Synthetic antigenically-active polypeptide and a process for its preparation

A synthetic antigenically active polypeptide, which includes the amino acid sequence:H-Asp-Ala-Glu-Gln-Arg-Gly-Glu-Leu-Ala-Ile-Arg-Asp-Ala-Asn-Ala-Arg -Leu-Ser-Glu-Leu-OH; a process for the preparation of such polypeptide; an antigenic agent comprising such polypeptide convalently bonded to an immunogenically active carrier; a pharmaceutical composition comprising such polypeptide or antigenic agent together with a pharmaceutically acceptable carrier or diluent; and a method for determining the presence or absence in a biological sample of antibodies monospecific to such polypeptide, which comprises introducing into the sample such polypeptide or such agent or such composition and determining the presence or absence of agglutination.

TECHNICAL FIELD 
The present invention relates to a synthetic antigenically active 
polypeptide which includes certain aminoacid sequences, and a process for 
the preparation of said polypeptide. 
BACKGROUND ART 
In published Swedish patent application 73- 08917-9 and in U.S. Pat. No. 
3,960,827 there is described a cancer-associated polypeptide antigen 
(CAPA), the technique for its isolation and its use in cancer diagnosis 
and in the preparation of antibodies. The CAPA is now commercialized under 
the designation TPA, which stands for "tissue polypeptide antigen". As is 
clear from the specification of the above-identified Swedish patent 
application and U.S. patent, the isolation of the natural antigen is a 
complicated procedure which, even if resulting in a practically useful 
product, still involves high production costs and moreover may involve 
difficulties in the provision of necessary starting materials, such as 
tumor tissue, etc. Against this background a synthetically prepared 
antigen would, of course, by very attractive, in view of the possibility 
of thereby obtaining a product exactly specified as to its composition, 
said product also being capable of being prepared at a more favorable 
price. 
SUMMARY OF THE INVENTION 
The main object of this invention is thus to provide a synthetic 
antigenically active polypeptide which reacts monospecifically with 
antibodies prepared by means of the polypeptide antigen described in the 
above-identified patent specifications. 
According to this invention there is thus provided a synthetic 
antigenically active polypeptide including the amino acid sequence: 
H-Asp-Ala-Glu-Gln-Arg-Gly-Glu-Leu-Ala-Ile-Arg-Asp-Ala-Asn-Ala-Arg-Leu-Ser-G 
lu-Leu-OH. 
According to one preferred embodiment of the instant invention, there is 
provided a synthetic antigenically active polypeptide containing as an 
active constituent or immunological determinative group the amino acid 
sequence: (I) 
H-Asp-Ala-Glu-Gln-Arg-Gly-Glu-Leu-Ala-Ile-Arg-Asp-Ala-Asn-Ala-Arg-Leu-Ser- 
Glu-Leu-Glu-Ala-Ala-Leu-Gln-Arg-Ala-Lys-Gln-Asp-OH. 
According to another preferred embodiment of the instant invention, the 
polypeptide contains the amino acid sequence: (II) 
H-Ala-Ser-Leu-Glu-Ala-Ala-Ile-Ala-Asp-Ala-Glu-Gln-Arg-Gly-Glu-Leu-Ala-Ile- 
Arg-Asp-Ala-Asn-Ala-Arg-Leu-Ser-Glu-Leu-OH. 
From hereon the following abbreviations for the amino acids in question are 
used: 
______________________________________ 
ALANINE Ala SERINE Ser 
ARGININE Arg LEUCINE Leu 
ASAGINE Asn TYROSINE Tyr 
ASTIC ACID Asp VALINE Val 
GLUTAMINE Gln THREONINE Thr 
GLUTAMIC ACID Glu PHENYLALANINE Phe 
GLYCINE Gly LYSINE Lys 
ISOLEUCINE Ile 
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The present invention also relates to a process for the preparation of the 
antigenically active polypeptide, and in this process an N-protected amino 
acid is attached to a resin by esterification, the N-protecting group is 
removed and a second N-protected amino acid is coupled to the amino group 
of the resin-bound amino acid, the N-protecting group is removed and the 
coupling step is repeated with a third N-protected amino acid. Protecting 
groups in the process are the usual groups, and are set forth more fully 
in the following. This procedure is repeated until the desired amino acid 
sequence is obtained, the polypeptide being then cleaved from the resin. 
It is preferred after each attachment of an N-protected amino acid to the 
resin-bound amino acid to wash away all by-products and unreacted soluble 
materials. 
