CN111973753A

CN111973753A - Anti-tumor combined medicine and application thereof

Info

Publication number: CN111973753A
Application number: CN202010440682.3A
Authority: CN
Inventors: 汤伟佳; 马健; 梅星星; 俞金泉; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2019-05-24
Filing date: 2020-05-22
Publication date: 2020-11-24
Anticipated expiration: 2040-05-22
Also published as: CN111973753B; WO2020238788A1

Abstract

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

Background

The incidence of tumors is increasing year by year, and the use of anti-tumor drugs is also increasing. In recent years, significant progress has been made in tumor therapy research, and particularly, some antibody drugs show good curative effects in treating malignant tumors. However, only a small fraction of patients can achieve long-term survival. In order to further improve the clinical curative effect of the medicine in antitumor treatment, combined medication can be adopted when the antitumor medicine is used.

The combination needs proper drug compatibility, but if the selection of the combined drugs is not proper, the ideal result can not be achieved, and even the opposite can be achieved. The main reason is that in the process of drug combination, the drugs have complex reactions in vivo, rather than simple actions of one plus one and equal to two. The drug effect can be strengthened or the side effect can be reduced by selecting proper drug combination. However, improper selection of the drugs can weaken the drug effect or cause undesirable toxic and side effects. The incidence rate of adverse reactions caused by drug interactions at present is reported to exceed 30%.

Therefore, there is a need to provide an anti-tumor drug combination, which can improve the drug effect and ensure the drug safety.

Disclosure of Invention

Based on the above, the invention aims to provide a safe and efficient anti-tumor combined medicament.

In order to achieve the purpose, the invention provides the following technical scheme:

an anti-tumor combination drug comprises 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-1antibody are each separate dosing units. In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1antibody 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 anti-HER2 antibody, 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) R⁵；

R¹Selected from-H, -OH, -OC (═ O) R⁵OR-OR⁵A group;

R²is-H or C1-C6 alkyl;

R³is methyl, -CH₂OH or-CH₂OC(＝O)R⁶；

R⁴is-OH or-SH;

R⁵is C1-C6 alkyl or benzyl;

R⁶is C1-C6 alkyl, phenyl or benzyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-6 alkyl;

z is independently-H or a C1-C4 hydrocarbyl group, or two Z together with the carbon atom to which they are attached form a carbonyl group;

l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH₂-the groups are independently optionally replaced by a group selected from: C3-C8 cycloalkylene, -O-, -S-, or-NR-⁸-﹑-C(＝O)-﹑-C(＝O)NR^8--﹑-NR⁸C(＝O)-﹑-SO₂NR⁸-or-NR⁸SO₂-。

The invention also provides application of the anti-tumor medicinal composition. The specific technical scheme is as follows:

the application of the anti-tumor combined medicine in preparing a kit, a medicine box, a medicine package or a medicine for inhibiting tumor cell proliferation.

The application of the anti-tumor combined medicine in the preparation of a kit, a medicine box, a medicine package or a medicine for preventing and treating tumors.

A method of treating a tumor, comprising: administering to a patient in need of treatment an effective dose of an anti-PD-1antibody and an anti-HER 2-antibody drug conjugate.

Based on the technical scheme, the invention has the following beneficial effects:

the inventor of the invention discovers in research that when the anti-PD-1antibody and the anti-HER 2-antibody drug conjugate are used together, the anti-PD-1antibody and the anti-HER 2-antibody drug conjugate are matched with each other, so that the synergistic effect can be exerted, and the effect of inhibiting tumors is improved. The anti-HER 2-antibody drug conjugate formed by the anti-HER2 antibody and one or more connected chemical drugs has the effect of jointly acting through the antibody-dependent cell-mediated cytotoxicity and the chemical drugs, and the anti-HER2 antibody can also realize the targeted delivery of the chemical drugs, so that the killing effect of the combined drug on tumor cells is further remarkably increased. Meanwhile, the anti-PD-1antibody serving as an immune examination site inhibitor can stimulate the tumor killing function of cells, so that the tumor inhibition effect is obviously improved. Moreover, the combined medicine provided by the invention has good tolerance for tumor patients while improving the tumor inhibition effect, and achieves the purpose of safe and efficient medication.

Drawings

FIG. 1 is a graph of the inhibitory effect of antibody C in combination with ADC1 and ADC1 alone on SK-BR-3 cell proliferation in the presence of T cells;

FIG. 2 is a graph of the inhibitory effect of antibody C in combination with ADC1 and ADC1 alone on NCI-87 cell proliferation in the presence of T cells;

FIG. 3 is a graph of the inhibitory effect of antibody C in combination with ADC1 and ADC1 alone on SK-BR-3 cell proliferation in the presence of PBMCs;

FIG. 4 shows the results of FACS analysis of the expression of the target antigen (HER2) by NCI-87 cells;

FIG. 5 shows the results of FACS analysis of the expression of the target antigen (HER2) by B16F10-hHER2 cells;

FIG. 6 is a graph of the inhibitory effect of ADC1 on B16F10-hHER2 cell proliferation;

FIG. 7 is a graph showing the inhibitory effect of the positive drug Cisplatin on B16F10-hHER2 cell proliferation;

FIG. 8 is a graph showing the trend of the effect of the combination of antibody C and ADC1 and the effect of ADC1 alone on tumor volume in mice;

FIG. 9 is a graph showing the trend of the effect of the combination of antibody C and ADC1 and the effect of ADC1 alone on body weight in mice.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. 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 is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

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.

As used herein, the term "and/or" 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, such as inhibiting the binding of a ligand to its receptor or by inhibiting ligand-induced receptor signaling. 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, also referred to as "monoclonal antibody", 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-HER2 antibody) in the present embodiment refers to an antibody that targets proto-oncogene human epidermal growth factor receptor 2 (HER2), and the expression Anti-PD-1antibody (Anti-PD-1antibody) refers to an antibody that targets programmed death protein-1 (PD-1).

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 chemically linked to one or more chemical drugs (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 drugs conjugated to the antibody. Drugs that may be included in the ADC are, but not limited to: mitotic inhibitors, antitumor antibiotics, immunomodulators, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, anti-hormonal 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 as described herein or a pharmaceutically acceptable salt thereof. In some embodiments, in the ADC, the antibody is conjugated to the drug via self-cysteine or an agluconated amino acid, such as an agluconated lysine, forming a disulfide bond.

The terms "alkyl" and "alkylene" as used herein are intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the 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 in a straight or branched chain arrangement. 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 the 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 a glycine side chain, is a substituent formed by substituting one hydrogen atom on a glycine methylene group. 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 a numerical value is within an acceptable error range for the particular value determined by one of ordinary skill in the art, which numerical value depends in part on how the value is measured or determined (i.e., the limits of the measurement system). Alternatively, "about" or "substantially comprising" may mean a range of up to ± 20%, such as a ± 10%, a ± 5% or a ± 1% range. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value.

