Methods of treatment and diagnosis of autoimmune diseases, especially arthritic conditions

A Mycobacterium bovis BCG polypeptide having a molecular mass of about 64 kD was found to be useful as an immunogen inducing resistance to autoimmune arthritis and similar autoimmune diseases. The invention relates to methods of treatment and diagnosis of autoimmune diseases especially arthritic conditions, in which said polypeptide is used. The invention also relates to a polypeptide comprising the epitope essential for this activity. The polypeptide has the formula ##STR1## Further, the invention relates to polypeptides showing sequential homology with said polypeptide, and to derivatives and multimers thereof. Also, microorganisms expressing the polypeptides either as such or as part of a fusion protein or as a multimer, form part of the invention. Finally, the invention relates to pharmaceutical compositions, diagnostic compositions and test kits comprising a compound according to the invention.

The present invention relates to a method of prophylaxis and treatment, and 
a method of diagnosis of autoimmune diseases, especially arthritic 
conditions. The invention further relates to a new peptide and compounds 
related to said peptide, to micro-organisms expressing said peptide and 
related compounds, and to pharmaceutical and diagnostic compositions 
comprising the new peptide or a compound related to said peptide, and to 
test kits for performing immunological tests. 
BACKGROUND OF THE INVENTION 
Millions of persons are afflicted with chronic forms of arthritis which are 
thought to involve autoimmunity to constituents of the joints or 
connecting tissues of the body. These conditions include rheumatoid 
arthritis, ankylosing spondylitis, Reiter's syndrome and other forms of 
reactive arthritis. The etiology of these diseases is not known, but 
previous infection with various microbes seems to act as an inciting 
circumstance in genetically susceptible individuals. For example, patients 
with rheumatoid arthritis may show unusual reactivity to mycobacterial 
antigens and immunization with the BCG strain of mycobacteria was found to 
lead to arthritis in 15 of 150 individuals. Ankylosing spondylitis has 
been associated with infection by Klebsiella or Yersinia species of 
bacteria and other cases of arthritis by Salmonella, Shigella, etc. There 
is no evidence of active infection of joints by these microbes in the vast 
majority of cases and it has been postulated that microbial infection may 
trigger an aberrant, autoimmune response of the individual against his own 
antigens present in the joints. Adjuvant arthritis (AA) is an experimental 
model of arthritis inducible by immunizing susceptible strains of rats to 
Mycobacteria. The disease which develops about 12 days after immunization 
has many of the features of rheumatoid arthritis and AA has been 
considered to be a model of rheumatoid arthritis. 
PRIOR ART 
EP A 0 181 364 discloses aqueous acetone soluble and insoluble fractions of 
certain mycobacteria, such as Mycobacterium H-37, M. kansasii and M. 
vaccae. The soluble fraction of Myc. H-37 was found to provoke an immune 
response leading to resistance to adjuvant arthritis. The insoluble 
fraction seemed to be responsible for induction of adjuvant arthritis. 
Micobacterium vaccae was shown to be substantially free of adjuvant 
arthritis inducing components. Further, EP A 0 181 364 describes certain 
lines and clones of T-lymphocytes selected for their reactivity to 
micobacteria. These can be used for producing arthritis upon inoculation 
into irradiated rats. One line, designated as A2 was found to induce 
arthritis upon intravenous injection into irradiated rats. The same line, 
A2 is effective in vaccinating unirradiated rats against subsequent 
autoimmune arthritis induced by active immunization to mycobacteria. Cell 
line A2 has been cloned. There were obtained two distinct clones, 
designated as A2b and A2c, respectively. A2b causes arthritis but does not 
vaccinate against it; clone A2c does not cause arthritis but vaccinates 
against it. In addition to preventing arthritis, clone A2c can be used to 
treat AA. Moreover, clones A2b and A2c can be used to identify antigens 
associated with arthritogenicity or with suppression of arthritogenicity. 
Both clones respond to whole mycobacteria as well as to cartilage 
proteoglycan. 
