WO2022002019A1 - 抗cd70抗体及其应用 - Google Patents

抗cd70抗体及其应用 Download PDF

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WO2022002019A1
WO2022002019A1 PCT/CN2021/102998 CN2021102998W WO2022002019A1 WO 2022002019 A1 WO2022002019 A1 WO 2022002019A1 CN 2021102998 W CN2021102998 W CN 2021102998W WO 2022002019 A1 WO2022002019 A1 WO 2022002019A1
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seq
antibody
variable region
chain variable
heavy chain
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PCT/CN2021/102998
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English (en)
French (fr)
Inventor
孙乐
叶鑫
陈雨潇
金薪盛
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to JP2022577166A priority Critical patent/JP2023532197A/ja
Priority to CA3185846A priority patent/CA3185846A1/en
Priority to AU2021298632A priority patent/AU2021298632A1/en
Priority to US18/013,231 priority patent/US20230257472A1/en
Priority to CN202180037216.3A priority patent/CN115803053A/zh
Priority to KR1020237002483A priority patent/KR20230030632A/ko
Priority to EP21834252.5A priority patent/EP4173638A1/en
Publication of WO2022002019A1 publication Critical patent/WO2022002019A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure belongs to the field of biomedicine, and particularly relates to an antibody that binds to CD70 and its application.
  • CD70 is a cell surface antigen, a member of the tumor necrosis factor (TNF) family, a type II membrane protein containing 193 amino acids and a molecular weight of about 50kD. In vivo, it interacts with its receptor CD27 in a homotrimeric form, and then the intracellular domain of CD27 binds to tumor necrosis factor receptor-associated factors (TRAF), such as TRAF2 and TRAF5, to activate NF ⁇ B and JNK pathways that ultimately lead to pro-survival and proliferative signals.
  • TRAF2 and TRAF5 tumor necrosis factor receptor-associated factors
  • CD70-induced CD27 signaling results in increased generation and activation of CD27-expressing regulatory T cells.
  • CD70 also promotes tumor growth by evading immune surveillance by inducing regulatory T cells.
  • CD70 is transiently expressed on activated T cells, B cells and dendritic cells, but hardly expressed on non-lymphoid normal tissues.
  • CD70 is highly expressed in a variety of hematological and solid tumors, such as B-cell lymphoma, renal cancer, and breast cancer, and is negatively correlated with prognosis.
  • CD27 is co-expressed with CD70 in hematological tumors, and the combination of the two leads to the cleavage of the extracellular domain of CD27 to form soluble CD27 (sCD27), which can be used as a diagnostic biomarker.
  • the present disclosure provides a novel anti-CD70 antibody.
  • the anti-CD70 antibodies described in this disclosure include anti-CD70 full-length antibodies and antigen-binding fragments thereof.
  • the present disclosure provides an anti-CD70 antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1 and HCDR3 as set forth in SEQ ID NO: 9 and SEQ ID NO: 11, respectively, and HCDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 42;
  • the The light chain variable region comprises LCDR1 and LCDR3 as set forth in SEQ ID NO: 12 and SEQ ID NO: 14, respectively, and LCDR2 as set forth in SEQ ID NO: 13 or SEQ ID NO: 43;
  • the heavy chain variable region comprises HCDR1 and HCDR3 as set forth in SEQ ID NO: 15 and SEQ ID NO: 17, respectively, and HCDR2 as set forth in SEQ ID NO: 16 or SEQ ID NO: 54;
  • the The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or
  • the heavy chain variable region comprises HCDR1 and HCDR3 as shown in SEQ ID NO: 21 and SEQ ID NO: 23, respectively, and HCDR2 as shown in SEQ ID NO: 22 or SEQ ID NO: 71;
  • the The light chain variable region comprises LCDR1 and LCDR3 as set forth in SEQ ID NO:24 and SEQ ID NO:25, respectively, and LCDR2 as set forth in SEQ ID NO:13 or SEQ ID NO:43.
  • the anti-CD70 antibodies of the present disclosure comprise a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the light chain variable region comprises SEQ ID NO:1, respectively: 12. LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 13 and SEQ ID NO: 14; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:42 and SEQ ID NO:11, respectively, and the light chain variable region comprises HCDR1, respectively, as SEQ ID NO:11: 12. LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 43 and SEQ ID NO: 14; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the light chain variable region comprises SEQ ID NO: 17, respectively; 18. LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 19 and SEQ ID NO: 20; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 54 and SEQ ID NO: 17, respectively; the light chain variable region comprises SEQ ID NO: 17, respectively; 18. LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 19 and SEQ ID NO: 20; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; the light chain variable region comprises SEQ ID NO: 23, respectively: 24.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 71 and SEQ ID NO: 23, respectively; the light chain variable region comprises SEQ ID NO: 24. LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:43 and SEQ ID NO:25.
  • the aforementioned anti-CD70 antibody is a murine antibody, a chimeric antibody, or a humanized antibody; in some embodiments, the aforementioned anti-CD70 antibody is a full-length antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, F(ab') 2 , F(ab) 2 , Fd, Fv, dsFv, scFv, and diabodies thereof.
  • the aforementioned anti-CD70 antibody is a humanized antibody comprising a framework region of a human antibody or a framework region variant of a human antibody relative to the light chain of a human antibody
  • the framework regions and/or the heavy chain framework regions have up to 11 amino acid backmutations, respectively.
  • the heavy chain framework region of the antibody comprises one or more amino acid backmutations selected from the group consisting of 4M, 37I, 38K, 48I, 67A, 69L, 71A, 73R, 78A, 80L, and 94T, And/or the light chain framework region of the antibody comprises one or more amino acid back mutations selected from 5S and 70N; in some embodiments, the light chain framework region of the antibody comprises 38R, 43S , 69R, 70Q and 71Y one or more amino acid back-mutations, and/or the heavy chain framework region of the antibody comprises one or more amino acids selected from 2I, 24T, 46K, 72E and 82aN backmutations; in some embodiments, the antibody comprises one or more amino acid backmutations on the heavy chain framework region selected from 27D, 30P, 37L, 38K, 48I, 66K, 67A, 69L, and 82aN, And/or the light chain framework region of the antibody comprises a 49S amino acid back mutation;
  • the anti-CD70 antibody comprises a heavy chain framework region variant of a human antibody comprising, relative to the heavy chain framework region of the human antibody, selected from the group consisting of 4M, 37I, 38K, 48I, 67A, 69L, 71A, 73R one or more amino acid back-mutations in , 78A, 80L and 94T, and/or the anti-CD70 antibody comprises a light chain framework region variant of a human antibody comprising a light chain framework region variant selected from the group consisting of 5S relative to the light chain framework region of the human antibody and one or more amino acid backmutations in 70N; in some embodiments, the anti-CD70 antibody comprises a light chain framework region variant of a human antibody comprising a light chain framework region variant selected from the group consisting of 38R, One or more amino acid back-mutations in 43S, 69R, 70Q and 71Y, and/or the anti-CD70 antibody comprises a variant of the heavy chain framework region of a human antibody comprising a
  • the humanized antibody comprises a light chain variable region and a heavy chain variable region selected from the group consisting of a), b) or c) below:
  • the humanized antibody comprises:
  • variable region comprises a heavy chain framework region variant of a human antibody, which relative to the heavy chain framework region of a human antibody comprises a group selected from the group consisting of 4M, 37I, 38K, 48I, 67A, 69L, 71A, 73R, 78A, 80L and 94T
  • the light chain variable region comprises a light chain framework region variant of a human antibody comprising one or more selected from the group consisting of 5S and 70N relative to the light chain
  • a heavy chain variable region comprising HCDR1 and HCDR3 as set forth in SEQ ID NO: 15 and SEQ ID NO: 17, respectively, and HCDR2 as set forth in SEQ ID NO: 16 or SEQ ID NO: 54; and a light A chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; and the light chain variable region comprises the light chain framework of a human antibody Region variants comprising one or more amino acid backmutations selected from the group consisting of 38R, 43S, 69R, 70Q and 71Y relative to the light chain framework region of a human antibody, and/or the heavy chain variable region comprising a human antibody A variant of the heavy chain framework region comprising one or more amino acid backmutations selected from the group consisting of 2I, 24T, 46K, 72E and 82aN relative to the heavy chain framework region of a human antibody; or
  • variable region comprises a heavy chain framework region variant of a human antibody comprising one or more selected from the group consisting of 27D, 30P, 37L, 38K, 48I, 66K, 67A, 69L and 82aN relative to the heavy chain framework region of the human antibody Multiple amino acid backmutations, and/or the light chain variable region comprises a light chain framework region variant of a human antibody comprising a 49S amino acid backmutation relative to the light chain framework region of the human antibody.
  • the mutation site corresponds to the Kab
  • the heavy chain variable region of the aforementioned anti-CD70 antibody is selected from the group consisting of 4M, 37I, 38K, 48I, 67A, 69L, 71A, SEQ ID NO: 26 or SEQ ID NO: 34.
  • a heavy chain variable region in which one or more amino acids in 73R, 78A, 80L, and 94T are backmutated, and the light chain variable region has a selection based on SEQ ID NO:32 or SEQ ID NO:40.
  • the aforementioned anti-CD70 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the amino acid sequence of the heavy chain variable region has at least 90%, 91% with SEQ ID NO: 3, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38 or 39, respectively , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and/or the amino acid sequence of the light chain variable region and SEQ ID NO: 4, 32, 33, 40 or 41 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, respectively;
  • the amino acid sequence of the variable region of the heavy chain has at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 5, 44, 45, 46, 51, 52 or 53, respectively , 96%, 97%, 98%, 99% or 100% sequence identity
  • the amino acid sequence of the light chain variable region has at least SEQ ID NO: 6, 47, 48, 49 or 50, respectively 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or
  • the amino acid sequence of the heavy chain variable region has at least 90%, 91% with SEQ ID NO: 7, 55, 56, 57, 58, 59, 60, 63, 64, 65, 66, 67 or 68, respectively , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and/or the amino acid sequence of the light chain variable region and SEQ ID NO: 8, 61, 62, 69 or 70 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, respectively.
  • the aforementioned anti-CD70 antibodies comprise heavy chain variable region and light chain variable region combinations as set forth in Table 1, Table 2, and Table 3 below:
  • variable region of the heavy chain of the antibody is SEQ ID NO: 26 in the same row
  • variable region of the light chain is SEQ ID NO: 40 in the same column
  • variable region of the heavy chain of the antibody is SEQ ID NO: 44 in the same row
  • variable region of the light chain is SEQ ID NO: 47 in the same column
  • variable region of the heavy chain of the antibody is SEQ ID NO: 55 in the same row
  • variable region of the light chain is SEQ ID NO: 69 in the same column, and so on.
  • the aforementioned anti-CD70 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region the amino acid sequence of which is shown in SEQ ID NO: 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38 or 39; and the light chain may be A variable region whose amino acid sequence is shown in SEQ ID NO: 32, 33, 40 or 41; or
  • the heavy chain variable region the amino acid sequence of which is shown in SEQ ID NO: 55, 56, 57, 58, 59, 60, 63, 64, 65, 66, 67 or 68; and the light chain can be The variable region, the amino acid sequence of which is shown in SEQ ID NO: 61, 62, 69 or 70.
  • the aforementioned anti-CD70 antibody comprises a heavy chain variable region and a light chain variable region as shown below:
  • the aforementioned anti-CD70 antibody comprises an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from the group consisting of human IgGl, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof,
  • the light chain constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof; more preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO:72 and a heavy chain constant region as shown in SEQ ID NO:73 Light chain constant region shown.
  • the aforementioned anti-CD70 antibody comprises:
  • the aforementioned anti-CD70 antibody comprises:
  • the present disclosure also provides an isolated anti-CD70 antibody, wherein the antibody competes with the anti-CD70 antibody of any of the foregoing for binding to human CD70, a human CD70 epitope, monkey CD70, or monkey CD70 antigenic epitopes. In some embodiments, the antibody binds the same epitope on human CD70 as the anti-CD70 antibody of any of the foregoing.
  • the anti-CD70 antibody of any of the preceding wherein the anti-CD70 antibody is a hypofucosylated antibody; in some embodiments, the hypofucosylated anti-CD70 antibody Are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the antibody heavy chains are free of fucose sugars Modified antibodies.
  • the hypofucosylated anti-CD70 antibody is an antibody whose heavy chain is at least 95%, 96%, 97%, 98%, 99%, or 100% unmodified by fucosylation.
  • the aforementioned anti-CD70 antibody is an IgG1 antibody whose 100% heavy chain is not modified by fucosylated glycosylation (also referred to as afucosylated IgG1 antibody).
  • the anti-CD70 antibody of any one of the preceding, the anti-CD70 antibody has at least one of the following characteristics:
  • the anti-CD70 antibody is less than 1 x 10-8 M, preferably less than 1 x 10 -9 M, or less than 1 x 10 -10 M, or less than 6 x 10 -11 M, or less than 5 x 10 -11 M, or a KD value of less than 4 ⁇ 10 ⁇ 11 M, or less than 3 ⁇ 10 ⁇ 11 M, binds to human CD70 as determined by surface plasmon resonance; for example, as described in Test Example 1 of the present disclosure method to detect;
  • the anti-CD70 antibody can bind to both human CD70 antigen and monkey CD70 antigen, but not to mouse CD70 antigen;
  • the anti-CD70 antibody can inhibit CD70-induced CD27 signaling, preferably, the anti-CD70 antibody inhibits the maximum percentage inhibition (Imax(%)) of IL-8 secretion by human CD27-expressing cells (eg, HT1080/CD27 cells) Greater than or equal to 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100%; more preferably greater than or equal to 90%, 91%, 93% or 98%, the IL-8 secretion is detected by the Elisa method, for example detected by the method described in Test Example 5 of the present disclosure;
  • the anti-CD70 antibody has one or more of the following effector functions: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cytotoxicity to cells expressing human CD70 Cell-mediated phagocytosis (ADCP); preferably, the anti-CD70 antibody has a maximal lysis rate greater than or equal to 70%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, or 100 %; more preferably greater than or equal to 74%, 78%, 84% or 86%; in some scenarios, CDC effector function is detected by the method described in Test Example 7 of the present disclosure; and
  • the anti-CD70 antibody can be internalized by cells expressing human CD70, preferably, the maximum lysis rate of cell internalization lysis is greater than or equal to 70%, 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; more preferably greater than or equal to 96% or 97%; in some embodiments, cellular internalization passes the test cases of the present disclosure 10 methods for detection.
  • the present disclosure also provides a nucleic acid molecule encoding the anti-CD70 antibody of any of the foregoing.
  • the present disclosure also provides a host cell comprising the aforementioned nucleic acid molecule.
  • the host cell can be selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells, preferably excluding human mammalian cells, wherein the mammalian cells include but are not limited to CHO, 293, NSO and gene editing in mammalian cells can alter the glycosylation modification of the antibody or its antigen-binding fragment, thereby altering the ADCC function of the antibody or its antigen-binding fragment, for example, knockout genes such as Fut8 or GnT-III for Glycosylation modification.
  • the present disclosure also provides a method for preparing the aforementioned anti-CD70 antibody, the method comprising the steps of culturing the aforementioned host cell, and then purifying and recovering the antibody.
  • the present disclosure also provides an immunoconjugate comprising the anti-CD70 antibody of any of the foregoing and an effector molecule coupled to the anti-CD70 antibody; preferably, the The effector molecule is selected from the group consisting of radioisotopes, antineoplastic agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the anti-CD70 antibody of any of the foregoing, or the foregoing nucleic acid molecule, or the foregoing immunoconjugate, and a or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure also provides a method for immunodetection or determination of CD70, the method comprising the step of contacting the anti-CD70 antibody of any of the foregoing with a subject or a sample from the subject .
  • the present disclosure also provides a kit comprising the anti-CD70 antibody or immunoconjugate of any of the foregoing.
  • the present disclosure also provides a method of preventing or treating a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of the anti-CD70 antibody of any of the foregoing, or the foregoing nucleic acid molecule , or the aforementioned pharmaceutical composition, or the aforementioned immunoconjugate.
  • the present disclosure also provides the anti-CD70 antibody described in any of the foregoing, or the foregoing nucleic acid molecule, or the foregoing pharmaceutical composition, or the foregoing immunoconjugate prepared for use in preventing or treating a disease or use in medicines for disorders.
  • the present disclosure provides the anti-CD70 antibody of any of the foregoing, or the foregoing nucleic acid molecule, or the foregoing pharmaceutical composition, or the foregoing immunoconjugate, as a medicament for use in To prevent or treat a disease or condition.
  • the disease or disorder of any of the foregoing is a CD70-related disease or disorder.
  • the disease or disorder is a disease or disorder in which high CD70 expression is detrimental to the subject.
  • the disease or disorder is a tumor, an autoimmune disease, or an infectious disease.
  • the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system Systemic cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, Chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Keukenberg's tumor, myeloproliferative
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriasis, joint psoriasis, psoriasis, dermatitis, systemic scleroderma, systemic scleroderma and sclerosis, inflammatory Intestinal disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, arteriosclerosis, leukocyte adhesion deficiency, multiple sclerosis Symptoms of immune-mediated thrombocytopenia (eg, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura idiopathic thrombocytopenic purpura), hemolytic anemia, myasthenia gravis, lupus
  • the disease or disorder is: acute myeloid leukemia, myelodysplastic syndrome, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, renal cell carcinoma, metastatic renal cell carcinoma, rheumatoid arthritis, and psoriasis.
  • the aforementioned therapeutically effective amount of the composition in a unit dose contains 0.1-3000 mg of the aforementioned anti-CD70 antibody, or the aforementioned nucleic acid molecule, or the aforementioned immunoconjugate, or the aforementioned pharmaceutical composition.
  • the treatment further comprises administering to the subject a therapeutically effective amount of a second therapeutic agent.
  • Figure 1A and Figure 1B show the experimental results of anti-CD70 antibody binding to CD70-positive 786-O cells;
  • Figure 1A shows the experimental results of anti-CD70 antibody binding to CD70-positive 786-O cells, and
  • Figure 1B is a non-fucosylated antibody Figure of the experimental results of CD70 antibody binding to CD70-positive 786-O cells.
  • Figures 2A and 2B show the experimental results of anti-CD70 antibody binding to CD70-positive Raji cells, wherein Figure 2A is the experimental results of anti-CD70 antibody binding to CD70-positive Raji cells, and Figure 2B is the binding of afucosylated anti-CD70 antibody to CD70-positive Experimental results of Raji cells.
  • Figure 3 shows the results of ELISA experiments of huB7002 binding to human, monkey and mouse CD70 proteins.
  • Figure 4 shows the results of ELISA experiments of huB1010 binding to human, monkey and mouse CD70 proteins.
  • Figure 5 shows the results of ELISA experiments of huF4011 binding to human, monkey and mouse CD70 proteins.
  • Figure 6 shows the experimental results of anti-CD70 antibody blocking CD27 binding to CD70 positive cells.
  • Figure 7 shows the results of experiments in which afucosylated anti-CD70 antibodies blocked CD27 binding to CD70 positive cells.
  • Figure 8 shows the results of the inhibition experiment of anti-CD70 antibody on IL-8 secretion of HT1080/CD27 cells.
  • Figure 9 shows the results of the inhibition experiment of afucosylated anti-CD70 antibody on IL-8 secretion by HT1080/CD27 cells.
  • Figure 10 shows the experimental results of anti-CD70 antibody on ADCC (NK92) of 786-O cells in vitro.
  • FIG 11 shows the experimental results of anti-CD70 antibody on ADCC (PBMC) of 786-O cells in vitro.
  • Figure 12 shows the experimental results of anti-CD70 antibody on Raji cell CDC in vitro.
  • Figure 13 shows the experimental results of in vitro CDC of Raji cells by afucosylated anti-CD70 antibody.
  • Figures 14A and 14B show the experimental results of in vitro ADCP of anti-CD70 antibody on 786-O and Raji cells, wherein Figure 14A is a graph of the ADCP experimental results of anti-CD70 antibody on 786-O cells, and Figure 14B is a graph of anti-CD70 antibody on Raji cells The results of ADCP experiments on cells.
  • Figure 15 shows the experimental results of the inhibition of Treg cells by anti-CD70 antibodies in vitro.
  • Figure 16 shows the results of internalization experiments of anti-CD70 antibodies in 786-O cells.
  • Figure 17 shows the results of in vivo efficacy experiments of anti-CD70 antibodies in mouse Raji model.
  • Figure 18 shows the results of in vivo efficacy experiments of afucosylated anti-CD70 antibodies in the mouse Raji model.
  • antibody in this disclosure is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies or antigen binding thereof Fragments (also referred to as "antigen-binding portions”) so long as they exhibit the desired antigen-binding activity.
  • Natural full-length antibodies are immunoglobulins (Ig) comprising at least two heavy chains and two light chains interconnected by disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region contains three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • the heavy and light chain variable regions include hypervariable regions (also called complementarity determining regions, abbreviated as CDRs or HVRs) and framework regions (also called framework regions, abbreviated as FRs) with relatively conserved sequences.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VL and VH consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies.
  • murine antibody in this disclosure is a murine monoclonal antibody directed against an antigen (eg, human CD70) prepared according to knowledge and skill in the art. For example, subjects are injected with the CD70 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-CD70 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a murine IgG1 , the heavy chain constant region of IgG2, IgG3 or variants thereof.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • a chimeric antibody first create a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the mouse variable region gene from the mouse hybridoma cell.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the CD70 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably comprises a human IgG1, IgG2 or IgG4 heavy chain constant region, or uses amino acid mutation (eg L234A and/or L235A mutation, and/or S228P mutation, 265A and/or 297A) IgG1, IgG2 or IgG4 variants.
  • amino acid mutation eg L234A and/or L235A mutation, and/or S228P mutation, 265A and/or 297A
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody antibodies produced in framework sequences.
  • the heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy and light chain variable region genes can be obtained in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the minimum reverse mutation or back mutation can be performed on the framework sequence of the variable region of the human antibody to maintain or enhance the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further subjected to affinity maturation mutation of the CDRs by yeast display.
