PENTAPEPTIDES AND METHODS

PROCEDES D'OBTENTION DE PENTAPEPTIDES

English Abstract

SK-205 CIP
042-010 NEW PENTAPEPTIDES AND METHODS
ABSTRACT OF THE DISCLOSURE
There are disclosed new polypeptide compositions having
the following amino acid sequence as the active site:
TYR-ASN-ILE-GLN-LYS
This polypeptide has the capability of inducing the
differentiation of both T-precursor cells as measured by the
acquisition of the thymic differentiation antigens TL and
THY-l (0), as well as B-precursor cells as measured by the
acquisition of receptors for complement, a distinctive marker of
B cells. The peptide is thus useful in thymic function and
immunity areas such as in treatment for a congenital absence of
thymus. The peptide is active in very low concentrations. Also
provided are derivatives of the pentapeptide, novel intermediate
polypeptides, methods of manufacture of the peptides, therapeutic
compositions, and methods for use of the compositions.
- 1 -

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for manufacture of an active peptide
fragment which has the capability of inducing differentiation
of both T-lymphocytes and complement receptor (CR+) B-
lymphocytes, which has the following active group therein:
R-NH-X-Y-Z-GLN-LYS-COR'
wherein X is TYR or ALA, Y is ASN or ALA, and Z is ILE or ALA,
and R and R' are terminal groups on said polypeptide which do
not substantially affect the biological capability thereof,
wherein R and R' are selected from the groups consisting of:
<IMG>

<IMG>
wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl,
C6-C20 aryl, C6-C20 aralkyl, or C6-C20 alkaryl,
which comprises esterifying L-lysine protected on its amino
groups, to an insoluble resin polymer by covalent bonding;
removing the .alpha.-amino protecting group from the L-lysine moiety,
reacting with an .alpha.-amino protected L-glutamine to couple L-
glutamine to the L-lysine resin; removing the .alpha.-amino protect-
ing group from the L-glutamine moiety, reacting with an .alpha.-amino
protected L-isoleucine or L- alanine to couple L- isoleucine or
L-alanine to the L-glutamine-L-lysine-resin; removing the a-
amino protecting group and reacting with an a-amino protected
L-asparagine or L-alanine to couple L-asparagine or L-alanine
to the L-isoleucine or L-alanine-L-glutamine-L-lysine-resin,
removing the a-amino protecting group and reacting with an a-
amino protected L-tyrosine or L-alanine to couple L-tyrosine
or L-alanine to L-asparagine or L-alanine-L-isoleucine or
L-alanine-L-glutamine-L-lysine-resin, removing all amino-
protecting groups from the peptide, separating from the resin
polymer, and reacting the resulting pentapeptide with an appro-
priate reagent so as to form the derivatives identified by R
and R' with protection of unreactive functional groups, if
necessary.
2. A process according to claim 1 wherein reactive side
chains on the reacting amino acids are protected during the
reaction.
31

3. A process according to claim 1 wherein the resin
polymer is selected from the group consisting of cellulose,
polyvinylalcohol, polymethacrylate, sulfonated polystyrene,
and a chloromethylated copolymer of styrene and divinylbenzene.
4. A polypeptide fragment which has the capability
of inducing differentiation of both T-lymphocytes and
complement receptor (CR+) B-lymphocytes, which has the
following active group therein:
R-NH-X-Y-Z-GLN-LYS-COR'
wherein X is TYR or ALA, Y is ASN or ALA, and Z is ILE or ALA,
and R and R' are terminal groups on said polypeptide which do
not substantially affect the biological capability thereof,
wherein R and R' are selected from the groups consisting of:
<IMG>
32

<IMG>
wherein R7 is C1-C7 alkyl, C2-C7 alkenyl, C2-C7 alkynyl,
C6-C20 aryl, C6-C20 aralkyl, or C6-C20 alkaryl, whenever
produced by the process of claim 1 or its obvious chemical
equivalent.
5. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein said polypeptide is a pentapep-
tide of the following sequence:
R-NH-TYR-ASN-ILE-GLN-LYS-COR'
wherein R is hydrogen, C1-C7 alkyl, C5-C12 aryl, or C1-C7
alkanoyl, and R' is OH, NH2, NHR7 or N(R7)2.
6. A pentapeptide of the following sequence:
R-NH-TYR-ASN-ILE-GLN-LYS-COR'
and the pharmaceutically acceptable salts thereof, wherein R
is hydrogen, C1-C7 alkyl, C5-C12 aryl, or C1-C7 alkanoyl, and
R' is OH, NH2, NHR7 or N(R7)2, whenever produced by the
process of claim 5 or its obvious chemical equivalent.
7. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is hydrogen and R' is OH.
8. A polypeptide according to claim 4 wherein R is
hydrogen and R' is OH, whenever produced by the process of
33

claim 7 or its obvious chemical equivalent.
9. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is CH3CO- and R' is OH.
10. A polypeptide according to claim 4 wherein R is
CH3CO- and R' is OH, whenever produced by the process of
claim 9 or its obvious chemical equivalent.
11. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is CH3 and R' is OH.
12. A polypeptide according to claim 4, wherein R
is CH3 and R' is OH, whenever produced by the process of
claim 11 or its obvious chemical equivalent.
13. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is H and R' is NH2.
14. A polypeptide according to claim 4 wherein R is
H and R' is NH2, whenever produced by the process of claim 13
or its obvious chemical equivalent.
15. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is H and R' is N(C2H5)2.
16. A polypeptide according to claim 4 wherein R is
H and R' is N(C2H5)2, whenever produced by the process of
claim 15 or its obvious chemical equivalent.
17. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is CH3CO- and R' is NH2.
18. A polypeptide according to claim 4 wherein R is CH3
CO- and R' is NH2, whenever produced by the process of claim
34

