CN111973753B - Anti-tumor combined medicine and application thereof - Google Patents

Anti-tumor combined medicine and application thereof Download PDF

Info

Publication number
CN111973753B
CN111973753B CN202010440682.3A CN202010440682A CN111973753B CN 111973753 B CN111973753 B CN 111973753B CN 202010440682 A CN202010440682 A CN 202010440682A CN 111973753 B CN111973753 B CN 111973753B
Authority
CN
China
Prior art keywords
antibody
tumor
alkyl
cells
drug conjugate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010440682.3A
Other languages
Chinese (zh)
Other versions
CN111973753A (en
Inventor
汤伟佳
马健
梅星星
俞金泉
李胜峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Thera Solutions Ltd
Original Assignee
Bio Thera Solutions Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bio Thera Solutions Ltd filed Critical Bio Thera Solutions Ltd
Publication of CN111973753A publication Critical patent/CN111973753A/en
Application granted granted Critical
Publication of CN111973753B publication Critical patent/CN111973753B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention provides an anti-tumor combined drug, which comprises an anti-HER 2-antibody drug conjugate and an anti-PD-1 antibody; the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody may each be separate dosing units, or the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody may be mixed to form one combined dosing unit.

Description

Anti-tumor combined medicine and application thereof
Technical Field
The invention relates to the field of medical biology, in particular to an anti-tumor combined drug and application thereof.
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 medicine in anti-tumor treatment, combined medication can be adopted when the anti-tumor medicine is used.
The combination requires proper drug compatibility, but if the combination is improperly selected, the ideal result may not be achieved, and even the combination is counterproductive. The main reason is that in the combined process, the medicines generate complex reactions in the body, and the effect of the medicines is not simply that one is added with two. The proper drug combination is selected, so that the drug effect is enhanced or the side effect is reduced. However, improper selection of the drugs can reduce the efficacy of the drugs or cause undesirable toxic and side effects. It is reported that the occurrence rate of adverse reactions caused by drug interactions is more than 30% at present.
Therefore, it is necessary to provide an antitumor drug combination, which can improve the efficacy and ensure the safety of the drug.
Disclosure of Invention
The present invention is based on the object of providing a safe and highly effective antitumor combination.
In order to achieve the above purpose, the present invention provides the following technical solutions:
an anti-tumor combination comprising an anti-HER 2-antibody drug conjugate and an anti-PD-1 antibody. In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody are separate dosing units. In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody together form a combined dosing unit.
In some embodiments, the anti-HER 2-antibody drug conjugate is a compound of formula I or a pharmaceutically acceptable salt thereof,
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or halogen;
y is selected from-H, C1-C6 alkyl, C3-C6 cycloalkyl or-C (=O) R 5 ；
R 1 Selected from-H, -OH, -OC (=O) R 5 OR-OR 5 A group;
R 2 is-H or C1-C6 alkyl;
R 3 is methyl, CH 2 OH or-CH 2 OC(＝O)R 6 ；
R 4 is-OH or-SH;
R 5 is C1-C6 alkyl or benzyl;
R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7 Is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-6 alkyl;
z is independently-H or a C1-C4 hydrocarbyl group, or two Z's form a carbonyl group with the carbon atom to which they are attached;
l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(＝O)-﹑-C(＝O)NR 8- -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
The invention also provides application of the anti-tumor pharmaceutical composition. The specific technical scheme is as follows:
the application of the combined anti-tumor medicament in preparing a kit, a medicine box, a medicine bag or a medicament for inhibiting proliferation of tumor cells.
The application of the combined anti-tumor medicament in preparing a kit, a medicine box, a medicine bag or a medicament for preventing and treating tumors.
A method of treating a tumor, comprising: an effective dose of an anti-PD-1 antibody and an anti-HER 2-antibody drug conjugate is administered to a patient in need of treatment.
Based on the technical scheme, the invention has the following beneficial effects:
the inventor discovers that when the anti-PD-1 antibody and the anti-HER 2-antibody drug conjugate are combined, the anti-PD-1 antibody and the anti-HER 2-antibody drug conjugate are matched with each other, so that a synergistic effect can be exerted, and the effect of inhibiting tumors is improved. Wherein, the anti-HER 2 antibody drug conjugate formed by the anti-HER 2 antibody and one or more chemical drugs connected with the anti-HER 2 antibody can realize targeted delivery of the chemical drugs through the combined action of the cell-mediated cytotoxicity of the antibody and the chemical drugs, thereby further obviously increasing the killing effect of the combined drugs on tumor cells. Meanwhile, the anti-PD-1 antibody is used as an immunodetection site inhibitor, and can stimulate the tumor killing function of cells, so that the inhibition effect on tumors is obviously improved. The combined medicine of the invention has good tolerance to tumor patients while improving the inhibition effect to tumors, thereby achieving the purposes of safe and efficient administration.
Drawings
FIG. 1 is a graph showing the inhibitory effect of combination of antibody C and ADC1 on SK-BR-3 cell proliferation in the presence of T cells and ADC1 alone;
FIG. 2 is a graph showing the inhibitory effect of combination of antibody C and ADC1 on NCI-87 cell proliferation in the presence of T cells, and ADC1 alone;
FIG. 3 is a graph showing the inhibition effect of combination of antibody C and ADC1 on SK-BR-3 cell proliferation in the presence of PBMC and ADC1 alone;
FIG. 4 shows the results of FACS analysis of NCI-87 cells for target antigen (HER 2) expression;
FIG. 5 shows the results of FACS analysis of target antigen (HER 2) expression by B16F10-hHER2 cells;
FIG. 6 is a graph showing the effect of ADC1 on inhibiting proliferation of B16F10-hHER2 cells;
FIG. 7 is a graph showing the effect of Cisplatin on inhibiting proliferation of B16F10-hHER2 cells;
FIG. 8 is a graph showing the trend of the effect of combination of antibody C and ADC1 on tumor volume in mice when ADC1 is administered alone;
fig. 9 is a graph showing the change in body weight effect of combination of antibody C and ADC1, and administration of ADC1 alone on mice.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions suggested by the manufacturer. The various reagents commonly used in the examples are all commercially available products.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The term "antibody" as used herein refers to any form of antibody that exhibits a desired biological activity (e.g., inhibits ligand binding to its receptor or receptor signaling induced by inhibition of ligand). Thus, "antibody" is administered in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially identical antibodies, the individual antibodies comprising the population being identical. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include a plurality of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. For example, the expression Anti-HER2 antibody (Anti-HER 2 antibody) in the examples of the present invention refers to an antibody using protooncogene HER2 (human epidermal growth factor receptor-2, HER 2) as a target protein, and the Anti-PD-1antibody (Anti-PD-1 antibody) refers to an antibody using programmed death protein-1 (PD-1) as a target protein.
The term "antibody-drug conjugate" or "ADC" in the present invention refers to a binding protein such as an antibody or antigen binding fragment thereof that is chemically linked to one or more chemical agents (which may optionally be therapeutic or cytotoxic agents). In some embodiments, the ADC comprises an antibody, a cytotoxic or therapeutic drug, and a linker that enables the drug to be linked or conjugated to the antibody. ADCs typically have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 numbers of drugs conjugated to an antibody. Drugs that may be included in an ADC are, but are not limited to: mitotic inhibitors, antitumor antibiotics, immunomodulators, carriers for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors and radiosensitizers. In some embodiments, the drug included in the ADC may be a maytansinoid drug. In some embodiments, the drug included in the ADC may be a compound of formula i or a pharmaceutically acceptable salt thereof as described herein. In some embodiments, in an ADC, an antibody is conjugated to a drug through self-cysteine or a thinned amino acid, such as a thinned lysine, to form a disulfide bond.
The terms "alkyl" and "alkylene" as used herein are meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, "C1-C6" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl (including 8 isomers), hexyl (including 23 isomers). The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and the like.
The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.
