Polypeptide competitor for immunoglobulin E

A competitor for human Immunoglobulin E (IgE) comprises a polypeptide which has a core sequence of seventy-six amino acids which is shown, together with the corresponding DNA sequence coding therefor, in FIG. 2. This amino acid sequence, numbered 1 to 76, corresponds to amino acids 301 to 376 of the epsilon heavy cain of IgE. The polypeptide may also include additional short sequences at the beginning and/or end of the core sequence which are physiologically harmless and do not contribute to the ability of the core sequence to bind compete with native IgE for the high-affinity receptor sites on human cells. The polypeptide is indicated for the treatment of Type I hypersensitivity reactions such as hay fever. The polypeptide may be produced synthetically or by expression from Escherichia coli containing a plasmid having a DNA segment coding for the polypeptide.

This invention relates to a polypeptide competitor for human Immunoglobulin 
E (IgE). More particularly the invention relates to a polypeptide which is 
capable of binding specifically to the high affinity Fc receptor sites for 
IgE which exist on human cells, particularly on mast cells and basophils, 
thereby inhibiting the biological responses, such as exocytosis or 
degranulation, which take place when antigen specific IgE binds to and 
crosslinks such receptor sites in the presence of antigen. The invention 
also relates to pharmaceutical preparations in which the polypeptide is an 
active constituent. The invention further relates to a method for the 
preparation of the polypeptide using genetically modified bacteria. 
In the human immune system, the principal role of IgE is believed to be to 
provide immunity to parasites. It also, however, mediates Type I 
hypersensitivity which is an allergic response leading to the 
manifestation of such symptoms as hay fever and asthma. Briefly, the 
mechanism of the allergic response is that on encountering a normally 
innocuous antigen such as pollen, synthesis of antigen-specific IgE by 
B-cells is initiated. The antigen-specific IgE then binds to mast cell 
receptor sites via its Fc region and thereafter any further encounter with 
the antigen triggers degranulation of the mast cells releasing mediators, 
principally histamine, resulting in the acute inflammatory symptoms 
typical of Type I hypersensitivity. 
Structurally, IgE, in common with the other immunoglobulins, comprises two 
heavy and two light chains, the epsilon heavy chain having five domains, a 
variable domain VH and constant domains CH1 to CH4. The molecular weight 
of IgE is in the region of 188,000 of which the heavy chain accounts for 
about 72,500, representing a sequence of approximately 550 amino acid 
residues. 
It has been reported (Nature, vol.315, 1985, No.6020, pp 577-578) that a 
peptide sequence of 330 amino-acids corresponding to amino acid residues 
218 to 547 (in accordance with the numbering given by Bennich, Progress in 
Immunology II, Vol I, July 1974, pp 49-58) of the epsilon heavy chain of 
IgE has an inhibitory effect on the release of mediators from human mast 
cells. The numbering is erroneously assigned in that paper in Nature; the 
more correct numbering would be 208 to 537. The 330 amino-acid sequence 
exists as a dimer consisting of two chains of amino-acids, each of 330 
amino-acids in length, linked by disulphide bonds. 
U.S. Pat. Nos. 4,171,299 and 4,161,522 disclose that an oligopeptide 
containing from three to ten amino acids in a sequence selected from a 
portion of amino acids 265 to 537 of the Bennich nomenclature (see 
reference above) of the Fc region of human IgE will block Fc receptors of 
mast cells thus inhibiting degranulation and release of mediators such as 
histamine. The most active of these oligopeptides is identified as the 
pentapeptide Asp-Ser-Asp-Pro-Arg (called HEPP: Human Immunoglobulin E 
Polypeptide) derived from the amino acid sequence 320 to 324 of the IgE 
heavy chain. In native IgE amino acid 322 is asparagine, but it is 
suggested in the Patents that substitution of asparagine by aspartic acid 
leads to a substantial enhancement of the blocking activity. 
In the Patents mentioned above the full sequence which is attributed to 
Bennich (Progress in Immunology II, Vol I, July 1974, pp 49-58) is quoted 
and shows aspartic acid at location 322. However, Bennich himself later 
asserts (Int. Arch. Allergy Appl. Immunol. 53, 459) that asparagine 
resides at that location. Bennich also reports that neither of the 
peptides Asp-Ser-Asp-Pro-Arg nor Asp-Ser-Asn-Pro-Arg has any blocking 
activity. Determination of the gene sequence has shown that amino acid 322 
is asparagine and not aspartic acid. In European Patent Application No. 
