WO2020143710A1 - 一种抗cd73单克隆抗体及其应用 - Google Patents

一种抗cd73单克隆抗体及其应用 Download PDF

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WO2020143710A1
WO2020143710A1 PCT/CN2020/071179 CN2020071179W WO2020143710A1 WO 2020143710 A1 WO2020143710 A1 WO 2020143710A1 CN 2020071179 W CN2020071179 W CN 2020071179W WO 2020143710 A1 WO2020143710 A1 WO 2020143710A1
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seq
antibody
heavy chain
light chain
cdr2
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徐刚
陈博
刘成菊
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康诺亚生物医药科技（成都）有限公司
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Priority to US17/422,028 priority Critical patent/US20220162331A1/en
Priority to EP20738682.2A priority patent/EP3901175A4/en
Publication of WO2020143710A1 publication Critical patent/WO2020143710A1/zh

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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • CD73 is a glycoprotein with 5'nucleotide hydrolase activity, also known as extracellular 5'nuclease (NT5E), which is anchored to the cell membrane surface by glycosyl-phosphatidylinositol (GPI). The CD73 on the cell membrane is cleaved by the GPI anchor, which will cause the CD73 to detach from the cell surface and be released into the blood to form soluble CD73 (sCD73). Both CD73 and sCD73 on the cell membrane have hydrolase activity, which can catalyze Adenosine monophosphate (AMP) in the extracellular matrix and dephosphorylation to generate Adenosine (ADO).
  • AMP Adenosine monophosphate
  • ADO Adenosine
  • the generated ADO can cause a series of biological effects through adenosine receptors (A1R, A2AR, A2BR, and A3R) on the cell membrane, such as dilating blood vessels, improving endothelial barrier function, stimulating angiogenesis, inhibiting platelet aggregation, and reducing freedom Base generation, etc.
  • adenosine receptors A1R, A2AR, A2BR, and A3R
  • CD73 is widely expressed in a variety of human tissues, including: intestine, kidney, brain, liver, heart, lung, respiratory epithelium, spleen, lymph nodes, and bone marrow. In the immune system, CD73 is expressed in lymphocytes, regulatory T cells (Treg), neutrophils, bone marrow-derived inhibitory cells (MDSC), dendritic cells, natural killer cells, and macrophages. As a rate-limiting enzyme in the production of ADO, CD73 is one of the determinants of ADO halo (ADO halo) surrounded by immune cells. ADO halo forms an immunosuppressive environment locally.
  • ADO halo forms an immunosuppressive environment locally.
  • CD73 is one of the key enzymes that determine the level of ADO in the tumor microenvironment (Augusto et al., 2013). Overexpression of tissue CD73 is usually associated with poor overall survival or tumor progression (Allard D et al., 2016; Wang R et al., 2017).
  • the ADO pathway is considered to be one of the major inhibitory pathways in the tumor microenvironment. As the content of ADO in the tumor microenvironment increases, ADO, which acts as an anti-inflammatory mediator, will lead to weakened infiltration of immune cells, resulting in chronic suppression of the immune response against tumors.
  • Changes in the tumor microenvironment produced by tumor cells further promote the growth of tumor cells through cytokines secreted by inflammatory cells, and suppress the anti-tumor activity of immune cells, and promote the occurrence and development of cancer.
  • Nucleotide hydrolase that blocks CD73 on the cell surface and sCD73 released into the blood helps to eliminate the inhibitory effect of high concentrations of adenosine on immune killing in the tumor microenvironment. Studies have shown that compared with normal mice, CD73 knockout mice significantly increase the anti-tumor immunity (Stagg et al., 2011, 2012).
  • CD73 Blocking the biological activity of CD73 through monoclonal antibodies or small molecules has been shown to slow the growth and proliferation of tumor cells (Stagg J et al 2010, Zhi X X et al 2010, Terp MG et al 2013), and reduce neovascularization (Koszalka P etc. 2014, Burghoff S et al. 2014), showing that CD73 has value as a potential therapeutic target for cancer treatment (Zhang B. 2010, Beavis, PA et al. 2012, Allard B et al. 2014, Hay CM et al. 2016).
  • the applicant has developed a novel monoclonal antibody targeting human CD73, which can not only inhibit the hydrolase activity of membrane-bound CD73 and sCD73, and after binding to CD73, it can be effectively reduced by receptor-mediated endocytosis
  • the abundance of CD73 on the surface of the cell membrane inhibits the generation of ADO.
  • Combining CD73 blockade therapy with other immune molecule modulators showed a significantly better efficacy than single drugs, showing that CD73 enzyme activity blocking antibody combined with other immune molecule modulators is a kind of treatment
  • An extremely attractive option is expected to become a new type of biological therapy in the field of anti-cancer.
  • the present disclosure relates to antibodies or antigen-binding portions that specifically bind CD73.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds to human CD73, which binds to human differentiation determinant (CD73), inhibits cell membrane CD73 hydrolase activity, and simultaneously inhibits soluble CD73 (sCD73) in serum and body fluids Hydrolase activity, and mediate the endocytosis of CD73 on the cell surface, effectively reducing the abundance of CD73 on the cell membrane surface.
  • CD73 human differentiation determinant
  • sCD73 soluble CD73
  • the antibody or antigen-binding portion thereof according to any of the preceding aspects, wherein the antibody is an antibody fragment.
  • the antibody or antigen-binding portion thereof according to any of the preceding aspects, wherein the antibody or antigen-binding portion blocks the hydrolase activity of CD73.
  • the antibody or antigen binding portion thereof according to any of the preceding aspects, wherein the antibody or antigen binding portion thereof is humanized.
  • a vector containing the nucleic acid according to any of the foregoing aspects.
  • a pharmaceutical composition or kit comprising an antibody or antigen-binding portion thereof or a nucleic acid encoding the same as in any preceding aspect and a pharmaceutically acceptable carrier.
  • a method of treating cancer comprising the steps of: administering to said mammal a therapeutically effective amount of an antibody or antigen-binding portion thereof or nucleic acid molecule or carrier or cell or pharmaceutical composition of any of the preceding aspects, optionally, said The method also includes administering to the mammal a therapeutically effective amount of anti-PD-1 antibody.
  • a method for treating a disease associated with the abnormal production of CD73 in a mammal comprising the steps of: administering to the mammal a therapeutically effective amount of the antibody or antigen-binding portion thereof or nucleic acid molecule or carrier or cell or drug of any of the foregoing aspects
  • the composition optionally, the method further comprises administering to the mammal a therapeutically effective amount of an anti-PD-1 antibody.
  • Figure 1 Expression of recombinant human, cynomolgus monkey and mouse CD73 extracellular domain proteins in HEK293 cells.
  • Figure 2 Flow cytometry results of human CD73 stably transfecting CHO cells.
  • Figure 3 Figure 3A. Binding of CD73 positive clones to Calu6 cells; Figure 3B. Binding of CD73 positive clones to hCD73-CHO-1C11 cells.
  • Figure 4 Figure 4A. Inhibition effect of CD73 antibody on CD73 enzyme activity on the surface of Calu6 cells; Figure 4B. Inhibition effect of CD73 antibody on CD73 enzyme activity in solution.
  • Figure 5 Figure 5A. Binding of CD73 chimeric antibody to recombinant human CD73 protein; Figure 5B. Binding of CD73 chimeric antibody to recombinant cynomolgus CD73 protein.
  • Figure 6 Figure 6A. Anti-CD73 antibody inhibits CD73 enzyme activity on the surface of Calu6 cells after humanization; Figure 6B. Anti-CD73 antibody inhibits soluble CD73 enzyme activity after humanization.
  • Figure 7 Humanized anti-CD73 antibody mediates CD73 endocytosis on the cell surface.
  • FIG 8 Peripheral blood PBMC mixed lymphatic reaction, the effect of humanized anti-CD73 antibody and PD-1 antibody alone or in combination on T lymphocyte activation, Figure 8A. T lymphocytes release INF- ⁇ after activation; Figure 8B. After activation of T lymphocytes, TNF- ⁇ is released.
  • antibodies that specifically bind to CD73 (eg, human CD73) and antigen-binding fragments thereof.
  • a monoclonal anti-CD73 antibody that specifically binds to human CD73, wherein the anti-CD73 antibody includes a variant of the parent antibody.
  • antibodies that specifically bind to CD73 eg, human CD73.
  • anti-CD73 antibodies that contain modifications in one or more amino acid residues (eg, 5-13 amino acid substitutions in the framework region of the heavy chain variable region), with and without the modifications Compared to its parent antibody, it maintains affinity for the antigen.
  • the hydrolase activity of such anti-CD73 antibody CD73 is in vivo or in vitro, or both in vivo and in vitro.
  • the antibodies or antigen-binding fragments described herein may contain sequences that are not naturally found in the germline repertoire of antibodies in animals or mammals (eg, humans).
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.
  • CD73 Human CD73 is also called "Differentiation Cluster 73" or “CD73” or extracellular 5'-nucleotidase or 5-pro-ribonucleotide phosphorohydrolase, EC3.1.3.5, encoded by the NT5E gene and displayed 5'-nucleotidase (particularly AMP-, NAD-, and NMN-nucleotidase) activity.
  • CD73 catalyzes the conversion of purine 5-pro-mononucleotides to nucleotides at neutral pH, and the preferred substrate is AMP.
  • the enzyme consists of two dimers of the same 70-kD subunit connected to the outer surface of the plasma membrane by glycosylphosphatidylinositol linkages.
  • the amino acid sequence of the human CD73 preprotein (monomer), including the signal sequence of amino acids 1-26, is shown in the GenBank as the accession number NP_00251, the entire disclosure of which is incorporated herein by reference.
  • neutralizing CD73 enzyme activity refers to the process of inhibiting CD73's 5'-nucleotidase (5'-extracellular nuclease) activity, including both CD73 on the cell surface and CD73 in free state.
  • the antibody preparation causes a reduction of AMP to adenosine conversion by at least 50%, AMP to adenosine conversion by at least 70%, or AMP to adenosine conversion by at least 80%, with reference to assays described herein, for example law.
  • the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, An antibody chain with 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequence the term can be understood as exhibiting at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 with reference nucleic acid sequence Nucleotide sequence with %, 99% or higher sequence identity.
  • sequence identity has an art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of the polynucleotide or polypeptide or along the region of the molecule (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic, Press, New York, 1993; Computer Analysis Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • phrases and terms such as "functional fragments, variants, derivatives or analogs" of antibodies or antigens and their various forms refer to compounds or molecules that have the same biological activity as the full-length antibody or antigen of interest.
  • a functional fragment or analog of an anti-CD73 antibody is a fragment or analog that can bind to a CD73 molecule, or a fragment or analog that can prevent or substantially reduce CD73 activity.
  • substitution variants are variants in which at least one amino acid residue in the natural sequence has been removed and inserted into its same position by a different amino acid.
  • the substitution may be single, in which only one amino acid is substituted in the molecule; or may be multiple, in which two or more amino acids of the same molecule are substituted. Multiple substitutions can be located at consecutive sites.
  • an amino acid can be substituted with multiple residues, where such variants include both substitutions and insertions.
  • An "insertion” variant is a variant in which one or more amino acids are inserted at a specific position immediately adjacent to a natural sequence. Immediately adjacent to an amino acid means attached to the ⁇ -carboxyl or ⁇ -amino functional group of the amino acid.
  • “Deletion” variants are those in which one or more amino acids have been removed from the natural amino acid sequence. Normally, a deletion variant has one or two amino acids deleted in a specific region of its molecule.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), carrying one or more CDRs or antibody fragments or synthetic polypeptides derived from CDR sequences, as long as these polypeptides exhibit the desired biological activity.
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics.
  • Antibody can also refer to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically produced (eg, recombinant), including full-length immunoglobulins that retain at least a portion of the variable regions of immunoglobulin molecules Any fragment with the ability to bind specificity.
  • antibodies include any protein that has a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site).
  • Antibodies include antibody fragments, such as anti-tumor stem cell antibody fragments.
  • antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (eg, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments , Such as but not limited to Fab fragments, Fab' fragments, F (ab') 2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies.
  • Fab fragments Fab' fragments, F (ab') 2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or anti
  • the antibodies provided herein include any immunoglobulin type (eg, IgG, IgM, IgD, IgE, IgA, and IgY), any class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (eg, IgG2a And IgG2b) ("type” and "class", as well as “subtype” and “subclass” are used interchangeably herein).
  • Natural or wild-type (i.e. from unmanipulated group members) antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, which consist of two identical light chains (L) and two identical Composed of the heavy chain (H).
  • Each heavy chain has a variable domain (V H ) at one end, followed by multiple constant domains.
  • Each light chain has a variable domain (V L ) at one end and a constant domain at the other end.
  • V H variable domain
  • V L variable domain
  • Wild type may refer to the most common allele or species found in a population or to antibodies obtained from unmanipulated animals, as compared to alleles or polymorphisms, or from some form of manipulation such as mutagenesis 3. Using recombinant methods or the like to change the amino acid variant or derivative of the antigen binding molecule.
  • anti-CD73 antibody means an antibody that can specifically bind to CD73 as defined herein or a polypeptide (derivative) derived from these antibodies, which includes but is not limited to inhibiting or substantially reducing the binding of CD73 to its receptor or Molecules that inhibit CD73 activity.
  • variable domains of antibodies refers to certain parts of related molecules that have extensive sequence differences between antibodies and are used for the specific recognition and binding of specific antibodies against their specific targets.
  • variability is not evenly distributed throughout the variable domains of antibodies.
  • the variability is concentrated in three segments called complementarity determining regions (CDRs; ie, CDR1, CDR2, and CDR3) or hypervariable regions, which are all located in the variable domains of the light chain and the heavy chain.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable domain of the natural heavy and light chains includes four FR regions, which mainly adopt a ⁇ -sheet configuration, they are connected by three CDRs, and the CDRs form a loop, and the loop connects the ⁇ -sheet structure and In some cases, a partial ⁇ -sheet structure is formed.
  • the CDRs of each chain are usually connected in the vicinity by the FR region, and with the help of CDRs from other chains, it helps to form the target binding site (epitope or determinant) of the antibody (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)).
  • the numbering of immunoglobulin amino acid residues is based on the Kabat et al. immunoglobulin amino acid residue numbering system, unless otherwise stated.
  • a CDR may have the ability to specifically bind related epitopes.
  • hinge or “hinge region” refers to a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any part of a full-length antibody that is less than the full-length, but at least includes a variable region (eg, one or more) of the antibody that binds to the antigen CDR and/or one or more antibody binding sites), and thus retain the binding specificity and at least part of the specific binding capacity of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may include multiple chains linked together, for example by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (eg, by replacing the corresponding region), obtains antibodies that immunospecifically bind (ie, exhibit a Ka of at least or at least about 10 7 -10 8 M -1 ) antigen .
  • a "functional fragment” or "an anti-CD73 antibody analog” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments", and in terms of antibodies, can refer to fragments that can prevent or substantially reduce the ability of the receptor to bind ligands or initiate signal transduction, such as Fv, Fab , F(ab′)2 and so on.
  • the "Fv" fragment is composed of a dimer (V H -V L dimer) formed by a variable domain of a heavy chain and a variable domain of a light chain by non-covalent bonding.
  • V H -V L dimer dimer
  • the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH- VL dimer, as is the case with intact antibodies.
  • the six CDRs together confer target binding specificity of the intact antibody.
  • a single variable domain or half of the F v comprising only three CDRs specific for a target), still having the ability to recognize and bind target.
  • Single-chain Fv Single-chain Fv
  • sF v single-chain Fv
  • scAb single-chain Fv
  • the F v polypeptide further comprises a polypeptide is located between the period of V H and V L, a linker region, which is typically a flexible molecule, can sFv to form the desired structure for target binding.
  • diabodies refers to antibody fragments with two antigen-binding sites, which fragments can comprise a heavy chain variable domain and a light chain variable domain in the same polypeptide chain (V L) is connected (V H ).
  • V L polypeptide chain
  • V H polypeptide chain
  • the Fab fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain.
  • the Fab' fragment differs from the Fab fragment in that the former adds several residues to the carboxyl terminus of the CH1 domain to include one or more cysteines from the hinge region of the antibody.
