Active combination for combined treatment of human immunodeficiency virus and its application

Abstract

The present invention provides an active combination for combined treatment of human immunodeficiency virus and its application. Specifically, a use of an active ingredient combination is provided, the active ingredient combination includes a first active ingredient, icotinib, and a second active ingredient, a human immunodeficiency virus neutralizing antibody, and the combination is used to prepare a pharmaceutical composition or a kit for synergistic treatment and prevention of HIV. icotinib and human immunodeficiency virus neutralizing antibody can act synergistically in anti-HIV treatment, thereby more significantly inhibiting the proliferation of HIV virus.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-HIV drugs, and more specifically to an active combination for treating human immunodeficiency virus and its application. Background Art

[0002] Aconin (Abovirta for injection, referred to as ABT) is the first long-acting injectable anti-AIDS drug independently developed by Frontier Biotechnology and is a national Class 1 new drug. On May 23, 2018, the National Medical Products Administration issued the new drug certificate and drug registration certificate for Abovirta and Abovirta for injection, approving it for use in combination with other antiviral drugs to treat HIV-1 adult infected people who have received antiviral treatment but still have viral replication. Abovirta has a new molecular mechanism of action and is an HIV-1 fusion inhibitor. It acts on the HIV-1 outer membrane glycoprotein gp41 to prevent the virus from entering cells. In vitro tests have shown that it has a broad spectrum of high-efficiency activity against European, American and Chinese epidemic strains and drug-resistant viruses. Abovirta is composed of 34 amino acids and a modified chemical group. It quickly forms a 1:1 covalent bond with albumin in the body, thereby extending the half-life to 12 days. Clinical Phase I-III trials have shown good safety and efficacy.

[0003] Human immunodeficiency virus neutralizing antibodies are a type of antibody isolated from HIV-infected people through biotechnology. This type of antibody can recognize HIV, inhibit the replication of the virus in the body by directly neutralizing the HIV virus strain, and reduce the level of HIV-1 virus in the body. This antibody can also stimulate other immune components in the body to kill the virus together, thereby enhancing immunity and clearing the virus in the body.

[0004] In the prior art, human immunodeficiency virus neutralizing antibodies and icotinib still have many deficiencies, such as insufficient virus coverage, and cannot be well used for the treatment and prevention of HIV virus. Therefore, the art is in urgent need of developing a more effective method for treating or preventing human immunodeficiency virus. Summary of the invention

[0005] The purpose of the present invention is to provide an active combination for the combined treatment of human immunodeficiency virus and its application, so as to solve the defect of insufficient coverage of HIV virus by using icotinib alone or using human immunodeficiency virus neutralizing antibodies (HIV neutralizing antibodies), provide broad-spectrum anti-HIV, and provide a more effective option for the treatment and prevention of HIV.

[0006] In the first aspect of the present invention, an active ingredient combination is provided, which includes a therapeutically effective amount of icotinib and a human immunodeficiency virus neutralizing antibody, wherein the human immunodeficiency virus neutralizing antibody includes a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2, or a long-acting mutant thereof.

[0007] In another preferred embodiment, the mass ratio of icotinib to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, and more preferably 1:20 to 10:1.

[0008] In another preferred embodiment, the administration concentration of the icotinib is selected from 0.1-1000 mg / ml; preferably, 0.5-500 mg / ml; more preferably, 2-320 mg / ml; and the administration concentration of the human immunodeficiency virus neutralizing antibody is 0.5-5000 mg / ml; preferably, 1-2000 mg / ml; more preferably, 5-1000 mg / ml.

[0009] In another preferred embodiment, the ratio of icotinib to human immunodeficiency virus neutralizing antibody (nmol / mg) is 0.2-4000nmol / mg, preferably 1-2000nmol / mg, more preferably 5-1000nmol / mg or 10-500nmol / mg.

[0010] In another preferred embodiment, the active ingredient combination further comprises an additional therapeutic drug.

[0011] In another preferred embodiment, the additional therapeutic drug is an antiviral agent, an anti-infective drug or a combination thereof.

[0012] In another preferred embodiment, the active ingredient combination is used to treat and / or prevent a mammal or is administered to a mammal. More preferably, the mammal is a rodent (eg, mouse, rat) or a human.

[0013] In another preferred embodiment, the long-acting mutant refers to a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid from the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and can retain binding affinity to human immunodeficiency virus.

[0014] In a second aspect of the present invention, a medicine kit is provided, comprising:

[0015] (a) a first preparation, wherein the first preparation contains icotinib and a pharmaceutically acceptable carrier;

[0016] (b) a second preparation comprising a human immunodeficiency virus neutralizing antibody and a pharmaceutically acceptable carrier;

[0017] (c) instructions describing a method of using icotinib in combination with human immunodeficiency virus neutralizing antibodies to treat and / or prevent HIV.

[0018] In another preferred embodiment, the first preparation and the second preparation are independent of each other.

[0019] In another preferred embodiment, the first preparation and the second preparation are freeze-dried preparations or liquid preparations.

[0020] In another preferred embodiment, the first preparation and the second preparation are injections.

[0021] In another preferred embodiment, the first preparation is administered before, during or after the second preparation or is mixed with the second preparation.

[0022] In another preferred embodiment, the human immunodeficiency virus neutralizing antibody comprises an antibody having a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2, or a long-acting mutant thereof.

[0023] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising the above-mentioned combination of active ingredients and a pharmaceutically acceptable carrier.

[0024] In the fourth aspect of the present invention, there is provided an application of an active ingredient combination in the preparation of a pharmaceutical composition or a medicine kit for treating and / or preventing HIV, wherein the active ingredient combination comprises a first active ingredient, icotinib, and a second active ingredient, a human immunodeficiency virus neutralizing antibody.

[0025] In another preferred embodiment, the mass ratio of icotinib to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, and more preferably 1:20 to 10:1.