17 or its obvious chemical equivalent.
19. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is H and R' is -OCH3.
20. A polypeptide according to claim 4 wherein R is
H and R' is -OCH3, whenever produced by the process of claim
19 or its obvious chemical equivalent.
21. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is H and R' is OC2H5.
22. A polypeptide according to claim 4 wherein R is
H and R' is OC2H5, whenever produced by the process of claim
21 or its obvious chemical equivalent.
23. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is C2H5 and R' is OC2H5.
24. A polypeptide according to claim 4 wherein R is
C2H5 and R' is OC2H5, whenever produced by the process of
claim 23 or its obvious chemical equivalent.
25. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is CH3 and R' is NH2.
26. A polypeptide according to claim 4 wherein R is
CH3 and R' is -NH2, whenever produced by the process of claim
25 or its obvious chemical equivalent.
27. A process for the manufacture of a polypeptide, as
claimed in claim 1, wherein R is ASP and R' is OH.
28. A polypeptide according to claim 4 wherein R is
ASP and R ' is OH, whenever produced by the process of claim

27 or its obvious chemical equivalent.
29. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is SER-ASP and R' is OH.
30. A polypeptide according to claim 4 wherein R is
SER-ASP and R' is OH, whenever produced by the process of
claim 24 or its obvious chemical equivalent.
31. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is LEU-SER-ASP and R' is OH.
32. A polypeptide according to claim 4 wherein R is
LEU-SER-ASP and R' is OH, whenever produced by the process of
claim 31 or its obvious chemical equivalent.
33. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is LEU-SER-ASP and R' is GLU.
34. A polypeptide according to claim 4 wherein R is
LEU-SER-ASP and R' is GLU, whenever produced by the process
of claim 33 or its obvious chemical equivalent.
35. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is LEU-SER-ASP and R' is
GLU-SER.
36. A polypeptide according to claim 4 wherein R is
LEU-SER-ASP and R' is GLU-SER, whenever produced by the
process of claim 35 or its obvious chemical equivalent.
37. A process for the manufacture of a polypeptide,
as claimed in claim 1 wherein R is LEU-SER-ASP and R' is
GLU-SER-THR.
36

38. A polypeptide according to claim 4 wherein R is
LEU-SER-ASP and R' is GLU-SER-THR, whenever produced by the
process of claim 37 or its obvious chemical equivalent.
39. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is LEU-SER-ASP and R' is
GLU-SER-THR-LEU.
40. A polypeptide according to claim 4 wherein R is
LEU-SER-ASP and R' is GLU-SER-THR-LEU, whenever produced by
the process of claim 39 or its obvious chemical equivalent.
41. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is LEU-SER-ASP and R' is
GLU-SER-THR-LEU-HIS.
42. A polypeptide according to claim 4 wherein R is
LEU-SER-ASP and R' is GLU-SER-THR-LEU-HIS, whenever produced
by the process of claim 41 or its obvious chemical equivalent.
43. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is ASP and R' is GLU.
44. A polypeptide according to claim 4 wherein R is
ASP and R' is GLU, whenever produced by the process of claim
43 or its obvious chemical equivalent.
45. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is ASP and R' is GLU-SER.
46. A polypeptide according to claim 4 wherein R is
ASP and R' is GLU-SER, whenever produced by the process of
claim 45 or its obvious chemical equivalent.
37

47. A process for the manufacture of a polypeptide,
as claimed in claim 1, wherein R is H and R' is GLU-SER-THR-
LEU-HIS-LEU-VAL-LEU-ARG-LEU-ARG.
48. A polypeptide according to claim 4 wherein R is
hydrogen and R' is GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU-ARG,
whenever produced by the process of claim 47 or its obvious
chemical equivalent.
38

Description

11~5923
It is well known that many polypeptides have been
isolated from various organs of animals. Until about the past
decade, however, very little was known about the thymus, an organ
which in man comprises about 0.8% of his body weight at birth,
although it has been previously hypothesized that a neuromuscular
blocking substance existed in the thymus. Despite keen interest
in possible functions of the thymus and early speculation and
experimentation, little was known of the function of the thymus
until recently. It is now realized, however, that the thymus is
a compound organ with both epithelial (endocrine) and lymphoid
(immunological) compounds and thus the thymus is involved in the
immunity functions of the body. The thymus is known to be a
compound organ consisting of an epithelial stroma derived from the
third branchial arch and lymphocytes derived from stem cells
originating in haemopoietic tissues, Goldstein et al, The Human
Thymus, Heinemann, London, 1969. Lymphocytes are differentiated
q~

5~23
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 but difficulties with bioassays had previously
hindered the complete isolation and structural characterization of
any hormones which may be present.
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 substances which
have been 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 Applicants have published a number
of articles which relate to research in this area. Pertinent
publications may be found for example in The Lancet, July 20, 1968,
pps. 119-122; Triangle, Vol. 11, No. 1, pps. 7-14, 1972; Annals
of the New York Academy of Sciences, Vol. 183, pps. 230-240, 1971;
and Clinical and Experimental Immunology, Vol. 4, No. 2, pps. 181-189,
1969; Nature, Vol. 247, pps. 11-14, 1974; Proceedings of the National
Academy of Sciences USA, Vol. 71, pps. 1474-1478, 1974; ell,
Vol. 5, pps 361-365 and 367-370, 1975; Lancet, Vol. 2, pps. 256-259,
1975; Proceedings of the National Academy of Sciences USA, Vol. 72,
pps. 11-15, 1975; Biochemistry, Vol. 14, pps. 2214-2218, 1974;
Nature, Vol. 255, pps. 423-424, 1975.
In the article by Goldstein and Manganaro in Annals of the
New York Academy of Sciences, Vol. 183, pps. 230-240, 1971, there
are disclosures regarding the presence of a thymic polypeptide

