WO2022242757A1 - 抗pd-1抗体的应用 - Google Patents

抗pd-1抗体的应用 Download PDF

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Publication number
WO2022242757A1
WO2022242757A1 PCT/CN2022/094181 CN2022094181W WO2022242757A1 WO 2022242757 A1 WO2022242757 A1 WO 2022242757A1 CN 2022094181 W CN2022094181 W CN 2022094181W WO 2022242757 A1 WO2022242757 A1 WO 2022242757A1
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Prior art keywords
antibody
seq
sequence
amino acid
weeks
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PCT/CN2022/094181
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English (en)
French (fr)
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黄贤明
岳睿
张慧
陈振埕
岳海涛
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百奥泰生物制药股份有限公司
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Priority to CN202280035707.9A priority Critical patent/CN117337300A/zh
Publication of WO2022242757A1 publication Critical patent/WO2022242757A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to the application of anti-PD-1 antibody in combination medicine.
  • PD-1 Programmed death receptor-1
  • ITIM immunosuppressive receptor expressed on activated T cells, B cells, and myeloid cells
  • ITSM immunosuppressive tyrosine switching motif
  • anti-PD-1 antibodies play an important role in anti-tumor drug combination.
  • the invention discloses a method or use of an anti-PD-1 antibody for combined treatment of tumor or cancer.
  • an anti-PD-1 antibody and a therapeutic agent are used in combination to treat a tumor or cancer.
  • the therapeutic agent is an anti-CD73 antibody.
  • the method or use comprises: administering an effective amount of an anti-PD-1 antibody and an anti-CD73 antibody to a patient in need thereof.
  • the use is the use of the anti-PD-1 antibody in the preparation of a drug combined with the anti-CD73 antibody for the treatment of tumor or cancer.
  • the use is the use of the anti-CD73 antibody in the preparation of a drug in combination with the anti-PD-1 antibody for the treatment of tumor or cancer.
  • the anti-PD-1 antibody at least comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, SEQ ID NO:4 One or more of LCDR1 shown, LCDR2 shown in SEQ ID NO:5, LCDR3 shown in SEQ ID NO:6.
  • the anti-PD-1 antibody comprises one or two CDRs selected from the amino acid sequences shown below: SEQ ID NO: 3 and 6; and optionally comprises one, two, three or Four CDRs selected from the amino acid sequences shown below: SEQ ID NO: 1, 2, 4 and 5.
  • the anti-PD-1 antibody comprises HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, and HCDR3 shown in SEQ ID NO:4.
  • the heavy chain variable region of the anti-PD-1 antibody comprises the sequence shown in SEQ ID NO: 7, a sequence at least 80% identical to the sequence shown in SEQ ID NO: 7, or a sequence with An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:7; and/or
  • the light chain variable region of the anti-PD-1 antibody comprises the sequence shown in SEQ ID NO: 8, a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 8, or a sequence with SEQ ID NO: 8 Amino acid sequences having one or more conservative amino acid substitutions compared to the indicated sequence.
  • the heavy chain variable region of the anti-PD-1 antibody comprises the sequence shown in SEQ ID NO: 7, and the light chain variable region of the anti-PD-1 antibody comprises SEQ ID NO: 8 sequence shown.
  • the heavy chain of the anti-PD-1 antibody comprises the sequence set forth in SEQ ID NO:9, a sequence at least 80% identical to the sequence set forth in SEQ ID NO:9, or a sequence identical to the sequence set forth in SEQ ID NO:9. : An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in 9; and/or
  • the light chain of the anti-PD-1 antibody comprises the sequence shown in SEQ ID NO: 10, a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 10, or a sequence similar to the sequence shown in SEQ ID NO: 10 Amino acid sequences having one or more conservative amino acid substitutions.
  • the anti-PD-1 antibody is antibody A
  • the heavy chain of antibody A comprises the sequence shown in SEQ ID NO:9
  • the light chain of antibody A comprises the sequence shown in SEQ ID NO:10
  • Antibody A contains two identical heavy chains and two identical light chains.
  • the anti-CD73 antibody comprises HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 12, HCDR3 as shown in SEQ ID NO: 13, HCDR3 as shown in SEQ ID NO: One or more of LCDR1 shown in 14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:16.
  • the anti-CD73 antibody comprises HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 12, HCDR3 as shown in SEQ ID NO: 13, HCDR3 as shown in SEQ ID NO: LCDR1 as shown in 14, LCDR2 as set forth in SEQ ID NO:15, and LCDR3 as set forth in SEQ ID NO:16.
  • the heavy chain variable region of the anti-CD73 antibody comprises the sequence set forth in SEQ ID NO: 17, a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 17, or a sequence identical to the sequence set forth in SEQ ID NO: 17 An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in NO:17; and/or
  • the light chain variable region of the anti-CD73 antibody comprises the sequence shown in SEQ ID NO: 18, a sequence having at least 80% identity to the sequence shown in SEQ ID NO: 18, or a sequence shown in SEQ ID NO: 18 Amino acid sequences having one or more conservative amino acid substitutions are compared.
  • the heavy chain variable region of the anti-CD73 antibody comprises the sequence shown in SEQ ID NO:17
  • the light chain variable region of the anti-CD73 antibody comprises the sequence shown in SEQ ID NO:18.
  • the heavy chain of the anti-CD73 antibody comprises the sequence set forth in SEQ ID NO: 19 or 20, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 19 or 20, or a sequence identical to that set forth in SEQ ID NO: 19 or 20.
  • the light chain of the anti-CD73 antibody comprises the sequence shown in SEQ ID NO: 21, a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 21, or having a sequence compared with the sequence shown in SEQ ID NO: 21 Amino acid sequence with one or more conservative amino acid substitutions.
  • the anti-CD73 antibody is antibody P59-L17
  • the heavy chain of antibody P59-L17 comprises the sequence shown in SEQ ID NO: 19
  • the light chain of antibody P59-L17 comprises the sequence shown in SEQ ID NO: 21 Sequences are shown; antibody P59-L17 contains two identical heavy chains and two identical light chains.
  • the anti-CD73 antibody is antibody P59-L17'
  • the heavy chain of antibody P59-L17' comprises the sequence shown in SEQ ID NO: 20
  • the light chain of antibody P59-L17' comprises the sequence shown in SEQ ID NO : the sequence shown in 21
  • antibody P59-L17' contains two identical heavy chains and two identical light chains.
  • Antibody proteins can be expressed in cells (such as CHO cells or HEK cells) through genetic engineering, and obtained by purification; purification can be performed by conventional methods, such as centrifuging the cell suspension first and collecting the supernatant, and centrifuging again to further remove impurities. Methods such as Protein A affinity column and ion exchange column can be used to purify antibody protein.
  • the method or use comprises: administering an effective amount of an anti-PD-1 antibody and an anti-CD73 antibody to a patient in need thereof.
  • the anti-PD-1 antibody is Antibody A.
  • the anti-CD73 antibody is antibody P59-L17 or P59-L17'.
  • the anti-PD-1 antibody is administered at an effective dose of about 50 mg to 600 mg per dose.
  • an anti-CD73 antibody (such as antibody P59-L17 or P59-L17') is administered at an effective dose of about 15 mg to 1200 mg per dose.
  • the anti-PD-1 antibody and the anti-CD73 antibody are separate administration units, and are administered in combination.
  • the anti-PD-1 antibody can be administered before applying the anti-CD73 antibody, or after applying the anti-CD73 antibody, or at the same time as applying the anti-CD73 antibody. medicine.
  • the anti-PD-1 antibody and the anti-CD73 antibody simultaneously form a combined administration unit and are administered in combination.
  • tumors and cancers include, but are not limited to: head and neck tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, precursor lymphoblastic lymphoma, small non-cleaved cell lymphoma, Burkitt's Lymphoma, non-Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, renal neoplasm, Wilms tumor, Wilms tumor, clear cell carcinoma of the kidney, rhabdoid tumor of the kidney, clear cell sarcoma of the kidney , renal primitive neuroectodermal tumor, neuroblastoma, ganglioneuroblastoma, ganglioneuroma, extracranial germ cell tumor, mature teratoma, immature teratoma, endodermal sinus tumor, yolk sac tumor, seminoma, dysgerminoma, choriocarcinom
  • the present invention discloses a method for treating a tumor or cancer in a patient in need thereof, comprising administering an effective amount of an anti-PD-1 antibody and an anti-CD73 antibody, wherein the effective amount of the anti-PD-1 administered From about 50 mg to 600 mg per treatment cycle.
  • a treatment cycle is 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values (including endpoint) or any value in it.
  • the anti-PD-1 antibody is Antibody A.
  • the anti-CD73 antibody is a fully human IgG1 antibody P59-L17, a fully human IgG4 antibody P59-L17', or a biosimilar thereof, or an ADCC effect-enhanced monoclonal antibody or afucosylated monoclonal antibody.
  • an effective amount of an anti-CD73 antibody (such as antibody P59-L17 or P59-L17') administered is about 15 mg to 1200 mg per treatment cycle.
