JP2001521908A - Encapsulated immunomodulators useful as vaccine adjuvants - Google Patents

Abstract

  (57) [Summary] The present invention relates to compositions comprising encapsulated immunomodulators such as cytokines and lymphokines, which are useful as adjuvants to stimulate an immune response when administered to a human or animal. For example, lymphokines such as IL-1α or IL-1β can be used as immunomodulators encapsulated in the matrices of the invention. The encapsulated composition can include vaccine antigens, such as whole inactivated or attenuated viruses, recombinant or synthetic peptides, and other antigenic substances. The encapsulated matrix may be natural or synthetic. The present invention also relates to the use of an immunomodulator encapsulated in a human or animal as an adjuvant to increase the immune response to an antigen or other substance against which an immune response is desired to occur.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The immune system is regulated in part by a complex network of chemical signals. These signals include interleukins such as IL-1α and IL-1β. IL-1β is a polypeptide hormone synthesized and secreted by stimulated monocytes. The primary translation product of IL-1β is a relatively low biological activity 31 kDa precursor polypeptide. After synthesis, the 31 kDa precursor of IL-1β is enzymatically cleaved to a size of approximately
A relatively active mature form of 17.5 kDa. Mature IL derived from human activated monocytes
The N-terminus of -1β is characterized by an N-terminal amino acid sequence beginning with Ala-Pro. Human mature IL-
The N-terminal Ala residue of 1β is located at position 117, counting from the N-terminus of the human precursor IL-1β polypeptide, and the Asp residue is at position 116. Mature IL-1β contains the C-terminal 153 residues of the precursor polypeptide.

[0002] Many physiological and biological activities have been identified for IL-1β. IL-1β biological activity is often measured by analyzing thymic growth stimulation. IL-1β
Activities include stimulation of B-lymphocyte maturation, lymphocyte proliferation, stimulation of fibroblast proliferation and induction of acute phase protein synthesis by hepatocytes.

[0003] Other biological activities are due to IL-1β polypeptides. These include controlling lymphocyte differentiation and activation, stimulating lymphokine and prostaglandin production, promoting inflammation, inducing acute phase proteins, stimulating bone resorption, and altering iron and zinc levels in the blood. Moreover, IL-1β was found to be able to stimulate the hypothalamus-pituitary-adrenal system, suggesting that IL-1β is incorporated into a complex neuroendocrine network that controls homeostasis. I have.

[0004] Maturation and release of mature IL-1β from macrophages does not proceed by the usual means normally associated with most secreted proteins, because the precursor IL-1β polypeptide lacks a hydrophobic signal sequence. . Furthermore, IL-1β is not associated with the membrane-bound compartment of monocytes. Most secreted proteins are characterized by the presence of a hydrophobic chain of amino acids called a signal sequence. The signal sequence moves the protein across the endoplasmic reticulum membrane during protein synthesis. The protein is then exported out of the cell through exocytosis. Most secreted proteins have a signal sequence at the amino terminus that is removed during translation. Other proteins, such as ovalbumin, have internal signal sequences that are not removed during translation. IL-1β precursors lack any region (amino terminal or internal) of sufficient hydrophobicity and length to qualify as a signal sequence.

[0005] Microencapsulation is the process of encapsulating certain drugs, enzymes, toxins, or other substances in a polymer matrix. This can be used for controlled or delayed release of the drug. Many applications for encapsulation, available matrices, and techniques for making and using encapsulation matrices are described in detail in other papers (eg, Chang, TMS [1977] "Immobilized enzymes and protein biomedical Biomedical applications of immobilized enzymes and proteins, Vol. 1-2, New York, Plenum Press; Deasy, PB (ed.)
[1984] "Microencapsulation and rel.
ated drug processes), Swarbrick (ed.), "Drugs and the pharmaceutical sciences", Volume 20, "Microencapsulation and related drug processes" New York: Marcel Decker, Inc .; McGinity
, JW) [1989] "Aqueous polymer coatings for pharmaceutical dosage forms (Aqueous p.
olymeric coatings for pharmaceutical dosage forms), Drugs and the Pha
rmaceutical Sciences 36; Nixon, JR (eds.) [1976], "Microencapsulation", Swarbrick (eds.), "Drugs and the pharmaceutical sciences," Volume 3, New York: Marcel Decker Inc.). U.S. Pat. No. 4,832,686 discloses microencapsulation of IL-2 in a biocompatible polymer dosage form.

