[go: up one dir, main page]

WO2024179470A1 - 抗dll3抗体、其抗体-药物偶联物及其医药用途 - Google Patents

抗dll3抗体、其抗体-药物偶联物及其医药用途 Download PDF

Info

Publication number
WO2024179470A1
WO2024179470A1 PCT/CN2024/078818 CN2024078818W WO2024179470A1 WO 2024179470 A1 WO2024179470 A1 WO 2024179470A1 CN 2024078818 W CN2024078818 W CN 2024078818W WO 2024179470 A1 WO2024179470 A1 WO 2024179470A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
antibody
acid sequence
cancer
Prior art date
Application number
PCT/CN2024/078818
Other languages
English (en)
French (fr)
Inventor
叶鑫
姚青青
贺峰
Original Assignee
苏州盛迪亚生物医药有限公司
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州盛迪亚生物医药有限公司, 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 苏州盛迪亚生物医药有限公司
Publication of WO2024179470A1 publication Critical patent/WO2024179470A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present disclosure relates to anti-DLL3 antibodies, antibody-drug conjugates thereof, and medical uses thereof.
  • Small cell lung cancer is a relatively malignant type of lung cancer, accounting for 10%-15% of all lung cancer cases. Small cell lung cancer tumors grow rapidly, are easy to metastasize, and have a 5-year survival rate of less than 7%.
  • Platinum/etoposide chemotherapy is often used to treat small cell lung cancer. Patients with small cell lung cancer respond well to chemotherapy in the early stages, but are very likely to develop drug resistance and relapse.
  • Immunotherapy in recent years, such as PD-L1 antibodies and PD1 antibodies has a certain effect on small cell patients, but the effective rate is about 15%. At present, specific targeted therapy drugs have not yet been developed.
  • DLL3 is a ligand that inhibits Notch. Under normal conditions, DLL3 is located on the Golgi apparatus. In cancer cells (such as small cell lung cancer cells), DLL3 goes to the cell surface and binds to Notch in a cis manner, hindering cell-cell binding and Notch endocytosis in target cells, thereby inhibiting the Notch signaling pathway and promoting the growth of tumor cells. DLL3 is mainly expressed in neural or neuroendocrine tumors, including SCLC, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, small cell bladder cancer, multiforme glioma, metastatic castration-resistant prostate cancer, melanoma, etc. Especially in SCLC, more than 80% of SCLC express DLL3, which is not expressed in normal lung cancer tissues and adjacent tissues. This difference in expression makes DLL3 a very potential therapeutic target for the treatment of SCLC.
  • Exotecan toxin is a camptothecin derivative that inhibits topoisomerase I and can selectively inhibit DNA replication in proliferating tumor cells.
  • exotecan toxin has good membrane permeability and can penetrate the killed cancer cells and continue to kill adjacent cancer cells, which has a clear bystander effect in clinical practice.
  • Antibodies are connected to biologically active drugs through a linker to obtain antibody-drug conjugates (ADCs).
  • ADCs fully utilize the specificity of antibodies for binding to surface antigens of normal cells and tumor cells and the high efficiency of drugs (such as cytotoxic agents), while avoiding the defects of low efficacy of antibodies and excessive toxic side effects of drugs.
  • drugs such as cytotoxic agents
  • antibody-drug conjugates can kill tumor cells more accurately and reduce the impact on normal cells.
  • the present disclosure relates to anti-DLL3 antibodies, antibody-drug conjugates thereof and medical uses thereof. More specifically, the present disclosure provides ADCs of anti-DLL3 antibodies of various novel sequences conjugated with exitecan toxoid.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof having a structure as shown in the general formula Pc-LYD:
  • Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 57; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27;
  • SEQ ID NO: 57 is represented by PLYX 1 YGRSYNX 2 VAY, wherein X 1 is Y or H; X 2 is A or G; or
  • the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 16; HCDR2 comprising the amino acid sequence of SEQ ID NO: 17; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 18; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 19; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 20; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 21;
  • Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-; wherein Ra and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen and a C 1-6 alkyl group;
  • R1 is a halogenated C1-6 alkyl or C3-6 cycloalkyl
  • R2 is selected from a hydrogen atom, a halogenated C1-6 alkyl group and a C3-6 cycloalkyl group;
  • R1 and R2 together with the carbon atom to which they are attached form a C3-6 cycloalkyl group
  • n is an integer from 0 to 4.
  • n 1 to 10 (including integers and decimals);
  • L is a connector
  • n is the average number of drug modules per antibody, which can be an integer or a decimal.
  • n is 1-10, or 2-10, or 3-10, or 4-10, or 5-10, or 6-10, or 7-10, or 8-10, or 1-9, or 2-9, or 3-9, or 4-9, or 5-9, or 6-9, or 7-9, or 1-8, or 2-8, or 3-8, or 4-8, In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 24, 30 or 31; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27.
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 24; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27.
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 30; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27.
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 31; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein
  • the anti-DLL3 antibody is a mouse antibody, a chimeric antibody or a humanized antibody.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a human immunoglobulin framework region (FR region).
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 22; HCDR2 comprising the amino acid sequence of SEQ ID NO: 23; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 57; wherein SEQ ID NO: 57 is represented by PLYX 1 YGRSYNX 2 VAY, wherein X 1 is Y or H; X 2 is A or G; and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 49A and 94S; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27; and the FR of the light chain variable region comprises a 43I back mutation.
  • the above back mutation sites are based on the Kabat numbering convention.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 16; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 17; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 18; and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 27Y, 30T, 38K, 43K, 48I, 67A, 68A, 69L, 71V, 73K, 75S, 76N and 93A; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 19; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 20; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 21; and the FR of the light chain variable region comprises one or more back mutations selected from 36L, 43S, 44F, 46G, 69A, 71Y and 85D.
  • the above back mutation sites are based on the Kabat numbering convention.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 16; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 17; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 18; and the FR of the heavy chain variable region comprises back mutations of 1E, 68A, 69L, 71V, 73K, 75S, and 76N; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 19; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 20; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 21; and the FR of the light chain variable region comprises back mutations of 36L, 46G, 69A, 71Y and 85D.
  • the above back mutation sites are based on the Kabat numbering convention.
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 24; and the FR of the heavy chain variable region comprises back mutations of 1E and 94S; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27.
  • LCDR1 which comprises the amino acid sequence of SEQ ID NO: 25
  • LCDR2 which comprises the amino acid sequence of SEQ ID NO: 26
  • LCDR3 which comprises the amino acid sequence of SEQ ID NO: 27.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 24, 30 or 31; and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 49A and 94S; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27; and the FR of the light chain variable region comprises a 43I back mutation.
  • the above back mutation sites are based on the Kabat numbering convention.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 24; and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 49A and 94S; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27; and the FR of the light chain variable region comprises a 43I back mutation.
  • the above back mutation sites are based on the Kabat numbering convention.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 30; and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 49A and 94S; and
  • the light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 25; LCDR2 comprising the amino acid sequence of SEQ ID NO: 26; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 27.
  • the amino acid sequence of the light chain variable region is as follows: and the FR of the light chain variable region comprises a 43I back mutation. The back mutation sites are numbered according to the Kabat convention.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 31; and the FR of the heavy chain variable region comprises one or more back mutations selected from 1E, 49A and 94S; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27; and the FR of the light chain variable region comprises a 43I back mutation.
  • the above back mutation sites are based on the Kabat numbering convention.
  • the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14, 50, 51, 52, 53 or 54; and/or
  • the light chain variable region comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15, 55 or 56; or
  • the heavy chain variable region comprises a sequence that has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44; and/or
  • the light chain variable region comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 13, 45, 46, 47, 48 or 49.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:
  • the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14; and/or
  • the light chain variable region comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15; or
  • the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 50, 51, 52, 53 or 54; and/or
  • the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 55 or 56; or
  • the heavy chain variable region comprises a heavy chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 11 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and/or
  • the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13; or
  • the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44; and/or
  • the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 45, 46, 47, 48 or 49.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:
  • the heavy chain variable region comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 50; and/or
  • the light chain variable region comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 55; or
  • the heavy chain variable region comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43; and/or
  • the light chain variable region comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 48.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or
  • the heavy chain variable region comprises any one of the amino acid sequences selected from SEQ ID NO: 50, 51, 52, 53 and 54, and/or the light chain variable region comprises the amino acid sequence of 55 or 56; or
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and/or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or
  • the heavy chain variable region comprises any amino acid sequence selected from SEQ ID NO: 43, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 44, and/or the light chain variable region comprises any amino acid sequence selected from SEQ ID NO: 48, 45, 46, 47 and 49.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55; or
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 43, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 48.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein
  • the anti-DLL3 antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof wherein the anti-DLL3 antibody is an antibody fragment, wherein the antibody fragment is Fab, Fab', F(ab') 2 , Fab'-SH, Fd, Fv, scFv, dsFv, diabody or domain antibody.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a light chain constant region and a heavy chain constant region.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a constant region of IgG1, IgG2, IgG3 or IgG4.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a constant region of a ⁇ chain or a ⁇ chain.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises a heavy chain constant region of IgG1 and a light chain constant region of a kappa chain.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:
  • the heavy chain constant region contains the sequence of SEQ ID NO: 28, and/or the light chain constant region contains the sequence of SEQ ID NO: 29.
  • the heavy chain comprises a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 60, and/or the light chain comprises a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 61; or
  • the heavy chain comprises a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 58, and/or the light chain comprises a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 59.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody comprises:
  • the heavy chain comprises the sequence shown in SEQ ID NO: 60, and/or the light chain comprises the sequence shown in SEQ ID NO: 61; or
  • the heavy chain comprises the sequence shown in SEQ ID NO: 58, and/or the light chain comprises the sequence shown in SEQ ID NO: 59.
  • the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the anti-DLL3 antibody has at least one of the following properties:
  • the KD value of the anti-DLL3 antibody binding to human DLL3 or its epitope is ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 0.9 nM, ⁇ 0.8 nM, ⁇ 0.7 nM, ⁇ 0.6 nM, ⁇ 0.5 nM or ⁇ 0.4 nM, the KD value is determined by Biacore;
  • the anti-DLL3 antibody binds to H1184 cells expressing DLL3 with an EC50 ⁇ 3 nM, EC50 ⁇ 2 nM, EC50 ⁇ 1 nM, EC50 ⁇ 0.5 nM, EC50 ⁇ 0.2 nM, EC50 ⁇ 0.1 nM, EC50 ⁇ 0.09 nM, EC50 ⁇ 0.08 nM, EC50 ⁇ 0.07 nM or EC50 ⁇ 0.06 nM, and the EC50 is detected by FACS;
  • the anti-DLL3 antibody is capable of being internalized by a cell expressing DLL3;
  • the anti-DLL3 antibody binds to DLL3 or an epitope thereof with an EC50 of ⁇ 0.1 nM, ⁇ 0.09 nM, ⁇ 0.08 nM, ⁇ 0.07 nM, ⁇ 0.06 nM, ⁇ 0.05 nM, or ⁇ 0.04 nM, and the EC50 is detected by ELISA;
  • the anti-DLL3 antibody recognizes a different DLL3 epitope than the positive antibody (eg, BI-764532).
  • the O-end of Y is connected to L.
  • the L is -L 1 -L 2 -L 3 -L 4 -, wherein:
  • L 1 is selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-6 alkylene, C 1-6 alkylene-C 3-6 cycloalkyl, wherein the C 1-6 alkylene, C 1-6 alkylene-C 3-6 cycloalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O) p CH 2 C(O)-, and a chemical bond, wherein p is an integer from 1 to 20;
  • L3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acids are selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH 2 ) t -, and a chemical bond, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group;
  • R6 and R7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.
  • the L is -L 1 -L 2 -L 3 -L 4 -, wherein:
  • L1 is s1 is an integer from 2 to 8;
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue; preferably, L 3 is a tetrapeptide residue comprising glycine-glycine-phenylalanine-glycine;
  • L 4 is -NH(CH 2 )t-, t is 1 or 2;
  • the L1 end is connected to Pc.
  • the L has the structure shown below:
  • Pc is an anti-DLL3 antibody as described in any of the preceding items; n is 1 to 10.
  • Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: HCDR1, which comprises the amino acid sequence of SEQ ID NO: 22; HCDR2, which comprises the amino acid sequence of SEQ ID NO: 23; and HCDR3, which comprises the amino acid sequence of SEQ ID NO: 24; and
  • the light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 25; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 26; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 27;
  • n 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.
  • Pc is an anti-DLL3 antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 55;
  • n 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61; or
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59;
  • n 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61;
  • n 1 to 10; preferably, n is 3 to 8; more preferably, n is 6 to 8.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61;
  • n 4.43.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61;
  • n 7.32.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, and the light chain comprises the amino acid sequence of SEQ ID NO: 61;
  • n 6.12.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59;
  • n 3.53.
  • Pc is an anti-DLL3 antibody comprising a heavy chain and a light chain, wherein:
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 58, and the light chain comprises the amino acid sequence of SEQ ID NO: 59;
  • n 7.43.
  • the present disclosure further provides a method for preparing an antibody-drug conjugate, which comprises reducing the anti-DLL3 antibody as described in any of the preceding items, and then performing a coupling reaction with the compound represented by (L-Y-D) to obtain the antibody-drug conjugate described in the present disclosure.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the unit dose of the pharmaceutical composition contains 0.1-3000 mg or 1-1000 mg of the antibody-drug conjugate as described above.
  • the present disclosure provides the use of the antibody-drug conjugate or its pharmaceutically acceptable salt as described in any of the preceding items, or a pharmaceutical composition comprising the same as a drug.
  • the antibody-drug conjugate or its pharmaceutically acceptable salt as described in any of the preceding items, or a pharmaceutical composition comprising the same is used as a drug for treating cancer or tumors.
  • the antibody-drug conjugate or its pharmaceutically acceptable salt as described in any of the preceding items, or a pharmaceutical composition comprising the same is used as a drug for treating cancer or tumors expressing DLL3.
  • the present disclosure provides a use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in any of the preceding items, in the preparation of a medicament for treating a DLL3-mediated disease or condition.
  • the DLL3-mediated disease or condition is a tumor or cancer.
  • the DLL3-mediated disease or condition is a disease or condition expressing DLL3.
  • the present disclosure provides the use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing a tumor or cancer; preferably, wherein the tumor or cancer is selected from:
  • Lung cancer e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer
  • head and neck squamous cell carcinoma head and neck cancer
  • brain cancer glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), Liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer,
  • the present disclosure further relates to a method for treating and/or preventing a tumor or cancer, the method comprising administering a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, to a subject in need thereof.
  • the tumor or cancer is a tumor or cancer expressing DLL3.
  • the present disclosure further relates to a method for treating or preventing a tumor or cancer, comprising administering a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition, to a subject in need thereof; wherein the tumor or cancer is selected from:
  • Lung cancer e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer
  • head and neck squamous cell carcinoma head and neck cancer
  • brain cancer glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, adrenal
  • the antibody-drug conjugate provided by the present disclosure has good affinity with cell surface antigens, can be effectively internalized by cells expressing DLL3, and has a strong effect of inhibiting tumor growth while having good safety.
  • Figures 1A to 1C show the binding of Hu6 and Hu100 antibodies to DLL3 of different species.
  • Figure 1A shows the FACS results of Hu6 and Hu100 antibodies binding to H1184 cells
  • Figure 1B shows the FACS results of Hu6 and Hu100 antibodies binding to cynoDLL3/CHO-s cells
  • Figure 1C shows the FACS results of Hu6 and Hu100 antibodies binding to ratDLL3/CHO-s cells.
  • Figure 2 shows the results of competition binding experiments with different anti-DLL3 antibodies. The results show that Hu6 and Hu100 do not compete with BI-764532, indicating that antibodies Hu6 and Hu100 bind to different epitopes than BI-764532.
  • FIG3 shows the results of cellular endocytosis of Hu6 and Hu100 antibodies; the results show that both Hu6 and Hu100 can be endocytosed by cells.
  • Figures 4A to 4B show the binding results of ADC-1, ADC-2 and ADC-3 to human and monkey DLL3 cells.
  • Figure 4A shows the binding results of ADC-1, ADC-2 and ADC-3 to human DLL3 cells;
  • Figure 4B shows the binding results of ADC-1, ADC-2 and ADC-3 to monkey DLL3 cells.
  • Figures 5A to 5D show the growth inhibition effects of ADC-1, ADC-2 and ADC-3 on different cells.
  • Figure 5A shows the growth inhibition results of ADC-1, ADC-2 and ADC-3 on H1184 cells that highly express DLL3
  • Figure 5B shows the growth inhibition results of ADC-1, ADC-2 and ADC-3 on DMS53 cells that highly express DLL3
  • Figure 5C shows the growth inhibition results of ADC-1, ADC-2 and ADC-3 on SK-MEL3 cells that lowly express DLL3
  • Figure 5D shows the growth inhibition results of ADC-1, ADC-2 and ADC-3 on CHO-K1 cells that do not express DLL3.
  • FIG6 shows the bystander cytotoxicity effect results of ADC-1, ADC-2, and ADC-3.
  • FIG. 7 shows the results of ADC-1, ADC-2, ADC-4 and ADC-5 inhibiting the growth of subcutaneously transplanted tumors of DMS53 cells in mice.
  • FIG. 8 shows the results of ADC-1 and ADC-3 inhibiting the growth of subcutaneously transplanted DMS53 cell tumors in mice.
  • FIG. 9 shows the results of ADC-2, ADC-3, ADC-6 and ADC-7 inhibiting the growth of H1184 cell subcutaneous transplanted tumors in mice.
  • Figures 10A to 10C show the pharmacokinetic results of ADC-1, ADC-2 and ADC-3 in rats.
  • Figure 10A shows the pharmacokinetic results of ADC-1 in rats;
  • Figure 10B shows the pharmacokinetic results of ADC-2 in rats;
  • Figure 10C shows the pharmacokinetic results of ADC-3 in rats.
  • FIG. 11 shows the pharmacokinetic results of ADC-2 in cynomolgus monkeys.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.
  • amino acid mutation includes amino acid substitution (also known as amino acid replacement), deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired properties, such as reduced or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain.
  • Specific amino acid mutations can be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, that is, an amino acid is replaced with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include replacement by non-natural amino acids or by derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering to change amino acid side chain groups, such as chemical modifications, may also be available. Various names can be used herein to indicate the same amino acid mutation.
  • the amino acid residue at a specific position can be represented by position+amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 is replaced by W instead of T.
  • antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • a complete antibody generally comprises two light chains and two heavy chains. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL).
  • full-length antibody “complete antibody” and “whole antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody or having an Fc region in the heavy chain.
  • the natural complete antibody light chain includes a light chain variable region VL and a constant region CL, VL is at the amino terminus of the light chain, and the light chain constant region includes a ⁇ chain and a ⁇ chain;
  • the heavy chain includes a variable region VH and a constant region (CH1, CH2 and CH3), VH is at the amino terminus of the heavy chain, and the constant region is at the carboxyl terminus, wherein CH3 is closest to the carboxyl terminus, and the heavy chain may belong to any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
  • IgG including IgG1, IgG2, IgG3 and
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in the antibody binding to an antigen.
  • the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved framework regions.
  • CDR complementarity determining region
  • VH contains three CDR regions: HCDR1, HCDR2 and HCDR3
  • VL contains three CDR regions: LCDR1, LCDR2 and LCDR3.
  • Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the amino acid sequence boundaries of a CDR can be determined by various well-known schemes, such as the "Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia” numbering convention, the "ABM” numbering convention, the "contact” numbering convention (see Martin, A.C. R. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art, and is exemplified as shown in Table 1 below.
  • variable region and CDR sequences in this disclosure are all based on the Kabat numbering convention.
  • antibody fragment refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and remodeled Fc regions.
  • the Fc region comprises two identical or different subunits.
  • the Fc region of the human IgG heavy chain is defined as the amino acid residue at the position of Cys226 or extending from Pro230 to its carboxyl terminus.
  • Suitable Fc regions for antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • the boundaries of the Fc region can also vary, such as the C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system) of the Fc region are missing.
  • the numbering convention of the Fc region is the EU numbering system, also known as the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from another different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the constant region and the framework region portion of the variable region).
  • human antibody “humanized antibody”, “fully human antibody”, and “fully human antibody” are used interchangeably to refer to antibodies whose variable and constant regions are human sequences.
  • the term encompasses antibodies that are derived from human genes but have, for example, sequences that reduce possible immunogenicity, increase affinity, eliminate cysteine or glycosylation sites that may cause undesirable folding, etc.
  • the term encompasses these antibodies that are recombinantly produced in non-human cells (which may confer glycosylation that is not characteristic of human cells).
  • the term also encompasses antibodies that have been raised in transgenic mice containing human immunoglobulin heavy and light chain loci.
  • the meaning of human antibodies explicitly excludes humanized antibodies that contain non-human antigen binding residues.
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, as used herein, binding “affinity” refers to internal binding affinity, which reflects the interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.
  • the term “kassoc” or “ka” refers to the association rate of a specific antibody-antigen interaction
  • kdis or “kd” refers to the dissociation rate of a specific antibody-antigen interaction
  • KD refers to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M).
  • M molar concentration
  • the KD value of an antibody can be measured using methods well known in the art. For example, a biosensor system such as a system measures surface plasmon resonance, or a solution equilibrium titration method (SET) is used to measure affinity in a solution.
  • SET solution equilibrium titration method
  • effector function refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutations) and that vary with the antibody isotype.
  • antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
  • the term “monoclonal antibody” refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for possible natural mutations that are present in small amounts.
  • polyclonal antibody preparations are typically comprised of a variety of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes.
  • “Monoclonal” should not be interpreted as requiring the production of antibodies by any particular method.
  • the antibody provided by the present disclosure is a monoclonal antibody.
  • antigen refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody).
  • An antigen may have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).
  • epitope refers to an area or region on an antigen that is capable of specific binding to an antibody or antigen-binding fragment thereof.
  • An epitope may be formed by a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example brought into spatial proximity by folding of the antigen (i.e., by tertiary folding).
  • Conformational epitopes and linear epitopes are The difference between a conformational epitope and a conformational epitope is that in the presence of a denaturing solvent, no antibody binding to a conformational epitope can be detected.
  • An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind to a specific epitope can be performed using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.
  • anti-DLL3 antibody and “antibody that binds to DLL3” refer to an antibody that is capable of binding to DLL3 or an epitope thereof with sufficient affinity.
  • the extent of binding of the anti-DLL3 antibody to an unrelated protein is less than at least about 10% of the binding of the antibody to DLL3, and the binding can be Surface plasmon resonance measurements.
  • the term “capable of specific binding”, “specific binding” or “binding” means that the antibody is able to bind to an antigen or its epitope with a higher affinity than other antigens or epitopes.
  • the antibody binds to the antigen or its epitope with an equilibrium dissociation constant (KD) of about 1 ⁇ 10-7 M or less (e.g., about 1 ⁇ 10-8 M or less).
  • KD equilibrium dissociation constant
  • the KD of the antibody binding to the antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein).
  • KD can be measured using known methods, such as by However, an antibody that specifically binds to an antigen or an epitope thereof may have cross-reactivity to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or marmoset (Callithrix jacchus) (common marmoset, marmoset).
  • homologous such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or marmoset (Callithrix jacchus) (common marmoset, marmoset).
  • nucleic acid is used interchangeably with the term “polynucleotide” herein, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections.
  • the nucleic acids are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides.
  • Isolated nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which cells typically contain the nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or are present at a different natural chromosome position.
  • the isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or a separate vector, and such one or more nucleic acid molecules present in one or more positions in a host cell.
  • a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as suitable Applicable to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity refers to the degree (percentage) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions; when two sequences are optimally aligned, gaps are introduced, if necessary, to obtain the maximum percent identity, and any conservative substitutions are not considered part of the sequence identity.
  • percent sequence identity alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 Megalign
  • vector means a polynucleotide molecule capable of transporting another polynucleotide connected thereto.
  • plasmid refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected.
  • viral vector such as an adeno-associated virus vector (AAV or AAV2), wherein additional DNA segments can be connected to the viral genome.
  • AAV adeno-associated virus vector
  • Some vectors can replicate autonomously in the host cell in which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins).
  • vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell and contains a nucleic acid sequence for guiding and/or controlling the expression of one or more heterologous coding regions operably connected thereto. Expression constructs may include, but are not limited to, affecting or controlling transcription, translation, and affecting the sequence of RNA splicing of the coding region operably connected thereto when introns are present.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and their derived progeny, without considering the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Such mutant progeny are included herein, which have the same function or biological activity as the cells screened or selected in the initial transformed cells.
  • Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells and fungal cells.
  • Exemplary host cells are as follows: Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells and HEK-293 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei.
  • ADC antibody-drug conjugate
  • Drug is any substance with biological or detectable activity (e.g., therapeutic agent, detectable marker, binding agent, etc.) and prodrugs that are metabolized into active agents in vivo.
  • therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulators.
  • Chemotherapeutic agents are agents that can be used to Chemical compounds for the treatment of cancer.
  • Representative therapeutic agents include cytotoxins, cytotoxic agents, and cytostatic agents.
  • a cytotoxic effect refers to the deletion, elimination and/or killing of target cells.
  • a cytotoxic agent refers to an agent that has a cytotoxic and/or cytostatic effect on cells.
  • a cytostatic effect refers to the inhibition of cell proliferation.
  • a cytostatic agent refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and/or expansion of a specific subset of cells.
  • Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents and cell lytic enzymes (e.g., RNAse). These drug descriptors are not mutually exclusive, and therefore, one or more of the above terms can be used to describe therapeutic agents.
  • the selected radioisotope is also a cytotoxin.
  • the therapeutic agent can be prepared as a pharmaceutically acceptable salt, acid or derivative of any of the above.
  • a conjugate with a radioisotope as a drug is referred to as a radioimmunoconjugate
  • a conjugate with a chemotherapeutic agent as a drug is referred to as a chemical immunoconjugate.
  • cytotoxic agents include, but are not limited to, exotecan, anthracyclines, auristatins, CC-1065, dolastatin, duocarmycin, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysin, hemiasterlin, eribulin, trabectedin, lurbinectedin, and stereoisomers, isosteres, analogs or derivatives thereof.
  • Chemotherapeutic agents, plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioactive isotopes, photosensitizers (i.e., for photodynamic therapy) may also be used.
  • label refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to generate a "labeled" antibody.
  • the label can be self-detectable (e.g., a radioisotope label or a fluorescent label), or in the case of an enzymatic label, the label can catalyze a detectable chemical change in a substrate compound or composition.
  • Radioisotope labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109.
  • the label can also be an undetectable entity, such as a toxin.