The resin used in the synthesis may consist of a copolymer of styrene and 
divinyl benzene, the styrene constituting the major part of the copolymer, 
for instance about 98% by weight of styrene and about 2% by weight of 
divinyl benzene. 
In order to provide a reactive group for coupling to the first amino acid, 
the benzene rings are suitably partially chloromethylated. When this 
chloromethylated resin is treated with the triethylamine salt of an 
N-protected amino acid, a bond of the benzyl ester type is formed. Such 
bond is stable under the synthesis steps, but can be cleaved with HBr in 
acetic acid or trifluoroacetic acid, the N-protecting group being 
simultaneously removed and the peptide separated from the resin. 
In order to protect the N-terminus of amino acid the t-butyloxycarbonyl 
group can suitably be used as it is conveniently cleaved by means of HCl 
in acetic acid. It is also possible to use the carbobenzoxy group for the 
purpose, but in this case HBr in acetic acid must be used for removal of 
the protecting group. As a protecting group there may also be used the 
o-nitrophenyl sulfenyl group. Other protecting groups may also be employed 
and will be apparent to one skilled in the art. 
The coupling reagent most frequently used in the synthesis is 
dicyclohexylcarbodiimide. If methylene chloride is used as a solvent and 
about 50% excess of t-butyloxy carbonyl aminoacid and dicyclohexyl 
carbodiimide, a quantitative reaction is obtained within a few minutes. 
Even dimethylformamide may be used as a solvent. Further details regarding 
the synthesis procedure may be found in "Protein Sequence Determination" 
summarized by Saul. B. Needleman, Springer-Verlag, Berlin-Heidelberg-New 
York, 1970, particularly at pp. 308-310. Other coupling agents are also 
suitable and will be apparent to one skilled in this art. 
EXAMPLES 
The present invention will now be illustrated further by means of 
non-limiting examples.

EXAMPLE 1 
By the process in solid phase according to Merrifield (cf. the above 
literature reference) the following product is prepared: 
##STR1## 
wherein: R denotes a resin; Boc is a t-butyloxy carbonyl group; 
##STR2## 
The amino acid sequence between the t-butyl-oxycarbonyl group and the resin 
part may be expressed in the following manner using the foregoing 
abbreviations regarding the amino acids: 
(I)-Asp-Ala-Glu-Gln-Arg-Gly-Glu-Leu-Ala-Ile-Arg-Asp-Ala-Asn-Ala-Arg-Leu-Se 
r-Glu-Leu-Glu-Ala-Ala-Leu-Gln-Arg-Ala-Lys-Gln-Asp-. 
0.4 mmole of Boc-aspartic acid .beta.-benzyl ester .alpha.-(resinbenzyl 
ester) was transferred to the cuvette of a Beckman peptide synthesizer and 
swollen in methylene chloride (CH.sub.2 Cl.sub.2). The protecting 
Boc-group was removed by treatment with trifluoroacetic acid in excess in 
methylene chloride. After washing with methylene chloride the resin was 
neutralized with triethylamine and again washed with methylene chloride. 
N-Boc-glutamic acid benzyl ester (coupling step 1) and 
cyclohexylcarbodiimide was added dissolved in CH.sub.2 Cl.sub.2, and the 
mixture was stirred for 30 minutes. The resin was washed and the coupling 
step repeated. This terminates the introduction of R.sub.2 in step 1 
according to the above. 
Step 2 of the synthesis starts by removal of an N-Boc from the N-terminated 
amino acid and repetition of the coupling step while using another 
N-Boc-amino acid. To build up the above amino acid sequence the following 
reagents are used in the subsequent steps: 
in 
step 2: N-Boc-glutamine-xanthydryl derivative 
3: .alpha.N-Boc-.epsilon.(o-chloro-carbobenzoxy)lysine 
4: N-Boc-alanine 
5: N-Boc-G-tosyl arginine 
6: As in step 2 
7: N-Boc-leucine 
8: As in step 4 
9: As in step 4 
10: N-Boc-glutamic acid .gamma.-benzyl ester 
11: As in step 7. 
12: As in step 10. 
13: N-Boc-serine-benzyl ether 
14: As in step 7. 
