CN119930816A - Anti-CD39 humanized antibody or antigen-binding fragment thereof and application thereof - Google Patents

Abstract

The present invention provides an anti-CD39 humanized antibody or antigen-binding fragment thereof and application thereof, and relates to the field of biomedical technology. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the CDR region of the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; and the CDR region of the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The humanized antibody or antigen-binding fragment thereof reduces the risk of antibody immunogenicity and maintains the functional activity of the antibody, and can be widely used in the preparation of drugs for diseases, disorders or conditions related to CD39.

Description

Anti-CD 39 humanized antibodies or antigen binding fragments thereof and uses thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to an anti-CD 39 humanized antibody or an antigen binding fragment thereof and application thereof.

Background

CD39, the ectonucleotide triphosphatehydrolase 1 (ENTPDase 1), is a single channel type II transmembrane protein that converts extracellular eATP or ADP to AMP, and then CD73 converts AMP to adenosine, a metabolite that exerts immunosuppressive effects in the tumor microenvironment. When the produced adenosine binds to adenosine receptors on the surfaces of CD 4T, CD T cells and Natural Killer (NK) cells, T cell and NK cell responses can be inhibited, thereby inhibiting the immune system and promoting tumor growth. In addition, adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, thereby inhibiting phagocytosis and antigen presentation, and increasing secretion of pro-cancer factors (e.g., VEGF, TGF, and IL-6). Whereas adenosine promotes immunosuppressive activity of tregs for tregs. Within TME, the adenosine pathway refers to the conversion of atp to adenosine by the action of an adenylate hydrolase, and the signaling of adenosine through A2A/A2B adenosine receptors on immune cells. Under normal conditions, CD39 maintains a balance of immunosuppressive adenosine and extracellular levels of immunostimulatory ATP. In healthy tissues, ATP is barely detectable in the extracellular environment because ATP is rapidly decomposed by CD39 to produce ADP or AMP, which is then converted to adenosine by CD 73. Under cellular stress conditions, including cancer, eATP levels rise significantly, but are converted to adenosine by successive hydrolysis by CD39 and CD73, resulting in the immune system's recognition of the tumor and its killing.

Therefore, the development and optimization of anti-CD 39 antibodies is of great importance for the prevention or treatment of tumor-related diseases.

Disclosure of Invention

The present invention aims at humanizing antibodies that specifically bind CD39 to reduce the risk of antibody immunogenicity while maintaining the functional activity of the antibodies.

In order to solve the technical problems, the invention adopts the following technical scheme:

in a first aspect, the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region set forth in any one of SEQ ID NOS.1-8, and

(B) A light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region of any one of SEQ ID nos. 9 to 16;

In alternative embodiments, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are defined according to IMGT definition, kabat definition, chothia definition, abM definition, or Contact definition.

In a second aspect, there is also provided a biomaterial selected from any one of (i) to (iii):

(i) A polynucleotide comprising a nucleotide sequence encoding an anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect.

(Ii) A vector carrying the polynucleotide of (i) above.

(Iii) A cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect.

In a third aspect, there is also provided an immunoconjugate comprising the anti-CD 39 humanized antibody or antigen binding fragment thereof of the first aspect conjugated to at least one diagnostic and/or therapeutic agent to form the immunoconjugate.

The diagnostic agent is selected from one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasonic contrast agent and a photosensitizer;

The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photoactive therapeutic agent, an immunoconjugate and an oligonucleotide.

In a fourth aspect, there is also provided the use of an anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect, or the biological material of the second aspect, or the immunoconjugate of the third aspect, in any one of the following:

(i) Detection of CD39 or cells expressing CD39 for non-diagnostic and therapeutic purposes;

(ii) Preparing a product for detecting CD39 or cells expressing CD 39;

(iii) Blocking the atpase hydrolytic activity of CD39 on cells for non-diagnostic and therapeutic purposes;

(iv) Preparing a blocking agent for blocking atpase hydrolytic activity of CD39 on a cell;

(v) Preparing a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD 39.

In a fifth aspect, there is also provided a pharmaceutical composition comprising an anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect, or a biological material of the second aspect, or an immunoconjugate of the third aspect, and optionally a pharmaceutically acceptable carrier and/or excipient.

Compared with the prior art, the invention has the following beneficial effects:

The invention reduces the risk of antibody immunogenicity and maintains the functional activity of the antibody by carrying out humanized transformation on the murine monoclonal antibody. Experiments prove that the anti-CD 39 humanized antibody or antigen binding fragment thereof provided by the invention has binding activity with CD39 on cells, can block the ATPase hydrolysis activity of the CD39 on the cell surface, can improve the tumor condition of mice when being applied to the mice, and shows that the anti-CD 39 humanized antibody or antigen binding fragment thereof provided by the invention can reduce adenosine in tumor microenvironment, play an anti-tumor role and provide new possibility for the treatment and/or prevention of cancers.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1a is the binding activity of humanized antibodies (R1801, R1802, R1803, R1804, R1805, R1806 and R1807) to Mino cells;

FIG. 1b is the binding activity of humanized antibodies (R1808, R1809, R1810, R1839, R1840, R1841 and R1842) to Mino cells;

FIG. 1c is the binding activity of humanized antibodies (R1843, R1844, R1845, R1846, R1847, R1848 and R1849) to Mino cells;

FIG. 1d shows the binding activity of humanized antibodies (R1850, R1851, R1852 and R1853) to Mino cells;

FIG. 2a is a graph showing the blocking of ATPase hydrolysis activity of CD39 on Mino cells by humanized antibodies (R1801, R1802, R1803, R1804, R1805, R1806 and R1807);

FIG. 2b is a graph showing the blocking of ATPase hydrolysis activity of CD39 on Mino cells by humanized antibodies (R1808, R1809, R1810, R1839, R1840, R1841 and R1842);

FIG. 2c is a graph showing the blocking of ATPase hydrolysis activity of CD39 on Mino cells by humanized antibodies (R1843, R1844, R1845, R1846, R1847, R1848 and R1849);

FIG. 2d is a graph showing the blocking of ATPase hydrolysis activity of CD39 on Mino cells by humanized antibodies (R1850, R1851, R1852 and R1853);

