WO2022143951A1 - 一种功能增强型抗体阻断剂的开发及其应用 - Google Patents

一种功能增强型抗体阻断剂的开发及其应用 Download PDF

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WO2022143951A1
WO2022143951A1 PCT/CN2021/143371 CN2021143371W WO2022143951A1 WO 2022143951 A1 WO2022143951 A1 WO 2022143951A1 CN 2021143371 W CN2021143371 W CN 2021143371W WO 2022143951 A1 WO2022143951 A1 WO 2022143951A1
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variant
variable region
chain variable
heavy chain
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徐刚
陈博
王常玉
于俊涛
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康诺亚生物医药科技（成都）有限公司
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    • 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
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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Definitions

  • the present disclosure relates to the development of a function-enhanced antibody blocking agent and its application, in particular to an antibody that binds to CD70 and its application.
  • CD70 is a type II transmembrane glycoprotein that belongs to the tumor necrosis factor superfamily.
  • the mutual binding of CD70 and CD27 is called the "CD70-CD27 signaling axis", which plays an important role in the process of cell proliferation and differentiation.
  • CD70-CD27 signaling axis plays an important role in the process of cell proliferation and differentiation.
  • Studies have shown that CD70 on the surface of acute myeloid leukemia cells interacts with CD27 on the surface of T cells to stimulate the expansion of CD4 + regulatory T reg cells in the tumor microenvironment or significantly increase exhausted effector CD8 + T cells in the bone marrow proportion, leading to immune escape of the tumor.
  • CD27 is also expressed on the surface of some tumor cells, and the CD70-CD27 activation loop formed between tumor cells can prolong the survival time of tumor cells or lead to proliferation.
  • CD34 + CD38 - malignantly proliferating tumor cells and leukemia stem cells
  • soluble CD27 was significantly increased in the serum of patients, which was negatively correlated with prognosis.
  • Blocking the CD70-CD27 interaction by antibodies can induce asymmetric cell division and differentiation of leukemia stem cells, inhibit the formation of tumor colonies, and significantly prolong the survival time of tumor model mice.
  • CD70 is not expressed in normal tissues and hematopoietic progenitor cells, but is highly expressed in T-cell lymphomas, B-cell lymphomas, and a considerable portion of solid tumors. And other hematological tumor targets such as CD33 or CD123, in addition to being highly expressed in tumor cells, are also expressed in normal hematopoietic progenitor cells or normal tissues (eg: lung, prostate, skin, peripheral blood T cells). Therefore, the development of a CD70 monoclonal antibody with killing and blocking activities can specifically remove tumor cells or leukemia stem cells in patients without affecting the hematopoietic progenitor cells and hematopoietic function after recovery.
  • Acute myeloid leukemia is a group of highly heterogeneous clonal diseases that originate from excessive proliferation or differentiation of bone marrow hematopoietic stem cells, resulting in the accumulation of a large number of undifferentiated cells in the bone marrow and the rejection of blood cells and immune cells with normal functions. Extramedullary organs such as liver, spleen, and lymph nodes may be extensively infiltrated. Patients eventually die from concurrent infections or other diseases due to anemia and immunocompromised conditions. Treatment of acute myeloid leukemia has progressed slowly over the past 40 years, and the standard clinical regimen is still cytarabine plus daunorubicin, introduced in the 1970s.
  • CN200680021851.8 describes CD70 antibody with antibody Fc effect, which can mediate ADCC, ADCP and/or CDC effect, can exercise cytostatic without conjugation to cytotoxic therapeutic agent or conjugated to cytotoxic therapeutic agent , cytotoxic or immunomodulatory effects.
  • CN200680035376.X describes a CD70 antibody that binds to human CD70 protein and inhibits tumor cell growth, binds to human CD70 with a KD of 1 x 10-7 M or less, the antibody also binds to CD70-expressing renal cell carcinoma tumor cell lines bind and be internalized by it.
  • CN201280013552.5 describes a llama-derived CD70 antibody that binds with high affinity to human CD70 and exhibits an off-rate of less than 7 ⁇ 10 -4 s -1 or less, and some preferred embodiments may exhibit a high affinity for CD70 and its The potent blocking or inhibition of the interaction of the ligand CD27 is superior to the CD70 antibodies described in CN200680021851.8 and CN200680035376.X.
  • the inventors immunized mice with CD70 recombinant protein, and obtained multiple high-affinity antibodies that recognize human and cynomolgus monkey CD70 recombinant protein.
  • the antibody of the present disclosure has extremely high ADCC and CDC activities, which is obviously better than that of the CD70 antibody in the prior art, and at the same time, it can effectively block the CD70-CD27 interaction and downstream signal transduction, and the inhibitory effect on tumors is significantly stronger than that of the existing antibody.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds CD70.
  • the present disclosure provides a bispecific antibody or antigen-binding portion thereof.
  • the present disclosure provides nucleic acid molecules encoding the aforementioned CD70-binding antibodies, or antigen-binding portions thereof, or bispecific antibodies, or antigen-binding portions thereof.
  • the present disclosure provides vectors comprising the aforementioned nucleic acid molecules.
  • the present disclosure provides cells comprising the aforementioned vectors.
  • compositions or kits comprising the aforementioned antibodies, or antigen-binding portions thereof, or nucleic acid molecules encoding them, and a pharmaceutically acceptable carrier.
  • the present disclosure provides antibody-drug conjugates comprising an antibody or antigen-binding portion thereof, bispecific or multispecific molecule of any of the foregoing aspects covalently attached to a therapeutic moiety.
  • the present disclosure provides a method of treating a CD70-related disorder comprising the step of administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, and /or pharmaceutical composition.
  • the present disclosure provides an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell and/or pharmaceutical composition of any of the preceding aspects in the manufacture of a medicament or kit for the treatment of a CD70-related disorder in a subject use in.
  • the present disclosure provides an antibody or antigen-binding fragment, nucleic acid molecule, vector, cell and/or pharmaceutical composition of any of the preceding aspects in the manufacture of a medicament or kit for treating a viral infection in a subject use.
  • Figure 1 shows the results of SDS-PAGE detection of human CD70 extracellular domain protein recombinantly expressed by HEK293 cells.
  • Figure 2-1 shows the detection results of CD70 expression in human CD70/CHO stably transfected cell lines detected by flow cytometry.
  • Figure 2-2 shows the detection results of CD70 expression in monkey CD70/CHO stably transfected cell line detected by flow cytometry.
  • Figure 3 shows FACS detection of antibody titers in the serum of immunized mice.
  • Figures 4-1 and 4-2 show the results of screening for CDC activity of CD70 monoclonal antibodies.
  • FIG. 5 shows CD70 monoclonal antibody ADCC activity.
  • Figure 6-1 shows the results of FACS detection of the binding of the chimeric antibody to CHO-human CD70.
  • Figure 6-2 shows the results of FACS detection of the binding of the chimeric antibody to CHO-cynomolgus CD70.
  • Figure 7-1 shows the results of the human CD70/CD27 blocking assay of chimeric antibodies.
  • Figure 7-2 shows the results of the chimeric antibody CD70/CD27 blockade assay in cynomolgus monkeys.
  • Figure 8 shows the CDC killing effect of different humanized antibodies on tumor cells Daudi.
  • Figure 9-1 shows the results of FACS detection of the binding of humanized antibodies to CHO-human CD70.
  • Figure 9-2 shows the results of FACS detection of the binding of humanized antibodies to CHO-cynomolgus CD70.
  • Figure 10-1 shows the results of human CD70/CD27 blockade assays for humanized antibodies.
  • Figure 10-2 shows the results of cynomolgus monkey CD70/CD27 blockade assays for humanized antibodies.
  • Figure 11-1 shows the CDC killing effect of humanized antibody on tumor cell Daudi.
  • Figure 11-2 shows the CDC killing effect of humanized antibodies on tumor cells MV-4-11-CD70.
  • Figure 11-3 shows the CDC killing effect of humanized antibody on tumor cell Raji.
  • Figure 12-1 shows the ADCC killing effect of humanized antibodies on tumor cells Daudi.
  • Figure 12-2 shows the ADCC killing effect of humanized antibody on tumor cell Raji.
  • Figure 13 shows the ADCP killing effect of humanized antibodies on tumor cells Daudi.
  • Figure 14 shows inhibition of CD70-mediated stimulation of IL-8 secretion by humanized antibodies.
  • Figure 15-1 shows the effect of humanized CD70 antibody on the growth of human Burkitt's lymphoma Raji subcutaneous xenografts.
  • Figure 15-2 shows the effect of humanized CD70 antibody on body weight of tumor-bearing mice.
  • Figure 16 shows the CDC killing activity of CDC-enhanced CD70 humanized antibody on tumor cells Daudi.
  • Figures 17-1 and 17-2 show ADCC killing activity mediated by CDC-enhanced CD70 humanized antibodies in PBMCs of different Fc ⁇ RIII alleles.
  • Figure 18 shows the antitumor effect of CDC activity-enhanced CD70 antibody in the subcutaneous xenograft NOD/SCID mouse model of Daudi cell line.
  • antibodies eg, monoclonal antibodies
  • antigen-binding fragments thereof that specifically bind CD70.
  • monoclonal anti-CD70 antibodies that specifically bind CD70, wherein the anti-CD70 antibodies comprise variants of the parent antibody.
  • antibodies that specifically bind CD70 eg, human CD70.
  • anti-CD70 antibodies comprising modifications in one or more amino acid residues (eg, 5-13 amino acid substitutions in the framework regions of the heavy chain variable regions), and no such modifications Compared to the parent antibody, it retains its affinity for the antigen.
  • CD70 includes variants, isoforms, homologs, orthologs, and intraspecific homologs.
  • antibodies specific for human CD70 protein can cross-react with CD70 protein from species other than human in certain instances.
  • an antibody specific for human CD70 protein may be completely specific for human CD70 protein and may not exhibit species or other types of cross-reactivity, or may interact with species from some other species but not All other species CD70 cross-reacted (eg, with primate CD70 but not with mouse CD70).
  • human CD70 refers to human sequence CD70, such as the complete amino acid sequence of human CD70 with Genbank accession number P32970.
  • mouse CD70 refers to mouse sequence CD70, such as the complete amino acid sequence of mouse CD70 with Genbank accession number NP_035747.
  • Human CD70 sequences may differ from human CD70 of Genbank Accession No. P32970, which has substantially the same biological function as human CD70 of Genbank Accession No. P32970, by having, for example, conservative mutations or mutations in non-conserved regions.
  • one biological function of human CD70 is to bind the cytokine receptor CD27.
  • cell surface is used according to its normal meaning in the art and thus includes the exterior of the cell accessible by binding to proteins and other molecules.
  • signal transduction pathway refers to a biochemical relationship between various signal transduction molecules that play a role in the transmission of signals from one part of a cell to another.
  • cell surface receptor as used herein includes, for example, molecules and complexes of molecules capable of receiving signals and transmitting such signals across the cell membrane of a cell.
  • An example of a “cell surface receptor” of the present disclosure is the CD70 receptor.
  • the term “about” or “approximately” means within plus or minus 10% of the given value or range. Where a whole number is required, the term refers to within plus or minus 10% of the given value or range, rounded up or down to the nearest whole number.
  • the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains of 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequences the term is to be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 A nucleotide sequence of %, 99% or greater sequence identity.
  • sequence identity has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule. While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • substitutional variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position.
  • the substitutions can be single, wherein only one amino acid is substituted in the molecule, or multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be made at consecutive sites.
  • one amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions.
  • An “insertional” variant is one in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in a native sequence. Immediately adjacent to an amino acid means attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid.
  • a “deletion” variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.
  • variable domains of antibodies refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs; ie CDR1, CDR2 and CDR3) or hypervariable regions, all located within the variable domains of light and heavy chains. The more conserved portions of the variable domains are referred to as framework (FR) regions or framework sequences.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable domain of native heavy and light chains includes four FR regions, predominantly in a beta-sheet configuration, linked by three CDRs that form loops that connect the beta-sheet structure and Partial ⁇ -sheet structures are formed in some cases.
  • the CDRs of each chain are usually linked in proximity by FR regions and, with the aid of CDRs from other chains, contribute to the formation of antibody target binding sites (epitopes or determinants).
  • the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • a CDR can have the ability to specifically bind to the cognate epitope.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable regions of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising 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, eg, 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, diabodies, Fd and Fd' fragments and other fragments, including modified fragments.
  • the fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally comprise 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 the antibody framework (eg, by substituting the corresponding region), results in an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 107-108 M -1 ) to an antigen .
  • a "functional fragment” or “analog of an anti-CD70 antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments” and, in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') 2 , and so on.
  • "Fv" fragments consist of a dimer ( VH - VL dimer) formed by non-covalent association of the variable domains of a heavy chain and the variable domains of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind targets.
  • BsAb Bispecific antibody
  • a bispecific antibody and/or antigen-binding molecule Contains two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by a number of 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, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV.
  • Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
  • hybridomas refers to a cell or cell line (usually myeloma or lymphoma cells) produced by fusing antibody-producing lymphocytes and non-antibody-producing cancer cells.
  • hybridomas can proliferate and provide continuous supply to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art.
  • hybridomas When referring to the term “hybridoma” or “hybridoma cell”, it also includes subclones and progeny cells of hybridomas.
  • a full-length antibody is one that has two full-length heavy chains (eg, 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 those naturally produced by antibody-secreting B cells and those produced synthetically with the same domains.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • telomeres can be mutated amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody .
  • PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • CDR refers to a complementarity-determining region
  • each of the heavy and light chains of antibody molecules is known to have 3 CDRs.
  • the CDRs are also referred to as hypervariable regions, and are present in the variable regions of each heavy and light chain of antibodies, with sites of very high variability in the primary structure of the CDRs.
  • the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDRs of the light chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the light chain.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants typically comprise chemically active surface profiles of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1x107 M -1 or 1x108 M -1 or greater (or 1x10-7 M or 1x A dissociation constant (Kd) of 10 ⁇ 8 M or lower binds the antigen.
  • Affinity constants can be determined by standard kinetic methods of antibody responses, eg, immunoassays, 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. Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27: 335).
  • nucleic acid molecules refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • an isolated nucleic acid molecule is one that is separated 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, can 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 when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • operably linked in reference 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 such that the promoter regulates or mediates transcription of the nucleic acid.
  • conservative sequence modifications of the sequences described in the Sequence Listing described herein, ie, nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody to the antigen encoded by the nucleotide sequence or containing the amino acid sequence.
  • 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 in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are 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
  • amino acids with uncharged polar side chains amino acids e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids with non-polar side chains e.g. alanine, valine
  • leucine, isoleucine, proline, phenylalanine, methionine amino acids with beta branched side chains
  • a predicted non-essential amino acid residue in an anti-CD70 antibody is 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 abolish antigen binding are well known in the art (for example, see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12 ( 10): 879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
  • mutations can be introduced randomly along all or a portion of the anti-CD70 antibody coding sequence, eg, by saturation mutagenesis, and the resulting modified anti-CD70 antibody can be screened for improved binding activity.
  • expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine 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 followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell.
  • References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • a vector also includes a "viral vector” or “viral vector.”
  • a viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an 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. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
  • treating an individual with a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment.
  • treatment includes prevention, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease.
  • Treatment also includes any provided antibodies or antigen-binding fragments thereof and any pharmaceutical uses of the compositions provided herein.
  • therapeutic effect refers to an effect resulting from treatment of an individual that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures the disease or condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms.
  • a fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
  • the term "patient” refers to a mammal, such as a human.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds CD70, comprising an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NOs: 5, 6, 7, 15, 16, 22, 29, 30, 31, 37, 38, 44, 45, 46, 54, 55, 56, 64, 65, 77, 83, 84, 85, 91, 97, 98, 106, 107, 114, 115, 116, 124, 131, 132, 135, 142, 160 or a heavy chain CDR of any variant thereof, and/or selected from the amino acid sequence SEQ ID NO: 10, 11, 12, 19, 25, 26, 34, 41, 49, 50, 51, 59, 60, 61, 68, 69, 74, 80, 88, 101, 102, 103, 110, 111, 119, 120, 121, 127, 128 or the light chain CDRs of any variant.
  • An antibody or antigen-binding portion thereof comprising a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO: 5, 15, 29, 44, 54, 83, 96, 114, 131 or any variant thereof, selected Heavy chain CDR2 from amino acid sequence SEQ ID NO: 6, 30, 37, 45, 55, 64, 77, 84, 91, 97, 106, 115, 124, 132, 135, 142 or any variant thereof, selected from Heavy chain CDR3 of amino acid sequence SEQ ID NO: 7, 16, 22, 31, 38, 46, 56, 65, 85, 98, 107, 116 or any variant thereof; and/or selected from amino acid sequence SEQ ID NO:
  • An antibody or antigen-binding portion thereof comprising a combination of CDRs of heavy and light chains selected from the group consisting of:
  • (1) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 5, 6 and 7, and/or comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 10, 11 and 12 respectively;
  • (10) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 44, 77 and 46, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 49, 80 and 51;
  • (11) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 83, 84 and 85, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 88, 11 and 12;
  • (12) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 5, 91 and 85, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 19, 11 and 12;
  • (13) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 96, 97 and 98, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 101, 102 and 103;
  • (14) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 15, 106, 107, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 110, 111, 103;
  • (16) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 15, 124 and 16, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 127, 128 and 103;
  • the antibody or antigen-binding portion thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 13, 20, 27, 35, 42, 52, 62, 70, 75, 81, 89, 94, 104,
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 3 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 8 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 13 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 17 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 20 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 23 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 27 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 32 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 35 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 39 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 42 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 47 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 52 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 57 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 62 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 66 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 70 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 72 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO:75 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO:78 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 81 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 86 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 89 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 92 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 94 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 99 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 104 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 108 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 112 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 117 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 122 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 125 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 129 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 117 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 133 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 147 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 133 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 150 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 133 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 152 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 136 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 147 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 136 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 150 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 136 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 152 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 138 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 147 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 138 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 150 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 138 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 152 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 140 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 154 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 140 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 156 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 140 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 158 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 143 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 154 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 143 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 156 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 143 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 158 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 145 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 154 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 145 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 156 or any variant thereof light chain variable region.
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO: 145 or any variant thereof, and the heavy chain variable region of amino acid sequence SEQ ID NO: 158 or any variant thereof light chain variable region.
  • the present disclosure provides a bispecific or multispecific molecule comprising the antibody or antigen-binding portion thereof of any of the preceding aspects.
  • a nucleic acid molecule encoding an antibody or antigen-binding portion thereof or bispecific or multispecific molecule according to any of the preceding aspects.
  • a nucleic acid molecule encoding an antibody or antigen-binding portion thereof, bispecific or multispecific molecule comprising a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 4, 14, 21, 28 , 36, 43, 53, 63, 71, 76, 82, 90, 95, 105, 113, 123, 130, 134, 137, 139, 141, 144, 146, or the antibody heavy chain nucleic acid sequence of any variant thereof, and/or selected from SEQ ID NO: 9, 18, 24, 33, 40, 48, 58, 67, 73, 79, 87, 93, 100, 109, 118, 126, 148, 151, 153, 155, 157 , 159 or any variant of the antibody light chain nucleic acid sequence.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 4 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 4 or any variant thereof Sequence of NO:9 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 14 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 14 or any variant thereof Sequence of NO: 18 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 21 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 21 or any variant thereof Sequence of NO:24 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 28 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 28 or any variant thereof Sequence of NO:33 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 36 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 36 or any variant thereof Sequence of NO:40 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 43 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO:43 or any variant thereof Sequence of NO:48 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 53 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 53 or any variant thereof Sequence of NO:58 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 63 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 63 or any variant thereof Sequence of NO:67 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 71 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 71 or any variant thereof Sequence of NO:73 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 76 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 76 or any variant thereof Sequence of NO:79 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 82 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 82 or any variant thereof Sequence of NO:87 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 90 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 90 or any variant thereof Sequence of NO:93 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 95 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 95 or any variant thereof Sequence of NO: 100 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 105 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 105 or any variant thereof Sequence of NO: 109 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 113 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 113 or any variant thereof Sequence of NO: 118 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 123 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 123 or any variant thereof Sequence of NO: 126 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 130 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 130 or any variant thereof Sequence of NO: 118 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 134 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 134 or any variant thereof Sequence of NO: 148 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 134 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 134 or any variant thereof Sequence of NO: 151 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 134 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 134 or any variant thereof Sequence of NO: 153 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 137 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 137 or any variant thereof Sequence of NO: 148 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 137 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 137 or any variant thereof Sequence of NO: 151 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 137 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 137 or any variant thereof Sequence of NO: 153 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 139 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 139 or any variant thereof Sequence of NO: 148 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 139 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 139 or any variant thereof Sequence of NO: 151 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 139 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 139 or any variant thereof Sequence of NO: 153 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 141 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 141 or any variant thereof Sequence of NO: 155 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 141 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 141 or any variant thereof Sequence of NO: 157 or any variant thereof.
