TETRAPEPTIDES AND METHODS

TETRAPEPTIDES ET METHODE DE PREPARATION

English Abstract

ORTH 333
ABSTRACT
NEW TETRAPEPTIDES AND METHODS
There are disclosed new biologically active
polypeptides containing the following polypeptide seg-
ment:
-ALA-LYS-SER-GLN-.
Biological activity is generally retained upon substi-
tution of a natural or non-natural amino acid residue
for either or both of L-alanyl in the first position
and L-seryl in the third position.
These polypeptides have the capability of in-
ducing the differentiation of T-lymphocytes as measured
by the acquisition of the thymic differentiation antigen
Th-1, as well as B-lymphocytes as measured by the acquisi-
tion of the differentiation antigen Bu-1. The polypeptides
are thus useful in thymic function and immunity areas such
as in treatment for congenital absence of thymus. Also
provided are substituted polypeptides, methods of manu-
facture of the polypeptides, therapeutic compositions,
and methods for use of the polypeptides.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for manufacture of the polypeptide
of sequence H-X-LYS-Y-GLN-OH, wherein X and Y are each
selected from the group consisting of L-seryl, L-alanyl,
L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl,
L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl,
D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-
alanyl, which comprises esterifying L-glutamine protected
on its amino group, to an insoluble resin polymer by co-
valent bonding; removing the .alpha.-amino protecting group from
the L-glutamine moiety, reacting with a Y amino acid pro-
tected on its .alpha.-amino group to couple the Y amino acid to
the L-glutamine-resin; removing the .alpha.-amino protecting
group from the Y amino acid moiety, reacting with an .alpha.-amino
protected L-lysine to couple L-lysine to the Y-amino acid-
L-glutamine-resin; removing the .alpha.-amino protecting group
from the L-lysine moiety, reacting with an X amino acid
protected on its .alpha.-amino group to couple the X amino acid to
the L-lysine-Y-amino acid L-glutamine-resin, and removing all
protecting groups and the resin from the peptide.
2. A method according to Claim 1, wherein
reactive side chains on the reacting amino acids are protected
during the reaction.
3. A method according to Claim 2, wherein
the resin polymer is selected from the group consisting
of cellulose, polyvinylalcohol, polymethacrylate, sul-
fonated polystyrene, and a chloromethylated copolymer
32

of styrene and divinylbenzene.
4. A method for manufacture of the polypeptide
of sequence H-X-LYS-Y-GLN-NH2, wherein X and Y are each
natural and non-natural amino acid residues selected from the
group consisting of L-seryl, L-alanyl, L-asparagyl, L-glutamyl,
L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl,
D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl,
sarcosyl, and 2-methylalanyl, which comprises esterifying L-
glutamine protected on its amino group, to an insoluble
33

ORTH 333
resin polymer by covalent bonding; removing the .alpha.-amino
protecting group from the L-glutamine moiety, reacting
with a Y amino acid protected on its .alpha.-amino group to
couple the Y amino acid to the L-glutamine-resin; re-
moving the .alpha.-amino protecting group from the Y amino acid
moiety, reacting with an .alpha.-amino protected L-lysine to
couple L-lysine to the Y-amino acid-L-glutamine-resin;
removing the .alpha.-amino protecting group from the L-lysine
moiety, reacting with an X amino acid protected on its
.alpha.-amino group to couple the X amino acid to the L-lysine-
Y-amino acid L-glutamine-resin, cleaving the resin from
the peptide with ammonia in dimethylformamide under
amidating conditions, and removing all protecting groups.
5. A method according to Claim 4, wherein
reactive side chains on the reacting amino acids are
protected during the reaction.
6. A method according to Claim 5, wherein
the resin polymer is selected from the group consisting
of cellulose, polyvinylalcohol, polymethacrylate,
sulfonated polystyrene, and a chloromethylated co-
polymer of styrene and divinylbenzene.
7. A method for manu-
facture of the polypeptide of sequence H-SAR-LYS-SAR-
GLN-NH2, which comprises esterifying L-glutamine pro-
tected on its amino group, to an insoluble resin polymer
by covalent bonding; removing the .alpha.-amino protecting
group from the L-glutamine moiety, reacting with an
.alpha.-amino protected sarcosine to couple sarcosine to the
L-glutamine-resin; removing the .alpha.-amino protecting
group from the sarcosine moiety, reacting with an .alpha.-
amino protected, .epsilon.-amino-protected L-lysine to couple
protected L-lysine to the sarcosine-L-glutamine-resin;
removing the .alpha.-amino protecting group from the L-lysine
moiety, reacting with an .alpha.-amino protected sarcosine
to couple sarcosine to the .epsilon.-amino-protected L-lysine-
sarcosine-L-glutamine-resin, cleaving the resin from the
peptide with ammonia in dimethylformamide under amidating
conditions, and removing all protecting groups.
34

ORTH 333
8. A method for manufacture of the poly-
peptide of sequence H-X-LYS-Y-GLN-NH2, wherein X and Y are each
natural and non-natural amino acid residues selected from the
group consisting of L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl,
glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-
glutamyl, D-threonyl, D-balyl, D-leucyl, sarcosyl, and
2-methylalanyl, which comprises amidating L-glutamine
protected on its amino group, to a benzhydrylamine
resin polymer by covalent bonding; removing the .alpha.-amino
protecting group from the L-glutamine moiety, reacting
with a Y amino acid protected on its .alpha.-amino group to
couple the Y amino acid to the L-glutamine-resin; re-
moving the .alpha.-amino protecting group from the Y amino acid
moiety, reacting with an a-amino protected L-lysine to
couple L-lysine to the Y-amino acid-L-glutamine-resin;
removing the .alpha.-amino protecting group from the L-lysine
moiety, reacting with an X amino acid protected on its
.alpha.-amino group to couple the X amino acid to the L-lysine-
Y-amino acid L-glutamine-resin, and removing all pro-
tecting groups and the resin from the peptide.
9. A method according to Claim 8, wherein
reactive side chains on the reacting amino acids are
protected during the reaction.
10. A method for manu-
facture of the polypeptide of sequence H-SAR-LYS-SAR-GLN-
NH2, which comprises amidating L-glutamine protected on
its amino group, to a benzhydrylamine resin polymer by
covalent bonding; removing the .alpha.-amino protecting group
from the L-glutamine moiety, reacting with an .alpha.-amino
protected sarcosine to couple sarcosine to the L-gluta-
mine-resin; removing the .alpha.-amino protecting group from
the sarcosine moiety, reacting with an .alpha.-amino pro-
tectad .epsilon.-amino-protected L-lysine to couple protected
L-lysine to the sarcosine-L-glutamine-resin; removing
the .alpha.-amino protecting group from the L-lysine moiety,
reacting with an .alpha.-amino protected sarcosine to couple

ORTH 333
sarcosine to the .epsilon.-amino-protected L-lysine-sarcosine-
L-glutamine-resin, and removing all protecting groups
and the resin from the peptide.
11. A method for manu-
facture of the polypeptide of sequence H-SAR-LYS-SAR-
GLN-OH, which comprises esterifying L-glutamine pro-
tected on its amino group, to an insoluble resin polymer
by covalent bonding; removing the .alpha.-amino protecting
group from the L-glutamine moiety, reacting with an
.alpha.-amino protected sarcosine to couple sarcosine to the
L-glutamine-resin; removing the a-amino protecting
group from the sarcosine moiety, reacting with an .alpha.-
amino protested, f-amino-protected L-lysine to couple
protected L-lysine to the sarcosine-L-glutamine-resin;
removing the a-amino protecting group from the L-lysine
moiety, reacting with an a-amino protected sarcosine to
couple sarcosine to the .epsilon.-amino-protected L-lysine-
sarcosine-L-glutamine-resin, and removing all protecting
groups and the resin from the peptide.
12. A method for manufacture of the polypeptide
of sequence R-X-LYS-Y-GLN-R' wherein X and Y are each natural
and non-natural amino acid residues selected from the group con-
sisting of L-seryl, L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl,
L-valyl, L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl,
D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-
alanyl and R and R' are each selected from the group
consisting of:
<IMG>
36

