HCDR2、HCDR3。
在本公开中，术语“抗CD39抗体”是指能够与CD39(例如，人类或猴CD39)特异性结合的抗体。
在本公开中，术语“氨基酸(amino acid)”是指是指天然存在的氨基酸和合成的氨基酸，以及与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸包括由遗传密码编码的氨基酸及其经修饰的氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。天然存在的氨基酸包括丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)；谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)；组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、甲硫氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝胺酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构(即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。氨基酸类似物通常具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但具有与天然存在的氨基酸类似的方式起作用的化学化合物。
在一些具体的实施方式中，上述HCDR1包括如SEQ ID NO.22、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20或者SEQ ID NO.21所示的氨基酸序列，上述HCDR2包括如SEQ ID NO.6、SEQ ID NO.23、SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.24所示的氨基酸序列，上述HCDR3包括如SEQ ID NO.28、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27或者SEQ ID NO.9所示的氨基酸序列，上述LCDR1包括如SEQ ID NO.32、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.29、SEQ ID NO.30或者SEQ ID NO.31所示的氨基酸序列，上述LCDR2包括如氨基酸残基YT、SA、GT或RM所示的氨基酸序列，上述LCDR3包括如SEQ ID NO.16、SEQ ID NO.33、SEQ ID NO.15、SEQ ID NO.34、SEQ ID NO.35或者SEQ ID NO.36所示氨基酸序列。
在一些具体的实施方式中，上述抗体的互补决定区选自如下(a)-(f)中的任意一种：
(a)：上述HCDR1包括SEQ ID NO.22所示的氨基酸序列，HCDR2包括SEQ ID NO.6所示的氨基酸序列，HCDR3包括SEQ ID NO.28所示的氨基酸序列，LCDR1包括SEQ ID NO.32 所示的氨基酸序列，LCDR2包括氨基酸残基YT所示的氨基酸序列，LCDR3包括SEQ ID NO.16所示的氨基酸序列；
(b)：上述HCDR1包括SEQ ID NO.17所示的氨基酸序列，HCDR2包括SEQ ID NO.23所示的氨基酸序列，HCDR3包括SEQ ID NO.25所示的氨基酸序列，LCDR1包括SEQ ID NO.12所示的氨基酸序列，LCDR2包括氨基酸残基GT所示的氨基酸序列，LCDR3包括SEQ ID NO.33所示的氨基酸序列；
(c)：上述HCDR1包括SEQ ID NO.21所示的氨基酸序列，HCDR2包括SEQ ID NO.4所示的氨基酸序列，HCDR3包括SEQ ID NO.9所示的氨基酸序列，LCDR1包括SEQ ID NO.31所示的氨基酸序列，LCDR2包括氨基酸残基SA所示的氨基酸序列，LCDR3包括SEQ ID NO.36所示的氨基酸序列；
(d)：上述HCDR1包括SEQ ID NO.18所示的氨基酸序列，HCDR2包括SEQ ID NO.24所示的氨基酸序列，HCDR3包括SEQ ID NO.26所示的氨基酸序列，LCDR1包括SEQ ID NO.13所示的氨基酸序列，LCDR2包括氨基酸残基RM所示的氨基酸序列，LCDR3包括SEQ ID NO.15所示的氨基酸序列；
(e)：上述HCDR1包括SEQ ID NO.19所示的氨基酸序列，HCDR2包括SEQ ID NO.5所示的氨基酸序列，HCDR3包括SEQ ID NO.27所示的氨基酸序列，LCDR1包括SEQ ID NO.29所示的氨基酸序列，LCDR2包括氨基酸残基YT所示的氨基酸序列，LCDR3包括SEQ ID NO.34所示的氨基酸序列；
(f)：上述HCDR1包括SEQ ID NO.20所示的氨基酸序列，HCDR2包括SEQ ID NO.4所示的氨基酸序列，HCDR3包括SEQ ID NO.9所示的氨基酸序列，LCDR1包括SEQ ID NO.30所示的氨基酸序列，LCDR2包括SA所示的氨基酸序列，LCDR3包括SEQ ID NO.35所示的氨基酸序列；
上述CDR根据IMGT定义方式进行确定。
在本公开的第二方面，本公开提供了一种抗CD39抗体或其抗原结合片段，其包含：
(a)重链可变区，所述重链可变区包含SEQ ID NO.85、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84任一项所示重链可变区的HCDR1,HCDR2和HCDR3；以及，
(b)轻链可变区，所述轻链可变区包含SEQ ID NO.91、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、SEQ ID NO.90任一项所示轻链可变区的LCDR1,LCDR2和LCDR3；
上述HCDR1,HCDR2和HCDR3，及LCDR1,LCDR2和LCDR3按照Kabat定义方式、Chothia定义方式、AbM定义方式或Contact定义方式确定。
本公开中公开的抗体或其抗原结合片段的CDR边界根据IMGT、Kabat或者Chothia定义方式定义或鉴定，以其他方式例如AbM、Contact等定义方式定义的CDR也属于本公开的保护范围(Kaas,Q et al.IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains.Dev.Comp.Immunol.29,185-203,(2005)；
R.M.MacCallum et al.,.Antibody–antigen interactions:contact analysis and binding site topography J.Mol.Biol.(1996)；
Martin,A.C.R.Protein sequence and structure analysis of antibody variable domains(Book chapter).In Antibody engineering lab manual Eds.Duebel,S.and Kontermann,R.(2001)；
Marie-Paule Lefranc et al.IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,Developmental and Comparative Immunology 27(2003)55–77)。
在一些具体的实施方式中，上述抗CD39抗体或其抗原结合片段的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3可选自表1。
表1中展示了示例性抗体#2(重链可变区的氨基酸序列如SEQ ID NO.80所示，轻链可变区的氨基酸序列如SEQ ID NO.86所示)、#66(重链可变区的氨基酸序列如SEQ ID NO.81所 示，轻链可变区的氨基酸序列如SEQ ID NO.87所示)、#75(重链可变区的氨基酸序列如SEQ ID NO.82所示，轻链可变区的氨基酸序列如SEQ ID NO.88所示)、#95(重链可变区的氨基酸序列如SEQ ID NO.83所示，轻链可变区的氨基酸序列如SEQ ID NO.89所示)、#103(重链可变区的氨基酸序列如SEQ ID NO.84所示，轻链可变区的氨基酸序列如SEQ ID NO.90所示)和#110(重链可变区的氨基酸序列如SEQ ID NO.85所示，轻链可变区的氨基酸序列如SEQ ID NO.91所示)根据不同定义方式定义的CDR氨基酸序列。
表1示例性单克隆抗体的CDR氨基酸序列

在一些具体的实施方式中，上述抗体含有重链框架区和/或轻链框架区，上述重链框架区和/或轻链框架区来自鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
在本公开中，抗CD39抗体的三个CDR由被称为框架区(FR，轻链FR包含LFR1、LFR2、LFR3和LFR4，重链FR包含HFR1、HFR2、HFR3和HFR4)的侧翼部分间隔开，。
在一些具体的实施方式中，上述抗CD39抗体的重链可变区的框架区包括HFR1，HFR2，HFR3和HFR4，上述轻链可变区的框架区包括LFR1，LFR2，LFR3和LFR4，其中
