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United States Patent [191
[11]
4,107,298
Liining
[45]
Aug. 15, 1978
[54] ANTIGENICALLY ACTIVE POLYPEPTIDE
4,018,753
AND A PROCESS FOR ITS PREPARATION
[75] Inventor:
Bj'tirn Erik Gustav Bertilsson Liining,
Stockholm, Sweden
[73] Assignee:
AB Bonnierforetagen, Stockholm,
Sweden
4/1977
Inouye et a1. ............... .. 260/1125 R
FOREIGN PATENT DOCUMENTS
787,918
728,129
6/1968
2/1966
Canada ........................... .. 260/1125 R
Canada ........................... .. 260/1125 R
Primary Examiner-Delbert R. Phillips
Attorney, Agent, or Firm—-Gordon W. Hueschen
[21] Appl. No.: 789,665
[57]
[22] Filed:
Apr. 21, 1977
[30]
Foreign Application Priority Data
The invention provides for a synthetic antigenically
active polypeptide containing an immunologically inert
Apr. 29, 1976 [SE]
[51]
ABSTRACT
portion and as an active immunological determinant
Sweden .............................. .. 7604972
Int. Cl.2 ................... .. A61K 37/00; C07C 103/52
group the amino acid sequence A_3-A_2-A_1-A0-A1
A2-A3, wherein A0 is an arginine residue, A1, A2, A_l
[52]
US. Cl. . . . . . .
424/177; 260/ 112.5 R
and A_2 are the same or different and selected from
[58]
Field of Search ................ .. 260/ 1 12.5 R; 424/ 177
residues of: glycine, alanine, valine, leucine, lysine, and
[56]
. . . . . . . . . . . . ..
isoleucine and A3 and A_, are same or different and
References Cited
selected from glutamic acid and aspartic acid. The in
U.S. PATENT DOCUMENTS
3,715,190
2/1973
3,929,756
3,960,827
12/1975
6/1976
Won Kil Park et al.
vention also provides for a process for the preparation
260/1125 R
Leeman et al. ..... ..
260/1125 R
Bjorklund .......................... .. 424/177
of such polypeptide.
12 Claims, 1 Drawing Figure
1
4,107,298
2
wherein A4 and A_., are the same or different and se
lected from residues originating from glycine, alanine,
ANTIGENICALLY ACTIVE POLYPEPTIDE AND A
PROCESS FOR ITS PREPARATION
valine, leucine and isoleucine which, as indicated above,
are neutral aminoacids. In view of thecharacter of the
aminoacids in said respect, the fractions of the aminoa
The present invention relates to a synthetic antigeni
cally active polypeptide which is characterized by con
taining an immunologically inert portion and as an im
munologically determinant group, a certain aminoacid
sequence, and a process for the preparation of said poly
peptide.
'
cid sequence flanking the centrally positioned arginine
residue indicated A0 ‘are still‘, mirror-images of each
other. Other symbols in this preferred aminoacid se
quence have the meanings given above.
10
(III) According to a particularly preferred embodi
In Swedish Pat. application 73-08917-9 and in US.
Pat. No. 3,960,827 there is described a cancer-associated
ment of the present invention, a tyrosine residue is at
tached to the aminoacid residue A_4, said tyrosine resi
polypeptide antigen (CAPA), the technique for its isola
due, as is clear from the following description, playing
tion and its use in cancer diagnosis and in the prepara
a relatively essential role for the activity of the product.
tion of antibodies. The CAPA is now commercialized
The sequence A_4 through A0 is most important in this
under the designation TPA, which stands for “tissue
embodiment.
polypeptide antigen”. As is clear from the speci?cation
(IV) In the last-mentioned embodiment containing a
of the above-identi?ed patent application and US. pa
tyrosine residue Al and A4 are preferably alanine resi
tent, the isolation of the natural antigen is a complicated
dues, A2, A_; and A_., are leucine residues, and A3 a
procedure which, even if resulting in a practically use 20 glutamic acid residue, A_, being a valine residue and
ful product, still involves high production costs and
A_; an aspartic acid residue.
moreover may involve dif?culties in the provision of
From hereon the following abbreviations for the
necessary starting materials, such as tumor tissue, etc.