The term "amino acid side chain" as used herein refers to a substituent that replaces a group (e.g., a hydrogen atom) in an amino acid; such as glycine side chains, are substituents formed by substituting a hydrogen atom on glycine methylene. Examples of amino acid side chains include, but are not limited to, natural amino acid side chains.
The term "about" as used herein means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limits of the measurement system). Alternatively, "about" or "substantially comprising" may mean a range of up to ±20%, such as a range of ±10%, ±5% or ±1%. Unless otherwise indicated, when a particular value is found in this application and in the claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.
When referring to an animal, human, subject, cell, tissue, organ or biological fluid with "administration" and "treatment" it is meant that the exogenous drug, therapeutic, diagnostic agent or composition is contacted with the animal, human, subject, cell, tissue, organ or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cell includes contacting the agent with the cell and contacting the agent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of cells, e.g., by agents, diagnostic agents, binding compositions, or by other cells.
As used herein, "inhibiting" includes delaying the progression of symptoms associated with a disease and/or lessening the severity of those symptoms to which the disease is about to or is expected to progress. The term also includes alleviation of existing symptoms, prevention of additional symptoms, and alleviation or prevention of the underlying cause of such symptoms. Thus, the term means that beneficial results have been conferred to a vertebrate subject suffering from a disease.
The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount effective to prevent or slow down a disease or disorder to be treated when an anti-HER 2-antibody drug conjugate (anti-HER 2-ADC) and/or an anti-PD-1 antibody or fragment thereof, alone or in combination, is administered to a cell, tissue or subject. A therapeutically effective dose further refers to an amount of an Antibody Drug Conjugate (ADC) and/or antibody or fragment thereof sufficient to cause a alleviation of symptoms, such as treatment, cure, prevention, or alleviation of a relevant medical condition, or increase the rate of treatment, cure, prevention, or alleviation of the condition. The effective amount for a particular subject can vary depending upon a variety of factors, such as the disease to be treated, the overall health of the patient, the route of administration, and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects. When administered to a patient as an active ingredient administered alone, a therapeutically effective amount refers to the individual ingredient. When a combination is administered, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously. A therapeutically effective amount will alleviate symptoms, typically by at least 10%; typically at least 20%; at least about 30%; at least 40% or at least 50%.
The anti-HER 2-antibody drug conjugate or anti-PD-1 antibody can form a wide variety of pharmaceutically acceptable salts, including, but not limited to: acid addition salts with organic acids including, but not limited to, aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, amino acids, and the like, such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like; acid addition salts formed by reaction with inorganic acids including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like; and salts with metal ions (e.g., alkali metal ions (e.g., sodium or potassium), alkaline earth metal ions (e.g., calcium or magnesium), or aluminum ions) or with organic bases such as diethanolamine, triethanolamine, N-methylglucamine, and the like. The anti-HER 2-antibody drug conjugates or anti-PD-1 antibody antibodies described herein include pharmaceutically acceptable salts thereof.
The term "individual," "subject," or "patient" as used herein refers to an animal that is the target of a treatment, observation, or experiment. By way of example only, the patient may be, but is not limited to, a mammal, including, but not limited to, a human.
The invention provides an anti-tumor combined drug, which comprises the following components: anti-HER 2-antibody drug conjugates (anti-HER 2-ADC) and anti-PD-1 antibodies; . In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody are separate dosing units. In some embodiments, or the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody together form a combined dosing unit.
In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody are separate dosing units. On the one hand, the stability of the anti-HER 2-ADC and the anti-PD-1 antibodies can be improved when the anti-HER 2-ADC and the anti-PD-1 antibodies are stored respectively; on the other hand, in the actual use process, the dosage of the anti-HER 2-ADC and the anti-PD-1 antibody can be flexibly adjusted.
In some embodiments, the anti-HER 2-ADC and the anti-PD-1 antibody can be administered simultaneously, sequentially or separately when a combination is used. The anti-PD-1 antibody may be administered prior to administration of the anti-HER 2-ADC, and/or after administration of the anti-HER 2-ADC, and/or concurrently with administration of the anti-HER 2-ADC. The anti-HER 2-ADC and anti-PD-1 antibodies are therefore preferably separate dosing units for administration to a patient suffering from HER 2-positive cancer. By co-administration is meant that the anti-HER 2-ADC and the anti-PD-1 antibody are administered in the same treatment cycle such that an effective dose of the anti-HER 2-ADC and the anti-PD-1 antibody are simultaneously administered in the patient. The method comprises the steps of carrying out a first treatment on the surface of the The co-administration may be administration of the anti-HER 2-ADC and the anti-PD-1 antibody on the same day or on different days. When the actual operation allows, the administration may be performed at the same time.
In some embodiments the anti-HER 2-antibody drug conjugate is a compound as shown in formula I or a pharmaceutically acceptable salt thereof,
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or halogen;
y is selected from-H, C1-C6 alkyl, C3-C6 cycloalkyl or-C (=O) R 5 ；
R 1 Selected from-H, -OH, -OC (=O) R 5 OR-OR 5 A group;
R 2 is-H or C1-C6 alkyl;
R 3 is methyl, CH 2 OH or-CH 2 OC(＝O)R 6 ；
R 4 is-OH or-SH;
R 5 is C1-C6 alkyl or benzyl;
R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-6 alkyl;
z is independently-H or a C1-C4 hydrocarbyl group, or two Z's form a carbonyl group with the carbon atom to which they are attached;
l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(＝O)-﹑-C(＝O)NR 8 -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
Preferably, L is- (CH) 2 ) m -wherein m is an integer, m = 1-20, preferably 1-10, more preferably 5-10.
In some embodiments, the C1-C20 hydrocarbylene group in LIs 1 to 4-SO 3 H﹑-P(＝O)(OH) 2 Or R is 23 Substituted C1-C20 hydrocarbylene wherein R 23 Each independently is optionally independently selected from-SH, -S-C 1-4 Alkyl, -CONR 11 R 11 and-NR 11 R 11 C substituted by one or two groups 1-6 An alkyl group.
R 11 Selected from-H, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocycle, or two R 11 And nitrogen form a heterocyclic ring, wherein carbon in the heterocyclic ring may be optionally substituted with one or two oxygens.
In some embodiments, the anti-HER 2-antibody drug conjugate is a compound as shown in formula II:
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or-Cl;
y is-H or methyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-C6 alkyl;
l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(O)-﹑-C(＝O)NR 8 -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
Preferably, L is- (CH) 2 ) m -wherein m is an integer, m = 1-20, preferably 1-10, more preferably 5-10.
In some embodiments, the C1-C20 hydrocarbylene in L is substituted with 1 to 4-SO 3 H﹑-P(＝O)(OH) 2 Or R is 23 Substituted C1-C20 hydrocarbylene wherein R 23 Each independently is optionally independently selected from-SH, -S-C 1-4 Alkyl, -CONR 11 R 11 and-NR 11 R 11 C substituted by one or two groups 1-6 An alkyl group.
In some embodiments, the anti-HER 2-antibody drug conjugate is a compound as shown in formula iii:
Wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or-Cl;
y is-H or methyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-C6 alkyl;
m is selected from any integer from 1 to 20. m is preferably 1 to 10, more preferably 5 to 10.
In some embodiments, the anti-HER 2-antibody drug conjugate is a compound as shown in formula IV:
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10.
In some embodiments, the anti-HER 2 antibody in the anti-HER 2-antibody drug conjugate is one or more of antibody a, antibody B, trastuzumab (Trastuzumab), and Pertuzumab (Pertuzumab);
wherein the amino acid sequence of the light chain of the antibody A is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain of the antibody A is shown as SEQ ID NO. 2; or the amino acid sequence of the light or heavy chain of antibody a has one or more substitutions, but the biological activity is unchanged. The amino acid sequence of the light chain of the antibody B is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain of the antibody B is shown as SEQ ID NO. 4; or the amino acid sequence of the light or heavy chain of antibody B has one or more substitutions, but the biological activity is unchanged.
In some embodiments, in the anti-HER 2-antibody drug conjugate, the drug may be conjugated to the anti-HER 2 antibody Abu through a linker. Wherein the structure after the drug and linker are linked is selected from the group consisting of compounds described by formulas V, VI, VII, or VIII:
