CN117337300A - Use of anti-PD-1 antibodies - Google Patents

Use of anti-PD-1 antibodies Download PDF

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CN117337300A
CN117337300A CN202280035707.9A CN202280035707A CN117337300A CN 117337300 A CN117337300 A CN 117337300A CN 202280035707 A CN202280035707 A CN 202280035707A CN 117337300 A CN117337300 A CN 117337300A
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antibody
seq
sequence
amino acid
weeks
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黄贤明
岳睿
张慧
陈振埕
岳海涛
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Bio Thera Solutions Ltd
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Bio Thera Solutions Ltd
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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

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

anti-PD-1 antibodies and their use in the preparation of a medicament for the treatment of a tumor or cancer are provided.

Description

Use of anti-PD-1 antibodies Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of an anti-PD-1 antibody in combined medication.
Background
The incidence of tumors increases year by year, and the use of antitumor drugs is also increasing. In recent years, significant progress has been made in tumor therapy research, and in particular, some antibody drugs have shown good therapeutic effects in the treatment of malignant tumors. However, only a small fraction of patients can reach long-term survival. In order to further improve the clinical curative effect of the anti-tumor treatment of the medicine, the combined medicine can be adopted when the anti-tumor medicine is used.
Programmed death receptor-1 (PD-1) is an immunosuppressive receptor expressed on activated T cells, B cells and myeloid cells, a member of the CD28 immunoglobulin superfamily. PD-1 is a 55kDa type I transmembrane glycoprotein containing an Ig variable domain that binds to a ligand and a cytoplasmic tail responsible for binding a signal transduction molecule. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs: ITIM (immunoreceptor tyrosine inhibitory motif) and ITSM (immunoreceptor tyrosine switching motif).
Numerous studies to date have shown that the interaction between PD-1 and PD-L1 (programmed death ligand 1) results in reduced lymphocytes penetrating the tumor, reduced T cell receptor mediated proliferation and immune evasion of cancer cells. Blocking the interaction between PD-1 and PD-L1 increases T cell proliferation and cytokine production, and increases tumor-specific CD8 + T cell immunity.
Thus, anti-PD-1 antibodies play an important role in the combination of anti-tumor agents.
Disclosure of Invention
The present invention discloses methods or uses of anti-PD-1 antibodies for the combined treatment of tumors or cancers. In some embodiments, the anti-PD-1 antibody and the therapeutic agent are used in combination to treat a tumor or cancer. In some embodiments, the therapeutic agent is an anti-CD 73 antibody. In some embodiments, the method or use comprises: administering to a patient in need thereof an effective amount of an anti-PD-1 antibody and an anti-CD 73 antibody. In some embodiments, the use is the use of an anti-PD-1 antibody in the manufacture of a medicament for use in combination with an anti-CD 73 antibody in the treatment of a tumor or cancer. In some embodiments, the use is the use of an anti-CD 73 antibody in the manufacture of a medicament for use in combination with an anti-PD-1 antibody in the treatment of a tumor or cancer.
In some embodiments, the anti-PD-1 antibody comprises at least one or more of HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2, HCDR3 shown in SEQ ID NO. 3, LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5, LCDR3 shown in SEQ ID NO. 6.
In some embodiments, the anti-PD-1 antibody comprises one or two CDRs selected from the amino acid sequences set forth below: SEQ ID NOs 3 and 6; and optionally comprises one, two, three or four CDRs selected from the amino acid sequences shown below: SEQ ID NOs 1, 2, 4 and 5.
In some embodiments, 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, LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5, and LCDR3 shown in SEQ ID NO. 6.
In some embodiments, the heavy chain variable region of the anti-PD-1 antibody comprises a sequence set forth in SEQ ID No. 7, a sequence having at least 80% identity to the sequence set forth in SEQ ID No. 7, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 7; and/or
The light chain variable region of the anti-PD-1 antibody comprises a sequence as set forth in SEQ ID NO. 8, a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO. 8, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence as set forth in SEQ ID NO. 8.
In some embodiments, the heavy chain variable region of the anti-PD-1 antibody comprises the sequence set forth in SEQ ID NO. 7 and the light chain variable region of the anti-PD-1 antibody comprises the sequence set forth in SEQ ID NO. 8.
In some embodiments, the heavy chain of the anti-PD-1 antibody comprises the sequence set forth in SEQ ID No. 9, a sequence having at least 80% identity to the sequence set forth in SEQ ID No. 9, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 9; and/or
The light chain of the anti-PD-1 antibody comprises a sequence as set forth in SEQ ID NO. 10, a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO. 10, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence as set forth in SEQ ID NO. 10.
In some embodiments, the anti-PD-1 antibody is antibody A, the heavy chain of antibody A comprises the sequence set forth in SEQ ID NO. 9, and the light chain of antibody A comprises the sequence set forth in SEQ ID NO. 10; antibody a contains two heavy chains of identical sequence and two light chains of identical sequence.
In some embodiments, the anti-CD 73 antibody comprises one or more of HCDR1 as set forth in SEQ ID NO. 11, HCDR2 as set forth in SEQ ID NO. 12, HCDR3 as set forth in SEQ ID NO. 13, LCDR1 as set forth in SEQ ID NO. 14, LCDR2 as set forth in SEQ ID NO. 15, and LCDR3 as set forth in SEQ ID NO. 16.
In some embodiments, the anti-CD 73 antibody comprises HCDR1 as set forth in SEQ ID NO. 11, HCDR2 as set forth in SEQ ID NO. 12, HCDR3 as set forth in SEQ ID NO. 13, LCDR1 as set forth in SEQ ID NO. 14, LCDR2 as set forth in SEQ ID NO. 15, and LCDR3 as set forth in SEQ ID NO. 16.