所述HFR1包括如SEQ ID NO.41、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、和SEQ ID NO.40中任一项所示的氨基酸序列，或包括与SEQ ID NO.41、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、和SEQ ID NO.40中任一项所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述HFR2包括如SEQ ID NO.46、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、和SEQ ID NO.45中任一项所示的氨基酸序列，或包括与SEQ ID NO.46、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、和SEQ ID NO.45中任一项所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述HFR3包括如SEQ ID NO.52、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、和SEQ ID NO.51中任一项所示的氨基酸序列，或与SEQ ID NO.52、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、和SEQ ID NO.51中任一项所示的氨基酸序列具有至少65％(例如70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述HFR4包括如SEQ ID NO.55、SEQ ID NO.53、SEQ ID NO.54、和SEQ ID NO.56中任一项所示的氨基酸序列，或与SEQ ID NO.55、SEQ ID NO.53、SEQ ID NO.54、和SEQ ID NO.56中任一项所示的氨基酸序列具有至少85％(例如86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述LFR1包括如SEQ ID NO.62、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、和SEQ ID NO.61中任一项所示的氨基酸序列，或与SEQ ID NO.62、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、和SEQ ID NO.61中任一项所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述LFR2包括如SEQ ID NO.67、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、和SEQ ID NO.66中任一项所示的氨基酸序列，或与SEQ ID NO.67、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、和SEQ ID NO.66中任一项所示的氨基酸序列具有至少80％(例如82％、84％、86％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述LFR3包括如SEQ ID NO.73、SEQ ID NO.68、SEQ ID NO.69、SEQ ID NO.70、SEQ ID NO.71、和SEQ ID NO.72中任一项所示的氨基酸序列，或与SEQ ID NO.73、SEQ ID NO.68、SEQ ID NO.69、SEQ ID NO.70、SEQ ID NO.71、和SEQ ID NO.72中任一项所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；
所述LFR4包括如SEQ ID NO.79、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、和SEQ ID NO.78中任一项所示的氨基酸序列，或与SEQ ID NO.79、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、和SEQ ID NO.78中任一项所示的氨基酸序列具有至少92％(例如93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列。
在一些具体的实施方式中，上述抗CD39抗体或其抗原结合片段的HFR1包括如SEQ ID NO.41所示的氨基酸序列，或与SEQ ID NO.41所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；HFR2包括如SEQ ID NO.46所示的氨基酸序列，或与SEQ ID NO.46所示的氨基酸序 列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；HFR3包括如SEQ ID NO.52所示的氨基酸序列，或与SEQ ID NO.52所示的氨基酸序列具有至少65％(例如70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；HFR4包括如SEQ ID NO.55所示的氨基酸序列，或与SEQ ID NO.55所示的氨基酸序列具有至少85％(例如86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；LFR1包括如SEQ ID NO.62所示的氨基酸序列，或与SEQ ID NO.62所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；LFR2包括如SEQ ID NO.67所示的氨基酸序列，或与SEQ ID NO.67所示的氨基酸序列具有至少80％(例如82％、84％、86％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；LFR3包括如SEQ ID NO.73所示的氨基酸序列，或与SEQ ID NO.73所示的氨基酸序列具有至少75％(例如80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列；和LFR4包括SEQ ID NO.79所示的氨基酸序列具有至少92％(例如93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的序列。
上述FR可根据IMGT定义方式进行确定。
在本公开中，术语“同一性(identity)”百分比是指当两个序列最佳比对时，两个多肽的氨基酸在等效位置相同的程度。氨基酸序列同一性百分比的比对可以采用所属技术领域中各种方式进行，例如本领域熟知的BLAST、BLAST-2、ALIGN、MEGALIGN(DNASTAR)、CLUSTALW或CLUSTAL OMEGA等软件。本领域技术人员可确定比对序列的适当参数，包括要实现比较序列全长的最大比对所需的任何算法。
在一些具体的实施方式中，本公开所述的抗体包含适当的框架区(FR)序列，只要所述的抗体可以与CD39特异性结合。上述的表1中所示的CDR序列获取自小鼠抗体，但可使用本领域公知的合适方法(例如，重组技术)将其嫁接到任何合适物种(例如小鼠、人、大鼠、兔以及其他)的任何合适的FR序列。
在一些具体的实施方式中，上述抗CD39抗体的HFR1、上述HFR2、上述HFR3、上述HFR4、上述LFR1、上述LFR2、上述LFR3和上述LFR4选自表2。
以下的表2中展示了示例性抗体#2、#66、#75、#95、#103和#110的FR氨基酸序列。FR边界根据IMGT定义方式定义或鉴定，以其他方式例如Kabat、Chothia、AbM、Contact或者Lesk定义方式定义的FR也属于本公开的保护范围。
表2示例性单克隆抗体的FR氨基酸序列

在一些具体的实施方式中，上述的抗体具有如SEQ ID NO.85、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、和SEQ ID NO.84中任一项所示或与SEQ ID NO.85、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、和SEQ ID NO.84中任一项所示的氨基酸序列具有至少70％(例如75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的氨基酸序列的重链可变区；
在一些具体的实施方式中，上述抗体具有如SEQ ID NO.91、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、和SEQ ID NO.90中任一项所示或与SEQ ID NO.91、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、和SEQ ID NO.90中任一项所示的氨基酸序列具有至少70％(例如75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或100％)序列同一性的氨基酸序列的轻链可变区；
以下的表3中展示了示例性抗体#2、#66、#75、#95、#103和#110的重链可变区和轻链可变区的氨基酸序列。
表3示例性抗体的可变区氨基酸序列
在一些具体的实施方式中，上述抗CD39抗体含有重链框架区和/或轻链框架区，上述抗体的重链框架区和/或轻链框架区来自鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
在一些具体的实施方式中，上述抗体的重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD中的任一种；上述抗体的轻链恒定区为κ或λ链。
在一些具体的实施方式中，上述抗体为全长抗体或其抗原结合片段，所述抗原结合片段为F(ab’)2、Fab’、Fab、scFv、Fv、dsFV中的任一种或几种。
在本公开中，术语“抗原结合片段”是指具有抗原结合活性，含有一个或多个CDR的抗体的一部分形成的抗体片段，或者与抗原结合但不具有完整的天然抗体结构的任何其他抗体片段。抗原结合片段的例子包括但不限于Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds diabody)、单链抗体分子(scFv)、scFv二聚体(双价的双功能抗体)。上述抗原结合片段能够与亲本抗体结合相同的抗原。
在本公开中，术语抗体的“Fab”是指由单条轻链(包括可变区和恒定区)和单条重链的可变区和第一恒定区经二硫键结合起来组成的抗体的一部分。
“Fab’片段”是指包含了部分铰链区的Fab片段。
“F(ab’)2”是指Fab’的二聚体。
“Fv片段”由单条轻链的可变区和或单条重链的可变区结合组成。
“单链Fv抗体”或“scFv”是指由轻链可变区与重链可变区直接相互连接或通过肽接头序列连接而成的抗体片段。
在一些具体的实施方式中，上述抗CD39抗体或其抗原结合片段具有如下(a)-(l)中的一种或多种性质：
(a)特异性地与人类和或猴CD39结合，但不与小鼠CD39特异性地结合，上述结合特性通过流式细胞荧光分选技术的方法测定。该测定方法可参考实施方式2中第5项或第6项所述的步骤进行；
(b)以不超过7nM(例如，不超过6nM、不超过5nM、不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM、不超过0.4nM、不超过0.3nM、不超过0.2nM、不超过0.1nM、不超过0.09nM或者不超过0.08nM)的EC50与人CD39结合，上述EC50通过流式细胞荧光分选技术的方法测定。该测定方法可参考实施方式2中第4项所述的步骤进行；