  • the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine ⁇ , ⁇ chains or variants thereof, or further comprise human or murine IgG1 , heavy chain constant regions of IgG2, IgG3, IgG4 or variants thereof; may comprise human IgG1, IgG2 or IgG4 heavy chain constant regions, or use amino acid mutations (e.g. L234A and/or L235A mutation, and/or S228P mutation, 265A and/or IgG1, IgG2 or IgG4 variants of 297A).
  • amino acid mutations e.g. L234A and/or L235A mutation, and/or S228P mutation, 265A and/or IgG1, IgG2 or IgG4 variants of 297A.
  • inventions of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region
  • exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed reengineering and amino acid substitutions of the heavy chain constant region, specifically replacing YTE as known in the art Mutations, L234A and/or L235A mutations, S228P mutations, 265A (eg D265A) and/or 297A (eg N297A), and/or mutations to obtain a knob-into-hole structure (so that the antibody heavy chain has knob-Fc and hole- Fc combination), these mutations have been shown to confer novel properties of the antibody without altering the function of the variable region of the antibody.
  • Human antibody (HuMAb), "human antibody”, “fully human antibody”, “fully human antibody” are used interchangeably and can be an antibody derived from a human or an antibody obtained from a transgenic organism,
  • the transgenic organism is "engineered” to produce specific human antibodies in response to antigenic stimulation and can be produced by any method known in the art.
  • elemental elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines in which endogenous heavy and light chain loci are targeted to destruction.
  • Transgenic organisms can synthesize human antibodies specific for human antigens, and the organisms can be used to generate human antibody-secreting hybridomas.
  • a human antibody can also be one in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources.
  • Fully human antibodies can also be constructed by gene or chromosomal transfection methods and phage display techniques, or by in vitro activated B cells, all of which are known in the art.
  • full-length antibody intact antibody
  • complete antibody completely antibody
  • whole antibody whole antibody
  • antibodies of the present disclosure include “full-length antibodies” and “antigen-binding fragments” thereof.
  • the full-length antibody of the present disclosure includes a full-length antibody formed by the combination of light and heavy chain variable regions in Table 1, Table 2, and Table 3, respectively, linked to the light and heavy chain constant regions.
  • Those skilled in the art can select light chain constant regions and heavy chain constant regions derived from different antibodies according to actual needs, such as light chain constant regions and heavy chain constant regions derived from human antibodies.
  • the different light chain variable regions and heavy chain variable regions in Table 1, Table 2 and Table 3 can be combined to form single chain antibodies (scFv), Fab or other antigen binding fragments comprising scFv or Fab.
  • antigen-binding fragment or “functional fragment” or “antigen-binding portion” refers to one or more fragments of an intact antibody that retain the ability to specifically bind an antigen (eg, CD70). Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment” include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) a VH and Fv fragment composed of VL domains; (v) dsFv, a stable antigen-binding fragment formed by VH and VL via interchain disulfide bonds; (vi) diabodies and bispecific antibodies comprising scFv, dsFv, Fab and other fragments and multispecific antibodies.
  • Antigen binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • amino acid difference or “amino acid mutation” refers to an amino acid change or mutation in a variant protein or polypeptide compared to the original protein or polypeptide, including 1, 2, 3, or Insertion, deletion or substitution of more amino acids.
  • antibody framework or "FR region” refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • VH variable domain
  • VL variable domain
  • CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and amino acids in VL Residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 in human VH (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers in VL are approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align.
  • the CDR amino acids in VH are numbered 26-32 (HCDR1), 50-58 ( HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2 ) and 89-97 (LCDR3).
  • the antibody variable region and CDR sequences of the present disclosure correspond to the "Kabat" numbering convention.
  • epitopes refers to a site on an antigen to which an antibody specifically binds (eg, a specific site on a CD70 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody is less than about 10 -8 M, e.g. less than about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or smaller affinity (KD) binding.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind CD70 with a dissociation equilibrium constant (KD) of less than about 10-8 M, eg, less than about 10-9 M or 10-10 M, using surface plasmon resonance (SPR) technology Measured in a Biacore T200 instrument.
  • SPR surface plasmon resonance
  • the term “compete” in the context of antigen-binding proteins competing for the same epitope (eg, neutralizing antigen-binding proteins or neutralizing antibodies), it means competition between antigen-binding proteins, as determined by the following assay:
  • the antigen binding protein eg, antibody or immunologically functional fragment thereof
  • the antigen binding protein prevents or inhibits (eg reduces) the interaction of a reference antigen binding protein (eg ligand or reference antibody) with a common antigen (eg CD70 antigen or specific binding of its fragments).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, eg, Kirkland et al., 1986, J. Immunol. 137:3614-3619
  • solid phase Direct Labeling Assay Solid Phase Direct Labeling Sandwich Assay (see e.g.
  • Solid Phase Direct Labeling with I-125 Label RIA see, eg, Morel et al., 1988, Molec. Immunol. 25:7-15
  • solid-phase direct biotin-avidin EIA see, eg, Cheung, et al., 1990, Virology 176:546-552
  • directly labeled RIA Methyl et al., 1990, Scand. J. Immunol. 32:77-82
  • the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Antigen-binding proteins identified by competitive assays include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen-binding protein protein, the two epitopes sterically prevent each other from binding. Additional details regarding methods for determining competitive binding are provided in the Examples herein.
  • a competing antigen binding protein when present in excess, it will inhibit (eg decrease) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more specific binding of the reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • amino acid sequence identity means that when aligning amino acid sequences (gaps are introduced where necessary to achieve maximum percent sequence identity, and any conservative substitutions are not considered part of sequence identity), in a first sequence with a second sequence The percentage of amino acid residues in which the amino acid residues are identical.
  • alignment can be accomplished in a variety of ways known in the art, eg, using software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR).
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • fucosylated or "fucosylated” or “fucosylated modification” refers to the presence of a fucose residue within the oligosaccharide attached to the peptide chain of an antibody. Fucosylation is a common process of post-translational modification of glycoproteins. Representative enzyme genes related to core fucosylation include GMD (GDP-mannose 4,6-dehydratase) gene and Fut8 (fut8, FUT8) , ⁇ -1,6-fucosyltransferase) gene, the core fucosylation level can be effectively regulated by inhibiting the expression of these two genes or constructing Fut8 knockout CHO host cells (YAMANE-OHNUKI et al.
  • fucosylated antibodies comprise in the N-oligosaccharide of the Fc region, at a core N-acetylglucosamine (GlcNAc) residue (eg, position Asn297 of a human IgG1 Fc (corresponding to the EU numbering convention)) Alpha-1,6-fucose.
  • GlcNAc N-acetylglucosamine
  • an antibody that is "hypofucosylated” refers to an antibody in which the carbohydrate structure attached to the Fc region has a low glycosylation modification of fucose; "afucosylated” or “afucosylated” “refers to an antibody in which the carbohydrate structure attached to the Fc region lacks the glycosylation modification of fucose.
  • Antibody fucosylation levels can be determined by methods known in the art for all oligosaccharides to determine the percentage of fucosylated oligosaccharides. Methods known in the art for determining fucosylation include, but are not limited to, gel electrophoresis, liquid chromatography, mass spectrometry, and the like.
  • the level of fucosylation of the antibody is determined by hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC), eg by treatment of the sample with peptide-N-glycanase F to denature To cleave N-linked glycans, the N-linked glycans were then analyzed for fucose content.
  • hydrophilic interaction chromatography or hydrophilic interaction liquid chromatography, HILIC
  • the hypofucosylated antibodies of the present disclosure are antibodies in which at least 80% of the heavy chains are not modified by glycosylation of fucose, eg, at least 80-95%, 90-95% %, 95-100%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the antibody heavy chain is fucose free Glycosylation modification.
  • fucose eg. at least 80-95%, 90-95% %, 95-100%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the antibody heavy chain is fucose free Glycosylation modification.
  • fucose eg.g, at least 80-95%, 90-95% %, 95-100%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the antibody heavy chain is fucos
  • Hypofucosylated or afucosylated antibodies can be prepared by methods well known in the art. Prepared, for example, by adding, removing or deleting one or more carbohydrate moieties present on the antibody, eg, by cleavage of fucose residues of the antibody using fucosidase (see Tarentino et al. (1975) Biochem. 14:5516). Antibodies with reduced fucosylation can also be prepared by altering the glycosylation composition by altering the level of glycosylation, for example by modifying the glycan module attached to each Fc fragment at residue N297 (Natsume et al. (2009) Drug Des. Devel. Ther. 3:7).
  • Hypofucosylated or afucosylated antibodies can also be expressed without altering the sequence of the antibody, e.g., by cells that alter the glycosylation pattern of the antibody, including, e.g., by genetically engineered glycosylation engineering.
  • Various glycosylated engineered cells have been disclosed in the art, for example, cells lacking the fucosyltransferase gene (FUT8, ( ⁇ -(1,6)fucosyltransferase) cell lines Ms704, Ms705, and Ms709, etc.
  • carrying an enzyme encoding an enzyme that uses GDP-6-deoxy-D-lyxo-4-hexose as a substrate can also produce hypofucosylated or afucosylated antibodies (see US Pat. No. 8,642,292).
  • the afucosylated antibody was prepared by the method described in Example 5.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR-expressing non-specific cytotoxic cells eg, natural killer (NK) cells, neutrophils, and macrophages
  • NK cells Primary cells that regulate ADCC
  • monocytes express FcyRI, FcyRII and FCYRIII.
  • ADCC assays can be performed (such as described by Clynes et al. (PNASUSA 95:652-656 (1998)), US Pat. Nos. US5500362, US5821337, etc.).
  • the ADCC is detected by the method of Test Example 6 of the present disclosure.
  • ADCP antibody-dependent cellular phagocytosis
  • phagocytic cells eg, macrophages, neutrophils, and dendritic cells
  • Internalized antibody-coated target cells or virions are contained in vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes.
  • ADCP can be assessed by in vitro cytotoxicity assays and videomicroscopy using macrophages as effector cells (eg, van Bij et al. Journal of Hepatology Vol 53, Issue 4, October 2010, pp. 677–685 ).
  • the ADCP is detected by the method of Test Example 8 of the present disclosure.
  • CDC complement-dependent cytotoxicity
  • in vitro assays eg, using normal human serum as a complement source for CDC
  • C1q concentration series e.g., C1q concentration series
  • the assay can be performed by an assay as described by Romeuf et al. (Romeuf et al., Br J Haematol. 2008 Mar; 140(6):635-43).
  • the CDC is detected by the method of Test Example 7 of the present disclosure.
  • conjugate refers to a new type of drug in which a ligand is linked to a biologically active drug through a stable linking unit.
  • antibody drug conjugate antibody drug conjugate, ADC
  • ADC antibody drug conjugate
  • the antibody can be conjugated to the drug either directly or via a linker.
  • the average number of drug moieties per antibody which can range, for example, from about 0 to about 20 drug moieties per antibody, in some embodiments 1 to about 10 drug moieties per antibody, in certain embodiments is 1 to about 8 drug moieties per antibody.
  • Compositions of mixtures of antibody-drug conjugates of the present disclosure wherein the average drug loading per antibody is from about 2 to about 5 or from about 3 to about 4.
  • an immunoconjugate disclosed herein can be an antibody attached to an effector molecule, wherein the antibody can be an antibody comprising a heavy chain and a light chain.
  • the antibody may be an antibody fragment, such as a Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, minibody, diabody, bispecific antibody fragment, Multimers, and any combination thereof.
  • the effector molecule can be a radioisotope, antineoplastic agent, immunomodulatory agent, biological response modifier, lectin, cytotoxic drug, chromophore, fluorophore, chemiluminescent compound, enzyme, metal ions, and any combination thereof.
  • the antibodies or antibody fragments described in this disclosure can be coupled to effector molecules by any means.
  • the antibody or antibody fragment can be attached to the toxin by chemical or recombinant means.
  • Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare immunoconjugates.
  • the method for conjugating the antibody or antibody fragment and the toxin must be capable of linking the antibody to the toxin without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.
  • cytotoxic drug refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Including toxins, chemotherapy drugs and other compounds that can be used to kill tumor cells.
  • toxin refers to any substance capable of having a deleterious effect on the growth or proliferation of cells, which may be small molecule toxins from bacteria, fungi, plants or animals and their derivatives, including camptothecin derivatives such as ixatib Kang, maytansinoids and derivatives thereof (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and derivatives thereof, such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria Toxins, exotoxins, ricin (ricin) A chain, abrin (abrin) A chain, modeccin, ⁇ -brucellin (sarcin), aleutites fordii toxin, carnation (dianthin) ) Toxin, Pokeweed (Phytolaca americana) Toxin (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Soa
  • chemotherapeutic drug is a chemical compound that can be used to treat tumors. This definition also includes antihormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic therapy. They can themselves be hormones.
  • chemotherapeutic drugs include alkylating agents such as thiotepa; cyclosphamide (CYTOXAN TM ); alkyl sulfonates such as busulfan, improsulfan and piperidine piposulfan; aziridines such as benaodopa, carboquone, meturedopa and uredopa; aziridine and methylamelamine include Altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil , naphthalene mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxazolam hydrochloride; melphalan, new Nitrogen mustard (novembichin), cholesteryl phenylacetate mustard, prednimustine, trofosfamide, uracil mustard; nitrosureas such
  • anti-hormonal agents that modulate or inhibit the effect of hormones on tumors, such as anti-estrogen agents including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene (trioxifene), keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide (flutamide), nilutamide ( nilutamide), bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.
  • anti-estrogen agents including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene (trioxifene), keoxifene, LY117018, onapristone, and torem
  • both the antibody and the toxin are proteins and can be conjugated using techniques well known in the art.
  • crosslinkers There are hundreds of crosslinkers disclosed in the art that can couple two proteins. Crosslinkers are generally selected based on reactive functional groups available or inserted on the antibody or toxin. Additionally, if no reactive groups are present, a photoactivatable crosslinker can be used. In some cases, it may be desirable to include a spacer between the antibody and the toxin.
  • Crosslinking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyl adipimide, and bis(diazobenzidine), and heterobifunctional agents: m-maleimide Benzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.
  • Crosslinkers that can be used to couple effector molecules to antibody fragments include, for example, TPCH (S-(2-thiopyridyl)-L-cysteine hydrazide) and TPMPH (S-(2-thiopyridyl) ) mercapto-propionic hydrazide).
  • TPCH and TPMPH react on the carbohydrate portion of the glycoprotein that has previously been oxidized by mild periodate treatment, thereby forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodate-generated aldehyde.
  • Heterobifunctional crosslinkers GMBS N-( ⁇ -maleimidobutyryloxy)-succinimide
  • SMCC succinimidyl 4-(N-maleimido- Methyl)cyclohexane
  • GMBS N-( ⁇ -maleimidobutyryloxy)-succinimide
  • SMCC succinimidyl 4-(N-maleimido- Methyl)cyclohexane
  • a cross-linking agent can be used to introduce a long spacer arm between the components, such as 3-(2-pyridyldithio)propionic acid n - Succinimidyl ester (SPDP).
  • SPDP 3-(2-pyridyldithio)propionic acid n - Succinimidyl ester
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • mice can be immunized with human CD70 or a fragment thereof, and the resulting antibody can be renatured, purified, and amino acid sequenced using conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the disclosed antibodies or antigen-binding fragments are genetically engineered to add one or more human FR regions to non-human CDR regions.
  • Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells and NSO cells. To obtain afucosylated antibodies, Glu1 and Fut8 gene knockout host cells can be used, including but not limited to Glu1 and Fut8 gene knockout CHOK1 cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind human CD70. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment as it applies to human, veterinary or research subjects, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, eg, a composition comprising any of the binding compounds of the present disclosure, to a patient having one or more disease symptoms for which the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are as follows:
  • Trp(W) Tyr Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu
  • an “effective amount” or “effective dose” refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of disorders, including biochemical, tissue academic and/or behavioral symptoms.
  • beneficial or desired outcomes include clinical outcomes, such as reducing the incidence or amelioration of one or more symptoms of the various target antigen-related disorders of the present disclosure, reducing the amount of other agents required to treat the disorder dose, enhances the efficacy of another agent, and/or delays the progression of a disorder associated with the target antigen of the present disclosure in a patient.
  • Exogenous refers to a substance produced outside an organism, cell, or human body, as the case may be.
  • Endogenous refers to a substance produced in a cell, organism, or human body as the case may be.
  • Homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position . The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100.
  • sequences when sequences are optimally aligned, two sequences are 60% homologous if 6 matches or homology at 10 positions in the two sequences; if 95 matches at 100 positions in the two sequences or homologous, then the two sequences are 95% homologous.
  • comparisons are made when aligning two sequences to give the greatest percent homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are chosen to give the maximum match between the respective sequences over the entire length of the respective reference sequences.
  • the following references refer to BLAST algorithms frequently used in sequence analysis: BLAST Algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.
  • Polymerase chain reaction or "PCR” as used herein refers to a procedure or technique in which traces of a specified portion of nucleic acid, RNA and/or DNA are amplified as described in, eg, US Pat. No. 4,683,195. Generally, sequence information from the end of the target region or beyond is required to allow the design of oligonucleotide primers; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5'-terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Generate a specific portion of nucleic acid.
  • isolated refers to a purified state, and in this case means that the named molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as a physiologically/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of a drug such as an anti-CD70 antibody or antigen-binding fragment described herein.
  • the carrier can be an antiadherent, binder, coating, disintegrant, filler or diluent, preservative (eg, antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent agents, emulsifiers, buffers, etc.
  • suitable pharmaceutically acceptable carriers include water, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oils (eg, olive oil), saline, buffers, buffered saline, and the like Osmotic agents such as sugars, polyols, sorbitol and sodium chloride.
  • the present disclosure includes an agent for treating a disease associated with a target antigen (eg, CD70) positive cell comprising the anti-CD70 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.
  • the active ingredient is administered to a subject in a therapeutically effective amount to treat a disease associated with CD70 positive cells in the subject.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg of an antibody that specifically binds to human CD70 as previously described.
  • the disease or disorder associated with CD70 in the present disclosure is not limited as long as it is a disease or disorder associated with CD70.
  • the molecules of the present disclosure are very useful for some of the following diseases or disorders that express CD70: eg, rheumatoid arthritis, autoimmune demyelinating diseases (eg, multiple sclerosis, allergy encephalomyelitis), endocrine eye disease, uveretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatological disease, inflammatory bowel disease (eg, Crohn's disease) disease, ulcerative colitis, celiac disease), anaphylaxis, allergic reactions, Sjogren's syndrome, type I diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis , dermatomyositis
  • the molecules of the present disclosure are useful for some of the following CD70-expressing diseases (eg, cancer), including kidney cancer (eg, renal cell carcinoma), breast cancer, brain tumors, chronic or acute leukemia (including acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphomas (eg, Hodgkin lymphoma and non-Hodgkin lymphoma, lymphocytic lymphoma, primary CNS lymphoma tumor, T-cell lymphoma), nasopharyngeal cancer, melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck , skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anus, stomach cancer, testicular cancer, uterine cancer, fallopian
  • pairs of molecules of the present disclosure are characterized by the presence of tumor cells expressing CD70, including, for example, renal cell carcinoma (RCC) such as clear cell RCC, glioblastoma, breast cancer, brain tumor, Nasopharyngeal (nasopharyngal) carcinoma, non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, anaplastic large cell lymphoma (ALCL), multiple myeloma, cutaneous T-cell lymphoma, nodular cleft cell lymphoma, lymphocytic lymphoma, peripheral T-cell lymphoma, Lennert lymphoma, immunoblastic lymphoma, T-cell leukemia/lymphoma (ATLL) , adult T-cell leukemia (T-ALL), centroblastic/centrocytic (cb/cc) follicular lymphomas cancers
  • the present disclosure relates to methods for immunodetection or assay of a target antigen (eg CD70), reagents for immunodetection or assay of a target antigen (eg CD70), for immunodetection or assay of cells expressing a target antigen (eg CD70)
  • a method and a diagnostic agent for diagnosing a disease associated with a target antigen (eg, CD70) positive cell comprising the present disclosure that specifically recognizes the target antigen (eg, human CD70) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure the antibody or antibody fragment as the active ingredient.
  • the method for detecting or determining the amount of the target antigen can be any known method.
  • the target antigen eg, CD70
  • the method for detecting or determining the amount of the target antigen can be any known method.
  • immunoassays or assays can be any known method.
  • An immunodetection or assay method is a method for detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or assay methods include radioactive substance-labeled immunoantibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescence immunoassays, Western blotting, physicochemical methods Wait.
  • CD70-positive cells eg, high-expressing CD70 cells
  • Diseases associated with CD70-positive cells can be diagnosed by detecting or assaying CD70-expressing cells with the antibodies or antibody fragments of the present disclosure.
  • immunodetection methods In order to detect the cells expressing the polypeptide, known immunodetection methods can be used, and preferably, immunoprecipitation methods, fluorescent cell staining methods, immunohistochemical staining methods, and the like are used. In addition, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) and the like can be used.
  • the biological sample for detecting or measuring the target antigen eg CD70
  • the target antigen eg CD70
  • cells expressing the target antigen eg CD70
  • tissue cells blood, plasma , serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • Reagents for performing antigen-antibody reactions include buffers, salts, and the like.
  • Reagents for detection include those commonly used in immunodetection or assay methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label, and the like.
  • the amino acid sequence of the antigen and detection protein used in the present disclosure was designed, and different tags such as His tag or Fc were optionally fused on the basis of the CD70 protein. Wait. They were respectively cloned into pTT5 vector (Biovector, CAT#102762), transiently expressed in 293 cells, and purified to obtain the antigen and detection protein of the present disclosure.
  • His-tagged CD70 protein extracellular domain (abbreviation: His-TNC-CD70) sequence, as a detection reagent;
  • the underlined part is the 6 ⁇ His tag
  • the italic part is the TNC tag
  • the rest is the extracellular domain of CD70 protein.
  • the underlined part is the Human-IgG1-Fc part, and the ununderlined part is the CD70 protein extracellular domain.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse 2-5 column volumes. The replaced cell supernatant sample was applied to a Ni Sepharose excel column (GE, 17-3712-02). The column was rinsed with 5 mM imidazole in PBS until the A280 reading dropped to baseline. Then, the column was washed with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and the effluent was collected.
  • Ni Sepharose excel column GE, 17-3712-02
  • the target protein was eluted with a PBS solution containing 300 mM imidazole, and the elution peaks were collected.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE, 28-9893-35), and the mobile phase was PBS. Aggregate peaks were removed and eluted peaks were collected.
