CA2404111C - A polysaccharide-polypeptide conjugate - Google Patents
A polysaccharide-polypeptide conjugate Download PDFInfo
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- CA2404111C CA2404111C CA2404111A CA2404111A CA2404111C CA 2404111 C CA2404111 C CA 2404111C CA 2404111 A CA2404111 A CA 2404111A CA 2404111 A CA2404111 A CA 2404111A CA 2404111 C CA2404111 C CA 2404111C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to a method for producing a polysaccharide-polypeptide conjugate by reacting a polysaccharide with a polypeptide comprising at least one free amino group. A polysaccharide carrier having vicinal hydroxy groups is oxidized onto the polypeptide whereby the ring is opened so as to create vicinal aldehyde groups and is reacted with one or more base-instable antigen 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
A Polysaccharide-Polypeptide Conjugate The present invention relates to a new use of oxi-dized polysaccharides as a carrier material for compo-nents of vaccines, in particular to a method of producing,a polysaccharide-polypeptide conjugate by re-acting 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 an-tigens are administered in an immunogenic formulation in a small amount, mostly parenteral (subcutaneously or intramuscularly) so as to trigger a strong and protec-tive 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 mi-croorganism.
For years also the effectiveness of many experi-mental vaccines against other diseases has been inves-tigated. 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 _ 1 -or allogenic tumor cells, chemically or molecular-bio-logically modified autologous or allogenic tumor cells, isolated tumor-associated antigens (TAA) or tumor-asso-ciated antigens prepared by chemical or molecular-bio-logical 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 in-duce an immunity from months up to years, since the at-tenuation 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 simul-taneously administering endogenous proteins which play an important role in the build-up of an immune re-sponse, such as, e.g., granulocyte macrophages-stimu-lating factor (GM-CSF), interleukin 2 (IL-2), interleukin 12 (IL-12) or gamma interferon (IFNy).
US-5,554,730-B relates to polysaccharide-protein conjugates, wherein a particulate vaccine is to be cre-ated. For this purpose, a polysaccharide-protein conju-gate is created as a Schiff's base (azomethin), primarily by reacting a protein carrier with an oxi-dized 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 pre-cipitates 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 con-jugates 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 anti-gen 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-an-tigen conjugates, wherein a suitable crosslinker is bound in the polysaccharide by means of an azomethin bond to aldehyde functions obtained by periodate oxida-tion. In the cross-linker, a maleimido function is ad-ditionally provided, to which an -SH group of cysteine can add. The utilized antigens then are N- or C-termi-nally 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 polysaccha-ride-antigen conjugates described.
Finally, US-5,846,951 relates to polysaccharides comprising at least 5 sialic acid residues which poly-saccharides 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 struc-tures with primary amino groups. In particular, all protein antigens normally comprise at least one, but mostly several, Iysines 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 re-act 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 com-prising vicinal hydroxyl groups can be oxidized with the help of suitable oxidants, in particular with peri-odic 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 neighbor-ing 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 men-tioned as two non-limiting examples. Such polysaccha-rides thus can be oxidized with periodate in the above-described manner without the bands 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 ac-cording 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.
_ 5 _ 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 di-rectly to the polysaccharide carrier via at least one azomethine bond. Partially oxidized polysaccharides thus are a suitable carrier material for the formula-tion of vaccines if the utilized base-instable anti-genic 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 pre-ferred 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 polysaccha-rides 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 hav-ing a molecular weight of approximately 2000 kDa.
According to a preferred embodiment of the present invention, the vicinal hydroxyl groups originally pres-ent in the polysaccharide carrier are at least par-tially oxidized, preferably by at least 20~. By controlling the rate of oxidation, e.g. by a stoichio-metric smaller amount of oxidating agent, the amount of aldehyde groups available for an azornethine bond be-tween carrier and polypeptide can easily be adjusted.
Preferably, the base-instable antigenic polypep-tide 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), "Ver-wendung von Antikorpern zur Vakzinierung gegen Krebs"
("The Use of Antibodies for Vaccinating against Can-cer"), the disclosure of which is included herein by reference thereto. The monoclonal antibody HE2 de-scribed 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 polypep-tide has the same binding fine specificity as the anti-body HE2.
