WO2023061063A1 - 靶向cd70的抗体及其制备和用途 - Google Patents

靶向cd70的抗体及其制备和用途 Download PDF

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WO2023061063A1
WO2023061063A1 PCT/CN2022/114942 CN2022114942W WO2023061063A1 WO 2023061063 A1 WO2023061063 A1 WO 2023061063A1 CN 2022114942 W CN2022114942 W CN 2022114942W WO 2023061063 A1 WO2023061063 A1 WO 2023061063A1
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antibody
cells
region
antigen
cancer
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French (fr)
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田文志
李松
陈典泽
郭慧琴
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宜明昂科生物医药技术（上海）股份有限公司
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    • 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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Definitions

  • the present application relates to a monoclonal antibody specifically binding to CD70 or its antigen-binding part, its preparation and use, especially its use in tumor treatment.
  • Cancer cells have developed some mechanisms to evade the host's immune surveillance, including: 1) By highly expressing the membrane protein CD70 and binding to the Siglec-10 receptor on the surface of immune cells, thereby inhibiting immune activation to escape macrophages and T lymphocytes , immune surveillance of B lymphocytes and natural killer cells; 2) escape the immune surveillance of macrophages (M ⁇ ) through high expression of CD47, CD47 binds to signal regulatory protein ⁇ (SIRP ⁇ ) on the surface of macrophages, thereby triggering inhibition Generation of a sex signal that inhibits the phagocytosis of cancer cells by macrophages.
  • SIRP ⁇ signal regulatory protein ⁇
  • CD70 is a type II transmembrane protein (BFIsrael et al., 2005), a member of the tumor necrosis factor superfamily, mainly expressed in highly activated lymphocytes and dendritic cells, and CD70 expression is also found in antigen-presenting cells .
  • CD70 also known as CD27L, can bind to CD27, a receptor expressed on mature T cells, memory B cells, germinal center B cells, and natural killer cells (NK).
  • NK natural killer cells
  • the interaction of CD70 and CD27 plays an important role in the activation of lymphocytes and the maintenance of immune responses.
  • the CD70-CD27 signaling pathway can induce the proliferation of CD4 + T cells and CD8 + T cells and the secretion of cytokines.
  • CD70-CD27 can also promote the activation, proliferation and differentiation of B cells, promote the formation of germinal centers, and induce the secretion of antibodies.
  • CD70 is expressed in a variety of tumor tissues, including renal cell carcinoma, nasopharyngeal carcinoma, Epstein-Barr virus-induced cancer, malignant Hodgkin's lymphoma (HL) (H.J.Gruss et al., 1996), non-Hodgkin's lymphoma Nodular lymphoma (NHL), diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphocytic leukemia, Burkitt lymphoma (S.M.Lens et al., 1999), multiple myeloma (J.A.McEarchern et al.,2008), Waldenstrom's macroglobulinemia (A.W.Ho et al.,2008), nasopharyngeal carcinoma (A.Agathanggelou et al.,1995), thymus carcinoma (T.Hishima et al.,2000), renal cell
  • CD70 expression is associated with poor prognosis in B-cell lymphoma, renal cell carcinoma, and breast cancer.
  • NSCLC primary and metastatic non-small cell lung cancer
  • Tumor cells use their expressed CD70 to induce T cell apoptosis and T cell exhaustion, and to massively increase the amount of regulatory T cells (Treg), thereby promoting their own growth (Julie Jacobs et al., 2018).
  • Treg regulatory T cells
  • Tumor cells use their expressed CD70 to induce T cell apoptosis and T cell exhaustion, and to massively increase the amount of regulatory T cells (Treg), thereby promoting their own growth (Julie Jacobs et al., 2018).
  • AML acute myeloid leukemia
  • the CD70-CD27 pathway in AML cells activates a stem cell gene expression program that promotes symmetrical cell division and proliferation, while antibody blockade inhibits cell growth (C.Riether et al ., 2017).
  • Blocking the interaction of CD70 and CD27 by the antibody cusatuzumab significantly reduced leukemia stem cells (LSC) (C. Riether et al., 2020).
  • the present application provides a novel CD70 antibody or an antigen-binding portion thereof, which can bind to CD70 + cells and trigger antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis ( ADCP ), And/or complement-dependent cytotoxicity (CDC), also has a strong anti-tumor effect in vivo.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • the application relates to an isolated monoclonal antibody, or antigen-binding portion thereof, that binds to CD70 and may contain i) a heavy chain variable region comprising a VH-CDR1 region, VH-CDR2 district and VH-CDR3 district, wherein, VH-CDR1 district, VH-CDR2 district and VH-CDR3 district can comprise the aminoacid sequence shown in SEQ ID NOs:1, 2 and 3 respectively, or have with above-mentioned sequence Amino acid sequences of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; and/or ii) the light chain may Variable region, the light chain variable region contains VL-CDR1 region, VL-CDR2 region and VL-CDR3 region, wherein, VL-CDR1 region, VL-CDR2 region and VL-CDR3 region can respectively comprise such as S
  • the heavy chain variable region of the monoclonal antibody of the present application or its antigen-binding portion may comprise the amino acid sequence shown in SEQ ID NOs: 7 or 9, or have at least 80%, 85%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • a monoclonal antibody of the present application, or an antigen-binding portion thereof, may comprise a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region may be an IgGl, IgG2 or IgG4 heavy chain constant region, with or engineered to have FcR binding and/or complement protein (eg C1q) binding.
  • the heavy chain constant region may be a human IgG1 heavy chain constant region having, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, Amino acid sequences that are 95%, 96%, 97%, 98% or 99% identical.
  • the light chain constant region may be a kappa light chain constant region, such as a human kappa constant region, having, for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of SEQ ID NO: 12. Amino acid sequences that are %, 96%, 97%, 98% or 99% identical.
  • the N-terminal of the heavy chain constant region is connected to the C-terminal of the heavy chain variable region
  • the N-terminal of the light chain constant region is connected to the C-terminal of the light chain variable region.
  • the CD70 antibody of the present application is an IgG antibody in some embodiments, comprising two heavy chains and two light chains, or consisting of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region sequence , heavy chain variable region sequence or CDR sequence, and each light chain comprises the above-mentioned light chain constant region sequence, light chain variable region sequence or CDR sequence.
  • Antibodies of the present application may be, for example, IgG1, IgG2, or IgG4 full-length antibodies.
  • an antibody of the present application, or an antigen-binding portion thereof may be a single chain antibody, or be composed of an antibody fragment, such as a Fab or F(ab') 2 fragment.
  • Antibodies of the present application may be, for example, murine, human, chimeric or humanized antibodies.
  • the antibody of the present application or its antigen-binding portion can bind to human CD70, and can block CD70-CD27 binding/interaction, trigger ADCC, ADCP, and/or CDC on CD70+ cells, and have a strong anti-tumor effect in vivo.
  • the application also provides immunoconjugates comprising an antibody of the invention, or an antigen-binding portion thereof, linked to a therapeutic agent, such as a cytotoxin or an anticancer agent.
  • the application also provides a bispecific molecule comprising an antibody of the present application, or an antigen-binding portion thereof, to which a second functional group, e.g., a second antibody, has a different functional group than that of the present application. Claim the binding specificity of the antibody or binding portion thereof.
  • an antibody of the present application, or an antigen-binding portion thereof can be part of a chimeric antigen receptor (CAR) or a genetically engineered T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR genetically engineered T cell receptor
  • the present application also provides immune cells with the above-mentioned CAR and/or TCR, including T cells, NK cells, etc.
  • the present application also includes a nucleic acid molecule encoding the antibody of the present application or an antigen-binding portion thereof, as well as an expression vector comprising the nucleic acid and a host cell comprising the expression vector.
  • the present application also provides a method for preparing CD70 antibody using the host cell containing the above expression vector, comprising: (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell or its culture.
  • the present application also provides a pharmaceutical composition, which comprises the antibody or its antigen-binding portion, immunoconjugate, bispecific molecule, immune cell, nucleic acid molecule, expression vector, or host cell of the present application, and a pharmaceutically acceptable carrier .
  • the pharmaceutical composition may also contain other anticancer agents such as SIRP ⁇ D1-Fc protein, or anti-inflammatory agents.
  • the present application provides a method for treating or alleviating CD70-related diseases in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present application to the subject.
  • the CD70-associated disease is cancer.
  • the cancer can be solid or hematological, including, but not limited to, renal cancer, myelodysplastic syndrome, cutaneous T-cell lymphoma, nasopharyngeal carcinoma, Epstein-Barr virus-induced cancer, malignant Hodgkin's lymphoma (HL), Non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphoblastic leukemia, Burkitt lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, nasopharyngeal carcinoma, Thymic carcinoma, glioblastoma, brain cancer, osteosarcoma, melanoma, ovarian cancer, renal cell carcinoma, breast cancer, and non-small cell lung cancer.
  • the pharmaceutical composition of the present application can be administered together with at least one other anticancer agent, such as a CD47 targeting protein, such as SIRP ⁇ D1-Fc protein, which can have at least 80% of SEQ ID NO:17, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence having N80A at position 80 of SEQ ID NO: 17 Mutation, the mutation of this site can achieve the effect of deglycosylation of SIRP ⁇ D1.
  • a CD47 targeting protein such as SIRP ⁇ D1-Fc protein
  • the CD70-associated disease is an inflammatory disease.
  • the inflammatory disease can be an autoimmune disease.
  • the inflammatory disease is selected from arthritis, enteritis, and lupus.
  • the antibody of the present application can also be used for in vitro detection of CD70 and the like.
  • FIG. 1 is a schematic diagram of the structure of the CD70 antibodies IMM47H and IMM47M of the present application.
  • IMM40M has N85E mutation in the light chain variable region, which can eliminate the original glycosylation site.
  • FIGS. 2A-2F show IMM40C, IMM40H, and IMM40M with Chinese hamster ovary cells expressing human CD70 (A), CD47 + CD70 + U266 human multiple myeloma cells (B), CD47 + CD70 + Raji human B lymphoblastoid cells (C), binding activity of CD47 + CD70 + U266 human multiple myeloma cells (D), Chinese hamster ovary cells expressing human CD70 (E), and CD47 + CD70 + U266 human multiple myeloma cells (F).
  • A CD47 + CD70 + U266 human multiple myeloma cells
  • C CD47 + CD70 + Raji human B lymphoblastoid cells
  • D binding activity of CD47 + CD70 + U266 human multiple myeloma cells
  • E Chinese hamster ovary cells expressing human CD70
  • F CD47 + CD70 + U266 human multiple myeloma cells
  • CD70 antibody Cusatuzumab (heavy and light chain amino acids shown in SEQ ID NOs: 20 and 21), CD47 binding protein IMM01 (SIRP ⁇ D1-Fc, SEQ ID NO: 17), human hIgG1-Fc were used as controls.
  • Figure 3 shows that IMM40H blocks the binding ability of CD27(ECD)-Fc (SEQ ID NO:22) to CD70 on Chinese hamster ovary cells expressing human CD70, Cusatuzumab and hIgG1-Fc are used as controls.
  • Figure 4 shows the activity of IMM40H blocking CD70 + Raji human B lymphoblastoma cells interacting with human T lymphocytic leukemia cells Jurkat-CAR-CD27 (the sequence of CAR-CD27 is SEQ ID NO: 23), wherein CD69 is expressed
  • the level indirectly reflects the binding ability of CD70 to Jurkat-CAR-CD27, and IMM01, Cusatuzumab and hIgG1-Fc were used as controls.
  • FIG. 5A-5D show IMM40H, and IMM40H+IMM01 on CD47 + CD70 + Raji human B lymphoblastoma cells (A), CD47 + CD70 + U266 human multiple myeloma cells (B), CD47 + CD70 + Daudi human The ability of Burkitt lymphoma cells (C), and CD47 + CD70 + Jeko-1 human mantle cell lymphoma cells (D) to elicit antibody-dependent cellular cytotoxicity (ADCC), IMM01 and hIgG1-Fc were used as controls.
  • 6A-6D show IMM40H, and IMM40H+IMM01 on CD47 + CD70 + Raji human lymphoblastoid cells (A), U266 human multiple myeloma cells (B), Daudi human Burkitt lymphoma cells (C), and The ability of Jeko-1 human mantle cell lymphoma cells (D) to elicit antibody-dependent cellular phagocytosis (ADCP) IMM01 and hIgG1-Fc were used as controls.
  • ADCP antibody-dependent cellular phagocytosis
  • Figure 7 shows the ability of IMM40H, and IMM40H+IMM01 to elicit complement-dependent cytotoxicity (CDC) on CD47 + CD70 + Raji human lymphoblastoid cells, hIgG1-Fc and IMM01 were used as controls.
  • Figure 8 shows the in vivo anti-tumor effect of IMM40H in the SCID mouse U266 xenograft tumor model.
  • CD70 herein refers to Clusterin of Differentiation 70.
  • the term includes variants, homologs, orthologs and paralogs.
  • an antibody specific for human CD70 may under certain circumstances cross-react with the CD70 protein of another species, such as a monkey.
  • antibodies specific for human CD70 protein may be completely specific for human CD70 protein and not cross-react with proteins of other species or types, or may cross-react with CD70 proteins of some other species but not all other species .
  • antibody herein includes whole antibodies such as IgG, IgA, IgD, IgE and IgM, and any antigen-binding fragment (or antigen-binding portion) or single chain thereof.
  • Whole antibodies are glycoproteins comprising at least two heavy chains and two light chains linked by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region ( VH ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1 , CH2 and CH3 .
  • Each light chain comprises a light chain variable region (V L ) and a light chain constant region.
  • the light chain constant region comprises one domain, CL .
  • the VH and VL regions can be further divided into highly variable regions, that is, CDR regions, and relatively conserved framework regions (FRs) are distributed between the CDR regions.