15: As in step 5 
16: D:o as in step 4 
17: N-Boc-asparagine-xanthydryl derivative 
18: As in step 4 
19: N-Boc-aspartic acid .beta.-benzyl ester 
20: As in step 5 
21: N-Boc-isoleucine 
22: As in step 4 
23: As in step 7 
24: As in step 10 
25: N-Boc-glycine 
26: As in step 5 
27: As in Step 2 
28: As in step 10 
29: As in step 4 
30: As in step 19 
The protecting groups are removed from the protected resin-bond peptide, 
the resin is cleaved off using anhydrous hydrogen fluoride at 0.degree. C. 
for 30 minutes, and the peptide is extracted from the resin using a 10% by 
weight aqueous solution of acetic acid. After evaporation the residue is 
purified by gel filtration on Sephadex G 25 Fine in 0.1 M NH.sub.4 
HCO.sub.3. AFter lyophilization of the peptide a satisfactory amino acid 
analysis is obtained, together with a molecular weight of approximately 
3300, as determined by gel filtration. The theoretical MW value is 
3320.80. 
The polypeptide prepared according to the foregoing was investigated with 
regard to its ability to inhibit the hemagglutination reaction between 
tanned red sheep blood cells labelled with natural cancer antigen and 
antibodies prepared by using natural cancer antigen (CAPA or TPA). For 
details concerning this technique reference can be made to the 
above-identified Swedish patent application 73-08917-9 and U.S. Pat. No. 
3,960,827. The specific activity of the polypeptide was found to be about 
0.2 Units per milligram (U/mg). Said Unit for specific activity is defined 
as 1/6 of the quantity of active polypeptide required to label 10.sup.9 
sheep blood cells so as to be fully agglutinated by a minimum number or 
amount of antibodies (maximum dilution of antibodies). 
For the purpose of investigating which functions are critical for the 
activity of the polypeptide, the basic structure of which is given above, 
certain experiments were carried out. The arginines were thus blocked with 
cyclohexanedione at pH 13 resulting in almost complete loss of activity. 
If, however, the polypeptide is subjected only to a basic environment, 
wherein the pH is 13 for the same period of time, only a minor part of the 
activity is lost. This indicates the fact that the arginine presence in 
the polypeptide according to the present invention is of decisive 
importance for the antigenic activity. 
From the character of the structure, it is clear that the polypeptide has 
an acidic character, thus being negatively charged in a neutral solution 
while, however, maintaining the positive charge of the arginine part 
thereof. 
The chemical basis for the antigenic character of the polypeptide has thus 
been set forth in the foregoing description. The remarkable specific 
activity which has been obtained by means of a synthetically prepared 
product, the activity of which can be said to be of a haptenic nature, due 
to the small determinant group, constitutes a pioneering advance with 
regard to the application of immunology and testing, i.e. diagnosis, 
within the cancer area. 
Clearly and in accordance with what is known in immunology, antigenic 
activity is a function not only of the structure of the haptenic 
determinant group, but also of the size of the polypeptide. Said size does 
not, however, effect the specificity of the peptide but only the degree of 
activity, inasmuch as a larger molecule tends to be more active than a 
smaller one. Therefore, an improved activity is obtained if the 
polypeptide is arranged on a suitable immunogenically active carrier, for 
instance a protein, such as albumin, for instance egg white. Already the 
polypeptide in its basic structural form, i.e., having at least twenty 
(20) amino acid units, does, however, show an antigenic activity which is 
sufficient to enable the polypeptide to react with corresponding 
antibodies. 
In Example 1 as described above the antigenic activity of the synthesized 
polypeptide is determined in the following manner. 
For quantitative determination of the amount of polypeptide 7 mgs of the 
peptide are dissolved in 1.4 mls of buffer grade serum, i.e. a 
physiological saline solution containing 2% of inert human serum and 
buffered to a pH of about 7.5 with a phosphate buffer. Under serial 
dilution a series of samples of said solution having a decreasing 
polypeptide concentration is prepared and to each of said samples there is 
added a pre-determined amount of anti-serum containing antibodies specific 
to TPA. To each of the resulting samples there is then added, after 
incubation, a predetermined amount of the polypeptide carried on a 
particulate carrier, hemagglutination taking place to an extent 
corresponding to the amount of available antibodies. In parallel there is 
prepared a corresponding series of control samples containing known 
decreasing amounts of TPA. The diameters of the hemagglutination 
depositions of the control samples are then visually compared with those 
of the control samples to estimate activity. 
EXAMPLE 2 
In the same manner as given in Example 1 the following polypeptide is 
prepared and found to be about equally immunologically active as the 
polypeptide of Example 1: 
##STR3## 
wherein: R denotes the resin; Boc is a t-butyloxy carbonyl group; 
The amino acid sequence between the t-butyloxycarbonyl group and the resin 
part may be expressed in the following manner using the foregoing 
abbreviations regarding the amino acids: (II) 
-Ala-Ser-Leu-Glu-Ala-Ala-Ile-Ala-Asp-Ala-Glu-Gln-Arg-Gly-Glu-Leu-Ala-Ile-A 
rg-Asp-Ala-Asn-Ala-Arg-Leu-Ser-Glu-Leu-. 