FIG. 3a is a graph showing the blocking of ATPase hydrolysis activity of humanized antibodies (R1801, R1802, R1803, R1804, R1805, R1806 and R1807) against CD39 on MOLP-8 cells;

FIG. 3b is a graph showing the blocking of ATPase hydrolysis activity of CD39 on MOLP-8 cells by humanized antibodies (R1808, R1809, R1810, R1841, R1847, R1850 and R1853);

FIG. 4a is a graph of inhibition of CD39 hydrolysis ATP mediated T cell proliferation by humanized antibodies (R1801, R1802, R1803, and R1804);

FIG. 4b is a graph of inhibition of CD39 hydrolysis ATP mediated T cell proliferation by humanized antibodies (R1805, R1806, R1807, and R1808);

FIG. 4c is a graph of inhibition of CD39 hydrolysis ATP mediated T cell proliferation by humanized antibodies (R1809, R1810, R1841 and R1847);

FIG. 4d is a plot of inhibition of T cell proliferation mediated by humanized antibodies (R1850 and R1853) against CD39 hydrolyzed ATP;

FIG. 5a is the binding activity of humanized antibodies (R1803, R1804, R1806, R1807, R1841 and R1847) to CHO-K1-cynoCD39 cells;

FIG. 5b is the binding activity of humanized antibody (R1853) to CHO-K1-cynoCD39 cells;

FIG. 6 is the binding activity of humanized antibodies (R2122 and R2123) to Mino cells;

FIG. 7 is the binding activity of humanized antibodies (R2122 and R2123) to MOLP-8 cells;

FIG. 8 is a graph showing the blocking of ATPase hydrolysis activity of CD39 on Mino cells by humanized antibodies (R2122 and R2123);

FIG. 9 is a graph showing the blocking of ATPase hydrolysis activity of CD39 on MOLP-8 cells by humanized antibodies (R2122 and R2123);

FIG. 10 is a graph of inhibition of CD39 hydrolysis ATP mediated T cell proliferation by humanized antibodies (R2122 and R2123);

FIG. 11 is a graph showing tumor volume change after mice were injected with MOLP-8 cells and humanized antibodies (R2122 and R2123) were administered to the mice;

FIG. 12 is a graph showing the change in body weight of mice after administration of humanized antibodies (R2122 and R2123) after injection of MOLP-8 cells into the mice.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Definition:

throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising" or the like will be understood to include the stated element or component without excluding other elements or components.

The term "CD39", also referred to herein as ENTPD a or ENTPD a, is a membrane protein that converts ATP to AMP. CD39 contains two transmembrane domains, a smaller cytoplasmic domain and a larger extracellular hydrophobic domain.

An "antibody or antigen-binding fragment thereof" as used herein refers to a protein that binds a particular antigen, which broadly refers to all proteins and protein fragments that comprise complementarity determining regions (CDR regions). In addition, "antibody or antigen-binding fragment thereof" also includes naturally occurring antibodies as well as non-naturally occurring antibodies.

An "antigen binding fragment" in this context is a substance comprising the CDRs of an antibody which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab ') ₂, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) ₂, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies). The antigen binding fragments described above are capable of binding the same antigen as the parent antibody.

As used herein, the term "Fab" of an antibody refers to the portion of the antibody that is made up of a single light chain (including both variable and constant regions) and the variable and first constant regions of a single heavy chain joined by disulfide bonds. "Fab' fragment" refers to a Fab fragment which contains a portion of the hinge region. "F (ab ') ₂" refers to the dimer of Fab'. An "Fv fragment" consists of a combination of variable regions of a single light chain and/or variable regions of a single heavy chain. "Single chain Fv antibody" or "scFv" refers to an antibody fragment in which the light chain variable region and the heavy chain variable region are directly linked to each other or are linked by a peptide linker sequence. By "antibody minimal recognition unit" is meant a structure comprising only a single CDR in the variable region, which has the ability to bind to an antigen, although the minimal recognition unit has a low molecular weight and low affinity.

Herein, the "variable region" or "variable domain" of an antibody or antigen binding fragment thereof refers to the domain of an antibody that recognizes and binds to an antigen at the amino terminus of the heavy or light chain, the composition and arrangement of the amino acids of the segment determining the specificity of the antibody for recognizing the antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". The variable domain contains an antigen binding site. The variable regions of the heavy and light chains each consist of 3 complementarity-DETERMINING REGION (CDRs) (also known as hypervariable regions) connected by 4 Frameworks (FR). Typically, the variable regions VL/VH of the heavy and light chains are obtained by connecting the CDRs numbered from FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a combined arrangement with the FRs.

The CDR boundaries of an antibody or antigen binding fragment thereof herein may be defined or identified in accordance with IMGT, kabat, chothia, abM, contact definitions and CDRs defined in other ways acceptable in the art are also within the scope of the invention (Kaas,Q etal.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 variabledomains(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).

Herein, a "humanized antibody" is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, it may be constructed by retaining the CDR regions of a non-human antibody and replacing the remainder of the antibody with the human antibody counterparts (i.e., the framework portions of the constant and variable regions).

The terms "specifically recognizes," "selectively binds," and "specifically binds" or the like refer to the binding of a binding protein to an epitope on a predetermined antigen. Typically, the binding protein binds at a K _D value of about less than 10 ^-5 M, e.g., about less than 10 ^-5M、10^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less. The K _D value of an antibody can be determined using methods well established in the art. Other standard assays for evaluating the binding capacity of a ligand, such as an antibody, to a target are known in the art and include, for example, ELISA, western blot, RIA, and flow cytometry analysis.

The term "polynucleotide" herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and/or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double-or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The polynucleotide comprises a portion encoding the antibody or antigen binding fragment thereof described above, optionally encoding the sense or antisense strand. The polynucleotide may be naturally occurring, synthetic, recombinant, or any combination thereof.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell.

Such vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, and papilloma virus. In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include progeny. The offspring may not necessarily be identical to the primary cells due to natural, accidental or deliberate mutation, e.g. are morphologically and/or differ from the primary cells in genomic DNA.

As used herein, the term "Mino cell" is a human non-Hodgkin's lymphoma cell line whose cell surface naturally expresses human CD39 protein.

