U.S. patent number 4,478,823 [Application Number 06/456,976] was granted by the patent office on 1984-10-23 for immunological preparations containing purified mhc antigens bonded to other antigens.
This patent grant is currently assigned to National Research Development Corporation. Invention is credited to Arnold R. Sanderson.
United States Patent |
4,478,823 |
Sanderson |
October 23, 1984 |
Immunological preparations containing purified MHC antigens bonded
to other antigens
Abstract
An immunologically active preparation for human or veterinary
use comprises an antigenic material in combination with an MHC
antigen.
Inventors: |
Sanderson; Arnold R. (East
Grinstead, GB2) |
Assignee: |
National Research Development
Corporation (London, GB2)
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Family
ID: |
10414407 |
Appl.
No.: |
06/456,976 |
Filed: |
January 10, 1983 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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116677 |
May 29, 1979 |
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Foreign Application Priority Data
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Sep 28, 1977 [GB] |
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40339/77 |
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Current U.S.
Class: |
424/194.1;
424/193.1; 424/195.11; 424/196.11; 424/197.11; 424/198.1;
424/210.1; 424/236.1; 424/238.1; 424/243.1; 436/543; 530/350;
530/397; 530/398; 530/399; 530/405; 530/806; 530/807; 530/825;
530/826; 530/830; 530/838 |
Current CPC
Class: |
A61K
39/385 (20130101); A61K 2039/55516 (20130101); A61K
2039/605 (20130101); Y10S 530/806 (20130101); Y10S
530/838 (20130101); Y10S 530/825 (20130101); Y10S
530/83 (20130101); Y10S 530/807 (20130101); Y10S
530/826 (20130101) |
Current International
Class: |
A61K
39/385 (20060101); A61K 039/385 (); A61K 039/00 ();
C07G 007/00 () |
Field of
Search: |
;260/112R,112B
;424/88,89.91,92 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Gooding et al. J. of Experimental Med., 1974, 140, 61-77. .
Henning et al. Nature, vol. 263, 1976, pp. 689-691. .
Garrido et. al. Nature, vol. 261, 1976, pp. 705-707. .
Fujimoto et. al. Journal of Immunology, 1973, 111, 1093-1100. .
Sanderson et. al. Transplantation, vol. 16, No. 4, (1973), 304-312.
.
Sanderson, Nature, vol. 269, 1977, pp. 414-417..
|
Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Oblon, Fisher, Spivak, McClelland
& Maier
Parent Case Text
This is a continuation of application Ser. No. 116,677, filed as
PCT GB78/00016, Sept. 27, 1978, published as WO79/00160, Apr. 5,
1979 .sctn.102(e) date filed May 29, 1979, now abandoned.
Claims
I claim:
1. A synthetic immunogenic complex comprising a purified MHC
antigen bonded to a second antigen, with the provisos that said
second antigen is not a protein with which said MHC antigen is
normally associated in nature and is not another MHC antigen.
2. The immunogenic complex according to claim 1, wherein the second
antigen is a viral antigen.
3. The immunogenic complex according to claim 2, wherein the viral
antigen is an influenza virus antigen.
4. The immunogenic complex according to claim 2, wherein the viral
antigen is a foot and mouth disease virus antigen.
5. The immunogenic complex according to claim 4, wherein the MHC
antigen is bovine or porcine LA.
6. The immunogenic complex according to claim 1, wherein the MHC
antigen is an HLA antigen or an HLA-analogous MHC antigen of a
non-human animal species.
7. The immunogenic complex according to claim 1, wherein the second
antigen is bonded to the MHC antigen through hydrogen bonding,
ionic bonding, hydrophobic bonding or covalent bonding.
8. A synthetic immunogenic complex comprising a purified MHC
antigen bonded to a second antigen selected from the group
consisting of bacterial antigens and hormone peptide antigens.
9. The immunogenic complex according to claim 8, wherein the second
antigen is a bacterial antigen.