  • a treatment cycle is about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or a range between any two of these values ( inclusive) or any value in it.
  • the effective amount of anti-CD73 antibody (such as antibody P59-L17 or P59-L17') administered is about 0.3 mg/kg to 18 mg/kg once every 2 weeks or every 3 weeks. In some embodiments, the effective amount of anti-CD73 antibody (such as antibody P59-L17 or P59-L17') administered is about 0.3 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 6 mg/kg kg, about 12 mg/kg, about 13 mg/kg, or about 18 mg/kg every 2 weeks or every 3 weeks.
  • the effective amount of anti-CD73 antibody (such as antibody P59-L17 or P59-L17') administered is about 5 mg/kg every 2 weeks, about 6 mg/kg every 2 weeks, about 10 mg/kg Once every 2 weeks, about 3mg/kg once every 3 weeks, about 5mg/kg once every 3 weeks, about 6mg/kg once every 3 weeks, about 7.5mg/kg once every 3 weeks, about 12mg/kg kg every 3 weeks, about 15 mg/kg every 3 weeks, or about 18 mg/kg every 3 weeks.
  • the anti-PD-1 antibody and the anti-CD73 antibody can be formulated into pharmaceutical compositions respectively, and the multiplicity of antibodies suitable for the selected route of administration can be formulated into pharmaceutical compositions. It can be administered to a patient in one form, for example, by parenteral, intravenous (iv), intramuscular, topical or subcutaneous routes.
  • the anti-PD-1 antibody and anti-CD73 antibody can be infused intravenously separately.
  • anti-PD-1 antibody and anti-CD73 antibody will depend on the nature of the drug, the degree to which the cell surface triggers the internalization, transport and release of the drug, the disease being treated, and the condition of the patient (such as age, gender, weight, etc.).
  • the anti-PD-1 antibody is about 1 mg/kg to 10 mg/kg per administration or a formulation containing such dose of anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg per administration , about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, or a range between any two of these values (including endpoints) or any value therein, or a preparation containing such a dose of anti-PD-1 antibody.
  • therapeutically effective amounts of anti-CD73 antibody and anti-PD-1 antibody are administered separately or simultaneously to the test patient.
  • the administration cycle of anti-CD73 antibody and anti-PD-1 antibody can be the same or different.
  • the anti-CD73 antibody (e.g., antibody P59-L17 or P59-L17') is about 0.3 mg/kg to 18 mg/kg or formulations containing such doses of anti-CD73 antibody per administration. In some embodiments, the anti-CD73 antibody (such as antibody P59-L17 or P59-L17') is about 0.3 mg/kg, about 1 mg/kg, about 1.2 mg/kg, about 2 mg/kg, about 2.4 mg/kg per administration.
  • mg/kg about 3mg/kg, about 3.6mg/kg, about 4mg/kg, about 4.8mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.9mg/kg, about 7mg /kg, about 8.4 mg/kg, about 9 mg/kg, about 11 mg/kg, about 12 mg/kg, about 15 mg/kg, about 18 mg/kg, or the range between any two of these values (inclusive) or any value therein, or preparations containing such doses of anti-CD73 antibodies.
  • the present invention discloses a method of treating tumor or cancer, comprising administering an effective amount of an anti-PD-1 antibody (or preparation) and an anti-CD73 antibody (or preparation) to a patient in need; wherein, The effective dose of anti-PD-1 antibody is about 50 mg to 600 mg in a single administration (or a preparation containing this dose of anti-PD-1 antibody).
  • the anti-CD73 antibody is antibody P59-L17 or P59-L17'.
  • the effective amount of anti-CD73 antibody (such as antibody P59-L17 or P59-L17') is about 15 mg to 1200 mg in a single administration (or a preparation containing such dose of anti-CD73 antibody).
  • the dosing schedule and mode of administration depend on the benefit-risk assessment of the anti-PD-1 antibody (or formulation), anti-CD73 antibody (or formulation) in certain patient populations, and general clinical practice guidelines.
  • the anti-PD-1 antibody is administered in an effective amount of about 50 mg to 600 mg of anti-PD-1 antibody (or a preparation containing this dose of anti-PD-1 antibody) in each treatment cycle of the patient.
  • the effective amount of anti-CD73 antibody (such as antibody P59-L17 or P59-L17') administered in the cycle is about 15 mg to 1200 mg (or a preparation containing this dose of anti-CD73 antibody).
  • the effective amount of the anti-PD-1 antibody administered to the patient per treatment cycle is about 50 mg, about 60 mg, about 80 mg, about 120 mg, about 200 mg, about 250 mg, about 290 mg, about 300 mg, about 330 mg, about 380 mg, about 400 mg, about 434 mg, about 480 mg, about 500 mg, about 567 mg, about 580 mg, about 600 mg, or a range between any two of these values (including endpoints) or any value therein, or containing this dose of anti-PD -1 Preparation of antibodies.
  • a treatment cycle is administered once from 1 week to 7 weeks.
  • the effective amount of anti-PD-1 antibody administered in each treatment cycle is about 100 mg to 200 mg, or a preparation containing this dose of anti-PD-1 antibody; wherein, one treatment cycle is about 1 week, about 2 weeks weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or a range between any two of these values (inclusive) or any value therein. In some embodiments, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 200 mg to 300 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • the effective amount of the anti-PD-1 antibody administered to the patient per treatment cycle is about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190mg, about 200mg, or the range between any two values in these values (including the endpoint) or any value therein, or preparations containing such doses; wherein, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks week or about 4 weeks.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 90 mg to 110 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; wherein, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 100 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; for example, about 100 mg is administered once.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 100 mg to 140 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; wherein, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the effective amount of anti-PD-1 antibody administered to the patient in each treatment cycle is about 120 mg, or a preparation containing this dose of anti-PD-1 antibody; for example, about 120 mg is administered once.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 150 mg to 190 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; wherein, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 160 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; for example, about 160 mg is administered once.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 190 mg to 230 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-PD-1 antibody of about 200 mg in each treatment cycle, or a preparation containing this dose of anti-PD-1 antibody; for example, about 200 mg is administered once.
  • the effective amount of anti-PD-1 antibody is about 100 mg to 600 mg every 3 weeks. In some embodiments, the effective amount of an anti-PD-1 antibody is about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, or about 600 mg once every 3 weeks. In some embodiments, the effective amount of anti-PD-1 antibody is about 100 mg, about 300 mg, or about 600 mg once every 3 weeks.
  • the effective amount of the anti-CD73 antibody administered to the patient per treatment cycle is about 15 mg, about 18 mg, about 90 mg, about 120 mg, about 160 mg, about 180 mg, about 200 mg, about 230 mg, about 250 mg, about 280 mg, About 300mg, about 310mg, about 334mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 500mg, about 600mg, about 720mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg , or a range between any two of these values (inclusive), or any value therein, or a formulation containing such an amount of anti-CD73 antibody.
  • a treatment cycle is administered once from 1 week to 7 weeks.
  • the effective amount of anti-CD73 antibody administered in each treatment cycle is about 100 mg to 300 mg, or a preparation containing this dose of anti-CD73 antibody; wherein, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks weeks, about 4 weeks, or a range between any two of these values (inclusive), or any value therein.
  • the patient is administered an effective amount of anti-CD73 antibody of about 300 mg to 600 mg in each treatment cycle, or a preparation containing such a dose of anti-CD73 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
  • the effective amount of anti-CD73 antibody administered in each treatment cycle is about 700 mg to 1100 mg, or a preparation containing this dose of anti-CD73 antibody; wherein, one treatment cycle is about 1 week, about 2 weeks, about 3 weeks weeks, about 4 weeks, or a range between any two of these values (inclusive), or any value therein.
  • the effective amount of the anti-CD73 antibody administered to the patient per treatment cycle is about 60 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, About 190 mg, about 200 mg, about 360 mg, about 420 mg, about 720 mg, about 1080 mg, or a range between any two of these values (including endpoints) or any value therein, or a preparation containing such a dose of anti-CD73 antibody;
  • one treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-CD73 antibody of about 50 mg to 80 mg in each treatment cycle, or a preparation containing this dose of anti-CD73 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-CD73 antibody of about 60 mg per treatment cycle, or a preparation containing such a dose of anti-CD73 antibody; for example, about 60 mg is administered once.
  • the patient is administered an effective amount of anti-CD73 antibody of about 150 mg to 200 mg in each treatment cycle, or a preparation containing such a dose of anti-CD73 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the effective amount of anti-CD73 antibody administered to the patient is about 180 mg per treatment cycle, or a preparation containing such a dose of anti-CD73 antibody; for example, about 180 mg is administered once.
  • the patient is administered an effective amount of anti-CD73 antibody of about 345 mg to 380 mg in each treatment cycle, or a preparation containing this dose of anti-CD73 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-CD73 antibody of about 360 mg per treatment cycle, or a preparation containing such a dose of anti-CD73 antibody; for example, about 360 mg is administered once.