[0006] Liposomes are closed structures with a lipid bilayer membrane that can be used to encapsulate a substance. Liposomes can be prepared using standard materials and methods well known in the art. For example, US Pat. No. 5,059,421 discloses a method for preparing targeted liposomes of defined size distribution.

[0007] It is known that in many cases, the cellular and / or humoral immune response to an antigen administered to an animal can be enhanced or increased by immunizing the animal with the antigen together with some type of adjuvant. Adjuvants are broadly considered compounds or compositions that can enhance or increase the immune response of an animal to an antigen or immunogen (eg, increase antibody titer). Freund's (
Various adjuvants are known in the art, including (complete and incomplete) adjuvants, muramyl dipeptide (MDP) and aluminum sulfate.

[0008] More recently, polypeptides and small peptides have been used as adjuvants. US Patent No. 5,503,841 discloses the use of interleukin-2 (IL-2) as an adjuvant for vaccines. U.S. Pat. No. 5,206,014 discloses the use of a peptide fragment of human IL-1β as an adjuvant for a less immunogenic antigen. However, systemic administration of an immunomodulator such as IL-2 as an adjuvant can result in overstimulation or dysfunctional activation of the animal's immune system. Systemic administration of IL-1β in vivo resulted in unwanted side effects, including fever and nausea. Thus, there is still a need in the art for adjuvants that stimulate or activate appropriate cells of the immune system with minimal systemic exposure.

[0009] BRIEF SUMMARY OF THE INVENTION The present invention relates to useful novel compositions and methods as a vaccine adjuvant. The compositions of the present invention are immunomodulators encapsulated within a matrix. The immunomodulator may be, for example, a cytokine or a lymphokine. In a preferred embodiment, an interleukin compound such as IL-1α and / or IL-1β is utilized according to the present invention. In certain embodiments, the present invention provides an adjuvant composition comprising encapsulated IL-1β. The encapsulated IL-1β composition can optionally include vaccine antigens, such as whole inactivated or attenuated virus, recombinant or synthetic peptides, and other antigenic substances. The use of encapsulated IL-1β makes it possible to present IL-1β to antigen presenting cells with minimal systemic exposure.

[0010] The present invention also relates to the use of an encapsulated immunomodulator used as a means of increasing an immune response in a patient. In an exemplary embodiment of the invention, encapsulated IL-1α or IL-1β can be used as an immunomodulator to increase an immune response. Typically, the encapsulated IL-1β is used as an adjuvant to stimulate or increase an immune response to an immunogen or antigen, as used in vaccine preparations.

[0011] The encapsulated immunomodulator of the present invention can be administered in the presence or absence of a vaccine antigen. The antigen can be encapsulated with the immunomodulator,
Alternatively, it can be administered as a separate composition from the encapsulated immunomodulator. The encapsulated immunomodulator of the present invention can be administered either before or after administration of the vaccine antigen.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the amino acid sequence of mature human IL-1β.

[0013] DETAILED DISCLOSURE OF THE INVENTION The present invention relates to novel compositions comprising encapsulated immunomodulators used as an adjuvant. Specifically described herein is the use of interleukin compounds such as IL-1α or IL-1β as encapsulated immunomodulators. In a preferred embodiment, the immunomodulator is IL-1β. The encapsulated IL-1β composition can be used as an adjuvant.

[0014] The encapsulated immunomodulator composition of the present invention may comprise a whole inactivated or attenuated virus,
Vaccines such as recombinant or synthetic polypeptides or peptides, haptens, and other antigenic or immunogenic agents can be selectively included or used with. Using the methods and materials of the present invention, IL-1β is presented to antigen-presenting cells of the immune system in the presence or absence of a vaccine so that the patient's systemic exposure to IL-1β is minimized.