ORTH 333
wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl,
C6-C20 aryl, C6-C20 aralkyl, and C6-C20 alkaryl, which
comprises esterifying L-glutamine protected on its
amino group, to an insoluble resin polymer by covalent
bonding; removing the .alpha.-amino protecting group from the
L-glutamine moiety, reacting with a Y amino acid protected
on its .alpha.-amino group to couple the Y amino acid to the
L-glutamine-resin, removing the .alpha.-amino protecting group
from the Y amino acid moiety, reacting with an .alpha.- amino
protected L-lysine to couple L-lysine to the Y-amino
acid-L-glutamine-resin; removing the .alpha.-amino protecting
group from the L-lysine moiety, reacting with an N-R
substituted X amino acid protected on its .alpha.-amino group
to couple the N-R substituted X amino acid to the L-
lysine-Y-amino acid L-glutamine-resin, cleaving the
resin from the peptide with an acid (R'=OH), ammonia
(R'=NH2), a primary amine of formula NH2R7 (R'=NHR7)
a secondary amine of formula NH(R7)2 [R'=N(R7)2], or an
alcohol of formula HOR7 (R'=OR7), and removing all protecting groups.
13. A method according to Claim 12, wherein
reactive side chains on the reacting amino acids are
protected during the reaction.
14. A method according to Claim 12, wherein
the resin polymer is selected from the group consisting
of cellulose, polyvinylalcohol, polymethacrylate,
sulfonated polystyrene, and a chloromethylated co-
polymer of styrene and divinylbenzene.
37

ORTH 333
15, A polypeptide having the capability of in-
ducing the differentiation of both Th-1+ T-lymphocytes
and Bu-1+ B-lymphocytes, said polypeptide having the
following sequence:
R-X-LYS-Y-GLN-R'
wherein X and Y are each natural and non-natural amino
acid residues selected from the group consisting of
L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl,
L-leucyl, L-alanyl, L-seryl, sarcosyl, 2-methylalanyl,
D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl,
D-alanyl, and D-seryl and R and Rr are each selected from
the groups consisting of:
<IMG>
wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl,
C6-C20 aryl, C6-C20 aralkyl, and C6-C20 alkaryl, provided
that when R is GLN, R' is other than GLY-GLY-SER-ASN, and
the pharmaceutically acceptable salts thereof; further pro-
vided that said polypeptide induce the differentiation of
both Th-1+ T-lymphocytes and Bu-1+ B-lymphocytes in the
chicken induction assay at a concentration of about one
ng/ml or less whenever prepared or produced by the method of
claim 12 or by any obvious chemical equivalent thereof.
38

16. A polypeptide of the following sequence:
H-SAR-LYS-SAR-GLN-NH2
and the pharmaceutically acceptable salts thereof whenever
prepared or produced by the method of claim 10 or by any
obvious chemical equivalent thereof.
17. A polypeptide of the following sequence:
H-X-LYS-Y-GLN-OH
wherein X and Y are each natural and non-natural amino acid
residues selected from the group consisting of L-seryl, L-alanyl,
L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl,
L-leucyl, D-alanyl, D-seryl, D-asparagyl, D-glutamyl,
D-threonyl, D-valyl, D-leucyl, sarcosyl, and 2-methyl-
alanyl and the pharmaceutically acceptable salts thereof
whenever prepared or produced by the method of claim 1 or by
any obvious chemical equivalent thereof.
18. A polypeptide of the following sequence:
H-X-LYS-Y-GLN-NH2
wherein X and Y are each natural and non-natural amino acid
residues selected from the group consisting of L-seryl, L-alanyl,
L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl, L-leucyl,
D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl, D-valyl,
D-leucyl, sarcosyl and 2-methylalanyl and the pharmaceutically
acceptable salts thereof whenever prepared or produced by the
method of claim 4 or by any obvious chemical equivalent thereof.
39

19. A polypeptide of the following sequence:
H-SAR-LYS-SAR-GLN-NH2
and the pharmaceutically acceptable salts thereof whenever pre-
pared or produced by the method of claim 7 or by any obvious
chemical equivalent thereof.
20. A polypeptide of the following sequence
H-X-LYS-Y-GLN-NH2
wherein X and Y are each natural and non-natural amino acid
residues selected from the group consisting of L-seryl,
L-alanyl, L-asparagyl, L-glutamyl, L-threonyl, glycyl, L-valyl,
L-leucyl r D-alanyl, D-seryl, D-asparagyl, D-glutamyl, D-threonyl,
D-valyl, D-leucyl, Sarcosyl, and 2-methylalanyl and the pharmaceuti-
cally acceptable salts thereof whenever prepared or produced by
the method of claim 8 or by any obvious chemical equivalent thereof.
21. A polypeptide of the following sequence:
H-SAR-LYS-SAR-GLN-OH
and the pharmaceutically acceptable salts thereof whenever
prepared or produced by the method of claim 11 or by any
obvious chemical equivalent thereof.

Description

3~
Field of the Invention
This invention relates generally to new polypeptide
segments and polypeptides, to methods for the preparatlon
thereof, and uses thereof.
Description of the Prior Art
It is well-known that many polypeptides have been
isolated from various tissues and organs (including the blood)
of animals. Many of these polypeptides are related to immune
function in the body, as, for example, the various immune glob-
ulins, the thymic hormone thymopoietin, and the like. Indeed,
Applicant has isolated and synthesized several of these poly-
peptides, as described in United States Patents Nos. 4,002,602
and 4,002,740 as well as in several scientific articles.
Until about the past decade, little was known about
the thymus, although it is now understood that the thymus is
one of the organs principally responsible for immune functions
in mammals and birds. Despite keen interest in possible
functions o the thymus and early speculation and experimenta-
tion, little was known of the function of the thymus until20
recently. It is now realized, however, that the thymus is a
compound organ with both epithelial (endocrine) and lymphoid
(immunological) components and thus the thymus is involved
in the immunity functions of the body. The thymus consists
of an epithelial stroma derived from the third branchial arch
and lymphocytes derived from stem cells originating in
- ', ! ~

oR~rH 333
)03~
,
haemopoietic tissues, Goldstein, et al., The Human Th~nus,
Heinemann, London, 1969. Lymphocytes are differentiated
within the thymus and leave as mature thymus-derived
cells, called T cells, which circulate to the blood,
lymph, spleen and lymph nodes. The induction of stem
cell differentiation within the thymus appears to be
mediated by secretions of the epithelial cells of the
thymus.
It has been known for some time that the thymus
is connected with the immunity characteristics of the body
and, therefore, great interest has been indicated in sub-
stances which have baen isolated from the thymus. In
this regard, there have been published in recent years a
relatively large body of articles based on scientific
work relating to materials which are present in bovine
thymus. In fact, the Applicant has published a number
of articles which relate to research in this area. Per-
tinent publications may be found, for example, in
The Lancet, July 20, 1968, pp. 119-122; Triangle, Vol. II,
No. 1, pp. 7-14, 1972; Annals of the New York Academy of
Sciences, Vol. 183, pp. 230-240, 1971; and Clinical and
Experimental Immunolog~, Vol. 4, No. 2, pp. 181-189,
1969; Nature, Vol. 247, pp. 11-14, 1974; Proceedings of
the National Academy of Sciences USA, Vol. 7], pp. 1474-
1478, 1974; Cell, Vol~ 5, pp. 361-365 and 367-370, 1975;
Lancet, Vol. 2, pp. 256-259, 1975; Proceedings of the
National Academy of Sciences USA, Vol. 72, pp. 11-15,
1975; Biochemistry, Vol. 14, pp. 2214-2218, 1974;
Nature, Vol. 255, pp. 473-424, 1975.
A second class of lymphocytes having immune
function are the B lymphocytes or B cells. These are
differentiated in the Bursa of Fabricius in birds and
by an as-yet-unidentified organ in mammals. T-cells and
B-cells cooperate in many aspects of immunity. See, for
example, articles by the Ap~licant in Science, 193, 319
(July 23, 197~) and Cold Spring Harbor Symposia on
Quantitative Biology, Vol. XLI, 5 (1977).
- : , .
.
.

` ~RTH 337
3~
A nonapeptide material has recently been iso-
lated from porcine serum by J. F. Bach, e~ al. and iden-
tified as "facteur thymique serique" (FTS). The isola-
tion of this material and its structure are disclosed
in C. R. Acad. Sc. Paris, t. 283 (November 29, 19~6),
Series D-1605 and Nature 266, 55 (March 3, 1977). The
structure of this nonapeptide has been identified as
GL~-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN, where "GLX" repre-
sents either glutamine or pyroglutamic acid. The material
where GLX is glutamine or pyroglutamic acid has been syn-
thesized. In these articles, Bach disclosed that his
nonapeptide FTS selectively differentiated T cells (and
not B cells) by use of an E rosette assay. Bach, there-
fore, concluded that his material was a thymic hormone.
Recently, a more thorough investigation of the activity
of this nonapeptide by the prasent Applicant disclosed
that FTS differentiated both T cells and B cells and was,
therefore, more like ubiquitin in its activity than thymo-
poeitin. Brand, Gilmour and Goldstein, Nature,
20 269:597 (1977).
It has now'been discovered that a synthesized
4-amino acid polypeptida segment of this FTS nonapeptide
possessas many of the characteristics of the nonapeptide
discussed in the above publications.
Summary of the Invention
It is accordingly one objec~ of this invention
to provide new polypeptide segments and polypeptides which
are important biologically.
A further object of the invention is to provide
new polypeptide segments and polypeptides which have the
ability to induce differentiation of both T-lymphocytes
as well as B-lymphocytes and are, therefore, highly use-
ful in the immune systems of humans and animals.
A further object of the invention is to provide
methods for synthesizing the novel polypeptide segments
and polypeptides of this invention, as well as composi-
tions and methods for use in biological actions.
Gther objects and advantages of the invention
--' will become apparent from an examination of the presen-t
disclosure.
:, . ,
.
,.