10. The immunogenic complex according to claim 9, wherein the
bacterial antigen is a bacterial toxin or toxoid.
11. The immunogenic complex according to claim 10, wherein the
toxoid is diptheria toxoid.
12. The immunogenic complex according to claim 8, wherein the
second antigen is a hormone peptide.
13. The immunogenic complex according to claim 12, wherein the
hormone peptide is HCG.
14. A synthetic immunogenic complex comprising a purified
MHC-protein complex, wherein an MHC antigen is complexed with a
protein with which said MHC antigen is normally associated in
nature, said MHC-protein complex being bonded to a second antigen
through either the protein portion or the MHC portion of the
MHC-protein complex.
15. The immunogenic complex according to claim 14, wherein the
second antigen is a bacterial antigen.
16. The immunogenic complex according to claim 15, wherein the
bacterial antigen is a bacterial toxin or toxoid.
17. The immunogenic complex according to claim 16, wherein the
toxoid is diptheria toxoid.
18. The immunogenic complex according to claim 14, wherein the
second antigen is a viral antigen.
19. The immunogenic complex according to claim 18, wherein the
viral antigen is an influenza virus antigen.
20. The immunogenic complex according to claim 18, wherein the
viral antigen is a foot and mouth disease virus antigen.
21. The immunogenic complex according to claim 20, wherein the MHC
antigen is bovine or porcine LA.
22. The immunogenic complex according to claim 14, wherein the
second antigen is a hormone peptide.
23. The immunogenic complex according to claim 22, wherein the
hormone peptide is HCG.
24. The immunogenic complex according to claim 14, wherein the MHC
antigen is an HLA antigen or an HLA-analogous MHC antigen of a
non-human animal species.
25. The immunogenic complex according to claim 14, wherein the
second antigen is covalently attached to the protein portion of the
MHC-protein complex.
26. The immunogenic complex according to claim 14, wherein the
second antigen is attached to the protein portion of the
MHC-protein complex through a linking group.
27. The immunogenic complex according to claim 26, wherein the
linking group is derived from a heterobifunctional coupler.
28. The immunogenic complex according to claim 27, wherein the
heterobifunctional coupler is
m-maleimidobenzoyl-N-hydroxysuccinimide ester.
29. An immunogenic composition, comprising:
an immunogenically effective amount of a synthetic immunogenic
complex comprising a purified MHC antigen or a purified MHC-protein
complex, wherein an MHC antigen is complexed with a protein with
which said MHC antigen is normally associated in nature, said MHC
antigen or MHC-protein complex being bonded to a second antigen;
and
a pharmaceutically acceptable diluent.
30. The immunogenic composition according to claim 29, wherein said
MHC antigen or MHC-protein complex is a purified MHC-protein
complex and said second antigen is bonded to said MHC-protein
complex through either the protein portion or the MHC portion of
the MHC-protein complex.
31. The immunogenic composition according to claim 30, wherein said
bonding is through said protein portion.
32. The immunogenic composition according to claim 31, wherein the
second antigen is bonded to the protein portion of the MHC-protein
complex through a linking group.
33. The immunogenic composition according to claim 32, wherein the
linking group is derived from a heterobifunctional coupler.
34. The immunogenic composition according to claim 33, wherein the
heterobifunctional coupler is
m-maleimidobenzoyl-N-hydroxysuccinimide ester.
35. The immunogenic composition according to claim 29, wherein the
MHC-protein complex is HLA-B2M.
36. The immunogenic composition according to claim 29, wherein the
second antigen is a bacterial antigen.
37. The immunogenic composition according to claim 36, wherein the
bacterial antigen is a bacterial toxin or toxoid.
38. The immunogenic composition according to claim 29, wherein the
second antigen is a viral antigen.
39. The immunogenic composition according to claim 38, wherein the
viral antigen is an influenza virus antigen.
40. The immunogenic composition according to claim 34, wherein the
second antigen is a hormone peptide.