When referring to an animal, human, subject, cell, tissue, organ, or biological fluid by "administering" and "treatment," it is meant that the exogenous drug, therapeutic agent, diagnostic agent, or composition is contacted with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" can refer to, for example, methods of treatment, pharmacokinetics, diagnostics, research and experimentation. Treating the cells comprises contacting the agent with the cells and contacting the agent with a flow, wherein the flow contacts the cells. "administering" and "treatment" also mean in vitro and ex vivo treatment of cells, for example, by agents, diagnostic agents, binding compositions, or by other cells.

As used herein, "inhibiting" includes delaying the development of symptoms associated with a disease and/or lessening the severity of those symptoms at which the disease is about to or expected to develop. The term also includes alleviation of existing symptoms, prevention of additional symptoms, and alleviation or prevention of the underlying causes of these symptoms. Thus, the term refers to vertebrate subjects that have been assigned beneficial results to a disease.

The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount which is effective to prevent or alleviate the disease or disorder to be treated when the anti-HER 2-antibody drug conjugate (anti-HER 2-ADC) and/or the anti-PD-1antibody or fragment thereof is administered to a cell, tissue or subject, alone or in combination. A therapeutically effective dose further refers to an amount of Antibody Drug Conjugate (ADC) and/or antibody or fragment thereof sufficient to cause alleviation of symptoms such as treatment, cure, prevention, or alleviation of a related medical condition, or increase in the rate of treatment, cure, prevention, or alleviation of the symptoms. An effective amount for a particular subject may vary depending on a number of factors, such as the disease to be treated, the overall health of the patient, the method of administration and the dosage and severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. When administering an active ingredient administered alone to a patient, 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, sequentially or simultaneously. A therapeutically effective amount will reduce symptoms, typically by at least 10%; usually at least 20%; at least about 30%; at least 40% or at least 50%.

The anti-HER 2-antibody drug conjugate or anti-PD-1antibody can form a wide variety of pharmaceutically acceptable salts, including but not limited to: acid addition salts formed 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, hydrobromic, sulfuric, nitric, phosphoric, hydroiodic, hydrofluoric, phosphorous, 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-1antibody 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 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 medicament, which comprises the following components in part by weight: 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-1antibody are each separate dosing units. In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1antibody together form a combined dosing unit.

In some embodiments, the anti-HER 2-antibody drug conjugate and the anti-PD-1antibody are each separate dosing units. This can on the one hand improve the stability of the anti-HER 2-ADC and anti-PD-1 antibodies when stored separately; on the other hand, in the actual use process, the usage amount of the anti-HER 2-ADC and the anti-PD-1antibody can be flexibly adjusted.

In some embodiments, where a combination is used, the anti-HER 2-ADC and anti-PD-1antibody may be administered simultaneously, sequentially or separately. The anti-PD-1antibody may be administered prior to the application of the anti-HER 2-ADC, and/or after the application of the anti-HER 2-ADC, and/or simultaneously with the application of the anti-HER 2-ADC. The anti-HER 2-ADC and anti-PD-1antibody are therefore preferably separate dosage units for administration to patients with HER2 positive cancer. By co-administration, it is meant that the anti-HER 2-ADC and the anti-PD-1antibody are administered during the same treatment cycle such that an effective amount of both the anti-HER 2-ADC and the anti-PD-1antibody is present in the patient. (ii) a The administration of the combination may be the administration of the anti-HER 2-ADC and the anti-PD-1antibody on the same day or on different days. Administration may also be carried out at the same time as the actual practice permits.

In some embodiments the anti-HER 2-antibody drug conjugate is a compound according to formula I or a pharmaceutically acceptable salt thereof,

wherein, Abu is anti-HER2 antibody, 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) R⁵；

R¹Selected from-H, -OH, -OC (═ O) R⁵OR-OR⁵A group;

R²is-H or C1-C6 alkyl;

R³is methyl, -CH₂OH or-CH₂OC(＝O)R⁶；

R⁴is-OH or-SH;

R⁵is C1-C6 alkyl or benzyl;

R⁶is C1-C6 alkyl, phenyl or benzyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-6 alkyl;

l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH₂-the groups are independently optionally replaced by a group selected from: C3-C8 cycloalkylene, -O-, -S-, or-NR-⁸-﹑-C(＝O)-﹑-C(＝O)NR⁸-﹑-NR⁸C(＝O)-﹑-SO₂NR⁸-or-NR⁸SO₂-。

Preferably, L is- (CH)₂)_m-, where m is an integer, and m is 1 to 20, preferably 1 to 10, more preferably 5 to 10.

In some embodiments, the C1-C20 hydrocarbylene group in L is substituted with 1 to 4-SO₃H﹑-P(＝O)(OH)₂Or R²³Substituted C1-C20 hydrocarbylene wherein R²³Each independently is optionally independently selected from-SH, S-C_1-4Alkyl, -CONR₁₁R₁₁and-NR₁₁R₁₁C substituted by one or two radicals_1-6An alkyl group.

R₁₁Selected from-H, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl and heterocycle, or two R₁₁And nitrogen, wherein the carbons in the heterocycle may optionally be substituted with one or two oxygens.

In some embodiments, the anti-HER 2-antibody drug conjugate is a compound according to formula II:

wherein, Abu is anti-HER2 antibody, p is selected from 1-10;

x is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

l is selected from optionally substituted C1-C20 hydrocarbylene or C3-C8 cycloalkylene, wherein one or more-CH₂-the groups are independently optionally replaced by a group selected from: C3-C8 cycloalkylene, -O-, -S-, or-NR-⁸-﹑-C(O)-﹑-C(＝O)NR⁸-﹑-NR⁸C(＝O)-﹑-SO₂NR⁸-or-NR⁸SO₂-。

Preferably, L is- (C)H₂)_m-, where m is an integer, and m is 1 to 20, preferably 1 to 10, more preferably 5 to 10.

In some embodiments, the anti-HER 2-antibody drug conjugate is a compound according to formula iii:

wherein, Abu is anti-HER2 antibody, p is selected from 1-10;

x is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

m is selected from any integer of 1-20. m is preferably 1 to 10, more preferably 5 to 10.

In some embodiments, the anti-HER 2-antibody drug conjugate is a compound according to formula IV:

wherein, Abu is anti-HER2 antibody, and p is selected from 1-10.