[0026] In another preferred embodiment, the administration concentration of the icotinib is selected from 0.1-1000 mg / ml; preferably, 0.5-500 mg / ml; more preferably, 2-320 mg / ml; and the administration concentration of the human immunodeficiency virus neutralizing antibody is 0.5-5000 mg / ml; preferably, 1-2000 mg / ml; more preferably, 5-1000 mg / ml.

[0027] In another preferred embodiment, the ratio of icotinib to human immunodeficiency virus neutralizing antibody (nmol / mg) is 0.2-4000nmol / mg, preferably 1-2000nmol / mg, more preferably 5-1000nmol / mg or 10-500nmol / mg.

[0028] In another preferred embodiment, the pharmaceutical composition or the drug kit is used to treat and / or prevent mammals, and more preferably the mammals are rodents (such as mice, rats) or humans.

[0029] In another preferred embodiment, the HIV includes any one or a combination of HIV strains selected from the following groups:

[0030] 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 virus strains (synergistic effect).

[0031] In another preferred embodiment, the HIV includes any one or a combination of HIV strains selected from the following group: 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM235, 97TH-NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03 and CNE23 virus strains (additive effect).

[0032] In the fifth aspect of the present invention, a method for treating and preventing HIV is provided, comprising the steps of administering the active ingredient combination described in the second aspect of the present invention and / or the pharmaceutical composition described in the third aspect to a subject in need (such as a human), thereby treating and / or preventing HIV.

[0033] In another preferred embodiment, the administration of Aconin is performed before, during and / or after the subject is administered human immunodeficiency virus neutralizing antibodies, or is mixed with the antibodies.

[0034] In another preferred embodiment, the human immunodeficiency virus neutralizing antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2, or a long-acting mutant thereof.

[0035] In another preferred embodiment, the subject includes humans and non-human mammals.

[0036] In another preferred embodiment, the subject is an HIV patient.

[0037] In another preferred embodiment, the HIV is selected from any one or a combination of the following HIV strains:

[0038] 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 virus strains.

[0039] In another preferred embodiment, the HIV is selected from any one or a combination of the following HIV strains:

[0040] 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM235, 97TH-NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03 and CNE23 virus strains.

[0041] In a sixth aspect of the present invention, a kit for detecting HIV is provided, the kit comprising:

[0042] (1) a first container, wherein the first container contains icotinamide;

[0043] (2) a second container comprising HIV neutralizing antibodies; and

[0044] (3) Companion diagnostic reagents for HIV virus.

[0045] In another preferred embodiment, the HIV includes any one or a combination of HIV strains selected from the following groups:

[0046] 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27, and BJOX41 virus strains; or

[0047] The HIV includes any one or a combination of HIV strains selected from the following group: 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM235, 97TH-NP 1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, BCF03, CNE23.

[0048] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 A schematic diagram of sample loading for the neutralization assay is shown;

[0050] Figure 2 The HIV neutralizing antibody of the present invention showed neutralizing activity against HIV-1 Chinese epidemic strain;

[0051] Figure 3 ABT showed neutralizing activity against HIV-1 Chinese epidemic strain;

[0052] Figure 4 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against CNE1 are shown;

[0053] Figure 5 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against CNE15 are shown;

[0054] Figure 6 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against CNE17 are shown;

[0055] Figure 7 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against CNE19 are shown;

[0056] Figure 8 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against CNE23 are shown;

[0057] Fig. 9 The HIV neutralizing antibody of the present invention and ABT showed a synergistic test result of 01 against BJOX25;

[0058] Fig.10 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against BJOX27 are shown;

[0059] Fig.11 The results of the synergistic test of the HIV neutralizing antibody of the present invention and ABT against BJOX41 are shown;

[0060] Fig.12 Genotyping, catalog number, and tropism information for HIV-1 isolates from the NIH AIDS Reagent Program are shown. DETAILED DESCRIPTION

[0061] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that iconin and human immunodeficiency virus neutralizing antibodies have a good synergistic effect in treating viruses (especially HIV). The combined use of iconin and human immunodeficiency virus neutralizing antibodies can effectively inhibit the growth of HIV virus, and its effect is far better than the additive effect of the two. On this basis, the present invention was completed.

[0062] Preferably, the HIV virus includes viruses CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 from Chinese prevalent strains, and HIV strains 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92U G00 1, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, and ADA strains from NIH AIDS.

[0063] the term

[0064] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0065] As used herein, the term "first active ingredient" refers to icotinamide. It should be understood that the term includes derivatives having the same function as icotinamide.

[0066] As used herein, the term "second active ingredient" refers to human immunodeficiency virus neutralizing antibodies and long-acting mutants.

[0067] As used herein, the term "long-acting mutant" refers to a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid with respect to the amino acid sequence shown in SEQ ID NO: 1 or 2, and is capable of retaining binding affinity to human immunodeficiency virus. Further, the long-acting mutant of the present invention refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These long-acting mutants are preferably generated by amino acid substitution according to Table A.

[0068] Table A

[0069] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0070] As used herein, the terms "active ingredient combination of the present invention", "substance combination of the present invention" and "pharmaceutical ingredient combination of the present invention" are used interchangeably to refer to the combination of the first active ingredient and the second active ingredient described above.

[0071] As used herein, the term "pharmaceutical composition of the present invention" refers to a composition comprising or containing a first active ingredient (or a first preparation containing the first active ingredient) and a second active ingredient (or a second preparation containing the second active ingredient), wherein the first preparation and the second preparation may be the same or different preparations. In addition, the first preparation and the second preparation may be the same preparation, or separate preparations.

[0072] The HIV neutralizing antibody of the present invention can specifically bind to the CD4 binding site of HIV-1 outer membrane protein gp120. The HIV neutralizing antibody of the present invention is composed of 1318 amino acid residues, has a molecular weight of 146280 Daltons, and has a half-life of 17.6 days in a healthy human body. Preclinical and clinical results show that the antibody not only has a highly efficient and extensive virus neutralization ability, but also can activate the immune response and delay the time of viral rebound after drug withdrawal. In vitro tests show that the combination of Aconin and the antibody has synergistic or additive antiviral activity.