41. The immunogenic composition according to claim 40, wherein the
hormone peptide is HCG.
42. A method for the production of an immunogenic response in an
animal which comprises administering to said animal an
immunogenically effective amount of a synthetic immunogenic complex
comprising a purified MHC antigen or a purified MHC-protein
complex, wherein an MHC antigen is complexed with a protein with
which said MHC antigen is normally associated in nature, said MHC
antigen or MHC-protein complex being bonded to a second
antigen.
43. The method according to claim 42, wherein said MHC antigen or
MHC-protein complex is a purified MHC-protein complex and said
second antigen is bonded to said MHC-protein complex through either
the protein portion or the MHC portion of the MHC-protein
complex.
44. The method according to claim 43, wherein the MHC-protein
complex is HLA-B2M.
45. The method according to claim 43, wherein the second antigen is
attached to the protein portion of the MHC-protein complex through
a linking group.
46. The method according to claim 45, wherein the linking group is
derived from a heterobifunctional coupler.
47. The method according to claim 46, wherein the
heterobifunctional coupler is
m-maleimidobenzoyl-N-hydroxysuccinimide ester.
48. The method according to claim 42, wherein the second antigen is
a bacterial antigen.
49. The method according to claim 48, wherein the bacterial antigen
is a bacterial toxin or toxoid.
Description
This invention relates to immunological preparations, particularly
those containing antigenic materials.
Immunisation against infectious diseases is widely used in both a
human and a veterinary context. It is often the case, however, that
the antigenic material contained in the immunological preparation
is not as immunogenic as could be desired, necessitating the use of
a plurality of injections of the preparation. As an alternative, or
in addition to the use of repeated injections, as adjuvant may be
incorporated in the immunological preparation in order to increase
the immune response provoked by the antigenic material. However,
the effective adjuvants at present available, for example Freund's
incomplete and complete adjuvants, have disadvantages particularly
for human use. For some time, therefore, attempts have been made to
discover new forms of adjuvant which would simplify immunization
procedures and reduce the amounts of antigenic material required or
amplify the response to a given quantity of antigenic material.
Such an effect is applicable not only for protective immunization
but also for the production of therapeutic or diagnostic
immunological and serological reagents.
It has now been found that members of a group of naturally occuring
substances have the effect of increasing the immunogenicity of
antigenic materials when administered in combination therewith.
Accordingly, the present invention comprises an immunologically
active preparation comprising an antigenic material in combination
with a MHC (Major Histocompatability Complex) antigen.
MHC antigens have been found in all animal species (including man)
where they have been sought, being carried on the surface of
nucleated cells of tissues, and constitute a particularly
polymorphic systems within any species. Therefore, although it is
necessary in order for the MHC antigen to enhance the
immunogenicity of the antigen material with which it is combined,
that the MHC antigen should contain an antigenic determinant or
determinants foreign to the recipient, the extreme polymorphism of
these MHC antigens is generally sufficient to ensure that this is
the case between one member of a species and another, except in the
extreme case of members of a species which are genetically
identical. The MHC antigen may therefore be from either the same
species or another species, for example a phylogenetically similar
species such as a primate in the case of preparations for human
administration. MHC antigen from the same species may often be
preferred, however, unless there are reasons, particularly an
enhancement of effect, for using MHC antigen from another
species.
MHC antigens of particular interest in the context of the present
invention are the HLA (Human Lymphocyte Antigen) antigens in man
and the analagous antigens of other species. Such MHC antigens are
referred to by many workers as SD (Serologically Determined)
antigens and may be distinguished from other types of MHC antigen
such as the Ia antigens of mice and their equivalents in man (DRW)
and other animal species. Examples of the MHC antigens analagous to
HLA in other species are RLA in rabbits and H-2 in mice, etc. The
MHC antigens may be of various specificities. Moreover they may be
of the naturally occuring form or of derivatives therefrom which
retain the epitope intact, for example papain-solubilised MHC
antigens.
