Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-INFLAMMATORY, TOLEROGENIC AND
IMMUNOINHIBITING PROPERTIES
OF CARBOHYDRATE BINDING PEPTIDES
Background of the Invention
5 1. Field of the Invention.
This invention is directed to methods for inhibiting immune
responses or cellular interactions in mammals by the administration
thereto of one or more lectin derived carbohydrate binding peptides. In
particular, this invention is directed to methods for the suppression of
10 inflammatory responses, induction of tolerance to antigens, modulation of
the induction of immune responses to antigens, and the inhibition of cell
adhesion in mammals by the administration of one or more carbohydrate
binding peptides. The lectin derived carbohydrate binding peptides
employed herein are preferably fragments of the S2 or S3 subunits of the
15 pertussis toxin expressed by Bordetella pertussis or functionally equivalent
variants thereof.
2. References.
The following references are cited in this application as superscript
numbers at the relevant portion of the application:
1. Brandley, et al., J. Leukocyte Biol., 40:97-111 (1986).
2. Jacobson, Developmental Neurobiology, New York, Plenum
Press, p. 5-25 (1978).
3. Trinkaus, Cells into Organs, Englewood Cliffs, N.J.,
Prentice Hall, p. 44-68 (1984).
4. Frazier, et al., Ann~. Rev. Biochem., 48:491 (1979).
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5. Glaser, Mediator of Developmental Processes (Substency, S.
and Wessels, N.K., Eds.) New York, Academic Press, p.
79 (1980).
6. Paulson, In ~The Receptors", Vol. II (Comm., P.M., Ed.),
S New York Academic Press, p. 131 (1985) .
7. Sharon, Lectin-Like Bacterial Adherence to Animal Cells. In
"Attachment of Microorganisms to the Gut Mucosa"
(Boeheker, E.D., Ed.), Boca Raton, Florida CRC Press, p.
129 (1984).
8. Wassarman, Fertilization. In "Cell Interactions and
Development: Molecular Mech~nicms" (Y~m~ , K.M.,
Ed.), New York, John Wiley and Sons, p. 1 (1983)
9. Schwartz, et al., Immunol. Rev., 40:153 et seq. (1978).
10. Coutinho, et al., Immunol. Rev., 78:211 et seq. (1984).
11. Hoffmann, et al., Eds., Mem~ranes in Growth and
Development, New York, Alan R. Liss, p. 429-442, (1982).
12. Galeotti, et al., Eds., Membranes in Tumor Growth,
Amsterdam, Elsevier, p. 77-81 (1982).
13. Nicolson, et al ., Invas. Metas., 5: 144 et seq. (1985) .
14. Aplin, et al., Biochim. Biophys. Acta 694:375 et seq.
(1982).
lS. Barondes, Developmentally Regulated Lectins. In "Cell
Interactions and Development: Molecular Mech~nisrns"
(Yamada, D.M., Ed.) New York, John Wiley and Sons, p.
185 (1983).
16. Monisigny, M., Ed., Biol. Cell, 51 (Special Issue), 113 et
seq. (1984).
17. Springer, et al., Nature, 349: 196-197 (1991) .
18. Lowe, et al ., Cell, 63 :475-485 (1990) .
CA 02211563 1997-09-19
WO 94/07~17 PCIJCA93JûD4
19. Phillips, et al., Science, Vol. 250:1130-1132 (1990).
20. Walz, et al., Science, 250:1132 et seq. (1990).
21. Larsen, et al., Cell, 63:467-474 (1990).
22. Bevilacqua, et al., Endothelial-Leukocyte Adhesion Molecule
S - I (ELAM-I). A Vascular SEI ECTIN That Regulates
lnflammation. In "Cellular and Molecular Mech~nisms of
Inflammation" Vol. 2, Academic Press, p. 1-13 (1991).
23. McEver, Leukocyte Interactions Me~inte~l ~oy GMP-140. In
"Cellular and Molecular Mechanisms of Inflammation", Vol.
0 2, Academic Press, p. 1S-29 (1991).
24. Larsen, et al., J. Biol. Chem., 267: 11104-11110 (1992).
25. Heerze, et al., Biochem and Biophys. Res. Comm.
172:1224 1229 (1990).
26. Paulson, et al., International Palem Applicalion Publication
No. WO91/19502, (199l).
27. Ippolito, et al., U.S. Patent Application Serial No.
07/714,161, filed 10 June 1991.
28. Ippolito, et al., U. S. Patent Application Serial No.
07/889,017, filed 26 May 1992.
29. McEver, International Patent Application Publication No.
WO92/0171~ (1992).
30. Heerze, et al., Biochem and Biophys. Res. Comm.,
179:1464-1469 (1991).
31. Pearce-Pratt, et al., J. Imm. Methods, 140: lS9-165 (1991).
2s 32. Smith, et al., Cell. Imm., 89:20-29 (1984).
33. Munoz, Action of Pertussigen (Pertussis ~oxinJ on the Host
Immune System. In "Pathogenesis and Immunity in
Pertussis", John Wiley & Sons Ltd., p. 173-192 (1988).
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34. Reuter, et al, Glycoconjugates 5:133-135 (1988).
35. Gaeta, et al., U.S. Patent Application Serial No.
07/538,853, filed 15 June 1990.
36. Paulson, et al., U.S. Patent Application Serial No.
07/619,319, filed 28 November 1990.
37. Paulson, et al., U.S. Patent Application Serial No.
07/632,390, filed 21 December 1990.
38. Brandley, et al., International Patent Application Publication
No. W092/02527 (1992) .
39. Lowe, International Patent Application Publication No.
W092/07572 (1992).
40. Furie, et al., International Patent Application Publication
No. WO92/16612 (1992).
41. Seed, et al., International Patent Application Publication
No. W092/09293 (1992).
42. Karlsson, Annu. Rev. Biochem., 58:309-350 (1989).
43. Brennan, et al., J. Biol. Chem., 263:4895-4899 (1988).
44. Armstrong, et al., J. Biol. Chem., 263:8677-8684 (1988).
45. Smith, et al., U. S. Patent Application Serial No.
07/956,0431 filed 2 October 1992.
46. Shibuya, et al., J. Biol. Chem., 261:7755-7761 (1987).
47. Wang, et al., J. Biol. Chem., 263:4576-4585 (1988).
48. Gimbrone, et al., International Patent Application
Publication No. WO91/08231 (1991).
49. Ratcliffe, et al., U.S. Patent No. 5,079,353, issued 7
January 1992.
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wO 94/07517 PCI/CA93~0041
50. Ha}comori? Ad~. Cancer ~c'es., 52:257-331 (1989) .
51. Sia}ic Acids in " Cell Biology Monographs", Schauer. Editor,
Vol. 10 (1982).
52. Tyrrell, et al.~ Infect. Immun., 57:185~1857 (1989).
53. Bhav~n~ n, et al., J. Biol. Chem., 2~4:4000-4008 (1979).
54. Nogimo.~, et al., Biochemistry, 25: 13S5-1363 (1986).
SS. Sato, et al., Infec. ~mmun., 46:415-421 (1986).
56. Tamura, et al., Jr. Biol. Chem., 256:67~6-6761 (1983).
57. Tuomanen, et al., J. Exp. Med., 168:267-277 (1988).
58. Tuomm~nen, et al., 92nd Gener~l Mee~ing of the Amenc~n
Sociery of Microbiology, New Orleans, LA, Poster B-16.
59. Morgan, et al., Ann. Repts. Med. Chem., 24:243-252
(1989).
60. Chou, et al.t Annu. Rev. Biochem.7 47:251-276 (1978).
61. Hopp, et al., Proc. Natl. Acaa'. Sci. U.S.A., 78:3824-3828
(1981).
62. W~ ht, J. Mol. Biol., 215:635-651 (1990).
63. Armstrong, et al., Infea. Immun., 55:1294-1299 (1987).
64. Smith, et al., Immunology, 58:245 et seq. (1986).
65. Sleyter, et al., Arch. Microbiol., 146:19 et seq. (1986).
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3. State of the Art.
o~ t processes involving lT ~mm~ n cells, such as _rowth,
5 locomotion, mo~phological development, and dirr~,r~ tiation, are partially
controlled by extracellular signals acting upon the ceIls' surfaces' 3.
While some e~l~,".al stimuli reach the cell via extracellular fluids, other
signals are received from neighboring or appro~rhin~ cell surfaces and
exert their effects through direct cell-cell corlt~.~t45.
Evidence suggests that specific cell-surface l~,C~1 ~015 can "sense" a
m~lec.-i~r signal of an opposing cell via specific binflin~, and biochemical
cl-~nicrnc exist to translate that binding into a cellular response. For
eX~mplr~ complex cell-surface interactions are believed to help direct
processes such as binding of pathogens to target tissues6 7, sperm-egg
15 binding~, interactions among cells in the imm--nte system9 ~~, and
recognition of cells during embryonic develoyll~ ln addition, defects
in cell-cell recognition are thought to underiie the uncontrolled cell
growth and motility which characterize neoplastic transforrnation and
met~ct~cic"-'3,
Other evidence suggests that cell-recognition processes are
me~i~ttod by carbohydrate chains or glycan portions of glycoconjugates' '~
16, For example, the b~nding of the surface glycoconjugates of one cell to
the complementary carbohydrate-binding proteins (lectins) on another cell
can result in the ini~iation of a specific interaction.