  • Each VH and VL consists of three CDRs and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus.
  • the variable regions of the heavy and light chains contain binding domains that react with antigen.
  • the heavy chain constant regions of antibodies can mediate the binding of immune proteins to host tissues or factors, including various immune system cells (eg, effector cells) and the first component of the complement system (Clq).
  • the constant region of the heavy chain comprising the Fc region
  • the constant region of the heavy chain is the domain that determines the effector function of the antibody, ie, how the antibody associates with specific cellular receptors or other defense proteins.
  • Fc receptors FcRs
  • B lymphocytes follicular dendritic cells
  • natural killer cells e.g., B lymphocytes
  • macrophages e.g., B lymphocytes
  • neutrophils eosinophils
  • basophils eosinophils
  • mast cells Proteins on the surface of other.
  • the Fc region can interact with Fc receptors and some proteins of the complement system to activate the immune system and/or the complement system.
  • the "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, CD70 protein). It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • binding fragments contained in the "antigen-binding portion" of an antibody include (i) Fab fragments, a monovalent fragment composed of VL, VH , CL and CH1 ; ( ii) F(ab') 2 fragments, A bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment composed of VH and CH1 ; (iv) an Fv fragment composed of an antibody single arm VL and VH ; (v ) dAb fragments composed of VH (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) Nanobodies, a domain and two constant domains of the heavy chain variable region.
  • Fab fragments a monovalent fragment composed of VL, VH , CL and CH1 ;
  • F(ab') 2 fragments A bivalent fragment comprising two Fab fragments connected by a disulfide bridge in
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by recombinant methods via a synthetic linker making the two a single protein chain, in which the VL and VH regions pair to form a monovalent molecule, called single-chain Fc (scFv).
  • scFv single-chain Fc
  • These single chain antibodies are also intended to be included within the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • an isolated antibody that specifically binds CD70 is substantially free of antibodies that specifically bind antigens other than CD70 proteins.
  • An isolated antibody that specifically binds human CD70 protein may, however, have cross-binding properties to other antigens, such as CD70 proteins from other species.
  • an isolated antibody is substantially free of other cellular material and/or chemicals.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.
  • antibody of mouse origin refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, these also are derived from mouse germline immunoglobulin sequences. Antibodies of mouse origin may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, eg, mutations introduced by random or point mutations in vitro or by somatic mutation in vivo. However, the term “murine antibody” does not include antibodies with inserted CDR sequences from other mammalian species within the mouse framework sequences.
  • chimeric antibody refers to an antibody obtained by combining non-human genetic material with human genetic material. Or more generally, chimeric antibodies are antibodies that combine genetic material from one species with genetic material from another species.
  • humanized antibody refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to antibodies naturally produced by humans.
  • the CDRs of the heavy chain variable region and the CDRs of the light chain variable region of the antibody or antibody fragment thereof of the present application are determined by the IMGT numbering system.
  • heavy chain variable region and light chain variable region CDRs can be determined by, for example, Chothia, Kabat, AbM or Contact numbering systems/methods.
  • antibody-dependent cellular cytotoxicity refers to cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens to antibodies such as the CD70 antibody Lysis of target cells such as cancer cells.
  • antibody-dependent cellular phagocytosis refers to the binding of antibodies such as CD70 antibodies or antibody-like proteins such as SIRP ⁇ -Fc to the FcR of immune cells such as macrophages, causing immune cells such as macrophages to Phagocytosis of target cells bound to antibodies such as the CD70 antibody or to SIRP ⁇ by phagocytes results in endocytosis and degradation of the target cells.
  • complement-dependent cytotoxicity refers to the binding of antibodies such as CD70 antibodies or antibody-like proteins such as SIRP ⁇ -Fc to complement system proteins such as C1q, triggering the complement pathway to target cells crack.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, Examples include non-human primates, sheep, dogs, cats, cows and horses.
  • EC50 also known as the half-maximal effect concentration, refers to the antibody concentration that causes 50% of the maximum effect.
  • IC50 also known as the half-maximal inhibitory concentration, refers to the antibody concentration that causes 50% of the maximum inhibitory effect.
  • sequence identity refers to the percentage of nucleotides/amino acids in a sequence that are identical to the nucleotides/amino acid residues in the reference sequence after sequence alignment, if necessary, the sequence introduced in the sequence alignment spaces to achieve the maximum percent sequence identity between the two sequences.
  • sequence identity refers to the percentage of nucleotides/amino acids in a sequence that are identical to the nucleotides/amino acid residues in the reference sequence after sequence alignment, if necessary, the sequence introduced in the sequence alignment spaces to achieve the maximum percent sequence identity between the two sequences.
  • Those skilled in the art can use various methods, such as using computer software, to perform pairwise sequence alignment or multiple sequence alignment to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences.
  • Such computer software For example, ClustalOmega, T-coffee, Kalign, MAFFT and the like.
  • the antibody of the present application comprises heavy chain and/or light chain variable region sequences or CDR1, CDR2 and CDR3 sequences with one or more conservative modifications of the CD70 antibody of the present application. It is known in the art that some conservative sequence modifications do not abolish antigen binding.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as point mutations and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues with similar side chains. Groups of amino acid residues having similar side chains are known in the art.
  • amino acid residues include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (for example, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains ( For example, amino acids of tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • one or more amino acid residues in the CDR regions of the antibodies of the present application may be replaced with other amino acid residues from the same side chain group, and the resulting antibody may be functionally retained using the functional assays described herein (i.e., function above).
  • the antibody of the present application can be prepared as a genetically modified antibody by using an antibody having one or more VH / VL sequences of the CD70 antibody of the present application as a starting material.
  • Antibodies can be modified by modifying one or more residues within one or both variable regions (i.e., VH and/or VL ) (e.g., in one or more CDR regions and/or in one or more framework regions). Genetic modification to improve binding affinity and/or increase similarity to antibodies naturally produced in certain species.
  • framework regions are modified to provide humanized antibodies.
  • the antibody can be genetically modified by modifying residues in the constant region, eg, to alter the effector function of the antibody.
  • CDR region implants can be used to genetically modify the variable region of an antibody.
  • Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, amino acid residues within CDRs are more diverse among individual antibodies than sequences outside CDRs. Because the CDR sequences are responsible for the major antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody can be expressed by constructing expression vectors containing the CDR sequences of a specific natural antibody implanted into the backbone sequences of different antibodies with different properties.
  • another embodiment of the present application relates to an isolated monoclonal antibody or an antigen-binding portion thereof, the heavy chain variable region comprising CDR1, CDR2 and CDR3 having the above sequence of the application, and the light chain variable region comprising the above sequence of the application CDR1, CDR2 and CDR3, but may contain different framework sequences.
  • Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • germline DNA sequences for human heavy and light chain variable region genes are available, eg, at the Vbase database of human germline sequences (www.mrc-cpe.cam.ac.uk/vbase).
  • the germline DNA sequences for the human heavy and light chain variable region genes are available in the Genbank database.
  • Preferred framework sequences for the antibodies of the present application are those that are structurally similar to the framework sequences used for the antibodies of the present application.
  • the CDR1, CDR2, and CDR3 sequences can be inserted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence was derived, or the CDR sequence can be inserted into a gene containing one or more genes compared to the germline sequence. in a mutated framework region. For example, in some cases it may be beneficial to mutate residues in the framework regions, in order to maintain or enhance the antigen binding properties of the antibody, etc.
  • variable region modification is to mutate amino acid residues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest .
  • Mutations can be introduced by point mutation or PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties can be assessed in in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. Mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. In addition, typically no more than one, two, three, four or five residues within a CDR region are altered.
  • Genetically engineered antibodies of the present application include those in which genetic modifications have been made in the framework residues of the VH and/or VL , eg, to alter the properties of the antibody. Typically, these backbone modifications are used to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. These residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody was derived.
  • Another type of framework modification involves mutating one or more residues of the framework region, or even one or more CDR regions, to remove T-cell epitopes and thereby reduce the resulting immunogenicity of the antibody.
  • antibodies of the present application can be genetically engineered to include genetic modifications in the Fc region, typically to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation , Fc receptor binding, and/or antibody-dependent cytotoxicity.
  • the antibodies of the present application can be chemically modified (eg, one or more chemical functional groups can be added to the antibody), or modified to alter its glycosylation, to alter one or more functional properties of the antibody.
  • the hinge region of CH1 is modified, changing, eg increasing or decreasing, the number of cysteine residues in the hinge region. Cysteine residues in the hinge region of CH1 are altered, for example, to facilitate assembly of heavy and light chains or to increase/decrease antibody stability.
  • the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2 - CH3 junction region of the Fc hinge fragment such that the antibody has reduced SpA binding relative to native Fc-hinge domain SpA binding.
  • the glycosylation of the antibody is modified.
  • an aglycosylated antibody can be prepared (ie, the antibody lacks glycosylation).
  • Glycosylation can be altered, for example, to increase the affinity of the antibody for antigen.
  • Such glycosylation modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence.
  • one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that position.
  • Such deglycosylation can increase the affinity of the antibody for the antigen.
  • antibodies with altered glycosylation patterns such as low-fucosyl antibodies with reduced amounts of fucose residues, or antibodies with increased bisecting GlcNac structures can be prepared.
  • Altered glycosylation patterns have been shown to increase ADCC activity of antibodies.
  • Such glycosylation modification can be performed, for example, by expressing the antibody in a host cell with an altered glycosylation system.
  • Cells with altered glycosylation systems are known in the art and can be used as host cells for expressing recombinant antibodies of the present application to produce antibodies with altered glycosylation.
  • the monoclonal antibodies of the present application can be prepared using the somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495.
  • Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art.
  • Antibodies of the present application can also be produced in host cell transfectomas using, for example, recombinant DNA technology combined with gene transfection methods (eg Morrison, S. (1985) Science 229:1202).
  • DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences.
  • operably linked means that the antibody gene is linked into a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.
  • regulatory sequence includes promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of the antibody gene.
  • Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)).
  • Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus and/or enhancers, such as adenovirus major late promoter (AdMLP) and polyoma virus.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus major late promoter
  • non-viral regulatory sequences can be used, such as the ubiquitin promoter or the beta-globin promoter.
  • regulatory elements are composed of sequences from different sources, such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia type I virus (Takebe et al., (1988) Mol. Cell . Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.
  • Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors.
  • the variable region is constructed by inserting into an expression vector already encoding the heavy chain constant region and the light chain constant region of the desired subtype, so that the VH and the CH in the vector are operable
  • the V L is operably linked to the CL in the carrier.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell.
  • the antibody chain genes can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain genes.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin).
  • the recombinant expression vectors of the present application can carry other sequences, such as sequences that regulate the replication of the vector in host cells (eg, origin of replication) and selectable marker genes.
  • Selectable marker genes can be used to select host cells into which the vector has been introduced (see, eg, US Patents 4,399,216; 4,634,665 and 5,179,017).
  • a selectable marker gene confers drug resistance, such as G418, hygromycin, or methotrexate resistance, to host cells into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection/amplification of dhfr host cells) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques.
  • Various forms of the term "transfection” include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, for example, electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, and the like.
  • electroporation calcium phosphate precipitation
  • DEAE-dextrose transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells administered with a DHFR selectable marker, in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci.USA 77:4216-4220, DHFR selectable markers are described, for example, in R.J.Kaufman and P.A.Sharp (1982) J.Mol.Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells.
  • Another preferred expression system, particularly when using NSO myeloma cells is the GS gene expression system.
  • a recombinant expression vector encoding an antibody gene When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, it is prepared by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or preferably sufficient to allow secretion of the antibody into the growth medium of the host cell.
  • Antibody Antibodies can be recovered from the culture medium using protein purification methods.
  • Antibodies of the present application, or antigen-binding portions thereof, can be cross-linked with therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs).
  • Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA crosslinkers, histone sirtuin inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerases Inhibitors of I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents.
  • the antibody and therapeutic agent are preferably cross-linked by a linker, which is cleavable, such as a peptide, disulfide or hydrazone linker.
  • the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit , Val-Lys, Lys, Cit, Ser or Glu.
  • the present application relates to pharmaceuticals comprising one or more antibodies of the present application, or antigen-binding portions thereof, linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand).
  • Bispecific molecules to generate bispecific molecules that bind to at least two different binding sites or targeting molecules.
  • the term "bispecific molecule” includes molecules with three or more specificities.
  • the bispecific molecule has a third specificity in addition to the Fc binding specificity and the CD70 binding specificity.
  • a third specificity could eg be for CD47 to better target tumor cells while sparing other CD70 + cells such as immune cells.
  • Bispecific molecules can come in a variety of forms and sizes. At one end of the size spectrum, bispecific molecules remain in the traditional antibody format, except that they have two binding arms each with a different specificity, instead of having two binding arms with the same specificity. At the other extreme are bispecific molecules, consisting of two single-chain antibody fragments (scFv) linked by peptide chains, referred to as Bs(scFv) 2 constructs. An intermediate-sized bispecific molecule consists of two different F(ab) fragments linked by a peptide linker. These and other forms of bispecific molecules can be produced by genetic modification, somatic cell hybridization, or chemical methods.
  • the present application also provides a chimeric antigen receptor comprising a CD70 single-chain antibody scFv comprising the heavy and light chain CDRs, or the heavy and light chain variable regions described in the present application.
  • a CD70 chimeric antigen receptor can comprise (a) an extracellular antigen binding domain comprising a CD70 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.
  • the application provides nucleic acid molecules encoding the heavy/light chain variable regions or CDRs of an antibody of the application, or an antigen-binding portion thereof.
  • Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “substantially pure” when it has been purified by standard techniques from other cellular components or other contaminants, such as other cellular nucleic acids or proteins.
  • a nucleic acid of the present application may be, for example, DNA or RNA, and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids of the present application can be obtained using standard molecular biology techniques.