  • a Fab' fragment can be generated. Additional enzyme treatment and chemical treatment of antibodies can generate other functional fragments of interest.
  • linear Fab refers to the tetravalent antibody described by Miller et al. (Miller et al. (2003), JImmunol. 170:4854-4861). "Linear Fab” is composed of a series of identical CH1-VH domains, paired with the same light chain at each CH1-VH position. These molecules were developed to increase the titer of antibodies and enhance their functional affinity through affinity effects, however, they are monospecific.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the population of monoclonal antibodies is the same as other antibody molecules. This characteristic is opposite to that of polyclonal populations of antibodies, which contain antibodies with multiple different sequences.
  • Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117).
  • monoclonal antibodies can be prepared by immortalizing B cells, for example, by fusing with myeloma cells to produce a hybridoma cell line or by infecting B cells with a virus such as EBV.
  • Recombinant technology can also be used to prepare antibodies from cloned populations of host cells in vitro by transforming the host cells with plasmids carrying artificial sequences encoding nucleotides encoding the antibodies.
  • hybridomas refers to cells or cell lines (usually myeloma or lymphoma cells) produced by fusing antibody-producing lymphocytes and non-antibody-producing cancer cells.
  • hybridomas can proliferate and continue to be supplied to produce specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988).
  • hybridodoma or “hybridoma cell”
  • it also includes subclones and progeny cells of the hybridoma.
  • a full-length antibody has two full-length heavy chains (for example, VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region
  • Antibodies such as antibodies produced naturally by antibodies secreting B cells and synthetically produced antibodies with the same domain.
  • dsFv refers to Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.
  • an scFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order through a polypeptide linker.
  • the length of the linker allows the two variable domains to bridge without substantial interference.
  • An exemplary linker is a (Gly-Ser) n residue with some Glu or Lys residues dispersed to increase solubility.
  • chimeric antibody refers to an antibody where the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as where the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody Of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) antibody forms, which are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or Other antigen-binding subsequences of antibodies), containing minimal sequences derived from non-human immunoglobulins.
  • the humanized antibody is human immunoglobulin (recipient antibody), wherein the residue of the complementarity determining region (CDR) of the recipient antibody is derived from a non-human species with the desired specificity, affinity and ability ( Donor antibodies) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • the humanized antibody of the present invention also encompasses antibodies containing 1 or 2 amino acid mutations in the CDR.
  • epitope refers to any epitope on the antigen to which the paratope of the antibody binds.
  • Epitope determinants usually contain chemically active surface typing of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • variable domain or variable region is a specific Ig domain of an antibody heavy or light chain, which contains amino acid sequences that vary between different antibodies. Each light chain and each heavy chain have a variable domain VL and VH, respectively. The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region contains a CDR and a framework region (FR), which is part of the antigen binding site domain.
  • FR framework region
  • antigen binding domain and "antigen-binding site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with a cognate antigen.
  • Natural conventional full-length antibody molecules have two conventional antigen binding sites, each of which contains a heavy chain variable region portion and a light chain variable region portion.
  • Conventional antigen binding sites contain loops connecting antiparallel ⁇ chains within the variable domain domain.
  • the antigen binding site may contain other parts of the variable domain domain.
  • Each conventional antigen binding site contains 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain.
  • the hypervariable region is also called complementarity determining region (CDR).
  • a functional region of a VH domain is at least a portion of an intact VH domain that retains at least a portion of the binding specificity of the intact VH domain (eg, by retaining one or more CDRs of the intact VH domain), such that the VH
  • the functional region of the domain binds to the antigen alone or in combination with another antibody domain (eg, VL domain) or a region thereof.
  • the functional region of an exemplary VH domain is a region containing CDR1, CDR2 and/or CDR3 of the VH domain.
  • a functional region of a VL domain is at least a portion of an intact VL domain that retains at least a portion of the binding specificity of the intact VL domain (eg, by retaining one or more CDRs of the intact VL domain), such that the VL
  • the functional region of the domain binds to the antigen alone or in combination with another antibody domain (eg, VH domain) or a region thereof.
  • the functional region of an exemplary VL domain is a region containing CDR1, CDR2 and/or CDR3 of the VL domain.
  • an antibody that immunospecifically binds (or specifically binds) to an antigen has an affinity constant Ka (or 1x10 -7 M or 1 ⁇ or about 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater 10 -8 M or lower dissociation constant (Kd)) binds the antigen.
  • the affinity constant can be determined by standard kinetic methods of antibody reaction, for example, immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also US Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • the term "competition" with respect to antibodies means that the first antibody or antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to the second antibody or antigen-binding fragment thereof, whereby the first antibody and its associated epitope The binding result is detectably reduced in the presence of the second antibody compared to the absence of the second antibody.
  • the first antibody can inhibit the binding of the second antibody to its epitope without using the second antibody to inhibit the binding of the first antibody to its respective epitope.
  • each antibody can detectably inhibit the binding of another antibody to its associated epitope or ligand, whether at the same, higher, or lower degree, the antibodies are said to "cross-compete" for binding to each other Their respective epitopes.
  • Both competitive and cross-competitive antibodies are covered by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (eg, steric hindrance, conformational change, or binding to a common epitope or fragment thereof), those skilled in the art will be aware of such competition and/or cross-competitive antibodies based on the teaching provided by the present invention It is encompassed in the present invention and can be used in the methods disclosed in the present invention.
  • polypeptide refers to two or more amino acids covalently linked.
  • polypeptide and protein are used interchangeably herein.
  • isolated protein means that the protein, polypeptide or antibody (1) is not associated with its naturally-related components in its natural state, (2) does not come from the same species Of other proteins, (3) are expressed by cells from different species, or (4) do not occur in nature. Therefore, a chemically synthesized polypeptide or a polypeptide synthesized in a cell system different from the naturally derived cell of the polypeptide will be "separated” from its natural related components. It can also be separated so that the protein is substantially free of natural related components, even using protein purification techniques well known in the art.
  • Suitable conservative amino acid substitutions are known to those skilled in the art, and can generally be made without altering the biological activity of the resulting molecule.
  • those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub .co., p.224).
  • polynucleotide and “nucleic acid molecule” refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, including those that are usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components during chemical synthesis.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • operably linked with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide so that the promoter regulates or mediates the transcription of the nucleic acid.
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.
  • conservative sequence modifications of the sequences described in the sequence listing described herein, that is, nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions.
  • modifications can be introduced into the sequence listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative sequence modifications include conservative amino acid substitutions, where amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues with similar side chains is already defined in the art.
  • amino acids with basic side chains eg lysine, arginine, histidine
  • amino acids with acidic side chains eg aspartic acid, glutamic acid
  • side chains with no electrode Amino acids (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan)
  • amino acids with non-polar side chains eg alanine, valine Acids, leucine, isoleucine, proline, phenylalanine, methionine
  • amino acids with beta branched side chains e.g.
  • the predicted non-essential amino acid residues in the anti-CD73 antibody are preferably replaced by another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10) :879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).
  • mutations can be randomly introduced along all or part of the anti-CD73 antibody coding sequence by, for example, saturation mutagenesis, and the resulting modified anti-CD73 antibody can be screened for improved binding activity.
  • nucleic acids For nucleic acids, the term "substantial homology" means that two nucleic acids or their designated sequences have at least about 80% of the nucleotides during optimal alignment and comparison (where nucleotides are appropriately inserted or deleted), usually At least about 90% to 95%, and more preferably at least about 98% to 99.5% of nucleotides are identical. Alternatively, if the segment hybridizes to the complement of the strand under selective hybridization conditions, there is substantial homology.
  • expression refers to the process of producing a polypeptide by the transcription and translation of a polynucleotide.
  • the expression level of a polypeptide can be evaluated using any method known in the art, including, for example, a method of determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • host cell is a cell used to receive, maintain, replicate, and expand a vector.
  • the host cell can also be used to express the polypeptide encoded by the vector.
  • the host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include but are not limited to CHO cells, various COS cells, HeLa cells, HEK cells such as HEK293 cells.
  • Codon optimization refers to replacing at least one codon of the natural sequence by a codon used more frequently or most frequently in the gene of the host cell (eg, about or more than about 1, 2, 3, 4, 5 , 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence while modifying the nucleic acid sequence in order to enhance expression in the host cell of interest.
  • Codon preference the difference in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transport RNA
  • genes can be customized to codon optimization based on codon optimization in a given organism The best gene expression.
  • the codon usage tables can be easily obtained, for example, in the codon usage database (“Codon Usage Database”) available on www.kazusa.orjp/codon/, and these tables can be passed through different The method adjustment is applicable. See, Nakamura Y. et al., "Codon usage tabulated from the international DNA sequences: status for the year 2000. Nucl. Acids Res., 28:292 (2000)”.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • the vectors include those in which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced into it by restriction digestion and ligation.
  • the vectors also include those containing nucleic acids encoding polypeptides.
  • the vector is used to introduce the nucleic acid encoding the polypeptide into the host cell, to amplify the nucleic acid or to express/display the polypeptide encoded by the nucleic acid.
  • Vectors usually remain free, but can be designed to integrate genes or parts thereof into the chromosomes of the genome.
  • Vectors of artificial chromosomes are also considered, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
  • vectors also include “viral vectors” or “viral vectors.”
  • the vector of the virus is an engineered virus that is operably linked to a foreign gene to transfer the foreign gene (as a vehicle or shuttle) into the cell.
  • expression vector includes a vector capable of expressing DNA operably linked with regulatory sequences, such as a promoter region, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which when introduced into an appropriate host cell results in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells as well as expression vectors that remain free or are integrated into the genome of the host cell.
  • treatment includes prevention, treatment, and/or cure. Prevention refers to preventing the underlying disease and/or preventing the deterioration of symptoms or the development of the disease. Treatment also includes any antibody or antigen-binding fragment provided and any pharmaceutical use of the compositions provided herein.
  • efficacy means an effect caused by an individual's treatment that changes, usually improves or ameliorates the symptoms of the disease or disease condition, or cures the disease or disease condition.
  • terapéuticaally effective amount or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a disease or condition.
  • prophylactically effective amount or “prophylactically effective dose” refers to the amount of a substance, compound, material, or composition containing a compound that will have the desired prophylactic effect when administered to a subject, for example, to prevent or delay a disease or symptom Occurrence or recurrence of the disease reduces the possibility of the occurrence or recurrence of disease or symptoms.
  • Completely prophylactically effective doses do not have to occur through the administration of one dose, and can only occur after the administration of a series of doses. Therefore, the prophylactically effective amount can be administered in one or more applications.
  • the term "patient” refers to a mammal, such as a human.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds to human CD73, which comprises an amino acid sequence selected from SEQ ID NO: 6-8, 16-18, 26-28, 36-38, 46- 48, 56-58, 66-68, 76-78, 86-88, 96-98, 106-108, 116-118, 126-128, 131-133, 136-138, 141-143, 146-148 or The heavy chain CDR of any variant thereof, and/or selected from the amino acid sequence SEQ ID NO: 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81- Light chain CDRs of 83, 91-93, 101-103, 111-113, 121-123, 151-153, 156-158, 161-163, 166-168, 171-173, or any variant.
  • the antibody or antigen-binding portion thereof which comprises an amino acid sequence selected from SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 131, Heavy chain CDR1 of 136, 141, 146 or any variant thereof, selected from the amino acid sequence SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 132 , 137, 142, 147 or any variant heavy chain CDR2 selected from the amino acid sequence SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 133, 138, 143, 148 or heavy chain CDR3 of any variant thereof; and/or selected from the amino acid sequence SEQ ID NO: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, Light chain CDR1 of 121, 151
  • the antibody or antigen-binding portion thereof which comprises an amino acid sequence selected from SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 130 , 135, 140, 145 or any variant heavy chain variable region, and/or selected from the amino acid sequence SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 , 120, 150, 155, 160, 165, 170, or the light chain variable region of any variant thereof.
  • Nucleic acid molecule encoding an antibody or antigen-binding portion thereof according to any of the preceding aspects, which comprises SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129 , 134, 139, 144, 149 or any variant nucleic acid sequence, and/or selected from SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 154, 159, 164, 174 or a nucleic acid sequence of any variant thereof.
  • the nucleic acid molecule comprises a nucleic acid sequence SEQ ID NO: 149 and a nucleic acid sequence SEQ ID NO: 174.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 125 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 150 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 125 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 155 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 125 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 160 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 125 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 165 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 125 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 170 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 130 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 150 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 130 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 155 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 130 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 160 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 130 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 165 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 130 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 170 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 135 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 150 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 135 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 155 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 135 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 160 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 135 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 165 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 135 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 170 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 140 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 150 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 140 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 155 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 140 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 160 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 140 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 165 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 140 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 170 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 145 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 150 or any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 145 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 155 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 145 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 160 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 145 or any variant thereof, and/or from an amino acid sequence SEQ ID NO: 165 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human CD73, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 145 or any variant thereof, and/or from an amino acid sequence The light chain variable region of SEQ ID NO: 170 or any variant thereof.
  • An antibody or antigen-binding portion thereof that binds to human CD73 which includes three heavy chain variable region CDRs and three light chain variable region CDRs in a heavy chain and light chain variable region pair selected from the group consisting of : (A) SEQ ID NO: 125 and 150 (b) SEQ ID NO: 125 and 155; (c) SEQ ID NO: 130 and 150; (d) SEQ ID NO: 130 and 155; (e) SEQ ID NO: 135 and 160; (f) SEQ ID NO: 135 and 165; (g) SEQ ID NO: 140 and 160; (h) SEQ ID NO: 140 and 165; (i) SEQ ID NO: 145 and 160; ( j) SEQ ID NO: 145 and 170.
  • An antibody or antigen-binding portion thereof that binds to human CD73 which comprises (a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 126-128, and/or comprising SEQ ID NO: 151-153, respectively Light chain CDR1, CDR2 and CDR3 sequences; (b) heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 126-128, and/or light chain CDR1, CDR2 and SEQ ID NO: 156-158, respectively CDR3 sequence; (c) the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 131-133, and/or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 151-153 respectively; (d) SEQ ID NO: 131-133 heavy chain CDR1, CDR2 and CDR3 sequences respectively, and/or SEQ ID NO: 156-158 light chain CDR1, CDR2 and CDR3 sequences respectively; (e) SEQ
  • An antibody or antigen-binding portion thereof that binds to human CD73 has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the antibody or antigen-binding portion of any of the foregoing aspects , 96%, 97%, 98%, 99% or higher sequence identity.
  • the present disclosure also includes a kit, for example, the kit includes the antibodies, fragments, homologs, derivatives thereof, etc. of the present disclosure, such as labeled or cytotoxic conjugates, and instructions for use of the antibody , Killing conjugates of specific types of cells, etc.
  • the instructions may include instructions for using antibodies, conjugates, etc. in vitro, in vivo or ex vivo.
  • Antibodies can be in liquid form or solid, usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Consideration is given to combinations of reagents packaged in predetermined amounts and instructions for their use, such as for therapeutic use or for performing diagnostic assays.
  • the kit may include a substrate and cofactors required by the enzyme (eg, to provide a substrate precursor that can detect a chromophore or fluorophore).
  • cofactors required by the enzyme eg, to provide a substrate precursor that can detect a chromophore or fluorophore.
  • other additives such as stabilizers, buffers (such as blocking buffer or lysis buffer), etc. may also be included.
  • the relative amounts of multiple reagents can be changed to provide a concentrate of reagent solutions, which provides user flexibility, space saving, and reagent saving.
  • These reagents can also be provided in dry powder form, usually in lyophilized form, including excipients, which, when dissolved, can provide a reagent solution with an appropriate concentration.
  • a method of treating cancer comprising the steps of: administering to said mammal a therapeutically effective amount of an antibody or antigen-binding portion thereof or nucleic acid molecule or carrier or cell or pharmaceutical composition of any of the preceding aspects, optionally, said The method also includes administering to the mammal a therapeutically effective amount of anti-PD-1 antibody.