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One important group of carbohydrate-binding proteins are selectin
(LEC-CAM) proteins (Lectin + EGF + complementary Regulatory
Domain-Cell Adhesion Molecules). These or functionally similar proteins
or lectins are believed to play a critical role in immune responses
s (including inflammatory responses) through mediation of cell-cell contact
and through extra-vasation of leucocytesl'--'. Specific carbohydrate
ligands have been identified as part of the putative receptor structures for
selectin proteins and other lectins'7~'5. The structures identified include
oligosaccharide glycosides containing terminally linked ~-sialic
10 acid(2 ~6)~Gal- and ~-sialic acid(2 3)~Gal- groups. The use of
oligosaccharides and derivatives thereof having such terminally linked
groups for controlling inflammation, immunosuppression, etc. by
interacting with selectin proteins and/or other lectins has been disclosed26~
29,35 1 1
Likewise, peptides derived from the selectin GMP-140 which
inhibit binding of GMP-140 and other selectins to leukocytes, presumably
by interfering with the binding of the GMP-selectin protein with the
carbohydrate receptor on the leukocyte, have also been disclosed as being
useful in suppressing an immune response~9. Similarly, other peptides are
20 also known to be potent modulators of neutrophil functionsS8.
Pertussis toxin (PT)~', a virulence factor produced by the organism
Bordetella pertussis -- the etiological agent of whooping cough, is known
to bind to glycoprotein receptors which terminate in sialyllactosamine
sugar sequences43 44, and we have previously shown that this protein is
25 useful in suppressing mammalian immune responses and cellular
adhesion45. PT's binding specificity has been shown to be similar to that
of the plant lectins from Sambucus nigra (SNA) and Maacl~ia amure~lsis
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(MAL)~s, which bind with high affinity to sialic acid-containing
glycoconjugates4647 and which have also been shown as possessing
immunomodulating properties45.
However, the use of proteins or large molecular weight
S polypeptides in inhibiting immune responses or cellular interactions in
mammals suffers from several drawbacks, including the fact that they are
difficult to produce in large quantities in pure form, that they tend to
produce adverse effects when repeatedly administered to a mammal, that
they can contain infectious agents or toxic substances which are
10 contraindications to mammalian administration, and that it is difficult to
modify the pharmokinetic properties of such proteins to improve their
efficacy.
In view of the above, the use of peptides having lectin-like binding
properties for terminal ~NeuSAc(2 3)~Gal- and cYNeuSAc(2 ~6)~BGal-
lS groups would be particularly beneficial for use in inhibiting immuneresponses and cellular interactions in mammals as compared to the
administration of proteins such as the pertussis toxin and the lectins
derived from SNA and MAL because such peptides would mitigate the
problems associated with the administration of proteins and large
20 molecular weight polypeptides to mammals.
S~lmmary of the Invention
This invention is directed, in part, to the discovery that the binding
domains for the ~-sialic acid(2 3),BGal- andlor the ~-sialic
acid(2 6),BGal- terminally linked structures are found in certain peptide
25 fragments of lectins (e.g., proteins and polypeptides) such as pertussis
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toxin and that it is not necessary to employ the entire lectin to effect
binding to these carbohydrates (oligosaccharides).
This invention is further directed, in part, to the discovery that
when these lectin derived carbohydrate binding peptides are administered
5 to a mammal (e.g., human) in effective amounts, they inhibit specific
immune responses and cellular interactions. In particular, this invention
is directed to the discovery that such lectin derived carbohydrate binding
peptides may be adminis~ered to a mammal in order to inhibit
inflammatory responses, modulate the induction of an immune response to
10 an antigen, induce long term tolerance to an antigen, and suppress cell
adhesion.
This invention is particularly directed to the discovery that lectin
derived carbohydrate binding peptides capable of binding terminally
linked cY-sialic acid(2 3),BGal- and/or o~-sialic acid(2~6),~Gal- (e.g.,
15 cYNeu5Ac(2 ~3)~Gal-) groups present in molecules (e.g., oligosaccharides,
glycoproteins, glycolipids, etc.) which can be found on cell surfaces (e.g.,
leukocytes) may be administered to a mammal as a means for inhibiting
cell adhesion or cell-medi~te~l immune responses. Cell-mediated immune
responses inhibited by the peptides disclosed herein include inflammatory
20 responses, modulation of the induction of the immune response to an
antigen and the induction of long term tolerance to an antigen.
Preferably, the lectin derived carbohydrate binding peptides
employed herein are fragments of the S2 or S3 subunits of the pertussis
toxin expressed by Bordetella pertussis or functionally equivalent variants
25 thereof. These preferred peptides have the amino acid sequences set forth
in peptides of Figure 1 below.
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Accordingly, in one of its method aspects, this invention is directed
to a method of suppressing an inflammatory response in a mammal by the
administration of an effective amount of at least one lectin derived
carbohydrate binding peptide or derivative thereof capable of binding
S terminally linked cY-sialic acid(2 6),~Gal- and/or cY-sialic acid(2~3)~Gal-
groups on structures or molecules comprising such groups.
In another of its method aspects, this invention is directed to a
method for modulating the induction of an immune response to an antigen
in a mammal by administering the antigen in combination with an
10 effective amount of at least one lectin derived carbohydrate binding
peptide or derivative thereof capable of binding terminally linked ~-sialic
acid(2~6),l~Gal- and/or cY-sialic acid(2 ~3),BGal- groups on structures or
molecules comprising such groups.
In another of its method aspects, this invention is directed to a
15 method for inducing in a sensitized mammal long term tolerance to an
antigen by exposing (challenging) the mammal with the antigen followed
by the administration of an effective amount of at least one lectin derived
carbohydrate binding peptide or derivative thereof capable of binding
terminally linked cY-sialic acid(2 ~6)~Gal- and/or cY-sialic acid(2 3),BGal-
20 groups on structures or molecules comprising such groups.
In still another one of its method aspects, this invention is directedto a method for inhibiting cell adhesion events involved in metastasis of
tumor cells or in inflammation by the administration of an effective
amount of at least one lectin derived carbohydrate binding peptide or
25 derivative thereof capable of binding terminally linked cY-sialic
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acid(2~6),~Gal- and/or ~-sialic acid(2~3),BGal- groups on structures or
molecules comprising such groups.
In yet another one of its method aspects, this invention is directed
to a method for treating lung inflammation and/or lung injury in a
5 mammal by the administration of an effective amount of one or more
lectin derived carbohydrate binding peptides or derivatives thereof capable
of binding terminally linked ~-sialic acid(2~6),BGal- and/or ~-sialic
acid(2 ~3),BGal- groups on structures or molecules comprising such
groups.
In its composition aspects, this invention is directed to
pharmaceutical compositions comprising the subject lectin derived
carbohydrate binding peptides including pharmaceutically acceptable salts
of such lectin derived carbohydrate binding peptides.
Brief Description of the Drawin~s
Figure 1 illustrates the amino acid sequence of preferred lectin
derived carbohydrate binding peptides.
Figure 2 illustrates the increase in footpad swelling of immunized
Balb/c mice arising from a DTH inflammatory response measured 24
hours after challenge with 20 ~g of OVA antigen wherein some of the
20 mice have been treated at 5 hours after challenge with 100 ~g of a lectin
derived carbohydrate binding peptide.
Figure 3 illustrates the long term (2 week) effects on the DTH
responses determined by footpad swelling in groups of Balb/c mice which
are immunized with 100 ,ug of the OVA antigen, challenged 7 days later
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with OVA, and then treated about 5 hours thereafter with a lectin derived
carbohydrate binding peptide.
In Figures 2 and 3, peptide 2275 is ACS2Pl (amino acids 9-23 of
SEQ ID NO:3) and peptide 2283 is SPYGRC (amino acids 18-23 of SEQ
5 ID NO:3), both of which are illustrated in Figure 1.
Figures 4 through 7 illustrate the same test as Figures 2 and 3 for
other peptides.
The amino acid residues depicted in Figure 1 and used throughout
this application are designated by the conventional single letter code set
forth below:
A alanine I isoleucine R arginine
C cysteine K Iysine S serine
D aspartic acid L leucine T threonine
E glutamic acid M methionine V valine
F phenyl alanine N asparagine W tryptophane
G glycine P proline Y tyrosine
H histidine Q glutamine
Additionally, as conventionally indicated, the amino acid residue forming
the H~N- terminus of the peptide is set forth on the left side of the peptide
20 chain, and the -COOH terminus of the peptide is set forth on the right
side of the peptide chain.
Detailed Description of the Preferred Embodiments
This invention is directed, in part, to the discovery that certain
lectin derived carbohydrate binding peptides when administered to a
25 m~mm~l are effective in suppressing infl~mm~tory responses, inducing
tolerance to an antigen, modulating the induction of immune responses to
antigens, and inhibiting cell adhesion events, e.g., cell adhesion events
involved in metastasis of tumor cells.
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l. Definitions
As used herein, the following terms have the meanings set forth
below:
The term "inflammatory response" or "inflammatory disorder" will
5 refer to immune reactions involving specific and non-specific defense
systems. A specific defense system reaction is a specific immune system
reaction to an antigen. Examples of specific defense system reactions
include antibody responses to antigens such as viruses, allergens, and
delayed-type hypersensitivity. A non-specific defense system reaction is
10 an inflammatory response mecli~terl by leukocytes generally incapable of
immunological memory. Such cells include macrophages~ eosinophils and
neutrophils. Examples of non-specific reactions include the immediate
swelling after a bee sting, and the collection of polymorphonuclear
(PMN) leukocytes at sites of bacterial infection (e.g., pulmonary
15 infiltrates in bacterial pneumonias and pus formation in abscesses).
Other "inflammatory responses" or "inflammatory disorders"
within the scope of this invention include, e.g., autoimmune disorders
such as rheumatoid arthritis, lupus, multiple sclerosis, post-ischemic
leukocyte medi~tefi tissue damage (reperfusion injury), frost-bite injury or
20 shock, acute leukocyte-me~ terl lung injury (ARDS), asthma~ traumatic
shock, septic shock, nephritis, and acute and chronic inflammation
including atopic dermatitis, psoriasis, and inflammatory bowel disease.
Various platelet-mediated pathologies such as atherosclerosis and clotting
are also included within the definition of "inflammatory responses" or
25 "inflammatory disorders". In addition, "inflammatory responses" or
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-14--
"inflammatory disorders" may include the adhesion of circulating cancer
cells, with specific examples including carcinoma of the colon and
melanoma.