  • the nucleic acid molecule comprises the sequence of the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 141 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 141 or any variant thereof Sequence of NO: 159 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 144 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 144 or any variant thereof Sequence of NO: 155 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 144 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 144 or any variant thereof Sequence of NO: 157 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 144 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 144 or any variant thereof Sequence of NO: 159 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 146 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 146 or any variant thereof Sequence of NO: 155 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 146 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 146 or any variant thereof Sequence of NO: 157 or any variant thereof.
  • the nucleic acid molecule comprises the antibody heavy chain variable region nucleotide sequence of SEQ ID NO: 146 or any variant thereof, and/or the light chain variable region nucleotide sequence of SEQ ID NO: 146 or any variant thereof Sequence of NO: 159 or any variant thereof.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds CD70, which is at least greater than 60%, 65%, 70%, 75%, 80%, 85% of the antibody or antigen-binding portion thereof of any of the preceding aspects %, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the present disclosure provides nucleic acid molecules encoding, or having at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90% thereof, an antibody or antigen-binding portion thereof according to any of the preceding aspects , 95%, 96%, 97%, 98%, 99% or higher sequence identity of nucleic acid molecules.
  • the present disclosure provides a vector comprising the nucleic acid molecule of any of the preceding aspects.
  • the present disclosure provides cells comprising the vector of any of the preceding aspects.
  • the present disclosure is a composition comprising the aforementioned antibody or antigen-binding portion thereof, bispecific or multispecific molecule, nucleic acid molecule, entitlement vector and/or cell.
  • the antibodies of the present disclosure are useful as therapeutic or diagnostic tools in various diseases in which CD70 is unfavorably expressed or found.
  • the expression of CD70 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ.
  • To increase means to increase by at least 10%, especially at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.
  • expression is found only in diseased tissue, whereas expression in corresponding healthy tissue is suppressed.
  • CD70 is expressed in a variety of human cancers, including renal cell carcinoma, metastatic breast cancer, brain tumors, leukemia, lymphoma, and nasopharyngeal carcinoma.
  • Anti-CD70 antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, as described below, anti-CD70 antibodies can also be used with other immunogenic agents, standard cancer treatments, or other antibodies.
  • Preferred cancers whose growth can be inhibited with the antibodies of the present disclosure include cancers that are generally responsive to immunotherapy.
  • preferred cancers that can be treated include kidney cancer (eg, renal cell carcinoma), breast cancer, brain tumors, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Chronic lymphocytic leukemia, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma) and nasopharyngeal carcinoma.
  • kidney cancer eg, renal cell carcinoma
  • breast cancer e.g, brain tumors, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Chronic lymphocytic leukemia, lymphoma (eg, Hodgkin's and non-Hodgkin
  • melanoma eg, metastatic malignant melanoma
  • prostate cancer colon cancer
  • lung cancer bone cancer
  • pancreatic cancer skin cancer
  • head and neck cancer skin or intraocular malignant melanoma Tumor
  • Uterine Cancer Ovarian Cancer, Rectal Cancer
  • Anal Cancer Stomach Cancer
  • Testicular Cancer Uterine Cancer, Fallopian Tube Cancer
  • Endometrial Cancer Cervical Cancer
  • Vaginal Cancer Vaginal Cancer
  • Esophagus Cancer Small Intestine Cancer
  • Endocrine System Cancer thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, tumor angiogenesis, spinal tumors , brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal
  • CNS central nervous system
  • the human antibodies, antibody compositions and methods of the present disclosure can be used to treat subjects suffering from tumorigenic diseases, such as diseases characterized by the presence of CD70-expressing tumor cells , including, for example, renal cell carcinoma (RCC) such as clear cell RCC, glioblastoma, breast cancer, brain tumor, nasopharyngeal carcinoma, non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL) ), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, anaplastic large cell lymphoma (ALCL), multiple myeloma, cutaneous T-cell lymphoma, nodular small cleavage cell lymphoma, lymphocytic lymphoma Lymphoma, peripheral T-cell lymphoma, Lennart's lymphoma, immunoblastic lymphoma, T-cell leukemia/lymphoma (ATLL), adult T-
  • RCC renal cell carcinoma
  • NHL non-
  • CD70 has also been proposed to play a role in cell-mediated autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE).
  • EAE experimental autoimmune encephalomyelitis
  • This effect is thought to be mediated in part by inhibition of TNF-alpha production.
  • blockade of CD70 signaling inhibits CD40-mediated clonal expansion of CD8+ T- cells and reduces the generation of CD8+ memory T cells (Taraban et al. (2004) J. Immunol. 173:6542-6).
  • the human antibodies, antibody compositions, and methods of the present disclosure can be used to treat patients with autoimmune diseases, such as diseases characterized by the presence of CD70-expressing B cells, including, for example, experimental autoimmune cerebrospinal inflammation.
  • autoimmune diseases for which the antibodies of the present disclosure may be used include, but are not limited to, systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's disease, ulcerative colitis, and celiac disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis.
  • SLE systemic lupus erythematosus
  • IDDM insulin-dependent diabetes mellitus
  • IBD inflammatory bowel disease
  • MS multiple sclerosis
  • psoriasis autoimmune thyroiditis
  • RA rheumatoid arthritis
  • glomerulonephritis glomerulonephritis.
  • the antibody compositions of the present disclosure can be used to inhibit or prevent transplant rejection or to treat graft versus host disease (GVHD).
  • the interaction of CD70 with CD27 has also been proposed to play a role in CD4+ T cell signaling.
  • Certain viruses have been shown to signal the CD27 pathway leading to disruption of neutralizing antibody responses (Matter et al. (2006) J Exp Med 203:2145-55).
  • the human antibodies, antibody compositions and methods of the present disclosure can be used to treat patients suffering from viral infections including, for example, human immunodeficiency virus (HIV), hepatitis (A, B, C), herpes virus (e.g.
  • HIV human immunodeficiency virus
  • A, B, C hepatitis
  • herpes virus e.g.
  • VZV HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus
  • adenovirus adenovirus, influenza virus, arbovirus, echo virus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, Mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arbovirus encephalitis virus and lymphocytic choriomeningitis virus (LCMV), or for the treatment of HIV infection/AIDS.
  • the human antibodies, antibody compositions and methods of the present disclosure can be used to inhibit TNF-alpha production.
  • the methods of treating diseases and symptoms with the CD70 antibodies of the present disclosure comprise the steps of administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment or nucleic acid molecule or vector or cell or pharmaceutical composition of any of the preceding aspects.
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding CD70, wherein the antibody is administered to provide a serum level of at least 40 ⁇ g/ml.
  • the antibody is administered to provide serum levels of at least 50 ⁇ g/ml, at least 150 ⁇ g/ml, at least 300 ⁇ g/ml, at least 400 ⁇ g/ml, or at least 500 ⁇ g/ml.
  • the antibody is administered to provide a serum level of no more than 800 ⁇ g/ml, 700 ⁇ g/ml, 600 ⁇ g/ml, 550 ⁇ g/ml or 500 ⁇ g/ml.
  • the provided serum level is 40 ⁇ g/ml to 700 ⁇ g/ml, preferably 40 ⁇ g/ml to 600 ⁇ g/ml, preferably 50 ⁇ g/ml to 500 ⁇ g/ml, such as 150 ⁇ g/ml to 500 ⁇ g/ml or 300 ⁇ g /ml to 500 ⁇ g/ml.
  • serum level as used in this specification means the concentration of the substance in question in serum.
  • serum levels are provided for at least 7 days or for at least 14 days.
  • the method comprises administering an antibody dose of at least 300 mg/m 2 , such as at least 600 mg/m 2 , and preferably at most 1500 mg/m 2 , at most 1200 mg/m 2 or at most 1000 mg/m 2 .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding CD70 at a concentration of at least 300 mg/m 2 , such as at least 600 mg/m 2 , and preferably at most 1500 mg/m 2 , the aforementioned antibodies are administered at a dose of up to 1200 mg/m 2 or up to 1000 mg/m 2 .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding CD70, wherein at least 50%, preferably 60%, 70%, 80% or 90% of the patient has cancer
  • the cells are CD70 positive and/or at least 40%, preferably 50% or 60% of the cancer cells of the patient are positive for surface expression of CD70.
  • the present disclosure also provides a method of treating or preventing a cancer disease, the method comprising: a. identifying at least 50%, preferably 60%, 70%, 80% or 90% of CD70 positive cancer cells and/or at least 40%, preferably 50% or 60% of the patient with cancer cells that are positive for surface expression of CD70; and b. administering to the patient an antibody capable of binding CD70.
  • at least 95% or at least 98% of the cancer cells of the patient are CD70 positive.
  • at least 70%, at least 80% or at least 90% of the cancer cells of the patient are positive for surface expression of CD70.
  • the outcome of the treatment of the cancer disease is the achievement of stable disease.
  • disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.
  • the present disclosure provides methods of achieving stabilization of a cancer patient comprising administering to the patient an antibody capable of binding CD70.
  • disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.
  • the antibody is administered in a single dose or multiple doses.
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding CD70, wherein the antibody is administered in multiple doses.
  • the antibody is administered in multiple doses according to the present disclosure, preferably at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, or at least 9 doses
  • the antibody is administered in 10 doses and preferably up to 30 doses, 25 doses, 20 doses, 15 doses or 10 doses.
  • Doses of the antibody are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days.
  • Doses of antibody are preferably administered at intervals of 7 to 30 days, 10 to 20 days, and preferably about 14 days.
  • the antibody is administered so as to provide a serum level of at least 40 ⁇ g/ml. In various embodiments, the antibody is administered so as to provide serum levels of at least 50 ⁇ g/ml, at least 150 ⁇ g/ml, at least 300 ⁇ g/ml, at least 400 ⁇ g/ml, or at least 500 ⁇ g/ml. In various embodiments, the antibody is administered so as to provide serum levels of no more than 800 ⁇ g/ml, 700 ⁇ g/ml, 600 ⁇ g/ml, 550 ⁇ g/ml or 500 ⁇ g/ml.
  • the provided serum level is 40 ⁇ g/ml to 700 ⁇ g/ml, preferably 40 ⁇ g/ml to 600 ⁇ g/ml, preferably 50 ⁇ g/ml to 500 ⁇ g/ml, such as 150 ⁇ g/ml to 500 ⁇ g/ml or 300 ⁇ g /ml to 500 ⁇ g/ml.
  • serum levels are provided for at least 7 days or for at least 14 days.
  • the method comprises administering a dose of the antibody of at least 300 mg/m 2 , such as at least 600 mg/m 2 and preferably at most 1500 mg/m 2 , at most 1200 mg/m 2 or at most 1000 mg/m 2 .
  • the antibody is optionally conjugated to other drugs, such as labeled or cytotoxic conjugates.
  • the present disclosure provides a kit comprising the aforementioned antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells, compositions, and/or antibody-drug conjugates .
  • kits described in the present disclosure include antibodies, fragments, homologues, derivatives thereof, etc. of the present disclosure, such as labeled or cytotoxic conjugates, as well as instructions for use of the antibodies, and conjugates that kill specific types of cells and many more.
  • the instructions may include instructions for using the antibody, conjugate, 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. Packaged reagent combinations in predetermined quantities are contemplated along with instructions for their use, eg, for therapeutic use or for performing diagnostic assays.
  • the kit may include a substrate and cofactors required by the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore).
  • a substrate precursor that provides a detectable chromophore or fluorophore e.g., a substrate precursor that provides a detectable chromophore or fluorophore.
  • other additives such as stabilizers, buffers (eg, blocking buffers or lysis buffers), etc., may also be included.
  • the relative amounts of the various reagents can be varied to provide concentrates of reagent solutions, which provides user flexibility, space savings, reagent savings, and the like.
  • These reagents can also be provided in dry powder form, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagents of appropriate concentrations.
  • the present disclosure provides the aforementioned antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells, compositions and/or antibody-drug conjugates in preparation for use in diagnosis, therapy Or use in a medicament or kit for preventing CD70-related disorders.
  • diseases associated with CD70 include tumors and autoimmune diseases.
  • the tumor is a cancer disease; preferably, the cancer disease is selected from renal cell carcinoma (RCC), clear cell RCC, glioblastoma, non-Hodgkin's lymphoma (NHL), acute lymphoma cell leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, anaplastic large cell lymphoma (ALCL), multiple myeloma, cutaneous T-cell lymphoma, nodular small cleavage cell lymphoma , lymphocytic lymphoma, peripheral T-cell lymphoma, Lennart's lymphoma, immunoblastic lymphoma, T-cell leukemia/lymphoma (ATLL), adult T-cell leukemia (T-ALL), centriblast/ Centrocellular (cb/cc) follicular lymphoma, B-lineage diffuse large cell lymphoma, angioimmunoblastic lymphadenopathy (AILD)-
  • RCC renal
  • the autoimmune disease is lupus.
  • the present disclosure provides the aforementioned antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells, compositions and/or antibody-drug conjugates prepared for use in therapeutic subjects Use in drugs or kits for viral infections of patients.
  • the viral infection is selected from the group consisting of human immunodeficiency virus (HIV), hepatitis (A, B, C), herpes virus (eg VZV, HSV-1, HAV-6, HSV- II and CMV, Epstein-Barr virus), adenovirus, influenza virus, arbovirus, echo virus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arbovirus encephalitis virus, and lymphocytic choriomeningitis virus (LCMV).
  • HCV human immunodeficiency virus
  • A, B, C hepatitis
  • herpes virus eg VZV, HSV-1, HAV-6, HSV- II and C
  • antibodies of the present disclosure can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • compositions comprising the anti-CD70 antibodies or fragments thereof of the present disclosure, the antibodies being conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is routine in the art.
  • the term "pharmaceutical composition” refers to a formulation of various preparations. Formulations containing a therapeutically effective amount of the multivalent antibody are in sterile liquid solutions, liquid suspensions, or lyophilized forms, optionally containing stabilizers or excipients.
  • the antibodies of the present disclosure can be used as compositions for administration alone, or can be used in combination with other active agents.
  • the humanized antibodies of the present disclosure are conjugated to a therapeutic moiety (ie, a drug).
  • Therapeutic moieties can be, for example, cytotoxins, chemotherapeutic agents, cytokines, immunosuppressive agents, immunostimulatory agents, lytic peptides, or radioisotopes.
  • conjugates are referred to herein as "antibody-drug conjugates" or "ADCs".
  • the antibody is conjugated to a cytotoxic moiety.
  • Cytotoxic moieties may, for example, be selected from the following: paclitaxel; cytochalasin B; gramicidin D; ethidium bromide; ipecine; mitomycin; etoposide; teniposide; vincristine; vinblastine ; Colchicine; Doxorubicin; Daunorubicin; F or its analogues or derivatives; Dolastatin 10 or 15 or its analogues; Irinotecan or its analogues; Mitoxantrone; corticosteroids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogs or derivatives; antimetabolites such as methotrexate, 6-mercaptopurine , 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, sebadiazine, hydroxy
  • the antibody is conjugated to auristatin or a peptide analog, derivative or prodrug thereof.
  • Auristatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division and has anticancer and antifungal activity.
  • auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F) and MMAE (monomethyl auristatin E).
  • Suitable auristatin and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugating auristatin to the Ab are described, for example, in US Pat. Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publication WO02088172 , WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.
  • the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or a peptide analog, derivative or prodrug thereof.
  • PDB pyrrolo[2,1-c][1,4]-benzodiazepine
  • Suitable PDBs and PDB derivatives and related techniques are described, for example, in Hartley J.A. et al., Cancer Res 2010; 70(17):6849-6858; Antonow D. et al., Cancer J 2008; 14(3):154-169; Howard P.W. et al, Bioorg Med Chem Lett 2009;19:6463-6466 and Sagnou et al, Bioorg MedChem Lett 2000;10(18):2083-2086.
  • the antibody is conjugated to a cytotoxic moiety selected from the group consisting of anthracyclines, maytansines, calicheamicin, dukamycin, racheomycin (CC-1065), dolastatin 10.
  • a cytotoxic moiety selected from the group consisting of anthracyclines, maytansines, calicheamicin, dukamycin, racheomycin (CC-1065), dolastatin 10.
  • Dolastatin 15 irinotecan, monomethylauristatin E, monomethylauristatin F, PDB, or any analog, derivative or prodrug thereof.
  • the antibody is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to Dokamycin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to Rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof.
  • the antibody is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin F or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to a pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.
  • the antibody is conjugated with a cytokine (eg, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFN ⁇ , IFN ⁇ , IFN ⁇ , GM-CSF, CD40L, Flt3 ligand, stem cell factor, axigrastim and TNF ⁇ ) link.
  • a cytokine eg, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFN ⁇ , IFN ⁇ , IFN ⁇ , GM-CSF, CD40L, Flt3 ligand, stem cell factor, axigrastim and TNF ⁇
  • the antibody is conjugated to a radioisotope or radioisotope-containing chelate.
  • the antibody can be conjugated to a chelator linker (eg, DOTA, DTPA, or tiracetam) that allows complexation of the antibody with the radioisotope.
  • Antibodies may also or alternatively contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules.
  • radioisotopes include3H , 14C , 15N , 35S , 90Y , 99Tc , 125I , 131I , 186Re , 213Bi , 225Ac , and227Th .
  • radioisotopes that emit beta or alpha particle radiation such as131I , 90Y , 211At , 212Bi , 67Cu , 186Re , 188Re and212Pb , can be used.
  • nucleic acid molecules are covalently linked to lysine or cysteine on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively.
  • Conjugation methods using engineered cysteines or incorporating unnatural amino acids have been reported to improve the homogeneity of the conjugates.
  • acyl-donor glutamine-containing tags eg Gin peptide-containing tags or Q-tags
  • polypeptide engineering eg by amino acid deletions, insertions, substitutions or mutations on polypeptides
  • Transglutaminase can then be covalently cross-linked with an amine-donating agent (eg, a small molecule comprising or linked to a reactive amine) to form a stable and homogeneous population of engineered Fc-containing polypeptide conjugates, wherein Amine donor agents are site-specifically coupled to Fc-containing polypeptides via an acyl-donor glutamine-containing tag or an accessible/exposed/reactive endogenous glutamine (WO2012059882).
  • an amine-donating agent eg, a small molecule comprising or linked to a reactive amine
  • the therapeutic agents according to the embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • 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)), especially Chapter 87 in Blaug, Seymour.
  • Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (eg, Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable for use in treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated by the route of administration.
  • antibodies can be used as therapeutic agents. Such agents will typically be used to treat, alleviate and/or prevent a disease or pathology associated with aberrant CD70 expression, activity and/or signaling in a subject. Treatment regimens can be implemented using standard methods by identifying a subject, eg, a human patient having (or at risk or developing) a disease or disorder associated with aberrant CD70 expression, activity and/or signaling, eg, a CD70-related disorder.
  • An antibody preparation preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect due to its binding to the target.
  • the administered antibody may abrogate or inhibit or interfere with the expression, activity and/or signaling function of the target (eg, CD70).
  • Administered antibodies can eliminate or inhibit or prevent the target (eg, CD70) from binding to the endogenous ligand to which it naturally binds.
  • target eg, CD70
  • an antibody binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes and/or otherwise interferes with CD70 expression, activity and/or signaling.
  • an antibody having heavy and light chain CDRs can be administered to a subject.
  • antibodies directed against CD70 can be used in methods known in the art related to the localization and/or quantification of CD70 (eg, for determining the level of CD70 and/or CD70 in an appropriate physiological sample, for diagnostic methods, for protein imaging, etc.).
  • an antibody specific for CD70 or a derivative, fragment, analog or homolog thereof, comprising an antigen-binding domain derived from an antibody is used as a pharmaceutically active compound (hereinafter referred to as "Therapeutic Agent").