Against this background a synthetically prepared anti
aminoacids in question are used:
gen would, of course, be very attractive, in view of the 25
possibility of thereby obtaining a product exactly speci
ALANINE
Ala
?ed as to its composition, said product also being capa
ble of being prepared at a more favorable price.
Referring to the drawing, the FIGURE shows in the
ARGININE
Arg
ASPARAGINE
Asn
hemagglutination inhibition test, comparison between
the natural CAPA and the representative synthetic
antigen of this invention according to Example 1. This
FIGURE will be further explained hereinafter.
ASPARTIC ACID
Asp
GLUTAMINE
GLUTAMIC ACID
GLYCINE
ISOLEUCINE
vLEUCINE
TYROSINE
VALINE
Gln
Glu
GLY
Ile
Leu TI-IREONINE
Tyr PHENYLALANINE
Val LYSINE
Thr
Phe
Lys
The main object of this invention is thus to provide a
synthetic antigenically active polypeptide which reacts
monospeci?cally with antibodies prepared by means of
the polypeptide antigen described in the above-identi
?ed patent application.
Using the above abbreviations, in a particularly pre
ferred embodiment A5 is an alanine residue, and A6 and
A_6are the same or different and selected from residues
of glutamine and asparagine. In this aminoacid se
According to one aspect of the instant invention,
there is thus provided a synthetic antigenically active 40 quence, the following speci?c sequence is particularly
preferred: (V) Gln-Tyr-Leu-Asp-Leu-Val-Arg-Ala
polypeptide characterized by containing as an active
constituent or immunological determinative group the
amino acid sequence: (I) A_3-A_2-A_1-A0-Al-A2-A3,
wherein A0 indicates an arginine residue A1, A2, A_l
Leu-Glu-Ala-Ala-Asn.
Moreover, it is preferred in this sequence at both ends
thereof to connect as A7 a glycine residue and as A_7 an
and A_; are the same or different and are selected from 45 alanine residue, i.e., using the above abbreviations the
aminoacid residues derived from glycine, alanine, val
ine, leucine, lysine and isoleucine. A; and A_; are the
same or different and selected from residues derived
from glutamic acid and aspartic acid.
sequence: (VI) Ala-Gln-Tyr-Leu-Asp-Leu-Val-Arg
Ala-Leu-Glu-Ala-Ala-Asn-Gly.
In the foregoing formulas V and VI Gln may be
replaced equally well by lysine or Asn, or vice versa.
As is clear from the above aminoacid sequence, the
The present invention also relates to a process for the
two fractions ?anking the centrally positioned arginine
preparation of the antigenically active polypeptide, and
in this process an N-protected amino acid is attached to
a resin by esteri?cation, the N-protecting group is re
moved and a second N-protected amino acid is coupled
from neutral aminoacids, namely glycine, alanine, val
ine, leucine and isoleucine. Moreover, the aminoacid 55 to the amino group of the resin-bound amino acid, the
residue indicated A0 are mirror-images of each other, in
that the two aminoacid residues adjacent to A0 originate
residues indicated by A3 and A_;, respectively, are
derived from negative or acidic aminoacids, namely
N-protecting group is removed and the coupling step is
repeated with a third N-protected amino acid. Protect
ing groups in the process are the usual groups, and are
set forth more fully in the following. This procedure is
atory arginine residue is moreover derived from a posi
tive or basic aminoacid, namely arginine, the basicity of 60 repeated until the desired amino acid sequence is ob
glutamic acid and aspartic acid, respectively. The oblig
which, however, is superseded by the combined acidi
ties of the aminoacid residues indicated by A3 and A_;.
Thus, the aminoacid fraction imparting the desired anti
genic activity to the polypeptide will thus, taken as a
tained, the polypeptide being then cleaved from the
quence:
constituting the major part of the copolymer, for in
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 mate
65 rials.
whole, have a negative or acidic character.
The resin used in the synthesis may consist of a co
According to a preferred embodiment of the instant
polymer of styrene and divinyl benzene, the styrene
invention, the polypeptide contains the aminoacid se
A _4-A_ 3'A_ Z'A _1'A0'A1'A2'A3'A4,
4,107,298
3
dleman,
stance about 98% of styrene and about 2% of divinyl
agents are also suitable and will be apparent to one
skilled in this art.