As used herein, the term "MOLP-8 cell" is a human multiple myeloma cell line that naturally expresses human CD39 protein on its cell surface, and is used to test the in vivo anti-tumor effects of anti-CD 39 antibodies by inoculating the cell into a multiple myeloma model established in NOD-SCID mice.

In this context, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not comprise additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered. In some embodiments, the antibody or expressed antibody contained in the above pharmaceutical composition is capable of specifically targeting CD39, blocking the hydrolysis of ATP by CD 39.

In a first aspect, there is provided an anti-CD 39 humanized antibody or antigen-binding fragment thereof, comprising (a) and (b):

(a) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region set forth in any one of SEQ ID NOS.1-8, and

(B) A light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region of any one of SEQ ID nos. 9 to 16.

In alternative embodiments, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are defined according to IMGT definition, kabat definition, chothia definition, abM definition, or Contact definition.

Exemplary antibodies 1847 (the amino acid sequence of heavy chain variable region H6 is shown in SEQ ID NO.1, the amino acid sequence of light chain variable region L8 is shown in SEQ ID NO. 9) and 1853 (the amino acid sequence of heavy chain variable region H8 is shown in SEQ ID NO.2, the amino acid sequence of light chain variable region L8 is shown in SEQ ID NO. 9) are shown in Table 1 according to the CDR amino acid sequences defined in different definitions.

TABLE 1 CDR amino acid sequences of exemplary monoclonal antibodies

In alternative embodiments, the HCDR1, HCDR2 and HCDR3 of the anti-CD 39 humanized antibody or antigen binding fragment thereof, and the LCDR1, LCDR2 and LCDR3 are determined according to IMGT definition:

the HCDR1 comprises an amino acid sequence shown as SEQ ID NO.17, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO.22, the HCDR3 comprises an amino acid sequence shown as SEQ ID NO.27, the LCDR1 comprises an amino acid sequence shown as SEQ ID NO.30, the LCDR2 comprises an amino acid sequence shown as YT, and the LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 36.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD 39 humanized antibody or antigen binding fragment thereof is shown in any one of SEQ ID NO. 1-8.

In an alternative embodiment, the antibody or antigen binding fragment thereof having a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NO. 1-8, and the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO. 9-16.

In an alternative embodiment, the amino acid sequence of the light chain variable region of the anti-CD 39 humanized antibody or antigen binding fragment thereof is shown in any one of SEQ ID NOS.9-16.

In an alternative embodiment, the antibody or antigen binding fragment thereof has a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NO. 9-16, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO. 1-18.

In alternative embodiments, the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment thereof are selected from any one of the following combinations, see table 2:

TABLE 2

In alternative embodiments, the antigen binding fragment comprises one or more of F (ab ') ₂, fab', fab, fv, scFv, dsFv, bispecific antibodies, and antibody minimal recognition units.

In alternative embodiments, the antibody or antigen binding fragment thereof further comprises a heavy chain constant region and/or a light chain constant region;

The heavy chain constant region comprises the sequence of a portion or all of any one of IgG1, igG1.8, igG2, igG3, igG4, igA, igM, igE, or IgD;

the light chain constant region is a kappa or lambda chain;

In alternative embodiments, the constant region source species comprises one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

In an alternative embodiment, the heavy chain constant region is selected from IgG1, and the amino acid sequence of the heavy chain constant region is preferably as set forth in SEQ ID NO. 39.

In an alternative embodiment, the heavy chain constant region is selected from IgG1.8, and the amino acid sequence of the heavy chain constant region is preferably as shown in SEQ ID NO. 40;

In an alternative embodiment, the light chain constant region is a kappa chain, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 41.

In a second aspect, there is also provided a biomaterial selected from any one of (i) to (iii):

(i) A polynucleotide comprising a nucleotide sequence encoding an anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect.

(Ii) A vector carrying the polynucleotide of (i) above.

(Iii) A cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect.

In a third aspect, there is also provided an immunoconjugate comprising the anti-CD 39 humanized antibody or antigen binding fragment thereof of the first aspect conjugated to at least one diagnostic and/or therapeutic agent to form the immunoconjugate.

The diagnostic agent is selected from one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasonic contrast agent and a photosensitizer;

The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photoactive therapeutic agent, an immunoconjugate and an oligonucleotide.

Radionuclides include, but are not limited to, one or more of ¹¹⁰In、¹¹¹In、¹⁷⁷Lu、¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁹⁰Y、⁸⁹Zr、⁹⁴mTc、⁹⁴Tc、⁹⁹mTc、¹²⁰I、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、^154-158Gd、³²P、¹¹C、¹³N、¹⁵O、¹⁸⁶Re、¹⁸⁸Re、⁵¹Mn、⁵²mMn、⁵⁵Co、⁷²As、⁷⁵Br、⁷⁶Br、⁸²mRb and ⁸³ Sr.

Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

Fluorescent labels include, but are not limited to, alexa 350, alexa 405, alexa 430, alexa 488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethyl rhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oregon Green488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol purple, light cresol Blue, para-aminobenzoic acid, erythrosine one or more of phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptates, tripyridyl diamine europium, europium cryptates or chelates, diamines, bis anthocyanin, la Jolla Blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine and texas red.

Oligonucleotides include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

Drugs include, but are not limited to, methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecins, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxine, and L-asparaginase.

Immunomodulators include, but are not limited to, one or more of cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony Stimulating Factors (CSF), interferons, erythropoietin, thrombopoietin, tumor Necrosis Factor (TNF), interleukins (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and stem cell growth factors.