  • a method for treating a disease related to the abnormal production of CD73 in a mammal comprising the steps of: administering to the mammal a therapeutically effective amount of the antibody or antigen-binding portion thereof or nucleic acid molecule or carrier or cell or drug of any of the foregoing aspects
  • the composition optionally, the method further comprises administering to the mammal a therapeutically effective amount of an anti-PD-1 antibody.
  • inhibition of CD73 hydrolase activity refers to inhibition of membrane-bound CD73 hydrolase activity, or inhibition of free CD73 hydrolase activity, or both membrane-bound CD73 hydrolase activity and free The hydrolase activity of CD73.
  • the antibodies of the present invention can be used to treat mammals.
  • the antibody or equivalent of interest is administered to a non-human mammal.
  • exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents, and other mammals in which preclinical studies have been conducted.
  • Such mammals may be established animal models of diseases that need to be treated with antibodies, or may be used to study the toxicity of antibodies of interest.
  • dose escalation studies can be conducted in mammals.
  • Antibodies regardless of the presence or absence of the second component (such as the portion of the therapeutic agent conjugated thereto), whether administered alone or in combination with cytotoxic factors, can be used as therapeutic agents.
  • the present invention relates to antibody-based therapies, in which the antibody of the present invention is reserved for animals, mammals, or humans to treat CD73-mediated diseases, disorders, or conditions.
  • treatment refers to therapeutic treatment and prophylactic or preventative measures. It refers to the harmful effects of preventing, curing, reversing, weakening, improving, minimizing, inhibiting or stopping the disease state, disease process, disease-causing factors (such as bacteria or viruses) or other abnormal conditions.
  • the present invention also includes multivalent antibodies, including bispecific anti-CD73 antibodies, which have effector molecules, atoms, or other substances associated with the diagnostic or therapeutic functions.
  • the antibody may have a radioactive diagnostic tag or a radioactive cytotoxic atom or a metal or cytotoxic substance such as ricin chain, which is linked to it for in vivo diagnosis or treatment of cancer.
  • antibodies of the present invention can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • the present invention also provides a composition
  • a composition comprising the anti-CD73 antibody or fragment thereof of the present invention, the antibody is conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a common practice in the art.
  • the term "pharmaceutical composition” refers to the preparation of various preparations.
  • the formulation containing the therapeutically effective amount of the multivalent antibody is in a sterile liquid solution, liquid suspension, or lyophilized form, optionally containing stabilizers or excipients.
  • disorder refers to any condition that can benefit from the antibody treatment of the present invention. This includes chronic and acute disorders or diseases, including those pathological conditions that tend to infect mammals, especially humans, with the disorder.
  • disorders to be treated herein include cancer, inflammation, autoimmune diseases, infections, cardiovascular diseases, respiratory diseases, neurological diseases, and metabolic diseases.
  • cancer refers to or describes the physiological condition of mammals, especially humans, which is typically characterized by the unregulated growth of cells.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • the antibody of the present invention may be used as a composition administered alone, or may be used in combination with other active agents.
  • a vector containing the nucleic acid according to any of the foregoing aspects.
  • a pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to any of the foregoing aspects or a nucleic acid encoding the same and a pharmaceutically acceptable carrier.
  • a method of treating cancer comprising the step of administering to the mammal a therapeutically effective amount of the antibody or antigen-binding portion of any of the foregoing aspects or a nucleic acid encoding the same.
  • a method for treating a disease associated with the abnormal production of CD73 in a mammal comprising the step of administering to the mammal a therapeutically effective amount of the antibody or antigen-binding portion of any of the foregoing aspects or a nucleic acid encoding the same.
  • the therapeutic agent according to the described embodiments will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc.
  • suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc.
  • suitable formulations can be found in the pharmacopoeia known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), in particular Chapter 87 of Blaug, Seymour among them.
  • formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (such as Lipofectin TM ), DNA conjugates, anhydrous syrups, oil-in-water And water-in-oil emulsion, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gel and semi-solid mixture containing polyethylene glycol. Any of the foregoing mixtures may be suitable for treatment or therapy according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerates the route of administration.
  • Abnormal CD73 production or abnormal CD73 expression is used interchangeably herein.
  • the antibody can be used as a therapeutic agent.
  • Such agents will generally be used to treat, alleviate, and/or prevent a subject's disease or pathology associated with abnormal CD73 expression, activity, and/or signaling.
  • Standard methods can be used to perform treatment by identifying subjects, such as human patients suffering from (or at risk or developing) diseases or disorders associated with abnormal CD73 expression, activity, and/or signaling, such as cancer or other neoplastic disorders Program.
  • An antibody preparation preferably an antibody preparation with high specificity and high affinity for its target antigen, is administered to the subject and will usually produce an effect due to its binding to the target.
  • the administered antibody can eliminate or inhibit or hinder the expression, activity and/or signaling function of the target (eg CD73).
  • the administered antibody can eliminate or inhibit or prevent the binding of the target (eg CD73) to its endogenous ligand (eg SIRP ⁇ ) to which it naturally binds.
  • the antibody binds to the target and regulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with CD73 expression, activity, and/or signaling.
  • antibodies having heavy and light chain CDRs having amino acid sequences as described in Table 2 can be administered to a subject.
  • the disease or disorder associated with abnormal CD73 expression may be cancer.
  • diseases or disorders associated with abnormal CD73 expression, activity, and/or signaling include hematological cancers and/or solid tumors.
  • Hematological cancers include, for example, leukemia, lymphoma, and myeloma.
  • certain forms of leukemia include acute lymphocytic leukemia (ALL); acute myelogenous leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); myeloproliferative Diseases/neoplastics (MPDS); and myelodysplastic syndromes.
  • certain forms of lymphoma include Hodgkin's lymphoma, low-grade malignant and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and Large cells).
  • certain forms of myeloma include multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bence-Jones myeloma.
  • Solid tumors include, for example, breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanomas, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.
  • Symptoms associated with cancer and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, frequent local inflammation, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue, and abdomen Symptoms such as abdominal pain, dysentery, or constipation.
  • the therapeutically effective amount of the antibodies described herein generally relates to the amount required to achieve the therapeutic goal. As noted above, this may be a binding interaction between the antibody and its target antigen, which in some cases hinders the function of the target.
  • the amount to be administered also depends on the binding affinity of the antibody for its specific antigen, and also on the rate of clearance of the administered antibody from the body.
  • a common range of therapeutically effective doses of antibodies or antibody fragments described herein may be from about 0.1 mg/kg body weight to about 100 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 0.1 mg/kg body weight to about 0.3 mg/kg body weight.
  • the therapeutically effective dose of the antibodies described herein is from about 0.4 mg/kg body weight to about 0.6 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 0.7 mg/kg body weight to about 0.9 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 1.0 mg/kg body weight to about 2.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 2.0 mg/kg body weight to about 3.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 3.0 mg/kg body weight to about 4.0 mg/kg body weight.
  • the therapeutically effective dose of the antibodies described herein is from about 4.0 mg/kg body weight to about 5.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 5.0 mg/kg body weight to about 6.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 6.0 mg/kg body weight to about 7.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 7.0 mg/kg body weight to about 8.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 8.0 mg/kg body weight to about 9.0 mg/kg body weight.
  • the therapeutically effective dose of the antibodies described herein is from about 9.0 mg/kg body weight to about 10.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 10.0 mg/kg body weight to about 15.0 mg/kg body weight. In one embodiment, the therapeutically effective dose of the antibodies described herein is from about 15.0 mg/kg body weight to about 20.0 mg/kg body weight.
  • the general frequency of administration may range, for example, from once a day to twice a day to once every other day to once a week.
  • the effectiveness of the treatment can be determined in conjunction with any known methods for diagnosing or treating specific inflammatory-related disorders.
  • the reduction in one or more symptoms of the inflammatory-related disorder indicates that the antibody confers clinical benefit.
  • antibodies directed against CD73 can be used in methods known in the art related to CD73 localization and/or quantification (eg, for the determination of CD73 and/or both CD73 and SIRP ⁇ in appropriate physiological samples Level, for diagnostic methods, for protein imaging, etc.).
  • an antibody specific for CD73 or its derivatives, fragments, analogs or homologues, comprising an antigen-binding domain derived from an antibody is used as a pharmacologically active compound (hereinafter referred to as "Therapeutic").
  • antibodies specific for CD73 can be used to isolate CD73 polypeptides by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation.
  • Antibodies against CD73 protein (or fragments thereof) can be used to detect proteins in biological samples.
  • CD73 can be detected in a biological sample as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Coupling (ie, physically connecting) the antibody to a detectable substance can facilitate detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes Lu Minol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H.
  • the antibody according to the present disclosure can be used as a reagent for detecting the presence of CD73 or a protein fragment thereof in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Use intact antibodies or fragments thereof (eg Fab, scFv or F(ab')2).
  • label with respect to an antibody is intended to include directly labeling the antibody by coupling (ie, physically connecting) a detectable substance to the antibody, and indirectly labeling the antibody by reacting with another reagent directly labeled.
  • Examples of indirect labeling include detection of the first antibody using a fluorescently-labeled second antibody, and end-labeling of the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells and biological fluids isolated from the subject, as well as tissues, cells and fluids present in the subject. Therefore, the term “biological sample” is used to include blood and blood fractions or components, including serum, plasma, or lymph fluid.
  • the detection method of the embodiment can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo.
  • analyte mRNA in vitro detection techniques include Norhtern hybridization and in situ hybridization.
  • Analyte protein in vitro detection techniques include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence.
  • Analyte genomic DNA in vitro detection techniques include Southern hybridization. The process for performing the immunoassay is described in, for example, "ELISA: Theory and Practice: Methods in Molecular Biology", Volume 42, JRCrowther (Editor) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T .Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays", P.Tijssen, Elsevier Science Publishers, Amsterdam, 1985.
  • in vivo detection techniques for analyte proteins include the introduction of labeled anti-analyte protein antibodies into the subject.
  • the antibody can be labeled with a radioactive label, and then the presence and location of the radioactive label in the subject can be detected by standard imaging techniques.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • the principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art, see for example Remington's Pharmaceuticals: The Science and Practice and Pharmacy 19th Edition (edited by Alfonso R. Gennaro et al.) MackPub.Co., Easton,Pa.: 1995; Drug AbsorptionEnhancement: Concepts, Possibilities, Limitations, AndTrends, Harwood AcademyPublishers, Langhorne, Pa., 1994; and Peptide AndProteinDrugDelivery(AdvancesInS 4 volumes), 1991, M. Dekker, New York.
  • compositions generally include an antibody and a pharmaceutically acceptable carrier.
  • antibody fragments When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein binding domain may be preferred.
  • peptide molecules that retain the ability to bind the target protein sequence can be designed.
  • Such peptides can be chemically synthesized and/or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration .
  • Suitable carriers are described in the latest edition of Remington's Pharmaceuticals, which is a standard bibliography in the art, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to water, saline, Ringer's solution, glucose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous carriers, such as immobilized oil can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Except for any conventional media or reagents that are incompatible with the antibody, its use in the composition is envisaged.
  • the pharmaceutical composition of the embodiment is formulated to be compatible with its intended route of administration.
  • administration routes include parenteral, such as intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solutions, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate Or phosphates, and agents that regulate osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multi-dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (here water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under manufacturing and storage conditions and must be able to prevent contamination by microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • polyol eg, glycerin, propylene glycol, and liquid polyethylene glycol, etc.
  • suitable mixtures thereof for example, by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants, proper fluidity can be maintained.
  • Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sodium chloride.
  • Prolonged absorption of the composition for injection can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.
  • a sterile injectable solution can be prepared by incorporating the antibody in the required amount in a suitable solvent having one or a combination of the ingredients listed above (as needed), followed by filter sterilization.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle containing an alkaline dispersion medium and the required other ingredients from those listed above.
  • the method of preparation is vacuum drying and freeze-drying of the powder, which contains the active ingredient and any additional desired ingredients from the aforementioned sterile filtered solutions of these ingredients .
  • the compound is delivered as an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant gas such as carbon dioxide.
  • transmucosal or transdermal administration penetrants suitable for penetrating barriers are used in the formulation. Such penetrants are generally known in the art, and include detergents, bile salts, and fusidic acid derivatives as used for transmucosal administration.
  • Transmucosal administration can be achieved through the use of nasal sprays or suppositories.
  • one or more of the antibodies can be formulated as a cream, ointment, gel, or cream as is generally known in the art.
  • the compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retentive enema for transrectal delivery.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retentive enema for transrectal delivery.
  • the antibody can be prepared with a carrier that prevents it from being rapidly eliminated by the body, such as sustained/controlled release formulations, including implants and microencapsulated delivery systems.
  • a carrier that prevents it from being rapidly eliminated by the body
  • sustained/controlled release formulations including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be obvious to those skilled in the art.
  • these active ingredients can be encapsulated in microcapsules prepared by, for example, coagulation technology or by interfacial polymerization, for example, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and Nanocapsules) or hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in a large emulsion.
  • colloidal drug delivery systems eg, liposomes, albumin microspheres, microemulsions, nanoparticles and Nanocapsules
  • hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in a large emulsion eg, hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in a large emulsion.
  • Sustained-release preparations can be prepared.
  • suitable sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.
  • sustained-release bases include polyester, hydrogel (eg, poly(2-hydroxyethyl-methyl propionate), or poly(vinyl alcohol)), polylactide (US Patent No.
  • L -Copolymers of glutamic acid and ⁇ ethyl-L-glutamate non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (from lactic acid-glycolic acid copolymer and acetic acid Leuprolide composed of microspheres for injection), and poly-D-(-)-3-hydroxybutyric acid.
  • polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter time.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as described in US Patent No. 4,522,811.
  • a dosage unit form refers to a physically separable unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined amount calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier One or more of the antibodies.
  • the specification of the dosage unit form of the embodiment is indicated by and directly dependent on: the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the limitations inherent in the field of formulation of such antibodies for the treatment of individuals.
  • the pharmaceutical composition can be placed in a container, package, or dispenser together with instructions for administration.
  • compositions described herein may also contain more than one of the antibodies according to the specific conditions to be treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may, for example, contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors.
  • agents that enhance its function such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors.
  • Such molecules are suitably combined in an amount effective for the intended purpose. For example, it may be co-existed in the kit or may be co-existed in use.
  • one or more of the antibodies can be administered in combination therapy, ie, with other agents such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders In combination with inflammatory diseases).
  • agents such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders In combination with inflammatory diseases).
  • the term “combination” herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the treatment site when the second compound is started.
  • “combination” may also include the antibody of the present invention and other therapeutic agents in a kit.
  • the combination therapy may comprise one or more antibodies described herein and one or more additional therapeutic agents (eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , Enzyme inhibitors, and/or cytotoxins or cell growth inhibitors, as described in more detail below) co-formulated and/or co-administered.
  • additional therapeutic agents eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , Enzyme inhibitors, and/or cytotoxins or cell growth inhibitors, as described in more detail below.
  • Such combination therapy can advantageously utilize lower doses of the administered therapeutic agent, thus avoiding possible toxicity or complications associated with various monotherapy.
  • Preferred therapeutic agents for use in conjunction with the antibodies described herein are those agents that interfere with different stages in the inflammatory response.
  • one or more antibodies described herein can be combined with one or more additional agents such as other cytokines or growth factor antagonists (eg, soluble receptors, peptide inhibitors, small molecules, Fusions); or antibodies or antigen-binding fragments that bind other targets (eg, antibodies that bind other cytokines or growth factors, their receptors, or other cell surface molecules); and anti-inflammatory cytokines or their agonists together Formulation and/or co-administration.
  • the therapeutic agent may be a PD-1 antibody, including, but not limited to, nivolumab or pabolizumab.
  • the antibodies described herein are used as vaccine adjuvants against autoimmune disorders, inflammatory diseases, and the like.
  • the combination of adjuvants used to treat these types of disorders is suitable for use in combination with a variety of antigens derived from the targeted autoantigens, ie, self-antigens involved in autoimmunity, such as myelin basic protein; Inflammatory autoantigens, such as amyloid peptide proteins, or transplantation antigens, such as alloantigens.
  • Antigens can include peptides or polypeptides derived from proteins and fragments of any of the following: sugars, proteins, polynucleotides or oligonucleotides, autoantigens, amyloid peptide proteins, transplantation antigens, allergens or other macromolecular components . In some examples, more than one antigen is included in the antigenic composition.