The present invention will now be illustrated further
by means of non-limiting examples.
In order to provide a reactive group for coupling to
the ?rst amino acid, the benzene rings are suitably par
tially 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,
EXAMPLE 1
By the process in solid phase according to Merri?eld
but can be cleaved with HBr in acetic acid or tri?uoro
(cf. the above literature reference) the following prod
acetic acid, the N-protecting group being simulta
neously removed and the peptide separated from the
R25—R24 .
.
.
.
.
Berlin-Heidelberg-New
York, 1970, particularly pp. 308-310. Other coupling
benzene.
R26
4
Springer-Verlag,
uct is prepared:
. R4—R,
Rl
|
Boc-NH-CH-CONH-CH ——————— —— CH-CO- NH=CH—CO—NH-CH—COO—CHZ—@— R
wherein: R indicates the resin part; B00 is a t-butyloxy
resin.
In order to protect the amino acid the t-butyloxycar
carbonyl group;
CH3
CH3
NH
bonyl group can suitably be used as it is conveniently
cleaved by means of HCl in acetic acid. It is also possi
ble to use the carbobenzoxy group for the purpose, but
The amino acid sequence between the t-butyloxycar
in this case HBr in acetic acid must be used for removal 55 bonyl group and the resin part may be expressed in the
of the protecting group. As a protecting group there
following manner using the foregoing abbreviations
may also be used the o-nitrophenyl sulfenyl group.
regarding the amino acids:
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 60
synthesis is dicyclohexylcarbodiimide. If methylene
Ala Leu Leu Asn Asp Glu Leu Ala Gln Tyr Leu Asp
Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Gln
Leu Glu Val
(v11)
chloride is used as a solvent and about 50% excess of
0.4 mmole of Boc-valine (resinbenzyl ester) was
transferred to the cuvette of a Beckman peptide synthe~
diimide, a quantitative reaction is obtained within a few
minutes. Even dimethylformamide may be used as a 65 sizer and swollen in methylene chloride (CHZCIZ). The
protecting Boc-group was removed by treatment with
solvent. As regards further details regarding the synthe
tri?uoroacetic acid in excess in methylene chloride.
sis procedure reference is had to the book “Protein
After washing with methylene chloride the resin was
Sequence Determination” summarized by Saul. B. Nee
t-butyloxy carbonyl aminoacid and dicyclohexyl carbo
4,107,298
5
6
neutralized with triethylamine and again washed with
methylene chloride. N-Boc-glutamic acid benzyl ester
(coupling step 1) and cyclohexylcarbodiimide was
peptide according to the present invention is of decisive
added dissolved in CHZCIZ, and the mixture was stirred
for 30 minutes. The resin was washed and the coupling
tyrosine designated A_5, the polypeptide was treated
step repeated. This terminates the introduction of R2 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.
about 10000 times in relation to the tyrosine content of
importance for the antigenic activity.
For the purpose of investigating the effect of the
with iodine at pH 9.5. When using iodine in an excess of
the polypeptide, about 85% of the activity is lost, at
1000 times excess about 75% of the activity is lost,
whereas at 100 times excess 40-50% of the activity
disappears. At an excess of only about 10 times the
To build up the above amino acid sequence the follow
activity of the polypeptide is, however, not effected.