In some embodiments, the anti-HER2 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 antibody A has one or more substitutions of amino acids in the light or heavy chain sequence, 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 antibody B has one or more substitutions of amino acids in the light or heavy chain sequence, 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-HER2 antibody Abu through a linker. Wherein the structure of the drug linked to the linker is selected from the group consisting of compounds of formula 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) R⁵；

R¹Selected from-H, -OH, -OC (═ O) R⁵OR-OR⁵A group;

R²is-H or C1-C6 alkyl;

R³is methyl, -CH₂OH or-CH₂OC(＝O)R⁶；

R⁴is-OH or-SH;

R⁵is C1-C6 alkyl or benzyl;

R⁶is C1-C6 alkyl, phenyl or benzyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-6 alkyl;

X is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

In some embodiments, the C1-C20 hydrocarbylene group in L is substituted with 1 to 4-SO₃H﹑-P(＝O)(OH)₂Or R²³Substituted C1-C20 hydrocarbylene wherein R²³Each independently is optionally independently selected from-SH, S-C_1-4Alkyl, -CONR₁₁R₁₁and-NR₁₁R₁₁One ofOr C substituted by two radicals_1-6An alkyl group.

X is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

In some embodiments, the main steps of preparing an anti-HER 2-antibody drug conjugate comprise:

after dilution of the anti-HER2 antibody, the antibody was incompletely reduced with Dithiothreitol (DTT), and after incubation, the antibody was eluted and exchanged on Sephadex G25 resin to determine the number of thiol groups of the antibody. Adding the drug shown in the formula V, VI, VII or VIII into the reduced antibody, stirring at room temperature, adding the cysteine, and continuing stirring to obtain the drug; the molar equivalent ratio of the medicine of the formula V, VI, VII or VIII to the mercapto group number of the antibody is (1-2): 1, in some embodiments the molar equivalent ratio of the drug of formula V, VI, VII, or VIII to the thiol number of the antibody is 1.5: 1.

In some embodiments, the diluted anti-HER2 antibody is diluted to 7-9 mg/mL.

In some embodiments, the mixture of the above reaction is ultrafiltered and purified using a gel filtration column equilibrated with G25 in phosphate buffer at pH 7.4 ± 0.1.

In some embodiments, the anti-HER 2-antibody drug conjugate prepared is filtered through a 0.22 micron filter and stored at-80 ± 5 ℃.

In some embodiments, the drug/anti-HER 2 antibody ratio, i.e., the drug-antibody coupling ratio (DAR), is determined by measuring the number of unreacted sulfhydryl groups of a drug of formula V, VI, VII, or VIII using 5, 5' dithiobis (2 nitrobenzoic acid) (DTNB).

In some embodiments, the DAR in an anti-HER 2-antibody drug conjugate prepared as described above is 2-8; in some embodiments, the DAR is 3 to 5; in some embodiments, the DAR is 3.3 to 3.7; in some embodiments, the DAR is about 3.5.

In some embodiments, the drug of formula V, VI, VII, or VIII is concentrated by uv absorption, aggregation is determined by size exclusion chromatography, and residual free drug is determined by reverse phase high performance liquid chromatography.

In one embodiment, the main steps for preparing the anti-HER 2-antibody drug conjugate comprise: the anti-HER2 antibody was diluted to 8.0mg/mL with solution B (50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 8.0) and incompletely reduced with DTT (6 molar equivalents). After incubation at 37 ℃ for 60 minutes, the exchange was eluted with solution A (50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 6.5) on Sephadex G25 resin. The thiol antibody value was determined by measuring absorbance, the concentration of thiol was determined by reacting thiol with DTNB, and then measuring the absorbance at 412 nm. The concentration of Diethylformamide (DMA) at the coupling reaction was 10% (V/V). The ratio of the thiol number of the drug of formula V, VI, VII or VIII to the anti-HER2 antibody is 1.5: 1.0 (molar equivalent). Adding the drug shown in formula V, VI, VII or VIII into reduced anti-HER2 antibody, stirring at room temperature for 3 hr, adding 5mM cysteine, and stirring for 1 hr.

In some embodiments, the anti-HER 2-ADC of formula IV is ADC1, wherein the anti-HER2 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 the anti-HER2 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-HER2 antibody Abu is trastuzumab.

In some embodiments, the anti-HER 2-ADC of formula IV is ADC4, wherein the anti-HER2 antibody Abu is pertuzumab.

In some embodiments, the anti-PD-1antibody is an anti-PD-1antibody disclosed in U.S. patent US8,354,509.

In some embodiments, the anti-PD-1antibody is antibody C, or Nivolumab (Nivolumab), or Pembrolizumab (Pembrolizumab), or tereprinimab (JS001), or sillizumab (sintillizumab), or tirlizumab (tislellizumab), or camelizumab (Camrelizumab), or genimumab (genilizumab), or any combination of two or more of the foregoing anti-PD-1 antibodies.

In some embodiments, the anti-PD-1antibody is antibody C, the light chain sequence of antibody C being SEQ ID No.5 and the heavy chain sequence being SEQ ID No. 6.

The antibody A, the antibody B and the antibody C are antibodies with sequences shown in table 1, or antibodies with amino acid sequences shown in table 1 and one or more substituted amino acids, but unchanged biological activity.

TABLE 1

In some embodiments, the number of small molecule drug binding, i.e. the number of drug binding of an antibody, or referred to as the drug-antibody coupling ratio (DAR), of one anti-HER2 antibody in the anti-HER 2-antibody drug conjugate antibody-drug conjugate is selected from an integer between 1 and 10, or is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10; when the average binding number of small molecule drugs is considered, i.e. the average binding number of drugs of an antibody, or referred to as the average drug-antibody coupling ratio (average DAR), the value is the value of p, and is selected from natural numbers between 1 and 10. In some embodiments, p is selected from 2 to 8; in some embodiments, p is selected from 3-5; in some embodiments, p is selected from 3.3 to 3.7; in some embodiments, p is selected from 3.5.

In some embodiments, the anti-PD-1antibody and anti-HER 2-ADC are present in a weight part ratio of (1:10) - (10: 1). For example, 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 a range between any two ratios, including endpoints.

In some embodiments, the anti-PD-1antibody and the anti-HER 2-ADC are present in a weight part ratio of 1:1 to 1: 10. For example, 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 a range between any two ratios, including endpoints.

In some embodiments, the anti-PD-1antibody and the anti-HER 2-ADC are present in a weight part ratio of 1:1 to 1:2. E.g., 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 a range between any two ratios, including endpoints.

In the combination drug of the present invention, the anti-PD-1antibody and the anti-HER 2-ADC may be administered separately or as a combined unit for co-administration. When the combination is administered separately as separate units, the anti-PD-1antibody and the anti-HER 2-ADC in the combination may be administered simultaneously, separately or sequentially.

The invention also provides application of the combined medicament in preparation of a kit, a medicament box, a medicament package or a medicament for preventing and treating tumors.

In some embodiments, the kit, pack or medicament comprises separate dosage units of said anti-PD-1antibody and said anti-HER 2-ADC. Preferably, the separate administration units are administered in combination for the prevention or treatment of tumors. In some embodiments, each separate administration unit may be administered simultaneously, sequentially or separately.