Radionuclides include, but are not limited to, one or more of ¹¹¹In、¹¹¹At、¹⁷⁷Lu、²¹¹Bi、²¹²Bi、²¹³Bi、²¹¹At、⁶²Cu、⁶⁷Cu、⁹⁰Y、¹²⁵I、¹³¹I、¹³³I、³²P、³³P、⁴⁷Sc、¹¹¹Ag、⁶⁷Ga、¹⁵³Sm、¹⁶¹Tb、¹⁵²Dy、¹⁶⁶Dy、¹⁶¹Ho、¹⁶⁶Ho、¹⁸⁶Re、¹⁸⁸Re、¹⁸⁹Re、²¹¹Pb、²¹²Pb、²²³Ra、²²⁵Ac、⁷⁷As、⁸⁹Sr、⁹⁹Mo、¹⁰⁵Rh、¹⁴⁹Pm、¹⁶⁹Er、¹⁹⁴Ir、⁵⁸Co、⁸⁰mBr、⁹⁹mTc、¹⁰³mRh、¹⁰⁹Pt、¹¹⁹Sb、¹⁸⁹mOs、¹⁹²Ir、²¹⁹Rn、²¹⁵Po、²²¹Fr、²⁵⁵Fm、¹¹C、¹³N、¹⁵O、⁷⁵Br、¹⁹⁸Au、¹⁹⁹Au、²²⁴Ac、⁷⁷Br、¹¹³mIn、⁹⁵Ru、⁹⁷Ru、¹⁰³Ru、¹⁰⁵Ru、¹⁰⁷Hg、²⁰³Hg、¹²¹mTe、¹²²mTe、¹²⁵mTe、¹⁶⁵Tm、¹⁶⁷Tm、¹⁶⁸Tm、¹⁹⁷Pt、¹⁰⁹Pd、¹⁴²Pr、¹⁴³Pr、¹⁶¹Tb、⁵⁷Co、⁵⁸Co、⁵¹Cr、⁵⁹Fe、⁷⁵Se、²⁰¹Tl、⁷⁶Br and ¹⁶⁹ Yb.

In a fourth aspect, there is also provided the use of an anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect, or the biological material of the second aspect, or the immunoconjugate of the third aspect, in any one of the following:

(i) Detection of CD39 or cells expressing CD39 for non-diagnostic and therapeutic purposes;

(ii) Preparing a product for detecting CD39 or cells expressing CD 39;

(iii) Blocking the atpase hydrolytic activity of CD39 on cells for non-diagnostic and therapeutic purposes;

(iv) Preparing a blocking agent for blocking atpase hydrolytic activity of CD39 on a cell;

(v) Preparing a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD 39.

In alternative embodiments, the use of aspect (i) above may utilize an anti-CD 39 antibody or antigen binding fragment thereof capable of specifically targeting CD39, detection of CD39 or cells expressing CD39 may be achieved based on immunodetection techniques, e.g. where the antibody or antigen binding fragment chain is an immunoconjugate, e.g. attached to a fluorescent moiety, the localization or real-time detection of CD39 may be achieved using a fluorescent detection device. For example, it can be used in immunoblotting, immunoprecipitation, or flow cytometry, etc., involving the use of the specific binding properties of CD39 antigen and antibody to detect CD39 or CD39 expressing cells. Accordingly, in the above aspect (ii), the reagent composition in the kit may be selected by those skilled in the art according to the actual detection means, including but not limited to antagonists, anti-CD 39 antibodies or drug reference materials, protein purification columns, immunoglobulin affinity purification buffers, assay diluents for cells, instructions or literature, etc.

In an alternative embodiment, the use of (iii) above may allow sufficient contact between the cells to be blocked for ATPase hydrolysis activity and an anti-CD 39 antibody or antigen binding fragment thereof, such as, but not limited to, incubating the cells to be blocked for ATPase hydrolysis activity in a medium containing an anti-CD 39 antibody or antigen binding fragment thereof.

In alternative embodiments, the blocking agent used to block the ATPase hydrolysis activity of CD39 on the cells also contains adjuvants acceptable in the art, including, but not limited to, culture media, preservatives, buffer components, and the like.

In a fifth aspect, there is also provided a pharmaceutical composition comprising an anti-CD 39 humanized antibody or antigen-binding fragment thereof of the first aspect, or a biological material of the second aspect, or an immunoconjugate of the third aspect, and optionally a pharmaceutically acceptable carrier and/or excipient.

Wherein "optionally" means that the pharmaceutical composition may or may not contain pharmaceutically acceptable carriers and/or excipients. In alternative embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or excipient. The acceptable carrier and pharmaceutically acceptable excipient may be any carrier and/or excipient known in the art and conventional. Examples of carriers include, but are not limited to, any physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, for extending the shelf life or efficacy of the antibodies, and examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, co-solvents, emulsifiers, and the like.

In alternative embodiments, the pharmaceutical composition further comprises one or more pharmaceutically active ingredients having other therapeutic effects, including, but not limited to, one or more combinations of chemotherapeutic agents, anticancer agents, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cytotherapeutic agents, gene therapeutic agents, hormonal therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

In alternative embodiments, the pharmaceutical composition is for use in a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD 39.

The CD 39-associated disease, disorder or condition of the fourth and fifth aspects described above refers to any disease or condition caused, exacerbated or associated with an increase or decrease in expression or activity of CD 39. In some embodiments, the CD 39-associated disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as cancer. In some embodiments, the CD 39-associated disease or condition is characterized by expression or overexpression of CD39 and/or a CD 39-associated gene, such as ENTPD, 2,3, 4, 5, 6, 7, or 8 genes.

In alternative embodiments, a "CD 39-related" disease, disorder or condition includes, but is not limited to, cancer, autoimmune diseases, and infections.

In alternative embodiments, a "CD 39-related" cancer includes a cancer in which CD39 is expressed in cancer cells or tumor-infiltrating immune cells or immunosuppressive cells, and in which CD39 is expressed at a significantly higher level than would be expected in normal cells.

In alternative embodiments, examples of "CD 39-related" cancers include, but are not limited to, anal, appendicular, astrocytoma, basal cell carcinoma, gall bladder, gastric, lung, bronchus, bone, hepatobiliary, pancreatic, breast, liver, ovarian, testicular, renal pelvis and ureter, salivary gland, small intestine, urinary tract, bladder, head and neck, spine, brain, cervical, uterine, endometrial, colon, colorectal, rectal, esophageal, gastrointestinal, skin, prostate, pituitary, vaginal, thyroid, laryngeal, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), acute Myelogenous Leukemia (AML), hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, T or B cell lymphoma, gastrointestinal stromal, soft tissue, and liver cancer. In some embodiments, the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not specified in the embodiments and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are fully explained in the literature, e.g.molecular cloning, laboratory Manual (Molecular Cloning: ALaboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (OligonucleotideSynthesis) (M.J.Gait, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I.Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), mammalian cell gene transfer vectors (GENE TRANSFER Vectors forMammalian Cells) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (CurrentProtocols in Molecular Biology) (F.M.Ausubel, et al, 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis, et al, 1994), contemporary immunological methods (Current Protocols in Immunology) (J.E.Coligan, et al, 1991), each of which is further understood by the detailed description of the invention by the present document, but not specifically incorporated herein by reference.

