RU2536956C1 - Antiviral similar mini-antibody, nucleotide sequence, expressing recombinant viral vector, pharmaceutical composition and method for prevention or therapy of influenza type a - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to virology and implies using the similar mini-antibodies for the prevention and therapy of influenza. What is declared is a similar mini-antibody specifically binding to a certain epitope of influenza A (H5N2) hemagglutinin and suppressing the influenza A (H5N2) infection. What is created is an adenovirus viral vector expressing the similar mini-antibody, which can effectively bind to the certain epitope of influenza hemagglutinin and thereby block the influenza progression. What is presented is a composition containing an effective amount of the similar mini-antibody and the viral vector expressing this similar mini-antibody.

EFFECT: what is presented is a method for the prevention and therapy of the influenza A (H5N2) infection providing administering the preventive or therapeutic effective amount of the pharmaceutical composition intranasally in the form of drops or spray into the patient in need thereof.

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Description

The technical field of the present invention

The present invention relates to the field of immunology and virology, and involves the use of antibodies, which are single-domain mini-antibodies, for the prevention and treatment of influenza, as well as the use of the genes of these antibodies in combination with a viral vector (vector based on recombinant adenovirus) and their expression in the body mammals for the prevention and treatment of influenza.

BACKGROUND OF THE INVENTION

Influenza viruses can cause epidemic emergencies. These viruses are widespread in nature and can infect humans, many types of mammals (horses, pigs, seals, etc.), as well as all types of birds. Respiratory diseases caused by influenza viruses in some cases can occur with complications and result in death.

Influenza viruses are characterized by high antigenic variability. The surface virion glycoproteins, hemagglutinin and neuraminidase, are most susceptible to changes. Two ways to change them are known.

The first is antigenic drift. Under the pressure of population immunity, mutations that allow you to get out of control of the immune system give the virus a serious advantage and become fixed. As a result, new antigenic variants of hemagglutinin and neuraminidase are constantly replacing each other. As a result, epidemics arise, as protection against previous strains of viruses, including the same subtype, is insufficient to neutralize the new strain. Unfortunately, the use of modern subunit and inactivated vaccines does not solve this problem, because provides good protection only from the strain from which they were derived. Therefore, currently, to protect the population from new epidemic strains, it is constantly necessary to create new vaccines.

The second way to change influenza viruses is antigenic shift (shift), i.e. a change in the antigenic formula of the virus as a result of the replacement of the gene (s) and the corresponding protein (s). The mechanism of antigenic shift is based on reassortment or recombination of genes, which can occur during joint infection with two or more viruses [Kochergin-Nikitsky K.S. // Analysis of gene interaction when crossing a low pathogenic avian influenza virus subtype H5 and a highly productive strain of human influenza virus. M., GU Research Institute of Virology. DI. Ivanovo RAMS. 2007]. Shift changes, as a rule, affect the antigenic structure of hemagglutinin, less often neuraminidase. Thus, at irregular time intervals, pandemic variants of influenza viruses with new antigenic and biological properties that cause severe illness and death of people appear [Lvov D.K. // Sov. Med. Rev. E. Virol. Rev. 1987. V.2. P.15-37; Webster R.G., Bean W.J., German O.T., et al. // Microbiol. Rev. 1992. V. 56. P.152-179]. For example, the pandemic of the "Spanish woman" - the H1N1 flu virus, in 1918-1920. led to the death of about 50 million people around the world. In June 2009, the World Health Organization (WHO) assigned the sixth degree of a pandemic threat to the new H1N1 virus, cases of which began to be detected in early April 2009. This virus is a reassortant containing the genes of avian, human and swine viruses [Shinde V., Bridges S.V., Uyeki T.M. // The New England journal of medicine. 2009. V.360; 25. P.2616-2625]. With the emergence of a new pandemic strain, the creation of a vaccine takes too much time, which does not allow you to quickly stop the spread of a dangerous virus. Thus, in spite of the ongoing large-scale preventive measures, including vaccination, in many countries of the world, including Russia, seasonal outbreaks of influenza are recorded annually, covering all sectors of the population from children to the elderly. To solve this problem, new approaches to the prevention and treatment of influenza are needed.

In addition to flu vaccines, there is now a wide selection of medicines. However, it should be noted that prophylaxis with such drugs does not have sufficient effectiveness, in addition, many drugs have a wide range of contraindications and can cause adverse reactions.

Currently, several groups of drugs are used for the treatment and prevention of influenza, among which M2-channel inhibitors and neuraminidase inhibitors occupy a special place. Of the drugs of the first group in our country, rimantadine has been used for many years, which had a pronounced therapeutic and prophylactic effect in case of influenza caused by type A virus.However, the widespread use of M2-channel inhibitors led to the emergence of resistant strains of influenza viruses resistant to remantadine. Of the drugs of the second group, zanamivir and oseltamivir are registered in Russia. Zanamivir is not suitable for widespread use in clinical practice, as can be used only in the form of inhalation, which is unacceptable for preschool children and elderly patients. In addition, a number of adverse reactions are possible, including bronchospasm and laryngeal edema. Another drug, oseltamivir (Tamiflu), has established itself as a highly effective and safe drug, but one of the most fundamental drawbacks of this drug is the need for its early use (most effective when taken in the first 36 hours of the disease).

The problem of timely access to medical care and early treatment of respiratory viral infections is one of the most acute practical health problems. According to the Institute of Influenza, Russian Academy of Medical Sciences, the majority of patients seek medical help only on the 2nd-3rd day, when it becomes obvious that the patient is not ill with a simple cold and you can not stop with treatment only with antipyretic drugs, however, patients come with severe forms of influenza and viral pneumonia in the clinic of the Institute of influenza RAMS only on the 5th day. In this regard, neuraminidase inhibitors cannot be widely used in real conditions of an emerging epidemic.

Thus, we can conclude that today there are no effective and safe drugs for the treatment or prevention of influenza.

The solution to this problem can be the creation of drugs based on antibodies obtained against type A influenza virus. The action of these drugs is based on the introduction of antibodies to the protective antigen of the pathogen into the body, which can protect the host from infection. This approach has been applied since the first half of the 20th century. Until the late 1960s in the USSR, hyperimmune horse serum was relatively widely used for the prevention and treatment of influenza. However, often as an adverse reaction, this drug caused an allergy, and in this regard, he had to be abandoned. But the very idea of using antiviral antibodies in the blood of people is still being implemented as blood products. These drugs are called immunoglobulins. The most effective and common drug in Russia is “specific influenza immunoglobulin” from the blood of donors specially and repeatedly vaccinated against influenza. In addition to anti-influenza antibodies specifically stimulated by vaccination, donor immunoglobulin contains antibodies to many common infectious agents, including respiratory viruses. In recent years, there have been reports of the formation of antibodies to the introduced human protein. The drug is not without other side effects: fever, allergization: from the appearance of a rash to the development of a serious condition and anaphylactic shock.

Immunoglobulin preparations obtained by genetic engineering methods lack some of these drawbacks. Thus, the effectiveness of monoclonal single chain antibodies (scFv) obtained against anthrax protein toxin [Mabry R., Infect Immun. 2005; 73: 8362-8368.]. However, extremely high doses of protein are used, which is a significant limitation. Humanized single-stranded single-domain mini-antibodies are not rejected by the human body and do not cause an allergic reaction. However, this type of antibody has several disadvantages: the high cost of their production and the limitations of genetic engineering manipulations.

Currently, a technology has been developed to obtain, using the methods of genetic engineering, another type of antibody - recombinant single-domain mini-antibodies of a given specificity. It is based on the production of nucleotide sequences of the genes of noncanonical antibodies of animals of the camelidae family (Camelidae). These antibodies are a dimer of only one shortened (the first constant region of CH1 is absent) immunoglobulin heavy chain. For the specific recognition and binding of antigen proper, in this case, only one variable domain of this antibody is necessary and sufficient.