  • CD70 polypeptides can be isolated by standard techniques such as immunoaffinity, chromatography or immunoprecipitation using antibodies specific for CD70.
  • Antibodies to the CD70 protein (or fragments thereof) can be used to detect the protein in biological samples.
  • CD70 can be detected in a biological sample as part of a clinical testing procedure, eg, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. 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, beta-galactosidase or acetylcholinesterase;
  • suitable prosthetic complexes include streptavidin/biotin and avidin/ Biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansyl chloride, or phycoerythrin;
  • luminescent materials include Mino;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include125I, 131I , 35S , or3H .
  • antibodies according to the present disclosure can be used as reagents for detecting the presence of CD70 or protein fragments thereof in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Whole antibodies or fragments thereof (eg Fab, scFv or F(ab') 2 ) are used.
  • labeling in reference to an antibody is intended to include direct labeling of the antibody by conjugating (ie, physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reaction with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody, and end-labeling the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in a subject.
  • biological sample is used to include blood and fractions or components in blood, including serum, plasma, or lymph.
  • the detection methods of the embodiments can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo.
  • in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization.
  • Analyte protein in vitro detection techniques include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence.
  • In vitro detection techniques for analyte genomic DNA include Southern hybridization.
  • in vivo detection techniques for analyte proteins include introducing into a subject a labeled anti-analyte protein antibody.
  • an antibody can be labeled with a radiolabel, and the presence and location of the radiolabel in a subject can then 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, as well as guidelines for selecting components, are well known in the art, see, for example, Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th Ed. (Edited by Alfonso R. Gennaro et al.) Mack Pub. Co., Easton, Pa.: 1995.
  • compositions typically comprise the 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 can be designed that retain the ability to bind target protein sequences. Such peptides can be chemically synthesized and/or produced by recombinant DNA techniques (see, eg, 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, and the like, compatible with pharmaceutical administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, 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, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or reagents that are incompatible with the antibody, its use in compositions is contemplated.
  • compositions of the embodiments are formulated to be compatible with their intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile injectable diluents such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, 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 phosphate, and agents to adjust 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 multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable pharmaceutically acceptable 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 it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin to maintain the desired particle size in the case of dispersions, and by the use of surfactants.
  • Prevention of the action of microorganisms 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 in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation are vacuum drying and freeze-drying to obtain a powder containing the active ingredient and any additional desired ingredient from a sterile-filtered solution of those previously enumerated. .
  • the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a gas of a suitable propellant, such as carbon dioxide, or a nebulizer.
  • a gas of a suitable propellant such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the permeation barrier are used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • one or more antibodies can be formulated into an ointment, ointment, gel, or cream as generally known in the art.
  • the compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retention enemas e.g., retention enemas.
  • the antibody may be prepared with a carrier that will prevent its rapid elimination from the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation 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 apparent to those skilled in the art.
  • Dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier one or more antibodies.
  • the specifications for the dosage unit forms of the embodiments are dictated by and directly dependent upon the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and the limitations inherent in the art of formulation of such antibodies for the treatment of individuals.
  • compositions can be placed in a container, pack, or dispenser with instructions for administration.
  • compositions described herein may also contain more than one antibody, preferably those that have complementary activities but do not negatively affect each other, depending on the particular condition to be treated.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • an agent that enhances its function such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • Such molecules are suitably combined in amounts effective for the intended purpose. For example, it can be combined in a kit, and can also be combined in use.
  • one or more antibodies may be administered in combination therapy, ie, with other agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders and inflammation sexually transmitted diseases) combined.
  • agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders and inflammation sexually transmitted diseases
  • the term "combination” as used herein refers to the administration of the agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, at the start of administration of the second compound, the first of the two compounds is still preferably detected at an effective concentration at the treatment site. In one instance, “combination” can also be the simultaneous inclusion of an antibody of the present disclosure and other therapeutic agents in a kit.
  • a combination therapy can comprise one or more antibodies described herein with 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 cytostatics, as described in more detail below) are 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 e.g., enzyme inhibitors, and/or cytotoxins or cytostatics
  • cDNA plasmid of human CD70 (Sino Biological, Cat. No.: HG10780-M) as a template, PCR amplification of the human CD70 extracellular region fragment (Glu13-Pro155), by connecting the peptide GSGSGNGS (SEQ ID NO: 161) to construct the CD70 extracellular region Trimeric structure, C-terminal fusion of murine IgG Fc (knob) and Avi tag tag GSGLNDIFEAQKIEWHE (SEQ ID NO: 162) to obtain purified human CD70mIgG Fc (knob) (whose amino acid sequence is shown in SEQ ID NO: 1).
  • the plasmid comprising the human CD70mIgG Fc (knob) encoding gene and the mouse IgG Fc (hole) (the amino acid sequence of which is shown in SEQ ID NO: 2) were co-transfected into HEK293E cells, and after culturing for 7 days, the medium supernatant was collected. The solution was purified by Protein G to obtain human CD70 extracellular domain trimer recombinant protein, and SDS-PAGE was shown in Figure 1.
  • Human CD70 cDNA and monkey CD70 cDNA (Sino Biological, product number: CG90089-G) were used to construct lentiviral vector plasmids containing the full-length sequence of human and monkey CD70, according to the instructions of the lentivirus packaging kit (Lenti-Pac HIV Expression Packaging Kit, Gene Copoeia, product number: HPK-LvTR-20), the constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells for lentiviral packaging. 48 hours after transfection, the medium was collected, centrifuged at 500*g for 10 minutes to remove cell debris, and the culture supernatant containing lentiviral particles was obtained.
  • the lentivirus packaging kit Li-Pac HIV Expression Packaging Kit, Gene Copoeia, product number: HPK-LvTR-20
  • Human CD70 recombinant protein was mixed with equal amount of immune adjuvant (Freund's adjuvant) as antigen, and 6 female Balb/c mice of 6 weeks in each group were subcutaneously immunized. Two weeks after the initial immunization, a booster immunization was performed. After three immunizations, the orbital blood was collected, and the serum titer was detected by flow cytometry. The detection results are shown in Figure 3. It can be seen from Fig. 3 that high titer CD70 antibody can be detected in mouse serum.
  • mice were immunized by tail vein injection with recombinant human CD70 protein. Three days later, the mice were sacrificed by cervical dislocation, and the spleen and some peripheral lymph nodes of the mice were collected, ground in DMEM medium, and centrifuged; after the supernatant was poured, the cell mass was gently broken up, 5 mL of red blood cell lysis solution was added, and lysed for 50 seconds. , add 40 mL of DMEM medium and centrifuge to obtain a B cell suspension without red blood cells.
  • the cells were fused with an electrofusion apparatus, and the fused hybridoma cells were cultured in DMEM complete medium containing HAT in 5% CO 2 at 37°C. .
  • the remaining B cells were subjected to Trizol lysis to extract total RNA, and a reverse transcription kit (SuperScript First-Strand Synthesis System, Item No.: 18080051) was used to reverse-transcribe the heavy chain-specific primers to obtain an antibody heavy chain cDNA library; using this cDNA as a template,
  • the antibody heavy chain variable region fragment was amplified by PCR with heavy chain variable region primers, and then cloned into a phage plasmid after double digestion with NcoI and NheI; similarly, the light chain cDNA library was obtained by reverse transcription using light chain specific primers.
  • the light chain variable region primer was used to amplify the antibody light chain variable region fragment by PCR, and after double digestion with NcoI and BsiwI, it was cloned into a phage plasmid; then a mouse Fab phage based on filamentous phage M13 was constructed The library is displayed with a capacity of 1 ⁇ 10 10 .
  • Phage-displayed libraries were screened for positive clones by classical panning. Recombinantly expressed human CD70 protein was coated in Immunotube tubes, 1 ⁇ 10 12 phage was added for incubation and panning, phage particles with high binding affinity were eluted with 100 mM triethylamine, and 1 M Tris-HCl (pH 7.4) was used for neutralization and infection. into Escherichia coli TG1 bacteria, and the enriched phage obtained after repackaging can be used for the next round of panning. The clones obtained after 2 rounds of panning and enrichment were inoculated into a 96-well U-shaped plate for culture and IPTG-induced expression, and the supernatant was collected for ELISA detection.
  • the positive monoclonal binding to CD70 antigen was re-induced by IPTG, purified to obtain Fab antibody protein, and ELISA detection and screening were carried out.
  • 1 ⁇ g/mL human or cynomolgus monkey CD27 recombinant protein was coated on a 96-well ELISA plate, and incubated overnight at 4°C. The next day, PBS was washed 3 times, 200 ⁇ L of 2% nonfat dry milk/PBS was added to block for 2 hours at room temperature, PBS was washed once, 50 ⁇ L of Fab antibodies of different dilution gradients were added, and the cells were incubated at room temperature for 30 minutes.
  • the positive clones that bind to the CD70 antibody detected by ELISA were further verified for binding to CD70 positive cells.
  • the RNA of candidate hybridoma monoclonal cells was extracted by TRNzol lysis method, and then reverse transcription was used to synthesize single-stranded cDNA, which was used as a template to amplify the variable region sequence of the antibody in the hybridoma monoclonal cells; according to the phage
  • the positive monoclonal bacterial plasmid was extracted, and after sequencing, the sequence of the variable region of the heavy chain and light chain of the candidate positive clone was obtained:
  • amino acid sequence of 2B12 heavy chain VH is shown in SEQ ID NO: 3
  • its encoding nucleic acid is shown in SEQ ID NO: 4
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 5, 6, 7.
  • amino acid sequence of 2B12 light chain VK is shown in SEQ ID NO: 8
  • its encoding nucleic acid is shown in SEQ ID NO: 9
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 10, 11, 12.
  • amino acid sequence of 3B2 heavy chain VH is shown in SEQ ID NO: 13
  • its encoding nucleic acid is shown in SEQ ID NO: 14
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 15, 6, 16.
  • amino acid sequence of 3B2 light chain VK is shown in SEQ ID NO: 17, its encoding nucleic acid is shown in SEQ ID NO: 18, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 19, 11, 12.
  • amino acid sequence of 12A1 heavy chain VH is shown in SEQ ID NO: 20
  • its encoding nucleic acid is shown in SEQ ID NO: 21
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 5, 6, and 22, respectively.
  • amino acid sequence of 12A1 light chain VK is shown in SEQ ID NO: 23, its encoding nucleic acid is shown in SEQ ID NO: 24, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 25, 11, 26.
  • amino acid sequence of 14C12 heavy chain VH is shown in SEQ ID NO: 27, its encoding nucleic acid is shown in SEQ ID NO: 28, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 29, 30, 31.
  • amino acid sequence of 14C12 light chain VK is shown in SEQ ID NO: 32
  • its encoding nucleic acid is shown in SEQ ID NO: 33
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 34, 11, 26.
  • amino acid sequence of 23A11 heavy chain VH is shown in SEQ ID NO: 35
  • its encoding nucleic acid is shown in SEQ ID NO: 36
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 5, 37, 38.
  • amino acid sequence of 23A11 light chain VK is shown in SEQ ID NO: 39
  • its encoding nucleic acid is shown in SEQ ID NO: 40
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 41, 11, 12.
  • amino acid sequence of 25H7 heavy chain VH is shown in SEQ ID NO: 42
  • its encoding nucleic acid is shown in SEQ ID NO: 43
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 44, 45, 46.
  • amino acid sequence of 25H7 light chain VK is shown in SEQ ID NO:47
  • its encoding nucleic acid is shown in SEQ ID NO:48
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO:49, 50, 51.
  • amino acid sequence of 26G11 heavy chain VH is shown in SEQ ID NO: 52
  • its encoding nucleic acid is shown in SEQ ID NO: 53
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 54, 55, 56.
  • the amino acid sequence of 26G11 light chain VK is shown in SEQ ID NO: 57, its encoding nucleic acid is shown in SEQ ID NO: 58, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 59, 60, 61.
  • the amino acid sequence of 27A3 heavy chain VH is shown in SEQ ID NO: 62, its encoding nucleic acid is shown in SEQ ID NO: 63, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 5, 64, 65.
  • the amino acid sequence of 27A3 light chain VK is shown in SEQ ID NO: 66, its encoding nucleic acid is shown in SEQ ID NO: 67, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 68, 11, 69.
  • amino acid sequence of 32D1 heavy chain VH is shown in SEQ ID NO:70
  • its encoding nucleic acid is shown in SEQ ID NO:71
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO:29, 30, 31 respectively.
  • amino acid sequence of 32D1 light chain VK is shown in SEQ ID NO: 72
  • its encoding nucleic acid is shown in SEQ ID NO: 73
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 74, 11, 26.
  • amino acid sequence of 39H5 heavy chain VH is shown in SEQ ID NO: 75
  • its encoding nucleic acid is shown in SEQ ID NO: 76
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 44, 77, 46.
  • amino acid sequence of 39H5 light chain VK is shown in SEQ ID NO: 78
  • its encoding nucleic acid is shown in SEQ ID NO: 79
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 49, 80, 51.
  • amino acid sequence of 1H21-D5 heavy chain VH is shown in SEQ ID NO: 81
  • its encoding nucleic acid is shown in SEQ ID NO: 82
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 83, 84, 85.
  • amino acid sequence of 1H21-D5 light chain VK is shown in SEQ ID NO: 86
  • its encoding nucleic acid is shown in SEQ ID NO: 87
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 88, 11, 12.
  • amino acid sequence of 10C19-D11 heavy chain VH is shown in SEQ ID NO: 89
  • its encoding nucleic acid is shown in SEQ ID NO: 90
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 5, 91, 85.
  • amino acid sequence of 10C19-D11 light chain VK is shown in SEQ ID NO: 92
  • its encoding nucleic acid is shown in SEQ ID NO: 93
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 19, 11, 12.
  • amino acid sequence of 16J15-H3 heavy chain VH is shown in SEQ ID NO: 94
  • its encoding nucleic acid is shown in SEQ ID NO: 95
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 96, 97, 98 respectively.
  • amino acid sequence of 16J15-H3 light chain VK is shown in SEQ ID NO:99
  • its encoding nucleic acid is shown in SEQ ID NO:100
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO:101, 102, 103.
  • the amino acid sequence of 17A9-G5 heavy chain VH is shown in SEQ ID NO: 104, its encoding nucleic acid is shown in SEQ ID NO: 105, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 15, 106, 107.
  • the amino acid sequence of 17A9-G5 light chain VK is shown in SEQ ID NO: 108, its encoding nucleic acid is shown in SEQ ID NO: 109, and its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 110, 111, 103.
  • amino acid sequence of 36E7-D6 heavy chain VH is shown in SEQ ID NO: 112
  • its encoding nucleic acid is shown in SEQ ID NO: 113
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 114, 115, 116.
  • amino acid sequence of 36E7-D6 light chain VK is shown in SEQ ID NO: 117
  • its encoding nucleic acid is shown in SEQ ID NO: 118
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 119, 120, 121.
  • amino acid sequence of 4H6-A6 heavy chain VH is shown in SEQ ID NO: 122
  • its encoding nucleic acid is shown in SEQ ID NO: 123
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 15, 124, 16.
  • amino acid sequence of 4H6-A6 light chain VK is shown in SEQ ID NO: 125
  • its encoding nucleic acid is shown in SEQ ID NO: 126
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 127, 128, 103.
  • amino acid sequence of 42H19-G4 heavy chain VH is shown in SEQ ID NO: 129
  • its encoding nucleic acid is shown in SEQ ID NO: 130
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 131, 132, 116.
  • amino acid sequence of 42H19-G4 light chain VK is shown in SEQ ID NO: 117
  • its encoding nucleic acid is shown in SEQ ID NO: 118
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 119, 120, 121.
  • the heavy chain variable regions were cloned into vectors containing human heavy chain fixed regions and regulatory elements to express intact IgG heavy chains in mammalian cells.
  • light chain variable regions were cloned into vectors containing human light chain fixed regions and regulatory elements to express intact IgG light chains in mammalian cells. After correct sequencing, it was transfected into CHO-S mammalian cells, IgG was expressed and secreted into the medium, and the supernatant was collected and filtered for purification.
  • IgG was purified by Protein A chromatography, and the culture supernatant was loaded on a protein A column of appropriate size, washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl, and bound with 0.1 M Glycine-HCl (pH 3.0). IgG eluted. The protein was concentrated by ultrafiltration using a concentration tube, OD280 was detected, and the concentration of IgG was determined by spectrophotometry.
  • Daudi cells were resuspended and plated into 96-well plates at 50 ⁇ L/well, and then 50 ⁇ L of antibody was added to each well.
  • the chimeric antibody was diluted to 10 ⁇ g/mL, using this as the starting concentration, and the 3-fold serial dilution was carried out for a total of 3 or 4 gradients.
  • the 96-well plate was placed in a microplate reader (BioTek, Synergy HT), the OD450 value was read, and the data was collected and the results were calculated using GraphPad Prism 5 software. Results As shown in Figures 4-1 and 4-2 and Table 4, the chimeric antibody 39H5 showed extremely strong CDC activity, and the maximum killing rate to tumor cells at a concentration of 66.7 nM was close to 80%.
  • Daudi cells at a density of 1 ⁇ 10 6 cells/mL were plated into a U-shaped 96-well plate at a density of 50 ⁇ L per well, and 50 ⁇ L of chimeric antibody was added to each well, and incubated in a 37° C., 5% CO 2 incubator for 30 minutes.
  • NK92-MI-CD16a cells were taken out, added with RPMI 1640 medium, counted, and centrifuged at 300g for 10 minutes.
  • OD450nm was measured, data were collected, and the results were calculated using Graph Pad Prism 5 software. The results are shown in Figures 7-1, 7-2 and Table 7.
  • the chimeric antibody was able to block the interaction between human CD70 and CD27 and cynomolgus monkey CD70 and CD27. combine.
  • the murine antibodies 39H5 and 32D1 with stronger CDC activity were selected for humanization of variable region sequences.
  • the mouse antibody 39H5 sequence was compared with the human antibody germline sequence, and the key amino acid sequences that maintained the antibody structural core (Upper hydrophobic core) with good homology were found to be identical, and the human germline light sequence appeared frequently in the human body.
  • a total of 6 heavy chain variants h39H5-VHv1-v3, h32D1-VHv1-v3 and 6 light chain variants h39H5-VKv1-v3, h32D1-VKv1-v3 were obtained by design. , cloned into a eukaryotic expression vector containing antibody kappa chain fixed region Ckappa or human IgG1 fixed region CH1-CH3, combined and paired light and heavy chain plasmids, transfected into CHO-S cells, and expressed at 37°C for 5-6 days. The culture supernatant was collected and purified by Protein A column.
  • the humanized antibody heavy/light chain variable region sequences are as follows:
  • amino acid sequence of the heavy chain VH is shown in SEQ ID NO: 133
  • its encoding nucleic acid is shown in SEQ ID NO: 134
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 44, 135, 46.
  • amino acid sequence of the heavy chain VH is shown in SEQ ID NO: 136
  • its encoding nucleic acid is shown in SEQ ID NO: 137
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 44, 135, 46.
  • amino acid sequence of the heavy chain VH is shown in SEQ ID NO: 138
  • its encoding nucleic acid is shown in SEQ ID NO: 139
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 44, 135, 46.
  • amino acid sequence of heavy chain VH is shown in SEQ ID NO: 140
  • its encoding nucleic acid is shown in SEQ ID NO: 141
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 29, 142, 31 respectively.
  • amino acid sequence of the heavy chain VH is shown in SEQ ID NO: 143
  • its encoding nucleic acid is shown in SEQ ID NO: 144
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 29, 142, 31.
  • amino acid sequence of the heavy chain VH is shown in SEQ ID NO: 145
  • its encoding nucleic acid is shown in SEQ ID NO: 146
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 29, 142, 31.
  • amino acid sequence of the light chain VH is shown in SEQ ID NO: 147
  • its encoding nucleic acid is shown in SEQ ID NO: 148
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 149, 80, 51.
  • amino acid sequence of the light chain VH is shown in SEQ ID NO: 150
  • its encoding nucleic acid is shown in SEQ ID NO: 151
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 149, 80, 51.
  • amino acid sequence of the light chain VH is shown in SEQ ID NO: 152
  • its encoding nucleic acid is shown in SEQ ID NO: 153
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 149, 80, 51.