EXAMPLE 1 humanization of monoclonal antibodies directed against CD39

The recombinant human CD39 fusion protein and the constructed CHOK1-hCD39 are adopted as immunogens to generate an antibody against human CD39, and the #110 murine monoclonal antibody obtained after further screening has better binding affinity to CHO-hCD39, mino, molp-8 and cynoCD, can be detected on Mino and Molp-8 to well block the enzyme activity of CD39, can be seen in a functional experiment part to reduce the generation of ADO by blocking the enzyme activity of CD39 on T cells, so as to promote the proliferation of T cells (the weight variable region and the light chain variable region of the #110 murine monoclonal antibody are shown in Table 3), and therefore, the embodiment performs humanization on the #110 murine monoclonal antibody, which is specifically prepared by the following steps:

The humanization was performed by the complementarity determining region grafting (CDR-grafting) method. First, antibody homology modeling was performed on Fv regions of #110 murine mab using MOE (Molecular Operating Environment) software. Based on the model structure, key constituent amino acid residues that can affect conformational stability of the antigen binding region are analyzed. Subsequently, the human immunoglobulin database was searched for human IGVH and IGVK sequences with high homology to #110 in the human germline antibody library as humanized altered templates. And (3) respectively comparing the #110 sequence with the matched human IGVH sequence and the IGVK sequence, and mainly analyzing the sites on the original mouse monoclonal antibody, which can influence the conformation stability of an antigen binding region, of key composition amino acid residues inconsistent with the human IGVH sequence and the human IGVK sequence, and observing whether humanized substitution can be carried out in a model. Multiple humanized sequences can be generated simultaneously based on one maternal sequence according to different humanized substitution degrees.

For the #110 molecule, the humanized heavy chain sequences are shown in table 3 below:

TABLE 3 Table 3

For the #110 molecule, the humanized light chain sequences are shown in table 4 below:

TABLE 4 Table 4

Combining different humanized light and heavy chains can in turn yield different humanized antibody molecules. The different combinations mainly consider the balance between the degree of humanization and the maintenance of antibody activity after humanization. In brief, pairing light and heavy chains with the highest degree of humanization gives rise to antibody molecules with the highest degree of humanization, which means that there is a potential for lower immunogenicity, but high degrees of humanization may result in reduced activity of the engineered molecule (replacement of key amino acid residues affecting maternal antibody activity with humanized amino acids results in a change in the antigen binding region of the engineered antibody). In contrast, pairing the light and heavy chains with the lowest degree of humanization may result in more consistent functional activity with the parent antibody (as most of the critical amino acid residues are retained), but also lower degrees of humanization may be at risk for higher immunogenicity. This is a balanced process, and various light and heavy chain combination expression preparation needs to be carried out in the process of preparing humanized antibody, and further verification is carried out through in vivo and in vitro functional tests, so that proper humanized antibody molecules are finally screened.

For the #110 molecule, the humanized antibody sequences are shown in table 5 below:

TABLE 5

The constant region sequences used to construct the complete antibodies are shown in Table 6 below:

TABLE 6

EXAMPLE 2 humanized antibody expression and purification

Antibody preparation was performed by conventional methods and the expression supernatant was purified by ProA affinity chromatography. The procedure is as follows, using AKTA AVANT.sup.150 chromatography apparatus, the column (e.g. MabSelectSuRe LX, GE) is equilibrated with at least 5CV equilibration buffer (10 mM PBS), and the sample is loaded onto the column, allowing the target protein to adsorb onto the column while other impurities penetrate the column. After loading was completed, the column was again rinsed with at least 5CV of equilibration buffer (10 mM PBS), followed by elution of the target protein with elution buffer (20 mM naac, ph=3.4), and the collection tube was pre-loaded with neutralization buffer (1 m tris, ph 8.0) at a volume of 10% of the elution volume depending on the estimated amount of eluted sample.

The concentration of the antibody was measured by using a Biotek-Epoch-Take-3 method, and the concentration of the antibody was measured by using an A280 method, that is, using an extinction coefficient E.C.=1.37 and an optical path=0.05 mm (the optical paths of different wells of the Take-3 plate have slight differences and can be automatically corrected), the absorbance of the sample was measured by the apparatus, and the concentration of the antibody to be measured was calculated according to the Lambert-Beer law. If the concentration of the sample is too low, ultrafiltration concentration is needed, and ultrafiltration concentration tube is usedUltra-15Centrifugal Filter Devices,30kD) concentrating the sample to a concentration of >0.5mg/ml according to the general operation method provided in the specification, collecting the concentrated end sample, sterilizing and filtering (Kebaite, PES,0.22 μm, diameter 13 mm) by a sterile needle filter of 0.22 μm, and sub-packaging for freezing storage.

Preparation data for #110 humanized antibodies are shown in table 7 below.

TABLE 7

Remarks in the purity column, the leftmost value is percent of polymer, the rightmost value is percent of fragment, the middle value is percent of antibody monomer, "-" indicates a value of 0%, e.g. "8.98%/91.02%/-" indicating that in an antibody solution, the percent of polymer is 8.98%, the percent of antibody monomer purity is 91.02%, and the percent of fragment is 0%.

EXAMPLE 3 binding Activity of humanized antibodies to Mino cells

Mino cells were collected, centrifuged for 5min, resuspended in PBS, plated on V-96 well plates at 2E 5/well, and incubated at 4℃for 30 min with CD39 monoclonal antibody and control antibody, respectively. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, followed by PE Goat anti mouse IgG Fc (1:500 dilution) at 100. Mu.L/well, and incubation at 4℃for 30 minutes after resuspension of the cells. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, and 100. Mu.L of PBS was added to each well to resuspend the cells, followed by assessment of the difference in binding of CD39 antibody to Mino cells using Cytoflex flow cytometer (Beckman Countler). The binding curves are shown in FIG. 1 a-FIG. 1d, wherein R1839, R1840, R1842, R1843, R1844, R1845, R1846, R1848, R1849, R1851 and R1852 have no binding activity or weak binding activity to Mino cells, the affinity of the 11 humanized antibodies is significantly lower than that of chimeric antibody R1796 (# 110-hIgG 1) and positive control antibody R1800 (ES 002-hIgG 1), and the rest antibodies are superior or identical to the positive antibodies.