  • the CD73 cDNA (Sino Biological, HG10904) was amplified by PCR using the upstream primer 5'-ctgagaggtgccagatgttgggagcttacgatttg-3' and the downstream primer 5'-tccgcctccgccgctagccttgac-3' to obtain the extracellular domain encoding human CD73 (Trp27-Lys546) .
  • the gene fragment was cloned into the downstream of the promoter of the eukaryotic expression vector pCMV3, and the tag containing 6 histidines was fused and expressed at the C-terminus (see SEQ ID NO: 1 in the sequence table).
  • mice from 6 to 8 weeks were taken, purified recombinant human CD73 extracellular domain protein was fully mixed with Freund's complete adjuvant (Sigma, Catalog No. F5881), and immunized by subcutaneous multi-point injection at a dose of each Only 50 ⁇ g. Two weeks later, it was immunized with recombinant human CD73 protein mixed with TiterMax (Sigma, catalog number: T2684) adjuvant, and alternately immunized with an expression plasmid containing the human CD73 full-length cDNA gene.
  • Freund's complete adjuvant Sigma, Catalog No. F5881
  • TiterMax Sigma, catalog number: T2684
  • the recombinant human CD73 protein or CD73 stably transfected the cell line for tail vein immunization.
  • the mice were sacrificed, and the spleen, popliteal lymph nodes, inguinal lymph nodes, and iliac lymph nodes were collected and ground in DMEM medium to obtain a B-cell-rich suspension. Take an appropriate amount of lymph node and spleen cell suspension and SP2/0, and use an electric fusion device to perform cell fusion.
  • the fusion cells were seeded in 96-well plates, placed in 5% CO 2 in DMEM complete medium containing HAT, and cultured at 37°C. Start to observe the growth of hybridoma cells in about a week, and take out the supernatant for antibody detection when it grows to more than 60%.
  • 1 ⁇ g/mL recombinant human CD73 extracellular domain protein was prepared with 0.05M pH9.0 bicarbonate buffer, added to a 96-well microplate (Costar, catalog number: 9018) at a volume of 100 ⁇ L per well, and incubated at 4°C overnight. The next day was washed 3 times with PBS, blocked with 200 ⁇ L of 2% skimmed milk powder/PBS for 2 hours at room temperature, washed 3 times with PBS, and added 50 ⁇ L of hybridoma supernatant.
  • CD73 positive clone supernatants detected by ELISA were used to further verify the binding to hCD73-CHO-1C11 stable transfected cell line with high expression of CD73 and human lung degenerative cancer cells (Calu6, ATCC HTB-56) by FACS.
  • Add CD73 positive cells to 96-well U-shaped plate at 5 ⁇ 10 4 /well add 50 ⁇ L of hybridoma supernatant to each well, incubate at 4 °C for 60 minutes, centrifuge to aspirate the supernatant, and wash with 0.5% BSA/PBS.
  • CD73 monoclonal antibody that binds to the CD73 distal membrane domain through full-length IgG or F(ab')2, which can inhibit the membrane-bound type or bind to magnetic beads
  • the CD73 enzyme activity but only the monovalent binding ability of Fab can not inhibit CD73 enzyme activity.
  • variable region gene of antibody 11F11 was synthesized, cloned into a plasmid containing human IgG1 constant region, and transiently transfected in HEK293 Expression, purified antibody 11F11-huG1 was obtained by Protein A.
  • the anti-CD73 hybridoma supernatant and control antibody 11F11-huG1 obtained by immunization of mice were tested for the ability to inhibit the hydrolase activity of CD73 on the cell membrane surface and sCD73 in the solution through the following two experiments.
  • the CD73 antibody purification positive clones obtained at 25 ⁇ g / mL of the initial concentration, with TM buffer (25mM Tris, pH7. 5, 5mM MgCl2) 1:5 equal dilution of 5 concentrations, mix it with cells and incubate in a 37°C incubator for 20 minutes. After the incubation, the supernatant was discarded, washed twice with PBS, centrifuged at 1500 rpm for 5 minutes, the experimental group was added with 100 ⁇ L of 180 ⁇ M AMP (Sigma, Catalog No.
  • TM buffer to dilute 10 concentration gradients in equal ratio of 1:3.
  • incubate 75 ⁇ L of antigen hCD73-cHis and 25 ⁇ L of antibodies of different concentrations at 37°C for 1 hour then each Add 100 ⁇ L of AMP/TM buffer to the well and incubate at 37°C for 1 hour, then pipette 50 ⁇ L of supernatant and mix with an equal volume of 60 ⁇ M ATP/TM buffer in a 96-well black enzyme plate.
  • the recombinant human, cynomolgus monkey, and mouse CD73 extracellular domain proteins were diluted with the coating solution to a concentration of 1 ⁇ g/mL, added to the enzyme plate at 100 ⁇ L/well, and overnight at 4°C. The next day, PBS was washed 3 times, 200 ⁇ L of 2% skimmed milk powder/PBS was blocked at room temperature for 2 hours, and PBS was washed 3 times. Add 100 ⁇ L of anti-hCD73 antibody with different concentrations, and wash with PBST and PBS three times after incubating for 1 hour at room temperature. Add 100 ⁇ L of secondary antibody Anti-hIgG Fc HRP (brand Jackson Cat. No.
  • the above heavy chain and light chain variable region sequence fragments are amplified by PCR, and the heavy chain variable region is cloned into a vector containing a human heavy chain constant region and regulatory elements to express the complete IgG heavy chain in mammalian cells.
  • the light chain variable region was cloned into a vector containing a human light chain constant region and regulatory elements to express the complete kappa light chain in mammalian cells. After correct sequencing, it was transfected into HEK293-6E mammalian cells, IgG was expressed and secreted into the medium, and the supernatant was pooled, filtered, and purified.
  • IgG Purify IgG by Protein A chromatography, load the culture supernatant on a suitable size Protein A column, wash with 50mM Tris-HCl pH8.0, 250mM NaCl, and 0.1M Glycine-HCl (pH3.0) IgG eluted. The protein was ultrafiltered and concentrated using a concentration tube (Millipore), OD280 was detected, and the concentration of IgG was determined by spectrophotometry.
  • Table 1 CD73 antibody affinity and inhibition of CD73 hydrolase activity
  • Calu6 cells were seeded into 24-well plates at 1 ⁇ 10 5 cells/well and placed in a 37° C., 5% CO 2 incubator overnight. The next day, add the complete medium containing the antibody to be detected, incubate for 0, 30 minutes, 60 minutes, 120 minutes, 240 minutes, and 480 minutes respectively, then aspirate the supernatant, wash twice with pre-chilled PBS, and digest with trypsin Collect cells.
  • the CD73 abundance on the cell surface was detected by flow cytometry: first, the collected cells were blocked with 3% BSA/PBS for 30 minutes, and a 1:300 diluted secondary antibody solution (anti-human IgGFc-AF647, Jackson ImmunoResearch, Cat. No.
  • mAb22C12 mouse antibody light chain is mouse kappa IGKV10-96#01, select the human Vk1-39 with the highest homology to its framework region for CDR transplantation, the mouse antibody heavy chain is IGHV1-50#01, select the human germline gene IGHV1-2 for CDR transplantation;
  • mAb6G8 mouse source The antibody light chain is mouse kappa IGKV6-15#01, select human IGKV_1_33 or IGKV6-15 with high homology to its framework region for CDR transplantation, the mouse antibody heavy chain is IGHV8S9#01, select human germline gene IGHV2-70 Carry out CDR transplantation.
  • the computer was used to perform homology modeling to analyze the framework amino acid sequence of the CDR region and its surroundings to avoid the concentrated distribution of charge or hydrophobic regions on the surface of the molecule caused by the selected human germline genes; And entropy value, analyze the key amino acid individuals that may interact with CD73 and maintain the spatial framework in the gene sequence of each positive monoclonal antibody, and design the reversion mutation site on this basis.
  • the affinity of the CD73 antibody was measured on the Biacore T200 using plasma surface resonance.
  • dilute the antibody concentration to 0.5 ⁇ g/mL with 1 ⁇ HBS-EP buffer (prepared from HBS-EP+Buffer 10 ⁇ , GE, BR1008-26), and coat the Protein A chip (GE with a flow rate of 10 ⁇ L per minute , 29-1275-56), the capture time is about 40 seconds, and the control packet value is about 100.
  • Dilute the recombinant human CD73 extracellular domain protein to 3 ⁇ g/mL in 1 ⁇ HBS-EP buffer, make two-fold gradient dilution, a total of 6 concentrations, each concentration gradient is 200 ⁇ L.
  • TM buffer incubated at 37°C. Incubate in the box for 60 minutes. After incubation, centrifuge at 1500 rpm for 5 minutes, and 50 ⁇ L of supernatant was mixed with an equal volume of 60 ⁇ M ATP (brand: Sigma, catalog number: A6419-1G) solution in a 96-well black microtiter plate. After reacting at 37°C for 15 minutes, 100 ⁇ L of Celltiter Glo solution (Promega, Cat. No. 7570) was added, placed on a well-plate shaker and shaken at 300 g for 2 minutes, and kept in the dark for 10 minutes.
  • 60 ⁇ M ATP brand: Sigma, catalog number: A6419-1G
  • the fluorescence value was detected with a microplate reader (BioTek Synergy HT).
  • the results showed that all the humanized antibodies of the present invention significantly inhibited CD73 activity on the cell membrane surface compared to the control antibody 11F11-huG1 (FIG. 6A).
  • the purified humanized antibody was started at 60 ⁇ g/mL, diluted 1:3 with TM buffer (25 mM Tris, pH 7.5, 5 mM MgCl2) 1:5, and 75 ⁇ L of antigen hCD73-cHis was diluted in a 96-well plate. Mix with 25 ⁇ L antibodies of different concentrations, incubate at 37°C for 1 hour, then add 100 ⁇ L of AMP/TM buffer to each well and incubate at 37°C for 1 hour.
  • TM buffer 25 mM Tris, pH 7.5, 5 mM MgCl2
  • Calu6 cells were seeded into a 24-well plate at 1 ⁇ 10 5 cells/well and placed in a 37° C. 5% CO 2 incubator for overnight cultivation. The next day, add complete medium containing 10 ⁇ g/mL of antibody to be detected, incubate at 4°C for 60 minutes, discard the supernatant, wash twice with complete medium, transfer to 37°C 5% CO 2 incubator, and incubate 0 After 30 minutes, 120 minutes, and 240 minutes, the supernatant was aspirated, washed twice with cold PBS, and digested with trypsin to collect the cells.
  • the CD73 abundance on the cell surface was detected by flow cytometry: first, the collected cells were blocked with 3% BSA/PBS for 30 minutes, and a 1:300 diluted secondary antibody solution (anti-human IgGFc-AF647, Jackson ImmunoResearch, Cat. No. 109) was added -606-170), incubate on ice for 45 minutes, wash twice to wash away the excess secondary antibody, and finally resuspend the cells with 100 ⁇ L of PBS solution containing 5 ⁇ g/mL PI, and detect by flow cytometry.
  • the results show that the Anti-CD73 antibodies can effectively mediate CD73 endocytosis on the cell surface (see Table 4, Figure 7).
  • Dilute anti-human PD1 antibody Nivolumab and anti-human CD73 antibody (6G8 VHv8-VKv4) with X-vivo15 medium to the desired concentration add 50 ⁇ L/well to the cells separately, set up the combined drug test well, that is, add anti-human PD1 antibody at the same time 50 ⁇ L/well of anti-human CD73 antibody, only medium was added to the control wells, and the volume of all experimental wells was made up to 200 ⁇ L.
  • the well plate was placed in a 5% CO 2 37°C constant temperature incubator for 96 hours. According to the kit instructions, the content of TNF- ⁇ (Human TNFa ELISA Kit, BD OptEIA, Catalog No.