(c)以不超过11nM(例如，不超过10nM、不超过9nM、不超过8nM、不超过7nM、不超过6nM、不超过5nM、不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM或者不超过0.4nM)的EC50与表达CD39的人Mino细胞结合，上述EC50通过流式细胞荧光分选技术的方法测定。该测定方法可参照实施方式2中第12项所述的步骤进行；
(d)以不超过8nM(例如，不超过7nM、不超过6nM、不超过5nM、不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM或者不超过0.4nM)的EC50与人MOLP-8细胞结合，上述EC50通过流式细胞荧光分选技术的方法测定。该测定方法可参照实施方式2中第13项所述的步骤进行；
(e)以不超过10‐9M(例如，不超过5x10‐10M、不超过4x10‐10M、不超过3x10‐10M、不超过2x10‐10M、不超过1x10‐10M、不超过9x10‐11M、不超过8x10‐11M、不超过7x10‐11M、不超过6x10‐11M、不超过5x10‐11M、不超过4x10‐11M、不超过3x10‐11M、不超过2x10‐11M、不超过1x10‐11M、不超过9x10‐12M、不超过8x10‐12M、不超过7x10‐12M、不超过6x10‐12M、不超过5x10‐12M、不超过4x10‐12M、不超过3x10‐12M或不超过2x10‐12M)的KD值与人类CD39结合，上述KD值通过Biacore检测方法测定。该测定方法可参照实施方式2中的第10项所述的步骤进行；
(f)以不超过7nM(例如，不超过6nM、不超过5nM、不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM、不超过0.4nM、不超过0.3nM、不超过0.2nM、不超过0.1nM或者不超过0.09nM)的IC50抑制在表达CD39的Mino细胞中的ATP酶活性，上述IC50通过ATP酶活性分析的方法测定。该测定方法可参照实施方式2中的第14项所述；
(g)以不超过5nM(例如，不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6nM、不超过0.5nM、不超过0.4nM、不超过0.3nM或者不超过0.2nM)的IC50抑制在表达CD39的MOLP-8细胞中的ATP酶活性，上述IC50通过ATP酶活性分析的方法测定。该测定方法可参照实施方式2中的第15项所述；
(h)以不超过10nM(例如，不超过9nM、不超过8nM、不超过7nM、不超过6nM、不超过5nM、 不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6M、不超过0.5nM、不超过0.4nM、不超过0.3nM、不超过0.2M、不超过0.1M、不超过0.09nM或者不超过0.08nM)的IC50阻断ATP，抑制CD8T细胞增殖，上述IC50通过流式细胞荧光分选技术分析的方法测定。该测定方法可参照实施方式2中的第16项所述的步骤进行；
(i)以不超过10nM(例如，不超过9nM、不超过8nM、不超过7nM、不超过6nM、不超过5nM、不超过4nM、不超过3nM、不超过2nM、不超过1nM、不超过0.9nM、不超过0.8nM、不超过0.7nM、不超过0.6M、不超过0.5nM、不超过0.4nM或者不超过0.3nM)的IC50阻断ATP，抑制CD3T细胞增殖，上述IC50通过流式细胞荧光分选技术分析的方法测定。该测定方法可参照实施方式2中的第16项所述的步骤进行；
(j)特异性的与人CD39结合，而与CD39的同家族蛋白(例如ENTPD2、3、5、和6)无交叉结合；
(k)能够抑制哺乳动物中的肿瘤生长(例如能够抑制MOLP-8异种移植的小鼠中的肿瘤生长)；
(l)上述抗CD39抗体或其抗原结合片段中的CD39为人CD39或猴CD39。
在本公开中，术语“特异性结合”或“特异性地结合”是指两分子间的非随机结合反应，例如，抗体和抗原间的反应。在一些具体的实施方式中，例如，根据流式细胞荧光分选技术测定，本公开提供的抗体或其抗原结合片段与人类CD39特异性结合，但不与小鼠CD39特异性结合。在一些实施方式中，本公开提供的抗体或其抗原结合片段特异性地与食蟹猴CD39结合。
在本公开中，术语“亲和力”或“亲和性”是指免疫球蛋白分子(即，抗体)或其片段与抗原之间非共价相互作用的强度。免疫结合相互作用的强度或亲和力可用相互作用的平衡解离常数(KD)表示，其中KD值越小，表示亲和力越高。可通过使用本领域公知的任何传统方法测定KD，包括但不限于Biacore测定法、Octet方法、微量热泳法、HPLC‐MS方法和流式细胞荧光分选技术。
本公开提供的抗体或其抗原结合片段与人类CD39的结合也可以用“半最大有效浓度(EC50)”是指在特定暴露时间后，能达到50％最大生物效应对应的药物或抗体的浓度。一般EC50越小亲和力越好，表示更低的浓度下即可以与靶蛋白结合。可以通过本领域已知的结合检测法，例如直接或间接结合检测法(例如酶联免疫吸附检测法(ELISA)、流式细胞荧光分选技术和其他结合检测法)来测定EC50值。
在本公开中，术语“半最大抑制浓度”(IC50)是对指定的生物过程(或该过程中的某个组分比如酶、受体、细胞等)抑制一半时所需的药物或者抑制剂的浓度。一般IC50越小，抗体的阻断活性越好，即越小的IC50，表示在更低的浓度下就可以发挥阻断作用。
在一些实施方式中，本公开提供的抗体或其抗原结合片段以不超过5nM的IC50抑制在表达CD39的Mino细胞中的ATP酶活性，所述IC50通过ATP酶活性分析测定。以不超过5nM的IC50抑制在表达CD39的MOLP-8细胞中的ATP酶活性，所述IC50通过ATP酶活性分析测定。可以使用本领域已知的方法来确定ATP酶活性测定，例如通过比色检测作为ATP酶活性的结果而释放的磷酸盐。在一些实施方式中，通过本公开的实施方式2中的第7项中描述的那些方法确定ATP酶活性。
在一些实施例中，IC50还可以用来表示抗体对细胞增殖的抑制活性，本公开提供的抗体或其抗原结合片段能够以不超过10nM的IC50阻断ATP，抑制CD8T细胞增殖，所述IC50通过流式细胞术分析测定。以不超过10nM的IC50阻断ATP，抑制CD3T细胞增殖，所述IC50通过流式细胞术分析测定。可以使用本领域已知的方法(例如本公开的实施方式2中的第8项或16项中描述的那些方法)来确定T细胞的增殖。
在本公开中，术语“Mino细胞”是一种人非霍奇金淋巴瘤细胞系，其细胞表面天然表达人CD39蛋白。
在本公开中，术语“MOLP-8细胞”是一种人多发性骨髓瘤细胞系，其细胞表面天然表达人CD39蛋白，将该细胞接种到NOD-SCID小鼠上建立的多发性骨髓瘤模型，可用于检测抗CD39抗体的体内抗肿瘤效果。
在本公开的第三方面，本公开提供了一种抗CD39抗体或其抗原结合片段，其与第一或第二方面所述的抗CD39抗体竞争性结合CD39，或者其结合CD39抗原的表位与第一或第二方面所述的抗CD39抗体结合CD39抗原的表位相同。
在本公开中，术语“表位”是指抗原上被抗体的互补位结合的任何抗原决定簇。抗原决定簇通常是具有一定组成和结构的特殊化学基团。表位可以是线性(即连续的)或构象的(即包括间隔开的氨基酸残基，非连续的)。表位限定了抗体的最小结合位点，因此是抗体或其抗原结合片段的特异性靶标。表位可以通过本领域熟知的任何方法确定，例如常规免疫测定法、抗体竞争性结合测定法或X射线晶体学或相关结构测定方法(例如,核磁共振光谱)。
在本公开的第四方面，本公开提供了一种多特异性抗体，其含有前述任一项所示的抗CD39抗体或其抗原结合片段。
在本公开中，术语“多特异性抗体”是指可以与同一抗原的多个(两个或两个以上)不同抗原表位或者多个(两个或两个以上)不同抗原相结合的一种抗体分子。
在本公开的第五方面，本公开提供了一种生物材料，其特征在于，所述生物材料选自(ⅰ)～(v)中任一项：
(i)嵌合抗原受体(Chimeric Antigen Receptor，CAR)，所述嵌合抗原受体包括胞外区，所述胞外区包括抗原结合结构域；并且，所述抗原结合结构域含有前述任一方面所述的抗CD39抗体或其抗原结合片段；
(ii)工程化的免疫细胞，所述免疫细胞表达(i)所述的嵌合抗原受体或含有编码(i)所述的嵌合抗原受体的核酸；
(iii)分离的核酸分子，包括编码前述任一方面所述的抗体或其抗原结合片段的核苷酸序列；
(iv)载体，所述载体携带前述(iii)核酸分子；
(v)重组细胞，所述重组细胞携带(iii)中核酸分子，或含有(iv)中载体，或表达前述任一方面所述的抗体或其抗原结合片段。
本公开中的“嵌合抗原受体”，CAR是模拟TCR功能的人工受体，由胞外结构域、跨膜结构域及胞内结构域依次连接组成，肿瘤细胞表面的抗原(受体)与嵌合抗原受体的抗体(配体)结合时，可通过铰链区和跨膜区将信号传递至胞内，胞内结构域再将信号转化为活化信号，激活效应细胞，效应细胞通过分泌穿孔素或者产生细胞因子杀伤肿瘤细胞，同时效应细胞本身也发生扩增，进一步扩大免疫杀伤作用。胞外结构域一般由负责识别并结合抗原的单克隆抗体的单链可变片段(single-chain variable fragment，scFv)及一段起连接作用的铰链区(Hinge)构成。单链可变片段是CAR的抗原结合结构域，该结构域决定CAR-免疫细胞的特异性和功能。铰链区是连接单链可变片段和跨膜结构域的CAR细胞外结构区，它通常维持效应细胞中稳健的CAR表达和活性所需的稳定性，大多数CAR的铰链区由IgG的铰链或CD8α/CD28胞外区衍变而来。铰链区的类型和长度对CAR的功能活动有重要影响。跨膜结构域将CAR的细胞外结构域与细胞内信号转导结构域连接。常用的跨膜结构域来源于CD4，CD8，CD28和CD3ζ或它们的衍生物。跨膜结构域的选择影响CAR结构在细胞功能上的活化程度。胞内结构域由共刺激结构域和信号转导结构域构成。共刺激域通常来自CD28受体家族(CD28，ICOS)或肿瘤坏死因子受体家族(4-1BB、OX40、CD27)。信号转导结构域通常为T细胞受体TCR/CD3ζ链或免疫球蛋白Fc受体FcεRIγ链。
本公开的CAR(包括其功能部分和功能变体)可通过本领域已知的方法获得，例如可以通过制备多肽或蛋白质的任何合适的方法制备。
在本公开中，术语“工程化的免疫细胞”是指表达CAR的免疫细胞或经CAR修饰的免疫细胞，其中，免疫细胞包括但不限于T细胞、自然杀伤细胞(NK细胞)、巨噬细胞(M细胞)、Treg细胞。
在本公开中，术语“核酸分子”通常是RNA或DNA，核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸分子与另一个核酸序列置于功能关系中时，核酸分子是“有效连接的”。 例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时优选采用DNA。