102634 asparagine and not aspartic acid is correctly quoted at the 
equivalent location. 
Further, it is also reported that the specific activity of HEPP is low 
requiring excessively large doses for any significant physiological 
effect. 
It is knwon that IgE epsilon chain fragments may be synthesised in 
Escherichia coli by cloning and expression of the DNA sequences coding for 
the appropriate domains of the IgE chain (Eur. J. Immunol. 1985, 15: 
966-969 and Proc. Natl. Acad. Sc. USA, vol.81, 1984, 2955-2959). 
An object of the present invention is to provide a new peptide for use in 
anti-allergy treatment. 
According to the present invention there is provided a polypeptide 
competitor for human Immunoglobulin E (IgE), comprising a monomeric chain 
of amino acids having the having following sequence: 
Gln-Lys-His-Trp-Leu-Ser-Asp-Arg-Thr-Tyr- 
Thr-Cys-Gln-Val-Thr-Tyr-Gln-Gly-His-Thr- 
Phe-Glu-Asp-Ser-Thr-Lys-Lys-Cys-Ala-Asp- 
Ser-Asn-Pro-Arg-Gly-Val-Ser-Ala-Tyr-Leu- 
Ser-Arg-Pro-Ser-Pro-Phe-Asp-Leu-Phe-Ile- 
Arg-Lys-Ser-Pro-Thr-Ile-Thr-Cys-Leu-Val- 
Val-Asp-Leu-Ala-Pro-Ser-Lys-Gly-Thr-Val- 
Asn-Leu-Thr-Trp-Ser-Arg 
The core sequence of seventy-six amino acids defined above is capable of 
binding to human IgE high-affinity receptor sites. The said sequence, 
numbered in FIG. 2 as 1 to 76, corresponds to the amino acid sequence 
spanning residues 291 to 366 (Bennich nomenclature) of the heavy chain of 
human IgE. 
In addition the core sequence may have short sequences of amino acids 
initiating (X--) and terminating (--Y) the core sequence and covalently 
attached to its 3' and/or 5' ends, which do not participate in the binding 
to IgE receptors and which are not physiologically harmful. 
Further according to the invention there is provided a DNA having the 
following nucleotide sequence: 
CAG AAG CAC TGG CTG TCA GAC CGC ACC TAC 
ACC TGC CAG GTC ACC TAT CAA GGT CAC ACC 
TTT GAG GAC AGC ACC AAG AAG TGT GCA GAT 
TCC AAC CCG AGA GGG GTC AGC GCC TAC CTA 
AGC CGG CCC AGC CCG TTC GAC CTG TTC ATC 
CGC AAG TCG CCC ACG ATC ACC TGT CTG GTC 
GTC GAC CTG GCA CCC ACC AAG GGG ACC GTG 
AAC CTG ACC TGG TCC CGG. 
The present invention also provides a transformant in which the DNA 
contains the nucleotide sequence defined above. Preferably the 
transformant host is Escherichia coli 
Most preferably the transformant comprises Escherichia coli N4830 
harbouring the plasmid pE 2-3 (Accession Number NCTC 11993, deposited with 
the National Collection of Type Cultures, London on Jul. 1st. 1986). 
The invention further provides a vector in which the DNA is as defined 
above, said segment being oriented within said vector such that in a host 
said segment is expressed to produce a polypeptide. The invention also 
provides a host organism transformed by the aforesaid vector. 
Further according to the present invention there is provided a method of 
preparing the polypeptide defined above, comprising culturing the 
aforesaid host organism and isolating the polypeptide from the culture. 
Using this method the resultant polypeptide product may include the groups 
X and Y defined above. If thought necessary or desirable these groups may 
be removed from the core sequence of amino acids by standard degradation 
procedures but, being physiologically harmless, there is no compelling 
reason for removing them. In the specific example which will be given 
later, the group represented by X is NH.sub.2 -Met-Asp- and the group 
represented by Y is -Leu-Ile-Asn. 