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Abstract

本发明提供了抗CD73单克隆抗体及其制备方法和应用。

Description

一种抗CD73单克隆抗体及其应用
CD73是一种具有5’核苷酸水解酶活性的糖蛋白，也称为胞外5’核苷酸酶(NT5E)，通过糖基-磷脂酰肌醇(GPI)锚定于细胞膜表面。细胞膜上的CD73由于GPI锚被酶切，会导致CD73从细胞表面脱落，释放到血液中形成可溶性CD73(sCD73)。细胞膜上的CD73和sCD73均具有水解酶活性，可以催化细胞外基质中的单磷酸腺苷(Adenosine monophosphate，简称AMP)，脱磷酸生成腺苷(Adenosine，简称ADO)。而产生的ADO通过细胞膜上的腺苷受体(A1R，A2AR，A2BR和A3R)，可以引起一系列的生物学效应，如扩张血管、提高内皮屏障功能、刺激血管新生、抑制血小板聚集、减少自由基产生等。
CD73广泛表达于多种人体组织，包括：肠道、肾脏、脑、肝、心脏、肺、呼吸道上皮、脾、淋巴结和骨髓等。免疫系统中，CD73表达于淋巴细胞、调节T细胞(Treg)、中性粒细胞、骨髓来源的抑制性细胞(MDSC)、树突状细胞、自然杀伤细胞和巨噬细胞。作为ADO生成中的限速酶，CD73是免疫细胞周围环绕的ADO晕(ADO halo)的决定因素之一，ADO halo在局部形成了免疫抑制环境。
在肿瘤患者血液中sCD73浓度显著高于正常人(Huang Q等，2017；Hay C等2015)，CD73是决定肿瘤微环境中ADO的含量水平的关键酶之一(Augusto等，2013年)，肿瘤组织CD73的过表达通常与较差的总体生存率或肿瘤进展相关(Allard D等，2016；Wang R等，2017)。ADO途径被认为是肿瘤微环境中主要的抑制途径之一。随着肿瘤微环境中ADO含量的增加，作为抗炎症介质的ADO会导致免疫细胞的浸润减弱，从而产生了对抗肿瘤免疫反应的慢性抑制。肿瘤细胞产生的肿瘤微环境变化，进一步通过炎症细胞分泌的细胞因子促进肿瘤细胞生长，并抑制免疫细胞抗肿瘤活性，促进癌症的发生和发展。阻遏细胞表面CD73和释放到血液中的sCD73的核苷酸水解酶，有助于消除肿瘤微环境中高浓度腺苷对免疫杀伤的抑制效应。研究显示，与正常小鼠相比，CD73敲除小鼠显著增加了抗肿瘤的免疫能力(Stagg等人，2011年，2012年)。
通过单克隆抗体或小分子阻断CD73的生物学活性，已证实可以减缓肿瘤细胞的生长和增殖(Stagg J等2010，Zhi X等2010，Terp MG等2013)，降低新生血管生成(Koszalka P等2014，Burghoff S等2014)，显示CD73具有作为癌症治疗潜在治疗目标的价值(Zhang B.2010，Beavis,P.A.等2012，Allard B等2014，Hay CM等2016)。申请人开发了一种靶向人CD73的新型单克隆抗体，不仅能抑制膜结合型CD73和sCD73的水解酶活性，并且结合到CD73以后，可以通过受体介导的细胞内吞作用，有效降低细胞膜表面CD73丰度，从而抑制ADO的生成。将CD73阻断治疗与其它免疫分子调节剂(如PD-1抗体)联合后，表现出显著优于单药的疗效，显示CD73酶活阻断抗体与其它免疫分子调节剂的联合治疗是一种极具吸引力的选择，有望成为抗癌领域的新型生物疗法。
发明内容
在一方面，本公开涉及特异性结合CD73的抗体或其抗原结合部分。
在一方面，本公开提供了一种结合人CD73的抗体或其抗原结合部分，其结合人分化决定簇(CD73)，抑制细胞膜CD73水解酶活性，同时抑制血清、体液中可溶性CD73(sCD73)的水解酶活性，并且介导细胞表面CD73发生内吞，有效降低细胞膜表面CD73丰度。
根据前述任一方面的抗体或其抗原结合部分，其中所述抗体是抗体片段。
根据前述任一方面的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分阻断CD73的水解酶活性。
根据前述任一方面的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分是人源化的。
含有如前述任一方面的核酸的载体。
含有如前述任一方面的载体的细胞。
包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和可药用载体的药物组合物或试剂盒。
治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物，任选地，所述方法还包括向所述哺乳动物施用治疗有效量的抗PD-1抗体。
治疗哺乳动物中与CD73异常产生有关的疾病的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物，任选地，所述方法还包括向所述哺乳动物施用治疗有效量的抗PD-1抗体。
前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中与CD73异常产生有关的疾病的药物中的用途。
前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中癌症的药物中的用途。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于抑制CD73水解酶活性的药物中的用途。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于制备介导细胞表面CD73内吞的试剂中的用途。
附图说明
图1：重组人、食蟹猴和小鼠CD73胞外区蛋白在HEK293细胞中的表达。
图2：人CD73稳转CHO细胞的流式细胞仪检测结果。
图3：图3A.CD73阳性克隆与Calu6细胞的结合；图3B.CD73阳性克隆与hCD73-CHO-1C11细胞的结合。
图4：图4A.CD73抗体对Calu6细胞表面CD73酶活性的抑制作用；图4B.CD73抗体对溶液中CD73酶活性的抑制作用。
图5：图5A.CD73嵌合抗体与重组人CD73蛋白的结合；图5B.CD73嵌合抗体与重组食蟹猴CD73蛋白的结合。
图6：图6A.抗CD73抗体人源化之后抑制Calu6细胞表面CD73酶活；图6B.抗CD73抗体人源化后抑制可溶性CD73酶活。
图7：人源化抗CD73抗体介导细胞表面的CD73内吞。
图8：外周血PBMC混合淋巴反应中，人源化抗CD73抗体与PD-1抗体单独或联用对T淋巴细胞活化的作用，图8A.T淋巴细胞活化后释放INF-γ；图8Β.T淋巴细胞活化后释放TNF-α。
具体实施方式
I.定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在一个方面，本文提供的是特异性结合CD73(例如，人CD73)的抗体(例如，单克隆抗体)及其抗原结合片段。在具体的方面，本文提供的是特异性结合人CD73的单克隆抗CD73抗体，其中所述抗CD73抗体包括亲本抗体的变体。在具体的方面，本文提供的是特异性结合CD73(例如，人CD73)的抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗CD73抗体(例如，重链可变区的框架区中5-13个氨基酸取代)，与没有所述修饰的亲本抗体相比，其保持与抗原的亲和力。在某些方面，在体内或在体外、或在体内与在体外两者，这样的抗CD73抗体CD73的水解酶活性。
在某些实施方式中，本文描述的抗体或抗原结合片段可以包含不是天然存在于动物或哺乳动物(例如，人类)体内的抗体种系全集之内的序列。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
人CD73也称为“分化簇73”或“CD73”或胞外-5'-核苷酸酶或5-原-核糖核苷酸磷酸水解酶，EC3.1.3.5，由NT5E基因编码，展示5'-核苷酸酶(尤其是AMP-、NAD-、和NMN-核苷酸酶)活性。CD73催化嘌呤5-原单核苷酸在中性pH下向核苷酸的转化，优选的底物是AMP。该酶由通过糖基磷脂酰肌醇键连接到质膜外表面的2个相同70-kD亚基的二聚体组成。人CD73前蛋白(单体)的氨基酸序列，包括氨基酸1-26的信号序列，在基因库(Genbank)中以登录号NP_00251显示，其全部披露内容通过引用结合在此。
在本文的上下文中，“中和CD73的酶活性”是指抑制CD73的5'-核苷酸酶(5'-胞外核苷酸酶)活性的过程，既包括细胞表面的CD73，也包括游离状态的CD73。这尤其包括抑制CD73介导的腺苷产生，即抑制CD73介导的将AMP分解代谢成腺苷。这可以例如在无细胞测定中进行测量，该无细胞测定测量测试化合物抑制AMP直接或间接转化为腺苷的能力。在一个实施方式中，抗体制剂引起AMP向腺苷的转化降低至少50％，AMP向腺苷的转化降低至少70％，或AMP向腺苷的转化降低至少80％，参考例如本文所述的测定法。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性(参见，例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
抗体或抗原的“功能片段、变体、衍生物或类似物”等以及它们的多种形式等短语和术语是指具有与全长目的抗体或抗原在性质上相同的生物活性的化合物或分子。例如，抗CD73抗体的功能片段或类似物是可结合CD73分子的片段或类似物，或是可防止或基本上降低CD73活性的片段或类似物。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
如本文所用，术语“抗体”被用于最宽泛的含义，具体涵盖单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、携带一个或多个CDR或源自CDR序列的抗体片段或合成多肽，只要这些多肽表现出所需的生物活性。抗体(Abs)和免疫球蛋白(Igs)是具有相同结构特征的糖蛋白。“抗体”也可指免疫球蛋白和免疫球蛋白片段，无论天然的或者部分或全部合成(例如重组)产生的，包括其至少包含免疫球蛋白分子的部分可变区的保留全长免疫球蛋白的结合特异性能力的任何片段。因此，抗体包括具有与免疫球蛋白抗原结合结构域(抗体结合位点)同源或基本上同源的结合结构域的任何蛋白。抗体包括抗体片段，例如抗肿瘤干细胞抗体片段。如本文所用，因此术语抗体包括合成抗体、重组产生的抗体、多特异性抗体(例如双特异性抗体)、人抗体、非人抗体、人源化抗体、嵌合抗体、胞内抗体以及抗体片段，例如但不限于Fab片段、Fab'片段、F(ab’)2片段、Fv片段、二硫键连接的 Fv(dsFv)、Fd片段、Fd’片段、单链Fv(scFv)、单链Fab(scFab)、双抗体、抗独特型(抗Id)抗体、或者上述任何抗体的抗原结合片段。本文所提供的抗体包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)的成员(“类型”和“种类”、以及“亚型”和“亚类”在本文中可互换使用)。天然或野生型(即得自未人工操纵的群体成员)抗体和免疫球蛋白通常为约150,000道尔顿的异四聚体糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条重链的一端具有可变结构域(V H)，随后是多个恒定结构域。每条轻链的一端具有可变结构域(V L)，另一端具有恒定结构域。所谓“未人工操纵”意指未经旨在使其含有或表达外来抗原结合分子的处理。野生型可指一个群体中发现的最普遍的等位基因或种类或指得自未操纵动物的抗体，相比较于等位基因或多态型，或得自以某种形式的操纵例如诱变、使用重组方法等改变该抗原结合分子的氨基酸的变体或衍生物。
正如本文所使用，“抗CD73抗体”意指可特异结合本文所定义的CD73的抗体或源自这些抗体的多肽(衍生物)，其包括但不限于抑制或实质降低CD73与其受体的结合或抑制CD73活性的分子。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成(参看Kabat等人Sequences of Proteins of Immunological Interest，NationalInstitute of Health，Bethesda，MD(1987))。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
本发明所用的术语“绞链”或“绞链区”系指包含抗体的第一和第二恒定结构域之间的氨基酸的柔性多肽。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab’)2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd’片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology,Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M -1的Ka)抗原的抗体。“功能片段”或“抗CD73抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab′)2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(V H-V L二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定V H-V L二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的F v的一半)，仍可具有识别和结合靶的能力。
“单链Fv”、“sF v”或“scab”抗体片段包括抗体的V H和V L结构域，其中这些结构域位于单条多肽链上。一般而言，所述F v多肽还包括一段位于V H和V L区域之间的多肽连接体，其通常为柔性分子，可使sFv形成适合于靶结合的所需结构。
术语“双抗体(diabody)”系指具有两个抗原结合位点的抗体片段，这些片段可包括与同一多肽链的轻链可变结构域(V L)相连的重链可变结构域(V H)。通过使用过短的连接体使得同一条链上的两个可变结构域无法配对，所述双抗体结构域被迫与另一条链的结合结构域配对，生成两个抗原结合位点。
Fab片段含有轻链的可变结构域和恒定结构域以及重链的可变和第一个恒定结构域(CH1)。Fab′片段与Fab片段的不同之处在于前者在CH1结构域的羧基端加入数个残基，以包括一个或多个来自抗体铰链区的半胱氨酸。通过裂解位于F(ab′)2胃蛋白酶消化产物的铰链半胱氨酸处的二硫键，可生成F ab′片段。对抗体另外进行酶处理和化学处理可生成其它目的功能片段。
术语“线性Fab”系指Miller等所述之四价抗体(Miller等人(2003)，JImmunol.170：4854-4861)。“线性Fab”是由一连串相同的CH1-VH结构域组成的，在每一个CH1-VH位置与相同的轻链配对。研发了这些分子是为了增加抗体的效价并透过亲合力效应来增强其功能性亲和力，但是，它们是单特异性的。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57,912-917；和Nelson et al.,J Clin Pathol(2000),53,111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的(见例如，Harlow&Lane，1988)。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
如本文所用，dsFv指具有稳定VH-VL对的工程化分子间二硫键的Fv。
如本文所用，scFv片段指包含通过多肽接头以任何顺序共价连接的可变轻链(VL)和可变重链(VH)的抗体片段。接头长度使得两个可变结构域基本不干扰地桥接。示例性接头是分散有一些Glu或Lys残基以增加溶解性的(Gly-Ser)n残基。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab')2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不 超过一个或两个。因此，本发明所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，可变结构域或可变区是抗体重链或轻链的特定Ig结构域，其包含在不同抗体之间变化的氨基酸序列。每条轻链和每条重链分别具有一个可变区结构域VL和VH。可变结构域提供抗原特异性，并且因此负责抗原识别。每个可变区包含CDR和框架区(FR)，CDR是抗原结合位点结构域的部分。
如本文所用，“抗原结合结构域”和“抗原结合位点(antigen-binding site)”同义地用来指识别并与同种(cognate)抗原物理相互作用的抗体内的结构域。天然的常规全长抗体分子具有两个常规抗原结合位点，每个包含重链可变区部分和轻链可变区部分。常规抗原结合位点包含连接可变区结构域内反向平行的β链的环。抗原结合位点可以包含可变区结构域的其他部分。每个常规抗原结合位点包含3个来自重链的高变区和3个来自轻链的高变区。高变区也称为互补决定区(CDR)。
如本文所用，VH结构域的功能区是保留完整VH结构域的至少部分结合特异性(例如通过保留完整VH结构域的一个或多个CDR)的完整VH结构域的至少一部分，从而所述VH结构域的功能区单独地或者与另一抗体结构域(例如VL结构域)或其区域组合地结合抗原。示例性VH结构域的功能区是包含VH结构域的CDR1、CDR2和/或CDR3的区域。
如本文所用，VL结构域的功能区是保留完整VL结构域的至少部分结合特异性(例如通过保留完整VL结构域的一个或多个CDR)的完整VL结构域的至少一部分，从而所述VL结构域的功能区单独地或者与另一抗体结构域(例如VH结构域)或其区域组合地结合抗原。示例性VL结构域的功能区是包含VL结构域的CDR1、CDR2和/或CDR3的区域。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1x10 8M -1或更大的亲和常数Ka(或者1x10 -7M或1×10 -8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11:54；Englebienne(1998)Analyst.123:1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定(参见，例如，Paul,ed.,Fundamental Immunology,2nd ed.,Raven Press,New York,pages 332-336(1989)；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7,229,619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335)。
如本文所用，关于抗体的术语“竞争”是指第一抗体或其抗原结合片段以与第二抗体或其抗原结合片段足够相似的方式结合一个表位，由此第一抗体与其关联表位的结合结果在存在第二抗体的条件下与不存在第二抗体的条件下相比可检测地降低。或者，在第二抗体与其表位的结合在存在第一抗体条件下也可检测地降低的情况中，可以但不必需是这种情况。也就是说，第一抗体可以抑制第二抗体与其表位的结合，而不用第二抗体抑制第一抗体与其各自表位的结合。然而，在每个抗体均可检测地抑制另一抗体与其关联表位或配体的结合的情况中，无论是相同、更高或更低程度，所述抗体被称为彼此“交叉竞争”结合其各自的表位。竞争及交叉竞争抗体均涵盖在本发明中。无论这种竞争或交叉竞争发生的机制如何(例如位阻、构象改变或者结合共同表位或其片段)，本领域技术人员基于本发明提供的教导将意识到这种竞争和/或交叉竞争抗体涵盖在本发明中且可用于本发明揭示的方法中。
如本文所用，“多肽”指共价连接的两个或更多个氨基酸。术语“多肽”和“蛋白质”在本文中可交换使用。
“分离的蛋白质”、“分离的多肽”或“分离的抗体”指所述蛋白质、多肽或抗体(1)不与在其天然状态下伴随其天然相关成分关联，(2)不含来自相同物种的其它蛋白质，(3)由来自不同物种的细胞表达，或(4)不在天然中发生。因此，经化学合成的多肽或在不同于多肽的天然来源细胞的细胞系统中合成的多肽将会与其天然相关成分"分离"。还可通过分离以使蛋白质实质上不含天然相关成分，即使用本领域众所周知的蛋白质纯化技术。
在肽或蛋白中，合适的保守氨基酸取代是本领域技术人员已知的，并且一般可以进行而不改变所得分子的生物活性。通常，本领域技术人员认识到多肽的非必需区中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson et al.,Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。
如本文所用，关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而所述启动子调控或介导所述核酸的转录。
如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
亦提供本文所述序列表中所述序列的“保守序列修饰”，即不消除由核苷酸序列编码或含有氨基酸序列的抗体与抗原的结合的核苷酸及氨基酸序列修饰。这些保守序列修饰包括保守核苷酸及氨基酸取代以及核苷酸及氨基酸添加及缺失。例如，可通过本领域已知的标准技术(例如定点诱变及PCR介导的诱变)将修饰引入本文所述的序列表中。保守序列修饰包括保守氨基酸取代，其中氨基酸残基被替换为具有类似侧链的氨基酸残基。具有类似侧链的氨基酸残基的家族是本领域中已有定义的。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、具有β分枝侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及具有芳香族侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，抗CD73抗体中的预测的非必需氨基酸残基优选被来自同一侧链家族的另一氨基酸残基替代。鉴定不消除抗原结合的核苷酸及氨基酸保守取代的方法为本领域所熟知(例如，参见Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人，Protein Eng.12(10):879-884(1999)；及Burks等人，Proc.Natl.Acad.Sci.USA 94:412-417(1997))。
作为另一选择，在另一实施方案中，可通过例如饱和诱变沿抗CD73抗体编码序列的全部或一部分随机引入突变，且可针对改良的结合活性筛选所得经修饰抗CD73抗体。
对于核酸而言，术语“实质同源性”表示，两个核酸或其指定序列在最佳比对及比较(其中适当插入或缺失核苷酸)时有至少约80％的核苷酸、通常至少约90％至95％、且更优选至少约98％至99.5％的核苷酸相同。作为另一选择，当若区段在选择性杂交条件下与链的互补物杂交，则存在实质同源性。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