Wherein X is-H or halogen;
y is selected from-H, C1-C6 alkyl, C3-C6 cycloalkyl or-C (=O) R 5 ；
R 1 Selected from-H, -OH, -OC (=O) R 5 OR-OR 5 A group;
R 2 is-H or C1-C6 alkyl;
R 3 is methyl, CH 2 OH or-CH 2 OC(＝O)R 6 ；
R 4 is-OH or-SH;
R 5 is C1-C6 alkyl or benzyl;
R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-6 alkyl;
z is independently-H or a C1-C4 hydrocarbyl group, or two Z's form a carbonyl group with the carbon atom to which they are attached;
l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(＝O)-﹑-C(＝O)NR 8 -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
Preferably, L is- (CH) 2 ) m -wherein m is an integer, m = 1-20, preferably 1-10, more preferably 5-10.
In some embodiments, what is described in LThe C1-C20 alkylene is substituted with 1 to 4-SO groups 3 H﹑-P(＝O)(OH) 2 Or R is 23 Substituted C1-C20 hydrocarbylene wherein R 23 Each independently is optionally independently selected from-SH, -S-C 1-4 Alkyl, -CONR 11 R 11 and-NR 11 R 11 C substituted by one or two groups 1-6 An alkyl group.
X is-H or-Cl;
y is-H or methyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-C6 alkyl;
L is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(O)-﹑-C(＝O)NR 8 -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
Preferably, L is- (CH) 2 ) m -wherein m is an integer, m = 1-20, preferably 1-10, more preferably 5-10.
In some embodiments, the C1-C20 hydrocarbylene in L is substituted with 1 to 4-SO 3 H﹑-P(＝O)(OH) 2 Or R is 23 Substituted C1-C20 hydrocarbylene wherein R 23 Each independently is optionally independently selected from-SH, -S-C 1-4 Alkyl, -CONR 11 R 11 and-NR 11 R 11 C substituted by one or two groups 1-6 An alkyl group.
X is-H or-Cl;
y is-H or methyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-C6 alkyl;
m is selected from any integer from 1 to 20. m is preferably 1 to 10, more preferably 5 to 10.
In some embodiments, the main steps of preparing an anti-HER 2-antibody drug conjugate include:
after dilution of the anti-HER 2 antibody, incomplete reduction with Dithiothreitol (DTT), exchange by elution with Sephadex G25 resin after incubation, and the number of thiol groups of the antibody was determined. Adding the medicine shown in the formulas V, VI, VII or VIII into the reduced antibody, stirring at room temperature, adding half-messenging amino acid, and continuously stirring to obtain the medicine; the molar equivalent ratio of the drug described by the formula V, VI, VII or VIII to the number of thiol groups of the antibody is (1-2): 1, in some embodiments wherein the molar equivalent ratio of the drug of formula V, VI, VII or VIII to the number of thiol groups of the antibody is 1.5:1.
In some embodiments, the diluted anti-HER 2 antibody is diluted to 7-9 mg/mL.
In some embodiments, the above-described reaction mixture is purified by ultrafiltration followed by gel filtration column equilibration with a phosphate buffer solution of G25 at pH 7.4±0.1.
In some embodiments, the prepared anti-HER 2-antibody drug conjugate is filtered with a 0.22 micron filter and stored at-80+ -5deg.C.
In some embodiments, determining the unreacted thiol number of the drug described by formula V, VI, VII, or VIII with 5,5' dithiobis (2 nitrobenzoic acid) (DTNB) results in a drug/anti-HER 2 antibody ratio, i.e., a drug-antibody coupling ratio (DAR).
In some embodiments, the DAR in the anti-HER 2-antibody drug conjugate prepared by the method described above is 2-8; in some embodiments, DAR is 3-5; in some embodiments, DAR is 3.3-3.7; in some embodiments, DAR is about 3.5.
In some embodiments, the concentration of the drug of formula V, VI, VII or VIII can be measured by uv absorption, aggregation rate by size exclusion chromatography, and residual free drug by reverse-phase high performance liquid chromatography.
In one embodiment, the main steps of preparing an anti-HER 2-antibody drug conjugate include: the anti-HER 2 antibody was diluted to 8.0mg/mL with solution B (50 mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 8.0) and then incompletely reduced with DTT (6 molar equivalents). After incubation at 37℃for 60 min, the exchange was eluted with solution A (50 mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 6.5) via Sephadex G25 resin. Thiol antibody values were determined by measuring absorbance, thiol concentration was determined by the reaction of thiol with DTNB, and then measuring absorbance at 412 nm. The concentration of Diethylformamide (DMA) during the coupling reaction was 10% (V/V). The ratio of the number of thiol groups of the drug to the anti-HER 2 antibody described by formula V, VI, VII or VIII is 1.5:1.0 (molar equivalent). Adding the medicine shown in the formulas V, VI, VII or VIII into the reduced anti-HER 2 antibody, stirring for 3 hours at room temperature, adding 5mM of half-messenging acid, and stirring for 1 hour.
In some embodiments, the anti-HER 2-ADC of formula IV is ADC1, wherein anti-HER 2 antibody Abu is antibody a, the light chain sequence of antibody a is SEQ ID No.1, and the heavy chain sequence of antibody a is SEQ ID No.2.
In some embodiments, the anti-HER 2-ADC of formula IV is ADC2, wherein anti-HER 2 antibody Abu is antibody B, the light chain sequence of antibody B is SEQ ID No.3, and the heavy chain sequence of antibody a is SEQ ID No.4.
In some embodiments, the anti-HER 2-ADC of formula IV is ADC3, wherein the anti-HER 2 antibody Abu is trastuzumab.
In some embodiments, the anti-HER 2-ADC of formula IV is ADC4, wherein the anti-HER 2 antibody Abu is pertuzumab.
In some embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody disclosed in U.S. patent No. 8,354,509.
In some embodiments, the anti-PD-1 antibody is antibody C, or Nivolumab, or Pembrolizumab (Pembrolizumab), or terlipressin Li Shan antibody (JS 001), or sindilimab (sintillimab), or tirelizumab (tisselizumab), or karilizumab (Camrelizumab), or jenomab (Genolimzumab), or any combination of two or more of the foregoing anti-PD-1 antibodies.
In some embodiments, the anti-PD-1 antibody is antibody C, the light chain sequence of antibody C is SEQ ID No.5, and the heavy chain sequence is SEQ ID No.6.
Antibodies a, B and C are antibodies whose sequences are shown in table 1, or antibodies whose amino acid sequences are shown in table 1, and which have one or more substitutions in amino acids, but whose biological activity is not altered.
TABLE 1
In some embodiments, the number of binding of a small molecule drug corresponding to one anti-HER 2 antibody in the anti-HER 2-antibody drug conjugate antibody-drug conjugate, i.e., the drug binding number of the antibody, or referred to as the drug-antibody coupling ratio (DAR), is selected from an integer between 1 and 10, or is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; when considering the average number of binding of small molecule drugs, i.e. the average number of drug binding of an antibody, or the average drug-antibody coupling ratio (average DAR), the value is the value of p, selected from natural numbers between 1 and 10. In some embodiments, p is selected from 2-8; in some embodiments, p is selected from 3-5; in some embodiments, p is selected from 3.3-3.7; in some embodiments, p is selected from 3.5.
In some embodiments, the anti-PD-1 antibody and anti-HER 2-ADC are present in a weight fraction ratio of (1:10) - (10:1). For example, a range of 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1, or between any two ratios, including endpoints.
In some embodiments, the anti-PD-1 antibody and anti-HER 2-ADC are present in a weight fraction ratio of 1:1 to 1:10. For example, a range of 1:9.5, 1:8.5, 1:7.5, 1:6.5, 1:5.5, 1:4.5, 1:3.5, 1:2.5, or 1:1.5, or between any two ratios, including endpoints.
In some embodiments, the anti-PD-1 antibody and anti-HER 2-ADC are present in a weight fraction ratio of 1:1 to 1:2. Such as 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, or 1:1.9, or ranges between any two ratios, including endpoints.
In the combination of the present invention, the anti-PD-1 antibody and the anti-HER 2-ADC may be administered separately or together as a unit. When the pharmaceutical combination is formed as separate administration units, the anti-PD-1 antibody and the anti-HER 2-ADC in the pharmaceutical combination may be administered simultaneously, separately or sequentially.
The invention also provides application of the combined medicament in preparing a kit, a medicine box, a medicine bag or a medicament for preventing and treating tumors.
In some embodiments, the kit, pack or medicament contains separate dosing units for the anti-PD-1 antibody and the anti-HER 2-ADC, respectively. Preferably, each individual administration unit is administered in combination for the prevention or treatment of a tumor. In some embodiments, each individual administration unit may be administered simultaneously, sequentially or separately.
In some embodiments, the kit, pack or medicament further comprises instructions for the combination of anti-tumor agents.
In some embodiments, the tumor is a benign tumor or a malignant tumor. Wherein the malignant tumor is cancer.