  • the patient is administered an effective amount of anti-CD73 antibody of about 693 mg to 730 mg in each treatment cycle, or a preparation containing this dose of anti-CD73 antibody; wherein, a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • a treatment cycle is about 1 week, about 2 weeks, about 3 weeks or about 4 weeks.
  • the patient is administered an effective amount of anti-CD73 antibody of about 720 mg per treatment cycle, or a preparation containing such a dose of anti-CD73 antibody; for example, about 720 mg is administered once.
  • the patient is administered an anti-PD-1 antibody and an anti-CD73 antibody (or a combination of an anti-PD-1 antibody and an anti-CD73 antibody) once in each treatment cycle.
  • the anti-PD-1 antibody and the anti-CD73 antibody are administered multiple times in each treatment cycle, for example, 2 times, 3 times, 4 times or 5 times.
  • the patient can only be dosed 1 or 4 times per treatment cycle.
  • the patient is treated for one treatment cycle. In some embodiments, the patient is treated for multiple (eg, 2, 3, or 4) treatment cycles. In some embodiments, the patient is treated until the condition is in remission and no longer requires treatment.
  • the present invention discloses a method for treating tumor or cancer, said method comprising: administering about 50 mg to 200 mg, about 200 mg to 300 mg, about 300 mg to 400 mg, or about 400 mg to 600 mg, such as about 100 mg, about 120 mg, about 200 mg, or about 400 mg of anti-PD-1 antibody, or a preparation containing this dose of anti-PD-1 antibody; also administer about 15 mg to 180 mg, about 180 mg to 360 mg, About 360mg to 500mg, about 500mg to 720mg or about 730mg to 1200mg, such as about 60mg, about 120mg, about 180mg, about 300mg or about 400mg of anti-CD73 antibody, or a preparation containing such dose of anti-CD73 antibody.
  • the patient receives a single dose of an anti-PD-1 antibody and a single dose of an anti-CD73 antibody.
  • the patient is treated with a single dose of the anti-PD-1 antibody and anti-CD73 antibody combination.
  • about 200 mg of the anti-PD-1 antibody is administered every 3 weeks, and about 3 mg/kg of the anti-CD73 antibody is administered every 3 weeks. In some embodiments, about 300 mg of the anti-PD-1 antibody is administered every 3 weeks and about 5 mg/kg of the anti-CD73 antibody is administered every 3 weeks. In some embodiments, about 200 mg of the anti-PD-1 antibody is administered every 3 weeks and about 6 mg/kg of the anti-CD73 antibody is administered every 3 weeks.
  • the patient's symptoms are relieved.
  • the patient's symptoms are not relieved as expected, and then about 50 mg to 600 mg of anti-PD-1 antibody and about 15 mg to 1200 mg of anti-CD73 antibody are administered to the patient.
  • the patient's symptoms are not relieved as expected, and then the patient is administered a composition of about 50 mg to 600 mg of anti-PD-1 antibody and about 15 mg to 1200 mg of anti-CD73 antibody.
  • the anti-PD-1 antibody (or formulation), anti-CD73 antibody (or formulation) is injected subcutaneously (s.c.), intraperitoneally (i.p.), parenterally, intraarterially, or intravenously (i.v. ) administration by way of injection or the like.
  • anti-PD-1 antibody (or preparation) and anti-CD73 antibody (or preparation) are administered by infusion.
  • anti-PD-1 antibody (or preparation) and anti-CD73 antibody (or preparation) are administered by bolus injection.
  • the anti-PD-1 antibody (or formulation), anti-CD73 antibody (or formulation) is administered by intravenous (i.v.) infusion.
  • the duration of the intravenous infusion is about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 81 minutes, about 87 minutes, about 90 minutes, about 95 minutes minutes, or the range between any two of these values (inclusive), or any value therein.
  • anti-PD-1 antibody or preparation
  • anti-CD73 antibody or preparation
  • other treatment methods for the treatment of tumors or cancers such as chemotherapy, radiotherapy and surgery.
  • the present invention discloses the application of anti-PD-1 antibody and therapeutic agent in the preparation of medicine for treating tumor or cancer.
  • the therapeutic agent is an anti-CD73 antibody.
  • the anti-CD73 antibody is antibody P59-L17 or P59-L17'.
  • the present invention also discloses a kit comprising anti-PD-1 antibody (or preparation), anti-CD73 antibody (or preparation) and anti-PD-1 antibody (or preparation) for guiding patients in need to administer anti-PD-1 antibody (or preparation). preparation) and instructions for the anti-CD73 antibody (or preparation).
  • the present invention also discloses a kit, which comprises a composition of an anti-PD-1 antibody (or preparation) and an anti-CD73 antibody (or preparation) and is used to guide patients in need to administer anti-PD -1 Description of the composition of the antibody (or preparation) and the anti-CD73 antibody (or preparation).
  • the present invention also discloses a pharmaceutical composition suitable for injection comprising an anti-PD-1 antibody and an anti-CD73 antibody, such as a bolus injection type pharmaceutical composition or an infusion (drip) type pharmaceutical composition.
  • the pharmaceutical composition comprises at least 0.1% anti-PD-1 antibody and 0.1% anti-CD73 antibody.
  • the percentage of antibody can vary and is between about 2% and about 90% by weight of a given dosage form.
  • the amount of anti-PD-1 antibody and anti-CD73 antibody in this therapeutically useful pharmaceutical composition can be an effective amount for administration.
  • the present invention also discloses a preparation method of the above-mentioned pharmaceutical composition: respectively combining the anti-PD-1 antibody and anti-CD73 antibody (or the composition of anti-PD-1 antibody and anti-CD73 antibody) described herein with pharmaceutically Acceptable auxiliary materials suitable for injection (such as water for injection, physiological saline, etc.) are mixed.
  • pharmaceutically Acceptable auxiliary materials suitable for injection such as water for injection, physiological saline, etc.
  • the anti-PD-1 antibody (or preparation) and anti-CD73 antibody (or preparation) are used in tumor or cancer treatment to relieve symptoms.
  • Figure 1 shows the effect of the antibody on the tumor; wherein, the ordinate represents the tumor volume, and the abscissa represents the days after administration.
  • an entity refers to one or more such entities, for example "an antibody” should be understood as one or more antibodies, therefore, the term “a” (or “an” ), “one or more” and “at least one” may be used interchangeably herein.
  • compositions, methods, etc. include the listed elements, such as components or steps, but not exclude others.
  • Consisting essentially of means that the compositions and methods exclude other elements that substantially affect the characteristics of the combination, but do not exclude elements that do not substantially affect the composition or method.
  • Consisting of means excluding elements not specifically recited.
  • polypeptide is intended to encompass the singular as well as the plural “polypeptides” and refers to a molecule formed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein”, “amino acid chain” or any other term used to refer to a chain of two or more amino acids, and the term “polypeptide” may Used in place of, or interchangeably with, any of the above terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.
  • amino acid refers to an organic compound containing both amino and carboxyl groups, such as an ⁇ -amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called “degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine amino acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein.
  • classes of amino acids that contain chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, Arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid.
  • the number of amino acids in the "conservative amino acid substitution of VL, VH" can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range between any two of these values (inclusive), or any value therein.
  • the number of amino acids for "conservative amino acid substitutions in the heavy or light chain” can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10 about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, About 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (inclusive), or any value therein.
  • encoding when applied to a polynucleotide refers to a polynucleotide which is said to "encode” a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, is transcribed and/or Or translation may result in the polypeptide and/or fragments thereof.
  • polypeptides or polynucleotides refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not occur in nature, non-limiting examples may be produced by combination of polynucleotides or polynucleotides that do not normally exist or peptide.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the alignable positions in each sequence. When a position in the sequences being compared is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • At least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, About 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these A range (inclusive) between any two values in Numeric or any value therein.
  • a polynucleotide or polynucleotide sequence has a certain percentage (eg, 90%, 95%, 98%, or 99%) of "identity” or “sequence identity” with another sequence.
  • “Sex” means that when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are identical. This alignment percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al.eds. (2007) in Current Protocols in Molecular Biology. It is preferred to use the default parameters for the alignment.
  • Biologically equivalent polynucleotides are polynucleotides that share the above indicated percentages of identity and encode a polypeptide having the same or similar biological activity.
  • Antibody and antigen-binding fragment refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof.
  • the term “antibody” thus includes any protein or peptide whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity to bind an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand-binding portions thereof (CH), light chain constant region (CL), framework region (FR) or any portion thereof, or at least a portion of a binding protein.
  • CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3).
  • antibody includes a wide variety of polypeptides that can be distinguished biochemically.
  • classes of heavy chains include gamma, mu, alpha, delta or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), with some subclasses (eg ⁇ 1- ⁇ 4).
  • the nature of this chain determines the "class” of the antibody as IgG, IgM, IgA, IgG or IgE, respectively.
  • the immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are also known.
  • the immunoglobulin molecule is of the IgG class.
  • the four chains are linked by disulfide bonds in a "Y" configuration, with the light chain starting at the mouth of the "Y” and continuing through the variable region surrounding the heavy chain.