Alternatively the polypeptide may be synthesised by known chemical 
synthetic methods. 
This invention also includes a pharmaceutical preparation in which the 
active principle is the polypeptide defined above. 
The preparation may also include a pharmaceutical carrier permitting 
administration of the polypeptide in an appropriate manner, for example 
intranasally. 
The polypeptide of the invention may also be covalently linked to or 
associated with other therapeutic or diagnositic agents or other molecules 
with the effect that the polypeptide acts to target the therapeutic or 
diagnostic agent to cells bearing IgE high-affinity receptors. 
By way of explanation, the core sequence of the polypeptide of this 
invention bridges the second and third domains of the epsilon chain 
constant region of IgE. Previous work (J. Immunol 114, 1838, 1975; 
Immunol. Rev. 41, 3, 1978) concluded that both the second and fourth 
domains were required to form the binding site. It was therefore 
unexpected that in respect of polypeptides with a major deletion in the 
CH2 domain, a major deletion in the CH3 domain and deletion of the entire 
CH4 domain or a combination of these deletions a binding ability 
comparable to that of native IgE would result. 
That the monomeric polypeptide of the invention has the ability to bind to 
the high-affinity receptors of mast cells and basophils is quite 
surprising. Firstly, the fact that the chain is monomeric at all is 
unexpected since it would have been anticipated that after synthesis of 
the peptide chain there would be an immediate spontaneous dimerisation of 
peptide chains via their cysteines at location 318 (28 in FIG. 2). The 
cysteines at locations 231 and 318 have been previously implicated in the 
formation of the inter-epsilon chain disulphide bonding in IgE. The 
surprising existence of the monomeric polypeptide of this invention which 
includes the cysteine at location 318 suggests that one explanation of 
this unexpected occurrence is that the inter epsilon chain disulphide 
pairing in IgE is not, as previously believed, of the homotypic (AA,BB) 
type but of the heterotypic (2AB) type. Secondly, the binding activity of 
the monomeric polypeptide is quite unexpected since it has previously been 
believed that two epsilon chains were necessary for the binding at mast 
cell receptor sites to trigger exocytosis. In the same study, loss of 
binding activity was found to occur when the inter epsilon chain bonding 
at location 318 was broken even although the chains remained covalently 
linked via the more resistant disulphide interchain bond assumed to link 
the two cysteines at location 241. The polypeptide of the invention lacks 
sequences necessary for the formation of any of the three immunoglobulin 
domains which exist in human IgE and thus this invention indicates that 
this dimeric structural framework is not essential in its entirety for 
recognition by the high affinity receptors of the mast cells. 
The core sequence of the invention is less than a quarter of the size of 
the Fc region of the IgE heavy chain, having a molecular weight of around 
10,000. The amino acid sequence of the polypeptide spans the C-terminal 
end of the CH2 domain and the N-terminal end of the CH3 domain and 
incorporates a beta-turn from each of the two domains and the so-called 
"hinge" between them. 
A method for producing the polypeptide utilising genetically modified 
Escherichia coli containing a DNA insert coding for the core amino acid 
sequence will now be described in the following Example 1.

EXAMPLE 1 
Human myeloma cell line 266B1 which had previously been used by Bennich 
(Prog. in Immunol. Vol I, North Holland Publishing Company, pages 49 to 
58, 1974) contains a functional epsilon gene sequence that can easily be 
cloned. 
The known amino acid sequence of IgE provided all the information required 
to make an oligonucleotide probe for screening a cDNA library from the 
266B1 cell line. The original cell line synthesised from 2 to 7 micrograms 
of IgE per 10.sup.6 cells per 48 hours. During propagation of the line and 
adaptation to growth in suspension culture, the synthesis of IgE had 
evidently declined, the levels obtained being about 20 nanograms of IgE 
per 10.sup.6 cells per 48 hours. The synthesis of IgE was confirmed by 
labelling the protein in culture and SDS polyacrylamide gel 
electrophoresis of the fraction precipititated from the culture 
supernatants by anti-human IgE Fc anti-serum. Similar analysis of the 
fraction precipitated by anti- human lambda light chain antiserum 
demonstrated the presence of a twenty-fold excess of monomeric over 
IgE-associated lambda light chain in the secreted immunoglobulin. Despite 
the poor expression of the epsilon gene in 266B1, the level of mRNA was 
sufficient for the task of cDNA synthesis and cloning. 