ing reagents are used in the subsequent steps:
From the character of the basic structure, it is clear
that the polypeptide has an acidic character, thus being
negatively charged in a neutral solution while, how
in step
N-Boc-Leucine
I:
N-Boc-asparagine-xanthydryl derivative
u
N-Boc-alanine
Do as in step 6
ever, 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
Do as in step 1
D20 as in step 2
D20 as in step 6
20 been obtained by means of a synthetically prepared
D20 as in step l
N-Boc-glycine
n
description. The remarkable speci?c activity which has
N-Boc-G-tosylargine
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
N-Boc-valine
Dzo as in step 2
N-Boc-aspartic acid benzyl ester
Dzo as in step 2
N~Boc-tyrosine-3-chlorobenzyl ether
N-Boc-glutamine~xanthydryl derivative
Do
D20
Do
D20
Dzo
Dzo
Do
D20
as
as
as
as
as
as
as
as
in
in
in
in
in
in
in
in
step
step
step
step
step
step
step
step
6
2
1
14
5
2
2
6
application of immunology and testing, i.e. diagnosis,
25 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,
30
however, effect the speci?city 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
The protecting groups are removed from the pro
tected resin-bond peptide and the resin is cleaved off by
arranged on a suitable immunogenically active carrier,
using anhydrous hydrogen ?uoride at 0° C for 30 min 35 for instance a protein, such as albumin, for instance egg
white. Already the polypeptide in its basic structural
utes, and the peptide is extracted from the resin by
means of a 10% aqueous solution of acetic acid. After
form, i.e., having at least 7 amino acid units, does, how
ever, show an antigenic activity which is suf?cient to
evaporation the residue is puri?ed by gel ?ltration on
enable the polypeptide to react with corresponding
Sephadex G 25 Fine in 0.1 M NH4HCO3. After lyophili
zation of the peptide a satisfactory amino acid analysis is 40 antibodies. Moreover, the polypeptide may be used for
obtained, together with a molecular weight of approxi
generating antibodies; however, this calls for coupling
the polypeptide to a suitable protein acting as a carrier
mately 3000, as determined by gel ?ltration. The theo
retical MW value is 2959.71.
or so-called “Schlepperantigene,” for instance swine
The polypeptide prepared according to the foregoing
serum. Thus, the activity is imparted to the polypeptide
was investigated with regard to its ability to inhibit the 45 chain beginning at as low as 7 and especially 9 amino
hemagglutination reaction between tanned red sheep
acid units, appears to be maximized at about 26 amino
blood cells labelled with natural cancer antigen and
acid units, and is still present at in excess of thirty amino
antibodies prepared by using natural cancer antigen
acid units, the portion of the polypeptide chain not
being an immunological determinant group as hereinbe
(CAPA or TPA). With regard to details concerning this
fore de?ned being inert immunologically but serving to
technique reference is had to the above-identi?ed
Swedish patent application 73-08917-9 and US. Pat.
provide a “carrier” for the immunologically determi
No. 3,960,827. The speci?c activity of the polypeptide
nant groups.
The present invention is in no way limited to the
was found to be 0.024 Units per milligram (U/mg). Said
Unit for speci?c activity is de?ned as l/ 6 of the quantity
active polypeptide required to label 109 sheep blood
cells so as to be fully agglutinated by a minimum num
ber or amount of antibodies (maximum dilution of anti
foregoing speci?c embodiments. Thus, in preparing the
55
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 esteri
?cation. The ester bond must be relatively easy to hy
For the purpose of investigating what functions that
drolyse and, of course, must be easier to cleave than the
are critical for the activity of the polypeptide, the basic 60 peptide bonds in the polypeptide. However, the ester
bond must be suf?ciently stable to be able to withstand
structure of which is given above, certain experiments
were carried out. The centrally positioned arginine
the reaction conditions under the synthesis.
indicated by A0 was thus blocked with cyclohexanedi
As an alternative to the above mentioned coupling
one at pH 13 resulting in 99.5% loss of activity. If,
reagent, dicyclohexyl carbodiimide, when attaching
however, the polypeptide is subjected only to a basic 65 glutamine and asparagine units, coupling may instead be
carried out with so-called active esters, for instance
environment, wherein the pH is 13 for the same period
cyanomethyl, thiophenyl or nitrophenyl esters. As a
of time, the activity is lowered only by about 20%. This
indicates the fact that the arginine presence in the poly
solvent in the synthesis so-called aprotic solvents are
bodies).
7
4,107,298
suitable, particularly solvents that are somewhat hydrophilic or “semipolar.”
8
activity of the synthesized polypeptide is found to be
0.024 Units per milligram of peptide.
Regarding useful groups to protect the N-amino
EXAMPLE 2
acids, it is noted that t-butyloxy carbonyl groups,
wherein one or two methyl groups are replaced by 5
In the same manner as given in Example 1 the follow
phenyl, also may be advantageously used. In this con-
ing polypeptide is prepared and found to be about
nection it can be mentioned that the product obtained in
equally immunologically active as the polypeptide of
the synthesis is provided with N-protecting groups and
Example 1:
attached to a resin by esteri?cation, and that it advanta
geously can be stored for a long period of time without 10
Th
AS
Leu
AS
A
A
being destroyed. In connection with the use _of the poly-
A‘;
Ly‘;
Tyr
L63
A25
L;§
pept1de, the protectmg groups and the resin part may
then be removed.