The heavy chain variable region amino acid sequence of chimeric antibody R1796 (# 110-hIgG 1) is shown as SEQ ID NO.37, the heavy chain constant region amino acid sequence is shown as SEQ ID NO.39, the light chain variable region amino acid sequence is shown as SEQ ID NO.38, and the light chain constant region amino acid sequence is shown as SEQ ID NO. 41.

The positive control antibody R1800 (ES 002-hIgG 1) has the heavy chain variable region amino acid sequence shown in SEQ ID NO.42, the heavy chain constant region amino acid sequence shown in SEQ ID NO.39, the light chain variable region amino acid sequence shown in SEQ ID NO.43 and the light chain constant region amino acid sequence shown in SEQ ID NO. 41.

Example 4 detection of the ATPase Activity of humanized antibodies blocking CD39 on Mino cells

Mino cells were adjusted to 4E6 cells/mL and transferred to 96-well U-bottom cell culture plates (with fresh medium as necessary) at 50. Mu.l per well, and antibody was prepared starting at 60. Mu.g/mL with medium. Diluting 3 times, adding 100 μL of antibody into cells at 10 times concentration, mixing, incubating at 37deg.C for 60min, preparing 200 μM ATP culture medium, adding 50 μL of ATP culture medium into cells at 50 μL/well, incubating at 37deg.C for 45min, centrifuging 350G cells for 5min, collecting 80 μL of supernatant, transferring to whiteboard, adding CELL TITER-Glo with equal volume, incubating at room temperature in dark place for 10min, and detecting. The blocking curves are shown in FIG. 2 a-FIG. 2d, and are similar to the binding activity results on Mino cells, wherein R1839, R1840, R1842, R1843, R1844, R1845, R1846, R1848, R1849, R1851 and R1852 have no blocking activity or weak blocking activity on CD39 on Mino cells, and the affinity and blocking activity of the 11 humanized antibodies are significantly lower than that of the chimeric antibody R1796, and thus are excluded, and the 11 humanized antibodies will not be detected in subsequent assays.

Example 5 detection of the ATPase Activity of humanized antibodies to block CD39 on MOLP-8 cells

MOLP-8 cells were adjusted to a density of 3E6 cells/ml and transferred to 96-well U-bottom cell culture plates (with fresh medium being necessary) at 50. Mu.L per well, and antibody was prepared starting at 60. Mu.g/ml with medium. Diluting 3 times, adding 100 μL of antibody into cells at 10 times concentration, mixing, incubating at 37deg.C for 60min, preparing 200 μM ATP culture medium, adding 50 μL of ATP culture medium into cells at 50 μL/well, incubating at 37deg.C for 35min, centrifuging 350G cells for 5min, collecting 80 μL of supernatant, transferring to whiteboard, adding CELL TITER-Glo with equal volume, incubating at room temperature in dark place for 10min, and detecting. The blocking activity curves of the antibodies are shown in FIGS. 3a and 3b, wherein R1803, R1804, R1805, R1806, R1807, R1841, R1847, R1850 and R1853 have blocking activity on CD39 hydrolase on MOLP-8 cells similar to or better than that of chimeric antibody R1796, while the blocking activity of the remaining R1801, R1802, R1808, R1809 and R1810 is relatively poor. The 14 antibodies were subsequently tested by functional assays.

Example 6 humanized antibodies block CD39 hydrolysis ATP-mediated T cell proliferation inhibition assay

Firstly, adjusting the density of T cells to 5E6/mL, using DPBS as Buffer to mark the T cells with the final concentration of 0.5 mu M, carrying out shading marking for 20min in a 37 ℃ water bath, adding 12mL of complete culture medium (10% FBS,4 ℃ precooling) for stopping CFSE reaction, diluting antibody with cell culture medium, preparing the antibody with the concentration of 400nM, 10X gradient dilution, setting 5 points, marking the well-marked cells to 300G, centrifuging for 5min to remove supernatant, adding complete culture medium to resuspension and counting cells, adjusting the density to 2E6/mL (total cells are counted here, living cells are not counted), adding 50 mu L/well into 96-well U-shaped plates, namely the final cells to 1E 5/well, adding the prepared antibody into the cells according to 50 mu L/well, diluting the beads according to the number of the cells, ensuring that the beads is 20:1, adding 96-well U-shaped plates according to 50 mu L/well, diluting ATP into the culture medium after three days (72 h) to 1200 mu L, and adding the culture medium into 96 mu L/well-shaped plates, namely the final well plates to 200 mu L/well system. Cells were placed in an incubator for two days (48 h) with cell plates centrifuged to discard the supernatant, BSA was configured with flow antibody (anti-humanCD T,400 Xdiluted, not FITC fluorescent antibody), incubated at 4℃for 30min, and flow-tested for T cell proliferation (10000 cells were harvested, peak shape was guaranteed to be smooth) and on-machine tested according to the standard procedure for flow cytometry of CytoFLEX. The fitted curve is shown in FIG. 4 a-FIG. 4d, wherein R1847 and R1853 are superior to the chimeric antibody R1796.

EXAMPLE 7 binding Activity of humanized antibodies to CHO-K1-cynoCD39 cells

CHO-K1-cynoCD39 cells were collected, centrifuged for 5min, resuspended in PBS, plated in V-96-well plates according to 2E 5/well and antibody was initiated with medium configuration at 60 μg/mL. 3-fold dilutions, 10 total concentrations, were added with CD39 humanized and chimeric antibodies, respectively, and incubated at 4 ℃ for 30min. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, followed by PE Goat anti mouse IgG Fc (1:500 dilution) at 100. Mu.L/well, and incubation at 4℃for 30 minutes after resuspension of the cells. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, and 100. Mu.L of PBS was added to each well to resuspend the cells, followed by assessment of the difference in binding of CD39 antibody to CHO-K1-cynoCD cells using Cytoflex flow cytometer (Beckman Countler). The binding curves are shown in FIGS. 5a and 5b, and the humanized antibody of CD39 has similar binding activity to CHO-K1-cynoCD39 cells as the chimeric antibody.