The full equivalent of the term “single-domain mini-antibody” for the purposes of the present invention is the widely used designation “nanobody” introduced by ABLYNX (NANOBODY ™), as well as “nanoantibody” and “single-domain nanoantibody”.

Obtained by this technology single-domain mini-antibodies have the following properties [Tillib S.V. Camel Mini Antibodies is an effective tool for research, diagnosis and therapy. Molecular Biology 2011; 45 (1): 77-85.]:

1) High solubility and stability (in a wide range of temperatures and acidity of the medium).

2) Improved cell permeability due to small size (~ 2 × 4 nm, 13-15 kDa).

3) The ability to form paratypes that are unusual for classical antibodies, allowing them to bind to recesses and active centers of proteins.

4) The presence of a proven method for obtaining and selection of single-domain mini-antibodies.

5) The possibility of economical operating time in large quantities. Single domain mini-antibodies can be generated in the periplasm of E. coli bacteria.

6) The possibility of genetic engineering modifications for polyvalent single domain mini-antibodies.

7) Low immunogenicity.

Another positive property of these antibodies is the possibility of their "humanization" without a noticeable loss of their specific activity by conducting a small number of point substitutions of amino acids [Vincke C., Loris R., Saerens D., et al. // J. Biol. Chem. 2009. V.284. No. 5. P.3273-3284]. This opens up the potential for the widespread use of single-domain mini-antibodies as passive immunization agents to prevent the development of various dangerous infectious diseases [Wesolowski J., Alzogaray V., Reyelt J. et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med. Microbiol. Immunol. 2009; 198, 157-174].

Thus, it was shown (WO 2007/052242; Prendergast Patrick; Composition and method for the treatment of viral infection using camelid heavy chain antibodies) that antibodies obtained against the influenza virus and selected for their specificity for neuraminidase can protect birds from the development of diseases, caused by influenza viruses of various strains. This technical solution as the closest to the claimed in the composition of the active substance of the pharmaceutical composition and the method of introducing it into the body is selected by the authors of the present for the prototype.

The disadvantages of the prototype are:

1) Antibodies to neuraminidase of influenza virus have a weak neutralizing activity of the virus. Effective neutralization of the virus with such a drug can only be achieved if it is circulated for a long time in the body. This leads to the need to introduce large doses of drugs into the body to achieve a positive effect, as well as to the need for repeated administration of the drug.

2) To prevent the occurrence of various immune reactions to a foreign protein (antibody), introduced in large quantities due to its weak neutralizing virus activity, an administration scheme is used according to which the drug is administered twice, and the second time together with a drug that inhibits TNF-α. Moreover, repeated administration is often difficult for patients.

Thus, in the prior art there is an urgent need for the development of a pharmaceutical composition for the prevention and treatment of influenza based on specific (recombinant) antibodies, characterized by high virus-neutralizing activity, which must be maintained / reproduced in the body for a long time, which would ensure the sufficiency of a single administration of the composition.

The present invention was the creation of:

1) a modified single-domain mini-antibody, which is characterized by a high neutralizing activity of the virus and due to this can effectively bind a certain epitope of hemagglutinin of the influenza virus and thereby block the development of influenza virus infection in the body.

2) a vector that ensures the delivery and long-term expression of a single-domain mini-antibody gene in the body, which can efficiently bind a certain hemagglutinin epitope of influenza virus and is characterized by high virus neutralizing activity, which, once administered, can effectively block the development of influenza virus infection in the body.

3) a composition consisting of a single-domain mini-antibody and an adenoviral vector, which ensures the delivery and long-term expression of the single-domain mini-antibody gene in the body, which can efficiently bind a certain epitope of hemagglutinin of the influenza virus and is characterized by a high neutralizing activity of the virus, which, once administered, can block the development flu virus infection in the body.

Disclosure of the present invention

The above objective of the present invention is solved in that the preparation was carried out using specially prepared single-domain mini-antibodies that bind the surface protein of the hemagglutinin of the influenza virus in such a way that the infectious activity of the influenza A virus is violated (blocked and neutralized).

More specifically, the problem is solved by the present invention by creating a single-domain mini-antibody against influenza virus that specifically binds to a certain type A influenza virus hemagglutinin epitope and suppresses type A influenza virus infection. A viral vector is also created that expresses a single-domain mini-antibody against influenza virus , which can effectively bind a specific hemagglutinin epitope of influenza virus and thereby block the development of influenza virus infection in the body. Moreover, the viral vector may be an adenovirus vector expressing a single-domain mini-antibody against the influenza virus, which is able to efficiently bind a certain epitope of influenza hemagglutinin and thereby block the development of influenza virus infection in the body. Also provided is a composition consisting of an effective amount of a single-domain mini-antibody against influenza virus and a viral vector expressing a single-domain mini-antibody against influenza virus, capable of efficiently binding influenza hemagglutinin and thereby block the development of influenza virus infection in the body.

The following were selected as methods of application for the active substance (antibody): use of single-domain mini-antibodies in the form of a suspension in a pharmaceutically acceptable solvent; the use as a carrier for the active substance of a recombinant viral vector based on an adenovirus expressing a single-domain mini-antibody gene capable of binding and / or neutralizing the influenza virus; the use of compositions containing both a viral vector with a single-domain mini-antibody gene and a single-domain mini-antibody (in the form of a protein).

The choice of implementation routes in order to obtain a pharmaceutical composition with the claimed properties is due to the following factors:

The present invention provides for the use of a special single-domain mini-antibody as an active ingredient in the pharmaceutical composition, which is capable of efficiently binding to the surface protein of the hemoglutinin of the influenza virus in such a way that the infectious activity of the virus is violated (blocked, neutralized). Most of the antibodies currently used to neutralize the virus (classical antibodies consisting of 4 immunoglobulin chains) have an affinity for the influenza virus hemoglutinin protein. Antibodies against hemagglutinin are able to prevent the penetration of the virus into the cell and thus prevent the development of infection. In contrast, the effect of antibodies against neuraminidase is carried out at the level of exit of mature virus particles from the cell and therefore can only shorten the period of the disease and its intensity. Due to the fact that it is antibodies to hemagglutinin that can have pronounced neutralizing activity of the virus and exert their effect against the influenza virus immediately after it enters the body, that is, they can prevent the development of infection, single-domain mini-antibodies selected against influenza hemagglutinin and possessing at the same time neutralizing the virus activity, were used as an active substance to create a pharmaceutical composition intended for the prevention and treatment of influenza.

The present invention relates to a method of using a single-domain mini-antibody, comprising the use of viral vectors as a carrier for the active substance. Viral vectors are recombinant viruses whose genome includes the target gene (single-domain mini-antibody gene) with a set of regulatory elements. The following recombinant viruses are most often used as viral vectors: adenoviruses [Shmarov M.M., Tutykhina I.L., Logunov D.Yu. et al. Induction of a protective immune response in mice vaccinated with CELO recombinant avian adenovirus expressing rabies virus glycoprotein G. Journal of Microbiology, Epidemiology and Immunology 2006; 4: 69-71; Tutykhina I.L., Shulpin M.I., Chvala I.A. et al. Construction of recombinant CELO adenoviruses expressing the avian influenza A virus hemagglutinin gene and studying the possibility of their use as vaccines for protection against avian influenza A virus H5N1 and H7N1. Molecular Genetics, Microbiology and Virology 2011 .; Tutykhina I.L., Shmarov M.M., Logunov D.Yu. et al. Design and prospects for the use in medicine of recombinant adenoviral nanostructures. Russian nanotechnology. 2009; 4 (11-12): 82-92; Liu M.A. Immunologic basis of vaccine vectors. Immunity. 2010; 33 (4): 504-15; Lasaro M.O., Ertl H.C. New insights on adenovirus as vaccine vectors. Mol ther. 2009; 17 (8): 1333-9], retroviruses [Liu M.A. Immunologic basis of vaccine vectors. Immunity. 2010; 33 (4): 504-15; Pincha M., Sundarasetty B.S., Stripecke R. .. Lentiviral vectors for immunization: an inflammatory field. Expert Rev Vaccines. 2010; 9 (3): 309-321; Negri D.R., Michelini Z., Cara A. Toward integrase defective lentiviral vectors for genetic immunization. Curr HIV Res. 2010; 8 (4): 274-281.], Adeno-associated viruses [Sun J.Y., Anand-Jawa V., Chatterjee S., Wong K.K. Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther. 2003; 10 (11): 964-76]. Among the existing delivery systems of antigens, viral vectors occupy a special place, since they have the following properties:

- have a natural mechanism of interaction with the cell and penetration into the cell;

- transport foreign genetic material into the cell nucleus;

- able to provide long-term expression of antigen;

- the viral envelope performs a protective function for the genetic material encoding the antigen.