In the amino acid sequences given in the present
Arg
Leu
Glu
Ala
Ala
Ala
Gly
Glu
Leu
Glu
Val
L
Asp
preparation, the terminal groups (after removal of the 15
protecting groups and the resin part) are always amino
EXAMPLE 3
group and carboxyl group, respectively. The amino
In the same manner as given in Example 1 the follow
group is found at the end of the chain wherein the syming polypeptide is prepared and found to be about
bols A are designated minus, whereas the carboxyl
equally immunologically active as the polypeptide of
group is found at the opposite end of the amino acid 20 Example 1:
sequence.
the antigenic- activ. In Example 1 as. described above
.
Ala
Gln
Tyr
Leu
Asp
Leu
Val
Arg
Ala
ity of the synthesized polypeptlde was determlned 1n
Glu
Ala
Ala
Asn
Gly
Glu
Leu
Glu
val
Leu
the following manner.
For quantitative determination of the amount of poly- 25
When this determinant group is coupled to inert bo
peptide 7 mgs of the peptide are dissolved in 1.4 mls of
vine albumin in a molar ratio of about 1:1 by means of a
buffer grade serum, ie a physiological saline solution
water soluble carbodiimide (EDC) the immunological
containing 2% of inert human serum and buffered to a
activity of the peptide is maintained.
pH of about 7.5 with a phosphate buffer. Under serial
dilution a series of samples of said solution having a 30
EXAMPLE 4
decreasing polypeptide concentration is prepared and
In the same manner as given in Example 1 the follow
to each of said samples there is added a pre-determined
amount of anti-serum containing antibodies speci?c to
TPA. To each of the resulting samples there is then
added, after incubation, a predetermined amount of the
Leu
Arg
ing polypeptide is prepared and found to be about
equally immunologically active as the polypeptide of
Example 1:
Asn
Ala
Asp
Leu
Glu
Glu
Leu
Ala
Ala
Ala
Gln
Asn
Tyr
Gly
Leu
Arg
Asp
Leu
Leu
Glu
Val
Val
When coupled to inert albumin in the same way as
.
.
. .
.
.
descrlbed
in
Example 3, the actlvlty
was maintained.
p 01 yp e p tide carried on a p articulate carrier, hemagg lutination taking place to an extent corresponding to the
amount of available antibodies. In parallel there is pre-
EXAMPLE 5
pared a corresponding series of control samples con
In the same manner as given in Example 1 the follow
taining known decreasing amounts of TPA. The diame 45 ing polypeptide is prepared and found to be about
ters of the hemagglutination depositions of the control
equally immunologically active as the polypeptide of
samples are then measured in their respective cavities
Example 1:
on a serial dilution test plate, and the values of the diam
Asn
Arg
Asp
Ala
Ile
Leu
Leu
Glu
Ala
Ala
Gln
Ala
Tyr
Asn
Leu
Gly
Asp
Leu
Val
eters obtained are plotted against the TPA concentraEXAMPLE 6
tions, an S-shaped curve being obtained, the intermedi
In the same manner as given in Example 1 the follow
ate part of which including an in?exion point as a steep 55 ing polypeptide is prepared and found to be about
inclination. The corresponding curve of the diameter of
equally immunologically active as the polypeptide of
the depositions from the hemagglutination as a function
Example 1:
of the TPA-concentrations is shown in the diagram of
Asp
Arg
Asp
Ala
Arg
Leu
Leu
Glu
Ala
Ala
Lys
Ala
Tyr
Asp
Leu
Gly
Asp
Lys
Val
EXAMPLES 7-22.
the appended drawing, as curve B.
65
In the same manner as given in Example 1 the follow
In the same diagram the corresponding curve for the
ing polypeptides are prepared and found to be about
synthesized polypeptide is shown as A, and with knowledge of the speci?c activity of the TPA the speci?c
equally immunologically active as the polypeptide of
Example 1.