[0073] Both Aconin and the antibody have unique new mechanisms of action. They act on different envelope glycoproteins of HIV, respectively blocking the process of its entry into cells. Their combined use is expected to cover a wider range of virus strains, enhance and prolong the antiviral effect, and reduce the development of drug resistance. The two have the common characteristics of long half-life and intravenous administration. Their combined use can form a complete new formula of fully injectable long-acting anti-AIDS drugs. The frequency of dosing is expected to be reduced to once a week, every two weeks, every month, or every few months, changing the daily medication treatment method.

[0074] As used herein, "human immunodeficiency virus neutralizing antibodies of the present invention", "HIV neutralizing antibodies of the present invention", and "human immunodeficiency virus HIV neutralizing antibodies of the present invention" are used interchangeably and refer to human immunodeficiency virus neutralizing antibodies containing the heavy chain and light chain sequences shown below, which include a heavy chain (SEQ ID NO: 1) and a light chain (SEQ ID NO: 2):

[0075] Heavy chain (SEQ ID NO: 1):

[0076] QVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKTGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVW GSGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0077] Light chain (SEQ ID NO: 2):

[0078] DIQMTQSPSSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0079] As used herein, "Aconin", "Abovetate" and "ABT" are used interchangeably and refer to a compound having a structure as shown below. The structure of Aconin in the present invention is as follows:

[0080]

[0081] Expression vector

[0082] As used herein, the term "expression vector" refers to a vector that can transfer a polynucleotide sequence of interest to a target cell. Such vectors can replicate themselves or integrate into host cell chromosomes (host cells include, for example, prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals, and plant individuals, etc.), and may contain a promoter at a site suitable for transcription of the polynucleotide of the present invention. The expression vector may contain a structural gene and a promoter that regulates its expression, in addition, there are various regulatory elements that can function in host cells. It is well known in the art that the type of expression vector for a living organism (such as an animal) and the type of regulatory elements used can vary depending on the type of host cell used.

[0083] The viral vectors that can be used in the present invention are not particularly limited, and can be any viral vector that can utilize the characteristics of the virus to transmit its genome, carry genetic material into other cells, and infect. It can occur in a whole living body or in cell culture. It includes lentiviral vectors, adenoviral vectors, herpes virus vectors, and poxvirus vectors. In the present invention, a preferred expression vector is a lentiviral vector.

[0084] Active ingredient combination, pharmaceutical composition and administration method

[0085] The present invention also provides an active ingredient combination for synergistic treatment of HIV, which can be used to inhibit HIV virus replication. The active ingredient combination of the present invention comprises: an effective amount of icotinib and an effective amount of human immunodeficiency virus neutralizing antibody.

[0086] The present invention also provides a pharmaceutical composition, which comprises the active ingredient combination and a pharmaceutically acceptable carrier.

[0087] Unless otherwise specified, the "combination of active ingredients" herein can be mixed or separately configured into iconin and neutralizing antibodies, used sequentially or simultaneously. The "pharmaceutical composition" can be prepared in the form of a liquid preparation. The preparation mainly includes an effective amount of iconin and an effective amount of human immunodeficiency virus neutralizing antibodies, which can be mixed or separately configured into iconin and neutralizing antibodies, used separately or simultaneously.

[0088] Typically, the icotinib or human immunodeficiency virus neutralizing antibody of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, wherein the pH is generally about 5-9, preferably, about 6-8.

[0089] As used herein, "pharmaceutically acceptable carrier" refers to a carrier used for administration of therapeutic agents, including various excipients and diluents.

[0090] The term refers to pharmaceutical carriers that are not essential active ingredients and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, and buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers. The carriers may also contain cell transfection reagents.

[0091] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and / or cells and can be accepted by humans and / or animals.

[0092] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared as a sustained-release preparation.

[0093] Preferably, the pharmaceutical composition of the present invention can be prepared in the form of a liquid preparation. The preparation mainly includes an effective amount of icotinamide, and an effective amount of human immunodeficiency virus neutralizing antibody; a buffer; a stabilizer; and a surfactant. Among them, the buffer is an acidic buffer, and the acidic buffer can be a buffer of acetic acid, phosphoric acid, tartaric acid, ascorbic acid, malonic acid, maleic acid, fumaric acid, malic acid or oxalic acid; the stabilizer can be selected from one or a combination of two or more of sodium chloride, amino acids, and polyols; the stabilizer can contain a polyol and / or sodium chloride, and the polyol is selected from sucrose, trehalose or sorbitol; the surfactant can be Tween 20 or Tween 80.

[0094] The pharmaceutical composition of the present invention may include one or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutica1 Sciences 16th edition, Osol, A. Ed. (1980), as long as they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used, and include other co-solvents; antioxidants, including ascorbic acid and methionine; integrators, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; and / or salt-forming counterions.

[0095] The pharmaceutical composition of the present invention can be prepared by combining various components at certain concentrations using methods known in the art.

[0096] Furthermore, the active ingredient combinations of the present invention may also be used together with other therapeutic agents such as antiviral agents or immunomodulators.

[0097] When using a pharmaceutical composition, a safe and effective amount of a combination of active ingredients (including a first active ingredient (or its formulation) and / or a second active ingredient (or its formulation)) is administered to a mammal.

[0098] It should be understood that the effective amount of the first active ingredient (or its preparation) and / or the second active ingredient (or its preparation) in the active ingredient combination of the present invention may vary with the mode of administration and the severity of HIV. Preferably, the selection of the effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of HIV, the patient's weight, the patient's immune status, the route of administration, etc.

[0099] Typically, for the first active ingredient, its safe and effective amount is usually at least about 10 ng / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dosage is about 50 ng / kg body weight-about 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0100] For the second active ingredient, its safe and effective amount is usually at least about 10 ng / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dosage is about 50 ng / kg body weight-about 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0101] Specifically, the active ingredient combination of the present invention includes a first active ingredient, icotinib, and a second active ingredient, a human immunodeficiency virus neutralizing antibody.