3~
-3a-
There is thus provided, in accordance ~ith the
present teachings, a method for manufacture of the polypeptide
of se~uence R-X-LYS-Y-GLN-R' wherein X and Y are each natural
and non-natural amino acid residues selected from the group
consisting of L-seryl, L-alanyl, L-asparagyl, L-glutamyl
L-threonyl, glycyl, L-valyl, L-leucyl, D-alanyl, D-seryl,
D-asparagyl, D-glutamyl, D-threonyl, D-valyl, D-leucyl, sarco~yl,
and 2-methyl-alanyl, and R and R' are each selected from the
group consisting of:
_ R
Hydrogen OH
Cl-C7 alkyl NH2
6 12 aryl NHR7
C6-C20 alkaryl ( 7)2
C6-C20 aralkyl OR7
Cl-C7 alkanoyl
C2-~7 alkenyl ~LY
C2-C7 alkynyl GLY-GLY
GLN
SAR GLY-GLY-SER-ASN
wherein R7 is Cl-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl,
C6-C20 aryl, C6-C20 aralkyl, and C6-C20 alkaryl- The method
eomprises esterifying L~glutamine proteeted on its amino group,
to an insoluble resin polymer by eovalent bonding; removing
the ~-amino proteeting group from the L-glutamine moiety, reaeting
with a Y amino acid proteeted on its ~-amino group to eouple the
Y amino aeid to the L-glutamine-resin; removing the a-amino
proteeting group from the Y-amino aeid moiety, reaeting with
an ~-amino proteeted L-lysine to eouple L-lysine to the Y-amino
aeid L-glutamine-resin; removing the ~-amino protecting group
from the L-lysine moiety, reaeting with an N-R substituted X
amino acid protected on its ~-amino group to couple the N-R
substituted X amino aeid to the L-lysine-Y-amino aeid L-glutamine-
resin, eleaving the resin from the peptide with an aeid (R'=OH),
ammonia (R'=NH2), a primary amine of formula NH2R7 (R'=NHR7) a
seeondary amine of formula NH(R7~21R'=N(R7)2], or an aleohol of the
formula ~OR7(R'=OR7) and removing all proteeting groups.

i:)P~TH ~ 3 3
03~L
In satisfaction of the foregoing objects and
advanta~es, there is provided by this invention the novel
biologically active polypeptide segment having the follow-
ing amino acid sequence:
-ALA-LYS-SER-GLN-.
The biological activity of the subject polypeptide segment
is generally retained upon substitution of a natural or
non-natural amino acid residue for either or both of:
1) L-alanyl in the first position; and 2) L-seryl in the
third position. Certain of these substituted polypeptide
segments are strikingly potent. Terminal substitution of
the subject polypeptide segments yields the subject poly-
peptides.
Also provided is a procedure for preparation of
the polypeptide segments and polypeptides of the invention
by solid phase peptide synthesis, as well as thera~eutic
compositions containing the polypeptides, and methods for
administration thereof to humans and animals for effecting
biological actions thereon.
Description of Preerred Embodiments
As indicated above, this invention is concerned
with new polypeptide segments and polypeptides having
therapeutic value in various areas, therapeutic composi-
tions and method for their use utilizing the polypeptides
of this invention, and methods for manufacture thereofO
In the principal embodiment of the present in-
vention, there is provided a biologically active poly-
peptide segment which has the following amino acid sequence:
~ L~-LYS-SER-GLN-.
Since the biological activity of the subject polypeptide
segment is generally retained upon substitution of a
natural or non-natural amino acid residue for either or
both of: 1) alaninyl in the first position; and 2) serinyl
in the third position, the principal embodiment of the
present invention further includes biologically active
polypeptide segments which have the following amino acid
- sequence:
I~. -X-LYS-Y-GLN-
;.: : : .
:
,: ..
::
- : :

~ 3~ ~RTH 333
_ 5
wherein, X and Y are each selected from the group con-
sisting of natural and non-natural alpha-amino carboxylic
acid (hereafter "amlno acid") residues. While it is be-
lieved that the large majority of such substitutions of
X and Y groups will allow retention of biological activity,
it is possible that certain natural or non-natural amino
acid residues will interfere with the folding of the
molecule (as discussed more fully below) and thus sub-
stantially eliminate the biological activity. Such
activity-destroying substituents are specifically ex-
cluded from the scope of the present invention.
The subs-tituents X and Y are preferably selected
from such natural amino acid residues as L-asparagyl,
L-glutamyl, L-threonyl, glycyl, L-valyl, L leucyl, L-alanyl,
L,seryl, and the like; and from such non-naturai ~r.o acid residues
as sarcosyl, 2-methylalanyl, the D-forms of the L-amino
acids listed above, and the like. ~hether a particular
substitution allows retention of the biological activity
of the polypeptide may be readily established by testing
it for differentiation of Th-1+ T-lymphocytes and Bu-l+
B-lymphocytes in the chicken induction assay described
below. Compounds which are specifically active in nano-
gram (ng)/milliliter (ml) concentrations (about one ng/ml
or less) in this assay are considered to be biologically
active.
A list of natural amino acids may be found in
many reference books, e.g., ~. T. Morrison and R. N. 30yd,
"Organic Chemistry", Allyn and Bacon, 1959, Chapter 33.
In addition to the natural amino acids (which are those
found in proteins~, there are also a number of so-called
"non natural" amino acids which are not found in proteins
although they sometimes occur naturally as metabolic inter-
mediates or the like. Thesa non-natural amino acids may
be the D-isomers corresponding to the optically active
CL-form) natural amino acids or they may be entirely
different chemical entities such as sarcosine (N-methyl
glycine) or 2-methylalanine men~ioned above. Lists of such
non-natural a~ino acids are also found in many reference
works.
.

~0~31 ORTH 333
The polypeptide segment indicated in the prin-
cipal embodiment above as Formula IA must additionally
contain terminal substituents on the 4-amino acid sequence,
thus yielding the subject polypeptides. These terminal
substituents must not substantially affect the biological
activity of the active 4-amino acid segment, as measured
by the ability to induce the differentiation of Th-l+
T-lymphocytes and Bu-l+ B-lymphocytes in the chicken in-
duction assay described below. The subject polypeptides0 may be described by the following general formula:
II. R-X-LYS-Y-GLN-R~
wherein X and Y are as previously described and R and R'
are substituents on the terminal amino group and terminal
caxboxyl yroup, respectively, of the peptide segment which,
as described above, do not substantially affect the bio-
logical activity of the active amino acid segment. Since
the active tetrapeptide segment is contained within a
longer sequence in the naturally-occurring material iso-
lated by Bach, it should be understood that the terminal
amino and carboxylic acid groups are not essential to
the biological activity o the tetrapeptide segment, as
is the case for some polypeptides. It is, therefore,
considered that the scope of the present invention in-
cludes not only those tetrapeptide segments which are
substituted by H and OH respectively, but also those
which are terminally substituted by one or more other
functional groups which do not substantially affect the
biological activity disclosed herein. It should be
clearly understood, however, that the nonapeptide des~
cribed by Bach, et al., is specifically excluded fr¢m the
scope of -the present invention.
From this statement, it will be understood that
these functional groups inc].ude such normal substitution
as acylation on the free amino group and amidation on the
3S free carboxylic acid group, as well as the substitution
of additional amino acids and polypeptides. In these
aspects, the polypeptide segments of this invention
appear to be hishly unusual since they exhibit tha same