Without being limited to any theory, we believe that primary
5 events in the initiation of such inflammatory responses and inflammatory
disorders are the binding of leukocytes to selectins (e.g., ELAM-l,
PADGEM, etc.) through carbohydrate receptors comprising ~-sialic
acid(2~3),BGal- and/or ~-sialic acid(2~6),BGal- groups35-40 found on the
surface of the leukocytes. Likewise, it has been shown that mammalian
10 and, in particular, human cancer cells contain cY-sialic acid(2 3)~BGal-
and/or ~-sialic acid(2 6)~BGal- groups on the surface thereof~9 50, and it is
believed that binding of such circulating cancer cells to selectins is an
integral part of the metastatic process36 37. Accordingly, by interfering
with the binding of such carbohydrate receptors to these selectins,
15 ~uppl~ ssion of the infl~mm~tory immune responses as well as inhibition
of met~t~tic processes is achieved.
The term "antigen" refers to any protein, peptide, carbohydrate,
nucleic acid or other non-endogenous substance which when exposed to a
m~mm~l induces an immune response in that mammal.
Disease conditions believed to be caused by antigen exposure
include, by way of example, psoriasis, asthma, dermatitis, rheumatoid
arthritis, delayed type hypersensitivity, infl~mm~tory bowel disease,
multiple sclerosis, viral pneumonia, bacterial pneumonia, and the like.
The term "non-sensitized mammal" refers to those mammals which
have yet to be educated to a particular antigen.
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The term "sensitized mammal" refers to those mammals which
have been previously exposed to a particular antigen, and, accordingly,
their immune systems have become educated to that antigen. Typically,
initial exposure of an antigen to a mammal primes or educates the
5 mammal's immune response to later exposure to that antigen with
minimal inflammation during such initial exposure.
The term "secondary immune response" refers to the effector phase
of a mammal's immune response to an antigen to which it has previously
been sensitized. A mammal's secondary immune response is typically
10 accompanied by inflammation at the point of antigen exposure.
"Acute respiratory distress syndrome" or "ARDS" refers to an
inflammatory condition comprising leukocyte mediated lung injury.
Without being limited to any theory, it is believed that such lung injury is
exacerbated by infiltration and subsequent disruption of neutrophils into
15 the lungs. Specifically, the disruption of the neutrophils in the lungs
releases superoxides which results in severe vascular endothelial damage.
Accordingly, while this lung damage is not antigen based. the infiltration
of neutrophils into the lungs requires an adhesion event.
"Reperfusion injury" refers to an inflammatory condition
20 comprising leukocyte medi~tecl tissue damage. Reperfusion injury
commonly occurs after myocardial infarction wherein, in response to the
inflammation caused by the rnyocardial infarction, the endothelium cells
are activated and produce selectins (e.g., ELAM-l). In turn, neutrophils
are then capable of binding the selectins expressed on the vascular
25 endothelium and cause further damage.
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The term "pertussis toxin" or "PT" in this application refers to the
virulence factors produced by Bordetella pertu~sis, the etiological agent of
whooping cough. The ,~-oligomer of PT binds to both ~-sialic
acid(2~6),BGal- and cY-sialic acid(2 3),(~Gal- structures. PT also has
5 similar binding characteristics to those of wheat germ agglutinin (WGA),
which can recognize terminal N-acetylglucosamine (GlcNAc) saccharide
sequences in addition to sialic acid44 5' 53.
PT is a classical A-B type of toxin comprised of an A subunit
(designated SI) that contains an ADP-ribosyltransferase enzyme activity,
10 which is responsible for most of the biological effects of PT5~. The
lectin-like activity is found in the complex ~ oligomer of PT, which
consists of four heterogeneous subunits that are arranged in a pair of
dimers (S2-S4 (Dimer 1) and S3-S4 (Dimer 2)) joined by a smaller S5
subunit. The functioning of the ,(~ oligomer is in binding to host cell
I5 sialylated oligosaccharide receptors as well as providing a delivery system
for the A subunit through the cytoplasmic membrane54 55 56. The ~
oligomer itself can induce a mitogenic response in Iymphocytes and has
the ability to agglutinate erythrocytes. PT may also contribute to the
attachment of B. per~ussis to epithelial cells lining the upper respiratory
20 tract of humans, the only known host of B. pertussis57. In addition, the ,
oligomer also appears to share functional homology with the selectin
family of mammalian lectins that regulate leukocyte trafficking.58
The term "cell-mediated immune response" refers to those
mammalian immune responses which are mediated by cell-cell
25 interactions. Included within this term are cell-mediated inflammatory
responses to an antigen including, by way of example, such responses as
delayed-type hypersensitivity (DTH) responses, virus-induced pneumonia,
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allergic responses, and the like as well as cell-mediated inflammatory
responses arising from injuries such as myocardial infarction, shock and
sequelae (e.g., multiple organ failure), acute respiratory distress syndrome
(ARDS), and the like. Generally, the cell-mediated immune response is a
5 leukocyte-mediated response.
The term "humoral immune response" refers to mammalian
immune responses which involve antigen-antibody interactions.
The term "DTH inflammation response" or "delayed type
hypersensitivity response" is a T cell mediated reaction which results in a
10 mononuclear cell-rich inflammation and swelling which occurs after
antigenic ~.~1!eng~.
The term "tolerance" or "immunological tolerance" refers to a
reduced immunogenic response elicited in a sensitized mammal to a
particular antigen upon a second or subsequent antigenic challenge in
15 comparison to the primary immune response elicited by said antigen under
equivalent conditions (e.g., dosage). In the present invention such
"tolerance" will be obtained by administration of an anti~gen to the
sensitized mammal followed by administration of one or more lectin
derived carbohydrate binding peptides which bind to cY-sialic
20 acid(2~6)~Gal- and/or ~x-sialic acid(2~3),l~Gal- structures.
The term "period for maximal inflammation" refers to the period
of time typically required to achieve maximal inflammation in a mammal
due to a cell-medi~ted immune response including both cell-mediated
inflammatory responses in a sensitized mammal due to antigen exposure
25 (challenge) and cell-mediated inflammatory responses in a mammal due to
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injury (e.g., myocardial infarction). This period of time depends on
several factors such as the specific antigen/injury aMicting Lhe mammal,
the particular mammalian species exposed to the antigen/injury, etc.
Accordingly, the period of time required to effect maximal inflammation
S will vary for, by way of example, rheumatoid arthritis as opposed to
myocardial infarction.
Moreover, while the specific time required to effect maximal
inflammation will vary somewhat in a given mammalian species. the time
typically required to effect maximal inflammation for different afflictions
10 due to either antigen exposure or injury in human and other mammals is
known in the art or is readily ascertainable by the skilled artisan. For
example, in the case of a DTH response in mice, maximal inflammation
is typically 24 hours after antigen exposure.
The term "sialic acid" refers to all naturally occurrin~g structures of
15 sialic acid and analogues of sialic acid as well as derivatives thereof.
Naturally occurring structures of sialic acid include, by way of example,
5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosylonic acid
("NeuSAc"), N-glycoyl neuraminic acid (NeuSGc) and 9-O-acetyl
neuraminic acid (NeuS,9Ac.). A complete list of naturally occurring
20 sialic acids known to date is provided by Schauer5'.
Derivatives of sialic acid refers to derivatives of naturally
occurring structures of sialic acid including those wherein the sialic acid
unit has been chemically modified so as to introduce and/or remove one
or more functionalities from such structures. For example, such
25 modification can result in the removal of an -OH functionality, the
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- 1 9-
introduction of an amine functionality, the introduction of a halide
functionality, and the lilce.
Certain derivatives of sialic acid are known in the art and include
chemically modified sialic acid derivatives such as 9-azido-Neu5Ac, 9-
5 amino-Neu5Ac, 9-deoxy-Neu5Ac, 9-~luoro-Neu5Ac, 9-bromo-NeuSAc, 8-
deoxy-Neu5Ac, 8-epi-Neu5Ac, 7-deoxy-NeuSAc, 7-epi-NeuSAc, 7-8-bis-
epi-NeuSAc, 4-O-methyl-Neu~Ac, 4-N-acetyl-NeuSAc, 4,7-di-deoxy-
NeuSAc, 4-uno-NeuSAc, 3-hydroxy-NeuSAc, 3-fluoro-NeuSAc acid as
well as 6-thio analogues of NeuSAc. Methods for ~,epa~ing such sialic
10 acid derivatives are taugh~ in commonly assigned PCT Patent Application
No. PCT/CA92/00244.
The nomenclature describing denvatives of sialic acid derivatives
herein is as set forth by Reu~er et al34.
lS The term "~-sialic acid(2~6),BGal- structures or groups" refers to
molecules comprising the terminally linked ~x-sialic acid(2~6)~ ctose
sequence or derivatives thereof. Molecules cont~ining such terminal
structures have been identified as CG,~ ing part of the putative receptor
structure for the ELAM-l and PADGEM selectinc334o.
The term "cY-sialic acid(2 3)~Gal- structures or groups" refers to
molloculec comprising the terminally linked ~-sialic acid(2 ~3)~ tose-
sequence or derivatives thereof. Molecules comprising such terminal
structures have similarly been identified as comprising part of the putative
receptor structures for the ELAM-I selectins35~39.
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The term "lectin derived carbohydrate binding peptide" refers to
any peptide or derivative thereof (including pharmaceutically acceptable
salts) derived from a lectin and which is capable of binding to
~-sialic acid(2~6),BGal- and/or ~-sialic acid(2 3),~Gal- carbohydrate
5 structures which are preferably comprised on the surface of mammalian
cells. Generally, in the present application, "lectin derived carbohydrate
binding peptide" will refer to such peptides which, in monomeric form,
have no more than about 35 amino acids in the lectin-iike domain (i.e.,
the part of the peptide responsible for binding to such carbohydrate
10 structures). Suitable derivatives of lectin derived carbohydrate binding
peptides include those peptides which have the NH. and/or the COOH
terminal functionalities blocked by conventionally bloclcing groups as well
as derivatives which include modifications, deletions or derivatizations of
one or more of the amino acids that yield a peptide which is capable of
15 bin~ling to cY-sialic acid(2~6)~Gal- and/or ~-sialic acid(2 3),~Gal-
structures.