It is also suitable if in addition to the respec-tive base-instable antigenic polypeptide substances are conjugated which cause an enhancement of the immune re-sponse, 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 ad-ditionally adsorbed on aluminum hydroxide and/or mixed with pharmaceutically acceptable carriers.
Finally, it is preferred if the polysaccharide-polypeptide conjugate obtained according to the inven-tion is formulated as a vaccine formulation to be ad-ministered by subcutaneous, intradermal or intramuscular injection, e.g. by dissolving or suspend-ing 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 polysaccha-rides 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 _ g -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 sev-eral differing vaccine antigens, or vaccine antigens together with components enhancing the immune re-sponse, such as, e.g., the proteins GM-CSF, IL-2, IL-12 or gamma interferon .
The enclosed Fig. 1 shows the comparison of two formulations as regards the induction of antibodies against HE2 (ELISA) in rhesus monkeys.
E x a m p 1 a .
At first, dextrane having a molecular weight of 2000 kDa (SIGMA D-5376) is oxidized by 20~ with sodium metaperiodate. For this purpose, 324 mg of dextrane are _ g _ dissolved with stirring in 4 mI of distilled water. To this solution, 86 mg of sodium metaperiodate previously dissolved in 0.6 ml of distilled water are admixed, and incubated in the dark at 37°C for 30 minutes. 25 mg of the antibody HE2 (PCT/EP00/00174) are brought to pH 7.4 with 1 M NaaHP04, and 45 u1 of a thimerosal solution (20 mg/ml) are added.
To this solution, 1.675 1 of the above-obtained oxidized dextrane solution are added and incubated in the dark at 37°C for 2 days. The completeness of the reaction is analytically checked by chromatography on a molecular weight column (Zorbax 450). The signal corre-sponding to a molecular weight of 150 kDa (monomeric HE2) has disappeared, and in its place a signal occurs in the exclusion volume of the column which corresponds to a molecular weight of >2000 kDa.
The solution obtained is chromatographed by means of a preparative molecular weight column which is equilibrated with the final buffer (1 mM phosphate buffer in physiological saline, pH=5.5). The material obtained in the exclusion volume consists of the high-molecular conjugate of the antibody HE2 on partially oxidized dextrane. The content of conjugated HE2 can be determined by integration of the signal after analyti-cal chromatography on a molecular weight column as com-pared to monomeric HE2. The solution obtained is mixed with an aqueous aluminum hydroxide such that the final concentration is 0.5 mg of HE2 on 1.67 mg of aluminum hydroxide in 0.5 ml of buffer.
Four rhesus monkeys are subcutaneously immunized with 0.5 ml of the above formulation on days 1, 15, 29 and 57. The sera of various points of time were assayed by means of ELISA for an induction of antibodies against monomeric HE2. As a comparison, four rhesus monkeys were vaccinated in the same manner with a stan-dard formulation of 0.5 mg of monomeric HE2 adsorbed on 1.67 mg of aluminum hydroxide.
The ELISA was carried out as follows:
100 dal aliquots of the MAb HE2 (solution with 10 ug/ml in binding buffer) are incubated in the wells of a microtiter plate for 1 h at 37°C. After having washed the plate six times with washing buffer A, 200 u1 each of blocking buffer A are added and incu-bated at 37°C for 30 minutes. After washing the plate as described above, 100 u1 aliquots each of the monkey sera to be tested are incubated in dilutions of 2:100 to 1:1 000 000 in dilution buffer A at 37°C for 1 h.
After having washed the plate as described above, 100 u1 each of the peroxidase-conjugated goat anti-hu-man-Ig antibody (Zymed) is added in a dilution of 1:1000 in dilution buffer A and incubated at 37°C for 30 minutes. The plate is washed four times with washing buffer A, and twice with staining buffer. The antibody bond is detected by the addition of 100 u1 each of the specific substrate, and the staining reaction is stopped after 10 minutes by adding 50 u1 each of stop-ping solution. The evaluation is effected by measuring the optical density (OD) at 490 nm (wave length of the reference measurement is 620 nm).
