Patent 6,956,119

Disclosed is a method of producing a polysaccharide-polypeptide conjugate by reacting a polysaccharide with a polypeptide which contains at least one free amino group, wherein a polysaccharide carrier comprising vicinal hydroxyl groups is oxidized under ring opening to create vicinal aldehyde groups and is reacted with one or more base-instable antigenic polypeptide(s) containing at least one free amino group, the polypeptide(s) being bound directly to the polysaccharide carrier via at least one azomethine bond.

Description of the Invention

The present invention relates to a new use of oxidized polysaccharides as a carrier material for components of vaccines, in particular to a method of producing a polysaccharide-polypeptide conjugate by reacting a polysaccharide with a polypeptide comprising at least one free amino group, as well as to the use of such a conjugate as a vaccine.

Vaccines are characterized in that one or more antigens are administered in an immunogenic formulation in a small amount, mostly parenteral (subcutaneously or intramuscularly) so as to trigger a strong and protective immune response. At present, most vaccines are produced for protecting against microbial infections. In these instances, the antigens used are inactivated and altered microorganisms or parts thereof, or defined proteins from such microorganisms which are suitable to trigger an immune response against the respective microorganism.

For years also the effectiveness of many experimental vaccines against other diseases has been investigated. Among them are vaccines against cancer. In this case, the immune system of cancer patients is to be selectively activated so as to combat malignant cells. This is attempted by means of the most differing approaches. Among them are vaccinations with autologous or allogenic tumor cells, chemically or molecular-biologically modified autologous or allogenic tumor cells, isolated tumor-associated antigens (TAA) or tumor-associated antigens prepared by chemical or molecular-biological methods, peptides derived therefrom, anti-idiotypical antibodies as a surrogate of a TAA, lately also vaccinations with DNA which codes for TAA or for structures derived therefrom, etc. In principle, very small amounts of a suitable vaccine will suffice to induce an immunity from months up to years, since the attenuation can be boosted by booster vaccinations. Moreover, in an active immunization both a humoral and a cellular immunity can be induced the interaction of which can yield an effective protection against cancer.

To attain a strong immunity, antigens in vaccines mostly are administered together with an adjuvant. As examples of adjuvants the following may be mentioned, without, however, being restricted thereto: aluminum hydroxide (Alu-Gel), derivatives of lipopolysaccharide, Bacillus Calmette Guerin (BCG), liposome preparations, formulations with additional antigens against which the immune system has already produced a pronounced immune response, such as, e.g., tetanus toxoid, Pseudomonas exotoxin or components of influenza viruses, optionally in a liposome preparation. Furthermore, it is known that the immune response may also be enhanced by simultaneously administering endogenous proteins which play an important role in the build-up of an immune response, such as, e.g., granulocyte macrophages-stimulating factor (GM-CSF), interleukin 2 (IL-2), interleukin 12 (IL-12) or gamma interferon (IFNγ).

U.S. Pat. No. 5,554,730-B relates to polysaccharide-protein conjugates, wherein a particulate vaccine is to be created. For this purpose, a polysaccharide-protein conjugate is created as a Schiff's base (azomethin), primarily by reacting a protein carrier with an oxidized polysaccharide antigen in the presence of a "crowding agent" (water displacing agent), wherein the protein carrier is immediately denatured due to the presence of the crowding agent, and the conjugate precipitates in the form of microparticles. Although a dissolution of the precipitated microparticles in a strongly basic environment (0.1 N NaOH) for obtaining a vaccination solution as such is possible and has also been disclosed, it only makes sense if a polysaccharide antigen is used, because any antigenic protein would have lost its antigenic determinants as a consequence of denaturing, and thus would no longer be effective.

WO 99/55715 describes polysaccharide-antigen conjugates in which the antigen is either bound to the polysaccharide via a suitable bivalent linker, or via a terminal aldehyde group. A direct binding of the antigen to the polysaccharide via an azomethin bond thus is limited to the number of the terminal aldehyde groups present in the polysaccharide.

Also DE-198 21 859-A1 describes polysaccharide-antigen conjugates, wherein a suitable crosslinker is bound in the polysaccharide by means of an azomethin bond to aldehyde functions obtained by periodate oxidation. In the cross-linker, a maleimido function is additionally provided, to which an —SH group of cysteine can add. The utilized antigens then are N- or C-terminally provided with an additional Cys so as to allow for the addition of the terminal SH function with the cross-linker and thus the obtaining of the polysaccharide-antigen conjugates described.

Finally, U.S. Pat. No. 5,846,951 relates to polysaccharides comprising at least 5 sialic acid residues which polysaccharides can be provided with terminal aldehyde groups at the non-reducing ends of the polysialic acids by means of oxidation with sodium periodate. Terminal aldehyde groups created in this manner may then bind amino-group-containing medicaments, e.g proteins, via azomethine bonds.

Most antigens used for vaccines comprise structures with primary amino groups. In particular, all protein antigens normally comprise at least one, but mostly several, lysines in their amino acid sequence. The amino groups of these lysines are present in free form.

It has long been known that primary amines can react with aldehydes. The product of this reaction is called Schiff's base. Schiff's bases are not completely stable compounds, they can be hydrolyzed under suitable conditions and thus be returned into their starting substances.

Furthermore, it has been known that compounds comprising vicinal hydroxyl groups can be oxidized with the help of suitable oxidants, in particular with periodic acid or salts of periodic acid, such as sodium metaperiodate, such that two aldehyde functions are formed by breaking the C—C bond on which the neighboring hydroxyl groups are located.