More preferably, the lectin derived carbohydrate ~inding peptides
refer to the peptides set forth in Figure 1.
Still more preferably, the lectin derived carbohydrate binding
20 peptiries are peptides which have a high degree of homology with a lectin-
like binding domain for the cY-sialic acid(2~6),BGal- andlor cY-sialic
acid(2~3),~Gal- carbohydrate structures found in the pertussis toxin.
These peptides are represente-~ by formula I (SEQ ID NO~
SPX,GX~C
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where X, is selected from the group of amino acids Y, F, W, and
H or peptide mimetics thereof; and
X, is selected from the group consisting of amino acids Y, F, R,
W, and H or peptide mimetics thereof;
or by formula II (SEQ ID NO:2):
SPX,GX,CX3X4 II
where Xl is selected from the group of amino acids Y, F, W, and
H or peptide mimetics thereof;
X, is selected from the group consisting of amino acids Y, F, R,
10 W, and H or peptide mimetics thereof;
X3 is an amino acid sequence of 4-6 amino acids; and
X4 iS selected from the group consisting of amino acids Y, F, W,
and H or peptide mimetics thereof.
Most preferably, the lectin derived carbohydrate binding peptide
refers to the hexapeptide SPYGRC (amino acids 18-23 of SEQ ID NO:3).
In addition to their use as modulating agents for mammalian
immune and cellular adhesion processes, the lectin derived carbohydrate
binding peptides described herein can further be used in assay methods
for determining the presence of ~-sialic acid(2 ~3),BGal- and/or c~-sialic
20 acid(2 6),BGal- structures on molecules and/or cell surfaces such as in
suspected cancer cells, for determining the relative binding affinity of
peptides and/or proteins to cY-sialic acid(2~3)~BGal- andlor c~-sialic
acid(2 6)~Gal- structures, and the like. When so employed, the lectin
derived carbohydrate binding peptides typically will be derivatized to
25 include a label or a label binding moiety.
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Suitable labels are well known in the art and include, by way of
example, enzymes (e.g., horseradish peroxidase), radioisotopes (e.g.,
'~5I), fluorescent moieties, chemiluminscent moieties, and the like. The
particular label employed is not critical, and methods for attaching labels
5 to peptides are well known in the art.
Suitable label binding moieties are also well known in the art and
include, by way of example, biotin, avidin, streptavidin antibodies, etc.
A preferred label binding moiety is biotin which permits binding of up to
4 peptide/biotin adducts to avidin. The avidin can be appropriately
10 labeled so that the resulting peptide/biotin/avidin complex can be detected.
The term "lectins" refers to carbohydrate binding proteins of non-
immune origin often obtained from plants or bacterial or viral
microorganisms which comprise carbohydrate binding sites. These
binding proteins typically comprise the ability to agglutinate cells and to
15 precipitate complex carbohydrates. Lectins are classified based upon their
carbohydrate binding specificity and are well known in the art.
The term "lectin-like domain" refers to those fragment(s) of a
lectin responsible for binding the carbohydrate.
The term "pharmaceutically acceptable salts" includes the
20 pharmaceutically acceptable addition salts of lectin derived carbohydrate
binding peptides capable of binding to terminally linked cx-sialic
acid(2 ~6),BGal- and/or cY-sialic acid(2 ~3),~Gal- structures. Such
pharmaceutically acceptable addition salts may be derived from a variety
of organic and inorganic counter salts well known in the art and include.
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by way of example only, sodium, potassium, calcium, magnesium,
ammonium, tetralkyl-ammonium, and the like.
2. Utility
Without being limited to any theory, it is believed that the subject
5 lectin derived carbohydrate binding peptides affect the immune response
in a number of ways. Lectin derived carbohydrate binding peptides can
inhibit a mammal from becoming "educated" about a specific antigen
when the lectin derived carbohydrate binding peptide is administered
simultaneously with the first exposure of the immune system to the
10 antigen.
The lectin derived carbohydrate binding peptides can reduce cell-
me~ terl immune responses to injury such as inflammatory responses
arising from myocardial infarction, ARDS, frost-bite, etc. The lectin
derived carbohydrate binding peptides can also inhibit the effector phase
15 of a cell-mediated immune response (e.g., the inflammatory component of
a DTH response) when administered to a sensitized mammal after
exposure of the sensitized mammal's immune system to the antigen. In
either case, in order to effect reduction in the cell-mediated immune
response, it is necess~ry to administer the lectin derived carbohydrate
20 binding peptides after initiation of the mammal's immune response and at
or prior to one-half the period required for maximal inflammation induced
by the injury or the antigen exposure.
Additionally, the subject lectin derived carbohydrate bindin~
peptides can induce tolerance to antigens in sensitized mammals when
25 administered at the time of second or later exposures of the immune
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system to the antigen when administration is conducted after initiation of
the mammal's secondary immune response to the antigen and at or prior
to one-half the period required for maximal inflammation induced by the
antigen exposure.
Further, the administration of lectin derived carbohydrate binding
peptides that bind cY-sialic acid(2~6),BGal- and/or (x-sialic acid(2 3),~Gal-
structures inhibits the binding of LEC-CAM proteins and other select~ns
to their putative receptors therefor which include both the ~-sialic
acid(2 6),~Gal-3i 40 and the ~x-sialic acid(2~3),~Gal-35-39 structures.
Accordingly, the subject invention provides both pharmaceutical
compositions containing lectin derived carbohydrate binding peptides
capable of binding terminally linked ~-sialic acid(2 6),BGal- and the ~-
sialic acid(2 3),BGal- structures which are useful in inhibiting specific
immune responses or cellular interactions in m~mm~ls as well as methods
which include the administration of such lectin derived carbohydrate
binding peptides to a mammal for inhibiting a cell-mediated immune
response.
As noted above, lectin derived carbohydrate binding peptides useful
for modulating a cell-mediated immune response in a mammal include
any lectin derived peptide or derivative thereof capable of binding
terminally linked ~-sialic acid(2 ~6),BGal- and the o~-sialic acid(2~3),BGal-
structures. Suitable lectin derived carbohydrate binding peptides
preferably are peptides which, in monomeric form, have no more than
about 35 amino acids in the lectin-like domain ~i.e., the part of the
peptide responsible for binding to such carbohydrate structures).
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More preferably, the lectin derived carbohydrate binding peptides
refer to the peptides set forth in Figure 1.
Still more preferably, the lectin derived carbohydrate binding
peptides are peptides which have a high degree of homology with a lectin-
5 like binding domain for terminally linked ~-sialic acid(2 6)~Gal- and/or
c~-sialic acid(2 ~3),BGal- carbohydrate structures found in the pertussis
toxin. These peptides are represented by formula I (SEQ ID NO:1):
SPX,GX.C
where Xl is selected from the group of amino acids Y, F, W, and
lO H or peptide mimetics thereof; and
X. is selected from the group consisting of amino acids Y, F, R,
W, and H or peptide mimetics thereof;
or by formula II (SEQ ID NO:2):
SPX,GX.CX3X4 II
15where X, is selected from the group of amino acids Y, F, W, and
H or peptide mimetics thereof;
X, is selected from the group consisting of amino acids Y, F, R,
W, and H or peptide mimetics thereof;
X3 iS an amino acid sequence of 4-6 amino acids; and
20X4 is selected from the group consisting of amino acids Y, F, W,
and H or peptide mimetics thereof.
The preparation of such peptides is well known in the art and
includes, by way of example, standardized commercially available peptide
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synthesizers such as Model ABI 403A available from Applied Biosystems,
Inc., Foster City, California, U.S.A. The methods for preparing such
peptides do not form a part of this invention.
Peptide mimetics refers to groups which mimic an amino acid in
5 the peptide chain. Such mimetics and their synthesis are well known in
the art59.
The lectin derived carbohydrate binding peptides can be used either
in monomeric or polymeric form. Examples of suitable polymers include
the attachment of biotin to the lectin derived carbohydrate binding peptide
10 followed by complexing with avidin which results in up to a tetravalent
complex. Likewise, multivalent derivatives of lectin derived carbohydrate
binding peptides can be synthesized by attaching such peptides to
polymers such as polylysine or an inert protein such as human serum
albumin. The multivalent derivatives so formed can contain one or a
15 mixture of different-lectin derived carbohydrate binding peptides so as to
enhance efficacy.
In the case of protein peptide conjugates, they will be chemically
cross-linked with the carrier protein by known cross-linking agents using
art recognized methodology.
In yet another embodiment, multivalent lectin derived carbohydrate
binding peptides can be generated as a copolymer wherein the peptides
are linked together through a spacer arm to provide for a repeating
subunit represented by the groups:
[carbohydrate binding peptide-spacer arm]n
[carbohydrate binding peptide]n-spacer arm
.
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In these subunits, the lectin derived carbohydrate binding peptide can be
the same or different lectin derived carbohydrate binding peptide, and the
spacer arm is selected to provide for optimal distance to bind to the
carbohydrate.
However, the invention is not restricted to the use of lectin derived
carbohydrate binding peptides specifically exemplified in Figure 1 or to
multivalent derivatives thereof, but rather embraces the use of any lectin
derived peptide or derivative thereof which binds terminally linked ~x-
sialic acid(2~6),BGal- and/or ~-sialic acid(2 3)~Gal- carbohydrate
structures. As noted previously, peptides capable of binding such
structures, when administered to a mammal, result in the inhibition of
immune responses and cellular interactions, in particular, inflammatory
responses or conditions, tolerance to antigens, modulation of the
immunogenic response to antigens, and the inhibition of cell adhesion
1~ events, which are involved, e.g., in metastasis and inflammation.