DESCRIPTION OF THE INVENTION 
According to the present invention it was found that a polypeptide having a 
molecular mass of about 64 kD, the preparation of which is described in 
Infection and Immunity 1985, pages 800-806, is useful as an immunogen 
inducing resistance to autoimmune arthritis and similar autoimmune 
diseases. 
In the above-mentioned article the peptide in question is called Antigen A 
and this designation will be used here as well. Antigen A was obtained by 
constructing a gene bank of Mycobacterium bovis BCG DNA in Escherichia 
coli by cloning Sau3A-cleaved mycobacterium DNA fragments into the lambda 
vector EMBL3. The expression of mycobacterial antigens was analyzed by 
Western blotting with hyperimmune rabbit sera. The article states that 
among 770 clones tested, several were found that produced various 
mycobacterial antigens in low amounts, with concentrations generally close 
to the detection limit. One particular clone was chosen for further 
investigation. This clone produced a 64 kD antigen. By placing the lambda 
promoter P.sub.L in front of the structural gene of this antigen, an 
overproducing E. coli strain was obtained. The article shows that antigens 
cross-reacting with the 64 kD protein are present in a wide variety of 
mycobacteria and also in so-called purified protein derivatives which are 
routinely used for skin tests. Finally, it is stated in the article that 
preliminary experiments indicate the presence of antibodies against the 64 
kD antigen in sera from tuberculosis patients. 
According to the present invention, Antigen A was found to have the 
following amino acid sequence: 
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1 MAKTIAYDEE ARRGLERGLN ALADAVKVTL 
61 LEDPYEKIGA ELVKEVAKKT DDVAGDGTTT 
121 KAVEKVTETL LKGAKEVETK EQIAATAAIS 
181 FGLQLELTEG MRFDKGYISG YFVTDPERQE 
241 AGKPLLIIAE DVEGEALSTL VVNKIRGTFK 
301 EEVGLTLENA DLSLLGKARK VVVTKDETTI 
361 EKLQERLAKL AGGVAVIKAG AATEVELKER 
421 APTLDELKLE GDEATGANIV KVALEAPLKQ 
481 VYEDLLAAGV ADPVKVTRSA LQNAASIAGL 
______________________________________ 
1 GPKGRNVVLE KKWGAPTITN DGVSIAKEIE 
61 ATVLAQALVR EGLRNVAGA NPLGLKRGIE 
121 AGDQSIGDLI AEAMDKVGNE GVITVEESNT 
181 AVLEDPYILL VSSKVSTVKD LLPLLEKVIG 
241 SVAVKAPGFG DRRKAMLQDM AILTGGQVIS 
301 VEGAGDTDAI AGRVAQIRQE IENSDSDYDR 
361 KHRIEDAVRN AKAAVEEGIV AGGGVTLLQA 
421 IAFNSGLEPG VVAEKVRNLP AGHGLNAQTG 
481 FLTTEAVVAD KPEKEKASVP GGGDMGGMDF 
______________________________________

DETAILED DISCUSSION OF THE INVENTION 
As mentioned above clones A2b and A2c as disclosed in EP A 0 181 364 can be 
used to identify antigens associated with arthritogenicity or with 
suppression of arthritogenicity. Both clones respond to whole mycobacteria 
and both A2b and A2c respond to antigen A. 
T-cell clones A2b, A2c and control cell-line Cla (anti-ovalbumin) were 
assayed for in vitro proliferative responses to Micobacterium 
tuberculosis, Antigen A, E. coli control lysate, ovalbumin (OVA) and 
mitogen ConA in a standard test (20.times.10.sup.3 clone/line cells, 
2.times.10.sup.6 irradiated accessory cells and antigens in optimum 
concentrations per well, .sup.3 H-Thymidine incorporation for 18 hours 
after 48 hours of incubation). The following table A shows the test 
results which are expressed as stimulation indexes. 
TABLE A 
______________________________________ 
M. tub. Ant. A coli contr. 
OVA ConA 
______________________________________ 
A2b 180 500 2.9 -- 430 
A2c 304 516 1.5 -- 390 
C1a -- 1.5 1.2 45 64 
______________________________________ 
The in vivo potency of Antigen A was checked by immunizing rats with 
Antigen A before and after induction of arthritis with M. tuberculosis. 