  • the obtained protein was identified as correct by electrophoresis, peptide map and LC-MS. His-tagged His-TNC-CD70 was obtained and used as a detection reagent for the antibody of the present disclosure.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography.
  • the MabSelect Sure column was first regenerated with 0.2M NaOH, rinsed with pure water and equilibrated with PBS. After combining the supernatants, washed with PBS until the A280 reading dropped to the baseline.
  • the target protein was eluted with 0.1M acetate buffer at pH 3.5 and neutralized with 1M Tris-HCl.
  • the eluted samples were properly concentrated and further purified by gel chromatography Superdex200 (GE, 28-9893-35) equilibrated with PBS, and then concentrated to an appropriate concentration in the receiving tube where the target protein was collected.
  • This method was used to purify the CD70-Fc fusion protein, and this method can also be used to purify the antibody proteins of this disclosure.
  • Anti-human CD70 antibodies were produced by immunizing mice.
  • Balb/c white mice were used in the experiment, female, 6-8 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Breeding environment SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, the temperature was 20-25°C, and the humidity was 40-60%. The acclimated mice were immunized according to the following protocol.
  • the protein antigen was emulsified and then inoculated, and the cell antigen was resuspended in a phosphate buffer solution for inoculation.
  • the inoculation time was 0, 14, 28, 42, 56 and 70 days
  • blood was collected on the 21st, 49th and 82nd days
  • the antibody titer in the mouse serum was determined by ELISA method. Mice with high antibody titers in serum and titers tending to a plateau were selected, and their spleens were used to establish immune pools.
  • the primers for constructing the library were designed and synthesized according to the IMGT database (Goldwisdom Corporation). Through three rounds of PCR reactions, single-chain antibody fragments were obtained. All PCR reactions used LA Tag (Takara Cat No. RR02MB).
  • the first round of PCR uses cDNA as the template to amplify the heavy chain variable region and light chain variable region sequences respectively; the second round of PCR uses the first round product as the template, and the 5' end of the heavy chain variable region and the light chain variable region are amplified.
  • the Sfi1 restriction site sequence was introduced at the 3' end of the variable region, and a linker sequence was introduced at the 3' end of the heavy chain variable region and the 5' end of the light chain variable region;
  • the variable region and the variable region of the light chain are used together as a template, and bridging PCR is performed to obtain a single-chain antibody fragment with the variable region of the heavy chain in the front and the variable region of the light chain behind.
  • the single-chain antibody fragment and the transformed library building vector pCantab5E were digested with Sfi1 (NEB Cat No.#R0123L), and electrophoresed with Gel Extraction Kit (Omega Cat No. D2500-02) was used for purification and recovery. Then use T4 DNA ligase (NEB Cat No.#M0202L) to ligate at 16°C for 16-18 hours, then use the above kit for purification and recovery, and finally eluate with deionized water. 1 ⁇ g of the ligation product was mixed with one electrotransformation competent TG1 (Lucigen Cat No.
  • the phage library (1 ⁇ 10 12 to 1 ⁇ 10 13 /pfu) was suspended in 1 mL of 2% MPBS (PBS containing 2% nonfat dry milk), and 100 ⁇ L was added M-280 Streptavidin (Invitrogen Cat No. 11206D), placed on a turntable with repeated inversion, and sealed at room temperature for 1 hour. Place the tube on the magnetic rack for 2 minutes, remove the Dynabeads, and transfer the phage library to a new tube. 2 ⁇ g/mL of biotin-labeled His-TNC-CD70 was added to the blocked phage library and placed on a turntable for 1 hour.
  • M-280 Streptavidin Invitrogen Cat No. 11206D
  • Dynabeads were suspended in 1 mL of 2% MPBS, placed on a turntable and repeatedly turned, and blocked at room temperature for 1 hour. Place the tube on a magnetic stand for 2 minutes and aspirate the blocking solution. The blocked Dynabeads were added to the phage library and the His-TNC-CD70 mixture, and placed on a turntable for 15 minutes. Place the tube on a magnetic stand for 2 minutes and aspirate the mixture. Dynabeads were eluted with 1 mL of PBST (PBS containing 0.1% Tween-20), then 0.5 mL of 1 mg/mL trypsin (Sigma Cat No.
  • PBST PBS containing 0.1% Tween-20
  • T1426-250MG T1426-250MG was added, and the cells were placed on a turntable and incubated for 15 minutes.
  • the eluted phages were directly infected with log-phase E. coli TG1, and the titer was determined, amplified and concentrated for the next round of panning.
  • the concentration of biotin-labeled human His-TNC-CD70 was reduced to 1 ⁇ g/mL, and the number of PBST washings was increased to 15 times.
  • the eluted phages were infected with Escherichia coli TG1 and plated, and single clones were randomly picked for phage ELISA.
  • the clones were seeded in 96-well deep-well plates (Nunc Cat No. 260251) and cultured at 37°C for 16-18 hours. A small amount was inoculated into another 96-well deep-well plate until the OD600 reached about 0.5, and M13K07 helper phage (NEB Cat No.N0315S) was added for packaging. The cells were removed by centrifugation at 4000g for 10 minutes, and the culture medium was aspirated for human CD70 binding ELISA detection. The positive clones were frozen and preserved in time and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the DNA sequences of positive clones B1, B7, and F4 were measured as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the underline is the CDR sequence determined according to the Kabat numbering system, and the italics is the FR sequence.
  • the heavy chain and light chain CDR region sequences of murine antibodies B1, B7, and F4 are shown in Table 5 below:
  • CDRs in the table are CDRs determined according to the Kabat numbering system
  • the human His-TNC-CD70 protein was diluted to a concentration of 2 ⁇ g/mL with PBS buffer pH 7.4, added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) at a volume of 100 ⁇ L/well, and incubated at 4°C. Place in refrigerator for 16-18 hours. After discarding the liquid, 200 ⁇ L/well of blocking solution of 5% nonfat milk powder (Sangon Biotech, Product No. A600669-0250) diluted with PBS was added, and incubated in a 37°C incubator for 2 hours for blocking.
  • PBST buffer pH 7.4 PBS containing 0.1% tween-20
  • MPBS pH 7.4 PBS containing 2% skim milk powder
  • the heavy and light chain variable region germline genes with high homology to B1, B7 and F4 were selected as templates respectively.
  • the CDRs of the source antibody were respectively grafted into the corresponding human template to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDR amino acid residues in the following specific examples are identified and annotated by the Kabat numbering system.
  • the humanized light chain templates of murine antibody B1 are IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ1*01, and the CDRs of murine antibody B1 were transplanted into them respectively.
  • the amino acid of the FR part of the humanized antibody is further modified by back mutation, wherein the FR part of the light chain includes one or more back mutations in 5S or 70N (wherein the position of the back mutation site is numbered according to Kabat.
  • the FR part of the heavy chain includes one or more back mutations in 4M, 37I, 38K, 48I, 67A, 69L, 71A, 73R, 78A, 80L, 94T (wherein the position of the back mutation site is according to Kabat Numbering rules are determined), the back mutation design of the humanized antibody of antibody B1 is shown in Table 7 below:
  • Graft represents the CDR of murine antibody implanted into the human germline FR region; the position of the back mutation site is determined according to the Kabat numbering system, such as "T5S", which means that the 5th T is mutated to S according to the Kabat numbering system.
  • B1 humanized antibody light chain variable region/heavy chain variable region sequences are as follows:
  • the individual amino acids of the CDR portion of the light chain variable region and the heavy chain variable region were modified, wherein the amino acid sequence of HCDR2 was modified from DIYPGNGDASYNQKFRD (as shown in SEQ ID NO: 10) to DIYPGTGDASYNQKFRD (as shown in SEQ ID NO: 10) 42), the amino acid sequence of LCDR2 was transformed by LASNLES (as shown in SEQ ID NO: 13) into: LADNLES (as shown in SEQ ID NO: 43), after the transformation, the B1 humanized antibody light chain variable region/heavy
  • the chain variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in sequence
  • the italic in the sequence is the FR sequence
  • the underline is the CDR sequence determined according to the Kabat numbering system.
  • the humanized light chain templates of mouse antibody B7 are IGKV27-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV7-4-1*02 and IGHJ6*01.
  • the CDRs of mouse antibody B7 were transplanted respectively into its human template, and then back-mutate the amino acids of the FR portion of the humanized antibody, wherein the light chain FR portion includes one or more back-mutations in 38R, 43S, 69R, 70Q or 71Y (wherein the back The position of the mutation site is determined according to the Kabat numbering rule), and the FR part of the heavy chain includes one or more back mutations in 2I, 24T, 46K, 72E, 82aN (the position of the back mutation site is determined according to the Kabat numbering rule) ).
  • the back-mutation design of the humanized antibody variable region of antibody B7 is shown in Table 8 below:
  • Grafted represents the CDR of murine antibody implanted into the human germline FR region; the position of the mutation site is determined according to the Kabat numbering system, such as "S82a N” means according to the Kabat numbering system, the 82a (also called 82A) position S is mutated is N.
  • B7 humanized antibody light/heavy chain variable region sequences are as follows:
  • the individual amino acids of the CDR portion of the heavy chain variable region were also modified, wherein the amino acid sequence of HCDR2 was modified from the original WINTYTGEPTYADDFKG (as shown in SEQ ID NO: 16) to: WINTYTGEPTYADEFKG (as shown in SEQ ID NO: 54) ), the sequence of the heavy chain variable region of the B7 humanized antibody after the transformation is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in sequence
  • the italic in the sequence is the FR sequence
  • the underline is the CDR sequence determined according to the Kabat numbering system.
  • the humanized light chain templates of murine antibody F4 are IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-69*08 and IGHJ1*01, and the CDRs of murine antibody F4 were transplanted into them respectively.
  • the amino acid of the FR part of the humanized antibody is further modified by back mutation, wherein the FR part of the light chain includes a back mutation of 49S (where the position of the back mutation site is determined according to the Kabat numbering rule), and the FR part of the heavy chain includes a back mutation of 49S.
  • Grafted means that the CDR of murine antibody is implanted into the human germline FR region; the position of the back mutation site is determined according to the Kabat numbering rule.
  • S82a N means mutating S to N at position 82a (also called 82A) according to the Kabat numbering system
  • the individual amino acids of the CDR parts of the light chain variable region and the heavy chain variable region were also modified, wherein the amino acid sequence of HCDR2 was modified from the original AIFPGNGETSYNQNFKG (SEQ ID NO: 22) to: AIFPGTGETSYNQNFKG (such as SEQ ID NO: 71), the amino acid sequence of LCDR2 was transformed from the original LASNLES (SEQ ID NO: 13) to: LADNLES (as shown in SEQ ID NO: 43)
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in sequence
  • the italic in the sequence is the FR sequence
  • the underline is the CDR sequence determined according to the Kabat numbering system.
  • the heavy chain constant region of the antibody can be selected from the heavy chain constant regions of human IgGl, IgG2, IgG3, IgG4, or variants thereof, and the light chain constant regions can be selected from the light chain constant regions of human kappa, lambda chains or variants thereof.
  • the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region shown in SEQ ID NO: 72, and the light chain constant region is selected from the human light chain constant region shown in SEQ ID NO: 73 .
  • the carboxy terminus of the variable region of the heavy chain of the previously screened murine antibodies B1, B7, and F4 was connected to the amino terminus of the constant region of the human heavy chain as shown in SEQ ID NO: 72, and the carboxy terminus of the variable region of the variable region of the light chain of the murine antibody was linked.
  • the chimeric antibody of B1, B7, and F4 is represented as CHB1, CHB7, and CHF4, respectively.
  • huB1001 indicates that the heavy chain variable region is huB1VH1 (SEQ ID NO: 26), the light chain variable region is huB1VL1-1 (SEQ ID NO: 40), and the heavy chain constant region is SEQ ID NO: : 72, the light chain constant region is the humanized antibody shown in SEQ ID NO: 73, and so on.
  • huB7001 indicates that the heavy chain variable region is huB7VH1 (SEQ ID NO: 44), the light chain variable region is huB7VL1 (SEQ ID NO: 47), and the heavy chain constant region is SEQ ID NO: 72 As shown, the light chain constant region is the humanized antibody shown in SEQ ID NO: 73, and so on.
  • huF4001 indicates that the heavy chain variable region is huF4VH1 (SEQ ID NO: 55), the light chain variable region is huF4VL1-1 (SEQ ID NO: 69), and the heavy chain constant region is such as SEQ ID NO: 69 : 72, the light chain constant region is the humanized antibody shown in SEQ ID NO: 73, and so on.
  • Exemplary humanized antibody light/heavy chain full-length sequences are as follows:
  • the underlined part is the antibody variable region sequence
  • the ununderlined part is the antibody constant region sequence
  • monkey CD70-Fc Sequence of monkey CD70 protein extracellular domain and Human-IgG1-Fc fusion protein (abbreviation: monkey CD70-Fc):
  • the underlined part is the Human-IgG1-Fc part, and the ununderlined part is the extracellular domain of the monkey CD70 protein
  • mouse CD70-Fc Sequence of mouse CD70 protein extracellular domain and Human-IgG1-Fc fusion protein
  • the underlined part is the Human-IgG1-Fc part
  • the ununderlined part is the mouse CD70 protein extracellular domain human CD27 and Human-IgG1-Fc fusion protein (abbreviation: CD27-Fc) sequence:
  • the underlined part is the Human-IgG1-Fc part
  • the ununderlined part is the human CD27 part
  • the italicized part is the linker sequence
  • a double-gene vector encoding the heavy chain and light chain amino acid sequences of CD70 antibody was constructed, and the double-gene vector was stably transfected by electroporation using an electroporator (BioRad) to knock out CHOK1 (ECACC, Cat#85051005) -1VL, Lot#12G006), the electroporated cells were ice-bathed for 5 min, and then transferred to pre-warmed cells containing 1% SP4 (CAT#BESP1076E, Lonza) + 0.5% Anti-Clumping agent (CAT#01-0057DG, Gibco) Gently mix in CD CHO medium (CAT# 10743-029, Gibco), and culture in a cell shaker (36.5°C, 6.0% CO 2 , 120 rpm, 80% relative humidity).
  • the cells were counted and plated in a minipool (micro cell pool), 2000 cells per well were seeded into 96-well cell culture plates, and 100 ⁇ L of 0.5% ACF supplement (CAT#3820, STEMCELL) was added to each well. Technologies) + 0.5% Anti-Clumping agent CD CHO medium to start the screening of minipool; observe the cell formation after the 14th day of plating, and after the cell coverage is greater than 30%, the protein expression in the supernatant is determined by the Octet-based method.
  • the cell population with better expression was transferred from 96-well cell plate to 24-well cell plate, and cultured in 1mL CD CHO medium containing 25 ⁇ M MSX (CAT#M5379-500MG, SIGMA) + 0.5% Anti-Clumping agent, pressurized After 14 days of screening, the Octet-based method was used to determine the protein expression in the supernatant of the minipool cell population, and the cell population with better expression was transferred into a 125 mL cell culture flask, and placed in a cell culture shaker for culture (shaker culture conditions are: 36.5°C, 6.0% CO 2 , 80% relative humidity, 120 rpm); after culturing for 2-3 days, the cells were sampled and counted, and the cells were passaged by the dilution method until the cells were in the exponential growth phase, and then the cell lines were subjected to Fed-Batch culture.
  • shaker culture conditions are: 36.5°C, 6.0% CO 2 , 80% relative humidity, 120 rpm
  • afucosylated humanized antibodies are represented by the suffix "(afuc)", for example: huB7002(afuc), huB1010(afuc), huF4011(afuc) and 41D12(afuc) represent huB7002(afuc), Afucosylated antibodies to huB1010, huF4011 and 41D12.
  • Test Example 1 Biacore detection of anti-CD70 antibody affinity experiment
  • Human Fc capture molecules were covalently coupled to a CM5 biosensor chip (CAT#BR-1005-30, GE) according to the method described in the instructions of the human Fc capture kit (CAT#BR-1008-39, GE). , so as to affinity capture the antibody to be tested. Then, the human His-TNC-CD70 antigen (as shown in SEQ ID NO: 1) was flowed on the surface of the chip, and the reaction signal was detected in real time by the Biacore T200 instrument, thereby obtaining the binding and dissociation curves. After each cycle of dissociation in the experiment, the biochip was washed and regenerated with the regeneration solution prepared in the human Fc capture kit (GE). The data fitting model adopts 1:1 Model. The experimental results are shown in Table 13:
  • the experimental results show that the anti-CD70 antibody of the present disclosure has high affinity to human CD70 antigen.
  • the experimental results show that the anti-CD70 antibodies of the present disclosure can effectively bind to CD70-positive cells, and huB7002 and its afucosylated antibodies huB7002 (afuc) and huB1010 and their afucosylated antibodies huB1010 (afuc)
  • the binding capacity was stronger than that of the positive control.
  • Test Example 3 ELISA experiment of anti-CD70 antibody binding to human, monkey and mouse CD70 protein
  • the binding capacity of the anti-CD70 antibody was detected by the amount of binding of the antibody to the CD70 antigen protein immobilized on the ELISA plate.
  • Human CD70-Fc (as shown in SEQ ID NO: 2), monkey CD70-Fc (as shown in SEQ ID NO: 82) and mouse CD70-Fc (as shown in SEQ ID NO: 83) were coated at 1 ⁇ g/mL ), blocked after incubation.
  • the experimental results show that the anti-CD70 antibodies of the present disclosure can all bind to human CD70 antigen and monkey CD70 antigen, but not to mouse CD70 antigen.
  • Test Example 4 Experiment in which anti-CD70 antibody blocks CD27 binding to CD70 positive cells
  • the cell blocking activity of anti-CD70 antibody was detected by flow cytometry. After blocking 1 ⁇ 10 6 cells/mL CHO-S cells (Invitrogen, R80007) expressing human full-length CD70 with 1% BSA PBS buffer, anti-CD70 antibody samples diluted at different concentrations and biotin-labeled CD27-Fc were added (as shown in SEQ ID NO: 84) and incubated for 1 h. After two washes, Alexa Fluor 488-streptavidin (Invitrogen, CAT#S11223) was added and incubated for 1 h. After two washes, the fluorescence signal values were read using a flow cytometer. The experimental results are shown in Figures 6 and 7.
  • the experimental results show that the anti-CD70 antibody of the present disclosure has the ability to block the binding of CD27 to CD70-positive cells.
  • CD27 cells will secrete IL-8.
  • the effect of anti-CD70 antibody on the level of CD70-induced CD27 signaling was detected by detecting the secretion of IL-8 by CD27-expressing cells.
  • HT1080/CD27 cells HT1080 cells expressing human full-length CD27 (ATCC, CCL-121) were collected, resuspended in RPMI1640 containing 10% FBS, and the cells were diluted to 2 ⁇ 10 6 cells/mL.
  • U266 cells and different concentrations of antibodies were added in a ratio of 1:1 to 96-well plates (Corning, CAT#3599) by adding 50 ⁇ L each, and incubated for 60 minutes (37°C). , 5% CO 2 ), 50 ⁇ L of HT1080/CD27 cells were added.
  • CD70 antibody inhibits CD70/CD27 binding-mediated IL-8 secretion assay
  • Imax is the maximum inhibition rate of anti-CD70 antibody inhibiting the secretion of IL-8 by CD27 cells
  • the experimental results show that the anti-CD70 antibody of the present disclosure has better inhibitory ability on IL-8 secretion of HT1080/CD27 cells than the control antibodies 41D12 and 41D12(afuc). It is indicated that the anti-CD70 antibody of the present disclosure can effectively inhibit CD70-induced CD27 signaling by blocking CD70/CD27 binding.
  • Test Example 6 Experiment of anti-CD70 antibody on ADCC of 786-O cells in vitro
  • 786-O-Luc cells (luciferase-expressing 786-O cells (ATCC, CCL-86)) were collected and incubated with Assay Buffer in MEM ⁇ (Gibco, CAT#12561-056) basal medium (containing 2 mM L-Glutamine) 12.5% fetal bovine serum (Gibco, CAT#10099-141), 12.5% horse serum (Biyuntian, CAT#C0262), 0.2mM inositol (SIGMA, CAT#I7508), 0.02mM folic acid (SIGMA, CAT#) F8758), 0.1mM 2-mercaptoethanol (MERCK, CAT#M6250-10ML) and 200U/mL recombinant human IL-2 (Peprotech, CAT#200-02-100) were resuspended, and the cells were diluted to 2 ⁇ 10 5 cells/ mL.
  • MEM ⁇ Gibco, CAT#12561-056
  • NK92 cells (Nanjing Kebai, CBP60980) were collected, resuspended in Assay buffer, and the cells were diluted to 1 ⁇ 10 6 cells/mL.
  • 786-O-Luc cells were collected, resuspended in RPMI 1640 medium (Gibco, CAT#11875119) containing 10% ultra-low IgG fetal bovine serum (Gibco, CAT#1921005PJ), and the cells were diluted to 1 ⁇ 10 5 cells/mL .
  • PBMC Peripheral blood mononuclear cells
  • the experimental results show that the anti-CD70 antibody of the present disclosure has a strong effect on ADCC of 786-O cells in vitro, and the afucosylated humanized antibody significantly improves the effect of ADCC.
  • Test Example 7 Experiment of anti-CD70 antibody on Raji cell in vitro CDC
  • Raji cells (ATCC, CCL-86) were collected and resuspended, and the cells were resuspended at 1 ⁇ 10 6 cells/mL in phenol red-free RPMI 1640 containing 10% ultra-low IgG fetal bovine serum (Gibco, CAT#1921005PJ). base (Gibco, CAT# 11835-030). Subsequently, the cells at 5 ⁇ 10 4 cells / hole (50 ⁇ L / well) were plated in 96-well plates (Corning, CAT # 3903). Then, 50 ⁇ L of different concentrations of antibodies were added.
  • Test Example 8 Experiment of anti-CD70 antibody on ADCP in vitro of 786-O and Raji cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • CD14 + monocytes were sorted using CD14 magnetic beads (Miltenyi Biotec, CAT#130-050-201).