In some embodiments, the tumor includes, but is not limited to: hodgkin's lymphoma, non-hodgkin's lymphoma, pre-lymphoblastic lymphoma, small anaplastic lymphoma, burkitt's lymphoma, non-burkitt's lymphoma, diffuse large B-cell lymphoma, anaplastic large cell lymphoma, renal tumor, wilms ' tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectoblastoma, neuroblastoma, ganglion neuroblastoma, intracranial germ cell tumor, mature teratoma, immature teratoma, endoembryo sinus tumor, yolk sac tumor, seminoma, asexual cell tumor, chorionic epithelial carcinoma, embryo carcinoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, soft tissue sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, smooth myosarcoma, angiosarcoma, lymphocanal sarcoma, malignant neurosheath tumor, acinar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, ewing film, ewing sarcoma, fibroblastic sarcoma, osteogenic sarcoma, schlemma family of small cell sarcoma: ewing's sarcoma, primitive neuroectoblastoma, liver tumor, hepatoblastoma, retinoblastoma, posterior fossa medulloblastoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecum carcinoid, mesothelioma, melanoma, interstitial cell tumor, myeloma or astrocytoma, etc. Such cancers include, but are not limited to: nasopharyngeal carcinoma, papillary thyroid carcinoma, intestinal cancer, breast carcinoma, gastric cancer, hepatocarcinoma, prostatic cancer, breast cancer, lung cancer, cervical cancer, ovarian cancer, renal cancer, lymphoma, leukemia, skin cancer, etc.
In some embodiments, the tumor is a solid tumor.
In some embodiments, the tumor is a HER2 positive solid tumor.
In some embodiments, the tumor is a tumor with high HER2 expression, or a tumor with low HER2 expression.
In some embodiments, the solid tumor is: HER2 positive melanoma, HER2 positive breast cancer or HER2 positive gastric cancer.
The invention also provides application of the combined anti-tumor drug in preparing a kit, a medicine box, a medicine bag or a compound medicine for inhibiting proliferation of tumor cells. Wherein the kit, the medicine box, the medicine bag or the medicine contains independent administration units formed by the anti-PD-1 antibody and the anti-HER 2-antibody medicine conjugate shown in a formula I or pharmaceutically acceptable salts thereof. Preferably, each individual administration unit is used in combination for inhibiting proliferation of tumor cells. Alternatively, the individual administration units may be administered simultaneously, sequentially or separately.
In some embodiments, the kit, pack or medicament further comprises instructions for the combination of anti-tumor agents.
In some embodiments, the tumor cell is a HER2 expressing tumor cell.
Wherein the tumor cell expressing HER2 is a tumor cell with high HER2 expression or a tumor cell with low HER2 expression.
In some embodiments, the HER 2-highly expressing tumor cells are: breast cancer cells or gastric cancer cells; the tumor cells with low HER2 expression are: melanoma cells.
In some embodiments, the HER 2-highly expressing tumor cells are: breast cancer cells SK-BR-3 or gastric cancer cells NCI-N87; the tumor cells with low HER2 expression are: melanoma cells B16F10-hHER2.
Each administration unit in the combination drug is respectively prepared into a pharmaceutical preparation with a carrier acceptable in the drug. Optionally, the dosage form of the pharmaceutical preparation is a liquid preparation or a solid preparation. Optionally, the dosage form of the pharmaceutical preparation is a water injection or a freeze-dried powder injection. If the dosage form of the pharmaceutical preparation is a water injection, a pharmaceutically acceptable solvent (such as water for injection) can be used for diluting to the required administration concentration when the pharmaceutical preparation is used; if the pharmaceutical preparation is in the form of lyophilized powder for injection, dissolving with pharmaceutically acceptable solvent (such as water for injection), and diluting according to the required concentration.
Use of an anti-tumor combination as described above for simultaneous, sequential or separate administration of an anti-PD-1 antibody to a patient suffering from HER2 positive cancer during treatment of said patient with an anti-HER 2-antibody drug conjugate.
The invention also provides the use of an anti-HER 2-antibody drug conjugate for the manufacture of an anti-tumour medicament for the treatment of a patient receiving an anti-PD-1 antibody.
The invention also provides the use of an anti-PD-1 antibody for the manufacture of an anti-tumor medicament for treating a patient receiving an anti-HER 2-antibody drug conjugate.
A method of the invention for treating or inhibiting a tumor, comprising: an effective dose of an anti-PD-1 antibody and an anti-HER 2-ADC is administered to a patient.
A method of the invention for treating or inhibiting a tumor, comprising: an effective dose of an anti-PD-1 antibody is administered to a patient undergoing anti-HER 2-ADC treatment.
A method of the invention for treating or inhibiting a tumor, comprising: an effective dose of an anti-HER 2-ADC is administered to a patient undergoing anti-PD-1 antibody therapy.
In some embodiments, the anti-HER 2-ADC is a compound of formula I or a pharmaceutically acceptable salt thereof;
Wherein Abu is an anti-HER 2 antibody and p is selected from 1-10; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
x is-H or halogen;
y is selected from-H, C1-C6 alkyl, C3-C6 cycloalkyl or-C (=O) R 5 ；
R 1 Selected from-H, -OH, -OC (=O) R 5 OR-OR 5 A group;
R 2 is-H or C1-C6 alkyl;
R 3 is methyl, CH 2 OH or-CH 2 OC(＝O)R 6 ；
R 4 is-OH or-SH;
R 5 is C1-C6 alkyl or benzyl;
R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-6 alkyl;
z is independently-H or a C1-C4 hydrocarbyl group, or two Z's form a carbonyl group with the carbon atom to which they are attached;
l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(＝O)-﹑-C(＝O)NR 8 -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
Preferably, L is- (CH) 2 ) m -wherein m is an integer, m = 1-20, preferably 1-10, more preferably 5-10.
In some embodiments, the C1-C20 hydrocarbylene in L is substituted with 1 to 4-SO 3 H﹑-P(＝O)(OH) 2 Or R is 23 Substituted C1-C20 hydrocarbylene wherein R 23 Each independently is optionally independently selected from-SH, -S-C 1-4 Alkyl, -CONR 11 R 11 and-NR 11 R 11 C substituted by one or two groups 1-6 An alkyl group.
In some embodiments, the anti-HER 2-ADC is:
Wherein Abu is an anti-HER 2 antibody and p is selected from 1-10; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
x is-H or-Cl;
y is-H or methyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-C6 alkyl;
l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH 2 -the groups are independently optionally replaced by groups selected from: C3-C8 Cyclohydrocarbylene, -O-, -S-, and-NR 8 -﹑-C(O)-﹑-C(＝O)NR 8 -﹑-NR 8 C(＝O)-﹑-SO 2 NR 8 -or-NR 8 SO 2 -。
Preferably, L is- (CH) 2 ) m -wherein m is an integer, m = 1-20, preferably 1-10, more preferably 5-10.
In some embodiments, the C1-C20 hydrocarbylene in L is substituted with 1 to 4-SO 3 H﹑-P(＝O)(OH) 2 Or R is 23 Substituted C1-C20 hydrocarbylene wherein R 23 Each independently is optionally independently selected from-SH, -S-C 1-4 Alkyl, -CONR 11 R 11 and-NR 11 R 11 C substituted by one or two groups 1-6 An alkyl group.
In some embodiments, the anti-HER 2-ADC is:
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
x is-H or-Cl;
y is-H or methyl;
R 7 is a-H, C1-C6 alkyl or amino acid side chain;
R 8 is-H or C1-C6 alkyl;
m is selected from any integer from 1 to 20. m is preferably 1 to 10, more preferably 5 to 10.
In some embodiments, the anti-HER 2-ADC is:
Wherein Abu is an anti-HER 2 antibody and p is selected from 1-10. For example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
When p represents an average DAR, p may be a non-integer, such as about 3.3 to about 3.7, or about 3.5.
In some embodiments, the anti-PD-1 antibody and the anti-HER 2-ADC are each separate dosing units, in combination; or the anti-PD-1 antibody and the anti-HER 2-ADC form a combined administration unit and are combined for administration.
In some embodiments, the anti-PD-1 antibody and the anti-HER 2-ADC are administered simultaneously, separately or sequentially.
When administered in combination, the anti-PD-1 antibody may be administered prior to the administration of the anti-HER 2-ADC, after the administration of the anti-HER 2-ADC, or simultaneously with the administration of the anti-HER 2-ADC. The anti-HER 2-ADC and anti-PD-1 antibodies are preferably administered separately as separate dosing units, each for administration to a patient suffering from HER2 positive cancer.
Alternatively, in other embodiments, the anti-HER 2-ADC and the anti-PD-1 antibody can be mixed together to form a combined dosage unit, and then the patient is administered with an effective dose of the anti-HER 2-ADC and the anti-PD-1 antibody.
In some embodiments, a therapeutically effective amount of an anti-HER 2-ADC and an anti-PD-1 antibody are administered to a subject patient separately or simultaneously. The dosing cycle of the anti-HER 2-ADC and anti-PD-1 antibody may be the same or different. The anti-HER 2-ADC dose is typically 1mg/kg to 10mg/kg. The anti-PD-1 antibody dose is typically 1mg/kg to 10mg/kg, or 60mg to 600mg per patient.
In some embodiments, the unit dose of anti-HER 2-ADC is about 1.2mg/kg to about 6.0mg/kg; in some embodiments, the unit dose of anti-HER 2-ADC is about 1.2mg/kg, or 2.4mg/kg, or 3.6mg/kg, or 4.8mg/kg, or 6.0mg/kg; in some embodiments, the unit dose of anti-HER 2-ADC is about 4.8mg/kg. In some embodiments, the unit dose of anti-HER 2-ADC is about 3.6mg/kg.
In some embodiments, the period of anti-HER 2-ADC administration is 1-28 days. In some embodiments, the period of anti-HER 2-ADC administration is 1-14 days, such as 2-4 days, or 3.5 days, i.e., once per 1-14 days, such as every 2-4 days, or every 3.5 days, respectively, of the anti-HER 2-ADC unit dose is administered to the patient.