  • Antibodies, antigen-binding fragments or derivatives disclosed in the present invention include but are not limited to polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding Fragments (eg Fab, Fab', F(ab') 2 , single chain Fv (scFv)).
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be associated with a kappa or lambda light chain.
  • ⁇ kappa
  • lambda ⁇
  • Each heavy chain can be associated with a kappa or lambda light chain.
  • immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonding.
  • the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin kappa light chain is V ⁇ ; the variable region of the immunoglobulin lambda light chain is V ⁇ .
  • the terms “constant” and “variable” are used according to function.
  • the light chain variable (VL) and heavy chain variable (VH) portions determine antigen recognition and specificity.
  • the constant region (CL) of the light chain and the constant region (CH) of the heavy chain confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant regions increases as they become farther away from the antigen-binding site or amino terminus of the antibody.
  • the N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.
  • CDR complementarity determining regions
  • CDRs as defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to use either definition to refer to the CDRs of an antibody or variant thereof.
  • the exact residue numbers comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues are included in the CDRs based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independently of other experimental data other than the sequence itself.
  • “Kabat numbering” refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest” (1983).
  • Antibodies can also use the EU or Chothia numbering system.
  • Treatment means therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow down, ameliorate and stop undesirable physiological changes or disorders, such as the progression of disease, including but not limited to the following whether detectable or undetectable Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, improvement or palliation of disease state, alleviation or disappearance (whether partial or total), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include those who already have a condition or disorder, are susceptible to a condition or disorder, or are in need of prevention of the condition or disorder, and can or are expected to benefit from the administration of an antibody or composition disclosed herein for detection, Patients who benefit from the diagnostic process and/or treatment.
  • Patient refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and the like.
  • Effective amount means that amount of an active compound or agent that elicits a biological or medical response in a tissue, system, animal, individual or human; an effective amount is sought by a researcher, veterinarian, physician or other clinician.
  • EC 50 means half maximum effect concentration (concentration for 50% of maximal effect, EC50) refers to the concentration that can cause 50% of the maximum effect.
  • the phrase "in need thereof” means that a patient has been identified as being in need of a particular method or treatment. In some embodiments, identification may be by any diagnostic means. A patient may in need thereof during any of the methods and treatments described herein.
  • an antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail.
  • Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • High-efficiency transcription can be obtained through the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus, and other cellular promoters such as muscle Kinetin promoter.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc.
  • Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.
  • Embodiment 1 Preparation method of antibody
  • Biacore T200 surface plasmon resonance instrument was used to measure the affinity constant (K D ) of the antibody.
  • the main test process was as follows (refer to the standard operating procedure of Biacore T200): -H08H) Samples were serially diluted with HBS-EP buffer (150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P-20, and 10 mM HEPES, pH 7.4) (the initial concentration was 32 nM, then 2-fold dilution ); Dilute the antibody with sodium acetate solution (10mM, pH5.5) to a final concentration of about 20 ⁇ g/ml; use BiacoreT200, Protein A chip (GE healthcare, product number 29127556) for detection, the instrument settings are as follows: antibody capture binding The contact time is 180s, the flow rate is 30 ⁇ l/min; the sample contact time is 120s, the dissociation time is 300s, and the flow rate is 30 ⁇ l/min; the regeneration condition is: Glycine-HCl (
  • the data analysis software Evaluation Software3.1 was used to analyze the test results, and the sensor signals collected by the sample experimental flow path were subtracted from the reference flow path and the sample blank, and the kinetic "1:1" model was selected for fitting. Get the kinetic parameters (Ka is the association rate, Kd is the dissociation rate; KD is the binding-dissociation equilibrium constant)
  • antibody P59-L17 has good binding ability to hCD73-His.
  • ELISA method was used for detection: first use 2 ⁇ g/ml human source hCD73-His (Beijing Sino Biological Science and Technology Co., Ltd., Catalog is 10904-H08H), cynomolgus monkey cCD73-His (near shore organisms, Catalog is CD3- C52H9) or mouse mCD73-His (near shore organisms, Catalog is CD3-M52H9) coated 96-well ELISA plate, overnight at 4°C; buffered with PBS (phosphate buffered saline) containing 5% BSA (bovine serum albumin) After blocking, wash with PBS containing 0.05% TW-20, and then add antibody solution diluted with PBS containing 0.05% TW-20 and 0.5% BSA (the initial concentration of antibody is 4 ⁇ g/ml, 3 double dilution, 10 gradients); incubate at 37°C for 1 hour; after washing the plate, add HRP (horseradish peroxidas
  • antibody P59-L17 binds well to human and cynomolgus CD73, but does not bind to mouse CD73.
  • NA means no binding or substantially no binding.
  • MDA-MB-231 cells are human breast cancer cells with high expression of CD73 protein on the cell membrane surface (https://www.proteinatlas.org/ENSG00000101017-CD40/cell). In this example, MDA-MB-231 cells were used to detect the binding ability of anti-CD73 antibodies to CD73 molecules on the cell membrane surface.
  • test method is as follows: after the well-cultured MDA-MB-231 cells are collected and centrifuged, they are incubated with isotype control IgG1-Fc or serially diluted anti-CD73 antibody; after 30 minutes, the supernatant is washed by centrifugation, washed once with PBS buffer, Resuspend in PBS buffer and add fluorescently labeled anti-human IgG flow antibody anti-humanFc-PE (purchased from Invitrogen, Cat. No.
  • the detection principle of the detection kit (purchased from promega, the article number is G7570) is: excess AMP can inhibit ATP-dependent
  • the detection reagent emits fluorescence, and the cell membrane CD73 can decompose AMP to release the inhibitory effect of AMP.
  • the anti-CD73 antibody can inhibit the enzyme activity of CD73 so as to maintain the inhibition of AMP on the fluorescence process.
  • the final performance is: with the increase of antibody concentration, the fluorescence intensity corresponding weakened.
  • the inhibitory effect of anti-CD73 antibody on the enzymatic activity of CD73 on the cell membrane was detected.
  • the test method is: use DMEM medium containing 10% FBS to spread the well-growing MDA-MB-231 cells on a 96-well cell culture plate at 2.5 ⁇ 10 4 cells/well, and culture in a 37°C cell culture box overnight; The next day, remove the supernatant of the culture medium, add the antibody solution diluted in serum-free DMEM and AMP with a final concentration of 600 ⁇ M, and place it in a 37°C cell culture incubator to react for 3 hours; after the reaction, take 50 ⁇ l of the supernatant per well in a 96-well white plate 50 ⁇ l of ATP with a concentration of 200 ⁇ M was added, and finally 100 ⁇ l of detection reagent was added, and the fluorescence value was read immediately on a microplate reader.
  • Example 6 Combined administration of anti-PD-1 antibody and anti-CD73 antibody inhibits the proliferation of cancer cells
  • This example is used to evaluate the colon cancer cell tumor model subcutaneously inoculated with antibody A and antibody P59-L17' in immune checkpoint humanized mice (BALB/c-hPD1/hPDL1/hCD73, Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) The medicinal effect in.
  • Resuscitated mouse colon cancer cells CT26-hPDL1/hCD73 (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), collected colon cancer cells in the logarithmic growth phase, removed the culture medium, washed twice with PBS, and then inoculated subcutaneously under the armpit.
  • mice When the average volume of the tumor was 80-120mm 3 , the mice were randomly divided into 4 groups, 8 in each group; the day of grouping was defined as D0 day, and the administration was started according to the administration plan on the day of grouping, and the time of intraperitoneal administration (ip) was : D0, D3, D7, D10 and D14.
  • TGItv inhibition rate relative to tumor volume
  • RTV n V nt /V n0 ;
  • V nt the tumor volume of the mouse numbered on day t,
  • V n0 the tumor volume of the mouse numbered on day 0,
  • RTV n the tumor volume of the mouse numbered n Relative tumor volume of mice on day t
  • TGItv (1-(mean RTV administration group)/(mean RTV control group)) ⁇ 100%; mean RTV administration group: administration group RTV average value, mean RTV control group: control group RTV average value.