5~23
which causes a myasthenic neuromuscular block in animals,
which is analogous to the human disease of myasthenia gravis.
Further, in this article it was discovered that two distinct
! effects were caused by separate polypeptides in bovine thymus.
One of these polypeptides, named "thymotoxin", was believed
to cause myosltis but it was further indicated that this poly-
peptide had not been isolated although it appeared to be a
polypeptide of approximately 7,000 molecular weight, had a strong
net positive of approximately 7,000 molecular weight, had a
strong net positive charge and was retained on CM-Sephadex~ at
a pH of 8Ø
In the publication "Nature", 247, 11, January 4, 1975
there are described products indentified as Thymin I and Thymin
II which were found to be new polypeptides isolated from bovine
thymus which have particular uses in various therapeutic areas.
Because of the use of similar names for other products isolated
from the thymus in the prior art, these Thymin I and Thymln II
products are now named as Thymopoietin I and Tnymopoietin II.
These products and processes are described in United States
Patent 4,077,949 issued March 7, 1978.
In issued United States Patent 4,002,602 dated January
11, 1977, there are disclosed long chain polypeptieds described
in Ubiquitous Immunopoietic Polypeptide (UBIP). This peptide has
subsequently been renamed as Ubiquitin. This polypeptide is a
74-amino acid polypeptide characterized by its ability to induce
in vitro, in nanogram concentrations, the differentiation
B - 4 -
,: .. : , . .
.
. .:

1iLg} 5~23
of both T-cell and B-cell immunocytes from precursors present
in bone marrow or spleen. Thus, the polypeptide is useful in
therapeutic areas involving thymic or immunity deficiencies and
the like.
In issued U,S, Patent No. 4,002,740, dated January ll,
1977 there are disclosed synthesized tridecapeptide compositions
which have the capability of inducing the differentiation of T-
lymphocytes but not of complement receptor B-lymphocytes.
This polypeptide thus exhibited many of the characteristics of
the long chain polypeptides isolated and narned as thymopoietin
in above-mentioned ~.S. Pat~nt 4,077,949.
The present invention provides a synthesized ~ive-amino
acid polypeptide having a definite active site sequence which has
been found to exhibit many of the characteristics of the long
chain polypeptide isolated and named as Ubiquitous Immunopoietic
Polypeptide (UBIP) in the above publications and U.S. Patent No.
4,002,602, dated January 11, 1977.
SUMMARY OF THE INVENTION
It is accordingly one object of this invention to
provide new polypeptides which are important biologically.
A further object of the invention is to provide new
polypeptides which have the ability in nanogram concentrations
to induce differentiation of both T-precursor cells as well as
B-precursor cells and are thereby highly useful in the immunity
systems of humans and animals.
A further object of the invention is to provide novel
intermediate products, methods for synthesizing the novel poly-
peptides of this invention, as well as compositions and methods
for use in biological actions.
- 5

~5g2l3
Other objects and advantages of the invention will
become apparent as the description thereof proceeds.
In satisfaction of the foregoing objects and advantages
there are provided by this invention novel polypeptides having
the following sequence as the active site:
-X-Y-Z-GLN-LYS-
wherein X is TYR or ALA, Y is ASN or ALA, and Z is ILE or ALA.
There are also provided novel polypeptide-resin
intermediates formed in the preparation of the polypeptide of
this invention which intermediate has the following sequence:
~ -R3-X-Y-Z-GLN-LYS-Resin
as well as this peptide intermediate freed from the resin and
other protecting groups, wherein X, Y and Z are as above, and
Rl, R2, and R3 represent protecting groups on the amino acids
indicated if such groups are necessary, and the resin is a solid
phase polymer which acts as a support for the reaction. Also
provided is a procedure for preparation of the polypeptide of
the invention by solid phase peptide synthesis, as well as
therapeutic compositions containing the polypeptide, and methods
for administration of the polypeptide to humans and animals for
effecting biological actions thereon.
DESCRIPTION OF PREFERRED EMBODIMENTS
As indicated above, this invention is concerned with
new polypeptides having therapeutic value in various areas,
intermediates formed in the preparation of the polypeptides,
therapeutic compositions and methods for their use utilizing the
polypeptides of this invention, and methods for manufacture of
the polypeptides.
In the main embodiment of the present invention, there
are provided polypeptides which have the following amino acid
sequence as the active site:

Sg23
I. X-Y-Z-GLN-LYS
wherein X is TYR or ALA, Y is ASN or ALA and Z is ILE or ALA,
with X being TYR, Y being ASN and Z being ILE, especially
preferred.
In a further embodiment, there are provided pentapeptides
containing the above mentioned sequence as the active site and
which may be described by the following general formula:
II. R-NH-X-Y-Z-GLN-LYS-COR'
wherein X, Y and Z are as described above, and R and R' are
substituents on the pentapeptide sequence which do not substantially
affect the biological activity of the basic active sequence. By
this statement is meant that the terminal amino acids on this
pentapeptide chain may be modified without departing from the scope
of the invention when functional groups or derivatives (R and R')
are placed on these terminal amino acids without substantially
affecting the biological activity of the molecule. Thus it is
to be understood that the terminal amino and carboxylic acid
groups are not essential to the biological activity of the penta-
peptide as in some polypeptides. Therefore, it is considered that
the scope of the present invention is inclusive of these penta-
peptides which are terminally unsubstituted and which are terminally
substituted by one or more functional groups which do not
substantially affect the biological activity disclosed herein.
From this statement it will be understood that these
functional groups include such normal substitution as acylation
on the free amino group and amidation on the free carboxylic
acid group, as well as the substitution of additional amino
acids and polypeptides. In these aspects the pentapeptides
of this invention appear to be unique since the pentapeptides
exhibit the same biological activity as long chain natural
peptides in which this pentapeptide sequence forms a portion