在本公开中，术语“载体”是指可将遗传元件(例如前述核酸分子)操作性地插入其中并使该遗传元件获得表达的一种运载工具，例如产生由该遗传元件编码的蛋白质、RNA或DNA，或者复制所述遗传元件。载体可用于转化、转导或转染宿主细胞，使其携带的遗传元件在宿主细胞内得以表达。举例来说，载体包括：质粒、噬菌粒、柯斯质粒(cosmid)、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、噬菌体如λ噬菌体或M13噬菌体，以及动物病毒等。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于，病毒颗粒、脂质体或蛋白外壳。载体可以是表达载体或克隆载体。在一些实施方案中，本公开提供的载体(例如表达载体)含有本公开所述的编码抗体或其抗原结合片段的核酸序列、至少一个可操作地连接至所述核酸序列的启动子(例如，SV40、CMV、EF‐1α)，以及至少一个选择标记。
在本公开中，术语“重组细胞”是指可以或已经导入外源多核苷酸和/或载体的细胞。该重组细胞中包含有该载体，可以将载体导入到哺乳动物细胞中，构建获得重组细胞，然后利用这些重组细胞表达本公开提供的抗体或者抗原结合片段。对该重组细胞进行培养，即可以获得相应抗体。可用的哺乳动物细胞可以为CHO细胞等。
在本公开的第六方面，本公开提供了一种药物组合物，其含有前述的抗CD39抗体或其抗原结合片段、前述的多特异性抗体、前述的生物材料。
在本公开中，术语“药物组合物”是以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。在一些具体的实施方式中，上述药物组合物中包含的抗体或表达的抗体能够特异性的靶向结合CD39，阻断CD39水解ATP。
在一些实施方式中，上述药物组合物还包括药学上可接受的载体和/或赋形剂。
在本公开中，术语“药学上可接受的载体”可以包括生理学上相容的任何溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和延迟吸收剂等，用来延长抗体的保存限期或效力。
在本公开的第七方面，本公开提供了前述的抗CD39抗体或其抗原结合片段、前述的多特异性抗体、前述的生物材料或前述的药物组合物在制备用于预防、治疗、或诊断治疗与CD39相关的肿瘤疾病的药物中的应用。
在本公开的第八方面，本公开提供了一种用于产生前述抗CD39抗体或其抗原结合片段的方法，其包括以下步骤：
(a)在表达抗CD39抗体或其抗原结合片段的条件下培养前述的重组细胞；
(b)分离和纯化步骤(a)中获得的抗CD39抗体或其抗原结合片段。
在本公开的第九方面，本公开提供了一种治疗、预防或减轻与CD39相关的疾病、病症或状况的方法，其包括向受试者施用治疗有效量的前述的抗CD39抗体或其抗原结合片段和/或前述的药物组合物。本公开还提供了一种用作药物的前述任一方面所述的抗CD39抗体或抗原结合片段。
在本公开中，术语“受试者”或“患者”是指哺乳动物受试者或患者。示例性受试者包括但不限于人、猴、犬、猫、小鼠、大鼠、牛、马、骆驼、禽、山羊和绵羊。在某些实施方案，所述受试者是人。在一些实施方案，所述受试者是疑似患有癌症、自体免疫性疾病或病况、和/或感染的人。
在本公开中，术语“诊断”是指病理状态、疾病或状况的鉴定，例如CD39相关疾病的鉴定，或指患有CD39相关疾病的可能会受益于特定的治疗方案的受试者的鉴定。在一些实施方式中，诊断包含鉴定CD39的异常含量或活性。在一些实施方式中，诊断是指在受试者中鉴定癌症或自身免疫性疾病。
在本公开中，“CD39相关”的疾病、病症或状况是指由CD39的表达或活性的增加或减少引起的、加剧的或与之相关的任何疾病或状况。在一些实施方式中，CD39相关的疾病、病症或状况是与过度细胞增殖有关的病症，例如癌症。在一些实施方式中，CD39相关的疾病或状况的特征在于表达或过表达CD39和或CD39相关基因，例如ENTPD1、2、3、4、5、6、7或8基因。
在本公开中，术语“表达CD39的”癌症是指在癌细胞或浸润肿瘤的免疫细胞或免疫抑制细胞中表达CD39，并且在癌细胞或浸润肿瘤的免疫细胞或免疫抑制细胞中表达CD39的水平显著高于正常细胞所预期的水平的癌症。
在一些实施方式中，“CD39相关”的疾病、病症或状况包括：癌症、自身免疫性疾病和感染。
在一些实施方式中，上述癌症选自下组：肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆囊癌、胃癌、肺癌、支气管癌、骨癌、肝胆管癌、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、肾盂和输尿管癌、唾液腺癌、小肠癌、尿道癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、直肠癌、食道癌、胃肠道癌、皮肤癌、前列腺癌、垂体癌、阴道癌、甲状腺癌、喉癌、胶质母细胞瘤、黑素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、慢性淋巴细胞白血病(CLL)、慢性髓性白血病(CML)、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、霍奇金淋巴瘤、非霍奇金淋巴瘤、多发性骨髓瘤、T或B细胞淋巴瘤、胃肠道间质瘤、软组织肿瘤、肝细胞癌和腺癌。在一些实施方式中，所述癌症是白血病、淋巴瘤、膀胱癌、神经胶质瘤、胶质母细胞瘤、卵巢癌、黑素瘤、前列腺癌、甲状腺癌、食道癌或乳腺癌。
在本公开中，术语“有效量”是指治疗量，所述量足以降低或改善病症的严重性和/或持续时间或其一种或多种症状；防止疾病的进展；导致疾病消退；预防与疾病相关的一种或多种症状的复发、发展或进展；检测疾病；或增强或改善另一种疗法(例如预防剂或治疗剂)的预防或治疗效果。本公开所述的抗体或其抗原结合片段的治疗有效剂量依赖于本领域公知的多种因素，例如体重、年龄、过往病史、现用治疗、对象的健康状况和交叉感染的潜力、过敏、超敏和副作用，以及给药途径和肿瘤发展的程度。本领域技术人员(例如医生或兽医)可根据这些或其它条件或要求按比例降低或升高剂量。
在一些实施方式中，如本公开所述的抗体或抗原结合片段可以在治疗有效剂量约0.001mg/kg到约1000mg/kg之间给药。在一些实施方式中，给药剂量可随治疗进程变化。例如，在一些实施方式中，初始给药剂量可比后续给药剂量高。在一些实施方式中，给药剂量在治疗进程中根据给药对象的反应进行调整。
在一些实施方式中，本公开中公开的抗体或其抗原结合片段可以单独给药或与治疗有效量的第二治疗剂联合给药。例如，本公开公开的抗体或其抗原结合片段可以与第二治疗剂(例如，化疗剂、抗癌药、放疗剂、免疫治疗剂、抗血管生成剂、靶向治疗剂、细胞治疗剂、基因治疗剂、激素治疗剂、抗病毒剂、抗生素、镇痛药、抗氧化剂、金属螯合剂或细胞因子)联合给药。
在一些实施方式中，本公开公开的抗体或其抗原结合片段与一种或多种另外的治疗剂联用时，可与所述的一种或多种另外的治疗剂同时给药，在某些这样的实施方式中，所述的抗体或其抗原结合片段和所述另外的治疗剂可作为同一个药物组合物的一部分同时给药。但是，与其他治疗剂“联用”的抗体或其抗原结合片段不需要同时给药或与该治疗剂在同一组合物中给药。本公开中“联用”的含义还包括，在另一个治疗剂之前或之后给药的抗体或其抗原结合片段也被认为是与该治疗剂“联用”，即所述抗体或其抗原结合片段与第二种物质通过不同给药方式给药。
在本公开的第十方面，本公开提供了一种检测CD39的试剂盒，其包含前述的抗CD39抗体或其抗原结合片段。
在本公开的第十一方面，本公开提供了前述的抗体、前述的嵌合抗原受体、前述的核酸分子、前述的载体、前述的重组细胞或前述的药物组合物在制备试剂盒中的用途，上述试剂盒用于检测CD39或者诊断CD39相关的疾病。
在一些具体的实施方式中，前述的抗CD39抗体或其抗原结合片段能够特异性靶向结合CD39，根据本公开实施方式的试剂盒可以实现CD39的特异性检测，如当抗体结合有荧光基团时，可以采用荧光检测装置实现对CD39的定位或实时检测。例如可以用于免疫印迹、免疫沉淀等涉及到利用CD39抗原和抗体的特异性结合性能来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、抗CD39抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。所述试剂盒还可以用于检测与CD39相关的疾病、病症或状况。
本公开包括以下有益效果：
在一些实施方案中，本公开提供的抗CD39抗体能够特异性结合CD39蛋白，阻断CD39水解ATP，减少肿瘤微环境中的腺苷，发挥抗肿瘤作用，为癌症的治疗和/或预防提供新的可能。
具体实施方式
为使本公开实施方式的目的、技术方案和优点更加清楚，下面将对本公开实施方式中的技术方案进行清楚、完整地描述。实施方式中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非另有定义，否则本文使用的所有技术和科学术语与本公开内容所属领域的普通技术人员通常理解的含义相同。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试，但现在描述一些方法和材料。除非另有说明，否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明，否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术，所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术，如《分子克隆：实验室手册(Molecular Cloning:ALaboratory Manual)》，第二版(Sambrook等人，1989)；《寡核苷酸合成(OligonucleotideSynthesis)》(M.J.Gait编，1984)；《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编，1987)；《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press，Inc.)；《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编)；《哺乳动物细胞用基因转移载体(Gene Transfer Vectors forMammalian Cells)》(J.M.Miller和M.P.Calos编，1987)；《当代分子生物学方法(CurrentProtocols in Molecular Biology)》(F.M.Ausubel等人编，1987)；《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编，1994)；以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编，1991)，所述文献中的每个文献均通过引用明确并入本文中。