  • hybridomas e.g., hybridomas produced from transgenic mice carrying human immunoglobulin genes
  • the cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be amplified by standard PCR or cDNA cloned Technology acquired.
  • nucleic acid encoding such antibodies can be collected from the gene bank.
  • Preferred nucleic acid molecules of the present application include those encoding the VH and VL sequences or CDRs of CD70 monoclonal antibodies.
  • VH and VL encoding DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes to full-length antibody chain genes, Fab fragment genes or scFv genes.
  • a VH or VL - encoding DNA segment is operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operably linked" means that two DNA fragments are joined together such that the amino acid sequences encoded by both DNA fragments are in reading frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH -encoding DNA to another DNA molecule encoding heavy chain constant regions ( CH1, CH2 , and CH3 ).
  • the sequences of the human heavy chain constant region genes are known in the art, and DNA fragments including these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region may be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgGl or IgG4 constant region.
  • the DNA encoding the VH region can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene by operably linking the VL - encoding DNA with another DNA molecule encoding the light chain constant region, CL .
  • the sequences of the human light chain constant region genes are known in the art, and DNA fragments including these regions can be obtained by standard PCR amplification.
  • the light chain constant regions may be kappa and lambda constant regions.
  • a DNA segment encoding VH and VL can be operably linked to another segment encoding a flexible linker, such as a 5-20 amino acid peptide, so that the VH and VL sequences can be expressed as a contiguous single-chain protein , wherein the VH and VL domains are connected by this flexible linker.
  • a flexible linker such as a 5-20 amino acid peptide
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more antibodies or antigen-binding portions thereof, immune conjugates, immune cells, bispecific antibodies, and/or antibodies or antigens thereof
  • the encoding nucleic acid molecule, expression vector or host cell of the binding part is formulated together with a pharmaceutically acceptable carrier.
  • the composition may optionally contain one or more other pharmaceutically active ingredients, such as another anti-tumor protein, such as IMM01, a SIRP ⁇ D1-Fc fusion protein used in combination in this application.
  • compositions can contain any number of excipients.
  • Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solubilizers, coloring agents, flavoring agents, coatings, disintegrants, lubricants, Sweeteners, preservatives, isotonic agents and combinations thereof.
  • the pharmaceutical composition may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or bolus injection).
  • the active ingredient can be encapsulated in a material that protects it from acids and other natural conditions that may inactivate it.
  • Parenteral administration means methods other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, intracardiac, intradermal, Intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and boluses.
  • the antibodies of the present application may be administered by non-parenteral routes, such as topical, epidermal or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.
  • compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable to high drug concentration.
  • the amount of active ingredient to be prepared with the carrier materials to produce a single dosage form will vary with the subject treated and the particular mode of administration, and is essentially that amount of the composition which produces a therapeutic effect. As a percentage, the amount is from about 0.01 to about 99% of the active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a bolus may be administered, divided doses may be administered over time or the dose may be decreased or increased in proportion to the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited for single administration to a subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.
  • the antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
  • the dose may be about 0.001-100 mg/kg body weight of the host.
  • a “therapeutically effective amount” of the CD70 antibody of the present application causes a decrease in the severity of disease symptoms and an increase in the frequency and duration of asymptomatic periods.
  • a "therapeutically effective amount” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, and even more preferably, compared to untreated subjects. At least about 60%, and more preferably at least about 80% inhibition.
  • a therapeutically effective amount of a therapeutic antibody can reduce tumor size, or alleviate symptoms in a subject, which can be a human or another mammal.
  • the "therapeutically effective amount” preferably reduces inflammation by at least about 20%, more preferably by at least about 40%, and even more preferably reduced by at least about 60%, and more preferably by at least about 80%.
  • a therapeutically effective amount of a therapeutic antibody can reduce or eliminate inflammation, or alleviate symptoms in a subject, which can be a human or another mammal.
  • compositions can be sustained release formulations, including implants, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • compositions can be administered via medical devices such as (1) needle-free hypodermic injection devices (e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; 4,487,603); (3) transdermal delivery devices (US Patent 4,486,194); (4) bolus injection devices (US Patents 4,447,233 and 4,447,224); and (5) osmotic devices (US Patents 4,439,196 and 4,475,196).
  • needle-free hypodermic injection devices e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; 4,487,603
  • transdermal delivery devices US Patent 4,486,194
  • bolus injection devices US Patents 4,447,233 and 4,447,224
  • osmotic devices US Patents 4,439,
  • the mAbs of the present application can be formulated to ensure proper in vivo distribution.
  • the antibody can be formulated in liposomes, which can additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.
  • the pharmaceutical composition of the present application has a variety of in vitro and in vitro applications, related to, for example, the treatment of cancer, or more generally, for the immune enhancement of patients with cancer diseases.
  • Antibodies can be administered to a human subject, eg, to inhibit tumor growth in vivo.
  • the present application provides a method for inhibiting the growth of tumor cells in a subject, comprising administering the pharmaceutical composition of the present application to the subject, so that in the subject Tumor growth was inhibited in the patients.
  • Tumors that can be treated by the antibodies of the present application include solid tumors and hematological tumors, non-limiting examples include, but are not limited to, renal cancer, myelodysplastic syndrome, cutaneous T-cell lymphoma, nasopharyngeal carcinoma, Epstein-Barr virus-induced cancer, Malignant Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphocytic leukemia, Burkitt's lymphoma, multiple myeloma, Waldenstrom's giant Gammaglobulinemia, nasopharyngeal carcinoma, thymic carcinoma, glioblastoma, brain cancer, osteosarcoma, melanoma, ovarian cancer, renal cell carcinoma, breast cancer, and non-small cell lung cancer, whether primary or metastatic of.
  • HL Hodgkin's lymphom
  • the present application provides a method for reducing or eliminating inflammation in a subject, comprising administering the pharmaceutical composition of the present application to the subject.
  • Inflammatory diseases can include autoimmune diseases.
  • Non-limiting examples of inflammatory diseases include, but are not limited to, arthritis, colitis, and lupus.
  • the pharmaceutical composition of the present application can be administered together with one or more other antibody or non-antibody therapeutic agents, which can effectively inhibit tumor growth in a subject, and/or reduce or eliminate inflammation in a subject.
  • the present application provides a method for inhibiting tumor growth in a subject, comprising administering to the subject a CD70 pharmaceutical composition and IMM01, a SIRP ⁇ D1-Fc fusion protein.
  • combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.
  • FIG 1 shows the structures of the CD70 antibodies IMM40C, IMM40H, and IMM40M of the present application.
  • IMM40C is an IgG antibody, comprising a mouse-derived heavy chain variable region and a mouse-derived light chain variable region.
  • IMM40C comprises the heavy chain variable region of SEQ ID NO:7, the heavy chain constant region of SEQ ID NO:11, the light chain variable region of SEQ ID NO:8, and the light chain constant region of SEQ ID NO:12. district.
  • IMM01 is a CD47-binding SIRP ⁇ D1-Fc fusion protein, which is described in US 2021/0024598A1. It contains two mutant SIRP ⁇ D1s connected to Fc dimer fragments, and its monomer contains the amino acid sequence shown in SEQ ID NO:17.
  • SIRP ⁇ D1 in IMM01 compared with wild-type SIRP ⁇ D1, has an N80A mutation at position 80 of SEQ ID NO: 17, and the mutation at this site can achieve the effect of deglycosylation.
  • Cusatuzumab is an IgG antibody specifically binding to CD70, comprising heavy and light chains as shown in SEQ ID NOs: 20 and 21, respectively.
  • mice were inoculated with human CD70 protein, their serum was collected, and mice with high titers were screened out.
  • the mouse spleen cells were taken, and the hybridoma cell line producing CD70 antibody was prepared by hybridoma technology, and the cell clones producing monoclonal antibodies with high CD70 binding activity were screened out through the CD70 binding activity test, including the cell clone producing IMM40C.
  • the heavy chain variable region and the light chain variable region of IMM40C were sequenced, and the amino acid sequences were shown in SEQ ID NOs: 7 and 8, respectively.
  • IMM40C was humanized by CDR grafting method to obtain humanized antibody IMM40H.
  • the N85E mutation is carried out on the light chain variable region of IMM40H to obtain IMM40M, and the N85E mutation can eliminate the original glycosylation site.
  • IMM40C The coding sequences of IMM40C, IMM40H, and IMM47M were artificially designed.
  • the same signal peptide sequence and Kozak sequence are added to the 5' end of the IMM47C antibody light chain variable region-constant region coding sequence (SEQ ID NO: 14), and HindIII and XbaI restriction enzyme digestion sites were added to the 5' and 3' ends of the resulting sequence, respectively.
  • the obtained sequences were synthesized by GenScript Biotechnology Co., Ltd., and cloned into pMac-H and pMac-L vectors, respectively.
  • the obtained sequences were synthesized by GenScript Biotechnology Co., Ltd., and cloned into pMac-H and pMac-L vectors, respectively.
  • the obtained sequences were synthesized by GenScript Biotechnology Co., Ltd., and cloned into pMac-H and pMac-L vectors, respectively.
  • the constructed expression vector was used to transiently express the protein in CHO-S cells.
  • the cells were washed once with 1% BSA-PBS, and 100 ⁇ l of anti-human IgG (Fc)-FITC (diluted at 1:500, Cat#F9512, Sigma) was added, and refrigerated at 4 degrees for 45 minutes. After incubation, wash once with 1% BSA-PBS. Cells were resuspended in 200 ⁇ l 1% BSA-PBS, and flow cytometer ( easyCyte 5HT, Merck Millipore) for FACS analysis.
  • Fc anti-human IgG
  • Figure 2A shows the binding ability of IMM40C and IMM40H to CD70 + Chinese hamster ovary cells, hIgG1-Fc was used as negative control. It can be seen from the results that IMM47C and IMM40H specifically bind to the CD70 + cells, and there is no difference in their binding abilities.
  • Figure 2B shows the binding ability of IMM40C and IMM40H to CD47 + CD70 + U266 human multiple myeloma cells, hIgG1-Fc was used as a negative control. It can be seen from the results that IMM40C and IMM40H specifically bind to the CD47 + CD70 + cells, and there is no difference in their binding ability.
  • Figure 2C shows the binding ability of IMM40H to CD47 + CD70 + Raji human B lymphoblastocytes, hIgG1-Fc was used as a negative control. It can be seen from the results that IMM40H specifically binds to the CD47 + CD70 + cells, and the binding activity is significantly stronger than that of Cusatuzumab and IMM01.
  • Figure 2D shows the binding ability of IMM40H to CD47 + CD70 + U266 human multiple myeloma cells, hIgG1-Fc was used as a negative control. It can be seen from the results that IMM40H specifically binds to the CD47 + CD70 + cells, and the binding activity is stronger than that of Cusatuzumab and IMM01.
  • Figure 2E shows the binding ability of IMM40H and IMM40M to CD70 + Chinese hamster ovary cells, hIgG1-Fc was used as negative control. It can be seen from the results that IMM40H and IMM40M specifically bind to the CD70 + cells, and the binding activities are equivalent, and IMM40H is slightly stronger than IMM40M.
  • Figure 2F shows the binding ability of IMM40H and IMM40M to CD47 + CD70 + U266 human multiple myeloma cells, hIgG1-Fc was used as a negative control. It can be seen from the results that IMM40H and IMM40M specifically bind to the CD47 + CD70 + cells, and the binding activities of the two are equivalent, and IMM40H is slightly stronger than IMM40M.
  • IMM40H can inhibit the binding of CD70-CD27, and the inhibitory activity is slightly stronger than that of the traditional monospecific CD70 antibody Cusatuzumab.
  • CD70 Antibody Elicits Antibody-Dependent Cellular Cytotoxicity (ADCC) against Tumor Cells
  • Each tumor cell was labeled with 500-fold dilution of 1 mM CFSE (Cat#21888-25 mg, Sigma).
  • PI propidium iodide
  • % Lysis (% PI positive target cells treated with antibody/protein -% PI positive target cells treated with negative control protein hIgG1-Fc)/(100-% PI positive target cells treated with negative control protein hIgG1-Fc) ⁇ 100
  • the ADCC effect induced by IMM40H was comparable to that of the IMM40H+IMM01 mixture, and much stronger than that of the monospecific CD47-binding protein IMM01.
  • CD70 Antibody Elicits High Levels of Antibody-Dependent Cells against CD47 + CD70 + Cells Phagocytosis (ADCP)
  • Tumor cells namely CD47 + CD70 + human lymphoblastoid cells (Raji cells), human multiple myeloma cells (U266 cells), human Burkitt lymphoma cells (Daudi cells) and human mantle cell lymphoma cells ( Jeko-1 cells), as the target cells.
  • 1 mM CFSE was diluted 500 times, added to the cell suspension with adjusted density, mixed evenly, and stained for 30 min in a cell incubator. After staining, the blank 1640 medium was washed once, and the cell density was adjusted to 2x10 6 /ml. The cells were collected by centrifugation at 1000 rpm for 5 min, washed once with 1640 medium, and the cell density was adjusted to 1x10 6 /ml.
  • the recombinant fusion protein and the control protein were serially diluted with 1640 medium, wherein for the mixture of IMM01 and IMM40H, the concentration was calculated as a single substance, and the respective initial concentrations of IMM01 and IMM40H were consistent with the single substance group.
  • the 96-well plate was washed 5 times with PBS to remove free tumor-CFSE cells. After washing, 200 ⁇ l PBS was added to each well, and the THP-1 cells were suspended by pipetting repeatedly. The green fluorescence intensity of THP-1 cells was detected and analyzed by flow cytometry. Phagocytic activity was evaluated by analyzing the green fluorescence intensity of THP-1 cells.
  • the ADCP effect induced by IMM40H was much stronger than that of CD47-binding Fc fusion protein IMM01, and stronger than that of IMM01+IMM04H mixture at high concentrations.