In combination with the above results, we screened for antibodies R1847 and R1853 that performed better in the binding block and function experiments, and thus selected these 2 antibodies for subsequent antibody subtype engineering.

Example 8 Fc engineering of candidate humanized antibodies

In order that antibodies do not produce strong ADCC and ADCP effects in the body, we mutated the igg1-Fc of the candidate protein, and the correspondence between the molecular numbers before and after the engineering is shown in table 8 below:

TABLE 8

HIgG1 subtype	HIgG1.8 subtype
		R1847	R2122
R1853	R2123
		Positive control antibody R1800	R1289

Example 9 binding Activity of humanized antibodies to Mino cells after subtype changes

Mino cells were collected, centrifuged for 5 min, resuspended in PBS, plated on V-96 well plates at 2E 5/well, and incubated at 4℃for 30 min with CD39 monoclonal antibody and control antibody, respectively. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, followed by PE Goat anti mouse IgG Fc (1:500 dilution) at 100. Mu.L/well, and incubation at 4℃for 30 minutes after resuspension of the cells. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, and 100. Mu.L of PBS was added to each well to resuspend the cells, followed by assessment of the difference in binding of CD39 antibody to Mino cells using Cytoflex flow cytometer (Beckman Countler). The binding curves are shown in FIG. 6, where R2122 and R2123 have similar binding activity to Mino cells and the plateau is superior to the positive control antibody R1289.

Example 10 binding Activity of humanized antibodies to MOLP-8 cells after replacement of subtypes

MOLP-8 cells were collected, centrifuged at 350G for 5min, resuspended in PBS, plated onto V-96 well plates according to 2E 5/well, and incubated at 4℃for 30 min with CD39 monoclonal antibody and control antibody, respectively. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, followed by PE Goat anti mouse IgG Fc (1:500 dilution) at 100. Mu.L/well, and incubation at 4℃for 30 minutes after resuspension of the cells. After centrifugation, 200. Mu.L of PBS was added to each well to wash the cells through, and 100. Mu.L of PBS was added to each well to resuspend the cells, followed by assessment of the difference in binding of CD39 antibody to Mino cells using Cytoflex flow cytometer (Beckman Countler). The binding curves are shown in FIG. 7, where R2122 and R2123 have similar binding activity to MOLP-8 cells and the plateau is superior to the positive control antibody R1289.

EXAMPLE 11 humanized antibody blocking the ATPase hydrolysis Activity assay of CD39 on Mino cells

Mino cells were adjusted to a density of 4E6 cells/mL and transferred to 96-well U-bottom cell culture plates (with fresh medium being necessary) at 50. Mu.L per well, and antibody was prepared starting at 60. Mu.g/mL with medium. Diluting with 3 times, adding 100 μL of antibody and hybridoma supernatant into cells at 10 times concentration, mixing, incubating at 37deg.C for 60min, preparing ATP culture medium into 200 μM, adding into cells at 50 μL/well, incubating at 37deg.C for 45min, centrifuging cells at 350G for 5min, transferring 80 μL of supernatant into white board, adding CELL TITER-Glo at equal volume, incubating at room temperature in dark place for 10min, and detecting. The blocking curves are shown in FIG. 8, where R2122 and R2123 showed superior blocking activity on Mino cells.

Example 12 detection of the ATPase Activity of humanized antibodies to block CD39 on MOLP-8 cells

MOLP-8 cells were adjusted to a density of 3E6 cells/mL and transferred to 96-well U-bottom cell culture plates (with fresh medium being necessary) at 50. Mu.L per well, and antibody was initiated with medium configuration at 60. Mu.g/mL. Diluting with 3 times, adding 100 μL of antibody and hybridoma supernatant into cells at 10 times concentration, mixing, incubating at 37deg.C for 60min, preparing ATP culture medium into 200 μM, adding into cells at 50 μL/well, incubating at 37deg.C for 35min, centrifuging cells at 350G for 5min, transferring 80 μL of supernatant into white board, adding CELL TITER-Glo at equal volume, incubating at room temperature in dark place for 10min, and detecting. The blocking activity curve of the antibody is shown in FIG. 9, and the blocking activity IC50 of R2123 on MOLP-8 cells is similar to that of R2122, and the plateau value is superior to that of R2122 and positive antibody R1289.

Example 13 humanized antibodies block CD39 hydrolysis ATP-mediated T cell proliferation inhibition assay

Firstly, adjusting the density of T cells to 5E6/mL, using DPBS as Buffer to carry out CFSE (10 mM mother liquor) marking the T cells with the final concentration of 0.5 mu M, carrying out shading marking for 20min in a water bath kettle at 37 ℃, adding 12mL of complete culture medium (10% FBS,4 ℃ precooling) for stopping CFSE reaction, diluting antibody with cell culture medium, preparing the concentration of 400nM, 10X gradient dilution, setting 5 points, marking the well cells to 300G, centrifuging for 5min to remove supernatant, adding complete culture medium to resuspension and counting cells, adjusting the density to 2E6 (total cells are counted here, living cells are not counted), adding 50 mu L/well into 96-well U-shaped plates, namely, final cells 1E 5/well, adding the prepared antibody into the cells according to 50 mu L/well, diluting the beads according to the number of the cells, ensuring that the beads is 20:1 in a final system, adding 96-well U according to 50 mu L/well, diluting the culture medium into 1200 mu M after three days (72 h), adding the culture medium into 96 mu L/well plates, namely, and obtaining the final well plate system with the final cell density of 200 mu L/well. Cells were placed in an incubator for two days (48 h) with cell plates centrifuged to discard the supernatant, BSA was configured with flow antibody (anti-humanCD T,400 Xdiluted, not FITC fluorescent antibody), incubated at 4℃for 30min, and flow-tested for T cell proliferation (10000 cells were harvested, peak shape was guaranteed to be smooth) and on-machine tested according to the standard procedure for flow cytometry of CytoFLEX. The fitted curve is shown in fig. 10, and the activity of R2122 in blocking T cell proliferation inhibition detected in this experimental system is superior to R2123. Since there are only two candidate molecules and there are advantages in blocking activity and functional assays, both candidate antibodies remain for subsequent functional assay detection.