“密码子优化”是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个或更多个密码子同时维持该天然氨基酸序列而修饰核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此，可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在www.kazusa.orjp/codon/上可获得的密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。参见，Nakamura Y.等，“Codon usage tabulated from the international DNA sequence databases:status for the year 2000.Nucl.Acids Res.，28:292(2000)”。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”是指哺乳动物，例如人。
II.具体实施方式
在一个具体实施方式中，本公开提供了结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:6-8、16-18、26-28、36-38、46-48、56-58、66-68、76-78、86-88、96-98、106-108、116-118、126-128、131-133、136-138、141-143、146-148或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:11-13、21-23、31-33、41-43、51-53、61-63、71-73、81-83、91-93、101-103、111-113、121-123、151-153、156-158、161-163、166-168、171-173或任何变体的轻链CDR。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:6、16、26、36、46、56、66、76、86、96、106、116、126、131、136、141、146或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:7、17、27、37、47、57、67、77、87、97、107、117、127、132、137、142、147或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:8、18、28、38、48、58、68、78、88、98、108、118、128、133、138、143、148或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:11、21、31、41、51、61、71、81、91、101、111、121、151、156、161、166、171或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:12、22、32、42、52、62、72、82、92、102、112、122、152、157、162、167、172或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:13、23、33、43、53、63、73、83、93、103、113、123、153、158、163、168、173或其任何变体的轻链CDR3。
根据前述任一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:5、15、25、35、45、55、65、75、85、95、105、115、125、130、135、140、145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:10、20、30、40、50、60、70、80、90、100、110、120、150、155、160、165、170或其任何变体的轻链可变区。
编码根据前述任一方面的抗体或其抗原结合部分的核酸分子，其包含选自SEQ ID NO:9、19、29、39、49、59、69、79、89、99、109、119、129、134、139、144、149或其任何变体的核酸序列，和/或选自SEQ ID NO:14、24、34、44、54、64、74、84、94、104、114、124、154、159、164、174或其任何变体的核酸序列，优选地，所述核酸分子包含核酸序列SEQ ID NO:149和核酸序列SEQ ID NO:174。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:125或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:125或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:155或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:125或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:160或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:125或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:165或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:125或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:170或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:130或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:130或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:155或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:130或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:160或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:130或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:165或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:130或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:170或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:135或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:135或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:155或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:135或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:160或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:135或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:165或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:135或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:170或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:155或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:160或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:165或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:170或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:155或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:160或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:165或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:170或其任何变体的轻链可变区。
一种结合人CD73的抗体或其抗原结合部分，其包含分别处于选自下组的重链及轻链可变区对中的三个重链可变区CDR及三个轻链可变区CDR：(a)SEQ ID NO:125与150(b)SEQ ID NO:125与155；(c)SEQ ID NO:130与150；(d)SEQ ID NO:130与155；(e)SEQ ID NO:135与160；(f)SEQ ID NO:135与165；(g)SEQ ID NO:140与160；(h)SEQ ID NO:140与165；(i)SEQ ID NO:145与160；(j)SEQ ID NO:145与170。
一种结合人CD73的抗体或其抗原结合部分，其包含(a)分别包含SEQ ID NO:126-128的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:151-153的轻链CDR1、CDR2及CDR3序列；(b)分别包含SEQ ID NO:126-128的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:156-158的轻链CDR1、CDR2及CDR3序列；(c)分别包含SEQ ID NO:131-133的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:151-153的轻链CDR1、CDR2及CDR3序列；(d)分别包含SEQ ID NO:131-133的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:156-158的轻链CDR1、CDR2及CDR3序列；(e)分别包含SEQ ID NO:136-138的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:161-163的轻链CDR1、CDR2及CDR3序列；(f)分别包含SEQ ID NO:136-138的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:166-168的轻链CDR1、CDR2及CDR3序列；(g)分别包含SEQ ID NO:141-143的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:161-163的轻链CDR1、CDR2及CDR3序列；(h)分别包含SEQ ID NO:141-143的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:166-168的轻链CDR1、CDR2及CDR3序列；(i)分别包含SEQ ID NO:146-148的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:161-163的轻链CDR1、CDR2及CDR3序列；(j)分别包含SEQ ID NO:146-148的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:171-173的轻链CDR1、CDR2及CDR3序列。
结合人CD73的抗体或其抗原结合部分，其与前述任一方面的抗体或其抗原结合部分具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性。
编码如前述任一方面的抗体或其抗原结合部分的核酸分子，或与其具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸分子。
在一方面，本公开还包括试剂盒，例如所述试剂盒包括本公开的抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的辍合物，以及抗体使用说明书、杀死特定类型细胞的辍合物等等。该说明书可包括在体外、体内或离体使用抗体、辍合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。 这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物，任选地，所述方法还包括向所述哺乳动物施用治疗有效量的抗PD-1抗体。
治疗哺乳动物中与CD73异常产生有关的疾病的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物，任选地，所述方法还包括向所述哺乳动物施用治疗有效量的抗PD-1抗体。
前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中与CD73异常产生有关的疾病的药物中的用途。
前述任一方面的抗体或其抗原结合部分或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中癌症的药物中的用途。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于抑制CD73水解酶活性的药物中的用途。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于制备介导细胞表面CD73内吞的试剂中的用途。根据前述任一方面，抑制CD73水解酶活性是指抑制膜结合的CD73的水解酶活性，或者抑制游离的CD73的水解酶活性，或者既能抑制膜结合的CD73的水解酶活性，又能抑制游离的CD73的水解酶活性。
本发明之抗体可用于治疗哺乳动物。在一个实施方案中，例如，出于获得临床前数据的目的，将目的抗体或等效物施用给非人哺乳动物。示例性的待治疗非人哺乳动物，包括非人灵长类动物、狗、猫、啮齿类动物以及其它哺乳动物，其中进行了临床前研究。这种哺乳动物可为需用抗体治疗的疾病的建立的动物模型，或可用于研究目的抗体的毒性。在每个这样的实施方案中，可在哺乳动物中进行剂量递增研究。
抗体，无论有无第二个组分(如与之缀合的治疗剂部分)，无论是单独或与细胞毒性因子组合施用，均可作为治疗剂使用。本发明涉及以抗体为基础的疗法，其中保留给动物、哺乳动物或人施用本发明之抗体，以治疗CD73介导的疾病、障碍或状况。
本发明所使用的术语"治疗"是指治疗性治疗和预防性或预防措施。它指的是预防、治愈、逆转、减弱、改善、最小化、抑制或停止疾病状态、疾病进程、疾病致病因素(如细菌或病毒)或其它异常状况的有害效应。
因此，本发明还包含多价抗体，包括双特异抗CD73抗体，其具有与之相连的诊断或治疗功能的效应分子、原子或其它物质。例如，抗体可具有放射性诊断标签或放射性细胞毒性原子或金属或细胞毒性物质如蓖麻毒蛋白链，与之相连用于癌症的体内诊断或治疗。
此外，本发明的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。
相应地，本发明还提供包含本发明的抗CD73的抗体或其片段的组合物，所述抗体方便地和可药用的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本发明所使用的术语"药物组合物"系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。
本发明所使用的术语"障碍"系指任何能够得益于本发明抗体治疗的状况。这包括慢性和急性障碍或疾病，包括那些倾向于使哺乳动物，尤其是人染上所述障碍的病理状况。本文中非限制性的有待治疗的障碍之实例包括癌症、炎症、自身免疫疾病、感染、心血管疾病、呼吸疾病、神经疾病以及代谢性疾病。
本发明所使用的术语“癌症”系指或描述了哺乳动物尤其是人的生理状况，其典型特点是细胞无调控地生长。癌症的实例包括但不限于癌(carcinoma)、淋巴瘤、母细胞瘤、肉瘤和白血病。
本发明的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。
编码如前述任一方面的抗体或其抗原结合部分的核酸。
含有如前述任一方面的核酸的载体。
含有如前述任一方面的载体的细胞。
包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和可药用载体的药物组合物。
治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合部分或其编码核酸。
治疗哺乳动物中与CD73异常产生有关的疾病之方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合部分或其编码核酸。
应当理解，根据所述实施方案的治疗剂将与合适的载体、赋形剂、以及其它被掺入制剂中以提供改善的转移、递送、耐受性等的试剂一同施用。大量适当的制剂可见于所有药物化学工作者已知的药典中：Remington's Pharmaceutical Sciences(第15版，Mack Publishing Company,Easton,Pa.(1975))，特别是其中Blaug、Seymour的第87章。这些制剂包括例如粉末、糊剂、膏剂、凝胶剂、蜡、油、脂质、含脂质(阳离子或阴离子)载体(例如Lipofectin TM)、DNA缀合物、无水吸浆、水包油和油包水乳液、乳液聚乙二醇(各种分子量的聚乙二醇)、半固态凝胶以及含有聚乙二醇的半固态混合物。任何前述混合物均可适用于根据本发明的治疗或疗法，条件是制剂中的活性成分不被制剂灭活并且制剂在生理学上是相容的并耐受给药途径。
CD73异常产生或异常CD73表达在本文中互换使用。在一个实施方案中，可将所述抗体用作治疗剂。此类试剂将通常用于治疗、缓解和/或预防受试者的与异常CD73表达、活性和/或信号传导相关的疾病或病理。可使用标准方法通过鉴定受试者，例如患有(或处于风险或发展)与异常CD73表达、活性和/或信号传导相关的疾病或障碍，例如癌症或其它赘生性障碍的人患者来实施治疗方案。将抗体制剂，优选对其靶抗原有高特异性和高亲和性的抗体制剂施用给受试者并且将通常因其与靶标结合而产生效应。施用的抗体可消除或抑制或妨碍靶标(例如CD73)的表达、活性和/或信号传导功能。施用的抗体可消除或抑制或妨碍靶标(例如CD73)与其所天然结合的内源性的配体(例如SIRPα)结合。例如，抗体与靶标结合并调节、阻断、抑制、减少、拮抗、中和、或以其它方式妨碍CD73表达、活性和/或信号传导。在一些实施方案中，为治疗与异常CD73表达相关的疾病或障碍，可将具有重链和轻链CDR(具有如表2所述的氨基酸序列)的抗体施用给受试者。在一个实施方案中，与异常CD73表达相关的疾病或障碍可为癌症。
作为非限制性示例，与异常CD73表达、活性和/或信号传导相关的疾病或障碍包括血液学癌症和/或实体瘤。血液学癌症包括例如白血病、淋巴瘤和骨髓瘤。作为非限制性示例，某些形式的白血病包括急性淋巴细胞性白血病(ALL)；急性髓性白血病(AML)；慢性淋巴细胞性白血病(CLL)；慢性髓细胞性白血病(CML)；骨髓增生性疾病/赘生物(MPDS)；和骨髓增生异常综合征。作为非限制性示例，某些形式的淋巴瘤包括霍奇金氏淋巴瘤、低度恶性和侵袭性非霍奇金氏淋巴瘤、伯基特淋巴瘤、以及滤泡性淋巴瘤(小细胞和大细胞)。作为非限制性示例，某些形式的骨髓瘤包括多发性骨髓瘤(MM)、巨细胞性骨髓瘤、重链骨髓瘤和轻链或Bence-Jones骨髓瘤。实体瘤包括例如乳腺肿瘤、卵巢肿瘤、肺肿瘤、胰腺肿瘤、前列腺肿瘤、黑色素瘤、结直肠肿瘤、肺肿瘤、头颈肿瘤、膀胱肿瘤、食管肿瘤、肝肿瘤和肾瘤。
与癌症及其它赘生性障碍相关的症状包括例如发炎、发烧、全身不适、发烧、疼痛、经常局部发炎、食欲不振、体重减轻、浮肿、头痛、疲乏、皮疹、贫血、肌无力、肌肉疲劳和腹部症状例如腹痛、痢疾或便秘。
本文所述抗体的治疗有效量通常涉及实现治疗目标所需的量。如上指出，这可为抗体及其靶抗原之间的结合相互作用，在某些情况下妨碍靶标的功能。需要施用的量此外取决于抗体对其特异性抗原的结合亲和力，并且还取决于所施用抗体从体内清除的速率。作为非限制性的示例，本文所述抗体或抗体片段的治疗有效剂量的常见范围可为约0.1mg/kg体重至约100mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约0.1mg/kg体重至约0.3mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约0.4mg/kg体重至约0.6mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约0.7mg/kg体重至约0.9mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约1.0mg/kg体重至约2.0mg/kg体重。在一 个实施方案中，本文所述抗体的治疗有效剂量为约2.0mg/kg体重至约3.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约3.0mg/kg体重至约4.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约4.0mg/kg体重至约5.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约5.0mg/kg体重至约6.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约6.0mg/kg体重至约7.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约7.0mg/kg体重至约8.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约8.0mg/kg体重至约9.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约9.0mg/kg体重至约10.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约10.0mg/kg体重至约15.0mg/kg体重。在一个实施方案中，本文所述抗体的治疗有效剂量为约15.0mg/kg体重至约20.0mg/kg体重。一般的给药频率的范围可为例如从每日一次至每日两次到隔日一次到每周一次。
可结合任意已知的用于诊断或治疗特定炎性相关的障碍的方法确定治疗的有效性。炎性相关的障碍的一个或多个症状减轻指示抗体赋予了临床益处。
在另一个实施方案中，针对CD73的抗体可用于本领域中已知的与CD73定位和/或定量相关的方法(例如，用于测定适当生理样品中的CD73和/或CD73和SIRPα两者的水平，用于诊断方法，用于蛋白成像等等)。在一个给定实施方案中，对CD73或其衍生物、片段、类似物或同系物具有特异性的、包含源于抗体的抗原结合结构域的抗体，被用作药物学活性化合物(下文称为“治疗剂”)。