The results of the ELISA are illustrated in Fig.
1. The animals vaccinated with the conjugate of HE2 on dextrane developed comparable titers of antibodies against HE2 as the monkeys vaccinated with the standard formulation.
Moreover, it was investigated to which extent HE2 after application can be found again in the serum of the monkeys. For this purpose, again an ELISA was used, which was carried out as follows:
100 u1 aliquots of a purified polyclonal anti-idi-otypic goat antibody against HE2 (solution with 10 ug/ml in binding buffer) are incubated in the wells of a microtiter plate at 37°C for 1 h. After having washed the plate six times with washing buffer A, 200 u1 each of the blocking buffer are admixed and in-cubated at 37°C for 30 minutes. After having washed the plate as described above, 100 u1 aliquots each of the monkey sera to be tested are incubated in dilutions of 1:4 to 1:100 000 in dilution buffer A at 37°C for 1 h.
After having washed the plate as described above, 100 u1 each of a peroxidase-conjugated goat anti-mouse-IgG antibody (Zymed) are added in a dilution of 1:1000 in diluting buffer and incubated at 37°C for 30 min.
The plate is washed four times with washing buffer, and twice with staining buffer. The antibody bond is de-tected by the addition of 100 u1 each of the specific substrate, and the staining reaction is stopped after 10 minutes by adding 50 u1 each of stopping solution.
The evaluation is effected by measuring the optical density (OD) at 490 nm (wave length of the reference measurement is 620 nm).
the results are illustrated in the following ta-ble:
Time Conjugate vaccine Standard formulation 0 0;0;0;0 ng/ml 0;0;0;0 ng/ml 1 h 0;0;0;0 ng/ml 13;17;74;2$0 ng/ml 4 h 0;0;0;0 ng/ml 200,280,400,740 ng/ml !,24 h 0;0;0;0 ng/ml 960,960,1000,740 ng/ml~
After 24 h, a trace of a HE2 concentration can be recognized in the serum of those animals which had been vaccinated with the conjugate, yet this concentration is below the detection limit of approximately 10 ng of HE2/ml serum. Apparently, the desorption of the vaccine antigen has clearly been reduced by the conjugation to partially oxidized dextrane, as compared to the stan-Bard formulation. Thereby probably fewer vaccinations will suffice to obtain a comparable titer than with a standard formulation on aluminum hydroxide.
Materials used:
Microtiter Immuno Plate F96 MaxiSorp (Nunc) for plates: ELISA
Binding buf fer : 15 mM NazC03 35 mM NaHCOa 3 mM NaNs pH: 9.6 PBS deficient: 138 mM NaCl 1.5 mM KHzPOa 2.7 mM KCl 6.5 mM NazHP04 pH: 7.2 tnlashing buffer: 0.05 Tween 20 in PBS deficient Blocking buffer: 5~ fetal calf serum (heat-inactivated) in PBS deficient Dilution buffer: 2~ fetal calf serum (heat-inactivated) in PBS deficient Staining buffer: 24.3 mM citric acid 51.4 mM NazHP04 pH: 5.0 Substrate: 40 mg o-phenylene-diamine-dihydrochlo-ride 100 ml staining buffer 20 ~.t1 HzOz (30~) Stopping solu- 4 N HzS04 Lion:
Vaccines are characterized in that one or more an-tigens are administered in an immunogenic formulation in a small amount, mostly parenteral (subcutaneously or intramuscularly) so as to trigger a strong and protec-tive 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 mi-croorganism.
For years also the effectiveness of many experi-mental vaccines against other diseases has been inves-tigated. 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 _ 1 -or allogenic tumor cells, chemically or molecular-bio-logically modified autologous or allogenic tumor cells, isolated tumor-associated antigens (TAA) or tumor-asso-ciated antigens prepared by chemical or molecular-bio-logical 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 in-duce an immunity from months up to years, since the at-tenuation 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 simul-taneously administering endogenous proteins which play an important role in the build-up of an immune re-sponse, such as, e.g., granulocyte macrophages-stimu-lating factor (GM-CSF), interleukin 2 (IL-2), interleukin 12 (IL-12) or gamma interferon (IFNy).