ORT~ 333
biological activity as the natural nonapeptide of which
the active tetrapeptide segment forms a portion. It is
believed, therefore, that the activitv requirements of
the molecule are generated by its stereochemistrv, that
is, by the particular "folding" of the molecule. In
this regard, it should be understood that polypeptide
bonds are not rigid but ~lexible, and polypeptides mav
e~ist as sheets, helices, and the like. As a result, the
entire molecule is flexible and will "fold" in a certain
way. In the present invention, it has been discovered
that the novel tetrapeptide segments probably "fold"
in a similar manner to the corresponding tetrapeptide
segment in the natural nonapeptide in that they exhibit
the same biological characteristics. For this reason,
the tetrapeptide segments may be terminally substituted
by various functional groups so long as the substituents
do not substantially affect the biological activity or
interfere with the natural "folding" of the molecule.
The ability of the molecule to retain its
biological activity and natural folding is clearly illus-
trated by the fact that the tetrapeptide segments of this
invention exhibit the same biological characteristics as
the natural 9-amino acid peptide disclosed as FTS by
J. F. Bach in the above disclosed articles. In this
nonapeptide, one tetrapeptide sequence of this invention
may be identified within the molecule but onlv in com-
bination with the other amino acids described therein.
Since the tetrapeptide segments of this invention pro-
vide the same biological actlvity as the nonapeptide FTS,
it is clear that the amino acids and peptide chains sub-
stituted on the terminal amino acid residues of the
tetrapeptide segment do not affect the biological charac-
teristics thereof.
In view of this discussion, it will, therefore,
be understood that R and R' in Formula II can be any
substituent that does not substantially affect the bio-
logical activity of the active segment. Thus, ror pur-
poses of illustration R and R' may be any of the following
substituents:
:'`
- -
~ -
.
.
:

~?~3~
,....
R R'
Hydrogen OH
Cl-C7 alkyl NH2
C6 C12 arYl NHR7
C6-C20 alkaryl ( 7)2
C6-C20 aralkyl OR7
Cl-C7 alkanoyl
C2-C7 alkenyl
C2-C7 alkynyl
wherein R7 is Cl-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl,
C6-C20 aryl, C6-C20 alkaryl, or C6-C20 aralkyl.
As pointed out above, however, R and R' can also be
neutral amino acid groups or residues of polypeptide chains
having 1 to 20 carbon atoms The following are illustrative:
R R
GLN GLY
SAR GLY-GLY
GLY-GLY-SER
GLY-GLY-SER-ASN
provided that, when R is GLN, R' is other than GLY-GLY-SER-ASN.
One preferred embodiment of the invention is that
wherein X is L-alanyl, Y is L-seryl, R is hydrogen and R' is OH.
This preferred embodiment may be symbolized chemically as:
H2N-CH-CO~H~CH-CONH-CH-CONH-CH-COOH
CH3 (CH2)4 CIH2 ( 12)2
NH2 OH CONH2
H- ALA - LYS - SER - GLN - OH
A second preferred embodiment is that wherein X and Y are each
selected from the group consisting of sarcosyl, D-alanyl, and
2-methylalanyl; a more prefexred embodiment being that wherein
X is sarcosyl, Y is sarcosyl, ox D-alanyl, R is
hydrogen, and R' is NH2.
Also included within the scope o~ the invention are
--8--
.
,: :: : ,
- ,. : , .~ : . . :
~ ! , '

Q3~
the pharmaceutically acceptable salts of the polypeptides.
As acids which are able to form salts with the polypeptides,
there may be mentioned inorganic acids such as hydrochloric
acid, hydrobromic acid, perchloric acid, nitric acid, thio-
cyanic acid, sulfuric acid, phosphoric acid, etc. and organic
-8a-
, ~
- .
.
,
,

~ ORTH 333
acids such as formic acid, acetic acid, propionic acid,
glycolic acid, lactic acid, pyruvic acid, oxalic acid,
maloni~ acid, succinic acid, maleic acid, fumaric acid,
anthranilic acid, cinnamic acid, naphthalenesulfonic acid
or sulfanilic acid, for instance.
Throughout the present application, the amino
acid components of the peptide are identified by abbre-
viations for convenience. These abbreviations are as
follows. The D-amino acids are indicated by placing "D"
before the abbreviation, e.g., D-alanine is represented
by "D-ALA".
Abbreviated
Amino Acid Designation
L-Alanine ALA
L-Aspartic Acid ASP
L-Asparagine ASN
L-Serine SER
L-Glutamic Acid GLU
L-Glutamine GLN
L-Leucine LEU
L-Lysine LYS
L-Threonine THR
Glycine GLY
L-Valine V~L
Sarcosine SAR
2-Methylalanine 2-Me-ALA
The polypeptides of this invention are 4-amino
acid peptides (and their substituted derivatives) which
have been found to exhibit characteristics similar to the
9-amino acid polypeptide FTS isolated from porcine blood
as disclosed in the above-referenced ~ach, et al., articles.
The peptides of this invention are particularly charac-
terized in their ability to induce the differentiation of
T-precursor cells as well as B-precursor cells. Certain
of the subject polypeptides are active in a concentration
as low as one picogram (pg)/ml in the chicken induction
assay discussed below.
It has been found that the polypeptides of this
invention induce the differentiation of immunocyte-pre-
cursor cells in vitro in the same way as the nonape~tides
-
disclosed by Bach. Thus, the polypeptides of this invention
' ' ' ' ' : ,.

oRrr~ ,33
031
have been found to induce the differentiation of both
T-precursor cells, as measured by the acquisition of the
thymic differentiation antigen Th-l as well as B-pre-
cursor cells, as measured by the acquisition of the
differentiation antigen Bu-l. Stated another way, the
~ubject polypeptides have the capability of inducing
differentiation o~ both Th-l+ T-lymphocytes and Bu-l+
B-lymphocytes.
It has also been found that the subject poly-
peptides increase the capability of ln vivo production
of cytotoxic lymphocytes upon stimulation by allogenic
antigens. That is t administration of the subject poly-
peptides to, e.g., rats,promotes the production of
cytotoxic lymphocyte precursors as measured by an in vitro
assay of rat spleen cells. Since ~he generation of cyto-
toxic lymphocytes directly corresponds to the extent of
graft rejection in allogenic graft vs host reaction, the
above finding is further support for the immunologic
utility of the subject polypeptides.
To provide an understanding of the importance
of the differentiating biological characteristics of the
polypeptides of this invention, it should be noted that
the function of the thymus in relation to immunity may be
broadly stated as the production of thymus-derived cells,
or lymphocytes, which are called T cells. T cells form a
large proportion of th~ pool of recirculating small
lymphocytes. T cells have i~munological specificity and
are directly involved in cell-mediated immune responses
(such as homograft responses), as effector cells. T cells,
however, do not secrete humoral antibodies. These anti-
bodies are secreted by cells (termed ~ cells) derived
directly from the bone marrow independently of the thymic
influence. However, for many antigens, B cells require
the presence of appropriately reactive T cells before
they can produce antibodies. The mechanism of this pro-
cess of cell cooperation is not yet completely understood.
From this explanation, it may be said that in
operational terms, the thymus is necessary for the
development of cellular immunity and many humoral antibody
:.. . . ..
.,
,

(~P~T}~ 3 3 3
: 11
responses and it affects these systems by inducing, with-
in the thymus, the differentiation of haemopoietic stem
ceils to T cells. This inductive influence is mediated
by secretions of the epithelial cells of the thymus, that
is, the thymic hormones.
Further, to understand the operation of the
thymus and the cell system of lymphocytes, and the circula-
tion of lymphocytes in the body, it should be pointed out
that stem cells arise in the bone marrow and reach the
thymus by the blood stream. Within the thvmus, stem
cells become differentiated to immunologically competent
T cells, which migrate to the blood stream and, together
with B ~ells, circulate between the tissues, lymphatics,
and the blood stream.
The cells of the body which secrete antibody ~B
cells) also develop from haemopoietic stem cells, but their
differentiation is not determined by the th~mus. In birds,
they are differentiated in an organ analogous to the
thymus, called the Bursa of Fabricius. In mammals, no
equivalent organ has been discovered and it is thought
that these cells differentiate within the bone marrow.
Hence, they are termed bone marrow-derived cells or B
cells. The physiological substances dictating this
differentiation remain completely unknown.
As pointed out above, the polypeptides of this
invention are therapeutically useful in the treatment of
humans and animals. Since the new polypeptides have the
capahility of inducing the differentiation of lympho-
poietic stem cells originating in the haemopoietic tissues
to both thymus-derived lymphocytes (T cells) and immuno-
competent B cells which are capable of involvement in
the immune response of the body, the products of this
invention are considered to have multiple therapeutic uses.
Primarily, since the compounds have the capability of
carrying out certain of the indicated functiQns o~ the
thymus, they have application in various thymic function
and immunity areas. A primary field of application is
in the treatment of DiGeorge Syndrome, a condition in
which there is a congenital absence of thymus. Injection
.
~'
,~