A large number of high-molecular polysaccharides consist of monomeric sugar units which carry vicinal hydroxyl groups. Dextrane and mannan should be mentioned as two non-limiting examples. Such polysaccharides thus can be oxidized with periodate in the above-described manner without the bonds between the monomers being split. If, based on the number of monomeric units, a stoichiometric smaller amount of periodate is used, the oxidation will occur only partially, which means that only so many monomers will be oxidized according to the principle of random as corresponds to the amount of periodate.

The present invention is based on the object of providing further means and methods which will lead to immunogenic formulations of vaccines.

In a method of the initially defined type, this object is achieved in that a polysaccharide carrier comprising vicinal hydroxyl groups is oxidized under ring opening to create vicinal aldehyde groups, and is reacted with one or several base-instable antigenic polypeptide(s) containing at least one free amino group, wherein the polypeptide(s) is (are) bound directly to the polysaccharide carrier via at least one azomethine bond. Partially oxidized polysaccharides thus are a suitable carrier material for the formulation of vaccines if the utilized base-instable antigenic polypeptides comprise one or more free primary amino groups and thus, via an azomethine bond, can be connected with the vicinal aldehyde groups created in the carrier material by ring opening. Preferably, the base-instable antigenic polypeptides used according to the invention are stable up to a pH of approximately 11, preferably up to a pH of approximately 10, still more preferred up to a pH of approximately 9, most preferred up to a pH of approximately 8. If polypeptides are mentioned in the context of the present invention, proteins having at least 6 amino acids in the chain are to be understood. In the same way, polysaccharides are understood to be poly-sugars comprising at least 3 monomer units in the chain. Preferably used polysaccharides are mannan, e.g. having a molecular weight of at least 70 kDa, and dextrane, e.g. having a molecular weight of at least 70 kDa, particularly preferred having a molecular weight of approximately 2000 kDa.

According to a preferred embodiment of the present invention, the vicinal hydroxyl groups originally present in the polysaccharide carrier are at least partially oxidized, preferably by at least 20%. By controlling the rate of oxidation, e.g. by a stoichiometric smaller amount of oxidating agent, the amount of aldehyde groups available for an azomethine bond between carrier and polypeptide can easily be adjusted.

Preferably, the base-instable antigenic polypeptide is a vaccine antigen, particularly preferred an antibody, e.g. a monoclonal antibody, such as the murine monoclonal antibody HE2. A new method of cancer vaccination has been described in application PCT/EP00/00174 (priority date: Jan. 13, 1999), "Verwendung von Antikörpern zur Vakzinierung gegen Krebs" ("The Use of Antibodies for Vaccinating against Cancer"), the disclosure of which is included herein by reference thereto. The monoclonal antibody HE2 described there which is used as the vaccine antigen in a cancer vaccination serves as a non-limiting example for the formulation of a vaccine according to the method of conjugation to a partially oxidized high-molecular polysaccharide described here.

According to a further preferred embodiment of the present invention, the base-instable antigenic polypeptide has the same binding fine specificity as the antibody HE2.

It is also suitable if in addition to the respective base-instable antigenic polypeptide substances are conjugated which cause an enhancement of the immune response, e.g. GM-CSF, IL-2, IL-12 or Gamma-Interferon, or a mixture of these substances.

Moreover, it is preferred if the polysaccharide-polypeptide conjugate according to the invention is additionally adsorbed on aluminum hydroxide and/or mixed with pharmaceutically acceptable carriers.

Finally, it is preferred if the polysaccharide-polypeptide conjugate obtained according to the invention is formulated as a vaccine formulation to be administered by subcutaneous, intradermal or intramuscular injection, e.g. by dissolving or suspending the optionally, e.g., aluminum-hydroxide-adsorbed conjugate in a suitable physiological buffer and the like.

In general, the following advantages and specific properties of the conjugate according to the invention should be mentioned:

	The components coupled to the oxidized polysaccharides via primary amines (conjugate and adjuvants and additives, respectively) are slowly released in the presence of an excess of molecules with free primary amines, e.g. serum proteins. The slow release effect thus forming is desired for vaccines, since by this antigen-presenting cells are able to locally receive the vaccination antigens at the site of vaccination for a longer period of time.
	By the choice of the polysaccharide, the properties of the conjugate can be influenced. This applies both to the molecular size of the polysaccharide and to its chemical composition. If, e.g., mannan is chosen as the polysaccharide, the corresponding conjugate preferably will be taken up by cells of the immune system which carry the mannose receptor. Among them are, in particular, macrophages and dendritic cells as professional antigen-presenting cells. In this manner, an increased immune response is attained.
	Several components can simultaneously be bound to partially oxidized polysaccharides. These may be several differing vaccine antigens, or vaccine antigens together with components enhancing the immune response, such as, e.g., the proteins GM-CSF, IL-2, IL-12 or gamma interferon.

Claim 1 of 19 Claims

1. A method of producing a polysaccharide-polypeptide conjugate, comprising:

providing a polysaccharide carrier having vicinal hydroxyl groups,

providing at least one antigenic polypeptide having at least one free amino group,

partially oxidizing said polysaccharide carrier under ring opening so as to form vicinal aldehyde groups,

reacting said oxidized polysaccharide carrier with said at least one antigenic polypeptide having the at least one free amino group, said at least one antigenic polypeptide being bound directly to said polysaccharide carrier via at least one azomethin bond thereby forming said polysaccharide-polypeptide conjugate.

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