In a preferred embodiment, IL-1β is used as an immunomodulator. More preferably, the IL-1β used in the present invention is human IL-1β. IL-1β can be either the precursor form or the mature form. When administering the encapsulated IL-1β to a human, the IL-1β can be human mature IL-1β, or a biologically active fragment or variant thereof. Preferably, IL-1β used in the present invention has the amino acid sequence shown in SEQ ID NO: 1. IL-1β from other animal species can also be used when administering the invention to species other than human. IL-1β can be isolated from animal cells, synthesized, or produced by recombinant gene expression means.

In one embodiment, additional molecules and / or immunomodulators such as cytokines, interleukins, and bioactive peptides are also encapsulated with or administered in conjunction with the immunomodulators of the invention. Is done. Preferably, IL-1α and / or IL-1β is the first immunomodulator, which is encapsulated with a second, different immunomodulator. For example, IL-2, IL-4, IL-12 and others have I
It can be encapsulated with L-1α and / or IL-1β. Other suitable molecules are known in the art and will be appreciated by those skilled in the art as being useful in the present invention.

[0017] The immunomodulator can be incorporated into the encapsulation matrix using methods known in the art. The encapsulation matrix may be natural or synthetic. For example, immunomodulators such as IL-1β can be incorporated into biocompatible polymeric materials such as lactic acid, glycolide and glutamic acid. In a preferred embodiment, the encapsulation matrix is a liposome. Liposomes may be produced by any of the standard liposome preparation techniques that are well known and readily implemented. Such liposome preparation techniques are described, for example, in US Pat. No. 5,252,348.

[0018] The immunomodulator can be encapsulated in a matrix in a suitable buffer or carrier solution. The encapsulation matrix can include molecules that target the encapsulated substance to a particular tissue or cell type, eg, antigen presenting cells. For example, liposomes can have receptor molecules in the lipid bilayer to target specific desired cells. In one embodiment, liposomes can incorporate the Fc portion of an immunoglobulin in a lipid bilayer to target cells that express the Fc receptor on their surface. In another embodiment, the encapsulation matrix comprises antibodies that are immunoreactive with molecules expressed on the surface of target cells (eg, antibodies against MHC class II molecules can be used to target antigen presenting cells). May be included. Antigen presenting cells can include mononuclear phagocytic cells, B lymphocytes, dendritic cells, Langerhans cells, and endothelial cells.

The immunomodulator can be incorporated into an encapsulation matrix that provides for controlled and / or sustained release of the immunomodulator when administered to a patient. IL
-1β or IL-1α, or other immunomodulators, may be encapsulated in a matrix that is continuously released over time. Alternatively, the matrix may be specially adapted to release the immunomodulator upon any event, such as a change in pH caused by a local infection. See, for example, US Pat. No. 5,554,147 for a description of pH sensitive biopolymers.

The present invention also relates to a method of enhancing an immune response in an animal or human by administering an effective amount of an encapsulated immunomodulator to the human or animal in need of such treatment. In a preferred embodiment, the encapsulated IL-1β can be used as an adjuvant to increase the immune response to an immunogen or antigen, such as when used in a vaccine composition. Preferably, the vaccine composition can comprise a whole inactivated or attenuated virus, a subunit of a viral component, a recombinant or synthetic polypeptide or peptide, a hapten, and other antigenic or immunogenic substances. The encapsulated IL-1β can be administered in the presence or absence of a vaccine composition. Preferably, the encapsulated IL-1β is administered in a pharmaceutically acceptable carrier. The vaccine composition can be encapsulated with IL-1β or can be administered in a separate composition from the encapsulated IL-1β. The encapsulated IL-1β of the present invention can be administered either before or after vaccine administration.

The amount of IL-1β or other immunomodulator to be administered according to the method of the present invention is
It can be readily determined by one skilled in the art having the benefit of this disclosure.

The encapsulated compositions of the present invention can be administered to animals or humans parenterally, for example, by intramuscular or subcutaneous injection.

The methods and compositions of the present invention can be used with vaccines aimed at treating or immunizing animals and / or humans against bacteria, viruses, tumor cells, fungi, and parasites.

[0024] All references cited herein are incorporated by reference.

The following is an example illustrating a method for practicing the present invention. These examples should not be construed as limiting. Unless stated otherwise, all percentages are by weight and all solvent blends are by volume.