  • amino acid sequence of the light chain VH is shown in SEQ ID NO: 154
  • its encoding nucleic acid is shown in SEQ ID NO: 155
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 25, 11, 26.
  • amino acid sequence of the light chain VH is shown in SEQ ID NO: 156
  • its encoding nucleic acid is shown in SEQ ID NO: 157
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 25, 11, 26.
  • amino acid sequence of the light chain VH is shown in SEQ ID NO: 158
  • its encoding nucleic acid is shown in SEQ ID NO: 159
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO: 25, 160, 26.
  • Daudi cells were resuspended and plated into 96-well plates at 50 ⁇ L/well, and then 50 ⁇ L of antibody was added to each well.
  • the humanized antibody was diluted to 10 ⁇ g/mL, which was used as the starting concentration, and the 3-fold serial dilution was carried out for a total of 3 gradients.
  • the 96-well plate was placed in a microplate reader (BioTek, Synergy HT), the OD450 value was read, and the data was collected and the results were calculated using GraphPad Prism 5 software. The results are shown in Figure 8 and Table 10.
  • the humanized variants Ab001, Ab004 and Ab007 had the highest killing rate on Dauai cells, which was about 60%.
  • FACS detected that the humanized antibodies Ab001, Ab004 and Ab007 bound with high affinity to the CD70 molecules expressed on the stably transfected cell lines CHO-human CD70 and CHO-monkey CD70, respectively.
  • 96-well microtiter plate was coated with 1 ⁇ g/mL human CD27 recombinant protein and incubated at 4°C overnight. The next day, PBS was washed three times, 200 ⁇ L of 2% nonfat dry milk/PBS was added to block for 2 hours at room temperature, and PBS was washed once; the purified chimeric antibody was subjected to 12 concentration gradients with an initial concentration of 100 nM, followed by a 1:3 gradient. For dilution, add 30 ⁇ l to each well and incubate for 30 minutes at room temperature. Then, 30 ⁇ L of biotin-labeled CD70 recombinant protein was added to each well and incubated at room temperature for 60 minutes.
  • Example 9 CDC killing of different tumor cells by humanized CD70 antibody
  • Daudi cells were resuspended and plated into 96-well plates at 50 ⁇ L/well, and then 50 ⁇ L of antibody was added to each well.
  • the humanized antibody was diluted to 10 ⁇ g/mL, and this was used as the starting concentration, and the serial dilution was 3 times.
  • 50 ⁇ L of complement mixture was added, and the 96-well plate was placed in a 37°C 5% CO 2 incubator for 2 hours.
  • 50 ⁇ L of 40% CCK-8 solution was added to each well, shaken for 10 seconds, and incubated in a 5% CO 2 incubator for 3 hours.
  • Inoculate Daudi cells into U-shaped 96-well plates add different concentrations of humanized antibodies to each well, and place them in a CO 2 incubator for half an hour; remove NK92-MI-CD16a cells, and resuspend the cells in RPMI-1640 medium to a density of 2 ⁇ 10 6 /mL; then 50 ⁇ L of NK92-MI-CD16a cells were added to each well of a 96-well plate and cultured in a CO 2 incubator for 4 hours. 30 minutes before detection, add 2 ⁇ L of cell lysate (100 ⁇ ) to the largest well of the target cells, and continue to culture at 37°C.
  • Example 12 Inhibitory effect of humanized CD70 antibody on CD70-mediated stimulation of IL-8 secretion
  • Raji cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Raji cells in logarithmic growth phase were collected, resuspended in PBS to an appropriate concentration and mixed 1:1 with matrigel for subcutaneous tumor inoculation in NOD/SCID mice.
  • Female mice were inoculated with 1 ⁇ 10 7 Raji cells subcutaneously on the right side, and when the average tumor volume was 100 mm 3 , they were randomly divided into groups according to tumor size. There were 5 mice in each group. Mice in the treatment group were injected twice a week through the tail vein at doses of 1 mg/kg, 3 mg/kg and 10 mg/kg for two weeks, and tumor volume was monitored. The tumor volume was calculated as:
  • Tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long axis and b represents the short axis).
  • the humanized CD70 antibody 3 mg/kg and 10 mg/kg dose groups both significantly inhibited the growth of human Burkitt's lymphoma Raji mice subcutaneously transplanted tumor, causing partial regression of the tumor; the humanized CD70 antibody
  • the tumor inhibition rate of Ab004 at 10 mg/kg was 69.4%.
  • the humanized CD70 antibody did not cause the weight loss of mice in different dose groups, and had no obvious toxic effect.
  • Fc ⁇ RIII V158 and Fc ⁇ RIII F158 Two CD16a receptor alleles, Fc ⁇ RIII V158 and Fc ⁇ RIII F158 , widely exist in the population.
  • the allele Fc ⁇ RIII V158 encodes a receptor with a higher affinity for antibody IgG1
  • NK cells expressing Fc ⁇ RIII V158 mediate more effective antibody-dependent cell-mediated mediation cytotoxicity (ADCC), which is considered to be the reason for the ineffectiveness of antibody drugs against the low-affinity Fc ⁇ RIII F158 population.
  • Biacore results showed that mutant Ab004IgG1e5 had a significant affinity enhancement to the low-affinity Fc ⁇ RIII F158 , which was comparable to wild-type IgG1 to the high-affinity Fc ⁇ RIII V158 receptor (Table 17).
  • Peripheral blood PBMCs were isolated and collected from healthy volunteers with different Fc ⁇ RIIIa allele receptors, and the cells were resuspended in RPMI-1640 medium; Daudi cells were seeded into U-shaped 96-well plates, and different concentrations of humanized humanized cells were added to each well.
  • the antibody was placed in a CO 2 incubator for half an hour; then 5 ⁇ 10 5 PBMC cells were added to each well into a 96-well plate, and placed in a CO 2 incubator for 4 hours. 30 minutes before detection, add 2 ⁇ L of cell lysate (100 ⁇ ) to the largest well of the target cells, and continue to culture at 37°C.
  • Centrifuge at 300g for 3 minutes take 50 ⁇ L of supernatant to a black microtiter plate, add 50 ⁇ L of LDH detection substrate to each well (dissolve in advance at room temperature), gently shake and mix; after 10 minutes, add 25 ⁇ L of stop solution to each well to stop, shake After 10 seconds, select fluorescence (excitation wavelength 560 nm, emission wavelength 590 nm) to read the plate, and calculate the killing rate.
  • Example 15 Anti-tumor effect of CDC activity-enhanced CD70 antibody in Daudi cell line subcutaneous xenograft NOD/SCID mouse model
  • Daudi cells in the logarithmic growth phase were taken, the cell density was adjusted to 1.2 ⁇ 10 8 /ml, an equal volume of Matrigel Matrix was added to mix well with the cell suspension, and 100 ⁇ l of each was inoculated into the right limb of BALB/c-Nude mice subcutaneously.
  • the mice were randomly divided into groups of 5 mice, and the first administration was started on the day of grouping, once every three days, for a total of three administrations, each with 100 ⁇ l tail vein injection.
  • the day of subcutaneous tumor receiving was D0, and the tumor growth curve was drawn (tumor volume did not exceed 2000 mm 3 , mice were sacrificed when reaching 2000 mm 3 ).
  • both the CDC activity-enhanced CD70 antibody and the parent antibody can dose-dependently inhibit the growth of human lymphoma Daudi subcutaneously transplanted tumor.
  • the tumor inhibition rates of CDC activity-enhanced CD70 antibodies from high to low doses were 114%, 92%, and 67%, respectively; the tumor inhibition rates of CD70 parental antibodies were 81%, 36%, and 24%, respectively.
  • Tumor-bearing mice can tolerate the above doses well, and there are no adverse reactions such as weight loss.

Abstract

提供了一种结合CD70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、6、7、15、16、22、29、30、31、37、38、44、45、46、54、55、56、64、65、77、83、84、85、91、97、98、106、107、114、115、116、124、131、132、135、142、160或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:10、11、12、19、25、26、34、41、49、50、51、59、60、61、68、69、74、80、88、101、102、103、110、111、119、120、121、127、128或任何变体的轻链CDR，可阻断CD70-CD27相互作用及下游信号传导，抑制肿瘤。还提供了一种包含该CD70抗体或其抗原结合部分的恶性肿瘤治疗剂。

Description

一种功能增强型抗体阻断剂的开发及其应用 技术领域
本公开涉及一种功能增强型抗体阻断剂的开发及其应用，特别是一种结合CD70的抗体及其应用。
背景技术
CD70是一种II型跨膜糖蛋白，属于肿瘤坏死因子超家族。CD70与CD27的相互结合被称为“CD70-CD27信号轴”，在细胞增殖和分化过程中具有重要作用。研究显示，急性髓细胞白血病细胞表面的CD70与T细胞表面的CD27相互作用，可刺激肿瘤微环境中的CD4 +调节性T reg细胞扩增，或显著增加骨髓中发生衰竭的效应CD8 +T细胞比例，导致肿瘤的免疫逃逸。同时，CD27也表达于部分肿瘤细胞表面，肿瘤细胞间形成的CD70-CD27活化环路，可延长肿瘤细胞生存时间或导致增殖。
在白血病患者体内，检测到恶性增殖的肿瘤细胞和白血病干细胞(CD34 +CD38 -)均广泛表达CD70分子，而可溶性CD27在患者血清中显著升高，和预后呈负相关。而通过抗体阻断CD70-CD27相互作用，可诱导白血病干细胞产生不对称细胞分裂和分化，抑制肿瘤集落的形成，显著延长肿瘤模型小鼠的存活时间。
CD70在正常组织和造血祖细胞中不表达，但在T细胞淋巴瘤、B细胞淋巴瘤以及相当一部分实体瘤中高表达。而CD33或CD123等其他血液肿瘤靶点，除了在肿瘤细胞高表达，也表达于正常造血祖细胞或正常组织(如：肺、前列腺、皮肤，外周血T细胞)。因此，开发具有杀伤和阻断活性的CD70单克隆抗体，可以特异性清除患者体内的肿瘤细胞或白血病干细胞，而不影响患者的造血祖细胞和恢复后的造血功能。
急性髓细胞白血病是一组高度异质性的克隆性疾病，源于骨髓造血干细胞的过度增殖或分化障碍，导致大量未分化细胞在骨髓中蓄积，并排斥具有正常功能的血细胞和免疫细胞，也可能广泛浸润肝、脾、淋巴结等髓外脏器。患者由于贫血和免疫功能低下，最终死于并发感染或其他疾病。急性髓细胞白血病的治疗在过去近40年的时间里进展缓慢，临床采用的标准方案仍是上世纪70年代引入的阿糖胞苷加柔红霉素治疗。
CN200680021851.8描述了具有抗体Fc效应的CD70抗体，可介导ADCC、ADCP和/或CDC效应，可以在没有偶联于细胞毒性治疗剂或偶联于细胞毒性治疗剂的情况下，行使细胞抑制、细胞毒或免疫调节作用。
CN200680035376.X描述了结合人CD70蛋白并且抑制肿瘤细胞生长的CD70抗体，以1×10 -7M或更低的K D与人CD70结合，所述抗体还与表达CD70的肾细胞癌肿瘤细胞系结合并且被其内化。
CN201280013552.5描述了来源于大羊驼的CD70抗体，与人CD70高亲和力结合，表现出小于7×10 -4s -1或更少的解离速率，一些优选实施方案可表现出对CD70与其配体CD27之相互作用的强效阻断或抑制，优于CN200680021851.8和CN200680035376.X描述的CD70抗体。
发明内容
本发明人以CD70重组蛋白免疫小鼠，得到了多株识别人及食蟹猴CD70重组蛋白的高亲和力抗体。本公开抗体具有极高ADCC和CDC活性，明显优于现有技术中的CD70抗体，同时还可高效阻断CD70-CD27相互作用及下游信号传导，对肿瘤的抑制效果显著强于现有抗体。
在一方面，本公开提供了一种结合CD70的抗体或其抗原结合部分。
在一方面，本公开提供了一种双特异性抗体或其抗原结合部分。
在一方面，本公开提供了编码前述的结合CD70的抗体或其抗原结合部分或双特异性抗体或其抗原结合部分的核酸分子。
在一方面，本公开提供了包含前述的核酸分子的载体。
在一方面，本公开提供了包含前述的载体的细胞。
在一方面，本公开提供了包含前述的抗体或其抗原结合部分或其编码核酸分子和药学上可接受的载体的药物组合物或试剂盒。
在一方面，本公开提供了包含共价附着至治疗部分的前述任一方面抗体或其抗原结合部分、双特异性或多特异性分子的抗体-药物偶联物。
在一方面，本公开提供了治疗与CD70相关病症的方法，其包括下述步骤：向受试者施用治疗有效量的前述任一方面的抗体或其抗原结合片段、核酸分子、载体、细胞和/或药物组合物。
在一方面，本公开提供了前述任一方面的抗体或其抗原结合片段、核酸分子、载体、细胞和/或药物组合物在制备用于治疗受试者中与CD70相关病症的药物或试剂盒中的用途。
在一方面，本公开提供了前述任一方面的抗体或其抗原结合片段、核酸分子、载体、细胞和/或药物组合物在制备用于治疗受试者的病毒感染的药物或试剂盒中的用途。
附图说明
图1示出了HEK293细胞重组表达的人CD70胞外区蛋白SDS-PAGE检测结果。
图2-1示出了流式细胞术检测人CD70/CHO稳转细胞株的CD70表达的检测结果。
图2-2示出了流式细胞术检测猴CD70/CHO稳转细胞株的CD70表达的检测结果。
图3示出了FACS检测免疫小鼠血清中抗体的滴度。
图4-1和4-2示出了CD70单克隆抗体CDC活性筛选结果。
图5示出了CD70单克隆抗体ADCC活性。
图6-1示出了FACS检测嵌合抗体与CHO-人CD70的结合结果。
图6-2示出了FACS检测嵌合抗体与CHO-食蟹猴CD70的结合结果。
图7-1示出了嵌合抗体的人CD70/CD27阻断检测结果。
图7-2示出了嵌合抗体的食蟹猴CD70/CD27阻断检测结果。
图8示出了不同人源化抗体对肿瘤细胞Daudi的CDC杀伤效果。
图9-1示出了FACS检测人源化抗体与CHO-人CD70的结合结果。
图9-2示出了FACS检测人源化抗体与CHO-食蟹猴CD70的结合结果。
图10-1示出了人源化抗体的人CD70/CD27阻断检测结果。
图10-2示出了人源化抗体的食蟹猴CD70/CD27阻断检测结果。
图11-1示出了人源化抗体对肿瘤细胞Daudi的CDC杀伤效果。
图11-2示出了人源化抗体对肿瘤细胞MV-4-11-CD70的CDC杀伤效果。
图11-3示出了人源化抗体对肿瘤细胞Raji的CDC杀伤效果。
图12-1示出了人源化抗体对肿瘤细胞Daudi的ADCC杀伤效果。
图12-2示出了人源化抗体对肿瘤细胞Raji的ADCC杀伤效果。
图13示出了人源化抗体对肿瘤细胞Daudi的ADCP杀伤效果。
图14示出了人源化抗体对CD70介导刺激IL-8分泌的抑制作用。
图15-1示出了人源化CD70抗体对人Burkitt's淋巴瘤Raji皮下移植瘤生长的影响。
图15-2示出了人源化CD70抗体对荷瘤小鼠体重的影响。
图16示出了CDC增强型CD70人源化抗体对肿瘤细胞Daudi的CDC杀伤活性。
图17-1和17-2示出了CDC增强型CD70人源化抗体在不同FcγRIII等位基因PBMC中介导的ADCC杀伤活性。
图18示出了CDC活性增强型CD70抗体在Daudi细胞株皮下异种移植NOD/SCID小鼠模型中的抗肿瘤作用。
具体实施方式
I.定义
在本公开中，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，为了更好地理解本公开，下面提供相关术语的定义和解释。
本文提供的是特异性结合CD70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CD70的单克隆抗CD70抗体，其中所述抗CD70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CD70(例如，人CD70)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CD70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。
术语“CD70”包括变异体、同种型、同系物、直向同源物以及种内同源物。例如对人类CD70蛋白具有特异性的抗体在某些情况下可以与来自除人类之外的物种的CD70蛋白进行交叉反应。在另一个实施方案中，对人类CD70蛋白具有特异性的抗体对于人类CD70蛋白可能是完全特异的，并且可能不表现出物种或其他类型的交叉反应性，或者可能与来自某些其他物种但不是所有其他物种的CD70进行交叉反应(如与灵长类CD70进行交叉反应但不与小鼠CD70进行交叉反应)。术语“人类CD70”指人类序列CD70，如具有Genbank登录号P32970的人类CD70的完整氨基酸序列。术语“小鼠CD70”指小鼠序列CD70，如具有Genbank登录号NP_035747的小鼠CD70的完整氨基酸序列。通过具有例如保守突变或在非保守区的突变，人类CD70序列可能不同于Genbank登录号P32970的人类CD70，而该CD70具有基本上与Genbank登录号P32970的人类CD70相同的生物功能。例如，人CD70的一个生物功能是结合细胞因子受体CD27。
术语“细胞表面”的使用是根据其在本领域的正常含义，因此包括可通过与蛋白和其它分子结合而接近的细胞外部。
术语“信号转导途径”指在多种信号转导分子之间的生物化学关系，这些信号转导分子在信号从细胞的一部分传递至细胞的另一部分的转递中发挥作用。此处使用的短语“细胞表面受体”包括，例如，能够接受信号并将这种信号传递穿过细胞的细胞膜的分子及分子的复合物。本公开的“细胞表面受体”的一个实例是CD70受体。
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。
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。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))。
“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。
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。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。
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。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CD70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Fv的一半)，仍可具有识别和结合靶的能力。
如本文中所用，术语“双特异性”(Bispecific antibody，BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇，通常，双特异性抗体和/或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，所述双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
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。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。
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，本公开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“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
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，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。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，BiaCore2000,Biacore AB,Upsala,Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335)。
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。
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。
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CD70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 et al.,Biochem.32:1180-1187(1993)；Kobayashi et al.,Protein Eng.12(10):879-884(1999)；Burks et al.,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CD70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CD70抗体。
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。
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
在一方面，本公开提供了结合CD70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、6、7、15、16、22、29、30、31、37、38、44、45、46、54、55、56、64、65、77、83、84、85、91、97、98、106、107、114、115、116、124、131、132、135、142、160或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:10、11、12、19、25、26、34、41、49、50、51、59、60、61、68、69、74、80、88、101、102、103、110、111、119、120、121、127、128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:5、15、29、44、54、83、96、114、131或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:6、30、37、45、55、64、77、84、91、97、106、115、124、132、135、142或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:7、16、22、31、38、46、56、65、85、98、107、116或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:10、16、25、34、51、59、59、38、74、88、101、110、119、127、149或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:11、50、60、80、102、111、120、128、160或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:12、26、51、61、69、103、121或其任何变体的轻链CDR3。
根据前一方面的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
(1)分别包含SEQ ID NO:5、6、7的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:10、11、12的轻链CDR1、CDR2及CDR3序列；
(2)分别包含SEQ ID NO:15、6、16的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:19、11、12的轻链CDR1、CDR2及CDR3序列；
(3)分别包含SEQ ID NO:5、6、22的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:25、11、26的轻链CDR1、CDR2及CDR3序列；
(4)分别包含SEQ ID NO:29、30、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:34、11、26的轻链CDR1、CDR2及CDR3序列；
(5)分别包含SEQ ID NO:5、37、38的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:41、11、12的轻链CDR1、CDR2及CDR3序列；