5923
or occurs therein. It is believed therefore that the activity
requirements of the molecule are generated by stereochemistry
of the molecule, that is, the particular "folding" of the
molecule. In this regard, it should be understood that polypeptide
bonds are not rigid but flexible, and may exist 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 pentapeptide "folds" in
the same manner as the long chain natural polypeptide and therefore
exhibits the same biological characteristics. For this reason,
the pentapeptide may be substituted by various functional groups
so long as the substituents do not substantially affect the
biological activity or interfere with the natural "folds"
of the molecule.
The ability of the molecule to retain its biological
activity and natural folding is clearly illustrated by the fact
that the pentapeptide sequence of this invention exhibits the
same biological characteristics as the natura~l seventy-four amino
acid peptide disclosed as Ubiquitin in Patent No. 4,002,602. In
this long chain polypeptide, the pentapeptide of this invention may
be identified within the molecule but only in combination with the
other amino acids described therein. However, this patent is
direct evidence that the pentapeptide of this invention is
the active site since the biological activities are the same
and the amino acids and peptide chains substituted on the
terminal amino acids do not affect the biological characteristics
of the basic pentapeptide fragment.

923
In view of this discussion therefore, it will be
understood that R and R' in formula II can be any substituent
that does not substantially affect the biological activity of
the basic active sequence. Thus, for purposes of illustra-
tion R and R' may be any of the following substituents:
R R'
Hydrogen OH
Cl-C7 alkyl NH2
5 12 Y NHR7
C6-C20 alkaryl ( 7)2
C6-C20 aralkyl 7
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 aralkyl or C6-C20 alkaryl,
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'
ASP GLU
SER SER
LEU THR
SER-ASP LEU
LEU-SER-ASP HIS
LEU-ASP VAL
LEU-SER ARG
GLU-SER
h

~S9~3
R'
GLU-THR
GLU-SER-LEU
GLU-SER-THR
GLU-SER-THR-LEU
GLU-SER-THR-LEU-HIS
GLU-SER-THR-LEU-HIS-LEU
GLU-SER-THR-LEU-HIS-LEU-VAL
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU
GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG-LEU-ARG
In a more specific embodiment of the invention,
there are provided novel pentapeptides having the following
sequence:
III. R-HN-TYR-ASN-ILE-GLN-LYS-COR'
wherein R is hydrogen, Cl-C7 alkyl, e.g. methyl, ethyl,
C5-C12 aryl, e.g. phenyl, or Cl to C7 alkanoyl, e.g. acetyl
or propionyl and R' is OH, NH2, NHR7, N(R7)2. The most
preferred polypeptides are those wherein R is hydrogen and
R' is OH.
Also included within the scope of the invention are
the pharmaceutically acceptable salts of the pentapeptides.
As acids which are able to form salts with the pentapeptides,
there may be mentioned inorganic acids such as hydrochloric
acid, hydrobromic acid, perchloric acid, nitric acid,
thiocyanic acid, sulfuric acid, phosphoric acid, etc.
and organic acids such as formic acid, acetic acid,
propionic acid, glycolic acid, lactic acid, pyruvic
acid, oxalic acid, malonic acid, succinic acid,
, , -- 1 0

592'3
maleic acid, fumaric acid, anthranylic acid, cinnamic acid,
naphthalenesulfonic acid or sulfanylic acid, for instance.
In the above structure the amino acid components of
the peptide are identified by abbreviations for convenience.
These abbreviations are as follows:
Amino Acid Abbreviated Designation
L-Tyrosine TYR
L-Asparagine ASN
L-Aspartic acid ASP
L-Isoleucine ILE
L-Serine SER
L-Glutamine GLN
L-Leucine LEU
L-Lysine LYS
L-Glutamic GLU
L-Threonine THR
L-Histidine HIS
L-Valine VAL
L-Arginine ARG
L-Alananine ALA
The polypeptides of this invention are five-amino acid
peptides which have been found to exhibit characteristics similar
to the 74 amino acid polypeptide Ubiquitin (or UBIP) isolated from
bovine thymus as disclosed in Patent No. 4,002,602. The peptides
of this invention are particularly characterized in their ability
to induce the selective differentiation of T-precursor cells as
well as B-precursor cells in nanogram concentrations.
It has been found that the polypeptides of this invention
induce the differentiation of immunocyte-precursor cells in vitro
in the same way as the long chain polypeptides disclosed and

BS~Z3
described in Patent No. 4,002,602. Thus, the polypeptides of this
invention, even in nanogram concentrations, have been found
to induce the differentiation of both T-precursor cells as
measured by the acquisition of the thymic differentiation antigens
TL and THY-l (~), as well as B-precursor cells as measured by
the acquisition of receptors for complement, a distinctive marker
of B cells.
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 the pool of recirculating small
lymphocytes. T cells have immunological 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 as these antibodies are secreted
by cells derived directly from the bone marrow independently of
the thymic influence and these latter cells are termed B cells.
However, for many antigens, B cells require the presence of
appropriately reactive T cells before they can produce antibodies.
The mechanism of this process 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 responses and it affects these
systems by inducing, within the thymus, the differentiation of
haemopoietic stem cells to T cells. This inductive influence is
mediated by secretions of the epithelial cells of the thymus, that
is, the thymic hormones.