[0102] In another preferred embodiment, the mass ratio of icotinib to human immunodeficiency virus neutralizing antibody is 1:200 to 50:1, preferably 1:100 to 20:1, and more preferably 1:20 to 10:1.

[0103] In another preferred embodiment, the administration concentration of the icotinib is selected from 0.1-1000 mg / ml; preferably, 0.5-500 mg / ml; more preferably, 2-320 mg / ml; and the administration concentration of the human immunodeficiency virus neutralizing antibody is 0.5-5000 mg / ml; preferably, 1-2000 mg / ml; more preferably, 5-1000 mg / ml.

[0104] In another preferred embodiment, the ratio of icotinib to human immunodeficiency virus neutralizing antibody (nmol / mg) is 0.2-4000nmol / mg, preferably 1-2000nmol / mg, more preferably 5-1000nmol / mg or 10-500nmol / mg.

[0105] There is no particular limitation on the administration method of the pharmaceutical composition of the present invention. Representative examples include (but are not limited to): intravenous injection, subcutaneous injection, intramuscular injection, etc.

[0106] Pill Box

[0107] The present invention provides a medicine box, comprising:

[0108] Component (1): a preparation containing icotinamide;

[0109] Component (2): a preparation containing human immunodeficiency virus neutralizing antibodies;

[0110] Component (3): instructions.

[0111] The preparations containing icotinamide include (but are not limited to): lyophilized preparations, liquid preparations or intravenous injection solutions.

[0112] The preparation containing human immunodeficiency virus neutralizing antibodies includes (but is not limited to): lyophilized preparation, liquid preparation, tablet, capsule, suppository, or intravenous injection.

[0113] In the present invention, the protein preparation is usually a lyophilized preparation or an injection.

[0114] Typically, the kit contains one or more (eg, at least two) unit dosage forms containing icotinib and one or more (eg, at least two) unit dosage forms containing human immunodeficiency virus neutralizing antibodies; preferably, 2-20 of each.

[0115] As used herein, the term "unit dosage form" means that for the convenience of taking, the composition is prepared into a dosage form required for a single dose, including but not limited to various solid doses (such as tablets), liquid doses, capsules, and sustained-release preparations.

[0116] The instructions provided by the present invention may include the following description: the method of using the drug kit is to simultaneously use a unit dosage form containing icotinib and a unit dosage form containing human immunodeficiency virus neutralizing antibodies.

[0117] The medicine box provided by the present invention is prepared by the following steps: placing a preparation containing icotinib, a preparation containing human immunodeficiency virus neutralizing antibodies, and instructions together to form a medicine box.

[0118] The preparation containing icotinib preferably contains a unit dosage form of icotinib, and the preparation containing human immunodeficiency virus neutralizing antibodies preferably contains a unit dosage form of human immunodeficiency virus neutralizing antibodies.

[0119] The step preferably places at least one unit dosage form containing icotinib and at least one unit dosage form containing human immunodeficiency virus neutralizing antibodies, as well as instructions, together to form a medicine kit.

[0120] The main advantages of the present invention include

[0121] 1. Aconin and human immunodeficiency virus neutralizing antibodies can work synergistically in anti-HIV treatment, thereby more significantly inhibiting the proliferation of HIV virus.

[0122] 2. Since Abovirtadine and HIV neutralizing antibodies act on gp41 and gp120 of the viral glycoproteins of HIV virus entering human T cells, respectively, gp41 and gp120 form gp160. First, the gp120 part of HIV binds to human CD4, triggering the binding of auxiliary receptors in the human body, which leads to conformational changes and dissociation of gp120 from gp41, fully exposing gp41 and forming a trimer transition state, which then fuses to form a hexameric virus and successfully enters human T cells. The combined use of icotinib, which acts on gp41, and HIV neutralizing antibodies, which act on gp120, is beneficial to improving the defect of insufficient viral coverage of antibodies or ABTs; experiments have shown that the combined use of icotinib and the HIV neutralizing antibodies of the present invention has synergistic and broad-spectrum antiviral activity against a variety of HIV strains, thereby providing a new and more effective option for the treatment and / or prevention of HIV; and because of its synergy and broad-spectrum against many HIV viruses, it can more effectively detect HIV virus infection.

[0123] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0124] Reagents and sample preparation

[0125] 1) Ghost (3) X4 / R5 cells, 293T cells, HIV-1 membrane protein expression plasmid, HIV-1 backbone plasmid pNL4-3.Luc.RE- were all kept by the laboratory;

[0126] 2) Human serum albumin, Albumin human, batch number: SLBS2806V, Sigma, USA;

[0127] 3) DMEM cell culture medium, 8117156, Gibco, USA;

[0128] 4) Fetal bovine serum: FBS, batch number: 1856032, Gibco, USA;

[0129] 5) 0.25% trypsin: Trypsin-EDTA 0.25%, batch number: G1509200, Macgene, USA;

[0130] 6) Luciferase Assay Kit: Bright-Glo TM Luciferase Assay System, batch number: 173MB, Beijing Promage Company.

[0131] 7) HIV neutralizing antibody (Lot#201307A) and Abovirtide (Lot No. 151201) of the present invention were provided by Frontier Biopharmaceuticals (Nanjing) Co., Ltd.

[0132] 8) ABT-albumin preparation:

[0133] ① Prepare 25% human serum albumin (3.79 mM HSA) solution.

[0134] ② Prepare 10 mM (46.66 mg / mL) Abovirtide-dimethyl sulfoxide (DMSO) solution.

[0135] ③ Prepare 1 mM Abavirtide-HSA (molecular ratio of about 1:3) solution.

[0136] ④Incubate in 37℃ water bath for 3 hours. Aliquot and store at 4℃.

[0137] 9) Experimental instruments and equipment: Microplate luminometer, Berthold Technologies, Germany.

[0138] 10) Experimental data analysis software: Graphpad Prism 5.0, used to process, analyze and plot experimental data.