  • Monocytes were differentiated by culturing for 7 days in RPMI 1640 medium (Gibco, CAT#11875119) containing 10% FBS (Gibco, CAT#10091148) and 50 ng/mL M-CSF (Peprotech, CAT#300-25) for 7 days become macrophages. Macrophages were collected by scraping the macrophages with a cell scraper on the day of the experiment.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • the experimental results show that the anti-CD70 antibody of the present disclosure has a good in vitro ADCP effect on 786-O cells and Raji cells.
  • Test Example 9 In vitro inhibition experiment of anti-CD70 antibody on Treg cells
  • PBMC Peripheral blood mononuclear cells
  • U266 cells and PBMCs were added at a ratio of 1:1 to 96-well plates (U bottom plate, corning, CAT#3788) in 50 ⁇ L each, followed by 25 ⁇ L of different concentrations of antibodies and 25 ⁇ L of anti-CD3 (final concentration of 3ug/mL) ( ebioscience, CAT#16-0037-85) and anti-CD28 (ebioscience, CAT#16-0289-85) antibody were incubated for 48 h (37°C, 5% CO 2 ), and the cells were collected in 1.5 mL ep tubes (Axygen, CAT).
  • the experimental results show that the anti-CD70 antibody of the present disclosure has a good ability to inhibit Treg cells in vitro.
  • 786-O-Luc cells (786-O cells expressing luciferase (ATCC, CRL-1932)) were harvested and treated with RPMI 1640 medium (Gibco) containing 10% ultra-low IgG fetal bovine serum (Gibco, CAT#1921005PJ). , CAT#11875119), resuspend and dilute the cells to 2 ⁇ 10 4 cells/mL. Subsequently, cells were plated in 96-well plates (Corning, CAT #3903) at 1000 cells/well (50 ⁇ L/well). It was incubated for 16 hours (37 °C, 5% CO 2 ).
  • the RPMI 1640 medium containing 10% ultra-low IgG fetal bovine serum was used to prepare 4 times the concentration of DT3C (Fragment A of diphtheria toxin and the 3C fragment of group G Streptococcus were fused, and its molar concentration was 6 times the molar concentration of the antibody. ).
  • Use the same medium to prepare 4 times the concentration of antibody mix DT3C and antibody at a volume of 1:1, and incubate at room temperature for 30 min. Concentration gradient dilutions were then performed. The diluted antibody was added to the cells at a ratio of 1:1, 50 ⁇ L/well. Incubated for 3 days (37 °C, 5% CO 2 ).
  • the experimental results show that the anti-CD70 antibody of the present disclosure can be internalized by 786-O cells, and the maximum cleavage rate of cell internalization lysis exceeds 96%.
  • Test Example 11 In vivo efficacy experiment of anti-CD70 antibody in mouse Raji model
  • mice purchased from Viton Lever
  • bioluminescent substrate 15 mg/mL
  • mice were intraperitoneally injected with bioluminescent substrate (15 mg/mL), injected at a volume of 10 mL/kg, anesthetized with isoflurane, and photographed and imaged by a small animal imaging system 10 minutes after injection, and the body weight and bioluminescent signal (Total Flux ) value is too large or too small
  • the mice are randomly divided into 4 groups according to the bioluminescence signal, including the negative control IgG (IgG protein irrelevant to the CD70 target, the dose of 30 mg/kg) group, the positive control 41D12 (the dose of 10mg/kg), huB7002 (administration dose 10mg/kg), huF4011-10 (administration dose 10mg/kg), 8 animals
  • Photographs were imaged twice a week, body weights were weighed, and data were recorded.
  • Excel statistical software was used to record the data, the bioluminescence signal value was Total Flux (unit, p/s), and the mean value was calculated by avg; SD value was calculated by STDEV; SEM value was calculated by STDEV/SQRT (number of animals in each group);
  • GraphPad was used Prism software was used for graphing, and Two-way ANOVA was used for statistical analysis of the data.
  • T/C(%) (T-T0)/(C-C0) ⁇ 100, where T and C are the tumor photons in the treatment group and control group at the end of the experiment; T0 and C0 are at the beginning of the experiment of tumor photons.
  • TGI (%) 100-T/C (%).
  • the anti-CD70 antibody at a dose of 10 mg/kg could significantly inhibit the growth of tumor cells in the Luc-Raji tumor model (p ⁇ 0.001), and the tumor inhibition rate of huB7002 was as high as 81%.
  • the tumor inhibition rate of 41D12 was only 63%.
  • mice purchased from Viton Lever
  • Luc-Raji cells luciferase-expressing Raji cells (ATCC, CCL-86)
  • CB17 SCID mice purchased from Viton Lever
  • Each mouse was intraperitoneally injected with bioluminescent substrate (15 mg/mL), injected at a volume of 10 mL/kg, anesthetized with isoflurane, and photographed and imaged by a small animal imaging system 10 minutes after injection, and the body weight and bioluminescent signal (Total Flux ) value is too large or too small, the mice are randomly divided into groups according to the bioluminescence signal, 8 mice in each group, and the antibody is administered by intraperitoneal injection on the day of grouping (negative control is IgG-type protein irrelevant to the target), administered twice, Dosing on days 1 and 3. Images were taken on days 7 and 14, body weights were weighed, and data were recorded. The experimental results are shown in Figure 18.
  • the experimental results showed that compared with the negative control IgG group, the afucosylated antibody group could significantly inhibit the growth of tumor (p ⁇ 0.001).
  • the tumor inhibition rates of huB1010(afuc) at doses of 1.5, 5, and 15 mg/kg were 82%, 85%, and 84%, respectively.
  • the tumor inhibition rates were 87%, 85%, and 82%, respectively.
  • each administration group still had high tumor inhibitory activity.
  • huB1010 (afuc) 5mg/kg dose The tumor inhibition rate of the group was still 68% on the 14th day. There was no dose effect in each dose group, indicating that even the 1.5 mg/kg dose was sufficient to sufficiently inhibit tumor growth.
  • Test Example 12 Anti-CD70 Antibody Pharmacokinetic Experiment in Vivo
  • mice Male SD rats (purchased from Viton Lever) 3 rats/group were administered intravenously at a dose of 3mpk.
  • the administration group was administered 5min (min means minutes) before and after administration, and 8h (h means minutes) hours), 1d (d means day), 2d, 4d, 7d, 10d, 14d, 21d, 28d, collect 0.15 mL of whole blood without anticoagulation, place the blood at 4°C for 30 min, centrifuge at 1000g for 15 min, and take the supernatant. (serum) in EP tubes and stored at -80°C.

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Abstract

本披露涉及抗CD70抗体及其应用。具体地，本披露涉及一种抗CD70抗体，其包含所述抗体轻链可变区和重链可变区，及其作为药物的用途。

Description

抗CD70抗体及其应用 技术领域
本披露属于生物医药领域，具体涉及结合CD70的抗体及其应用。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
CD70是一种细胞表面抗原，属于肿瘤坏死因子(Tumor necrosis factor，TNF)家族成员，是含193个氨基酸，分子量约为50kD的II型膜蛋白。在体内以同源三聚体形式与其受体CD27相互作用，再由CD27的胞内域结合肿瘤坏死因子受体相关因子(TNF receptor-associated factor，TRAF)，如TRAF2和TRAF5来激活NFκB和JNK通路，最终导致促生存和增殖信号。CD70诱导的CD27信号传导引起表达CD27的调节性T细胞的产生和激活增加。CD70还通过诱导调节性T细胞，逃避免疫监视，从而促进肿瘤的生长。生理条件下，CD70在活化的T细胞、B细胞和树突状细胞上瞬时表达，而在非淋巴的正常组织上几乎不表达。但是，CD70在多种血液瘤和实体瘤中高表达，比如B细胞淋巴瘤，肾癌和乳腺癌等，并与预后呈负相关性。CD27在血液瘤中与CD70共表达，两者结合会导致CD27的胞外域被切下，形成可溶性CD27(sCD27)，可作为诊断的生物标识物。
目前WO2012123586A1、WO2006044643A3、WO2007038637A3、WO2017138471A1等多篇文献已公开了多种抗CD70抗体。目前仍无有效抗CD70抗体药物应用于临床，仍需研发新的有效的抗CD70抗体药物。
发明内容
本披露提供一种新的抗CD70抗体。本披露所述的抗CD70抗体包括抗CD70全长抗体及其抗原结合片段。
在一些实施方案中，本披露提供抗CD70抗体，其包含重链可变区和轻链可变区，其中：
i)所述重链可变区包含分别如SEQ ID NO:9和SEQ ID NO:11所示的HCDR1和HCDR3，以及如SEQ ID NO:10或SEQ ID NO:42所示的HCDR2；所述轻链可变区包含分别如SEQ ID NO:12和SEQ ID NO:14所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；
ii)所述重链可变区包含分别如SEQ ID NO:15和SEQ ID NO:17所示的HCDR1和HCDR3，以及如SEQ ID NO:16或SEQ ID NO:54所示的HCDR2；所述轻链可变区包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；或
iii)所述重链可变区包含分别如SEQ ID NO:21和SEQ ID NO:23所示的 HCDR1和HCDR3，以及如SEQ ID NO:22或SEQ ID NO:71所示的HCDR2；所述轻链可变区包含分别如SEQ ID NO:24和SEQ ID NO:25所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2。
在一些实施方案中，本披露的抗CD70抗体包含重链可变区和轻链可变区，其中，
所述重链可变区包含分别如SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含分别如SEQ ID NO:9、SEQ ID NO:42和SEQ ID NO:11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:12、SEQ ID NO:43和SEQ ID NO:14所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含分别如SEQ ID NO:15、SEQ ID NO:54和SEQ ID NO:17所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:22和SEQ ID NO:23所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:24、SEQ ID NO:13和SEQ ID NO:25所示的LCDR1、LCDR2和LCDR3；或者
所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:71和SEQ ID NO:23所示的HCDR1、HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:24、SEQ ID NO:43和SEQ ID NO:25所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，前述的抗CD70抗体是鼠源抗体、嵌合抗体或人源化抗体；在一些实施方案中，前述的抗CD70抗体是全长抗体或其抗原结合片段。在一些实施方案中，所述抗原结合片段选自：Fab、F(ab') 2、F(ab) 2、Fd、Fv、dsFv、scFv，及其双抗体。
在一些实施方案中，前述的抗CD70抗体为人源化抗体，所述人源化抗体包含人抗体的框架区或人抗体的框架区变体，所述框架区变体相对于人抗体的轻链框架区和/或重链框架区分别具有至多11个氨基酸的回复突变。
在一些实施方案中，所述抗体的重链框架区上包含选自4M、37I、38K、48I、67A、69L、71A、73R、78A、80L和94T中的一个或更多个氨基酸回复突变，和/或所述抗体的轻链框架区上包含选自5S和70N中的一个或更多个氨基酸回复突变；在一些实施方案中，所述抗体的轻链框架区上包含选自38R、43S、69R、70Q和71Y中的一个或更多个氨基酸回复突变，和/或所述抗体的重链框架区上包含选自2I、24T、46K、72E和82a N中的一个或更多个氨基酸回复突变；在一些实施方 案中，所述抗体的重链框架区上包含选自27D、30P、37L、38K、48I、66K、67A、69L和82a N中的一个或更多个氨基酸回复突变，和/或所述抗体的轻链框架区上包含49S氨基酸回复突变；其中，所述突变位点根据Kabat编号规则编号，例如，“4M”表示重链可变区第4位(对应于Kabat编号规则编号)残基为“M”。
在一些实施方案中，所述抗CD70抗体包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自4M、37I、38K、48I、67A、69L、71A、73R、78A、80L和94T中的一个或更多个氨基酸回复突变，和/或所述抗CD70抗体包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含选自5S和70N中的一个或更多个氨基酸回复突变；在一些实施方案中，所述抗CD70抗体包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含选自38R、43S、69R、70Q和71Y中的一个或更多个氨基酸回复突变，和/或所述抗CD70抗体包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自2I、24T、46K、72E和82aN中的一个或更多个氨基酸回复突变；在一些实施方案中，所述抗CD70抗体包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自27D、30P、37L、38K、48I、66K、67A、69L和82a N中的一个或更多个氨基酸回复突变，和/或所述抗CD70抗体包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含49S氨基酸回复突变；其中，所述突变位点根据Kabat编号规则编号，例如，“4M”表示重链可变区第4位(对应于Kabat编号规则编号)残基为“M”。
在一些实施方案中，所述人源化抗体包含选自以下a)、b)或c)所述的轻链可变区和重链可变区：
所述人源化抗体包含：
a)重链可变区，其包含分别如SEQ ID NO:9和SEQ ID NO:11所示的HCDR1和HCDR3，以及如SEQ ID NO:10或SEQ ID NO:42所示的HCDR2；和轻链可变区，其包含分别如SEQ ID NO:12和SEQ ID NO:14所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；而且所述重链可变区包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自4M、37I、38K、48I、67A、69L、71A、73R、78A、80L和94T中的一个或更多个氨基酸回复突变，和/或所述轻链可变区包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含选自5S和70N中的一个或更多个氨基酸回复突变；或
b)重链可变区，其包含分别如SEQ ID NO:15和SEQ ID NO:17所示的HCDR1和HCDR3，以及如SEQ ID NO:16或SEQ ID NO:54所示的HCDR2；和轻链可变区，其包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；而且所述轻链可变区包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含选自38R、43S、69R、70Q和71Y中的一个或更多个氨基酸回复突变，和/或所述重链可变区包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自2I、24T、46K、72E和82a N中的一个或更多个氨基 酸回复突变；或
c)重链可变区，其包含分别如SEQ ID NO:21和SEQ ID NO:23所示的HCDR1和HCDR3，以及如SEQ ID NO:22或SEQ ID NO:71所示的HCDR2；和轻链可变区，其包含分别如SEQ ID NO:24和SEQ ID NO:25所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；而且所述重链可变区包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自27D、30P、37L、38K、48I、66K、67A、69L和82a N中的一个或更多个氨基酸回复突变，和/或所述轻链可变区包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含49S氨基酸回复突变。其中，所述突变位点对应于Kabat编号规则编号。
在一些实施方案中，前述的抗CD70抗体的重链可变区为在SEQ ID NO:26或SEQ ID NO:34的基础上具有选自4M、37I、38K、48I、67A、69L、71A、73R、78A、80L和94T中的一个或更多个氨基酸回复突变的重链可变区，和所述轻链可变区为在SEQ ID NO:32或SEQ ID NO:40的基础上具有选自5S和70N中的一个或更多个氨基酸回复突变的轻链可变区；在一些实施方案中，前述的抗CD70抗体的重链可变区为在SEQ ID NO:44或SEQ ID NO:51的基础上具有选自2I、24T、46K、72E和82a N中的一个或更多个氨基酸回复突变的重链可变区，和所述轻链可变区为在SEQ ID NO:47的基础上具有选自38R、43S、69R、70Q和71Y中的一个或更多个氨基酸回复突变的轻链可变区；在一些实施方案中，前述的抗CD70抗体的重链可变区为在SEQ ID NO:55或SEQ ID NO:63的基础上具有选自27D、30P、37L、38K、48I、66K、67A、69L和82a N中的一个或更多个氨基酸回复突变的重链可变区，和所述轻链可变区为在SEQ ID NO:61或SEQ ID NO:69的基础上具有49S氨基酸回复突变的轻链可变区。其中，所述突变位点对应于Kabat编号规则编号。
本领域技术人员应当理解，前述氨基酸回复突变位点，当采用Kabat以外的其他编号规则时，在功能和/或结构上具有等同地位的氨基酸残基可能被赋予不同的编号，但仍对应于本披露中所限定的位点。
在一些实施方案中，前述的抗CD70抗体包含重链可变区和轻链可变区，其中：
d)所述重链可变区的氨基酸序列与SEQ ID NO:3、26、27、28、29、30、31、34、35、36、37、38或39分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，和/或所述轻链可变区的氨基酸序列与SEQ ID NO:4、32、33、40或41分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性；
e)所述重链可变区的氨基酸序列与SEQ ID NO:5、44、45、46、51、52或53分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，和/或所述轻链可变区的氨基酸序列与SEQ ID NO:6、47、48、49或50分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、 99％或100％的序列同一性；或