  • the ADCP effect induced by IMM40H was comparable to that of the CD47-binding Fc fusion protein IMM01, and the mixture of IMM01+IMM04H.
  • PI propidium iodide
  • IMM40H induced a high level of complement-dependent cytotoxicity (CDC), and its activity was comparable to that of IMM01+IMM40H mixture and much higher than that of IMM01.
  • Example 8.CD70 antibody shows strong anti-tumor activity in vivo
  • mice Thirty SCID mice aged 5-6 weeks were inoculated with 100 ⁇ L of culture medium containing 5 ⁇ 10 6 human multiple myeloma U266 cells and 100 ⁇ L of Matrigel mixture on the right side of the back near the armpit.
  • the mice were randomly divided into 5 groups with 6 mice in each group, and the day of grouping was defined as D0. From day D0, mice in each group were injected intraperitoneally with PBS, IMM40H (0.3mg/kg), IMM40H (1.0mg/kg), IMM01 (0.5mg/kg), and IMM01+IMM40H (0.5mg/kg+1.0mg /kg), for 4 weeks, once a week. After 4 weeks, the administration was terminated, and the experiment was terminated after 3.5 weeks of continuous observation. Tumor volume and mouse body weight were measured every 3-4 days.
  • Tumor volume (V) was calculated as (length x width 2 )/2.
  • Relative tumor volume (RTV) V t /V 0 , where V t is the tumor volume at day t (Dt) and V 0 is the tumor volume at the beginning of treatment (D0).
  • Relative tumor inhibition rate TGI (%) 1-average relative tumor volume of the treatment group/average relative tumor volume of the negative control group ⁇ 100%.
  • the average tumor volume of the PBS control group on day 52 was 2715.13 ⁇ 107.27 mm 3
  • the average relative tumor volume (RTV) was 14.01 ⁇ 0.67.
  • the tumor volume was 61.42 ⁇ 27.64 mm 3 , and the RTV was 0.30 ⁇ 0.13.
  • the tumor volume and relative tumor volume were significantly decreased during D3-D28 (p ⁇ 0.05).
  • the tumor volume was 487.78 ⁇ 233.37mm 3 , and the RTV was 2.43 ⁇ 1.16.
  • the tumor volume and relative tumor volume continued to significantly decrease within three and a half weeks of drug withdrawal (p ⁇ 0.01).
  • the tumor volume on D28 was 0.00 ⁇ 0.00mm 3
  • the RTV was 0.00 ⁇ 0.00.
  • the tumor volume and relative tumor volume were significantly decreased during D3-D28 (p ⁇ 0.01).
  • the tumor volumes were all 0.00 ⁇ 0.00mm 3
  • the RTV was 0.00 ⁇ 0.00.
  • the tumor volume and relative tumor volume continued to significantly decrease within three and a half weeks of drug withdrawal (p ⁇ 0.01).
  • Anti-CD70 antibodies a potential treatment for EBV+CD70-expressing lymphomas, Molecular cancer therapeutics, 4(2005) 2037 -2044.
  • Low density lipoprotein receptor-related protein is a calreticulin coreceptor that signals focal adhesion disassembly. J Cell 13 Biol. 20: 1189
  • Membrane proteomic analyzes of ovarian cancer identifies the immune modulators CD70 and B7- H2 as candidate markers of cisplatin response, in, AACR, 2008.

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Abstract

提供一种与CD70特异结合的单克隆抗体或其抗原结合部分。还提供编码该抗体或其抗原结合部分的核酸分子、包含该核酸分子的表达载体和宿主细胞、制备该抗体或其抗原结合部分的方法，以及使用该抗体或其抗原结合部分来治疗与CD70过表达相关的疾病的方法。

Description

靶向CD70的抗体及其制备和用途 发明领域
本申请涉及一种特异结合CD70的单克隆抗体或其抗原结合部分、及其制备和用途，特别是其在肿瘤治疗中的用途。
背景技术
癌细胞已经发展出一些逃避宿主免疫监视的机制，包括：1)通过高表达膜蛋白CD70，与免疫细胞表面的Siglec-10受体结合，从而抑制免疫激活，来逃避巨噬细胞、T淋巴细胞、B淋巴细胞和自然杀伤细胞的免疫监视；2)通过高表达CD47来逃避巨噬细胞(Mφ)的免疫监视，CD47与巨噬细胞表面上的信号调节蛋白α(SIRPα)结合，从而引发抑制性信号的生成，该抑制信性号抑制巨噬细胞对癌细胞的吞噬作用。可以看出，癌细胞相当聪明，可以基于它们发展出的逃避机制而迅速增殖。因此，有效杀灭癌细胞的抗癌药物的开发可以针对这些机制。
CD70
CD70为II形跨膜蛋白(B.F.Israel et al.,2005)，属于肿瘤坏死因子超家族成员，主要表达于高度活化的淋巴细胞、以及树突细胞，在抗原递呈细胞中也发现了CD70的表达。CD70又称为CD27L，可以结合于CD27，一种表达于成熟T细胞、记忆B细胞、生发中心B细胞和自然杀伤细胞(NK)的受体。CD70与CD27的相互作用在激活淋巴细胞和维持免疫应答中发挥重要作用。CD70-CD27信号通路可以诱导CD4 +T细胞和CD8 +T细胞的增殖以及细胞因子的分泌。CD70-CD27还可促进B细胞活化、增殖及分化，促进生发中心的形成，诱导抗体的分泌。
大量研究表明，CD70在多种肿瘤组织中表达，包括肾细胞癌、鼻咽癌、Epstein-Barr病毒诱导的癌症、恶性霍金奇淋巴瘤(HL)(H.J.Gruss et al.,1996)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤、滤泡状淋巴瘤、B细胞淋巴细胞白血病、Burkitt淋巴瘤(S.M.Lens et al.,1999)、多发性骨髓瘤(J.A.McEarchern et al.,2008)、Waldenstrom巨球蛋白血症(A.W.Ho et al.,2008)、鼻咽癌(A.Agathanggelou et al.,1995)、胸腺癌(T.Hishima et al.,2000)、肾细胞癌(C.L.Law et al.,2006)、胶质母细胞瘤(J.Wischhusen et al.,2002)、脑癌(J.Held‐Feindt et al.,2002)、骨肉瘤(J.H.Pahl et al.,2015)、黑色素瘤(C.Pich et al.,2016)、和卵巢癌(S.Aggarwal et al.,2008)。在B细胞淋巴瘤、肾细胞癌和乳腺癌中，CD70的表达与不良预后相关。一些研究还发现CD70在原发性和转移性非小细胞肺癌(NSCLC)肿瘤细胞表面以及肿瘤微环境中表达，从而使得CD70成为了治疗NSCLC的潜在靶点(J.Jacobs et al.,2015)。
肿瘤细胞利用其表达的CD70，诱导T细胞凋亡和T细胞衰竭，并大量增加 调节性T细胞(Treg)的量，从而促进其自身生长(Julie Jacobs et al.,2018)。例如，在急性髓性细胞样白血病(AML)中，AML细胞中的CD70-CD27通路可激活干细胞基因表达程序，促进对称细胞***和增殖，而抗体阻断可抑制细胞生长(C.Riether et al.,2017)。通过抗体cusatuzumab阻断CD70与CD27相互作用，可大幅减少白血病干细胞(LSC)(C.Riether et al.,2020)。
另有研究表明，CD70的过量表达还与关节炎、肠炎、以及狼疮有关(Bobby Kwanghoon Han et al.,2015)。
领域内需要更多具有所需特性的CD70抗体。
对于本申请中任何文件的引用，并不等同于承认这些文件是本申请的现有技术。
发明内容
本申请提供一种新的CD70抗体或其抗原结合部分，其可以结合CD70 +细胞，引发对于CD70 +细胞的抗体依赖性细胞介导的细胞毒性(ADCC)、抗体依赖性细胞吞噬(ADCP)、和/或补体依赖性细胞毒性(CDC)，还具有强劲的体内抗肿瘤效果。
因此，在一个方面，本申请涉及一种分离的单克隆抗体、或其抗原结合部分，其与CD70结合，可以含有i)重链可变区，该重链可变区含有VH-CDR1区、VH-CDR2区和VH-CDR3区，其中，VH-CDR1区、VH-CDR2区和VH-CDR3区可以分别包含如SEQ ID NOs:1、2和3所示的氨基酸序列，或与上述序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；和/或ii)轻链可变区，该轻链可变区含有VL-CDR1区、VL-CDR2区和VL-CDR3区，其中，VL-CDR1区、VL-CDR2区和VL-CDR3区可以分别包含如SEQ ID NOs:4、5、和6所示的氨基酸序列，或与上述序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
本申请单克隆抗体或其抗原结合部分的重链可变区可以包含如SEQ ID NOs:7或9所示的氨基酸序列，或与上述序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
本申请的单克隆抗体或其抗原结合部分的轻链可变区可以包含如SEQ ID NOs:8、或10(X＝N或E)所示的氨基酸序列，或与上述序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
在一些实施方式中，本申请单克隆抗体或其抗原结合部分可以包含重链可变区和轻链可变区，其中重链可变区和轻链可变区可以分别包含如(i)SEQ ID NOs:7和8；(ii)SEQ ID NOs:9和10(X＝N)；或(iii)SEQ ID NOs:9和10(X＝E) 所示的氨基酸序列；或与上述具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，其中该抗体或其抗原结合部分与CD70结合。
本申请的单克隆抗体或其抗原结合部分可以包含重链恒定区和/或轻链恒定区。重链恒定区可以是IgG1、IgG2或IgG4重链恒定区，具有或经改造具有FcR结合力和/或补体蛋白(如C1q)结合力。在一些实施方式中，重链恒定区可以是人IgG1重链恒定区，具有例如与SEQ ID NO:11具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。轻链恒定区可以是κ轻链恒定区，例如人κ恒定区，具有例如与SEQ ID NO:12具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。其中，重链恒定区的N端与重链可变区的C端连接，轻链恒定区的N端与轻链可变区的C端连接。