The present invention is widely applicable to a whole variety of
antigenic materials which are of use in vaccines. The term
antigenic material as used herein thus covers any substance that
will elicit a specific immune response when administered in
combination with a MHC antigen and includes antigenic determinants
such as peptides (small or large), oligo or polysaccharides,
alloepitopes, haptens and the like.
In the context of vaccines for human administration, a variety of
microbial antigens may be used as the antigenic material. Examples
include bacterial antigens, for instance toxins such as
Staphylococcus enterotoxin and particularly toxoids such as
diphtheria and tetanus toxoid, and viral antigens such as those
derived from the influenza and the rabies viruses. The invention is
also of interest, however, in relation to vaccines against other
forms of pathogen such as protozoa and fungi and also in relation
to the field of contraception, for example for vaccination against
HCG when a hormone peptide is used as the antigenic material, as
well as in the immunotherapy of cancer. The invention is also of
considerable interest and of similar wide applicability in the
context of veterinary vaccines for both mammalian and avian
administration, for example in the treatment of the viral foot and
mouth disease in cattle and pigs. For the treatment of cattle and
pigs bovine LA and porcine LA are preferred as the MHC antigen. As
indicated above, however, there may be an advantage in using the
MHC antigen of a closely related species so that the use of bovine
LA in pigs and of porcine LA in cattle may be considered in
addition to the more usual intraspecies usage.
MHC antigens may be readily obtained from natural sources and
procedures for doing this are described in the literature. The HLA
antigens, for example, may conveniently be obtained by extraction
of the membranes of human lymphoblastoid cells cultured in a
suitable medium to provide sufficient quantities thereof for
extraction. Alternatively, a human non-lymphoid cell line may be
used as the source. In their natural form MHC antigens are
generally bound to a protein molecule, for example HLA and the
equivalent MHC antigens of other species being bound to
B2-microglobulin (B2M). The nature of the binding between the two
molecules is not yet known for any species, although present
indications are that it is not of the form of a covalent bond.
However, the bonding is strong enough so that in several species
including man B2M is co-isolated with MHC antigen under normal
circumstances. It is not necessary to remove the B2M molecule from
the MHC molecule before the latter is used in the present invention
and indeed it is preferred to use the MHC antigen in the form of a
complex with the appropriate B2M.
Accordingly the present invention excludes known naturally
occurring combinations consisting of a MHC antigen with a B2M or
like protein as the only immunologically active species, but
particularly includes such combinations, for example a MHC-B2M
complex, when combined with a further antigenic material.
The bonding between the MHC antigen and the antigenic material may
be of a variety of types including bonds of a noncovalent type such
as hydrogen bonds, ionic bonds, and hydrophobic bonds, as well as
covalent bonds. The preparation of the MHC antigen-antigenic
material combination may require varying degrees of manipulation
ranging from simple mixing of the two components to the deliberate
construction of a covalent bond between the MHC antigen and the
antigenic material used. It will be appreciated that when, as is
usually the case, the antigenic material is combined with a MHC-B2M
complex, then the material may be attached through either the B2M
component or a suitable modification thereof (such modifications
being included by such terms as MHC-B2M complex and B2M protein
etc. when used herein in a general sense), or the MHC epitope
bearing protein. Methods of effecting combination include various
of the techniques described in the literature for attaching
antigens or haptens to proteins, for example the use of a coupling
agent such as chromic chloride, divinyl sulphone, cyanogen bromide,
bisdiazotised benzidine (BDB) and especially glutaraldehyde, tannic
acid and carbodiimides, for example
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC).
One particularly convenient approach which is based upon a
procedure described, for example, by Kitagawa and Aikawa in the
Journal of Biochemistry, 1976, 79, 233, involves the use of a
heterobifunctional coupler. Such a coupler contains a group which
will bond to the antigenic material and a different group which
will bond to the MHC antigen or MHC-B2M complex. Conveniently, the
antigenic material is attached through the B2M portion of the
complex and a preferred approach comprises the addition of the
coupler to the B2M protein followed by exchange of the modified B2M
protein with the B2M protein in the naturally occurring MHC-B2M
complex and the addition of the desired antigenic material to the
complex through the free end of the coupler.