~1~59;~3
Further, to understand the operation of the thymus and
the cell system of lymphocytes, and the circulation 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
thymus, stem cells become differentiated to immunologically
competent T cells, which migrate to the blood stream and together
with B cells, circulate between the tissues, lymphatics, and
the blood stream.
The cells of the body which secrete antibody also
develop from hemopoietic stem cells but their differentiation
is not determined by the thymus. Hence, they are termed bone
marrow-derived cells or B cells. In birds they are differentiated
in an organ analogous to the thymus, which is called the Bursa
of Fabricius. In mammals no equivalent organ has been discovered
and it is thought that B cells differentiate within the bone
marrow. 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 capability of inducing the
differentiation of lymphopoietic stem cells originating in the
hemopoietic tissues to thymus-derived cells or T cells which are
capable of involvement in the immune response of the body and also
of inducing the differentiation of B cells, 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 functions of the thymus they have application
in various thymic function and immunity areas. A primary field of

11~5923
application is in the treatment of DiGeorge Syndrome, a condition
in which there is a congenital absence of thymus. Injection of
the polypeptides will overcome this deficiency. Another
application is in agammaglobulinemia which is due to a defect
of the putative s cell differentiative hormone of the body.
Injection of the polypeptides will overcome this defect. Because
of its biological characteristics, the polypeptides being extremely
active at low concentrations, are useful in assisting the collective
immunity of the body in that they increase or assist in
therapeutic stimulation of cellular immunity and humoral immunity
and thereby become 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 peptides are considered to be useful in any
area in which cellular or humoral immunity is an issue and
particularly where there are deficiencies in immunity such as in
the DiGeorge Syndrome mentioned above. Further, because of the
characteristics of the polypeptides, they have in vitro usefulness
in inducing the development of surface antigens of T cells, in
inducing the development of the functional capacity to achieve
responsiveness to mitogens and antigens and cell collaborativity
in enhancing the ability of B cells to produce antibodies. They
have ln vitro usefulness in inducing the development of B cells
as measured by the development of surface receptors for complement.
The peptides are also useful in inhibiting the uncontrolled
proliferation of lymphocytes which are responsive to Ubiquitin.
An important characteristic of the polypeptide is its in vivo
ability to restore cells with the characteristics of T cells and
also its in vivo ability to restore cells with the characteristics
of B cells.
- 14 -

59~:3
A further important property of the peptides of this
invention are that they are highly active in very low concentrations.
Thus, it has been found that the peptides are active in
concentrations ranging from 10 nanogram per ml, and are maximally
active at concentrations from about 0.05-1 microgram per 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 concentrations. The peptides of this
invention are active at a range of above about 0.1 mg/kg of
body weight. For the treatment of DiGeorge Syndrome, the poly-
peptides may be administered at a rate of about 0.1 to 10 mg/kg
of body weight. Generally, the same range of dosage amounts may
be used in treatment of the other conditions or diseases mentioned.
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, pps. 2149-2154, 1963.
The synthesis involved the stepwise addition of protected amino
acids to a growing peptide chain which was bound by covalent bonds
to a solid resin particle. By this procedure, reagents and by-
products were removed by filtration and the recrystallization of
intermediates were eliminated. The general concept of this method
depends on attachment of the first 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 manner until the desired
sequence is assembled. Finally the peptide is removed from the
solid support and protective groups removed. This method provides
a growing peptide chain attached to a completely insoluble solid
particle so that it is in a convenient form to be filtered and
washed free of reagents and by-products.
- 15 -

~S~23
The amino acids may be attached to any suitable polymer
which merely has to be insoluble in the solvents used and have a
stable physical form permitting ready filtration. It must contain
a functional group to which the first protected amino acid can be
firmly linked by a covalent bond. Various polymers are suitable for
this purpose such as cellulose, polyvinyl alcohol, polymethacrylate
and sulfonated polystyrene but in the synthesis of this invention,
there was used a chloromethylated copolymer of styrene and
divinylbenzene.
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 groups on
tyrosine and lysine were protected by protecting groups which could
be removed on completion of the sequence without adversely
affecting the polypeptide final product. The synthesis was
performed by a modification of the solid synthesis method in that
fluorescamine was used to determine if coupling was complete by
an indication of positive fluorescence (see Felix et al, Analyt,
Biochem., 52, 377, 1973). If complete coupling was not indicated,
the coupling was repeated with the same protected amino acid
before deprotection.
The general procedure involved initially esterifying
L-lysine, protected on its amino groups, to the resin in absolute
alcohol containing an amine. The coupled amino acid resin was then
filtered, washed with alcohol and water and dried. The protecting
group on the a-amino group of the lysine amino acid (e.g. t-BOC, i.e.,
t -butyloxycarbonyl), was then removed without affecting other
protecting groups. The resulting coupled amino acid resin, having
- 16 -

5i923
the free amino group, was then reacted with a protected L-glutamine,
preferably alpha-tBOC-L-glutamine to couple the L-glutamine. The
reactions were then repeated with protected L-isoleucine, L-
asparagine and L-tyrosine until the complete molecule was prepared.
The sequence of reactions was carried out as follows:
Resin lR2
1~_BOC_LYS-COOH
~2
~-BOC-Lys-Resin
Remove ~-amino protecting group
lR2
H2N-Lys-Resin
~-BOC-L-Gln
, IR2
~-BOC-Gln-Lys-Resin
¦ Remove ~-amino protecting group
~lR2
H2N-Gln-Lys-Resin
¦ ~-BOC-Ile-COOH
~1 lR2
~-BOC-Ile-Gln-Lys-Resin
¦ Remove ~-amino protecting group
J, IR2
H2N-Ile-Gln-Lys-Resin
¦ ~-BOC-Asn-COOH
~, R2
~-BOC-Asn-Ile-Gln-Lys-Resin
¦ Remove ~-amino protecting group
~ IR2
H2N-Asn-Ile-Gln-Lys-Resin
Rl
C~-R3-Tyr-COOH
R2
~-R3-Tyr-Asn-Ile-Gln-Lys-Resin
~ Remove all protecting groups
H2N-Tyr-Asn-Ile-Gln-Lys-COOH