f)所述重链可变区的氨基酸序列与SEQ ID NO:7、55、56、57、58、59、60、63、64、65、66、67或68分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，和/或所述轻链可变区的氨基酸序列与SEQ ID NO:8、61、62、69或70分别具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性。
在一些实施方案中，前述抗CD70抗体包含如下表1、表2和表3所示的重链可变区和轻链可变区组合：
表1.huB1人源化抗体轻/重链可变区组合表
Figure PCTCN2021102998-appb-000001
备注：表中例如“huB1V001”表示抗体的重链可变区为与其同行的SEQ ID NO：26，轻链可变区为与其同列的SEQ ID NO：40，其它以此类推。
表2.huB7人源化抗体重轻链可变区组合表
Figure PCTCN2021102998-appb-000002
备注：表中例如“huB7V001”表示抗体的重链可变区为与其同行的SEQ ID NO:44，轻链可变区为与其同列的SEQ ID NO:47，其它以此类推。
表3.huF4人源化抗体重轻链可变区组合表
Figure PCTCN2021102998-appb-000003
Figure PCTCN2021102998-appb-000004
备注：表中例如“huF4V001”表示抗体的重链可变区为与其同行的SEQ ID NO:55，轻链可变区为与其同列的SEQ ID NO:69，其它以此类推。
在一些实施方案中，前述的抗CD70抗体包含重链可变区和轻链可变区，其中：
g)所述重链可变区，其氨基酸序列如SEQ ID NO:3所示；和所述轻链可变区，其氨基酸序列如SEQ ID NO:4所示；或
h)所述重链可变区，其氨基酸序列如SEQ ID NO:26、27、28、29、30、31、34、35、36、37、38或39所示；和所述轻链可变区，其氨基酸序列如SEQ ID NO:32、33、40或41所示；或
i)所述重链可变区，其氨基酸序列如SEQ ID NO:5所示；和所述轻链可变区，其氨基酸序列如SEQ ID NO:6所示；或
j)所述重链可变区，其氨基酸序列如SEQ ID NO:44、45、46、51、52或53所示；和所述轻链可变区序列，其氨基酸序列如SEQ ID NO:47、48、49或50所示；或
k)所述重链可变区，其氨基酸序列如SEQ ID NO:7所示；和所述轻链可变区，其氨基酸序列如SEQ ID NO:8所示；或
l)所述重链可变区，其氨基酸序列如SEQ ID NO:55、56、57、58、59、60、63、64、65、66、67或68所示；和所述轻链可变区，其氨基酸序列如SEQ ID NO:61、62、69或70所示。
在一些实施方案中，前述的抗CD70抗体包含如下所示的重链可变区和轻链可变区：
n)所述重链可变区，其氨基酸序列如SEQ ID NO:35所示；和所述轻链可变区，其氨基酸序列如SEQ ID NO:40所示；或
o)所述重链可变区，其氨基酸序列如SEQ ID NO:51所示；和所述轻链可变区序列，其氨基酸序列如SEQ ID NO:47所示；或
p)所述重链可变区，其氨基酸序列如SEQ ID NO:65所示；和所述轻链可变区序列，其氨基酸序列如SEQ ID NO:70所示。
在一些实施方案中，前述的抗CD70抗体包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含如SEQ ID NO:72所示的重链恒定区和如SEQ ID NO:73所示的轻链恒定区。
在一些实施方案中，前述的抗CD70抗体包含：
q)与SEQ ID NO:74具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链，和/或与SEQ ID NO:75具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链；或
r)与SEQ ID NO:76具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链，和/或与SEQ ID NO:77具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链；或
s)与SEQ ID NO:78具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链，和/或与SEQ ID NO:79具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链。
在一些实施方案中，前述的抗CD70抗体包含：
t)如SEQ ID NO:74所示的重链和如SEQ ID NO:75所示的轻链；或
u)如SEQ ID NO:76所示的重链和如SEQ ID NO:77所示的轻链；或
v)如SEQ ID NO:78所示的重链和如SEQ ID NO:79所示的轻链。
在一些实施方案中，本披露还提供一种分离的抗CD70抗体，其中所述抗体与前面任一项所述的抗CD70抗体竞争性结合人CD70、人CD70抗原表位、猴CD70或猴CD70抗原表位。在一些实施方案中，所述抗体与前面任一项所述的抗CD70抗体结合人CD70上相同表位。
在一些实施方案中，前面任一项所述的抗CD70抗体，其中所述抗CD70抗体为低岩藻糖基化抗体；在一些实施方案中，所述低岩藻糖基化的抗CD70抗体是至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的抗体重链未被岩藻糖糖基化修饰的抗体。在一些实施方案中，所述低岩藻糖基化抗CD70抗体是至少95％、96％、97％、98％、99％或100％重链未被岩藻糖糖基化修饰的抗体。在一些实施方案中，前述抗CD70抗体为100％重链未被岩藻糖糖基化修饰的IgG1抗体(也称非岩藻糖基化IgG1抗体)。
在一些实施方案中，前面任一项所述的抗CD70抗体，所述抗CD70抗体具有以下特征中的至少一种：
A.所述抗CD70抗体以小于1×10 -8M，优选小于1×10 -9M、或小于1×10 -10M、或小于6×10 -11M、或小于5×10 -11M、或小于4×10 -11M、或小于3×10 -11M的KD值与人CD70结合，所述KD值通过表面等离子共振技术所测定；例如，通过本披露的测试例1所述方法进行检测；
B.所述抗CD70抗体既能与人CD70抗原结合，也能与猴CD70抗原结合，但不与小鼠CD70抗原结合；
C.所述抗CD70抗体能抑制CD70诱导的CD27信号传导，优选地，所述抗 CD70抗体抑制表达人CD27细胞(例如HT1080/CD27细胞)分泌IL-8的最大抑制百分比(Imax(％))大于或等于72％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％；更优选大于或等于90％、91％、93％或98％，所述IL-8分泌通过Elisa方法检测，例如通过本披露测试例5所述方法检测；
D.所述抗CD70抗体具有以下的一个或更多个效应子功能：对表达人CD70细胞的抗体依赖性细胞介导的细胞毒性(ADCC)、补体依赖性细胞毒性(CDC)和抗体依赖性细胞介导的吞噬作用(ADCP)；优选地，所述抗CD70抗体通过CDC效应子功能使表达人CD70细胞(例如Raji细胞)裂解的最大裂解率大于或等于70％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％、或100％；更优选大于或等于74％、78％、84％或86％；在一些方案中，CDC效应子功能通过本披露的测试例7所述方法进行检测；和
E、所述抗CD70抗体能被表达人CD70细胞内化，优选地，细胞内化裂解的最大裂解率大于或等于70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％；更优选大于或等于96％或97％；在一些方案中，细胞内化通过本披露的测试例10所述方法进行检测。
在一些实施方案中，本披露还提供一种核酸分子，其编码前面任一项所述的抗CD70抗体。
在一些实施方案中，本披露还提供一种宿主细胞，其包含前述的核酸分子。所述宿主细胞可选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞，优选不包括人类的哺乳动物细胞，其中所述的哺乳动物细胞包括但不限于CHO，293，NSO以及在哺乳动物细胞中进行基因编辑可改变抗体或其抗原结合片段的糖基化修饰，进而改变抗体或其抗原结合片段的ADCC功能的细胞，例如，敲除Fut8或GnT-III等基因进行糖基化修饰。
在一些实施方案中，本披露还提供一种制备前述抗CD70抗体的方法，所述方法包括培养前述的宿主细胞，然后纯化回收抗体的步骤。
在一些实施方案中，本披露还提供一种免疫偶联物，其包含前面任一项所述的抗CD70抗体和效应分子，所述效应分子偶联至所述抗CD70抗体；优选地，所述效应分子选自放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
在一些实施方案中，本披露还提供一种药物组合物，其含有治疗有效量的前面任一项所述的抗CD70抗体，或前述的核酸分子，或前述的免疫偶联物，以及一种或更多种药学上可接受的载体、稀释剂或赋形剂。
在一些实施方案中，本披露还提供一种用于免疫检测或测定CD70的方法，所 述方法包括使前面任一项所述的抗CD70抗体接触受试者或来自受试者的样品的步骤。
在一些实施方案中，本披露还提供一种试剂盒，其包含前面任一项所述的抗CD70抗体或免疫偶联物。
在一些实施方案中，本披露还提供一种预防或治疗疾病或病症的方法，所述方法包括向受试者施用治疗有效量的前面任一项所述的抗CD70抗体，或前述的核酸分子，或前所的药物组合物，或前述的免疫偶联物。
在一些实施方案中，本披露还提供前面任一项所述的抗CD70抗体，或前述的核酸分子，或前所的药物组合物，或前述的免疫偶联物在制备用于预防或治疗疾病或病症的药物中的用途。
在一些实施方案中，本披露提供作为药物的前面任一项所述的抗CD70抗体，或前述的核酸分子，或前所的药物组合物，或前述的免疫偶联物，所述药物用于预防或治疗疾病或病症。
在一些实施方案中，前面任一项所述的疾病或病症是CD70相关的疾病或病症。在一些实施方案中，所述疾病或病症为CD70高表达对受试者是有害的疾病或病症。在一些实施方案中，所述疾病或病症为肿瘤、自身免疫性疾病或感染性疾病。在一些实施方案中，其中所述肿瘤选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在另一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞性白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。在一些实施方案中，所述自身免疫性疾病选自类风湿性关节炎、牛皮癣、关节银屑病、银屑病、皮炎、系统性硬皮病、系统性硬皮病及硬化症、炎症性肠病(IBD)、Crohn病、溃疡性结肠炎、呼吸窘迫综合征、脑膜炎、脑炎、葡萄膜炎、肾小球肾炎、湿疹、哮喘、动脉硬化、白细胞粘附缺陷病、多发性硬化症、Raynaud症候群、Sjogren症候群、青少年糖尿病、Reiter病、Behcet病、免疫复合物性肾炎、IgA肾病、IgM多发性神经病、免疫介导的血小板减少症状(如急性特发性血小板减少性紫癜、慢性特发性血小板减少 性紫癜)、溶血性贫血、重症肌无力、狼疮性肾炎、系统性红斑狼疮、风湿关节炎(RA)、异位性皮炎、天疱疮、Graves病、桥本甲状腺炎、Wegener肉芽肿、Omenn症候群、慢性肾功能衰竭、急性传染性单核细胞增多征、多发性硬化症、HIV和疱疹病毒相关的疾病、严重急性呼吸综合征和脉络视网膜炎(choreoretinitis)、以及病毒感染引起的免疫性疾病(如伊波病毒(EBV)感染B细胞引起或介导的疾病)。在一些实施方式中，疾病或病症为：急性髓细胞性白血病、骨髓增生异常综合症、鼻咽癌、非霍奇金淋巴瘤、肾细胞癌、转移性肾细胞癌、类风湿性关节炎和银屑病。
在一些实施方案中，前述治疗有效量为单位剂量的组合物中含有0.1-3000mg的前述的抗CD70抗体、或前述的核酸分子、或前述的免疫偶联物、或前所的药物组合物。在一些实施方案中，所述治疗进一步包括向受试者施用治疗有效量的第二治疗剂。
附图说明
图1A和图1B显示抗CD70抗体结合CD70阳性786-O细胞的实验结果；其中，图1A为抗CD70抗体结合CD70阳性786-O细胞的实验结果图，图1B为非岩藻糖基化抗CD70抗体结合CD70阳性786-O细胞实验结果图。
图2A和图2B显示抗CD70抗体结合CD70阳性Raji细胞的实验结果，其中图2A为抗CD70抗体结合CD70阳性Raji细胞的实验结果图，图2B为非岩藻糖基化抗CD70抗体结合CD70阳性Raji细胞的实验结果图。
图3显示huB7002结合人、猴和小鼠CD70蛋白的ELISA实验结果。
图4显示huB1010结合人、猴和小鼠CD70蛋白的ELISA实验结果。
图5显示huF4011结合人、猴和小鼠CD70蛋白的ELISA实验结果。
图6显示抗CD70抗体阻断CD27结合CD70阳性细胞的实验结果。
图7显示非岩藻糖基化抗CD70抗体阻断CD27结合CD70阳性细胞的实验结果。
图8显示抗CD70抗体对HT1080/CD27细胞IL-8分泌的抑制实验结果。
图9显示非岩藻糖基化抗CD70抗体对HT1080/CD27细胞IL-8分泌的抑制实验结果。
图10显示抗CD70抗体对786-O细胞体外ADCC(NK92)的实验结果。
图11显示抗CD70抗体对786-O细胞体外ADCC(PBMC)的实验结果。
图12显示抗CD70抗体对Raji细胞体外CDC的实验结果。
图13显示非岩藻糖基化抗CD70抗体对Raji细胞体外CDC的实验结果。
图14A和图14B显示抗CD70抗体对786-O和Raji细胞的体外ADCP的实验结果，其中，图14A为抗CD70抗体对786-O细胞的ADCP实验结果图，图14B为抗CD70抗体对Raji细胞的ADCP实验结果图。
图15显示抗CD70抗体对Treg细胞体外的抑制实验结果。
图16显示786-O细胞对抗CD70抗体的内化实验结果。
图17显示抗CD70抗体在小鼠Raji模型的体内药效实验结果。
图18显示非岩藻糖基化的抗CD70抗体在小鼠Raji模型的体内药效实验结果。
发明详述
术语(定义)
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本披露的术语“抗体”以最广义使用，其涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体或其抗原结合片段(也称“抗原结合部分”)，只要它们展现出期望的抗原结合活性。天然全长抗体是包含由二硫键互相连接的至少两条重链和两条轻链的免疫球蛋白(Ig)。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(缩写为Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。每条重链由重链可变区(缩写为VH)和重链恒定区(缩写为CH)组成。重链恒定区包含CH1、CH2和CH3三个结构域。每条轻链由轻链可变区(缩写为VL)和轻链恒定区(缩写为CL)组成。重链可变区和轻链可变区包括高变区(也称为互补性决定区，缩写为CDR或HVR)和序列相对保守的骨架区(也称框架区，缩写为FR)。每个VL和VH由从氨基末端排到羧基末端按以下顺序排列的3个CDR 4个FR组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本披露的抗体包括鼠源抗体、嵌合抗体和人源化抗体。
术语“鼠源抗体”在本披露中为根据本领域知识和技能制备的针对抗原(例如人CD70)的鼠源性单克隆抗体。例如，用CD70抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本披露的一个优选的实施方案中，所述的鼠源抗CD70抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体 的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。通常，建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本披露一个优选的实施方案中，所述嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的CD70嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变，265A和/或297A)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库获得，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免抗体免疫原性下降的同时，引起活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持或增强活性。本披露的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
在本披露一个的实施方案中，所述的抗体或其抗原结合片段，可进一步包含人源或鼠源κ、λ链或其变体的轻链恒定区，或进一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区；可以包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变、和/或S228P突变，265A和/或297A)的IgG1、IgG2或IgG4变体。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，265A(例如D265A)和/或297A(例如N297A)，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体并且可以通过本领域已知的任何 方法产生。在某些技术中，将人重链和轻链基因座的元素元件引入到源于胚胎干细胞系的生物体的细胞株中，这些细胞系中的内源性重链和轻链基因座被靶向破坏。转基因生物可以合成对人抗原特异的人抗体，并且该生物可以用于产生人抗体-分泌杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或更多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指轻链和重链包含恒定区的抗体。
本披露的“抗体”包含“全长抗体”及其“抗原结合片段”。
在一些实施方案中，本披露的全长抗体包括由前述表1、表2和表3中轻、重链可变区组合分别与轻、重链恒定区连接后所形成的全长抗体。本领域技术人员可以根据实际需要选择不同的抗体来源的轻链恒定区、重链恒定区，例如人抗体来源的轻链恒定区和重链恒定区。同时，表1、表2和表3中不同轻链可变区和重链可变区组合可以形成单链抗体(scFv)、Fab或其他包含scFv或Fab的抗原结合片段形式。
术语“抗原结合片段”或“功能片段”或“抗原结合部分”是指保持特异性结合抗原(例如，CD70)的能力的完整抗体的一个或更多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。示例性的，涵盖在术语“抗原结合片段”的结合片段的实例包括：(i)Fab片段，一种由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，一种包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法将这两个结构域通过能够使它们形成为单条蛋白质链的人工肽接头来接合，其中VL和VH配对形成单价分子，称为单链Fv(scFv)(参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883))。此类单链抗体也包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或 多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的序列上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。遵循AbM规则，VH中的CDR氨基酸编号为26-32(HCDR1)、50-58(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。除非另有说明，本披露的抗体可变区和CDR序列均对应于“Kabat”编号规则。
术语“表位”或“抗原决定簇”是指抗原上抗体特异性结合的部位(例如，CD70分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M、10 -12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体以小于大约10 -8M，例如小于大约10 -9M或10 -10M的解离平衡常数(KD)结合CD70，所述KD值使用表面等离子体共振(SPR)技术在Biacore T200仪器中测定。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如CD70抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指在比对氨基酸序列(必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分)，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过本领域已知的多种方式来实现，例如使用诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)等软件。本领域 技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“岩藻糖基化”或“岩藻糖基化的”或“岩藻糖糖基化修饰”指附着于抗体的肽链的寡糖内存在岩藻糖残基。岩藻糖基化是糖蛋白翻译后修饰的一个普遍过程，与核心岩藻糖基化相关的代表性酶基因有GMD(GDP-甘露糖4,6-脱水酶)基因和Fut8(fut8，FUT8，α-1,6-岩藻糖基转移酶)基因，通过抑制这两个基因的表达或者构建Fut8敲除型CHO宿主细胞可有效调节核心岩藻糖基化水平(YAMANE-OHNUKI et al.,“Establishment of FUT8knockout Chinese hamster ovary cells:an ideal host cellline for producing completely defucosylated antibodies with enhancedantibody-dependent cellular cytotoxicity.”,BIOTECHNOLOGY AND BIOENGINEERING,2004,p614～622)。通常，岩藻糖基化抗体在Fc区的N-寡糖中，在核心N-乙酰基葡糖胺(GlcNAc)残基处(例如人IgG1 Fc的Asn297位置(对应于EU编号规则))包含α-1,6-岩藻糖。
“低岩藻糖基化”的抗体是指附着于Fc区的碳水化合物结构具有低的岩藻糖的糖基化修饰的抗体；“非岩藻糖基化”或“无岩藻糖基化”是指附着于Fc区的碳水化合物结构缺乏岩藻糖的糖基化修饰的抗体。抗体岩藻糖基化水平可通过本领域已知方法测定所有寡糖，进而确定岩藻糖基化寡糖的百分比。本领域已知的测定岩藻糖基化方法包括但不限于凝胶电泳、液相色谱法、和质谱分析法等。例如，通过亲水性相互作用色谱法(或亲水性相互作用液相色谱法，HILIC)测定抗体的岩藻糖基化的水平，例如用肽-N-聚糖酶F对样品进行处理变性以切割N-连接的聚糖，然后分析N-连接的聚糖的岩藻糖含量。本披露中，在一些实施方案中，本披露的低岩藻糖基化抗体是至少80％的重链未被岩藻糖的糖基化修饰的抗体，例如至少80-95％、90-95％、95-100％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％抗体重链未被岩藻糖糖基化修饰。本披露中，除非另有说明，否则“非岩藻糖基化”是指100％重链未被岩藻糖糖基化修饰的抗体。
低岩藻糖基化或非岩藻糖基化抗体可通过本领域公知方法制备。例如通过添加、移动或缺失抗体上存在的一个或多个碳水化合物部分，例如使用岩藻糖苷酶切割抗体的岩藻糖残基(参见Tarentino等(1975)Biochem.14:5516)来制备。具有减少的岩藻糖基化的抗体还可通过改变糖基化的组成来改变糖基化水平，例如通过修饰在N297残基处与每个Fc片段附接的聚糖模块来制备(Natsume等(2009)Drug Des.Devel.Ther.3:7)。也可以在不改变抗体序列的情况下，例如，可以通过改变抗体糖基化模式的细胞表达低岩藻糖基化或非岩藻糖基化抗体，包括例如经遗传工程改变的糖基化工程的细胞来制备(参见例如，Hse等，(1997)J.Biol.Chem.272:9062-9070；Yang等，(2015)Nature Biotechnology 33,842-844)。各种糖基化工程的细胞已经在本领域中公开，例如，缺乏岩藻糖转移酶基因(FUT8，(α-(1,6)岩藻糖转移酶)细胞系Ms704、Ms705和Ms709等(参见Yamane-Ohnuki 等(2004)Biotechnol.Bioeng.87:614；美国专利20040110704)，具有使岩藻糖附接至Asn(297)连接的糖类的能力降低CHO细胞系Lec13(参见WO 03/035835)，具有很小或没有添加岩藻糖至N-乙酰葡萄糖胺(其结合抗体的Fc区)的活性的大鼠骨髓瘤细胞系YB2/0(参见EP 1176195)；用于生产具有经修饰的糖基化模式的抗体的植物细胞(参见美国专利US20120276086)。另外，携带编码使用GDP-6-脱氧-D-来苏-4-己糖作为底物的酶(诸如GDP-6-脱氧-D-来苏-4-己糖还原酶(RMD))的重组基因细胞也可生产低岩藻糖基化或非岩藻糖基化抗体(参见美国专利US8642292)。本披露的一些具体实施方案中，通过例如实施例5所述方法制备获得非岩藻糖基化抗体。
“抗体依赖性的细胞介导的细胞毒性”或“ADCC”指的是细胞介导的反应，其中表达FcR的非特异性细胞毒性细胞(例如天然杀伤(NK)细胞、嗜中性粒细胞、和巨噬细胞)识别靶细胞上结合的抗体，并且随后引起靶细胞的裂解。调节ADCC的原代细胞、NK细胞仅表达FcyRIII，而单核细胞表达FcyRI、FcyRII和FCYRIII。为了评估目的分子的ADCC活性，可进行体内外ADCC测定(诸如Clynes等人(PNASUSA 95:652-656(1998))、美国专利号US5500362、US5821337等所述)。本披露中，在一些实施例中，所述ADCC通过本披露的测试例6方法进行检测。
“抗体依赖性细胞吞噬作用”或“ADCP”指通过吞噬细胞(例如，巨噬细胞、嗜中性粒细胞和树突细胞)内化来消除经抗体包被的靶细胞或病毒体的机制。内化的抗体包被的靶细胞或病毒体被包含在称为吞噬体的囊泡中，所述囊泡随后与一个或多个溶酶体融合，以形成吞噬溶酶体。ADCP可以通过使用巨噬细胞作为效应细胞的体外细胞毒性测定和电视显微术(videomicroscopy)来评价(如van Bij等于Journal of Hepatology第53卷,第4期,2010年10月,第677–685)。本披露中，在一些实施例中，所述ADCP通过本披露的测试例8方法进行检测。
“补体依赖性细胞毒性”或“CDC”是指补体参与的细胞毒作用，即通过抗体与细胞或病毒体上的相应抗原结合，形成复合物而激活补体经典途径，所形成的攻膜复合物对靶细胞发挥裂解效应。CDC可以通过体外测定(如使用正常人血清作为补体源测定CDC)或在C1q浓度系列中评价。CDC活性的降低(例如由于在多肽或抗体中引入第二突变而导致的CDC活性的降低)可以通过在相同的测定法内比较多肽或抗体的CDC活性与其不具有第二突变的亲本多肽或抗体的CDC活性来测量。为了评估抗体诱导CDC的能力，可通过如Romeuf等人所述的测定方法(Romeuf等人,Br J Haematol.2008Mar；140(6):635-43)进行测定。本披露中，在一些实施例中，所述CDC通过本披露的测试例7方法进行检测。