4,107,298
9
Ex. 7
Ex. 8
Ex. 9
Ex.l0
Ex.ll
Ex.l2
Ex.l3
Ex.l4
Ex.l5
Ex,l6
Ex.l7
Ex.l3
Ex.l9
Ex.20
Ex.2l
Ex.22
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Leu Asn
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
10
Arg Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Glu Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Arg Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Glu Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Arg Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Glu Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Arg Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Glu Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Glu Leu Glu Val
Arg Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Glu Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Arg Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Glu Leu Ala Gln Tyr Leu Asp Lys Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Arg Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Glu Leu Ala Lys Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Arg Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
Glu Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu Ala Ala Asn Gly Arg Leu Glu Val
residues of: glycine, alanine, valine, leucine, and isoleu
EXAMPLE 23
In the same manner as given in Example 1 the follow
cine and A3 and A_; are same or different and selected
from glutamic acid and aspartic acid.
ing polypeptide is prepared and found to be about
3. Synthetic antigenically active polypeptide, charac
equally immunologically active as the polypeptide of
terized by containing an immunologically inert portion
Example 1
20 and as an active immunological determinant group the
Thr
Val
Asp
Arg
Phe
Leu
Ala
Asp
Asp
Asp
Arg
Leu
Ala
Lys
Tyr
Leu
Asp
Lys
Leu
Glu
Glu
Leu
Ala
Ala
Asp
Gly
Glu
Leu
Glu
Val
Asn
Leu
Gln
amino acid sequence: A_4-A_3-A,2-A_l-AO-Al-A2-A3
A4, wherein A0 is an arginine residue, Al, A2, A_, and
EXAMPLE 24
In the same manner as given in Example 1 the follow
A“ 2 are same or different and selected from residues of:
ing polypeptide is prepared and found to be about
glycine, alanine, valine, leucine, lysine, and isoleucine
equally immunologically active as the polypeptide of 30 and A3 and A_; are same or different and selected from
Example 1
glutamic acid and aspartic acid, and A4 and A_,, are
Ala
Arg
Glu
Leu
Ala
Leu
Leu
Leu
Asn
Asn
Glu
Leu
Asp
Ala
Gln
Glu
Ala
Leu
Asn
Ala
Gly
Gln
Arg
Tyr
Leu
Leu
Glu
Asp
Val
Leu
Phe
Val
Asp
same or different and selected from g1 ycine ’ alanine ’
EXAMPLE 25
In the same manner as given in Example 1 the follow
ing polypeptide is prepared and found to be about
equally immunologically active as the polypeptide of
Example 1
valine, leucine, and isoleucine.
4. Synthetic antigenically active polypeptide, charac
terized by containing an immunologically inert portion
and as an active immunological determinant group the
amino acid sequence: A_5-A_4-A_3-A_2-A_FAQ-A,
Ala
Leu
Leu
Asn
Asp
Ile
Leu
Ala
Gln
Tyr
Arg
Ala
Leu
Glu
Ala
Ala
Asn
Gly
Arg
Leu
Leu
Glu
Asp
Leu
Val
Val
Various modifications and substitutions of equiva
lents may be made in the products, process, conditions,
and procedures of the present invention and will be 50 AZ-A3-A4, wherein A_5 is a tyrosine residue and
immediately apparent to one skilled in the art, where
wherein A0 is an arginine residue, A], A2, A_, and A_2
fore the invention is to be limited only by the full scope
are same or different and selected from residues of:
of the appended claims, including application of the
doctrine of equivalents thereto.
glycine, alanine, valine, leucine, lysine, and isoleucine
and A3 and A_, are same or different and selected from
55 glutamic acid and aspartic acid, and wherein A4 and
1. Synthetic antigenically active polypeptide, charac
A_., are same or different and selected from glycine,
What is claimed is:
terized by containing an immunologically inert portion
and as an active immunological determinant group the
alanine, valine, leucine and isoleucine.