ORTH 333
3~
12
of one of the subject polypeptides, as further set out
below, will overcome this deficiency. Another applica-
tion is in agammaglobulinemia, which is due to a defect
of the putative B cell differentiative hormone o the
body. Injection of one of the subject polypeptides will
overcome this defect. Since the subject polypeptides
are extremely active at low concentrations, they are
useful in augmenting the collective immunity of the
body in that they increase therapeutic stimulation of
cellular immunity and humoral immunity and are thereby
useful in the treatment of diseases involving chronic
infection in vivo, such as fungal or mycoplasma infections,
tuberculosis, leprosy, acute and chronic viral infections,
and the like. Further, the subject peptides are con-
sidered to be useful in any area in which cellular orhumoral immunity is an issue and particularly where
there are deficiencies in immunity such as in the DiGeorge
Syndrome mentioned above. Further, because of the charac-
teristics of the polypeptides, they have ln vitro use-
fulness in inducing the development of surface antigensof T cells, in inducing the development of the functional
capacity to achieve responsiveness to mitogens and anti-
gens, and cell collaborativity in enhancing the ability
of B cells to produce antibodies. Thev have ln vitro
usefulness in inducing the development of B cells as
measured by the developmen~ of surface receptors for
complement. The subject peptides are also useful in in-
hibiting the uncontrolled proliferation of lymphocytes
which are responsive to ubiquitin ~described in Applicant's
3~ United States Patent No. 4,002,602). An important charac-
teristic of the subject polypeptides is their in vlvo
ability to restore cells with the characteristics of T
cells and also their in ivo ability to restore cells
with the characteristics of B cells~ They are, therefore,
useful in the treatmen~ of relative or absolute B cell
deficiencies as well as relative or absolute T cell de-
ficiencies, whether or not these deficiencies are due to
deficiencies in the tissue differentiating B cells or the
thymus, respectively, or to some other cause.
,

~2~3~ ORTH 33~
13
A further important property of the polypeptides
of this invention is that they are highly active in very
low concentrations. Thus, it has been found that the
polypeptides are generally active in concentrations of
about 1 ng/ml, while certain strikingly potent polypeptides
(H-SAR-LYS-D-ALA-GLN NH~ and H-SAR-LYS-SAR-GLN-NH2) are
active in concentrations ranging from about 0.1 pg/ml. The
carrier may be any of the well-known carriers for this
purpose including normal saline solutions, preferably
with a protein diluent such as bovine serum albumin to
prevent adsorptive losses to glassware at these low con-
centrations. The polypeptides of this invention are
generally active at a range of above about 1 ~g/kg of body
weight, while certain strikingly potent polypeptides are
active from about 1 ng/kg of body weigh~. For the treatment
of DiGeorge Syndrome, the polypeptides may be administered
at a rate of about 1 to about 100 ~g/kg of body weight,
while the strikingly potent polypeptides may be adminis-
tered at a rate of about 1 to about 1~0 ng/kg of body
weight. Generally, the same range of dosage amounts may
be used in treatment of the other conditions or diseases
mentioned. While the above discussion has been given with
respect to parenteral administration, it should be under-
stood that oral administration is also possible at dosage
ranges generally about 100 to 1000 times greater than those
for injection.
The polypeptides of this invention were prepared
using the concepts similar to those described by Merrifield
as reported in Journal of'American Chemical Society, 85,
.
pp. 2149-2154, 1~63. The synthesi3 involved the stepwise
addition of protected amino acids to a ~ro~ing peptide
chain which was bound by covalent bonds to a solid resin
particle. By this procedure, reagents and by-prGducts
were removed by iltration and the recrystallization of
intermediates were eliminated. The general concept o
this method depends on attachment of theC-ten~nal amino acid
of the chain to a solid polymer by a covalent bond and
the addition of the succeeding amino acids one at a time
in a stepwise manne- until the desired sequence is assem-
~0 bled. Finally, the peptide is removed from the soiidsupport and protective aroups removed. This method provi~es
,
, ~
; " , ,

ORTH 333
~2~)~)3~
a growing peptide chain attached to a completely in-
soluble solid particle so that it is in a convenient
form to be iltered and washed free of reagents and
by-products.
The amino acids may be attached to any suitable
polymer which merely has to be readily separable from the
unreacted reagents. The polymer may be insoluble in the
solvents us~d or may be soluble in certain solvents and
insoluble in others. The polymer should have a stable
physical form permitting ready filtration. It must con-
tain a functional group to which the first protected
amino acid can be firmly linked by a covalent bond.
Various insoluble polymers suitable for this purpose are
those such as cellulose, polyvinyl alcohol, poly~eth-
acrylate and sulfonated polystyrene but in the synthesis
of this invention, there was generally used a chlorc~iethylated co--
polymer of styrene and divinylbenzene. Polymers which
are soluble in organic solvents while being insoluble
in aqueous solvents may also be used. One such polymer
is a polyethylene/glycol having a molecular weight of
about 20,000, which is soluble in methylene chloride
but insoluble in water. ~he use of this poly~er in
peptide synthesis is descrlbed in F. Bayer and M~ Mutter,
Nature 237, 512 (1972) and references contained therein.
The various functional groups on the amino acid
which were active, but which were not to enter into the
reactions, were protected by conventional protecting
groups as used in the polypeptide art throughout the
reaction. Thus, the functional group on lysine was pro-
tected by protecting groups w~ich could be removed on
completion of the sequence without adversel~ affecting
the polypeptide final product. In the synthesis
fluorescamine was used to determine if coupling was com-
plete by an indication of positive fluorescence (see
Felix, et al., ~G~ " ~b~ h~., 52, 377, 1973). If com-
plete coupling was not indicated, the coupling was re-
peated with the same protected amino acid before depro-
tection.
.
,: ~ . . . . .

O~ 33
The C-terminal amino acid may ~e attached to the
polymer in a variety of well-known ways. Summaries of
methods for attachment to halomethyl resins are given in
Horiki, et al., Chem Letters, pp 165-168 ~1978) and
Gisin, Helv. Chim. Acta, 56, 1475 (1973), and references
given therein.
The general procedure involved initially esteri-
fying L-glutamine, protected on its amino groups, to ~he
resin in absolute alcohol containing an amine. The
coupled ami~o acid resin was then filtered, washed with
alcohol and water and dried. The protecting group on the
a-amino group of the glutamine amino acid (e.g., t-BOC,
i.e., t-butyloxycarbonyl), was then removed. The resulting
coupled amino acid resin, having the free amino group, was
then reacted with a protected L-serine, preferably alpha-
t-BOC-O benzyl-L-serine to couple the L-serine. The reactions
were then repeated with protected L-lys.ine and L-alamne until
the complete molecule was prepared. The sequence of
reactions was carried out as follows:
Resin
1 a Rl-Gln-OH
a-Rl-Gln-Resin
Remove a-amino
I protecting group
I*-Gln-Resin l2
R2 1 a Rl~L-Ser-OH
a-Rl-Ser-Gln-Resin
Re~ove a-amino
R12~ protectins group
H-Ser-Gln-Resin
R1 3
a-Rl-Lys-OH
l3 R12~ ,
a-R1-Lys-Ser-Gln-Resin
Remove a-amino
R R2~ protecting group
13
E-Lys-Ser-Gln-Resin
'' '.
' ~ :
'~ . ,' ' . ' '
, I , ' ' ' ' '
'
~: `

V~ ", OP.T~I 333
16
R ¦,R
a-Rl-Ala-Lys--Ser-Gln-Resin
¦ Remo~-e all protecting
~ groups and resin
H-Ala-Lys-Ser-Gln-OH
In the above sequence of reactions Rl is a pro-
tecting group of the a-amino group and R2 and R3 are pro-
tecting groups on the reactive side chains of the L-serine
and L-lysine, respectively, which are not affected or re-
moved ~Ihen Rl is removed to permit further reaction.Preferably, in the above intermediate pentapeptide
resin, the term Rl stands for a protective grouping
such as t-butyloxycarbonyl, R2 stands for benzyl
or substituted benzyl (e.g., 4 chlorobenzyl), and R3
stands for substituted benzyloxycarbonyl (e.g., 2,6-di-
chlorobenzyloxycarbonyl). The resin is any of the resins
men~ioned above as being useful in the process.
After the final intermediate was prepared, the
peptide resin was cleaved to remove the Rl, R2, and R3 pro-
2~ tecting groups thereon and the resin. The protectinggroups were removed by conventional means, e.g., by
treatment with anhydrous hydrogen fluoride, and the re-
sulting free peptide was then recovered.
As pointed out above, in conducting the process,
it is necessary to protect or block the amino groups in
order to control the reaction and oDtain the products
desired. Suitable amino protecting groups which may be
usefully employed include salt formation for protecting
strongly-basic amino groups, or urethane protecting sub-
stitutes such as p-methoxy benzyloxycarbonyl and t-butyl-
oxycarbonyl. It is preferred to utilize t-butylo~ycarbonyl
~BOC) or t-amyloxycarbonyl (AOC) for protecting the a-
amino group in the amino acids undergoing reaction at the
carboxyl end of the molecule, since the BOC and AOC
(t-amyloxycarbonyl) protecting groups are readily removed
following such reaction and prior to the subse~uent step
~ (wherein such a-amino group itself undergoes reaction)
. .
..
:
,,, . . - :
,
' '