(6)分别包含SEQ ID NO:44、45、46的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:49、50、51的轻链CDR1、CDR2及CDR3序列；
(7)分别包含SEQ ID NO:54、55、56的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:59、60、61的轻链CDR1、CDR2及CDR3序列；
(8)分别包含SEQ ID NO:5、64、65的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:68、11、69的轻链CDR1、CDR2及CDR3序列；
(9)分别包含SEQ ID NO:29、30、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:74、11、26的轻链CDR1、CDR2及CDR3序列；
(10)分别包含SEQ ID NO:44、77、46的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:49、80、51的轻链CDR1、CDR2及CDR3序列；
(11)分别包含SEQ ID NO:83、84、85的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:88、11、12的轻链CDR1、CDR2及CDR3序列；
(12)分别包含SEQ ID NO:5、91、85的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:19、11、12的轻链CDR1、CDR2及CDR3序列；
(13)分别包含SEQ ID NO:96、97、98的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:101、102、103的轻链CDR1、CDR2及CDR3序列；
(14)分别包含SEQ ID NO:15、106、107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:110、 111、103的轻链CDR1、CDR2及CDR3序列；
(15)分别包含SEQ ID NO:114、115、116的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:119、120、121的轻链CDR1、CDR2及CDR3序列；
(16)分别包含SEQ ID NO:15、124、16的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:127、128、103的轻链CDR1、CDR2及CDR3序列；
(17)分别包含SEQ ID NO:131、132、116的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:119、120、121的轻链CDR1、CDR2及CDR3序列；
(18)分别包含SEQ ID NO:44、135、46的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:149、80、51的轻链CDR1、CDR2及CDR3序列；
(19)分别包含SEQ ID NO::29、142、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:25、11、26的轻链CDR1、CDR2及CDR3序列；
(20)分别包含SEQ ID NO::29、142、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:25、160、26的轻链CDR1、CDR2及CDR3序列。
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:3、13、20、27、35、42、52、62、70、75、81、89、94、104、112、122、129、133、136、138、140、143、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:8、17、23、32、39、47、57、66、72、78、86、92、99、108、117、125、147、150、152、154、156、158或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:3或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:8或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:13或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:17或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:20或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:23或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:27或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:32或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:39或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:42或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:47或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:52或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:57或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:62或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:66或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:70或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:72或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:75或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:78或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:81或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:86或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:89或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:92或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:94或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:99或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:104或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:108或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:112或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:117或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:122或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:125或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:129或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:117或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:133或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:147或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:133或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:133或其任何变体的重 链可变区，和氨基酸序列SEQ ID NO:152或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:136或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:147或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:136或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:136或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:152或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:138或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:147或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:138或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:138或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:152或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:154或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:158或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:143或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:154或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:143或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:143或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:158或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:154或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区。
在本公开的一个优选实施方案中，抗体或其抗原结合部分包含氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:158或其任何变体的轻链可变区。
在一方面，本公开提供了一种双特异性或多特异性分子，其包含前述任一方面的抗体或其抗原结合部分。
编码根据前述任一方面的抗体或其抗原结合部分或双特异性或多特异性分子的核酸分子。
在本公开的一个优选实施方案中，提供了一种编码抗体或其抗原结合部分、双特异性或多特异性分子的核酸分子，核酸分子包含选自SEQ ID NO:4、14、21、28、36、43、53、63、71、76、82、90、95、105、113、123、130、134、137、139、141、144、146或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO:9、18、24、33、40、48、58、67、73、79、87、93、100、109、118、126、148、151、153、155、157、159或其任何变体的抗体轻链核酸序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:4或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:9或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:14或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:18或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:21或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:24或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:28或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:33或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:36或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:40或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:43或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:48或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:53或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:58或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:63或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:67或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:71或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:73或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:76或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:79或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:82或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:87或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:90或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:93或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:95或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:100或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:105或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:109或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:113或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:118或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:123或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:126或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:130或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:118或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:134或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:148或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:134或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:151或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:134或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:153或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:137或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:148或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:137或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:151或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:137或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:153或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:139或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:148或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:139或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:151或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:139或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:153或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:141或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:155或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:141或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:157或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:141或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:159或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:144或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:155或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:144或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:157或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:144或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:159或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:146或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:155或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:146或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:157或其任何变体的序列。
在本公开的一个优选实施方案中，核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:146或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:159或其任何变体的序列。
在一方面，本公开提供了结合CD70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。
在一方面，本公开提供了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，本公开提供了包含如前述任一方面的核酸分子的载体。
在一方面，本公开提供了包含如前述任一方面的载体的细胞。
在一方面，本公开一种组合物，其包含前述的抗体或其抗原结合部分、双特异性或多特异性分子、核酸分子、权利载体和/或细胞。
本公开的抗体在各种CD70被不利地表达或发现的疾病中可用作治疗或诊断工具。
在一个与CD70相关的疾病实施方案中，CD70在患病组织或器官的细胞中的表达与在健康组织或器官中的状态相比有所增加。增加是指增加至少10％、特别是至少20％、至少50％、至少100％、至少200％、至少500％、至少1000％、至少10000％或甚至更多。在一个实施方案中，表达仅在患病组织中发现，而在相应健康组织中的表达受阻抑。
CD70在多种人类癌症中表达，癌症包括肾细胞癌、转移性乳腺癌、脑瘤、白血病、淋巴瘤和鼻咽癌。抗CD70抗体可以单独使用，用来抑制癌性肿瘤的生长。或者，如下文所述，抗CD70抗体也可以与其他免疫原性剂、标准癌症治疗或其他抗体一起使用。
其生长可以用本公开的抗体抑制的优选癌症包括一般对免疫治疗有应答的癌症。可治疗的优选癌症的非限制性的例子包括肾癌(例如肾细胞癌)、乳腺癌、脑瘤、慢性或急性白血病，包括急性髓性白血病、慢性髓性白血病、急性淋巴母细胞性白血病、慢性淋巴细胞性白血病、淋巴瘤(例如何杰金氏和非何杰金氏淋巴瘤、淋巴细胞淋巴瘤、原发性CNS淋巴瘤、T细胞淋巴瘤)和鼻咽癌。可以用本公开的方法治疗的其他癌症的例子包括黑色素瘤(例如转移性恶性黑色素瘤)、前列腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈癌、皮肤或眼内恶性黑色素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、阴户癌、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、儿童实体瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、环境诱发的癌症，包括石棉诱发的癌症，例如间皮瘤，和癌症的组合。
此外，鉴于CD70在各种肿瘤细胞上表达，本公开的人抗体、抗体组合物和方法能够用来治疗患有致肿瘤疾病的受试者，疾病例如是以存在表达CD70的肿瘤细胞为特征的疾病，包括，例如，肾细胞癌(RCC)，如透明细胞RCC、胶质母细胞瘤、乳腺癌、脑瘤、鼻咽癌、非何杰金氏淋巴瘤(NHL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病(CLL)、伯基特淋巴瘤、间变性大细胞淋巴瘤(ALCL)、多发性骨髓瘤、皮肤T细胞淋巴瘤、结节性小卵裂细胞淋巴瘤、淋巴细胞性淋巴瘤、外周T细胞淋巴瘤、伦纳特淋巴瘤、免疫母细胞性淋巴瘤、T细胞白血病/淋巴瘤(ATLL)、成人T细胞白血病(T-ALL)、中心母细胞/中心细胞性(cb/cc)滤泡性淋巴瘤、B系弥漫性大细胞淋巴瘤、血管免疫母细胞性淋巴结病(AILD)-样T细胞淋巴瘤、HIV相关体腔淋巴瘤、胚胎性癌、未分化鼻咽癌(例如施明克瘤)、卡斯尔曼病、卡波西肉瘤、多发性骨髓瘤、沃尔登斯特伦巨球蛋白血症和其他B细胞淋巴瘤。
另外，也已经提出CD70与CD27的相互作用在细胞介导的自身免疫病如实验性自身免疫脑脊髓炎(EAE)中起作用。这种作用被认为部分是由TNF-α产生的抑制介导的。而且，CD70信号的阻断抑制了CD40介导的CD8+T-细胞的克隆扩增，并且减少了CD8+记忆T细胞的产生(Taraban等.(2004)J.Immunol.173：6542-6)。这样，本公开的人抗体、抗体组合物和方法可以用来治疗患有自身免疫病的患者，自身免疫病例如是以存在表达CD70的B细胞为特征的疾病，包括，例如实验性自身免疫脑脊髓炎。可以使用本公开的抗体的其他自身免疫病包括但不限于系统性红斑狼疮(SLE)、胰岛素依赖性糖尿病(IDDM)、炎性肠病(IBD)(包括克罗恩病、溃疡性结肠炎和乳糜泻)、多发性硬化症(MS)、牛皮癣、自身免疫性甲状腺炎、类风湿性关节炎(RA)和肾小球肾炎。此外，本公开的抗体组合物能够用于抑制或预防移植排斥或用于治疗移植物抗宿主疾病(GVHD)。
另外，也已经提出CD70与CD27的相互作用在CD4+T细胞信号传导中起作用。已经证实某些病毒向CD27途径发出信号，导致中和抗体应答的破坏(Matter等.(2006)J Exp Med 203：2145-55)。这样，本公开的人抗体、抗体组合物和方法可以用来治疗患有病毒感染的患者，病毒包括，例如，人类免疫缺陷病毒(HIV)、肝炎(甲、乙、丙)、疱疹病毒(例如VZV、HSV-1、HAV-6、HSV-II和CMV、EB病毒)、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、JC病毒和虫媒病毒脑炎病毒和淋巴细胞性脉络丛脑膜炎病毒(LCMV)，或者用于治疗HIV感染/AIDS。另外，本公开的人抗体、抗体组合物和方法可以用来抑制TNF-α产生。
用本公开的CD70抗体治疗的疾病和症状方法包括下述步骤：向受试者施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合CD70的抗体，其中施用抗体以提供至少40μg/ml的血清水平。在不同实施方案中，施用抗体以提供至少50μg/ml、至少150μg/ml、至少300μg/ml、至少400μg/ml或至少500μg/ml的血清水平。在不同实施方案中，施用抗体以提供不超过800μg/ml、700μg/ml、600μg/ml、550μg/ml或500μg/ml的血清水平。在一个实施方案中，所提供的血清水平为40μg/ml至700μg/ml，优选为40μg/ml至600μg/ml，优选为50μg/ml至500μg/ml，如150μg/ml至500μg/ml或者300μg/ml至500μg/ml。如本说明书中使用的术语“血清水平”，其意指所讨论的物质在血清中的浓度。在一个实施方案中，提供血清水平至少7天或至少14天。在一个实施方案中，方法包括施用至少300mg/m 2的抗体剂量，如至少600mg/m 2，且优选至多1500mg/m 2，至多1200mg/m 2或至多1000mg/m 2。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合CD70的抗体，其中 以至少300mg/m 2，如至少600mg/m 2，且优选至多1500mg/m 2，至多1200mg/m 2或至多1000mg/m 2的剂量施用前述抗体。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合CD70的抗体，其中患者的至少50％，优选为60％、70％、80％或90％的癌细胞为CD70阳性和/或患者的至少40％，优选为50％或60％的癌细胞为CD70的表面表达阳性。在该方面，本公开还提供治疗或预防癌症疾病的方法，该方法包括：a.鉴定显示至少50％，优选为60％、70％、80％或90％的CD70阳性癌细胞和/或至少40％，优选为50％或60％的癌细胞的患者，癌细胞为CD70的表面表达阳性；以及b.向患者施用能够结合CD70的抗体。在一个实施方案中，患者的至少95％或至少98％的癌细胞为CD70阳性的。在一个实施方案中，患者的至少70％、至少80％或至少90％的癌细胞为CD70的表面表达阳性。
在本文所述的任何方面的方法的一个实施方案中，癌症疾病的治疗结果是实现病情稳定。在一个实施方案中，病情稳定实现至少2个月、至少3个月或至少6个月。
在一些实施方案中，本公开提供实现癌症患者的病情稳定的方法，其包括向患者施用能够结合CD70的抗体。在一个实施方案中，病情稳定实现至少2个月、至少3个月或至少6个月。
在本文所述的任何方面的方法的一个实施方案中，以单剂量或多剂量施用抗体。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合CD70的抗体，其中以多剂量施用抗体。
如果根据本公开以多次剂量施用抗体，则优选以至少3次剂量、至少4次剂量、至少5次剂量、至少6次剂量、至少7次剂量、至少8次剂量、至少9次剂量或至少10次剂量且优选至多30次剂量、25次剂量、20次剂量、15次剂量或10次剂量施用抗体。优选以至少7天、至少10天、至少14天或至少20天的时间间隔施用抗体的剂量。优选以7至30天、10至20天且优选为约14天的时间间隔施用抗体的剂量。
在一个实施方案中，施用抗体以便提供至少40μg/ml的血清水平。在不同实施方案中，施用抗体以便提供至少50μg/ml、至少150μg/ml、至少300μg/ml、至少400μg/ml或至少500μg/ml的血清水平。在不同实施方案中，施用抗体以便提供不超过800μg/ml、700μg/ml、600μg/ml、550μg/ml或500μg/ml的血清水平。在一个实施方案中，所提供的血清水平为40μg/ml至700μg/ml，优选为40μg/ml至600μg/ml，优选为50μg/ml至500μg/ml，如150μg/ml至500μg/ml或300μg/ml至500μg/ml。在一个实施方案中，提供血清水平至少7天或至少14天。在一个实施方案中，方法包括施用至少300mg/m 2，如至少600mg/m 2且优选至多1500mg/m 2、至多1200mg/m 2或至多1000mg/m 2的抗体的剂量。
前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物在制备用于治疗受试者中CD70相关病症的药物中的用途。
根据前述任一方面，任选地，抗体偶联其他药物，例如带标记或具有细胞毒性的缀合物。
在一方面，本公开提供了一种试剂盒，其包含前述抗体或其抗原结合部分、双特异性或多特异性分子、核酸分子、载体、细胞、组合物和/或抗体-药物偶联物。
本公开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缀合物，以及抗体使用说明书、杀死特定类型细胞的缀合物等等。该说明书可包括在体外、体内或离体使用抗体、缀合物等的指导。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。
在一方面，本公开提供了前述抗体或其抗原结合部分、双特异性或多特异性分子、核酸分子、载体、细胞、组合物和/或抗体-药物偶联物在制备用于诊断、治疗或预防与CD70相关病症的药物或试剂盒中的用途。
在一些实施方案中，与CD70相关的疾病包括肿瘤和自身免疫疾病。
在一些优选的实施方案中，肿瘤为癌症疾病；优选地，癌症疾病选自肾细胞癌(RCC)、透明细胞RCC、胶质母细胞瘤、非何杰金氏淋巴瘤(NHL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病(CLL)、伯基特淋巴瘤、间变性大细胞淋巴瘤(ALCL)、多发性骨髓瘤、皮肤T细胞淋巴瘤、结节性小卵裂细胞淋巴瘤、淋巴细胞性淋巴瘤、外周T细胞淋巴瘤、伦纳特淋巴瘤、免疫母细胞性淋巴瘤、T细胞白血病/淋巴瘤(ATLL)、成人T细胞白血病(T-ALL)、中心母细胞/中心细胞性(cb/cc)滤泡性淋巴瘤、B系弥漫性大细胞淋巴瘤、血管免疫母细胞性淋巴结病(AILD)-样T细胞淋巴瘤、HIV相关体腔淋巴瘤、胚胎性癌、未分化鼻咽癌、施明克瘤、卡斯尔曼病、卡波西肉瘤、多发性骨髓瘤、沃尔登斯特伦巨球蛋白血症和B细胞淋巴瘤。
在一些优选的实施方案中，自身免疫疾病是狼疮。
在一方面，本公开提供了前述抗体或其抗原结合部分、双特异性或多特异性分子、核酸分子、载体、细胞、组合物和/或抗体-药物偶联物在制备用于治疗受试者的病毒感染的药物或试剂盒中的用途。
在一些优选的实施方案中，病毒感染选自以下病毒的感染：人类免疫缺陷病毒(HIV)、肝炎(甲、乙、丙)、疱疹病毒(例如VZV、HSV-1、HAV-6、HSV-II和CMV、EB病毒)、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、 柯萨奇病毒、冠状病毒、呼吸道合胞病毒、腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、JC病毒和虫媒病毒脑炎病毒、和淋巴细胞性脉络丛脑膜炎病毒(LCMV)。
此外，本公开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，本公开还提供包含本公开的抗CD70的抗体或其片段的组合物，抗体方便地和药学上可接受的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本公开所使用的术语“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。