In some embodiments, the kit, pack or medicament further comprises instructions directing the anti-tumor combination.

In some embodiments, the tumor is a benign tumor or a malignant tumor. Among them, malignant tumor is cancer.

In some embodiments, the tumor includes, but is not limited to: hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic lymphoma, small non-dividing cell lymphoma, Burkitt's lymphoma, non-Burkitt's lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, kidney tumor, nephroblastoma, Wilms ' tumor, clear cell carcinoma of the kidney, rhabdoid tumor of the kidney, clear cell sarcoma of the kidney, renal primitive neuroblastoid phylloma, neuroblastoma of the node cell, extracranial germ cell tumor, mature teratoma, immature teratoma, endodontic sinus tumor, yolk sac tumor, seminoma, dysgerminoma of the cell, chorioepithelioma, embryo carcinoma, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, soft tissue sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwanoma, malignant schwannoma, malignant neurothecoma, neuroblastoma, hemangiosarcoma, Alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, fibroproliferative small round cell tumor, ewing family sarcoma: ewing's sarcoma, primitive neuroectoblastoma, liver tumor, hepatoblastoma, retinoblastoma, posterior fossa medulloblastoma, thymoma, pneumocoblastoma, pancreatoblastoma, islet cell tumor of pancreas, ileocecal carcinoid, mesothelioma, melanoma, mesenchymal cell tumor, myeloma, or brain astrocytoma, and the like. Such cancers include, but are not limited to: nasopharyngeal carcinoma, papillary thyroid carcinoma, intestinal cancer, breast cancer, gastric cancer, liver cancer, prostate 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 expression of HER2, or a tumor with low expression of HER 2.

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 anti-tumor combined medicine in preparation of a kit, a medicine box, a medicine package or a compound medicine for inhibiting tumor cell proliferation. Wherein the kit, the medicine package or the medicine contain independent administration units formed by the anti-PD-1antibody and the anti-HER 2-antibody medicine conjugate which are respectively represented by the formula I or pharmaceutically acceptable salts thereof. Preferably, the separate administration units are administered in combination for inhibiting the proliferation of tumor cells. Alternatively, the separate administration units may be administered simultaneously, sequentially or separately.

In some embodiments, the tumor cell is a HER 2-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-high expressing tumor cell is: breast or gastric cancer cells; the tumor cells with low HER2 expression are: a melanoma cell.

In some embodiments, the HER 2-high expressing tumor cell is: breast cancer cell SK-BR-3 or gastric cancer cell NCI-N87; the tumor cells with low HER2 expression are: melanoma cells B16F10-hHER 2.

The administration units in the combined medicine are respectively prepared with carriers acceptable in medicines to obtain the medicinal preparation. 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 water injection or lyophilized powder injection. When the pharmaceutical preparation is in the form of an aqueous injection, the pharmaceutical preparation can be diluted to a required administration concentration by using a pharmaceutically acceptable solvent (such as water for injection) when in use; if the dosage form of the pharmaceutical preparation is lyophilized powder for injection, the pharmaceutical preparation is dissolved by using a pharmaceutically acceptable solvent (such as water for injection), and then diluted according to the required concentration.

Use of an anti-tumor combination as described above for simultaneous, sequential or separate administration to a patient suffering from a HER2 positive cancer with an anti-HER 2-antibody drug conjugate.

The invention also provides the use of an anti-HER 2-antibody drug conjugate for the preparation of an anti-tumour drug for the treatment of a patient receiving an anti-PD-1 antibody.

The invention also provides the use of an anti-PD-1antibody for the manufacture of a medicament for the treatment of a patient receiving an anti-HER 2-antibody drug conjugate.

The invention provides a method for treating or inhibiting tumor, which comprises the following steps: administering to the patient an effective dose of an anti-PD-1antibody and an anti-HER 2-ADC.

The invention provides a method for treating or inhibiting tumor, which comprises the following steps: administering an effective dose of an anti-PD-1antibody to a patient undergoing anti-HER 2-ADC therapy.

The invention provides a method for treating or inhibiting tumor, which comprises the following steps: administering to a patient being treated with an anti-PD-1antibody an effective dose of anti-HER 2-ADC.

In some embodiments, the anti-HER 2-ADC is a compound of formula I or a pharmaceutically acceptable salt thereof;

wherein, Abu is anti-HER2 antibody, p is selected from 1-10; e.g., 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) R⁵；

R¹Selected from-H, -OH, -OC (═ O) R⁵OR-OR⁵A group;

R²is-H or C1-C6 alkyl;

R³is methyl, -CH₂OH or-CH₂OC(＝O)R⁶；

R⁴is-OH or-SH;

R⁵is C1-C6 alkyl or benzyl;

R⁶is C1-C6 alkyl, phenyl or benzyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-6 alkyl;

In some embodiments, the anti-HER 2-ADC is:

x is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

In some embodiments, the anti-HER 2-ADC is:

x is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

In some embodiments, the anti-HER 2-ADC is:

wherein, Abu is anti-HER2 antibody, and p is selected from 1-10. Such as 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 from about 3.3 to about 3.7, or about 3.5.

In some embodiments, the anti-PD-1antibody and the anti-HER 2-ADC are each separate dosage units, administered in combination; or the anti-PD-1antibody and the anti-HER 2-ADC together form a combined administration unit for combined administration.

In some embodiments, the anti-PD-1antibody and the anti-HER 2-ADC are administered simultaneously, separately or sequentially.

In combination, the anti-PD-1antibody may be administered before 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-1antibody are preferably administered as separate administration units to a patient suffering from HER2 positive cancer.

Alternatively, in other embodiments, the anti-HER 2-ADC and the anti-PD-1antibody are combined to form a combined unit and the patient is administered an effective amount of both the anti-HER 2-ADC and the anti-PD-1 antibody.

In some embodiments, a therapeutically effective amount of the anti-HER 2-ADC and anti-PD-1antibody are administered separately or simultaneously to a subject patient. The dosing cycles of the anti-HER 2-ADC and anti-PD-1antibody may be the same or different. The dose of anti-HER 2-ADC is generally 1mg/kg-10 mg/kg. The dose of anti-PD-1antibody is generally 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 6.0 mg/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 6.0 mg/kg; in some embodiments, the unit dose of anti-HER 2-ADC is about 4.8 mg/kg. In some embodiments, the unit dose of anti-HER 2-ADC is about 3.6 mg/kg.

In some embodiments, the anti-HER 2-ADC is administered for a period of 1-28 days. In some embodiments, the anti-HER 2-ADC is administered to the patient in a unit dose of anti-HER 2-ADC administered once every 1 to 14 days, such as 2 to 4 days, or 3.5 days, i.e. every 1 to 14 days, such as every 2 to 4 days, or every 3.5 days, respectively.