以下结合实施方式对本公开的特征和性能作进一步的详细描述。
实施方式1.产生针对CD39的小鼠单克隆抗体
1、免疫原
在免疫过程中，采用携带his标签的人CD39胞外结构域融合蛋白hCD39-his(Acro，Cat.CD9-H52H4)作为免疫原，用于产生抗人CD39的抗体。
2、免疫流程
采用常用的小鼠杂交瘤体系产生抗人CD39鼠单抗。过程如下：
将重组人CD39重组蛋白粉末配置成浓度为0.5mg/mL的溶液，然后与titermax佐剂(sigma，Cat.T2684)等体积混合，得到油状乳液。以0.2mL的剂量皮下施给6周龄雌性BALB/c小鼠(广东省医学实验动物中心)背部位点。第一次免疫7天后，采用重组人CD39重组蛋白与alum 佐剂(Thermo，Cat.77161)等体积混合后进行腹腔免疫，免疫至4-5针后，采集尾血进行效价检测，待效价达到既定滴度后，采用0.1mL用生理盐水稀释的免疫原进行免疫加强，3天后进行脾脏融合。免疫方案如表4所示：
表4用于产生单克隆抗体的免疫方案
3、杂交瘤细胞的制备
将经免疫过BALb/c小鼠的脾细胞与小鼠骨髓瘤细胞融合，然后对得到的杂交瘤细胞进行抗原特异性抗体的筛选。小鼠的脾细胞基于标准方案进行分离，将免疫脾细胞与小鼠骨髓瘤SP2/0细胞按照1:1细胞数量比例混合，转移至50mL离心管中，用DMEM基础培养基洗涤一次。弃上清，用40mL电融合液(BTX，Cat.47-0001)将细胞混匀，加入电融合槽，按照既定参数进行电融合。融合后，加入200mL含HAT的DMEM完全培养基轻轻混悬，平分于96孔板中，共40块，50μL/孔，37℃、5％CO2细胞培养箱静置培养。培养至第六天，更换含HT的DMEM完全培养基一次。
4、抗人CD39杂交瘤上清的检测
用0.05M碳酸缓冲液稀释携带人his标签的人CD39重组蛋白至终浓度为1μg/mL，按100μL/孔加入96孔ELISA检测板中，2℃-8℃包被过夜。弃上清，按200μL/孔加入封闭液(1×PBS+1％BSA)，37℃封闭0.5h。将融合第7天的上清按100μL/孔加入96孔板中，37℃静置孵育30min。用1×PBS洗涤3次。按100μL/孔加入HRP标记的羊抗鼠IgG(sigma,Cat.A0168-1ML)，37℃静置孵育30min，用1×PBS洗涤3次后，进行显色反应。
用CHO-K1-hCD39细胞(表达人源CD19蛋白的人源化CHO-K1中国仓鼠卵巢癌细胞系)进行抗人CD39抗体的结合活性筛选。使用2E5个细胞/孔，按100μL/孔加入96孔流式检测板中，按100μL/孔加入细胞上清，4℃孵育1h。用200μL/孔2％BSA洗涤3次后，按100μL/孔加入荧光标记的羊抗鼠IgG抗体，4℃静置孵育1h，用2％BSA洗涤3次后，用100μL/孔PBS重悬后上机检测。
5、亚克隆的筛选
挑选人CD39阳性结合的融合子，采用有限稀释法进行克隆化。采用以上步骤4的实验流程进行杂交瘤克隆检测。所得的阳性克隆经体外培养进行保种和表达。
6、产生单克隆抗体
以2×106个细胞/mL将杂交瘤细胞接种到基于透析的生物反应器中，每周收获一次含有抗体的上清液。借助FPLC使用Protein A(GE-MabSelectSuRe LX，Cat#17-5438-03)纯化小鼠单克隆抗体。通过BCA试剂盒或A280吸光值来确定抗体浓度，抗体纯度通过SEC(尺寸排阻色谱)确定，并通过SDS(十二烷基硫酸钠)凝胶电泳和考马斯亮蓝染色来检查纯度。
7、抗体亚型鉴定
进行同种型ELISA试验，以确定抗体的同种型。用ELISA试剂盒
(IgG1\IgG2a\IgG2b\IgG3\IgM\IgA)(博奥龙，Cat#BF06001)测定CD39单克隆抗体的Ig亚类。在整个免疫小鼠免疫中共产生131个抗CD39的单克隆抗体，经过多重筛选最终选择出了6个单克隆抗体，所产生抗体同种型结果如表5所示：
表5鼠单克隆抗体亚型

实施方式2.针对CD39的单克隆抗体的结合特征
1.转染细胞的质量控制
为了产生稳定表达猴或鼠CD39蛋白的细胞，将编码有食蟹猴CD39(食蟹猴(Cynomolgus)CD39蛋白的示例性序列公开于NCBI Ref SeqNo.XP_015311944.1中，其氨基酸序列如SEQ ID NO.127所示)或者小鼠CD39(小鼠(Mus musculus)CD39蛋白的示例性序列公开于NCBI Ref SeqNo.NP_033978.1中，其氨基酸序列如SEQ ID NO.128所示)的质粒转染CHO-K1细胞，然后通过靶向小鼠CD39蛋白的抗体(Biolegend，Cat#143804)以及靶向人CD39的抗体(R1048-mIgG1(D265A)，其重链可变区以及轻链可变区序列如本公开的序列表中SEQ ID NO.129、130所示)和PE goat anti-mouse Fc，用流式细胞术以CHO细胞作为阴性对照，筛选稳定细胞系。
分别收集CHO细胞和稳定表达小鼠CD39的CHO-K1-mCD39细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，加入PE anti-mCD39，4℃孵育30分钟。离心后用PBS洗一遍细胞，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估细胞系中CD39表达量的差异。由图1可以看到，转染并稳定表达mCD39的CHO-K1细胞在结合相应的抗体后，流式峰图呈现三种右移趋势，代表三种mCD39表达量的细胞株。
采用相同的步骤将CHO-K1-cynoCD39以及阴性对照CHO细胞分别与R1048-mIgG1(D265A)共孵育30分钟后，再与PE goat anti-mouse Fc共孵育30分钟，同样使用Cytoflex流式细胞仪评估结合。结果如图1所示，转染并稳定表达cynoCD39的CHO-K1细胞在结合相应的抗体后，流式峰图同样呈现三种右移趋势，代表三种cynoCD39表达量的细胞株。
根据流式结果挑选高表达小鼠CD39的CHO-K1-mCD39和高表达食蟹猴CD39的CHO-K1-cynoCD39细胞系用于后续的扩培。
2.CD39单克隆抗体与Mino细胞的结合活性
收集Mino细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，CD39单克隆抗体和阳性对照抗体R1048-mIgG1(D265A)分别用培养基配制为40μg/mL，然后分别进行3倍梯度稀释，共设置10个浓度点，iso组(mIgG1，Biolegend，Cat#401411)则只配置40μg/mL一个浓度，将抗体按每孔50μL加入到孔板中，抗体，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti mouse IgG Fc(1：500稀释)，重悬细 胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对Mino细胞的结合差异。
结合曲线如图2所示，其中，抗体#75、#2、#103、#110分别以0.2492nM、0.9503nM、0.6152nM、0.4378nM的EC50与Mino细胞结合，具有较优的亲和力。
3.CD39单克隆抗体与MOLP-8细胞的结合活性
收集MOLP-8细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，CD39单克隆抗体和阳性抗体R1048分别用培养基配制为40μ/mL，然后分别进行3倍梯度稀释，共设置10个浓度点，iso组只配置40μg/mL一个浓度，将抗体按每孔50μL加入到孔板中，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti mouse IgG Fc(1：500稀释)，重悬细胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对MOLP-8细胞的结合差异。
结合曲线如图3所示，其中，抗体#75、#95、#110、#103分别以0.4398nM、0.5699nM、1.067nM、0.891nM的EC50与MOLP-8细胞结合，具有较优的亲和力。
4.CD39单克隆抗体与CHO-K1-hCD39细胞的结合活性
收集CHO-K1-hCD39细胞，350g,离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，CD39单克隆抗体和阳性抗体R1048分别用培养基配制为40μg/mL，然后分别进行3倍梯度稀释，共设置10个浓度点，iso组只配置40μg/mL一个浓度，将抗体按每孔50μL加入到孔板中，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti mouse IgG Fc(1：500稀释)，重悬细胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对CHO-K1-hCD39细胞的结合差异。
结合活性如图4所示，其中，抗体#2、#66、#95分别以2.048nM、3.769nM、3.606nM的EC50与CHO-K1-hCD39细胞结合。
5.CD39单克隆抗体与CHO-K1-cynoCD39细胞的结合活性
收集CHO-K1-cynoCD39细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，CD39单克隆抗体和阳性抗体R1048分别用培养基配制为40μg/mL，然后分别进行3倍梯度稀释，共设置10个浓度点，iso组只配置40μg/mL一个浓度，将抗体按每孔50μL加入到孔板中，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti mouse IgG Fc(1：500稀释)，重悬细胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对CHO-K1-cynoCD39细胞的结合差异。
结合情况如图5所示，其中抗体#66与CHO-K1-cynoCD39细胞无结合活性；抗体#2，#75，#95，#103和#110均与CHO-K1-cynoCD39细胞有结合活性，表明这些CD39抗体均与猴的CD39蛋白有结合活性。