本公开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。
在一些实施方案中，本公开的人源化抗体与治疗部分(即药物)偶联。治疗部分可以是例如细胞毒素、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素。此类偶联物在本文中称为“抗体-药物偶联物”或“ADC”。
在一些实施方案中，抗体与细胞毒性部分偶联。细胞毒性部分可以例如选自以下：紫杉醇；细胞松弛素B；短杆菌肽D；溴化乙锭；吐根碱；丝裂霉素；依托泊苷；替尼泊苷；长春新碱；长春碱；秋水仙碱；多柔比星；柔红霉素；二羟基蒽二酮；微管蛋白抑制剂如美登素或其类似物或衍生物；抗有丝分裂剂如单甲基奥瑞他汀E或F或其类似物或衍生物；海兔毒素10或15或其类似物；伊立替康或其类似物；米托蒽醌；光辉霉素；放线菌素D；1-脱氢睾酮；糖皮质激素；普鲁卡因；丁卡因；利多卡因；普萘洛尔；嘌呤霉素；卡奇霉素或其类似物或衍生物；抗代谢药，如甲氨喋呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷、氟达拉滨、5氟尿嘧啶、癸二嗪、羟基脲、天冬酰胺酶、吉西他滨或克拉屈滨；烷化剂，如二氯甲基二乙胺、硫代嘌呤、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲佐菌素、达卡巴嗪(DTIC)、丙卡巴嗪、丝裂霉素C；铂类衍生物，如顺铂或卡铂；多卡霉素A、多卡霉素SA、雷切霉素(CC-1065)或其类似物或衍生物；抗生素，如放线菌素、博来霉素、柔红霉素、多柔比星、伊达比星、光霉素、丝裂霉素、米托蒽醌、普力霉素、安定霉素(AMC)；吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)；白喉毒素及相关分子如白喉A链及其活性片段和杂合分子、蓖麻毒素如蓖麻毒素A或去糖基化蓖麻毒素A链毒素、霍乱毒素、志贺样毒素如SLT I、SLT II、SLT IIV、LT毒素、C3毒素、志贺毒素、百日咳毒素、破伤风毒素、大豆Bowman-Birk蛋白酶抑制剂、假单胞菌外毒素、阿罗林、皂草素、蒴莲根毒素、胶凝蛋白、相思豆毒素A链、蒴莲根毒素A链、α-sarcin、油桐(Aleurites fordii)蛋白、石竹素蛋白、美洲商陆蛋白如PAPI、PAPII和PAP-S、苦瓜(momordica charantia)抑制剂、泻果素、巴豆毒素、肥阜草(sapaonaria officinalis)抑制剂、白树毒素、丝裂霉素、局限曲菌素、酚霉素和依诺霉素毒素；核糖核酸酶(RNase)；DNase I、葡萄球菌内毒素A；商陆抗病毒蛋白；白喉毒素和假单胞菌内毒素。
在一些实施方案中，抗体与奥瑞他汀或其肽类似物、衍生物或前药偶联。已经表明，奥瑞他汀干扰微管动力学、GTP水解以及核和细胞分裂并具有抗癌和抗真菌活性。例如，奥瑞他汀E可以与对乙酰基苯甲酸或苯甲酰基戊酸反应，分别产生AEB和AEVB。其他典型的奥瑞他汀衍生物包括AFP、MMAF(单甲基奥瑞他汀F)和MMAE(单甲基奥瑞他汀E)。合适的奥瑞他汀和奥瑞他汀的类似物、衍生物和前药，以及用于将奥瑞他汀与Ab偶联的合适接头描述于例如美国专利号5,635,483、5,780,588和6,214,345以及国际专利申请公开WO02088172、WO2004010957、WO2005081711、WO2005084390、WO2006132670、WO03026577、WO200700860、WO207011968和WO205082023。
在一些实施方案中，抗体与吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)或其肽类似物、衍生物或前药偶联。合适的PDB以及PDB衍生物和相关技术描述于例如Hartley J.A.等,Cancer Res 2010；70(17):6849-6858；Antonow D.等,Cancer J 2008；14(3):154-169；Howard P.W.等,Bioorg Med Chem Lett 2009；19:6463-6466和Sagnou等,Bioorg MedChem Lett 2000；10(18):2083-2086。
在一些实施方案中，抗体与选自以下的细胞毒性部分偶联：蒽环类抗生素、美登素、卡奇霉素、多卡霉素、雷切霉素(CC-1065)、海兔毒素10、海兔毒素15、伊立替康、单甲基奥瑞他汀E、单甲基奥瑞他汀F、PDB，或它们任何的类似物、衍生物或前药。
在一些实施方案中，抗体与蒽环类抗生素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与美登素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与卡奇霉素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与多卡霉素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与雷切霉素(CC-1065)或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与海兔霉素10或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与海兔霉素15或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与单甲基奥瑞他汀E或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与单甲基奥瑞他汀F或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与吡咯并[2,1-c][1,4]-苯并二氮杂卓或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与伊立替康或其类似物、衍生物或前药偶联。
在一些实施方案中，抗体与细胞因子(例如IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、IL-24、IL-27、IL-28a、IL-28b、IL-29、KGF、IFNα、IFNβ、IFNγ、GM-CSF、CD40L、Flt3配体、干细胞因子、安西司亭和TNFα)偶联。
在一些实施方案中，抗体与放射性同位素或含放射性同位素的螯合物偶联。例如，抗体可以与允许抗体与放射性同位素络合的螯合剂接头(例如DOTA、DTPA或噻西坦)偶联。抗体还可以或可选地包含一个或多个放射性标记的氨基酸或其他放射性标记的分子或与之偶联。放射性同位素的非限制性实例包括 3H、 14C、 15N、 35S、 90Y、 99Tc、 125I、 131I、 186Re、 213Bi、 225Ac和 227Th。为了治疗目的，可以使用发射β或α颗粒辐射的放射性同位素，例如 131I、 90Y、 211At、 212Bi、 67Cu、 186Re、 188Re和 212Pb。
将分子与抗体偶联的技术是本领域熟知的。通常，核酸分子分别通过N-羟基琥珀酰亚胺酯或马来酰亚胺官能团与抗体上的赖氨酸或半胱氨酸共价连接。据报道，使用工程化半胱氨酸或整合非天然氨基酸的偶联方法可改进偶联物的同质性。特别地，本领域技术人员还可以预期用含酰基供体谷氨酰胺的标签(例如含有Gin肽的标签或Q-标签)或通过多肽工程(例如通过多肽上的氨基酸缺失、插入、取代或突变)产生反应性的内源性谷氨酰胺工程化的含Fc多肽。然后，转谷氨酰胺酶可与胺供体剂(例如，包含或连接于反应性胺的小分子)共价交联，以形成稳定且均质的工程化含Fc多肽偶联物群，其中胺供体剂通过含酰基供体谷氨酰胺标签或可接近/暴露/反应性的内源性谷氨酰胺位点特异性地与含Fc多肽偶联(WO2012059882)。
应当理解，根据实施方案的治疗剂将与合适的药学上可接受的载体、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公开的治疗或疗法，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。
在一个实施方案中，可将抗体用作治疗剂。此类试剂将通常用于治疗、缓解和/或预防受试者的与异常CD70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CD70表达、活性和/或信号传导相关的疾病或障碍，例如CD70相关病症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CD70)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CD70)与其所天然结合的内源性的配体结合。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CD70表达、活性和/或信号传导。在一些实施方案中，为治疗与异常CD70表达相关的疾病或障碍，可将具有重链和轻链CDR的抗体施用给受试者。
在另一个实施方案中，针对CD70的抗体可用于本领域中已知的与CD70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CD70和/或CD70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CD70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CD70具有特异性的抗体来分离CD70多肽。针对CD70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CD70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CD70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，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。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。
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。合适的药学上可接受的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，合适的药学上可接受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，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。
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抗体。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。
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公开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。
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，本公开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
实施例1、CD70重组蛋白的制备
以人CD70的cDNA质粒(Sino Biological，货号：HG10780-M)为模板，PCR扩增人CD70胞外区片段(Glu13-Pro155)，通过连接肽GSGSGNGS(SEQ ID NO:161)构建CD70胞外区三聚体结构，C端融合鼠IgG Fc(knob)和Avi tag标签GSGLNDIFEAQKIEWHE(SEQ ID NO:162)以便获得纯化的人CD70mIgG Fc(knob)(其氨基酸序列如SEQ ID NO:1所示)。将包含所述人CD70mIgG Fc(knob)编码基因的质粒与鼠IgG Fc(hole)(其氨基酸序列如SEQ ID NO:2所示)质粒共转染HEK293E细胞，培养7天后，收集培养基上清液，经Protein G纯化得到人CD70胞外区三聚体重组蛋白，SDS-PAGE见图1。
实施例2、构建CD70-CHO稳转细胞株
分别以人CD70cDNA和猴CD70cDNA(Sino Biological，货号：CG90089-G)，构建含有人和猴CD70全长序列的慢病毒载体质粒，按照慢病毒包装试剂盒说明书(Lenti-Pac HIV Expression Packaging Kit，Gene Copoeia，货号：HPK-LvTR-20)，将构建好的慢病毒质粒和包装质粒共转染HEK293T细胞，进行慢病毒包装。转染48小时后收集培养基，以500*g离心10分钟去除细胞碎片，获得含慢病毒颗粒的培养上清，0.45μm的PES膜过滤后，分装至1.5mL EP管中，分别取10μL用于感染1×10 6的CHO细胞，在培养基中加入10μg/mL嘌呤霉素(puromycin)进行筛选。获得阳性克隆后，通过有限稀释，最终分别得到表达人和猴全长CD70的稳转细胞CHO-人CD70-B07和CHO-猴CD70-D01，结果见图2-1和图2-2。
实施例3、抗人CD70单克隆抗体的制备
将人CD70重组蛋白作为抗原与等量免疫佐剂(弗氏佐剂)混合，取每组6只6周雌性Balb/c小鼠进行皮下免疫。在初次免疫两周后，进行加强免疫。三次免疫后，眼眶取血，流式细胞仪检测血清效价。检测结果如图3所示。由图3可见，在小鼠血清中均能检测到高滴度CD70抗体。
用人CD70重组蛋白对免疫小鼠进行尾静脉注射冲击免疫。三天后，断颈处死小鼠，取小鼠脾和部分外周淋巴结，在DMEM培养基中研磨后离心；倾倒上清后，轻轻将细胞团打散，加入5mL红细胞裂解液，裂解50秒后，加入40mL DMEM培养基离心，得到不含红细胞的B细胞悬浮液。取适量的B细胞悬浮液和SP2/0混合后，用电融合仪进行细胞融合，将融合后的杂交瘤细胞在含有HAT的DMEM完全培养基中置于5％CO 2，37℃条件下培养。剩余B细胞用Trizol裂解法提取总RNA，用逆转录试剂盒(SuperScript First-Strand Synthesis System，货号：18080051)，利用重链特异性引物逆转录得到抗体重链cDNA文库；以该cDNA为模板，利用重链可变区引物PCR扩增抗体重链可变区片段，经NcoI和NheI双酶切后，克隆到噬菌体质粒；以此类似，利用轻链特异性引物逆转录得到轻链cDNA库，以该cDNA为模板，利用轻链可变区引物PCR扩增抗体轻链可变区片段，经NcoI和BsiwI双酶切后，克隆到噬菌体质粒；然后构建基于丝状噬菌体M13的小鼠Fab噬菌体展示文库，库容为1×10 10。
实施例4、抗人CD70单克隆抗体的制备
1.抗体噬菌体展示库的筛选
噬菌体展示文库采用经典的淘选方式，进行阳性克隆筛选。在Immunotube管包被重组表达的人CD70蛋白，加入1×10 12噬菌体进行孵育和淘洗，用100mM三乙胺洗脱结合亲和力高的噬菌体颗粒，1M Tris-HCl(pH 7.4)中和后感染入大肠杆菌TG1细菌中，再次包装后得到富集的噬菌体可用于下一轮淘洗。经过2轮淘洗富集得到的克隆，由单克隆接种至96孔U形孔板中进行培养和IPTG诱导表达，取上清进行ELISA检测。
1)酶联免疫吸附实验(ELISA)筛选结合CD70的Fab抗体
在96孔酶标板上包被1μg/mL的CD70重组蛋白，4℃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1次，加入30μL诱导上清，室温孵育1小时后，用PBST(PBS+0.05％Tween-20)和PBS重复洗涤3次，加入100μL/孔HRP标记的Anti-Flag二抗溶液，室温孵育60分钟。用PBST和PBS各洗三遍后，加入100μL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，立即用酶联检测仪(BioTek，Synergy HT)测定OD450nm。
2)ELISA筛选阻断CD70与CD27相互作用的Fab抗体
将与CD70抗原结合的阳性单克隆重新进行IPTG诱导，纯化得到Fab抗体蛋白，进行ELISA检测筛选。分别在96孔酶标板上包被1μg/mL的人或者食蟹猴CD27重组蛋白，4℃孵育过夜。次日PBS洗涤3次，加入200μL 2％脱脂奶粉/PBS室温封闭2小时，PBS洗涤1次，加入50μL不同稀释梯度的Fab抗体，室温孵育30分钟。然后每孔加入50μL Biotin标记的CD70重组蛋白，室温孵育60分钟。用PBST和PBS重复洗涤3次，甩干。用0.5％BSA按1:8000比例稀释二抗溶液链霉亲和素偶联辣根过氧化物酶SA-HRP(BD bioscience，货号：6222697)，每孔加入100μl，室温孵育30分钟。用PBST和PBS重复洗涤3次，甩干。每孔加入100μl显色液(TMB溶液，Sigma，货号：T2885)，37℃避光反应，待底物显色适中，每孔加入50μl 2M浓硫酸溶液终止反应，30分钟内用酶联检测仪测定OD450nm，收集数据，结果如表1所示。
3)流式细胞仪筛选特异性结合细胞表面CD70的Fab抗体
将ELISA检测到的结合CD70抗体的阳性克隆，进一步验证与CD70阳性细胞的结合。准备空白对照细胞CHO与稳转细胞株CHO-CD70，分别按5×10 4/孔细胞数，加入96孔板中，每孔再加入50μL不同稀释梯度的Fab抗体，4℃孵育60分钟，离心弃去上清，用0.5％BSA/PBS洗涤，再加入50μL二抗溶液(Anti-human IgG，F(ab')2-AF647，Jackson Immuno Research)，4℃孵育45分钟，之后用0.5％BSA洗去多余的二抗，加入PBS溶液重悬细胞，进行流式细胞仪检测，收集数据，结果如表1所示。
4)Octet筛选特异性结合CD70的Fab抗体
利用生物膜干涉技术(BLI)将10μg/mL生物素标记的CM352抗原固定在SA探针上，设置时间300s，然后结 合不同浓度的CM352Fab抗体，设置结合时间200s，解离时间300s，得到解离速率kdis(1/s)，结果如表1所示。
表1、抗人CD70抗体噬菌体展示库的筛选
Figure PCTCN2021143371-appb-000001
2.杂交瘤的筛选
培养7天后，取杂交瘤细胞培养上清进行ELISA检测，得到多个能特异性识别CD70，并高效阻断CD70-CD27结合的阳性克隆。进一步利用流式细胞术，排除不能结合细胞表面CD70的克隆，结果如表2所示：
表2、抗人CD70抗体杂交瘤的筛选
Figure PCTCN2021143371-appb-000002
3.抗人CD70嵌合抗体的基因克隆及表达
根据杂交瘤筛选的结果，采用TRNzol裂解法提取候选杂交瘤单克隆细胞的RNA，然后逆转录合成单链cDNA，以此为模板扩增杂交瘤单克隆细胞中抗体的可变区序列；根据噬菌体筛选的结果，提取阳性单克隆细菌质粒，经测序后，得到候选阳性克隆重链和轻链可变区序列为：
表3、BCMA鼠源抗体的可变区序列
Figure PCTCN2021143371-appb-000003
克隆：2B12
2B12重链VH的氨基酸序列如SEQ ID NO:3所示，其编码核酸如SEQ ID NO:4所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:5、6、7所示。
Figure PCTCN2021143371-appb-000004
核酸序列
Figure PCTCN2021143371-appb-000005
2B12轻链VK的氨基酸序列如SEQ ID NO:8所示，其编码核酸如SEQ ID NO:9所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:10、11、12所示。
Figure PCTCN2021143371-appb-000006
核酸序列
Figure PCTCN2021143371-appb-000007
克隆：3B2
3B2重链VH的氨基酸序列如SEQ ID NO:13所示，其编码核酸如SEQ ID NO:14所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:15、6、16所示。
Figure PCTCN2021143371-appb-000008
核酸序列
Figure PCTCN2021143371-appb-000009
3B2轻链VK的氨基酸序列如SEQ ID NO:17所示，其编码核酸如SEQ ID NO:18所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:19、11、12所示。
Figure PCTCN2021143371-appb-000010
核酸序列
Figure PCTCN2021143371-appb-000011
克隆：12A1
12A1重链VH的氨基酸序列如SEQ ID NO:20所示，其编码核酸如SEQ ID NO:21所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:5、6、22所示。
Figure PCTCN2021143371-appb-000012
核酸序列
Figure PCTCN2021143371-appb-000013
12A1轻链VK的氨基酸序列如SEQ ID NO:23所示，其编码核酸如SEQ ID NO:24所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:25、11、26所示。
Figure PCTCN2021143371-appb-000014
核酸序列
Figure PCTCN2021143371-appb-000015
克隆：14C12
14C12重链VH的氨基酸序列如SEQ ID NO:27所示，其编码核酸如SEQ ID NO:28所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:29、30、31所示。
Figure PCTCN2021143371-appb-000016
核酸序列
Figure PCTCN2021143371-appb-000017
14C12轻链VK的氨基酸序列如SEQ ID NO:32所示，其编码核酸如SEQ ID NO:33所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:34、11、26所示。
Figure PCTCN2021143371-appb-000018
核酸序列
Figure PCTCN2021143371-appb-000019
克隆：23A11
23A11重链VH的氨基酸序列如SEQ ID NO:35所示，其编码核酸如SEQ ID NO:36所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:5、37、38所示。
Figure PCTCN2021143371-appb-000020
核酸序列
Figure PCTCN2021143371-appb-000021
23A11轻链VK的氨基酸序列如SEQ ID NO:39所示，其编码核酸如SEQ ID NO:40所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:41、11、12所示。
Figure PCTCN2021143371-appb-000022
Figure PCTCN2021143371-appb-000023
核酸序列
Figure PCTCN2021143371-appb-000024
克隆：25H7
25H7重链VH的氨基酸序列如SEQ ID NO:42所示，其编码核酸如SEQ ID NO:43所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:44、45、46所示。
Figure PCTCN2021143371-appb-000025
核酸序列
Figure PCTCN2021143371-appb-000026
25H7轻链VK的氨基酸序列如SEQ ID NO:47所示，其编码核酸如SEQ ID NO:48所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:49、50、51所示。
Figure PCTCN2021143371-appb-000027
核酸序列
Figure PCTCN2021143371-appb-000028
克隆：26G11
26G11重链VH的氨基酸序列如SEQ ID NO:52所示，其编码核酸如SEQ ID NO:53所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:54、55、56所示。
Figure PCTCN2021143371-appb-000029
核酸序列
Figure PCTCN2021143371-appb-000030
26G11轻链VK的氨基酸序列如SEQ ID NO:57所示，其编码核酸如SEQ ID NO:58所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:59、60、61所示。
Figure PCTCN2021143371-appb-000031
核酸序列
Figure PCTCN2021143371-appb-000032
克隆：27A3
27A3重链VH的氨基酸序列如SEQ ID NO:62所示，其编码核酸如SEQ ID NO:63所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:5、64、65所示。
Figure PCTCN2021143371-appb-000033
核酸序列
Figure PCTCN2021143371-appb-000034
27A3轻链VK的氨基酸序列如SEQ ID NO:66所示，其编码核酸如SEQ ID NO:67所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:68、11、69所示。
Figure PCTCN2021143371-appb-000035
核酸序列
Figure PCTCN2021143371-appb-000036
克隆：32D1
32D1重链VH的氨基酸序列如SEQ ID NO:70所示，其编码核酸如SEQ ID NO:71所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:29、30、31所示。
Figure PCTCN2021143371-appb-000037
核酸序列
Figure PCTCN2021143371-appb-000038
32D1轻链VK的氨基酸序列如SEQ ID NO:72所示，其编码核酸如SEQ ID NO:73所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:74、11、26所示。
Figure PCTCN2021143371-appb-000039
核酸序列
Figure PCTCN2021143371-appb-000040
克隆：39H5
39H5重链VH的氨基酸序列如SEQ ID NO:75所示，其编码核酸如SEQ ID NO:76所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:44、77、46所示。
Figure PCTCN2021143371-appb-000041
核酸序列
Figure PCTCN2021143371-appb-000042
Figure PCTCN2021143371-appb-000043
39H5轻链VK的氨基酸序列如SEQ ID NO:78所示，其编码核酸如SEQ ID NO:79所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:49、80、51所示。
Figure PCTCN2021143371-appb-000044
核酸序列
Figure PCTCN2021143371-appb-000045
克隆：1H21-D5
1H21-D5重链VH的氨基酸序列如SEQ ID NO:81所示，其编码核酸如SEQ ID NO:82所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:83、84、85所示。
Figure PCTCN2021143371-appb-000046
核酸序列
Figure PCTCN2021143371-appb-000047
1H21-D5轻链VK的氨基酸序列如SEQ ID NO:86所示，其编码核酸如SEQ ID NO:87所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:88、11、12所示。
Figure PCTCN2021143371-appb-000048
核酸序列
Figure PCTCN2021143371-appb-000049
克隆：10C19-D11
10C19-D11重链VH的氨基酸序列如SEQ ID NO:89所示，其编码核酸如SEQ ID NO:90所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:5、91、85所示。
Figure PCTCN2021143371-appb-000050
核酸序列
Figure PCTCN2021143371-appb-000051
10C19-D11轻链VK的氨基酸序列如SEQ ID NO:92所示，其编码核酸如SEQ ID NO:93所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:19、11、12所示。
Figure PCTCN2021143371-appb-000052
核酸序列
Figure PCTCN2021143371-appb-000053
克隆：16J15-H3
16J15-H3重链VH的氨基酸序列如SEQ ID NO:94所示，其编码核酸如SEQ ID NO:95所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:96、97、98所示。
Figure PCTCN2021143371-appb-000054
核酸序列
Figure PCTCN2021143371-appb-000055
16J15-H3轻链VK的氨基酸序列如SEQ ID NO:99所示，其编码核酸如SEQ ID NO:100所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:101、102、103所示。
Figure PCTCN2021143371-appb-000056
核酸序列
Figure PCTCN2021143371-appb-000057
克隆：17A9-G5
17A9-G5重链VH的氨基酸序列如SEQ ID NO:104所示，其编码核酸如SEQ ID NO:105所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:15、106、107所示。
Figure PCTCN2021143371-appb-000058
核酸序列
Figure PCTCN2021143371-appb-000059
17A9-G5轻链VK的氨基酸序列如SEQ ID NO:108所示，其编码核酸如SEQ ID NO:109所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:110、111、103所示。
Figure PCTCN2021143371-appb-000060
核酸序列
Figure PCTCN2021143371-appb-000061
克隆：36E7-D6
36E7-D6重链VH的氨基酸序列如SEQ ID NO:112所示，其编码核酸如SEQ ID NO:113所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:114、115、116所示。
Figure PCTCN2021143371-appb-000062
Figure PCTCN2021143371-appb-000063
核酸序列
Figure PCTCN2021143371-appb-000064
36E7-D6轻链VK的氨基酸序列如SEQ ID NO:117所示，其编码核酸如SEQ ID NO:118所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:119、120、121所示。
Figure PCTCN2021143371-appb-000065
核酸序列
Figure PCTCN2021143371-appb-000066
克隆：4H6-A6
4H6-A6重链VH的氨基酸序列如SEQ ID NO:122所示，其编码核酸如SEQ ID NO:123所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:15、124、16所示。
Figure PCTCN2021143371-appb-000067
核酸序列
Figure PCTCN2021143371-appb-000068
4H6-A6轻链VK的氨基酸序列如SEQ ID NO:125所示，其编码核酸如SEQ ID NO:126所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:127、128、103所示。
Figure PCTCN2021143371-appb-000069
核酸序列
Figure PCTCN2021143371-appb-000070
克隆：42H19-G4
42H19-G4重链VH的氨基酸序列如SEQ ID NO:129所示，其编码核酸如SEQ ID NO:130所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:131、132、116所示。
Figure PCTCN2021143371-appb-000071
核酸序列
Figure PCTCN2021143371-appb-000072
42H19-G4轻链VK的氨基酸序列如SEQ ID NO:117所示，其编码核酸如SEQ ID NO:118所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:119、120、121所示。
Figure PCTCN2021143371-appb-000073
核酸序列
Figure PCTCN2021143371-appb-000074
将重链可变区克隆入含有人重链固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固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轻链。经测序正确后转染入CHO-S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。利用浓缩管将蛋白超滤浓缩，检测OD280，通过分光光度法测定IgG的浓度。