本申请的CD70抗体在一些实施方式中为IgG抗体，包含两条重链和两条轻链，或由两条重链和两条轻链构成，其中各重链包含上述的重链恒定区序列、重链可变区序列或CDR序列，且各轻链包含上述的轻链恒定区序列、轻链可变区序列或CDR序列。本申请的抗体可以是例如IgG1、IgG2、或IgG4的全长抗体。在一些实施方式中，本申请的抗体或其抗原结合部分可以是单链抗体，或由抗体片段构成，例如Fab或F(ab') 2片段。
本申请的抗体可以是例如鼠源、人源、嵌合或人源化抗体。
本申请的抗体或其抗原结合部分可以与人CD70结合，并能够阻断CD70-CD27结合/相互作用，引发对于CD70+细胞的ADCC、ADCP、和/或CDC，并具有强劲的体内抗肿瘤效果。
本申请还提供含有本申请抗体或其抗原结合部分的免疫交联物，该抗体或其抗原结合部分与治疗剂例如细胞毒素或抗癌剂相连接。本申请还提供含有本申请抗体或其抗原结合部分的双特异性分子，该抗体或其抗原结合部分连接有第二功能基团，例如，第二抗体，该第二功能基团具有不同于本申请抗体或其结合部分的结合特异性。在另一方面，本申请的抗体或其抗原结合部分可以是嵌合抗原受体(CAR)或基因改造T细胞受体(TCR)的一部分。本申请还提供具有上述CAR和/或TCR的免疫细胞，包括T细胞、NK细胞等。
本申请还包括编码本申请抗体或其抗原结合部分的核酸分子，以及包含该核酸的表达载体以及包含该表达载体的宿主细胞。本申请还提供使用含有上述表达载体的宿主细胞来制备CD70抗体的方法，包括：(i)在宿主细胞中表达抗体，以及(ii)从宿主细胞或其培养物中分离抗体。
本申请还提供药物组合物，其包含本申请的抗体或其抗原结合部分、免疫交联物、双特异性分子、免疫细胞、核酸分子、表达载体、或宿主细胞，以及药学上可接受的载体。该药物组合物还可以包含其他的抗癌剂如SIRPαD1-Fc蛋白、 或抗炎性剂。
在一个方面，本申请提供在受试者中治疗或减缓CD70相关疾病的方法，包括向受试者施用治疗有效量的本申请药物组合物。
在一些实施方式中，CD70相关疾病为癌症。癌症可以为实体癌或血液癌，包括，但不限于肾癌、骨髓增生异常综合征、皮肤T细胞淋巴瘤、鼻咽癌、Epstein-Barr病毒诱导的癌症、恶性霍金奇淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤、滤泡状淋巴瘤、B细胞淋巴细胞白血病、Burkitt淋巴瘤、多发性骨髓瘤、Waldenstrom巨球蛋白血症、鼻咽癌、胸腺癌、胶质母细胞瘤、脑癌、骨肉瘤、黑色素瘤、卵巢癌、肾细胞癌、乳腺癌、和非小细胞肺癌。在一些实施方式中，本申请药物组合物可以与至少一种其他抗癌剂一起施用，例如CD47靶向蛋白，如SIRPαD1-Fc蛋白，该蛋白可以具有与SEQ ID NO:17具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，在SEQ ID NO:17的第80位具有N80A突变，该位点的突变可实现SIRPαD1去糖基化的效果。
在一些实施方式中，CD70相关疾病为炎性疾病。在一些实施方式中，炎性疾病可以为自免疫疾病。在一些实施方式中，炎性疾病选自关节炎、肠炎、和狼疮。
本申请的抗体还可以用于CD70的体外检测等。
基于以下的详细描述和实施例，当前公开的其他特征和有利之处将是非常明晰可见的，详细描述和实施例不应当解读为是限制性的。所有在说明书中引用的文献、Genbank登记号、专利和公开专利申请均通过引用的方式并入本文。
附图说明
详细描述在以下以示例方式给出但不意在将本申请限制于所述具体实施方式，可以结合附图更好地进行理解。
图1是本申请CD70抗体IMM47H、和IMM47M的结构示意图。其中IMM47H为IgG抗体，包含人源化的重链可变区、和人源化的轻链可变区，具体包含SEQ ID NO:9的重链可变区、SEQ ID NO:11的重链恒定区、SEQ ID NO:10(X＝N)的轻链可变区、以及SEQ ID NO:12的轻链恒定区。相比于IMM40H，IMM40M在轻链可变区具有N85E突变，该突变可消除原有的糖基化位点。
图2A-2F示出IMM40C、IMM40H、和IMM40M与表达人CD70的中国仓鼠卵巢细胞(A)、CD47 +CD70 +U266人多发性骨髓瘤细胞(B)、CD47 +CD70 +Raji人B淋巴母细胞(C)、CD47 +CD70 +U266人多发性骨髓瘤细胞(D)、表达人CD70的中国仓鼠卵巢细胞(E)、以及CD47 +CD70 +U266人多发性骨髓瘤细胞(F)的结合活性。CD70抗体Cusatuzumab(重链和轻链氨基酸示于SEQ ID NOs:20和21)、CD47结合蛋白IMM01(SIRPαD1-Fc，SEQ ID NO:17)、人hIgG1-Fc 用作对照。
图3示出IMM40H阻断CD27(ECD)-Fc(SEQ ID NO:22)与表达人类CD70的中国仓鼠卵巢细胞上的CD70的结合能力，Cusatuzumab和hIgG1-Fc用作对照。
图4示出IMM40H阻断CD70 +Raji人B淋巴母细胞瘤细胞与人T淋巴细胞白血病细胞Jurkat-CAR-CD27(CAR-CD27的序列为SEQ ID NO:23)相互作用的活性，其中CD69表达水平间接反映CD70与Jurkat-CAR-CD27的结合能力，IMM01、Cusatuzumab和hIgG1-Fc用作对照。
图5A-5D示出IMM40H、以及IMM40H+IMM01对CD47 +CD70 +Raji人B淋巴母细胞瘤细胞(A)、CD47 +CD70 +U266人多发性骨髓瘤细胞(B)、CD47 +CD70 +Daudi人伯基特淋巴瘤细胞(C)、以及CD47 +CD70 +Jeko-1人套细胞淋巴瘤细胞(D)引发抗体依赖性细胞毒性(ADCC)的能力，IMM01和hIgG1-Fc用作对照。
图6A-6D示出IMM40H、以及IMM40H+IMM01对CD47 +CD70 +Raji人淋巴母细胞(A)、U266人多发性骨髓瘤细胞(B)、Daudi人伯基特淋巴瘤细胞(C)、以及Jeko-1人套细胞淋巴瘤细胞(D)引发抗体依赖性细胞吞噬(ADCP)的能力IMM01和hIgG1-Fc用作对照。
图7示出IMM40H、以及IMM40H+IMM01对CD47 +CD70 +Raji人淋巴母细胞引发补体依赖性细胞毒性(CDC)的能力，hIgG1-Fc与IMM01用作对照。
图8示出IMM40H在SCID小鼠U266异种移植肿瘤模型中的体内抗肿瘤效果。
具体实施方式
为更好理解本申请，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
本文中的“CD70”是指分化簇蛋白70。该术语包括变体、同源物、直向同源物和平行同源物。例如，对人CD70特异的抗体可以在某些情况下与另一物种例如猴的CD70蛋白交叉反应。在其他实施方式中，对人CD70蛋白特异的抗体可以完全地对人CD70蛋白特异而不与其他物种或其他类型的蛋白交叉反应，或者可以与一些其他物种而非所有其他物种的CD70蛋白交叉反应。
本文中的术语“抗体”包括例如IgG、IgA、IgD、IgE和IgM的全抗体、及其任意的抗原结合片段(或抗原结合部分)或单链。全抗体是包含至少两条重链和两条轻链的糖蛋白，重链和轻链间经二硫键连接。每一重链包含重链可变区(V H)和重链恒定区。重链恒定区包含三个结构域，C H1、C H2和C H3。每一轻链包含轻链可变区(V L)和轻链恒定区。轻链恒定区包含一个结构域C L。V H和V L区域还可以再分成高度变化区，即CDR区，CDR区之间分布有较为保守的框架区(FR)。每一V H和V L由三个CDR和四个FR区构成，从氨基端到羧基端以FR1、CDR1、 FR2、CDR2、FR3、CDR3、FR4的顺序排列。重链和轻链的可变区包含与抗原反应的结合域。抗体的重链恒定区可以介导免疫蛋白与宿主组织或因子的结合，包括多种免疫***细胞(例如效应细胞)和补体***第一成分(C1q)。特别地，重链恒定区包含Fc区，是决定抗体的效应子功能，即，抗体如何与特定细胞受体或其他防御蛋白建立关系，的结构域。Fc受体(FcR)是在某些细胞，包括B淋巴细胞、滤泡树突状细胞、自然杀伤细胞、巨噬细胞、中性粒细胞、嗜酸粒细胞、嗜碱粒细胞、和肥大细胞等的表面上的蛋白。Fc区可以与Fc受体以及补体***的一些蛋白相互作用，激活免疫***和/或补体***。
本文中的术语，抗体的“抗原结合部分”(或简称为抗体部分)，是指抗体的保持有特异结合抗原(例如，CD70蛋白)能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。包含在抗体的“抗原结合部分”中的结合片段的例子包括(i)Fab片段，由V L、V H、C L和C H1构成的单价片段；(ii)F(ab') 2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由V H和C H1构成的Fd片段；(iv)由抗体单臂V L和V H构成的Fv片段；(v)由V H构成的dAb片段(Ward et al.,(1989)Nature 341:544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域V L和V H由不同的基因编码，它们可以通过重组法经由使两者成为单蛋白链的合成接头而连接，其中V L和V H区配对形成单价分子，称为单链Fc(scFv)。这些单链抗体也意在包括在术语涵义中。这些抗体片段可以通过本领域技术人员已知的常用技术而得到，且片段可以通过与完整抗体相同的方式进行功能筛选。
本文所用的术语“分离的抗体”是指基本不含具有不同抗原特异性的其他抗体的抗体。例如，与CD70特异结合的分离抗体基本不含特异结合CD70蛋白之外抗原的抗体。但是，特异结合人CD70蛋白的分离抗体可能对其他抗原例如其他物种的CD70蛋白具有交叉结合性。此外，分离的抗体基本不含其他细胞材料和/或化学物质。
术语“单克隆抗体”或“单抗”或“单克隆抗体组成”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
术语“小鼠源抗体”是指可变区框架和CDR区得自小鼠种系免疫球蛋白序列的抗体。此外，如果抗体包含恒定区，其也得自小鼠种系免疫球蛋白序列。小鼠源抗体可以包含不由小鼠种系免疫球蛋白序列编码的氨基酸残基，例如通过体外随机突变或点突变或通过体内体细胞突变而导入的突变。然而，术语“鼠源抗体”不包括在小鼠框架序列中***得自其他哺乳动物物种的CDR序列的抗体。
术语“嵌合抗体”是指通过组合非人源遗传物质与人源遗传物质而得来的抗体。或者更笼统地说，嵌合抗体是指组合有一个物种的遗传物质与另一物种遗传物质的抗体。
术语“人源化抗体”是指来源于非人物种但其蛋白序列已经被修改以增加其与人天然生成抗体的相似度的抗体。
本申请抗体或其抗体片段的重链可变区CDR和轻链可变区CDR通过IMGT编号***确定。领域内熟知，重链可变区和轻链可变区CDR可以通过例如Chothia、Kabat、AbM或Contact编号***/方法确定。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫***效应细胞主动地将细胞膜表面抗原与抗体如CD70抗体结合的靶细胞例如癌细胞裂解。
术语“抗体依赖的细胞吞噬”、“抗体依赖性细胞吞噬”或“ADCP”是指抗体如CD70抗体或类抗体蛋白如SIRPα-Fc与免疫细胞如巨噬细胞的FcR结合，引起免疫细胞如巨噬细胞对与抗体如CD70抗体或与SIRPα结合的靶细胞的吞噬，引起靶细胞的内吞和降解。
术语“补体依赖的细胞毒性”、“补体依赖性细胞毒性”或“CDC”是指抗体如CD70抗体或类抗体蛋白如SIRPα-Fc与补体***蛋白如C1q结合，激发补体通路，对靶细胞进行裂解。
术语“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳类和非哺乳类，例如非人灵长类、羊、狗、猫、牛、马、鸡、两栖类、和爬行类，尽管优选哺乳动物，例如非人灵长类、羊、狗、猫、牛和马。
术语“EC50”，又叫半最大效应浓度，是指引起50％最大效应的抗体浓度。
术语“IC50”，又叫半最大抑制浓度，是指引起50％最大抑制效应的抗体浓度。
本文中提到的“序列一致度”是指在进行序列比对后，一条序列中与参照序列中核苷酸/氨基酸残基相同的核苷酸/氨基酸百分比，如果需要的话，在序列对比中引入空格来达到两条序列间最大的序列一致性百分比。本领域技术人员可以通过多种方法，例如使用计算机软件，来进行两两序列对比或多序列比对，以确定两条或多条核酸或氨基酸序列之间的序列一致性百分比，此类计算机软件为例如ClustalOmega、T-coffee、Kalign和MAFFT等。
本申请的抗体包含与本申请CD70抗体存在一个或多个保守修饰的重链和/或轻链可变区序列或CDR1、CDR2和CDR3序列。本领域知道，一些保守序列修改不会使抗原结合性消失。
本文所用的术语“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请抗体中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(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)、非极性侧链(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)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，本申请抗体的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换，且得到的抗体可以使用本文所述的功能检测对其进行保留功能(即，上述的功能)的测试。
本申请的抗体可以以具备本申请CD70抗体的一个或多个V H/V L序列的抗体作为起始材料，制备成基因修饰的抗体。抗体可以通过修饰一个或两个可变区(即，V H和/或V L)内(例如，在一个或多个CDR区和/或一个或多个骨架区)的一个或多个残基来进行基因修饰，以改善结合亲和力和/或增加与某些物种天然产生的抗体的相似性。例如，骨架区经修饰成提供人源化的抗体。此外，或者抗体可以通过修饰恒定区中的残基进行基因修饰，例如改变抗体的效应功能。