Total RNA was extracted from 266B1 cells and mRNA was purified by oligo-dT 
chromatography. The presence of intact epsilon chain mRNA was demonstrated 
by translation into polypeptide chains immunoprecipitable by goat 
anti-human IgE and having the expected electrophoretic mobility in SDS 
polyacrylamide gels corresponding to the 66,000 dalton unglycosylated 
human epsilon chain. The epsilon chain mRNA was enriched by a factor of 
ten by sucrose gradient centrifugation, the relative concentrations of 
epsilon chain mRNA in the different fractions being monitored both by the 
translation assay and by oligonucleotide-primed synthesis of cDNA of the 
expected length. 
Double-stranded cDNA was enzymatically synthesised using routine procedures 
and the cDNA was recombined by means of linkers into a appropriate 
restriction site in a plasmid vector and transformed into E. coli. 
An oligonucleotide probe of eleven nucleotides was designed on the basis of 
the amino acid sequence of the protein previously determined by 
conventional amino acid sequencing techniques and was chemically 
synthesised. The probe itself failed to detect any cDNA clones but served 
as a satisfactory primer for cDNA synthesis, permitting the acquisition of 
additional sequence information by DNA sequencing. A new 22 
nucleotide-long probe was constructed, based on this sequence and the 
larger probe detected five positive cDNA clones out of a total of 500. The 
cDNA inserts of the positive clones were excised by digestion with the 
appropriate restriction endonuclease and the sizes were found to be in the 
range of from 0.6 to 2.0 kb; only the largest, 2 kb clone, designated 
pJJ71, was extensively characterised. It contains the sequences 
correponding to the 5' and 3' untranslated regions of the mRNA, plus those 
encoding the amino terminal secretion peptide of twenty amino acids and 
the entire mature epsilon chain. 
Reference is now made to the accompanying drawing which shows the 
derivation of the plasmid pE2-3, the expression plasmid directing the 
synthesis of the polypeptide of the invention. The human epsilon DNA 
coding sequence is represented by the box V indicating the variable 
region, and C1 to C4, the four constant domains. The solid arrows denote 
the inducible promoters mediating transcription of sequences cloned 
downstream. In ptac-85 and its derivative pE49 the tac promoter is 
present: the lambda P1 promoter is used by vector pAS1 and recombinants 
pASE1 and pE2-3. The synthetic DNA translation terminator in pE2-3 has the 
sequence 5'-GCTTAATTAATTAAGC-3'. 
Expression of the polypeptide in E. coli was achieved via three subclonings 
of epsilon Fc cDNA cloned in pJJ71. First, the Sa1I-PvuII fragment 
corresponding to epsilon Fc and some forty base pairs of untranslated 
sequences, after digestion with S1 nuclease, was ligated into the filled 
NcoI site of ptac-85. The resulting plasmid, pe49, directs the expression 
of epsilon Fc and introduces a unique Sa1I site at the 3' end of the 
truncated flanking sequences. Second, pe49, linearised by SacI and treated 
with the double stranded exonuclease Ba131, was recleaved by Sa1I and the 
DNA fragment corresponding to amino acids 291 to 537 of epsilon Fc was 
subcloned into pASI. The pASI had been treated with BamHI and Sa1I 
restriction enzymes (the BamHI site having been made blunt-ended using DNA 
polymerase) in order to have compatible termini to those bounding the 
fragment from pe49. The resulting plasmid pASe1 directed the synthesis of 
an epsilon fragment comprising the third and fourth domains and part of 
the second domain from amino acid 291. Third, the expression product of 
pASE1 was foreshortened at its carboxy terminus by introducing a 
translation termination signal into the cloned DNA at a SmaI site in the 
position corresponding to amino acid 365. The construct, pE2-3, was 
generated by blunt ligation of a synthetic DNA fragment which contains 
translational stop codons in all three reading frames to pASE1 DNA 
linearised with SmaI. 