  • linker unit refers to a chemical structure fragment or bond that is connected to an antibody at one end and to a drug at the other end, and may also be connected to other linkers before being connected to an antibody or a drug. Attachment of a linker to an antibody can be accomplished in a variety of ways, such as via surface lysine, reductive coupling to oxidized carbohydrates, cysteine residues released by reduction of interchain disulfide bonds, reactive cysteine residues engineered at specific sites, and tags containing acyl donor glutamine or by engineering a polypeptide in the presence of transglutaminase and an amine to provide reactive endogenous glutamine.
  • a variety of ADC linking systems are known in the art, including hydrazone-, disulfide-, and peptide-based linkages.
  • the linker may comprise one or more linker elements.
  • exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (“SMCC”, also referred to herein as "MCC”), and N-succinimidyl (4-iodo- acetyl)aminobenzoate (“SIAB”).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • val-cit valine-citrulline
  • the linker can be selected from the following elements or combinations thereof: an extender, a spacer, and an amino acid unit.
  • the linker can be synthesized by methods known in the art, such as those described in US20050238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • an acid-labile linker e.g., a hydrazone
  • a protease-sensitive e.g., a peptidase-sensitive
  • a photolabile linker a dimethyl linker
  • disulfide-containing linker can be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).
  • Connector components include but are not limited to:
  • MC 6-maleimidocaproyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker),
  • Citrulline 2-amino-5-ureidopentanoic acid
  • PAB p-aminobenzyloxycarbonyl (an example of a "self-immolative” linker element)
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B),
  • MC(PEG)6-OH Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine),
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • L-D is the linker-drug moiety resulting from the attachment of a drug (D) to a linker (L).
  • Drug loading also known as the drug-to-antibody ratio (DAR) is the average number of drugs coupled to each antibody in the ADC. It can be, for example, in the range of about 1 to about 10 drugs coupled to each antibody, and in certain embodiments, in the range of about 1 to about 8 drugs coupled to each antibody, preferably in the range of 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8 and 6-8.
  • the ADC formula disclosed herein includes a collection of antibody-drug conjugates within the aforementioned certain range.
  • the drug loading can be expressed as n, which is a decimal or an integer.
  • the drug loading can be determined by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA assay, HIC and RP-HPLC.
  • the drug is coupled to a reactive group (eg, sulfhydryl) of the antibody via a linker.
  • a reactive group eg, sulfhydryl
  • the drug loading of ADC can be controlled by the following non-limiting methods, including:
  • alkyl refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g.
  • the alkyl group preferably has 1 to 12 carbon atoms (i.e. , C 1-12 alkyl), and more preferably has 1 to 6 carbon atoms (i.e., C 1-6 alkyl).
  • Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl
  • the alkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituent is preferably selected from one or more of a D atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.
  • alkylene refers to a divalent alkyl group, wherein the alkyl group is as defined above, and has 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., C1-20 alkylene).
  • the alkylene group is preferably an alkylene group having 1 to 12 carbon atoms (i.e., C1-12 alkylene), and more preferably an alkylene group having 1 to 6 carbon atoms (i.e., C1-6 alkylene).
  • Non-limiting examples include: -CH2- , -CH( CH3 )-, -C( CH3 ) 2- , -CH2CH2-, -CH(CH2CH3)-, -CH2CH(CH3 ) - , -CH2C ( CH3 )2-, -CH2CH2CH2-, -CH2CH2CH2- , -CH2CH2CH2CH2- , etc.
  • the alkylene group may be substituted or unsubstituted, and when substituted , it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a deuterium atom, a halogen, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.
  • alkenyl refers to an alkyl group containing at least one carbon-carbon double bond in the molecule, wherein the definition of alkyl is as described above, and it has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms (i.e., C2-12 alkenyl).
  • the alkenyl group preferably has an alkenyl group of 2 to 6 carbon atoms (i.e., C2-6 alkenyl).
  • Non-limiting examples include: vinyl, propenyl, isopropenyl, butenyl, etc.
  • the alkenyl group can be substituted or unsubstituted, and when substituted, it can be substituted at any available point of attachment, and the substituent is preferably selected from one or more of a deuterium atom, an alkoxy group, a halogen, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.
  • alkynyl refers to an alkyl group containing at least one carbon-carbon triple bond in the molecule, wherein alkyl is as defined above.
  • the alkynyl group is preferably an alkynyl group having 2 to 6 carbon atoms (i.e. , C2-6 alkynyl).
  • Non-limiting examples include: ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc.
  • the alkynyl group may be substituted or unsubstituted, and when substituted, it may be substituted at any available point of attachment, and the substituent is preferably selected from one or more of a deuterium atom, an alkoxy group, a halogen, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.
  • alkoxy refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples include: methoxy, ethoxy, propoxy and butoxy, etc.
  • Alkoxy can be substituted or unsubstituted, and when substituted, it can be substituted at any available point of attachment, and the substituent is preferably selected from one or more of a deuterium atom, a halogen, an alkoxy, a haloalkyl, a haloalkoxy, a cycloalkyloxy, a heterocyclyloxy, a hydroxyl, a hydroxyalkyl, a cyano, an amino, a nitro, a cycloalkyl, a heterocyclyl, an aryl and a heteroaryl.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic all-carbon ring (i.e., monocyclic cycloalkyl) or polycyclic ring system (i.e., polycyclic cycloalkyl) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 3 to 20-membered cycloalkyl).
  • the cycloalkyl is preferably a cycloalkyl having 3 to 12 ring atoms (i.e., 3 to 12-membered cycloalkyl), more preferably a cycloalkyl having 3 to 8 ring atoms (i.e., 3 to 8-membered cycloalkyl), and most preferably a cycloalkyl having 3 to 6 ring atoms (i.e., 3 to 6-membered cycloalkyl, C3-6cycloalkyl ).
  • Non-limiting examples of the monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl and cyclooctyl.
  • the polycyclic cycloalkyl group includes: spirocycloalkyl group, fused cycloalkyl group and bridged cycloalkyl group.
  • spirocycloalkyl refers to a polycyclic system in which one carbon atom (called spiro atom) is shared between the rings, and the rings may contain one or more double bonds, or the rings may contain one or more heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but not including -O-O-, -O-S- or -S-S-), provided that at least one all-carbon ring is contained and the connection point is on the all-carbon ring, and it has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 5 to 20-membered spirocycloalkyl).
  • nitrogen may be optionally oxidized, i.e., to form nitrogen oxides
  • the spirocycloalkyl preferably has 6 to 14 ring atoms (i.e., 6 to 14-membered spirocycloalkyl), and more preferably has 7 to 10 ring atoms (i.e., 7 to 10-membered spirocycloalkyl).
  • the spirocycloalkyl includes monospirocycloalkyl and polyspirocycloalkyl (such as bispirocycloalkyl, etc.), preferably monospirocycloalkyl or bispirocycloalkyl, more preferably 3-yuan/4-yuan, 3-yuan/5-yuan, 3-yuan/6-yuan, 4-yuan/4-yuan, 4-yuan/5-yuan, 4-yuan/6-yuan, 5-yuan/3-yuan, 5-yuan/4-yuan, 5-yuan/5-yuan, 5-yuan/6-yuan, 5-yuan/7-yuan, 6-yuan/3-yuan, 6-yuan/4-yuan, 6-yuan/5-yuan, 6-yuan/6-yuan, 6-yuan/7-yuan, 6-yuan/3-yuan, 6-yuan/4-yuan, 6-yuan/5
  • connection point can be at any position
  • fused cycloalkyl refers to a polycyclic system in which two adjacent carbon atoms are shared between the rings, which is a monocyclic cycloalkyl fused to one or more monocyclic cycloalkyls, or a monocyclic cycloalkyl fused to one or more heterocyclyls, aryls or heteroaryls, wherein the point of attachment is on the monocyclic cycloalkyl, which may contain one or more double bonds within the ring, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 5 to 20-membered fused cycloalkyl).
  • the fused cycloalkyl preferably has 6 to 14 ring atoms (i.e., 6 to 14-membered fused cycloalkyl), and more preferably has 7 to 10 ring atoms (i.e., 7 to 10-membered fused cycloalkyl).
  • the fused cycloalkyl includes bicyclic fused cycloalkyl and polycyclic fused cycloalkyl (such as tricyclic fused cycloalkyl, tetracyclic fused cycloalkyl, etc.), preferably bicyclic fused cycloalkyl or tricyclic fused cycloalkyl, more preferably 3 yuan/4 yuan, 3 yuan/5 yuan, 3 yuan/6 yuan, 4 yuan/4 yuan, 4 yuan/5 yuan, 4 yuan/6 yuan, 5 yuan/3 yuan, 5 yuan/4 yuan, 5 yuan/5 yuan, 5 yuan/6 yuan, 5 yuan/7 yuan, 6 yuan/3 yuan, 6 yuan/4 yuan, 6 yuan/5 yuan, 6 yuan
  • connection points can be at any position
  • bridged cycloalkyl refers to a full carbon polycyclic system that shares two carbon atoms that are not directly connected between the rings, which may contain one or more double bonds in the ring and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) carbon atoms (i.e., 5 to 20-membered bridged cycloalkyl).
  • the bridged cycloalkyl preferably has a bridged cycloalkyl of 6 to 14 carbon atoms (i.e., 6 to 14-membered bridged cycloalkyl), and more preferably has a bridged cycloalkyl of 7 to 10 carbon atoms (i.e., 7 to 10-membered bridged cycloalkyl).
  • the bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic bridged cycloalkyl or tricyclic bridged cycloalkyl.
  • Non-limiting examples include:
  • connection point can be at any position.
  • the cycloalkyl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of D atoms, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., a monocyclic heterocyclyl) or a polycyclic heterocyclic ring system (i.e., a polycyclic heterocyclyl), which contains at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -O-O-, -O-S- or -S-S-), and has 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 3- to 20-membered heterocyclyl).
  • the heterocyclic group is preferably a heterocyclic group having 3 to 12 ring atoms (i.e., a 3- to 12-membered heterocyclic group); further preferably a heterocyclic group having 3 to 8 ring atoms (i.e., a 3- to 8-membered heterocyclic group); more preferably a heterocyclic group having 3 to 6 ring atoms (i.e., a 3- to 6-membered heterocyclic group); and most preferably a heterocyclic group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heterocyclic group).
  • Non-limiting examples of the monocyclic heterocyclic group include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and homopiperazinyl.
  • the polycyclic heterocyclic group includes a spiro heterocyclic group, a fused heterocyclic group and a bridged heterocyclic group.
  • spiroheterocyclyl refers to a polycyclic heterocyclic ring system in which the rings share one atom (called a spiro atom), which may contain one or more double bonds in the ring and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -O-O-, -O-S- or -S-S-), provided that it contains at least one monocyclic heterocyclic group and the point of attachment is on the monocyclic heterocyclic group, which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., a 5- to 20-membered spiroheterocyclyl
  • the spiro heterocyclic radical preferably has a spiro heterocyclic radical of 6 to 14 ring atoms (i.e., a 6 to 14-membered spiro heterocyclic radical), and more preferably has a spiro heterocyclic radical of 7 to 10 ring atoms (i.e., a 7 to 10-membered spiro heterocyclic radical).
  • the spiro heterocyclic radical includes a monospiro heterocyclic radical and a polyspiro heterocyclic radical (such as a bispiro heterocyclic radical, etc.), preferably a monospiro heterocyclic radical or a bispiro heterocyclic radical, more preferably a 3-yuan/4-yuan, 3-yuan/5-yuan, 3-yuan/6-yuan, 4-yuan/4-yuan, 4-yuan/5-yuan, 4-yuan/6-yuan, 5-yuan/3-yuan, 5-yuan/4-yuan, 5-yuan/5-yuan, 5-yuan/6-yuan, 5-yuan/7-yuan, 6-yuan/3-yuan, 6-yuan/4-yuan, 6-yuan/5-yuan, 6-yuan/6-yuan, 6-yuan/7-yuan, 6-yuan/3-yuan, 6-yuan/4-yuan
  • fused heterocyclyl refers to a polycyclic heterocyclic ring system that shares two adjacent atoms between the rings, which may contain one or more double bonds within the ring, and which contains at least one (e.g., 1, 2, 3 or 4) selected from nitrogen, Oxygen and sulfur heteroatoms (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxoed, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-), which are monocyclic heterocyclic groups fused to one or more monocyclic heterocyclic groups, or monocyclic heterocyclic groups fused to one or more of cycloalkyl, aryl or heteroaryl groups, wherein the connection point is on the monocyclic heterocyclic group, and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring
  • the fused heterocyclic group is preferably a fused heterocyclic group having 6 to 14 ring atoms (i.e., 6 to 14-membered fused heterocyclic groups), and more preferably a fused heterocyclic group having 7 to 10 ring atoms (i.e., 7 to 10-membered fused heterocyclic groups).
  • the fused heterocyclic group includes bicyclic and polycyclic fused heterocyclic groups (such as tricyclic fused heterocyclic groups, tetracyclic fused heterocyclic groups, etc.), preferably bicyclic fused heterocyclic groups or tricyclic fused heterocyclic groups, more preferably 3-membered/4-membered, 3-membered/5-membered, 3-membered/6-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/3-membered, 5-membered/4-membered, 5-membered/5-membered, 5-membered/6-membered, 5-membered/7-membered, 6-membered/3-membered, 6-membered/4-membered, 6-membered/5-membered, 6-membered/6-membered, 6-membered/7-membered, 7-membered/5-membered or 7-membered/6-member
  • bridged heterocyclic group refers to a polycyclic heterocyclic ring system that shares two atoms that are not directly connected between the rings, which may contain one or more double bonds in the ring, and which contains at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -OO-, -OS-, or -SS-), and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 5 to 20-membered bridged heterocyclic groups).
  • the bridged heterocyclic group is preferably a bridged heterocyclic group having 6 to 14 ring atoms (i.e., 6 to 14-membered bridged heterocyclic groups), and more preferably a bridged heterocyclic group having 7 to 10 ring atoms (i.e., 7 to 10-membered bridged heterocyclic groups). According to the number of constituent rings, it can be divided into bicyclic bridged heterocyclic groups and polycyclic bridged heterocyclic groups (such as tricyclic bridged heterocyclic groups, tetracyclic bridged heterocyclic groups, etc.), preferably bicyclic bridged heterocyclic groups or tricyclic bridged heterocyclic groups.
  • Non-limiting examples include:
  • the heterocyclic group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of a deuterium atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclic groupoxy group, a hydroxyl group, a hydroxyalkyl group, an oxo group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclic group, an aryl group and a heteroaryl group.
  • aryl refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aromatic group) or a polycyclic aromatic ring system (i.e., a polycyclic aromatic group) having a conjugated ⁇ electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., a 6- to 14-membered aromatic group).
  • the aryl group is preferably an aromatic group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aromatic group).
  • the monocyclic aromatic group is, for example, phenyl.
  • Non-limiting examples of the polycyclic aromatic group include: naphthyl, anthracenyl, phenanthryl, etc.
  • the polycyclic aromatic group also includes a phenyl group fused with one or more heterocyclic groups or cycloalkyl groups, or a naphthyl group fused with one or more heterocyclic groups or cycloalkyl groups, wherein the connection point is on the phenyl group or the naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, and non-limiting examples include:
  • the aryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituents are preferably selected from one or more of D atoms, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • heteroaryl refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl) having a conjugated ⁇ electron system, which contains at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur in the ring (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but does not include -O-O-, -O-S-, or -S-S-), and has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl).
  • a monocyclic heteroaromatic ring i.e., a monocyclic hetero
  • the heteroaryl is preferably a heteroaryl having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl), and more preferably a heteroaryl having 5 or 6 ring atoms (i.e., a 5- or 6-membered heteroaryl).
  • the monocyclic heteroaryl group includes, but is not limited to, furanyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazanyl, pyrrolyl, N-alkylpyrrolyl, pyridyl, pyrimidinyl, pyridonyl, N-alkylpyridone (e.g. etc.), pyrazinyl, pyridazinyl, etc.
  • the polycyclic heteroaryl groups include, but are not limited to, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, phthalazinyl, benzimidazolyl, benzothiophenyl, quinazolinyl, benzothiazolyl, Carbazolyl, etc.
  • the polycyclic heteroaryl group also includes a monocyclic heteroaryl group fused to one or more aromatic groups, wherein the point of attachment is on the aromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system.
  • the polycyclic heteroaryl group also includes a monocyclic heteroaryl group fused to one or more cycloalkyl or heterocyclic groups, wherein the point of attachment is on the monocyclic heteroaromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaromatic ring system.
  • Non-limiting examples include:
  • the heteroaryl group may be substituted or unsubstituted. When substituted, it may be substituted at any available point of attachment, and the substituent is preferably selected from one or more of a deuterium atom, a halogen, an alkyl group, an alkoxy group, a haloalkyl group, a haloalkoxy group, a cycloalkyloxy group, a heterocyclyloxy group, a hydroxyl group, a hydroxyalkyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.
  • amino protecting group refers to a group that is easily removed and introduced on the amino group in order to keep the amino group unchanged when other parts of the molecule are reacted.
  • Non-limiting examples include: (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), methyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl, ethoxycarbonyl, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), trityl (Trt), 2,4-dimethoxybenzyl (DMB), acetyl, benzyl, allyl, p-methoxybenzyl, etc.
  • hydroxy protecting group refers to a group that is introduced on a hydroxy group and is easily removed, and is used to block or protect the hydroxy group while reacting on other functional groups of the compound.
  • Non-limiting examples include: trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), formyl, acetyl, benzoyl, p-nitrobenzoyl, etc.
  • cycloalkyloxy refers to a cycloalkyl-O- group wherein cycloalkyl is as defined above.
  • heterocyclyloxy refers to heterocyclyl-O-, wherein heterocyclyl is as defined above.
  • aryloxy refers to an aryl-O- group in which aryl is as defined above.
  • heteroaryloxy refers to heteroaryl-O-, wherein heteroaryl is as defined above.
  • alkylthio refers to an alkyl-S- group in which alkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
  • haloalkoxy refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxyalkyl refers to an alkyl group substituted with one or more hydroxy groups, wherein alkyl is as defined above.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • hydroxy refers to -OH.
  • thiol refers to -SH.
  • amino refers to -NH2 .
  • cyano refers to -CN.
  • nitro refers to -NO2 .
  • carboxylate refers to -C(O)O(alkyl), -C(O)O(cycloalkyl), (alkyl)C(O)O-, or (cycloalkyl)C(O)O-, wherein alkyl and cycloalkyl are as defined above.
  • THF tetrahydrofuran
  • EtOAc refers to ethyl acetate
  • MeOH refers to methanol
  • DMF N,N-dimethylformamide
  • DIPEA diisopropylethylamine
  • TFA trifluoroacetic acid
  • MeCN refers to acetonitrile
  • DMA refers to N,N-dimethylacetamide.
  • DCE 1,2 dichloroethane
  • DIPEA N,N-diisopropylethylamine
  • NBS N-bromosuccinimide
  • NIS N-iodosuccinimide
  • Cbz-Cl refers to benzyl chloroformate
  • Pd 2 (dba) 3 refers to tris(dibenzylideneacetone)dipalladium.
  • Dppf refers to 1,1'-bisdiphenylphosphinoferrocene.
  • HATU refers to 2-(7-oxybenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
  • KHMDS refers to potassium hexamethyldisilazide
  • LiHMDS refers to lithium bistrimethylsilylamide.
  • MeLi refers to methyllithium
  • n-BuLi refers to n-butyllithium
  • NaBH(OAc) 3 refers to sodium triacetoxyborohydride.
  • DCM dichloromethane
  • DMAP refers to 4-dimethylaminopyridine.
  • DMBOH refers to 2,4-dimethoxybenzyl alcohol.
  • EDCI refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
  • MTBE means methyl tert-butyl ether
  • DMF N,N-dimethylformamide
  • DTMM refers to 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride.
  • EtOAc refers to ethyl acetate
  • stereoisomer refers to isomers with identical structures but different arrangements of atoms in space. It includes cis and trans (or Z and E) isomers, (-)- and (+)-isomers, (R)- and (S)-enantiomers, diastereomers, (D)- and (L)-isomers, tautomers, atropisomers, conformers and mixtures thereof (such as racemates, mixtures of diastereomers).
  • the substituents in the compounds disclosed herein may have additional asymmetric atoms. All of these stereoisomers and their mixtures are included within the scope of the disclosure.
  • Optically active (-)- and (+)-isomers, (R)- and (S)-enantiomers and (D)- and (L)-isomers may be prepared by chiral synthesis, chiral reagents or other conventional techniques.
  • An isomer of a compound disclosed herein can be prepared by asymmetric synthesis or chiral auxiliary, or, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereoisomer salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art to obtain pure isomers.
  • the separation of enantiomers and diastereomers is usually completed by chromatography.
  • the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or include both Two configurations.
  • tautomer or tautomeric form refers to a structural isomer that exists in equilibrium and is easily converted from one isomeric form to another isomeric form. It includes all possible tautomers, that is, in the form of a single isomer or in the form of a mixture of any proportions of the tautomers. Non-limiting examples include: keto-enol, imine-enamine, lactam-lactim, etc. The equilibrium of lactam-lactim is shown below:
  • the compounds disclosed herein include all suitable isotopic derivatives of their compounds.
  • isotopic derivative refers to a compound in which at least one atom is replaced by an atom having the same atomic number but different atomic masses.
  • isotopes that can be introduced into compounds disclosed herein include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 2 H (deuterium, D), 3 H (tritium, T), 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 33 P, 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I, 129 I and 131 I , etc., preferably deuterium.
  • deuterated drugs Compared with non-deuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds disclosed herein, whether radioactive or not, are included in the scope of the disclosure.
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom, wherein the replacement of deuterium can be partial or complete, and partial deuterium replacement means that at least one hydrogen is replaced by at least one deuterium.
  • the position is understood to have an abundance of deuterium that is at least 1000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 15% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 1000 times greater than the natural abundance of deuterium (i.e., at least 15% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 2000 times greater than the natural abundance of deuterium (i.e., at least 30% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 3000 times greater than the natural abundance of deuterium (i.e., at least 45% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3340 times greater than the natural abundance of deuterium (i.e., at least 50.1% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 3500 times greater than the natural abundance of deuterium (i.e., at least 52.5% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 4000 times greater than the natural abundance of deuterium (i.e., at least 60% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 4500 times greater than the natural abundance of deuterium (i.e., at least 67.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5000 times greater than the natural abundance of deuterium (i.e., at least 75% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 5500 times greater than the natural abundance of deuterium (i.e., at least 82.5% deuterium incorporation). At least 6000 times (i.e., at least 90% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6333.3 times greater than the natural abundance of deuterium (i.e., at least 95% deuterium incorporation).
  • the abundance of deuterium for each designated deuterium atom is at least 6466.7 times greater than the natural abundance of deuterium (i.e., at least 97% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6600 times greater than the natural abundance of deuterium (i.e., at least 99% deuterium incorporation). In some embodiments, the abundance of deuterium for each designated deuterium atom is at least 6633.3 times greater than the natural abundance of deuterium (i.e., at least 99.5% deuterium incorporation).
  • substitution refers to one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, which are replaced independently of each other by a corresponding number of substituents.
  • substitutions by experiment or theory) without undue effort.
  • an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated bond (such as an alkene).
  • the present disclosure also includes various deuterated forms of antibody-drug conjugates of formula (Pc-L-Y-D).
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom.
  • Those skilled in the art can synthesize deuterated forms of antibody-drug conjugates of formula (Pc-L-Y-D) with reference to relevant literature.
  • deuterated starting materials can be used when preparing deuterated forms of antibody-drug conjugates of formula (Pc-L-Y-D), or they can be synthesized using deuterated reagents using conventional techniques, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.
  • alkyl optionally (optionally) substituted with halogen or cyano includes the situation in which alkyl is substituted with halogen or cyano and the situation in which alkyl is not substituted with halogen and cyano.
  • composition refers to a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • the pharmaceutical composition can be in the form of a sterile injectable aqueous solution.
  • Acceptable vehicles and solvents are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation can be a sterile injectable water-in-oil microemulsion in which the active ingredient is dissolved in the oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion.
  • the injection or microemulsion can be injected into the subject's bloodstream by local mass injection.
  • the solution and microemulsion are administered in a manner that maintains a constant circulating concentration of the disclosed compound.
  • a continuous intravenous drug delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration.
  • the suspension may be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents such as those described above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a nontoxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol.
  • sterile fixed oils may be conveniently used as solvents or suspending agents. For this purpose, any fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may also be used to prepare injectables.
  • pharmaceutically acceptable salt refers to a salt of the antibody-drug conjugate of the present disclosure, which is safe and effective when used in a subject and has the desired biological activity.
  • the antibody-drug conjugate of the present disclosure contains at least one amino group, and thus can form a salt with an acid
  • pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • pharmaceutically acceptable carrier refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, stabilizers, or preservatives.
  • excipient refers to the additives in pharmaceutical preparations other than the active ingredients, and can also be called excipients.
  • the binders, fillers, disintegrants, and lubricants in tablets can also be called excipients.
  • the matrix part in semi-solid preparations such as ointments and creams
  • the preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.
  • diluent is also called filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients and improves the compression molding of the drug.
  • an absorbent needs to be added to absorb the oily substance and keep it in a "dry” state to facilitate tableting.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus), sheep, dogs, cattle, birds, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cynomolgus or “cynomolgus” refers to cynomolgus (Macaca fascicularis).
  • the individual or subject is a human.
  • administering refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.
  • sample refers to a collection of fluid, cells, or tissue separated from a subject, as well as fluid, cells, or tissue present in a subject.
  • samples are biological fluids, such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs
  • Treatment and “treat” refer to an attempt to change the condition being treated.
  • Clinical intervention of the pathological process of an individual and can be implemented for prevention or during the course of clinical pathology.
  • the desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.
  • the antibodies disclosed herein are used to delay the formation of the disease or slow the progression of the disease.
  • an effective amount is generally enough to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or potential causes, prevent symptoms and/or their potential causes from occurring and/or improve or ameliorate the amount of damage caused by or associated with a disease state.
  • an effective amount is a therapeutically effective amount or a preventive effective amount.
  • “Therapeutically effective amount” is enough to treat a disease state or symptom, a state or symptom particularly associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way.
  • Preventive effective amount is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the onset (or recurrence) possibility of the disease state or related symptoms.
  • a complete treatment or preventive effect may not occur after administering one dose, but may occur after administering a series of doses.
  • a therapeutically or preventive effective amount may be administered in one or more administrations.
  • a “therapeutically effective amount” and a “prophylactically effective amount” may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.
  • the antibodies provided herein are full-length antibodies.
  • the antibodies provided herein are antibody fragments.
  • the antibody fragment is a Fab, Fab', Fab'-SH or F(ab') 2 fragment, in particular a Fab fragment.
  • Fab which is a monovalent fragment consisting of VL, VH, CL and CH1 domains.
  • Fab fragment can be produced by papain cleavage of an antibody.
  • Fab' contains VL, CL as well as VH and CH1, and also contains the region between the CH1 and CH2 domains so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab') 2 molecule.
  • Fab'-SH is a Fab' fragment in which the cysteine residues of the constant region have free sulfhydryl groups.
  • F(ab') 2 is a bivalent fragment comprising two Fab fragments connected by a disulfide bond at the hinge region.
  • the antibody fragment is a diabody, a triabody or a tetrabody.
  • a diabody is an antibody fragment with two antigen binding sites, which contains a linked VH and VL in the same polypeptide chain (VH-VL).
  • the antibody fragment is a single-chain Fab fragment.
  • a "single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL and a linker, wherein the domains and the linker are at N In the C-terminal to C-terminal direction, there is one of the following sequences: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL.
  • the linker is a polypeptide having at least 30 amino acids.
  • the linker is a polypeptide having between 32 and 50 amino acids.
  • the single-chain Fab fragment is stabilized via the natural disulfide bond between CL and CH1.
  • these single-chain Fab molecules can be further stabilized by inserting cysteine residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to produce interchain disulfide bonds.
  • the antibody fragment is a single chain variable fragment (scFv).
  • scFv is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short flexible peptide linker, capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • scFv herein can have VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.
  • the antibody fragment is a Fd fragment consisting of the VH and CH1 domains.
  • the antibody fragment is a Fv fragment consisting of the VH and VL domains of a single arm of an antibody.
  • the antibody fragment is a dsFv obtained by linking polypeptides in which one amino acid residue in each of VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residue substituted with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method (Protein Engineering. 7: 697 (1994)).
  • the antibody fragment is a single domain antibody, which is an antibody fragment that comprises all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • the antibody fragment is a domain antibody (dAb); see, e.g., U.S. Patent No. 6,248,516.
  • Domain antibodies are functional binding domains of antibodies that correspond to the variable regions of the heavy (VH) or light (VL) chains of human antibodies.
  • dABs have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a complete antibody.
  • dABs express well in a variety of hosts including bacteria, yeast, and mammalian cell systems.
  • dAbs are highly stable and remain active even under harsh conditions, such as lyophilization or heat denaturation. See, for example, U.S.
  • the antibodies provided herein are chimeric antibodies.
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from the class or subclass of the parent antibody.
  • the antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody.
  • the antibody comprises one or more variable regions, wherein CDR or its part is derived from non-human antibody, and FR or its part is derived from human antibody.
  • humanized antibody also comprises a part of human constant region.
  • some FR residues in humanized antibody can be replaced with corresponding residues from non-human antibody (for example, antibody providing CDR sequence).
  • Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light chain variable regions or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623). (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.