In some embodiments, the anti-HER 2-ADC is administered to the patient for a period of 14-28 days, such as 20-22 days, or 21 days, i.e., once per 14-28 days, such as 20-22 days, or 21 days, respectively.
In some embodiments, the unit dose of anti-PD-1 antibody is about 100mg-200mg. In some embodiments, the unit dose of the anti-PD-1 antibody is about 100mg,150mg, or 200mg. In some embodiments, the unit dose of the anti-PD-1 antibody is about 1mg/kg to about 2mg/kg, to about 200mg; in some embodiments, the unit dose of the anti-PD-1 antibody is about 2mg/kg to 200mg.
In some embodiments, the anti-PD-1 antibody is administered for a period of 1-49 days. In some embodiments, the anti-PD-1 antibody is administered to the patient once per unit dose for an anti-PD-1 antibody for a period of 1-14 days, such as 6-8 days, or 7 days, i.e., every 1-14 days, such as every 6-8 days, or every 7 days, respectively.
In some embodiments, the anti-PD-1 antibody is administered to the patient for a period of 14-28 days, such as 20-22 days, or 21 days, i.e., once a unit dose of anti-PD-1 antibody is administered every 14-28 days, such as every 20-22 days, or every 21 days, respectively.
In some embodiments, the anti-PD-1 antibody is administered to the patient once per unit dose for an anti-PD-1 antibody for a period of 28-49 days, such as 35-49 days, or 41-43 days, or 42 days, i.e., every 28-49 days, such as every 35-49 days, or every 41-43 days, or every 42 days, respectively.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of anti-HER 2-ADC and 100mg to 200mg of anti-PD-1 antibody are administered to the patient once every 21 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of anti-HER 2-ADC and 200mg of anti-PD-1 antibody are administered to the patient once every 21 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of anti-HER 2-ADC is administered to the patient once every 21 days, and 100mg to 200mg of anti-PD-1 antibody is administered to the patient once every 42 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of anti-HER 2-ADC is administered to the patient once every 21 days, and 200mg of anti-PD-1 antibody is administered to the patient once every 42 days.
In some embodiments, the anti-HER 2-ADC and the anti-PD-1 antibody are administered separately or simultaneously in intravenous infusion.
When the invention adopts the anti-HER 2-ADC to treat patients with HER2 positive cancers, the anti-PD-1 antibody is added to improve the treatment effect. In some embodiments, the efficacy of the anti-HER 2-ADC of the invention has a positive correlation with the level of HER2 expression, but the addition of anti-PD-1 antibody increases the therapeutic effect or reduces the effective dose of anti-HER 2-ADC, whether the level of HER2 expression in HER2 positive cancers is high or not. For cancer patients with low HER2 expression level, when the anti-HER 2-ADC alone cannot achieve better treatment effect, the combined medicine provided by the invention can treat the cancer patients, and the application range of the anti-HER 2-ADC is widened.
In some embodiments, the tumor treated with the combination of the invention is a HER2 positive solid tumor.
In some embodiments, the solid tumor is: HER2 positive melanoma, HER2 positive breast cancer or HER2 positive gastric cancer.
In some embodiments, the anti-PD-1 antibody and anti-HER 2-ADC exert an inhibitory effect on tumor cell proliferation and/or an anti-tumor effect in coordination with Peripheral Blood Mononuclear Cells (PBMCs) or T cells of the patient. Further, the anti-PD-1 antibody and the anti-HER 2-ADC are combined according to the weight part ratio of (1:10) - (10:1).
In some embodiments, the anti-PD-1 antibody and anti-HER 2-ADC are administered in a ratio of 1:1 to 1:10 by weight parts.
In some embodiments, the anti-PD-1 antibody and anti-HER 2-ADC are administered in a weight ratio of 1:1 to 1:2.
In some embodiments, the anti-HER 2-ADC is a compound of formula IV, for use in combination with an anti-PD-1 antibody (e.g., antibody C):
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In some embodiments, p has an average value of 3.3 to 3.7.
In some embodiments, the light chain sequence of Abu is SEQ ID No.1 and the heavy chain sequence is SEQ ID No.2; or Abu light chain sequence is SEQ ID NO.3, heavy chain sequence is SEQ ID NO.4.
In some embodiments, the anti-HER 2-ADC of formula IV is ADC1, wherein Abu has a light chain sequence of SEQ ID No.1 and a heavy chain sequence of SEQ ID No.2; the average value of p is about 3.5.
In some embodiments, the anti-HER 2-ADC of formula IV is ADC2, wherein Abu has a light chain sequence of SEQ ID No.3 and a heavy chain sequence of SEQ ID No.4; the average value of p is about 3.5.
In some embodiments, the anti-PD-1 antibody is antibody C, or nivolumab, or pembrolizumab, or terlipressin Li Shan, or signal di Li Shan, or tirelizumab, or karilizumab, or jernomaab, or any combination of the foregoing PD-1 antibodies.
In some embodiments, the anti-PD-1 antibody is antibody C, the light chain sequence of which is SEQ ID No.5 and the heavy chain sequence of which is SEQ ID No.6.
In some embodiments, the unit dose of ADC1 is about 1.2mg/kg to about 6.0mg/kg; in some embodiments, the unit dose of ADC1 is about 1.2mg/kg, or 2.4mg/kg, or 3.6mg/kg, or 4.8mg/kg, or 6.0mg/kg; in some embodiments, the unit dose of ADC1 is about 4.8mg/kg. In some embodiments, the unit dose of ADC1 is about 3.6mg/kg.
In some embodiments, the ADC1 dosing cycle is 14-28 days, such as 20-22 days, or 21 days, i.e., once a unit dose of ADC1 is administered to the patient every 14-28 days, such as every 20-22 days, or every 21 days, respectively.
In some embodiments, the unit dose of antibody C is about 100mg-200mg. In some embodiments, the unit dose of antibody C is about 100mg,150mg, or 200mg. In some embodiments, the unit dose of antibody C is about 1mg/kg-2mg/kg, to 200mg; in some embodiments, the unit dose of antibody C is about 2mg/kg to 200mg.
In some embodiments, antibody C is administered for a period of 14-49 days. In some embodiments, antibody C is administered to the patient for a period of 14-28 days, such as 20-22 days, or 21 days, i.e., once every 14-28 days, such as every 20-22 days, or every 21 days, respectively. In some embodiments, antibody C is administered to the patient once a unit dose of antibody C for a period of 28-49 days, such as 35-49 days, every 41-43 days, or 42 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of ADC1, and 100mg to 200mg of antibody C, are administered to the patient once every 21 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of ADC1, and 200mg of antibody C, are administered to the patient once every 21 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of ADC1 is administered to the patient once every 21 days, and 100mg to 200mg of antibody C is administered once every 42 days.
In some embodiments, 1.2mg/kg to 4.8mg/kg or 2.4mg/kg to 3.6mg/kg of ADC1 is administered to the patient once every 21 days, and 200mg of antibody C is administered once every 42 days.
In some embodiments, ADC1 is administered separately or simultaneously with antibody C in the form of an intravenous infusion.
The sources of materials used in the specific embodiments of the present invention are as follows:
CCK8 kit: derived from Dojindo.
Staphylococcal enterotoxin B (staphylococcal enterotoxin B, SEB): military medical science institute SL008.
Cell type: SK-BR-3 (breast cancer cells), NCI-N87 (gastric cancer cells), B16F10/hHER2 (melanoma cells).
PBMC or T cells: reed Biotech Co.
Example 1
The concentration of antibody C was fixed (25. Mu.g/mL), the concentration of ADC1 was varied (10 nM initiation, 3-fold gradient dilution), PBMC or T cells were added, and SEB was added simultaneously to activate PBMC or T cells, and after 72h incubation the proliferation inhibition of HER2 positive and PDL1 biscationic cells by in vitro combination was determined by CCK.
1. 96-well plate cells were plated, SK-BR3, N87 were plated at 6000/well and 10000/well, respectively, at 50. Mu.L per well.
2. After 3-5h of cell culture, PMBC or T cells are added, the ratio of PBMC to target cells is 5:1, the ratio of T cells to target cells is 10:1, and 100ng/mL of SEB is added, and the volume is 50 mu L.
3. Administration: gradient dilution ADC1, 10nM was initiated, 3-fold gradient dilution, 9 gradients total, and 10 th well concentration was 0. Antibody C was added at 25. Mu.g/mL to the top three rows and the plates were as follows.
4. 37 ℃ incubator 5% CO 2 Culturing for about 72h, removing supernatant, and adding culture medium containing 10% of CCK 8.
5. Incubate at 37℃for 2-3h.
6. The microplate reader reads the plate at absorbance 450.
Results and analysis:
1. t cells and SK-BR-3 cells 10:1 mixture, SK-BR-3 cells 1X 10 4 /well.
The results are shown in FIG. 1, where the IC50 value of ADC1 was 0.055nM in the antibody C+ADC 1-combined group compared to the ADC 1-alone group. In the ADC1 alone group, the IC50 value of ADC1 was 0.065nM. It can be seen that the addition of antibody C overall enhanced the inhibitory effect of ADC1 on SK-BR-3 in the presence of T cells.
2. T cells and NCI-87 cells at 10:1 mixing
As a result, as shown in fig. 2, the IC50 value of the antibody c+adc1-combined group, in which the IC50 value of ADC1 was 0.075nM, was significantly reduced as compared to the ADC1 alone-administered group. In the ADC1 alone dosing group, the IC50 value of ADC1 was 0.261nM. It can be seen that the addition of antibody C overall enhanced the inhibition of NCI-87 by ADC1 in the presence of T cells.