[0139] General Methods

[0140] 1. HIV-1 pseudovirus packaging

[0141] (1) Resuspend 1×10 7 293T cells were inoculated into one dish and cultured overnight at 37°C, 5% CO2. On the day of transfection, the coverage of the monolayer of adherent cells should reach 80%-90%.

[0142] (2) Add appropriate amount of HIV-1 membrane protein expression plasmid and pNL4-3.Luc.RE- to an EP tube containing serum-free DMEM medium. Then add appropriate amount of PEI to the above mixture, mix well, and incubate at room temperature to form a transfection complex. Add the transfection complex to the culture dish of 293T cells and mix well.

[0143] (3) Culture at 37°C, 5% CO2 for 6-8 hours to allow the plasmid to be transfected into the cells. Carefully remove the culture medium containing the DNA-PEI transfection complex, add an appropriate amount of fresh DMEM medium containing 10% FBS, and continue culturing.

[0144] (4) After continued cultivation, the culture supernatant containing pseudovirus particles was collected from the culture dish, centrifuged to remove cell debris and impurities, and the supernatant was collected and stored in aliquots at -80°C.

[0145] 2. Determination of pseudovirus titer using Ghost(3)X4 / R5 cells

[0146] (1) 1×10 4 Ghost(3)X4 / R5 cells were cultured overnight (at least 15 hours).

[0147] (2) Dilute the virus with DMEM at a ratio of 1:2 and 1:10, respectively, and add 100 μl of the diluted virus solution to each well of the 96-well plate inoculated with cells, and incubate.

[0148] (3) After incubation, remove all culture supernatants from the cell culture wells, add appropriate amounts of PBS and luciferase detection reagent, and place in the dark for 5 min. After pipetting, transfer the cell lysis liquid to a 96-well white-bottom plate, and measure the luciferase activity using a chemiluminescence instrument.

[0149] 3. Pseudovirus Neutralization Test

[0150] (1) Add 300 μl PBS to the surrounding wells of the 96-well plate (the surrounding wells evaporated severely and were not used for the experiment). See the schematic diagram of the neutralization test sample addition for details. Figure 1 Each sample was tested in triplicate.

[0151] HIV neutralizing antibody neutralization test of the present invention: 100 μl of 10% FBS DMEM medium was added to each of the 60 wells in the middle, and the two leftmost columns were virus control (Virus Control, VC, only virus and cells were added) and cell control (Cell Control, CC, only cells were added). 50 μl of medium was added to the CC column (a total of 150 μl), and 39 μl of medium was added to the 11th column (a total of 139 μl). Among them, S1 and S2 are the wells of different concentrations in the sample addition column.

[0152] Peptide Abovirtide Neutralization Test: Add 100 μl 10% FBS DMEM medium to each well in column VC, add 150 μl 10% FBS DMEM medium to each well in column CC, and do not add medium to columns 4 to 11 until the peptide is diluted in multiples.

[0153] (2) Concentration gradient setting

[0154] HIV neutralizing antibody neutralization test of the present invention: add 11 μL of antibody (11 μg, 1 mg / ml) to each well in the 11th column, and the total volume is 150 μL. After mixing with a dispenser, aspirate 50 μL to the 11th column, and after mixing, aspirate 50 μl and transfer to the 10th column, and repeat this process to the left for 3-fold serial dilution, and finally aspirate 50 μL from the 4th column and discard. The final concentrations of antibodies from the 4th column to the 11th column are 0.022, 0.067, 0.201, 0.604, 1.811, 5.432, 16.296, 48.889 (μg / ml), respectively. The final volume of each well is 100 μl. The cell control and virus control wells do not participate in the serial dilution.

[0155] Peptide Abovirte neutralization test: The peptide ABT was diluted 8-fold with DMEM medium containing 10% FBS. The final concentrations of ABT-albumin (in terms of ABT) from column 4 to column 11 were 0.0233, 0.186, 1.491, 11.936, 95.486, 763.889, 6111.111, 48888.89 (nM), respectively. First, the concentrations of each column were configured and then 100 μl of peptide was added in sequence. The final volume of each well was 100 μl. Cell control and virus control wells were not involved in the dilution.

[0156] (3) Except for the cell control well, add 50 μl of pseudovirus with a titration reading of more than 100,000 to each well. Place the 96-well plate in a 37°C, 5% CO2 incubator to allow the antibody to fully bind to the virus particles. Then, remove the 96-well plate and add Ghost(3)X4 / R5 cells to all the middle 60 wells. Continue culturing for 48-72 hours.

[0157] (4) After 48-72 hours, remove the culture supernatant, add PBS, and then add luciferase detection reagent to measure luciferase activity and calculate the inhibition percentage. Fit the neutralization curve and calculate IC 50 The calculation formula of neutralization inhibition rate is: inhibition rate = [1-(luminescence intensity mean value of sample group-blank control CC mean value) / (luminescence intensity VC mean value of negative group-blank control CC mean value)] × 100%.

[0158] 4. Neutralization synergy test of HIV neutralizing antibodies and ABT of the present invention

[0159] IC obtained by neutralization test of HIV neutralizing antibody and ABT alone according to the present invention 50 The maximum concentrations of the HIV neutralizing antibodies and ABT of the present invention are set to 3 4 IC 50 value, perform 3-fold serial dilution, a total of 8 dilutions (the concentration of the 5th dilution is approximately IC 50Value), 3 wells were measured for each sample. The above test system was used to evaluate the neutralizing activity of the HIV neutralizing antibody of the present invention and ABT in combination. CompuSyn software (ComboSyn Inc., Paramus, NJ) was used to evaluate whether the HIV neutralizing antibody of the present invention and ABT combined have synergistic, additive or antagonistic antiviral activity according to the median effect analysis method (Chou and Talalay). The three main parameters used to evaluate the synergistic test are fractional effects (FA), combined index (CI), and logCI. In this experiment, FA refers to the neutralization percentage of the HIV neutralizing antibody, ABT, etc. of the present invention on the virus; CI is the combined use index, and logCI is the log value of CI. When CI>1, that is, logCI>0, it indicates that the two drugs have an antagonistic effect; when CI=1, that is, logCI=0, it indicates that the two drugs have an additive effect; when CI<1, that is, logCI<0, it indicates that the two drugs have a synergistic effect.