The test with challenge after immunization was carried out as follows: 
Groups of 4 Lewis rats were treated by intraperitoneal inoculation of 
water, Antigen A (50 .mu.g) and E. coli control lysate (amount equivalent 
to coli content of 50 .mu.g Antigen A) in oil. 35 Days later, 
susceptibility to induction of adjuvant arthritis was tested by 
inoculating the rats intracutaneously with M. tuberculosis (1 mg) in oil. 
Occurrence of arthritis was checked by daily inspection of the rat joints. 
The results are shown in table B. 
TABLE B 
______________________________________ 
Secondary challenge (35 days later) 
Primary immunization 
with M. tuberculosis in oil. 
Arthritis Arthritis 
Inoculum in oil 
incidence incidence Clinical grade 
______________________________________ 
Water 0/4 4/4 severe 
Antigen A 0/4 2/4 very mild 
E. coli contr. 
0/4 4/4 severe 
______________________________________ 
The tests involving inoculation after induction of autoimmune arthritis 
were carried out as follows: 
Arthritis was induced by inoculating groups of 3 Lewis rats with M. 
tuberculosis (1 mg) in oil intracutaneously. 3 Days later the rats were 
treated by intraperitoneal inoculation of water, Antigen A (200 .mu.g) and 
E. coli control lysate (amount equivalent to coli content of 200 .mu.g 
Antigen A) in oil. Occurrence of arthritis was checked by daily inspection 
of the rat joints. 
The results are shown in table C. 
TABLE C 
______________________________________ 
Inoculum administered at 
Arthritis 
day 3 after disease induction 
incidence clinical grade 
______________________________________ 
Water 3/3 severe 
Antigen A 1/3 very mild 
E. coli contr. 3/3 severe 
______________________________________ 
It is seen that Antigen A is not arthritogenic by itself but reduces the 
incidence of arthritis after active induction disease with 50%, and also 
reduces the severity of remaining disease remarkably. A similar reduction 
of disease incidence and severity is seen when Antigen A is administered 
three days after disease is induced. E. coli itself has no effect. Thus, 
Antigen A is arthritis suppressive, while not being arthritogenic. 
Further, it was found that Antigen A cross-reacts with similar proteins 
present in various other mycobacteria and E. coli and with Treponema and 
gram-negative enterobacteria. This cross-reactivity is shown in the 
following table D. 
TABLE D 
__________________________________________________________________________ 
Cross-reactivity between Antigen A and antigens 
present in other bacteria. 
64kD of 
Antig. A 
mycobact. 
E. coli 60kD 
Trep. poll 
Shig. 
Salmon. 
Klebsiella 
__________________________________________________________________________ 
MCA HATR 
1-24 + - + + + + + 
F47-10 + + + + + + 
Polycl. anti 
+ + + + + + + 
comm. ag. 
Legion/ 
Pseudom. 
__________________________________________________________________________ 
Serological cross-reactivity as shown by Western-blot analysis. HATR 1-24 
and F47-10 are monoclonal antibodies raised against Treponema and 
Mycobacterium tuberculosis respectively. The polyclonal serum was raised 
against the common antigen of Legionella and Pseudomonas. 
This indicates that epitopes present on Antigen A are similarly present on 
presumably equivalent proteins of various bacterium species, such as from 
Mycobacterium, Escherichia, Treponema, Shigella, Salmonella, Yersinia, 
Nocardia, Campylobacter, or Klebsiella species. Particularly, antigen A 
amino acid sequence 190-213 is also present in a corresponding 65 KD 
protein from Mycobacterium leprea, with the exception that, in the M. 
leprae protein, amino acid 206 is not proline, but alanine. 
Further, it was found that only part of the Antigen A sequence is 
responsible for the stimulating activity upon T-cell clones A2b and A2c. 
This was determined by testing Antigen A fragments, namely truncated 
derivatives produced by deletion mutants of the gene, fusion proteins with 
.beta.-galactosidase and proteolysis products of Antigen A, for their 
ability to stimulate said T-cell clones. These fragments were obtained by 
means of recombinant-DNA techniques, by incorporating parts of the Antigen 
A gene, in some cases fused to the .beta.-galactosidase gene, into a 
plasmide and expressing in E. coli K12 M1070. 