5. A polypeptide according to claim 4, characterized
in that Al and A4 are alanine residues, A2, A_; and A_4
amino acid sequence: A_3-A_2-A_1-A0-Al-A2-A3,
wherein A0 is an arginine residue, A1, A2, A_, and A_; 60 are leucine residues and A3 is a glutamic acid residue,
are same or different and selected from residues of:
glycine, alanine, valine, leucine, lysine, and isoleucine
A_, is a valine residue and A_; is an aspartic acid resi
due.
and A3 and A_;, are same or different and selected from
glutamic acid and aspartic acid.
'
6. Synthetic antigenically active polypeptide, charac
terized by containing an immunologically inert portion
2. A polypeptide according to claim 1, characterized 65 and as an active immunological determinant group the
in that the amino acid sequence is: A_3-A_2-A_1-A0
amino acid sequence: A_b-A_5-A_,,-Ag_,-A_Z-A_l-Ao
ArAz-As, wherein A0 is an arginine residue, A‘, A2,
A_, and A_2 are same or different and selected from
A,-A2-A3~A4-A5-A6, characterized in that Al and A4 and
A5 are alanine residues, A2, A_2 and A_,, are leucine
11
4,107,298
12
10. Synthetic antigenically active polypeptide, char
residues and A3is a glutamic acid residue, A_ 1 is a valine
residue and A_; is an aspartic acid residue, A0 is an
acterized by containing an immunologically inert por
arginine residue, A_5 is a tyrosine residue, and A6 and
tion and as an active immunological determinant group
the amino acid sequence: Ala Leu Leu Asn Asp Glu
5 Leu Ala Gln Tyr Leu Asp Leu Val Arg Ala Leu Glu
glutamine, asparagine, and lysine.
Ala Ala Asn Gly Glu Leu Glu Val.
7. A polypeptide according to claim 6, wherein A6
A_6 are same or different and selected from residues of
11. Synthetic antigenically active polypeptide, char
and A_6 are same or different and selected from residues
acterized by containing an immunologically inert por
of glutamine and asparagine.
tion and as an active immunological determinant group
8. A polypeptide according to claim 6, characterized
in that A6 is an asparagine residue and A ,,a glutamine
residue.
the amino acid sequence: Thr Asp Leu Asp Asp Arg
Leu Ala Lys Tyr Leu Asp Lys Val Arg Ala Leu Glu
Ala Ala Asp Gly Glu Leu Glu Val.
9. Synthetic antigenically active polypeptide, charac
terized by containing an immunologically inert portion
12. Synthetic antigenically active polypeptide, char
and as an active immunological determinant group the
acterized by containing an immunologically inert por
amino
acid
sequence:
A_7-A_(,-A_5
'A_4'A _ 3'A_ 2"A _1‘Ag-A]-A2"A3-A4'A5-A6'A7, Char
acterized in that Al and A4 and A5 are alanine residues,
A2, A_2 and A_4 are leucine residues and A3 is a glu
tamic acid residue, A_l is a valine residue and A_, is an
aspartic acid residue, A0 is an arginine residue, A_5 is a
tyrosine residue, A6 is an asparagine residue and A_,, a
tion and as an active immunological determinant group
the amino acid sequence: Leu Asn Asp A_9 Leu Ala
A_6 Tyr Leu Asp A_Z Val Arg Ala Leu Glu Ala Ala
Asn Gly A8 Leu Glu Val, wherein A_,, and A8 are same
or different and selected from residues of glutamic acid
and arginine; A_(,is selected from residues of glutamine
and lysine; and A_2 is selected from residues of leucine
and lysine.
glutamine residue, and wherein A7 is a glycine residue
i
and A_7 an alanine residue.
25
35
40
45
55
60
65
II‘
II‘
*
*
UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION
PATENT N0.
DATED
:
:
4,107,298
August 15, 1978
INVENTOR(S) : Liining
It is certified that error appears in the above-identified patent and that said Letters Patent
are hereby corrected as shown below:
\
Col. 5, line 21, Step 11 in the chart; "tosylargine" should read
—-tosylarginineCol. 11, line 10; "A 6"
(2nd occurrence) should read --A_6--.
Signal and Scaled this
Sixth Day Of February 1979
lsmu
A ttest:
RUTH a MASON
Arresting Oj?cer
DONALD W. BANNER
Commissioner of Patents and Trademarks