3. PBMC with SK-BR-3 cell 5:1 mixture, SK-BR-3 cells 0.6X10 4 /well.
As shown in fig. 3, the antibody c+adc1 combination group showed significantly lower readings than the ADC1 alone group because ADC1 had stronger antibody-dependent cell-mediated cytotoxicity, and overall values were lower. It can be seen that the addition of antibody C overall enhanced the inhibition of SK-BR-3 by ADC1 in the presence of PBMC.
4. When the ADC1 is subjected to gradient dilution, the concentration of the ADC1 in the 10 th column is 0, and the antibody C (25 mug/ml) is added in the first three rows, namely the detection result of the antibody C used alone in the first 3 rows of the 10 th column is the detection result of the antibody C used alone, and the blank control is the blank control in the last three rows of the 10 th column. By comparison, antibody C alone had no inhibitory effect on the proliferation of SK-BR-3 and NCI-87.
5. The SK-BR-3 cells and NCI-87 cells are HER2 high-expression cells, and experimental results show that when the antibody C is combined, the ADC1 shows better tumor cell inhibition effect. ADC1 and antibody C have a synergistic effect.
Example 2
Instrument: flow cytometer (BD Accuri C6); bench top centrifuge (Thermo ST 16R); biological safety cabinet (Antai Airtech BHC-1300IIA 2); a cytometer (Nexcelom Bioscience CelloMer Auto T4);
reagent: FACS buffer; 3% FBS (Grnco CAT # 100991-148); 2mM EDTA (Grnco CAT # 15575-038); sterI Ca 2+ and Mg 2+ PBS (1X, stock); murine Fc blocking reagent Purified Rat Anti-Mouse CDI6/CD32 (Mouse BD Fc Block) TM Cat# 553141); human Fc blocking reagent FcR Blocking Reagent, human (Miltenyi Biotec CAT #130-059-901); PBS (Hyclone CAT#SH 0256.01); ultraCompeBeads (eBioscience CAT#01-2222-42).
FACS antibody: hCD340 (hHER 2), fluorescein APC, clone number 24D2, isotype control Mouse IgG1, κ, manufacturer Biolegend.7-AAD, manufacturer BD.
FACS experiment procedure:
1. washing cells with pre-chilled FACS buffer once, centrifuging, re-suspending cells, counting cells, adding FACS buffer to adjust the concentration to 1×10 6 1100. Mu.L.
2. Transfer 100 μl of cells to flow tubes, add 2 μl of murine Fc blocking reagent or 10 μl of human Fc blocking reagent to B16F10-hHER2 or NCI-N87 cells, respectively.
3. The cells were mixed and incubated at 4℃for 10 minutes in the dark.
4. Anti-hCD340 antibody or corresponding isotype control antibody (5. Mu.L/sample) was added to the corresponding cell well.
5. The cells were mixed and incubated at 4℃for 30 minutes in the absence of light.
6. 2mL of pre-chilled FACS buffer was added to the cell wells and the cells resuspended and centrifuged at 1200rpm at 4℃for 5 min.
7. The supernatant was discarded, 2mL of pre-chilled FACS buffer was added to the cell wells and the cells resuspended, and centrifuged at 1200rpm at 4℃for 5 min. Repeating once.
8. The supernatant was discarded, cells were resuspended in 200. Mu.LFACS buffer, 7-AAD (5. Mu.L/sample) was added to the corresponding wells, incubated at room temperature for 10 minutes in the dark, and the samples were analyzed by flow cytometry.
9. Electronic data generated by flow cytometry was analyzed using Kaluza software.
The results are shown in fig. 4 and 5, and the statistical results are shown in table 2. It can be seen that hHER2 (hCD 340) was expressed on NCI-N87, B16F10-hHER2 cell lines. However, the intensity of HER2 expression of B16F10-hHER2 was much lower than NCI-N87, indicating that HER2 on B16F10-hHER2 was not highly expressed.
TABLE 2
Example 3
IC50 of ADC1 was determined in the B16F10/hHER2 cell line (9 concentrations, 3 multiplex wells). The 9 concentration gradients were 240nM,48nM,9.6nM,1.92nM,0.384nM,76.8pM,15.36pM,3.07pM,0.61pM, respectively. Cisplatins served as positive controls.
Cell resuscitation and culture prior to experiment
1) Resuscitates the B16F10-hHER2 cell line with DMEM+10% FBS medium, maintains the growth of the cells to the logarithmic phase after 16 hours after the adherence of the cells, digests with the membrane enzyme, adds the medium and mixes, centrifugates at 1000rpm for 5 minutes at room temperature, removes the supernatant, and collects the cells. Appropriate amounts of medium were added to resuspend the cells.
Inhibition of B16F10-hHER2 cells by drug to be tested and positive control Cisplatin
The first day: cell seeding
2) Taking the cell suspension and performing cell counting by using a cell counter:
3) Cell plating was performed according to the required number of tests, 100 μl of cell suspension was added to each well of a 96-well plate, the final cell density was 1000 cells/well, and 3 multiplex wells were set for each drug concentration.
4) At 37℃with 5% CO 2 Culturing cells under 95% humidity;
the following day: drug treatment
The stock solution concentration of the ADC drug to be tested was 20mg/ml, 133330nM. 5-fold gradient dilutions were performed with medium.
5) 50 μl of 3 Xdrug solution was added to each well. The highest concentration of the drug to be tested is 240nM,9 concentrations and 5-fold dilution.
6) At 37℃with 5% CO 2 Culturing cells under 95% humidity;
fifth day: data reading
7) After 72 hours of drug treatment, CTG reagent was thawed and cell plates equilibrated to room temperature for 30 minutes;
8) CTG solution (e.g., 100. Mu.LCTG/150. Mu.L of cell culture solution in 96-well plates) was added to each well in equal volumes as the culture solution;
9) Vibrating on an orbital shaker for 2 minutes to lyse cells;
10 Placing the cell plate at room temperature for 10 minutes to stabilize the luminescence signal;
11 Luminescence values were read with EnVision.
The results are shown in fig. 6 and 7. Wherein, FIG. 6 is an IC50 curve of ADC1 in B16F10-hHER2 cells, and the IC50 value of ADC1 is calculated to be 61nM. FIG. 7 shows the IC50 profile of the positive control Cisplatins in B16F10-hHER2 cells, calculated to be 38.7. Mu.M.
In vitro efficacy test results show that ADC1 has an inhibitory effect on B16F10/hHER2 proliferation, but the IC50 is only 61nM, which is 700-fold or more lower than the NCI-N87 proliferation inhibitory capacity of ADC1 (ic50=0.08 nM).
Example 4
1. Experimental method
hPD1/hPDL1 KI HuGEMM mice were inoculated subcutaneously with B16F10-hHER2 cells to establish a melanoma subcutaneous engrafting tumor model. The tests were divided into high dose (10 mg/kg, QW), medium dose (3 mg/kg, QW) and low dose (1 mg/kg, QW) of test agent ADC1, test agent antibody C (10 mg/kg, BIW), and high, medium and low doses of test agent ADC1 were administered in combination with test agent antibody C, respectively. Saline control group, 6 in each group, were administered by tail vein injection for two weeks. Efficacy was evaluated according to relative tumor inhibition (TOI), and safety was evaluated according to weight changes and mortality of animals. Wherein QW: the administration period is once a week; BIW refers to the dosing cycle twice a week. The route of administration is i.v (tail vein injection).
TABLE 3 Table 3
And (3) injection:
1. day of grouping is day0 and day of dosing is day0.
2. In the combination group, antibody C was administered first, followed by ADC1, at intervals of more than 2 hours.
3. Within the test period (dosing or observation), if death of the mice occurs, drug-related death requires sample collection and dissection to make the pathology.
2. Experimental materials
1. Experimental animals: HPD1/hPDL 1K 1 HuGEMM mice, females, 6-8 weeks (mice weeks of age at tumor cell inoculation), 83 mice were used for transfer. 48 mice were grouped into groups, and the weights of the mice were 19-26.5g. Purchased from Shanghai, mode Biotech, inc. The feeding environment is SPF grade.
2. Test article and reference article
Sample ADC1: white yellowish freeze-dried powder injection, packing specification: 100 mg/bottle, 1 bottle, sealing and preserving at 4 ℃.
Test antibody C: clear or milky white injection, package specification: 2 mg/ml/bottle, 1 bottle, 4ml, sealing and preserving at 4 ℃.
3. Cell culture
B16F10-hHER2 cells were cultured in a medium containing 10% fetal bovine serum and 0.5. Mu.g/ml Puromycin DMEM. The exponentially growing B16F10-hHER2 cells were collected and PBS was resuspended to a suitable concentration for subcutaneous tumor inoculation in mice.
4. Animal modeling and random grouping
The collected exponential growth phase B16F10-hHER2 (1X 10) 5 ) Cells were resuspended in 0.1ml PBS (0.1 ml/mouse), inoculated subcutaneously on the right back of experimental mice, and tumor growth was observed periodically.
When the average tumor volume of tumor-bearing mice reached about 75mm 3 When mice were randomly grouped according to table 3. The Day of grouping was set to Day 0 and dosing started at Day 0 (tumor volume was measured on the Day of grouping and no repeated measurements of tumor volume were made at the time of dosing). The detailed methods of administration, dosages and routes of administration are shown in Table 3.
5. Preparation of test and control
The preparation of the test sample, the control sample and the positive control sample solution is carried out in a biosafety cabinet or an ultra-clean workbench.
Dosing volume = dosing volume adjusted according to body weight (dosing volume = 10 μl/g)
The preparation method of the medicine comprises the following steps:
ADC1：
in the first step, 5ml of water for injection and 100mg of ADC1 are added to be recombined into mother liquor, and the final concentration is 20mg/ml. Avoiding vibration and vortex, and storing mother liquor at 2-8 ℃.