When introduced into the body, viral vectors penetrate into various types of cells, where they express the genes integrated into the DNA. In this regard, viral vectors are a widely used tool for delivering genes to human and animal cells [R. Harrop, et al., Advanced Drug Delivery Reviews, V.58, I.8, 2006, P.931-947].

Along with the above, viral vectors are highly efficient in expressing the target gene in various types of cells, and are safe for humans and animals.

Thus, the use of viral vectors as a carrier for the active substance of the claimed composition allows delivery of target protein genes to the body, which, in turn, provides an extension of the circulation period of the target gene product in the body.

As a carrier, viral vectors obtained on the basis of recombinant human adenovirus of the 5th serotype are used. Adenoviral vectors are known to be used most often in practice. Currently, vaccines against various bacterial (tularemia, tuberculosis, brucellosis, etc.) and viral (human immunodeficiency virus, human papillomavirus, rabies virus, Ebola, etc.) human pathogens [Wang J, Thorson L, Stokes RW, Santosuosso M, Huygen K, Zganiacz A, Hitt M, Xing Z. Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis. J Immunol. 2004 Nov 15; 173 (10): 6357-65; Richardson JS, Yao MK, Tran KN, Croyle MA, Strong JE, Feldmann H, Kobinger GP. Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine. Plos one. 2009; 4 (4): e5308. Epub 2009 Apr 23; Li WH, Zhang Y, Wang SH, Liu L, Yang F. Recombinant replication-defective adenovirus based rabies vaccine. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2003 Dec; 25 (6): 650-4; Lees CY, Briggs DJ, Wu X, Davis RD, Moore SM, Gordon C, Xiang Z, Ertl HC, Tang DC, Fu ZF. Induction of protective immunity by topic application of a recombinant adenovirus expressing rabies virus glycoprotein. Vet Microbiol. 2002 Apr 2; 85 (4): 295-303]. Thus, adenoviral vectors containing the nucleotide sequence of human influenza virus hemagglutinin were obtained for further use as a vaccine for humans and animals against a pathogenic strain of influenza virus (Van Kampen KR, Shi Z, Gao P, Zhang J, Foster KW, Chen DT, Marks D, Elmets CA, Tang DC. Vaccine. 2005, V 23, p. 1029-1036). The H5N1 avian influenza virus hemagglutinin gene was included in the DNA of recombinant pseudo adenovirus nanoparticles (RPAN) based on human adenovirus of the 5th serotype also with the aim of using the resulting product as a vaccine for humans, mammals and birds (Toro H, Tang DC, Suarez DL, Sylte MJ , Pfeiffer J, Van Kampen KR. Vaccine 2007; Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, Robbins PD, Swayne DE, Donis RO, Katz JM, Barratt-Boyes SM, Gambotto AJ of Virology , 2006, V.80, No. 4, p. 1959-1964). Adenoviral vectors were also used for gene therapy of tumors when the tumor suppressor gene p53 was cloned into their DNA (US 7,033,750). In addition, two drugs, which are adenoviral vectors based on human adenovirus of the 5th serotype, are approved for use in oncology.

The use of viral vectors as carriers gives the pharmaceutical composition the following advantages:

- Using viral vectors, the problem of instability of antibody preparations (introduced into the body in the form of proteins) can be solved.

- Viral vectors are stable, can be lyophilized, can be stored at 4 ° C for a long time without loss of activity.

- Technologies for producing viral vectors on an industrial scale are economically viable and allow you to get drugs with a high titer. The process of obtaining a new type of viral vector takes several weeks, which can allow you to quickly respond to changing epidemiological conditions in the shortest possible time [R. Harrop, et al., Advanced Drug Delivery Reviews, V.58, I.8, 2006, P.931-947].

- The use of viral vectors allows to ensure the development of a protective level of antibodies in 96 hours after administration. Moreover, the protective level of antibodies is maintained in the body for at least 2 weeks [Kasuya K., Mol Ther. 2005 Feb; 11 (2): 237-44; Sofer-Podesta C., Infect Immun. 2009 Apr; 77 (4): 1561-8; Chiuchiolo M.J., J Infect Dis. 2006 Nov 1; 194 (9): 1249-57] in the case of adenoviral vectors.

The use of drugs, which are a viral vector that carries a single-domain mini-antibody gene in the DNA, which can neutralize the influenza virus of the type of the current strain, makes it possible to protect the body from the currently circulating strain of influenza virus after 48-96 hours. Moreover, the use of a pharmaceutical composition consisting of a single type of viral vector carrying a single-domain mini-antibody gene and protein preparations of the corresponding single-domain mini-antibody allows protecting the body in the period immediately after administration of the drug, which lasts at least 3-4 weeks.

Thus, it is advisable to use a complex preparation having two components: single-domain mini-antibodies against type A influenza virus and a recombinant viral vector expressing the single-domain mini-antibodies against type A influenza virus

Such a drug will have the following advantageous characteristics:

1) The basis of the drug are antibodies - the most effective natural antiviral substances.

2) A short course of administration (1 time), which reduces the risk of side effects and intolerance.

3) Effectiveness of action at various stages of the development of the infectious process.

4) The possibility of using as a supplement to late vaccination of people at risk in the first 2 weeks after vaccination (for the period of antibody production).

5) The possibility of use for persons with immunodeficiency who may give an insufficient immune response to vaccination.

6) The possibility of use for the elderly, for whom the effectiveness of vaccination is reduced and reaches 50-70%, as an addition to vaccination.

7) The possibility of use for unvaccinated persons who are in contact with sick relatives and neighbors.

8) The possibility of use for those who, for whatever reason, were not vaccinated on time.

9) The cost of the course of use of such a drug is comparable to the cost of one dose of vaccine.

The present invention combines the advantages of the various approaches mentioned and relates to single-domain mini-antibodies that bind / neutralize the influenza virus, as well as to a method for using such antibodies as using a recombinant viral vector carrying an expression cassette containing a nucleotide sequence as a carrier of an active substance (antibody) , which encodes a single-domain mini-antibody that binds to the influenza virus, and by creating a pharmaceutical composition consisting of a single-domain mini-antibody against influenza virus and a recombinant viral vector expressing this single-domain mini-antibody.

Thus, the present invention relates to a single domain mini-antibody characterized by the amino acid sequence of SEQ ID NO: 2, specifically binding to type A (H5N2) influenza hemagglutinin and suppressing type A (H5N2) influenza virus infection, and its functionally active variants. A single domain mini-antibody characterized by the amino acid sequence of SEQ ID NO: 2 is sometimes referred to in this application by its trade name Neogrippab.

The present invention also relates to a nucleotide sequence encoding a single domain mini-antibody characterized by the amino acid sequence of SEQ ID NO: 2, and its functionally active variants. Special cases of this sequence include, without limitation, SEQ ID NO: 1 and its genetically degenerate variants.