O~T~I 333
17
by relatively mild action of acids ~e.g., trifluoro-
acetic acid~, which treatment does not otherwise affect
groups used to protect other reactive side chains. It
will thus be understood that the a-amino groups may be
protected by reaction with any material which will pro-
tect the amino groups for the subsequent reaction(s)
but which may later be removed under conditions which
will not otherwise affect the molecule. Illustrative
of such materials are organic carboxylic acid derivatives
which will acylate the amino group.
In general, any of the amino groups can be pro-
tected by reaction with a compound containing a grouping
of the formula:
O
R4 - O - C -
wherein R4 is any grouping which will prevent the amino
group from entering into subsequent coupling reactions and
which can be removed without destruction of the molecule.
Thus, R4 is a straight or branched chain alkyl which may
be unsaturated, preferably of 1 to 10 carbon atoms, and
prefexably halo- or cyano-substituted; aryl, preferably
of 6 to 15 carbons; cycloalkyl, preferably of 5 to 8
carbon atoms; aralkyl, preferably of 7 to 18 carbon
atoms; alkaryl, preferably of 7 to 18 carbon atoms; or
heterocyclic, e.g., isonicotinyl. The aryl, aralk~l and
alkaryl moieties may also be ~urther substituted as by
one or more alkyl groups of 1 to about 4 carbon atoms.
Preferred groupings for R include t-butyl, t-amyl, tolyl,
xylyl and benzyl. Highly preferred specific amino-pro-
tecting groups include benzyloxycarbonyl; substituted
benzyloxycarbonyl, wherein the phenyl ring is substltuted
by one or more halogens, e.g., Cl or Br; nitro; loweralkoxy,
e.g., methoxy; loweralkyl; t-butyloxycarbonyl, t-amyloxy-
carbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl;
adamantyloxycarbonyl; biphenylisopropoxycarbonyl; and
the like. Other protecting groups which can be used
include isonicotinyloxycarbonyl, phthaloyl, p-tolyl-
sulfonyl, formyl and the like.
.,, . . ~ . ~
.

ORTH 333
18
In conducting the general process of the in-
vention, the peptide is built by reaction of the free
~-amino group with a compound possessing protected amino
groups. For reaction or coupling, the compound being
attac~ed is activated at its carboxyl group so that the
carboxyl group can then react with the free ~-amino
group on the attached peptide chain. To achieve activa-
tion the carboxyl group can be converted to any reactive
group such as an ester, anhydride, a~ide, acid chloride,
or the like. Alternately, a suitable coupling reagent
may be added during the reaction. Suitable coupling re
agents are disclosed, e.g., in Bodanszky, et al. -
Peptide Synthesis, Interscience, second edition, 1976,
chapter five, including carbodiimides ~e.g.-, dicyclo-
carbodiimide), carbonyldiimidizole, and the like.
It should also be understood that during thesereactions, the amino acid moieties contain both amino
groups and carboxyl groups and usually one grouping
enters into the reaction while the other is protected.
Prior to the coupling step, the protecting group on the
alpha or terminal amino group of the attacked peptide is
removed under conditions which will not substantially
affect other protecting groups, e.g., the group on the
epsilon-amino of the lysine molecule. The preferred
procedure for effecting this step is mild acidolysis, as
~y reaction at room temperature with trifluoroacetic acid.
As may be appreciated, ~he above-described
series of process steps results in the production of
the tetrapeptide of Formula III as follows:
III. H-AL~-LYS-SER-&LN-OH
~ his tetrapeptide contains one tetrapeptide
segment of this invention necessary for biological activ-
ity. The substitution of a natural or non-natural amino
acid residue for either or both of L-alanyl and L-seryl
_ ~ .
- . . - .
.: . ..
., ,
. ' .

~RT~ 333
1~
may be effected ~y replacing either or hoth.of alanine
and se.rine by the appropriately protected natural or
non-natural amino acid in the above synthetic scheme,
thus yielding the tetrapeptide of the following formula:
IIIA. H-X-LYS-Y-GLN-OH
wherein X and Y are as previously described. The sub-
stituted tetrapeptide of Formula II, wherein the terminal
amino acid groups may be further substituted as described
above, may then be prepared by reaction of the tetra-
peptide of ~ormula (.IIIA) or the protected peptide resin
precursor with suitable reagents to prepare the desired
derivatives. Reactions of this type such as acylation,
esterification, amidation and the like, are, of course,
well-known in the art. Further, other amino acids, that
is amino acid groups which do not affect the biological
activity of the tetrapeptide sequence, may be added to
either end or the peptide chain by the same sequence of
reactions by which the tetrapeptide itself was synthesized.
Still further, substitution for either or both the ala-
nine or the serine moieties may be accomplished by em-
ploying the desired substituent (suitably protected) in
place of alanine or serine in the preceding sequence
of reactions by which the unsubstituted tetrapepti.de was
synthesized.
While the solid phase technique of Merrifield
has been used to prepare the subject polypeptide~, it is
clearly contemplated that classical techniques described
in, for example, M. Bodanszky and M. A. Ondetti, Peptide
Synthesis, Interscience, 1966, may also be employed.
Identity and purity of the subject peptides
T~ere determined by such well known methods as thin layer
chromatography, electrophDre.sis~ amino acid anal~sïs ?
and the like.
. : . : . : . :
, , : - ~ ~
::, . . . ..

~ ORT~ 3~3
3~L
The following Examples are precented fo illus-
trate the invention, but it is not to be considered as
limited thereto. In the Examples, and throughout the
specification, parts are by weight unless otherwise
indicated.
EXAMPLE I
In preparation of the polypeptide of this
invention, the following materials were purchased
commercially:
Alpha-BOC-L-Glutamine-o-nitrophenyl-ester
Alpha-BOC~-2-chloro~benzyloxycarbonyl-L-lysine
Alpha-BOC-O-benzyl-L-serine
Alpha-BOC-L-Alanine.
In these reagents, BOC is t-butyloxycarbonyl.
"Sequenal" grade reagents for amino acid sequence deter-
minations, dicyclohexyl carbodiimide, fluorescamine, and
the resin were also purchased commercially. The resin
used was a polystyrene divinyl benzene resin, 200-400 mesh
size containing 1~ divinyl benæene and .75 mM o~ chloride
per gram o~ resin.
In preparation of the polypeptide, 2 mmoles of
a-BOC-L-Glutamine were esterified to 2 mmoles of chloro-
methylated resin in absolute alcohvl containing lmM
triethylamine for 24 hours at 80C. The resulting amQno acid
resin ester was filtered, washed with absolute alcohol and
dried. Thereafter, the other ~-BOC-amino acids were
similarly coupled to the deprotected a-amino group of
the peptide-resin in the correct sequence to result in
the polypeptide of this invention using equivalent amounts
of dicyclohexyl carbodiimide. After each coupling reac-
tion, an aliquot of resin was tested with fluorescamine
and if positive fluorescence was found, coupling was
taken to be incomplete and was repeated with the same
protective amino acid. As a result of the several coupling
reactions, the intermediate tetrapeptide-resin was prepared.
This peptide-resin was cleaved and the protective
groups removed in a Kel--F cleavage apparatus (Peninsula
Labora-cories, Inc.) using anhydrous hydrogen fluoride a~
. ~ , .. , ; : -
- . . . . :,
. : ; ~-
. ~ , .

` OP~T~ 333
V3~ -
21
0C for 60 minutes with 1.2 ml anisole per gram peptide-
resin as scavenger. The peptide mixture was washed ~ith
anhydrous ether and extracted with aqueous acid. The
extract was lyophilized and the peptide was chromatographed
on P-6 Bio-Gel in 1 N acetic acid. The resulting poly-
peptide was determined to be 9~% pure and was determined
to have the following sequence:
H-ALA-L~S-SER-GLN-OH
For identification, thin layer chromatography
and electrophoresis were performed as follows.
Thin layer chromatography was performed on
a 30 ~g sample on silica gel tBrinkman Silica Gel with
fluorescent indicator, 20 x 20 cm, 0.1 mm thick) using
the following eluents.
Rf : _-butanol:pyridine:acetic acid:waterl 30:15:3:12
Rf : ethyl acetate:pyridine:actlc acid:water; 5:5:1:3
Rf : ethyl acetate:_-butanol:actic acid:water; 1:1:1:1
Electrophoresis was performed on 100 ~g sample
on Whatman 3 mm paper (11.5 x 56.5 cm) using a pH 5.6
pyridine-acetate buffer solution and 1000 volts potential
for one hour.
Spray reagents for both thin layer chromatography
and electrophoresis were Pauly and Ninhydrin.
The following results were obtained: Rfl =
immobile, Rf = immobile, and Rf = 0.336~ Electrophoresis
resulted in a migration of 9.4 cm toward cathode.
EXAMPLE II
To determine the activity and characteristics
of the tetrapeptide produced in Example I, the following
chicken induction assay was employed. This assay is des-
cribed in greater detail in Brand, et al., Science, 193
319-321 (~uly 23, 1976) and references contained therein.
Bone marrow from newly-hatched chickens was
selected as a source of inducible cells because it lacks
an appreciable number of Bu-l+ or Th-l~ cells. Pooled
:.. , , - ~ , .:
.