It is well within the level of ordinary skill in the art to identify
other lectin derived peptides capable of binding terminally linked ~-sialic
acid(2 6)~Gal- and/or c~-sialic acid(2~3),BGal- structures by conventional
methods for assaying binding between ligands. Such methods include,
e.g., competitive binding assays and receptor binding assays. The subject
application, in particular, sets forth one method in the examples which
illustrates rather simple assaying techniques that are capable of
deterrnining binding to terminally linked ~x-sialic ac;d(2 6),l~Gal- and/or
c~-sialic acid(2~3),l~Gal- structures. Other methods for determining the
binding of a candidate peptide with such terminally linked structures are
known in the art. See, for example, Pearce-Pratt et al.3l
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The subject invention accordingly further provides a method by
which lectin derived peptides capable of inducing or suppressing various
immune responses and cellular interactions, e.g., inflammation, antigenic
tolerance, modulation of antigenic response, or cell adhesion, may be
5 putatively identified on the basis of their ability to bind to terminally
linked ~-sialic acid(2 6),BGal- and/or cY-sialic acid(2 3),BCial- structures.
In regard to the above, the subject invention contemplates the
attachment of labels or label binding groups to the lectin derived
carbohydrate binding peptides and/or to candidate lectin derived
lO carbohydrate binding peptides in order to facilitate the assays described
above. Such labels are conventionally formed on the peptides by methods
well known in the art. Suitable labels include, by way of example,
enzymes (e.g., horseradish peroxidase), radioisotopes (e.g., l25I),
fluorescent moieties, chemiluminscent moieties, and the like.
Suitable label binding moieties are also well known in the art and
include, by way of example, biotin, avidin~ antibodies, etc. A preferred
label binding moiety is biotin which permits binding of the peptide/biotin
adduct to avidin. The avidin can be a~pro~uriately labeled so that the
resulting peptide/biotin/avidin complex can be detected.
The subject invention also contemplates kits for use in conducting
such assays. Such kits would comprise the labelled lectin derived
carbohydrate binding peptide or the lectin derived carbohydrate binding
peptide attached to label binding groups.
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.
-29-
Suitable lectin derived carbohydrate binding peptides for use herein
are those which are capable of binding terminally linked ~-sialic
acid(2 ~6)~Gal- and/or ~x-sialic acid(2~3)~Gal- structures. However, an
additional prerequisite of efficacious lectin derived carbohydrate binding
5 peptides will include suitability for in vivo administration. In particular,
the lectin derived carbohydrate binding peptide should not be toxic, and
should be sufficiently soluble at the required dosages, which will typically
range from about 0.5-50 mg/kg of body weight. In this regard, it is art
recognized that the solubility of lectin derived carbohydrate binding
10 peptides can be enhanced by attaching hydrophilic amino acid groups and
can be reduced by attaching hydrophobic amino acid groups from the
carboxyl terminal and/or amino terminal positions of the peptide.
The invention further contemplates fragments or derivatives of
peptides capable of binding terminally linked (x-sialic acid(2~6),BGal-
15 and/or c~-sialic acid(2 3),l~Gal- structures which peptides have been
modified to render them non-toxic, e.g., by chemical derivatization,
mutagenesis, etc. while still retaining the ability to bind such terminally
linked structures.
The subject invention provides, in particular, methods for
20 suppressing cell-mediated immune responses in mammals including cell-
mefli~ted inflammatory responses or disorders by the administration of an
effective amount of one or more lectin derived carbohydrate binding
peptides capable of binding terminally linked ~-sialic acid(2 ~6)~Gal-
and/or ~-sialic acid(2~3),BGal- structures or molecules/cell surfaces
25 comprising such structures.
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The cell-me~ A immune responses or disorders treatable by the
subject invention include inflammatory immune reactions involving
specific and non-specific defense systems. As discussed above, such
conditions include antibody responses to antigens, such as viruses,
5 allergens, delayed-type hypersensitivity, autoimmune disorders such as
rheumatoid arthritis and lupus, post-ischemic leukocyte medi~te~ tissue
damage (reperfusion injury), frost-bite injury or shock-acute leukocyte-
mediated lung injury (e.g., acute respiratory distress syndrome), asthma,
traumatic shock, septic shockt nephritis, and acute and chronic
10 inflammation, including atopic dermatitis, psoriasis, and inflammatory
bowel disease. Further, inflammatory disorders treatable by the subject
invention may include platelet-mediated pathologies such as
atherosclerosis and clotting disorders.
Inflammatory conditions of special interest include delayed type
15 hypersensitivity reactions, reperfusion, and acute leukocyte-mediated lung
injury (ARDS).
This invention provides a generic method by which cell-mediated
immune responses such as cell-mediated inflammatory responses or
disorders in mammals (e.g., humans) may be suppressed by the
20 administration of an effective amount of one or more lectin derived
carbohydrate binding peptides or fragments or derivatives thereof capable
of binding terminally linked (x-sialic acid(2 ~6)~Gal- and/or ~-sialic
acid(2 3)~BGal- structures or molecules/cell surfaces comprising such
structures. In a preferred embodiment, the invention provides methods by
25 which inflammatory responses or disorders may be treated or suppressed
by the administration of an effective amount of one or more peptides
selected from the group of peptides set forth in Figure 1.
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The subject invention further provides a general method for
inhibiting immune responses and cell adhesion events in mammals by the
administration of an effective amount of one or more lectin derived
carbohydrate binding peptides or fragments or derivatives thereof capable
S of binding terminally linked c~-sialic acid(2 ~6),l~Gal- and/or c~-sialic
acid(2 3),BGal- structures or molecules/cell surfaces comprising such
structures. Such immune responses include cell medi~tecl and humoral
immune responses. As has been discussed, such immune responses
inelt~de, in particular, inflammatory responses or inflammatory disorders.
The invention further provides methods for affecting the induction
of immune responses to antigens comprising administering to a mammal
an antigen in conjunction with one or more lectin derived carbohydrate
binding peptides capable of binding terminally linked c~-sialic
acid(2~6),BGal- and/or ~-sialic acid(2 ~3),BGal- structures or
15 molecules/cell surfaces comprising such structures. For example,
administration of an effective amount of the SPYGRC (amino acids 18-23
of SEQ ID NO:3) hexapeptide to a mammal with an antigen will
modulate the induction of the immune response in the mammal to the
antigen. Accordingly, the subject lectin derived carbohydrate binding
20 peptides may comprise applicability as immune modulators, which may be
administered in conjunctlon with vaccines, artificial organs or tissue
transplants, and allogeneic organ and tissue transplants as a means for
modulating the immune response to foreign antigens comprised therein.
It has further been found that the subject lectin derived
25 carbohydrate binding peptides capable of binding terminally linked
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~-sialic acid(2~6),BGal- and/or ~-sialic acid(2~3),~Gal- structures or
molecules/cell surfaces comprising such structures, when administered in
an effective amount to a mammal which has been immunized with a
particular antigen, result in the induction of long term tolerance to said
5 antigen. In this regard, administration is conducted after onset of the
secondary immune response but at or prior to one-half the period required
for maximal inflammation.
In particular, it has been found that administration, during the
critical period set forth above, of an effective amount of the S3P9a (SEQ
ID NO:9), the ACS2PI (2275) (amino acids 9-23 of SEQ ID NO:3), and
the SPYGRC (2283) (amino acids 18-23 of SEQ ID NO:3~ peptides
illustrated in Figure 1 to m~mnl~ls that have been immunized with an
antigen results in said mammals exhibiting a reduced immune response
upon subsequent challenge(s) with said antigen (Figure 3). Thus, the
15 subject lectin derived carbohydrate binding peptides have applicability as
tolerogens. Given this property, such lectin derived carbohydrate binding
peptides or fragments or derivatives thereof may be especially suitable for
use in the treatment of allergic disorders since administration of
"tolerogenic" derivatized allergens is a known means for treating allergic
20 disorders.
The subject invention further provides methods for inhibiting the
adhesion of certain cell types [e.g., tumor cells and polymorphonuclear
cells (PMN's)] to endothelial cells. In this regard, the art suggests that
tumor metastasis involves tumor cell adhesion to selectin bearing cells. In
25 this regard, circulating cancer cells apparently take advantage of the
body's normal inflammatory mechanisms and bind to areas of blood
vessel walls were the endothelium is activated and, accordingly. contains
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selectins. As noted previously, the putative receptors for such selectins
contain terminally linked cY-sialic acid(2~6)~BGal- and/or cY-sialic
acid(2 ~3),l~Gal- structures. Therefore, administration of lectin derived
carbohydrate binding peptides capable of binding terminally linked ~-
S sialic acid(2 6),BGal- andlor (x-sialic acid(2 ~3)~BGal- structures should
provide a method for inhibiting metastasis. For example, the subject
lectin derived carbohydrate binding peptides or fragments or derivatives
thereof may be administered before, during or after cancer surgery or
biopsy as a means for inhibiting metastasis of tumor cells which may be
10 released into the circulatory system during surgery. In these methods, the
subject lectin derived carbohydrate binding peptides are administered
either prior to, at the time of surgery or biopsy, or shortly thereafter.
Prior administration is typically no more than about S hours prior to
surgery or biopsy, and administration and subsequent administration is
15 typically no more than about lS hours after surgery or biopsy. In either
case, administration is either continuous or intermittent, but preferably is
continuous.
In the methods pertaining to suppression of cell-mediated
inflammatory reactions or disorders arising from injury or antigen
20 exposure, the subject lectin derived carbohydrate binding peptides or
fragments or derivatives thereof are administered after initiation of the
mammal's immune response but at or prior to one-half the period
required for maximal inflammation to the antigen exposure or injury.