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Abstract

提供了抗PD-1抗体及其在制备治疗肿瘤或癌症的药物中的应用。

Description

抗PD-1抗体的应用 技术领域
本发明属于生物医药领域，尤其涉及抗PD-1抗体在联合用药中的应用。
背景技术
肿瘤的发病率逐年上升，抗肿瘤药物的使用也逐渐增多。近年来，肿瘤治疗研究取得了重大进展，尤其是一些抗体药物在恶性肿瘤治疗中显示出了良好疗效。然而，只有少部分患者可达到长期生存。为了进一步提高药物的抗肿瘤治疗的临床疗效，在使用抗肿瘤药物时，可采用联合用药。
程序性死亡受体-1(PD-1)是在活化的T细胞、B细胞和髓样细胞上表达的免疫抑制性受体，是CD28免疫球蛋白超家族成员。PD-1为55kDa的I型跨膜糖蛋白，其含有与配体结合的Ig可变型结构域和负责结合信号转导分子的胞质尾区。PD-1胞质尾区含有两个基于酪氨酸的信号转导模体：ITIM(免疫受体酪氨酸抑制基序)和ITSM(免疫受体酪氨酸转换基序)。
迄今有大量的研究显示PD-1和PD-L1(程序性死亡配体1)之间的相互作用导致渗入肿瘤的淋巴细胞减少、T细胞受体介导的增殖减少和癌细胞的免疫逃避。阻断PD-1与PD-L1之间的相互作用可增加T细胞增殖和细胞因子产生，提高肿瘤特异性CD8 +T细胞的免疫性。
因此，抗PD-1抗体在抗肿瘤的联合用药中发挥着重要作用。
发明内容
本发明公开了抗PD-1抗体用于联合治疗肿瘤或癌症的方法或用途。在一些实施方案中，抗PD-1抗体和治疗剂联合用于治疗肿瘤或癌症。在一些实施方案中，所述治疗剂为抗CD73抗体。在一些实施方案中，所述方法或用途包括：向有需要的患者施用有效量的抗PD-1抗体和抗CD73抗体。在一些实施方案中，所述用途为抗PD-1抗体在制备和抗CD73抗体联合用于治疗肿瘤或癌症的药物的用途。在一些实施方案中，所述用途为抗CD73抗体在制备和抗PD-1抗体联合用于治疗肿瘤或癌症的药物的用途。
在一些实施方案中，所述抗PD-1抗体至少包含SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3、SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2、SEQ ID NO:6所示的LCDR3中一个或多个。
在一些实施方案中，所述抗PD-1抗体包含一个或两个选自以下所示的氨基酸序列的CDRs：SEQ ID NO:3和6；并且可选地包含一个、两个、三个或四个选自以下所示的氨基酸序列的CDRs：SEQ ID NO:1、2、4和5。
在一些实施方案中，所述抗PD-1抗体包含SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3、SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3。
在一些实施方案中，所述抗PD-1抗体的重链可变区包含SEQ ID NO:7所示的序列，与SEQ ID NO:7所示序列具有至少80％同一性的序列，或与SEQ ID NO:7所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述抗PD-1抗体的轻链可变区包含SEQ ID NO:8所示的序列，与SEQ ID NO:8所示序列具有至少80％同一性的序列，或与SEQ ID NO:8所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗PD-1抗体的重链可变区包含SEQ ID NO:7所示的序列，所述抗PD-1抗体的轻链可变区包含SEQ ID NO:8所示的序列。
在一些实施方案中，所述抗PD-1抗体的重链包含SEQ ID NO:9所示的序列，与SEQ ID NO:9所示序列具有至少80％同一性的序列，或与SEQ ID NO:9所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述抗PD-1抗体的轻链包含SEQ ID NO:10所示的序列，与SEQ ID NO:10所示序列具有至少80％同一性的序列，或与SEQ ID NO:10所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗PD-1抗体为抗体A，抗体A的重链包含如SEQ ID NO:9所示序列，所述抗体A的轻链包含如SEQ ID NO:10所示序列；抗体A含有两条序列相同的重链和两条序列相同的轻链。
在一些实施方案中，所述抗CD73抗体包含如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:12所示的HCDR2、如SEQ ID NO:13所示的HCDR3、如SEQ ID NO:14所示的LCDR1、如SEQ ID NO:15所述的LCDR2和如SEQ ID NO:16所示的LCDR3中一个或多个。
在一些实施方案中，所述抗CD73抗体包含如SEQ ID NO:11所示的HCDR1、如SEQ ID NO:12所示的HCDR2、如SEQ ID NO:13所示的HCDR3、如SEQ ID NO:14 所示的LCDR1、如SEQ ID NO:15所述的LCDR2和如SEQ ID NO:16所示的LCDR3。
在一些实施方案中，所述抗CD73抗体的重链可变区包含SEQ ID NO:17所示的序列，与SEQ ID NO:17所示序列具有至少80％同一性的序列，或与SEQ ID NO:17所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述抗CD73抗体的轻链可变区包含SEQ ID NO:18所示的序列，与SEQ ID NO:18所示序列具有至少80％同一性的序列，或与SEQ ID NO:18所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗CD73抗体的重链可变区包含SEQ ID NO:17所示的序列，所述抗CD73抗体的轻链可变区包含SEQ ID NO:18所示的序列。
在一些实施方案中，所述抗CD73抗体的重链包含SEQ ID NO:19或20所示的序列，与SEQ ID NO:19或20所示序列具有至少80％同一性的序列，或与SEQ ID NO:19或20所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述抗CD73抗体的轻链包含SEQ ID NO:21所示的序列，与SEQ ID NO:21所示序列具有至少80％同一性的序列，或与SEQ ID NO:21所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗CD73抗体为抗体P59-L17，抗体P59-L17的重链包含如SEQ ID NO:19所示序列，抗体P59-L17的轻链包含如SEQ ID NO:21所示序列；抗体P59-L17含有两条序列相同的重链和两条序列相同的轻链。
在一些实施方案中，所述抗CD73抗体为抗体P59-L17’，抗体P59-L17’的重链包含如SEQ ID NO:20所示序列，抗体P59-L17’的轻链包含如SEQ ID NO:21所示序列；抗体P59-L17’含有两条序列相同的重链和两条序列相同的轻链。
抗体蛋白可以通过基因工程在细胞(如CHO细胞或HEK细胞)中表达，并通过纯化获得；纯化可以采用常规方法进行，例如先离心细胞悬液并收集上清液，再次离心进一步去除杂质。Protein A亲和柱和离子交换柱等方法可以用于纯化抗体蛋白。
在一些实施方案中，所述方法或用途包括：向有需要的患者施用有效量的抗PD-1抗体和抗CD73抗体。在一些实施方案中，所述抗PD-1抗体为抗体A。在一些实施方案中，所述抗CD73抗体为抗体P59-L17或P59-L17’。在一些实施方案中，抗PD-1抗体施用的有效剂量为每剂约50mg至600mg。在一些实施方案中，抗CD73抗体(如抗体P59-L17或P59-L17’)施用的有效剂量为每剂约15mg至1200mg。
在一些实施方案中，所述抗PD-1抗体和所述抗CD73抗体分别为独立的给药单元，联合用药。在一些实施方案中，所述抗PD-1抗体可以在施加所述抗CD73抗体之前给药，也可以在施加所述抗CD73抗体之后给药，也可以在施加所述抗CD73抗体的同 时给药。在一些实施方案中，所述抗PD-1抗体和所述抗CD73抗体同时形成组合给药单元，联合用药。
在一些实施方案中，患者患有肿瘤或癌症。在一些实施方案中，肿瘤和癌症包括但不限于：头颈部肿瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、前体淋巴母细胞淋巴瘤、小无裂细胞淋巴瘤、伯基特淋巴瘤、非伯基特淋巴瘤、弥漫性大B细胞淋巴瘤、间变性大细胞淋巴瘤、肾脏肿瘤、肾母细胞瘤、Wilms瘤、肾透明细胞癌、肾横纹肌样瘤、肾透明细胞肉瘤、肾原始神经外胚叶瘤、神经母细胞瘤、节细胞神经母细胞瘤、节细胞神经瘤、颅外生殖细胞瘤、成熟畸胎瘤、未成熟畸胎瘤、内胚窦瘤、卵黄囊瘤、精原细胞瘤、无性细胞瘤、绒毛膜上皮癌、胚胎癌、骨肉瘤、软骨肉瘤、横纹肌肉瘤、软组织肉瘤、纤维肉瘤、恶性纤维组织细胞瘤、脂肪肉瘤、平滑肌肉瘤、血管肉瘤、淋巴管肉瘤、恶性神经鞘瘤、腺泡状软组织肉瘤、上皮样肉瘤、透明细胞肉瘤、恶性黑色素瘤、滑膜肉瘤、促纤维增生性小圆细胞瘤、尤文氏肉瘤、原始神经外胚叶瘤、肝脏肿瘤、肝母细胞瘤、视网膜母细胞瘤、后颅窝髓母细胞瘤、胸腺瘤、肺母细胞瘤、胰母细胞瘤、胰岛细胞瘤、回盲部类癌、间皮瘤、黑色素瘤、间质细胞瘤、骨髓瘤、脑星形细胞瘤、鼻咽癌、甲状腺乳头状癌、肠癌、乳癌、胃癌、肝癌、前列腺癌、乳腺癌、肺癌、宫颈癌、卵巢癌、肾癌、淋巴瘤、白血病、皮肤癌和食管鳞癌等。
在一些实施方案中，本发明公开了一种用于治疗有需要患者的肿瘤或癌症的方法，其包括施用有效量的抗PD-1抗体和抗CD73抗体，其中抗PD-1施用的有效量为约50mg至600mg每个治疗周期。在一些实施方案中，一个治疗周期为1周、2周、3周、4周、1个月、5周、6周、7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，所述抗PD-1抗体为抗体A。
在一些实施方案中，所述抗CD73抗体为全人源IgG1型抗体P59-L17、全人源IgG4型抗体P59-L17’或其生物类似物或ADCC效应增强单抗或去岩藻糖基化单抗。在一些实施方案中，抗CD73抗体(如抗体P59-L17或P59-L17’)施用的有效量为约15mg至1200mg每个治疗周期。在一些实施方案中，一个治疗周期为约1周、2周、3周、4周、1个月、5周、6周、7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，抗CD73抗体(如抗体P59-L17或P59-L17’)施用的有效量为约0.3mg/kg至18mg/kg每2周或每3周1次。在一些实施方案中，抗CD73抗体(如抗体P59-L17或P59-L17’)施用的有效量为约0.3mg/kg，约1mg/kg，约3mg/kg，约5mg/kg，约6mg/kg，约12mg/kg，约13mg/kg，或约18mg/kg每2周或每3周1次。在一些实施方案中，抗CD73抗体(如抗体P59-L17或P59-L17’)施用的有效量为约5mg/kg每2周1次，约6mg/kg每2周1次，约10mg/kg每2周1次，约3mg/kg每3周1次，约5mg/kg每3周1次，约6mg/kg每3周1次，约7.5mg/kg每3周1次，约12mg/kg每3周1次，约15mg/kg每3周1次，或约18mg/kg 每3周1次。