5923
In the above sequence of reactions Rl and R2 are
protecting groups on the various reactive side chains on the
amino acids which are not affected or removed when the
protective group on the a-amino group is removed to permit
further reaction and a-R3 is a protecting group of the a-
amino group. Preferably in the above intermediate pentapep-
tide resin, the term Rl stands for a protective grouping such
as 0-2,6-dichlorobenzyl, R2 stands for ~-2-chlorobenzyloxy-
carbonyl and R3 stands for t-butyloxycarbonyl. The resin is
any of the resins mentioned above as being useful in the
process. In the above series of reactions, the peptides
where ALA is substituted for TYR, ASN, or ILE, are prepared
in the same manner.
After the final intermediate is prepared, the
peptide-resin is cleaved to remove the Rl, R2 and R3
protecting groups thereon and the resin. The protecting
groups are removed by conventional means, e.g., by treat-
ment with anhydrous hydrogen fluoride, and the resulting
free peptide was then recovered.
While the preferred method for production of the
polypeptides of this invention is by the use of an insoluble
solid polymer as described in the method of Merrifield, it
is also to be understood that other methods for preparation
may also be used. For example, a different solid support
may be employed such as an N-methyl-benzhydrylamine resin
or benzhydrylamine resin, which is advantageous under certain
conditions. In this procedure the C-terminal amino acid is
attached directly to the resin and the finished peptide maybe
cleaved from the substrate in HF to form C-terminal amides.
,.,_ " ~."
1 8 -
.. .. .

S~23
Techniques for use of an N-methyl-benzhydrylamine resin are
described by Monahan et al in Biochemical and Biophysical
Research Communications, _, 1100-1105 (1972). TechniqueS for
use of a benzhydrylamine resin are described by J. Rivier, et
al, in Journal of Medicinal Chemistry, 1973, vol. 16, 545-549.
Various derivatives of the basic pentapeptide may
also be produced using methods known to the art. Obviously, in
the production of such derivatives it will be necessary to block
functional groups which might interfere with the reaction
sequence in order to produce the desired product. For example,
the alpha-carboxylic acid group of aspartic acid or the alpha-
amino group of lysine should be blocked during preparation of
these derivatives.
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 obtain the products desired. Suitable
amino-protecting groups which may be usefully employed include
salt formation for protecting strongly-basic amino groups, or
urethane protecting substitutes such as benzyloxycarbonyl and
t-butyloxycarbonyl. It is preferred to utilize tert-butyloxy-
carbonyl (tBOC) 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-amyloxy-
carbonyl) protecting groups are readily removed following such
reaction and prior to the subsequent step (wherein such a-amino
group itself undergoes reaction) by relatively mild action of
acids (e.g., trifluoroacetic acid) which treatment does not
otherwise affect groups used to protect other reactive side
chains. It will thus be understood that the a-amino
- 18A -

~5~23
groups may be protected by reaction with any material which will
protect 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 protected by
reaction with a compound containing a grouping of the formula:
1l
R - O - C -
wherein R 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 R is a straight
or branched chain alkyl which may be unsaturated, preferably of 1
to 10 carbon atoms, 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
hetercyclic, e.g., isonicotinyl. The aryl, aralkyl and alkaryl
moieties may also be further substituted as by one or more alkyl
groups of 1 to about 4 carbon atoms. Preferred groupings for R
include tertiary-butyl, tertiary-amyl, phenyl, tolyl, xylyl and
benzyl. Highly preferred specific amino-protecting groups include
benzyloxycarbonyl; substituted benzyloxycarbonyl wherein the phenyl
ring is substituted by one or more halogens, e.g., Cl or Br; nitro;
lower alkoxy, e.g., methoxy; lower alkyl; tertiary-butyloxycarbonyl,
tertiary-amyloxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl;
adamantyloxycarbonyl; biphenylisopropoxycarbonyl; and the like.
Other protecting groups which can be used include isonicotinyloxy-
carbonyl, phthaloyl, para-tolylsulfonyl, formyl and the like.
-- 19 --

~S923
In conducting the general process of the invention, 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 attacked 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
activation the carboxyl group can be converted to any reactive
group such as an ester, anhydride, azide, acid chloride, or the
like.
It should also be understood that during these
reactions,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 epsilon-amino of the lysine molecule. The preferred procedure
for effecting this step is mild acidolysis, as by reaction at
room temperature with trifluroacetic acid.
As may be appreciated, the above-described series
of process steps results in the production of the specific
pentapeptide in the following formula:
H2N-TYR-ASN-ILE-GLN-LYS-COOH
This pentapeptide also includes the basic active-site
sequence of the polypeptide of this invention. The substituted
pentapeptide of Formula II, wherein the terminal TYR and LYS amino
acid groups may be further substituted as described above, are then
prepared by reaction of this basic pentapeptide with suitable
reagents to prepare the desired derivatives. Reactions of this
- 20 -

S923
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 basic pentapeptide molecule, are added to the
peptide chain by the same sequence of reactions by which the
pentapeptide was synthesized to either end of the peptide chain.
The following examples are presented to illustrate 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-asparagine
Alpha-BOC-L-Isoleucine
Alpha-BOC-0-2,6-dichlorobenzyl-I,-tyrosine
In these reagents, BOC is t-butyloxycarbonyl. "Sequenal"
grade reagents for amino acid sequence determinations, 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 benzene and .75 mM
of chloride per gram of resin.
In preparation of the polypeptide, 2m moles of ~-BOC-~-2-
chlorobenzyloxycarbonyl-L-lysine were esterified to 2m moles
of chloromethylated resin in absolute alcohol containing lmM
triethylamine for 24 hours at 80C. The resulting coupled amino
acid resin was filtered, washed with absolute alcohol and dried.
- 21 -