Preferably, the subject lectin derived carbohydrate binding peptides are
25 administered about l-lO hours after initiation of the immune response,
and more preferably about 1-5 hours after initiation of the immune
response. However, the specific time for administration will vary
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dependent upon the particular antigen/injury and the lectin derived
carbohydrate binding peptide which is administered.
In the methods pertaining to modulating the induction of an
immune response to an antigen in a non-sensitized mammal, an effective
S amount of the subject lectin derived carbohydrate binding peptides or
derivatives thereof will be ~lministered in conjunction with the antigen.
Typically, such conjunctive administration is simultaneous with antigen
administration but can be up to +3 hours from the time of antigen
administration .
In the methods pertaining to induction of long term tolerance to an
antigen, an effective amount of the subject lectin derived carbohydrate
binding peptides or fragments or derivatives thereof will generally be
administered after antigen challenge to a sensitized mammal. In
particular, aclrnini~tration is after initiation of the mammal's secondary
15 immune response to the antigen challenge but at or prior to one-half the
period required for maximal inflammation to the antigen challenge.
Preferably, the subject lectin derived carbohydrate binding peptides are
administered about 1-10 hours after initiation of the immune response to
the antigen challenge, and more preferably about 1-5 hours after initiation
20 of the immune response to the antigen challenge. However, the specific
time for administration will vary dependent upon the particular antigen
and the lectin derived carbohydrate binding peptide which is administered.
Generally, the subject lectin derived carbohydrate binding peptides
or derivatives thereof will be administered parenterally, e.g., by
25 intramuscular or intravenous routes. However, other dosage forms should
also be suitable including, e.g., oral, transdermal, rectal, intratracheal,
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and intranasal formulations. For example, intranasal and intratracheal
formulations may be preferred if the inflammatory condition treated
involves lung inflammation, e.g., acute respiratory distress syndrome
(ARDS). In contrast, an oral formulation would likely be preferred if the
5 inflammatory condition treated involves the digestive tract, e.g.,
inflammatory bowel disease.
Pharmaceutical compositions for use in the subject invention will
generally comprise an effective amount of one or more of the subject
peptides or derivatives thereof capable of binding terminally linked
10 cY-sialic acid(2~6),BGal- and/or ~-sialic acid(2 ~3),~Gal- structures in
combination with a pharmaceutically acceptable carrier and/or excipients.
The particular pharmaceutically acceptable carrier and excipients will vary
dependent upon the dosage form. In one embodiment, several of the
subject peptides or derivatives thereof are mixed into the pharmaceutical
15 composition to form a "cocktail" having enhanced activity.
Parenteral dosage forms may contain phosphate buffered saline as a
carrier, while intranasal formulations will comprise inhalants, and oral
dosage forms may comprise enteric coatings. The selection of suitable
carriers and excipients and formulation of different dosage forms is well
20 within the level of ordinary skill in the pharmaceutical art.
As noted above, the subject lectin derived carbohydrate binding
peptides or derivatives thereof are administered in effective amounts. An
effective amount is an amount sufficient to obtain the desired therapy
without causing undue toxicity to the mammal. Preferably, the subject
25 peptides are administered at dosages ranging from about 0.5 to 50mg/kg
body weight, with 5-lO mg/kg being most preferred for each of the
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above-cited methods. The specific dose employed is regulated by the
particular cell-mediated immune response being treated as well as by the
judgement of the attending clinician depending upon factors such as the
severity of the adverse immune response, the age and general condition of
5 the patient, and the like.
Generally, the methods of the present invention will involve
administration of a single dose of the subject lectin derived carbohydrate
binding peptides. However, the invention further contemplates repeated
administration of the subject lectin derived carbohydrate binding peptides
10 or derivatives thereof. Repeated administration of these peptides may be
desirable, e.g., in the treatment of chronic or sustained inflammatory
disorders such as rheumatoid arthritis, acute and chronic inflammation,
psoriasis, inflammatory bowel disorders, and autoimmune disorders
associated with inflammatory responses such as lupus, multiple sclerosis
15 or rheumatoid arthritis.
It is also contemplated that the subject peptides and derivatives are
useful as receptor-targeted antibacterial and anti-viral drugs wherein the
bacteria, virus, or toxin produced therefrom employs a terminally linked
cY-sialic acid(2 ~6),l~Gal- and/or ~-sialic acid(2~3),~Gal- structure as the
20 receptor site on a cell of the targeted mammalian host.
Such bacteria/virus and/or toxins include, by way of example,
influenza virus, pertussis toxin, cholera toxin, and the like. Such methods
are illustrated in the examples hereinbelow wherein in vitro assays
demonstrate the ability of two of the subject lectin derived carbohydrate
25 binding peptides to neutralize the effects of pertussis toxin on Chinese
Hamster Ovary cells.
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Accordingly, when administered in effective amounts, the subject
lectin derived carbohydrate binding peptides are useful in methods for
inhibiting inoculation in mammalian hosts of bacterial/viral a~ents and/or
their toxins which employ a terminally linked ~-sialic acid(2~6)~Gal-
5 and/or ~-sialic acid(2 ~3),BGal- structure as the receptor site on a cell of
the targeted mammalian host thereby inhibiting the likelihood that the
mammalian host will become afflicted with the dise~e produced by the
bacterial/viral agent and/or its toxin.
Effective amounts of the subject lectin derived carbohydrate
10 binding peptides or derivatives thereof will preferably be dosages ranging
from about 0.5 to 50mg/kg body weight, with S-10 mg/kg being most
preferred.
3. Examples
In order to fully illustrate the present invention and the advantages
15 thereof, the following specific examples are given, it being understood
that these examples are intended to be illustrative only and in nowise
limitative of the scope of the present invention.
In these examples as well as in the application, all sugars disclosed
herein are in their D form, except for fucose which is in its L form, and
20 all amino acids are conventional.
In these examples, unless otherwise defined below, the
abbreviations employed herein have their generally accepted meaning:
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ABTS = 2,2'-azino-bis(3-ethylbe~7~thi~7oline-6-sulfonic acid)
BSA = Bovine serum albumin
cm = centimeter
HPLC = High Performance Liquid Chromatography
5 MAL = Maackia amurensis
mg = milligram
mM = millimolar
mm = millimeter
ng = nanogram
10 nm = nanometer
PBS = phosphate buffered saline
PT = Pertussis toxin
SNA = Sambucus nigra
~g = microgram
15 ~1 = microliter
,uM = micromolar
,umol = micromole
v/v = volume/vol~me
Unless otherwise indicated, all temperatures are in degrees Celsius
20 (~C). Also, as noted previously, all amino acid residues recited herein
employ their conventional one-letter abbreviation.
General Procedures
All of the reagents used in Examples 1-10 were obtained from
Sigma Chemical Company, St. Louis, Missouri, U.S.A., except for
25 pertussis toxin (PT) which was obtained from Connaught Center for
Biotechnology Research, Willowdale, Ontario, Canada; SNA-, WGA- and
MAL-biotin, which were obtained from Boehringer Mannheim, Dorval,
Quebec, Canada; and IODO-GEN which was obtained from Pierce
Chemical Co., St. Louis, Missouri, U.S.A. The acetylated and
30 biotinylated analogs of the peptide S2Pl (amino acids 9-23 of SEQ ID
NO:~) were prepared using conventional methods. PT-biotin as well as
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-39-
asialo- and asialoagalactofetuin were prepared as described earlier30~4.
Removable flat-bottomed microtiter well strips of Immunulon 2 were from
Dynatech, Alexandria, Virginia, U.S.A.
F.xample I -- Synthesis of Synthetic Peptides
Peptides corresponding to amino acid sequences found in the S2
and S3 subunits of PT were synthesized using an ABI 403A peptide
synthesizer (Applied Biosystems, Inc., Foster City, California, U.S.A.),
then cleaved from the resin by HF and purified by reversed-phase HPLC
on a Vydac C4 semipreparative column. All synthetic peptides used in
ELISA inhibition assays were >95~ pure as judged by analytical HPLC,
and their amino acid analyses were in good agreement with the theoretical
compositions.
Regions in the S2 and S3 subunits of PT that correspond to the
variable amino acid sequences distinguishing the S2 from the S3 subunit
were secured as above. These peptide sequences were chosen, in part,
based on their high index of hydrophilic ~-turns as judged by secondary
structure prediction analysis6~6'. Upon careful examination of the PT
sequences, 20 peptides were synthesized including those containing amino
acid residues 62-73 of WGA (SEQ ID NO:l 1). The acetylated version of
S2Pl (amino acids 9-23 of SEQ ID NO:3) was also prepared in order to
better mimic the native peptide backbone. The synthesized peptides are
set forth in Table I below:
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TABLE I
Posilion Sc~u~ cr SEQ ID NO
1. S''PI 9-'~3 PQEQITQHGSPYGRC 3 (aa 9-"3)
''. ACS~PI-b 9-~3 BIOTIN-PQEQITQHGSPYGRC-CO-NH~ 3 (aa 9-"3)
3. S'~ (14-~3) 14-:!3 TQHGSPYGRC 3 (aa 14-"3)
4. S'7-b 14-~3 BIOTIN-TQHGSPYGRC 3 (aa 14-''3)
5. S"P~ 1-''3 STPGIVIPPQEQITQHGSPYGRC 3
6. SPYGRC 18-"3 SPYGRC-CO-NH. 3 (aa 18-13)
7. SPYGRC-b 18-~3 BloTlN-spyGRc-co-NH2 3 (aa 18-"3)
8. S"P3 78-98 GAFDLKl-rFCIMl-rRNTGQPA 4
9. S7P4 138-154 YDGKYWSMYSRLRKMLY S (aa 16-3'')
10. S"P6 1''3-154 FVRSGQPVIGACTSPYDGKYWSMYSRLRKML 5
Il. S3P1 9-'~3 PKALFTQQGGAYGRC 6 (aa 9-73)
1'~. S3P~ 1-''3 VAPGlVlPPKALFrQQGGAYGRC 6
13. S3P3 87-108 Cll-rIYKTGQPAADHYYSKVTA 7 (aa 10-31)
14. S3P4 78-108 AGFlYRkl~ IYKTGQPAADHYYSKVTA 7
15. S3PS 134-154 CASPYEGRYRDMYDALRRLLY 8
16. S3P9a 110-1''7 RLLASTNSRLCAVFVRDG 9
17. S''(WGA)~ - PQEQITQHGSQYGYC 10
18. S"(WGA)-b - BIOTIN-PQEQITQHGSQYGYC 10
19. WGA(6~-73) - SQYGYCGFGAEY 11
''0.WGA(6''-73)-b - BIOTIN-SQYGYCGFGAEY 11
* Underlined sequences correspond to sequences found in the PT
peptide that showed homology to the sequence SQYGHC (SEQ ID
NO: 12) found in the binding site of WGA isolectin 2 (24).