在一些实施方案中，可以分别将抗PD-1抗体和抗CD73抗体(或抗PD-1抗体和抗CD73抗体的组合物)配制成药物组合物，并以适合于所选给药途径的多种形式向患者给药，例如通过肠胃外、静脉内(iv)、肌肉内、局部或皮下途径。在一些实施方案中，可以分别将抗PD-1抗体和抗CD73抗体(或抗PD-1抗体和抗CD73抗体的组合物)静脉输注。抗PD-1抗体和抗CD73抗体的量将取决于药物的性质，细胞表面触发药物的内在化、运输和释放的程度，所治疗的疾病，患者的状况(如年龄，性别，体重等)。
在一些实施方案中，每次施用的抗PD-1抗体约1mg/kg至10mg/kg或含此剂量抗PD-1抗体的制剂。在一些实施方案中，每次施用的抗PD-1抗体为约1mg/kg，约2mg/kg，约3mg/kg，约4mg/kg，约5mg/kg，约6mg/kg，约7mg/kg，约8mg/kg，约9mg/kg，约10mg/kg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗PD-1抗体的制剂。
在一些实施方案中，采用治疗有效量的抗CD73抗体和抗PD-1抗体分别或者同时施加在受试患者上。抗CD73抗体和抗PD-1抗体的给药周期可以相同或者不同。
在一些实施方案中，每次施用的抗CD73抗体(如抗体P59-L17或P59-L17’)为约0.3mg/kg至18mg/kg或含此剂量抗CD73抗体的制剂。在一些实施方案中，每次施用的抗CD73抗体(如抗体P59-L17或P59-L17’)为约0.3mg/kg，约1mg/kg，约1.2mg/kg，约2mg/kg，约2.4mg/kg，约3mg/kg，约3.6mg/kg，约4mg/kg，约4.8mg/kg，约5mg/kg，约5.5mg/kg，约6mg/kg，约6.9mg/kg，约7mg/kg，约8.4mg/kg，约9mg/kg，约11mg/kg，约12mg/kg，约15mg/kg，约18mg/kg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CD73抗体的制剂。
在一些实施方案中，本发明公开了一种治疗肿瘤或癌症的方法，其包括向有需要的患者施用有效量的抗PD-1抗体(或制剂)和抗CD73抗体(或制剂)；其中，抗PD-1抗体的有效量为单次给药约50mg至600mg(或含此剂量抗PD-1抗体的制剂)。在一些实施方案中，抗CD73抗体为抗体P59-L17或P59-L17’。在一些实施方案中，抗CD73抗体(如抗体P59-L17或P59-L17’)的有效量为单次给药约15mg至1200mg(或含此剂量抗CD73抗体的制剂)。剂量时间表和给药方式取决于某些患者群中的抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)的获益风险评估和一般临床实践指南。
在一些实施方案中，患者每个治疗周期内抗PD-1抗体施用的有效量为约50mg至600mg的抗PD-1抗体(或含此剂量抗PD-1抗体的制剂)，患者每个治疗周期内抗CD73抗体(如抗体P59-L17或P59-L17’)施用的有效量为约15mg至1200mg(或含 此剂量抗CD73抗体的制剂)。
在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约50mg、约60mg、约80mg、约120mg、约200mg、约250mg、约290mg、约300mg、约330mg、约380mg、约400mg、约434mg、约480mg、约500mg、约567mg、约580mg、约600mg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗PD-1抗体的制剂。在一些实施方案中，一个治疗周期为1周至7周给药1次。在一些实施方案中，每个治疗周期内施用抗PD-1抗体的有效量为约100mg至200mg，或含此剂量抗PD-1抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、约4周、约5周、约6周、约7周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约200mg至300mg，或含此剂量抗PD-1抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、或约4周。在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约100mg、约110mg、约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg、约190mg、约200mg，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值，或含此剂量的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。
在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约90mg至110mg，或含此剂量抗PD-1抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约100mg，或含此剂量抗PD-1抗体的制剂；比如约100mg给药1次。
在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约100mg至140mg，或含此剂量抗PD-1抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约120mg，或含此剂量抗PD-1抗体的制剂；比如约120mg给药1次。
在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约150mg至190mg，或含此剂量抗PD-1抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约160mg，或含此剂量抗PD-1抗体的制剂；比如约160mg给药1次。
在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约190mg至230mg，或含此剂量抗PD-1抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗PD-1抗体的有效量为约200mg，或含此剂量抗PD-1抗体的制剂；比如约200mg给药1次。
在一些实施方案中，抗PD-1抗体的有效量为约100mg到600mg每3周一次。在一些实施方案中，抗PD-1抗体的有效量为约100mg、约200mg、约300mg、约400mg、约500mg或约600mg每3周一次。在一些实施方案中，抗PD-1抗体的有效量为约100mg、约300mg或约600mg每3周一次。
在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约15mg、约18mg、约90mg、约120mg、约160mg、约180mg、约200mg、约230mg、约250mg、约280mg、约300mg、约310mg、约334mg、约350mg、约360mg、约370mg、约380mg、约390mg、约400mg、约500mg、约600mg、约720mg、约800mg、约900mg、约1000mg、约1100mg、约1200mg，或这些数值中任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CD73抗体的制剂。在一些实施方案中，一个治疗周期为1周至7周给药1次。在一些实施方案中，每个治疗周期内施用抗CD73抗体的有效量为约100mg至300mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、约4周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约300mg至600mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、或约4周。在一些实施方案中，每个治疗周期内施用抗CD73抗体的有效量为约700mg至1100mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周、约4周，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约60mg、约100mg、约110mg、约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg、约190mg、约200mg、约360mg、约420mg、约720mg、约1080mg，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。
在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约50mg至80mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约60mg，或含此剂量抗CD73抗体的制剂；比如约60mg给药1次。
在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约150mg至200mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约180mg，或含此剂量抗CD73抗体的制剂；比如约180mg给药1次。
在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约345mg 至380mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约360mg，或含此剂量抗CD73抗体的制剂；比如约360mg给药1次。
在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约693mg至730mg，或含此剂量抗CD73抗体的制剂；其中，一个治疗周期为约1周、约2周、约3周或约4周。在一些实施方案中，患者每个治疗周期内施用抗CD73抗体的有效量为约720mg，或含此剂量抗CD73抗体的制剂；比如约720mg给药1次。
在一些实施方案中，患者每个治疗周期内分别给药一次抗PD-1抗体和抗CD73抗体(或给药一次抗PD-1抗体和抗CD73抗体的组合物)。在一些实施方案中，每个治疗周期内多次分别给药抗PD-1抗体和抗CD73抗体(或抗PD-1抗体和抗CD73抗体的组合物)，例如2次、3次、4次或5次。在一些实施方案中，患者每个治疗周期只能给药1次或4次。
在一些实施方案中，患者接受一个治疗周期治疗。在一些实施方案中，患者接受多个(例如2个、3个或4个)治疗周期治疗。在一些实施方案中，患者接受治疗直至病症得到缓解而不再需要治疗。