The peptide with Antigen A amino acid sequence 234-540 was shown not to 
stimulate clones A2b and A2c. However, the fragment lacking amino acid 
sequence 481-540 did. .beta.-Galactosidase-fused peptides with Antigen A 
amino acid sequences 61-540, 109-540 and 171-540 were reactive, those with 
amino acid sequences 272-540 and 280-540 were not reactive. 
.beta.-Galactosidase alone was not reactive. 
Therefore, the epitope responsible for the stimulation of T-cell clones A2b 
and A2c resides in amino acid sequence 171-234. 
In order to further characterize the area which is essential for the T-cell 
epitopes, protease digests of Antigen A were tested for their stimulating 
activity on both T-cell clones. Digesting Antigen A with clostripain 
yielded only one reactive mixture of two peptides. The mixture is called 
CP15. The two peptides, which were not separated, are designated as CP15a 
and CP 15b. The CP15a sequence begins with amino acid 193 and that of 
CP15b starts with amino acid 197. Digesting CP15 with trypsin, again, 
yielded a reactive mixture of two peptides (CP-TP-T12a and b) with 
sequences beginning with amino acid 193, and 196, respectively, as well as 
a non-reactive peptide, the sequence of which starts with amino acid 209. 
The carboxy ends of the peptides were not determined. 
It may be concluded from these results that the epitope responsible for the 
stimulation of T-cell clones A2b and A2c resides in Antigen A amino acid 
sequence 193-234, and more specifically in the amino acid sequence 
193-208. 
Finally, it was found that a synthetic peptide having Antigen A amino acid 
sequence 180-196 is also recognized by T-cell clones A2b and A2c. The 
overlap between this synthetic peptide and the above-discussed digests is 
only 4 amino acids, namely Antigen A amino acids 193-196 designated as 
FDKG. Therefore, at least one of these amino acids seems to be essential 
in the T-cell epitope. It is possible that one or more of these amino 
acids is seen by the T-cells in conjunction with additional amino acids 
having lower or higher numbers or lower and higher numbers in the 
sequence. Therefore, the polypeptide having Antigen A amino acid sequence 
171-240, and polypeptides showing sequential homology with this peptide 
will comprise the epitope of T-cell clones A2b and A2c. In this 
specification, polypeptides showing sequential homology with the 
polypeptide having Antigen A amino acid sequence 171-240 are polypeptides 
composed of 4 to 70 amino acid residues, in the amino acid sequence of 
which at least 4 amino acid residues are in the same relative position as 
the same amino acid residues are in the polypeptide having Antigen A amino 
acid sequence 171-240. 
Consequently, the invention relates to a method of prophylaxis or treatment 
of autoimmune diseases, especially arthritic conditions, in which an 
effective amount of Antigen A, that is the polypeptide having the sequence 
of 540 amino acids mentioned earlier in this specification, is 
administered to a patient. 
Preferably, Antigen A is administered orally, intracutaneously or 
intramuscularly in the form of a suitable pharmaceutical composition which 
may be prepared in a way known in the art. 
Further, the invention relates to a method for the diagnosis of autoimmune 
diseases, especially arthritic conditions in which Antigen A is injected 
intracutaneously into a patient, and the occurrence of a detectable skin 
reaction is observed, or in which Antigen A is contacted with a patient's 
blood or blood component, and the occurrence of any reaction is detected 
by means of immunological methods known per se. 
In the in vivo skin test the skin reaction at the site of the injection is 
measured after a sufficient time period, for example 24 to 72 hours after 
administration. Swelling and/or redness is due to a delayed 
hypersensitivity-like reaction. 
In the in vitro tests with blood or blood components, Antigen A may be 
contacted, for example, with peripheral blood cells. Lymphocytes of 
positive patients will be stimulated by Antigen A in that they will 
proliferate and/or produce biologically active factors, such as 
interleukines or products involved in the degradation of cartilage. Such 
reactions may be detected by methods known in the art. 