According to another aspect, the present invention relates to a recombinant viral vector containing such a nucleotide sequence and expressing a single domain mini-antibody characterized by the amino acid sequence of SEQ ID NO: 2, or its functionally active variants. According to one preferred embodiment of this aspect, the viral vector is an adenoviral vector. However, it will be apparent to one of ordinary skill in the art that several other viruses, such as adeno-associated viruses, lentiviruses, can be used as viral vectors.

According to a further aspect, the present invention relates to a pharmaceutical composition for suppressing the development of an influenza A (H5N2) virus infection, comprising an effective amount of the single-domain mini-antibody disclosed above and / or a viral vector expressing such a single-domain mini-antibody. It will be apparent to one of ordinary skill in the art that for various purposes (therapeutic, prophylactic, combined), it may be preferable to use pharmaceutical compositions containing a single domain antibody or a viral vector encoding it, either individually or in combination. The present invention covers every such option.

Thus, the present invention relates to a pharmaceutical composition for the prevention or treatment of influenza, which is a suspension of the above-disclosed recombinant viral vectors and / or the above-disclosed single-domain mini-antibodies in a pharmaceutically acceptable solvent or excipient. Such pharmaceutically acceptable solvents and excipients are well known in the art (Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]).

The use of such a pharmaceutical composition makes it possible to achieve transient expression of single-domain mini-antibodies in a patient, which, due to their unique properties described in detail above, are capable of providing sufficiently reliable immune defense against the influenza virus. One of ordinary skill in the art will understand that the use of such a pharmaceutical composition is effective mainly for prevention, but also for the treatment of infection.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of influenza, which is a suspension of the above disclosed recombinant viral vector and single domain mini antibodies that bind to the influenza virus of the same type and antigenic formula as a single domain mini antibody encoded by an expression cassette contained in the indicated recombinant viral vector, in a pharmaceutically acceptable solvent or excipient.

The use of such a pharmaceutical composition provides a sufficiently reliable immune defense against the influenza virus, not only as a preventive measure, but also after the patient is infected with the influenza virus. Of course, the earlier the pharmaceutical composition of the present invention is applied at an earlier stage of infection, the more effective such treatment will be.

In addition, the present invention relates to a method for the prevention or treatment of influenza, comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of one of the pharmaceutical compositions described above.

According to one preferred embodiment of the present invention, said pharmaceutical composition is administered intranasally.

In accordance with more specific embodiments of the present invention, intranasal administration is in the form of drops or in the form of a spray (aerosol).

The authors of the present invention proceed from the fact that, as is well known to the average person skilled in the art, the primary, initially obtained sequences of single-domain mini-antibodies can serve as source modules for more complex multi-module preparations. It is possible to combine in one multivalent derivative two, three or more monovalent primary single-domain mini-antibodies. These single-domain mini-antibodies combined into one construct can bind both to the same epitope of the target antigen, and to its different epitopes, or even to different target antigens. It is also possible to combine single domain mini-antibodies and other molecules or drugs into one construct to produce multifunctional drugs [Conrath KE, Lauwereys M, Wyns L, Muyldermans S. Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem. 2001 Mar 9; 276 (10): 7346-50; Zhang J, Tanha J, Hirama T, Khieu NH, To R, Tong-Sevinc H, Stone E, Brisson JR, MacKenzie CR. Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents. J Mol Biol. 2004 Jan 2; 335 (1): 49-56; Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, Revets H. Efficient cancer therapy with a nanobody-based conjugate. Cancer Res. 2004 Apr 15; 64 (8): 2853-7; Baral TN, Magez S, Stijlemans B, Conrath K, Vanhollebeke B, Pays E, Muyldermans S, De Baetselier P. Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor. Nat Med. 2006 May; 12 (5): 580-4; Coppieters K, Dreier T, Silence K, Haard HD, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Wiele CV, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis Rheum. 2006 Jun; 54 (6): 1856-66]; multimerization by introducing additional amino acid sequences of interacting protein domains, such as leucine zippers [Harbury R.V., Zhang T., Kim P.S., et al. A switch between two-, three- and four-stranded coiled coils in GCN4 leucine zipper mutants. Science, 1993, 262: 1401-1407; Shirashi T., Suzuyama k., Okamoto H. et al. Increased cytotoxicity of soluble Fas ligand by fusing isoleucine zipper motif. Biochem. Biophys. Res. Communic. 2004, 322: 197-202; Chenchik A., Gudkov A., Komarov A., Natarajan V. Reagents and methods for producing bioactive secreted peptides. 2010. US Patent Application 20100305002], sequences of small proteins forming stable complexes [Deyev SM, Waibel R, Lebedenko EN, Schubiger AP, Plückthun A. Design of multivalent complexes using the barnase * barstar module. Nat Biotechnol. 2003, 21 (12): 1486-92.].

To modulate the properties of the preparation of a single-domain mini-antibody, for example, increasing the in vivo lifetime (in the patient’s blood), additional amino acid sequences, protein sequences (such as, for example, serum albumin), or another single-domain mini-antibody, can be introduced into the final compound specifically binding to a major and long-lived protein in human blood (eg, albumin or immunoglobulin) [Gibbs WW. Nanobodies. Sci am. 2005 Aug; 293 (2): 78-83; Harmsen MM, Van Solt CB, Fijten HP, Van Setten MC. Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins. Vaccine 2005 Sep 30; 23 (41): 4926-34; Coppieters K, Dreier T, Silence K, Haard HD, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Wiele CV, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis Rheum. 2006 Jun; 54 (6): 1856-66].

It will be apparent to one of ordinary skill in the art that such modifications and other antibody variants that underlie the present invention fall within the scope of the present invention because they are structural and functional variants of single domain mini antibodies. Thus, the authors of the present invention understand the term "single-domain mini-antibodies" as the primary, initially obtained, "minimum" amino acid sequences of single-domain mini-antibodies, and their modifications obtained as a result of the mentioned adaptation or "formatting" and their variants.

The term "antibody variant" for the purposes of the present invention means a polypeptide that contains changes in the amino acid sequence, namely deletion, insertion, addition or replacement of amino acids, provided that this maintains the necessary level of protein activity, for example, at least 10% of the activity original single domain mini antibody. A number of changes in the variant of the protein depends on the position or type of amino acid residue in the three-dimensional structure of the protein. The number of changes can be, for example, from 1 to 30, more preferably from 1 to 15, and most preferably from 1 to 5 changes in the sequence of the original single-domain mini-antibodies. These changes can occur in areas of the polypeptide that are not critical to its function. This is possible due to the fact that some amino acids have high homology with each other, and therefore the tertiary structure or activity of the protein is not violated by such a change. Therefore, as a variant of the protein can be a protein that is characterized by a homology of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with respect to the amino acid sequence of the original single domain mini-antibody, provided that polypeptide activity. Homology between amino acid sequences can be established using well-known methods, for example, using sequence alignment in the BLAST 2.0 computer program, which calculates three parameters: count, identity, and similarity.

Substitution, deletion, insertion, addition or replacement of one or more amino acid residues will be a conservative mutation or conservative mutations, provided that the protein activity is maintained. An example of a conservative mutation (s) is a conservative substitution (s). A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acids having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, series, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine , fe nilalanine, tryptophan, histidine). Thus, examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gln, His or Lys, replacing Asn with Glu, Gln, Lys, His or Asp, replacing Asp with Asn, Glu or Gln, replacing Cys with Ser or Ala, replacing Gln with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gln, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gln, Arg or Tyr, replacing Ile with Leu , Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met , Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, Ile or Leu.

DNA fragments that encode essentially the same functional polypeptide can be obtained, for example, by modifying the nucleotide sequence of a DNA fragment encoding the original single-domain mini-antibody, for example, by the method of site-directed mutagenesis, so that one or more amino acid residues in a particular site will be deleted, replaced, inserted or added. DNA fragments modified as described above can be obtained using conventional processing methods to obtain a mutation.

DNA fragments that encode essentially the same functional polypeptide of the original single domain mini-antibody can be obtained by expressing DNA fragments having the mutation described above in the corresponding cell and establishing the activity of the expressed product.