在某些实施方式中，CDR区植入可以用来基因修饰抗体的可变区。抗体主要通过位于六个重链和轻链互补决定区(CDR)中的氨基酸残基与靶标抗原进行相互作用。出于这个原因，CDR内的氨基酸残基比起CDR外的序列在个体抗体之间更加地多样。因为CDR序列负责主要的抗体-抗原相互作用，可以通过构建含有特定天然抗体的CDR序列植入到不同特性的不同抗体的骨架序列的表达载体，来表达模拟特定天然抗体的特性的重组抗体。
因此，本申请的另一实施方式涉及分离的单克隆抗体或其抗原结合部分，重链可变区包含具有本申请上述序列的CDR1、CDR2和CDR3，轻链可变区包含具有本申请上述序列的CDR1、CDR2和CDR3，而可以含有不同的骨架序列。骨架序列可以从包括种系抗体基因序列的公开DNA数据库或公开参考文献中获得。例如，用于人重链和轻链可变区基因的种系DNA序列可以在例如Vbase人种系序列数据库(www.mrc-cpe.cam.ac.uk/vbase)获得。作为另一实施方式，用于人重链和轻链可变区基因的种系DNA序列可以在Genbank数据库中得到。用于本申请抗体的优选骨架序列是结构上与本申请抗体所用的骨架序列相似的那些。CDR1、CDR2、和CDR3序列可以植入到与得到该骨架序列的种系免疫球蛋白基因具有相同序列的骨架区中，或者CDR序列可以植入到包含有与种系序列相比具有一个或多个突变的骨架区中。例如，在一些情况下，将骨架区中的残基进行突变是有益的，以保持或增强抗体的抗原结合性等。
另一类的可变区修饰是将V H和/或V LCDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，从而改进目标抗体的一种或多种结合特性(例如，亲和力)。可以进行点突变或PCR介导的突变来引入突变，且其对于抗体结合或其他功能特性的影响可以在本领域所知的体外或体内检测中进行评价。优选地，引入本领域所知的保守修饰。突变可以是氨基酸替换、添加或缺失，但优选为替换。此外，通常改变CDR区内的不多于一个、两个、三个、四个或五个的残基。
本申请的基因改造抗体包括在V H和/或V L的骨架残基中做出基因修饰以例如改变抗体特性的那些。通常而言，这些骨架修饰用来降低抗体的免疫原性。例如，一种方法是将一个或多个骨架残基“回复突变”成相应的种系序列。更加具体而言，经历体细胞突变的抗体可能包含不同于得到抗体的种系序列的骨架残基。这些残基可以通过将抗体骨架序列与得到抗体的种系序列相比较而识别出来。
另一类的骨架修饰包括对骨架区的、或者甚至一个或多个CDR区的一个或多个残基进行突变，以去除T细胞表位，从而减少抗体的可能导致的免疫原性。
此外，作为骨架或CDR区内修饰之外的另一种选择，本申请的抗体可以基因改造成在Fc区包括基因修饰，通常来改变抗体的一个或多个功能特性，例如血清半衰期、补体结合、Fc受体结合、和/或抗体依赖的细胞毒性。此外，本申请的抗体可以进行化学修饰(例如，可以向抗体附加一个或多个化学功能基团)，或者修饰成改变其糖基化，来改变抗体的一个或多个功能特性。
在一个实施方式中，C H1的铰链区进行修饰，改变，例如增加或减少铰链区的半胱氨酸残基的数量。改变C H1铰链区的半胱氨酸残基，来例如促进重链轻链的组装或增加/降低抗体的稳定性。
在另一个实施方式中，对抗体的Fc铰链区进行突变，以降低抗体的生物半衰期。更加具体地，将一个或多个氨基酸突变引入Fc铰链片段的C H2-C H3连接区，从而抗体相对于天然Fc-铰链结构域SpA结合而言，具有减弱的SpA结合力。
在另一实施方式中，修饰抗体的糖基化。例如，可以制备去糖基化的抗体(即，抗体缺少糖基化)。可以改变糖基化，来例如增加抗体对抗原的亲和性。这样的糖化修饰可以通过例如改变抗体序列中的一个或多个糖基化位点来达成。例如，可以做出一个或多个氨基酸替换，以消除一个或多个可变区骨架糖基化位点，从而消除该位置的糖基化。这样的去糖基化可以增加抗体对抗原的亲和性。此外，可以制备具有改变的糖基化类型的抗体，例如岩藻糖残基量减少的低岩藻糖基抗体，或者具有增加的平分型GlcNac结构的抗体。改变的糖基化形式被证明能增加抗体的ADCC活性。这样的糖化修饰可以通过例如在糖基化***改变的宿主细胞中表达抗体而进行。具有改变的糖基化***的细胞在领域中已知，且可以用作表达本申请重组抗体的宿主细胞，以制备具有改变的糖基化的抗体。
本申请的单克隆抗体可以使用Kohler and Milstein(1975)Nature 256:495的体细胞杂交(杂交瘤)技术进行制备。制备单克隆抗体的其他实施方式包括B淋巴细胞的病毒或致癌性转化以及噬菌体展示技术。嵌合或人源化抗体也在领域内熟知。
本申请的抗体还可以使用例如重组DNA技术结合基因转染方法，在宿主细胞转染瘤中生成(例如Morrison,S.(1985)Science 229:1202)。在一个实施方式中，将由标准分子生物技术得到的编码部分或全长轻链和重链的DNA***一个或多个表达载体中，从而基因与转录和翻译调控序列可操作地连接。在该情况下， 术语“可操作地连接”是指抗体基因连接到载体中，从而载体内的转录和翻译控制序列行使它们既定的调控抗体基因转录和翻译的功能。
术语“调控序列”包括控制抗体基因转录或翻译的启动子、增强子和其他表达控制元件(例如，多腺苷酸化信号)。这样的调控序列在例如Goeddel(Gene Expression Technology.Methods in Enzymology 185,Academic Press,San Diego,Calif.(1990))中有过描述。优选的用于哺乳动物宿主细胞表达的调控序列包括引导在哺乳动物细胞中的高水平蛋白表达的病毒元件，例如得自巨细胞病毒(CMV)、猿猴病毒40(SV40)、腺病毒的启动子和/或增强子，如腺病毒主要晚期启动子(AdMLP)和多瘤病毒。或者，可以使用非病毒调控序列，例如泛素启动子或β-珠蛋白启动子。另外，调控元件由不同来源的序列构成，例如SRα启动子***，其包含来自SV40早期启动子的序列和人T细胞白血病I型病毒的长末端重复(Takebe et al.,(1988)Mol.Cell.Biol.8:466-472)。表达载体和表达控制序列选为与所使用的表达宿主细胞相容。
抗体轻链基因和抗体重链基因可以***到同一或不同的表达载体中。在优选实施方式中，可变区通过***到已经编码所需亚型的重链恒定区和轻链恒定区的表达载体中而构建全长抗体基因，从而V H与载体中的C H可操作地连接，V L与载体中的C L可操作地连接。或者，重组表达载体可以编码促进抗体链从宿主细胞分泌的信号肽。抗体链基因可以克隆到载体中，从而信号肽在阅读框内连接到抗体链基因的氨基端。信号肽可以是免疫球蛋白信号肽或异源信号肽(即，来自非免疫球蛋白的信号肽)。
除抗体链基因和调控序列外，本申请的重组表达载体可以携带其他序列，例如调控载体在宿主细胞中复制的序列(例如，复制起始点)和可选择标记物基因。可选择标记物基因可用于选择已导入载体的宿主细胞(参见，例如，美国专利4,399,216；4,634,665和5,179,017)。例如，通常可选择标记物基因赋予已导入载体的宿主细胞以药物抗性，例如G418、潮霉素、或氨甲喋呤抗性。优选的可选择标记物基因包括二氢叶酸还原酶(DHFR)基因(用于dhfr宿主细胞的氨甲喋呤选择/扩增)和neo基因(用于G418选择)。
对于轻链和重链的表达，编码重链和轻链的表达载体通过标准技术转染到宿主细胞中。多个形式的术语“转染”包括多种常用于将外源DNA导入原核或真核宿主细胞的技术，例如，电穿孔、磷酸钙沉淀、DEAE-右旋糖转染等。尽管在原核或真核宿主细胞中表达本申请抗体在理论上是可行的，优选抗体在真核细胞中表达，最优选在哺乳动物宿主细胞中表达，因为真核细胞，特别是哺乳动物细胞，比原核细胞更可能组装并分泌适当折叠且有免疫活性的抗体。
优选的用于表达本申请重组抗体的哺乳动物宿主细胞包括中国仓鼠卵巢(CHO细胞)(包括与DHFR可选择标记物一起施用的dhfr-CHO细胞，在Urlaub and Chasin,(1980)Proc.Natl.Acad.Sci.USA 77:4216-4220中有过描述，DHFR 可选择标记物在例如R.J.Kaufman and P.A.Sharp(1982)J.Mol.Biol.159:601-621中描述)、NSO骨髓瘤细胞、COS细胞和SP2细胞。特别在使用NSO骨髓瘤细胞时，另一优选的表达***是GS基因表达***。当编码抗体基因的重组表达载体导入哺乳动物宿主细胞时，通过将宿主细胞培养足以使得宿主细胞中抗体表达、或优选地足以使得使抗体分泌到宿主细胞生长的培养基中的一段时间，从而制备抗体。抗体可以使用蛋白纯化方法从培养基中回收。
本申请的抗体或其抗原结合部分可以与治疗剂交联，形成免疫交联物，例如抗体-药物交联物(ADC)。合适的治疗剂包括细胞毒素、烷化剂、DNA小沟结合分子、DNA嵌入剂、DNA交联剂、组蛋白去乙酰化酶抑制剂、核输出抑制剂、蛋白酶体抑制剂、拓扑异构酶I或II的抑制剂、热激蛋白抑制剂、酪氨酸激酶抑制剂、抗生素和抗有丝***剂。在ADC中，抗体和治疗剂优选地通过接头交联，该接头可切割，例如肽类接头、二硫类接头或腙类接头。更优选地，接头是肽类接头，例如Val-Cit、Ala-Val、Val-Ala-Val、Lys-Lys、Ala-Asn-Val、Val-Leu-Lys、Ala-Ala-Asn、Cit-Cit、Val-Lys、Lys、Cit、Ser或Glu。
另一方面，本申请涉及包含与至少一个其他功能分子如另一种肽或蛋白(例如，另一抗体或受体配体)相连接的一种或多种本申请抗体或其抗原结合部分的双特异性分子，以生成与至少两个不同结合位点或靶向分子结合的双特异性分子。术语“双特异性分子”包括具有三种或更多种特异性的分子。在一些实施方式中，除Fc结合特异性和CD70结合特异性外，双特异性分子还具有第三特异性。第三特异性可以例如针对CD47，以更好地靶向肿瘤细胞，而免除其他CD70 +细胞如免疫细胞。
双特异性分子可以以多种形式和尺寸出现。在尺寸谱的一端，双特异性分子保持传统抗体形式，除其具有两个结合臂且各臂具有不同特异性外，替代具有两个特异性相同的结合臂的情况。在另一极端的是双特异性分子，由两个经肽链连接的单链抗体片段(scFv)构成，称为Bs(scFv) 2构建体。中间尺寸的双特异性分子包括由肽类接头连接的两个不同的F(ab)片段。这些和其他形式的双特异性分子可以通过基因改造、体细胞杂交或化学法进行制备。
本申请还提供包含CD70单链抗体scFv的嵌合抗原受体，该scFv包含本申请中所述的重链和轻链CDR、或重链和轻链可变区。CD70嵌合抗原受体可以包含(a)含有CD70scFv的胞外抗原结合域；(b)跨膜结构域；和(c)胞内信号转导结构域。
在另一方面，本申请提供编码本申请抗体或其抗原结合部分的重链/轻链可变区或CDR的核酸分子。核酸可以存在整细胞中，在细胞裂解液中，或处于部分纯化或基本纯的形式。当通过标准技术从其他细胞组分或其他污染物例如其他细胞核酸或蛋白中纯化出来后，核酸是“分离的”或“基本纯的”。本申请的核酸可以为例如DNA或RNA，且可能包含或可能不包含内含子序列。在优选实施方式中， 核酸是cDNA分子。
本申请的核酸可以使用标准的分子生物学技术获得。对于由杂交瘤(例如，由携带人免疫球蛋白基因的转基因小鼠制备的杂交瘤)表达的抗体，编码杂交瘤制备的抗体的轻链和重链的cDNA可以通过标准PCR扩增或cDNA克隆技术获得。对于(例如使用噬菌体展示技术)从免疫球蛋白基因库获得的抗体，编码这类抗体的核酸可以从基因库中收集。
优选的本申请核酸分子包括编码CD70单克隆抗体的V H和V L序列或CDR的那些。一旦获得了编码V H和V L的DNA片段，这些DNA片段可以进一步通过标准的重组DNA技术进行操作，例如将可变区基因转变为全长抗体链基因、Fab片段基因或scFv基因。在这些操作中，编码V H或V L的DNA片段与编码另一蛋白的另一DNA片段，例如抗体恒定区或柔性接头，可操作地连接。术语“可操作地连接”是指两个DNA片段连接在一起，从而两个DNA片段编码的氨基酸序列都在阅读框内。
编码V H区的分离DNA可以通过可操作地连接V H编码DNA与编码重链恒定区(C H1、C H2和C H3)的另一DNA分子而转变成全长重链基因。人重链恒定区基因的序列在领域内已知，且包括这些区域的DNA片段可以通过标准PCR扩增而获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是优选为IgG1或IgG4恒定区。对于Fab片段重链基因，编码V H区的DNA可以可操作地与仅编码重链C H1恒定区的另一DNA分子连接。
编码V L区的分离DNA可以通过可操作地连接V L编码DNA与编码轻链恒定区C L的另一DNA分子而转变成全长轻链基因。人轻链恒定区基因的序列在领域内已知，且包括这些区域的DNA片段可以通过标准PCR扩增而获得。在优选实施方式中，轻链恒定区可以是κ和λ恒定区。
为创建scFv基因，编码V H和V L的DNA片段可以可操作地与编码柔性接头例如5-20氨基酸肽的另一片段连接，从而V H和V L序列可以作为连续的单链蛋白进行表达，其中V H和V L区域通过该柔性接头连接。
在另一方面，本申请提供一种药物组合物，其包含本申请的一种或多种抗体或其抗原结合部分、免疫交联物、免疫细胞、双特异抗体、和/或抗体或其抗原结合部分的编码核酸分子、表达载体或宿主细胞，与药学上可接受的载体配制在一起。组合物可以任选地包含一种或多种其他药学上的有效成分，例如另一抗肿瘤蛋白，如本申请中联用的IMM01，一种SIRPαD1-Fc融合蛋白。
药学组合物可以包含任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠或乳化剂、固体粘合剂、分散或混悬剂、增溶剂、染色剂、矫味剂、涂层、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂及其组合。