# PT S2Pl sequence (amino acids 9-23 of SEQ ID NO:3) inserted
with the WGA hexapeptide sequence (SQYGYC) (amino acids 10-
15 of SEQ ID NO:10)
aa amino acid b biotin
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. ~ -41 -
The above peptides were tested for their ability to bind to
terminally linked cY-sialic acid(2 6),~'Gal- and cY-sialic acid(2 3)~Gal-
structures in the examples below. In these examples, the peptides were
first screened for their ability to inhibit binding of different lectins (PT,
5 SNA, and MAL) known to bind to fetuin, a carbohydrate containing
multiple copies of the c~NeuSAc(2~3),BGal(l 4)~BGlc- structure.
Example 2 -- Binding Inhibition Assays (Initial Peptide Screening)
Microtiter wells were coated with 100 ~1 of fetuin or asialofetuin
(50 ~g/ml) in 50 mM sodium phosphate buffer (pH 6.8) containing 5 mM
MgCl. and 15 mM NaN3 for 16 hours at 4~C. The solution was removed
by aspiration and replaced with 100 ~1 of 1% BSA in PBS containing
Q.05~ T~R ~ ~PBS~. Af~er incubation for 2-4 hours at roo~m
temperature, the microtiter wells were washed four times with 300 ,ul of
PBST. Peptides ranging in concentration from 0.5 to 4.5 mg/ml in PBS
(40 ,ul) were added to each well, and PT-biotin (10 ,ul cont~ining 10 ng in
PBS) was then added to the microtiter wells. After incubating for 1 hour,
the binding reaction was stopped by aspirating the solutions, and the plate
was washed with PBST (300 ,ul). Horseradish peroxidase-conjugated
avidin (100 ,ul, 1/3000 dilution in PBST to a concentration of 0.3 ,ug/ml)
20 was then added to the wells, and the plates were incubated at ambient
temperature for 1 hour. After washing the wells as described above, the
substrate solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2,
containing 0.1 % hydrogen peroxide, v/v) was added, and the plates were
incubated for 30 minutes. Color development was recorded at 405 nm
25 using a Titertek Multiskan MC plate reader. Maximum binding was
determined in the absence of peptide, and background binding was
measured in wells coated with BSA only. Binding assays for each peptide
were done in duplicate. Binding inhibition experiments utilizin SNA-,
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.
-42-
WGA-, and MAL-biotin were performed as described above using 10 ng
of each biotinylated lectin in PBS.
The panel of 20 peptides was assayed for the ability to inhibit PT-,
WGA-, MAL-, and SNA-biotin binding to fetuin or asialofetuin. The
5 biotinylated plant lectins have proved to be useful controls, since we have
previously showed that these lectins possess similar binding specificities
as Pr-5 46. The results from this example are set forth in Table II below.
These results indicate that peptides S2P3 (SEQ ID NO:4) and S2P6 (SEQ
ID NO:5) inhibited PT biotin binding by 15-20~ both to fetuin as well as
10 to asialofetuin. Two additional peptides derived from the S3 subunit
[S3P3 (amino acids 10-31 of SEQ ID NO:7) and S3PS (SEQ ID NO:8)]
were found to reduce binding of PT to asialofetuin only. The majority of
the other peptides displayed in Table II exhibited either marginal
inhibition or enhancement in binding of biotinylated PT to fetuin or
asialofetuin. Peptides S2Pl (amino acids 9-23 of SEQ ID NO:3),
ACS2Pl (amino acids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO:3)
showed nearly a 2-fold enhancement of PT-biotin binding relative to
control experiments done in the absence of peptide.
Peptide S2P3 (SEQ ID NO:4) was found to inhibit binding to
20 fetuin of all the biotinylated lectins. However, it is noted that peptides
S2Pl (amino acids 9-23 of SEQ ID NO:3) and S2P2 (SEQ ID NO:3),
which showed enhancement of binding of PT to fetuin, inhibited WGA
binding activity.
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Table II
Pçrcent Changes in Binding Activitv
Pq~tide SEQ ID NO Concen- Pl/ MALI SN~I WGA/ Pl/
~liOD fcb~- fd~ fduin fdu~ ~i~lo-
(m~/mL~ fauin
S2P1 3(~9-23) 1.2 +85i4 +8i4 -Sil -18i2 +113(1)
ACS2P1 3 (~ 9-23) 1.0 +89i3 +16i9 -3il -22i6 +190i73
S2 (14-23) 3 ( -14-23) 1.0 +68+9 +32i1 +1$2 +7+1 ND
S2P2 3 ~.1 +58i4 +14+2 -1+1 -15i2 +33(1
S2P3 4 0.51 -21iO -18i2 -ISi4 ~56i8 -15(1)
S2P4 5 (-~ 16-32) 0.9~ +~4i22 +2(1) +3(1) +15il -3(1)
0 S2P6 5 0.85 -28+6 +18il6 +2~2 +57+0 -18(1)
53P1 6 (~ 9-23) 3.2 +54i25 +4i6 -2+1 -9+4 +~5il6
S3P2 6 3.1 +77iS9 +12il6 +3i3 +9i4 +82ill
S3P3 7 (~10-31) 4.5 +19i67 +l3il5 +3+0 +6_3 -28+7
S3P4 7 3.3 +12i2 +7+1 +1(1) +13+1 +75~50
S3PS ~ 0.48 +~4i6 +3i6 0(1) +35i8 -26ilO
S3P9~ 9 1.7 +328+10 +18+6 - li3 ND ND
SPYGRC 3 (~-18-23) 4.4 +34+2 -16iO -2+2 +7+3 +7_9
S2(WGA) 10 1.0 +79+15 +22i3 ~il 4+3 ND
WGA 11 1.0 -32iS -li4 -3i2 -9il ND
(62-73)
* Biotinylated lectin/glycoprotein bound to
microtiter wells. Positive signs preceding
the numbers in this table indicate enhancement
and negative signs indicate inhibition of
binding.
(1) One determination only.
aa amino acid
ND Not determined.
SUBSTITUTE SHEET
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Example 3 -- Binding Inhibition Assays (Determination
of IC 50 Values)
Peptides found to have inhibitory activity in the initial screening
experiments were further analyzed in binding inhibition experiments to
5 determine the concentration of peptide that was required to reduce binding
by 50% (IC50 values).
Inhibition experiments were performed by a method similar to that
described in Example 2 with 2-fold dilutions of PT peptide in PBS, except
that microtiter wells were coated with 3 ,ug/ml fetuin or asialofetuin.
10 Binding assays for each inhibitor concentration were done at least in
duplicate, and the average value varied less than 15%. The concentration
of peptide required for 50% inhibition (IC50) was determined by plotting
the amount of binding observed in the presence of peptide inhibitor as a
percent of the maximum binding achieved without inhibitor.
Two of the peptides derived from the S2 subunit we~e able to
inhibit PT-biotin binding to fetuin at submillimolar concentrations, but
they were unable to inhibit PT-biotin binding to asialofetuin in subsequent
experiments (Table III). The two peptide sequences from the S3 subunit
(S3P3 [amino acids 10-31 of SEQ ID NO:7] and S3P5 [SEQ ID NO: 8])
20 were found to inhibit PT-biotin's interaction with asialofetuin in a
concentration-dependent manner, but their IC50 values were above the
solubility limits of the peptide in PBS. The peptide S2P3 (SEQ ID NO:
4) was also very active at inhibiting the interaction of MAL- and WGA-
biotin with fetuin. Upon closer examination this peptide proved to be
25 non-inhibitory for SNA-biotin. Two additional peptides from the S2
subunit (S2PI [amino acids 9-23 of SEQ ID NO: 3] and S2P2 [SEQ ID
NO: 3]) were active at inhibiting WGA-biotin binding at millimolar
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concentrations, suggesting that these peptide sequences may also be
important for interacting with oligosac~haride in the binding site of WGA.
Table III
Concentrations of PT Peptides and WGA Peptides Resulting
5in 50% Inhibition of Biotinylated PT and T ec~ins Binding
to Fetuin or Asialofetuin
Biotinylaled Lectin ~Iy.~prulc;.~) Pcptide SEQ ID NO IC,O(mM)
Pl'-b (retuin) S1P3 4 0.19iO.09
PT-b ~feluin) S2P6 5 0.'~ 0.06
10 WGA-b (fetuin) ACS"PI 3 (aa 9-"3) 3.'~5:~:0.45*
WGA-b (fetuin) S'~P' 3 Sl
WGA-b (fetuin) S''P3 4 014+0 03
MAL-b (fduin) S'~P3 4 0.86iO.0"
Pl-b (nsialofetuin) S3P3 7 (aa 10-31) Sl
15 Pl'-b (asialofetuin) S3P5 8 Sl
SNA-b (fetuin) S3P9a 9 ".74iO.86
Pl-b (fetuin) WGA16''-73) 11 1.5+0
WGA-b (fetuin) WGA(6~-73) 11 3.4iO
SI Slightly inhibitory, but the concentration of
20peptide required for 50% inhibition of binding is
above the solubility limit of the peptide.