The organization of the variable domains of noncanonical antibodies (VHH, nanoantibodies) is largely similar to that of the variable domains (VH) of classical antibodies (in humans, the VH domains of immunoglobulins of the IgG3 subclass have a particularly pronounced homology with camelids VH and VHH). In both cases, V-domains consist of four conservative framework regions (FR, “framework regions”) surrounding three hypervariable regions (complementarity determining, CDR, from “complementarity determining regions). In both cases, the domains form a spatial structure typical of the immunoglobulin V domain of two beta layers (sheets), one of four amino acid chains, and the second of five [Padlan E.A. X-Ray crystallography of antibodies. Adv. Protein Chem. 1996; 49: 57-133. Muyldermans S., Cambillau C., Wyns L. Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. TIBS 2001; 26: 230-235]. In this structure, all three hypervariable regions are clustered on one side of the V domain (where they are involved in antigen recognition) and are located in loops connecting the beta structures. However, there are important differences associated with the functioning of VHH in a single domain format. Thus, the hypervariable regions of CDR1 and CDR3 are markedly increased in the case of VHH. Often, cysteine residues are found in the hypervariable regions of VHH, moreover, they are present in two regions at once (most often in CDR1 and CDR3, less often in CDR2 and CDR3). When studying the crystal structures of VHH, it was shown that these cysteine residues form disulfide bonds, which leads to additional stabilization of the loop structure of this antigen. The most obvious and reproducible hallmark of VHH are four hydrophobic amino acid residues replaced by hydrophilic ones in the second frame region (Val37Phe, Gly44Glu, Leu45Arg, Trp47Gly, according to Kabat numbering). In the case of the VH domain, this framework region is highly conserved, enriched with hydrophobic amino acid residues, and is especially important for linking to the variable domain of the VL light chain. The VHH domain in this regard is very different: the indicated substitutions of hydrophobic amino acids for hydrophilic ones make the association of VHH and VL impossible. These substitutions also explain the high solubility of VHH, a nanoantibody, when it is obtained as a recombinant protein [Tillib S.V. “Camel Nanoantibodies” is an effective tool for research, diagnosis and therapy. Molecular Biology 2011; 45 (1): 77-85].

It is the hypervariable regions of mini-antibodies that determine their specific interaction with the antigen, and it is the homologous substitutions of amino acids in these areas that can lead to the production of slightly different sequence mini-antibodies that have identical or similar properties. Thus, it will be apparent to one of ordinary skill in the art that the scope of the present invention encompasses not only the sequence of mini-antibodies indicated in the appendix, but also those that can be obtained by substituting amino acids in the hypervariable regions (indicated in the sequence listing as CDR) to other, but very similar in properties, amino acids (conservative substitutions).

Substitution, deletion, insertion or addition of nucleotides described above also includes mutations that occur in nature and, for example, are due to variability.

The single-domain mini-antibody polypeptides of the present invention can be encoded by a large number of nucleic acid molecules, which is the result of the degeneracy of the genetic code well known in the art. The essence of the phenomenon is that any amino acid (with the exception of tryptophan and methionine) that is part of natural peptides can be encoded by more than one triplet nucleotide codon (see table 1). Any of these degenerate coding nucleic acid molecules may be included in the expression cassettes contained in the recombinant pseudo-adenovirus nanoparticles of the invention that are within the scope of the present invention.

Table 1 Universal Amino Acid Coding Table Ala gcu gcc gca gcg alanine Arg aga agg cgu cgc cga cgg arginine Asn aau aac aspargin Asp gau gac aspartic acid Cys ugu ugc cysteine Gln caa cag glutamine Glu gaa gag glutamic acid Gly ggu ggc gga ggg glycine His cau cac histidine Ile auu auc aua isoleucine Leu uua uug cuu cuc cua cug leucine Lys aaa aag lysine Met aug methionine Phe uuu uuc phenylalanine Pro ccu ccc cca ccg proline Ser agu agc ucu ucc uca ucg series Thr acu acc asa acg threonine trm uaa uag uga stop codon Trp ugg tryptophan Tyr uau uac tyrosine Val guu guc gua gug valine

Brief Description of the Drawings

Figure 1 shows the amino acid sequence of a selected single-domain mini-antibody SEQ ID NO: 2, capable of specifically binding and blocking influenza A virus, and the nucleotide sequence encoding it, SEQ ID NO: 1. In the indicated amino acid sequence are underlined respectively (from left to right, from N - to the C-terminus) the hypervariable regions of CDR1, CDR2 and CDR3, which in fact are the main determining specificity of binding by regions of similar antigen-knowing molecules.

Figure 2 presents additional amino acid sequences that were attached (by molecular cloning and genetic engineering insertion of additional DNA sequences encoding the corresponding amino acid sequences) to the C-terminus of the initially selected antibody. Four additional amino acid sites (domains) are indicated: (1) - a linear linker site (corresponding to the upper elongated hinge site of non-canonical camel IgG2a immunoglobulins), (2) - a special trimerization domain (highlighted in gray) - "ILZ" ("isoleucine zipper domain, isoleucine lightning "), as well as (3) the HA-tag (a peptide from hemagglutinin protein to which there are commercial antibodies) and (4) the His-tag (six histidine residues at the C-terminus of the protein for efficient protein purification using metal chelate chromatography).

Figure 3 illustrates the results of an enzyme-linked immunosorbent assay for recognition of a bottom-sampled mini-antibody SEQ ID NO: 2 of both the hemagglutinin protein preparation immobilized in the wells of the immunological plate (at the top of the figure) and the influenza virus preparation (at the bottom of the drawing). Cells with immobilized chicken ovalbumin protein were used as controls.

Figure 4 illustrates the results of an enzyme-linked immunosorbent assay for recognition of a selected influenza virus preparation immobilized in the wells of an immunological plate of a selected single-domain mini-antibody SEQ ID NO: 2. As a control, a cell with immobilized chicken ovalbumin protein was used.

Figure 5 presents a graph of the survival of mice after intranasal administration of a single-domain mini-antibody against the influenza virus and their subsequent infection with the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection. The solid line with a black square marker is 20 μl of an intranasal solution of a single domain mini-antibody against influenza virus in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a white square marker is 20 μl of an intranasal phosphate buffer solution.

Figure 6 presents a graph of the change in body weight of mice after intranasal administration of a single-domain mini-antibody against influenza virus and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection. The solid line with a black square marker is 20 μl of an intranasal solution of a single domain mini-antibody against influenza virus in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a marker in the form of a cross is 20 μl of an intranasal solution of phosphate buffer.

Figure 7 presents a graph of the survival of mice after infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) and subsequent intranasal administration of a solution of single domain mini-antibodies against influenza virus in phosphate buffer at a dose of 2.5 μg / mouse. The solid line with a black square marker is 20 μl of an intranasal solution of a single domain mini-antibody against influenza virus in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a white square marker is 20 μl of an intranasal phosphate buffer solution.

On Fig presents a graph of the changes in body weight of mice after infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) and subsequent intranasal administration of a solution of single domain mini-antibodies against influenza virus in phosphate buffer at a dose of 2.5 mcg / mouse. The solid line with a black square marker is 20 μl of an intranasal solution of a single domain mini-antibody against influenza virus in phosphate buffer at a dose of 2.5 μg / mouse, the solid line with a marker in the form of a cross is 20 μl of an intranasal solution of phosphate buffer.

Figure 9 shows a graph of the survival of mice after intranasal administration of the recombinant adenoviral vector Ad5-anti-flu and their subsequent infection with the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection. The solid line with the marker in the form of a black square is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad5-anti-flu at a dose of 10 ⁷ PFU / mouse, the solid line with the marker in the form of a cross is 20 μl of the intranasal preparation of the recombinant adenoviral vectors Ad-null, not containing the insertion of the target gene in the expression cassette at a dose of 10 ⁷ PFU / mouse, a solid line with a marker in the form of a white square - 20 μl intranasally 1 × PBS.