The polypeptide of the invention was obtained when E. coli strain N4830 
harbouring pE2-3 was grown under inducing conditions. Expression is 
controlled by the lambda cI repressor which shuts off transcription from 
the lambda PL promoter. E. coli strain N4830 contains a thermolabile cI 
repressor which is active at 30 degrees and inactive at 42 degrees 
Centigrade. A culture of N4830/pE2-3 was thus grown under non-inducing 
conditions at 30 degrees Centigrade to an A.sub.600 of 0.8 then 
heat-shocked at this density by addition of an equal volume of medium 
preheated to 65 degrees Centigrade. After repressor inactivation the 
culture was grown at 42 degrees Centigrade for a further three hours and 
then harvested. Electrophoresis of a lysate of this culture showed the 
presence of a 10K peptide (not present in the absence of induction) 
visible on Coomassie staining, and shown immunologically to be an epsilon 
derivative by Western blotting. The expected size of the product of the 
gene fragment is 9,500 daltons. 
The polypeptide was present in the lysate as insoluble material which was 
recovered by dissolution in 8M urea. The peptide remained soluble after 
removal of urea by dialysis and was purified to near homogeneity by 
anti-human epsilon affinity chromatography. Polyacrylamide gel 
electrophoresis under non-reducing (as well as reducing) conditions showed 
that the purified polypeptide had a molecular weight of about 10,000, 
indicating that unreduced peptide was monomeric. 
EXAMPLE 2 
The effectiveness of the polypeptide of the invention was compared with 
natural IgE and various fragments thereof in a series of tests using the 
passive cutaneous anaphylaxis (PCA) reaction [described in Nature 315: 
577-578 (1985)]. The results are presented below in Table I. 
TABLE I 
______________________________________ 
Heavy-chain 
Amino Domains Ac- 
Source acids VH CH1 CH2 CH3 CH4 tivity 
______________________________________ 
Myeloma IgE 
1-457 + + + + + + 
(PS) 
pSC213 208-537 - - + + + + 
pES1 300-537 - - p + + + 
PE delta 4 
209-429 - - + + - .+-. 
pE2-3 291-366* - - p p - + 
______________________________________ 
p = part of domain 
*amino acid sequence 1 to 76 shown in FIG. 2 
The approach to intervention in the allergic response adopted in the 
present invention is to block IgE high affinity receptor sites by 
administration to the patient of an amount of the polypeptide of the 
invention. This approach is believed to leave the low-affinity receptors 
unaffected and free to participate in their apparent immunological role. 
From the results summarised in Table I, it can be seen that a positive 
effect is attained with all the sequences quoted, thus narrowing down the 
binding sites of IgE to mast cells to the seventy-six amino acid sequence 
of this invention. This sequence displayed an affinity constant for the 
human basophil receptor (5 .times.10.sup.9 /mol) which was 
indistinguishable from that of a myeloma IgE. 
Inhibition of the Prausnitz-Kustner reaction was also displayed by the 
fragments listed in Table 1 above, that is, by amino acid sequences which 
contain the sequence 1 to 76 shown in FIG. 2, but, no inhibition was found 
for three other fragments of IgE, namely: 
(i) amino acids spanning locations 430 to 537 of the IgE sequence, and 
therefore containing no residues in common with the polypeptide of the 
invention, 
(ii) amino acids spanning residues 208 to 326, and therefore containing the 
residues 1 to 35 of the polypeptide of this invention; and, 
(iii) amino acids spanning residues 329 to 537, and therefore containing 
the residues 38 to 76 of the polypeptide of this invention. 
The results of the P-K reaction tests were as follows: 
Inhibition of the P-K Reaction by IgE Fragments 
A single subject was used for passive sensitisation. The serum IgE of this 
subject was 4 IU/ml (approximately 10 ng/ml). The sensitising serum (E.C.) 
contained 380 IU/ml of IgE (912 ng/ml serum E.C.), of which 8.7% was 
directed against ragweed antigen, as determined by the specific drop in 
serum IgE following absorption of the serum over a Sepharose 4B ragweed 
antigen column compared with a control Sepharose 4B human serum albumin. 