术语“缀合物”、“药物偶联物”、“免疫偶联物”指配体通过稳定的连接单元与具有生物活性的药物相连而成的新型药物。例如，“抗体药物偶联物”(antibody drug conjugate，ADC)，其是将单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。抗体可直接地或经接头地偶联至药物。每个抗体的平 均药物模块数，其范围可以是例如每个抗体约0到约20个药物模块，在一些实施方案中是每个抗体1个到约10个药物模块，在某些实施方案中是每个抗体1个到约8个药物模块。本披露的抗体-药物偶联物的混合物的组合物，其中每个抗体的平均药物载荷是约2个至约5个或约3个至约4个。
在一些实施方案中，提供了免疫偶联物。在一些实施方案中，本文所公开的免疫偶联物可以是附接至效应分子的抗体，其中抗体可以是包含重链和轻链的抗体。在一些实施方案中，抗体可以是抗体片段，诸如Fab、Fab'、F(ab')2、scFv、dsFv、ds-scFv、二聚体、微型抗体、双功能抗体、双特异性抗体片段、多聚体，以及其任何组合。
在本文所描述的实施方案中，效应分子可以是放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
本披露所描述的抗体或抗体片段可以通过任何方式偶联至效应分子。举例来说，抗体或抗体片段可以通过化学或重组方式附接至毒素。制备融合物或偶联物的化学方式在本领域中是已知的并且可以用于制备免疫偶联物。用于偶联抗体或抗体片段和毒素的方法必须能够连接抗体与毒素而不会干扰抗体或抗体片段结合至标靶分子的能力。
术语“细胞毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。包括毒素、化疗药物等能用于肿瘤细胞杀伤的化合物。
术语“毒素”是指能够对细胞的生长或增殖产生有害效果的任何物质，可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4，auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1，化合物7)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“化疗药物”是可用于治疗肿瘤的化学化合物。该定义还包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的抗激素剂，且常常是系统或全身治疗的形式。它们自身可以是激素。化疗药物实例包括烷化剂，如噻替哌(thiotepa)；环磷酰胺(cyclosphamide)(CYTOXAN TM)；烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)如苯并多巴(benaodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和 methylamelamine包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，authramycin，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin,洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin,嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)；抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)；喋吟类似物氟达拉滨(f1udarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine),依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮(calusterone),丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)；抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如frolinic acid；醋葡内脂；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；defofamine；秋水仙胺；地吖醌(diaziquone)；elfomithine；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；硝呋旦(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；
Figure PCTCN2021102998-appb-000005
雷佐生(razoxane)；西索菲兰(sizofiran)；锗螺胺(spirogermanium)；细交链孢菌酮酸；三亚胺醌；2，2'，2"-三氯二乙胺(trichlorrotriethylamine)；乌拉坦(urethan)；长春碱酰胺；达卡巴嗪(dacarbazine)；甘露醇氮芥；二溴甘露醇(mitobronitol)；二溴卫矛醇；哌溴烷坑(pipobroman)；gacytosine；阿拉伯糖苷("Ara-C")；环磷酰胺；三胺硫磷(thiotepa)；紫杉烷，如紫杉醇(
Figure PCTCN2021102998-appb-000006
Bristol-Myers Squibb Oncology,Princeton,NJ)和docetaxel(
Figure PCTCN2021102998-appb-000007
Rhone-Poulenc Rorer,Antony,France)；苯丁酸氮芥；吉西他滨(gemcitabine)；6-硫代鸟嘌呤；巯基嘌呤；氨甲蝶呤；铂类似物如顺铂和卡铂；长春花碱；铂；依托泊甙(etoposide)(VP-16)；异环磷航胶；丝裂霉素C；米托蒽醌；长春新碱；长春瑞宾(vinorelbine)；新霉酰胺(navelbine)；novantrone；替尼泊甙(teniposide)；柔红霉素；氨基蝶呤；xeloda；伊拜磷酸盐(ibandronate)；CPT-11；拓扑异构酶抑制剂RFS2000；二氟甲基鸟氨酸(DMFO)；维甲酸esperamicins；capecitabine；以及上述任何物质的可药用盐，酸或衍生物。此定义还包括能调节或抑制激素对肿瘤的作用的抗激素制剂，如抗雌激素制剂包括他莫昔芬(tamoxifen)，雷洛昔芬(raloxifene)，芳香酶抑制剂4(5)-咪唑，4-羟基他莫昔芬，曲沃昔芬(trioxifene),keoxifene，LY117018,onapristone，和托瑞米芬(Fareston)；和抗雄激素制剂如氟他氨(flutamide)，尼鲁米特(nilutamide)，bicalutamide，亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)；和上述任何物质的可药用盐，酸或衍生物。
在一个实施方案中，抗体和毒素都是蛋白质并且可以使用本领域中熟知的技术偶联。存在本领域中所公开的数百种交联剂，其可以偶联两种蛋白质。交联剂一般基于在抗体或毒素上可用或插入的反应官能团来选择。另外，如果不存在反应基团，那么可以使用光可活化交联剂。在某些情况下，可能需要在抗体与毒素之间包括间隔子。本领域中已知的交联剂包括同型双功能剂：戊二醛、二甲基己二亚酰胺化物和双(重氮基联苯胺)，以及异型双功能剂：间马来酰亚胺基苯甲酰基-N-羟基琥珀酰亚胺和磺基-间马来酰亚胺基苯甲酰基-N-羟基琥珀酰亚胺。
可以用于使效应分子偶合至抗体片段的交联剂包括例如TPCH(S-(2-硫代吡啶基)-L-半胱氨酸酰肼)和TPMPH(S-(2-硫代吡啶基)巯基-丙酰肼)。TPCH和TPMPH在先前已经通过温和高碘酸盐处理而氧化的糖蛋白的碳水化合物部分反应，由此在交联剂的酰肼部分与高碘酸盐产生的醛之间形成腙键。异型双功能交联剂GMBS(N-(γ-马来酰亚胺基丁酰氧基)-琥珀酰亚胺)和SMCC(琥珀酰亚胺基4-(N-马来酰亚胺基-甲基)环己烷)与伯胺反应，由此将马来酰亚胺基引入组分上。这个马来酰亚胺基随后可以与另一种组分上的可以由交联剂引入的硫氢基反应，由此在组分之间形成稳定的硫醚键。如果组分之间的位阻干扰任一种组分的活性，那么可以使用交联剂，将长的间隔臂引入组分之间，诸如3-(2-吡啶基二硫基)丙酸n-琥珀酰亚胺酯(SPDP)。因此，存在许多适合的交联剂，其可以被使用并且各自取决于其对最佳免疫偶联物产量的影响来选择。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加 型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人CD70或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。为了获得非岩藻糖基化的抗体，可使用敲除Glul和Fut8基因的宿主细胞，包括但不限于敲除Glul和Fut8基因的CHOK1细胞。
本披露工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人CD70特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给予”和“处理”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”，当其应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种结合化合物 的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代如下：
表4.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本披露靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本披露靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4，683，195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物； 这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必然发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送药物(例如本披露所述的抗CD70抗体或抗原结合片段)的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本披露包括用于治疗与目标抗原(例如CD70)阳性细胞相关的疾病的药剂，所述药剂包含本披露的抗CD70抗体或其抗原结合片段作为活性成分。活性成分以治疗有效量施与受试者，可以治疗受试者CD70阳性细胞相关疾病。所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的特异性结合人CD70抗体。
本披露中与CD70相关的疾病或病症没有限制，只要它是与CD70相关的疾病或病症即可。例如，在一些实施方案中，本披露的分子对于以下一些表达CD70的疾病或病症是非常有用的：例如，类风湿性关节炎、自身免疫性脱髓鞘疾病(例如，多发性硬化、变应性脑脊髓炎)、内分泌性眼病、葡萄膜视网膜炎、系统性红斑狼疮、重症肌无力、Grave病、肾小球性肾炎、自身免疫性肝(hepatological)病、炎性肠病(例如，Crohn病、溃疡性结肠炎、乳糜泻病)、过敏反应、变应性反应、 Sjogren综合征、I型糖尿病、原发性胆汁性肝硬化、韦格纳肉芽肿病、纤维肌痛、多肌炎、皮肌炎、多内分泌不足、Schmidt综合征、自身免疫性葡萄膜炎、Addison病、肾上腺炎、甲状腺炎、Hashimoto甲状腺炎、自身免疫性甲状腺疾病、恶性贫血症、胃萎缩、慢性肝炎、狼疮样肝炎、动脉粥样硬化、亚急性皮肤性红斑狼疮、甲状旁腺功能减退、Dressier综合征、自身免疫性血小板减少症、特发性血小、板减少性紫癜、溶血性贫血、寻常性天疱疮、天疱疮、疱疹样皮炎、斑秃、类天疱疮、硬皮病、进行性系统性硬化症、CREST综合征(钙质沉着、Raynaud现象、食管蠕动障碍、硬指症和毛细管扩张)、男性和女性自身免疫性不育症、强直性脊柱炎、溃疡性结肠炎、混合性结缔组织病、结节性多动脉炎(polyateritis nodosa)、系统性坏死性血管炎、特应性皮炎、特应性鼻炎、Goodpasture综合征、Chagas病、结节病、风湿热、哮喘、复发性流产、抗磷脂综合征、农民肺、多形性红斑、心切开术后综合征、Gushing综合征、自身免疫性慢性活动性肝炎、养鸟人肺、中毒性表皮坏死性溶解症、Alport综合征、肺泡炎、变应性肺泡炎、纤维化肺泡炎、间质性肺病、结节性红斑、坏疽性脓皮病、输血反应、Takayasu动脉炎、风湿性多肌痛、颞动脉炎、血吸虫病、巨细胞动脉炎(giant cell arthritis)、蛔虫病、曲霉病、Sampter综合征、湿疹、淋巴瘤样肉芽肿病、白塞氏病、Caplan综合征、川崎病、登革热、脑脊髓炎、心内膜炎、心内膜心肌纤维化、眼内炎、持久性隆起性红斑(erythema elevatum et diutinum)、银屑病、胎儿成红细胞增多症、嗜酸性筋膜炎、Shulman综合征、Felty综合征、丝虫病、睫状体炎、慢性睫状体炎、异色性睫状体炎、Fuch睫状体炎、IgA肾病、Henoch-Schonlein紫癜、移植物抗宿主病、移植排斥反应、心肌病、Eaton-Lambert综合征、复发性多软骨炎、冷沉球蛋白血症、Evan综合征和自身免疫性性腺衰竭，B淋巴细胞的病症(例如，系统性红斑狼疮、Goodpasture综合征、类风湿性关节炎和I型糖尿病)、Th1淋巴细胞的病症(例如，类风湿性关节炎、多发性硬化症、银屑病、Sjorgren综合征、Hashimoto甲状腺炎、Grave病、原发性胆汁性肝硬化、韦格纳肉芽肿病、肺结核或移植物抗宿主病)、或者Th2淋巴细胞的病症(例如，特应性皮炎、系统性红斑狼疮、特应性哮喘、鼻结膜炎、变应性鼻炎、Omenn综合征、系统性硬化症或慢性移植物抗宿主病)，Churg Strauss综合征、显微多血管炎、以及Takayasu动脉炎。在另外一些方案中，本披露的分子对于以下一些表达CD70的疾病(例如癌症)是非常有用，包括肾癌(例如、肾细胞癌)、乳腺癌、脑肿瘤，慢性或急性白血病(包括急性髓性白血病、慢性髓性白血病，急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、淋巴瘤(例如、霍奇金淋巴瘤和非霍奇金淋巴瘤、淋巴细胞性淋巴瘤、原发性CNS淋巴瘤、T细胞淋巴瘤)、鼻咽癌、黑素瘤(例如、转移性恶性黑素瘤)、前列腺癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头部或颈部的癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛门区的癌症、胃癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、宫颈癌、阴道癌、外阴癌、食管癌、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、儿童实体肿瘤、膀胱癌、肾脏或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波济氏肉瘤、类上皮癌、鳞状细胞癌、间皮瘤。在另外一些方案中，本披露的分子对表征为存在表达CD70的肿瘤细胞，包括，例如肾细胞癌(RCC)如透明细胞(clear cell)RCC、胶质母细胞瘤、乳腺癌、脑肿瘤、鼻咽(nasopharangeal)癌、非霍奇金淋巴瘤(NHL)、急性淋巴细胞性白血病(ALL)、慢性淋巴细胞性白血病(CLL)、Burkitt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、结节性小裂细胞淋巴瘤、淋巴细胞性淋巴瘤、外周T细胞淋巴瘤、Lennert淋巴瘤、免疫母细胞淋巴瘤、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)滤泡性淋巴瘤癌(entroblastic/centrocytic(cb/cc)follicular lymphomas cancers)、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相关的基于体腔的淋巴瘤、胚胎癌、鼻咽的未分化癌(例如，Schmincke瘤)、Castleman病、Kaposi肉瘤、多发性骨髓瘤、Waldenstrom巨球蛋白血症、套细胞淋巴瘤和其他的B细胞淋巴瘤，具有良好作用。
此外，本披露涉及用于免疫检测或测定目标抗原(例如CD70)的方法、用于免疫检测或测定目标抗原(例如CD70)的试剂、用于免疫检测或测定表达目标抗原(例如CD70)的细胞的方法和用于诊断与目标抗原(例如CD70)阳性细胞相关的疾病的诊断剂，其包含本披露的特异性识别目标抗原(例如人CD70)并与胞外区的氨基酸序列或其三维结构结合的抗体或抗体片段作为活性成分。
在本披露中，用于检测或测定目标抗原(例如CD70)的量的方法可以是任何已知方法。例如，免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
与CD70阳性细胞(例如高表达CD70细胞)相关的疾病可以通过用本披露的抗体或抗体片段检测或测定表达CD70的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本披露中，对用于检测或测定目标抗原(例如CD70)的活体样品没有特别限制，只要它具有包含表达目标抗原(例如CD70)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本披露的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体 反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本披露一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方式。这些实施例不应以任何方式理解为限制本披露的范围。
具体实施方式
以下结合实施例和测试例进一步描述本披露，但这些实施例和测试例并非限制着本披露的范围。本披露实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件；未注明具体来源的试剂材料，为市场购买获得。
实施例
实施例1：CD70抗原的制备
以UniProt CD70抗原(人CD70蛋白，Uniprot号：P32970)作为CD70的模板，设计本披露使用的抗原及检测用蛋白的氨基酸序列，可选的在CD70蛋白基础上融合不同的标签如His标签或Fc等。分别克隆到pTT5载体上(Biovector，CAT#102762)，在293细胞中瞬转表达，纯化，获得本披露抗原及检测用蛋白。
带His标签的CD70蛋白胞外域(简写：His-TNC-CD70)序列，作为检测试剂；
Figure PCTCN2021102998-appb-000008
注释：下划线部分为6×His标签，斜体部分为TNC标签，其余部分为CD70蛋白胞外域。
CD70蛋白胞外域与Human-IgG1-Fc融合蛋白(简写：CD70-Fc)序列，作为免疫原；
Figure PCTCN2021102998-appb-000009
注释：下划线部分为Human-IgG1-Fc部分，无下划线部分为CD70蛋白胞外域。
实施例2：CD70相关重组蛋白的纯化
1.带His标签的重组蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，并将缓冲液置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的细胞上清样品上Ni Sepharose excel柱(GE，17-3712-02)。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE，28-9893-35)进一步纯化，流动相为PBS。去除聚体峰，收集洗脱峰。所得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。得到带His标签的His-TNC-CD70，用作本披露抗体的检测试剂。
2.CD70-Fc融合蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，上清进行MabSelect Sure(GE，17-5438-01)亲和层析。MabSelect Sure层析柱先用0.2M NaOH再生，利用纯水冲洗后用PBS平衡柱子，将上清结合后，利用PBS进行洗涤至A280读数降至基线。用0.1M醋酸缓冲液在pH3.5条件下洗脱目的蛋白，用1M Tris-HCl中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE，28-9893-35)进一步纯化，合并收集目的蛋白所在接收管浓缩至适当浓度。此方法用来纯化CD70-Fc融合蛋白，该方法也可以用来纯化本披露中的抗体蛋白。
实施例3：鼠抗CD70噬菌体库抗体的筛选
1.小鼠免疫：
抗人CD70抗体通过免疫小鼠产生。实验用Balb/c白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。
用蛋白抗原(CD70-Fc，SEQ ID NO：2)与细胞抗原(表达人全长CD70的CHO-S细胞(Invitrogen,R80007))交叉免疫，其中蛋白抗原交替使用
Figure PCTCN2021102998-appb-000010
Gold Adjuvant(Sigma Cat No.T2684)与Thermo
Figure PCTCN2021102998-appb-000011
Alum(Thermo Cat No.77161)佐剂。蛋白抗原与佐剂(
Figure PCTCN2021102998-appb-000012
Gold Adjuvant)比例为1:1，蛋白抗原与佐剂(Thermo
Figure PCTCN2021102998-appb-000013
Alum)比例为3:1，50μg/只/次(蛋白抗原免疫)，1×10 7cells/只/次(细胞抗原免疫)。蛋白抗原乳化后进行接种，细胞抗原由磷酸缓冲溶液重悬后接种。接种时间为第0、14、28、42、56和第70天，于第21，49，82天取血，用ELISA方法确定小鼠血清中的抗体滴度。选择血清中抗体滴度高并且滴度趋于平台的小鼠，取其脾脏用于建立免疫库。
2.鼠源噬菌体单链抗体库构建：
取小鼠脾细胞，用Trizol(Invitrogen Cat No.15596-018)提取总RNA。用PrimeScript TM II 1st Strand cDNA Synthesis Kit试剂盒(Takara Cat No.6210A)进行反转录获得cDNA。根据IMGT数据库设计并合成构建文库的引物(金唯智公司)。通过三轮PCR反应，获得单链抗体片段。PCR反应均使用LA Tag(Takara Cat No.RR02MB)。第一轮PCR以cDNA为模板，分别扩增得到重链可变区和轻链可变区序列；第二轮PCR以第一轮产物为模板，在重链可变区5’端和轻链可变区3’端引入Sfi1酶切位点序列，在重链可变区3’端和轻链可变区5’端引入连接序列；第三轮PCR以第二轮PCR产物的重链可变区和轻链可变区共同为模板，进行搭桥PCR，获得重链可变区在前，轻链可变区在后的单链抗体片段。
将单链抗体片段和经过改造的建库载体pCantab5E(Amersham Biosciences/GE Cat No.27-9400-01)用Sfi1(NEB Cat No.#R0123L)进行酶切，电泳后用
Figure PCTCN2021102998-appb-000014
Gel Extraction Kit(Omega Cat No.D2500-02)进行纯化回收。然后用T4 DNA连接酶(NEB Cat No.#M0202L)16℃连接16-18小时，再用上述试剂盒进行纯化回收，最后用去离子水洗脱。取1μg连接产物与1支电转化感受态TG1(Lucigen Cat No.60502-2)混合，电转化仪(Bio Rad Micropulser)进行电转化。重复转化20次，涂平板，37℃倒置培养16-18小时。将所有菌落刮洗下来混和在一起，加入终浓度为15％的甘油，-80度保存备用。
3.筛选噬菌体单链抗体库得到抗CD70的阳性单克隆序列：
噬菌体单链抗体库进行包装浓缩后，首先进行两轮淘洗。将噬菌体库(1×10 12～1×10 13/pfu)悬浮于1mL 2％MPBS(含有2％脱脂奶粉的PBS)中，并加入100μL
Figure PCTCN2021102998-appb-000015
M-280 Streptavidin(Invitrogen Cat No.11206D)，置于转台上反复翻转，室温封闭1小时。将管子置于磁力架上2分钟，去除Dynabeads，噬菌体库转移至新的管子中。向封闭后的噬菌体库中加入2μg/mL生物素标记的His-TNC-CD70，置于转台上反复翻转1小时。同时取100μL Dynabeads悬浮于1mL 2％MPBS中，置于转台上反复翻转，室温封闭1小时。将管子置于磁力架上2分钟，吸去封闭液。将封闭后的Dynabeads加入到噬菌体库和His-TNC-CD70混合液中，置于转台上反复翻转15分钟。将管子置于磁力架上2分钟，吸去混合液。用1mL PBST(含0.1％Tween-20的PBS)洗脱Dynabeads，再加入0.5mL 1mg/mL胰蛋白酶(Sigma  Cat No.T1426-250MG)，置于转台上反复翻转孵育15分钟，进行洗脱。洗脱下来的噬菌体直接感染对数期的大肠杆菌TG1，测定滴度并进行扩增浓缩，用于下一轮淘洗。第二轮淘洗噬菌体库(1×10 11～1×10 12/pfu)，生物素标记的人His-TNC-CD70浓度降为1μg/mL，PBST洗涤次数增加至15次。将洗脱下来的噬菌体感染大肠杆菌TG1涂平板，随机挑取单克隆，用于噬菌体ELISA。
将克隆接种于96孔深孔板(Nunc Cat No.260251)37度培养16～18小时。取少量接种至另一个96孔深孔板，至OD600达到0.5左右，加入M13K07辅助噬菌体(NEB Cat No.N0315S)进行包装。4000g离心10分钟去除菌体，吸取培养液进行人CD70结合ELISA检测。阳性克隆菌种及时冻存保种并送测序公司测序。测得阳性克隆B1、B7、F4的DNA序列对应的氨基酸序列如下：
>B1重链可变区序列(B1mVH)：
Figure PCTCN2021102998-appb-000016
>B1轻链可变区序列(B1 mVL)：
Figure PCTCN2021102998-appb-000017
>B7重链可变区序列(B7 mVH)：
Figure PCTCN2021102998-appb-000018
>B7轻链可变区序列(B7 mVL)：
Figure PCTCN2021102998-appb-000019
>F4重链可变区序列(F4 mVH)：
Figure PCTCN2021102998-appb-000020
>F4轻链可变区序列(F4 mVL)：
Figure PCTCN2021102998-appb-000021
Figure PCTCN2021102998-appb-000022
注：上述序列中，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为依照Kabat编号系统确定的CDR序列，斜体为FR序列。
鼠源抗体B1、B7、F4的重链及轻链CDR区序列如下表5所示：
表5.抗体重链轻链CDR序列
Figure PCTCN2021102998-appb-000023
备注：表中CDR为依照Kabat编号系统确定的CDR
4.噬菌体展示单链抗体结合人CD70蛋白的ELISA实验：