Figure 10 presents graphs of the dependence of the change in body weight of mice after intranasal administration of the recombinant adenoviral vector Ad5-anti-flu and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) versus time after infection. The solid line with a black square marker is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad5-anti-flu at a dose of 10 ⁷ PFU / mouse, the solid line with a marker in the form of a cross is 20 μl of the intranasal preparation of recombinant adenoviral vectors Ad-null, not containing the insertion of the target gene in the expression cassette at a dose of 10 ⁷ PFU / mouse, a solid line with a marker in the form of a white square - 20 μl intranasally 1 × PBS.

Examples of the implementation of the present invention

Example 1

Obtaining a library of variable domains of single-chain antibodies

Immunization

Bactrian camel Camelus bactrianus was subsequently immunized 4 times by subcutaneous administration of antigenic material mixed with an equal volume of incomplete Freund's adjuvant. The antigen used was the preparation of the inactivated avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) from the Museum of FSBI NIIEM named after N.F. Gamalei ”of the Ministry of Health of Russia and the preparation of the bird flu virus H5N2 (strain A / 17 / Utka / Potsdam / 86/92) obtained from the Federal State Unitary Enterprise NPO Microgen of the Ministry of Health of Russia, Irkutsk. The second immunization was carried out 3 weeks after the first, then two more immunizations were carried out with an interval of two weeks. Blood sampling (150 ml) was performed 6 days after the last injection. To prevent coagulation of the taken blood, 50 ml of standard saline solution (PBS) containing heparin (100 units / ml) and EDTA (3 mM) was added.

Blood was diluted 2 times with standard saline (PBS) containing 1 mm EDTA. 35 ml of a diluted blood solution was layered on a step of a special medium (Histopaque-1077, Sigma) with a density of 1.077 g / ml and a volume of 15 ml and centrifugation was performed for 20 min at 800 × g. Mononuclear cells (lymphocytes and monocytes) were selected from the plasma / Histopaque interphase, and then washed with a PBS solution containing 1 mM EDTA.

Total RNA from B lymphocytes was isolated using TRIzol reagent (Invitrogen). Then, on a column of oligo (d-T) cellulose, poly (A) -containing RNA was isolated from total RNA. RNA concentration was determined using a biophotometer (Eppendorf) and the quality of the isolated RNA was checked by electrophoresis in 1.5% formaldehyde agarose gel.

The reverse transcription reaction was carried out according to the standard protocol [Sambrook et al., 1989] using reverse transcriptase H ^- M-MuLV and oligo primer (dT) ₁₅ as a seed.

Reverse transcription products were used as a template in a two-step polymerase chain reaction and the obtained amplification products were cloned at the NcoI (PstI) and NotI sites into a phagemid vector as previously described [Hamers-Casterman et al., 1993; Nguyen et al., 2001; Saerens et al., 2004; Rothbauer et al., 2006]. The selection procedure was also carried out similarly to those described in these works. It was based on the phage display method, in which the bacteriophage M13KO7 (New England Biolabs, USA) was used as an assistant phage.

Example 2

Selection of single-domain mini-antibodies that specifically recognize influenza A virus, their formatting and operating time

Single domain mini-antibodies were selected by phage display using the inactivated avian influenza virus preparation A / Mallard duck / PA / 10218/84 (H5N2) immobilized on the bottom of the wells of a 96-well ELISA plate. Used polystyrene immunological plates with high sorption MICROLON 600 (Greiner Bio-One). For blocking, 1% BSA (Sigma-Aldrich, USA) and / or 1% skim milk (Bio-Rad, USA) in PBS were used. The selection and subsequent amplification of the selected phage particles (containing the single-domain mini-antibody gene inside, and the expressed single-domain mini-antibody as part of the surface phage protein pIII) was repeated, as a rule, three times in succession. All manipulations were performed as described in the publications [Tillib S.V., Ivanova T.I., Vasiliev L.A. 2010. Fingerprint analysis of the selection of "single-domain mini-antibodies" by the phage display method using two variants of assistant phages. Acta Naturae 2010; 2 (3): 100-108; Hamers-Casterman C., Atarhouch T., Muyldermans S. et al. Nature 1993; 363: 446-448 .; Nguyen V.K., Desmyter A., Muyldermans S. Adv. Immunol. 2001; 79: 261-296; Saerens D., Kinne J., Bosmans E., Wernery U., Muyldermans S., Conrath K. J Biol Chem. 2004; 279: 51965-51972; Rothbauer U., Zolghadr K., Tillib S., et al. Nature Methods 2006; 3: 887-889].

Clone sequences of selected single-domain mini-antibodies were grouped according to the similarity of their fingerprints obtained by electrophoretic separation of the hydrolysis products of amplified single-domain mini-antibodies in parallel with three often-digested restriction endonucleases (HinfI, MspI, RsaI). Then, selected clones representing each of the fingerprint-different groups were used for analytical production of single-domain mini-antibodies, the activity of which was checked by enzyme-linked immunosorbent assay for effective binding of the inactivated avian influenza virus drug A / Mallard duck / PA / 10218 immobilized in the cell of an immunological tablet / 84 (H5N2).

As a result, a sequence encoding a single-domain mini-antibody Neogrippab was selected (Fig. 1).

In order to increase the efficiency of isolation and detection of a single-domain mini-antibody, as well as to significantly enhance the biological activity of the initially selected monovalent single-domain antibody, we used the previously efficient formatting (modification of the amino acid sequence of the antibody) procedure developed by us, which was described in detail by Tillib et al. (Tillib et al., 2013, Antiviral Res. 97: 245-254. Formatted single-domain antibodies can protect mice against infection with influenza virus (H5N2). // Formatted single-domain antibodies can protect the mouse from infection with influenza virus (H5N2). ) This procedure included attaching to the C-terminus of the initially selected antibody four additional amino acid sites (domains) shown in FIG. 2: (1) a linear linker site (corresponding to the upper elongated hinge site of the camel’s special immunoglobulins), (2) a special trimerizing domain - "ILZ" ("isoleucine zipper domain", or, in water, “isoleucine zipper”), (3) - the HA tag (a peptide from the hemagglutinin protein to which there are commercial antibodies) and (4) the His tag (six histidine residues at the C-terminus of the protein for effective protein purification using metal chelate chromatography).

Single domain mini antibody production

The coding DNA sequences of the selected Neogrippab single-domain mini-antibody, formatted as described above, were cloned into an expression plasmid vector — a modified pHEN6 vector [Conrath KE, Lauwereys M, Galleni M, Matagne A, Frère JM, Kinne J, Wyns L, Muyldermans S Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother. 2001; 45: 2807-12]. Due to the presence of the signal peptide (pelB) sequence at the N-terminus of the expressed sequence, the produced recombinant protein (single-domain mini-antibody) accumulates in the periplasm of bacteria, which allows it to be efficiently isolated by the osmotic shock method without destroying the actual bacterial cells. The production of single domain mini antibodies was performed in E. coli (strain BL21). Expression was induced by adding 1 mM indolyl-beta-D-galactopyranoside and the cells were incubated with vigorous stirring for 7 hours at 37 ° C or overnight at 29 ° C. A single domain mini antibody was isolated from the periplasmic extract using affinity chromatography on Ni-NTA agarose using the QIAExpressionist purification system (QIAGEN, USA).