One example of such a heterobifunctional coupler which has been
used with some success is m-maleimidobenzoyl N-hydroxysuccinimide
ester (MBS). This coupler will couple with free amino groups
through active ester acylation by the m-maleimidobenzoyl groups and
also with sulphydryl groups, for example of cysteine, by addition
at the double bond of the maleimido group. For the sake of
simplicity the symbol MBS has also been used hereinafter in
identifying products linked through the use of this reagent
although such products will, of course, only contain a residue of
the reagent. By blocking sulphydryl groups in the MHC antigen, for
example by the use of N-ethyl maleimide, linking to the MHC-B2M
couplex is effected through free amino groups only (the B2M protein
containing no sulphydryl groups) and coupling of the antigenic
material may then be effected through the sulphydryl groups which
are naturally present therein or are inserted for this purpose.
The amounts of antigenic material incorporated as the
immunologically effective agent to provide pharmaceutical
preparations according to the present invention may be similar to
those used in existing vaccines incorporating such material, but it
may be possible in view of the increased immunogenicity to reduce
these amounts. Similarly, although additional adjuvants may be
incorporated into the preparations, this may likewise be
unnecessary. The proportion of MHC antigen to antigenic material
may be varied according to the particular circumstances. However,
as a guide it may be indicated that in primates three dosages
containing about 10, 10 and 5 micrograms of HLA have proved
effective and that broadly similar unit dosage levels are generally
applicable in humans, for example in the range from 5 to 100
micrograms, particularly from 10 to 50 microgram, for example about
25 micrograms. A similar basis may be used for veterinary
applications with due consideration for variation in body
weight.
In other respects, the preparations may be formulated in a similar
manner to conventional vaccines, for example in a medium such as
isotonic saline, and may be administered similarly, often by a
parenteral route, for example intravenously, intramuscularly or
subcutaneously.
The present invention thus includes a method for the immunization
of a mammal or a bird which comprises administering thereto as an
immunologically effective agent an antigenic material which is in
combination with an MHC antigen.
The invention is illustrated by the following examples. Also
provided are the details of an experiment illustrating the ability
of MHC antigens to enhance the immunogenic effect of antigenic
materials by reference to the immunogenicity of B2M in an HLA-B2M
combination (such a known combination, however, being excluded from
the scope of the invention).
EXPERIMENT
Comparison of Immunogenicity of Free and HLA Antigen
B2-Microglobulin
(1) Isolation of materials.
Papain-solubilized HLA antigens (in combination with B2M) were
prepared from typed cadaver human spleen as described by Sanderson
and Welsh in Transplantation, Volume 16, 304-312, (1973), or from
cultured human cell lines as described by Turner, Cresswell,
Parham, Strominger, Mann, and Sanderson, Journal of Biological
Chemistry, Volume 250, 4512-4518, (1975). In this procedure cells
were disrupted by freeze-thawing, and membranes prepared by
differential centrifugation and washing. HLA-antigen was
solubilized by papain, and purified by DEAE chromatography, CM
chromatography, and Sephadex filtration. Assessment of the degree
of purification was made at each stage by a standard assay
involving the inhibition of cytolosis of .sup.51 chromium-labelled
human peripheral lymphocytes by specific HLA antibodies and
complement.
Human B2M protein was isolated according to the method of Berggard
and Bearn, Journal of Biological Chemistry, Volume 243, 4095-4100
(1968).