In some embodiments, the anti-HER 2-ADC is administered to the patient in a cycle of 14-28 days, such as 20-22 days, or 21 days, i.e. once per unit dose of anti-HER 2-ADC every 14-28 days, such as every 20-22 days, or every 21 days, respectively.

In some embodiments, the anti-PD-1antibody unit dose is about 100mg-200 mg. In some embodiments, the unit dose of the anti-PD-1antibody is about 100mg, 150mg, or 200 mg. In some embodiments, the unit dose of the anti-PD-1antibody is from about 1mg/kg to about 2mg/kg to about 200 mg; in some embodiments, the unit dose of the anti-PD-1antibody is about 2mg/kg to about 200 mg.

In some embodiments, the anti-PD-1antibody is administered for a period of 1-49 days. In some embodiments, the anti-PD-1antibody is administered to the patient in a cycle of 1-14 days, such as 6-8 days, or 7 days, i.e., once per unit dose of anti-PD-1antibody every 1-14 days, such as every 6-8 days, or every 7 days, respectively.

In some embodiments, the anti-PD-1antibody is administered to the patient in a cycle of 14-28 days, such as 20-22 days, or 21 days, i.e., once per unit dose of anti-PD-1antibody every 14-28 days, such as every 20-22 days, or every 21 days, respectively.

In some embodiments, the anti-PD-1antibody is administered to the patient in a cycle of 28-49 days, such as 35-49 days, or 41-43 days, or 42 days, i.e., one unit dose of anti-PD-1antibody is administered to the patient every 28-49 days, such as every 35-49 days, or 41-43 days, or every 42 days, respectively.

In some embodiments, 1.2mg/kg-4.8mg/kg or 2.4mg/kg-3.6mg/kg of anti-HER 2-ADC, and 100mg-200mg of anti-PD-1antibody are administered to the patient once every 21 days.

In some embodiments, 1.2mg/kg-4.8mg/kg or 2.4mg/kg-3.6mg/kg of anti-HER 2-ADC, and 200mg of anti-PD-1antibody are administered to the patient once every 21 days.

In some embodiments, 1.2mg/kg-4.8mg/kg or 2.4mg/kg-3.6mg/kg of anti-HER 2-ADC is administered to the patient once every 21 days, and 100mg-200mg of anti-PD-1antibody is administered to the patient once every 42 days.

In some embodiments, 1.2mg/kg-4.8mg/kg or 2.4mg/kg-3.6mg/kg of anti-HER 2-ADC is administered to the patient once every 21 days, and 200mg of anti-PD-1antibody is administered to the patient once every 42 days.

In some embodiments, the anti-HER 2-ADC and the anti-PD-1antibody are administered separately or simultaneously as an intravenous infusion.

The invention adds anti-PD-1antibody to improve the treatment effect when treating a patient with HER2 positive cancer by using anti-HER 2-ADC. In some embodiments, the efficacy of the anti-HER 2-ADC of the invention has a positive correlation with the expression level of HER2, but whether the expression level of HER2 is high or not in HER2 positive cancers, the addition of the anti-PD-1antibody increases the therapeutic effect or reduces the effective dose of the anti-HER 2-ADC. For cancer patients with low HER2 expression level, when the anti-HER 2-ADC is used alone for treatment and cannot achieve better treatment effect, the combined medicament provided by the invention can be used for treating 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 anti-PD-1antibody and anti-HER 2-ADC function to inhibit tumor cell proliferation and/or anti-tumor in conjunction with Peripheral Blood Mononuclear Cells (PBMCs) or T cells of the patient. Furthermore, the anti-PD-1antibody 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-1antibody and the anti-HER 2-ADC are administered in combination in a weight part ratio of 1:1 to 1: 10.

In some embodiments, the anti-PD-1antibody and the anti-HER 2-ADC are administered in combination in a weight ratio of 1:1 to 1:2.

In some embodiments, the anti-HER 2-ADC is a compound of formula IV, used in combination with an anti-PD-1antibody (e.g., antibody C):

wherein Abu is an anti-HER2 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 the light chain sequence of the Abu is SEQ ID NO.3, and the heavy chain sequence is SEQ ID NO. 4.

In some embodiments, the anti-HER 2-ADC of formula IV is ADC1, wherein the light chain sequence of Abu is SEQ ID No.1 and the heavy chain sequence is SEQ ID No. 2; the mean value of p is about 3.5.

In some embodiments, the anti-HER 2-ADC of formula IV is ADC2, wherein the light chain sequence of Abu is SEQ ID No.3 and the heavy chain sequence is SEQ ID No. 4; the mean value of p is about 3.5.

In some embodiments, the anti-PD-1antibody is antibody C, or nivolumab, or pembrolizumab, or tereprinizumab, or sillimumab, or tirezlizumab, or carprilizumab, or geminzelizumab, or any combination of the aforementioned PD-1 antibodies.

In some embodiments, the anti-PD-1antibody is antibody C having a light chain sequence of SEQ ID No.5 and a heavy chain sequence of SEQ ID No. 6.

In some embodiments, the unit dose of ADC1 is about 1.2mg/kg to about 6.0 mg/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 6.0 mg/kg; in some embodiments, the unit dose of ADC1 is about 4.8 mg/kg. In some embodiments, the unit dose of ADC1 is about 3.6 mg/kg.

In some embodiments, the ADC1 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 ADC1 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-200 mg. In some embodiments, the unit dose of antibody C is about 100mg, 150mg, or 200 mg. In some embodiments, the unit dose of antibody C is from about 1mg/kg to about 2mg/kg, to about 200 mg; in some embodiments, the unit dose of antibody C is about 2mg/kg, to about 200 mg.

In some embodiments, antibody C is administered for a period of 14-49 days. In some embodiments, the administration cycle of antibody C is 14-28 days, such as 20-22 days, or 21 days, i.e., one unit dose of antibody C is administered to the patient every 14-28 days, such as every 20-22 days, or every 21 days, respectively. In some embodiments, the administration cycle of antibody C is 28-49 days, such as 35-49 days, every 41-43 days, or 42 days, with one unit dose of antibody C being administered to the patient.

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 as an intravenous infusion.

The sources of materials used in the specific examples of the invention are as follows:

CCK8 kit: derived from Dojindo.

Staphylococcal Enterotoxin B (SEB): military medical academy of sciences SL 008.

Cell types: SK-BR-3 (breast cancer cells), NCI-N87 (gastric cancer cells), B16F10/hHER2 (melanoma cells).

PBMC or T cells: reid Biotechnology Ltd.

Example 1

The concentration of antibody C (25. mu.g/mL) was fixed, the concentration of ADC1 was varied (10nM start, 3-fold gradient dilution), PBMC or T cells were added, and SEB was added simultaneously to activate PBMC or T cells, and the inhibition of proliferation of HER2 positive and PDL1 double positive cells by the in vitro combination was determined by CCK method after 72h of culture.