4.嵌合抗体对Daudi细胞的CDC活性
Daudi细胞重悬后，按照50μL/孔铺入96孔板中，然后每孔加入50μL抗体。将嵌合抗体稀释至10μg/mL，以此为起始浓度，3倍梯度稀释，共3或者4个梯度。每孔再加入50μL补体混合液，将96孔板放至37℃5％CO 2培养箱中培养2小时。然后每孔加入50μL 40％CCK-8溶液，震荡10秒，放于5％CO 2培养箱培养3小时。将96孔板置于酶标仪(BioTek，Synergy HT)中，读取OD450的值，收集数据使用GraphPad Prism 5软件计算结果。结果如图4-1及4-2、表4所示，嵌合抗体39H5显示了极强的CDC的活性，66.7nM浓度下对肿瘤细胞的最大杀伤率接近80％。
表4、CD70单克隆抗体CDC活性筛选
Figure PCTCN2021143371-appb-000075
5.嵌合抗体对Duadi细胞的ADCC杀伤活性
按照每孔50μL将密度为1×10 6个/mL Daudi细胞铺至U型96孔板中，每孔加入50μL嵌合抗体，放至37℃、5％CO 2培养箱培养30分钟。取出NK92-MI-CD16a细胞，加入RPMI 1640培养基，计数，300g离心10分钟。弃上清，RPMI-1640培养基重悬细胞密度至2×10 6/mL；取出96孔板，每孔加入50μL NK92-MI-CD16a细胞至孔板中，置于5％CO 2培养箱中培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，继续37℃培养。300*g离心3分钟，取50μL上清至黑色酶标板，加入50μL乳酸脱氢酶(LDH)检测底物(放置室温提前溶解)，轻轻震荡混匀；10分钟后，加入25μL终止液进行终止，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板。结果如图5和表5所示，嵌合抗体对Daudi细胞有明显的ADCC杀伤作用，并且呈剂量依赖关系。
表5、CD70单克隆抗体ADCC活性筛选
Figure PCTCN2021143371-appb-000076
ND：未检测。
6.嵌合抗体FACS细胞亲和力测定
取对数生长期的不同细胞，3％BSA封闭30分钟以5×10 4细胞/100μL铺U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从30μg/mL起，3倍稀释5个梯度)，4℃孵育1小时。孵育结束后，每孔加入180μL 0.5％BSA洗3次，加30μL/孔二抗AlexaFluro647抗人IgG(Jackson Immuno Research，货号：109-606-170)，4℃孵育45分钟。孵育结束后，每孔加入180μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行iQue(Intellicyt，USA)检测。如图6-1、6-2和表6所示，FACS检测到嵌合抗体分别高亲和力结合稳转细胞株CHO-人CD70和CHO-食蟹猴CD70上表达的CD70分子。
表6、FACS细胞亲和力测定
Figure PCTCN2021143371-appb-000077
ND：未检测。
7.嵌合抗体对CD70与CD27结合的阻断作用
在96孔酶标板上包被1μg/mL的CD27重组蛋白，4℃孵育过夜。次日PBS洗涤3次，加入200μL 2％脱脂奶粉/PBS室温封闭2小时，PBS洗涤1次；将纯化的嵌合抗体做12个浓度梯度，起始浓度为100nM，按照1:3的梯度进行稀释，每孔加入30μl，室温孵育30分钟。然后每孔加入30μL生物素标记的CD70重组蛋白，室温孵育60分钟。用PBST和PBS重复洗涤3次，甩干。用0.5％BSA按1:8000比例稀释二抗链霉亲和素偶联辣根过氧化物酶SA-HRP(BD bioscience，货号：6222697)，每孔加入100μ，室温孵育30分钟。用PBST和PBS重复洗涤3次，甩干。每孔加入100μl显色液(TMB溶液，Sigma，货号：T2885)，37℃避光反应，待底物显色适中，每孔加入50μl H 2SO 4终止反应，30分钟内用酶联检测仪测定OD450nm，收集数据，使用Graph Pad Prism 5软件计算结果，结果如图7-1、7-2和表7所示，嵌合抗体能够阻断人CD70和CD27以及食蟹猴CD70和CD27的相互结合。
表7、对人CD70与CD27结合的阻断作用测定
Figure PCTCN2021143371-appb-000078
Figure PCTCN2021143371-appb-000079
ND：未检测。
实施例5、Anti-CD70抗体的人源化
选择CDC活性较强的鼠抗体39H5和32D1进行可变区序列人源化改造。首先将鼠抗体39H5序列与人抗体胚系序列进行比对，找出同源性好、维持抗体结构核心(Upper hydrophobic core)的关键氨基酸序列完全相同、同时在人体出现频率高的人胚系轻链基因IMGT_hVK1-33、IGKJ1*01和人胚系重链基因IMGT_hVH7-4、IGHJ4*01，进行鼠抗体CDR移植；同时将鼠抗体32D1序列与人抗体胚系序列进行比对，找出同源性好、维持抗体结构核心的关键氨基酸序列完全相同、同时在人体出现频率高的人胚系轻链基因IMGT_hVK7-3和IMGT_hVK3-20、IGKJ1*01和人胚系重链基因IMGT_hVH1-69、IGHJ4*01，进行鼠抗体CDR移植；利用计算机进行同源建模，分析CDR区及其周边框架氨基酸序列，避免分子表面电荷或疏水区域集中分布。
设计共得到6个重链变体h39H5-VHv1-v3、h32D1-VHv1-v3和6个轻链变体h39H5-VKv1-v3、h32D1-VKv1-v3，将轻、重链分别进行全序列合成后，克隆到含有抗体kappa链固定区Ckappa或人IgG1固定区CH1-CH3的真核表达载体，将轻、重链质粒进行组合配对后，转染CHO-S细胞，37℃表达5-6天，收取培养上清，通过Protein A柱进行纯化。
人源化抗体重链/轻链可变区序列如下：
表8、CD70人源化抗体的重链可变区序列
Figure PCTCN2021143371-appb-000080
表9、CD70人源化抗体的轻链可变区序列
Figure PCTCN2021143371-appb-000081
h39H5 VHv1
重链VH的氨基酸序列如SEQ ID NO:133所示，其编码核酸如SEQ ID NO:134所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:44、135、46所示。
Figure PCTCN2021143371-appb-000082
核酸序列
Figure PCTCN2021143371-appb-000083
h39H5 VHv2
重链VH的氨基酸序列如SEQ ID NO:136所示，其编码核酸如SEQ ID NO:137所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:44、135、46所示。
Figure PCTCN2021143371-appb-000084
核酸序列
Figure PCTCN2021143371-appb-000085
h39H5 VHv3
重链VH的氨基酸序列如SEQ ID NO:138所示，其编码核酸如SEQ ID NO:139所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:44、135、46所示。
Figure PCTCN2021143371-appb-000086
核酸序列
Figure PCTCN2021143371-appb-000087
h32D1 VHv1
重链VH的氨基酸序列如SEQ ID NO:140所示，其编码核酸如SEQ ID NO:141所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:29、142、31所示。
Figure PCTCN2021143371-appb-000088
核酸序列
Figure PCTCN2021143371-appb-000089
h32D1 VHv2
重链VH的氨基酸序列如SEQ ID NO:143所示，其编码核酸如SEQ ID NO:144所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:29、142、31所示。
Figure PCTCN2021143371-appb-000090
核酸序列
Figure PCTCN2021143371-appb-000091
h32D1 VHv3
重链VH的氨基酸序列如SEQ ID NO:145所示，其编码核酸如SEQ ID NO:146所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:29、142、31所示。
Figure PCTCN2021143371-appb-000092
Figure PCTCN2021143371-appb-000093
核酸序列
Figure PCTCN2021143371-appb-000094
h39H5 VKv1
轻链VH的氨基酸序列如SEQ ID NO:147所示，其编码核酸如SEQ ID NO:148所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:149、80、51所示。
Figure PCTCN2021143371-appb-000095
核酸序列
Figure PCTCN2021143371-appb-000096
h39H5 VKv2
轻链VH的氨基酸序列如SEQ ID NO:150所示，其编码核酸如SEQ ID NO:151所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:149、80、51所示。
Figure PCTCN2021143371-appb-000097
核酸序列
Figure PCTCN2021143371-appb-000098
h39H5 VKv3
轻链VH的氨基酸序列如SEQ ID NO:152所示，其编码核酸如SEQ ID NO:153所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:149、80、51所示。
Figure PCTCN2021143371-appb-000099
核酸序列
Figure PCTCN2021143371-appb-000100
h32D1 VKv1
轻链VH的氨基酸序列如SEQ ID NO:154所示，其编码核酸如SEQ ID NO:155所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:25、11、26所示。
Figure PCTCN2021143371-appb-000101
核酸序列
Figure PCTCN2021143371-appb-000102
h32D1 VKv2
轻链VH的氨基酸序列如SEQ ID NO:156所示，其编码核酸如SEQ ID NO:157所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:25、11、26所示。
Figure PCTCN2021143371-appb-000103
核酸序列
Figure PCTCN2021143371-appb-000104
h32D1 VKv3
轻链VH的氨基酸序列如SEQ ID NO:158所示，其编码核酸如SEQ ID NO:159所示，其CDR1、CDR2和CDR3分别如SEQ ID NO:25、160、26所示。
Figure PCTCN2021143371-appb-000105
核酸序列
Figure PCTCN2021143371-appb-000106
实施例6、不同人源化CD70抗体对肿瘤细胞Daudi的CDC杀伤
Daudi细胞重悬后，按照50μL/孔铺入96孔板中，然后每孔加入50μL抗体。将人源化抗体稀释至10μg/mL，以此为起始浓度，3倍梯度稀释，共3个梯度。每孔再加入50μL补体混合液，将96孔板放至37℃5％CO 2培养箱中培养2小时。然后每孔加入50μL 40％CCK-8溶液，震荡10秒，放于5％CO 2培养箱培养3小时。将96孔板置于酶标仪(BioTek，Synergy HT)中，读取OD450的值，收集数据使用GraphPad Prism 5软件计算结果。结果如图8和表10所示，在不同人源化CD70抗体中，人源化变体Ab001、Ab004和Ab007对Dauai细胞的最大杀伤率最高，为60％左右。
表10、不同人源化CD70抗体对肿瘤细胞Daudi的CDC杀伤
Figure PCTCN2021143371-appb-000107
实施例7、人源化CD70抗体的亲和力测定
分别取稳转细胞CHO-人CD70和CHO-食蟹猴CD70，3％BSA封闭30分钟，以5×10 4细胞/100μL铺于U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，加入50μL梯度稀释的抗体(抗体浓度从30μg/mL起，3倍稀释8个梯度)，4℃孵育1小时。孵育结束后，每孔加入200μL 0.5％BSA洗3次，加50μL/孔二抗溶液Alexa Fluro647抗人IgG(Jackson ImmunoResearch，货号：109-606-170)，4℃孵育45分钟。孵育结束后，每孔加入180μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行iQue(Intellicyt)检测。如图9-1、9-2和表11所示，FACS检测到人源化抗体Ab001、Ab004和Ab007分别高亲和力结合稳转细胞株CHO-人CD70和CHO-猴CD70上表达的CD70分子。
表11、FACS细胞亲和力测定
Figure PCTCN2021143371-appb-000108
实施例8、人源化CD70抗体对CD70与CD27结合的阻断作用
在96孔酶标板上包被1μg/mL的人CD27重组蛋白，4℃孵育过夜。次日PBS洗涤3次，加入200μL 2％脱脂奶粉/PBS室温封闭2小时，PBS洗涤1次；将纯化的嵌合抗体做12个浓度梯度，起始浓度为100nM，按照1:3的梯度进行稀释，每孔加入30μl，室温孵育30分钟。然后每孔加入30μL生物素标记的CD70重组蛋白，室温孵育60分钟。用PBST和PBS重复洗涤3次，甩干。用0.5％BSA按1:8000比例稀释二抗溶液链霉亲和素偶联辣根过氧化物酶SA-HRP(BD bioscience，货号：6222697)，每孔加入100μL，室温孵育30分钟。用PBST和PBS重复洗涤3次，甩干。每孔加入100μl显色液(TMB溶液，Sigma，货号：T2885)，37℃避光反应，待底物显色适中，每孔加入50μL H 2SO 4终止反应，30分钟内用酶联检测仪测定OD450nm，收集数据，使用Graph Pad Prism 5软件计算结果，结果如图10-1、10-2和表12，人源化抗体Ab001、Ab004和Ab007可有效阻断人或食蟹猴CD70受体和配体CD27的相互结合。
表12、对人CD70与CD27结合的阻断作用测定
Figure PCTCN2021143371-appb-000109
实施例9、人源化CD70抗体对不同肿瘤细胞的CDC杀伤
Daudi细胞重悬后，按照50μL/孔铺入96孔板中，然后每孔加入50μL抗体。将人源化抗体稀释至10μg/mL，以此为起始浓度，3倍梯度稀释。再加入50μL补体混合液，将96孔板放至37℃5％CO 2培养箱中培养2小时。然后每孔加入50μL 40％CCK-8溶液，震荡10秒，放于5％CO 2培养箱培养3小时。将96孔板置于酶标仪(BioTek，Synergy HT)中，读取OD450的值，收集数据使用GraphPad Prism 5软件计算结果。结果如图11-1、11-2、11-3和表13所示，人源化抗体Ab001、Ab004和Ab007对肿瘤细胞Daudi、MV-4-11-CD70、Raji均具有明显的杀伤作用。
表13、不同人源化CD70抗体对不同肿瘤细胞的CDC杀伤
Figure PCTCN2021143371-appb-000110
ND：未检测
实施例10、人源化CD70抗体对不同肿瘤细胞的ADCC杀伤
接种Daudi细胞至U型96孔板中，每孔加入不同浓度的人源化抗体，放至CO 2培养箱培养半小时；取出NK92-MI-CD16a细胞，RPMI-1640培养基重悬细胞密度至2×10 6/mL；然后在96孔板的每孔中加入50μL NK92-MI-CD16a细胞，置于CO 2培养箱中培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，继续37℃培养。300g离心3分钟，取50μL上清至黑色酶标板，每孔加入50μL LDH检测底物(放置室温提前溶解)，轻轻震荡混匀；10分钟后，每孔加入25μL终止液进行终止，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板，并计算杀伤率。杀伤率计算公式如下：
Figure PCTCN2021143371-appb-000111
结果如图12-1、12-2和表14所示，人源化抗体Ab001、Ab004和Ab007对肿瘤细胞Daudi、Raji均具有明显的杀伤作用。
表14、不同人源化CD70抗体对不同肿瘤细胞的ADCC杀伤
Figure PCTCN2021143371-appb-000112
ND：未检测
实施例11、人源化CD70抗体对肿瘤细胞的ADCP杀伤
用10nM荧光染料CFSE标记的Daudi细胞作为CD70+靶细胞，接种至96孔板中，加入梯度稀释的人源化抗体，置于CO 2培养箱中培养，最后加入GM-CSF1诱导成熟的巨噬细胞，比例为巨噬细胞：Daudi＝1:4，于37℃、5％CO 2条件下；加入Anti-CD14APC抗体稀释溶液，于冰上避光20分钟；流式细胞仪检测收集细胞，以单独巨噬细胞孔为对照，圈出CD14单阳性细胞群，实验孔圈出CD14单阳性细胞及CD14和CSFE双阳性细胞群，统计两群细胞数量。结果如图13和表15所示，人源化抗体Ab001、Ab004和Ab007对肿瘤细胞Daudi均有明显的ADCP吞噬作用。
表15、不同人源化CD70抗体对肿瘤细胞Daudi的ADCP杀伤
Figure PCTCN2021143371-appb-000113
实施例12、人源化CD70抗体对CD70介导刺激IL-8分泌的抑制作用
取3×10 6个HT1080-人CD27细胞，300g离心10分钟，弃上清，加入10mL PBS清洗一次，用DMEM完全培养基重悬细胞密度至2×10 5/mL，每孔加入100μL，置于5％CO 2培养箱中培养过夜。第二天，在96孔U型孔板中按照摩尔比1:1，加入实施例1制备的人CD70重组蛋白和Anti-mouse IgG Fc(Jackson Immuno Research)，总体积为110μL，充分混匀后，于37℃中预孵育30分钟形成CD70聚体。取新的96孔板，每孔加入110μL不同梯度稀释的人源化CD70抗体和110μL上一步预孵育样品，混匀后，37℃孵育30分钟。取出细胞培养孔板，离心弃去上清，每孔加入200μL抗体和重组蛋白预混合样品，置于5％CO 2培养箱中培养6小时，然后300g离心10分钟，收集上清，用人IL-8细胞因子检测试剂盒(BD，货号：555244)检测上清中IL-8的含量。结果如图14和表16所示，人源化抗体Ab001、Ab004和Ab007均能抑制CD70介导刺激IL-8的分泌。
表16、不同人源化CD70抗体对CD70介导刺激IL-8分泌的抑制作用
Figure PCTCN2021143371-appb-000114
实施例13、人源化CD70抗体在Raji细胞株皮下异种移植NOD/SCID小鼠模型中的抗肿瘤作用
在含10％胎牛血清的RPMI1640培养液中培养Raji细胞。收集对数生长期的Raji细胞，PBS重悬至适合浓度并与基质胶(matrigel)1:1混合后用于NOD/SCID小鼠皮下肿瘤接种。雌性小鼠右侧皮下接种1×10 7Raji细胞，待肿瘤平均体积为100mm 3时，根据肿瘤大小随机分组。每组5只小鼠，治疗组的小鼠以1mg/kg、3mg/kg和10mg/kg的剂量每周尾静脉注射二次，共给药两周，监控肿瘤体积。肿瘤体积计算方法为：
肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。
结果如图15-1所示，人源化CD70抗体3mg/kg和10mg/kg剂量组均明显抑制人Burkitt's淋巴瘤Raji小鼠皮下移植瘤的生长，引起肿瘤部分消退；其中人源化CD70抗体Ab004在10mg/kg给药剂量下对肿瘤的抑制率为69.4％。
同时如图15-2所示，人源化CD70抗体在不同剂量组均不引起小鼠体重的下降，无明显的毒性作用。
实施例14、CDC活性增强型CD70人源化抗体
基于IgG1和补体C1的结构模型(Ugurlar et al.,2017)，在IgG1和补体C1结合区域的第一结合位点引入突变以降低极性(R 292和/或Y 300)，减弱对C1q的R129和R114的排斥，或同时在CH3引入突变P 396L，优化CH2和CH3之间的角度。含有Fc突变的CD70抗体在CHO细胞表达，纯化后检测各突变对CD70抗体的生物学活性影响。对照抗体41D12按文献CN201280013552.5合成，并在CHO细胞表达和纯化。
结果显示，CD70人源化抗体Ab004和Ab007的CDC活性均显著优于对照抗体41D12，其中含Fc突变的CD70人源化抗体(突变位点如表17所示)，Ab004IgG1e1、Ab004IgG1e3和Ab004IgG1e5三种抗体CDC杀伤作用得到显著增强，对Daudi细胞的最大杀伤率大于90％，中效杀伤浓度EC50低于野生型约2-3倍(图16)。
人群中广泛存在FcγRIII V158和FcγRIII F158两种CD16a受体等位基因，其中等位基因FcγRIII V158编码受体与抗体IgG1亲和力较高，表达FcγRIII V158的NK细胞介导更有效的抗体依赖细胞介导的细胞毒作用(ADCC)，被认为是抗体药物对低亲和力FcγRIII F158群体疗效不显著的原因。Biacore结果显示，突变体Ab004IgG1e5与低亲和力FcγRIII F158有显著亲和力增强，与野生型IgG1与高亲和力FcγRIII V158受体亲和力相当(表17)。
表17、不同Fc突变体的生物学活性评价
Figure PCTCN2021143371-appb-000115
Figure PCTCN2021143371-appb-000116
ND：未检测
从含不同FcγRIIIa等位基因受体的健康志愿者，分离收集外周血PBMC，以RPMI-1640培养基重悬细胞；接种Daudi细胞至U型96孔板中，每孔加入不同浓度的人源化抗体，放至CO 2培养箱培养半小时；然后每孔加入5×10 5个PBMC细胞至96孔板中，置于CO 2培养箱中培养4小时。检测前30分钟，向靶细胞最大孔加入2μL细胞裂解液(100×)，继续37℃培养。300g离心3分钟，取50μL上清至黑色酶标板，每孔加入50μL LDH检测底物(放置室温提前溶解)，轻轻震荡混匀；10分钟后，每孔加入25μL终止液进行终止，震荡10秒，选择荧光(激发波长560nm，发射波长590nm)进行读板，并计算杀伤率。
结果如图17-1、17-2和表18所示，以FcγRIII V158/V158志愿者PBMC或FcγRIII F158/F158志愿者PBMC为效应细胞，Ab004IgG1e5对肿瘤细胞Daudi杀伤作用相对野生型Ab004IgG1均有明显增强。其中，Ab004IgG1e5在低亲和力受体FcγRIII F158/F158志愿者PBMC介导的ADCC杀伤作用，与野生型Ab004IgG1在高亲和力受体FcγRIII V158/V158志愿者PBMC中介导的ADCC作用相当，提示Fc采用突变体IgG1e5的抗体对广泛人群均有相当的ADCC药效作用。
表18、不同人源化CD70抗体对不同肿瘤细胞的ADCC杀伤
Figure PCTCN2021143371-appb-000117
实施例15、CDC活性增强型CD70抗体在Daudi细胞株皮下异种移植NOD/SCID小鼠模型中的抗肿瘤作用
取对数生长期的Daudi细胞，调整细胞密度为1.2×10 8/ml，加入等体积Matrigel Matrix与细胞悬液充分混匀，每只100μl接种于BALB/c-Nude小鼠右肢皮下。待肿瘤平均体积为100-200mm 3时，随机分组，每组5只小鼠，分组当天开始第一次给药，每三天一次，共给药三次，每只100μl尾静脉注射给药。皮下接瘤当天为D0，绘制肿瘤生长曲线(肿瘤体积不超过2000mm 3，到达2000mm 3时处死小鼠)。
结果如图18所示，CDC活性增强型CD70抗体以及母本抗体均能剂量依赖性地抑制人淋巴瘤Daudi皮下移植瘤的生长。CDC活性增强型CD70抗体按照剂量由高到低的抑瘤率分别为114％、92％、67％；CD70母本抗体的抑瘤率分别为81％、36％、24％。荷瘤小鼠对以上给药剂量均能很好耐受，没有体重减轻等不良反应。

Claims (14)

  1. 一种结合CD70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、6、7、15、16、22、29、30、31、37、38、44、45、46、54、55、56、64、65、77、83、84、85、91、97、98、106、107、114、115、116、124、131、132、135、142、160或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO:10、11、12、19、25、26、34、41、49、50、51、59、60、61、68、69、74、80、88、101、102、103、110、111、119、120、121、127、128或任何变体的轻链CDR。
  2. 根据权利要求1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、29、44、54、83、96、114、131或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO:6、30、37、45、55、64、77、84、91、97、106、115、124、132、135、142或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO:7、16、22、31、38、46、56、65、85、98、107、116或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO:10、16、25、34、51、59、59、38、74、88、101、110、119、127、149或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO:11、50、60、80、102、111、120、128、160或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO:12、26、51、61、69、103、121或其任何变体的轻链CDR3。
  3. 根据权利要求1或2的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
    (1)分别包含SEQ ID NO:5、6、7的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:10、11、12的轻链CDR1、CDR2及CDR3序列；
    (2)分别包含SEQ ID NO:15、6、16的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:19、11、12的轻链CDR1、CDR2及CDR3序列；
    (3)分别包含SEQ ID NO:5、6、22的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:25、11、26的轻链CDR1、CDR2及CDR3序列；
    (4)分别包含SEQ ID NO:29、30、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:34、11、26的轻链CDR1、CDR2及CDR3序列；
    (5)分别包含SEQ ID NO:5、37、38的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:41、11、12的轻链CDR1、CDR2及CDR3序列；
    (6)分别包含SEQ ID NO:44、45、46的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:49、50、51的轻链CDR1、CDR2及CDR3序列；
    (7)分别包含SEQ ID NO:54、55、56的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:59、60、61的轻链CDR1、CDR2及CDR3序列；
    (8)分别包含SEQ ID NO:5、64、65的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:68、11、69的轻链CDR1、CDR2及CDR3序列；
    (9)分别包含SEQ ID NO:29、30、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:74、11、26的轻链CDR1、CDR2及CDR3序列；
    (10)分别包含SEQ ID NO:44、77、46的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:49、80、51的轻链CDR1、CDR2及CDR3序列；
    (11)分别包含SEQ ID NO:83、84、85的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:88、11、12的轻链CDR1、CDR2及CDR3序列；
    (12)分别包含SEQ ID NO:5、91、85的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:19、11、12的轻链CDR1、CDR2及CDR3序列；
    (13)分别包含SEQ ID NO:96、97、98的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:101、102、103的轻链CDR1、CDR2及CDR3序列；
    (14)分别包含SEQ ID NO:15、106、107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:110、111、103的轻链CDR1、CDR2及CDR3序列；
    (15)分别包含SEQ ID NO:114、115、116的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:119、120、121的轻链CDR1、CDR2及CDR3序列；
    (16)分别包含SEQ ID NO:15、124、16的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:127、128、103的轻链CDR1、CDR2及CDR3序列；
    (17)分别包含SEQ ID NO:131、132、116的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:119、120、121的轻链CDR1、CDR2及CDR3序列；
    (18)分别包含SEQ ID NO:44、135、46的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:149、80、51的轻链CDR1、CDR2及CDR3序列；
    (19)分别包含SEQ ID NO::29、142、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:25、11、26的轻链CDR1、CDR2及CDR3序列；
    (20)分别包含SEQ ID NO::29、142、31的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:25、160、26的轻链CDR1、CDR2及CDR3序列。