[0160] Example 1 Neutralizing activity of HIV neutralizing antibodies against HIV-1 Chinese epidemic strains

[0161] The test evaluated the neutralizing activity of the HIV neutralizing antibody of the present invention against 22 pseudoviruses of HIV-1 epidemic strains in acute and chronic phases in China. VRC01 was used as a positive control, and the neutralizing activity was consistent with the laboratory's previous data, and the methodology was reliable.

[0162] The test results show that the HIV neutralizing antibody of the present invention can neutralize 77.2% of the viruses prevalent in China, and its IC 50 and IC 90 The median values ​​were 0.06 and 0.78 μg / ml, respectively, and the average IC 50 and IC 90 The HIV neutralizing antibody of the present invention cannot neutralize CNE6, CNE7, CNE15, CNE23 and BJOX41 virus strains, and its IC 50 >50μg / ml. See Table 1. Figure 2 .

[0163] Table 1. Neutralizing activity of the HIV neutralizing antibodies of the present invention against HIV-1 Chinese epidemic strains

[0164]

[0165] *:Except IC 50 or IC90>50μg / ml

[0166] Example 2 Neutralizing activity of ABT against HIV-1 pseudovirus of Chinese epidemic strain

[0167] The test evaluated the neutralizing activity of ABT against 22 pseudoviruses of HIV-1 prevalent in China in the acute and chronic phases. VRC01 was used as a positive control, and the neutralizing activity was consistent with the laboratory's previous data. The test results showed that the median IC of ABT against the prevalent virus in China was 50 and IC 90 The average IC 50 and IC 90 71.41±116.26 and 2101.42±2258.81 nM, see Table 2, Figure 3 .

[0168] Neutralization activity test is mainly used to evaluate the neutralization ability of monoclonal antibodies against HIV-1 virus. Abovirta is a synthetic peptide of 34 amino acids. The results of this test show that Abovirta has a certain neutralization ability against HIV virus. However, individual differences in the test are large, and IC 50 From 0.00343 to 254.849nM.

[0169] Table 2. Neutralizing activity of ABT against HIV-1 epidemic strains in China

[0170]

[0171] Example 3 Neutralization synergy test of HIV neutralizing antibodies and ABT

[0172] The experiment evaluated the neutralizing activity of the HIV neutralizing antibody of the present invention and ABT in combination against 8 pseudoviruses of HIV-1 epidemic strains in China in the acute and chronic phases.

[0173] As shown in Tables 3 and 4, the experiments show that the HIV neutralizing antibody of the present invention and ABT used in combination have synergistic anti-disease activity against six virus strains CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41; an additive effect on the CNE23 virus; and an antagonistic effect on CNE19.

[0174] Table 3. Synergistic effect of neutralizing activity of HIV neutralizing antibodies and ABT of the present invention

[0175]

[0176] Table 4. Neutralization activity results of HIV neutralizing antibodies of the present invention and ABT synergistic test

[0177]

[0178] (1) Synergistic effect on CNE1

[0179] The HIV neutralizing antibody of the present invention and ABT both have neutralizing activity against CNE1. When the HIV neutralizing antibody of the present invention is used in combination with ABT, the neutralizing effect is significantly improved, and a synergistic relationship exists (Table 5, Figure 4 ).

[0180] Table 5. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against CNE1

[0181]

[0182] (2) Synergistic effect on CNE15

[0183] The HIV neutralizing antibody of the present invention cannot neutralize CNE15, but the combination of the HIV neutralizing antibody of the present invention and ABT can improve the neutralizing activity of ABT against CNE15, and there is a synergistic effect (Table 6, Figure 5 ).

[0184] Table 6. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against CNE15

[0185]

[0186] (3) Synergistic effect on CNE17

[0187] The HIV neutralizing antibody and ABT of the present invention both have neutralizing activity against CNE17. 50 The neutralizing activity of the combination of the two drugs increased above the concentration, indicating a synergistic effect at the therapeutic concentration. The neutralizing activity of the combination of the two drugs decreased at lower concentrations, indicating possible antagonism (Table 7, Figure 6 ).

[0188] Table 7. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against CNE17

[0189]

[0190] (4) Antagonism against CNE19

[0191] ABT and the HIV neutralizing antibody of the present invention both have neutralizing activity against CNE19. The combined use showed no significant change in neutralizing activity. The combined use index was greater than 1, which may indicate a certain antagonistic effect (Tables 8-9, Figure 7 ).

[0192] Table 8. Results of the synergistic test of the HIV neutralizing antibodies and ABT against CNE19 of the present invention

[0193]

[0194] Table 9. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against CNE19

[0195]

[0196] (5) Additive effect on CNE23

[0197] The HIV neutralizing antibody of the present invention has no neutralizing activity against CNE23. When the HIV neutralizing antibody of the present invention is used in combination with ABT, the neutralizing effect is similar to that of ABT alone, and there is an additive effect (Table 10, Figure 8 ).

[0198] Table 10. IC values ​​of neutralizing activity of HIV neutralizing antibodies and ABT against CNE23 50

[0199]

[0200] (6) Synergistic effect on BJOX25.01

[0201] The neutralizing activity of the HIV neutralizing antibody of the present invention on BJOX25.01 is relatively weak, but the neutralizing effect is improved after the combination of ABT and the HIV neutralizing antibody of the present invention, and there is a synergistic effect (Table 11, Fig. 9 ).

[0202] Table 11. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against BJOX25.01

[0203]

[0204] (7) Synergistic effect on BJOX27

[0205] Both ABT and the HIV neutralizing antibody of the present invention have a neutralizing effect on BJOX27. When the HIV neutralizing antibody of the present invention is used in combination with ABT, the neutralizing effect of the single drug is improved, and a synergistic effect exists (Table 12, Fig.10 ).