In some embodiments, the heavy chain variable region of the anti-CD 73 antibody comprises the sequence set forth in SEQ ID No. 17, a sequence having at least 80% identity to the sequence set forth in SEQ ID No. 17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID No. 17; and/or
The light chain variable region of the anti-CD 73 antibody comprises the sequence shown as SEQ ID NO. 18, a sequence having at least 80% identity to the sequence shown as SEQ ID NO. 18, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 18.
In some embodiments, the heavy chain variable region of the anti-CD 73 antibody comprises the sequence shown in SEQ ID NO. 17 and the light chain variable region of the anti-CD 73 antibody comprises the sequence shown in SEQ ID NO. 18.
In some embodiments, the heavy chain of the anti-CD 73 antibody comprises a 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 an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO. 19 or 20; and/or
The light chain of the anti-CD 73 antibody comprises a sequence as set forth in SEQ ID NO. 21, a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO. 21, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence as set forth in SEQ ID NO. 21.
In some embodiments, the anti-CD 73 antibody is antibody P59-L17, the heavy chain of antibody P59-L17 comprises the sequence shown in SEQ ID NO. 19, and the light chain of antibody P59-L17 comprises the sequence shown in SEQ ID NO. 21; antibodies P59-L17 contain two heavy chains of identical sequence and two light chains of identical sequence.
In some embodiments, the anti-CD 73 antibody is antibody P59-L17', the heavy chain of antibody P59-L17' comprises the sequence shown in SEQ ID NO. 20, and the light chain of antibody P59-L17' comprises the sequence shown in SEQ ID NO. 21; antibodies P59-L17' contain two heavy chains of identical sequence and two light chains of identical sequence.
Antibody proteins can be expressed in cells (e.g., CHO cells or HEK cells) by genetic engineering and obtained by purification; purification can be carried out by conventional methods, for example, centrifugation of the cell suspension followed by collection of the supernatant, and centrifugation again to further remove impurities. The Protein A affinity column and ion exchange column can be used to purify antibody proteins.
In some embodiments, the method or use comprises: administering to a patient in need thereof an effective amount of an anti-PD-1 antibody and an anti-CD 73 antibody. In some embodiments, the anti-PD-1 antibody is antibody a. In some embodiments, the anti-CD 73 antibody is antibody P59-L17 or P59-L17'. In some embodiments, the effective dose for administration of an anti-PD-1 antibody is about 50mg to 600mg per dose. In some embodiments, an effective dose of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is administered at about 15mg to 1200mg per dose.
In some embodiments, the anti-PD-1 antibody and the anti-CD 73 antibody are each independently administered units, in combination. In some embodiments, the anti-PD-1 antibody may be administered prior to administration of the anti-CD 73 antibody, may be administered after administration of the anti-CD 73 antibody, or may be administered concurrently with administration of the anti-CD 73 antibody. In some embodiments, the anti-PD-1 antibody and the anti-CD 73 antibody form a combination dosing unit, in combination.
In some embodiments, the patient has a tumor or cancer. In some embodiments, tumors and cancers include, but are not limited to: head and neck tumors, hodgkin's lymphoma, non-hodgkin's lymphoma, precursor lymphoblastoma, small non-split cell lymphoma, burkitt's lymphoma, non-burkitt's lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, kidney tumor, wilms 'tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroblastoma, ganglion neuroblastoma, extracranial germ cell tumor, mature teratoma, immature teratoma, inner embryo tumor, yolk sac tumor, sperm cell tumor, asexual cell tumor, chorionic epithelial carcinoma, embryo carcinoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, soft tissue sarcoma, fibrosarcoma, malignant fibrous histiocytoma liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, acinar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, pro-fibrotic small round cell tumor, ewing's sarcoma, primitive neuroectoblastoma, hepatoma, hepatoblastoma, retinoblastoma, posterior fossa medulloblastoma, thymoma, pneumoblastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoma, mesothelioma, melanoma, interstitial cell tumor, myeloma, cerebral astrocytoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, intestinal carcinoma, breast carcinoma, stomach carcinoma, liver carcinoma, prostate carcinoma, breast carcinoma, lung carcinoma, cervical carcinoma, ovarian carcinoma, renal carcinoma, lymphoma, leukemia, skin carcinoma, esophageal squamous carcinoma, and the like.
In some embodiments, the 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-CD 73 antibody, wherein the effective amount of anti-PD-1 administration is about 50mg to 600mg per treatment cycle. In some embodiments, one 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 (inclusive) or any value therein. In some embodiments, the anti-PD-1 antibody is antibody a.
In some embodiments, the anti-CD 73 antibody is a fully human IgG 1-type antibody P59-L17, a fully human IgG 4-type antibody P59-L17', or a biological analog thereof, or an ADCC effect enhancing mab or defucosylamab. In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is administered between about 15mg and 1200mg per treatment cycle. In some embodiments, one 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 therein. In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is administered from about 0.3mg/kg to 18mg/kg 1 time every 2 weeks or every 3 weeks. In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is administered at about 0.3mg/kg, about 1mg/kg, about 3mg/kg, about 5mg/kg, about 6mg/kg, about 12mg/kg, about 13mg/kg, or about 18mg/kg 1 time every 2 weeks or every 3 weeks. In some embodiments, an effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is administered about 5mg/kg 1 time every 2 weeks, about 6mg/kg 1 time every 2 weeks, about 10mg/kg 1 time every 2 weeks, about 3mg/kg 1 time every 3 weeks, about 5mg/kg 1 time every 3 weeks, about 6mg/kg 1 time every 3 weeks, about 7.5mg/kg 1 time every 3 weeks, about 12mg/kg 1 time every 3 weeks, about 15mg/kg 1 time every 3 weeks, or about 18mg/kg 1 time every 3 weeks.