US-5,554,730-B relates to polysaccharide-protein conjugates, wherein a particulate vaccine is to be cre-ated. For this purpose, a polysaccharide-protein conju-gate is created as a Schiff's base (azomethin), primarily by reacting a protein carrier with an oxi-dized 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 pre-cipitates 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 con-jugates 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 anti-gen 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-an-tigen conjugates, wherein a suitable crosslinker is bound in the polysaccharide by means of an azomethin bond to aldehyde functions obtained by periodate oxida-tion. In the cross-linker, a maleimido function is ad-ditionally provided, to which an -SH group of cysteine can add. The utilized antigens then are N- or C-termi-nally 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 polysaccha-ride-antigen conjugates described.
Finally, US-5,846,951 relates to polysaccharides comprising at least 5 sialic acid residues which poly-saccharides 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 struc-tures with primary amino groups. In particular, all protein antigens normally comprise at least one, but mostly several, Iysines 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 re-act 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 com-prising vicinal hydroxyl groups can be oxidized with the help of suitable oxidants, in particular with peri-odic 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 neighbor-ing 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 men-tioned as two non-limiting examples. Such polysaccha-rides thus can be oxidized with periodate in the above-described manner without the bands 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 ac-cording 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.
_ 5 _ 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 di-rectly to the polysaccharide carrier via at least one azomethine bond. Partially oxidized polysaccharides thus are a suitable carrier material for the formula-tion of vaccines if the utilized base-instable anti-genic 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 pre-ferred 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 polysaccha-rides 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 hav-ing a molecular weight of approximately 2000 kDa.
According to a preferred embodiment of the present invention, the vicinal hydroxyl groups originally pres-ent in the polysaccharide carrier are at least par-tially oxidized, preferably by at least 20~. By controlling the rate of oxidation, e.g. by a stoichio-metric smaller amount of oxidating agent, the amount of aldehyde groups available for an azornethine bond be-tween carrier and polypeptide can easily be adjusted.
Preferably, the base-instable antigenic polypep-tide 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), "Ver-wendung von Antikorpern zur Vakzinierung gegen Krebs"
("The Use of Antibodies for Vaccinating against Can-cer"), the disclosure of which is included herein by reference thereto. The monoclonal antibody HE2 de-scribed 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 polypep-tide has the same binding fine specificity as the anti-body HE2.
It is also suitable if in addition to the respec-tive base-instable antigenic polypeptide substances are conjugated which cause an enhancement of the immune re-sponse, 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 ad-ditionally adsorbed on aluminum hydroxide and/or mixed with pharmaceutically acceptable carriers.
Finally, it is preferred if the polysaccharide-polypeptide conjugate obtained according to the inven-tion is formulated as a vaccine formulation to be ad-ministered by subcutaneous, intradermal or intramuscular injection, e.g. by dissolving or suspend-ing 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 polysaccha-rides 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 _ g -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 sev-eral differing vaccine antigens, or vaccine antigens together with components enhancing the immune re-sponse, such as, e.g., the proteins GM-CSF, IL-2, IL-12 or gamma interferon .
The enclosed Fig. 1 shows the comparison of two formulations as regards the induction of antibodies against HE2 (ELISA) in rhesus monkeys.
E x a m p 1 a .
At first, dextrane having a molecular weight of 2000 kDa (SIGMA D-5376) is oxidized by 20~ with sodium metaperiodate. For this purpose, 324 mg of dextrane are _ g _ dissolved with stirring in 4 mI of distilled water. To this solution, 86 mg of sodium metaperiodate previously dissolved in 0.6 ml of distilled water are admixed, and incubated in the dark at 37°C for 30 minutes. 25 mg of the antibody HE2 (PCT/EP00/00174) are brought to pH 7.4 with 1 M NaaHP04, and 45 u1 of a thimerosal solution (20 mg/ml) are added.