  • amino acid sequence variants of antibodies in the antibody conjugates provided herein are contemplated.
  • the amino acid sequence variants of the antibody may be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis.
  • Such modifications may include, for example, deletions, and/or insertions, and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, as long as the final construct possesses desired characteristics, such as antigen binding.
  • antibody variants with one or more amino acid substitutions are provided.
  • Interested sites of substitution mutagenesis include CDR and FR.
  • Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions”. More substantial changes are provided in Table 2 under the heading of "exemplary substitutions", and are further described below with reference to amino acid side chain categories.
  • Amino acid substitutions can be introduced into the antibody of interest, and the product is screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen.
  • conservative changes e.g., conservative substitutions, as provided herein
  • Such changes may be, for example, outside the antigen contact residues in the CDRs.
  • each CDR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.
  • a method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • neutral or negatively charged amino acids e.g., Ala or polyalanine
  • Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitutions.
  • the crystal structure of the antigen-antibody complex can be studied to identify Contact points between antibody and antigen. These contact residues and neighboring residues can be targeted or eliminated as replacement candidates.
  • Variants can be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include polypeptides ranging in length from 1 residue to 100 or more residues at the amino and/or carboxyl termini, and intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with an N-terminal methionyl residue.
  • Other insertion variants of antibody molecules include fusions of the N or C termini of antibodies with enzymes or polypeptides that extend the serum half-life of the antibodies.
  • one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein.
  • the one or more amino acid modifications can reduce the binding of Fc to Fc receptors, such as its binding to Fc ⁇ receptors, and reduce or eliminate effector functions.
  • the modified Fc region has a binding affinity for Fc receptors that is reduced by 50%, 80%, 90% or 95% or more compared to the native Fc region.
  • the Fc receptor is a human Fc ⁇ receptor, such as Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the modified Fc region also has a reduced binding affinity for complement, such as C1q, compared to the native Fc region.
  • the modified Fc region has an enhanced binding affinity for the neonatal Fc receptor (FcRn) compared to the native Fc region; for example, the M252Y/S254T/T256E mutation is introduced into the Fc region.
  • FcRn neonatal Fc receptor
  • the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody dependent cell-mediated cytotoxicity (ADCC), reduced antibody dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.
  • CDC complement dependent cytotoxicity
  • ADCC reduced antibody dependent cell-mediated cytotoxicity
  • ADCP reduced antibody dependent cellular phagocytosis
  • reduced cytokine secretion reduced immune complex-mediated antigen uptake by antigen presenting cells
  • reduced binding to NK cells reduced binding to macrophages
  • monocytes reduced binding to monocytes
  • polymorphonuclear cells reduced direct signaling
  • substitutions of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • substitution of amino acid residues at positions such as 228 may reduce effector function.
  • the antibodies comprise one or more amino acid substitutions with improved ADCC, such as substitutions at positions 298, 333, and/or 334 (using the EU numbering system) of the Fc region.
  • the Fc domain of the antibodies herein comprises a "knob-in-hole” mutation.
  • "Knob-in-hole” is a design strategy for engineering antibody heavy chain homodimers to undergo heterodimerization (e.g., to efficiently generate bispecific antibodies, multispecific antibodies, or one-armed antibodies).
  • such techniques involve introducing a protrusion ("knob") at the interface of a first polypeptide (such as the first CH3 domain in the first antibody heavy chain) and introducing a cavity ("hole”) at the corresponding interface of a second polypeptide (such as the second CH3 domain in the second antibody heavy chain), so that the protrusion can be placed in the cavity to promote heterodimer formation and hinder homodimer formation.
  • the protuberance is constructed by replacing a larger side chain (e.g., arginine, phenylalanine, tyrosine, or tryptophan).
  • a compensatory cavity of the same or similar size as the protuberance is created in the interface of a second polypeptide (such as a second CH3 domain in a second antibody heavy chain) by replacing a larger amino acid side chain with a smaller side chain (e.g., alanine, serine, valine, or threonine).
  • the protuberance and cavity can be generated by altering the nucleic acid encoding the polypeptide (e.g., by site-specific mutagenesis) or by peptide synthesis.
  • the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain
  • the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain.
  • the subunit of the Fc domain comprising the knob modification further comprises the amino acid substitution S354C
  • the subunit of the Fc domain comprising the hole modification further comprises the amino acid substitution Y349C.
  • knob-to-hole mutations include, but are not limited to, those described in Table 3.
  • the C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed.
  • the C-terminus of the heavy chain is a truncated C-terminus ending with PG. Therefore, in some embodiments, the composition of the complete antibody can include an antibody population from which all K447 residues and/or G446+K447 residues have been removed. In some embodiments, the composition of the complete antibody can include an antibody population from which K447 residues and/or G446+K447 residues have not been removed. In some embodiments, the composition of the complete antibody comprises a mixture of antibodies with and without K447 residues and/or G446+K447 residues.
  • Anti-DLL3 antibodies can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antibodies are provided.
  • the disclosure provides an isolated nucleic acid encoding an antibody as described above. Such nucleic acid can be independently encoded by any of the aforementioned polypeptide chains.
  • the disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acid.
  • the disclosure provides a host cell comprising such nucleic acid.
  • a method for preparing an anti-DLL3 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression. cells, as provided above, and optionally recovering the antibody from the host cells (or host cell culture medium).
  • nucleic acids encoding antibodies are separated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • Such nucleic acids can be easily separated and sequenced using conventional procedures, or produced by recombinant methods or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, they can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. After expression, they can be separated from the bacterial cell paste in a soluble fraction and can be further purified.
  • the antibody drug conjugates provided herein can be identified, screened or characterized for their physical/chemical characteristics and/or biological activity by a variety of assays known in the art.
  • the antibody drug conjugate activity of the present disclosure is tested, for example, by known methods such as ELISA, Western blotting, etc.
  • Any of the aforementioned antibody-drug conjugates or pharmaceutically acceptable salts thereof provided herein can be used to treat a disease.
  • the present disclosure provides the use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament. In some embodiments, the present disclosure provides the use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor or cancer.
  • the tumor or cancer includes but is not limited to lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung cancer), head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, thyroid cancer (e.g., medullary thyroid cancer), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colorectal cancer (e.g., colon cancer and rectal cancer), kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer
  • lung cancer
  • the present disclosure provides use of an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a DLL3-related disease.
  • the use further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent (eg, one, two, three, four, five, or six additional therapeutic agents).
  • at least one additional therapeutic agent eg, one, two, three, four, five, or six additional therapeutic agents.
  • a pharmaceutical composition comprising the anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof is provided, for example, for use in any of the above pharmaceutical uses or methods of treatment.
  • the pharmaceutical composition comprises any polypeptide or drug conjugate provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure can be used alone or in combination with other agents for treatment.
  • the anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof of the present disclosure can be co-administered with at least one additional therapeutic agent.
  • the anti-DLL3 antibody-drug conjugates of the present disclosure or pharmaceutically acceptable salts thereof can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is desired, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.
  • a variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration and pulse infusions.
  • the anti-DLL3 antibody-drug conjugates or pharmaceutically acceptable salts thereof disclosed herein will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners.
  • the anti-DLL3 antibody or drug conjugate thereof may be formulated with one or more other agents. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used in the same dosages and administration routes as described herein, or in about 1 to 99% of the dosages described herein, or in other dosages and by any route determined empirically/clinically to be appropriate.
  • an anti-DLL3 antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof (when used alone or in combination with one or more other additional therapeutic agents)
  • the appropriate dosage of an anti-DLL3 antibody-drug conjugate of the present disclosure will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether it is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician.
  • the therapeutic molecule is appropriately administered to the patient at one time or over a series of treatments.
  • an article e.g., a kit
  • the article comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like.
  • the container can be formed from a variety of materials such as glass or plastic.
  • the container contains an anti-DLL3 antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, alone or in combination with another composition.
  • the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper).
  • At least one active agent in the composition is an anti-DLL3 antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt thereof.
  • the label or package insert indicates that the composition is used to treat the selected condition.
  • the article of manufacture may comprise: (a) a first container having an anti-DLL3 antibody-drug conjugate or a pharmaceutically acceptable salt thereof contained therein; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.
  • the product may further comprise a second (or third) container, wherein the second (or third) container comprises a pharmaceutically acceptable buffer.
  • the second (or third) container comprises a pharmaceutically acceptable buffer.
  • it may further comprise other materials required, including other buffers, diluents, filters, needles and syringes.
  • the DLL3 genes of different species and human DLL1 and DLL4 genes were transfected into Chinese hamster ovary cells CHO-s cells (Invitrogen, R80007) to construct CHO-s cell lines expressing DLL3 proteins of different species for subsequent antibody screening and identification.
  • the amino acid sequences of the relevant proteins are as follows:
  • Cynomolgus monkey DLL3 full-length protein (Uniprot, A0A2K5WSR4):
  • Rat DLL3 full-length protein (Uniprot, O88671):
  • the pCDH lentiviral expression vector plasmid containing SEQ ID NO: 1-6 (synthesized by GENEWIZ), pCDH plasmid and pVSVG, pCMV lentiviral packaging vector were transfected into 293T cells (Chinese Academy of Sciences Cell Bank, GNHu17) using Lipofectamine 3000 (Invitrogen, L3000015) transfection reagent, and the culture supernatant containing the virus was collected, filtered and subjected to ultra-high-speed centrifugation. After discarding the supernatant, it was resuspended in 0.2 mL sterile PBS.
  • the concentrated virus was used to infect Chinese hamster ovary cells CHO-s (Invitrogen, R80007), DMS53 (ATCC, CRL-2062) and H82 (ATCC, HTB-175), respectively, and selected with puromycin for two to three weeks, and then single-cell sorting was performed by FACS. The selected monoclonal cell lines were expanded and frozen.
  • DLL3ECD fusion proteins containing different tags were designed and cloned into pTT5 vectors, respectively. After expression in 293E cells, antigens were obtained.
  • the amino acid sequences of the relevant proteins are as follows:
  • the dot-dashed line represents part of the signal peptide sequence
  • the single-dash line represents the his tag and linker
  • the double-dash line represents the DLL3 extracellular region.
  • the dot-dashed part is the signal peptide sequence
  • the single-dash part is the Fc tag and linker
  • the double-dash part is the DLL3 extracellular region.
  • the dot-dashed part is the signal peptide sequence
  • the double-dashed part is the DLL3 extracellular region
  • the single-dashed part is the strep twin tag.
  • the dot-dashed part is the signal peptide sequence
  • the double-dashed part is the DLL3 extracellular region
  • the single-dashed part is the strep twin tag.
  • the dot-dashed part is the signal peptide sequence
  • the double-dashed part is the DLL3 extracellular region
  • the single-dashed part is the strep twin tag.
  • Anti-human DLL3 monoclonal antibodies were produced by immunizing mice.
  • the SJL mice used in the experiment were female, 6-8 weeks old (Shanghai Slake Laboratory Animal Co., Ltd., Animal Production License No.: SCXK (Shanghai) 2017-0005). Housing environment: SPF grade. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12/12 hour light/dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The mice that had been adapted to the environment were immunized according to the following scheme.
  • the first group of mice was immunized with His-hDLL3 (ECD) (SEQ ID NO: 7).
  • Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) adjuvant cross immunization.
  • Antigen and adjuvant The ratio of Gold Adjuvant is 1:1, antigen and adjuvant Thermo Alum ratio is 3:1, 50 ⁇ g/animal/time (first immunization), 25 ⁇ g/animal/time (boosting Immunization).
  • Antigen emulsification was performed for inoculation, and the immunization time was 0, 7, 14, and 21 days. Blood was collected on the 7th and 21st days, and the antibody titer in the mouse serum was determined by ELISA.
  • mice with high antibody titers in serum and titers tending to a plateau were selected for spleen cell fusion.
  • booster immunization was performed, and 25 ⁇ g/mouse of antigen solution prepared with physiological saline was injected intraperitoneally (ip).
  • mice The second group of mice were immunized with DLL3CHO-s and Fc-hDLL3 (ECD) (SEQ ID NO: 8) by alternating immunization with cell and protein antigens.
  • Gold Adjuvant (Sigma Cat No. T2684) 0.1 mL/mouse was injected intraperitoneally. Half an hour later, each mouse was intraperitoneally injected with 0.1 mL of cell solution diluted with saline to a concentration of 10 8 /mL. The cells were evenly dispersed and inoculated on days 0, 14, 28, and 42.
  • Fc-hDLL3 (ECD) antigen was used Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) adjuvant cross immunization.
  • Gold Adjuvant Sigma Cat No.T2684
  • Thermo Alum Thermo Cat No.77161 adjuvant cross immunization.
  • the ratio of Gold Adjuvant is 1:1, antigen and adjuvant Thermo
  • the alum ratio was 3:1, 50 ⁇ g/mouse/time (first immunization), 25 ⁇ g/mouse/time (boost immunization).
  • the hDLL3-Fc immunization time was 7, 21, 35, and 49 days. Blood was collected on days 14, 35, and 49, and the antibody titer in the mouse serum was determined by ELISA.
  • mice with high antibody titers in serum and titers tending to a plateau were selected for spleen cell fusion.
  • boost immunization was performed, and 50 ⁇ g/mouse of hDLL3-Fc protein antigen solution prepared in physiological saline was injected intraperitoneally (ip).
  • the spleen lymphocytes were fused with myeloma cells Sp2/0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 3-4 ⁇ 10 5 /mL, and 150 ⁇ L/well was seeded in a 96-well plate. After incubation at 37°C, 5% CO 2 for 3-4 days, the supernatant was removed, and 200 ⁇ L/well of HT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI) was added, and screening was performed after incubation at 37°C, 5% CO 2 for 3 days.
  • complete medium IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • hybridoma culture supernatant was detected by ELISA method binding to DLL3 protein and FACS method binding to DLL3 CHO-s cells.
  • the clones that bind to human DLL3 protein, monkey DLL3 protein and DLL3 CHO-s cells, and do not bind to wild-type CHO-s cells were selected for timely freezing, amplification, seed preservation and one or two subcloning until a single cell clone was obtained.
  • Hybridoma clones mAb100 and mAb6 were obtained through the above experimental screening.
  • the hybridoma clones were expanded and cultured, RNA was extracted, and reverse transcription amplification (RT-PCR) was performed using degenerate primers of mouse-Ig to finally obtain the variable region sequence of the antibody.
  • the heavy chain variable region and light chain variable region of the mouse antibody were cloned into the pTT 5 vector plasmid containing the human IgG1 heavy chain constant region shown in SEQ ID NO:28 and the ⁇ light chain constant region shown in SEQ ID NO:29, respectively, and then transfected into HEK293 cells to obtain the anti-DLL3 chimeric antibodies M6CHI and M100CHI.
  • variable region germline genes By comparing the Kabat human antibody heavy and light chain variable region germline gene database, the heavy and light chain variable region germline genes with high homology were selected as templates, and the CDRs of the mouse antibody were grafted into the corresponding human templates to form a variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acids in the variable region were reverse mutated and then recombined with the constant region (exemplarily, with the human IgG1 heavy chain constant region shown in SEQ ID NO: 28 and the human ⁇ light chain constant region shown in SEQ ID NO: 29) to obtain a full-length antibody.
  • the human germline light chain variable region FR1, FR2, FR3 template of mAb6 antibody is IGKV1-16*01
  • the light chain FR4 region template is IGKJ4*01
  • the human germline heavy chain variable region FR1, FR2, FR3 template is IGHV1-3*01, IGHV7-4-1*02 or IGHV3-73*01
  • the heavy chain FR4 region template is IGHJ6*01.
  • the human germline light chain variable region FR1, FR2, FR3 template of mAb100 antibody is IGKV1-27*01
  • the light chain FR4 region template is IGKJ4*01
  • the human germline heavy chain variable region FR1, FR2, FR3 template is IGHV3-11*01
  • the heavy chain FR4 region template is IGHJ6*01.
  • amino acid residue at position 4 in HCDR3 PLYYYGRSYNAVAY (SEQ ID NO: 24) of the heavy chain variable region of mAb100 was mutated from Y to H, and the amino acid residue at position 11 was mutated from A to G to obtain new HCDR3: PLY H YGRSYNAVAY (SEQ ID NO: 30) and PLYYYGRSYN G VAY (SEQ ID NO: 31).
  • sequences of the obtained humanized antibody variable regions are as follows:
  • hAb6 VL2 (F36L, S46G, T69A, F71Y)
  • the single-lined part is the CDR region, and the double-lined part is the mutation site.
  • X1 is Y or H; X2 is A or G.
  • Exemplary humanized antibody heavy and light chain variable region combinations are as follows:
  • hAb6L1H1 indicates that the antibody comprises a heavy chain variable region hAb6VH1 and a light chain variable region hAb6VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 28, and the sequence of its light chain constant region is SEQ ID NO: 29, and so on.
  • hAb100L1H1 indicates that the antibody comprises a heavy chain variable region hAb100VH1 and a light chain variable region hAb100VL1, and the sequence of its heavy chain constant region is SEQ ID NO: 28, and the sequence of its light chain constant region is SEQ ID NO: 29, and so on.
  • the above antibodies were cloned, expressed, and purified, and humanized antibodies with better activity were finally selected through protein binding experiments (Test Example 1), cell binding experiments (Test Example 2), and Biacore (Test Example 4).
  • the heavy and light chain amino acid sequences of exemplary humanized antibodies are as follows:
  • Hu6 also known as hAb6L4H12 heavy chain
  • Hu6 also known as hAb6L4H12
  • Hu100 also known as hAb100L1H1 heavy chain
  • Hu100 also known as hAb100L1H1 light chain
  • the positive control antibody used in this disclosure is BI-764532 (constructed with reference to WO2019234220A1), and the negative control is C25 (wherein the VH/VL sequence is from patent US6114143A), and their sequences are as follows:
  • TCEP.HCl tris(2-carboxyethyl)phosphine hydrochloride
  • TCEP.HCl tris(2-carboxyethyl)phosphine hydrochloride
  • TCEP.HCl tris(2-carboxyethyl)phosphine hydrochloride
  • TCEP.HCl tris(2-carboxyethyl)phosphine hydrochloride
  • TCEP.HCl tris(2-carboxyethyl)phosphine hydrochloride
  • TCEP.HCl tris(2-carboxyethyl)phosphine hydrochloride
  • ADC is an antibody-drug conjugate, and its mechanism of treating diseases is to rely on the targeting of antibodies to deliver drugs into cells, thereby killing cells or inhibiting cell growth.
  • the drug loading plays a decisive role in the efficacy of the drug.
  • This disclosure uses RP-HPLC to analyze drug loading, and the process is basically as follows:
  • Trifluoroacetic acid produced by Sigma, 100 mL/bottle; Acetonitrile: LC grade, 4 L/bottle, produced by Thermo Fisher; DTT: produced by Sigma, 1 g/bottle.
  • Preparation example Take 5.78 mg of DTT, add 150 ⁇ L of purified water to fully dissolve it, then prepare 0.25 M DTT solution and store it at -20°C.
  • Preparation example Measure 1000 mL of purified water into a measuring cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.
  • Preparation example Measure 1000 mL of acetonitrile into a measuring cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.
  • Naked antibody and test sample (concentration of 1 mg/mL, about 200 ⁇ L) were reduced by adding 4 ⁇ L DTT and placed in a 37°C water bath for 1 hour. After reduction, the samples were taken out and placed in the inner tube for injection.
  • DAD detector detection wavelength 280nm; sample chamber temperature: 4°C; flow rate: 1mL/min;
  • Injection volume 40 ⁇ L
  • Total HC peak area HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area
  • LC DAR ⁇ (number of connected drugs * peak area percentage)/total LC peak area
  • HC DAR ⁇ (number of connected drugs * peak area percentage)/total HC peak area
  • Test Example 1 ELISA to detect protein level binding of antibodies
  • the DLL3-expressing small cell lung cancer cell lines H1184 (ATCC, catalog number CRL-5858), DLL3/H82, cynoDLL3/CHO-s and RatDLL3/CHO-s were prepared into a cell suspension of 1 ⁇ 10 6 /mL using FACS buffer (1% BSA + pH 7.4 PBS), and 100 ⁇ L/well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the test antibody at 100 ⁇ L/well. Incubate in a 4°C refrigerator in the dark for 1 hour.
  • the antibodies disclosed herein can specifically bind to DLL3 expressed in cells, among which Hu6 has a high binding ability to cells expressing DLL3 of different species.
  • Hu100 has excellent binding ability to cells expressing human and cynomolgus monkey DLL3, but Hu100 does not bind to cells expressing rat DLL3.
  • BI-764532 only has binding ability to cells expressing human and cynomolgus monkey DLL3, and its binding activity is weaker than that of Hu6 and Hu100.
  • Test Example 3 FACS detection of antibody binding to DLL1 and DLL4 cells
  • Stably transfected human DLL1/CHO-s and human DLL4/CHO-s cells were prepared into a cell suspension of 1 ⁇ 10 6 /mL using FACS buffer (containing 1% BSA and pH 7.4 PBS), and 100 ⁇ L/well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add the antibody to be tested, 100 ⁇ L/well, and incubate in a 4°C refrigerator in the dark for 1 hour.
  • the antibody to be tested was captured by affinity using a Protein A biosensor chip (Cat.#29127556, Cytiva) for 18 seconds, and then the antigens human DLL3 (ACRO, DLL3-H52H4), monkey DLL3 (KACTUS, DLL-RM103) and mouse DLL3 (KACTUS, DLL-MM103) were passed over the chip surface for 180 seconds, and then dissociated for 600 seconds.
  • the binding and dissociation curves were obtained by real-time detection of the reaction signal using a Biacore 8K (Cytiva) instrument.
  • BI-764532 antibody (1 ⁇ g/mL) was coated on the plate at 100 ⁇ L/well at 4°C overnight. PBST solution 250 ⁇ L/well was added and the plate was washed 3 times. 5% milk was blocked at 250 ⁇ L/well at 37°C for 2 hours. PBST solution 250 ⁇ L/well was added and the plate was washed 3 times. Biotinylated DLL3-Strep (0.1 ⁇ g/mL, SEQ ID NO: 9) was added. Competing antibodies, BI-764532, Hu6 and Hu100 (maximum concentration 100 ⁇ g/mL, 4-fold gradient dilution) were prepared and incubated at 37°C for 1 hour.
  • PBST solution 250 ⁇ L/well was added and the plate was washed 6 times.
  • Streptavidin-peroxidase (1:2000 dilution) (Jackson Immuno Research, 016-030-084) was added at 100 ⁇ L/well and incubated at 37°C for 1 hour.
  • PBST solution 250 ⁇ L/well wash the plate 6 times.
  • TMB KPL, 5120-0077) colorimetric solution 100 ⁇ L/well, colorimetric at room temperature for 5-10min.
  • 1M H 2 SO 4 100 ⁇ L/well to stop colorimetric, read at 450nm on a microplate reader. The results are shown in Figure 2.
  • DT3C is a recombinantly expressed fusion protein, which is formed by the fusion of the fragment A of diphtheria toxin (toxin part only) and the 3C fragment of group G streptococcus (IgG binding part). This protein has a high affinity with the IgG part of the antibody, and enters the cell together when the antibody is internalized. Under the action of intracellular furin protease, toxic DT is released. DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and eventually lead to cell death. DT3C that has not entered the cell does not have the activity of killing cells. The activity of antibody internalization is evaluated based on the cell killing situation.
  • Test Example 7 FACS detection of ADC binding experiment at the cellular level
  • hDLL3/CHO-s and cynoDLL3/CHO-s cells expressing DLL3 were prepared into a cell suspension of 1 ⁇ 10 6 /mL using FACS buffer (1% BSA + pH 7.4 PBS), and 100 ⁇ L/well was added to a 96-well round-bottom plate (Corning, 3795). Centrifuge at 300g for 5 minutes and remove the supernatant. Add different concentrations of the ADC to be tested, 100 ⁇ L/well. Incubate in a 4°C refrigerator in the dark for 1 hour.
  • the antibody to be tested was captured by affinity using a Protein A biosensor chip (Cat.#29127556, Cytiva) for 18 seconds. Then, antigens human DLL3 (ACRO, DLL3-H52H4) and monkey DLL3 (KACTUS, DLL-RM103) flowed over the chip surface for 180 seconds, and then dissociated for 600 seconds. The reaction signal was detected in real time using a Biacore 8K (Cytiva) instrument to obtain binding and dissociation curves. After each experimental cycle of dissociation was completed, the biosensor chip was washed and regenerated with a 10mM glycine-hydrochloric acid solution (pH 1.5) (Cat.#BR-1003-54, Cytiva). The data fitting model used was a 1:1 model. The results are shown in Table 15.
  • a Prepare a cell suspension with fresh cell culture medium containing 10% FBS and add 135 ⁇ L per well to a 96-well cell culture plate (Corning, 3903). Do not plate cells in the 1st and 12th columns. Only add 135 ⁇ L of culture medium and culture at 37°C with 5% carbon dioxide for 16 hours.
  • the ADC sample was prepared into the first well working solution (10 ⁇ concentration) with PBS, and this was used as the first concentration, and gradient dilution was performed with PBS at the corresponding multiples. 15 ⁇ L of 10 ⁇ concentration ADC solution was added to each well, and cultured at 37°C with 5% carbon dioxide for 6 days.
  • the cells used in the experiment are as follows:
  • H1184 (+++) was purchased from ATCC, CRL-5858;
  • DMS53(++) was purchased from ATCC, CRL-2062;
  • SK-MEL3(+) was purchased from ATCC, HTB-69;
  • CHO-K1(-) was purchased from ATCC, CCL-61.
  • ADC-1, ADC-2 and ADC-3 had strong target cell killing activity and could kill SK-MEL3, DMS53 and H1184 cells expressing DLL3, but had no killing effect on DLL3-negative CHO-K1 cells.
  • DMS53/DLL3high DMS53 cells stably transfected with DLL3
  • U-2OS ATCC, HTB-96 cells
  • DMS53/DLL3high DMS53 cells stably transfected with DLL3
  • U-2OS ATCC, HTB-96 cells
  • the cells were trypsinized, neutralized with fresh culture medium, and centrifuged at 1000rpm for 3 minutes. The supernatant was discarded and the cells were resuspended in RPMI1640+20% FBS+1 ⁇ Glutamax.
  • the cell density of DMS53/DLL3high was adjusted to 9 ⁇ 10 4 cells/mL
  • U-2OS was adjusted to 3 ⁇ 10 4 cells/mL.
  • 500 ⁇ L of DMS53/DLL3high cells and 500 ⁇ L of U-2OS cells were added to each well of the corresponding wells of the 12-well plate.
  • 500 ⁇ L of U-2OS cells and 500 ⁇ L of RPMI1640+20% FBS+1 ⁇ Glutamax culture medium were added to the corresponding wells of the 12-well plate.
  • 5% carbon dioxide was cultured at 37°C for 24 hours.
  • the samples were prepared into an intermediate solution (200 nM) of 40 ⁇ concentration.
  • 25 ⁇ L of ADC samples were added to the corresponding wells of the 12-well plate.
  • a solvent control group was set up. 5% carbon dioxide was cultured at 37°C for 6 days.
  • the cells in the 12-well plate were digested with trypsin, neutralized with fresh culture medium, 20 ⁇ L of cells were added with 20 ⁇ L of trypan blue, and counted. The cells were centrifuged at 1000 rpm for 3 minutes and the supernatant was discarded. The cells were washed once with 100 ⁇ L of FACS buffer, centrifuged at 1500 rpm for 3 minutes, and the supernatant was discarded. Resuspended with 100 ⁇ L of FACS buffer, 2 ⁇ g/mL of anti-DLL3 positive antibody was added, and incubated on ice for 60 minutes.
  • Test Example 11-1 In vivo efficacy evaluation of DMS53 cell CDX mouse model
  • DMS53 cells 200 ⁇ L/mouse of human small cell lung cancer DMS53 cells (5 ⁇ 10 6 , containing 50% matrigel/mouse; ATCC, CRL-2062) were inoculated subcutaneously in the right rib of Balb/c. After 7 days of inoculation, when the tumor volume reached ⁇ 220 mm 3 , the weight, tumors that were too large and too small were removed, and the mice were randomized according to the tumor volume, with 8 mice in each group, and drug administration began on the same day. ADC was injected intraperitoneally at a dose of 1.5 mg/kg, once a week, for a total of 2 times. After the second administration, the drug administration was stopped and the tumor growth was continued to be observed.
  • the tumor volume and body weight were measured twice a week and the data were recorded. Data were recorded using Excel statistical software: the average value was calculated as avg; the SD value was calculated as STDEV; the SEM value was calculated as STDEV/SQRT (number of animals in each group); GraphPad Prism software was used for plotting, and Two-way ANOVA or One-way ANOVA was used for statistical analysis of the data.
  • V 1/2 ⁇ L long ⁇ L short 2
  • T/C (%) (TT 0 )/(CC 0 ) ⁇ 100%, wherein T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • TGI (%) 1-T/C (%).
  • Test Example 11-2 In vivo efficacy evaluation of DMS53 cell CDX mouse model
  • DMS53 cells 5 ⁇ 10 6 cells, containing 30% matrigel/mouse; ATCC, CRL-2062
  • mice were randomized according to the tumor volume, with 8 mice in each group, and medication began on the same day.
  • ADC was injected intraperitoneally at a dose of 1.5 mg/kg or 0.5 mg/kg, once a week, for a total of 2 times. The medication was stopped after the second dose, and the tumor growth was continuously observed. The tumor volume and body weight were measured twice a week, and the data were recorded.
  • V 1/2 ⁇ L long ⁇ L short 2
  • T/C (%) (TT 0 )/(CC 0 ) ⁇ 100%, wherein T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • TGI (%) 1-T/C (%).
  • Test Example 12 In vivo efficacy evaluation of NCI-H1184 cell CDX mouse model
  • NCI-H1184 cells (6 ⁇ 10 6 cells, containing 50% MatrixGel/mouse; ATCC, CRL-5858) 200 ⁇ L/mouse were inoculated subcutaneously in the right ribs of 80 NDG mice. After 13 days of inoculation, when the average tumor volume reached ⁇ 185 mm 3 , the weight, tumor size and tumor size were removed, and the mice were randomly divided into 7 groups according to the tumor volume, with 9 mice in each group. The drug was administered on the same day, as shown in Table 19. The antibody was injected intraperitoneally for a total of 21 days. The tumor volume was measured twice a week, the body weight was weighed, and the data were recorded.
  • the data were recorded using Excel statistical software: the average value was calculated as avg; the SD value was calculated as STDEV; the SEM value was calculated as STDEV/SQRT (number of animals in each group); GraphPad Prism software was used for drawing, and Two-way ANOVA or One-way ANOVA was used for statistical analysis of the data.
  • V 1/2 ⁇ L long ⁇ L short 2
  • T/C (%) (TT 0 )/(CC 0 ) ⁇ 100%, wherein T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • TGI (%) 1-T/C (%).
  • the collected blood samples were placed at room temperature for half an hour to coagulate, and then centrifuged at 1000g for 15 minutes at 4°C to collect serum.
  • the HTRF method was used for bioanalysis measurement to measure the rat serum samples, biotin-labeled DLL3 capture monoclonal antibody, and biotin-labeled anti-toxin antibody capture toxin.
  • the quantitative analysis of the content of the test product was performed using the four-parameter model curve of the standard product.
  • the pharmacokinetic parameters were analyzed using the standard non-compartmental model of WinNonlin software (6.4). The experimental results are shown in Table 21 and Figures 10A to 10C.
  • the collected blood samples were placed at room temperature for half an hour to coagulate, and then centrifuged (4°C, 2600g, 10 min), and the upper serum was taken and immediately stored at -60°C. Serum was collected for bioanalysis and measurement.
  • the DELFIA method was used to measure serum samples, and the four-parameter model curve of the standard was used to quantitatively analyze the content of the test product.
  • the pharmacokinetic parameters were analyzed using the standard non-compartmental model of WinNonlin software (6.4). The experimental results are shown in Table 22 and Figure 11.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种抗DLL3抗体、其抗体-药物偶联物及其医药用途。具体涉及如通式Pc-L-Y-D所示的抗DLL3抗体-依喜替康类药物偶联物，其中Pc为抗DLL3抗体，L、Y和n如说明书中所定义。