(2) In vivo Tests
Two groups of Macaca irus primates were treated by intramuscular
administration with (a) 50-100 .mu.g doses of the free B2M protein
in incomplete Freund's adjuvant, and (b) 0.5 to 1.0 .mu.g doses of
the papain-solubilised HLA-B2M complex (protein content based on an
assumed final purity of 10% validated by radio immunoassay of B2M
content), respectively. In the former instance six booster
injections were required to produce a detectable response and in
the latter case though six booster injections were given, a
detectable response was present after two of these had been
given.
Anti-B2M titres were determined as follows. 5 .mu.l of .sup.125
I-B2M solution was added to 8 .mu.l volumes of five fold dilutions
of the animal's serum in diluent, followed by incubation at room
temperature for 1 hour, then the addition of 0.180 ml of 19%
Na.sub.2 So.sub.4 solution and further incubation at room
temperature for 1 hour. After centrifugation at 1,000 g for 5
minutes, 50% of the supernatant was sampled and the results were
plotted as the percentage radionuclide remaining in solution
against antiserum dilution in the reaction volume. The titre of
antiserum was the dilution at which 50% of the nuclide was
precipitated. Control samples containing diluent in place of
antiserum were run and the results were corrected according to the
titration value given by a reference chicken anti-human B2M
serum.
The results obtained for anti-B2M titre and other tests are shown
in Table 1 below.
TABLE 1 ______________________________________ Human Lymophocy-
totoxin: 50% Injected Anti-B2M titre cytotoxic Protein 3 boosts 6
boosts Titre (1) SR.sup.(2) ______________________________________
50-100 .mu.g nil 93 .+-. 13 8 .+-. 2 1.0 Free B2M 0.5-1.0 .mu.g
1100 .+-. 275 2,406 .+-. 1295 3250 .+-. 1673 4.8 .+-. 1.1 Papain-
solubilised HLA Antigens ______________________________________
(1) Estimated in microtitre plates using 25 .mu.l volumes of
lymphocytes, antiserum dilutions, and rabbit complement, and a
Trypan blue indication of cell death.
(2) Specificity Ratio: the ratio of titre with cells positive or
negative for a HLA specificity, based on analysis of a population
of 20 cell types covering the relevant specificities.
EXAMPLES
(Products are isolated in solid form by freeze drying. As an
alternative, for storage their solutions may be frozen at
-20.degree. C.).
EXAMPLE 1
Preparation of Dinitrophenyl-B2M-HLA
(1) Human B2M prepared as described in the Experiment is reacted
with fluorodinitrobenzene essentially by the general procedure for
the introduction of dinitrophenyl (DNP) groups which is described
by Hudson and Hay in Practical Immunology (Blackwell) Scientific
Publications, London, 1976). A low level of 3H labelled
fluorodinitrobenzene is incorporated into the bulk of unlabelled
fluorodinitrobenzene to provide a convenient means of identifying
the complex. The DNP-B2M complex is purified by gel filtration
chromatography on G25 Sephadex using 0.05M tris chloride buffer
(tris is an abbreviation for tris-hydroxymethylamino methane) of pH
7.3, a yield of about 50% typically being obtained of a complex
containing 1 to 2 DNP residues per protein molecule.
(2) Papain solubilised HLA [4 nm; in combination with B2M- prepared
as described in Experiment] is incubated with DNP-B2M complex [54
nm; prepared as described in (1) above] in a total volume of 0.65
ml phosphate buffered saline for 16-20 hours. The reaction mixture
is purified by gel filtration on G75 Sephadex using 0.05M tris
chloride buffer of pH 7.3, typically giving a near quantitative
yield of DNP-B2M-HLA.
EXAMPLE 2
Immunization Experiments with Dinitrophenyl-B2M-HLA
Macaca irus primates are immunized intramuscularly with 10 to 50
microgram quantities of the dinitrophenyl hapten coupled to B2M-HLA
(prepared as described in Example 1) or coupled to ovalbumin to
provide a control, ovalbumin being of comparable size to B2M-HLA;
the DNP-ovalbumin containing 1-2 DNP residues per protein molecule
is prepared by an analagous procedure to that described in Example
1 (1) for DNP-B2M. The hapten is administered in incomplete
Freund's adjuvant and booster injections are given at 2 to 3 week
intervals, bleeds being taken before and during the immunization
protocol.