The resulting protein fractions were concentrated (to a final concentration of about 1-5 mg / ml) using ultrafiltration devices (with a cut-off size of 10 kDa) from Millicore Amicon, and then layered in a volume of 0.5-2 ml onto a gel column filtration chromatography (Sephacryl S-100 HR; 1.5 × 50 cm). Chromatography was performed using a BioLogic LP system from Bio-Rad at a constant flow rate of 0.3 ml / min. A standard saline buffer solution (PBS) was used as the buffer solution. Fractions were collected according to the absorption profile (at a wavelength of 280 nm) of the eluted protein. The size of the trimerized formatted antibody approximately corresponded to the size (and peak elution) of the marker protein — bovine serum albumin (BSA, 67 kDa). The combined selected formatted antibodies (from fractions of the corresponding elution peaks) were again concentrated (as described above), further purified from endotoxin on an Thermo Scientific affinity column (Detoxi-Gel Endotoxin Removing Gel), and then sterilized by filtration through a cellulose acetate membrane (Nalgene company) ) with pores of 0.2 μm. The purified antibodies were stored in aliquots at 4 ° C or, after adding 50% glycerol, at -20 ° C. For longer storage, antibodies were lyophilized.

Demonstration of the binding of a single domain mini-antibody to the influenza virus

The ability of the Neogrippab single-domain mini-antibody to bind hemagglutinin and the whole influenza virus was tested using an enzyme-linked immunosorbent assay with immobilized hemagglutinin protein (H5 strain A / American green-winged teal / California / HKWF609 / 07), Cat. No. 11699-V08H, Sino Biological Inc.) and and inactivated recombinant avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2), according to the standard protocol. As a control, wells with immobilized chicken ovalbumin protein (non-specific protein) were used. A formatted single-domain mini-antibody bound to hemagglutinin or influenza virus was detected using mouse anti-HA antibodies, secondary antibodies to mouse immunoglobulins conjugated to horseradish peroxidase, and the ABTC chromogenic substrate (2,2'-azino-bis (3-ethylbenzothiazoline) ) -6-sulfonic acid, Sigma).

Figures 3 and 4 show the results of the analysis, from which it follows that the single-domain mini-antibody NEOGRIPPAB specifically binds to the immobilized hemagglutinin protein (H5 from strain A / American green-winged teal / California / HKWF609 / 07), Cat. No. 11699-V08H, Sino Biological Inc.), and with the preparation of the influenza virus, but does not bind to ovalbumin (or BSA), used as a blocking agent.

The selected sequence of the single-domain mini-antibody Neogrippab was then cloned into the genome of the recombinant adenoviral vector.

Example 3

Creation of the plasmid construct pShuttle-CMV-Neogrippab with portions of the genome of human adenovirus 5th serotype to obtain a recombinant adenovirus vector carrying an expression cassette containing a nucleotide sequence that encodes a single-domain mini-antibody "Neogrippab" that binds to the influenza virus

To obtain the pShuttle-CMV-Neogrippab plasmid construct, the pShuttle-CMV plasmid construct with portions of the 5th serotype human adenovirus genome for the production of recombinant adenoviral vectors included in the AdEasy Adenoviral vector system (Stratagene Cat) kit was used as a vector. No. 240009). The pShuttle-CMV plasmid construct was hydrolyzed at the EcoRV restriction endonuclease site, and then the nucleotide sequence of a single domain mini-antibody was inserted to obtain, respectively, the pShuttle-CMV-Neogrippab construct. The nucleotide sequence of a single-domain mini-antibody Neogrippab for insertion into pShuttle-CMV was obtained by hydrolysis of the plasmid construct pAL-Neogrippab at EcoRV restriction endonuclease sites. The presence of the neogrippab single domain mini-antibody gene in the pShuttle-CMV-Neogrippab plasmid construct was confirmed by restriction analysis using endonucleases EcoRI, NotI and EcoRV and PCR.

Example 4

Obtaining a recombinant adenoviral vector Ad5-Neogrippab carrying an expression cassette containing a nucleotide sequence that encodes a single-domain mini-antibody "Neogrippab" that binds to the influenza virus

The preparation of the recombinant adenoviral vector Ad5-Neogrippab was carried out according to the method of "AdEasy Adenoviral vector system" ("Stratagene" Cat. No. 240009). The presence of the single domain mini-antibody neogrippab gene in the corresponding recombinant adenoviral vector Ad5-Neogrippab was confirmed by PCR.

Next, the titer of the preparation of the recombinant adenoviral vector Ad5-Neogrippab was determined by plaque formation on a 293 cell culture (human embryonic kidney cells).

Example 5

Neutral Influenza Virus Neutralization Mini-Antibody Neogrippab in Cell Culture

The virus neutralization reaction was performed using the MDCK cell line cultured in 96-well culture plates (Costar, USA) until the confluence of the cell monolayer was 80%. Before carrying out the reaction in culture plates with cells, the medium was replaced with serum-free. For the reaction, the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) was used at a dose of 100 TCD50 per well. Dilutions of the virus and the Neogrippab antibody tested were performed in serum-free DMEM. 50 μl of a virus suspension was added to the wells of a 96-well plate, then equal volumes of two-fold dilutions of serum were added to each well, starting at 1:10. The plate was incubated at 37 ° C for 1 hour. After incubation, a mixture of virus and sera was added to each well of the cell plate to adsorb the virus on the cells. Cells were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 2 hours. Then, serum-free medium was taken from the plates and replaced with MEM embryonic serum-containing cattle serum with 0.2% BSA and trypsin at a concentration of 1 μg / ml. The results of the virus neutralization reaction were taken into account after 72 h after the onset of CPD in the cell culture according to the method of Reed and Mench. The studied Neogrippab antibody neutralized the influenza virus in the concentration range from 0.009 μg / ml to 2.5 μg / ml.

Example 6

Neutralization of influenza virus with a single-domain mini-antibody Neogrippab expressed by the recombinant adenoviral vector Ad5-Neogrippab in an in vitro cell culture

Expression of a single-domain mini-antibody “Neogrippab” was performed in cells of the A549 line. For this, A549 cells were scattered into the wells of a 48-well plate (Costar, USA) in the amount of 104 cells per well, after which serum-free medium 293AGT (Invitrogen, USA) was added to the cells and incubated at 37 ° C in an atmosphere of 5% CO ₂ within 20-24 hours. Then, the cells were transduced with the preparation of the recombinant adenoviral vector Ad5-Neogrippab at a dose of 5 PFU / cell. Cells were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 2 h, after which the medium was taken from the wells and replaced with a new portion of 293AGT medium. Transduced cells were incubated for 72 hours, then culture medium was selected. Thus, preparations of a culture medium containing Ad5-Neogrippab recombinant adenoviral vector single-domain mini-antibody Neogrippab were obtained.

The virus neutralization reaction was performed using the MDCK cell line cultured in 96-well culture plates (Costar, USA) until the confluence of the cell monolayer was 80%. Before carrying out the reaction in culture plates with cells, the medium was replaced with serum-free. For the reaction, the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) was used at a dose of 100 TCD50 per well. Dilutions of the virus and culture medium containing the Neogrippab test antibody were performed in serum-free DMEM. 50 μl of a virus suspension was added to the wells of a 96-well plate, then equal volumes of two-fold dilutions of the culture medium were added to each well, starting at 1:10. The plate was incubated at 37 ° C for 1 h. After incubation, a mixture of the virus and culture medium was added to each well of the cell plate to adsorb the virus on the cells. Cells were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 2 hours. Then, serum-free medium was taken from the plates and replaced with MEM embryonic serum-containing cattle serum with 0.2% BSA and trypsin at a concentration of 1 μg / ml. The results of the virus neutralization reaction were taken into account after 72 h after the onset of CPD in the cell culture according to the method of Reed and Mench. Culture medium preparations containing the Ad5-Neogrippab recombinant adenoviral vector single-domain mini-antibody Neogrippab provided neutralization of the influenza virus at a 1: 100 dilution.

Example 7

The use of single-domain mini-antibodies "Neogrippab" for the prevention of influenza

In order to prevent type A influenza disease, female Balb / c mice weighing 18 g were divided into 2 groups of 50 mice in each group:

- the first group of mice was injected with 20 μl of a solution of single-domain mini-antibodies "Neogrippab" in a phosphate buffer dose of 2.5 μg / ml,

- the second group of mice was intranasally injected with 20 μl of phosphate buffer.