In in vitro serological tests serum of a patient is contacted with Antigen 
A. If the serum contains antibodies against antigenic determinants of 
Antigen A an immunological reaction will occur which may be detected and 
assayed by means of standard techniques such as ELISA, agglutination, etc. 
The invention also relates to the polypeptide having Antigen A amino acid 
sequence 171-240 which is 
##STR2## 
as well as to polypeptides composed of 4-70 amino acid residues, and 
showing sequential homology with said polypeptide having Antigen A amino 
acid sequence 171-240 in the sense that in its amino acid sequence at 
least 4 of the amino acid residues are in the same relative position as 
the same amino acid residues are in the polypeptide having Antigen A amino 
acid sequence 171-240. 
More specifically, the invention relates to polypeptides showing sequential 
homology with the polypeptide having Antigen A amino acid sequence 
171-240, which are further characterized by the fact that they comprise in 
their amino acid sequence at least one of amino acid residues F, D, K and 
G corresponding to positions 193, 194, 195 and 196. Preferably, these 
polypeptides comprise in their molecule amino acid sequences 193-234, 
193-208 or 180-196. 
Although T-cell clones A2b and A2c respond to all of the above-defined 
polypeptides, the antigenicity and immunogenicity of the polypeptides may 
be enhanced by coupling thereto at least one radical capable of improving 
the presentation of the antigenic determinants of the polypeptides. Such 
radicals are known in the art, and comprise, for example, radicals of 
peptides, tetanus toxoid, diphtheria toxoid, .beta.-galactosidase, and 
microbial outer membrane proteins. Multimers of the polypeptides in 
question are also contemplated. These modified polypeptides also form part 
of the invention. 
All of the polypeptides of the invention, namely the polypeptide having 
Antigen A amino acid sequence 171-240, the polypeptides showing sequential 
homology with that polypeptide, the above-defined modified peptides 
including the multimers, can be used as immunogens in pharmaceutical 
compositions, especially vaccines for the alleviation and treatment of 
autoimmune diseases, especially arthritic conditions, and also as antigens 
in diagnostic compositions for the diagnosis of these diseases. These 
pharmaceutical and diagnostic compositions, which may be prepared in a way 
known in the art, also form part of the invention. 
Another way to improve the immunogenicity of the polypeptides according to 
the invention is to construct, by known genetical engineering methods, 
microorganisms expressing a polypeptide according to the invention either 
as such or as part of a fusion protein or as a multimer thereof. These 
microorganisms can be used for the preparation of a live vaccine which 
will provoke not only the production of antibodies against the 
micro-organism in question, but will also be useful for the alleviation 
and treatment of autoimmune diseases. These genetically engineered 
microorganisms, and pharmaceutical compositions containing these, also 
form part of the invention. Examples of suitable genetically engineered 
microorganisms are Vaccinia and Salmonella strains. 
Finally, the invention provides kits for performing immunological tests 
comprising a container with at least one of the antigenic compounds 
discussed above, or a container with the diagnostic composition mentioned 
above. 
The antigenic compounds and diagnostic compositions as well as the 
diagnostic kits according to the invention may be used for various types 
of assays, such as: 
a.1. a lymphocyte proliferation test, or determination of any entity 
indicative of such proliferation; 
a.2. indicative of the measure of lymphocyte activation are also changes 
which can be assayed by standard means so as to establish the presence and 
degree of lymphocyte activation: amongst these there may be mentioned: 
a. production of lymphokines (such as interleukin-2 (IL-2)); 
b. gamma interferon; 
c. migration inhibition factor (MIF); 
d. expression of membrane markers, such as IL-2 receptor; peanut 
agglutination receptor; 
e. expression of enzymes such as heparanase. 
b. determination of antibody titer in absolute terms or as a ratio of the 
values obtained by different compositions, said values or ratios being 
indicative of the presence or absence of the disease. Quantitative values 
obtained are of use in establishing the severity of the disease. 
The diagnostic compositions according to the invention may be prepared by 
combining one or more antigenic compounds according to the invention as 
above-defined with suitable adjuvants and auxiliary components. 
Standardized kits with reference and calibration means are of value in the 
rapid and convenient determination of arthritic disease and its stage 
and/or severity.