To this solution, 1.675 1 of the above-obtained oxidized dextrane solution are added and incubated in the dark at 37°C for 2 days. The completeness of the reaction is analytically checked by chromatography on a molecular weight column (Zorbax 450). The signal corre-sponding to a molecular weight of 150 kDa (monomeric HE2) has disappeared, and in its place a signal occurs in the exclusion volume of the column which corresponds to a molecular weight of >2000 kDa.
The solution obtained is chromatographed by means of a preparative molecular weight column which is equilibrated with the final buffer (1 mM phosphate buffer in physiological saline, pH=5.5). The material obtained in the exclusion volume consists of the high-molecular conjugate of the antibody HE2 on partially oxidized dextrane. The content of conjugated HE2 can be determined by integration of the signal after analyti-cal chromatography on a molecular weight column as com-pared to monomeric HE2. The solution obtained is mixed with an aqueous aluminum hydroxide such that the final concentration is 0.5 mg of HE2 on 1.67 mg of aluminum hydroxide in 0.5 ml of buffer.
Four rhesus monkeys are subcutaneously immunized with 0.5 ml of the above formulation on days 1, 15, 29 and 57. The sera of various points of time were assayed by means of ELISA for an induction of antibodies against monomeric HE2. As a comparison, four rhesus monkeys were vaccinated in the same manner with a stan-dard formulation of 0.5 mg of monomeric HE2 adsorbed on 1.67 mg of aluminum hydroxide.
The ELISA was carried out as follows:
100 dal aliquots of the MAb HE2 (solution with 10 ug/ml in binding buffer) are incubated in the wells of a microtiter plate for 1 h at 37°C. After having washed the plate six times with washing buffer A, 200 u1 each of blocking buffer A are added and incu-bated at 37°C for 30 minutes. After washing the plate as described above, 100 u1 aliquots each of the monkey sera to be tested are incubated in dilutions of 2:100 to 1:1 000 000 in dilution buffer A at 37°C for 1 h.
After having washed the plate as described above, 100 u1 each of the peroxidase-conjugated goat anti-hu-man-Ig antibody (Zymed) is added in a dilution of 1:1000 in dilution buffer A and incubated at 37°C for 30 minutes. The plate is washed four times with washing buffer A, and twice with staining buffer. The antibody bond is detected by the addition of 100 u1 each of the specific substrate, and the staining reaction is stopped after 10 minutes by adding 50 u1 each of stop-ping solution. The evaluation is effected by measuring the optical density (OD) at 490 nm (wave length of the reference measurement is 620 nm).
The results of the ELISA are illustrated in Fig.
1. The animals vaccinated with the conjugate of HE2 on dextrane developed comparable titers of antibodies against HE2 as the monkeys vaccinated with the standard formulation.
Moreover, it was investigated to which extent HE2 after application can be found again in the serum of the monkeys. For this purpose, again an ELISA was used, which was carried out as follows:
100 u1 aliquots of a purified polyclonal anti-idi-otypic goat antibody against HE2 (solution with 10 ug/ml in binding buffer) are incubated in the wells of a microtiter plate at 37°C for 1 h. After having washed the plate six times with washing buffer A, 200 u1 each of the blocking buffer are admixed and in-cubated at 37°C for 30 minutes. After having washed the plate as described above, 100 u1 aliquots each of the monkey sera to be tested are incubated in dilutions of 1:4 to 1:100 000 in dilution buffer A at 37°C for 1 h.
After having washed the plate as described above, 100 u1 each of a peroxidase-conjugated goat anti-mouse-IgG antibody (Zymed) are added in a dilution of 1:1000 in diluting buffer and incubated at 37°C for 30 min.
The plate is washed four times with washing buffer, and twice with staining buffer. The antibody bond is de-tected by the addition of 100 u1 each of the specific substrate, and the staining reaction is stopped after 10 minutes by adding 50 u1 each of stopping solution.
The evaluation is effected by measuring the optical density (OD) at 490 nm (wave length of the reference measurement is 620 nm).
the results are illustrated in the following ta-ble:
Time Conjugate vaccine Standard formulation 0 0;0;0;0 ng/ml 0;0;0;0 ng/ml 1 h 0;0;0;0 ng/ml 13;17;74;2$0 ng/ml 4 h 0;0;0;0 ng/ml 200,280,400,740 ng/ml !,24 h 0;0;0;0 ng/ml 960,960,1000,740 ng/ml~
After 24 h, a trace of a HE2 concentration can be recognized in the serum of those animals which had been vaccinated with the conjugate, yet this concentration is below the detection limit of approximately 10 ng of HE2/ml serum. Apparently, the desorption of the vaccine antigen has clearly been reduced by the conjugation to partially oxidized dextrane, as compared to the stan-Bard formulation. Thereby probably fewer vaccinations will suffice to obtain a comparable titer than with a standard formulation on aluminum hydroxide.
Materials used:
Microtiter Immuno Plate F96 MaxiSorp (Nunc) for plates: ELISA
Binding buf fer : 15 mM NazC03 35 mM NaHCOa 3 mM NaNs pH: 9.6 PBS deficient: 138 mM NaCl 1.5 mM KHzPOa 2.7 mM KCl 6.5 mM NazHP04 pH: 7.2 tnlashing buffer: 0.05 Tween 20 in PBS deficient Blocking buffer: 5~ fetal calf serum (heat-inactivated) in PBS deficient Dilution buffer: 2~ fetal calf serum (heat-inactivated) in PBS deficient Staining buffer: 24.3 mM citric acid 51.4 mM NazHP04 pH: 5.0 Substrate: 40 mg o-phenylene-diamine-dihydrochlo-ride 100 ml staining buffer 20 ~.t1 HzOz (30~) Stopping solu- 4 N HzS04 Lion:
Claims (19)
1. A method of producing a polysaccharide-polypeptide conjugate by reacting a polysaccharide with a polypep-tide which comprises at least one free amino group, characterized in that a polysaccharide carrier compris-ing vicinal hydroxyl groups is oxidized under ring opening to create vicinal aldehyde groups and reacted with one or more base-instable antigenic polypeptide(s) containing at least one free amino group, the polypep-tide(s) being bound directly to the polysaccharide car-rier via at least one azomethine bond.
2. A method according to claim 1, characterized in that the antigenic polypeptide(s) is (are) stable up to a pH of approximately 11.
3. A method according to claim 1, characterized in that the antigenic polypeptide(s) is (are) stable up to a pH of approximately 10.
4. A method according to claim 1, characterized in that the antigenic polypeptide(s) is (are) stable up to a pH of approximately 9.
5. A method according to claim 1, characterized in that the antigenic polypeptide(s) is (are) stable up to a pH of approximately 8.
6. A method according to any one of claims 1 to 5, characterized in that mannan, preferably having a mo-lecular weight of at least 70 kDa, is used as the poly-saccharide.
7. A method according to any one of claims 1 to 5, characterized in that dextrane, preferably having a mo-lecular weight of at least 70 kDa, particularly pre-ferred having a molecular weight of approximately 2000 kDa, is used as the polysaccharide.
8. A method according to any one of claims 1 to 7, characterized in that the vicinal hydroxyl groups originally present in the polysaccharide carrier are at least partially oxidized, preferably by at least 20%.
9. A method according to any one of claims 1 to 8, characterized in that a vaccine antigen is used as the base-instable antigenic polypeptide.
10. A method according to claim 9, characterized in that an antibody is used as the vaccine antigen.
11. A method according to claim 10, characterized in that a monoclonal antibody is used as the antibody.
12. A method according to claim 11, characterized in that the antibody HE2 is used as the monoclonal anti-body.
13. A method according to any one of claims 10 to 12, characterized in that the antibody has the same binding fine specificity as the antibody HE2.
14. A method according to any one of claims 1 to 13, characterized in that in addition to the respective base-instable antigenic polypeptides, further sub-stances are conjugated which cause an enhancement of the immune response.
15. A method according to claim 14, characterized in that these substances are GM-CSF, IL-2, IL-12 or gamma-interferon or a mixture of these substances.
16. A method according to any one of claims 1 to 15, characterized in that the conjugate additionally is ad-sorbed on aluminum hydroxide.
17. A method according to any one of claims 1 to 16, characterized in that the conjugate is mixed with a pharmaceutically acceptable carrier.
18. A conjugate obtainable according to any one of claims 1 to 17.
19. A conjugate according to claim 18, characterized in that it is formulated to be administered by subcuta-neous, intradermal or intramuscular injections.
Applications Claiming Priority (3)
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| ATA471/2000 | 2000-03-21 | ||
| AT4712000 | 2000-03-21 | ||
| PCT/AT2001/000079 WO2001070272A1 (en) | 2000-03-21 | 2001-03-21 | Polysaccharide-polypeptide conjugate |
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| CA2404111A1 CA2404111A1 (en) | 2002-09-19 |
| CA2404111C true CA2404111C (en) | 2010-05-25 |
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| CA2404111A Expired - Fee Related CA2404111C (en) | 2000-03-21 | 2001-03-21 | A polysaccharide-polypeptide conjugate |
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| AT (1) | ATE429251T1 (en) |
| AU (1) | AU2001242084A1 (en) |
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| DE10209821A1 (en) | 2002-03-06 | 2003-09-25 | Biotechnologie Ges Mittelhesse | Coupling of proteins to a modified polysaccharide |
| AT502293B1 (en) * | 2002-05-15 | 2008-03-15 | Igeneon Krebs Immuntherapie | IMMUNOGENERAL, MONOCLONAL ANTIBODY |
| WO2005014655A2 (en) | 2003-08-08 | 2005-02-17 | Fresenius Kabi Deutschland Gmbh | Conjugates of hydroxyalkyl starch and a protein |
| WO2005044861A1 (en) * | 2003-10-31 | 2005-05-19 | Wyeth Holdings Corporation | Polysaccharides of helicobacter pylori |
| ES2390885T3 (en) * | 2004-03-11 | 2012-11-19 | Fresenius Kabi Deutschland Gmbh | Conjugates of hydroxyalkylamidone and a protein |
| DE102006035899A1 (en) | 2006-07-31 | 2008-02-07 | Dade Behring Marburg Gmbh | New polysaccharide-peptide-conjugate, where the peptide portion contains a sequence having a signal group that is splittable by thrombin at its carbon-terminal end, useful in a procedure for determining the thrombin generation in a sample |
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| SU730694A1 (en) * | 1976-07-05 | 1980-04-30 | Всесоюзный Научно-Исследовательский Технологический Институт Антибиотиков И Ферментов Медицинского Назначения | Terrilitine-modified dextran possessing fibrinolytic activity |
| WO1996020012A2 (en) * | 1994-12-23 | 1996-07-04 | Middlesex Sciences, Inc. | Methods for preparing and purifying macromolecular conjugates |
| US6011008A (en) * | 1997-01-08 | 2000-01-04 | Yissum Research Developement Company Of The Hebrew University Of Jerusalem | Conjugates of biologically active substances |
| CA2233725A1 (en) * | 1998-03-31 | 1999-09-30 | Hemosol Inc. | Hemoglobin-hydroxyethyl starch complexes |
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2001
- 2001-03-21 EP EP01914811A patent/EP1267937B1/en not_active Expired - Lifetime
- 2001-03-21 WO PCT/AT2001/000079 patent/WO2001070272A1/en active Application Filing
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| WO2001070272A1 (en) | 2001-09-27 |
| CA2404111A1 (en) | 2002-09-19 |
| AU2001242084A1 (en) | 2001-10-03 |
| MXPA02008247A (en) | 2004-04-05 |
| DK1267937T3 (en) | 2009-08-03 |
| EP1267937A1 (en) | 2003-01-02 |
| EP1267937B1 (en) | 2009-04-22 |
| DE50114852D1 (en) | 2009-06-04 |
| ATE429251T1 (en) | 2009-05-15 |
| ES2325528T3 (en) | 2009-09-08 |
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