5~3!Z3
Thereafter~ the other ~-BOC amino acids were similarly coupled to
the deprotected ~-amino group of the peptide-resin in the correct
sequence to result in the polypeptide of this invention using
equivalent amounts of dicyclohexyl carbodiimide except for ~-BOC-L-
glutamine-O-nitrophenyl ester which was coupled directly. After
each coupling reaction, an aliquot of resin was tested with
fluorescamine and if positive fluorescence was found, coupling was
taken to be incomple-te and was repeated with the same protective
amino acid. As a result of the several coupling reactions, the
intermediate pentapeptide-resin was prepared.
This peptide-resin was cleaved and the protective
groups removed in a Kelf cleavage apparatus (Peninsula Laboratories,
Inc.) using anhydrous hydrogen fluoride at 0C. for 60 minutes
with 1.2 ml anisole per gram peptide-resin as scavenger. The
peptide mixture was lyophilized and washed with anhydrous ether
and the peptide was chromatographed on P-6 Bio-Gel in 1 N acetic
acid. The resulting polypeptide was determined to be 94% pure
and was determined to have the following sequence.
H2N-TYR-ASN-ILE-GLN-LYS-COOH
EXAMPLE II
To determine the activity and characteristics of the
polypeptide, determinations were carried out on healthy 5-6 week
old nu/nu mice of both sexes, the mice being bred on a BALBtc
background (thymocytes expressing Thy-1.2 surface antigen) and
maintained under conventional conditions. For the antisera, anti
Thy-1.2 sera were prepared in Thy-l congenic mice.
For the induction of in vitro of Thy-l T cell or
-
CR B cell differentiation, the induction of thymocyte
-- - 22 -
:

~5923
differentiation from prothymocytes ln vltro was performed as
described by Komuro and Boyse, (Lancet, 1, 740, 1973~, using the
acquisition of Thy-1.2 as a marker of T cell differentiation.
The induction of CR B cell differentiation from CR B cell
precursors _ vitro was performed under similar conditions using
as the assay criterion, the capacity of CR s cells to bind sheep
erythrocytes coated with subagglutinating quantities of rabbit
antibody and nonlytic complement. Spleen cell populations from
health nu/nu mice fractionated on discontinuous bovine serum
albumin gradients were used as the source of both precursor
types (Thy-l and CR ) because they have very few or no Thy-l
cells and low numbers of CR cells.
As a result of this determination it was found that
the polypeptide displayed a selectivity of actions similar to
that of Ubiquitin in inducing the differentiation of T-
lymphocytes and of complement receptors (CR+) B-lymphocytes.
The pentapeptide induced differentiation of Thy-l+ T cells in
concentrations ranging from lOng to 1 ~g/ml, and also induced ,~
the differentiation of CR B cells in concentrations of 10 ng to
1 ~g/ml.
- 23 -

S9Z3
EXAMPLES III and IV
Example III
The pentapeptide prepared as in Example I, while
still coupled to the resin, is treated with trifluoroacetic
acid in dichloromethane to remove the t-BOC protecting group
from the tryosine moiety. The resulting peptide is then
acylated with acetic anhydride, followed by cleavage from the
resin substxate and removal of all protective groups with HF.
The following acrylated derivative is thus produced:
A. CH3CONH-TYR-ASN-ILE-GLN-LYS-COOH
Example IV
To produce the amidated derivative of the pentapep-
tide of Example I, the protected pentapeptide is Prepared using
a benzhydrylamine resin as the substrate, following the method
of J. Rivier, et al, referred to above. The amidated deriva-
tive is produced by cleaving the protected pentapeptide attached
to the basic resin by reaction with hydrogen fluoride. The
amidated pentapeptide has the following formula:
B. H2N-TYR-ASN-ILE-GLN-LYS-CONH2
For identification, thin layer chromatography and
electrophoresis were employed which provided the following data:
Thin Layer Chromatography:
Sample: 30 ~g
Silica Gel (Brinkman glass plate, 5x20cm, 0.25mm thickness)
Rl : n-BuOH : Pyridine : HOAc : H2O 30 : 15 : 3 : 12
Rf : EtOAc : Pyridine : HOAc : H2O 5 : 5 : 1 : 3
R : EtOAc : n-BuOH HOAc : H2O 1 : 1 : 1 :
f
Spray reagent: Pauly, Ninhydrin & I2
- 24 -
.

~l~iS~23
TLC Electrophoresis
R R2 R3 Peptide moved
Compounds: f f f to cathode
Ac-Tyr-Asn-Ile-Gln-Lys 0.47 0.87 0.54 - 1.35 cm
Tyr-Asn-Ile-Gln-Lys-NH2 0.48 0.87 0.37 - 6.60 cm
Electrophoresis:
Whatman 3 mm paper (11.5cm x 56.5cm)
Sample : 100 ~g
pH 5.6, Pyridine-Acetate buffer solution
1000 V, 1 hour
Spray reagent: Pauly & Ninhydrin
The acetylated pentapeptide of Example III, when
utilized in a concentration of 1 ~g/ml in 14% Twomey solution,
showed maximum and minimum activities comparable to the basic
pentapeptide of Example I.
The amidated pentapeptide of Example IV, when
utilized in a concentration of 1 ~g/ml in 6~ Twomey solution,
showed activity comparable to the basic pentapeptide of
Example I.
EXAMPLE V
The basic pentapeptide prepared according to Example
I, while still attached to the resin, is cleaved from the resin
by transesterification with sodium methoxide in methanol under
transesterification conditions. Removal of the protecting
groups yields the esterified product of the following formula:
H2N-TYR-ASN-ILE-GLN-LYS-COOCH3
EXAMPLE VI
The diethylamine derivative of the pentapeptide
prepared as in Example I is produced from the methyl ester
produced in Example V. In this reaction the methyl ester of
B - 25 -