Description

抗DLL3抗体、其抗体-药物偶联物及其医药用途 技术领域
本披露涉及抗DLL3抗体、其抗体-药物偶联物及其医药用途。
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。
小细胞肺癌(Small Cell Lung Cancer，SCLC)属于一种比较恶性的肺癌类型，占所有肺癌病例的10％-15％。小细胞肺癌的肿瘤生长速度快，容易转移，5年生存率低于7％。对小细胞肺癌的治疗，常采用铂类/依托泊苷联用的化疗。小细胞肺癌病人对化疗初期响应良好，但极易产生耐药和复发。近几年的免疫治疗，如PD-L1抗体和PD1抗体，对小细胞患者有一定的效果，但是有效率约为15％。目前仍然还没有开发出特异性的靶向治疗药物。
DLL3是一种抑制Notch的配体。在正常状态下，DLL3处于高尔基体上，在癌细胞(如小细胞肺癌细胞)中，DLL3会到细胞表面，以顺式方式结合Notch，阻碍细胞和细胞结合以及Notch在目标细胞的内吞，从而抑制Notch信号通路，促进肿瘤细胞的生长。DLL3主要表达于神经或神经内分泌肿瘤，包括SCLC、大细胞神经内分泌癌、胃肠道神经内分泌瘤、小细胞膀胱癌、多形性胶质细胞瘤、转移性去势性前列腺癌、黑色素瘤等。尤其是SCLC，超过80％的SCLC均表达DLL3，而在正常肺癌组织及癌旁组织中不表达。这种表达的差异性使得DLL3成为治疗SCLC的一种极具潜力的治疗靶点。
依喜替康类毒素是喜树碱衍生物，抑制拓卜异构酶I，可选择性抑制增殖期肿瘤细胞DNA复制。此外，依喜替康类毒素还具有良好的透膜性，可以穿透被杀死的癌细胞，继续杀死旁边的癌细胞，在临床上具有明确的旁观者效应。
将抗体通过接头与具有生物活性的药物相连，获得抗体-药物偶联物(antibody drug conjugate，ADC)。ADC充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和药物(如细胞毒性剂)的高效性，同时又避免了抗体的疗效偏低和药物的毒副作用过大等缺陷。与以往传统的化疗药物相比，抗体-药物偶联物能更精准地杀伤肿瘤细胞并降低将对正常细胞的影响。
发明内容
本披露涉及抗DLL3抗体、其抗体-药物偶联物及其医药用途。更具体的，本披露提供多种全新序列的抗DLL3抗体与依喜替康类毒素偶联的ADC。
在一些实施方案中，本披露提供一种抗体-药物偶联物或其药学上可接受的盐，其具有如通式Pc-L-Y-D所示的结构：
其中：
Pc为抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：57的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；
其中，SEQ ID NO：57如PLYX1YGRSYNX2VAY所示，其中X1为Y或H；X2为A或G；或
ii)所述重链可变区包含：HCDR1，其包含SEQ ID NO：16的氨基酸序列；HCDR2，其包含SEQ ID NO：17的氨基酸序列；和HCDR3，其包含SEQ ID NO：18的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：19的氨基酸序列；LCDR2，其包含SEQ ID NO：20的氨基酸序列；和LCDR3，其包含SEQ ID NO：21的氨基酸序列；
Y为-O-(CRaRb)m-CR1R2-C(O)-；其中，Ra和Rb相同或不同，且各自独立地选自氢原子、氘原子、卤素和C1-6烷基；
R1为卤代C1-6烷基或C3-6环烷基；
R2选自氢原子、卤代C1-6烷基和C3-6环烷基；
或者，R1和R2与其相连接的碳原子一起形成C3-6环烷基；
m为0至4的整数；
n为1至10(包括整数和小数)；
L为接头。
在一些实施方案中，如前所述的抗体-药物偶联物或其药学上可接受的盐，其中n是每个抗体的平均药物模块数，可以是整数或小数。在一些实施方案中，n为1-10，或2-10，或3-10，或4-10，或5-10，或6-10，或7-10，或8-10，或1-9，或2-9，或3-9，或4-9，或5-9，或6-9，或7-9，或1-8，或2-8，或3-8，或4-8， 或5-8，或6-8，或1-7，或2-7，或3-7，或4-7，或5-7，或1-6，或2-6，或3-6，或4-6，或1-5，或2-5，或3-5，或1-4，或2-4，或1-3。在一些实施方案中，n为1，2，3，4，5，6，7，8，9或10。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24、30或31的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：30的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：31的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所 述的抗DLL3抗体为鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体为人源化抗体。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含人免疫球蛋白框架区(FR区)。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：57的氨基酸序列；其中，SEQ ID NO：57如PLYX1YGRSYNX2VAY所示，其中X1为Y或H；X2为A或G；且所述重链可变区的FR包含选自1E、49A和94S中的一个或多个回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；且所述轻链可变区的FR包含43I回复突变。上述的回复突变位点依据Kabat编号规则。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：16的氨基酸序列；HCDR2，其包含SEQ ID NO：17的氨基酸序列；和HCDR3，其包含SEQ ID NO：18的氨基酸序列；且所述重链可变区的FR包含选自1E、27Y、30T、38K、43K、48I、67A、68A、69L、71V、73K、75S、76N和93A中的一个或多个回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：19的氨基酸序列；LCDR2，其包含SEQ ID NO：20的氨基酸序列；和LCDR3，其包含SEQ ID NO：21的氨基酸序列；且所述轻链可变区的FR包含选自36L、43S、44F、46G、69A、71Y和85D中的一个或多个回复突变。上述的回复突变位点依据Kabat编号规则。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：16的氨基酸序列；HCDR2，其包含SEQ ID NO：17的氨基酸序列；和HCDR3，其包含SEQ ID NO：18的氨基酸序列；且所述重链可变区的FR包含1E、68A、69L、71V、73K、75S、76N的回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：19的氨基酸序列；LCDR2，其包含SEQ ID NO：20的氨基酸序列；和LCDR3，其包含SEQ ID NO：21的氨基酸序列；且所述轻链可变区的FR包含36L、46G、69A、71Y和85D的回复突变。上述的回复突变位点依据Kabat编号规则。
在一个具体的实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24的氨基酸序列；且所述重链可变区的FR包含1E和94S的回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列。上述的回复突变位点依据Kabat编号规则。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24、30或31的氨基酸序列；且所述重链可变区的FR包含选自1E、49A和94S中的一个或多个回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；且所述轻链可变区的FR包含43I回复突变。上述的回复突变位点依据Kabat编号规则。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24的氨基酸序列；且所述重链可变区的FR包含选自1E、49A和94S中的一个或多个回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；且所述轻链可变区的FR包含43I回复突变。上述的回复突变位点依据Kabat编号规则。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：30的氨基酸序列；且所述重链可变区的FR包含选自1E、49A和94S中的一个或多个回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨 基酸序列；且所述轻链可变区的FR包含43I回复突变。上述的回复突变位点依据Kabat编号规则。
在一个实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：31的氨基酸序列；且所述重链可变区的FR包含选自1E、49A和94S中的一个或多个回复突变；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；且所述轻链可变区的FR包含43I回复突变。上述的回复突变位点依据Kabat编号规则。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体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与SEQ ID NO：14、50、51、52、53或54具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：15、55或56具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的轻链可变区；或
ii)所述重链可变区包含与SEQ ID NO：12、32、33、34、35、36、37、38、39、40、41、42、43或44具有至少90％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：13、45、46、47、48或49具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的轻链可变区。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含：
i)所述重链可变区包含与SEQ ID NO：14具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：15具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的轻链可变区；或
ii)所述重链可变区包含与SEQ ID NO：50、51、52、53或54具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：55或56具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；或
iii)所述重链可变区包含与SEQ ID NO：12具有至少90％、91％、92％、93％、 94％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：13具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；或
iv)所述重链可变区包含与SEQ ID NO：32、33、34、35、36、37、38、39、40、41、42、43或44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：45、46、47、48或49具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含：
i)所述重链可变区包含与SEQ ID NO：50具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：55具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；或
ii)所述重链可变区包含与SEQ ID NO：43具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；和/或
所述轻链可变区包含与SEQ ID NO：48具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体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SEQ ID NO：14的氨基酸序列，和/或所述轻链可变区包含SEQ ID NO：15的氨基酸序列；或
ii)所述重链可变区包含选自SEQ ID NO：50、51、52、53和54中的任一氨基酸序列，和/或所述轻链可变区包含55或56的氨基酸序列；或
iii)所述重链可变区包含SEQ ID NO：12的氨基酸序列，和/或所述轻链可变区包含SEQ ID NO：13的氨基酸序列；或
iv)所述重链可变区包含选自SEQ ID NO：43、32、33、34、35、36、37、38、39、40、41、42和44中的任一氨基酸序列，和/或所述轻链可变区包含选自SEQ ID NO：48、45、46、47和49中的任一氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含：
所述重链可变区包含SEQ ID NO：50的氨基酸序列，和所述轻链可变区包含SEQ ID NO：55的氨基酸序列；或
所述重链可变区包含SEQ ID NO：43的氨基酸序列，和所述轻链可变区包含SEQ ID NO：48的氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所 述的抗DLL3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SEQ ID NO：50的氨基酸序列，和所述轻链可变区包含SEQ ID NO：55的氨基酸序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体是抗体片段，其中所述的抗体片段为Fab、Fab'、F(ab')2、Fab'-SH、Fd、Fv、scFv、dsFv、双抗体或结构域抗体。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含轻链恒定区和重链恒定区。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含IgG1、IgG2、IgG3或IgG4的恒定区。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含λ链或κ链的恒定区。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含IgG1的重链恒定区和κ链的轻链恒定区。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含：
所述重链恒定区包含SEQ ID NO：28的序列，和/或轻链恒定区包含SEQ ID NO：29的序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含重链和轻链，其中：
所述重链包含与SEQ ID NO：60具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，和/或所述轻链包含与SEQ ID NO：61具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列；或
所述重链包含与SEQ ID NO：58具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列，和/或所述轻链包含与SEQ ID NO：59具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含：
所述重链包含如SEQ ID NO：60所示的序列，和/或所述轻链包含如SEQ ID NO：61所示的序列；或
所述重链包含如SEQ ID NO：58所示的序列，和/或所述轻链包含如SEQ ID NO：59所示的序列。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体具有以下特性中的至少一种：
a)所述抗DLL3抗体与人DLL3或其表位结合的KD值≤3nM、≤2nM、≤ 1nM、≤0.9nM、≤0.8nM、≤0.7nM、≤0.6nM、≤0.5nM或≤0.4nM，所述KD值通过Biacore测定；
b)所述抗DLL3抗体与表达DLL3的H1184细胞结合的EC50≤3nM、EC50≤2nM、EC50≤1nM、EC50≤0.5nM、EC50≤0.2nM、EC50≤0.1nM、EC50≤0.09nM、EC50≤0.08nM、EC50≤0.07nM或EC50≤0.06nM，所述EC50通过FACS检测；
c)所述抗DLL3抗体能够被表达DLL3的细胞内吞；和
d)所述抗DLL3抗体与DLL3或其表位结合EC50≤0.1nM、≤0.09nM、≤0.08nM、≤0.07nM、≤0.06nM、≤0.05nM、或≤0.04nM，所述EC50通过ELISA检测；
e)所述抗DLL3抗体与阳性抗体(例如BI-764532)识别不同的DLL3表位。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的Y选自：
其中Y的O-端与L相连。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的Y选自：(包括)；其中Y的O-端与L相连。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的L为-L1-L2-L3-L4-，其中：
L1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH2-C(O)-NR3-W-C(O)-和-C(O)-W-C(O)-，其中W选自C1-6亚烷基、C1-6亚烷基-C3-6环烷基，其中所述的C1-6亚烷基、C1-6亚烷基-C3-6环烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L2选自-NR4(CH2CH2O)pCH2CH2C(O)-、-NR4(CH2CH2O)pCH2C(O)-和化学键，其中p为1至20的整数；
L3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
L4选自-NR5(CR6R7)t-、-C(O)NR5、-C(O)NR5(CH2)t-和化学键，其中t为1至6的整数；
R3、R4和R5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R6和R7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的L为-L1-L2-L3-L4-，其中：
L1为s1为2至8的整数；
L2为化学键；
L3为四肽残基；优选地，L3为包含甘氨酸-甘氨酸-苯丙氨酸-甘氨酸的四肽残基；
L4为-NH(CH2)t-，t为1或2；
其中所述的L1端与Pc相连。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的L具有如下所示的结构：
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有如下所示的结构：