用pH7.4的PBS缓冲液将人His-TNC-CD70蛋白稀释至2μg/mL浓度，以100μL/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于4℃冰箱中放置16-18小时。弃去液体后，加入用PBS稀释的5％脱脂奶粉(生工生物，产品编号A600669-0250)封闭液200μL/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.1％tween-20)洗板3次后，加入100μL/孔用2％MPBS(pH7.4 PBS含2％脱脂奶粉)1:1稀释的噬菌体培养液，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板6次，加入100μL/孔用2％MPBS稀释的抗M13 Antibody(HRP)二抗(SB，货号11973-MM05T-H)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用VERSAmax酶标仪(Molecular Devices)在波 长450nm处读取吸收值。实验结果如下表：
表6.单链抗体与人CD70抗原结合ELISA实验结果
抗体 酶标仪OD450nm处读值
B1 1.16
F4 1.09
B7 1.97
实施例4：抗CD70鼠源抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与B1、B7和F4同源性高的重轻链可变区种系基因作为模板，将这三个鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。示例性地，以下具体实施例中CDR氨基酸残基由Kabat编号系统确定并注释。
1.B1鼠源抗体的人源化
鼠源抗体B1的人源化轻链模板为IGKV4-1*01和IGKJ4*01，人源化重链模板为IGHV1-46*01和IGHJ1*01，将鼠源抗体B1的CDR分别移植到其人源模板中，进而对人源化抗体的FR部分氨基酸进行回复突变改造，其中轻链FR部分包括5S或70N中的一个或更多个回复突变(其中的回复突变位点的位置根据Kabat编号规则确定)，重链FR部分包括4M,37I,38K,48I,67A,69L,71A,73R,78A,80L,94T中的一个或更多个回复突变(其中的回复突变位点的位置根据Kabat编号规则确定)，抗体B1的人源化抗体的回复突变设计见下表7：
表7.B1人源化抗体的回复突变设计
Figure PCTCN2021102998-appb-000024
注：Graft代表鼠源抗体CDR植入人种系FR区；其中回复突变位点的位置根据 Kabat编号规则确定，如“T5S”表示依照Kabat编号系统，将第5位T突变为S。
B1人源化抗体轻链可变区/重链可变区序列如下：
>huB1VH1(huB1VH-graft)
Figure PCTCN2021102998-appb-000025
>huB1VH2
Figure PCTCN2021102998-appb-000026
>huB1VH3
Figure PCTCN2021102998-appb-000027
>huB1VH4
Figure PCTCN2021102998-appb-000028
>huB1VH5
Figure PCTCN2021102998-appb-000029
>huB1VH6
Figure PCTCN2021102998-appb-000030
>huB1VL1(huB1VL-graft)
Figure PCTCN2021102998-appb-000031
>huB1VL2
Figure PCTCN2021102998-appb-000032
另外，对轻链可变区和重链可变区的CDR部分的个别氨基酸进行改造，其中，HCDR2的氨基酸序列由 DIYPGNGDASYNQKFRD(如SEQ ID NO:10所示)改造为 DIYPGTGDASYNQKFRD(如SEQ ID NO:42所示)，LCDR2的氨基酸序列由 LASNLES(如SEQ ID NO:13所示)改造为： LADNLES(如SEQ ID NO:43所示)，改造后B1人源化抗体轻链可变区/重链可变区序列如下：
>huB1VH1-1
Figure PCTCN2021102998-appb-000033
>huB1VH2-1
Figure PCTCN2021102998-appb-000034
>huB1VH3-1
Figure PCTCN2021102998-appb-000035
>huB1VH4-1
Figure PCTCN2021102998-appb-000036
>huB1VH5-1
Figure PCTCN2021102998-appb-000037
>huB1VH6-1
Figure PCTCN2021102998-appb-000038
Figure PCTCN2021102998-appb-000039
>huB1VL1-1
Figure PCTCN2021102998-appb-000040
>huB1VL2-1
Figure PCTCN2021102998-appb-000041
其中，上述序列中，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为依照Kabat编号系统确定的CDR序列。
2.抗体库huB7人源化构架选择
鼠源抗体B7的人源化轻链模板为IGKV27-1*01和IGKJ4*01，人源化重链模板为IGHV7-4-1*02和IGHJ6*01，将鼠源抗体B7的CDR分别移植到其人源模板中，进而对人源化抗体的FR部分氨基酸进行回复突变改造，其中轻链FR部分包括38R、43S、69R、70Q或71Y中的一个或更多个回复突变(其中的回复突变位点的位置根据Kabat编号规则确定)，重链FR部分包括2I、24T、46K、72E、82a N中的一个或更多个回复突变(其中的回复突变位点的位置根据Kabat编号规则确定)。抗体B7的人源化抗体可变区的回复突变设计见下表8：
表8.B7人源化抗体的回复突变设计
Figure PCTCN2021102998-appb-000042
注：Grafted代表鼠源抗体CDR植入人种系FR区；其中突变位点的位置根据Kabat编号规则确定，如“S82a N”表示依照Kabat编号系统，将第82a(也称82A)位S突变为N。
B7人源化抗体轻/重链可变区序列如下：
>huB7VH1(huB7VH-graft)
Figure PCTCN2021102998-appb-000043
>huB7VH2
Figure PCTCN2021102998-appb-000044
>huB7VH3
Figure PCTCN2021102998-appb-000045
>huB7VL1(huB7VL-graft)
Figure PCTCN2021102998-appb-000046
>huB7VL2
Figure PCTCN2021102998-appb-000047
>huB7VL3
Figure PCTCN2021102998-appb-000048
>huB7VL4
Figure PCTCN2021102998-appb-000049
另外，还对重链可变区的CDR部分的个别氨基酸进行改造，其中，HCDR2的氨基酸序列由原 WINTYTGEPTYADDFKG(如SEQ ID NO:16所示)改造为：WINTYTGEPTYADEFKG(如SEQ ID NO:54所示)，改造后B7人源化抗体重链可变区序列如下：
>huB7VH1-1
Figure PCTCN2021102998-appb-000050
Figure PCTCN2021102998-appb-000051
>huB7VH2-1
Figure PCTCN2021102998-appb-000052
>huB7VH3-1
Figure PCTCN2021102998-appb-000053
其中，上述序列中，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为依照Kabat编号系统确定的CDR序列。
3.抗体库huF4人源化构架选择
鼠源抗体F4的人源化轻链模板为IGKV4-1*01和IGKJ2*01，人源化重链模板为IGHV1-69*08和IGHJ1*01，将鼠源抗体F4的CDR分别移植到其人源模板中，进而对人源化抗体的FR部分氨基酸进行回复突变改造，其中轻链FR部分包括49S的回复突变(其中的回复突变位点的位置根据Kabat编号规则确定)，重链FR部分包括27D、30P、37L、38K、48I、66K、67A、69L、82a N中的一个或更多个回复突变(其中的回复突变位点的位置根据Kabat编号规则确定)。抗体F4的人源化抗体可变区的回复突变设计见下表9：
表9.F4人源化抗体的回复突变设计
Figure PCTCN2021102998-appb-000054
Figure PCTCN2021102998-appb-000055
注：Grafted代表鼠源抗体CDR植入人种系FR区；其中的回复突变位点的位置根据Kabat编号规则确定。如“S82a N”表示依照Kabat编号系统，将第82a(也称82A)位S突变为N
>huF4VH1(huF4VH-graft)
Figure PCTCN2021102998-appb-000056
>huF4VH2
Figure PCTCN2021102998-appb-000057
>huF4VH3
Figure PCTCN2021102998-appb-000058
>huF4VH4
Figure PCTCN2021102998-appb-000059
>huF4VH5
Figure PCTCN2021102998-appb-000060
>huF4VH6
Figure PCTCN2021102998-appb-000061
>huF4VL1(huF4VL-graft)
Figure PCTCN2021102998-appb-000062
>huF4VL2
Figure PCTCN2021102998-appb-000063
另外，还对轻链可变区和重链可变区的CDR部分的个别氨基酸进行改造，其中，HCDR2的氨基酸序列由原 AIFPGNGETSYNQNFKG(SEQ ID NO：22)改造为： AIFPGTGETSYNQNFKG(如SEQ ID NO:71所示)，LCDR2的氨基酸序列由原 LASNLES(SEQ ID NO：13)改造为：LADNLES(如SEQ ID NO:43所示)
>huF4VH1-1
Figure PCTCN2021102998-appb-000064
>huF4VH2-1
Figure PCTCN2021102998-appb-000065
>huF4VH3-1
Figure PCTCN2021102998-appb-000066
>huF4VH4-1
Figure PCTCN2021102998-appb-000067
>huF4VH5-1
Figure PCTCN2021102998-appb-000068
Figure PCTCN2021102998-appb-000069
>huF4VH6-1
Figure PCTCN2021102998-appb-000070
>huF4VL1-1
Figure PCTCN2021102998-appb-000071
>huF4VL2-1
Figure PCTCN2021102998-appb-000072
其中，上述序列中，顺序依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为依照Kabat编号系统确定的CDR序列。
4.构建和表达抗CD70人源化抗体IgG1形式
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pTT5(带信号肽及恒定区基因(CH1-FC/CL)片段，实验室构建)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pTT5/VK-CL-pTT5。抗体的重链恒定区可选自人IgG1、IgG2、IgG3、IgG4、或其变体的重链恒定区，轻链恒定区可选自人κ、λ链或其变体的轻链恒定区。示例性的，以下实施例中，抗体重链恒定区选自如SEQ ID NO：72所示的人IgG1重链恒定区，轻链恒定区选自如SEQ ID NO：73所示的人轻链恒定区。
人IgG1重链恒定区序列：
Figure PCTCN2021102998-appb-000073
人轻链恒定区序列：
Figure PCTCN2021102998-appb-000074
Figure PCTCN2021102998-appb-000075
将前面筛选的鼠源抗体B1、B7、F4的重链可变区羧基端与如SEQ ID NO：72所示的人重链恒定区氨基端连接，同时将鼠源抗体轻链可变区羧基端与如SEQ ID NO：73所示的人轻链恒定区氨基端连接，可获得其对应的嵌合抗体，B1、B7、F4的嵌合抗体分别表示为CHB1、CHB7、CHF4。
将前面构建的B1、B7、F4的人源化抗体重链可变区羧基端与如SEQ ID NO：72所示的人重链恒定区氨基端连接形成抗体全长重链，将人源化抗体轻链可变区羧基端与如SEQ ID NO：73所示的人轻链恒定区氨基端连接形成抗体全长轻链，可得到如下表10-12所示的人源化抗体：
表10.B1的人源化抗体
可变区 huB1VL1-1 huB1VL2-1 huB1VL1 huB1VL2
huB1VH1 huB1001 huB1015 huB1025 huB1037
huB1VH2 huB1002 huB1016 huB1026 huB1038
huB1VH3 huB1003 huB1017 huB1027 huB1039
huB1VH4 huB1004 huB1018 huB1028 huB1040
huB1VH5 huB1005 huB1019 huB1029 huB1041
huB1VH6 huB1006 huB1007 huB1030 huB1042
huB1VH1-1 huB1009 huB1008 huB1031 huB1043
huB1VH2-1 huB1010 huB1020 huB1032 huB1044
huB1VH3-1 huB1011 huB1021 huB1033 huB1045
huB1VH4-1 huB1012 huB1022 huB1034 huB1046
huB1VH5-1 huB1013 huB1023 huB1035 huB1047
huB1VH6-1 huB1014 huB1024 huB1036 huB1048
备注：表中，“huB1001”表示重链可变区为huB1VH1(SEQ ID NO：26)，轻链可变区为huB1VL1-1(SEQ ID NO：40)，且重链恒定区如SEQ ID NO：72所示，轻链恒定区如SEQ ID NO：73所示的人源化抗体，其它以此类推。
表11.B7的人源化抗体
可变区 huB7VL1 huB7VL2 huB7VL3 huB7VL4
huB7VH1 huB7001 huB7007 huB7013 huB7019
huB7VH1-1 huB7002 huB7008 huB7014 huB7020
huB7VH2 huB7003 huB7009 huB7015 huB7021
huB7VH2-1 huB7004 huB7010 huB7016 huB7022
huB7VH3 huB7005 huB7011 huB7017 huB7023
huB7VH3-1 huB7006 huB7012 huB7018 huB7024
备注：表中，“huB7001”表示重链可变区为huB7VH1(SEQ ID NO：44)，轻链可变区为huB7VL1(SEQ ID NO：47)，且重链恒定区如SEQ ID NO：72所示，轻链恒定区如SEQ ID NO：73所示的人源化抗体，其它以此类推。
表12.F4的人源化抗体
可变区 huF4VL1-1 huF4VL2-1 huF4VL1 huF4VL2
huF4VH1 huF4001 huF4014 huF4025 huF4037
huF4VH2 huF4002 huF4015 huF4026 huF4038
huF4VH3 huF4003 huF4016 huF4027 huF4039
huF4VH4 huF4004 huF4017 huF4028 huF4040
huF4VH5 huF4005 huF4018 huF4029 huF4041
huF4VH6 huF4006 huF4019 huF4030 huF4042
huF4VH1-1 huF4007 huF4020 huF4031 huF4043
huF4VH2-1 huF4008 huF4021 huF4032 huF4044
huF4VH3-1 huF4009 huF4011 huF4033 huF4045
huF4VH4-1 huF4010 huF4022 huF4034 huF4046
huF4VH5-1 huF4012 huF4023 huF4035 huF4047
huF4VH6-1 huF4013 huF4024 huF4036 huF4048
备注：表中，“huF4001”表示重链可变区为huF4VH1(SEQ ID NO：55)，轻链可变区为huF4VL1-1(SEQ ID NO：69)，且重链恒定区如SEQ ID NO：72所示，轻链恒定区如SEQ ID NO：73所示的人源化抗体，其它以此类推。
示例性的人源化抗体轻链/重链全长序列如下：
huB1010重链序列：
Figure PCTCN2021102998-appb-000076
huB1010轻链序列：
Figure PCTCN2021102998-appb-000077
huB7002重链序列：
Figure PCTCN2021102998-appb-000078
Figure PCTCN2021102998-appb-000079
huB7002轻链序列：
Figure PCTCN2021102998-appb-000080
huF4011重链序列：
Figure PCTCN2021102998-appb-000081
huF4011轻链序列：
Figure PCTCN2021102998-appb-000082
备注：上述抗体全长序列中，下划线部分为抗体可变区序列，无下划线部分为抗体恒定区序列。
另外，本披露实施例中的阳性对照抗体41D12(参见WO2012123586)的轻重链序列如下：
41D12重链序列：
Figure PCTCN2021102998-appb-000083
Figure PCTCN2021102998-appb-000084
41D12轻链序列：
Figure PCTCN2021102998-appb-000085
本披露实施例中使用的其它蛋白序列：
猴CD70蛋白胞外域与Human-IgG1-Fc融合蛋白(简写：猴CD70-Fc)序列：
Figure PCTCN2021102998-appb-000086
注释：下划线部分为Human-IgG1-Fc部分，无下划线部分为猴CD70蛋白胞外域
鼠CD70蛋白胞外域与Human-IgG1-Fc融合蛋白(简写：鼠CD70-Fc)序列：
Figure PCTCN2021102998-appb-000087
注释：下划线部分为Human-IgG1-Fc部分，无下划线部分为鼠CD70蛋白胞外域人CD27与Human-IgG1-Fc融合蛋白(简写：CD27-Fc)序列：
Figure PCTCN2021102998-appb-000088
注释：下划线部分为Human-IgG1-Fc部分，无下划线部分为人CD27部分，斜体字部分为连接序列
实施例5：非岩藻糖基化的人源化抗体制备
构建编码CD70抗体的重链和轻链氨基酸序列的双基因载体，采用电转仪(BioRad)将双基因载体通过电穿孔的方式稳定地转染敲除Glul和Fut8基因的CHOK1(ECACC，Cat#85051005-1VL，Lot#12G006)，将电转后的细胞冰浴5min后，转移至预热的含1％SP4(CAT#BESP1076E，Lonza)+0.5％Anti-Clumping agent(CAT#01-0057DG，Gibco)的CD CHO培养基(CAT#10743-029，Gibco)中轻轻混匀，细胞摇床中培养(36.5℃、6.0％CO 2、120rpm、80％相对湿度)。
转染后24h，将细胞进行计数，并进行minipool(微细胞池)铺板，以每孔2000个细胞接种至96孔细胞培养板中，每孔加入100μL含0.5％ACF supplement(CAT#3820，STEMCELL Technologies)+0.5％Anti-Clumping agent CD CHO培养基，开始minipool的筛选；铺板第14天后观察细胞形成状况，细胞覆盖率大于30％以后采用基于Octet的方法测定上清中蛋白表达量，将其中表达量较好的细胞群从96孔细胞板转移至24孔细胞板，培养至1mL含25μM MSX(CAT#M5379-500MG，SIGMA)+0.5％Anti-Clumping agent的CD CHO培养基中，加压筛选14天后，采用基于Octet的方法测定minipool细胞群上清中蛋白表达量，取表达量较好的细胞群转入125mL细胞培养瓶中，置于细胞培养摇床中培养(摇床培养条件为36.5℃、6.0％CO 2、80％相对湿度、120rpm)；培养培养2-3天后细胞取样计数，采用稀释法进行细胞传代，直至细胞处于指数生长期，接着对其中细胞株进行Fed-Batch培养，培养14天左右后取上清样品采用Protein A纯化，纯化后的样品进行SEC、CE-SDS纯度检测以及糖型检测。最终获得非岩藻糖基化的人源化抗体。以下测试例中，非岩藻糖基化的人源化抗体以“(afuc)”后缀表示，例如：huB7002(afuc)、huB1010(afuc)、huF4011(afuc)和41D12(afuc)分别表示huB7002、huB1010、huF4011和41D12的非岩藻糖基化抗体。
测试例1：Biacore检测抗CD70抗体亲和力实验
用Biacore仪器测试本披露嵌合抗体及人源化抗体对人CD70抗原的亲和力。方法如下：
按照人Fc捕获试剂盒(CAT#BR-1008-39，GE)说明书中所述的方法，将人Fc捕获分子共价偶联于CM5生物传感芯片(CAT#BR-1005-30，GE)上，从而亲和捕获待测抗体。然后于芯片表面流经人His-TNC-CD70抗原(如SEQ ID NO：1所示)，利用Biacore T200仪器实时检测反应信号，从而获得结合和解离曲线。在实验中每个循环解离完成后，用人Fc捕获试剂盒(GE)里配置的再生溶液将生物芯片洗净再生。数据拟合模型采用1:1Model。实验结果见表13：
表13.抗CD70抗体对人CD70抗原的亲和力检测结果
抗体 ka(1/Ms) kd(1/s) KD(M)
CHB1 2.26E+06 7.54E-05 3.34E-11
CHF4 2.23E+06 8.37E-05 3.75E-11
CHB7 5.66E+06 1.70E-04 3.00E-11
huF4025 2.15E+06 8.68E-05 4.03E-11
huF4026 2.44E+06 9.56E-05 3.92E-11
huF4027 2.73E+06 9.13E-05 3.35E-11
huF4028 2.56E+06 1.06E-04 4.12E-11
huF4009 2.51E+06 1.14E-04 4.55E-11
huF4011 3.12E+06 1.45E-04 4.65E-11
huB1025 3.33E+06 1.54E-04 4.62E-11
huB1026 1.91E+06 9.62E-05 5.03E-11
huB1037 3.93E+06 1.60E-04 4.07E-11
huB1038 1.80E+06 1.01E-04 5.61E-11
huB1010 3.67E+06 1.30E-04 3.55E-11
huB7002 6.09E+06 1.74E-04 2.86E-11
huB7004 5.58E+06 1.57E-04 2.82E-11
huB7006 6.41E+06 1.58E-04 2.46E-11
huB7008 4.87E+06 1.69E-04 3.47E-11
huB7010 4.36E+06 1.72E-04 3.96E-11
huB7012 5.99E+06 1.54E-04 2.57E-11
huB7014 4.92E+06 1.82E-04 3.71E-11
huB7016 4.43E+06 1.85E-04 4.18E-11
huB7018 4.12E+06 1.91E-04 4.64E-11
huB7020 5.76E+06 1.59E-04 2.76E-11
huB7022 4.41E+06 1.95E-04 4.42E-11
huB7024 4.41E+06 2.05E-04 4.64E-11
实验结果显示，本披露的抗CD70抗体与人CD70抗原具有高亲和力。
测试例2：抗CD70抗体结合CD70阳性细胞实验
通过流式细胞仪检测抗CD70抗体结合CD70阳性细胞结合活性。方法如下：
将1×10 6cells/mL 786-O细胞(ATCC，CRL-1932)或Raji细胞(ATCC，CCL-86)用1％BSA PBS缓冲液封闭后，加入不同浓度稀释的抗CD70抗体样品孵育1小时。洗两次后，再加入Alexa Fluor 488-羊抗人(H+L)抗体(Invitrogen，CAT#A11013)孵育1h。洗两次后，使用流式细胞仪读取荧光信号值。实验结果见附图1A、1B、2A和2B。
实验结果显示，本披露的抗CD70抗体均能与CD70阳性细胞有效结合，且huB7002及其非岩藻糖基化抗体huB7002(afuc)和huB1010及其非岩藻糖基化抗体huB1010(afuc)的结合能力均比阳性对照更强。
测试例3：抗CD70抗体结合人、猴和小鼠CD70蛋白的ELISA实验
抗CD70抗体的结合力通过抗体与固定在ELISA板上CD70抗原蛋白的结合的量来检测。包被1μg/mL的人CD70-Fc(如SEQ ID NO：2所示)、猴CD70-Fc(如SEQ ID NO：82所示)和小鼠CD70-Fc(如SEQ ID NO：83所示)，孵育后封闭。洗板后加入不同浓度稀释的抗CD70抗体，再洗板后加入辣根过氧化物酶-羊抗人F(ab’)2抗体(Jackson，CAT#109-036-097)，再洗板加入四甲基联苯胺溶液显色，最后加入终止液，在酶标仪上测量OD450值。实验结果见附图3、4、5。
实验结果显示，本披露的抗CD70抗体均能与人CD70抗原和猴CD70抗原结合，而不与小鼠CD70抗原结合。
测试例4：抗CD70抗体阻断CD27结合CD70阳性细胞的实验
抗CD70抗体的细胞阻断活性通过流式细胞仪来检测。将1×10 6cells/mL表达人全长CD70的CHO-S细胞(Invitrogen,R80007)用1％BSA PBS缓冲液封闭后，加入不同浓度稀释的抗CD70抗体样品和生物素标记的CD27-Fc(如SEQ ID NO:84所示)孵育1h。洗两次后，再加入Alexa Fluor 488-streptavidin(Invitrogen，CAT#S11223)孵育1h。洗两次后，使用流式细胞仪读取荧光信号值。实验结果见附图6、7。
实验结果显示，本披露的抗CD70抗体具有对CD27结合CD70阳性细胞的阻断能力。
测试例5：抗CD70抗体抑制CD70/CD27结合介导的IL-8报告系统实验
CD70和CD27结合后，CD27细胞会分泌IL-8，本实验通过检测表达CD27细胞的IL-8分泌情况来检测抗CD70抗体对CD70诱导的CD27信号传导的水平的影响。
收集U266细胞(ATCC，TIB-196)，用含10％FBS(Gibco，CAT#10099-141)的RPMI1640(Gibco，CAT#11875119)重悬，稀释细胞至1×10 6cells/mL。收集HT1080/CD27细胞(表达人全长CD27的HT1080细胞(ATCC，CCL-121))， 用含10％FBS的RPMI1640重悬，稀释细胞至2×10 6cells/mL。
将U266细胞与不同浓度的抗体(其中阴性对照为与CD70抗原无关的IgG蛋白)按1:1的比例，在96孔板(Corning,CAT#3599)中各加入50μL，孵育60分钟(37℃，5％CO 2)后，加入50μL HT1080/CD27细胞。
孵育18小时(37℃，5％CO2)后，收集上清，并进行10倍稀释，然后用IL-8 Elisa试剂盒(欣博盛，CAT#EHC007.96)进行Elisa检测，在酶标仪上测量OD450值。实验结果见附图8、9以及表14，