6.CD39单克隆抗体与CHO-K1-mCD39细胞的结合活性
收集CHO-K1-mCD39细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，CD39单克隆抗体和阳性抗体R1048分别用培养基配制为40μg/mL，然后分别进行3倍梯度稀释，共设置10个浓度点，iso组只配置40μg/mL一个浓度，将抗体按每孔50μL加入到孔板中，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti mouse IgG Fc(1：500稀释)，重悬细胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后，用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对CHO-K1-mCD39细胞的结合差异。
结合情况如图6所示，所有的CD39抗体均与CHO-K1-mCD39细胞无结合活性，表明这些CD39抗体与鼠的CD39蛋白无结合活性。
7.CD39单克隆抗体阻断Mino细胞上CD39的ATP酶水解活性检测
将Mino细胞调整密度为4E6cells/mL后，按每孔50μL转移至96孔U底细胞培养板中；抗体用培养基配置为20μg/mL起始。3倍稀释，共10个浓度，将抗体按每孔100μL添加到细胞当中，混匀后，37℃，孵育60min；将ATP用培养基配置成200μM，按50μL/孔加入细胞当中，混匀后，37℃，孵育45min；将细胞350G离心，5min，取上清80μL，转移到白板中，再加入等体积的Cell Titer-Glo，室温避光孵育10min，上机检测。
抗体的阻断活性曲线如图7所示，其中，抗体#75以0.2102nM的IC50阻断Mino细胞上CD39的ATP酶水解，优于阳性对照抗体R1048；抗体#95、#110、#103分别以0.3216nM、0.4047nM、0.3696nM的IC50阻断Mino细胞上CD39的ATP酶水解；#2、#75、#66的平台值高于阳性对照抗体R1048。
8.CD39单克隆抗体阻断MOLP-8细胞上CD39的ATP酶水解活性检测
将MOLP-8细胞调整密度为4E6cells/mL后按每孔50μL转移至96孔U底细胞培养板中；抗体用培养基配置为20μg/mL起始。3倍稀释，共10个浓度，将抗体按每孔100μL添加到细胞当中，混匀后，37℃，孵育60min；将ATP用培养基配置成200μM，按50μL/孔加入细胞当中，混匀后，37℃，孵育45min；将细胞350G离心，5min，取上清80μL，转移到白板中，再加入等体积的Cell Titer-Glo，室温避光孵育10min，上机检测。
抗体的阻断活性曲线如图8所示，抗体#75、#95、#103、#110分别以0.506nM、0.6498nM、0.2488nM、0.3138nM的IC50阻断MOLP-8细胞上CD39的ATP酶水解。
抗体#95的重链可变区的氨基酸序列如SEQ ID NO.83所示，轻链可变区的氨基酸序列如SEQ ID NO.89所示。
9.CD39单克隆抗体测序以及亚型替换
首先从6株单克隆抗体对应的杂交瘤细胞中分离RNA，通过商业化试剂盒进行反转录，得到各自的cDNA。然后以cDNA为模板，用Mouse Ig-Primer Set(Novagen)的引物分别扩增出重链可变区以及轻链可变区。对分子大小正确的PCR产物进行胶回收，然后连接到合适的载体上。连接产物转化至DH5α感受态细胞。经过筛选克隆、DNA测序分析得到这6株抗体的重链可变区序列以及轻链可变区序列；然后再将各自的可变区序列与鼠IgG1亚型的恒定区连接，产生亚型均为mIgG1的抗体，编号对应关系以及可变区序列如表6所示。
表6鼠单克隆抗体换mIgG1亚型后对应编号

表6中为替换mIgG1亚型后的鼠单克隆抗体，对照抗体R1048替换mIgG1亚型后的编号为R1444，后续在包括人的CD39结合活性、阻断实验以及体外免疫细胞活化实验在内的功能实验中对这些抗体进行进一步的评估。
10.通过Biacore测定法检测CD39抗体对人CD39的结合亲和力
使用Biacore表征CD39抗体以及参考抗体对人CD39的结合亲和力。简而言之，使用1列Octet HIS1K Biosensor(SARTORIUS)浸入至固化溶液中捕获适量带有His标签的人CD39ECD抗原(ACRO，Cat#CD9-H52H4)，将固化完抗原的探针浸入至缓冲液中做基线，再分别浸入至不同浓度的待测抗体溶液中结合180秒，然后浸入至缓冲液中解离240秒(动力学模式)。使用10mM甘氨酸溶液(pH＝1.7)进行探针再生。将结合和解离曲线与1：1结合模型拟合，并计算每种抗体的Kon/Kd/KD值。结果如表7所示。
表7鼠单克隆抗体亲和力
11.检测mIgG1亚型的CD39抗体与CD39蛋白的结合活性
将CD39蛋白按照1μg/mL的浓度加入到50mM的CB包被液中，按照每孔100μL的体积加入酶标板，4℃，过夜包被；PBST洗2次后，每孔加入200μL的1％BSA，37℃，60min，用1％的BSA稀释样品，将受试抗体初始浓度设为10μg/mL，每个受试抗体稀释十个点，将稀释好的样品按照每孔100μL加入到酶标板中，37℃，1h；加二抗，按照1：15K的稀释比例用1％BSA稀释二抗酶，酶标板PBST洗5次；将稀释好的二抗按照每孔100μL加入到酶标板中，37℃，0.5h，酶标板用PBST洗5次，加入显色液A,B液各50μL，37℃，10min，然后加入终止液50μL，酶标仪读数。
结合曲线如图9所示，R1448、R1476、R1478分别以0.4509nM、0.1654nM、0.07216nM的EC50与CD39蛋白结合，具有较优的亲和力。
12.检测mIgG1亚型的CD39抗体与Mino细胞的结合活性
收集Mino细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，分别加入CD39单克隆抗体和对照抗体，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti moμse IgG Fc(1：500稀释)，重悬细胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对Mino细胞的结合差异。
结合曲线如图10A所示，其中，R1476以0.3307nM的EC50与Mino细胞结合，具有较优的亲和力；如图10B所示，R1448的平台值高于阳性对照R1444。
13.检测mIgG1亚型的CD39抗体与MOLP-8细胞的结合活性
收集MOLP-8细胞，350g，离心5分钟，用PBS重悬，按照2E5/孔铺板到V型96孔板，分别加入CD39单克隆抗体和对照抗体，4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后按照100μL/孔加入PE Goat anti mouse IgG Fc(1：500稀释)，重悬细胞后4℃孵育30分钟。离心后每孔加入200μL的PBS洗一遍细胞，然后每孔加入100μL的PBS，将细胞重悬后用Cytoflex流式细胞仪(Beckman Countler)评估CD39抗体对Molp细胞的结合差异。
结合曲线如图11A所示，其中R1476以0.3064nM的EC50与MOLP-8细胞结合，具有较优的亲和力；如图11B所示，R1448的平台值高于阳性对照R1444。
14.mIgG1亚型的CD39抗体阻断Mino细胞上CD39的ATP酶水解活性检测
将Mino细胞调整密度为4E6cells/mL后按每孔50μL转移至96孔U底细胞培养板中(用新鲜的培养基重悬)；抗体用培养基配置为60μg/mL起始。3倍稀释，共10个浓度，将抗体以及杂交瘤上清按每孔添加100μL到细胞当中，混匀后，37℃，孵育60min；将ATP用培养基配置成200μM，按50μL/孔加入细胞当中，混匀后，37℃，孵育45min；将细胞350G离心，5min，取上清80μL，转移到白板中，再加入等体积的Cell Titer-Glo，室温避光孵育10min，上机检测。
阻断曲线如图12所示，其中，R1448、R1478、R1446、R1476分别以0.404Nm、0.187nM、0.8614nM、0.08273nM的IC50阻断Mino细胞上CD39的ATP酶水解，具有较优的阻断活性。
15.mIgG1亚型的CD39抗体阻断MOLP-8细胞上CD39的ATP酶水解活性检测
将MOLP-8细胞调整密度为4E6cells/mL后按每孔50μL转移至96孔Μ底细胞培养板中(务用新鲜的培养基重悬)；抗体用培养基配置为60μg/mL起始。3倍稀释，共10个浓度，将抗体以及杂交瘤上清按每孔添加100μL到细胞当中，混匀后，37℃，孵育60min；将ATP用培养基配置成200μM，按50μL/孔加入细胞当中，混匀后，37℃，孵育45min；将细胞350G离心，5min，取上清80μL，转移到白板中，再加入等体积的Cell Titer-Glo，室温避光孵育10min，上机检测。
抗体的阻断活性曲线如图13所示，其中R1476、R1446分别以0.1473nM、0.2404nM的IC50阻断MOLP-8细胞上CD39的ATP酶水解，优于阳性对照抗体R1444；R1448、R1445、R1446、R1476的平台值高于阳性对照抗体R1444。
16.CD39阻断ATP介导的T细胞增殖抑制分析
首先将T细胞密度调整为5E6/mL，用DPBS为Buffer进行CFSE(10mM母液)标记T细胞，终浓度为0.5μM，在37℃水浴锅中避光标记20min；加入12mL完全培养基(10％FBS，4℃预冷)，用于终止CFSE反应；用细胞培养基稀释抗体，配置浓度为400nM，10倍梯度稀释，设5个点；标记好的细胞300g、离心5min去上清，加完全培养基重悬并细胞计数，调整密度为2E6(此处计总细胞，不计活细胞)，50μL/well加入96孔U形板，即最终细胞1E5/孔；将配置好的抗体按照50μL/well加入到细胞中；根据细胞的数量稀释beads，保证最终体系中细胞：beads为20：1，50μL/well加入96孔U形板；三天(72h)后培养基稀释ATP为1200μM，加入至96孔板，50μL/孔，即最终体系为200μL。将细胞置于培养箱静置培养两天(48h)细胞板离心弃上清，BSA配置流式抗体(APC anti-humanCD8T，400X稀释)，4℃孵育30min，流式检测T细胞增殖情况(收10000细胞，保证峰形平滑)，按照《CytoFLEX流式细胞仪标准操作规程》上机检测。在Flowjo软件中进行数据处理，分别圈出CD3T和CD8T的细胞群，并分析各自的细胞增殖。