药物组合物可适合于静脉内、肌内、皮下、肠道外、脊柱或表皮施用(例如通过注射或推注)。基于施用途径的不同，有效成分可以包在材料中，以保护其 不受酸和可能使其失活的其他自然条件的影响。“肠道外施用”是指不同于肠道和局部外用的方式，通常通过注射进行，包括但不限于静脉内、肌内、动脉内、膜内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬脑膜上和胸骨内注射和推注。或者，本申请的抗体可以通过非肠道外路径施用，例如外用、表皮施用或粘膜施用，例如鼻内、经口、***、直肠、舌下、或局部外用。
药物组合物可以为无菌水溶液或分散液的形式。它们也可以配制在微乳剂、脂质体或其他适于高浓度药物的有序结构中。
与载体材料一起制备成单剂型的有效成分的量将随着治疗主体和特定施用模式而变，且基本上而言是产生疗效的组合物的量。以百分比计，该量为约0.01-约99％的与药学上可接受载体结合的有效成分。
给药方案经调整提供最佳的所需反应(例如，治疗反应)。例如，可以施用快速灌注剂，可以随时间推移施用多个分剂量，或者剂量可以随治疗情况的危急程度成比例降低或提高。特别有利的是，以方便施用和剂量均匀的剂量单位型配置肠道外组合物。剂量单位型是指物理上分开的单位，适于治疗主体的单次给药；各单位包含计算出来与药学载体一起产生所需疗效的预定量的有效成分。或者，抗体可以以缓释剂施用，这种情况下所需的施用频率降低。
对于抗体的施用，剂量可以为约0.001-100mg/kg宿主体重。
“治疗有效量”的本申请CD70抗体引起疾病症状严重程度的降低、无症状期频率和持续时间的增加。例如，对于荷肿瘤受试者的治疗，“治疗有效量”优选地，与未治疗受试者相比，将肿瘤生长抑制至少约20％、更优选抑制至少约40％，甚至更优选地抑制至少约60％，且更优选地抑制至少约80％。治疗有效量的治疗抗体可以减小肿瘤尺寸，或者减轻受试者的症状，受试者可以是人或另一哺乳动物。例如，对于罹患炎性疾病的受试者的治疗，“治疗有效量”优选地，与未治疗受试者相比，将炎症减少至少约20％、更优选减少至少约40％，甚至更优选地减少至少约60％，且更优选地减少至少约80％。治疗有效量的治疗抗体可以减轻或消除炎症，或者减轻受试者的症状，受试者可以是人或另一哺乳动物。
药物组合物可以是缓释试剂，包括植入体、和微胶囊递送***。可以使用生物可降解、生物相容的聚合物，例如乙烯-醋酸乙烯、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯、和聚乳酸。参见，例如，Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978。
药学组合物可以经医学设备来给药，例如(1)无针皮下注射设备(例如，美国专利5,399,163；5,383,851；5,312,335；5,064,413；4,941,880；4,790,824；和4,596,556)；(2)微量输液泵(美国专利4,487,603)；(3)经皮给药设备(美国专利4,486,194)；(4)推注设备(美国专利4,447,233和4,447,224)；和(5)渗透设备(美国专利4,439,196和4,475,196)。
在某些实施方式中，本申请的单抗可以经配制，以确保合适的体内分布。例如，为确保本申请的治疗抗体穿越血脑屏障，抗体可以配制在脂质体中，其还可以额外地包含靶向功能基团，以增强对特定细胞或器官的选择性输送。
本申请的药物组合物具有多种体外和外内应用，涉及例如癌症的治疗，或者更笼统地讲，用于癌症疾病患者的免疫增强。抗体可以施用至人受试者，以例如体内抑制肿瘤生长。
考虑到本申请药物组合物的抑制肿瘤细胞增殖和存活的能力，本申请提供抑制受试者中肿瘤细胞生长的方法，包括向该受试者施用本申请的药物组合物，从而在该受试者中肿瘤生长被抑制。可以由本申请抗体治疗的肿瘤包括实体瘤和血液瘤，非限制性例子包括但不限于，肾癌、骨髓增生异常综合征、皮肤T细胞淋巴瘤、鼻咽癌、Epstein-Barr病毒诱导的癌症、恶性霍金奇淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤、滤泡状淋巴瘤、B细胞淋巴细胞白血病、Burkitt淋巴瘤、多发性骨髓瘤、Waldenstrom巨球蛋白血症、鼻咽癌、胸腺癌、胶质母细胞瘤、脑癌、骨肉瘤、黑色素瘤、卵巢癌、肾细胞癌、乳腺癌、和非小细胞肺癌，不管是原发或是转移的。
鉴于CD70与炎性疾病的关联，本申请提供减轻或消除受试者中炎症的方法，包括向该受试者施用本申请的药物组合物。炎性疾病可包括自免疫疾病。炎性疾病的非限制性例子包括但不限于，关节炎、肠炎、和狼疮。
本申请的药物组合物可以与一种或多种其他抗体或非抗体类治疗剂一起施用，其能有效抑制受试者中的肿瘤生长、和/或减轻或消除受试者中的炎症。在一个实施方式中，本申请提供在受试者中抑制肿瘤生长的方法，包括向受试者施用CD70药物组合物以及IMM01，一种SIRPαD1-Fc融合蛋白。
本文讨论的治疗剂的组合可以作为在药学可接受载体中的单一组合物同时施用，或者作为分开的组合物同时施用，其中各药剂处于药学可接受载体中。在另一个实施方式中，治疗剂的组合可以按序施用。
此外，如果进行多次联合疗法施用，且药剂按序施用，则在各时间点的按序施用的次序可以反转或保持相同，按序施用可以与同时施用或其任何组合相结合。
本申请将参照以下非限制性实施例进行进一步说明。
实施例
图1示出本申请CD70抗体IMM40C、IMM40H、和IMM40M的结构。
其中，IMM40C为IgG抗体，包含小鼠源重链可变区、和小鼠源轻链可变区。具体地，IMM40C包含SEQ ID NO:7的重链可变区、SEQ ID NO:11的重链恒定区、SEQ ID NO:8的轻链可变区、以及SEQ ID NO:12的轻链恒定区。
IMM40H为IgG抗体，包含人源化重链可变区、和人源化轻链可变区。具体地，IMM40H包含SEQ ID NO:9的重链可变区、SEQ ID NO:11的重链恒定区、 SEQ ID NO:10(X＝N)的轻链可变区、以及SEQ ID NO:12的轻链恒定区。
IMM40M为IgG抗体，包含人源化重链可变区、和人源化轻链可变区。相比于IMM40H，IMM40M在轻链可变区具有N85E突变，该突变可消除原有的糖基化位点。具体地，IMM40M包含SEQ ID NO:9的重链可变区、SEQ ID NO:11的重链恒定区、SEQ ID NO:10(X＝E)的轻链可变区、以及SEQ ID NO:12的轻链恒定区。
IMM01为结合CD47的SIRPαD1-Fc融合蛋白，记载于US 2021/0024598A1，包含两个突变SIRPαD1，与Fc二聚体片段连接，其单体含有SEQ ID NO:17所示的氨基酸序列。IMM01中的SIRPαD1，相比于野生型SIRPαD1，在SEQ ID NO:17的第80位处存在N80A突变，该位点的突变可实现去糖基化的效果。
Cusatuzumab为特异结合CD70的IgG抗体，包含分别如SEQ ID NOs:20和21所示的重链和轻链。
实施例1.CD70抗体的生成、人源化、表达载体构建及表达
对小鼠接种人CD70蛋白，取其血清，筛选出滴度高的小鼠。取小鼠脾脏细胞，经杂交瘤技术制备出生产CD70抗体的杂交瘤细胞系，并经CD70结合活性测试筛选出生产CD70结合活性高的单克隆抗体的细胞克隆，包括生产IMM40C的细胞克隆。IMM40C的重链可变区和轻链可变区经测序得知，氨基酸序列分别如SEQ ID NOs:7和8所示。
用CDR移植法对IMM40C进行人源化改造，获得人源化的抗体IMM40H。IMM40H包含SEQ ID NO:9的重链可变区、和SEQ ID NO:10(X＝N)的轻链可变区。对IMM40H的轻链可变区进行N85E突变，获得IMM40M，N85E突变可消除原有的糖基化位点。IMM40M包含SEQ ID NO:9的重链可变区、和SEQ ID NO:10(N＝E)的轻链可变区。
人工设计出IMM40C、IMM40H、和IMM47M的编码序列。
具体地，对于IMM47C的重链，将编码小鼠IgG1重链信号肽的57个核苷酸(SEQ ID NO:18)加至IMM47C重链可变区-恒定区编码序列(SEQ ID NO:13)的5’端，并将Kozak序列(SEQ ID NO:19)加至信号肽序列的5’端。最后，将HindIII和NheI限制性酶切位点分别加至所得序列的5’和3’端。对于IMM47C的轻链，将相同的信号肽序列以及Kozak序列加至IMM47C抗体轻链可变区-恒定区编码序列(SEQ ID NO:14)的5’端，并将HindIII和XbaI限制性酶切位点分别加至所得序列的5’和3’端。所得的序列经金斯瑞生物科技股份有限公司合成，并分别克隆到pMac-H和pMac-L载体中。
对于IMM47H的重链，将编码小鼠IgG1重链信号肽的57个核苷酸(SEQ ID NO:18)加至IMM47H重链可变区-恒定区编码序列(SEQ ID NO:15)的5’端，并将Kozak序列(SEQ ID NO:19)加至信号肽序列的5’端。最后，将HindIII和 NheI限制性酶切位点分别加至所得序列的5’和3’端。对于IMM47H的轻链，将相同的信号肽序列以及Kozak序列加至IMM47H抗体轻链可变区-恒定区编码序列(SEQ ID NO:16，N1＝A，N2＝C)的5’端，并将HindIII和XbaI限制性酶切位点分别加至所得序列的5’和3’端。所得的序列经金斯瑞生物科技股份有限公司合成，并分别克隆到pMac-H和pMac-L载体中。
对于IMM47M的重链，将编码小鼠IgG1重链信号肽的57个核苷酸(SEQ ID NO:18)加至IMM47H重链可变区-恒定区编码序列(SEQ ID NO:15)的5’端，并将Kozak序列(SEQ ID NO:19)加至信号肽序列的5’端。最后，将HindIII和NheI限制性酶切位点分别加至所得序列的5’和3’端。对于IMM47M的轻链，将相同的信号肽序列以及Kozak序列加至IMM47M抗体轻链可变区-恒定区编码序列(SEQ ID NO:16，N1＝G，N2＝A)的5’端，并将HindIII和XbaI限制性酶切位点分别加至所得序列的5’和3’端。所得的序列经金斯瑞生物科技股份有限公司合成，并分别克隆到pMac-H和pMac-L载体中。
构建好的表达载体，利用CHO-S细胞瞬转表达蛋白。大致过程如下：1)瞬转前一天将CHO-S细胞按照1×10 6个/ml密度接种至含6mM谷氨酰胺的瞬转培养基(TransFx-CTMCHO瞬转培养基，Hyclone)；2)重/轻链表达载体质量比为1：1，按照1μg/ml准备所需DNA，加入至瞬转体积1/20的OPTI-MEM培养基(Gibco)；3)PEI(MW 40,000聚乙烯亚胺盐酸盐，polysciences)配置成1mg/ml，按照PEI:DNA＝4:1质量比例准备所需PEI，加入至瞬转体积1/20的OPTI-MEM培养基(Gibco)；4)将PEI稀释液缓慢加入至DNA稀释液中，混匀，室温孵育20分钟；5)将PEI/DNA混合液加入至细胞液中，并将细胞置于37度5％CO 2，转速110rpm培养箱振荡培养；6)隔天添加转染增强剂(1mM丁酸钠，0.25％V/V DMSO),同时将培养温度降至33度；7)当细胞活率降至50％以下时，3000rpm，离心5分钟收集上清利用Protein A填料进行亲和纯化。
实施例2.CD70抗体与与细胞表面的CD70结合
将表达人CD70的中国仓鼠卵巢细胞、CD47 +CD70 +U266人多发性骨髓瘤、CD47 +CD70 +Raji人B淋巴母细胞、以及CD47 +CD70 +U266人多发性骨髓瘤细胞1000rpm离心5min，收集细胞，PBS清洗1次，细胞密度调整为1×10 6/ml。取100μl上述各细胞，分别在100μl用1％BSA-PBS梯度稀释的IMM40C、IMM40H和IMM40M中4℃孵育45min。细胞用1％BSA-PBS清洗1遍，加入100μl抗人IgG(Fc)-FITC(1:500稀释，Cat#F9512，Sigma)，4度冰箱45min。孵育结束后，1％BSA-PBS清洗1遍。细胞重悬于200μl 1％BSA-PBS，用细胞用流式细胞仪(
Figure PCTCN2022114942-appb-000001
easyCyte 5HT，Merck Millipore)进行FACS分析。
图2A示出IMM40C和IMM40H与CD70 +中国仓鼠卵巢细胞的结合能力，hIgG1-Fc用作阴性对照。由结果可知，IMM47C和IMM40H特异结合该CD70 + 细胞，且两者的结合能力没有差异。
图2B示出IMM40C和IMM40H与CD47 +CD70 +U266人多发性骨髓瘤细胞的结合能力，hIgG1-Fc用作阴性对照。由结果可知，IMM40C和IMM40H特异结合该CD47 +CD70 +细胞，且两者的结合能力没有差异。
图2C示出IMM40H与CD47 +CD70 +Raji人B淋巴母细胞的结合能力，hIgG1-Fc用作阴性对照。由结果可知，IMM40H特异结合该CD47 +CD70 +细胞，且结合活性明显强于Cusatuzumab和IMM01。
图2D示出IMM40H与CD47 +CD70 +U266人多发性骨髓瘤细胞的结合能力，hIgG1-Fc用作阴性对照。由结果可知，IMM40H特异结合该CD47 +CD70 +细胞,且结合活性强于Cusatuzumab和IMM01。
图2E示出IMM40H和IMM40M与CD70 +中国仓鼠卵巢细胞的结合能力，hIgG1-Fc用作阴性对照。由结果可知，IMM40H和IMM40M特异结合该CD70 +细胞,且结合活性相当，IMM40H稍强于IMM40M。
图2F示出IMM40H和IMM40M与CD47 +CD70 +U266人多发性骨髓瘤细胞的结合能力，hIgG1-Fc用作阴性对照。由结果可知，IMM40H和IMM40M特异结合该CD47 +CD70 +细胞，且两者的结合活性相当，IMM40H稍强于IMM40M。
实施例3.CD70抗体阻断CD27(ECD)-Fc与表达人CD70的中国仓鼠卵巢细 胞上的CD70的结合
将50μl密度为5×10 5/ml的CHO-hCD70细胞分别与50μl起始浓度为900nM、3倍梯度稀释的IMM40H、Cusatuzumab、和hIgG-Fc混合。将上述混合物添加到含50μl 2.5μg/ml生物素-CD27(ECD)-Fc(SEQ ID NO:22，氨基酸1-172为CD27胞外段)的96孔板中，板于4℃孵育45分钟。细胞用PBS洗，之后加入100μl PE标记的针对生物素的链霉亲和素二抗(Cat#554061，Biolegend)，孵育45分钟。细胞清洗两遍，并重悬浮于200μl PBS，并经FACS分析CD27-Fc-CD70的相互作用。