O~TH 3~3
_ ~%~03~
22
cells from femur and tibiotarsus of five newly-hatched
chicks of strain SC (Hy-Line) -~ere fractionated by ultra-
centrlfugation on a five-layer discontinuous bovine serum
albumin (BSA) gradi~nt. Cells fro~ the two lighter layers
were combined, washed, and suspended for incubation at a
concentration of 5 x 106 cells per milliliter with the
appropriate concentration of test polypeptide in RPMI
1~30 medium supplemented with 15 mM hepes, 5 percent
y-globulin-free fetal calf serum, deoxyribonuclease (14 to
18 unit/ml), heparin (5 unit/ml), penicillin (lO0 unit/ml),
and streptomycin (100 ~g/ml~. Controls were incubated
with BSA (l ~g/ml) or medium alone. After incubation,
the cells were tested in the cytotoxicity assay using
chicken Cl and guinea pig C2 to C9 complement fractions
as described in the reference article. The proportion of
Bu-l+ or Th-l+ cells in each layer was calculated as a
cytotoxicity index, lO0 ~a b)/a, where a and b are the
percentages of viable cells in the complement control and
test preparation, respectively. The percentage of cells
induced was obtained by subtracting the mean values in the
control incubations without inducing agents (usually l to
3 percent) ~rom those of the test inductions.
The specificity of the action of the test poly-
peptide and its similarity to ubiquitin were demonstrated
by the inhibition of induction of Bu-l+ B cells and Th-l+
T cells by the test polypeptide upon addition o ubiquitin
in a concentration of 100 ~g/ml. This high dose of ubiqui-
tin inactivates the ubiquitin receptors and thus prevents
the induction of cells by any agent which acts through
these receptors.
As a result of this assay, it W2S discovered
that the tetrapeptide of Example I displayed biological
activity similar to that of ubiquitin in inducing the
differentiation of both Th-l+ T and Bu-l+ B lymphocytes in
ng/ml concentrations.
.. ..
; ; .
. : , ..
~ ' ` . , ,
, . . ; : ~,

O~TH 333
23
EXAMPLE III
A. The assay of Example II was repeated, using
as the test polypeptide one of the following:
H-GLN-ALA-LYS-SER-GIN-GLY-GLY-SER-ASN-OH
H-GL~-ALA-LYS-SER-GLN-OH
H-SAR-ALA-L~S-SER-GLN-OH
In each case, biological activity similar to that of
ubiquitin was observed.
B. The assay of Example II was repeated, using
as the test polypeptide one of the following:
H-SAR-LYS-D-ALA-GLN-NH2
H-SAR-LYS-SAR-GLN-NH2
H-D-ALA-LYS-D--ALA--GLN-NH2
In each case, biological activity similar to that of
ubiquitin was observed. For the first of these polypeptides,
this activity was observed in the range of concentration
from about 1 pg/ml to about 100 pg/ml. For the second
polypeptide, activity was observed at a concentration as
low as 0.1 pg/ml.
EXA~IPLES IV - VI
Using the reaction techniques described herein-
ab~ve for preparing substituted polypeptides, these are
prepared polypeptides of the following formula:
R-X-LYS-Y-GLN-R'
These peptide amides were prepared on a benzhydrylamine
resin by solid phase synthesis techniqu~s known in the art.
EXAMPLE
NUMBER_ R X Y R'
IV HSAR D-ALA NH2
IVA HSAR SAR NH2
30 V H D-ALA D-ALA NH2
VA H D-ALA SAR NH~
VI H SAR 2-Me-ALA NH~
VIA H SAR SAR OH
.; . . .;, ' ! ~ ~
~ '.'~ . . ` ` "'.' ' ' ' ' "'
.: ' ` ` .. ' `

` ORTH 333
3~
24
The polypeptides prepared in Examples IY-VI re-
tain the biological activity as described herein for the
active polypeptide segment.
For identification, thin layer chromatography
and electrophoresis were performed as follows.
Thin layer chromatography was performed on 20 ~g
samples on silica gel (Kieselgel, 5 x 20 cm) using as
eluent n-butanol:acetic acid:ethyl acetate:water in pro-
portions of 1:1:1:1 tRfl) and on cellulose 6064 (Eastman,
20 x 20 cm) using as eluent n-butanol:pyridine:acetic acid:
water in proportions of 15:10:3:12 (Rf2).
Electrophoresis was performed on 50 ~g samples
on Whatman No. 3 paper (5.7 x 55 cm) using a pH 5.6
pyridine-acetate buffer solution and 1000 volts potential
for one hour. The compounds migrate toward the cathode.
Spray reagents for both thin layer chromatography
and electrophoresis were Pauly and Ninhydrin.
The following results were obtained ~both Rf
values and electrophoresis are given relative to H-ARG-
LYS-ASP-VAL-TYR-OH):
1 2 Electrophoresis
Example Rf Rf mi~ration purity
IV 0.44 0.68 2.07 98%
IVA 0.88 0.60 1.78 98
V 0.56 0.71 2.10 9
Following the thin layer chromatography and
electrophoresis procedure of Example I, the following re-
sults are obtained for the compound of Example VI: Rfl =
(0.155), Rf2 = immobile, Rf3 = 0.265 and electrophoresis
migration is 13.1 cm toward cathode.
EXAMPLE VII
The tetrapeptide resins having protected LYS and
SER prepared as in Examples I and VIA are each acylated by
reaction with acetic anhydride under acetylating conditionc,
followed by removal of the protecting groups and the resin
to prepare the following acylated derivatives:
CH CO-ALA-LYS-SER-GLN-OH
CH3CO-SAR-LYS-SAR-GLN-OH
:
, .: . , . ~ .
'

ORTH 3~3
3~
EXAMPLE VIII
The protected tetrapeptide resins prepared as in
Examples I and VIA are each transesterified from the resin
by reaction with sodium methoxide in methyl alcohol under
transesterification conditions, followed by removal of
the protecting groups, to prepar~ the esterified deriva-
tives of the following formulas:
H-ALA-LYS-SER-GLN-OCH3
H-SAR-LYS-SAR GLN-OCH3
EXAMPLE IX
The protected tetrapeptide resins prepared as in
Examples I and VIA are each cleaved from the resin with
diethyl amine under reaction conditions known in the art,
followed by removal of the protecting groups, to prepare
the following amino substituted derivatives:
H-ALA-LYS-SER-GLN-N(C2H5)2
H-SAR-LYS-SAR GLN-N(C2H5)2
EXAMPLE X
Following the methods of Examples I and VIA but
substituting for the ALA or SAR used to add the N-terminal
amino acid residue, an equivalent amount of suitably pro-
tected N-a-ethyl-L-alanine or ~ ethyl-sarcosine, respec-
tively, there are prepared the following:
C2H5-ALA-LYS-SER-GLN-OH
C2H5-SAR-LYS-SAR-GLN-OH
EXAMPLE XI
Cleaving the protected resin tetrapeptides ormed
in Example X rom the resin using ammonia in dimethyl-
formamide under amidation conditions, followed by removal
of the protecting groups yields peptide amides of the
formulas:
C2H5-ALA-LYS-SER-GLN--NH2
C H -SAR-LYS-SAR-GLN-NH2
,,.
.
~ , , .
,

ORTH 333
?Q3~l
26
EXAMPLE XII
The protected acetylated tetrapeptide resins
prepared as in Example VII are each reacted with ammonia in
dimethylformamide under amidation conditions, followed
S by removal of the protective groups, to prepare the
following peptide amides:
CH3CO-ALA-LYS-SER-GLN-NH2
CH3CO-SAR-LYS-SAR-GLN-NH2
EXAMPLES XIII - XXVIII
Using the reaction techniques described herein-
above ~or the lengthening o~ the polypeptide chain, the
following polypeptides are prepared which contain the
active amino acid sequence but which are su~stituted on
the terminal amino and carboxylic groups R and R' to
lS provide the polypeptide of formula:
R-ALA-LYS-SER-GLN-R'
which is substituted by the amino acids given in the
followina Table as indicated.
EXAMPLE
NUMBER _ R
XIII GLN OH
XIV SAR OH
XV. E GLY
XVI ~ GLY-GLY
X~7II H GLY-GLY-SER
XVIII H GLY GLY-SER-ASN
XIX GLN GLY
XX GLN GLY-GLY
XXI GLN GLY-GLY-SER
XXII SAR GLY
XXIII SAR GLY-GLY
XXIV SAR GLY-GLY-SER
XXV SAR GLY-GLY-SER-ASN
XXVI GLN GLY-GLY-SER-ASN
The polypeptide derivatives prepared in Examples
IV-XXVI retain the biological activity as described herein
for the active polypeptide segment.
.
.
-, ;
' ' ' ' . ' ~ : .