在GraphPad中拟合曲线如图14所示，可以看到每个抗体对CD3T细胞(图14A，14B)和CD8T细胞(图14C，14D)增殖的阻断效果是相似的。其中，R1446分别以1.202nM、1.112nM的IC50阻断CD3T细胞、CD8T细胞的增殖；R1476以0.08791nM、0.07378nM的IC50分别阻断CD3T细胞、CD8T细胞的增殖；R1448以0.2983nM、0.2448nM的IC50分别阻断CD3T细胞、CD8T细胞的增殖；R1478分别以0.3913nM、0.3801nM的IC50阻断CD3T细胞、CD8T细胞的增殖。
17.通过ELISA检测CD39抗体与人CD39蛋白以及其同家族蛋白的结合
将CD39以及同家族蛋白分别按照1μg/mL的浓度加入到50mM的CB包被液中，按照每孔100μL的体积加入酶标板，4℃，过夜包被；PBST洗2次后，每孔加入200μL的1％BSA，37℃，60min；用1％的BSA稀释样品，将受试抗体初始浓度设为10μg/mL，每个受试抗体稀释十个点；将稀释好的样品按照每孔100μL加入到酶标板中，37℃，1h；加二抗，按照1：15K 的稀释比例用1％BSA稀释二抗酶；酶标板PBST洗5次，将稀释好的二抗按照每孔100μL加入到酶标板中，37℃，0.5h；酶标板用PBST洗5次，加入显色液A,B液各50μL，37℃，10min；然后加入终止液50μL，酶标仪读数。
结合统计结果如图15所示，所有的CD39单克隆抗体均特异性的与人CD39(ENTPD1)有结合，而与人CD39的同家族蛋白(ENTPD2、ENTPD3、ENTPD5、ENTPD6)无结合。
18.在MOLP-8异种移植的小鼠上检测mIgG1亚型的CD39抗体的抑瘤作用
在第0天给6-8周的雌性NOD SCID小鼠皮下注射5x106个MOLP-8细胞，分别在第1天，第5天，第8天和第12天按照30mg/kg的剂量分别给与CD39抗体或者对照抗体R0859(mIgG1，其重链氨基酸序列如SEQ ID NO.131所示，其轻链氨基酸序列如SEQ ID NO.132所示)。
结果如图16A所示，曲线描绘了平均肿瘤体积，误差条表示SEM。X轴的三角形标注的是给药的时间点，X轴表示植入后的天数，Y轴表示肿瘤的体积，与对照组相比，所有的CD39抗体均有显著抑瘤作用，其中R1445，R1478以及R1448的抑瘤效果均优于阳性对照抗体R1444。图16B显示的是小鼠的体重，可以看到在给药的过程中小鼠的体重在组间没有显著差异。
以上所述仅为本公开的优选实施方式而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内
工业实用性
本公开提供了一种抗CD39抗体及其应用，该抗体能够特异性结合CD39蛋白，阻断CD39水解ATP，减少肿瘤微环境中的腺苷，发挥抗肿瘤作用，具备优异的工业实用性。

Claims (18)

  1. 一种抗CD39抗体或其抗原结合片段，其特征在于，所述抗体含有重链可变区和轻链可变区，其中，所述重链可变区的互补决定区包括HCDR1、HCDR2和HCDR3，所述轻链可变区的互补决定区包括LCDR1、LCDR2和LCDR3；
    其中，所述HCDR1包括如SEQ ID NO.2(GX2-2TFX2-5NX2-7G)或SEQ ID NO.1(X1-1X1-2SX1-4TX1-6YX1-8)所示的氨基酸序列，其中，X2-2是Y或F；X2-5是T或S；X2-7是Y或F；X1-1是G或D；X1-2是Y、F或L；X1-4是F或L；X1-6是D或N；X1-8是N或A；
    所述HCDR2包括如SEQ ID NO.6(INTNTGEP)、SEQ ID NO.3(IDPYNGX3-7T)、SEQ ID NO.4(IWRRGST)或SEQ ID NO.5(ISSGSNTI)所示的氨基酸序列，其中X3-7是A或V；
    所述HCDR3包括如SEQ ID NO.7(ARX7-3X7-4IYYDYVWFFDV)、SEQ ID NO.8(AX8-2YGYDX8-7X8-8X8-9X8-10YFDY)或SEQ ID NO.9(AKGGTATSWFAY)所示的氨基酸序列，其中X7-3是R或S；X7-4是A或G；X8-2是I或V；X8-7是G或E；X8-8是G或V；X8-9是A或P；X8-10是Y或N；
    所述LCDR1包括如SEQ ID NO.10(QDINX10-5Y)、SEQ ID NO.12(SSVNY)、SEQ ID NO.11(QNVDTX11-6)或SEQ ID NO.13(KSLLHSNGNTY)所示的氨基酸序列，其中X10-5是N或K；X11-6是N或T；
    所述LCDR2包括如氨基酸残基YT、GT、SA或RM所示的氨基酸序列；
    所述LCDR3包括如SEQ ID NO.16(LQYHDLIT)、SEQ ID NO.14(X14-1QX14-3X14-4X14-5X14-6X14-7X14-8T)或SEQ ID NO.15(MQHLEYPFT)所示的氨基酸序列，其中X14-1是Q或L；X14-3是R或Y；X14-4是S、D或N；X14-5是S或N；X14-6是Y或L；X14-7是P或缺失；X14-8是F或L。
  2. 根据权利要求1所述抗CD39抗体或其抗原结合片段，其特征在于，所述HCDR1包括如SEQ ID NO.22、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20或者SEQ ID NO.21所示的氨基酸序列；
    所述HCDR2包括如SEQ ID NO.6、SEQ ID NO.23、SEQ ID NO.4、SEQ ID NO.5或者SEQ ID NO.24所示的氨基酸序列；
    所述HCDR3包括如SEQ ID NO.28、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27或者SEQ ID NO.9所示的氨基酸序列；
    所述LCDR1包括如SEQ ID NO.32、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.29、SEQ ID NO.30或者SEQ ID NO.31所示的氨基酸序列；
    所述LCDR2包括如氨基酸残基YT、GT、SA或RM所示的氨基酸序列；
    所述LCDR3包括如SEQ ID NO.16、SEQ ID NO.33、SEQ ID NO.15、SEQ ID NO.34、SEQ ID NO.35或者SEQ ID NO.36所示氨基酸序列。
  3. 根据权利要求1或权利要求2所述的抗CD39抗体或其抗原结合片段，其特征在于，所述抗体的互补决定区选自如下(a)-(f)中的任意一种：
    (a)：所述HCDR1包括SEQ ID NO.22所示的氨基酸序列，所述HCDR2包括SEQ ID NO.6所示的氨基酸序列，所述HCDR3包括SEQ ID NO.28所示的氨基酸序列，所述LCDR1包括SEQ ID NO.32所示的氨基酸序列，所述LCDR2包括氨基酸残基YT所示的氨基酸序列，所述LCDR3包括SEQ ID NO.16所示的氨基酸序列；
    (b)：所述HCDR1包含如SEQ ID NO.17所示的氨基酸序列，所述HCDR2包含SEQ ID NO.23所示的氨基酸序列，所述HCDR3包含SEQ ID NO.25所示的氨基酸序列，所述LCDR1包括SEQ ID NO.12所示的氨基酸序列，所述LCDR2包括氨基酸残基GT所示的氨基酸序列，所述LCDR3包括SEQ ID NO.33所示的氨基酸序列；
    (c)：所述HCDR1包括SEQ ID NO.21所示的氨基酸序列，所述HCDR2包括SEQ ID NO.4所示的氨基酸序列，所述HCDR3包括SEQ ID NO.9所示的氨基酸序列，所述LCDR1包括SEQ ID NO.31所示 的氨基酸序列，所述LCDR2包括氨基酸残基SA所示的氨基酸序列，所述LCDR3包括SEQ ID NO.36所示的氨基酸序列；
    (d)：所述HCDR1包括SEQ ID NO.18所示的氨基酸序列，所述HCDR2包括SEQ ID NO.24所示的氨基酸序列，所述HCDR3包括SEQ ID NO.26所示的氨基酸序列，所述LCDR1包括SEQ ID NO.13所示的氨基酸序列，所述LCDR2包括氨基酸残基RM所示的氨基酸序列，所述LCDR3包括SEQ ID NO.15所示的氨基酸序列；
    (e)：所述HCDR1包括SEQ ID NO.19所示的氨基酸序列，所述HCDR2包括SEQ ID NO.5所示的氨基酸序列，所述HCDR3包括SEQ ID NO.27所示的氨基酸序列，所述LCDR1包括SEQ ID NO.29所示的氨基酸序列，所述LCDR2包括氨基酸残基YT所示的氨基酸序列，所述LCDR3包括SEQ ID NO.34所示的氨基酸序列；
    (f)：所述HCDR1包括SEQ ID NO.20所示的氨基酸序列，所述HCDR2包括SEQ ID NO.4所示的氨基酸序列，所述HCDR3包括SEQ ID NO.9所示的氨基酸序列，所述LCDR1包括SEQ ID NO.30所示的氨基酸序列，所述LCDR2包括氨基酸残基SA所示的氨基酸序列，所述LCDR3包括SEQ ID NO.35所示的氨基酸序列。
  4. 根据权利要求1-3任一项所述抗CD39抗体或其抗原结合片段，其特征在于，所述抗体含有重链框架区和/或轻链框架区，所述重链框架区和/或轻链框架区来自鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  5. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其特征在于，所述重链可变区的框架区包括HFR1，HFR2，HFR3和HFR4，所述轻链可变区的框架区包括LFR1，LFR2，LFR3和LFR4，其中
    所述HFR1包括如SEQ ID NO.41、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、和SEQ ID NO.40中任一项所示的氨基酸序列，或包括与SEQ ID NO.41、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、和SEQ ID NO.40中任一项所示的氨基酸序列具有至少75％序列同一性的序列；
    所述HFR2包括如SEQ ID NO.46、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、和SEQ ID NO.45中任一项所示的氨基酸序列，或包括与SEQ ID NO.46、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、和SEQ ID NO.45中任一项所示的氨基酸序列具有至少75％序列同一性的序列；