In some embodiments, the anti-PD-1 antibody and the anti-CD 73 antibody (or a combination of the anti-PD-1 antibody and the anti-CD 73 antibody) may be formulated separately into a pharmaceutical composition and administered to a patient in a variety of forms suitable for the chosen route of administration, for example by parenteral, intravenous (iv), intramuscular, topical or subcutaneous routes. In some embodiments, the anti-PD-1 antibody and the anti-CD 73 antibody (or a combination of the anti-PD-1 antibody and the anti-CD 73 antibody) may be infused intravenously, respectively. The amount of anti-PD-1 antibody and anti-CD 73 antibody will depend on the nature of the drug, the extent of internalization, transport and release of the cell surface triggering drug, the disease being treated, the condition of the patient (e.g., age, sex, weight, etc.).
In some embodiments, the anti-PD-1 antibody administered at a time is about 1mg/kg to 10mg/kg or a formulation containing such a dose of anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody administered at a time is about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, or a range between any two of these values (inclusive) or any value therein, or a formulation comprising such a dose of anti-PD-1 antibody.
In some embodiments, a therapeutically effective amount of an anti-CD 73 antibody and an anti-PD-1 antibody are employed for separate or simultaneous administration to a subject patient. The administration cycles of the anti-CD 73 antibody and the anti-PD-1 antibody may be the same or different.
In some embodiments, each administration of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is about 0.3mg/kg to 18mg/kg or a formulation containing such a dose of anti-CD 73 antibody. In some embodiments, each administration of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is about 0.3mg/kg, about 1mg/kg, about 1.2mg/kg, about 2mg/kg, about 2.4mg/kg, about 3mg/kg, about 3.6mg/kg, about 4.8mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 6.9mg/kg, about 7mg/kg, about 8.4mg/kg, about 9mg/kg, about 11mg/kg, about 12mg/kg, about 15mg/kg, about 18mg/kg, or a range between any two of these values (inclusive), or a formulation comprising such a dose of an anti-CD 73 antibody.
In some embodiments, the invention discloses a method of treating a tumor or cancer comprising administering to a patient in need thereof an effective amount of an anti-PD-1 antibody (or formulation) and an anti-CD 73 antibody (or formulation); wherein the effective amount of the anti-PD-1 antibody is about 50mg to 600mg (or a formulation containing such a dose of the anti-PD-1 antibody) per single administration. In some embodiments, the anti-CD 73 antibody is antibody P59-L17 or P59-L17'. In some embodiments, the effective amount of an anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') is about 15mg to 1200mg (or a formulation containing such a dose of an anti-CD 73 antibody) per single administration. The dosage schedule and mode of administration will depend on the risk assessment of benefit of the anti-PD-1 antibody (or formulation), the anti-CD 73 antibody (or formulation), and general clinical practice guidelines in certain patient populations.
In some embodiments, the effective amount of anti-PD-1 antibody administered per treatment cycle to a patient is about 50mg to 600mg of anti-PD-1 antibody (or a formulation containing such a dose of anti-PD-1 antibody), and the effective amount of anti-CD 73 antibody (e.g., antibody P59-L17 or P59-L17') administered per treatment cycle to a patient is about 15mg to 1200mg (or a formulation containing such a dose of anti-CD 73 antibody).
In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 50mg, about 60mg, about 80mg, about 120mg, about 200mg, about 250mg, about 290mg, about 300mg, about 330mg, about 380mg, about 400mg, about 434mg, about 480mg, about 500mg, about 567mg, about 580mg, about 600mg, or a range between any two of these values (inclusive), or any value therein, or a formulation containing such dose of anti-PD-1 antibody. In some embodiments, one treatment cycle is 1 to 7 weeks of administration. In some embodiments, the effective amount of anti-PD-1 antibody administered per treatment cycle is about 100mg to 200mg, or a formulation containing such dose of anti-PD-1 antibody; wherein one treatment cycle is about 1 week, about 2 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. In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 200mg to 300mg, or a formulation containing such a dose of anti-PD-1 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-PD-1 antibody administered per treatment cycle to the patient is about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, or a range between any two of these values (inclusive), or any value therein, or a formulation containing such a dose; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 90mg to 110mg, or a formulation containing such a dose of anti-PD-1 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 100mg, or a formulation containing such dose of anti-PD-1 antibody; such as about 100mg administered 1 time.
In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 100mg to 140mg, or a formulation containing such a dose of anti-PD-1 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 120mg, or a formulation containing such a dose of anti-PD-1 antibody; such as about 120mg administered 1 time.
In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 150mg to 190mg, or a formulation containing such a dose of anti-PD-1 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 160mg, or a formulation containing such dose of anti-PD-1 antibody; such as about 160mg, for 1 administration.
In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 190mg to 230mg, or a formulation containing such a dose of anti-PD-1 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-PD-1 antibody administered to the patient per treatment cycle is about 200mg, or a formulation containing such dose of anti-PD-1 antibody; such as about 200mg administered 1 time.
In some embodiments, the effective amount of the anti-PD-1 antibody is about 100mg to 600mg once every 3 weeks. In some embodiments, the effective amount of the anti-PD-1 antibody is about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, or about 600mg once every 3 weeks. In some embodiments, the effective amount of the anti-PD-1 antibody is about 100mg, about 300mg, or about 600mg once every 3 weeks.