And a second step of dilution: the solution was stored at 2-8 ℃ for no more than 4 hours after dilution with water for injection, depending on the mother liquor reagent concentration of 20mg/ml.
The solution is obtained after configuration, is ready for use and is discarded after use.
Antibody C:
according to the actual concentration of the mother liquor of 25mg/ml, the corresponding multiple is diluted by water for injection to obtain the administration concentration of 1mg/ml. The diluted solution is stored at 2-8deg.C for no more than 4 hr.
The solution is obtained after configuration, is ready for use and is discarded after use.
6. Experimental observations and data collection
Conventional monitoring after tumor inoculation includes the effect of tumor growth and treatment on normal animal behavior, specifically the activity of experimental animals, feeding and drinking conditions, weight gain or loss (measured daily weight), eyes, hair and other abnormalities. Clinical symptoms observed during the course of the experiment are all recorded in the raw data. Tumor volume (mm) according to the calculation formula of tumor volume 3 )＝1/2×(a×b 2 ) (wherein a represents a long diameter and b represents a short diameter). Studiodirector was used in the experiment TM (version number 3.1.399.19, vendor students System, inc., S.SanFrancisco, CA, USA) software collects data, including measurements of the major and minor diameters of tumors and weighing of animal body weight.
When the weight of a single mouse drops by >20%, it was euthanized according to animal welfare.
7. Efficacy evaluation criteria
Relative tumor proliferation rate, T/C, i.e., the percentage value of the treated and control groups relative to tumor volume or tumor weight at a time point. The calculation formula is as follows:
T/C％＝T RTV /C RTV ×100％(T RTV : treatment group mean RTV; c (C) RTV : mean RTV in vehicle control group;
RTV＝V t /V 0 ，V 0 tumor volume, V, of the animals when grouped t Tumor volume for the animal after treatment);
or T/C% = T TW /C TW ×100％(T TW : average tumor weights at the end of treatment group experiments; c (C) TW : average tumor weight at the end of vehicle control experiments).
Relative tumor inhibition rate, TGI (%), was calculated as follows: TGI% = (1-T/C) ×100%. (T and C are the Relative Tumor Volume (RTV) or Tumor Weight (TW) of the treatment and control groups, respectively, at a particular time point).
8. Statistical analysis
All statistical analyses and graphic renderings were performed in the R language environment (version 3.3.1). Without being particularly bound, all tests were two-tailed tests, with p-values less than 0.05 being considered statistically significant.
9. Experimental results
(1) Test agent ADC1 and antibody C anti-tumor effect study results in melanoma B10F16/hHER2 HuGEMM (hPD/hPDL 1K 1) model
On day 10 after the initial administration, the low dose group (G02 1 mg/kg), the medium dose group (G03 3 mg/kg) and the high dose group (G04 10 mg/kg) of the test drug ADC1 showed no significant tumor suppression (p > 0.05) as compared to the control group (physiological saline (G01)) average tumor volume 2719.61 ± 508.94, and the medium dose group (medium dose group) and 2335.15 ± 276.99, respectively, having average tumor volumes of 1784.89 ± 349.96 (low dose group), 2355.02 ± 345.36 (medium dose group) and 2335.15 ± 276.99 (high dose group). The average tumor of the test agent antibody C (G0510 mg/kg) group is 1587.86 +/-470.78, the TGI is 38.97 percent, and the difference of the tumor inhibition effect is not obvious (p > 0.05) through statistical analysis. Test agent ADC1 and test agent BAT13 06 were co-administered (G06 ADC1 (1 mg/kg) +antibody C (10 mg/kg), G07 ADC1 (3 mg/kg) +antibody C (10 mg/kg), G08 ADC1 (10 mg/kg) +antibody C (10 mg/kg)), G08 (ADC 1 (10 mg/kg) +IBATI306 (10 mg/kg)) showed a significant tumor inhibition (p < 0.05) with an average tumor volume of 1292.59.+ -. 314.22, TGI of 49.62%.
By comparison between treatment groups, the combination administration group G08 (ADC 1 (10 mg/kg) +BAT 1306 (10 mg/kg)) showed significantly inhibitory effect on tumor (p < 0.05) compared to the ADC1 high dose group G04 (10 mg/kg).
The HER2 expression level of the tumor model has a positive correlation with the efficacy of ADC1, and since the HER2 expression level of B16F10/hHER2 is not high, the in vivo and in vitro efficacy of ADC1 alone is not obvious. But showed significant tumor inhibition after combination with antibody C.
The tumor growth of each of the treatment group and the control group is shown in tables 4, 5 and 8.
Wherein, the data are expressed as "mean ± standard error"; all groups were tested using 6 mice and mean and standard deviation calculated.
Drug administration record (arrow in fig. 8): all groups of day 0; day3 (antibody C BIW): g01 (veccle)/G05/G06/G07/G08; all groups of day 7; day10 (antibody C BIW): g01 (vecicle) G05/G06/G07/G08.
TABLE 4 tumor volume changes over time of treatment in mice of each group in animal models
Table 5 table of pharmacodynamic analyses of groups in animal models
Safety study results and discussion of test agent ADC1 and antibody C in melanoma B10F16/hHER2 HuGEMM (hPD/hPDL 1K 1) model.
The changes in body weight after the treatment group and the control group were administered are shown in Table 6 and FIG. 9.
Wherein, the data are expressed as "average soil standard error"; the rate of change of body weight was compared to the body weight on the day of administration.
TABLE 6 variation of body weight of groups in animal models
The high dose (10 mg/kg, QW), the medium dose (3 mg/kg, QW) and the low dose (1 mg/kg, QW) of the test drug ADC1, the test drug antibody C (10 mg/kg, BIW), and the high dose, the medium dose and the low dose of the test drug ADC1 are respectively combined with the test drug antibody C, animals do not die due to obvious drug toxicity in each treatment group, the treatment period is well tolerated, the combined application of the ADC1 and the antibody C does not bring adverse reaction to mice, and the administration is safe.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. An anti-tumor combination comprising an anti-HER 2-antibody drug conjugate and an anti-PD-1 antibody; the combined medicine is used for treating HER2 positive tumors;
the anti-HER 2-antibody drug conjugate is a compound shown in a formula I or pharmaceutically acceptable salt thereof,
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or halogen;
y is selected from-H or C1-C6 alkyl;
R 1 selected from-H;
R 2 is-H or C1-C6 alkyl;
R 3 is methyl;
R 4 is-OH or-SH;
R 7 is-H or C1-C6 alkyl;
R 8 is-H or C1-6 alkyl;
two Z forms a carbonyl group with the carbon atom to which they are attached;
l is selected from C1-C20 hydrocarbylene;
the anti-PD-1 antibody is an antibody C, and the antibody C has a light chain shown as SEQ ID NO.5 and a heavy chain shown as SEQ ID NO. 6.
2. The anti-tumor combination according to claim 1, wherein the anti-HER 2-antibody drug conjugate is a compound represented by formula ii:
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or-Cl;
y is-H or methyl;
R 7 is-H or C1-C6 alkyl;
R 8 is-H or C1-C6 alkyl;
l is selected from C1-C20 hydrocarbylene.
3. The anti-tumor combination according to claim 2, wherein the anti-HER 2-antibody drug conjugate is a compound represented by formula iii:
Wherein Abu is an anti-HER 2 antibody and p is selected from 1-10;
x is-H or-Cl;
y is-H or methyl;
R 7 is-H or C1-C6 alkyl;
R 8 is-H or C1-C6 alkyl;
m is selected from any integer from 1 to 20.
4. The anti-tumor combination according to claim 3, wherein the anti-HER 2-antibody drug conjugate is a compound represented by formula iv:
wherein Abu is an anti-HER 2 antibody and p is selected from 1-10.
5. The anti-tumor combination according to any one of claims 1 to 4, wherein the anti-PD-1 antibody is used as an immunodetection site inhibitor.
6. The anti-tumor combination according to any one of claims 1-4, wherein the anti-HER 2 antibody in the anti-HER 2-antibody drug conjugate is selected from one or more of antibody a, antibody B, trastuzumab and pertuzumab;
the antibody A has a light chain shown as SEQ ID NO.1 and a heavy chain shown as SEQ ID NO. 2;
the antibody B has a light chain shown as SEQ ID NO.3 and a heavy chain shown as SEQ ID NO. 4.
7. The anti-tumor combination according to claim 6, wherein the weight ratio of the anti-PD-1 antibody to the anti-HER 2-antibody drug conjugate is (1:10) - (10:1).
8. The anti-tumor combination according to any one of claims 1 to 4, wherein the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody are each separate administration units; alternatively, the anti-HER 2-antibody drug conjugate and the anti-PD-1 antibody together form a combined dosing unit.
9. The use of an anti-tumor co-agent according to any one of claims 1-8 in the manufacture of a kit, pack or medicament for inhibiting proliferation of tumor cells, or in the manufacture of a kit, pack or medicament for preventing and treating tumors, said tumor cells being HER2 expressing tumor cells; and/or the tumor is a HER2 positive solid tumor.
10. The use according to claim 9, wherein the tumour cells are melanoma cells, breast cancer cells or gastric cancer cells; and/or the tumor is HER2 positive melanoma, HER2 positive breast cancer or HER2 positive gastric cancer.
CN202010440682.3A 2019-05-24 2020-05-22 Anti-tumor combined medicine and application thereof Active CN111973753B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2019104380568 2019-05-24
CN201910438056 2019-05-24