  4. 根据权利要求1或2的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO:3、13、20、27、35、42、52、62、70、75、81、89、94、104、112、122、129、133、136、138、140、143、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:8、17、23、32、39、47、57、66、72、78、86、92、99、108、117、125、 147、150、152、154、156、158或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:3或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:8或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:13或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:17或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:20或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:23或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:27或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:32或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:35或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:39或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:42或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:47或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:52或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:57或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:62或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:66或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:70或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:72或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:75或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:78或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:81或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:86或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:89或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:92或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:94或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:99或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:104或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:108或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:112或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:117或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:122或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:125或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:129或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:117或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:133或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:147或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:133或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:133或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:152或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:136或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:147或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:136或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:136或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:152或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:138或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:147或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:138或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:150或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:138或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:152或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:154或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其 任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:140或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:158或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:143或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:154或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:143或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:143或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:158或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:154或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:156或其任何变体的轻链可变区；
    优选地，其包含氨基酸序列SEQ ID NO:145或其任何变体的重链可变区，和氨基酸序列SEQ ID NO:158或其任何变体的轻链可变区。
  5. 一种双特异性或多特异性分子，其包含权利要求1-4任一项所述的结合CD70的抗体或其抗原结合部分。
  6. 根据权利要求1-4任一项所述的抗体或其抗原结合部分或权利要求5所述的双特异性或多特异性分子，所述抗体或其抗原结合部分是人源化的。
  7. 编码根据权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子或权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子的核酸分子；
    优选地，所述核酸分子包含选自SEQ ID NO:4、14、21、28、36、43、53、63、71、76、82、90、95、105、113、123、130、134、137、139、141、144、146或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO:9、18、24、33、40、48、58、67、73、79、87、93、100、109、118、126、148、151、153、155、157、159或其任何变体的抗体轻链核酸序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:4或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:9或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:14或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:18或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:21或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:24或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:28或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:33或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:36或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:40或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:43或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:48或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:53或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:58或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:63或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:67或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:71或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:73或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:76或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:79或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:82或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:87或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:90或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:93或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:95或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:100或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:105或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:109或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:113或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:118或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:123或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:126或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:130或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:118或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:134或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:148或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:134或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:151或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:134或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:153或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:137或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:148或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:137或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:151或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:137或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:153或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:139或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:148或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:139或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:151或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:139或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:153或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:141或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:155或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:141或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:157或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:141或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:159或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:144或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:155或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:144或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:157或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:144或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:159或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:146或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:155或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:146或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:157或其任何变体的序列；
    优选地，所述核酸分子包含抗体重链可变区核苷酸序列SEQ ID NO:146或其任何变体的序列，和/或轻链可变区核苷酸序列SEQ ID NO:159或其任何变体的序列。
  8. 包含权利要求7所述核酸分子的载体。
  9. 包含权利要求7所述的核酸分子或权利要求8所述载体的细胞。
  10. 一种组合物，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸分子、权利要求8所述的载体和/或权利要求9所述的细胞。
  11. 一种抗体-药物偶联物，其包含共价附着至治疗部分的权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子；
    优选地，所述治疗部分选自细胞毒性部分、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素；
    优选地，所述细胞毒性部分选自以下：紫杉醇；细胞松弛素B；短杆菌肽D；溴化乙锭；吐根碱；丝裂霉素；依托泊苷；替尼泊苷；长春新碱；长春碱；秋水仙碱；多柔比星；柔红霉素；二羟基蒽二酮；微管蛋白抑制剂如美登素或其类似物或衍生物；抗有丝分裂剂如单甲基奥瑞他汀E或F或其类似物或衍生物；海兔毒素10或15或其类似物；伊立替康或其类似物；米托蒽醌；光辉霉素；放线菌素D；1-脱氢睾酮；糖皮质激素；普鲁卡因；丁卡因；利多卡因；普萘洛尔；嘌呤霉素；卡奇霉素或其类似物或衍生物；抗代谢药，如甲氨喋呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷、氟达拉滨、5氟尿嘧啶、癸二嗪、羟基脲、天冬酰胺酶、吉西他滨或克拉屈滨；烷化剂，如二氯甲基二乙胺、硫代嘌呤、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲佐菌素、达卡巴嗪(DTIC)、丙卡巴嗪、丝裂霉素C；铂类衍生物，如顺铂或卡铂；多卡霉素A、多卡霉素SA、雷切霉素(CC-1065)或其类似物或衍生物；抗生素，如放线菌素、博来霉素、柔红霉素、多柔比星、伊达比星、 光霉素、丝裂霉素、米托蒽醌、普力霉素、安定霉素(AMC)；吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)；白喉毒素及相关分子如白喉A链及其活性片段和杂合分子、蓖麻毒素如蓖麻毒素A或去糖基化蓖麻毒素A链毒素、霍乱毒素、志贺样毒素如SLT I、SLT II、SLT IIV、LT毒素、C3毒素、志贺毒素、百日咳毒素、破伤风毒素、大豆Bowman-Birk蛋白酶抑制剂、假单胞菌外毒素、阿罗林、皂草素、蒴莲根毒素、胶凝蛋白、相思豆毒素A链、蒴莲根毒素A链、α-sarcin、油桐(Aleurites fordii)蛋白、石竹素蛋白、美洲商陆蛋白如PAPI、PAPII和PAP-S、苦瓜(momordica charantia)抑制剂、泻果素、巴豆毒素、肥阜草(sapaonaria officinalis)抑制剂、白树毒素、丝裂霉素、局限曲菌素、酚霉素和依诺霉素毒素；核糖核酸酶(RNase)；DNase I、葡萄球菌内毒素A；商陆抗病毒蛋白；白喉毒素和假单胞菌内毒素；
    优选地，所述细胞因子选自IL-2、IL-4、IL-6、IL-7、IL-10、IL-12、IL-13、IL-15、IL-18、IL-23、IL-24、IL-27、IL-28a、IL-28b、IL-29、KGF、IFNa、IFN3、IFNy、GM-CSF、CD40L、Flt3配体、干细胞因子、安西司亭和TNFa；
    优选地，所述放射性同位素选自 3H、 14C、 15N、 35S、 67Cu、 90Y、 99Tc、 125I、 131I、 186Re、 188Re、 211At、 212Bi、 212Pb、 213Bi、 225Ac和 227Th。
  12. 一种试剂盒，其包含权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的细胞、权利要求10所述的组合物和/或权利要求11的抗体-药物偶联物。
  13. 权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的细胞、权利要求10所述的组合物和/或权利要求11的抗体-药物偶联物在制备用于诊断、治疗或预防与CD70相关病症的药物或试剂盒中的用途；
    优选地，与CD70相关的疾病包括肿瘤和自身免疫疾病；
    优选地，所述肿瘤为癌症疾病；优选地，所述癌症疾病选自肾细胞癌(RCC)、透明细胞RCC、胶质母细胞瘤、非何杰金氏淋巴瘤(NHL)、急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病(CLL)、伯基特淋巴瘤、间变性大细胞淋巴瘤(ALCL)、多发性骨髓瘤、皮肤T细胞淋巴瘤、结节性小卵裂细胞淋巴瘤、淋巴细胞性淋巴瘤、外周T细胞淋巴瘤、伦纳特淋巴瘤、免疫母细胞性淋巴瘤、T细胞白血病/淋巴瘤(ATLL)、成人T细胞白血病(T-ALL)、中心母细胞/中心细胞性(cb/cc)滤泡性淋巴瘤、B系弥漫性大细胞淋巴瘤、血管免疫母细胞性淋巴结病(AILD)-样T细胞淋巴瘤、HIV相关体腔淋巴瘤、胚胎性癌、未分化鼻咽癌、施明克瘤、卡斯尔曼病、卡波西肉瘤、多发性骨髓瘤、沃尔登斯特伦巨球蛋白血症和B细胞淋巴瘤；
    优选地，所述自身免疫疾病是狼疮。
  14. 权利要求1-4任一项所述的抗体或其抗原结合部分、权利要求5所述的双特异性或多特异性分子、权利要求6所述的抗体或其抗原结合部分或双特异性或多特异性分子、权利要求7所述的核酸分子、权利要求8所述的载体、权利要求9所述的细胞、权利要求10所述的组合物和/或权利要求11的抗体-药物偶联物在制备用于治疗受试者的病毒感染的药物或试剂盒中的用途；
    优选地，所述病毒感染选自以下病毒的感染：人类免疫缺陷病毒(HIV)、肝炎(甲、乙、丙)、疱疹病毒(例如VZV、HSV-1、HAV-6、HSV-II和CMV、EB病毒)、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、腮腺炎病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革热病毒、乳头瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、JC病毒和虫媒病毒脑炎病毒、和淋巴细胞性脉络丛脑膜炎病毒(LCMV)。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635483A (en) 1992-12-03 1997-06-03 Arizona Board Of Regents Acting On Behalf Of Arizona State University Tumor inhibiting tetrapeptide bearing modified phenethyl amides
US5780588A (en) 1993-01-26 1998-07-14 Arizona Board Of Regents Elucidation and synthesis of selected pentapeptides
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
WO2002088172A2 (en) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
WO2003026577A2 (en) 2001-09-24 2003-04-03 Seattle Genetics, Inc. P-amidobenzylethers in drug delivery agents
WO2004010957A2 (en) 2002-07-31 2004-02-05 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
WO2005082023A2 (en) 2004-02-23 2005-09-09 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
WO2005081711A2 (en) 2003-11-06 2005-09-09 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2005084390A2 (en) 2004-03-02 2005-09-15 Seattle Genetics, Inc. Partially loaded antibodies and methods of their conjugation
WO2006132670A2 (en) 2004-11-12 2006-12-14 Seattle Genetics, Inc. Auristatins having an aminobenzoic acid unit at the n terminus
WO2007000860A1 (ja) 2005-06-28 2007-01-04 Pioneer Corporation 放送受信装置、妨害検出装置および妨害検出方法
WO2007011968A2 (en) 2005-07-18 2007-01-25 Seattle Genetics, Inc. Beta-glucuronide-linker drug conjugates
CN101203241A (zh) * 2005-04-19 2008-06-18 西雅图基因公司 人源化抗-cd70结合物和其应用
US20090028872A1 (en) * 2005-09-26 2009-01-29 Jonathan Alexander Terret Human monoclonal antibodies to cd70
CN101370830A (zh) * 2005-09-26 2009-02-18 米德列斯公司 抗cd70的人单克隆抗体
US20100150950A1 (en) * 2006-12-14 2010-06-17 Medarex, Inc. Human antibodies that bind cd70 and uses thereof
WO2012059882A2 (en) 2010-11-05 2012-05-10 Rinat Neuroscience Corporation Engineered polypeptide conjugates and methods for making thereof using transglutaminase
CN103596979A (zh) * 2011-03-16 2014-02-19 阿尔金-X公司 针对cd70的抗体

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101605906A (zh) * 2006-12-14 2009-12-16 梅达雷克斯公司 结合cd70的人类抗体及其用途
BR112013032899A2 (pt) * 2011-06-22 2017-01-24 Inserm Inst Nat De La Santé Et De La Rech Médicale anticorpos anti-axl e utilizações dos mesmos
EP3137501B1 (en) * 2014-05-02 2021-09-29 Medimmune Limited Ion channel modulators and uses thereof

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635483A (en) 1992-12-03 1997-06-03 Arizona Board Of Regents Acting On Behalf Of Arizona State University Tumor inhibiting tetrapeptide bearing modified phenethyl amides
US5780588A (en) 1993-01-26 1998-07-14 Arizona Board Of Regents Elucidation and synthesis of selected pentapeptides
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
WO2002088172A2 (en) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
WO2003026577A2 (en) 2001-09-24 2003-04-03 Seattle Genetics, Inc. P-amidobenzylethers in drug delivery agents
WO2004010957A2 (en) 2002-07-31 2004-02-05 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
WO2005081711A2 (en) 2003-11-06 2005-09-09 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2005082023A2 (en) 2004-02-23 2005-09-09 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
WO2005084390A2 (en) 2004-03-02 2005-09-15 Seattle Genetics, Inc. Partially loaded antibodies and methods of their conjugation
WO2006132670A2 (en) 2004-11-12 2006-12-14 Seattle Genetics, Inc. Auristatins having an aminobenzoic acid unit at the n terminus
CN101203241A (zh) * 2005-04-19 2008-06-18 西雅图基因公司 人源化抗-cd70结合物和其应用
WO2007000860A1 (ja) 2005-06-28 2007-01-04 Pioneer Corporation 放送受信装置、妨害検出装置および妨害検出方法
WO2007011968A2 (en) 2005-07-18 2007-01-25 Seattle Genetics, Inc. Beta-glucuronide-linker drug conjugates
US20090028872A1 (en) * 2005-09-26 2009-01-29 Jonathan Alexander Terret Human monoclonal antibodies to cd70
CN101370830A (zh) * 2005-09-26 2009-02-18 米德列斯公司 抗cd70的人单克隆抗体
US20100150950A1 (en) * 2006-12-14 2010-06-17 Medarex, Inc. Human antibodies that bind cd70 and uses thereof
WO2012059882A2 (en) 2010-11-05 2012-05-10 Rinat Neuroscience Corporation Engineered polypeptide conjugates and methods for making thereof using transglutaminase
CN103596979A (zh) * 2011-03-16 2014-02-19 阿尔金-X公司 针对cd70的抗体

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. NP 035747
"Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy", 1995, MACK PUB. CO.
ANTONOW D. ET AL., CANCER J, vol. 14, no. 3, 2008, pages 154 - 169
BLAUGSEYMOUR: "Remington's Pharmaceutical Sciences", 1975, MACK PUBLISHING COMPANY
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC.NATL.ACAD.SCI.USA, vol. 94, 1997, pages 412 - 417
CARRILLO, H.LIPMAN, D., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073
HARTLEY J.A, CANCER RES, vol. 70, no. 17, 2010, pages 6849 - 6858
HOWARD P.W. ET AL., BIOORG MED CHEM LETT, vol. 19, 2009, pages 6463 - 6466
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
MARASCO ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 7889 - 7893
MATTER ET AL., J EXP MED, vol. 203, 2006, pages 2145 - 55
NELSON ET AL., J CLIN PATHOL, vol. 53, 2000, pages 111 - 117
SAGNOU ET AL., BIOORG MEDCHEM LETT, vol. 10, no. 18, 2000, pages 2083 - 2086
SMITH ET AL., J.CLIN.PATHOL., vol. 57, 2004, pages 912 - 917
TARABAN, J.IMMUNOL., vol. 173, 2004, pages 6542 - 6

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

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