在另一个实施方案中，可通过标准技术例如免疫亲和、色谱或免疫沉淀，使用对CD73具有特异性的抗体来分离CD73多肽。针对CD73蛋白质的抗体(或其片段)可用于检测生物样品中的蛋白质。在一些实施方案中，在生物样品中可检测CD73作为临床测试过程的一部分，例如，用于确定给定治疗方案的功效。将抗体偶联(即物理连接)到可检测物质可有利于检测。可检测物质的示例包括各种酶、辅基、荧光材料、发光材料、生物发光材料和放射性材料。合适的酶的示例包括辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶；合适的辅基复合物的示例包括链霉亲和素/生物素和亲和素/生物素；合适的荧光材料的示例包括伞形酮、荧光素、异硫氰酸荧光素、罗丹明、二氯三嗪氨荧光素、丹磺酰氯或藻红蛋白；发光材料的一个示例包括鲁米诺；生物发光材料的示例包括荧光素酶、荧光素和水母蛋白，并且合适放射性材料的示例包括 125I、 131I、 35S或 3H。
在另一个实施方案中，根据本公开的抗体可用作检测样品中CD73或其蛋白质片段)存在的试剂。在一些实施方案中，抗体包含可检测标记。抗体为多克隆抗体，或更优选单克隆抗体。使用完整的抗体或其片段(例如Fab、scFv或F(ab')2)。关于抗体的术语“标记”旨在包括通过将可检测物质偶联(即物理连接)到该抗体来直接标记该抗体，以及通过与直接标记的另一种试剂反应来间接标记该抗体。间接标记的示例包括使用荧光标记的第二抗体检测第一抗体，以及用生物素进行末端标记抗体，以便能够用荧光标记的链霉亲和素进行检测。术语“生物样品”旨在包括从受试者分离的组织、细胞和生物学流体，以及受试者体内存在的组织、细胞和流体。因此，使用的术语“生物样品”包括血液和血液中的级分或组分，包括血清、血浆、或淋巴液。换言之，所述实施方案的检测方法可用于在体外及体内检测生物样品中的分析物mRNA、蛋白质或基因组DNA。例如，分析物mRNA体外检测技术包括Norhtern杂交和原位杂交。分析物蛋白质体外检测技术包括酶联免疫吸附测定(ELISA)、Western印迹、免疫沉淀、以及免疫荧光。分析物基因组DNA体外检测技术包括Southern杂交。用于进行免疫测定的过程描述于例如“ELISA:Theory and Practice:Methods in Molecular Biology”，第42卷，J.R.Crowther(编辑)Human Press,Totowa,N.J.,1995；“Immunoassay”,E.Diamandis和T.Christopoulus,Academic Press,Inc.,San Diego,Calif.,1996；以及“Practice and Theory of Enzyme Immunoassays”,P.Tijssen,Elsevier Science Publishers,Amsterdam,1985。此外，分析物蛋白质的体内检测技术包括向受试者体内导入标记的抗分析物蛋白抗体。例如，可以用放射性标记标记抗体，然后可以通过标准成像技术检测受试者体内该放射性标记物的存在和位置。
可将本文所述抗体和其衍生物、片段、类似物和同系物掺入适于施用的药物组合物中。制备此类组合物所涉及的原理和考虑事项以及选择组分的指南在本领域中是熟知的，例如参见Remington's Pharmaceutical Sciences:The Science And Practice Of Pharmacy第19版(Alfonso R.Gennaro等人编辑)Mack Pub.Co.,Easton,Pa.:1995；Drug Absorption Enhancement:Concepts,Possibilities,Limitations,And Trends,Harwood Academic Publishers,Langhorne,Pa.,1994；以及Peptide And Protein Drug Delivery(Advances In Parenteral Sciences，第4卷),1991,M.Dekker,New York。
此类组合物通常包含抗体和药学上可接受的载体。当使用抗体片段时，与靶蛋白结合结构域特异性结合的最小抑制片段可为优选的。例如，基于抗体的可变区序列，可以设计保留结合靶蛋白质序列能力的肽分子。此类肽可化学合成和/或通过重组DNA技术产生(参见例如Marasco等人，Proc.Natl.Acad.Sci.USA,90:7889-7893(1993))。
如本文所用，术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适载体描述于最新版的Remington's Pharmaceutical Sciences中，这是本领域的标准参考书目，其以引用方式并入本文。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
将所述实施方案的药物组合物配制成与其预期施用途径相容。给药途径的示例包括肠胃外，例如静脉内、皮内、皮下、经口(例如吸入)、经皮(即局部的)、经粘膜和直肠给药。用于肠胃外、皮内或皮下施用的溶液或悬浮液可包括以下组分：注射用无菌稀释剂例如水、盐溶液、固定油、聚乙二醇类、甘油、丙二醇或其它合成溶剂；抗细菌剂，例如苄醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如乙二胺四乙酸(EDTA)；缓冲剂，例如乙酸盐、柠檬酸盐或磷酸盐、以及调节渗透压的试剂，例如氯化钠或右旋糖。pH可用酸或碱进行调节，例如盐酸或氢氧化钠。可将肠胃外制剂包装在安瓿、一次性注射器或玻璃或塑料制多剂量小瓶内。
适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的载体包括生理盐水、抑菌水、Cremophor EL TM(BASF，Parsippany,N.J.)或磷酸盐缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的并且应当为流动性达到易于注射的程度。其在制造和储存条件下必须是稳定的并且必须能防止微生物例如细菌和真菌的污染作用。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。例如通过利用涂层例如卵磷脂，在分散体情况下维持所需颗粒尺寸，以及利用表面活性剂，可以保持适宜的流动性。对微生物作用的防止可以通过各种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在许多情况下，将优选在组合物中包含等渗剂，例如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。注射用组合物的延长吸收可通过在所述组合物中包含延缓吸收的试剂例如单硬脂酸铝和明胶来达到。
根据需要，可以通过将抗体以所需量掺入具有上文所列成分中的一种或组合(按需要)的合适溶剂中来制备无菌注射溶液，然后过滤消毒。一般来讲，通过将抗体掺入含有碱性分散介质和上文所列那些中的所需其它成分的无菌载体中来制备分散体。就用于制备无菌注射溶液的无菌粉末而言，制备方法是获得粉末的真空干燥和冷冻干燥，该粉末包含活性成分和任何另外的期望成分，它们来自前述的这些成分的无菌过滤溶液。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、 胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种所述抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，所述抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
例如，这些活性成分可胶囊包封于例如通过凝聚技术或通过界面聚合法制备的微胶囊中，例如分别在胶体给药系统(例如，脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊剂)或大乳液中的羟甲基纤维素或明胶微胶囊和聚(甲基丙烯酸甲酯)微胶囊。
可制备缓释制剂。适宜的缓释制剂的示例包括含有抗体的固体疏水聚合物的半透性基质，该基质是成型制品形式例如膜或微胶囊。缓释基质的示例包括聚脂、水凝胶(例如，聚(2-羟乙基-甲基丙酸酯)、或聚(乙烯醇))、聚交酯(美国专利No.3,773,919)、L-谷氨酸和γ乙基-L-谷氨酸盐的共聚物、不可降解的乙烯-乙酸乙烯酯、可降解乳酸-乙醇酸共聚物例如LUPRON DEPOT TM(由乳酸-乙醇酸共聚物和醋酸亮丙瑞林构成的注射用微球)、和聚-D-(-)-3-羟基丁酸。虽然聚合物例如乙烯-乙酸乙烯酯和乳酸-乙醇酸能够释放分子100天以上，但是一些水凝胶释放蛋白质的时间却较短。
脂质体混悬剂(包括用针对病毒抗原的单克隆抗体靶向受感染细胞的脂质体)也可以用作药学上可接受的载体。这些可根据本领域技术人员已知的方法制备，例如在美国专利No.4,522,811中有所描述。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种所述抗体。所述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
所述药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种所述抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种所述抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症、自身免疫性障碍和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本发明的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
与本文所述抗体联合使用的优选治疗剂是干扰炎症应答中不同阶段的那些试剂。在一个实施方案中，可将本文所述一种或多种抗体与一种或多种附加试剂例如其它细胞因子或生长因子拮抗剂(例如，可溶受体、肽抑制剂、小分子、配体融合物)；或结合其它靶标的抗体或抗原结合片段(例如，结合其它细胞因子或生长因子、其受体、或其它细胞表面分子的抗体)；以及抗炎性细胞因子或其激动剂共同配制和/或共同施用。在一些实施方案中，所述治疗剂可以是PD-1抗体，包括但不限于，纳武单抗或帕博利珠单抗。
在其它实施方案中，本文所述抗体被用作针对自身免疫性障碍、炎性疾病等的疫苗佐剂。用于治疗这些类型障碍的佐剂的组合适于与各种各样的抗原联合使用，所述抗原来自于所靶向的自体抗原，即参与自身免疫的自身抗原，例如髓磷脂碱性蛋白；炎性自体抗原，例如淀粉状肽蛋白、或移植抗原，例如同种抗原。抗原可包括衍生自蛋白质的肽或多肽以及以下任一项的片段：糖、蛋白质、多核苷酸或寡核苷酸、自身抗原、淀粉样肽蛋白、移植抗原、过敏原或其它大分子组分。在一些例子中，多于一种抗原被包含在抗原性组合物中。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本发明的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
1、CD73重组蛋白在真核细胞的表达
以上游引物5’-ctgagaggtgccagatgttgggagcttacgattttg-3’，下游引物5’-tccgcctccgccgctagccttgatccgaccttcaac-3’，对CD73的cDNA(Sino Biological，HG10904)进行PCR扩增，获得编码人CD73胞外域(Trp27-Lys546)的基因片段。将该基因片段克隆到真核表达载体pCMV3的启动子下游，同时将含6个组氨酸的标签融合表达在C端(参见序列表中SEQ ID NO:1)。由于食蟹猴CD73胞外区和人CD73胞外区同源性非常高，采用突变引物和overlapping PCR，得到表达食蟹猴CD73胞外区序列(Trp27-Lys546)的真核表达载体(参见序列表中SEQ ID NO:2)。同样，以购买的小鼠CD73cDNA质粒基因pMD18-CD73(Sino Biological，MG50231)为模板，设计上游引物5’-ctgagaggtgccagatgttgggagctcacgatcctg-3’，下游引物5’-tccgcctccgccgctagccttgatccgcccttcaacg-3’，将编码小鼠CD73基因(Trp29-Lys549)克隆到真核表达质粒系统(参见序列表中SEQ ID NO:3)。上述质粒分别转染HEK293.6E细胞5-7天后，收集培养基上清液，经镍螯合柱纯化得到重组人CD73胞外区(hCD73)、重组猴CD73胞外区(cynoCD73)或重组小鼠CD73胞外区蛋白(muCD73)。SDS-PAGE结果见图1。
2.人CD73稳转CHO细胞株的构建
使用Lenti-Pac HIV慢病毒包装试剂盒(广州复能基因有限公司，货号：HPK-LvTR-20)，将含有人CD73cDNA全长序列(参见SEQ ID NO:4)的慢病毒载体，和包装质粒共转染293T细胞，进行慢病毒包装。转染48小时后收集上清，以10000*g离心10分钟去除细胞碎片，获得含慢病毒的培养上清，以0.45μm的PES过滤膜过滤后，取10μL用于感染1x10 6CHO细胞，在培养基中加入10μg/mL puromycin进行筛选。两周后检测CHO转化细胞的CD73表达，收集1x10 5细胞，加入0.5μg/mL抗-hCD73抗体(R&D system，货号NBP2-48480)，冰上孵育45分钟，用0.5％BSA/PBS洗2次，加入50μL 1:300稀释的二抗溶液(抗-小鼠IgGFc-AF647，Jackson ImmunoResearch)，冰上孵育45分钟后洗去多余二抗，用100μL PBS重悬细胞用流式细胞仪检测FL-4通道荧光信号。将得到的CHO-CD73阳性克隆进行有限稀释，最终得到稳定表达人全长CD73分子的单克隆细胞株hCD73-CHO-1C11。结果见图2。
3、小鼠免疫和杂交瘤克隆筛选
取6～8周雌性Balb/c小鼠，以纯化得到的重组人CD73胞外区蛋白与弗式完全佐剂(Sigma，货号：F5881)充分混合后，皮下多点注射进行免疫，剂量为每只50μg。两周后采用重组人CD73蛋白混合TiterMax(Sigma，货号：T2684)佐剂免疫，与自主构建的含有人CD73全长cDNA基因的表达质粒交替进行免疫。检测小鼠血清中产生的CD73抗体滴度达到1:50000后，用重组人CD73蛋白或CD73稳转细胞株进行尾静脉冲击免疫。三天后处死小鼠，收集小鼠脾脏、腘淋巴结、腹股沟淋巴结和髂淋巴结，在DMEM培养基中研磨得到富含B细胞的悬浮液。取适量的淋巴结和脾细胞的混悬液和SP2/0混合，用电融合仪进行细胞融合。将融合细胞接种于96孔板中，在含有HAT的DMEM完全培养基中置于5％CO 2，37℃条件下培养。一周左右开始观察杂交瘤细胞的生长情况，待其长至60％以上时取出上清供抗体检测。
4.杂交瘤上清的筛选
用0.05M pH9.0碳酸氢盐缓冲液配制1μg/mL重组人CD73胞外区蛋白，按照100μL每孔的体积加入96孔酶标板中(Costar，货号：9018)，4℃中孵育过夜，次日PBS洗涤3次，用200μL 2％脱脂奶粉/PBS室温封闭2小时，PBS洗涤3次，加入50μL杂交瘤上清，室温孵育1小时后，用PBST和PBS重复洗涤3次，加入二抗(抗-mouse IgG-Fc-HRP，Jackson ImmunoResearch，货号：115-035-071)孵育1小时，重复洗涤3次后加入50μL显色液(TMB溶液，Sigma货号：T2885)，37℃放置5分钟后加入50μL 2M浓硫酸溶液终止反应，立即放于酶标仪中读取OD450的值。
将ELISA检测得到的约1200个CD73阳性克隆上清，进一步用FACS验证与高表达CD73的hCD73-CHO-1C11稳转细胞株和人肺退行性癌细胞(Calu6，ATCC HTB-56)的结合。将CD73阳性细胞分别按5×10 4/孔加入96孔U型板中，每孔再加入50μL杂交瘤上清，4℃孵育60分钟，离心吸弃上清，用0.5％BSA/PBS洗涤，再加入50μL二抗溶液(抗-mouse IgG-Fc-AF647，Jackson ImmunoResearch货号：115-606-071)，4℃孵育45分钟，之后用0.5％BSA/PBS洗去多余的二抗，最后用60μL含有1μg/mL荧光染料(碘化丙啶，PI，Sigma货号：P4170)的PBS溶液重悬细胞，上流式细胞仪检测。结果见图3A、图3B。
5、CD73抗体对CD73水解酶活性的抑制作用
Bryan C等(AACR 2016，US9605080)发现了一种CD73的单克隆抗体，通过全长IgG或F(ab’)2结合到CD73远膜端的结构域，可以抑制膜结合型或结合在磁珠上的CD73酶活性，而只有单价结合能力的Fab不能抑制CD73酶活性。根据公开发表的序列(序列135，GenBank:ATL10689.1；序列8，GenBank:ATL10563.1)，合成抗体11F11的可变区基因，并克隆到含有人IgG1恒定区的质粒，在HEK293进行瞬转表达，通过Protein A得到纯化的抗体11F11-huG1。
小鼠免疫得到的抗CD73杂交瘤上清和对照抗体11F11-huG1，通过下面两个实验，分别验证对细胞膜表面CD73和溶液中sCD73的水解酶活性抑制能力。
细胞膜表面CD73水解酶活性的抑制实验
在96孔U型板中按照3x10 4个细胞/孔接种人肺退行性癌细胞Calu6，将纯化得到的CD73阳性克隆抗体以25μg/mL为起始浓度，用TM缓冲液(25mM Tris，pH7.5，5mM MgCl2)1：5等比稀释5个浓度，将其与细胞混合后在37℃培养箱中孵育20分钟。孵育结束后，弃上清，并用PBS洗2次，1500rpm，离心5分钟，实验组每孔加入100μL 180μM AMP(Sigma，货号01930-5G)溶液，对照组加入等量TM缓冲液，37℃培养箱中孵育60分钟。孵育结束后，1500rpm，离心5分钟，取50μL上清与等体积60μM ATP(品牌：Sigma，货号：A6419-1G)溶液在96孔黑色酶标板中混合。37℃反应15分钟后加入100μL Celltiter Glo溶液(Promega，货号7570)，置于孔板振荡器上300g震荡2分钟，避光静置10分钟，用酶标仪(BioTek Synergy HT)检测荧光值，结果见图4A。
可溶性CD73水解酶活性的抑制实验
将抗CD73抗体从60μg/mL为开始用TM buffer按照1:3等比稀释10个浓度梯度，在96孔板中将75μL抗原hCD73-cHis和25μL不同浓度抗体在37℃孵育1小时，然后每孔加入100μL AMP/TM缓冲液37℃孵育1小时，然后吸取50μL上清与等体积60μM ATP/TM缓冲液在96孔黑色酶标板中混合。37℃反应15分钟后加入100μL Celltiter Glo溶液，置于孔板振荡器300g震荡2分钟，避光静置10分钟，最后用酶标仪检测荧光值。结果见图4B。
6、CD73抗体的种属特异性
用包被液分别稀释重组人、食蟹猴和小鼠的CD73胞外区蛋白至浓度为1μg/mL，以100μL/孔加入酶标板，4℃过夜。次日PBS洗涤3次，用200μL 2％脱脂奶粉/PBS室温封闭2小时，PBS洗涤3次。加入100μL不同稀释浓度的抗-hCD73抗体，室温孵育1小时后用PBST和PBS重复洗涤3次。加入100μL二抗Anti-hIgG Fc HRP(品牌Jackson货号109-035-098)室温孵育1小时。重复洗涤3次后加入50μL显色液(底物缓冲液9.5mL，TMB溶液0.5mL，3％H 2O 210μL，混匀，现配现用)，37℃放置5分钟后立即加入50μL 2M浓硫酸溶液终止反应，立即放于酶标仪(BioTek Synergy HT)中读取OD450的值。结果见图5A、图5B。
7、CD73抗体DNA序列的克隆
选取10个CD73阳性克隆(表1)，采用TRNzol裂解法提取候选杂交瘤单克隆细胞的RNA，然后利用逆转录试剂盒(Invitrogen，18080051)合成单链cDNA后，以此为模板扩增杂交瘤单克隆细胞中抗体的可变区序列。将扩增产物测序，得到候选杂交瘤重链和轻链可变区序列如下：
克隆2D4：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000001
重链可变区核酸序列
Figure PCTCN2020071179-appb-000002
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000003
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000004
克隆2H2：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000005
重链可变区核酸序列
Figure PCTCN2020071179-appb-000006
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000007
Figure PCTCN2020071179-appb-000008
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000009
克隆6A8：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000010
重链可变区核酸序列
Figure PCTCN2020071179-appb-000011
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000012
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000013
克隆6G8：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000014
重链可变区核酸序列
Figure PCTCN2020071179-appb-000015
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000016
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000017
克隆7E10：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000018
重链可变区核酸序列
Figure PCTCN2020071179-appb-000019
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000020
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000021
克隆8A8：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000022
重链可变区核酸序列
Figure PCTCN2020071179-appb-000023
Figure PCTCN2020071179-appb-000024
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000025
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000026
克隆10D9：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000027
重链可变区核酸序列
Figure PCTCN2020071179-appb-000028
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000029