Publications (2)

Publication Number Publication Date
CN111973753A CN111973753A (en) 2020-11-24
CN111973753B true CN111973753B (en) 2024-03-29

Family

ID=73442093

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010440682.3A Active CN111973753B (en) 2019-05-24 2020-05-22 Anti-tumor combined medicine and application thereof

Country Status (2)

Country Link
CN (1) CN111973753B (en)
WO (1) WO2020238788A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022073515A1 (en) * 2020-10-11 2022-04-14 百奥泰生物制药股份有限公司 Use of anti-pd-1 antibody in combination medication

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103648523A (en) * 2011-05-09 2014-03-19 弗吉尼亚大学专利基金会 Compositions and methods for treating cancer
WO2018160538A1 (en) * 2017-02-28 2018-09-07 Mersana Therapeutics, Inc. Combination therapies of her2-targeted antibody-drug conjugates

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103288957B (en) * 2012-12-21 2015-01-28 百奥泰生物科技(广州)有限公司 Antibody drug derivative inhibiting tumor growth, its preparation method and use
WO2018217227A1 (en) * 2017-05-24 2018-11-29 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103648523A (en) * 2011-05-09 2014-03-19 弗吉尼亚大学专利基金会 Compositions and methods for treating cancer
WO2018160538A1 (en) * 2017-02-28 2018-09-07 Mersana Therapeutics, Inc. Combination therapies of her2-targeted antibody-drug conjugates

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A HER2-Targeting Antibody–Drug Conjugate,Trastuzumab Deruxtecan (DS-8201a), Enhances Antitumor Immunity in a Mouse Model;Iwata T.N,et al;Mol Cancer Ther;第17卷(第7期);1494-1503 *
Emens,et al.Abstract PD3-01: Results from KATE2, a randomized phase 2 study of atezolizumab (atezo)+trastuzumab emtansine (T-DM1) vs placebo (pbo)+T-DM1 in previously treated HER2+ advanced breast cancer (BC).Cancer Res.2019,1-5. *
Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade;Müller.P, et al;Science Translational Medicine;第7卷(第315期);1-16 *

Also Published As

Publication number Publication date
CN111973753A (en) 2020-11-24
WO2020238788A1 (en) 2020-12-03

Similar Documents

Publication Publication Date Title
JP7342701B2 (en) Pharmaceutical composition for cancer treatment and/or prevention
EP3442584B1 (en) Combinations of pbd-based antibody drug conjugates with bcl-2 inhibitors
JP2023085465A (en) Anti-trop2 antibody-drug conjugate
AU2007281684C1 (en) Methods of treating multiple myeloma using combination therapies based on anti-CS1 antibodies
CN108136218A (en) For treating 8 antibody of AntiCD3 McAb of light chain amyloidosis and other CD38 positive bloods malignant tumours
JP2022515077A (en) CD70 and Venetoclax, BCL-2 Inhibitors, Combination Therapy for the Treatment of Acute Myeloid Leukemia
CN107365385A (en) The antibody of AntiCD3 McAb 8
CN108025070A (en) The compositions for cancer therapy that anti-CD 26 antibodies are composed with other anticancers
AU2016204335A1 (en) EMP2 Regulates Angiogenesis in Cancer Cells Through Induction of VEGF
RU2682161C2 (en) Antitumour agent and antitumour effect enhancer
BR112013033919B1 (en) USE OF A SPECIFIC ANTIBODY FOR CD19
EP2766045B1 (en) Treatment of breast cancer with companion diagnostic
CN111973753B (en) Anti-tumor combined medicine and application thereof
RU2011146339A (en) COMBINED THERAPY USING AGENT (S) AGAINST IGFR AND SPECIFIC IGF-1R INHIBITORS
JP2003505432A (en) Combination of anti-EP-CAM antibody and chemotherapeutic agent
JP2022533009A (en) Therapeutic combination including anti-CD123 immune complex
Smyth et al. Management of indolent B-cell Lymphomas: A review of approved and emerging targeted therapies
WO2022154044A1 (en) Pharmaceutical composition
CN114246864A (en) CSF1R kinase inhibitors and uses thereof
CN115998900A (en) anti-TROP-2 antibody drug conjugate and medical application thereof
KR20240058146A (en) Antibody-drug conjugates, methods for their preparation and medicinal uses
WO2012121958A2 (en) Combination

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant