Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1997-05-07
2001-10-30
Wessendorf, T. D. (Department: 1627)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S194100, C424S193100, C514S054000, C514S023000, C514S008100, C530S402000, C436S528000, C436S529000, C436S530000, C436S532000, C436S822000, C436S823000, C436S828000
Reexamination Certificate
active
06309646
ABSTRACT:
BACKGROUND OF THE INVENTION
Vaccines have been very effective in protecting people from a wide variety of diseases, whether caused by viruses, bacteria, or fungi. The ability of vaccines to induce specific protection to such a wide range of pathogenic organisms results from their ability to stimulate specific humoral antibody responses, as well as cell-mediated responses. This invention relates to such vaccines, and particularly to a process for making conjugates, such as protein/polysaccharide conjugates, that are used in the preparation of vaccines and other valuable immunological reagents. The invention further relates to the vaccines and immunological reagents that are produced from the conjugates made in accordance with the invention.
Certain agents can stimulate an immune response with minimal chemical modifications, such as, for example, tetanus toxoid, which is immunogenic even in the absence of adjuvant. Other important agents are either non-immunogenic or poorly immunogenic, but they can be converted into immunogenic molecules or constructs, in which form they can induce vigorous immune responses. For example, most polysaccharides are poorly immunogenic. After they are coupled to proteins, however, the resulting construct becomes immunogenic. The conjugation of proteins to polysaccharides converts the polysaccharide from a weakly immunogenic T-cell independent antigen to a T-cell dependent antigen that recruits T-cell help, and thus stimulates heightened immune responses. Note the discussion by J. M. Cruse, et al. (Editors.),
Conjugate Vaccines,
Karger, Basel, (1989); and R. W. Ellis, et al. (Editors),
Development and Clinical Uses of Haemophilus B Conjugate Vaccines,
Marcel Dekker, New York (1994). These books are entirely incorporated herein by reference.
Conjugation of a protein and a polysaccharide may provide other advantageous results. For example, Applicant has found that a protein/polysaccharide conjugate enhances the antibody response not only to the polysaccharide component, but also to the protein component. This effect is described, for example, in the dual conjugate patent application of Mond and Lees, U.S. patent application Ser. No. 08/402,565 (filed Mar. 13, 1995, now U.S Pat. No. 5,585,100); application Ser. No. 08/444,727 (filed May 19, 1995, now abandoned); and application Ser. No. 08/468,060 (filed Jun. 6, 1995, now abandoned). These patent applications each are entirely incorporated herein by reference. This effect also is described in A. Lees, et al., “Enhanced Immunogenicity of Protein-Dextran Conjugates: I. Rapid Stimulation of Enhanced Antibody Responses to Poorly Immunogenic Molecules,”
Vaccine,
Vol. 12, No. 13 (1994), pp. 1160-1166. This article is entirely incorporated herein by reference.
Techniques have been developed to facilitate coupling of proteins and polysaccharides. Note W. E. Dick, et al., “Glyconjugates of Bacterial Carbohydrate Antigens: A Survey and Consideration of Design and Preparation Factors,”
Conjugate Vaccines
(Eds. Cruse, et al.,), Karger, Basel, 1989, beginning at page 48. This excerpt also is entirely incorporated herein by reference. Many techniques for activation of carbohydrates, however, are not suitable for use in aqueous media because the activating or functional reagents are not stable in water. For example, the use of N,N′-carbonyldiimidazole is described in Marburg et al., U.S. Pat. No. 4,695,624 (which patent is entirely incorporated herein by reference). This reagent must be used in organic media.
For use in aqueous media, applicant has developed the use of 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate, also called “CDAP” in this patent application, to activate polysaccharides. These activated polysaccharides may be directly or indirectly coupled to proteins. The use of CDAP is described in the following U.S. patent applications of Andrew Lees: U.S. patent application Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned), U.S. patent application Ser. No. 08/408,717 (filed Mar. 22, 1995), and U.S. patent application Ser. No. 08/482,666 (filed Jun. 7, 1995). These U.S. patent applications each are entirely incorporated herein by reference. The use of CDAP also is described in Lees, et al., “Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate For Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents,”
Vaccine,
Vol. 14, No. 3 (1996), pp. 190-198. This article also is entirely incorporated herein by reference.
Some polysaccharides have few or cryptic hydroxyls. Thus, these polysaccharides are not suitable for direct derivatization with vinylsulfone, nor for activation by other common methods, such as CNBr activation. Examples of such polysaccharides are Vi antigen and
Neisseria meningiditis
polysaccharide type C (“Neisseria PsC”). Additionally, some polysaccharides are pH sensitive. Thus, they are unsuitable for direct derivatization with vinylsulfone. Examples of such polysaccharides are
Haemophilus influenzae
type B (“PRP”), and Vi. Thus, the ability to perform the entire derivatization process at a lower pH may be important for derivatizing certain polysaccharides.
Often, however, the process of coupling a protein and a polysaccharide may lead to undesirable effects. In some cases, direct coupling can place the protein and polysaccharide in very close proximity to one another and encourage the formation of excessive crosslinks between the protein and the polysaccharide. Under the extreme of such conditions, the resultant material can become very thick (e.g., in a gelled state). Such a material would not be useful as a vaccine formulation.
Over-crosslinking also can result in decreased immunogenicity of the protein and polysaccharide components. In addition, the crosslinking process can result in the introduction of foreign epitopes into the conjugate or can otherwise be detrimental to production of a useful vaccine. The introduction of excessive crosslinks exacerbates this problem.
To limit the probability of excess crosslinking between the protein and polysaccharide, a spacer may be provided between the protein and polysaccharide. The spacer helps maintain physical separation between the protein and polysaccharide molecules, and it can be used to limit the number of crosslinks between the protein and polysaccharide. As an additional advantage, spacers also can be used to control the structure of the resultant conjugate. If a conjugate does not have the correct structure, problems can result that can adversely affect the immunogenicity of the conjugate material. The speed of coupling, either too fast or too slow, also can affect the overall yield, structure, and immunogenicity of the resulting conjugate product. Note Schneerson et al.,
Journal of Experimental Medicine,
Vol. 152, beginning at pg. 361 (1980). This article is entirely incorporated herein by reference. Spacers help regulate the kinetics of the conjugation reaction.
In view of the potential advantages of using spacers, it is desirable to provide a process where a protein is coupled to a polysaccharide via a spacer. In this coupling procedure, spacers are used in the chemical reaction that is needed to join the protein with the polysaccharide. Spacers facilitate this chemical reaction by providing a functional group on one of the molecules that will react with a group present on the other molecule. Either the polysaccharide molecule or the protein molecule may be derivatized with the spacer molecule including the reactive functional group. If necessary, the other molecule also may be separately derivatized with a reactive functional group (e.g., a thiol, hydrazide, or amine) that will facilitate reaction with the spacer during conjugation.
The possible use of homobifunctional vinylsulfones has been considered for certain conjugation reaction processes. One member of this group is divinylsulfone, which has the following structure:
Divinylsulfone has been used to crosslink proteins and to derivatize proteins with haptens. Note, for example, “Conjuga
The Henry M. Jackson Foundation for the Advancement of Military
Wessendorf T. D.
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