Example 1 IL-1β Inclusion and Vaccine Experiment In this test, four groups of female Sprague-Dawley rats were used. The study was performed for 58 days and the purpose was to evaluate the use of IL-1β as an adjuvant. Vaccines used in these experiments was the influenza A / Beijing type H ₃ N ₃ (Park Davis). After recording the body weight of each animal on day 0 before administration, the animals were intramuscularly administered with 100 μl of a sample containing liposomes encapsulating IL-1β. Thereafter, the animals were weighed again and the body weight was recorded every 7 days during the test before blood was collected. 30
On day, animals received a booster dose of the test sample. Blood was collected on days 0, 7, 14, 28, 37, and 58 to determine serum IgG ELISA titers. Animals were observed daily for sickness or death. On day 58, the rats were weighed and euthanized.

Group: I. It was mixed with mature IL-l [beta] Influenza A / Beijing type H ₃ N ₃ vaccine (encapsulated tooth) II. Influenza A / Beijing type H ₃ N ₃ vaccine alone (without encapsulation) III. Influenza A / Beijing type H ₃ N ₃ vaccines and mature IL-l [beta] which together encapsulated in liposomes

As shown in FIG. 1, IL-1 was mixed with influenza A / Beijing type H ₃ N ₃ Vaccine
Animals receiving β-encapsulated liposomes showed a significant increase in anti-influenza A antibody titers compared to animals administered vaccine alone or unencapsulated vaccine / IL-1β.

Example 2-Vaccine Composition The encapsulated immunomodulator composition described herein can be conveniently used with an antigenic or immunogenic composition for preparing a vaccine. Such compositions, when administered to a human or animal, increase the immune response to the administered vaccine antigen as compared to the vaccine antigen when administered alone.

[0030] Vaccines can be prepared by methods well known in the art. For example,
Such vaccines can be prepared as injectables, for example, as liquid solutions or suspensions. Solid dosage forms can also be prepared in solutions or suspensions prior to injection. Alternatively, it is possible to emulsify the preparation. The encapsulated immunomodulator composition and one or more active antigenic ingredients can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents or adjuvants such as aluminum hydroxide or muramyl dipeptide or a variant thereof. Similarly, cholera toxin subunit B or other substances that stimulate antibody production at mucosal sites can be used. In the case of peptides, coupling to larger molecules such as KLH or tetanus toxoid sometimes increases immunogenicity. Vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include suppositories, and optionally include oral formulations. For suppositories, traditional binders and carriers include, for example, polyalkylenes (polyalka
lene) glycols or triglycerides. Suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, containing from about 10% to about 95% of active ingredient, preferably from about 25% to About 70% can be included.

The compounds may be formulated into a vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed at the free amino groups of the peptide), for example, inorganic acids such as hydrochloric or phosphoric acid, or acetic, oxalic, tartaric,
It is formed with an organic acid such as mandelic acid. Salts formed with free carboxyl groups also include, for example, sodium, potassium, ammonium, calcium,
Or it can be derived from inorganic bases such as iron hydroxide and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

The vaccines of the present invention can be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The dosage will depend on the subject to be treated and the degree of protection desired. Advantageously, methods known to enhance mucosal immunity can be combined with systemic immunity enhancers to maximize the immune response. Suitable regimes for the first dose and the booster dose also vary, but are usually followed by one or two weeks after the first dose and subsequent injections or other doses.

The examples and embodiments described herein are for illustrative purposes only, and in view of them, various modifications or alterations will occur to those skilled in the art and It is to be understood that within the spirit and scope of the present invention and within the scope of the appended claims.

[Sequence list]

[Brief description of the drawings]

1 shows the following anti-influenza A IgG antibodies obtained after immunization of rats by one: mature IL-l [beta] Influenza were mixed with A / Beijing type H ₃ N ₃ vaccines (without encapsulation), Influenza A / Beijing type H ₃ N ₃ vaccines (without encapsulation) alone or influenza a / Beijing type H ₃ N ₃ vaccine and mature IL-l [beta] encapsulated together liposomes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/39 A61P 31/12 A61P 31/12 C07K 14/54 C07K 14/54 C12N 7/00 C12N 7 / 00 C07K 14/545 // C07K 14/545 A61K 37/02 (72) Inventor Casey Leslie S. New York, New York, USA. 88th Street 119 Apartment 3B F-term (Reference) 4B065 AA95X BC41 BD06 CA45 4C076 AA19 BB16 CC06 CC07 EE24 EE25 FF70 4C084 AA02 DA13 MA24 MA66 NA05 ZB071 ZB072 4C085 AA03 AA13 AA38 DD86 EE06 A110 CA03 A03 A03 A04 A10 CAA EA30 EA31 FA74