Serum E.C. was free of detectable hepatitis B antigen and of antibodies to 
that antigen and to human immunodeficiency virus (HIV). Serum E.C. was 
obtained in 1983 and its donor is currently (1987) healthy and HIV 
antibody negative. Epsilon chaim fragments were injected into skin sites 
one hour before the injection of serum E.C. Skin sites were challenged 48 
hours later with ragweed antigen (1,000 protein nitrogen units/ml of a 
mixture of giant and short ragweed). Twenty minutes later the skin sites 
were examined for the presence of wheal and erythema. The surface area of 
the reaction was estimated as follows: Transparent tape was used to 
transfer the outline for the reactions to paper which was then cut out and 
weighed on an analytical balance. The area was read from a standard curve. 
All injections were intradermal and 0.02 ml in volume. In each experiment 
a set of skin sites was also sensitised with diluent. None of these sites 
showed wheal or flare when challenged with ragweed antigen. The diluent 
consisted of 0.15M sodium chloride and 0.03% human serum albumin. In both 
experiments, reported in Table 2 below, skin sites were sensitised with a 
1:100 dilution of serum E.C. containing 5.times.10.sup.-11 M IgE. 
TABLE 2 
______________________________________ 
Area of Wheal & Flare 
Expt. 1 Expt. 2 
Inhibitor 10 ug/ml 1 ug/ml 
______________________________________ 
Diluent 65/380 92/455 
IgE (P.S.) 0/0 0/0 
aa 208-537 0/0 0/0 
aa 291-537 0/0 0/0 
aa 209-429 0/0 0/0 
aa 291-366* 0/0 0/0 
aa 430-537 60/416 85/438 
aa 208-326 70/350 80/405 
aa 329-537 69/375 78/398 
______________________________________ 
* = aa 1 to 76 in FIG. 2. 
Relative Activity of Recombinant IgE (ND) Peptides in the Inhibition of the 
P-K Reaction 
The molarities of the epsilon chain fragments were calculated taking into 
account the proportion of dimers versus monomers in each preparation. 
Monomers were included in the calculations because the polypeptide of the 
invention has never existed in detectable dimeric or oligomeric forms and 
it was a potent inhibitor of the P-K reaction (see Table 2 above). Each 
fragment was used over a range of 10.sup.-13 to 10.sup.-6 M in ten-fold 
increments. The results are presented in Table 3 below. 
TABLE 3 
______________________________________ 
Molarity required for 50% inhibition 
of the P-K reaction reduced by 
Expt 1 Expt 2 
Serum E.E. dil 1:100 
Serum E.C. dil 1:20 
= 5 .times. 10.sup.-11 M IgE 
= 2.5 .times. 10.sup.-10 M IgE 
Source Molarity % Potency Molarity 
% Potency 
______________________________________ 
IgE (P.S.) 
2 .times. 10.sup.-10 
100 2 .times. 10.sup.-9 
100 
aa 208-537 
4 .times. 10.sup.-10 
50 4 .times. 10.sup.-9 
50 
aa 291-537 
5 .times. 10.sup.-10 
40 4 .times. 10.sup.-9 
50 
aa 209-429 
5 .times. 10.sup.-10 
40 6 .times. 10.sup.-9 
33 
aa 291-366* 
6 .times. 10.sup.-10 
33 5 .times. 10.sup.-9 
40 
______________________________________ 
* = 1 to 76 in FIG. 2. 
Duration of the Inhibition of the P-K Reaction 
In Table 4 below, values are given in days elapsed following the injection 
of the inhibitor before a successful P-K reaction could be achieved. 
Multiple skin sites of a normal subject were injected at day 0 with IgE 
(P.S.) the polypeptide of the invention or diluent. At intervals (days 
0,4,9,12,14,17,19,21) individual skin sites were sensitised with a 1:100 
dilution of serum E.C. (5.times.10.sup.11 M IgE) then challenged 48 hours 
later with ragweed antigen. Sites pretreated with diluent always gave a 
positive wheal and flare reaction with a mean standard deviation of the 
flare of 392.+-.58 mm for the eight successive determinations. The days 
shown in Table 4 represent the time of the first appearance of flare 
and/or erythema at the challenged skin sites. 
TABLE 4 
______________________________________ 
Inhibitor Concentration 
Inhibitor 10.sup.-7 M 
10.sup.-6 M 
______________________________________ 
IgE (P.S.) 12 19 
aa 301-376* 9 14 
______________________________________ 
*= 1 to 76 in FIG. 2.