Pc为如前任一项所述的抗DLL3抗体；n为1至10。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链可变区和轻链可变区，其中：
所述重链可变区包含：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24的氨基酸序列；和
所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；
n为1至10；优选地，n为3至8；更优选地，n为6至8。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链可变区和轻链可变区，其中：
所述重链可变区包含SEQ ID NO：50的氨基酸序列，和所述轻链可变区包含SEQ ID NO：55的氨基酸序列；
n为1至10；优选地，n为3至8；更优选地，n为6至8。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；或
所述重链包含SEQ ID NO：58的氨基酸序列，和所述轻链包含SEQ ID NO：59的氨基酸序列；
n为1至10；优选地，n为3至8；更优选地，n为6至8。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；
n为1至10；优选地，n为3至8；更优选地，n为6至8。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；
n为4.43。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；
n为7.32。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；
n为6.12。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：58的氨基酸序列，和所述轻链包含SEQ ID NO：59的氨基酸序列；
n为3.53。
在一些实施方案中，前述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有选自如下所示的结构：
其中：
Pc为抗DLL3抗体，其包含重链和轻链，其中：
所述重链包含SEQ ID NO：58的氨基酸序列，和所述轻链包含SEQ ID NO：59的氨基酸序列；
n为7.43。
本披露进一步提供一种制备抗体-药物偶联物的方法，其包括将如前任一项所述的抗DLL3抗体还原，然后与(L-Y-D)所示的化合物进行偶联反应，得到本披露所述的抗体-药物偶联物。
另一方面，本披露提供一种药物组合物，其包含如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。在一些实施方案中，所述单位剂量的药物组合物中含有0.1-3000mg或1-1000mg如前所述的抗体-药物偶联物。
另一方面，本披露提供如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或包含其的药物组合物作为药物的用途。在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或包含其的药物组合物作为治疗癌症或肿瘤的药物。在一些实施方案中，如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或包含其的药物组合物作为治疗表达DLL3的癌症或肿瘤的药物。
另一方面，本披露提供如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或前述的药物组合物在制备用于治疗DLL3介导的疾病或病症的药物中的用途。在一些实施方案中，其中所述DLL3介导的疾病或病症为肿瘤或癌症。在一些实施方案中，其中所述的DLL3介导的疾病或病症为表达DLL3的疾病或病症。
另一方面，本披露提供如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或前述的药物组合物在制备用于治疗或预防肿瘤或癌症的药物中的用途；优选地，其中所述肿瘤或癌症选自：
肺癌(例如小细胞肺癌、非小细胞肺癌、大细胞肺癌)、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、咽鳞癌、口腔鳞癌、鼻咽癌、食管癌、甲状腺癌(例如甲状腺髓样癌)、恶性胸膜间皮瘤、乳腺癌(例如三阴性乳腺癌)、 肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结直肠癌(例如结肠癌和直肠癌)、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、肾上腺癌、胶质母细胞瘤、皮肤癌和黑色素瘤；优选地，其中所述肺癌为小细胞肺癌。在一些实施方案中，所述的肿瘤或癌症为表达DLL3的肿瘤或癌症。
另一方面，本披露进一步涉及一种用于治疗和/或预防肿瘤或癌症的方法，该方法包括向需要其的受试者施用治疗有效剂量的如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或前述的药物组合物。在一些实施方案中，其中所述的肿瘤或癌症为表达DLL3的肿瘤或癌症。
另一方面，本披露进一步涉及一种用于治疗或预防肿瘤或癌症的方法，该方法包括向需要其的受试者施用治疗有效剂量的如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或前述的药物组合物；其中所述肿瘤或癌症选自：
肺癌(例如小细胞肺癌、非小细胞肺癌、大细胞肺癌)、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、咽鳞癌、口腔鳞癌、鼻咽癌、食管癌、甲状腺癌(例如甲状腺髓样癌)、恶性胸膜间皮瘤、乳腺癌(例如三阴性乳腺癌)、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结直肠癌(例如结肠癌和直肠癌)、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、肾上腺癌、胶质母细胞瘤、皮肤癌和黑色素瘤；优选地，其中所述肺癌为小细胞肺癌。
本披露提供的抗体-药物偶联物与细胞表面抗原具有良好的亲和力，可有效的被表达DLL3的细胞内吞，并具有很强的抑制肿瘤生长的效果，同时具有良好的安全性。
附图说明
图1A至图1C显示Hu6和Hu100抗体与不同种属的DLL3的结合。其中：图1A显示Hu6和Hu100抗体对H1184细胞结合的FACS结果；图1B显示Hu6和Hu100抗体对cynoDLL3/CHO-s细胞结合的FACS结果；图1C显示Hu6和Hu100抗体对ratDLL3/CHO-s细胞结合的FACS结果。
图2显示不同的抗DLL3抗体竞争结合实验结果。结果显示，Hu6和Hu100与BI-764532不竞争，说明抗体Hu6和Hu100与BI-764532结合不同的表位。
图3显示Hu6和Hu100抗体被细胞内吞的结果；结果显示，Hu6和Hu100均可被细胞内吞。
图4A至图4B显示ADC-1、ADC-2和ADC-3对人和猴DLL3细胞的结合结果。其中：图4A显示ADC-1、ADC-2和ADC-3对人DLL3细胞的结合结果；图4B显示ADC-1、ADC-2和ADC-3对猴DLL3细胞的结合结果。
图5A至图5D显示ADC-1、ADC-2和ADC-3对不同细胞的生长抑制作用。其中：图5A显示ADC-1、ADC-2和ADC-3对高表达DLL3的H1184细胞的生长抑制结果；图5B显示ADC-1、ADC-2和ADC-3对高表达DLL3的DMS53细胞的生长抑制结果；图5C显示ADC-1、ADC-2和ADC-3对低表达DLL3的SK-MEL3细胞的生长抑制结果；图5D显示ADC-1、ADC-2和ADC-3对不表达DLL3的CHO-K1细胞的生长抑制结果。
图6显示ADC-1、ADC-2和ADC-3的旁观者细胞毒性效应结果。
图7显示ADC-1、ADC-2、ADC-4和ADC-5抑制小鼠的DMS53细胞皮下移植肿瘤生长的结果。
图8显示ADC-1和ADC-3抑制小鼠的DMS53细胞皮下移植肿瘤生长的结果。
图9显示ADC-2、ADC-3、ADC-6和ADC-7抑制小鼠的H1184细胞皮下移植肿瘤生长的结果。
图10A至图10C显示ADC-1、ADC-2和ADC-3大鼠药代动力学结果。其中：图10A显示ADC-1大鼠药代动力学结果；图10B显示ADC-2大鼠药代动力学结果；图10C显示ADC-3大鼠药代动力学结果。
图11显示ADC-2食蟹猴药代动力学结果。
具体实施方式
一、术语
为了更容易理解本披露，以下对某些技术和科学术语进行了描述。除非在本文中另有明确定义，本文使用的全部技术和科学术语具有与本领域的普通技术人员通常所理解的相同含义。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
说明书和权利要求书中所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。
除非上下文另外清楚要求，否则在专利说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为“包括但不仅限于”的意义，而不是排他性或穷举性意义。
术语“和/或”，意指包含“和”与“或”两种含义。例如短语“A、B和/或C”旨在涵盖以下方面中的每一个：A、B和C；A、B或C；A或C；A或B；B或C；A和C；A和B；B和C；A(单独)；B(单独)；和C(单独)。当本披露中使用商品名时，旨在包括该商品名产品的制剂、该商品名产品的药物和活性药物部分。
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。
术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗 传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同的基本化学结构(即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但是以与天然存在的氨基酸类似的方式起作用的化学化合物。
术语“氨基酸突变”包括氨基酸取代(也称氨基酸替换)、缺失、插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体，只要最终构建体拥有期望的特性，例如降低或对Fc受体的结合。氨基酸序列缺失和插入包括在多肽链的氨基端和/或羧基端的缺失和插入。具体的氨基酸突变可以是氨基酸取代。在一个实施方案中，氨基酸突变是非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸取代包括由非天然存在的氨基酸或由20种天然氨基酸的衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变。遗传方法可以包括定点诱变、PCR，基因合成等。预计基因工程以外的改变氨基酸侧链基团的方法，如化学修饰也可能是可用的。本文中可使用各种名称来指示同一氨基酸突变。本文中，可采用位置+氨基酸残基的方式表示特定位点的氨基酸残基，例如366W，表示在366位点上的氨基酸残基为W。T366W则表示第366位点上的氨基酸残基由W取代了原来的T。
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。完整抗体通常包含两条轻链和两条重链。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域、重链可变区，接着是三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻域(轻链恒定区、CL)。
术语“全长抗体”、“完整抗体”和“全抗体”在本文可互换使用，指具有与天然抗体结构基本类似的结构或重链具有Fc区的的抗体。天然完整抗体轻链包括轻链可变区VL及恒定区CL，VL处于轻链的氨基末端，轻链恒定区包括κ链及λ链；重链包括可变区VH及恒定区(CH1、CH2及CH3)，VH处于重链的氨基末端，恒定区处于羧基末端，其中CH3最接近羧基末端，重链可属于任何同种型，包括IgG(包括IgG1、IgG2、IgG3及IgG4亚型)、IgA(包括IgA1及IgA2亚型)、IgM及IgE。
术语抗体“可变区”或“可变域”指抗体重链或轻链中参与抗体结合抗原的域。本文中，抗体重链可变区(VH)和轻链可变区(VL)各包含四个保守的框架 区(FR)和三个互补决定区(CDR)。其中，术语“互补决定区”或“CDR”指可变结构域内主要促成与抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2和LCDR3。每个VH和VL由从氨基末端(也称N末端)排到羧基末端(也称C末端)按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“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、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018Oct 16；9:2278)等；各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表1中所示。
表1.CDR编号系统之间的关系
除非另有说明，本披露中的可变区和CDR序列均适用Kabat编号规则。
术语“抗体片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体所结合的抗原相结合。抗体片段的实例包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab′)2、单域抗体、单链Fab(scFab)、双抗体、线性抗体、单链抗体分子(例如scFv)；以及由抗体片段形成的多特异性抗体。
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。在一些实施方案中，Fc区包含了相同或不同的两个亚基。在一些实施方案中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。用于本文所述抗体的合适Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4的Fc区。在一些实施方案中，Fc区的边界还可以变化，例如缺失Fc区的C末端赖氨酸(根据EU编号系统的残基447)或缺失Fc区的C末端甘氨酸和赖氨酸(根据EU编号系统的残基446和447)。除非另有说明，Fc区的编号规则为EU编号系统，又称作EU索引。
术语“嵌合抗体”指抗体中的重和/或轻链的一部分由特定的来源或物种衍生，而重和/或轻链的剩余部分由另外的不同来源或物种衍生的抗体。
术语“人源化抗体”是保留非人抗体的反应性同时在人中具有较低免疫原性的抗体。例如，可以通过保留非人CDR区并用其人对应物(即，恒定区以及可变区的框架区部分)替换抗体的其余部分来实现。
术语“人抗体”、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，意指可变区及恒定区是人序列的抗体。该术语涵盖源自人基因但具有，例如，降低可能的免疫原性、增加亲和力、消除可能会引起不期望的折叠的半胱氨酸或糖基化位点等序列的抗体。该术语涵盖这些在非人细胞(其可能会赋予不具人细胞特征的糖基化)中重组产生的抗体。该术语亦涵盖已在含有人免疫球蛋白重链及轻链基因座的转基因小鼠中饲养的抗体。人抗体的含义明确排除包含非人抗原结合残基的人源化抗体。
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，结合“亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间相互作用。分子X对其配体Y的亲和力通常可以由解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些方法)测量。
如本文所使用的，术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，术语“kdis”或“kd”指特定抗体-抗原相互作用的解离速率。术语“KD”指解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域公知的方法测定抗体的KD值。例如，使用生物传感系统例如系统测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。
术语“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列突变的Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括但不限于：C1q结合和补体依赖性细胞毒性、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、吞噬作用、细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“单克隆抗体”指基本上均质的抗体的群，即在该群中包含的抗体分子的氨基酸序列是相同的，除了可能少量存在的天然突变以外。相比之下，多克隆抗体制剂通常包含在其可变结构域具有不同氨基酸序列的多种不同抗体，其通常特异性针对不同表位。“单克隆”不应解释为要求通过任何特定方法来生产抗体。在一些实施方案中，本披露提供的抗体是单克隆抗体。
术语“抗原”是指能够由诸如抗原结合蛋白(包括例如抗体)的选择性结合剂结合的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
术语“表位”指能够与抗体或其抗原结合片段特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸串(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过三级折叠)而变成空间接近。构象表位和线 性表位的差别在于：在变性溶剂的存在下，不能检测到抗体对构象表位的结合。表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描，肽印迹，肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496)，和交叉阻断。
术语“抗DLL3抗体”和“结合DLL3的抗体”是指能够以足够的亲和力结合DLL3或其表位的抗体。在一个实施方案中，抗DLL3抗体与无关蛋白的结合程度小于该抗体与DLL3结合的至少约10％，所述结合可通过表面等离子体共振测定法测量。
术语“能够特异性结合”、“特异性结合”或“结合”是指相比其他抗原或表位，抗体能够以更高的亲和力结合至某个抗原或其表位。通常地，抗体以约1×10-7M或更小(例如约1×10-8M或更小)的平衡解离常数(KD)结合抗原或其表位。在一些实施方案中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％或更低(例如1％)。可使用已知的方法来测量KD，例如通过表面等离子体共振测定法所测量的。然而，特异性结合至抗原或其表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相应抗原具有交叉反应性。
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸。所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置处。编码多肽或融合蛋白的分离的核酸指编码多肽或融合蛋白的一个或更多个核酸分子，包括在单一载体或分开的载体中的这样的一个或更多个核酸分子，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个核酸分子。除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代。
术语“多肽”和“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物，其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学模拟物，以及适 用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。除非另外说明，否则特定的多肽序列还隐含地涵盖其保守修饰的变体。
术语序列“同一性”指两条序列的氨基酸/核酸在等价位置相同的程度(百分比)；当对两条序列进行最佳比对时，必要时引入间隙以获取最大的同一性百分比，且不将任何保守性取代视为序列同一性的一部分。为测定序列同一性百分比，比对可以通过本领域技术已知的技术来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)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“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。术语“表达载体”或“表达构建体”是指可对宿主细胞进行转化，且含有指导和/或控制与其可操作地连接的一个或多个异源编码区的表达的核酸序列的载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及其衍生的后代，而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，可以含有突变。本文中包括这样的突变体后代，其与初始转化细胞中筛选或选择的细胞具有相同的功能或生物学活性。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系植物细胞和真菌细胞。示例性的宿主细胞如下：中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，HepG2)、A549细胞、3T3细胞和HEK-293细胞、巴氏毕赤酵母(Pichiapastoris)、芬兰毕赤酵母(Pichia finlandica)、白色念珠菌(Candida albicans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)。
“抗体-药物偶联物”(antibody drug conjugate，ADC)，是将抗体(或其抗原结合片段)直接或通过接头与药物相连后得到的偶联物。
“药物(Drug，缩写：D)”是任何具有生物或可检测活性的物质(例如治疗剂、可检测标记、结合剂等)和在体内代谢成活性剂的前药。治疗剂的实例包括细胞毒性剂、化学治疗剂、细胞生长抑制剂和免疫调节剂。化学治疗剂是可用于 治疗癌症的化学化合物。代表性治疗剂包括细胞毒素、细胞毒性剂和细胞生长抑制剂。
细胞毒性效应是指缺失、消除和/或杀死目标细胞。细胞毒性剂是指对细胞具有细胞毒性和/或细胞生长抑制效应的药剂。细胞生长抑制效应是指抑制细胞增殖。细胞生长抑制剂是指对细胞具有细胞生长抑制效应、由此抑制细胞的特定亚组的生长和/或扩增的药剂。
额外代表性治疗剂包括放射性同位素、化学治疗剂、免疫调节剂、抗血管生成剂、抗增殖剂、促细胞凋亡剂和细胞裂解酶(例如，RNAse)。这些药物描述词并不互斥，且因此，可使用一个或多个上述术语描述治疗剂。例如，所选放射性同位素也是细胞毒素。治疗剂可制备为上述任一种的药学上可接受的盐、酸或衍生物。通常，具有放射性同位素作为药物的偶联物被称为放射性免疫偶联物，具有化学治疗剂作为药物的被称为化学免疫偶联物。
细胞毒性剂的实例包括，但不限于，依喜替康、蒽环霉素、奥里斯他汀、CC-1065、尾海兔素(dolastatin)、多卡米星、烯二炔、格尔德霉素(geldanamycin)、美登素(maytansine)、嘌呤霉素、紫杉烷、长春花生物碱、SN-38、微管溶素(tubulysin)、hemiasterlin、艾日布林、Trabectedin、Lurbinectedin和其立体异构体、等排物、类似物或衍生物。也可使用化学治疗剂、植物毒素、其他生物活性蛋白质、酶(即，ADEPT)、放射性同位素、光敏剂(即，用于光动力学治疗)。
术语“标记”当本文中使用时是指直接或间接缀合至抗体以便生成“标记的”抗体的可检测化合物或组合物。标记可以是自身可检测(例如，放射性同位素标记或荧光标记)，或在酶促标记的情况下，标记可催化底物化合物或组合物可检测的化学变化。放射性同位素标记包括，例如，I-131、I-123、I-125、Y-90、Re-188、Re-186、At-211、Cu-67、Bi-212、和Pd-109。标记也可以是不可检测的实体，诸如毒素。
术语“接头单元”、“接头”是指一端与抗体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与抗体或药物相连。接头附接至抗体可以多种方式完成，诸如经由表面赖氨酸、还原性偶联至氧化碳水化合物、通过还原链间二硫键释放的半胱氨酸残基、在特定位点处改造的反应性半胱氨酸残基、和含有酰基供体谷氨酰胺的标签或通过在转谷氨酰胺酶和胺存在下，对多肽改造，使其具有反应性的内源性谷氨酰胺。多种ADC连接系统是本领域已知的，包括基于腙-、二硫化物-和肽-的连接。
接头可以包含一种或多种接头元件。示例性的接头元件包括6-马来酰亚氨基己酰基(“MC”)、马来酰亚氨基丙酰基(“MP”)、缬氨酸-瓜氨酸(“val-cit”或“vc”)、丙氨酸-苯丙氨酸(“ala-phe”)、对氨基苄氧羰基(“PAB”)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(“SMCC”，在本文中也称作“MCC”)和N-琥珀酰亚氨基(4-碘- 乙酰基)氨基苯甲酸酯(“SIAB”)。
接头可以选自以下的元件或其组合：延伸物、间隔物和氨基酸单元。可以通过本领域已知方法合成接头，诸如US20050238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
接头元件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的示例二肽)，
瓜氨酸＝2-氨基-5-脲基戊酸，
PAB＝对氨基苄氧羰基(“自我牺牲”接头元件的示例)，
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)，
MC(PEG)6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)，
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯，
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯，
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯，
IT＝亚氨基硫烷。
“L-D”是药物(D)连接至接头(L)产生的接头-药物部分。
“载药量”，也称药物抗体比例(Drug-to-Antibody Ratio，DAR)，即ADC中每个抗体所偶联的药物的平均数量。其可在例如每个抗体偶联约1至约10个药物的范围内，并且在某些实施例中，在每个抗体偶联约1至约8个药物的范围内，优选自2-8，2-7，2-6，2-5，2-4，1-3，3-4，3-5，3-6，3-7，3-8，4-5，4-6，4-7，4-8，5-6，5-7，5-8和6-8的范围。本披露的ADC通式包括前述一定范围内的抗体-药物偶联物的集合。在本披露的实施方案中，载药量可表示为n，是小数或整数。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、HIC和RP-HPLC测定载药量。
本披露的一个实施方案中，药物通过接头偶联在抗体的反应性基团(如巯基)上。
可以用以下非限制性方法控制ADC的载药量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
术语“烷基”指饱和的直链或带有支链的脂肪族烃基，其具有1至20个(例 如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)碳原子(即C1-20烷基)。所述烷基优选具有1至12个碳原子的烷基(即C1-12烷基)，更优选具有1至6个碳原子的烷基(即C1-6烷基)。非限制性的实例包括：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。烷基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自D原子、卤素、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“亚烷基”指二价烷基，其中烷基如上所定义，其具有1至20个(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)碳原子(即C1-20亚烷基)。所述亚烷基优选具有1至12个碳原子的亚烷基(即C1-12亚烷基)，更优选具有1至6个碳原子的亚烷基(即C1-6亚烷基)。非限制性的实例包括：-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH2CH3)-、-CH2CH(CH3)-、-CH2C(CH3)2-、-CH2CH2CH2-、-CH2CH2CH2CH2-等。亚烷基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自氘原子、卤素、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“烯基”指分子中含有至少一个碳碳双键的烷基，其中烷基的定义如上所述，其具有2至12个(例如2、3、4、5、6、7、8、9、10、11或12个)碳原子(即C2-12烯基)。所述烯基优选具有2至6个碳原子的烯基(即C2-6烯基)。非限制性的实例包括：乙烯基、丙烯基、异丙烯基、丁烯基等。烯基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自氘原子、烷氧基、卤素、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“炔基”指分子中含有至少一个碳碳三键的烷基，其中烷基的定义如上 所述，其具有2至12个(例如2、3、4、5、6、7、8、9、10、11或12个)碳原子(即C2-12炔基)。所述炔基优选具有2至6个碳原子的炔基(即C2-6炔基)。非限制性的实例包括：乙炔基、丙炔基、丁炔基、戊炔基、己炔基等。炔基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自氘原子、烷氧基、卤素、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“烷氧基”指-O-(烷基)，其中烷基的定义如上所述。非限制性的实例包括：甲氧基、乙氧基、丙氧基和丁氧基等。烷氧基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自氘原子、卤素、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“环烷基”指饱和或部分不饱和的单环全碳环(即单环环烷基)或多环系统(即多环环烷基)，其具有3至20个(例如3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即3至20元环烷基)。所述环烷基优选具有3至12个环原子的环烷基(即3至12元环烷基)，更优选具有3至8个环原子的环烷基(即3至8元环烷基)，最优选具有3至6个环原子的环烷基(即3至6元环烷基，C3-6环烷基)。
所述的单环环烷基，非限制性的实例包括：环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基和环辛基等。
所述的多环环烷基包括：螺环烷基、稠环烷基和桥环烷基。
术语“螺环烷基”指环之间共用一个碳原子(称螺原子)的多环系统，其环内可以含有一个或多个双键，或其环内可以含有一个或多个选自氮、氧和硫的杂原子(所述的氮可任选被氧化，即形成氮氧化物；所述的硫可任选被氧代，即形成亚砜或砜，但不包括-O-O-、-O-S-或-S-S-)，条件是至少含有一个全碳环且连接点在该全碳环上，其具有5至20个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即5至20元螺环烷基)。所述螺环烷基优选具有6至14个环原子的螺环烷基(即6至14元螺环烷基)，更优选具有7至10个环原子的螺环烷基(即7至10元螺环烷基)。所述螺环烷基包括单螺环烷基和多螺环烷基(如双螺环烷基等)，优选单螺环烷基或双螺环烷基，更优选3元/4元、3元/5元、3元/6元、4元/4元、4元/5元、4元/6元、5元/3元、5元/4元、5元/5元、5元/6元、5元/7元、6元/3元、6元/4元、6元/5元、6元/6元、6元/7元、7元/5元或7元/6元单螺环烷基。非限制性的实例包括：
其连接点可在任意位置；
等。
术语“稠环烷基”指环之间共享毗邻的两个碳原子的多环系统，其为单环环烷基与一个或多个单环环烷基稠合，或者单环环烷基与杂环基、芳基或杂芳基中的一个或多个稠合，其中连接点在单环环烷基上，其环内可以含有一个或多个双键，且具有5至20个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即5至20元稠环烷基)。所述稠环烷基优选具有6至14个环原子的稠环烷基(即6至14元稠环烷基)，更优选具有7至10个环原子的稠环烷基(即7至10元稠环烷基)。所述稠环烷基包括双环稠环烷基和多环稠环烷基(如三环稠环烷基、四环稠环烷基等)，优选双环稠环烷基或三环稠环烷基，更优选3元/4元、3元/5元、3元/6元、4元/4元、4元/5元、4元/6元、5元/3元、5元/4元、5元/5元、5元/6元、5元/7元、6元/3元、6元/4元、6元/5元、6元/6元、6元/7元、7元/5元或7元/6元双环稠环烷基。非限制性的实例包括：
，其连接点可在任意位置；
等。
术语“桥环烷基”指环之间共用两个不直接连接的碳原子的全碳多环系统，其环内可以含有一个或多个双键，且具有5至20个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)碳原子(即5至20元桥环烷基)。所述桥环烷基优选具有6至14个碳原子的桥环烷基(即6至14元桥环烷基)，更优选具有7至10个碳原子的桥环烷基(即7至10元桥环烷基)。所述桥环烷基包括双环桥环烷基和多环桥环烷基(如三环桥环烷基、四环桥环烷基等)，优选双环桥环烷基或三环桥环烷基。非限制性的实例包括：
其连接点可在任意位置。
环烷基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自D原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氧代基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“杂环基”指饱和或部分不饱和的单环杂环(即单环杂环基)或多环杂环系统(即多环杂环基)，其环内至少含有一个(例如1、2、3或4个)选自氮、氧和硫的杂原子(所述的氮可任选被氧化，即形成氮氧化物；所述的硫可任选被氧代，即形成亚砜或砜，但不包括-O-O-、-O-S-或-S-S-)，且具有3至20个(例如3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即3至20元杂环基)。所述杂环基优选具有3至12个环原子的杂环基(即3至12元杂环基)；进一步优选具有3至8个环原子的杂环基(即3至8元杂环基)；更优选具有3至6个环原子的杂环基(即3至6元杂环基)；最优选具有5或6个环原子的杂环基(即5或6元杂环基)。
所述的单环杂环基，非限制性的实例包括：吡咯烷基、四氢吡喃基、1,2,3,6-四氢吡啶基、哌啶基、哌嗪基、吗啉基、硫代吗啉基和高哌嗪基等。
所述的多环杂环基包括螺杂环基、稠杂环基和桥杂环基。
术语“螺杂环基”指环之间共用一个原子(称螺原子)的多环杂环系统，其环内可以含有一个或多个双键，且其环内至少含有一个(例如1、2、3或4个)选自氮、氧和硫的杂原子(所述的氮可任选被氧化，即形成氮氧化物；所述的硫可任选被氧代，即形成亚砜或砜，但不包括-O-O-、-O-S-或-S-S-)，条件是至少含有一个单环杂环基且连接点在该单环杂环基上，其具有5至20个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即5至20元螺杂环基)。所述螺杂环基优选具有6至14个环原子的螺杂环基(即6至14元螺杂环基)，更优选具有7至10个环原子的螺杂环基(即7至10元螺杂环基)。所述螺杂环基包括单螺杂环基和多螺杂环基(如双螺杂环基等)，优选单螺杂环基或双螺杂环基，更优选3元/4元、3元/5元、3元/6元、4元/4元、4元/5元、4元/6元、5元/3元、5元/4元、5元/5元、5元/6元、5元/7元、6元/3元、6元/4元、6元/5元、6元/6元、6元/7元、7元/5元或7元/6元单螺杂环基。非限制性的实例包括：
等。
术语“稠杂环基”指环之间共享毗邻的两个原子的多环杂环系统，其环内可以含有一个或多个双键，且其环内至少含有一个(例如1、2、3或4个)选自氮、 氧和硫的杂原子(所述的氮可任选被氧化，即形成氮氧化物；所述的硫可任选被氧代，即形成亚砜或砜，但不包括-O-O-、-O-S-或-S-S-)，其为单环杂环基与一个或多个单环杂环基稠合，或者单环杂环基与环烷基、芳基或杂芳基中的一个或多个稠合，其中连接点在单环杂环基上，且具有5至20个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即5至20元稠杂环基)。所述稠杂环基优选具有6至14个环原子的稠杂环基(即6至14元稠杂环基)，更优选具有7至10个环原子的稠杂环基(即7至10元稠杂环基)。所述稠杂环基包括双环和多环稠杂环基(如三环稠杂环基、四环稠杂环基等)，优选双环稠杂环基或三环稠杂环基，更优选3元/4元、3元/5元、3元/6元、4元/4元、4元/5元、4元/6元、5元/3元、5元/4元、5元/5元、5元/6元、5元/7元、6元/3元、6元/4元、6元/5元、6元/6元、6元/7元、7元/5元或7元/6元双环稠杂环基。非限制性的实例包括：
等。
术语“桥杂环基”指环之间共用两个不直接连接的原子的多环杂环系统，其环内可以含有一个或多个双键，并且其环内至少含有一个(例如1、2、3或4个)选自氮、氧和硫的杂原子(所述的氮可任选被氧化，即形成氮氧化物；所述的硫可任选被氧代，即形成亚砜或砜，但不包括-O-O-、-O-S-或-S-S-)，其具有5至20个(例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个)环原子(即5至20元桥杂环基)。所述桥杂环基优选具有6至14个环原子的桥杂环基(即6至14元桥杂环基)，更优选具有7至10个环原子的桥杂环基(即7至10元桥杂环基)。根据组成环的数目可以分为双环桥杂环基和多环桥杂环基(如三环桥杂环基、四环桥杂环基等)，优选双环桥杂环基或三环桥杂环基。非限制性的实例包括：
等。
杂环基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自氘原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氧代基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“芳基”指具有共轭的π电子体系的单环全碳芳环(即单环芳基)或多环芳环系统(即多环芳基)，其具有6至14个(例如6、7、8、9、10、11、12、13或14个)环原子(即6至14元芳基)。所述芳基优选具有6至10个环原子的芳基(即6至10元芳基)。所述的单环芳基，例如苯基。所述的多环芳基，非限制性的实例包括：萘基、蒽基、菲基等。所述多环芳基还包括苯基与杂环基或环烷基中的一个或多个稠合，或萘基与杂环基或环烷基中的一个或多个稠合，其中连接点在苯基或萘基上，并且在这种情况下，环原子个数继续表示多环芳环系统中的环原子个数，非限制性的实例包括：
等。
芳基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自D原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氧代基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“杂芳基”指具有共轭的π电子体系的单环杂芳环(即单环杂芳基)或多环杂芳环系统(即多环杂芳基)，其环内至少含有一个(例如1、2、3或4个)选自氮、氧和硫的杂原子(所述的氮可任选被氧化，即形成氮氧化物；所述的硫可任选被氧代，即形成亚砜或砜，但不包括-O-O-、-O-S-或-S-S-)，其具有5至14个(例如5、6、7、8、9、10、11、12、13或14个)环原子(即5至14元杂芳基)。所述杂芳基优选具有5至10个环原子的杂芳基(即5至10元杂芳基)，更优选具有5或6个环原子的杂芳基(即5或6元杂芳基)。
所述的单环杂芳基，非限制性的实例包括：呋喃基、噻吩基、噻唑基、异噻唑基、噁唑基、异噁唑基、噁二唑基、噻二唑基、咪唑基、吡唑基、三唑基、四唑基、呋咱基、吡咯基、N-烷基吡咯基、吡啶基、嘧啶基、吡啶酮基、N-烷基吡啶酮(如等)、吡嗪基、哒嗪基等。
所述的多环杂芳基，非限制性的实例包括：吲哚基、吲唑基、喹啉基、异喹啉基、喹喔啉基、酞嗪基、苯并咪唑基、苯并噻吩基、喹唑啉基、苯并噻唑基、 咔唑基等。所述多环杂芳基还包括单环杂芳基与一个或多个芳基稠合，其中连接点在芳香环上，并且在这种情况下，环原子个数继续表示多环杂芳环系统中的环原子个数。所述多环杂芳基还包括单环杂芳基与环烷基或杂环基中的一个或多个稠合，其中连接点在单环杂芳环上，并且在这种情况下，环原子个数继续表示多环杂芳环系统中的环原子个数。非限制性的实例包括：
等。
杂芳基可以是取代的或非取代的，当被取代时，其可以在任何可使用的连接点被取代，取代基优选选自氘原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个。
术语“氨基保护基”是指为了使分子其它部位进行反应时氨基保持不变，在氨基上引入的易于脱去的基团。非限制性的实例包括：(三甲基硅)乙氧基甲基、四氢吡喃基、叔丁氧羰基(Boc)、苄氧羰基(Cbz)、笏甲氧羰基(Fmoc)、烯丙氧羰基(Alloc)、三甲基硅乙氧羰基(Teoc)、甲氧羰基、乙氧羰基、邻苯二甲酰基(Pht)、对甲苯磺酰基(Tos)、三氟乙酰基(Tfa)、三苯甲基(Trt)、2,4-二甲氧基苄基(DMB)、乙酰基、苄基、烯丙基、对甲氧苄基等。
术语“羟基保护基”是指在羟基上引入的易于脱去的基团，用于阻断或保护羟基而在化合物的其它官能团上进行反应。非限制性的实例包括：三甲基硅基(TMS)、三乙基硅基(TES)、三异丙基硅基(TIPS)、叔丁基二甲基硅基(TBS)、叔丁基二苯基硅基(TBDPS)、甲基、叔丁基、烯丙基、苄基、甲氧基甲基(MOM)、乙氧基乙基、2-四氢吡喃基(THP)、甲酰基、乙酰基、苯甲酰基、对硝基苯甲酰基等。
术语“环烷基氧基”指环烷基-O-，其中环烷基如上所定义。
术语“杂环基氧基”指杂环基-O-，其中杂环基如上所定义。
术语“芳基氧基”指芳基-O-，其中芳基如上所定义。
术语“杂芳基氧基”指杂芳基-O-，其中杂芳基如上所定义。
术语“烷硫基”指烷基-S-，其中烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“卤代烷氧基”指烷氧基被一个或多个卤素取代，其中烷氧基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟烷基”指烷基被一个或多个羟基取代，其中烷基如上所定义。
术语“甲叉基”指＝CH2。
术语“卤素”指氟、氯、溴或碘。
术语“羟基”指-OH。
术语“巯基”指-SH。
术语“氨基”指-NH2。
术语“氰基”指-CN。
术语“硝基”指-NO2。
术语“氧代”或“氧代基”指“＝O”。
术语“羰基”指C＝O。
术语“羧基”指-C(O)OH。
术语“羧酸酯基”指-C(O)O(烷基)、-C(O)O(环烷基)、(烷基)C(O)O-或(环烷基)C(O)O-，其中烷基和环烷基如上所定义。
化学式中简称“Me”为甲基。
化学式中简称“Ph”为苯基。
术语“THF”指四氢呋喃。
术语“EtOAc”指乙酸乙酯。
术语“MeOH”指甲醇。
术语“DMF”指N,N-二甲基甲酰胺。
术语“DIPEA”指二异丙基乙胺。
术语“TFA”指三氟乙酸。
术语“MeCN”指乙腈。
术语“DMA”指N,N-二甲基乙酰胺。
术语“Et2O”指乙醚。
术语“DCE”指1,2二氯乙烷。
术语“DIPEA”指N,N-二异丙基乙胺。
术语“NBS”指N-溴代琥珀酰亚胺。
术语“NIS”指N-碘代丁二酰亚胺。
术语“Cbz-Cl”指氯甲酸苄酯。
术语“Pd2(dba)3”指三(二亚苄基丙酮)二钯。
术语“Dppf”指1,1’-双二苯基膦二茂铁。
术语“HATU”指2-(7-氧化苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯。
术语“KHMDS”指六甲基二硅基胺基钾。
术语“LiHMDS”指双三甲基硅基胺基锂。
术语“MeLi”指甲基锂。
术语“n-BuLi”指正丁基锂。
术语“NaBH(OAc)3”指三乙酰氧基硼氢化钠。
术语“DCM”指二氯甲烷。
术语“DMAP”指4-二甲氨基吡啶。
术语“DMBOH”指2,4-二甲氧基苄醇。
术语“EDCI”指1-(3-二甲基氨基丙基)-3-乙基碳二亚胺。
术语“MTBE”指甲基叔丁基醚。
术语“DMF”指N,N-二甲基甲酰胺。
术语“DMTMM”指4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐。
术语“EtOAc”指乙酸乙酯。
本披露的化合物可以存在特定的立体异构体形式。术语“立体异构体”是指结构相同但原子在空间中的排列不同的异构体。其包括顺式和反式(或Z和E)异构体、(-)-和(+)-异构体、(R)-和(S)-对映异构体、非对映异构体、(D)-和(L)-异构体、互变异构体、阻转异构体、构象异构体及其混合物(如外消旋体、非对映异构体的混合物)。本披露化合物中的取代基可以存在另外的不对称原子。所有这些立体异构体以及它们的混合物，均包括在本披露的范围内。可以通过手性合成、手性试剂或者其他常规技术制备光学活性的(-)-和(+)-异构体、(R)-和(S)-对映异构体以及(D)-和(L)-异构体。本披露某化合物的一种异构体，可以通过不对称合成或者手性助剂来制备，或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，得到纯的异构体。此外，对映异构体和非对映异构体的分离通常是通过色谱法完成。
本披露所述化合物的化学结构中，键表示未指定构型，即如果化学结构中存在手性异构体，键可以为或者同时包含 两种构型。
本披露的化合物可以以不同的互变异构体形式存在，并且所有这样的形式包含在本披露的范围内。术语“互变异构体”或“互变异构体形式”是指平衡存在并且容易从一种异构形式转化为另一种异构形式的结构异构体。其包括所有可能的互变异构体，即以单一异构体的形式或以所述互变异构体的任意比例的混合物的形式存在。非限制性的实例包括：酮-烯醇、亚胺-烯胺、内酰胺-内酰亚胺等。内酰胺-内酰亚胺的平衡如下所示：
如当提及吡唑基时，应理解为包括如下两种结构中的任何一种或两种互变异构体的混合物：
所有的互变异构形式在本披露的范围内，且化合物的命名不排除任何互变异构体。
本披露的化合物包括其化合物的所有合适的同位素衍生物。术语“同位素衍生物”是指至少一个原子被具有相同原子序数但原子质量不同的原子替代的化合物。可引入到本披露化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、氯、溴和碘等的稳定和放射性的同位素，例如分别为2H(氘，D)、3H(氚，T)、11C、13C、14C、15N、17O、18O、32p、33p、33S、34S、35S、36S、18F、36Cl、82Br、123I、124I、125I、129I和131I等，优选氘。
相比于未氘代药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本披露的化合物的所有同位素组成的变换，无论放射性与否，都包括在本披露的范围之内。与碳原子连接的各个可用的氢原子可独立地被氘原子替换，其中氘的替换可以是部分或完全的，部分氘的替换是指至少一个氢被至少一个氘替换。
当一个位置被特别地指定为“氘”或“D”时，该位置应理解为氘的丰度比氘的天然丰度(其为0.015％)大至少1000倍(即至少15％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少1000倍(即至少15％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少2000倍(即至少30％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少3000倍(即至少45％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少3340倍(即至少50.1％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少3500倍(即至少52.5％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少4000倍(即至少60％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少4500倍(即至少67.5％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少5000倍(即至少75％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少5500倍(即至少82.5％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大 至少6000倍(即至少90％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6333.3倍(即至少95％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6466.7倍(即至少97％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6600倍(即至少99％的氘掺入)。在一些实施方案中，每个被指定的氘原子的氘的丰度比氘的天然丰度大至少6633.3倍(即至少99.5％的氘掺入)。
“取代”或“取代的”指基团中的一个或多个氢原子，优选1至6个，更优选1至3个氢原子彼此独立地被相应数目的取代基取代。本领域技术人员能够在不付出过多努力的情况下(通过实验或理论)确定可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和键的碳原子(如烯)结合时可能是不稳定的。
本披露还包括各种氘化形式的式(Pc-L-Y-D)抗体-药物偶联物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(Pc-L-Y-D)抗体-药物偶联物。在制备氘代形式的式(Pc-L-Y-D)抗体-药物偶联物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
“任选地”或“任选”是指随后所描述的事件或环境可以但不必然发生，其包括该事件或环境发生或不发生两种情形。例如“任选地(任选)被卤素或者氰基取代的烷基”包括烷基被卤素或者氰基取代的情形和烷基不被卤素和氰基取代的情形。
术语“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，以及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。
药物组合物可以是无菌注射水溶液形式。可接受的溶媒和溶剂有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入受试者的血流中。或者，按可保持本披露化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的示例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬 浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
术语“药学上可接受的盐”或“可药用盐”是指本披露的抗体-药物偶联物的盐，这类盐用于受试者时具有安全性和有效性，且具有应有的生物活性。作为一个示例，本披露抗体-药物偶联物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性示例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“药学上可接受的载体”指药学配制剂中与活性成分不同的，且对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂、稳定剂或防腐剂。
术语“赋形剂”是在药物制剂中除活性成分以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
术语“受试者”或“个体”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、羊、狗、牛、禽、两栖动物和爬行动物。除非指出时，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。
“施用”或“给予”，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。
术语“样本”是指从受试者分离的流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样本为生物流体，诸如血液、血清和浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、排泄物、痰、分泌组织和器官的粘膜分泌物、阴道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液，例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样本、细针穿刺、手术切除的组织、器官培养物或细胞培养物。
“治疗(treatment或treat)”和“处理”(及其语法变型)指试图改变所治疗 个体的病理过程的临床干预，并且可以为了预防或者在临床病理学的过程期间实施。治疗的期望效果包括但不限于预防疾病的发生或再发生，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。在一些实施方案中，使用本披露的抗体来延迟疾病的形成或减缓疾病的进展。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤的量。在一些实施方案中，有效量是治疗有效量或预防有效量。“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效未必在给予一个剂量之后便发生，可能在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于多种因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如患者改善的健康状况。
二、具体实施方式的描述
A.抗体药物偶联物中抗体的结构
在某些实施方案中，本文中提供的抗体是全长抗体。
在某些实施方案中，本文中提供的抗体是抗体片段。
在一个实施方案中，抗体片段是Fab、Fab'、Fab'-SH或F(ab')2片段，特别是Fab片段。“Fab”，其是由VL、VH、CL和CH1结构域组成的单价片段。“Fab片段”可以是抗体经木瓜蛋白酶裂解产生的。“Fab'”含有VL、CL以及VH和CH1，还含有CH1和CH2结构域之间的区域，以使得在两个Fab'片段的两条重链之间可以形成链间二硫键，以形成F(ab')2分子。“Fab'-SH”是其中恒定区的半胱氨酸残基具有游离巯基的Fab'片段。“F(ab')2”包含在铰链区通过二硫键连接的两个Fab片段的二价片段。
在另一个实施方案中，抗体片段是双抗体，三抗体或四抗体。双抗体是具有两个抗原结合位点的抗体片段，该片段在同一条多肽链(VH-VL)中包含相连的VH和VL。通过使用过短的接头使得同一条链上的两个结构域之间不能配对，迫使这些结构域与另一条链的互补结构域配对，从而产生两个抗原结合位点，两个抗原可以是相同或不同的。
在另一个实施方案中，抗体片段是单链Fab片段。“单链Fab片段”或“scFab”是由VH，CH1，VL，CL和接头组成的多肽，其中所述各结构域和所述接头在N 端至C端方向具有以下顺序之一：a)VH-CH1-接头-VL-CL，b)VL-CL-接头-VH-CH1，c)VH-CL-接头-VL-CH1或d)VL-CH1-接头-VH-CL。在一个实施方案中，所述接头是具有至少30个氨基酸的多肽。在另一个实施方案中，所述接头是具有32至50个氨基酸之间的多肽。所述单链Fab片段经由CL和CH1之间的天然二硫键而被稳定化。另外，通过插入半胱氨酸残基(例如在重链可变区中的位置44和轻链可变区中的位置100，根据Kabat编号)产生链间二硫键，这些单链Fab分子可以进一步被稳定化。
在另一个实施方案中，抗体片段是单链可变片段(scFv)。“scFv”是包含至少一个含有轻链可变区的抗体片段和至少一个含有重链可变区的抗体片段的融合蛋白，其中轻链可变区和重链可变区通过短的柔性肽接头连续连接，能够表达为单链多肽，并且其中scFv保持其所源自的完整抗体的特异性。除非特别指出，否则在本文中scFv可以以任何一种顺序具有VL和VH可变区，例如相对于多肽的N端和C端，scFv可以包含VL-接头-VH或可以包含VH-接头-VL。
在另一个实施方案中，抗体片段是由VH和CH1结构域组成的Fd片段。
在另一个实施方案中，抗体片段是由抗体的单臂的VH和VL结构域组成的Fv片段。
在另一个实施方案中，抗体片段是dsFv，dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering.7:697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
在另一个实施方案中，抗体片段是单域抗体，单域抗体是包含抗体的整个或部分重链可变域或整个或部分轻链可变域的抗体片段。
在另一个实施方案中，抗体片段是结构域抗体(domain antibody,dAb)；参见例如美国专利No.6,248,516。结构域抗体(dAb)为抗体的功能结合结构域，其对应于人抗体的重(VH)或轻(VL)链的可变区。dAB具有约13kDa的分子量、或者为小于完整抗体大小十分之一。dAB良好地表达于包括细菌、酵母和哺乳动物细胞体系的各种宿主中。此外，即使经历严厉条件下，例如冻干或者热变性，dAb高度稳定和保持活性。参见例如，美国专利6,291,158；6,582,915；6,593,081；6,172,197；美国系列No.2004/0110941；欧洲专利0368684；美国专利6,696,245；WO04/058821；WO04/003019和WO03/002609。
在某些实施方案中，本文中提供的抗体是嵌合抗体。在一个例子中，嵌合抗体包含非人可变区(例如自小鼠、大鼠、仓鼠、家兔、或非人灵长类，诸如猴衍生的可变区)和人恒定区。在又一个例子中，嵌合抗体是“类转换的”抗体，其中类或亚类已经自亲本抗体的类或亚类改变。
在某些实施方案中，抗体是人源化抗体。通常，将非人抗体通过人源化以降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。一般地，人源化 抗体包含一个或多个可变区，其中CDR或其部分衍生自非人抗体，而FR或其部分衍生自人抗体。任选地，人源化抗体还会包含人恒定区的一部分。在一些实施方案中，可将人源化抗体中的一些FR残基用来自非人抗体(例如提供CDR序列的抗体)的相应残基替代。
人源化抗体及其生成方法综述于如Almagro and Fransson,Front.Biosci.13:1619-1633(2008)，并且进一步记载于如Riechmann等，Nature 332:323-329(1988)；Queen等，Proc.Nat'l Acad.Sci.USA 86:10029-10033(1989)；美国专利No.5,821,337,7,527,791,6,982,321和7,087,409；Kashmiri等，Methods 36:25-34(2005)(描述了特异性决定区(SDR)嫁接)；Padlan,Mol.Immunol.28:489-498(1991)(描述了“再表面”(resurfuacing))；Dall’Acqua等，Methods 36:43-60(2005)(描述了“FR改组”)；和Osbourn等，Methods 36:61-68(2005)和Klimka等，Br.J.Cancer 83:252-260(2000)(描述了FR改组的“引导选择”方法)。
可以用于人源化的人框架区包括但不限于：使用“最佳拟合(best-fit)”方法选择的框架区(见例如Sims等，J.Immunol.151:2296(1993))；衍生自轻链可变区或重链可变区的特定亚组的人抗体的共有序列的框架区(见例如Carter等Proc.Natl.Acad.Sci.USA,89:4285(1992)；和Presta等，J.Immunol.,151:2623(1993))；人成熟的(体细胞突变的)框架区或人种系框架区(见例如Almagro and Fransson,Front.Biosci.13:1619-1633(2008))；和通过筛选FR文库获得的框架区(见例如Baca等，J.Biol.Chem.272:10678-10684(1997)和Rosok等，J.Biol.Chem.271:22611-22618(1996))。
B.抗体药物偶联物中抗体的修饰
在某些实施方案中，涵盖本文中提供的抗体偶联物中抗体的氨基酸序列变体。例如，可以期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗体的氨基酸序列内的残基的删除、和/或插入、和/或替代。可以进行删除、插入、和替代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如抗原结合。
a)替代、插入和删除变体
在某些实施方案中，提供了具有一处或多处氨基酸替代的抗体变体。替代诱变感兴趣的位点包括CDR和FR。保守替代在表2中在“优选的替代”的标题下显示。更实质的变化在表2中在“示例性替代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸替代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。
表2