表14.CD70抗体抑制CD70/CD27结合介导的IL-8分泌实验
抗体 IC50(ng/mL) Imax(％)
huB7002(afuc) 19.8 91.7
huF4011(afuc) 13.0 92.3
huB7002 21.0 98.7
huF4011 14.2 93.8
41D12 23.3 70.9
41D12(afuc) 24.2 71.1
备注：Imax为抗CD70抗体抑制CD27细胞分泌IL-8的最大抑制率
实验结果显示，本披露的抗CD70抗体对HT1080/CD27细胞的IL-8分泌的抑制能力优于对照抗体41D12和41D12(afuc)。说明本披露的抗CD70抗体能通过阻断CD70/CD27结合，有效抑制CD70诱导的CD27信号传导。
测试例6：抗CD70抗体对786-O细胞体外ADCC的实验
收集786-O-Luc细胞(表达荧光素酶的786-O细胞(ATCC，CCL-86))，用Assay Buffer在MEMα(Gibco，CAT#12561-056)基础培养基(含2mM L-Glutamine)中添加12.5％胎牛血清(Gibco，CAT#10099-141)，12.5％马血清(碧云天，CAT#C0262)，0.2mM肌醇(SIGMA，CAT#I7508)，0.02mM叶酸(SIGMA，CAT#F8758)，0.1mM 2-巯基乙醇(MERCK，CAT#M6250-10ML)以及200U/mL重组人IL-2(Peprotech，CAT#200-02-100)重悬，稀释细胞至2×10 5cells/mL。
收集NK92细胞(南京科佰，CBP60980)，用Assay buffer重悬，稀释细胞至1×10 6cells/mL。
将786-O-Luc与不同浓度的抗体按1:1的比例，在96孔板(Corning,CAT#3903)中各加入25μL，孵育30分钟(37℃，5％CO 2)后，加入25μL效应细胞NK92。孵育4小时(37℃，5％CO 2)后，向每孔加入50μL One-Glo reagent(Promega，CAT#E6120)，常温孵育10min，用酶标仪检测发光强度(Luminescence)，实验结果见附图10及表15。
表15.CD70抗体对786-O细胞体外ADCC的实验
Figure PCTCN2021102998-appb-000089
Figure PCTCN2021102998-appb-000090
收集786-O-Luc细胞，用含10％超低IgG胎牛血清(Gibco，CAT#1921005PJ)的RPMI 1640培养基(Gibco，CAT#11875119)重悬，稀释细胞至1×10 5cells/mL。
使用Ficoll(GE，CAT#17-5442-02)从新鲜人血液中分离外周血单核细胞(PBMC)，于RPMI 1640培养基中重悬，稀释细胞至2×10 6cells/mL。
将786-O-Luc与不同浓度的抗体按1:1的比例，在96孔板(Corning,CAT#3903)中各加入50μL，孵育30分钟(37℃，5％CO 2)后，加入50μL效应细胞PBMC。孵育4小时(37℃，5％CO 2)后，向每孔加入50μL One-Glo reagent(Promega，CAT#E6120)，常温孵育10min，用酶标仪检测发光强度。实验结果见附图11及表16。
表16.CD70抗体对786-O细胞体外ADCC的实验
  huB7002(afuc) huB7002 huB1010(afuc) huB1010 huF4011(afuc)
EC50(ng/mL) 0.11 8.32 0.07 7.21 0.08
最大裂解(％) 89.79 78.10 92.03 82.03 90.29
实验结果显示，本披露的抗CD70抗体对786-O细胞体外ADCC的作用较强，非岩藻糖基化的人源化抗体显著提高ADCC的作用。
测试例7：抗CD70抗体对Raji细胞体外CDC的实验
收集Raji细胞(ATCC，CCL-86)后重悬，将细胞以1×10 6cells/mL重悬在含10％超低IgG胎牛血清(Gibco，CAT#1921005PJ)的无酚红RPMI 1640培养基(Gibco，CAT#11835-030)中。随后，将细胞以5×10 4cells/孔(50μL/孔)铺于96孔板(Corning,CAT#3903)中。然后，加入50μL不同浓度的抗体。孵育30分钟(37℃，5％CO 2)后，向每个孔中加入50μL人血清(新鲜提取)。孵育2小时(37℃，5％CO 2)后，向每个孔中加入16.6μL Alamar Blue reagent(Thermo，CAT#DAL1025)，孵育20小时(37℃，5％CO 2)，用FlexStation 3(Molecular Devices)检测发射波长585nm(激发波长570nm)。实验结果见表17以及附图12和附图13所示。
表17.抗CD70抗体对Raji细胞体外CDC的实验结果
Figure PCTCN2021102998-appb-000091
实验结果显示，本披露涉及的抗CD70抗体对Raji细胞体外CDC的作用比对 照抗体41D12更强。
测试例8：抗CD70抗体对786-O和Raji细胞体外ADCP的实验
PBMC使用Ficoll(GE，CAT#17-5442-02)分离自人血，从中使用CD14磁珠(Miltenyi Biotec，CAT#130-050-201)分选CD14 +单核细胞。在含10％FBS(Gibco，CAT#10091148)和50ng/mL M-CSF(Peprotech，CAT#300-25)的RPMI 1640培养基(Gibco，CAT#11875119)中培养7天，使单核细胞分化成为巨噬细胞。实验当天用细胞刮刮下巨噬细胞，收集巨噬细胞。1％BSA PBS缓冲液重悬后以1:4的E:T比例添加使用0.1μM CFSE(羧基荧光素二乙酸丁二酰亚胺酯(carboxyfluorescein diacetate succinimidyl ester)，BD，CAT#565082)标记的786-O细胞(ATCC，CRL-1932)或Raji细胞(ATCC，CCL-86)，并加入不同浓度稀释的抗CD70抗体样品，对靶细胞进行1.5h的吞噬作用。吞噬结束后，用PBS洗两次，加入偶联APC的抗人CD14抗体(Ebioscience，CAT#17-0149-42)，冰上孵育30min后，用PBS洗2次。最后通过流式细胞术进行分析。通过在CD14 +阳性的活细胞门上评估CFSE +阳性细胞的百分比的方式，测量吞噬作用。实验结果见附图14A和图14B。
实验结果显示，本披露的抗CD70抗体对786-O细胞和Raji细胞具有良好的体外ADCP作用。
测试例9：抗CD70抗体对Treg细胞体外的抑制实验
收集U266细胞(ATCC，TIB-196)，用含10％FBS(Gibco，CAT#10099-141)的RPMI1640(Gibco，CAT#11875119)重悬，稀释细胞至2×10 6cells/mL。
使用Ficoll(GE，CAT#17-5442-02)从新鲜人血液中分离外周血单核细胞(PBMC)，于RPMI 1640培养基中重悬，稀释细胞至2×10 6cells/mL。
将U266细胞与PBMC按1:1的比例，在96孔板(U底板，corning，CAT#3788)中各加入50μL，随后加入25μL不同浓度的抗体与25μL终浓度为3ug/mL的抗CD3(ebioscience，CAT#16-0037-85)与抗CD28(ebioscience，CAT#16-0289-85)抗体，孵育48h(37℃，5％CO 2)后，收集细胞于1.5mL ep管(Axygen，CAT#MCT-150-C-S)，用500μL流式缓冲液清洗一次，加入PerCP-Cy TM5.5Mouse抗人CD4(BD Pharmingen TM，CAT#560650)，CD25单克隆抗体,PE(eBioscience，CAT#12-0257-42)，CD127单克隆抗体,Alexa Fluor 647(eBioscience，CAT#51-1278-42)流式抗体染色(4℃，20min)，用BD FACSVerseTM流式细胞仪(BD Biosciences，651154)分析CD4 +CD25 +CD127 low的Treg细胞比例。实验结果见附图15。
实验结果显示，本披露的抗CD70抗体对Treg细胞具有良好的体外抑制能力。
测试例10：786-O细胞对抗CD70抗体的内化实验
收集786-O-Luc细胞(表达荧光素酶的786-O细胞(ATCC，CRL-1932))， 用含10％超低IgG胎牛血清(Gibco，CAT#1921005PJ)的RPMI 1640培养基(Gibco，CAT#11875119)重悬，稀释细胞至2×10 4细胞/mL。随后，将细胞以1000细胞/孔(50μL/孔)铺于96孔板(Corning,CAT#3903)中。孵育16小时(37℃，5％CO 2)。
用含10％超低IgG胎牛血清的RPMI 1640培养基配制成4倍浓度的DT3C(白喉毒素的Fragment A和G群链球菌的3C片段融合而成，其摩尔浓度为抗体摩尔浓度的6倍)。用相同的培养基配制4倍浓度的抗体，将DT3C和抗体按照1:1的体积混匀，室温下静置孵育30min。然后进行浓度梯度稀释。将稀释好的抗体按照1：1的比例加入细胞中，50μL/孔。孵育3天(37℃，5％CO 2)。向每孔加入50μL CellTiter-Glo Luminescent Cell Viability Assay(Promega，CAT#G7573)，常温孵育10min，用酶标仪检测发光强度。实验结果见附图16及表18。
表18. 786-O细胞对抗CD70抗体的内化实验结果
抗体 huB7002(afuc) huB7002 41D12(afuc)
EC50(nM) 8.65 13.26 55.46
最大裂解％ 96.45 97.18 63.59
实验结果显示，本披露的抗CD70抗体能被786-O细胞内化，细胞内化裂解的最大裂解率超过96％。
测试例11：抗CD70抗体在小鼠Raji模型的体内药效实验
1、抗CD70抗体在小鼠Raji模型的体内药效实验
将Luc-Raji细胞(表达荧光素酶的Raji细胞(ATCC，CCL-86))(1×10 6cells)200μL通过尾静脉注射CB17 SCID小鼠(购自维通利华)，接种7天后，每只小鼠腹腔注射生物发光底物(15mg/mL)，按照10mL/kg体积注射，通过异氟烷麻醉，注射10分钟后通过小动物成像系统拍照成像，去除体重、生物发光信号(Total Flux)值过大和过小的，按生物发光信号将小鼠随机分为4组，包括阴性对照IgG(CD70靶点无关的IgG蛋白，给药剂量30mg/kg)组、阳性对照41D12(给药剂量10mg/kg)、huB7002(给药剂量10mg/kg)、huF4011-10(给药剂量10mg/kg)，每组8只，分组当天开始通过腹腔注射抗体给药，每周给药2次，共2周。每周拍照成像2次，称体重，记录数据。使用Excel统计软件记录数据，生物发光信号值为Total Flux(单位，p/s)，平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT(每组动物数)计算；采用GraphPad Prism软件作图，采用Two-way ANOVA对数据进行统计学分析。
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100，其中T、C为实验结束时治疗组和对照组的肿瘤光子数；T0、C0为实验开始时的肿瘤光子数。
抑瘤率TGI(％)＝100-T/C(％)。
实验结果见表19和附图17。
表19.抗CD70抗体在小鼠Raji模型的体内药效实验
分组 总发光(p/s) 抑瘤率 p
   D0 SEM D14 SEM TGI(％) (vs blank)
IgG-30mg/kg 1.33E+07 2.00E+06 4.73E+09 6.64E+08 - -
41D12-10mg/kg 1.33E+07 2.05E+06 1.75E+09 3.81E+08 63％ p<0.001
huB7002-10mg/kg 1.34E+07 2.07E+06 8.92E+08 2.32E+08 81％ p<0.001
huF4011-10mg/kg 1.33E+07 2.25E+06 1.11E+09 4.28E+08 77％ p<0.001
实验结果表明，与阴性对照IgG相比，10mg/kg剂量的抗CD70抗体均能显著抑制Luc-Raji肿瘤模型中肿瘤细胞的生长(p<0.001)，其中huB7002抑瘤率高达81％，阳性对照41D12抑瘤率仅为63％。
2、非岩藻糖基化的抗CD70抗体在小鼠Raji模型的体内药效实验
将Luc-Raji细胞(表达荧光素酶的Raji细胞(ATCC，CCL-86))(1×10 6cells)200μL通过尾静脉注射CB17 SCID小鼠(购自维通利华)，接种7天后，每只小鼠腹腔注射生物发光底物(15mg/mL)，按照10mL/kg体积注射，通过异氟烷麻醉，注射10分钟后通过小动物成像系统拍照成像，去除体重、生物发光信号(Total Flux)值过大和过小的，按生物发光信号将小鼠随机分组，每组8只，分组当天开始通过腹腔注射抗体给药(阴性对照为靶点无关的IgG型蛋白)，给药2次，在第1天和第3天给药。第7天和第14天拍照成像，称体重，记录数据。实验结果见附图18。
实验结果表明，与阴性对照IgG组相比，非岩藻糖基化抗体组均能显著抑制肿瘤的生长(p＜0.001)。在给药后第7天时，huB1010(afuc)在1.5、5、15mg/kg剂量的抑瘤率分别为82％、85％、84％，huB7002(afuc)在1.5、5、15mg/kg剂量的抑瘤率分别为87％、85％、82％，给药后第14天时，各给药组仍具有高的抑瘤活性，例如与阴性对照IgG组相比，huB1010(afuc)5mg/kg剂量组第14天的抑瘤率仍达68％。各剂量组未体现剂量效应，这表明即使1.5mg/kg剂量已经足够充分抑制肿瘤的生长。
测试例12：抗CD70抗体体内药代动力学实验
雄性SD大鼠(购自维通利华)3只/组，静脉注射给药，给药剂量为3mpk，给药组于给药前及给药后5min(min表示分钟)，8h(h表示小时)，1d(d表示天)，2d，4d，7d，10d，14d，21d，28d采集全血0.15mL，不加抗凝，取血后在4℃放置30min，1000g离心15min，取上清(血清)置于EP管中，于-80℃保存。根据测试例3中CD70抗体结合CD70蛋白的ELISA实验中的方法作不同样品的标准曲线，由OD450值换算不同时间CD70抗体于血清中的浓度，所得数据由Phoenix WinNonlin软件分析计算药代动力学相关参数。实验结果见表20。
表20.抗CD70抗体体内药代动力学实验结果
   huB7002 huB1010 41D12
t1/2(d) 17.2 16.2 13.3
Cmax(ug/mL) 58.4 60.6 57.6
AUC 0-t(ug/mL*h) 7568 9444 7005
AUC 0-∞(ug/mL*h) 10788 13401 8851
CL(mL/day/kg) 6.7 5.4 8.2
MRT 0-∞(h) 549.1 542.3 413.3
实验结果显示，huB7002和huB1010均具有良好的大鼠体内药代药代动力学表现：平均t1/2分别为17.2d和16.2d，优于阳性对照41D12的13.3d，提示大鼠体内抗体稳定性良好。

Claims (16)

  1. 一种抗CD70抗体，其包含重链可变区和轻链可变区，其中：
    i)所述重链可变区包含分别如SEQ ID NO:15和SEQ ID NO:17所示的HCDR1和HCDR3，以及如SEQ ID NO:54或SEQ ID NO:16所示的HCDR2；所述轻链可变区包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；
    ii)所述重链可变区包含分别如SEQ ID NO:9和SEQ ID NO:11所示的HCDR1和HCDR3，以及如SEQ ID NO:10或SEQ ID NO:42所示的HCDR2；所述轻链可变区包含分别如SEQ ID NO:12和SEQ ID NO:14所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；或
    iii)所述重链可变区包含分别如SEQ ID NO:21和SEQ ID NO:23所示的HCDR1和HCDR3，以及如SEQ ID NO:22或SEQ ID NO:71所示的HCDR2；所述轻链可变区包含分别如SEQ ID NO:24和SEQ ID NO:25所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；
    优选地，
    i)所述重链可变区包含分别如SEQ ID NO:15、SEQ ID NO:54和SEQ ID NO:17所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的LCDR1、LCDR2和LCDR3；
    ii)所述重链可变区包含分别如SEQ ID NO:9、SEQ ID NO:42和SEQ ID NO:11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:12、SEQ ID NO:43和SEQ ID NO:14所示的LCDR1、LCDR2和LCDR3；或
    iii)所述重链可变区包含分别如SEQ ID NO:21、SEQ ID NO:71和SEQ ID NO:23所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:24、SEQ ID NO:43和SEQ ID NO:25所示的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗CD70抗体，其中所述抗CD70抗体是鼠源抗体、嵌合抗体或人源化抗体。
  3. 根据权利要求1或2所述的抗CD70抗体，其中所述抗CD70抗体为人源化抗体，所述人源化抗体包含人抗体的框架区或人抗体的框架区变体，所述框架区变体相对于人抗体的轻链框架区和/或重链框架区分别具有至多11个氨基酸的回复突变；
    优选地，所述人源化抗体包含：
    a)重链可变区，其包含分别如SEQ ID NO:15和SEQ ID NO:17所示的HCDR1和HCDR3，以及如SEQ ID NO:54或SEQ ID NO:16所示的HCDR2；和轻链可变区，其包含分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的 LCDR1、LCDR2和LCDR3；且
    所述轻链可变区包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含选自38R、43S、69R、70Q和71Y中的一个或更多个氨基酸回复突变，和/或所述重链可变区包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自2I、24T、46K、72E和82a N中的一个或更多个氨基酸回复突变；或
    b)重链可变区，其包含分别如SEQ ID NO:9和SEQ ID NO:11所示的HCDR1和HCDR3，以及如SEQ ID NO:10或SEQ ID NO:42所示的HCDR2；和轻链可变区，其包含分别如SEQ ID NO:12和SEQ ID NO:14所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；且
    所述重链可变区包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自4M、37I、38K、48I、67A、69L、71A、73R、78A、80L和94T中的一个或更多个氨基酸回复突变；和/或所述轻链可变区包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含选自5S和70N中的一个或更多个氨基酸回复突变；或
    c)重链可变区，其包含分别如SEQ ID NO:21和SEQ ID NO:23所示的HCDR1和HCDR3，以及如SEQ ID NO:22或SEQ ID NO:71所示的HCDR2；和轻链可变区，其包含分别如SEQ ID NO:24和SEQ ID NO:25所示的LCDR1和LCDR3，以及如SEQ ID NO:13或SEQ ID NO:43所示的LCDR2；且
    所述重链可变区包含人抗体的重链框架区变体，其相对于人抗体的重链框架区包含选自27D、30P、37L、38K、48I、66K、67A、69L和82a N中的一个或更多个氨基酸回复突变，和/或所述轻链可变区包含人抗体的轻链框架区变体，其相对于人抗体的轻链框架区包含49S氨基酸回复突变。
  4. 根据权利要求1或2所述的抗CD70抗体，其包含重链可变区和轻链可变区，其中：
    d)所述重链可变区，其氨基酸序列与SEQ ID NO:5、44、45、46、51、52或53具有至少90％的序列同一性，和/或所述轻链可变区，其氨基酸序列与SEQ ID NO:6、47、48、49或50具有至少90％的序列同一性；或
    e)所述重链可变区，其氨基酸序列与SEQ ID NO:3、26、27、28、29、30、31、34、35、36、37、38或39具有至少90％的序列同一性，和/或所述轻链可变区，其氨基酸序列与SEQ ID NO:4、32、33、40或41具有至少90％的序列同一性；或
    f)所述重链可变区，其氨基酸序列与SEQ ID NO:7、55、56、57、58、59、60、63、64、65、66、67或68具有至少90％的序列同一性，和/或所述轻链可变区，其氨基酸序列与SEQ ID NO:8、61、62、69或70具有至少90％的序列同一性；
    优选地，所述的抗CD70抗体包含重链可变区和轻链可变区，其中：
    g)所述重链可变区如SEQ ID NO:44、45、46、51、52或53所示；和所述轻链可变区序列如SEQ ID NO:47、48、49或50所示；或
    h)所述重链可变区如SEQ ID NO:26、27、28、29、30、31、34、35、36、37、38或39所示；和所述轻链可变区如SEQ ID NO:32、33、40或41所示；或
    i)所述重链可变区如SEQ ID NO:5所示；和所述轻链可变区如SEQ ID NO:6所示；或
    j)所述重链可变区如SEQ ID NO:3所示；和所述轻链可变区如SEQ ID NO:4所示；或
    k)所述重链可变区如SEQ ID NO:7所示；和所述轻链可变区如SEQ ID NO:8所示；或
    l)所述重链可变区如SEQ ID NO:55、56、57、58、59、60、63、64、65、66、67或68所示；和所述轻链可变区如SEQ ID NO:61、62、69或70所示；
    更优选地，所述抗CD70抗体包含如下所示的重链可变区和轻链可变区：
    n)所述重链可变区如SEQ ID NO:51所示；和所述轻链可变区如SEQ ID NO:47所示；或
    o)所述重链可变区如SEQ ID NO:35所示；和所述轻链可变区如SEQ ID NO:40所示；或
    p)所述重链可变区如SEQ ID NO:65所示；和所述轻链可变区如SEQ ID NO:70所示。
  5. 根据权利要求1至4中任一项所述的抗CD70抗体，其中所述抗CD70抗体包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含如SEQ ID NO:72所示的重链恒定区和如SEQ ID NO:73所示的轻链恒定区。
  6. 根据权利要求1至5中任一项所述的抗CD70抗体，其包含：
    q)与SEQ ID NO:74具有至少85％序列同一性的重链，和/或与SEQ ID NO:75具有至少85％序列同一性的轻链；或
    r)与SEQ ID NO:76具有至少85％序列同一性的重链，和/或与SEQ ID NO:77具有至少85％同一性的轻链；或
    s)与SEQ ID NO:78具有至少85％序列同一性的重链，和/或与SEQ ID NO:79具有至少85％同一性的轻链；
    优选地，所述的抗CD70抗体包含：
    t)如SEQ ID NO:74所示的重链和如SEQ ID NO:75所示的轻链；或
    u)如SEQ ID NO:76所示的重链和如SEQ ID NO:77所示的轻链；或
    v)如SEQ ID NO:78所示的重链和如SEQ ID NO:79所示的轻链。
  7. 一种分离的抗CD70抗体，其中所述抗CD70抗体与权利要求1至6中任一项所述的抗CD70抗体竞争性结合人CD70或猴CD70。
  8. 根据权利要求1至7中任一项所述的抗CD70抗体，其中所述的抗CD70抗体为低岩藻糖基化抗体；优选地，所述低岩藻糖基化抗体为至少80％、85％、90％、95％或100％的抗体重链未被岩藻糖糖基化修饰的抗体；更优选地，所述抗体为100％的抗体重链未被岩藻糖糖基化修饰的IgG1抗体。
  9. 根据权利要求1至8中任一项所述的抗CD70抗体，所述抗CD70抗体具有以下特征中的至少一种：
    A.所述抗CD70抗体以小于1×10 -8M，优选小于1×10 -9M，或小于1×10 -10M，或小于6×10 -11M的KD值与人CD70结合，所述KD值通过表面等离子共振技术测定；
    B.所述抗CD70抗体既能与人CD70抗原结合，也能与猴CD70抗原结合，但不与小鼠CD70抗原结合；
    C.所述抗CD70抗体能抑制CD70诱导的CD27信号传导，优选地，所述抗CD70抗体抑制表达人CD27细胞分泌IL-8的最大抑制百分比大于72％、90％、91％、93％或98％；
    D.所述抗CD70抗体具有以下的一个或更多个效应子功能：对表达人CD70细胞的抗体依赖性细胞介导的细胞毒性、补体依赖性细胞毒性和抗体依赖性细胞介导的吞噬作用；或
    E.所述抗CD70抗体能被表达人CD70的细胞内化。
  10. 核酸分子，其编码权利要求1至9中任一项所述的抗CD70抗体。
  11. 宿主细胞，其包含权利要求10所述的核酸分子，优选地，所述宿主细胞为微生物、植物或动物细胞宿主细胞；更优选地，所述宿主细胞为敲除了Glul和Fut8基因的宿主细胞。
  12. 一种药物组合物，其含有治疗有效量的根据权利要求1至9中任一项所述的抗CD70抗体，或根据权利要求10所述的核酸分子，以及一种或更多种药学上可接受的载体、稀释剂或赋形剂。
  13. 一种免疫偶联物，其包含：权利要求1至9中任一项所述的抗CD70抗体 和效应分子，其中，所述效应分子偶联至所述抗CD70抗体；优选地，所述效应分子选自由放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶和金属离子组成的组中的一种或更多种。
  14. 一种用于免疫检测或测定CD70的方法，所述方法包括使权利要求1至9中任一项所述的抗CD70抗体接触受试者或来自受试者的样品的步骤。
  15. 一种试剂盒，其包含根据权利要求1至9中任一项所述的抗CD70抗体、或根据权利要求13所述的免疫偶联物。
  16. 一种预防或治疗疾病或病症的方法，所述方法包括向受试者施用治疗有效量的以下物质：
    A.权利要求1至9中任一项所述的抗CD70抗体，
    B.权利要求10所述的核酸分子，
    C.权利要求12所述的药物组合物，或
    D.权利要求13所述的免疫偶联物，
    其中所述疾病或病症优选为肿瘤、自身免疫性疾病或感染性疾病；优选地，所述疾病或病症为与CD70有关的疾病或病症。
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WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024054992A1 (en) 2022-09-09 2024-03-14 Bristol-Myers Squibb Company Methods of separating chelator

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