OR~H 333
3~L
,. . .
27
Following the thin layer chromatography and
electrophoresis procedure of Example I for the compound
of Examples XVI and XVII, the following results are ob-
tained:
electrophoresis
1 2 3migration
5 Example R R R toward cathode
f f f
XII immobile immobile 0.303 7.4 cm
XIV immobile immobile 0.186 8.3 cm
Following the thin layer chromatography andelectrophoresis procedures of Examples IV-V for the
compound of Example XXVI, the following results are ob-
tained: Rfl = 0.23, Rf2 = 0.25, electrophore~is migration
toward cathode = 0.88.
EX~MPLE XXVII
To further illustrate the utility of the subject
polypeptides, this example describes a microculture assay
for estimating the fre~uencies of the cytotoxic lympho-
cytes produced upon stimulation by allogenic antigens.
The frequencies of cytotoxic precursors between control
animals and animals injected with various concentrations
of the drug were compared by a limiting dilution assay.
Materials and Methods
~ice
Inbreed C57 BL/6J (female, 8 weeks) were obtained from
Jackson Laboratory, Bar Har~or, Maine.
Inbreed DBA/2J (male or female) were also obtained from
Jackson Laboratory, Bar Harbor, Maine.
Media
Phosphate buffered saline (PBS), RPMI 1640, fetal calf
serum (FCS) - ~lot number R776116), N-2-hydroxyethyl-
piperazine-N'-2-ethanesulfonic acid (HEPES) buffer were
obtained from Gibol Grand Island; 2-mercaptoethanol was
from Eastman Kodak, Rochester, N~Yo Cells were washed
with PBS and cultured in RPMI containing 10~ ~CS, 10
mM HEPES buffer and 5xlO 5M 2-mercaptoethanol.
....
,

ORT~ 333
3~
28
Drug treatment
The test animals (C57 BL/6J) were injected (i.v. or
i.p.) with various concentrations of H-SAR-LYS-SAR-
GLN-NH2 (the drug; identification no. GO40) in 0.2 ml
volume 24 hours befors they were sacrificed for the
experimentsO
Cell preparations
Cell suspensions from spleens of C57 BL/6J mice or
DBA/2J mice were prepared by mincing the organ and
pressing them through a wire mesh (60 gauge) with the
plunger of a 5 c.c. syringe into a falcon petri dish
(falcon 3002, 15x60 mm). The cell suspensions were
allowed to stand at room temperature for 10 minutes
to let the big chunks of tissue settle. The cell
suspensions were then transferred to 15 ml Corning
centrifuge tubes (Corning 25310) and spun for 10
minutas at 1500 RPM in the Beckmen TJ-6 centrifuge.
All cell suspensions were washed at least three more
times with PBS. After the third wash, the responder
(C57 BL/6J) cells were resuspended in culture medium
and counted in the Coulter counter. DBA/2J (stimu-
lator cells) were resuspended in RPMI to 107 cells/
ml. 30 ~g of mitomycin C was added to each ml of the
DBA/2J spleen cells and the mixtures were incubated
at 37 for 30 minutes. After mitomycin C treatment,
the spleen cells were washed three times wi-th PBS
to remove any excess mitomycin C. The DBA cells
were then resuspended in the culture medium and
counted in the Coulter counter.
Mixed lymphocyte cultures (MLC)
MLC were set up in microtiter trays (Linbro Cnemicals,
New Haven, Conn , IS-~IC~96). Each tray contained
95-V bottom wells. The outside wells surrounding the
edge of the plate were not used for cell culture but
filled with PBS to avoid evaporation from the culture
wells. 60 samples were set up in each V-bottom tray.
Usually each tray contained three responder cell con-
_ centrations (20 replicates of each) and one stimulator
. . .

ORT~ 33~
3~
29
cell concentration. The responder cells were usually
suspended to concentrations of 7.5x105, 5x105 and
2. sxlo5 per ml and 0.1 ml was added to each well. The
stimulator cell concentrations used were 106, 2.5x106,
5X106 per ml, also 0.1 ml was added to each well. The
same stimulator cell concentration was used throughout
the whole plate. Control plate containing only re-
sponder cells with no stimulator cells was also set
up or estimating background stimulation due to the
medium. The cells were cultured for six days at 37C
in a humidiied incubator containing 5% CO2.
Target cells
The target cell used in the cytotoxic assay wa~ a ~BA
mastocytoma cell line P815. The cell line was main-
~ained by routine passage through DBA/2J mice. 5X108
P815 cells were used for each passage and the tumor
cells from the peritoneal cavity of the carriers
were used four to five days after passage. The tumor
cells from the peritoneal cavity were washed three
times with PBS and then labeled with CrSl at a con-
centration of 100 ~ci per 107 cells. Labeling was
done for an hour at 37C in a humidified incubator.
The labeled target cells were then washed for three
times with PBS to remove any excess label.
C~totoxic assay
After six days of culture, 0.1 ml of medium was removed
from each well witho~t disturbing the cell pellet.
Then, using an automatic micropipet (MLA pipet), 100
~l o~ ta~get cells, containing 2.5x104 Cr51 labeled
target cells were pipetted into each well, resuspend-
ing the cell pellet during the process (a new pipet
tip has to be used for each well). The microtiter
trays were then spun at room tem~erature at 1000 R~M
for seven minutes in the Sorvall GLC-2B. The trays
were then incubated for four hours at 37C. 100 micro-
liters of supernatant were then removed into Gamma
counting tubes (~mershen 196271). The tubes were
then counted in the Beck~en Gamma Counter (Beckmen 31n)~
The tubes were usually counted ~or one minute.
: ~ ; .: , , : -
. . .
:,

ORT~ 3~1
~.
Determination of the frequencies of
the precursors of cytotoxic lymphocytes (CLP)
The limiting dilution analysis is an all or none
response assay described by the Poisson probability
distribution. The probability of a non-response is
given by the zero order term Po=e ~N where ~ = fre-
quency of CLP and N = the number of lymphocytes per
well. Thus a plot of the logarithm of the proportion
of non-responding cultures vs cell dose should yield
a straight line with a slope of -~, the frequency of
CLP.
In the Example, the background chromium release
~ (spontaneous release) from 20 wells containing just
responder cells (C57 BL/6J) with no stimulator cells
~ere averaged. Test wells were scored as positive
if their counts were greater than the mean spon-
taneous value by more than 2.07 standard deviations
(P<0.05); The spontaneous lysis usually ranged from
9-15% of the toal counts incorporated into the tar-
get cells. According to the Poisson equation Po=e
when Po=e 1 _ 0.37 (corresponds to 37% non-responding
cultures), ~ = l/N, thus the reciprocal of the respond-
ing cell number corresponding to 37% non-responding
cultures is the CLP fre~uency. Usually the number of
cells per well and their corresponding value for per-
cent non-responders were fitted into the computer which
compute the best fit regression line through these
points and the number of cells per well which corre-
spond to 37% non-responding cultures, the reciprocal
of that value is the frequency of the CLP.
Results
The frequencies of cytoto~ic lymphocyte precursors for
each stimulator cell concentration were plotted as a
function of drug dose. The mean and standard deviation
of the twenty replicates were also computed for com-
parison. The three stimulator cell concentrationsused were 105 (suboptimal stimulation), 2.5x105
(optimal stimulation) and 5x105 (over optimal stimu-
lation). It was found that the test drug promoted
.
.
,
:.: . :

OP~TH 333
3~
31
the production of cytotoxic lymphocyte precursors
at concentrat~ons of from about l pg/mouse to about
100 ng/mouse (equivalent to about 50 pg/kg to about
5 ~g/kg body weight) in the presence of suboptimal
stimulator cell concentrations. The test drug is
therefore acting as an immuno-regulator at these con-
centrations to increase the cellular immune response
of the treated mice.
The invention has been described herein with
reference to certain preferred embodiments. However, as
obvious varïations will appear to those skilled in the
art, the invention is not to be considered as limited
thereto.