[0206] Table 12. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against BJOX27

[0207]

[0208] (8) Synergistic effect on BJOX41

[0209] The HIV neutralizing antibody of the present invention cannot neutralize BJOX41, but the combination of the HIV neutralizing antibody of the present invention and ABT can improve the neutralizing activity of ABT against BJOX41, and there is a synergistic effect (Table 13, Fig.11 ).

[0210] Table 13. Neutralizing activity of HIV neutralizing antibodies and ABT of the present invention against BJOX41

[0211]

[0212] Example 4 Activity test of HIV neutralizing antibodies and ABT against American HIV virus

[0213] Using methods similar to those in Examples 1-3, the combined administration of the HIV neutralizing antibodies of the present invention and ABT was evaluated for the effects of 32 HIV strains ( Fig.12 ) neutralizing activity.

[0214] Determination of the combined effect:

[0215] Combination effects were determined using Chou and Talalay and CompuSyn software (CalcuSyn 2.0, BIOSOFT PO Box 1013, Great Shelford, Cambridge CB22 5WQ GB-United Kingdom, http: / / www.biosoft.com / w / calcusyn.htm). Raw relative light unit data were expressed as a fraction of the maximum signal control (cells + virus) after subtracting the background signal control (cells only) using the equation: 1-(experimental value / average virus control value). The 50% HIV inhibition (CI 50 ) under the combination index value. The combination index (CI) value represents the combined effect of two or more compounds. CI values ​​<0.5 indicate strong compound combination synergy; >0.5 to 0.75 indicate synergy; ~0.76-1.25 indicate additivity; 1.26-1.5 indicate antagonism, and >1.5 indicates high antagonism. The dose reduction index (DRI) can also be calculated and indicates how much the compound concentration can be reduced to produce the same effect when acting alone (not in combination) when acting in combination.

[0216]

[0217] Where, CI = combination index, IC = inhibition concentration, x = percentage parameter of effect, A = dose of drug A at time x, B = dose of drug B at time x, A+B = dose of combined drug at time x.

[0218]

[0219] Where DRI = dose reduction index, IC = inhibitory concentration, x = percentage parameter of effect, A = dose of drug A at time x, B = dose of drug B at time x, A+B = dose of the combination drug at time x.

[0220] Experimental results: The results of the combination index, inhibition concentration value and dose reduction index at half HIV replication are shown in Table 14 below. Among them, CI values ​​<0.5 indicate strong compound combination synergy; >0.5 to 0.75 indicate synergy; ~0.76-1.25 indicate additivity; 1.26-1.5 indicates antagonism, and >1.5 indicates high antagonism.

[0221] The results showed that the HIV neutralizing antibody of the present invention and ABT in combination had a synergistic effect on 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, and ADA strains; in particular, it had a strong synergistic effect on 931N101 and 02ET14 strains (CI 50 <0.5); it showed an additive effect on 92UG037, 00KE_KER2008, 91US001, 91US004, 96TH_NP1538, JR-CSF, 98US_MSC5016, 02ET288, 00UG_D26830M4, 93BR019, 93BR029, JV1083, 90TH_CM235, 97TH-NP1525, 91DJ263, 02CM_0014BBY, BCF01, BCF02, and BCF03.

[0222] Table 14. Summary of Combination Index, Inhibitory Concentration Values ​​and Dose Reduction Index at Half-Maximal HIV Replication

[0223]

[0224]

[0225] 1 ABT-HSA:3BNC1 17(μg / mL:μg / mL)

[0226] 2 Half-maximal inhibition combination index (C1 50 ) + / - standard deviation (SD), N = 3

[0227] 3 CI 50Interpretation of values: <0.5 indicates strong synergistic effect; 0.5 to 0.75 indicates synergistic effect; ~0.76-1.25 indicates additive effect; 1.26-1.5 indicates antagonistic effect; >1.5 indicates highly antagonistic effect (Syn = Synergistic, Add = Additive)

[0228] 4 Two isolates out of three experiments were not susceptible to 3BNC117

[0229] 5 It cannot be calculated; it can be inferred that the combined effect is additive;

[0230] 6 Taking into account the error (SD), DRI ~ 1.

[0231] It is noteworthy that the HIV neutralizing antibodies of the present invention, when used alone, are insensitive to HIV strains 97TH-NP 1525, BCF01, BCF02, and BCF03, but the neutralization effect is improved after combined use with ABT, and the effect on the strains is additive;

[0232] The HIV neutralizing antibody of the present invention has low activity against HIV strains 92UG001 and 01CM_0002BBY when used alone, but the neutralization effect is improved after combination with ABT, and has a synergistic effect on the strain. The above results show that the combination of the HIV neutralizing antibody of the present invention and ABT expands the broad spectrum of antiviral activity of the HIV neutralizing antibody of the present invention and shows a synergistic effect against a large number of viruses.

[0233] in conclusion

[0234] The present invention evaluated the neutralizing activity of the HIV neutralizing antibody and ABT against 22 HIV-1 pseudoviruses of acute and chronic epidemic strains in China through experiments. The results showed that the HIV neutralizing antibody of the present invention has a broad-spectrum neutralizing activity against the Chinese epidemic strains and can neutralize 77.2% of the viruses. The median IC of the HIV neutralizing antibody of the present invention against 17 Chinese epidemic strains is 50 The median IC 90 The HIV neutralizing antibody of the present invention cannot neutralize CNE6 of B' subtype, BJOX41 of B subtype, CNE7 and CNE15 of B'C subtype, and CNE23 of CRF_08BC subtype. Its IC 50 >50μg / ml. Abovir has neutralizing activity against the Chinese epidemic strain, with a median IC 50 The average IC 50 It is 71.41±116.26nM.

