Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-05-13
2002-10-22
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S827000, C530S850000, C530S402000, C530S403000, C435S069100, C435S252300, C435S471000
Reexamination Certificate
active
06469139
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to substances, in particular to modified human chorionic gonadotropin (&bgr;-hCG) proteins/genes, and their medical use, for example as immunological contraceptives having improved specificity and/or which in vivo avoid producing antibodies having undesirable cross-reactivity, for example with other natural hormones.
BACKGROUND OF THE INVENTION
The principle of immunising the female with &bgr;-hCG or its C-terminal peptide to induce antibodies which neutralise hCG and therefore inhibit pregnancy has been proposed
1
and has been the subject of trials by the World Health Organization
2
and the Indian Health Authorities
3
.
Shortly after fertilization of the ovum, the hormone hCG which at other times is essentially absent from the body, is produced and acts on the corpus luteum in the ovary to promote synthesis of progesterone. Progesterone is vital for the maintenance of the fertilized egg in the uterus and so the production of antibodies to neutralise the hCG will effectively prevent the pregnancy from proceeding. This strategy has been successfully employed to block fertility in baboons
1
and marmosets
4
and more recently in humans
3
.
hCG itself is composed of two chains, &agr; and &bgr;. The &agr;-chain is common to other hormones (FSH, TSH and LH), which contribute to normal physiological function, so that autoantibodies made to this chain would be highly undesirable. The &bgr;-chain of hCG is far more specific, but a major problem still remains in that there is an 85% homology of &bgr;-hCG with the &bgr;-chain of luteinizing hormone (LH) which is present continually in the potentially fertile female. A strategy adopted by the W.H.O. has been to prepare a vaccine based on the &bgr;-hCG C-terminal peptide (residues 109-145) which is unique to hCG. This is made immunogenic by linking to the carrier proteins tetanus or diphtheria toxoids to provide T-cell help. This has not produced adequately high responses in high frequency within the cohorts tested
5
partly because of the relatively weak immunogenicity of the peptide and the fact that antibodies to a peptide fragment of a protein do not usually bind with high affinity to the parent protein
6
.
Talwar adopted a less cautious approach by using the whole &bgr;-hCG chain (together with ovine &agr;-chain as a carrier) in the hope that the antibodies produced which cross-reacted with LH would not prove to be troublesome. However, not enough experience has been gained so far to confirm this hope and in principle, where possibly millions of people could be immunized with the vaccine for several years, it would seem prudent, to devise a vaccine which did not cross-react with LH.
SUMMARY OF THE INVENTION
It is known that the epitopes specific for &bgr;-hCG other than the C-terminus are discontinuous, i.e. the residues making up the epitope may be separate from each other in primary structure but are brought together by the protein folding. However, the contact residues forming these discontinuous epitopes are very difficult to identify and even if they could be, the “floppiness” of any synthetic peptide formed from these residues would make it a poor immunogen with respect to the generation of antibodies with high affinity.
In the present invention, we have adopted a strategy
7
which relies upon the natural folding of the protein to form the specific discontinuous epitope, while at the same time mutating the parent gene in such a way that the amino acid residues forming the LH cross-reacting epitopes are altered without affecting the more distant folding of the hCG-specific epitope(s). The retention of the desired epitope(s) and the loss of the unwanted epitopes can be monitored by reaction of the mutants with monoclonal antibodies specific for hCG and others giving cross-reaction between hCG and LH.
Broadly, the present invention provides a substance which has the property of inducing a neutralising antibody response to &bgr;-hCG in vivo, said antibodies not substantially cross-reacting with LH, the substance comprising one or more of the conformational epitopes specific to native &bgr;-hCG, or functional equivalents or mimetics of these epitopes.
In one aspect, the substance is a modified &bgr;-hCG protein having one or more conformational epitopes specific to native &bgr;-hCG, the protein being modified at one or more amino acid residues forming epitope(s) of native &bgr;-hCG that cross-react with LH, to reduce the cross-reactivity the &bgr;-hCG protein with LH, as defined by the ability of both proteins to react with the same antibody. The present invention also includes substances which are variants, derivatives, functional equivalents or mimetics of these above proteins.
Preferably, the substance includes two or more epitopes that are specific to native &bgr;-hCG. This helps to induce the production of antibodies specific for these epitopes, which will form complexes of the &bgr;-hCG with two or more antibody molecules, so helping to improve the in vivo neutralising activity caused by the substance.
Preferably, the modified amino acid residues are selected from the following residues of native &bgr;-hCG; Lys20, Glu21, Gly22, Pro24, Val25, Glu65, Arg68, Gly71, Arg74, Gly75 and/or Val79.
There are other residues common to &bgr;-hCG and &bgr;-LH which lie on the outside of the protein molecule accessible to the aqueous solvent phase, which might potentially be immunogenic and give rise to cross-reacting antibodies. This would have to be established following immunisation with the mutant &bgr;-hCG and a similar further mutation procedure would then be required to abolish the epitopes reacting with these new antibodies.
The rationale for selecting the residues to replace the native residues is set out below in more detail. Preferred modifications are set out in table 2.
In a further aspect, the present invention provides nucleic acid encoding the above proteins, vectors incorporating the nucleic acid and host cells transformed with the vectors.
In a further aspect, the present invention includes compositions comprising one or more of the above substances, in combination with a physiologically acceptable carrier. Preferably, the compositions will be contraceptive compositions in a form suitable for immunisation. However, the substances, proteins or compositions described herein may prove useful in hCG-specific immunoassays and for applications where hCG is active, such as the inhibition of Kaposi sarcoma.
In a further aspect, the present invention provides a method of contraception, more strictly in this context contragestative, for a female mammal comprising immunising the female mammal with a contraceptively effective amount of one or more of the substances.
In a further aspect, the present invention includes the use of the substances in the manufacture of a contraceptive composition.
Conveniently, the immunogenicity of the substance may be enhanced by linking it to a carrier such as tetanus toxoid, or to appropriate sequences from such a carrier acting as T-helper epitopes. Additionally the substance may be engineered as a fusion protein with an appropriately immunogenic partner. Engineered DNA constructs containing nucleotide sequences encoding the substance together with, for example, additional sequences encoding T-helper epitopes or cytokine adjuvants, may be directly administered as a nucleic acid, preferably DNA, vaccine.
It will be appreciated that the nucleic acid construct encoding the modified &bgr;-hCG protein can be used as an initial vaccine to prime an immune response. This initial response can then be boosted by subsequent injection of the modified &bgr;-hCG protein itself. Likewise, the modified &bgr;-hCG protein could be used first followed by the nucleic acid to boost the immune response.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitabl
Delves Peter John
Lund Torben
Roitt Ivan Maurice
Kunz Gary L.
Landsman Robert S.
University College London
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