EXAMPLE 14 detection of humanized antibody Effect on MOLP-8 xenograft mice

Female NOD SCID mice from 6 to 8 weeks were subcutaneously injected with 5X 10 ⁶ MOLP-8 cells on day 0, and CD39 antibody was administered at 30mg/kg doses on days 1, 5,8 and 12, respectively. FIG. 11 is a graph depicting average tumor volume, error bars represent SEM, red triangles on the X-axis indicate time points of compression, X-axis indicate days after implantation, Y-axis indicate tumor volume, FIG. 12 is a graph depicting average mouse weight, error bars represent SEM, arrows on the X-axis indicate time points of compression, X-axis indicate days after implantation, Y-axis indicate mouse weight. The results show that CD39 humanized antibodies all have tumor inhibiting effects compared to the placebo, wherein the tumor inhibiting effect of R2123 is superior to that of R2122, and that the body weight of mice does not significantly differ between groups during administration.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.

Claims (13)

1. An anti-CD 39 humanized antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region set forth in any one of SEQ ID NOS.1-8, and

(B) A light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region of any one of SEQ ID nos. 9 to 16;

Preferably, the HCDR1, the HCDR2 and the HCDR3, and the LCDR1, the LCDR2 and the LCDR3 are determined according to an IMGT definition mode, a Kabat definition mode, a Chothia definition mode, an AbM definition mode, or a Contact definition mode.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the CDR regions of the heavy chain variable region comprise HCDR1, HCDR2 and HCDR3;

the HCDR1 comprises an amino acid sequence shown as SEQ ID NO.17, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO.22, the HCDR3 comprises an amino acid sequence shown as SEQ ID NO.27, the LCDR1 comprises an amino acid sequence shown as SEQ ID NO.30, the LCDR2 comprises an amino acid sequence shown as YT, and the LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 36.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region is as set forth in any one of SEQ ID nos. 1 to 8.

4. An antibody or antigen-binding fragment thereof according to claim 3, wherein the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID nos 9 to 16.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the amino acid sequence of the light chain variable region is as set forth in any one of SEQ ID nos. 9 to 16.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID nos. 1 to 8.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are selected from any combination of:

Combination of two or more kinds of materials Heavy chain variable region Light chain variable region H6L8 SEQ ID NO.1 SEQ ID NO.9 H8L8 SEQ ID NO.2 SEQ ID NO.9 H1L1 SEQ ID NO.6 SEQ ID NO.16 H1L2 SEQ ID NO.6 SEQ ID NO.10 H1L3 SEQ ID NO.6 SEQ ID NO.11 H1L4 SEQ ID NO.6 SEQ ID NO.12 H1L5 SEQ ID NO.6 SEQ ID NO.13 H2L2 SEQ ID NO.8 SEQ ID NO.10 H3L2 SEQ ID NO.3 SEQ ID NO.10 H3L3 SEQ ID NO.3 SEQ ID NO.11 H3L4 SEQ ID NO.3 SEQ ID NO.12 H3L5 SEQ ID NO.3 SEQ ID NO.13 H4L6 SEQ ID NO.4 SEQ ID NO.14 H4L7 SEQ ID NO.4 SEQ ID NO.15 H4L8 SEQ ID NO.4 SEQ ID NO.9 H5L6 SEQ ID NO.5 SEQ ID NO.14 H5L7 SEQ ID NO.5 SEQ ID NO.15 H5L8 SEQ ID NO.5 SEQ ID NO.9 H6L6 SEQ ID NO.6 SEQ ID NO.14 H6L7 SEQ ID NO.6 SEQ ID NO.15 H7L6 SEQ ID NO.7 SEQ ID NO.14 H7L7 SEQ ID NO.7 SEQ ID NO.15 H7L8 SEQ ID NO.7 SEQ ID NO.9 H8L6 SEQ ID NO.2 SEQ ID NO.14 H8L7 SEQ ID NO.2 SEQ ID NO.15

8. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antigen-binding fragment comprises one or more of F (ab ') ₂, fab', fab, fv, scFv, dsFv, bispecific antibody, and antibody minimal recognition unit.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and/or a light chain constant region;

The heavy chain constant region comprises the sequence of a portion or all of any one of IgG1, igG1.8, igG2, igG3, igG4, igA, igM, igE, or IgD;

the light chain constant region is a kappa or lambda chain;

Preferably, the constant region source species comprises one or more of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human;

preferably, the heavy chain constant region is selected from IgG1, and the amino acid sequence of the heavy chain constant region is preferably as shown in SEQ ID NO. 39;

preferably, the heavy chain constant region is selected from IgG1.8, and the amino acid sequence of the heavy chain constant region is preferably as shown in SEQ ID NO. 40;

preferably, the light chain constant region is a kappa chain, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 41.

10. A biomaterial, characterized in that the biomaterial is selected from any one of (i) to (iii):

(i) A polynucleotide comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9;

(ii) A vector carrying the polynucleotide of (i) above;

(iii) A cell carrying the polynucleotide of (i), or comprising the vector of (ii), or expressing the antibody or antigen-binding fragment thereof of any one of claims 1-9.

11. An immunoconjugate characterized in that the anti-CD 39 humanized antibody or antigen-binding fragment thereof of any one of claims 1-9 is conjugated to at least one diagnostic and/or therapeutic agent to form an immunoconjugate;

The diagnostic agent is selected from one or more of a radiocontrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer;

The therapeutic agent is selected from one or more of a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photoactive therapeutic agent, an immunoconjugate and an oligonucleotide.

12. Use of an antibody or antigen binding fragment thereof according to any one of claims 1 to 9, or a biomaterial according to claim 10, or an immunoconjugate according to claim 11, in any one of the following:

(i) Detection of CD39 or cells expressing CD39 for non-diagnostic and therapeutic purposes;

(ii) Preparing a product for detecting CD39 or cells expressing CD 39;

(iii) Blocking the atpase hydrolytic activity of CD39 on cells for non-diagnostic and therapeutic purposes;

(iv) Preparing a blocking agent for blocking atpase hydrolytic activity of CD39 on a cell;

(v) Preparing a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD 39.

13. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or a biomaterial according to claim 10, or an immunoconjugate according to claim 11, and optionally a pharmaceutically acceptable carrier and/or excipient.