由图3可知，IMM40H对CD70-CD27的阻断活性，在CD70 +CD47 -中国仓鼠卵巢细胞上，与Cusatuzuma相当。
实施例4.CD70抗体阻断CD70 +Raji人B淋巴母细胞瘤细胞与人T淋巴细 胞白血病细胞Jurkat-CAR-CD27的相互作用
取50μl密度为2×10 5个/ml的CD70 +Raji人B淋巴母细胞瘤细胞，分别与50μl起始浓度16ng/ml、4倍梯度稀释的IMM40H、Cusatuzumab、IMM01和hIgG1-Fc，37℃孵育45分钟。之后加入50μl密度为1×10 6个/ml的人T淋巴细胞白血病细胞Jurkat-CAR-CD27(公司自制，CAR-CD27的氨基酸序列如SEQ ID NO:23所示，氨基酸1-172为CD27胞外段)，于37℃孵育过夜。将0.5％BSA-PBS 加入到细胞混合液后，离心，并将细胞重悬于50μl 1：100稀释的PE标记抗人CD69抗体稀释液(Cat#sc-373799，Santa Cruz)，4℃孵育45分钟，细胞用0.5％BSA-PBS清洗一次，并重悬于0.5％BSA-PBS。之后，细胞用流式细胞仪(
Figure PCTCN2022114942-appb-000002
easyCyte 5HT，Merck Millipore)进行FACS分析。CD69表达水平能间接反映Raji人B淋巴母细胞瘤细胞上CD70与Jurkat-CAR-CD27的结合度。
如图4所示，IMM40H能够抑制CD70-CD27的结合，抑制活性稍强于传统的单特异CD70抗体Cusatuzumab。
实施例5.CD70抗体针对肿瘤细胞引发抗体依赖性细胞毒性(ADCC)
用1mM CFSE(Cat#21888-25mg，Sigma)500倍稀释标记各肿瘤细胞。
取50μl密度为6×10 5/ml的各肿瘤细胞(用作靶向细胞)，与100μl密度为6×10 5/ml的稳定表达FcγRIIIa的NK92MI细胞(效应细胞)，按照效靶比2：1混合，且混合的细胞在5％CO 2下分别与50μl起始浓度为1nM、4倍梯度稀释的抗体/蛋白(对于IMM01与IMM40H的混合物，浓度按单物质算，IMM01与IMM40H各自初始浓度1nM)以及对照蛋白于37℃培养4小时。之后，向细胞培养液中加入碘化丙啶(PI)(Cat#P4170，Sigma)，浓度5μg/ml，细胞经FACS分析PI信号。由ADCC造成的细胞裂解百分比基于以下公式进行计算：
％裂解＝(％抗体/蛋白处理的PI阳性靶细胞-％阴性对照蛋白hIgG1-Fc处理的PI阳性靶细胞)/(100-％阴性对照蛋白hIgG1-Fc处理的PI阳性靶细胞)×100
如图5A所示，对于CD47 +CD70 +Raji人B淋巴母细胞瘤细胞，IMM40H引发的ADCC效应与IMM40H+IMM01混合物相当，并远强于单特异性CD47结合蛋白IMM01。
如图5B所示，对于CD47 +CD70 +U266人多发性骨髓瘤细胞，IMM40H引发的ADCC效应与IMM40H+IMM01混合物相当，并远强于单特异性CD47结合蛋白IMM01。
如图5C所示，对于CD47 +CD70 +Daudi人伯基特淋巴瘤细胞，IMM40H引发的ADCC效应与IMM40H+IMM01混合物相当，并远强于单特异性CD47结合蛋白IMM01。
如图5D所示，对于CD47 +CD70 +Jeko-1人套细胞淋巴瘤细胞，IMM40H引发的ADCC效应与IMM40H+IMM01混合物相当，并远强于单特异性CD47结合蛋白IMM01。
实施例6.CD70抗体针对CD47 +CD70 +细胞引发高水平的抗体依赖性细胞 吞噬(ADCP)
用0.25％胰酶37度消化巨噬细胞THP-1细胞2min后，用完全培养基(1640+10％FBS)终止，并将细胞吹打悬浮，1000rpm离心5min，收集THP-1 细胞，并加入到96孔板中，3x10 5个/孔，每孔含100μl完全培养基，200ng/ml PMA刺激过夜培养。
用CFSE标记肿瘤细胞，即CD47 +CD70 +人淋巴母细胞(Raji细胞)、人多发性骨髓瘤细胞(U266细胞)、人伯基特淋巴瘤细胞(Daudi细胞)和人套细胞淋巴瘤细胞(Jeko-1细胞)，作为靶向细胞。具体地，1mM CFSE稀释500倍，加入到调整好密度的细胞悬液中，混匀，细胞培箱染色30min。染色结束后，空白1640培养基清洗1次，并将细胞密度调整为2x10 6/ml。细胞1000rpm离心5min，收集，1640培养基清洗1次，将细胞密度调整为1x10 6/ml。
用1640培养基倍比梯度稀释重组融合蛋白和对照蛋白，其中对于IMM01和IMM40H混合物，浓度按单物质算，IMM01与IMM40H各自的起始浓度同单物质组一致。将100μl上述处理好的肿瘤细胞与100μl上述稀释好的重组融合蛋白或对照蛋白混合，每个浓度2个复孔，于培养箱孵育45min。将接种THP-1细胞的孔板上清吸尽，加入上述肿瘤细胞-融合蛋白混合物200μl，同时设置对照组，即单独THP-1细胞、及THP-1与肿瘤细胞，于培养箱孵育2小时。
PBS清洗96孔板洗涤5次，去除游离的肿瘤-CFSE细胞。洗涤结束后，每孔加入200μl PBS，反复吹打将THP-1细胞悬浮。流式检测并分析THP-1细胞绿色荧光强度。通过分析THP-1细胞的绿色荧光强度评价吞噬活性。
如图6A所示，对于CD47 +CD70 +Raji人淋巴母细胞，IMM40H引发的ADCP效应远强于结合CD47的Fc融合蛋白IMM01，在高浓度时强于IMM01+IMM04H混合物。
如图6B所示，对于CD47 +CD70 +U266人多发性骨髓瘤细胞，IMM40H引发的ADCP效应远强于结合CD47的Fc融合蛋白IMM01、以及IMM01+IMM04H混合物。
如图6C所示，对于CD47 +CD70 +Daudi人伯基特淋巴瘤细胞，IMM40H引发的ADCP效应与结合CD47的Fc融合蛋白IMM01、以及IMM01+IMM04H混合物相当。
如图6D所示，对于CD47 +CD70 +Jeko-1人套细胞淋巴瘤细胞，IMM40H引发的ADCP效应与IMM01+IMM04H混合物相当，并强于结合CD47的Fc融合蛋白IMM01。
实施例7.CD70抗体针对CD47 +CD70 +人淋巴母细胞(Raji细胞)引发高水 平的补体依赖性细胞毒性(CDC)
制备无血清Raji细胞悬浮液，细胞密度调整为3×10 5/ml，取50μl接种至96孔U形底部细胞培养板中，并分别与30μl起始浓度为25nM、4倍梯度稀释的IMM01、IMM40H、IMM01+IMM40H混合液(IMM01与IMM40H混合物的浓度按照单物质浓度算，各单物质起始浓度25nM)、以及hIgG1-Fc孵育30分钟。 向混合物中加入20μl 13倍稀释的兔子血清补体(Cat#CL3111，Accurate Chemical)，继续孵育4小时。之后向细胞培养液中加入碘化丙啶(PI)(Cat#P4170，Sigma)，浓度5μg/ml，细胞经FACS分析PI信号计算细胞裂解率。
如图7所示，对于人CD47和CD70双阳性的Raji人淋巴母细胞，IMM40H引发高水平的补体依赖性细胞毒性(CDC)，其活性与IMM01+IMM40H混合物相当，远高于IMM01。
实施例8.CD70抗体显示出强劲的体内抗肿瘤活性
30只5-6周龄的SCID小鼠背部右边靠近腋下接种含5×10 6人多发性骨髓瘤U266细胞的培养基100μL和100μL Matrigel混匀物。当肿瘤体积达到～200mm 3时，小鼠随机分成5组，每组6只小鼠，分组当天定义为D0。从D0天开始，各组小鼠分别腹膜注射PBS、IMM40H(0.3mg/kg)、IMM40H(1.0mg/kg)、IMM01(0.5mg/kg)、以及IMM01+IMM40H(0.5mg/kg+1.0mg/kg)，持续4周，每周1次。在4周后结束给药，持续观察3.5周后实验结束。每3-4天测量一次肿瘤体积和小鼠体重。
肿瘤体积(V)计算为(长×宽 2)/2。相对肿瘤体积(RTV)＝V t/V 0，其中V t为第t天(Dt)的肿瘤体积，V 0为治疗开始(D0)时的肿瘤体积。
相对肿瘤抑制率TGI(％)＝1-治疗组相对肿瘤体积平均值/阴性对照组相对肿瘤平均值平均值×100％。
表1.IMM40H和其他治疗剂实验终点(D52)抗肿瘤效果
Figure PCTCN2022114942-appb-000003
如图8所示，实验期间，PBS对照组在第52天(D52)的肿瘤体积均值为2715.13±107.27mm 3，相对肿瘤体积(RTV)均值14.01±0.67。
在IMM40H(0.3mg/kg)给药4周后，即第28天(D28)时，肿瘤体积为61.42±27.64mm 3，RTV为0.30±0.13。与同期溶媒对照组相比，肿瘤体积和相对肿瘤体积在D3～D28期间显著降低(p<0.05)。D52时，肿瘤体积为487.78±233.37mm 3，RTV为2.43±1.16。与同期溶媒对照组相比，肿瘤体积和相对肿瘤体积在停药三周半内持续显著降低(p<0.01)。
在IMM40H(1.0mg/kg)、IMM01(0.5mg/kg)、以及IMM01+IMM40H(0.5 mg/kg+1.0mg/kg)组，D28时肿瘤体积均为0.00±0.00mm 3，RTV为0.00±0.00。与同期溶媒对照组相比，肿瘤体积和相对肿瘤体积在D3～D28期间均显著降低(p<0.01)。D52时，肿瘤体积均为0.00±0.00mm 3，RTV为0.00±0.00。与同期溶媒对照组相比，肿瘤体积和相对肿瘤体积在停药三周半内持续显著降低(p<0.01)。
以上数据表明，IMM40H体现了强大的抗肿瘤效果，在1.0mg/kg的剂量下肿瘤完全清除。
本申请的序列信息总结如下。
Figure PCTCN2022114942-appb-000004
Figure PCTCN2022114942-appb-000005
Figure PCTCN2022114942-appb-000006
Figure PCTCN2022114942-appb-000007
Figure PCTCN2022114942-appb-000008
Figure PCTCN2022114942-appb-000009
Figure PCTCN2022114942-appb-000010
尽管本申请已经结合一个或多个实施方式进行了描述，应当理解的是，本申请并不受限于这些实施方式。本申请中的描述意在涵盖所有变体形式以及等同物，均包含在所附权利要求的主旨和范围内。所有在本文中引用的文献通过引用的方式全部并入本文。
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Claims (16)

  1. 一种分离的单克隆抗体或其抗原结合部分，与CD70结合，包含重链可变区和轻链可变区，该重链可变区包含HV-CDR1区、HV-CDR2区和HV-CDR3区，该轻链可变区包含LV-CDR1区、LV-CDR2区和LV-CDR3区，其中HV-CDR1区、HV-CDR2区、HV-CDR3区、LV-CDR1区、LV-CDR2区和LV-CDR3区的氨基酸序列分别如SEQ ID NOs:1、2、3、4、5和6所示。
  2. 根据权利要求1所述的分离的单克隆抗体或其抗原结合部分，其中所述重链可变区包含SEQ ID NOs:7或9所示的氨基酸序列。
  3. 根据权利要求1所述的分离的单克隆抗体或其抗原结合部分，其中所述轻链可变区包含SEQ ID NOs:8、或10(X＝N或X＝E)所示的氨基酸序列。
  4. 根据权利要求2所述的分离的单克隆抗体或其抗原结合部分，其中所述重链可变区和所述轻链可变区分别包含i)SEQ ID NOs:7和8；ii)SEQ ID NOs:9和10(X＝N)；或iii)SEQ ID NOs:9和10(X＝E)所示的氨基酸序列。
  5. 根据权利要求1所述的分离的单克隆抗体或其抗原结合部分，其为IgG1、IgG2、或IgG4亚型。
  6. 根据权利要求1所述的分离的单克隆抗体或其抗原结合部分，包含重链恒定区和轻链恒定区，其中所述重链恒定区和轻链恒定区分别包含如SEQ ID NOs:11和12所示的氨基酸序列。
  7. 根据权利要求1所述的分离的单克隆抗体或其抗原结合部分，其为鼠源、嵌合或人源化抗体。
  8. 一种核酸分子，其编码权利要求1-7中任一项所述的分离的单克隆抗体或其抗原结合部分。
  9. 一种表达载体，其包含权利要求8所述的核酸分子。
  10. 一种宿主细胞，其包含权利要求9所述的表达载体。
  11. 一种药物组合物，其包含权利要求1-7中任一项所述的分离的单克隆抗 体或其抗原结合部分、权利要求8所述的核酸分子、权利要求9所述的表达载体、或权利要求10所述的宿主细胞，以及至少一种药学上可接受的载体。
  12. 权利要求11所述的药物组合物在制备用于治疗与CD70过表达相关的疾病的药物中的用途。
  13. 根据权利要求12所述的用途，其中所述疾病选自癌症、和炎性疾病。
  14. 根据权利要求13所述的用途，其中癌症为实体癌或血液癌。
  15. 根据权利要求14所述的用途，其中癌症选自肾癌、骨髓增生异常综合征、皮肤T细胞淋巴瘤、鼻咽癌、Epstein-Barr病毒诱导的癌症、恶性霍金奇淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、弥漫性大B细胞淋巴瘤、滤泡状淋巴瘤、B细胞淋巴细胞白血病、Burkitt淋巴瘤、多发性骨髓瘤、Waldenstrom巨球蛋白血症、鼻咽癌、胸腺癌、胶质母细胞瘤、脑癌、骨肉瘤、黑色素瘤、卵巢癌、肾细胞癌、乳腺癌、和非小细胞肺癌。
  16. 根据权利要求13所述的用途，其中炎性疾病选自关节炎、肠炎、和狼疮。
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