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000030
克隆14E4：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000031
重链可变区核酸序列
Figure PCTCN2020071179-appb-000032
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000033
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000034
克隆14H10：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000035
重链可变区核酸序列
Figure PCTCN2020071179-appb-000036
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000037
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000038
克隆22C12：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000039
Figure PCTCN2020071179-appb-000040
重链可变区核酸序列
Figure PCTCN2020071179-appb-000041
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000042
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000043
克隆30A11：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000044
重链可变区核酸序列
Figure PCTCN2020071179-appb-000045
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000046
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000047
克隆36A10：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000048
重链可变区核酸序列
Figure PCTCN2020071179-appb-000049
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000050
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000051
上述重链和轻链可变区序列片段经PCR扩增，将重链可变区克隆入含有人重链恒定区和调节元件的载体，以在哺乳动物细胞中表达完整的IgG重链。类似地，将轻链可变区克隆入含有人轻链恒定区和调节元件的载体，以在哺乳动物细胞中表达完整的kappa轻链。经测序正确后转染入HEK293-6E哺乳动物细胞中，IgG经表达分泌入培养基中，合并收集上清，过滤后纯化。采用Protein A层析纯化IgG，将培养上清液加载于大小合适的Protein A柱子上，用50mM Tris-HCl pH8.0，250mM NaCl洗涤，用0.1M Glycine-HCl(pH3.0)将结合的IgG洗脱下来。利用浓缩管(Millipore)将蛋白超滤浓缩，检测OD280，通过分光光度法测定IgG的浓度。
Figure PCTCN2020071179-appb-000052
表1：CD73抗体的亲和力以及对CD73水解酶活性的抑制
8、CD73抗体介导的CD73细胞内吞
将Calu6细胞按照1x10 5细胞/孔接种至24孔板中，置于37℃，5％CO 2培养箱培养过夜。次日，加入含有待检测抗体的完全培养基，在分别孵育0、30分钟、60分钟、120分钟、240分钟、480分钟后吸除上清，用预冷PBS清洗2次后用胰酶消化收集细胞。细胞表面CD73丰度经流式细胞仪进行检测：首先将收集的细胞用3％BSA/PBS封闭30分钟，加入1:300稀释的二抗溶液(抗-人IgGFc-AF647，Jackson ImmunoResearch，货号109-606-170)，冰上孵育45分钟，洗涤2次将多余二抗洗去，最后用100μL含5μg/mL PI的PBS溶液重悬细胞，用流式细胞仪检测。结果见表2。
Figure PCTCN2020071179-appb-000053
表2：CD73抗体介导的CD73细胞内吞
9、抗CD73抗体的人源化
把选择的鼠源单克隆抗体可变区序列与人抗体序列进行比对，找出适合的同源性高的人胚系基因序列进行CDR移植；例如，mAb22C12鼠源抗体轻链为小鼠kappa IGKV10-96#01，选择与其框架区同源性最高的人Vk1-39进行CDR移植，鼠源抗体重链为IGHV1-50#01，选择人胚系基因IGHV1-2进行CDR移植；mAb6G8鼠源抗体轻链为小鼠kappa IGKV6-15#01，选择与其框架区同源性高的人IGKV_1_33或IGKV6-15进行CDR移植，鼠源抗体重链为IGHV8S9#01，选择人胚系基因IGHV2-70进行CDR移植。随后同时利用计算机进行同源建模，分析CDR区及其周边的框架氨基酸序列，避免选择的人胚系基因造成分子表面电荷或疏水区域集中分布；同时，通过计算静电力，范德华力，亲疏水性和熵值，分析各阳性单克隆抗体基因序列中可能与CD73作用以及维护空间构架的关键氨基酸个体，在此基础上设计回复突变位点。
hu22C12VHv9：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000054
重链可变区核酸序列
Figure PCTCN2020071179-appb-000055
hu22C12VHv10：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000056
重链可变区核酸序列
Figure PCTCN2020071179-appb-000057
hu6G8VHv1：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000058
重链可变区核酸序列
Figure PCTCN2020071179-appb-000059
hu6G8VHv2：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000060
重链可变区核酸序列
Figure PCTCN2020071179-appb-000061
hu 6G8VHv8：
重链可变区氨基酸序列
Figure PCTCN2020071179-appb-000062
重链可变区核酸序列
Figure PCTCN2020071179-appb-000063
hu22C12Vkv2：
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000064
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000065
hu22C12Vkv3：
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000066
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000067
hu 6G8Vkv1：
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000068
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000069
hu 6G8Vkv2：
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000070
Figure PCTCN2020071179-appb-000071
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000072
hu 6G8VkV4：
轻链可变区氨基酸序列
Figure PCTCN2020071179-appb-000073
轻链可变区核酸序列
Figure PCTCN2020071179-appb-000074
将设计得到的多个轻、重链衍生物分别进行DNA合成(苏州金唯智生物科技公司)，克隆到含有抗体kappa链恒定区或人IgG1恒定区CH1-CH3的真核表达载体，质粒轻重链配对后转染HEK293.6E细胞，表达5-7天，收取培养上清，Protein A柱纯化后测定浓度和纯度。纯化得到的人源化抗体用ELISA、FACS测定与CD73阳性细胞的结合、用Biacore测定与CD73的亲和力、抗体对CD73酶活的抑制等，以检测人源化抗体的亲和力及生物学活性。
10、等离子表面共振测定人源化抗CD73抗体的结合特性
CD73抗体的亲和力测定采用等离子表面共振在Biacore T200上测定。室温条件下，用1×HBS-EP缓冲液(从HBS-EP+Buffer 10×配制，GE，BR1008-26)稀释抗体浓度至0.5μg/mL，以每分钟10μL流速包被Protein A芯片(GE，29-1275-56)，捕获时间约40秒，控制包被RU值100左右。以1×HBS-EP缓冲液稀释重组人CD73胞外区蛋白至3μg/mL，做两倍梯度稀释，共6个浓度，每个浓度梯度200μL。以1×HBS-EP缓冲液作为对照，流速30μl/min，结合时间120秒，解离时间600秒。亲和力采用Langmuir 1:1 Kinetic理论模型拟合，由Biacore T200 Evaluation Software进行分析(表3)。
CD73人源化抗体 ka(1/Ms) kd(1/s) KD(M)
6G8嵌合体 6.17E+05 1.11E-04 1.80E-10
6G8 VH-v1/VK-v1 3.89E+05 1.06E-04 2.72E-10
6G8 VH-v8/VK-v4 3.30E+05 8.48E-05 2.57E-10
22C12 VH-v9/Vk-v2 1.22E+06 1.65E-04 1.35E-10
22C12 VH-v10/VL-v2 1.68E+06 2.25E-04 1.34E-10
22C12 VH-v10/VL-v3 1.87E+06 1.79E-04 9.56E-11
表3.人源化抗体亲和力
11.人源化抗体对CD73水解酶活性的抑制
人源化抗体对细胞膜表面CD73水解酶活性的抑制实验
在96孔U型板中按照3x10 4个细胞/孔接种人肺退行性癌细胞Calu6，将纯化得到的人源化抗体以25μg/mL为起始浓度，用TM缓冲液(25mM Tris，pH7.5，5mM MgCl2)1：5等比稀释3个浓度。将其与细胞混合后在37℃培养箱中孵育20分钟。孵育结束后，弃上清，并用PBS洗2次，1500rpm，离心5分钟，实验组每孔加入100μL 180μM AMP(Sigma，货号01930-5G)溶液，对照组加入等量TM缓冲液，37℃培养箱中孵育60分钟。孵育结束后，1500rpm，离心5分钟，取50μL上清与等体积60μM ATP(品牌：Sigma，货号：A6419-1G)溶液在96孔黑色酶标板中混合。37℃反应15分钟后加入100μL Celltiter Glo溶液(Promega，货号7570)，置于孔板振荡器上300g震荡2分钟，避光静置10分钟，用酶标仪(BioTek Synergy HT)检测荧光值，结果表明，所有的本发明人源化抗体相比对照抗体11F11-huG1都显著抑制细胞膜表面CD73活性(图6A)。
人源化抗体对可溶性CD73水解酶活性的抑制实验
将纯化得到的人源化抗体从60μg/mL开始，用TM缓冲液(25mM Tris，pH7.5，5mM MgCl2)1：5等比稀释3个浓度，在96孔板中将75μL抗原hCD73-cHis和25μL不同浓度抗体混合，在37℃孵育1小时，然后每孔加入100μL AMP/TM缓冲液37℃孵育1小时。吸取50μL上清与等体积60μM ATP/TM缓冲液在96孔黑色酶标板中混合，37℃反应15分钟，加入100μL Celltiter Glo溶液，置于孔板振荡器300g震荡2分钟，避光静置10分钟。用酶标仪检测荧光值，结果表明，所有的本发明人源化抗体都可以抑制可溶性CD73活性，而对照抗体11F11-huG1不能抑制(图6B)。
12.人源化抗体介导的CD73细胞内吞
将Calu6细胞按照1x10 5细胞/孔接种至24孔板中，置于37℃5％CO 2培养箱培养过夜。次日，加入含有10μg/mL待检测抗体的完全培养基，在4℃孵育60分钟，弃去上清，用完全培养基洗涤2次，转入37℃5％CO 2培养箱，分别孵育0、30分钟120分钟、240分钟后吸除上清，用预冷PBS清洗2次后用胰酶消化收集细胞。细胞表面CD73丰度经流式细胞仪进行检测：首先将收集的细胞用3％BSA/PBS封闭30分钟，加入1:300稀释的二抗溶液(抗-人IgGFc-AF647，Jackson ImmunoResearch，货号109-606-170)，冰上孵育45分钟，洗涤2次将多余二抗洗去，最后用100μL含5μg/mL PI的PBS溶液重悬细胞，用流式细胞仪检测，结果表明，本发明的抗CD73抗体可以有效介导细胞表面的CD73内吞(见表4、图7)。
Figure PCTCN2020071179-appb-000075
表4：人源化抗体介导的CD73内吞
13.外周血PBMC混合淋巴反应实验
取健康志愿者30mL新鲜血液加入至15mL PBS中，混匀备用。取20mL Ficoll Paque Plus(GE Healthcare，货号17-1440-02)于50mL离心管中，然后取30ml稀释过的新鲜血液轻轻铺于Ficoll Paque Plus表面。2000rpm离心30分钟，将离心管中最上层血清吸除，吸取PBMC至一个新的50mL离心管，加入40mL PBS混匀，于4℃1300rpm离心10分钟，将上清液吸除后，再加入15mL PBS重悬细胞，计数，于4℃1300rpm离心10分钟。用X-vivo15培养基(Lonza，货号04-418Q)调整PBMC密度至4×10 6/mL，50μL/孔加入平底96孔板中。将抗人PD1抗体Nivolumab、抗人CD73抗体(6G8 VHv8-VKv4)用X-vivo15培养基稀释至目的浓度，以50μL/孔分别加入细胞中，设置联合用药实验孔，即同时加入抗人PD1抗体、抗人CD73抗体各50μL/孔，对照孔只加入培养基，所有实验孔补足体积至200μL。孔板放于5％CO 2 37℃恒温培养箱培养，培养96小时。按照试剂盒说明书，测定细胞上清中TNF-α(Human TNFa ELISA Kit，BD OptEIA，货号555212)、IFN-γ(Human IFN-γELISA Kit， BD OptEIA，货号555142)含量。结果表明，抗CD73抗体与抗PD-1抗体联合使用可以显著刺激淋巴细胞产生TNF-α(见图8A、图8B)。

Claims (11)

  1. 结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:6-8、16-18、26-28、36-38、46-48、56-58、66-68、76-78、86-88、96-98、106-108、116-118、126-128、131-133、136-138、141-143、146-148或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:11-13、21-23、31-33、41-43、51-53、61-63、71-73、81-83、91-93、101-103、111-113、121-123、151-153、156-158、161-163、166-168、171-173或任何变体的轻链CDR。
  2. 抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:6、16、26、36、46、56、66、76、86、96、106、116、126、131、136、141、146或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:7、17、27、37、47、57、67、77、87、97、107、117、127、132、137、142、147或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:8、18、28、38、48、58、68、78、88、98、108、118、128、133、138、143、148或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:11、21、31、41、51、61、71、81、91、101、111、121、151、156、161、166、171或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:12、22、32、42、52、62、72、82、92、102、112、122、152、157、162、167、172或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:13、23、33、43、53、63、73、83、93、103、113、123、153、158、163、168、173或其任何变体的轻链CDR3，任选地，所述抗体或其抗原结合部分抑制CD73水解酶活性，并介导细胞表面CD73发生内吞。
  3. 结合人CD73的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:5、15、25、35、45、55、65、75、85、95、105、115、125、130、135、140、145或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO:10、20、30、40、50、60、70、80、90、100、110、120、150、155、160、165、170或其任何变体的轻链可变区，任选地，所述抗体或其抗原结合部分抑制CD73水解酶活性，并介导细胞表面CD73发生内吞。
  4. 一种结合人CD73的抗体或其抗原结合部分，其包含分别处于选自下组的重链及轻链可变区对中的三个重链可变区CDR及三个轻链可变区CDR：(a)SEQ ID NO:125与150(b)SEQ ID NO:125与155；(c)SEQ ID NO:130与150；(d)SEQ ID NO:130与155；(e)SEQ ID NO:135与160；(f)SEQ ID NO:135与165；(g)SEQ ID NO:140与160；(h)SEQ ID NO:140与165；(i)SEQ ID NO:145与160；(j)SEQ ID NO:145与170，任选地，所述抗体或其抗原结合部分抑制CD73水解酶活性，并介导细胞表面CD73发生内吞。
  5. 一种结合人CD73的抗体或其抗原结合部分，其包含(a)分别包含SEQ ID NO:126-128的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:151-153的轻链CDR1、CDR2及CDR3序列；(b)分别包含SEQ ID NO:126-128的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:156-158的轻链CDR1、CDR2及CDR3序列；(c)分别包含SEQ ID NO:131-133的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:151-153的轻链CDR1、CDR2及CDR3序列；(d)分别包含SEQ ID NO:131-133的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:156-158的轻链CDR1、CDR2及CDR3序列；(e)分别包含SEQ ID NO:136-138的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:161-163的轻链CDR1、CDR2及CDR3序列；(f)分别包含SEQ ID NO:136-138的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:166-168的轻链CDR1、CDR2及CDR3序列；(g)分别包含SEQ ID NO:141-143的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:161-163的轻链CDR1、CDR2及CDR3序列；(h)分别包含SEQ ID NO:141-143的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:166-168的轻链CDR1、CDR2及CDR3序列；(i)分别包含SEQ ID NO:146-148的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID  NO:161-163的轻链CDR1、CDR2及CDR3序列；(j)分别包含SEQ ID NO:146-148的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:171-173的轻链CDR1、CDR2及CDR3序列，任选地，所述抗体或其抗原结合部分抑制CD73水解酶活性，并介导细胞表面CD73发生内吞。
  6. 根据前述权利要求任一项的抗体或其抗原结合部分，其包含选自SEQ ID NO:146的重链CDR1，SEQ ID NO:147的重链CDR2和SEQ ID NO:148的重链CDR3序列，以及选自SEQ ID NO:171的轻链CDR1、SEQ ID NO:172的轻链CDR2和SEQ ID NO:173的轻链CDR3序列，优选地，所述抗体或其抗原结合部分包含选自SEQ ID NO:145的重链可变区和选自SEQ ID NO:170的轻链可变区。
  7. 编码如前述权利要求任一项的抗体或其功能片段的核酸分子，和/或含有如前述任一方面的核酸的载体，和/或含有如前述任一方面的载体的细胞，和/或包含如权利要求任一项的抗体或其功能片段或其编码核酸和可药用载体的药物组合物。
  8. 一种药物组合物或试剂盒，其包含结合人CD73的抗体或其抗原结合部分和抗PD-1抗体，任选地，所述结合人CD73的抗体或其抗原结合部分是根据前述权利要求任一项的抗体或其功能片段，任选地，所述抗PD-1抗体选自纳武单抗或帕博利珠单抗。
  9. 前述权利要求任一项的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于治疗哺乳动物中与CD73异常产生有关的疾病的药物中的用途，优选地，所述哺乳动物中与CD73异常产生有关的疾病是癌症。
  10. 前述权利要求任一项的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于抑制CD73水解酶活性的药物中的用途。
  11. 前述权利要求任一项的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于制备介导细胞表面CD73内吞的试剂中的用途。
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