0.4 mmole of Boc-leucine (resinbenzyl ester) was transferred to the cuvette 
of a Beckman peptide synthesizer and swollen in methylene chloride 
(CH.sub.2 Cl.sub.2). The protecting Boc-group was removed by treatment 
with trifluoroacetic acid in excess in methylene chloride. After washing 
with methylene chloride the resin was neutralized with triethylamine and 
again washed with methylene chloride. N-Boc-glutamic acid benzyl ester 
(coupling step 1) and cyclohexylcarbodiimide was added dissolved in 
CH.sub.2 Cl.sub.2, and the mixture was stirred for 30 minutes. The resin 
was washed and the coupling step repeated. This terminates the 
introduction of R.sub.2 in step 1 according to the above. 
Step 2 of the synthesis starts by removal of an N-Boc from the N-terminated 
amino acid and repetition of the coupling step while using another 
N-Boc-amino acid. To build up the above amino acid sequence the following 
reagents are used in the subsequent steps: 
in 
step 2: N-Boc-glutamic acid .gamma.-benzylester 
3: N-Boc-serine-benzyl ether 
4: N-Boc-leucine 
5: N-Boc-G-tosylarginine 
6: N-Boc-alanine 
7: N-Boc-asparagine-xanthydryl derivative 
8: D:o as in step 6 
9: N-Boc-aspartic acid .beta.-benzyl ester 
10: D:o as in step 5 
11: N-Boc-isoleucine 
12: D:o as in step 6 
13: D:o as in step 4 
14: D:o as in step 2 
15: N-Boc-glycine 
16: D:o as in step 5 
17: N-Boc-glutamine-xanthydryl derivative 
18: D:o as in step 2 
19: D:o as in step 6 
20: D:o as in step 9 
21: D:o as in step 6 
22: D:o as in step 11 
23: D:o as in step 6 
24: D:o as in step 6 
25: D:o as in step 2 
26: D:o as in step 4 
27: D:o as in step 3 
28: D:o as in step 6 
The resulting protected resin-bond peptide is then treated and analyzed in 
accordance with the procedure of Example 1. 
The molecular weight as determined by gel filtration is approximately 3000, 
whereas the theoretical molecular weight is 2936.38. 
The results of amino acid analyses made on the polypeptides of examples 1 
and 2 above are summarized in the table below. The figures given therein 
refer to mole percent of each amino acid, i.e. the numbers of the 
respective amino acids per 100 amino acids of the peptide. The figures 
given in the table correspond satisfactorily to the theoretical values. 
TABLE 
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Ex. 
No. Asp Ser Glu Gly Ala Ile Leu Lys Arg 
______________________________________ 
1 6.99 4.11 20.94 
2.53 26.61 
3.28 16.87 
5.39 13.27 
2 10.45 7.03 17.09 
2.75 30.43 
6.74 15.48 
-- 10.02 
______________________________________ 
The present invention is in no way limited to the foregoing specific 
embodiments. Thus, in preparing the polypeptide, the resin may be any 
material containing benzene rings and in addition at least one group 
having the ability of binding N-protected amino acids by esterification. 
The ester bond must be relatively easy to hydrolyse and, of course, must 
be easier to cleave than the peptide bonds in the polypeptide. However, 
the ester bond must be sufficiently stable to be able to withstand the 
reaction conditions under the synthesis. 
As an alternative to the above mentioned coupling reagent, dicyclohexyl 
carbodiimide, when attaching glutamine and asparagine units, coupling may 
instead be carried out with so-called active esters, for instance 
cyanomethyl, thiophenyl or nitrophenyl esters. As a solvent in the 
synthesis so-called aprotic solvents are suitable, particularly solvents 
that are somewhat hydrophilic or "semipolar". 
Regarding useful groups to protect the N-amino acids, it is noted that 
t-butyloxy carbonyl groups, wherein one or two methyl groups are replaced 
by phenyl, also may be advantageously used. In this connection it can be 
mentioned that the product obtained in the synthesis is provided with 
N-protecting groups and attached to a resin by esterification, and that it 
advantageously can be stored for a long period of time without being 
destroyed. In connection with the use of the polypeptide, the protecting 
groups and the resin part may then be removed. 
In the amino acid sequences given in the present preparation, the terminal 
groups (after removal of the protecting groups and the resin part) are 
always amino group and carboxyl group, respectively. The amino group is 
found at the left end of the chain, whereas the carboxyl group is found at 
the opposite end of the amino acid sequence.