依照常见的侧链特性，氨基酸可以如下分组:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性，亲水性的：Cys，Ser，Thr，Asn，Gln；
(3)酸性的：Asp，Glu；
(4)碱性的：His，Lys，Arg；
(5)影响链取向的残基：Gly，Pro；
(6)芳香族的：Trp，Tyr，Phe。
非保守替代会需要用这些类别之一的成员替换另一个类别的成员。
在某些实施方案中，替代、插入或缺失可以在一个或多个CDR内发生，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对CDR做出保守变化(例如保守替代，如本文中提供的)，其不实质性降低结合亲和力。此类变化可以例如在CDR中的抗原接触残基外部。在上文提供的变体VH和VL序列的某些实施方案中，每个CDR是未改变的，或者含有不超过1、2或3处氨基酸替代。
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”，如由Cunningham and Wells(1989)Science,244:1081-1085所描述的。在这种方法中，鉴定一个残基或残基组(例如带电荷的残基，诸如Arg、Asp、His、Lys和Glu)，并且用中性或带负电荷的氨基酸(例如，Ala或聚丙氨酸)替换以确定该抗体与抗原的相互作用是否受影响。可以在对初始替代显示功能敏感性的氨基酸位置引入进一步的替代。此外，可通过研究抗原-抗体复合物的晶体结构来鉴定 抗体与抗原间的接触点。这些接触残基及邻近残基可以作为替代候选物被打靶或消除。可以筛选变体以确定它们是否含有期望的特性。
氨基酸序列插入包括在氨基和/或羧基端融合长度范围为1个残基至100或更多个残基的多肽，和单个或多个氨基酸残基的序列内插入。末端插入的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它插入变体包括抗体的N或C端与酶或延长抗体的血清半衰期的多肽的融合物。
b)Fc区修饰
在某些实施方案中，可以将一个或多个氨基酸修饰引入本文所提供的抗体的Fc区中。
在一些实施方案中，所述一个或多个氨基酸修饰可减少Fc与Fc受体的结合，例如其与Fcγ受体的结合，并且降低或消除效应子功能。在一些实施方案中，改造的Fc区与天然Fc区相比，对Fc受体的结合亲和力下降50％、80％、90％或95％以上。在一些实施方案中，所述Fc受体是人Fcγ受体，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些实施方案中，改造的Fc区与天然Fc区相比，对补体，如C1q的结合亲和力也降低。在一些实施方案中，改造的Fc区与天然Fc区相比，对新生儿Fc受体(FcRn)的结合亲和力增强；比如在Fc区引入M252Y/S254T/T256E突变。在一些实施方案中，改造的Fc区具有降低的效应子功能，所述降低的效应子功能可以包括但不限于以下中的一个或多个：降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。对于IgG1Fc区，在238、265、269、270、297、327和329等位置的氨基酸残基取代可降低的效应子功能。在一些实施方案中，所述Fc区是人IgG1Fc区，并且在234和235位置的氨基酸残基为A，编号依据为EU索引。对于IgG4Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。
在某些实施方案中，抗体包含具有改善ADCC的一处或多处氨基酸替代，例如在Fc区的位置298、333、和/或334(采用EU编号系统)的替代。
在某些实施方案中，本文中的抗体的Fc域包含“杵臼”突变。“杵臼”是一种用于工程化改造抗体重链同二聚体进行异二聚化(例如为了有效生成双特异性抗体，多特异性抗体，或独臂抗体)的设计策略。一般地，此类技术牵涉在第一多肽(诸如第一抗体重链中的第一CH3域)的界面处引入隆起(“杵”)和在第二多肽(诸如第二抗体重链中的第二CH3域)的界面对应处引入空腔(“臼”)，使得隆起能安置在空腔中从而促进异二聚体形成，并阻碍同二聚体形成。通过将来自第一多肽(诸如第一抗体重链中的第一CH3域)的界面的较小氨基酸侧链用 较大侧链(例如精氨酸，苯丙氨酸，酪氨酸或色氨酸)替换来构建隆起。通过将较大氨基酸侧链用较小侧链(例如丙氨酸，丝氨酸，缬氨酸，或苏氨酸)替换在第二多肽(诸如第二抗体重链中的第二CH3域)的界面中创建与隆起相同或相似尺寸的补偿性空腔。可以通过改变编码多肽的核酸(例如通过位点特异性诱变)或通过肽合成来生成隆起和空腔。在一些实施方案中，杵修饰包含Fc域的两个亚基之一中的氨基酸替代T366W，而臼修饰包含Fc域的两个亚基之中的另一中的氨基酸替代T366S、L368A和Y407V。在一些实施方案中，Fc域的包含杵修饰的亚基另外包含氨基酸替代S354C，而且Fc域的包含臼修饰的亚基另外包含氨基酸替代Y349C。引入这两个半胱氨酸残基导致形成Fc区的两个亚基之间的二硫桥，如此进一步稳定二聚体(Carter,J.Immunol.Methods 248:7-15(2001))。杵臼突变的例示性组合包括但不限于表3中所述。
表3
关于杵臼技术的细节描述于例如美国专利No.5,731,168；美国专利No.7,695,936；WO 2009/089004；US 2009/0182127；Marvin and Zhu,Acta Pharmacologica Sincia(2005)26(6):649-658；Kontermann,Acta Pharmacologica Sincia(2005)26:1-9；Ridgway等，Prot Eng 9:617-621(1996)；和Carter,J Immunol Meth 248:7-15(2001)。
Fc区的C末端可以是以氨基酸残基PGK结束的完整C末端；也可以是截短的C末端，例如在所述截短的C末端中已经去除了一个或两个C末端氨基酸残基。在一个优选的方面中，重链的C末端是以PG结束的截短的C末端。因此，在一些实施方案中，完整抗体的组合物可以包括去除了所有K447残基和/或G446+K447残基的抗体群体。在一些实施方案中，完整抗体的组合物可以包括没有去除K447残基和/或G446+K447残基的抗体群体。在一些实施方案中，完整抗体的组合物包含带有和不带有K447残基和/或G446+K447残基的抗体混合物。
C.重组方法
抗DLL3抗体可以使用重组方法来产生。对于这些方法，提供编码抗体的一个或更多个分离的核酸。
在一个实施方案中，本披露提供了编码如前所述的抗体的分离的核酸。此类核酸可以给自独立的编码前述的任一多肽链。在另一方面中，本披露提供了包含此类核酸的一种或多种载体(例如表达载体)。在另一方面中，本披露提供了包含此类核酸的宿主细胞。在一个实施方案中，提供制备抗DLL3抗体的方法，其中所述方法包括，在适合表达的条件下，培养包含编码所述抗体的核酸的宿主细 胞，如上文所提供的，和任选地从宿主细胞(或宿主细胞培养基)回收所述抗体。
为了重组产生抗体，将编码抗体的核酸分离并插入一个或更多个载体中，用于在宿主细胞中进一步克隆和/或表达。此类核酸可以使用常规程序容易地分离和测序，或者通过重组方法产生或通过化学合成获得。
用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，可在细菌中产生，特别是当不需要糖基化和Fc效应子功能时。在表达后，可以在可溶级分中从细菌细胞糊状物分离，并且可进一步纯化。
D.测定
本文提供的抗体药物偶联物可以通过本领域已知的多种测定法对其物理/化学特征和/或生物学活性进行鉴定、筛选或表征。在一个方面中，例如通过已知方法如ELISA、蛋白印迹法等，测试本披露的抗体药物偶联物活性。
E.治疗方法与施用途径
本文提供的前述任何抗体-药物偶联物或其药学上可接受的盐可用于治疗疾病。
在一个方面，本披露提供抗DLL3抗体-药物偶联物或其药学上可接受的盐在制备药物中的用途。在一些实施方案中，本披露提供的抗DLL3抗体-药物偶联物或其药学上可接受的盐在制备用于治疗肿瘤或癌症的药物中的用途。在一些实施方案中，所述的其中所述的肿瘤或癌症包括但不限于肺癌(例如小细胞肺癌、非小细胞肺癌、大细胞肺癌)、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、咽鳞癌、口腔鳞癌、鼻咽癌、食管癌、甲状腺癌(例如甲状腺髓样癌)、恶性胸膜间皮瘤、乳腺癌(例如三阴性乳腺癌)、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结直肠癌(例如结肠癌和直肠癌)、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、肾上腺癌、胶质母细胞瘤、皮肤癌和黑色素瘤；优选地，其中所述肺癌为小细胞肺癌。
在一些实施方案中，本披露提供的抗DLL3抗体-药物偶联物或其药学上可接受的盐在制备用于治疗DLL3相关的疾病的药物中的用途。
在一个此类实施方案中，所述用途进一步包括向受试者施用治疗有效量的至少一种另外的治疗剂(例如一种、两种、三种、四种、五种或六种另外的治疗剂)。
在又一个的方面，提供包含所述抗DLL3抗体-药物偶联物或其药学上可接受的盐的药物组合物，例如，其用于以上任何制药用途或治疗方法。在一个实施方案中，药物组合物包含本文提供的任何多肽或药物偶联物和药学上可接受的载体。在另一个实施方案中，药物组合物还包含至少一种另外的治疗剂。
本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐可单独使用或与其他试剂联合用于治疗。例如，本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐可与至少一种另外的治疗剂共同施用。
本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐可通过任何合适的手段施用，包括肠胃外、肺内和鼻内，并且如果需要局部治疗，则病灶内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。给药可以通过任何适当的途径，例如，通过注射，诸如静脉内或皮下注射，这部分取决于施用是短期的还是长期的。本文考虑多种给药时间方案，包括但不限于，单次或在多个时间点多次施用，推注施用和脉冲输注。
本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐将以符合良好医疗实践的方式配制、给药和施用。在此背景下考虑的因素包括所治疗的具体病症、所治疗的具体哺乳动物、个体患者的临床状况、病症的起因、试剂的递送部位、施用方法、施用时间安排以及医学从业者已知的其他因素。抗DLL3抗体或其药物偶联物可以与一种或更其它试剂一起配制。此类其它试剂的有效量取决于药物组合物中存在的量、病症或治疗的类型以及其它因素。这些通常以与本文所述相同的剂量和施用路径使用，或以本文所述剂量的约1至99％使用，或以其它剂量使用，并通过经验/临床确定为合适的任何途径使用。
为了预防或治疗疾病，本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐(当单独使用或与一种或更多种其他另外的治疗剂组合使用时)的适当的剂量将取决于待治疗的疾病的类型，治疗分子的类型，疾病的严重性和病程，是为预防还是治疗目的施用，之前的治疗，患者的临床病史和对治疗分子的响应，和主治医师的判断。治疗分子恰当地以一次或经过一系列治疗施用于患者。
F.制品
在本披露的另一方面中，提供一种制品(如药盒)，所述制品包含可用于治疗、预防和/或诊断上述病症的材料。该制品包含容器和在容器上或与容器联合的标签或包装插页(package insert)。合适的容器包括，例如，瓶子、管形瓶、注射器、IV溶液袋等。容器可以自各种材料诸如玻璃或塑料形成。
容器单独装有本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐、或与另一种组合物组合。容器可具有无菌的存取口(例如，容器可以是具有塞子的静脉内溶液袋或管形瓶)。组合物中的至少一种活性试剂是本披露的抗DLL3抗体-药物偶联物或其药学上可接受的盐。标签或包装插页指示使用该组合物是来治疗选择的病况。
此外，制品可以包含：(a)其中装有抗DLL3抗体-药物偶联物或其药学上可接受的盐的第一容器；和(b)其中装有组合物的第二容器，其中所述组合物包含另外的细胞毒性剂或其他方面的治疗剂。
备选地，或另外地，制品可进一步包含第二(或第三)容器，所述第二(或第三)容器包含药学上可接受的缓冲液。从商业和用户立场，它可进一步包括所需的其他材料，包括其他缓冲剂、稀释剂、滤器、针头和注射器。
实施例
以下结合实施例进一步描述本披露，但这些实施例并非限制本发明的范围。
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，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，为市场购买的常规试剂。
一、抗体的制备
实施例1：DLL3抗原及检测用蛋白和稳转细胞株的制备
将不同种属的DLL3基因和人DLL1、DLL4基因转染中国仓鼠卵巢细胞CHO-s细胞(Invitrogen，R80007)，构建表达不同种属的DLL3蛋白的CHO-s细胞株，用于后续抗体的筛选和鉴定。相关蛋白氨基酸序列如下：
人DLL3全长蛋白(Uniprot，Q9NYJ7)：
食蟹猴DLL3全长蛋白(Uniprot，A0A2K5WSR4)：
大鼠DLL3全长蛋白(Uniprot，O88671)：
小鼠DLL3全长蛋白(Uniprot，O88516)：
人DLL1全长蛋白(Uniprot，O00548)：