One hour after the administration of the above preparations, the mice were infected with 25 LD50 adapted for their virulent properties for the mouse model of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection was performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of the mice and the change in their body weight were taken into account.

The protection of mice against the A / Mallard duck / PA / 10218/84 (H5N2) virus was 100% in the group to which the neogrippab single-domain mini-antibody preparation was administered (see FIG. 5). Also, a decrease in body weight was not observed in these mice. Curves of weight loss in mice of each of the two above groups after infection with the influenza virus are presented in Fig.6.

Thus, the intranasal administration of the Neogrippab single-domain mini-antibody completely protects mice from infection with the influenza virus at a dose of 25 LD50.

Example 8

The use of single-domain mini-antibodies "Neogrippab" for the treatment of influenza

For the treatment of type A influenza, 100 female Balb / c mice weighing 18 g were infected with 25 LD50 adapted by their virulent properties for the mouse model of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection was performed intranasally under mild ether anesthesia.

4 hours after the administration of the above preparations, the mice were divided into 2 groups of 50 mice in each group:

- the first group of mice was injected with 20 μl of a solution of single-domain mini-antibodies "Neogrippab" in phosphate buffer at a dose of 2.5 μg / ml,

- the second group of mice was injected with 20 μl of intranasal phosphate buffer.

Within 14 days after infection, the survival rate of the mice and the change in their body weight were taken into account.

The protection of mice against the A / Mallard duck / PA / 10218/84 (H5N2) virus was 100% in the group to which the neogrippab single-domain mini-antibody preparation was administered (see Fig. 7). Also, a decrease in body weight was not observed in these mice. Weight loss curves in mice of each of the two above groups are shown in FIG.

Thus, the intranasal administration of a single-domain mini-antibody Neogrippab fully protects mice from infection with the influenza virus at a dose of 25 LD50.

Example 9

The use of the recombinant adenoviral vector Ad5-Neogrippab for the prevention of influenza

In order to prevent influenza infection, female Balb / c mice weighing 18 g were divided into 3 groups of 50 mice in each group:

- the first group of mice was intranasally injected with 20 μl of a colloidal solution of the recombinant adenoviral vector Ad5-Neogrippab in phosphate buffer at a dose of 10 ⁷ PFU / mouse,

- the second group of mice was intranasally injected with 20 μl of a colloidal solution of a recombinant adenoviral vector Ad-null that does not contain the insert of the target gene in the expression cassette in a phosphate buffer at a dose of 10 ⁷ PFU / mouse,

- the third group of mice was intranasally injected with 20 μl of phosphate buffer.

72 hours after the administration of the above preparations, the mice are infected with 25 LD50 adapted for their virulent properties for the mouse model of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection was performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of the mice and the change in their body weight were taken into account.

The protection of mice against virus A / Mallard duck / PA / 10218/84 (H5N2) was 100% in the group to which the preparation of the recombinant adenoviral vector Ad5-Neogrippab was administered (see Fig. 9). Also, a decrease in body weight was not observed in these mice. Curves of weight loss in mice of each of the three above groups after infection with influenza virus are presented in figure 10.

Thus, the intranasal administration of the recombinant adenoviral vector Ad5-Neogrippab fully protected mice from infection with influenza virus at a dose of 25 LD50.

Example 10

The use of a pharmaceutical composition consisting of a single-domain mini-antibody "Neogrippab" and a recombinant adenoviral vector Ad5-Neogrippab for the prevention and treatment of influenza

In order to prevent and treat influenza in female Balb / c mice weighing 18 g, they were divided into 18 groups of 20 mice in each group:

Group 1, 2 and 3 - each animal was intranasally injected with 20 μl of the preparation containing 3 μg of single-domain mini-antibodies Neogrippab and 10 ⁷ PFU of recombinant adenoviral vector Ad5-Neogrippab,

Group 4, 5 and 6 — each animal was intranasally injected with 20 μl of a preparation containing 3 μg BSA and 10 ⁷ PFU of recombinant adenoviral vector Ad5-Neogrippab,

7, 8 and 9 group - each animal was intranasally injected with 20 μl of the preparation containing 3 μg of single-domain mini-antibodies Neogrippab and 10 ⁷ PFU of recombinant adenoviral vector Ad5-null,

10, 11 and 12 group - each animal was intranasally injected with 20 μl of a preparation containing 3 μg BSA and 10 ⁷ PFU of recombinant adenoviral vector Ad5-null,

13, 14 and 15 group - each animal was intranasally injected with 20 μl of phosphate buffer,

Group 16, 17 and 18 — intact mice.

In groups 1, 4, 7, 10, 13, 16, 1 hour after the administration of the above preparations, the mice were infected with 25 LD50 adapted by their virulent properties for the mouse model of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection was performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of the mice and the change in their body weight were taken into account.

In groups 2, 4, 8, 11, 14, 17, 72 hours after the administration of the above preparations, the mice were infected with 25 LD50 adapted in their virulent properties for the mouse model of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection was performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of the mice and the change in their body weight were taken into account.

In groups 3, 6, 9, 12, 15, 18, 14 days after the administration of the above preparations, the mice were infected with 25 LD50 adapted in their virulent properties for the mouse model of the avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2). Infection was performed intranasally under mild ether anesthesia. Within 14 days after infection, the survival rate of the mice and the change in their body weight were taken into account.

The protection of mice against the virus A / Mallard duck / PA / 10218/84 (H5N2) was 100% in groups No. 1-3, 5-8. Also, a decrease in body weight was not observed in these mice (see table 2).

table 2 Survival and change in body weight of mice after intranasal administration of a pharmaceutical composition consisting of a single domain mini-antibody Neogrippab and a recombinant adenoviral vector Ad5-Neogrippab, and subsequent infection with avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) Group number The maximum decrease in body weight in relation to the initial body weight, on average per group,% The number of surviving mice,% one 4,5 one hundred 2 4,5 one hundred Group number The maximum decrease in body weight in relation to the initial body weight, on average per group,% The number of surviving mice,% 3 5 one hundred four 22 0 5 6 90 6 5 one hundred 7 4,5 one hundred 8 4,5 thirty 9 40 0 10 40 0 eleven 40 0 12 40 0 13 40 0 fourteen 40 0 fifteen 40 0 16 40 0 17 45 0 eighteen 40 about

Thus, the intranasal administration of a pharmaceutical composition consisting of Neogrippab single-domain mini-antibodies and Ad5-Neogrippab recombinant adenoviral vectors fully protects against type A influenza virus infection in all selected infection modes, unlike using Neogrippab antibodies alone or only vector Ad5-Neogrippab.

Claims (9)

1. A single domain mini-antibody characterized by the amino acid sequence of SEQ ID NO: 2, specifically binding to type A (H5N2) influenza hemagglutinin and suppressing type A (H5N2) influenza virus infection, or a functionally active variant thereof. 2. The nucleotide sequence encoding a single-domain mini-antibody according to claim 1 or its functionally active variant. 3. The recombinant viral vector expressing a single-domain mini-antibody according to claim 1 or its functionally active variant containing the nucleotide sequence according to claim 2. 4. The viral vector according to claim 3, characterized in that it is an adenoviral vector. 5. A pharmaceutical composition for preventing or treating an influenza A (H5N2) virus infection, comprising a prophylactically or therapeutically effective amount of a single domain mini-antibody according to claim 1 or a functionally active variant thereof and / or a viral vector according to claim 3 and a pharmaceutically acceptable carrier. 6. A method for the prevention or treatment of influenza A (H5N2) virus infection, comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of a pharmaceutical composition according to claim 5. 7. The method according to claim 6, in which the specified pharmaceutical composition is administered intranasally. 8. The method according to claim 7, in which the intranasal administration is carried out in the form of drops. 9. The method according to claim 7, in which the intranasal administration is carried out in the form of a spray.

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