1. 96-well plates were plated with cell plating, SK-BR3, N87 plated at 6000/well and 10000/well, 50. mu.L per well, respectively.

2. After 3-5h of cell culture, PMBC or T cells were added, the ratio of PBMC to target cells was 5:1, the ratio of T cells to target cells was 10:1, and SEB was added at 100ng/mL in a volume of 50. mu.L.

3. Administration: gradient dilution ADC1, 10nM start, 3-fold gradient dilution, 9 gradients total, and 0 concentration in well 10. Antibody C was added to the top three rows at 25. mu.g/mL and plated as follows.

4. 37 ℃ incubator 5% CO₂After culturing for about 72 hours, the supernatant was removed and a medium containing 10% CCK8 was added.

5. Incubation was carried out at 37 ℃ for 2-3 h.

6. The plate reader reads the plate at absorbance 450.

Results and analysis:

1. t cells and SK-BR-3 cells 10:1 hybrid, SK-BR-3 cells 1X 10⁴A hole.

The results are shown in fig. 1, and the readings of the antibody C + ADC1 combination group are significantly reduced compared with the ADC1 single administration group, wherein the IC50 value of the ADC1 in the antibody C + ADC1 combination group is 0.055 nM. In the group administered with ADC1 alone, IC50 value of ADC1 was 0.065 nM. 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 were differentiated at 10:1 mixing

As shown in fig. 2, the IC50 value of the antibody C + ADC1 combination group was significantly lower than that of the ADC 1-alone group, wherein the IC50 value of ADC1 in the antibody C + ADC1 combination group was 0.075 nM. In the group administered with ADC1 alone, IC50 value of ADC1 was 0.261 nM. It can be seen that the addition of antibody C overall enhanced the inhibitory effect of ADC1 on NCI-87 in the presence of T cells.

3. PBMC and SK-BR-3 cells 5:1 Mixed, SK-BR-3 cells 0.6X 10⁴A hole.

As shown in fig. 3, the antibody C + ADC1 combination group showed significantly lower readings than the ADC1 alone group, because ADC1 had a stronger antibody-dependent cell-mediated cytotoxicity effect, and the overall values were lower. It can be seen that the addition of antibody C in the presence of PBMC enhances overall the inhibitory effect of ADC1 on SK-BR-3.

4. When ADC1 was diluted in a gradient, the concentration of ADC1 in column 10 was 0, and antibody C (25 μ g/ml) was added to the first three rows, i.e., antibody C alone was detected in the first 3 rows of column 10, and no drug was added to the last three rows of column 10, which was a blank control. As shown by comparison, the antibody C has no inhibition effect on the proliferation of SK-BR-3 and NCI-87 when being singly administered.

5. The SK-BR-3 cells and the NCI-87 cells are both cells with high HER2 expression, and the experimental result shows that when the antibody C is combined, the ADC1 shows better tumor cell inhibition effect. ADC1 and antibody C have synergistic effects.

Example 2

The instrument comprises the following steps: flow cytometry (BD Accuri C6); bench centrifuge (Thermo ST 16R); biological safety cabinets (Antai Airtech BHC-1300IIA 2); cell counter (Nexcelom Bioscience CelloMer Auto T4);

reagent: FACS buffer; 3% FBS (Grnco CAT # 100991-148); 2mM EDTA (Grnco CAT # 15575-038); SterI Ca²⁺and Mg²⁺PBS (1X, Stock); mouse Fc blocking reagent, Purified Rat Anti-Mouse CDI6/CD32(Mouse BD Fc Block)^TMCAT # 553141); human Fc Blocking Reagent FcR Blocking Reagent, human (Miltenyi Biotec CAT # 130-; PBS (Hyclone CAT # SH 0256.01); UltraComp eBeads (eBioscience CAT # 01-2222-42).

FACS antibody: hCD340(hHER2), fluorescein APC, clone No. 24D2, isotype control Mouse IgG1, κ, manufacturer Biolegend. 7-AAD, manufacturer BD.

FACS experiment steps:

1. washing the cells once with pre-cooled FACS buffer, centrifuging, resuspending the cells, counting the cells, adding FACS buffer to adjust the concentration to 1X 10⁶1100. mu.L each.

2. Transfer 100. mu.L of cells to flow tube, add 2. mu.L of murine Fc blocking reagent or 10. mu.L of human Fc blocking reagent to B16F10-hHER2 or NCI-N87 cells, respectively.

3. The cells were mixed well and incubated at 4 ℃ for 10 min in the dark.

4. Anti-hCD340 antibody or the corresponding isotype control antibody (5. mu.L/sample) was added to the corresponding wells.

5. The cells were mixed well and incubated at 4 ℃ for 30 minutes in the dark.

6. Add 2mL of pre-cooled FACS buffer to the cell wells and resuspend the cells, centrifuge at 1200rpm, 4 ℃ for 5 min.

7. The supernatant was discarded, 2mL of pre-cooled FACS buffer was added to the cell wells and the cells resuspended, and centrifuged at 1200rpm for 5 minutes at 4 ℃. And repeating the steps once.

8. The supernatant was discarded, 200. mu.L LFACS buffer was added to resuspend the cells, 7-AAD (5. mu.L/sample) was added to the corresponding cell wells, incubated at room temperature in the dark for 10 min, and the samples were analyzed by flow cytometry.

9. The flow cytometer generated electronic data that was analyzed using Kaluza software.

The results are shown in FIGS. 4 and 5, and the statistical results are shown in Table 2. It can be seen that hHER2(hCD340) was expressed on NCI-N87 and B16F10-hHER2 cell lines. However, the expression intensity of HER2 of B16F10-hHER2 is much lower than that of NCI-N87, which indicates that HER2 on B16F10-hHER2 is not highly expressed.

TABLE 2

Example 3

The IC50 of ADC1 was determined in the B16F10/hHER2 cell line (9 concentrations, 3 duplicate wells). The 9 concentration gradients were 240nM, 48nM, 9.6nM, 1.92nM, 0.384nM, 76.8pM, 15.36pM, 3.07pM, 0.61pM, respectively. Cisplatin served as a positive control.

Cell recovery and culture before experiment

1) Recovering the B16F10-hHER2 cell line by using DMEM + 10% FBS culture medium, maintaining the growth of the cells to a growth log phase by using the culture medium added with puromycin (puromycin) 16 hours after the cells are attached, digesting by using membrane enzyme, adding the culture medium, uniformly mixing, centrifuging at room temperature for 5 minutes at 1000rpm, removing supernatant, and collecting the cells. The cells were resuspended by adding the appropriate amount of medium.