Claims (34)

[Claims] 1. A composition for modulating an immune response in an animal or human comprising an immunomodulator encapsulated in a matrix. 2. The composition of claim 1, wherein the immunomodulator comprises an IL-1α or IL-1β polypeptide, or a fragment or variant thereof. 3. The composition according to claim 2, wherein the immunomodulator is an IL-1β polypeptide. 4. The composition of claim 3, wherein said IL-1β polypeptide is human mature IL-1β. 5. The composition of claim 4, wherein the IL-1β polypeptide has the amino acid sequence shown in SEQ ID NO: 1. 6. The composition of claim 1, further comprising a second immunomodulator. 7. The composition of claim 6, wherein the second immunomodulator is selected from the group consisting of IL-2, IL-4, IL-12 and biologically active fragments and variants thereof. 8. The composition of claim 1, further comprising an antigenic composition. 9. The composition of claim 8, wherein the vaccine composition is selected from the group consisting of a whole inactivated virus, an attenuated virus, a recombinant or synthetic polypeptide, a hapten, an antigen, and an immunogen. 10. The composition according to claim 1, wherein the encapsulation matrix is a liposome. 11. The composition of claim 1, wherein the encapsulation matrix comprises a biocompatible polymer. 12. The composition according to claim 11, wherein the biocompatible polymer is selected from the group consisting of lactic acid, glycolide, and glutamic acid. 13. The composition of claim 1, wherein the matrix comprises a molecule that targets the encapsulation composition to a target tissue or cell. 14. The composition of claim 13, wherein the targeting molecule is the Fc portion of an immunoglobulin. 15. The composition of claim 13, wherein the targeting molecule is an antibody that is immunoreactive with a molecule expressed on the surface of the target tissue or cell. 16. The composition of claim 15, wherein the antibody binds to an MHC class II molecule. 17. A method of modulating an animal's immune response to an antigen, immunogen, or vaccine, comprising administering to the animal a composition comprising an immunomodulator encapsulated in a matrix. 18. The immunomodulator may be an IL-1β polypeptide, an IL-1α polypeptide,
18. The method of claim 17, comprising a fragment or variant thereof. 19. The method of claim 17, wherein the encapsulated immunomodulator is administered prior to administering the antigen, immunogen, or vaccine. 20. The method of claim 17, wherein the encapsulated immunomodulator is administered following administration of the antigen, immunogen, or vaccine. 21. The method of claim 19, wherein said polypeptide is human mature IL-1β. 22. The method of claim 17, wherein a second encapsulated immunomodulator is administered. 23. The method of claim 22, wherein the second immunomodulator is selected from the group consisting of IL-2, IL-4, IL-12 and biologically active fragments and variants thereof. 24. The method of claim 17, further comprising administering a vaccine composition. 25. The method of claim 24, wherein the vaccine composition is selected from the group consisting of a whole inactivated virus, an attenuated virus, a recombinant or synthetic polypeptide, a hapten, an antigen and an immunogen. 26. The method of claim 17, wherein the encapsulation matrix is a liposome. 27. The method of claim 17, wherein the encapsulation matrix comprises a biocompatible polymer. 28. The method of claim 27, wherein the biocompatible polymer is selected from the group consisting of lactic acid, glycolide, and glutamic acid. 29. The method of claim 17, wherein the matrix comprises a molecule that targets the encapsulation composition to a target tissue or cell. 30. The method of claim 29, wherein the targeting molecule is the Fc portion of an immunoglobulin. 31. The method of claim 29, wherein the targeting molecule is an antibody that is immunoreactive with a molecule expressed on the surface of the target tissue or cell. 32. The method of claim 31, wherein the antibody binds to an MHC class II molecule. 33. The encapsulated immunomodulator is administered parenterally.
The described method. 34. The method of claim 17, wherein the encapsulated immunomodulator is administered by intramuscular or subcutaneous injection.