In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 15mg, about 18mg, about 90mg, about 120mg, about 160mg, about 180mg, about 200mg, about 230mg, about 250mg, about 280mg, 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 formulation containing such dose of anti-CD 73 antibody. In some embodiments, one treatment cycle is 1 to 7 weeks of administration. In some embodiments, the effective amount of anti-CD 73 antibody administered per treatment cycle is about 100mg to 300mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or a range between any two of these values (inclusive) or any value therein. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 300mg to 600mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered per treatment cycle is about 700mg to 1100mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein a treatment cycle is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or a range between any two of these values (inclusive) or any value therein. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 60mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 360mg, about 420mg, about 720mg, about 1080mg, or a range between any two of these values (inclusive), or any value therein, or a formulation comprising such a dose of anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.
In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 50mg to 80mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 60mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 60mg administered 1 time.
In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 150mg to 200mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 180mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 180mg administered 1 time.
In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 345mg to 380mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 360mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 360mg administered 1 time.
In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 693mg to 730mg, or a formulation containing such a dose of anti-CD 73 antibody; wherein one treatment cycle is about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the effective amount of anti-CD 73 antibody administered to the patient per treatment cycle is about 720mg, or a formulation containing such a dose of anti-CD 73 antibody; such as about 720mg administered 1 time.
In some embodiments, the anti-PD-1 antibody and the anti-CD 73 antibody (or a combination of the anti-PD-1 antibody and the anti-CD 73 antibody) are administered once per treatment cycle to the patient. In some embodiments, the anti-PD-1 antibody and the anti-CD 73 antibody (or a combination of the anti-PD-1 antibody and the anti-CD 73 antibody) are administered separately, e.g., 2, 3, 4, or 5 times, multiple times per treatment cycle. In some embodiments, the patient can only be administered 1 or 4 times per treatment cycle.
In some embodiments, the patient receives one treatment cycle. In some embodiments, the patient receives multiple (e.g., 2, 3, or 4) treatment cycles. In some embodiments, the patient receives treatment until the condition is alleviated without further treatment.
In some embodiments, the invention discloses a method for treating a tumor or cancer, the method comprising: administering about 50mg to 200mg, about 200mg to 300mg, about 300mg to 400mg, or about 400mg to 600mg, such as about 100mg, about 120mg, about 200mg, or about 400mg, of an anti-PD-1 antibody, or a formulation comprising such a dose of an anti-PD-1 antibody, to a patient in need thereof; about 15mg to 180mg, about 180mg to 360mg, 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 an anti-CD 73 antibody, or a formulation containing such a dose of an anti-CD 73 antibody, is also administered to a patient in need thereof. In some embodiments, the patient is treated with a single dose of an anti-PD-1 antibody, and a single dose of an anti-CD 73 antibody. In some embodiments, the patient is treated with a single dose of the anti-PD-1 antibody and anti-CD 73 antibody composition.
In some embodiments, about 200mg of anti-PD-1 antibody is administered once every 3 weeks and about 3mg/kg of anti-CD 73 antibody is administered once every 3 weeks. In some embodiments, about 300mg of anti-PD-1 antibody is administered once every 3 weeks and about 5mg/kg of anti-CD 73 antibody is administered once every 3 weeks. In some embodiments, about 200mg of anti-PD-1 antibody is administered once every 3 weeks and about 6mg/kg of anti-CD 73 antibody is administered once every 3 weeks.
In some embodiments, the symptoms of the patient are alleviated after a single dose administration. In some embodiments, the symptoms after a single dose of administration are not expected to be alleviated after the patient is further administered about 50mg to 600mg of the anti-PD-1 antibody and about 15mg to 1200mg of the anti-CD 73 antibody, respectively. In some embodiments, symptoms after a single dose of administration are not expected to be alleviated after the patient is administered a composition of about 50mg to 600mg of the anti-PD-1 antibody and about 15mg to 1200mg of the anti-CD 73 antibody.
In some embodiments, the anti-PD-1 antibody (or formulation), anti-CD 73 antibody (or formulation) is administered by subcutaneous (s.c.) injection, intraperitoneal (i.p.) injection, parenteral injection, intraarterial injection, or intravenous (i.v.) injection, among others. In some embodiments, the anti-PD-1 antibody (or formulation), anti-CD 73 antibody (or formulation) is administered by infusion. In some embodiments, the anti-PD-1 antibody (or formulation), anti-CD 73 antibody (or formulation) is administered as a bolus.
In some embodiments, the anti-PD-1 antibody (or formulation), anti-CD 73 antibody (or formulation) is administered by intravenous (i.v.) infusion. In some embodiments, the intravenous infusion duration 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, or a range between any two of these values (inclusive) or any value therein.
In some embodiments, the anti-PD-1 antibody (or formulation), anti-CD 73 antibody (or formulation) is used in combination with other therapeutic methods for treating tumors or cancers, such as chemotherapy, radiation therapy, and surgical therapies, among others.
In another aspect, the invention discloses the use of an anti-PD-1 antibody and a therapeutic agent in the manufacture of a medicament for the treatment of a tumor or cancer. In some embodiments, the therapeutic agent is an anti-CD 73 antibody. In some embodiments, the anti-CD 73 antibody is antibody P59-L17 or P59-L17'.
In another aspect, the invention also discloses a kit comprising an anti-PD-1 antibody (or formulation), an anti-CD 73 antibody (or formulation), and instructions for administering the anti-PD-1 antibody (or formulation) and the anti-CD 73 antibody (or formulation) to a patient in need thereof. In some embodiments, the invention also discloses a kit comprising a composition of an anti-PD-1 antibody (or formulation) and an anti-CD 73 antibody (or formulation) and instructions for directing administration of the composition of the anti-PD-1 antibody (or formulation) and the anti-CD 73 antibody (or formulation) to a patient in need thereof.