Inhibition of B16F10-hHER2 cells by test drug and positive control Cisplatin

The first day: cell seeding

2) Taking the cell suspension, and counting the cells by using a cell counter:

3) cell plating was performed according to the number of required assays, and 100. mu.L of cell suspension was added per well in a 96-well plate with a final cell density of 1000 per well and 3 replicate wells per drug concentration.

4) At 37 deg.C, 5% CO₂Culturing the cells under the condition of 95% humidity;

the next day: drug treatment

The stock solution concentration of the ADC drug to be tested was 20mg/ml, i.e., 133330 nM. A 5-fold gradient dilution was performed with the medium.

5) Add 50. mu.L of 3X drug solution per well. The highest concentration of the drug to be tested is 240nM, 9 concentrations, 5-fold dilution.

6) At 37 deg.C, 5% CO₂Culturing the cells under the condition of 95% humidity;

the fifth day: data reading

7) After 72 hours of drug treatment, the CTG reagent was thawed and the cell plates were equilibrated to room temperature for 30 minutes;

8) adding a CTG solution in an equal volume to the culture medium to each well (e.g., adding 100. mu. LCTG/150. mu.L of cell culture medium to a 96-well plate);

9) the cells were lysed by shaking on an orbital shaker for 2 minutes;

10) the plate was left at room temperature for 10 minutes to stabilize the luminescence signal;

11) the cold light values were read with EnVision.

The results are shown in FIGS. 6 and 7. FIG. 6 is a graph of the IC50 of ADC1 in B16F10-hHER2 cells, calculated as 61nM IC50 for ADC 1. FIG. 7 is an IC50 curve of positive control Cisplatin in B16F10-hHER2 cells, calculated to have an IC50 value of 38.7. mu.M for Cisplatin.

In vitro pharmacodynamic test results show that ADC1 has an inhibitory effect on B16F10/hHER2 proliferation, but IC50 is only 61nM, and is 700 times or more lower than the inhibitory effect of ADC1 on NCI-N87 proliferation (IC50 is 0.08 nM).

Example 4

First, experiment method

hPD1/hPDL1 KI HuGEMM mice are inoculated with B16F10-hHER2 cells subcutaneously to establish a melanoma subcutaneous transplantation tumor model. The assay was divided into high (10mg/kg, QW), medium (3mg/kg, QW) and low (1mg/kg, QW) doses of test agent ADC1, test agent antibody C (10mg/kg, BIW), and high, medium and low doses of test agent ADC1 administered in combination with test agent antibody C, respectively. Saline control groups of 6 individuals were administered by tail vein injection for two weeks. The evaluation of the efficacy was carried out on the basis of the relative tumor inhibition (TOI) and the safety was carried out on the basis of the change in body weight and the death of the animals. Wherein QW: the dosing cycle is once per week; BIW refers to the dosing cycle twice weekly. The route of administration was i.v (tail vein injection).

TABLE 3

Note:

1. day of grouping was day0 and day of dosing was day 0.

2. In the combination group, antibody C was administered first, followed by ADC1, with an interval of more than 2 hours.

3. Within the test period (dosing or observation), if mouse death occurs, drug-related death requires sampling and dissection for pathology.

Second, Experimental materials

1. Experimental animals: HPD1/hPDL 1K 1 HuGEMM mice, female, 6-8 weeks (week old mice at tumor cell inoculation), 83 mice were used for the transfer. 48 mice were divided into groups weighing 19-26.5 g. Purchased from Shanghai, Square model Biotech, Inc. The breeding environment is SPF grade.

2. Test and reference substances

Test sample ADC 1: white yellowish freeze-dried powder injection, and the packaging specification is as follows: 100 mg/bottle, 1 bottle in total, and sealed and stored at 4 ℃.

Test antibody C: clear or milky white injection, and the packaging specification is as follows: 2 mg/ml/bottle, 1 bottle and 4ml in total, and is stored in a sealed manner at 4 ℃.

3. Cell culture

B16F10-hHER2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 0.5. mu.g/ml Puromycin. The exponentially growing B16F10-hHER2 cells were collected and PBS resuspended to appropriate concentration for subcutaneous tumor inoculation in mice.

4. Animal modeling and random grouping

Collecting B16F10-hHER2 (1X 10) in exponential growth phase⁵) The cells were resuspended in 0.1ml of PBS (0.1 ml/mouse), and inoculated subcutaneously in the right dorsal side of experimental mice, and the growth of the tumor was observed periodically.

When the average tumor volume of the tumor-bearing mice reaches about 75mm³At time, mice were randomly grouped according to table 3. Day of grouping was set to Day0 and dosing was started at Day0 (tumor volume was measured on Day of grouping, tumor volume was not repeatedly measured at dosing). The details of the administration method, the administration dose and the administration route are shown in Table 3.

5. Preparation of test and reference substances

The preparation of the test sample, the reference substance and the positive reference substance solution is carried out in a biological safety cabinet or an ultra-clean workbench.

Dose volume adjusted according to body weight (dose volume 10. mu.L/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 re-melted into a mother solution with a final concentration of 20 mg/ml. Avoid shaking and vortex, and the mother liquor is stored at 2-8 ℃.

And (3) second-step dilution: the solution was stored at 2-8 ℃ for no more than 4h after dilution with water for injection, depending on the mother liquor reagent concentration of 20 mg/ml.

The solution is obtained after the preparation, is ready for use and is discarded after use.

Antibody C:

according to the actual concentration of 25mg/ml of the mother liquor, the mother liquor is diluted by corresponding times by using water for injection to obtain the administration concentration of 1 mg/ml. The diluted solution is stored for no more than 4h at 2-8 ℃.

6. Experimental observations and data Collection

After tumor inoculation, routine monitoring includes the effect of tumor growth and treatment on the normal behavior of the animal, including activity, feeding and drinking, weight gain or loss (daily body weight measurement), eye, hair coat and other abnormalities in the experimental animal. Clinical symptoms observed during the experiment were recorded in the raw data. Tumor volume calculation formula tumor volume (mm)³)＝1/2×(a×b²) (wherein a represents a long diameter and b represents a short diameter). studyDirector was used in the experiment^TM(version number 3.1.399.19, supplier studio System, inc., s.sanfrancisco, CA, USA) software data were collected including measurements of the length and length of the tumor and weighing of the animal body weight.

When individual mice lost > 20% weight, they were euthanized according to animal welfare.

7. Criteria for evaluating therapeutic effects

Relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight of the treated and control groups at a certain time point. The calculation formula is as follows:

T/C％＝T_RTV/C_RTV×100％(T_RTV: treatment group mean RTV; c_RTV: vehicle control mean RTV;

RTV＝V_t/V₀，V₀is the tumor volume of the animal in the group, V_tIs the tumor volume of the animal after treatment);

or T/C%_TW/C_TW×100％(T_TW: mean tumor weights at the end of treatment group experiments; c_TW: mean tumor weight at the end of the vehicle control experiment).