在一些实施方案中，本发明公开了一种用于治疗肿瘤或癌症的方法，所述方法包括：向有需要的患者给予约50mg至200mg、约200mg至300mg、约300mg至400mg或约400mg至600mg，比如约100mg、约120mg、约200mg或约400mg的抗PD-1抗体，或含此剂量抗PD-1抗体的制剂；还向有需要的患者给予约15mg至180mg、约180mg至360mg、约360mg至500mg、约500mg至720mg或约730mg至1200mg，比如约60mg、约120mg、约180mg、约300mg或约400mg的抗CD73抗体，或含此剂量抗CD73抗体的制剂。在一些实施方案中，患者接受单剂量抗PD-1抗体的治疗，以及单剂量抗CD73抗体的治疗。在一些实施方案中，患者接受单剂量抗PD-1抗体和抗CD73抗体组合物的治疗。
在一些实施方案中，每3周一次给药抗PD-1抗体约200mg，每3周一次给药抗CD73抗体约3mg/kg。在一些实施方案中，每3周一次给药抗PD-1抗体约300mg，每3周一次给药抗CD73抗体约5mg/kg。在一些实施方案中，每3周一次给药抗PD-1抗体约200mg，每3周一次给药抗CD73抗体约6mg/kg。
在一些实施方案中，单剂量给药后，患者的症状得到缓解。在一些实施方案中，单剂量给药后，患者后的症状未得到预期缓解，再对患者分别给药约50mg至600mg抗PD-1抗体和约15mg至1200mg抗CD73抗体。在一些实施方案中，单剂量给药后，患者后的症状未得到预期缓解，再对患者给药约50mg至600mg抗PD-1抗体和约15 mg至1200mg抗CD73抗体的组合物。
在一些实施方案中，抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)是通过皮下(s.c.)注射、腹膜内(i.p.)注射、肠胃外注射、动脉内注射或静脉内(i.v.)注射等方式进行给药。在一些实施方案中，抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)是输液方式进行给药。在一些实施方案中，抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)是推注方式进行给药。
在一些实施方案中，抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)是通过静脉内(i.v.)输液方式进行给药。在一些实施方案中，静脉内输液持续时间为约50分钟、约55分钟、约60分钟、约65分钟、约70分钟、约75分钟、约81分钟、约87分钟、约90分钟、约95分钟，或这些数值中任何两个值之间的范围(包括端点)或其中任何值。
在一些实施方案中，抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)与其他治疗方法联合使用用于治疗肿瘤或癌症，例如化疗、放疗和手术治疗等。
另一方面，本发明公开了抗PD-1抗体和治疗剂在制备用于治疗肿瘤或癌症的药物中的应用。在一些实施方案中，所述治疗剂为抗CD73抗体。在一些实施方案中，抗CD73抗体为抗体P59-L17或P59-L17’。
另一方面，本发明还公开了一种试剂盒，试剂盒包含抗PD-1抗体(或制剂)、抗CD73抗体(或制剂)和用于指导有需要患者给药抗PD-1抗体(或制剂)和抗CD73抗体(或制剂)的说明书。在一些实施方案中，本发明还公开了一种试剂盒，试剂盒包含抗PD-1抗体(或制剂)和抗CD73抗体(或制剂)的组合物以及用于指导有需要患者给药抗PD-1抗体(或制剂)和抗CD73抗体(或制剂)的组合物的说明书。
另一方面，本发明还公开了包含抗PD-1抗体和抗CD73抗体的适合注射用的药物组合物，如推注型药物组合物或输液(滴注)型药物组合物。在一些实施方案中，药物组合物至少包含0.1％的抗PD-1抗体和0.1％的抗CD73抗体。抗体的百分比可以变化，并且为给定剂型重量的约2％约90％之间。这种治疗上有用的药物组合物中抗PD-1抗体和抗CD73抗体的量可以为给药的有效量。
另一方面，本发明还公开了上述药物组合物的制备方法：分别将本文所述的抗PD-1抗体和抗CD73抗体(或抗PD-1抗体和抗CD73抗体的组合物)与药学上可接受的适合注射用的辅料(例如注射用水，生理盐水等)混合。上述抗PD-1抗体和抗CD73抗体与药学上可接受的辅料的混合方法是本领域通常已知的。
本发明将抗PD-1抗体(或制剂)和抗CD73抗体(或制剂)用于肿瘤或癌症治疗中，减缓症状。
附图说明
图1示抗体对肿瘤的影响；其中，纵坐标表示肿瘤体积，横坐标表示给药后天数。
术语
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。
定义
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。
本文所用的术语“包含”或“包括”意味着组合物和方法等包括所列举的元素，例如组份或步骤，但不排除其它。“基本上由……组成”意味着组合物和方法排除对组合的特征有根本影响的其它元素，但不排除对组合物或方法无本质上影响的元素。“由……组成”意味着排除未特别列举的元素。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体形成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
“VL、VH的保守氨基酸取代”的氨基酸数目可为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。“重链或轻链的保守氨基酸取代”的氨基酸数目可为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约18个、约19个、约22个、约24个、约25个、约29个、约31个、约35个、约38个、约41个、约45个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
术语“编码”应用于多聚核苷酸时，是指被称为“编码”多肽的多聚核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
“至少80％同一性”为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性”或“序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对同一性百分比或序列同一性，比如Ausubel et al.eds.(2007) 在Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。抗体和抗原结合片段包括但不局限重链或轻链或其配体结合部分的互补决定区(CDR)、重链可变区(VH)、轻链可变区(VL)、重链恒定区(CH)、轻链恒定区(CL)、框架区(FR)或其任何部分，或结合蛋白的至少一部分。CDR区包括轻链的CDR区(LCDR1-3)和重链的CDR区(HCDR1-3)。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。
本发明公开的抗体、抗原结合片段或衍生物包括但不限于多克隆、单克隆、多特异性、全人源、人源化、灵长类化、嵌合抗体、单链抗体、表位结合片段(例如Fab、Fab'、F(ab') 2、单链Fv(scFv))。
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为V λ。
术语“恒定的”和“可变的”根据功能被使用。轻链可变区(VL)和重链可变区(VH)部分决定了抗原识别和特异性。轻链的恒定区(CL)和重链的恒定区(CH)赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可 变区，C端部分是恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基端。
在本领域中使用和/或接受的术语有两个或多个定义的情况下，除非明确地对立指出，否则本文使用的术语的定义包括所有这些含义。一个具体的例子是使用“互补决定区”(“CDR”)一词来描述在重链和轻链多肽的可变区内发现的非连续的抗原结合位点。这一特定区域在Kabat et al.,U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)和Chothia等在J.Mol.Biol.196:901-917(1987)有相关描述，其通过引用全部并入本文。
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在“Sequence of Proteinsof Immunological Interest”(1983)提出的编号系统。抗体还可以用EU或Chothia编号系统。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善和停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善或缓和，减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或组合物用于检测、诊断过程和/或治疗中受益的患者。
“患者”指需要诊断、预后或治疗的任何哺乳动物，包括人类、狗、猫、豚鼠、兔子、大鼠、小鼠、马、牛等。
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
“有效量”是指活性化合物或药剂的量，其能引起组织、系统、动物、个体或人类的生物学或医学反应；有效量由研究人员、兽医、医生或其他临床医生寻求的。
“EC 50”即半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
如本文所用，短语“有需要”是指已将患者鉴定为需要特定方法或治疗。在一些实施例中，可以通过任何诊断方式进行识别。在本文描述的任何方法和治疗中，患者可能需要。
可以按常规方法根据本文所述抗体氨基酸序列设计合成编码抗体的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生单克隆抗体。在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，或者Plncx，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。常使用的哺乳动物细胞包括293细胞，Cos1细胞，Cos7细胞，CV1细胞，鼠L细胞和CHO细胞等。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1 抗体的制备方法
1)根据抗体的重链和轻链氨基酸序列设计并合成轻链和重链的DNA序列。用PCR引物修饰DNA序列的5’和3’端，所述引物设计成为各链增加合适的前导序列，再克隆到现有重组抗体表达载体上，通过测序分析验证载体构建正确。将表达载体导入CHO细胞中进行表达，并通过纯化获得；抗体A的制备方法可参照PCT专利申请WO2020207432；抗体A相关序列见表1和表3，抗体P59-L17和P59-L17’相关序列见表2和表4。
表1抗体A的氨基酸序列
Figure PCTCN2022094181-appb-000001
Figure PCTCN2022094181-appb-000002
表2抗体P59-L17和P59-L17’的氨基酸序列
Figure PCTCN2022094181-appb-000003
Figure PCTCN2022094181-appb-000004
表3抗体A的核酸序列
Figure PCTCN2022094181-appb-000005
Figure PCTCN2022094181-appb-000006
表4抗体P59-L17和P59-L17’的核酸序列
Figure PCTCN2022094181-appb-000007
Figure PCTCN2022094181-appb-000008
实施例2 抗CD73抗体亲和力的测定
采用Biacore T200表面等离子体共振仪测定抗体的亲和力常数(K D)，主要试验过程如下(可参考Biacore T200的标准操作规程)：将hCD73-His(北京义翘神州科技股份有限公司，Catalog为10904-H08H)样品采用HBS-EP缓冲液(150mM NaCl，3mM EDTA，0.005％(v/v)表面活性剂P-20，和10mM HEPES，pH 7.4)梯度稀释(初始浓度为32nM，然后2倍稀释)；将抗体采用醋酸钠溶液(10mM，pH5.5)分别稀释至最终浓度约为20μg/ml；使用BiacoreT200，Protein A芯片(GE healthcare，货号为29127556)进行检测，仪器设置如下：抗体捕获结合时间(contact time)为180s，流速为30μl/min；样品结合时间(contact time)为120s，解离时间(dissociation time)为300s，流速为30μl/min；再生(regeneration)条件为：Glycine-HCl(pH值为2.5)，再生时间为60s，流速为30μl/min。采用数据分析软件Evaluation Software3.1对试验结果进行分析，将样品实验流路采集所得传感信号进行参比流路、样品空白双扣减，并选用动力学“1:1”模型进行拟合，得出动力学参数(K a为结合速率，K d为解离速率；K D为结合解离平衡常数)
如表5所示，抗体P59-L17与hCD73-His具有良好的结合能力。
表5抗CD73抗体与hCD73-His结合的亲和力常数
抗体 K a(1/Ms) K d(1/s) K D(M)
P59-L17 2.21E+05 1.26E-04 5.69E-10
实施例3 抗CD73抗体与不同种属CD73结合情况的检测
采用ELISA的方法进行检测：先用2μg/ml的人源hCD73-His(北京义翘神州科技股份有限公司，Catalog为10904-H08H)、食蟹猴cCD73-His(近岸生物，Catalog为CD3-C52H9)或鼠源mCD73-His(近岸生物，Catalog为CD3-M52H9)包被96孔ELISA板，4℃过夜；用含5％BSA(牛血清白蛋白)的PBS(磷酸盐缓冲液)缓冲液封闭2小时；封闭结束后，用含0.05％TW-20的PBS洗涤，之后加入用含0.05％TW-20和0.5％BSA的PBS稀释的抗体溶液(抗体的初始浓度为4μg/ml，3倍稀释，10个梯度)；37℃孵育1小时；洗板后，加入HRP(辣根过氧化物酶)标记的二抗anti-h-Kappa-HRP(购自Sigma，货号为A7164)孵育1h；洗板，加入TMB(四甲基联苯胺)显色液进行显色读取OD450。
如表6所示，抗体P59-L17与人、食蟹猴CD73进行良好地结合，与小鼠CD73不结合。
表6抗体P59-L17与CD73-His结合的EC 50(ng/ml)
抗体 hCD73-His cCD73-His mCD73-His
EC 50 33.9 42.7 NA
NA表示不结合或基本不结合。
实施例4 抗CD73抗体与表达CD73细胞的结合力的检测
MDA-MB-231细胞为人乳腺癌细胞，其细胞膜表面高表达CD73蛋白(https://www.proteinatlas.org/ENSG00000101017-CD40/cell)。本实施例采用MDA-MB-231细胞用于检测抗CD73抗体与细胞膜表面CD73分子的结合能力。
试验方法为：培养状态良好的MDA-MB-231细胞收集离心后，用同型对照IgG1-Fc或梯度稀释的抗CD73抗体进进行孵育；30min后离心洗去上清，用PBS缓冲液洗涤一次，PBS缓冲液重悬加入荧光标记的抗人IgG流式抗体anti-humanFc-PE(购自Invitrogen，货号为12-4998-82)；孵育30min后离心洗去上清，PBS缓冲液洗涤两次，PBS缓冲液重悬细胞后进行流式检测，统计MFI并用SoftMax Pro处理数据。
结果显示，抗体P59-L17结合MDA-MB-231细胞的EC 50为438.1ng/ml。
实施例5 抗CD73抗体抑制细胞膜上CD73的活性
Figure PCTCN2022094181-appb-000009
检测试剂盒(购自promega，货号为G7570)的检测原理为：过量 AMP能够抑制ATP依赖性的
Figure PCTCN2022094181-appb-000010
检测试剂发荧光，而细胞膜CD73能够分解AMP从而解除AMP的抑制作用，抗CD73抗体能够抑制CD73酶活性从而保持AMP对荧光发光过程的抑制，最终表现为：随着抗体浓度的提高，荧光强度相应地减弱。本实施例检测抗CD73抗体对细胞膜CD73酶活性的抑制情况。
试验方法为：用含10％FBS的DMEM培养基将生长状态良好的MDA-MB-231细胞以2.5×10 4cells/孔铺于96孔细胞培养平板，置于37℃细胞培养箱培养过夜；第二天去掉培养基上清，加入无血清DMEM梯度稀释的抗体溶液和终浓度为600μM的AMP，置于37℃细胞培养箱反应3小时；反应结束后取上清50μl每孔于96孔白板中，加入50μl浓度为200μM的ATP，最后加入100μl检测试剂，立即在酶标仪读取荧光值。
结果显示，抗体P59-L17浓度梯度依赖地抑制CD73的酶活性，其IC 50为126.8ng/ml。
实施例6 抗PD-1抗体与抗CD73抗体联合给药抑制癌细胞的增殖
本实施例用于评价抗体A和抗体P59-L17’在免疫检查点人源化小鼠(BALB/c-hPD1/hPDL1/hCD73，江苏集萃药康生物科技有限公司)皮下接种结肠癌细胞肿瘤模型中的药效。
1)肿瘤细胞接种
复苏小鼠结肠癌细胞CT26-hPDL1/hCD73(江苏集萃药康生物科技有限公司)，收集对数生长期的结肠癌细胞，去除培养液后采用PBS清洗两次后进行腋下皮下接种。
2)分组给药
当肿瘤平均体积在80-120mm 3，小鼠随机分成4组，每组8只；分组当天定义为D0天，并于分组当天根据给药方案开始给药，腹腔给药(i.p.)的时间为：D0、D3、D7、D10和D14。
3)实验观察和数据采集
细胞接种后，每周常规监测肿瘤对动物正常行为的影响。具体指标包括小鼠的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。肿瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
TGItv(相对肿瘤体积的抑制率)计算公式：
RTV n＝V nt/V n0；V nt：编号为n的小鼠在第t天的肿瘤体积，V n0：编号为n的小鼠在第0天的肿瘤体积，RTV n：编号为n的小鼠在第t天的肿瘤相对体积
TGItv＝(1-(mean RTV给药组)/(mean RTV对照组))×100％；mean RTV给药组：给药 组RTV平均值，mean RTV对照组：对照组RTV平均值。
表7给药方案
Figure PCTCN2022094181-appb-000011
结果显示，1)各组之间的小鼠体重未发现明显差异，表明小鼠对当前体系中的药物耐受性良好；2)如图1所示，抗体A和抗体P59-L17’联合给药组有显著的抗肿瘤药效，且抗肿瘤效果优于其他组，联合给药组中TGItv为88.4％。

Claims (10)

  1. 一种用于治疗肿瘤或癌症的方法，所述方法包括：向有需要的患者给药有效量的抗PD-1抗体和抗CD73抗体；
    所述抗PD-1抗体包含SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3、SEQ ID NO：4所示的LCDR1、SEQ ID NO：5所示的LCDR2和SEQ ID NO：6所示的LCDR3。
  2. 如权利要求1所述的方法，所述抗PD-1抗体的重链包含SEQ ID NO：9所示的序列，与SEQ ID NO：9所示序列具有至少80％同一性的序列，或与SEQ ID NO：9所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗PD-1抗体的轻链包含SEQ ID NO：10所示的序列，与SEQ ID NO：10所示序列具有至少80％同一性的序列，或与SEQ ID NO：10所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  3. 如权利要求1或2所述的方法，所述抗CD73抗体至少包含SEQ ID NO：11所示的HCDR1、SEQ ID NO：12所示的HCDR2、SEQ ID NO：13所示的HCDR3、SEQ ID NO：14所示的LCDR1、SEQ ID NO：15所示的LCDR2和SEQ ID NO：16所示的LCDR3。
  4. 如权利要求1或2所述的方法，所述抗CD73抗体的重链可变区包含SEQ ID NO：17所示的序列，与SEQ ID NO：17所示序列具有至少80％同一性的序列，或与SEQ ID NO：17所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗CD73抗体的轻链可变区包含SEQ ID NO：18所示的序列，与SEQ ID NO：18所示序列具有至少80％同一性的序列，或与SEQ ID NO：18所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  5. 如权利要求1或2所述的方法，所述抗CD73抗体的重链包含SEQ ID NO：19或20所示的序列，与SEQ ID NO：19或20所示序列具有至少80％同一性的序列，或与SEQ ID NO：19或20所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗CD73抗体的轻链包含SEQ ID NO：21所示的序列，与SEQ ID NO：21所示序列具有至少80％同一性的序列，或与SEQ ID NO：21所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  6. 如权利要求1-5任一项所述的方法，每个治疗周期内抗PD-1抗体给药的有效量为50mg至600mg。
  7. 如权利要求1-5任一项所述的方法，抗PD-1抗体的每次给药量为1-10mg/kg。
  8. 如权利要求1-7任一项所述的方法，每个治疗周期内抗CD73抗体给药的有效量为15mg至1200mg。
  9. 如权利要求1-7任一项所述的方法抗CD73抗体的每次给药量为0.3-18mg/kg。
  10. 一种试剂盒，其包含抗PD-1抗体、抗CD73抗体和用于指导有需要患者给药PD-1抗体和抗CD73抗体的说明书；
    所述抗PD-1抗体包含SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3、SEQ ID NO：4所示的LCDR1、SEQ ID NO：5所示的LCDR2和SEQ ID NO：6所示的LCDR3。
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