In another aspect, the invention also discloses pharmaceutical compositions suitable for injection, such as bolus or infusion (instillation), comprising an anti-PD-1 antibody and an anti-CD 73 antibody. In some embodiments, the pharmaceutical composition comprises at least 0.1% anti-PD-1 antibody and 0.1% anti-CD 73 antibody. The percentage of antibody may vary and is between about 2% and about 90% of the weight of a given dosage form. The amount of anti-PD-1 antibody and anti-CD 73 antibody in such a therapeutically useful pharmaceutical composition may be an effective amount for administration.
On the other hand, the invention also discloses a preparation method of the pharmaceutical composition, which comprises the following steps: the anti-PD-1 antibodies and anti-CD 73 antibodies (or the anti-PD-1 antibody and anti-CD 73 antibody compositions) described herein are separately admixed with pharmaceutically acceptable excipients suitable for injection (e.g., water for injection, physiological saline, etc.). Methods of mixing the above-described anti-PD-1 antibodies and anti-CD 73 antibodies with pharmaceutically acceptable excipients are generally known in the art.
The invention uses anti-PD-1 antibodies (or formulations) and anti-CD 73 antibodies (or formulations) in tumor or cancer treatment to alleviate symptoms.
Drawings
FIG. 1 shows the effect of antibodies on tumors; wherein the ordinate indicates the tumor volume and the abscissa indicates the number of days after administration.
Terminology
Unless otherwise indicated, each term below shall have the meaning described below.
Definition of the definition
It should be noted that the term "an" entity refers to one or more of the entity, e.g. "an antibody" should be understood as one or more antibodies, and thus the terms "one" (or "one"), "one or more" and "at least one" can be used interchangeably herein.
The terms "comprising" or "including" as used herein mean that the compositions and methods, etc., include the recited elements, e.g., components or steps, but do not exclude the others. By "consisting essentially of … …" it is meant that the compositions and methods exclude other elements that have a fundamental impact on the characteristics of the combination, but do not exclude elements that have no essential impact on the compositions or methods. "consisting of … …" means that elements not specifically recited are excluded.
The term "polypeptide" is intended to encompass both the singular and the plural of "polypeptides" and refers to molecules formed from amino acid monomers that are linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to a particular length of product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or in addition to, any of the terms described above. The term "polypeptide" is also intended to refer to products of modification of the polypeptide after expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from the specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.
"amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid directly or in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, a so-called codon or base triplet. Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons 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 (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).
"conservative amino acid substitution" refers to the substitution of one amino acid residue with another amino acid residue that contains a side chain (R group) that is similar in chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. Examples of classes of amino acids containing chemically similar side chains include: 1) Aliphatic side chain: 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 chain: phenylalanine, tyrosine, and tryptophan; 5) Basic side chain: lysine, arginine, and histidine; 6) Acidic side chain: aspartic acid and glutamic acid.
The number of amino acids of a "conservative amino acid substitution of VL, VH" may 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 acid substitutions of "conservative amino acid substitutions of a heavy or light chain" may 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.
The term "encoding" when applied to a polynucleotide refers to a polynucleotide referred to as "encoding" a polypeptide, which polypeptide and/or fragment thereof may be produced by transcription and/or translation in its natural state or when manipulated by methods well known to those skilled in the art.
The term "recombinant" refers to a polypeptide or polynucleotide, meaning a form of the polypeptide or polynucleotide that does not exist in nature, and non-limiting examples can be combined to produce a polynucleotide or polypeptide that does not normally exist.
"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences 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 matched or homologous positions shared by the sequences.
"at least 80% identical" is about 80% identical, about 81% identical, about 82% identical, about 83% identical, about 85% identical, about 86% identical, about 87% identical, about 88% identical, about 90% identical, about 91% identical, about 92% identical, about 94% identical, about 95% identical, about 98% identical, about 99% identical, or a range between any two of these values (inclusive) or any value therein.
A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity" or "sequence identity" with another sequence, meaning that when sequences are aligned, the percentage of bases (or amino acids) in the two sequences that are compared are identical. The percentage of alignment identity or sequence identity may be determined using visual inspection or software programs known in the art, such as the software program described in Ausubel et al eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: genetics code = standard; filter = none; strand = both; cutoff = 60; expect=10; matrix = BLOSUM62; descriptive = 50sequences; sortby=highscore; databases = non-redundants; genbank+embl+ddbj+pdb+genbank cdstransplation+swissprotein+spldate+pir. Biologically equivalent polynucleotides are those that have the indicated percent identity and encode polypeptides having the same or similar biological activity.
An "antibody" or "antigen-binding fragment" refers to a polypeptide or complex of polypeptides that specifically recognizes and binds an antigen. The antibody may be an intact antibody, any antigen-binding fragment thereof, or a single chain thereof. The term "antibody" thus includes any protein or peptide comprising at least a portion of an immunoglobulin molecule having biological activity for binding to an antigen in a molecule. 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 (CH), light chain constant regions (CL), framework Regions (FR) or any portion thereof, or at least a portion of a binding protein, of a heavy chain or light chain or ligand binding portion thereof. The CDR regions include the CDR regions of the light chain (LCDR 1-3) and the heavy chain (HCDR 1-3).
The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the heavy chain classes include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), some of which are also subclasses (e.g., γ1- γ4). The nature of this chain determines the "class" of antibody as IgG, igM, igA, igG or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igG5, etc., have been well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class. The four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain starts at the "Y" mouth and continues through the variable region surrounding the heavy chain.
Antibodies, antigen binding fragments or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope-binding fragments (e.g., fab ', F (ab') 2 Single chain Fv (scFv)).
Light chains can be classified as kappa (kappa) or lambda (lambda). Each heavy chain may be associated with a kappa or lambda light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains thereof are bound by covalent bonds, and the "tail" portions of the two heavy chains are bound by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y-configuration to the C-terminus of the bottom of each chain. Immunoglobulin kappa light chain variable region is vkappaThe method comprises the steps of carrying out a first treatment on the surface of the Immunoglobulin lambda light chain variable region V λ 。
The terms "constant" and "variable" are used in terms of function. The light chain variable region (VL) and heavy chain variable region (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. Conventionally, the numbering of constant regions increases as they become farther from the antigen binding site or amino terminus of an antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.
Where there are two or more definitions of terms used and/or accepted in the art, the definitions of terms used herein include all such meanings unless explicitly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al, U.S. Dept. Of Health and Human Services, sequences of Proteins of Immunological Interest (1983) and Chothia et al, J.mol. Biol.196:901-917 (1987), which are incorporated herein by reference in their entirety.
CDRs defined according to 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 apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue number comprising a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can generally determine which specific residues a CDR comprises based on the variable region amino acid sequence of an antibody.
Kabat et al also define a numbering system for variable region sequences suitable for use with any antibody. The "Kabat numbering" system can be applied to any variable region sequence by one of ordinary skill in the art independent of other experimental data than the sequence itself. "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. Of Health and Human Services at "Sequence of Proteinsof Immunological Interest" (1983). Antibodies may also use EU or Chothia numbering systems.
"treatment" refers to therapeutic treatment and prophylactic or preventative measures, with the object of preventing, slowing, ameliorating, and halting an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to, a decrease in symptoms, a decrease in the degree of a disease, stabilization (i.e., not worsening) of a disease state, a delay or slowing of disease progression, an amelioration or palliation of a disease state, a decrease or disappearance (whether partial or total), an extension and an expected lifetime without treatment, and the like, whether detectable or undetectable. Patients in need of treatment include those already with the condition or disorder, those prone to the condition or disorder, or those in need of prophylaxis of the condition or disorder, for whom the administration of the disclosed antibodies or compositions for detection, diagnostic procedures, and/or treatment would be expected to benefit.
"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.
"about" refers to a conventional error range of corresponding numerical values as readily known to one of ordinary skill in the relevant art. In some embodiments, references herein to "about" refer to the values described and ranges thereof of ± 10%, ± 5% or ± 1%.
An "effective amount" refers to an amount of an active compound or agent that is capable of eliciting a biological or medical response in a tissue, system, animal, individual or human; an effective amount is sought by a researcher, veterinarian, medical doctor or other clinician.
“EC 50 "half maximal effect concentration (concentration for 50%of maximal effect,EC50) refers to the concentration that causes 50% of the maximal effect.
As used herein, the phrase "in need of" means that the patient has been identified as in need of a particular method or treatment. In some embodiments, the identification may be performed by any diagnostic means. In any of the methods and treatments described herein, the patient may need.
The DNA encoding the antibody may be synthesized according to the amino acid sequence design of the antibody described herein by conventional methods, placed into an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a medium to produce monoclonal antibodies. In some embodiments, the expression antibody vector comprises 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 insertion. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeats from retroviruses such as the early promoters of RSV, HTLV1, HIVI and cytomegalovirus, and other cellular promoters such as actin promoters may be used. 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, among others. Commonly used mammalian cells include 293 cells, cos1 cells, cos7 cells, CV1 cells, murine L cells, CHO cells and the like.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of antibodies
1) The DNA sequences of the light and heavy chains were designed and synthesized from the heavy and light chain amino acid sequences of the antibodies. And (3) modifying the 5 'and 3' ends of the DNA sequence by using PCR primers, wherein the primers are designed to add proper leader sequences for each chain, then cloning the primer sequences onto the existing recombinant antibody expression vector, and verifying that the vector is constructed correctly by sequencing analysis. Introducing the expression vector into CHO cells for expression, and purifying to obtain the recombinant strain; the preparation of antibody A can be found in PCT patent application WO2020207432; the antibody A-related sequences are shown in tables 1 and 3, and the antibodies P59-L17 and P59-L17' related sequences are shown in tables 2 and 4.
TABLE 1 amino acid sequence of antibody A
TABLE 2 amino acid sequences of antibodies P59-L17 and P59-L17
TABLE 3 nucleic acid sequences of antibody A
TABLE 4 nucleic acid sequences of antibodies P59-L17 and P59-L17
Example 2 determination of anti-CD 73 antibody affinity
Determination of affinity constant (K) of antibodies using Biacore T200 surface plasmon resonance D ) The main test procedure is as follows (reference may be made to the standard protocol for Biacore T200): hCD73-His (catalyst 10904-H08H, available from the scientific company limited, beggar, seemed) samples were diluted gradient (initial concentration 32nM, then 2-fold dilution) with HBS-EP buffer (150mM NaCl,3mM EDTA,0.005% (v/v) surfactant P-20, and 10mM HEPES,pH 7.4); the antibodies were diluted with sodium acetate solution (10 mM, pH 5.5) to a final concentration of about 20. Mu.g/ml, respectively; the tests were performed using a Biacore T200, protein A chip (GE healthcare, cat. No. 29127556), the instrument set up as follows: antibody capture binding time (contact time) was 180s with a flow rate of 30 μl/min; sample binding time (contact time) of 120s, dissociation time (dissociation time) of 300s, flow rate of 30 μl/min; regeneration (regeneration) conditions are: glycine-HCl (pH 2.5), regeneration time 60s, flow rate 30. Mu.l/min. Analyzing the test result by adopting data analysis software Evaluation software software3.1, carrying out double deduction on a reference flow path and a sample blank by using a sensing signal acquired by a sample experiment flow path, and fitting by using a dynamics '1:1' model to obtain a dynamics parameter (K a For the binding rate, K d Is the dissociation rate; k (K) D For binding dissociation equilibrium constants
As shown in Table 5, the antibody P59-L17 had good binding ability to hCD 73-His.
TABLE 5 affinity constants for binding of anti-CD 73 antibodies to hCD73-His
Antibodies to K a (1/Ms) K d (1/s) K D (M)
P59-L17 2.21E+05 1.26E-04 5.69E-10
Example 3 detection of binding of anti-CD 73 antibodies to different species of CD73
Detection was performed by ELISA: 96-well ELISA plates were coated with 2. Mu.g/ml human hCD73-His (Catalog 10904-H08H, available from Techno, gmbH, yinqiao), cynomolgus monkey cCD73-His (off-shore organism, catalog CD3-C52H 9) or murine mCD73-His (off-shore organism, catalog CD3-M52H 9) overnight at 4 ℃; blocking with PBS (phosphate buffer) containing 5% BSA (bovine serum albumin) for 2 hours; after blocking was completed, washing with PBS containing 0.05% TW-20, followed by addition of antibody solution diluted with PBS containing 0.05% TW-20 and 0.5% BSA (initial concentration of antibody 4. Mu.g/ml, 3-fold dilution, 10 gradients); incubation at 37 ℃ for 1 hour; after washing the plates, HRP (horseradish peroxidase) -labeled secondary antibody anti-h-Kappa-HRP (purchased from Sigma, cat. A7164) was added and incubated for 1h; plates were washed and stained with TMB (tetramethylbenzidine) staining solution to read OD450.
As shown in Table 6, the antibodies P59-L17 bound well to human and cynomolgus monkey CD73 and did not bind to mouse CD 73.
TABLE 6 EC of antibodies P59-L17 binding to CD73-His 50 (ng/ml)
Antibodies to hCD73-His cCD73-His mCD73-His
EC 50 33.9 42.7 NA
NA means unbound or substantially unbound.
Example 4 detection of binding force of anti-CD 73 antibody to CD 73-expressing cells
MDA-MB-231 cells are human breast cancer cells, and the surface of the cell membrane of the MDA-MB-231 cells is highly expressed with CD73 protein (https:// www.proteinatlas.org/ENSG00000101017-CD 40/cell). This example uses MDA-MB-231 cells for testing the binding capacity of anti-CD 73 antibodies to CD73 molecules on the surface of the cell membrane.
The test method comprises the following steps: after the MDA-MB-231 cells with good culture state are collected and centrifuged, incubation is carried out by using isotype control IgG1-Fc or gradient diluted anti-CD 73 antibody; after 30min the supernatant was washed off by centrifugation, washed once with PBS buffer, resuspended in PBS buffer and added with fluorescent-labeled anti-human IgG flow antibody anti-humanFc-PE (from Invitrogen, cat. No. 12-4998-82); after 30min incubation, the supernatant was washed off by centrifugation, washed twice with PBS buffer, flow-tested after cell resuspension with PBS buffer, MFI was counted and data were processed with SoftMax Pro.
The results show that antibody P59-L17 binds to the EC of MDA-MB-231 cells 50 438.1ng/ml.
Example 5 anti-CD 73 antibodies inhibit CD73 Activity on cell membranes
The detection principle of the detection kit (purchased from promega, cat# G7570) is: excess AMP can inhibit ATP dependency The detection reagent fluoresces, while cell membrane CD73 can decompose AMP to release the inhibition of AMP, and anti-CD 73 antibody can inhibit CD73 enzymatic activity to maintain inhibition of AMP on the fluorescent process, which finally appears as: as the concentration of antibody increases, the fluorescence intensity correspondingly decreases. This example detects inhibition of cell membrane CD73 enzymatic activity by anti-CD 73 antibodies.
The test method comprises the following steps: MDA-MB-231 cells well grown at 2.5X10 s were cultured in DMEM medium containing 10% FBS 4 cells/wells were plated in 96-well cell culture plates and incubated overnight in a 37℃cell incubator; the next day the culture supernatant was removed, and serum-free DMEM gradient diluted antibody solution and AMP at a final concentration of 600 μm were added and allowed to react in a 37 ℃ cell incubator for 3 hours; after the reaction, 50. Mu.l of the supernatant was taken in 96-Kong Baiban, 50. Mu.l of ATP was added at a concentration of 200. Mu.M, and finally 100. Mu.l of a detection reagent was added, and the fluorescence value was immediately read on an ELISA reader.
The results showed that the concentration gradient of antibody P59-L17 dependently inhibited the enzymatic activity of CD73, IC thereof 50 126.8ng/ml.
Example 6 administration of anti-PD-1 antibodies in combination with anti-CD 73 antibodies inhibits proliferation of cancer cells
This example was used to evaluate the efficacy of antibody A and antibody P59-L17' in subcutaneously vaccinating immune checkpoint humanized mice (BALB/c-hPD 1/hPDL1/hCD73, jiangsu Jiugao Kangsu Biotech Co., ltd.) against colon cancer cell tumor models.
1) Tumor cell inoculation
The colon cancer cells CT26-hPDL1/hCD73 (Jiangsu Jiugang Biotech Co., ltd.) of the mice were recovered, colon cancer cells in the logarithmic growth phase were collected, and after the culture solution was removed, the cells were washed twice with PBS and then were inoculated under the armpit.