Relative tumor inhibition, TGI (%), calculated as follows: TGI% (1-T/C) × 100%. (T and C are the Relative Tumor Volume (RTV) or Tumor Weight (TW) at a particular time point for the treated and control groups, respectively).

8. Statistical analysis

All statistical analysis and graphical rendering was performed in the R language environment (version 3.3.1). All tests are two-tailed tests, not specifically stated, and a p-value of less than 0.05 is considered statistically significant.

9. Results of the experiment

(1) Results of research on antitumor effects of test drugs ADC1 and antibody C in melanoma B10F16/hHER2 HuGEMM (hPD1/hPDL 1K 1) model

On day10 after the initial dose, the treatment group tested drugs ADC1 in the low dose group (G021 mg/kg), the medium dose group (G033 mg/kg) and the high dose group (G0410 mg/kg) showed mean 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), respectively, compared to the mean tumor volume 2719.61 ± 508.94 in the control group (saline (G01)) and no significant tumor inhibition (p > 0.05). The mean tumor of test drug antibody C (G0510mg/kg) group was 1587.86 + -470.78, TGI was 38.97%, and the difference of tumor inhibition was not significant by statistical analysis (p > 0.05). In the test agent group (G06 ADC1(1mg/kg) + antibody C (10mg/kg), G07 ADC1(3mg/kg) + antibody C (10mg/kg), G08 ADC1(10mg/kg) + antibody C (10mg/kg)), G08(ADC1(10mg/kg) + IBATI306(10mg/kg)) average tumor volume 1292.59. + -. 314.22, TGI 49.62%, showed significant tumor suppression (p <0.05) in the test agent BAT 1306 combined administration group.

By comparison between treatment groups, the combination group G08(ADC1(10mg/kg) + BAT 1306(10mg/kg)) showed significant tumor suppression (p <0.05) compared to the ADC1 high dose group G04(10 mg/kg).

The expression level of HER2 of the tumor model has positive correlation with the drug effect of ADC1, and the in vivo and in vitro drug effect of the single drug of ADC1 is not obvious because the expression level of HER2 of B16F10/hHER2 is not high. But showed significant tumor suppression when combined with antibody C.

The tumor growth in each treatment group and control group is shown in table 4, table 5 and figure 8.

Wherein, the data are expressed as "mean ± standard error"; all groups were tested in 6 mice and the mean and standard deviation were calculated.

Dosing record (shown by arrows in fig. 8): day0 all groups; day3 (antibody C BIW): g01 (vehicle)/G05/G06/G07/G08; day7 all groups; day10 (antibody C BIW): g01(vehicle) G05/G06/G07/G08.

TABLE 4 tumor volume as a function of treatment time in animal models for groups of mice

TABLE 5 Table for the analysis of the drug effects of each group in animal models

The results and discussion of the safety of the test drugs ADC1 and antibody C in the model melanoma B10F16/hHER2 HuGEMM (hPD1/hPDL 1K 1).

The body weight changes after the treatment group and the control group were administered are shown in Table 6 and FIG. 9.

Wherein, the data is expressed as the 'standard error of the mean soil'; the rate of body weight change was compared to the body weight on the day of administration.

TABLE 6 body weight changes in animal models

The test drug ADC1 is high dose (10mg/kg, QW), medium dose (3mg/kg, QW) and low dose (1mg/kg, QW), the test drug antibody C (10mg/kg, BIW), and the test drug ADC1 is high dose, medium dose and low dose which are respectively combined with the test drug antibody C for administration, no animal dies due to obvious drug toxicity in each treatment group, the tolerance during treatment is good, 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 embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. An anti-tumor combination drug, which is characterized by comprising an anti-HER 2-antibody drug conjugate and an anti-PD-1 antibody.

2. The anti-tumor combination according to claim 1, wherein the combination is a combination for the treatment of HER2 positive tumors.

3. The anti-tumor combination drug according to claim 1, wherein the anti-HER 2-antibody drug conjugate is a compound represented by formula I or a pharmaceutically acceptable salt thereof,

wherein, Abu is anti-HER2 antibody, 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) R⁵；

R¹Selected from-H, -OH, -OC (═ O) R⁵OR-OR⁵A group;

R²is-H or C1-C6 alkyl;

R³is methyl, -CH₂OH or-CH₂OC(＝O)R⁶；

R⁴is-OH or-SH;

R⁵is C1-C6 alkyl or benzyl;

R⁶is C1-C6 alkyl, phenyl or benzyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-6 alkyl;

4. The anti-tumor combination drug according to claim 3, wherein the anti-HER 2-antibody drug conjugate is a compound represented by formula II:

wherein, Abu is anti-HER2 antibody, p is selected from 1-10;

x is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

5. The anti-tumor combination according to claim 4, wherein the anti-HER 2-antibody drug conjugate is a compound of formula III:

wherein, Abu is anti-HER2 antibody, p is selected from 1-10;

x is-H or-Cl;

y is-H or methyl;

R⁷is-H, C1-C6 alkyl or an amino acid side chain;

R⁸is-H or C1-C6 alkyl;

m is selected from any integer of 1-20.

6. The anti-tumor combination drug according to claim 5, wherein the anti-HER 2-antibody drug conjugate is a compound represented by formula IV:

wherein, Abu is anti-HER2 antibody, and p is selected from 1-10.

7. The anti-tumor combination according to any one of claims 1 to 6, wherein the anti-HER2 antibody of the anti-HER 2-antibody drug conjugate is selected from one or more of antibody A, antibody B, trastuzumab and pertuzumab; and/or

The anti-PD-1antibody is one or more of antibody C, nivolumab, pembrolizumab, terepril monoclonal antibody, Cedilizumab, tirezlizumab, Carrilizumab and Jennuzumab;

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;

the antibody C has a light chain shown as SEQ ID NO.5 and a heavy chain shown as SEQ ID NO. 6.

8. The anti-tumor combination according to claim 7, wherein the weight ratio of the anti-PD-1antibody and the anti-HER 2-antibody drug conjugate is (1:10) - (10: 1).

9. The anti-tumor combination according to any one of claims 1 to 6, wherein the anti-HER 2-antibody drug conjugate and the anti-PD-1antibody are each present as separate administration units; alternatively, the anti-HER 2-antibody drug conjugate and the anti-PD-1antibody together form a combined dosing unit.

10. Use of the anti-tumor combination according to any one of claims 1 to 9 for the preparation of a kit, a pack or a medicament for inhibiting tumor cell proliferation or for the preparation of a kit, a pack or a medicament for the prevention and treatment of tumors; preferably, the tumor cell is a HER 2-expressing tumor cell; and/or the tumor is a HER2 positive solid tumor.