[0235] The in vitro combined antiviral activity showed that the HIV neutralizing antibody and ABT of the present invention had synergistic antiviral activity against six virus strains, namely, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41; an additive effect on the CNE23 virus; and an antagonistic effect on CNE19.

[0236] The test in Example 4 evaluated the neutralizing activity of the HIV neutralizing antibody and ABT of the present invention against 32 HIV strains from the NIH AIDS reagent project, showing broad-spectrum antiviral activity against multiple HIV virus subtypes including A, B, C, D, F, G, BF, CRF01_AE, CRF02_AG, Group O, etc. The in vitro combined antiviral activity showed that the HIV neutralizing antibody and ABT of the present invention had synergistic or additive antiviral activity against the above 32 HIV strains.

[0237] This shows that the combined use of ABT and HIV neutralizing antibodies can help improve the insufficient viral coverage of antibodies or ABT, and has synergistic and broad-spectrum antiviral activity against multiple HIV strains, thus providing a new and more effective option for the treatment and prevention of HIV.

[0238] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Frontier Biopharmaceuticals (Nanjing) Co., Ltd. <120> Active combination for combined treatment of human immunodeficiency virus and its application <130> P2022-1082 <150> 202110872475X <151> 2021-07-30 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> HIV neutralizing antibody heavy chain <400> 1 Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr 20 25 30 Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val 35 40 45 Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe 50 55 60 Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr 65 70 75 80 Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala 85 90 95 Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val 100 105 110 Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 2 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> HIV neutralizing antibody light chain <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp Tyr 20 25 30 Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly Ser 35 40 45 Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp Gly 50 55 60 Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala 65 70 75 80 Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg Leu 85 90 95 Asp Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 100 105 110 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 115 120 125 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 130 135 140 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 145 150 155 160 Light Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Light Ala 165 170 175 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 180 185 190 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 195 200 205

Claims

1. An active ingredient combination, characterized in that The active ingredient combination comprises an effective therapeutic amount of icotinib and a human immunodeficiency virus neutralizing antibody, wherein the human immunodeficiency virus neutralizing antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO:

2.

2. The active ingredient combination according to claim 1, characterized in that The mass ratio of icotinib to human immunodeficiency virus neutralizing antibody is 1:200 to 50:

1.

3. The active ingredient combination according to claim 1, characterized in that The mass ratio of icotinib to human immunodeficiency virus neutralizing antibody is 1:20 to 10:

1.

4. The active ingredient combination according to claim 1, characterized in that The administration concentration of the icotinib is selected from 0.1-1000 mg / ml; and the administration concentration of the human immunodeficiency virus neutralizing antibody is 0.5-5000 mg / ml.

5. The active ingredient combination according to claim 1, characterized in that The administration concentration of the icotinib is selected from 2-320 mg / ml; and the administration concentration of the human immunodeficiency virus neutralizing antibody is 5-1000 mg / ml.

6. The active ingredient combination according to claim 1, characterized in that The ratio of icotinin to human immunodeficiency virus neutralizing antibody is 0.2-4000nmol / mg.

7. The active ingredient combination according to claim 1, characterized in that The ratio of icotinib to human immunodeficiency virus neutralizing antibody is 5-1000nmol / mg.

8. The active ingredient combination according to claim 1, characterized in that The ratio of icotinib to human immunodeficiency virus neutralizing antibody is 10-500nmol / mg.

9. The active ingredient combination according to any one of claims 1 to 8, characterized in that The active ingredient combinations may also include additional therapeutic agents.

10. The active ingredient combination according to claim 9, characterized in that The additional therapeutic agents include anti-infective drugs.

11. The active ingredient combination according to claim 10, characterized in that The anti-infective drugs include antiviral agents.

12. A medicine box, characterized in that: The medicine kit comprises: (a) a first preparation, wherein the first preparation contains icotinib and a pharmaceutically acceptable carrier; (b) a second preparation comprising a human immunodeficiency virus neutralizing antibody and a pharmaceutically acceptable carrier; (c) instructions describing a method of using icotinib in combination with a human immunodeficiency virus neutralizing antibody to treat and / or prevent HIV, and a method of using icotinib in combination with other antiviral drugs to treat and / or prevent HIV; The human immunodeficiency virus neutralizing antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO:

2.

13. The kit according to claim 12, wherein: The medicine box also includes anti-infective drugs.

14. A pharmaceutical composition comprising the active ingredient combination according to any one of claims 1 to 11, and further comprising a pharmaceutically acceptable carrier.

15. Use of an active ingredient combination in the preparation of a pharmaceutical composition or a drug kit for treating HIV, the active ingredient combination comprising a first active ingredient, icotinib, and a second active ingredient, a human immunodeficiency virus neutralizing antibody; the HIV comprises any one or a combination of HIV virus strains selected from the following group: 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27, and BJOX41 virus strains; The human immunodeficiency virus neutralizing antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO:

2.

16. The use according to claim 15, characterized in that The mass ratio of iconin to human immunodeficiency virus neutralizing antibodies is 1:200 to 50:1; the administration concentration of iconin is selected from 0.1-1000 mg / ml; and the administration concentration of human immunodeficiency virus neutralizing antibodies is 0.5-5000 mg / ml; the ratio of iconin to human immunodeficiency virus neutralizing antibodies is 0.2-4000 nmol / mg.

17. The use according to claim 15, characterized in that The HIV includes any one or a combination of HIV strains selected from the following groups: 92US159, 99KE_KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 virus strains.

18. A kit for detecting HIV, characterized in that: The kit comprises: (1) a first container, the first container containing icotinamide; and (2) a second container, wherein the second container contains an HIV neutralizing antibody; the neutralizing antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2; (3) Companion diagnostic reagents for HIV virus; The HIV includes any one or a combination of HIV strains selected from the following groups: 92US159, 99KE KNH1088, 92BR025, 931N101, 02ET14, 92UG001, 00KENKU3006, 93BR020, G3, 96TH NI1149, 01CM_0002BBY, SF162, ADA, CNE1, CNE15, CNE17, BJOX25.01, BJOX27 and BJOX41 virus strains.

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