    所述HFR3包括如SEQ ID NO.52、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、和SEQ ID NO.51中任一项所示的氨基酸序列，或包括与SEQ ID NO.52、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、和SEQ ID NO.51中任一项所示的氨基酸序列具有至少65％序列同一性的序列；
    所述HFR4包括如SEQ ID NO.55、SEQ ID NO.53、SEQ ID NO.54、和SEQ ID NO.56中任一项所示的氨基酸序列，或包括与SEQ ID NO.55、SEQ ID NO.53、SEQ ID NO.54、和SEQ ID NO.56中任一项所示的氨基酸序列具有至少85％序列同一性的序列；
    所述LFR1包括如SEQ ID NO.62、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、和SEQ ID NO.61中任一项所示的氨基酸序列，或包括与SEQ ID NO.62、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、和SEQ ID NO.61中任一项所示的氨基酸序列具有至少75％序列同一性的序列；
    所述LFR2包括如SEQ ID NO.67、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、和SEQ ID NO.66中任一项所示的氨基酸序列，或包括与SEQ ID NO.67、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、和SEQ ID NO.66中任一项所示的氨基酸序列具有至少80％序列同一性的序列；
    所述LFR3包括如SEQ ID NO.73、SEQ ID NO.68、SEQ ID NO.69、SEQ ID NO.70、SEQ ID NO.71、和SEQ ID NO.72中任一项所示的氨基酸序列，或包括与SEQ ID NO.73、SEQ ID NO.68、SEQ ID NO.69、SEQ ID NO.70、SEQ ID NO.71、和SEQ ID NO.72中任一项所示的氨基酸序列具有至少75％序列同一性的序列；
    所述LFR4包括如SEQ ID NO.79、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、和SEQ ID NO.78中任一项所示的氨基酸序列，或包括与SEQ ID NO.79、SEQ ID NO.74、SEQ ID NO.75、 SEQ ID NO.76、SEQ ID NO.77、和SEQ ID NO.78中任一项所示的氨基酸序列具有至少92％序列同一性的序列。
  6. 一种抗CD39抗体或其抗原结合片段，其特征在于，所述抗体包含：
    (a)重链可变区，其包含SEQ ID NO.85、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、和SEQ ID NO.84中任一项所示重链可变区的HCDR1,HCDR2和HCDR3；以及，
    (b)轻链可变区，其包含SEQ ID NO.91、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、和SEQ ID NO.90中任一项所示轻链可变区的LCDR1,LCDR2和LCDR3；
    优选地，所述HCDR1，所述HCDR2和所述HCDR3，及所述LCDR1,所述LCDR2和所述LCDR3按照IMGT定义方式、Kabat定义方式、Chothia定义方式、AbM定义方式或Contact定义方式确定。
  7. 根据权利要求6所述的抗CD39抗体或其抗原结合片段，其特征在于，所述抗体含有重链框架区和/或轻链框架区，所述重链框架区和/或轻链框架区来自鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  8. 根据权利要求1-7任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体的重链可变区具有如SEQ ID NO.85、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、和SEQ ID NO.84中任一项所示的氨基酸序列；并且，所述抗体的轻链可变区具有如SEQ ID NO.91、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.88、SEQ ID NO.89、和SEQ ID NO.90中任一项所示的氨基酸序列；
    优选地，所述抗体包括如下(1)-(6)中任一项所示的重链可变区和轻链可变区：
    (1)：如SEQ ID NO.85所示的重链可变区序列和如SEQ ID NO.91所示的轻链可变区序列；
    (2)：如SEQ ID NO.80所示的重链可变区序列和如SEQ ID NO.86所示的轻链可变区序列；
    (3)：如SEQ ID NO.81所示的重链可变区序列和如SEQ ID NO.87所示的轻链可变区序列；
    (4)：如SEQ ID NO.82所示的重链可变区序列和如SEQ ID NO.88所示的轻链可变区序列；
    (5)：如SEQ ID NO.83所示的重链可变区序列和如SEQ ID NO.89所示的轻链可变区序列；
    (6)：如SEQ ID NO.84所示的重链可变区序列和如SEQ ID NO.90所示的轻链可变区序列。
  9. 根据权利要求1-8任一项所述的抗CD39抗体或其抗原结合片段，其特征在于，所述抗体包含恒定区，所述恒定区来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一；
    优选地，所述抗体的重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD中的任一种；所述抗体的轻链恒定区为κ或λ链。
  10. 根据权利要求1-9任一项所述抗CD39抗体，其特征在于，所述抗体为全长抗体或其抗原结合片段，所述抗原结合片段为F(ab’)2、Fab’、Fab、scFv、Fv、dsFV中的任一种或几种。
  11. 根据权利要求1-10任一项所述抗CD39抗体或其抗原结合片段，其特征在于，所述抗体具有如下(a)-(l)中的一种或多种性质：
    (a)特异性地与人类和/或猴CD39结合，但不与小鼠CD39特异性地结合，所述结合通过流式细胞荧光分选技术测定；
    (b)以不超过7nM的EC50与人CD39结合，所述EC50通过流式细胞荧光分选技术测定；
    (c)以不超过11nM的EC50与表达人CD39蛋白的Mino细胞结合，所述EC50通过流式细胞荧光分选技术测定；
    (d)以不超过8nM的EC50与表达人CD39蛋白的MOLP-8细胞结合，所述EC50通过流式细胞荧光分选技术测定；
    (e)以不超过10‐9M的KD值与人类CD39结合，所述KD值通过Biacore检测法测定；
    (f)以不超过7nM的IC50抑制在表达CD39的Mino细胞中的ATP酶活性，所述IC50通过ATP酶活性分析测定；
    (g)以不超过5nM的IC50抑制在表达CD39的MOLP-8细胞中的ATP酶活性，所述IC50通过ATP酶活性分析测定；
    (h)以不超过10nM的IC50阻断ATP，抑制CD8T细胞增殖，所述IC50通过流式细胞荧光分选技术分析测定；
    (i)以不超过10nM的IC50阻断ATP，抑制CD3T细胞增殖，所述IC50通过流式细胞荧光分选技 术分析测定；
    (j)特异性的与人CD39结合，而与人CD39的同家族蛋白无交叉结合；
    (k)能够抑制哺乳动物中的肿瘤生长；
    (l)所述抗CD39抗体或其抗原结合片段中的CD39为人CD39或猴CD39。
  12. 一种抗CD39抗体或其抗原结合片段，其特征在于，其与权利要求1-11任一项所述的抗CD39抗体竞争性结合CD39，或者其结合CD39抗原的表位与权利要求1-11任一项所述的抗CD39抗体结合CD39抗原的表位相同。
  13. 一种多特异性抗体，其特征在于，所述多特异性抗体含有权利要求1-12任一项所示的抗CD39抗体或其抗原结合片段。
  14. 生物材料，其特征在于，所述生物材料选自(ⅰ)～(v)中任一项：
    (i)嵌合抗原受体(CAR)，所述嵌合抗原受体包括胞外区，所述胞外区包括抗原结合结构域；并且，所述抗原结合结构域含有权利要求1-12任一项所述的抗CD39抗体或其抗原结合片段；
    (ii)工程化的免疫细胞，所述免疫细胞表达(i)所述的嵌合抗原受体或含有编码(i)所述的嵌合抗原受体的核酸；
    (iii)分离的核酸分子，包括编码权利要求1～12任一项所述的抗体或其抗原结合片段的核苷酸序列；
    (iv)载体，所述载体携带前述(iii)核酸分子；
    (v)重组细胞，所述重组细胞携带(iii)中核酸分子，或含有(iv)中载体，或表达权利要求1～12任一项所述的抗体或其抗原结合片段。
  15. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-12任一项所述抗CD39抗体或其抗原结合片段、权利要求13所述的多特异性抗体、权利要求14所述的生物载体；
    优选地，所述药物组合物还包括药学上可接受的载体。
  16. 权利要求1-12任一项所述抗CD39抗体或其抗原结合片段、权利要求13所述的多特异抗体、权利要求14所述的生物载体或权利要求15所述药物组合物在制备用于预防、治疗、或诊断与CD39相关的肿瘤疾病的药物中的应用。
  17. 一种用于产生如权利要求1-13任一项所述抗CD39抗体或其抗原结合片段的方法，其特征在于，所述方法包括以下步骤：
    (a)在表达抗CD39抗体或其抗原结合片段的条件下培养如权利要求14的重组细胞；
    (b)分离和纯化步骤(a)中获得的抗CD39抗体或其抗原结合片段。
  18. 一种治疗、预防或减轻与CD39相关的疾病、病症或状况的方法，其特征在于，其包括向受试者施用治疗有效量的如权利要求15所述的药物组合物。
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