For the assessment of immunity in each case the hapten is attached
to the wells of a soft plastic plate (Cooke Microtitre Plate) by
incubation for 1 hour at 37.degree. C. of 0.05 ml of a solution
containing 0.02 mg/ml of protein in phosphate buffered saline.
After washing, 0.025 ml dilutions of serum are incubated in each
well for 1 hour at 37.degree. C. After further washing, the wells
are each incubated with 0.01 ml .sup.125 I-labelled F(ab).sub.2
fragments of immunopurified goat anti human F(ab).sub.2. The wells
are then counted in a well type gamma spectrometer, appropriate
controls being included. Association of nuclide with a well
indicates the presence of the radio labelled anti F(ab).sub.2
protein and thus of primate antibodies which cross react with human
immunoglobulins and are detected by the goat reagent. Positive and
negative controls typically differ by at least a factor of 3 to 4
fold and typical titres are shown in Table 2 below.
TABLE 2 ______________________________________ Titre Antigenic
material after boost 1 after boost 2
______________________________________ DNP-ovalbumin nil nil
DNP-B2M-HLA 32 >128 ______________________________________
EXAMPLE 3
Preparation of Fab-B2M-HLA
(1) Human B2M [8.3 mg, 714 nm; prepared as described in the
Experiment] is incubated in 1.0 ml of 0.05M tris chloride of pH 7.3
with m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) for 1.5
hours at room temperature. The acylation reaction is terminated by
the addition of excess lysine (0.05 ml of a 20 mg/ml aqueous
solution of lysine) and the m-maleimidobenzoyl derivative of B2M
(MBS-B2M) is separated from low molecular weight contaminants by
gel filtration on Sephadex G50 using 0.05M tris chloride of pH 7.3
as eluant. Emergence of the protein peak is accompanied
simultaneously by the SH coupling capacity as determined by
reaction with cysteine resulting in an increase in the Ellman
calorimetric assay for SH groups. The MBS-lysine emerges from the
column much later than the MBS-B2M and is detected by the Ellman
test. The fractions containing B2M-MBS are pooled and lyophilised,
this procedure resulting in a negligible deterioration of the SH
coupling capacity.
(2) Papain solubilised HLA [0.125 mg, 2.8 nm; in combination with
B2M- prepared as in the Experiment and treated with 0.01M
N-ethylmaleimide for 1 hour at room temperature, followed by
reisolation by gel filtration, to block reactive SH groups] is
reacted with MBS-B2M [0.83 mg, 62 nM prepared as in (1) above] in
phosphate buffered saline for 1 hour and the reaction is purified
by gel filtration chromatography on G75 Sephadex using 0.05M tris
chloride buffer of pH 7.3 to give MBS-B2M-HLA, typically in
quantitative or near quantitative yield.
(3) MB 4-B2M-HLA [the whole of the product of (2) above] is reacted
with human Fab (11.6 mg; 232 nm) in 0.1 ml of 0.15M tris chloride
for 5 hours at room temperature. The products are then separated by
chromatography on Sephadex G200 using 0.05M tris chloride of pH 7.3
as eluant. The Fab-MBS-B2M-HLA product emerges from the column at a
position corresponding to a molecular weight of about
90,000-100,000 and is established to contain B2M and Fab by radio
immunoassay, and also to be specifically inhibitory for HLA
antisera. The yield as determined by specific inhibitory capacity
with HLA alloantisera typically corresponds to a recovery of about
50% of the total HLA antigen activity. The remainder of the
activity corresponds to a product emerging in the anticipated
position for unreacted MBS-B2M-HLA as established using
radioiodinated HLA alloantigen.