2) Group administration
When the average tumor volume is 80-120mm 3 Mice were randomly divided into 4 groups of 8 mice each; the day of the group was defined as D0 and the administration was started on the day of the group according to the dosing regimen, the time of intraperitoneal administration (i.p.) was: d0, D3, D7, D10 and D14.
3) Experimental observation and data acquisition
Following cell inoculation, the effect of the tumor on normal behavior of the animals was routinely monitored weekly. Specific indicators include the activity, ingestion and drinking status, weight gain or loss status, eye, hair and other abnormalities of the mice. The tumor volume calculation mode is as follows: tumor volume (mm) 3 ) =0.5× (tumor long diameter×tumor short diameter 2 )。
TGItv (inhibition ratio against tumor volume) calculation formula:
RTV n ＝V nt /V n0 ；V nt : tumor volume, V, on day t of mice numbered n n0 : tumor volume, RTV, on day 0 of mice numbered n n : tumor relative volume of mice numbered n on day t
Tgitv= (1- (mean RTV dosing group)/(mean RTV control group)) ×100%; mean RTV dosing group: mean RTV for dosing group, mean RTV control group: RTV mean of control group.
Table 7 dosing regimen
The results showed that 1) no significant differences in mouse body weight were found between groups, indicating that mice were well-tolerated for the drugs in the current system; 2) As shown in fig. 1, the combination administration group of antibody a and antibody P59-L17' had remarkable antitumor effect, and the antitumor effect was superior to that of the other groups, and TGItv in the combination administration group was 88.4%.

Claims (10)

  1. A method for treating a tumor or cancer, the method comprising: administering to a patient in need thereof an effective amount of an anti-PD-1 antibody and an anti-CD 73 antibody;
    the anti-PD-1 antibody comprises SEQ ID NO:1, HCDR1, SEQ ID NO:2, HCDR2, SEQ ID NO:3, HCDR3, SEQ ID NO:4, LCDR1, SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO: LCDR3 as shown in fig. 6.
  2. The method of claim 1, wherein the heavy chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO:9, and SEQ ID NO:9, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:9 having one or more conservative amino acid substitutions; and/or
    The light chain of the anti-PD-1 antibody comprises the amino acid sequence of SEQ ID NO:10, and SEQ ID NO:10, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:10, and an amino acid sequence having one or more conservative amino acid substitutions.
  3. The method of claim 1 or 2, wherein the anti-CD 73 antibody comprises at least the amino acid sequence of SEQ ID NO:11, HCDR1, SEQ ID NO:12, HCDR2, SEQ ID NO:13, HCDR3, SEQ ID NO:14, LCDR1, SEQ ID NO:15 and LCDR2 as set forth in SEQ ID NO:16, LCDR3.
  4. The method of claim 1 or 2, wherein the heavy chain variable region of the anti-CD 73 antibody comprises the amino acid sequence of SEQ ID NO:17, and SEQ ID NO:17, or a sequence having at least 80% identity 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 set forth in seq id no; and/or
    The light chain variable region of the anti-CD 73 antibody comprises SEQ ID NO:18, and SEQ ID NO:18, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:18, and an amino acid sequence having one or more conservative amino acid substitutions.
  5. The method of claim 1 or 2, wherein the heavy chain of the anti-CD 73 antibody comprises the amino acid sequence of SEQ ID NO:19 or 20, and SEQ ID NO:19 or 20, or a sequence having at least 80% identity to a sequence set forth in SEQ ID NO:19 or 20, and an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence set forth in seq id no; and/or
    The light chain of the anti-CD 73 antibody comprises SEQ ID NO:21, and SEQ ID NO:21, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO:21, and an amino acid sequence having one or more conservative amino acid substitutions.
  6. The method of any one of claims 1-5, wherein the effective amount of anti-PD-1 antibody administered per treatment cycle is 50mg to 600mg.
  7. The method of any one of claims 1-5, wherein the amount of anti-PD-1 antibody administered is 1-10mg/kg per administration.
  8. The method of any one of claims 1-7, wherein the effective amount of anti-CD 73 antibody administered per treatment cycle is 15mg to 1200mg.
  9. The method of any one of claims 1-7, wherein the amount of anti-CD 73 antibody administered is 0.3-18mg/kg per administration.
  10. A kit comprising an anti-PD-1 antibody, an anti-CD 73 antibody, and instructions for directing administration of the PD-1 antibody and the anti-CD 73 antibody to a patient in need thereof;
    the anti-PD-1 antibody comprises SEQ ID NO:1, HCDR1, SEQ ID NO:2, HCDR2, SEQ ID NO:3, HCDR3, SEQ ID NO:4, LCDR1, SEQ ID NO:5 and LCDR2 as shown in SEQ ID NO: LCDR3 as shown in fig. 6.
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TWI595006B (en) * 2014-12-09 2017-08-11 禮納特神經系統科學公司 Anti-pd-1 antibodies and methods of use thereof
AU2018290237A1 (en) * 2017-06-22 2020-01-16 Novartis Ag Antibody molecules to CD73 and uses thereof
BR112020020826A2 (en) * 2018-04-12 2021-01-19 Bristol-Myers Squibb Company ANTICANCER COMBINATION THERAPY WITH CD73 ANTAGONIST ANTIBODY AND PD-1 / PD-L1 AXIS ANTIBODY
WO2019232244A2 (en) * 2018-05-31 2019-12-05 Novartis Ag Antibody molecules to cd73 and uses thereof
US11034771B2 (en) * 2018-07-25 2021-06-15 I-Mab Biopharma Us Limited Anti-CD73 anti-PD-L1 bispecific antibodies

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