人DLL4全长蛋白(Uniprot，Q9NR61)：
1.1高表达DLL3、DLL1和DLL4细胞株构建
包含SEQ ID NO：1-6的pCDH慢病毒表达载体质粒(由GENEWIZ公司合成)，pCDH质粒分别与pVSVG，pCMV慢病毒包装载体用Lipofectamine 3000(Invitrogen，L3000015)转染试剂转染至293T细胞(中科院细胞库，GNHu17)中，收集含有病毒的培养基上清，过滤并进行超高速离心，弃去上清后，用0.2mL无菌PBS重悬。使用浓缩后的病毒分别感染中国仓鼠卵巢细胞CHO-s(Invitrogen，R80007)，DMS53(ATCC，CRL-2062)和H82(ATCC，HTB-175)，经嘌呤霉素筛选两至三周，再进行FACS单细胞分选。将挑选出的单克隆细胞株扩大培养，冻存。
1.2抗原的制备
以人DLL3(Uniprot，Q9NYJ7)、食蟹猴DLL3(Uniprot，A0A2K5WSR4)和小鼠DLL3(Uniprot，O88516)序列为模板，设计含有不同标签的DLL3ECD融合蛋白，分别克隆到pTT5载体上，经293E细胞表达后，获得抗原。相关蛋白氨基酸序列如下：
1)His-hDLL3(ECD)：

注：点划线为部分信号肽序列，单划线部分为his标签和连接子，双划线部分为DLL3胞外区。
2)Fc-hDLL3(ECD)：
注：点划线部分为信号肽序列，单划线部分为Fc标签和连接子，双划线部分为DLL3胞外区。
3)hDLL3(ECD)-strep twin：
注：点划线部分为信号肽序列，双划线部分为DLL3胞外区，单划线部分为strep twin标签。
4)cynoDLL3(ECD)-strep twin：

注：点划线部分为信号肽序列，双划线部分为DLL3胞外区，单划线部分为strep twin标签。
5)mouDLL3(ECD)-strep twin：
注：点划线部分为信号肽序列，双划线部分为DLL3胞外区，单划线部分为strep twin标签。
实施例2：小鼠抗人DLL3单克隆抗体的制备
1.免疫
抗人DLL3单克隆抗体通过免疫小鼠产生。实验用SJL小鼠，雌性，6-8周龄(上海斯莱克实验动物有限公司，动物生产许可证号：SCXK(沪)2017-0005)。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。
免疫方案：
第一组小鼠的免疫抗原为His-hDLL3(ECD)(SEQ ID NO：7)。用Gold Adjuvant(Sigma Cat No.T2684)与ThermoAlum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂Gold Adjuvant比例为1:1，抗原与佐剂ThermoAlum比例为3:1，50μg/只/次(首次免疫)，25μg/只/次(加强 免疫)。抗原乳化后进行接种，免疫时间为0、7、14、21天。于第7、第21天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(i.p.)注射25μg/只的生理盐水配制的抗原溶液。
第二组小鼠的免疫抗原为DLL3CHO-s和Fc-hDLL3(ECD)(SEQ ID NO：8)，免疫方式为细胞和蛋白抗原交替免疫。首次免疫DLL3CHO-s细胞前，提前用Gold Adjuvant(Sigma Cat No.T2684)0.1mL/只注射小鼠腹膜内，半小时后每只小鼠腹膜内注射0.1mL用生理盐水稀释至108/mL浓度的细胞液。细胞吹散均匀后进行接种，时间为第0、14、28、42天。Fc-hDLL3(ECD)抗原用Gold Adjuvant(Sigma Cat No.T2684)与ThermoAlum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂Gold Adjuvant比例为1:1，抗原与佐剂ThermoAlum比例为3:1，50μg/只/次(首次免疫)，25μg/只/次(加强免疫)。hDLL3-Fc免疫时间为7、21、35、49天。于第14、35、49天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第8次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(i.p.)注射50μg/只的生理盐水配制的hDLL3-Fc蛋白抗原溶液。
2.脾细胞融合
采用优化的电融合方法将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(CRL-8287TM)进行融合得到杂交瘤细胞。
融合的杂交瘤细胞以3-4×105/mL的密度用完全培养基(含20％FBS、1×HAT、1×OPI的IMDM培养基)重悬，150μL/孔种于96孔板中。37℃，5％CO2孵育3-4天后，去除上清，加入200μL/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，37℃，5％CO2培养3天后进行筛选检测。
3.杂交瘤细胞筛选及抗体序列测定
根据杂交瘤细胞生长密度，用结合DLL3蛋白的ELISA方法和结合DLL3CHO-s细胞FACS方法进行杂交瘤培养上清检测。选择结合人DLL3蛋白，猴DLL3蛋白以及DLL3CHO-s细胞，同时不结合野生型CHO-s细胞的克隆及时进行冻存、扩增、保种和一到二次亚克隆直至获得单细胞克隆。通过以上实验筛选得到杂交瘤克隆mAb100和mAb6。
将杂交瘤克隆扩培养，提取RNA，利用mouse-Ig的简并引物进行反转录扩增(RT-PCR)，最后得到抗体的可变区序列。
mAb6重链可变区：
mAb6轻链可变区：
mAb100重链可变区：
mAb100轻链可变区：
表4.鼠源抗体的CDR序列
注：VH/VL的CDR的氨基酸残基由Kabat编号系统确定并注释。
将鼠抗体的重链可变区和轻链可变区分别克隆到包含SEQ ID NO:28所示的人IgG1重链恒定区和SEQ ID NO:29所示的κ轻链恒定区的pTT 5载体质粒，然后转染HEK293细胞，得到了抗DLL3的嵌合抗体M6CHI和M100CHI。
人IgG1重链恒定区：

人κ轻链恒定区：
实施例3：鼠源抗DLL3单克隆抗体的人源化
通过比对Kabat人类抗体重轻链可变区种系基因数据库，分别挑选同源性高的重、轻链可变区种系基因作为模板，将鼠源抗体的CDR分别嫁接(graft)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。并对可变区的氨基酸进行回复突变，再与恒定区重组(示例性的，与SEQ ID NO：28所示的人IgG1重链恒定区和SEQ ID NO：29所示的人κ轻链恒定区)，获得全长抗体。
mAb6抗体的人种系轻链可变区FR1，FR2，FR3模板为IGKV1-16*01，轻链FR4区模板为IGKJ4*01，人种系重链可变区FR1，FR2，FR3模板为IGHV1-3*01、IGHV7-4-1*02或IGHV3-73*01，重链FR4区模板为IGHJ6*01。mAb100抗体的人种系轻链可变区FR1，FR2，FR3模板为IGKV1-27*01，轻链FR4区模板为IGKJ4*01，人种系重链可变区FR1，FR2，FR3模板为IGHV3-11*01，重链FR4区模板为IGHJ6*01。
此外，将mAb100重链可变区的HCDR3：PLYYYGRSYNAVAY(SEQ ID NO：24)中的第4位氨基酸残基由Y突变为H，第11位由A突变为G，获得新的HCDR3：PLYHYGRSYNAVAY(SEQ ID NO：30)和PLYYYGRSYNGVAY(SEQ ID NO：31)。
表5.mAb6和mAb100抗体的人源化模板及相应点突变

注：以表中的A100EG为例，表示kabat编号下的第100E位的A突变为G。
获得的人源化抗体可变区的序列如下：
hAb6 VH1(Q1E,R71V,T73K)
hAb6 VH2(Q1E,I69L,R71V,T73K,S76N)
hAb6 VH3(Q1E,M48I,V67A,I69L,R71V,T73K,S76N)
hAb6 VH4(Q1E,Q43K，I69L,R71V,T73K,S76N)
hAb6 VH5(Q1E,F69L,L71V,T73K)
hAb6 VH6(Q1E,F69L,L71V,T73K,V75S,S76N)
hAb6 VH7(F27Y,S30T,I69L,R71V,D73K,T93A)
hAb6VH8(GraftIGHV1-3*01)
hAb6VH9(GraftIGHV7-4-1*02)
hAb6VH10(GraftIGHV3-73*01)
hAb6VH11(Q1E，R38K,F69L,L71V,T73K,V75S,S76N)
hAb6VH12(Q1E，V68A，F69L,L71V,T73K,V75S,S76N)
hAb6VH13(Q1E,R38K，V68A，F69L,L71V,T73K,V75S,S76N)
hAb6 VL1(F36L,S46G)
hAb6 VL2(F36L,S46G,T69A,F71Y)
hAb6 VL3(F36L,A43S,P44F,S46G,T69A,F71Y)
hAb6 VL4(F36L,S46G,T69A,F71Y,T85D)
hAb6VL5(GraftIGKV1-16*01)
hAb100 VH1(Q1E,R94S)

hAb100 VH2(Q1E,S49A,R94S)
hAb100 VH3(Q1E,S49A,R94S,Y98H)
hAb100 VH4(Q1E,S49A,R94S,A100EG)
hAb100VH5(Graft IGHV3-11*01)
hAb100 VL1(Graft IGKV1-27*01)
hAb100 VL2(V43I)
注：单划线部分为CDR区，双划线处为突变位点。
表6.hAb100抗体的CDR区的通式
其中，X1为Y或H；X2为A或G。
示例性的人源化抗体的重、轻链可变区组合如下：
表7.mAb6的人源化抗体
注：hAb6L1H1表示该抗体包含重链可变区hAb6VH1和轻链可变区hAb6VL1，且其重链恒定区的序列为SEQ ID NO：28，轻链恒定区的序列为SEQ ID NO：29，其他类推。
表8.mAb100的人源化抗体
注：hAb100L1H1表示该抗体包含重链可变区hAb100VH1和轻链可变区hAb100VL1，且其重链恒定区的序列为SEQ ID NO：28，轻链恒定区的序列为SEQ ID NO：29，其他类推。
分别克隆、表达、纯化上述抗体，经蛋白结合实验(测试例1)、细胞结合实验(测试例2)、Biacore(测试例4)，最终选出活性较好的人源化抗体。示例性的人源化抗体的重、轻链氨基酸序列如下：
Hu6(也称hAb6L4H12)重链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
SEQ ID NO：58
Hu6(也称hAb6L4H12)轻链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
SEQ ID NO：59
Hu100(也称hAb100L1H1)重链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
SEQ ID NO：60
Hu100(也称hAb100L1H1)轻链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
SEQ ID NO：61。
本披露中使用的阳性对照抗体为BI-764532(参照WO2019234220A1构建)，阴性对照为C25(其中VH/VL序列来自专利US6114143A)，其序列分别如下：
BI-764532重链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
SEQ ID NO：62
BI-764532轻链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
SEQ ID NO：63
C25重链：
C25轻链：
注：序列中下划线部分为可变区，斜体部分为恒定区。
二、ADC的制备
实施例4.ADC-1的制备
在37℃条件下，向抗体Hu100的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3mL，203nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，50.7μL，507nmol)，置于水浴振荡器，于37℃下振 荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(2.18mg，2030nmol，参照专利申请WO2020063676A1中的实施例9制备)溶解于150μL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-1的PBS缓冲液(1.61mg/mL，15.1mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝4.43。
实施例5.ADC-2的制备
在37℃条件下，向抗体Hu100的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3mL，203nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，121.7μL，1217nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(3.27mg，3042nmol)溶解于150μL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-2的PBS缓冲液(1.42mg/mL，15.8mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝7.32。
实施例6.ADC-3的制备
在37℃条件下，向抗体Hu100的PBS缓冲水溶液(pH＝6.5的0.05M的PBS 缓冲水溶液；10.0mg/mL，6.5mL，440nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，159.4μL，1594nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(5.66mg，5265nmol)溶解于330μL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-3的PBS缓冲液(2.72mg/mL，19.2mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝6.12。
实施例7.ADC-4的制备
在37℃条件下，向抗体Hu6的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，76mL，5140nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，1.14mL，11.4μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(49.0mg，45.6μmol)溶解于3.8mL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-4的PBS缓冲液(4.07mg/mL，167mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝3.53。
实施例8.ADC-5的制备
在37℃条件下，向抗体Hu6的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，68mL，4595nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，2.74mL，27.4μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(82.75mg，77.04μmol)溶解于3.4mL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-5的PBS缓冲液3.12mg/mL，174.1mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝7.43。
实施例9.ADC-6的制备
在37℃条件下，向抗体C25的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.6mL，108nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，35.6μL，356nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.39mg，1.294μmol)溶解于80μL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-6的PBS缓冲液(1.06mg/mL，12.3mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝6.76。
实施例10.ADC-7的制备
在37℃条件下，向抗体C25的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.6mL，108nmol)加入配制好的三(2-羧乙基)膦盐酸盐(TCEP.HCl)的水溶液(10mM，64.8μL，648nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.74mg，1.62μmol)溶解于80μL二甲亚砜中，逐滴加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-7的PBS缓冲液(1.03mg/mL，11.9mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝7.75。
ADC原液药物载药量分析
ADC是一种抗体-药物偶联物，其治疗疾病的机理是依赖抗体的靶向性将药物运送到细胞中，进而将细胞杀死或抑制细胞生长。药物的载药量对药效起着决定性的作用。
本披露采用RP-HPLC法分析药物载药量，过程基本如下：
试剂和仪器：
三氟乙酸(TFA)：sigma生产，100mL/瓶；乙腈：LC级，4L/瓶，Thermo Fisher生产；DTT：sigma生产，1g/瓶。
高效液相色谱仪：Agilent 1200。
溶液配制:
1)0.25M DTT溶液：
配制示例：取DTT 5.78mg，加入150μL纯化水充分溶解后，配得0.25M DTT溶液，-20℃保存。
2)流动相A(0.1％TFA水溶液)：
配制示例：量筒量取1000mL纯化水，加入1mL TFA，充分混匀后使用，2-8℃保存14天。
3)流动相B(0.1％TFA乙腈溶液)：
配制示例：量筒量取1000mL乙腈，加入1mL TFA，充分混匀后使用，2-8℃保存14天。
裸抗体和供测试样品(浓度为1mg/mL，约200μL)，加入4μLDTT还原，37度水浴1小时，结束后取出到内插管中，待进样使用。
色谱条件：
色谱柱：Agilent PLRP-S 1000A 8μm 4.6*250mm；柱温：80℃；
DAD检测器：检测波长280nm；样品室温度：4℃；流速：1mL/分；
进样量为：40μL；
色谱梯度见表9-1：
表9-1
数据分析：
通过样品与裸抗体的谱图比对，区分出轻重链的位置，然后对检测样品的谱图进行积分，计算出DAR值。计算公式如下所示：
表9-2
LC峰面积总和＝LC峰面积+LC+1峰面积
HC峰面积总和＝HC峰面积+HC+1峰面积+HC+2峰面积+HC+3峰面积
LC DAR＝Σ(连接药物数*峰面积百分比)/LC峰面积总和
HC DAR＝Σ(连接药物数*峰面积百分比)/HC峰面积总和
DAR＝LC DAR+HC DAR
以下用生化测试方法验证本披露的抗体及ADC的活性。
测试例1：ELISA检测抗体的蛋白水平结合实验
将链酶亲和素(abcam，ab136200，1μg/mL)包板，100μL/孔，4℃过夜。用PBST溶液(PBS含0.1％吐温20)250μL/孔，洗板3次。5％牛奶封闭，250μL/孔，37℃封闭2小时。用PBST溶液250μL/孔，洗板3次。加入生物素化的DLL3抗原(1μg/mL)(SEQ ID NO：9)，37℃孵育1小时。使用PBST溶液250μL/孔，洗板3次。配制抗体Hu6，Hu100(最高浓度100nM，4倍梯度稀释)，37℃孵育1小时。使用PBST溶液250μL/孔，洗板6次。加入工作浓度的human IgG(H+L)-HRP(Jackson,109-035-003，1：4000稀释)抗体，100μL/孔，37℃孵育1小时。使用PBST溶液250μL/孔，洗板6次。加入TMB(KPL,5120-0077)显色液100μL/孔，室温显色5-10分钟。加入1M H2SO4，100μL/孔，中止显色，酶标仪(Molecular Devices,VERSA max)450nm读数。
表10.抗体对蛋白的结合活性
结果显示：抗体Hu6和Hu100均对DLL3具有优异的结合能力。
测试例2：FACS检测抗体细胞水平的结合实验
将表达DLL3的小细胞肺癌细胞系H1184(ATCC，货号CRL-5858)、DLL3/H82、cynoDLL3/CHO-s和RatDLL3/CHO-s细胞用FACS缓冲液(1％BSA+pH 7.4PBS)制备成1×106/mL的细胞悬液，100μL/孔加入96孔圆底板(Corning，3795)中。300g离心5分钟，去除上清。加入不同浓度待测抗体，100μL/孔。放于4℃冰箱中避光孵育1小时。300g离心洗涤3次后，加入工作浓度的APC抗人IgG Fc(BioLegend，410712)或者PE F(ab')2-羊抗人IgG(invitrogen,H10104)，放于4℃冰箱中避光孵育40分钟。300g离心洗涤3次后，在Invitrogen流式细胞仪上检测几何平均数荧光强度，计算抗体对表达DLL3的细胞的结合EC50值。结果见表11-1、表11-2、表11-3和图1A至图1C。
表11-1.抗体与细胞表达的DLL3的结合活性
表11-2.抗体与表达人DLL3的DLL3/H82细胞的结合活性

表11-3.抗体与H1184细胞的结合活性
结果显示：本披露中的抗体均能与细胞表达的DLL3特异性结合，其中，Hu6与表达不同种属的DLL3细胞均具有较高的结合能力。Hu100对表达人和食蟹猴的DLL3的细胞均具有优异的结合能力，但Hu100不结合表达大鼠DLL3的细胞。BI-764532仅与表达人和食蟹猴DLL3的细胞具有结合能力，且其结合活性比Hu6和Hu100弱。
测试例3：FACS检测抗体对DLL1和DLL4细胞的结合实验
将稳转人DLL1/CHO-s和人DLL4/CHO-s细胞用FACS缓冲液(包含1％BSA和pH7.4PBS)制备成1×106/mL的细胞悬液，100μL/孔加入96孔圆底板(Corning，3795)中。300g离心5分钟，去除上清。加入待检测的抗体，100μL/孔，放于4℃冰箱中避光孵育1小时。300g离心洗涤3次后，加入工作浓度的PE F(ab')2-羊抗人IgG Fc二抗(invitrogen,H10104)，放于4℃冰箱中避光孵育40分钟。300g离心洗涤3次后，在Invitrogen流式细胞仪上检测几何平均数荧光强度。
结果显示：抗体Hu6和Hu100均不结合人DLL1和DLL4。
测试例4：Biacore检测抗体亲和力实验
用Protein A生物传感芯片(Cat.#29127556，Cytiva)亲和捕获待测抗体18秒，然后于芯片表面流经抗原人DLL3(ACRO，DLL3-H52H4)，猴DLL3(KACTUS，DLL-RM103)和小鼠DLL3(KACTUS，DLL-MM103)180秒，然后解离600秒。用Biacore 8K(Cytiva)仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用10mM甘氨酸-盐酸溶液(pH 1.5)(Cat.#BR-1003-54，Cytiva)将生物传感芯片洗净再生。数据拟合模型采用1:1Model。结果见表12-1、表12-2和表12-3。
表12-1.抗体亲和力
表12-2.抗体与人DLL3的亲和力
表12-3.抗体与人DLL3的亲和力