EXAMPLE 4
Preparation of HCG-B2M-HLA
MBS-B2M-HLA is prepared as described in Example 3(2) above using
the same quantities of reactants. The whole of the product obtained
is reacted with HCG (0.075 mg; 20.8 nM) in 0.1 ml of 0.05M tris
chloride buffer of pH 7.3 for 3 hours at room temperature. The
products are separated by G75 Sephadex chromatography using 0.005M
tris chloride buffer of pH 7.3. The HCG-MBS-B2M-HLA is eluted from
the column just ahead of the position expected for HLA protein on
this column, typically being obtained in 1 to 2% yield. The low
yield obtained as compared with that of Example 2(3) is believed to
be due to the smaller amount of the antigenic material used in this
instance, the reaction yield based upon HLA being very much higher
when a greater excess of the antigenic material is used.
EXAMPLE 5
Preparation of B2M-HLA Linked Influenza Neuraminidase and
Haemagglutinin
MBS-B2M-HLA is prepared as described in Example 3(2) above using
the same quantities of reactants. A suspension (5 ml) of a mixture
of influenza virus neuraminidase and haemagglutinin (Strain
A/Victoria/3/75; 5,222 IU/0.5 ml: Lowry protein 1 mg/ml;
neuraminidase 5650 U/ml; supplied by Evans Medical Ltd.) is freeze
dried and the product reconstituted in 0.2 ml of water. To the
aqueous neuraminidase/haemagglutinin is added the MBS-B2M-HLA and
the mixture is kept overnight at room temperature. The resulting
neuraminidase/haemagglutinin-MBS-B2M-HLA is used is crude form or
isolated by gel filtration chromatography in a similar fashion to
the products of Examples 3(3) and 4.
EXAMPLE 6
Preparation of B2M-HLA Linked Staphylococcus Enterotoxin B
MBS-B2M-HLA is prepared as described in Example 3(2) above using
the same quantities of reactants. Staphylococcus enterotoxin B (10
mg) obtained in solid form by freeze drying is reconstituted in
0.25 ml water. To the aqueous enterotoxin is added the MBS-B2M-HLA
and the mixture is kept overnight at room temperature. The
resulting enterotoxin-MBS-B2M-HLA is used in crude form (after
extensive dilution) or is isolated by gel filtration chromatography
in a similar fashion to the products of Examples 3(3) and 4.
EXAMPLE 7
Immunization Experiments with B2M-HLA Linked Staphylococcus
Enterotoxin B
Macaca irus primates are immunized intramuscularly with about 1 to
5 microgram quantities of antigen or antigen linked to B2M-HLA
(prepared as described in Example 6) in incomplete Freund's
adjuvant. Three closely spaced injections are given and the
primates are then bled and the serum evaluated.
For the assessment of immunity in each case the S. enterotoxin is
attached to the wells of a soft plastic plate (Cooke Microtitre
Plate) by incubation for 1 hour at 37.degree. C. of 0.05 ml of a
solution containing 0.01 mg/ml of protein in phosphate buffered
saline. After washing, 0.025 ml dilutions of serum are incubated in
each wall for 1 hour at 37.degree. C. After further washing, the
wells are each incubated with 0.01 ml .sup.125 I-labelled
F(ab).sub.2 fragments of immunopurified goat anti human
F(ab).sub.2. The wells are then counted in a well type gamma
spectrometer, appropriate controls being included. Association of
nuclide with a well indicates the presence of the radio labelled
anti F(ab).sub.2 protein and thus of primate antibodies, which
cross-react with human immunoglobulins and are detected by the goat
reagent. Negative controls typically differ by at least a factor of
3 to 4 fold and typical titres are shown in Table 3 below.
TABLE 3 ______________________________________ Antigenic material
Titre ______________________________________ S. enterotoxin nil S.
enterotoxin-B2M-HLA >128
______________________________________
The following claims are subject to the proviso that no claim is
made relating to the combination of an MHC antigen with a protein
with which it occurs naturally as the only immunologically active
species in the combination.
* * * * *