Lgl 5~23
Example V is reacted with diethylamine in dimethylformamide
solution to produce the following diamino-substituted amide:
H2N-TyR-AsN-ILE-GLN-Lys-coN(c2H5)2
This diethylamine derivative can also be prepared
by reaction of the basic pentapeptide of Example I, while
still attached to the resin substrate, by cleavage of the
peptide from the resin by reaction with diethylamine. The
resulting product after removal from the resin and removal
of protecting groups is the diethylamide derivative.
EXAMPLE VII
In this example the N-ethyl tyrosine derivative
of the basic pentapeptide of Example I is prepared by reaction
with ethyl bromide. To carry out the reaction, the alpha-
amino group on the lysine moiety remains blocked by ~-2-chloro-
benzyloxycarbonyl group but the TYR terminal amino group is
freed by reaction with trifluoroacetic acid in dichloromethane.
The blocked intermediate is then reacted with a stoichiometric
amount of ethyl bromide under alkylation conditions. The pro-
tective group is then removed from the LYS alpha-amino acid to -
form the substituted polypeptide of the following formula:
C2H5NH-TYR-ASN-ILE-GLN-LYS-COOH
EXAMPLE VIII
In this example, the amidated derivative of the
ethylamino polypeptide of Example VII is produced. The
reactions of Example VII are carried out while permitting
the basic pentapeptide to remain coupled to the resin sub-
strate and the N-ethyl derivative is produced without cleav-
ing from the substrate. Thereafter, this intermediate product
attached to the resin substrate is cleaved from the resin with
';'
- 26 -

5923
anhydrous ammonia in dimethylformamide solvent to form the
amido polypeptide of the following formula:
C2H5NH-TYR-ASN-ILE-GLN-LYS-CONH2
EXAMPLE IX
In this example, the acylated pentapeptide prepared
as in Example III, while still attached to the resin substrate,
is cleaved by reaction with anhydrous ammonia under amidation
conditions to free the compound and, following removal of the
protecting groups, form the following polypeptide:
CH3CONH-TYR-ASN-ILE-GLN-LYS-CONH2
EXAMPLES X-XXI
Using the reaction techniques described hereinabove
for the lengthening of the polypeptide chain, the following
polypeptides are prepared which contain the active amino acid
sequence but which are substituted on the terminal amino and
carboxylic groups by R and R' to provide the basic amino acid
of the formula:
R-NH-TYR-ASN-ILE-GLN-LYS-COR'
which is substituted by the amino acids given in the following
Table as indicated.
EXAMPLE NO. _ R'
X ASP OH
XI SER-ASP OH
XII LEU-SER-ASP OH
XIII LEU-SER-ASP GLU
XIV LEU-SER-ASP GLU-SER
XV LEU-SER-ASP GLU-SER-THR
XVI LEU-SER-ASP GLU-SER-THR-LEU
XVII LEU-SER-ASP GLU-SER-THR-LEU-HIS
- 27 -

~5~23
EXAMPLE NO. R R'
XVIII LEU-SER-ASP GLu-sER-THR-LEu-HIs-LEu
XIX ASP GLU
XX ASP GLU-SER
XXI LEU-SER-ASP GLU-SER-THR-LEU-HIS-LEU-VAL-LEU-ARG
The polypeptide derivatives prepared in Examples
V-XXI retain the biological activity as described herein for
the basic amino acid sequence.
EXAMPLES XXII, XXIII and XXIV
Using the sequencing procedure described for
Example I, the followingpentapeptides are prepared:
Ex. XXII H2N-ALA-ASN-ILE-GLN-LYS-COOH
Ex. XXIII H2N-TYR-ALA-ILE-GLN-LYS-COOH
: Ex. XXIV H2N-TYR-ASN-ALA-GLN-LYS COOH.
To determine the pharmacological activity and
characteristics of these pentapeptides, use was made of the
induction of Th-l (T-cell) antigen on chicken bone marrow for
use as an assay of the peptides at a concentration of 1 ~g/mil.
This method is described in Science, Brand et al, Vol. 193,
pp. 319-321, July, 1976. In this method pooled cells from
femur and tibiotarsus of newly hatched chicks of strain SC
(Hy-Line) are fractionated by ultracentrifugation on a five-
layer discontinuous bovine serum albumin (BSA) gradient (11).
Cells from each interface are washed and suspended for incubation
at a concentration of 5 x 106 cells per milliliter with the
peptide (0.1 ~g/ml) in RPMI 1630 medium supplemented with 15 mM
Hepes, 5 percent y-globulin-free fetal calf serum, deoxyribonu-
clease (14 to 18 unit/ml), heparin (5 unit/ml), penicillin
(100 unit/ml), and streptomycin (100 ~g/ml). Controls are
1.,~,
- 28 -

~l~S923
incubated with BSA (l ~g/ml) or medium alone. After incubation,
the cells are tested in the cytotoxicity assay using chicken
and guinea pig complement fractions. The proportion of Bu-l
or Th-l cells in each layer are 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, respec-
tively. The percentage of cells induced is obtained by sub-
tracting the mean values in the control incubations without
inducing agents (usually l to 3 percent) from those of the test
inductions.
As a result of the characterizations of this type,
the pentapeptides of Examples XXII, XXIII and XXIV were
found to show the following percentages of inductions of
Th-l (T-cell) antigen on chicken bone marrow.
Example No. ~ Induction
XXII 12%
XXIII 15%
XXIV 21%
The invention has been described herein with
reference to certain preferred embodiments. However, as
obvious variations will appear to those skilled in the art,
the invention is not to be considered as limited thereto.
- 29 -
, :