结果显示，mAb100和mAb6的人源化抗体和嵌合抗体可特异性结合人DLL3，且亲和力较高。其中抗体Hu6与人、猴和小鼠的DLL3均具有较高的亲和力；Hu100与人和猴DLL3具有较高的亲和力，但不结合小鼠的DLL3。
测试例5：抗体的表位竞争结合实验
将BI-764532抗体(1μg/mL)包板，100μL/孔，4℃过夜。PBST溶液250μL/孔，洗板3次。5％牛奶封闭，250μL/孔，37℃封闭2小时。PBST溶液250μL/孔，洗板3次。加入生物素化的DLL3-Strep(0.1μg/mL，SEQ ID NO：9)。配制竞争抗体，BI-764532、Hu6和Hu100(最高浓度100μg/mL，4倍梯度稀释)，37℃孵育1小时。PBST溶液250μL/孔，洗板6次。加入链霉亲和素-过氧化物酶(1:2000稀释)(Jackson Immuno Research,016-030-084)，100μL/孔，37℃孵育1小时。PBST溶液250μL/孔，洗板6次。加入TMB(KPL,5120-0077)显色液100μL/孔，室温显色5-10min。加入1M H2SO4，100μL/孔，中止显色，酶标仪450nm读数。结果如图2所示。
结果显示，抗体Hu6和Hu100与BI-764532之间不竞争，说明抗体Hu6和Hu100与BI-764532结合的表位不同。
测试例6：抗DLL3抗体的内吞活性检测
DT3C是重组表达的融合蛋白，由白喉毒素的片段A(仅毒素部分)和G群链球菌的3C片段(IgG结合部分)融合而成，该蛋白能够与抗体的IgG部分高度亲和，在抗体被内吞时一同进入细胞，在胞内弗林蛋白酶的作用下，释放出具有毒性的DT，DT能够抑制EF2-ADP核糖基化的活性，阻断蛋白翻译过程，最终导致细胞死亡。而未进入细胞的DT3C则不具有杀伤细胞的活性。根据对细胞杀伤情况评价抗体的被细胞内吞的活性。
实验步骤
a.用含20％FBS的新鲜细胞培养基RPMI1640(GE，SH30809.01)制取DMS53/DLL3细胞悬液，以2000个细胞/50μL/孔加入96孔细胞培养板中，第1 和12列不铺细胞只加入50μL的培养基，5％二氧化碳37℃16小时培养。
b.用无血清培养基配制成4×浓度的DT3C(9600nM，由上海磐超生物科技有限公司表达纯化)溶液，并使用0.22μm滤器过滤。用无血清培养基配制4×浓度的抗体(1600nM)，将80μL DT3C溶液和80μL抗体溶液按照1:1的体积混匀，室温下静置孵育30分钟。
c.用无血清培养基5倍梯度稀释该混合物，共9个浓度，第10个点为纯培养基。
d.取50μL稀释好的抗体加入细胞中，培养箱中孵育三天。
e.每孔加入50μL CTG(Luminescent Cell Viability Assay，Promega，G7573)，室温下避光孵育10分钟，Victor3上读取化学发光。
结果见下表13和图3。
表13.抗体的内吞引起的细胞杀伤
结果显示：抗体Hu6和Hu100均可被细胞内吞。
测试例7：FACS检测ADC的细胞水平的结合实验
将表达DLL3的hDLL3/CHO-s和cynoDLL3/CHO-s细胞用FACS缓冲液(1％BSA+pH 7.4PBS)制备成1×106/mL的细胞悬液，100μL/孔加入96孔圆底板(Corning，3795)中。300g离心5分钟，去除上清。加入不同浓度待测ADC，100μL/孔。放于4℃冰箱中避光孵育1小时。300g离心洗涤3次后，加入工作浓度的APC抗人IgG Fc(BioLegend，410712)或者PE F(ab')2-羊抗人IgG(invitrogen,H10104)，放于4℃冰箱中避光孵育40分钟。300g离心洗涤3次后，在Invitrogen流式细胞仪上检测几何平均数荧光强度，计算抗体对表达DLL3的细胞的结合EC50值。结果见表14、图4A和图4B。
表14.抗体与细胞表达的DLL3的结合活性
结果显示：本披露中的ADC均能与细胞表达的人和猴DLL3特异性结合，其中随着DAR值增加，ADC的结合基本无影响。
测试例8：Biacore检测ADC亲和力实验
用Protein A生物传感芯片(Cat.#29127556，Cytiva)亲和捕获待测抗体18秒， 然后于芯片表面流经抗原人DLL3(ACRO，DLL3-H52H4)和猴DLL3(KACTUS，DLL-RM103)180秒，然后解离600秒。用Biacore 8K(Cytiva)仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用10mM甘氨酸-盐酸溶液(pH 1.5)(Cat.#BR-1003-54，Cytiva)将生物传感芯片洗净再生。数据拟合模型采用1:1Model。结果见表15。
表15.ADC对不同种属DLL3的亲和力
结果显示：本披露中的ADC均能与人和猴DLL3蛋白特异性结合。其中随着DAR值增加，ADC的结合基本无影响。
测试例9：ADC对不同DLL3表达水平细胞毒性实验
实验步骤如下：
a.用含10％FBS的新鲜细胞培养基制取细胞悬液，以每孔135μL加入到96孔细胞培养板(Corning，3903)中，第1和第12列不铺细胞，只加入135μL的培养基，5％二氧化碳37℃培养16小时。
b.ADC样品用PBS配制成首孔工作液(10×浓度)，以此为首浓度，用PBS按相应的倍数进行梯度稀释。每孔加入15μL的10×浓度ADC溶液，5％二氧化碳37℃培养6天。
c.每孔加入70μL CTG(Promega，G7573)，室温下避光孵育10分钟，Victor3上读取化学发光，用GraphPad Prism5对数据处理，以抗体或ADC浓度为X轴，光强度值为Y轴作图。
不同细胞的铺板密度、首孔工作液浓度(10×浓度)，稀释倍数见表16。
表16.细胞的铺板密度和首孔工作液浓度
实验使用的细胞如下：
H1184(+++)购于ATCC，CRL-5858；
DMS53(++)购于ATCC，CRL-2062；
SK-MEL3(+)购于ATCC，HTB-69；
CHO-K1(-)购于ATCC，CCL-61。
其中“+”表示DLL3的表达量，“-”表示不表达DLL3。
结果见表17和图5A至图5D。
表17.ADC对不同DLL3表达量细胞的杀伤活性
结果显示，ADC-1，ADC-2和ADC-3有较强的靶细胞杀伤活性，可杀伤表达DLL3的SK-MEL3，DMS53和H1184，但对DLL3阴性的CHO-K1细胞无杀伤。
测试例10：旁观杀伤活性实验
DMS53/DLL3high(稳转DLL3的DMS53细胞)和U-2OS(ATCC，HTB-96)细胞分别用RPMI1640+20％FBS+1×Glutamax和McCoy's 5A+10％FBS培养，细胞用胰酶消化，新鲜培养基中和，1000rpm离心3分钟。弃上清，细胞用RPMI1640+20％FBS+1×Glutamax重悬。细胞计数后，将DMS53/DLL3high细胞密度调整为9×104个/mL，将U-2OS细胞密度调整为3×104个/mL。12孔板对应孔中每孔加入500μL的DMS53/DLL3high细胞和500μL的U-2OS细胞。12孔板对应孔中加入500μL的U-2OS细胞和500μL的RPMI1640+20％FBS+1×Glutamax培养液。5％二氧化碳37℃培养24小时。将样品配制成40×浓度的中间溶液(200nM)。各取25μL ADC样品加入到12孔板相应孔中。设置溶剂对照组。5％二氧化碳37℃培养6天。12孔板中的细胞用胰酶消化，新鲜培养基中和，取20μL的细胞加入20μL的台盼蓝，计数。细胞1000rpm离心3分钟,弃上清。细胞用100μL的FACS缓冲液洗一遍，1500rpm离心3分钟，弃上清。用100μL的FACS缓冲液重悬，加入2μg/mL的抗DLL3的阳性抗体，冰上孵育60分钟。FACS缓冲液洗1遍，1500rpm离心3分钟，加入二抗APC anti-human IgG Fc(100x)孵育30分钟，FACS缓冲液洗1遍。加入200μLFACS缓冲液重悬细胞，用FACS检测。用FlowJo对流式数据进行分析得到DMS53/DLL3high比例并计算出DMS53/DLL3high及U-2OS的总数量。用GraphPad Prism5对数据作图，以不同样品为X轴，计算得 到的细胞数为Y轴作图，结果见图6。
结果显示ADC-1，ADC-2和ADC-3具有明显的旁观者细胞毒性效应。
体内活性生物学评价
测试例11-1：DMS53细胞CDX小鼠模型体内药效评价
将人小细胞肺癌DMS53细胞(5×106个，含50％matrigel/只；ATCC，CRL-2062)200μL/只，接种Balb/c右肋部皮下。接种7天后，待肿瘤体积在～220mm3后去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机，每组8只，当天开始给药。通过腹腔注射ADC，剂量为1.5mg/kg，每周给药1次，共给药2次。第2次给药后停止给药，继续观察肿瘤生长情况。每周测量2次瘤体积和体重，记录数据。使用Excel统计软件记录数据：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或One-way ANOVA对数据进行统计学分析。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
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体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
结果见表18和图7。
表18.ADC对荷瘤裸鼠DMS53移植瘤的疗效
结果显示在1.5mpk剂量下，ADC-1和ADC-4均能显著抑制DMS53细胞皮下移植肿瘤的生长。在0.5mpk和1.5mpk剂量下，ADC-2和ADC-5均能显著抑制DMS53细胞皮下移植肿瘤的生长。
测试例11-2：DMS53细胞CDX小鼠模型体内药效评价
将人小细胞肺癌DMS53细胞(5×106个，含30％matrigel/只；ATCC，CRL-2062)200μL/只，接种Balb/c右肋部皮下。接种15天后，待肿瘤体积在～180mm3后去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机，每组8只，当天开始给药。通过腹腔注射ADC，剂量为1.5mg/kg或0.5mg/kg，每周给药1次，共给药2次。第2次给药后停止给药，继续观察肿瘤生长情况。每周测量2次瘤体积和体重，记录数据。使用Excel统计软件记录数据：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或One-way ANOVA对数据进行统计学分析。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
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体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
结果见表19和图8。
表19.ADC对荷瘤裸鼠DMS53移植瘤的疗效
结果显示在ADC-3在0.5mpk和1.5mpk剂量下，ADC-1在1.5mpk剂量下，均能显著抑制DMS53细胞皮下移植肿瘤的生长。
测试例12：NCI-H1184细胞CDX小鼠模型体内药效评价
将NCI-H1184细胞(6×106个，含50％MatrixGel/只；ATCC，CRL-5858)200μL/只，接种于80只NDG小鼠右肋部皮下，接种13天后，待肿瘤体积平均在～185mm3后去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为7组，每组9只，当天开始给药，如表19所示。通过腹腔注射抗体，共21天。每周测2次瘤体积，称体重，记录数据。使用Excel统计软件记录数据：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或One-way ANOVA对数据进行统计学分析。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
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体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
结果见表20和图9。
表20.ADC对荷瘤裸鼠H1184移植瘤的疗效
结果显示，ADC-2和ADC-3均能显著抑制NCI-H1184肿瘤的生长。
测试例13：大鼠PK研究
SD大鼠(北京维通利华实验动物技术有限公司)中研究ADC分子的单剂量药代动力学。
静脉注射ADC分子3mg/kg(n＝4/组)，给药后5min、8h、1d、2d、4d、7d、10d、14d、21d和28d，于大鼠眼底静脉取血。收集的血液样本在室温下置放半小时至凝集，然后4℃下1000g离心15分钟，收集血清。进行生物分析测量采用HTRF的方法进行大鼠血清样品的测定，生物素标记DLL3捕捉单抗，生物素标记抗毒素抗体捕捉毒素。通过标准品的四参数模型曲线进行待测品含量的定量分析。药代参数使用WinNonlin软件(6.4)的标准非房室模型进行分析。实验结果如表21和图10A至图10C。
表21.ADC分子的大鼠药代动力学
结果显示，ADC分子在体内测定的总抗体和完整ADC的AUC相似，可见三个ADC分子的完整ADC结构均稳定。
测试例14：猴PK研究
食蟹猴(苏州西山中科实验动物有限公司)中研究ADC分子的单剂量药代动力学。
静脉注射ADC分子10mg/kg(n＝3/组)，给药后5min、8h、1d、1.5d、2d、3d、4d、7d、10d、14d、21d和28d。收集的血液样本在室温下置放半小时至凝集，然后进行离心(4℃，2600g，10min)，取上层血清，立即放置-60℃贮存。收集血清进行生物分析测量采用DELFIA的方法进行血清样品的测定，通过标准品的四参数模型曲线进行待测品含量的定量分析。药代参数使用WinNonlin软件(6.4)的标准非房室模型进行分析。实验结果如表22和图11。
表22.ADC分子的猴药代动力学

结果显示，静脉给予食蟹猴ADC-2 10mpk，总抗体和完整ADC的半衰期分别为9.9±1.1天、8.5±0.1天；总抗体和完整ADC的AUC分别为27405μg/mL*h，25702μg/mL*h。总抗体和完整ADC在食蟹猴体内的半衰期基本一致，完整ADC与总抗体的AUC比值为94％，ADC-2在食蟹猴体内稳定性较好。

Claims (11)

  1. 一种抗体-药物偶联物或其药学上可接受的盐，其具有如通式Pc-L-Y-D所示的结构：
    其中：
    Pc为抗DLL3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：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：57的氨基酸序列；和
    所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；
    其中，SEQ ID NO：57如PLYX1YGRSYNX2VAY所示，其中X1为Y或H；X2为A或G；或
    ii)所述重链可变区包含：HCDR1，其包含SEQ ID NO：16的氨基酸序列；HCDR2，其包含SEQ ID NO：17的氨基酸序列；和HCDR3，其包含SEQ ID NO：18的氨基酸序列；和
    所述轻链可变区包含：LCDR1，其包含SEQ ID NO：19的氨基酸序列；LCDR2，其包含SEQ ID NO：20的氨基酸序列；和LCDR3，其包含SEQ ID NO：21的氨基酸序列；
    优选地，
    所述重链可变区包含：HCDR1，其包含SEQ ID NO：22的氨基酸序列；HCDR2，其包含SEQ ID NO：23的氨基酸序列；和HCDR3，其包含SEQ ID NO：24、30或31的氨基酸序列；和
    所述轻链可变区包含：LCDR1，其包含SEQ ID NO：25的氨基酸序列；LCDR2，其包含SEQ ID NO：26的氨基酸序列；和LCDR3，其包含SEQ ID NO：27的氨基酸序列；
    Y为-O-CR1R2-C(O)-，其中，R1为卤代C1-6烷基或C3-6环烷基；R2选自氢原 子、卤代C1-6烷基和C3-6环烷基；或者，R1和R2与其相连接的碳原子一起形成C3-6环烷基；
    n为1至10；
    L为接头。
  2. 根据权利要求1所述抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体为鼠源抗体、嵌合抗体或人源化抗体；优选为人源化抗体。
  3. 根据权利要求1或2所述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含：
    i)所述重链可变区包含与SEQ ID NO：50、14、51、52、53或54具有至少90％序列同一性的氨基酸序列；和/或
    所述轻链可变区包含与SEQ ID NO：55、15或56具有至少90％序列同一性的氨基酸序列；或
    ii)所述重链可变区包含与SEQ ID NO：43、12、32、33、34、35、36、37、38、39、40、41、42或44具有至少90％序列同一性的氨基酸序列；和/或
    所述轻链可变区包含与SEQ ID NO：48、13、45、46、47或49具有至少90％序列同一性的氨基酸序列；
    优选地，
    i)所述重链可变区包含选自SEQ ID NO：50、51、52、53和54中的任一氨基酸序列，和/或所述轻链可变区包含SEQ ID NO：55或56的氨基酸序列；或
    ii)所述重链可变区包含选自SEQ ID NO：43、32、33、34、35、36、37、38、39、40、41、42和44中的任一氨基酸序列，和/或所述轻链可变区包含选自SEQ ID NO：48、45、46、47和49中的任一氨基酸序列；或
    iii)所述重链可变区包含SEQ ID NO：14的氨基酸序列，和/或所述轻链可变区包含SEQ ID NO：15的氨基酸序列；或
    iv)所述重链可变区包含SEQ ID NO：12的氨基酸序列，和/或所述轻链可变区包含SEQ ID NO：13的氨基酸序列；
    更优选地，
    所述重链可变区包含SEQ ID NO：50的氨基酸序列，和所述轻链可变区包含SEQ ID NO：55的氨基酸序列；或
    所述重链可变区包含SEQ ID NO：43的氨基酸序列，和所述轻链可变区包含SEQ ID NO：48的氨基酸序列。
  4. 根据权利要求1至3中任一项所述抗体-药物偶联物或其药学上可接受的盐，其中所述抗DLL3抗体是抗体片段；优选地，其中所述抗体片段为Fab、Fab'、 F(ab')2、Fab'-SH、Fd、Fv、scFv、dsFv、双抗体或结构域抗体。
  5. 根据权利要求1至3中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含重链恒定区和轻链恒定区；优选地，所述重链恒定区包含SEQ ID NO：28的氨基酸序列，和/或所述轻链恒定区包含SEQ ID NO：29的氨基酸序列。
  6. 根据权利要求5所述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗DLL3抗体包含重链和轻链，其中：
    所述重链包含与SEQ ID NO：60具有至少85％序列同一性的氨基酸序列，和/或所述轻链包含与SEQ ID NO：61具有至少85％序列同一性的氨基酸序列；或
    所述重链包含与SEQ ID NO：58具有至少85％序列同一性的氨基酸序列，和/或所述轻链包含与SEQ ID NO：59具有至少85％序列同一性的氨基酸序列；
    优选地，
    所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；或
    所述重链包含SEQ ID NO：58的氨基酸序列，和所述轻链包含SEQ ID NO：59的氨基酸序列。
  7. 根据权利要求1至6中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述的Y选自：
    其中Y的O-端与L相连。
  8. 根据权利要求1至7中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述的L为-L1-L2-L3-L4-，其中：
    L1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH2-C(O)-NR3-W-C(O)-和-C(O)-W-C(O)-，其中W为C1-6亚烷基或C1-6亚烷基-C3-6环烷基，其中所述的C1-6亚烷基或C1-6亚烷基-C3-6环烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L2选自-NR4(CH2CH2O)pCH2CH2C(O)-、-NR4(CH2CH2O)pCH2C(O)-和化学键，其中p为1至20的整数；
    L3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    L4选自-NR5(CR6R7)t-、-C(O)NR5、-C(O)NR5(CH2)t-和化学键，其中t为1至6的整数；
    R3、R4和R5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R6和R7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基；
    优选地，
    L1为s1为2至8的整数；
    L2为化学键；
    L3为四肽残基；优选地，L3为包含甘氨酸-甘氨酸-苯丙氨酸-甘氨酸的四肽残基；
    L4为-NH(CH2)t-，t为1或2；
    其中所述的L1端与Pc相连；
    更优选地，所述的L为以下结构所示：
  9. 根据权利要求1至8中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述的抗体-药物偶联物具有如下所示的结构：

    其中：
    Pc为如权利要求1所述的抗DLL3抗体；n为1至10；
    优选地，所述抗体-药物偶联物具有如下所示的结构：
    其中：
    Pc为抗DLL3抗体，其包含重链和轻链，其中：
    所述重链包含SEQ ID NO：60的氨基酸序列，和所述轻链包含SEQ ID NO：61的氨基酸序列；或
    所述重链包含SEQ ID NO：58的氨基酸序列，和所述轻链包含SEQ ID NO：59的氨基酸序列；
    n为3至8；更优选地，n为6至8。
  10. 一种药物组合物，其包含：
    权利要求1至9中任一项所述的抗体-药物偶联物或其药学上可接受的盐，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。
  11. 根据权利要求1至9中任一项所述的抗体-药物偶联物或其药学上可接受的盐，或权利要求10所述的药物组合物在制备用于治疗肿瘤或癌症的药物中的用途；优选地，其中所述肿瘤或癌症选自：
    肺癌、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、咽鳞癌、口腔鳞癌、鼻咽癌、食管癌、甲状腺癌、甲状腺髓样癌、恶性胸膜间皮瘤、乳腺癌、三阴性乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结直肠癌、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、肾上腺癌、胶质母细胞瘤、皮肤癌和黑色素瘤；更优选地，其中所述肺癌为小细胞肺癌。
PCT/CN2024/078818 2023-02-27 2024-02-27 抗dll3抗体、其抗体-药物偶联物及其医药用途 WO2024179470A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202310172129 2023-02-27
CN202310172129.X 2023-02-27
CN202311485274.X 2023-11-09
CN202311485274 2023-11-09

Publications (1)

Publication Number Publication Date
WO2024179470A1 true WO2024179470A1 (zh) 2024-09-06

Family

ID=92589881

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/078818 WO2024179470A1 (zh) 2023-02-27 2024-02-27 抗dll3抗体、其抗体-药物偶联物及其医药用途

Country Status (1)

Country Link
WO (1) WO2024179470A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150297748A1 (en) * 2012-10-11 2015-10-22 Daiichi Sankyo Company, Limited Antibody-drug conjugate
WO2017031458A2 (en) * 2015-08-20 2017-02-23 Abbvie Stemcentrx Llc Anti-dll3 antibody drug conjugates and methods of use
CN114456186A (zh) * 2020-10-12 2022-05-10 四川百利药业有限责任公司 一种喜树碱类衍生物及其配体-药物偶联物
WO2022153195A1 (en) * 2021-01-13 2022-07-21 Memorial Sloan Kettering Cancer Center Anti-dll3 antibody-drug conjugate
CN116271079A (zh) * 2021-07-30 2023-06-23 上海复旦张江生物医药股份有限公司 一种抗dll3抗体及其制备方法、其药物偶联物和应用
CN117693365A (zh) * 2021-06-25 2024-03-12 同宜医药（苏州）有限公司 配体药物偶联物及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150297748A1 (en) * 2012-10-11 2015-10-22 Daiichi Sankyo Company, Limited Antibody-drug conjugate
WO2017031458A2 (en) * 2015-08-20 2017-02-23 Abbvie Stemcentrx Llc Anti-dll3 antibody drug conjugates and methods of use
CN114456186A (zh) * 2020-10-12 2022-05-10 四川百利药业有限责任公司 一种喜树碱类衍生物及其配体-药物偶联物
WO2022153195A1 (en) * 2021-01-13 2022-07-21 Memorial Sloan Kettering Cancer Center Anti-dll3 antibody-drug conjugate
CN117693365A (zh) * 2021-06-25 2024-03-12 同宜医药（苏州）有限公司 配体药物偶联物及其应用
CN116271079A (zh) * 2021-07-30 2023-06-23 上海复旦张江生物医药股份有限公司 一种抗dll3抗体及其制备方法、其药物偶联物和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DESAI AAKASH; ABDAYEM PAMELA; ADJEI ALEX A.; PLANCHARD DAVID: "Antibody-drug conjugates: A promising novel therapeutic approach in lung cancer", LUNG CANCER., ELSEVIER, AMSTERDAM., NL, vol. 163, 15 December 2021 (2021-12-15), NL , pages 96 - 106, XP086927600, ISSN: 0169-5002, DOI: 10.1016/j.lungcan.2021.12.002 *
KONG, C. ET AL.: "MTX-13, a Novel PTK7-Directed Antibody–Drug Conjugate with Widened Therapeutic Index Shows Sustained Tumor Regressions for a Broader Spectrum of PTK7-Positive Tumors", AACR JOURNALS, vol. 22, no. 10, 31 December 2023 (2023-12-31) *
RICCIUTI BIAGIO, LAMBERTI GIUSEPPE, ANDRINI ELISA, GENOVA CARLO, DE GIGLIO ANDREA, BIANCONI VANESSA, SAHEBKAR AMIRHOSSEIN, CHIARI : "Antibody–drug conjugates for lung cancer in the era of personalized oncology", SEMINARS IN CANCER BIOLOGY., SAUNDERS SCIENTIFIC PUBLICATIONS, PHILADELPHIA, PA., US, vol. 69, 1 February 2021 (2021-02-01), US , pages 268 - 278, XP093206255, ISSN: 1044-579X, DOI: 10.1016/j.semcancer.2019.12.024 *

Similar Documents

Publication Publication Date Title
CN114845739B (zh) 艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用
WO2021147993A1 (zh) 抗trop-2抗体-依喜替康类似物偶联物及其医药用途
WO2022228406A1 (zh) 抗Nectin-4抗体和抗Nectin-4抗体-药物偶联物及其医药用途
KR20190021244A (ko) 항-axl 길항성 항체
WO2021115426A1 (zh) 抗密蛋白抗体药物偶联物及其医药用途
AU2019262953A1 (en) Antibodies, and bispecific antigen-binding molecules that bind HER2 and/or APLP2, conjugates, and uses thereof
CN115368461A (zh) 抗trop-2抗体、其抗体-药物偶联物及其医药用途
TW202341984A (zh) Ｈｅｒ３抗體藥物偶聯物及其用途
WO2023125619A1 (zh) 抗ror1抗体和抗ror1抗体药物偶联物及其医药用途
CN116942845A (zh) 抗psma抗体、其药物偶联物及其医药用途
WO2024179470A1 (zh) 抗dll3抗体、其抗体-药物偶联物及其医药用途
KR20240134329A (ko) 글루코코르티코이드의 약물 접합체
US20250057968A1 (en) Anti-dll3 antibody and pharmaceutical use thereof, and antibody-drug conjugate containing anti-dll3 antibody
JP2023506805A (ja) 抗ｃｅａ抗体－エキサテカン類似体複合体及びその医薬用途
WO2025031285A1 (zh) 抗psma抗体-药物偶联物及其医药用途
RU2830242C1 (ru) Лекарственный конъюгат производного эрибулина, способ его получения и его применение в медицине
WO2024140917A1 (zh) 抗cd40抗体药物偶联物、其制备方法及其医药用途
WO2024213106A1 (zh) 特异性结合egfr和muc1的抗原结合分子、其药物偶联物及其医药用途
WO2024222868A1 (zh) 抗tf抗体和抗tf抗体-药物偶联物及其医药用途
WO2024227426A1 (zh) hROR1的抗原结合蛋白及其用途
CN117242097A (zh) 一种抗egfr/vegf双功能融合蛋白及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24763151

Country of ref document: EP

Kind code of ref document: A1