* Unacetylated form of S2P1 (amino acids 9-23 of SEQ
ID NO: 3) inhibited to the same extent as the
acetylated form.
aa amino acid
Since three of the peptides derived from the S2
subunit of PT were able to inhibit WGA binding, the
amino acid sequences which constitute the sialic acid
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-46-
binding site of WGA were closely ~X~m; ned to determine
if there were any homologies with the inhibitory PT S2
subunit peptide sequences. One short 6-amino acid
sequence (SQYGHC) (SEQ ID NO: 12) corresponding to
amino acids 62-67 in WGA isolectin 2 displayed
reasonable homology with a sequence found both in the
S2Pl (amino acids 9-23 of SEQ ID NO: 3) and S2P2
peptides (SPYGRC, amino acids 18-23 of SEQ ID NO: 3)
from PT. This short sequence in WGA is responsible for
binding with the carbonyl of the N-acetyl ~roup of
sialic acid or N-acetyl-glucosamine (i.e., serine 62)
through a hydrogen bond. Non-polar interactions
between the aromatic side chains of tyrosine 64 as well
as histidine 66 interact with the glycerol side chain
of sialic acid and the pyranose ring of sialic acid or
N-acetyl-glucosamine, respectively62. The other
inhibitory peptide, S2P3 (SEQ ID NO: 4), di.d not
display any good homology with sequences responsible
for interacting with sialic acid in WGA. This
indicates that other motifs may also be functionally
important for interaction with sialic acid.
Exam~le 4 -- Binding Inhibition Studies Utilizing
Biotinylated and Acetylated S2Pl (amino
acids 9-23 of SEQ ID NO: 3)
From the initial peptide screening results (Table
II), peptides S2Pl (amino acids 9-23 of SEQ ID NO: 3)
and S2P2 (SEQ ID NO: 4) showed a 2-fold enhancement in
binding of PT-biotin to fetuin relative to control
experiments. One possible explanation for the
enhancement may be the ability of the peptide to form a
bridge between PT-biotin and fetuin. In order for the
peptide to act as a bridging molecule, the peptide must
contain both a recognition site for fetuin as well as a
sequence which binds to PT itself. To answer this
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-47-
question an acetylated and biotinylated form of the
peptide S2Pl (amino acids 9-23 of SEQ ID NO: 3)
(biotinylated at the terminal proline) was prepared to
determine if we could measure direct binding of the
S peptide to PT and fetuin.
Binding assays were done as described in Example 2
in PBS by using ACS2Pl-biotin (amino acids 9-23 of SEQ
ID NO: 3) at a concentration of lO ~g/ml in PBS.
Assays were carried out for l hour at room temperature,
and the amount of biotinylated peptide bound to fetuin
was determined by using avidin-peroxidase.
From these direct binding studies, a
concentration-dependent binding both to fetuin as well
a~ to PT ir~.ruobilized in micro~i~er wells was observed.
Furthermore, the binding of ACS2Pl-biotin (amino acids
~-23 of SEQ ID NO: 3) to PT could be inhibited by
fetuin (IC50 = 50 ~M; n=2) indicating that the
biotinylated peptide bound at or adjacent to the fetuin
binding site in PT.
In view of the above, it was concluded that
because of potential bridging by the peptide between
the lectin and the fetuin, any of the peptide sequences
set forth in Table I which caused reduction in the
binding of any of the lectins to fetuin were able to
bind to terminally linked ~-sialic acid(2-6)~Gal- and
~-sialic acid(2-3)~Gal- structures.
ExamPle 5 -- Binding Inhibition Assays Using
Oligosaccharides to Inhibit the Binding of
ACS2Pl-biotin (amino acids 9-23 of SEQ ID
NO: 3)
The binding results obtained for ACS2Pl-biotin
(amino acids 9-23 of SEQ ID NO: 3) described in Example
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-48-
4 above indicate a direct involvement of carbohydrate
moiety with the lectin through a biotinylated peptide
bridge.
In order to assess the binding to ~-sialic
acid(2-6)~Gal- and ~-sialic acid(2-3)~Gal- structures
by this peptide as well as the hexapeptide SPYGRC
(amino acids 18-23 of SEQ ID NO: 3) which only
exhibited reduction in the binding of one lectin to
fetuin (Table II), inhibition experiments with simple
saccharides were conducted to determine if the
interaction was carbohydrate-dependent. The simple
sugars chosen were sialic acid, sialyllactose from
bovine colostrum (contains a mixture of ~(2-6) and
~(2-3) linked sialic acid structures), lactose, and N-
acetyl-glucosamine.
Binding inhibition assays were done using sialic
acid, sialyllactose, lactose, or N-acetyl-glucosamine
at a concentration of 8 mM in PBS. Inhibition assays
were done for 1 hour as described above (e.g., Example
2) at room temperature, and the amount of biotinylated
peptide bound to fetuin (3~g/ml) quantitated by using
avidin-peroxidase. The pH of free sialic acid was
carefully monitored and adjusted to physiological pH
values with dilute sodium hydroxide. The results of
this example are set forth in Table IV below:
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49
Table IV
Inhibition of Binding of ACS2Pl-biotin (amino acids
9-23 of SEQ ID NO. 3) and SPYGRC-biotin (amino
acids 18-23 of SEQ ID NO. 3) to Fetuin by Simple
Saccharides*
S~cchAride Percent Change~ in Binding
Activity
ACS2P1-biotin SPYGRC-biotin
(n=3) (n~3)
Sialic acid -19+6 +9+8
Sialyllacto~e-46+3 -24~6
Lacto~e ~-74+7 +20~11
0 N-Acetylgluco~amine -10+2 +6+5
Sucrose 0+3 ND
* Inhibition experiments were carried out as
outlined above using O.l~g or 0.5~g of ACS2Pl-
biotin (amino acids 9-23 of SEQ ID NO. 3) and
SPYGRC-biotin (amino acids 18-23 of SEQ ID
NO.3) respectively.
Positive signs prpr~ ng the number in this
table indicate enhancement and negative signs
indicate inhibition of binding to fetuin.
ND Not determined
The above results indicate that both peptides
tested were able to bind to terminally linked ~-
sialic acid(2~6)~Gal- and ~-sialic acid(2~3)~Gal-
structures. Control experiments indicated that
sialic acid was necessary for high affinity
interaction since lactose failed to reduce the
binding of ACS2Pl-biotin (amino acids 9-23 of SEQ
ID NO. 3) on N-acetyl-glucosamine was also found
to marginally inhibit the binding of biotinylated
peptide (lO + 2%, n=3) suggesting that the ACS2Pl-
biotin (amino acids 9-23 of SEQ ID NO. 3) may have
weak affinity for the N-acetylglucosamine
consistent with the finding described below, which
shows enhanced binding of the peptide to
SV~STITVTE SHEEl~
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WO94/07517 PCT/CA93/00415
-50-
asialoagalactofetuin relative to asialofetuin. This is
also in good agreement with previous results which
indicate PT's binding specificities to be similar to
those of WGA~52.
In view of the above, it was concluded that any of
the peptides which inhibit the binding of fetuin to any
of the lectins set forth in Table II above are
effective in binding to terminally linked ~-sialic
acid(2-6)~Gal- and/or ~-sialic acid(2~3)~Gal-
structures. Such peptides are set forth in Figure l.
ExamPle 6 -- Screening of Subunit S2 and S3 Peptides
from PT for the Ability to Inhibit ACS2Pl-
biotin Binding to Fetuin and Asialofetuin
Binding inhibition assays were conducted using
subunit S2 and S3 peptides from PT in PBS as
competitors for binding of ACS2Pl-biotin (amino acids
9-23 of SEQ ID NO: 3) (lO ~g/ml) to fetuin or
asialofetuin. Inhibition assays were done for l hour
at room temperature, as described above, and the amount
of biotinylated peptide bound to fetuin or asialofetuin
(3 ~g/ml) quantitated by using avidin-peroxidase.
The specificity and relative affinity of ACS2Pl-
biotin (amino acids 9-23 of SEQ ID NO: 3) for fetuin
was determined by performing binding inhibition
experiments with the peptides shown in Table I.
Binding inhibition experiments (Table V) indicated that
the ACS2Pl peptide (amino acids 9-23 of SEQ ID NO: 3)
bound to fetuin with high affinity (IC50 = 4.l ~M; n-2)
when the unbiotinylated form of ACS2Pl (amino acids 9-
23 of SEQ ID NO: 3) was used as competitor. Thepeptide S2P2 (SEQ ID NO: 3), an extended version of
S2Pl (amino acids 9-23 of SEQ ID NO: 3), was also found
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-51-
to inhibit ACS2P1-biotin (amino acids 9-23 of SEQ ID
NO: 3) binding to fetuin but with a 10-fold decrease in
affinity (IC50 = 42.5 ~M). Both peptides were una~le to
compete for the binding of ACS2Pl-biotin (amino acids
9-23 of SEQ ID NO: 3) to asialofetuin, indicating the
importance of sialic acid for high affinity interaction
with the peptide. Similar hexapeptide sequences to
those found within the S2P1 (amino acids 9-23 of SEQ ID
NO: 3) and S2P2 (SEQ ID NO: 3) peptides are also
present in a number of the peptides from Table I (see
underlined segments). Each of these peptides were
analyzed for their ability to inhibit ACS2P1-biotin
(amino acids 9-23 of SEQ ID NO: 3) binding to fetuin
and asialofetuin at peptide concentrations
10-fold higher than the IC50 determined for the S2Pl
(amino acids 9-23 of SEQ ID NO: 3) peptide. None of
the other peptides examined from Table I had the
ability to inhibit binding of ACS2P1-biotin (amino
acids 9-23 of SEQ ID NO: 3) to fetuin or asialofetuin
to the same extent indicating that the peptide sequence
SPYGRC may play a crucial role for binding sialic acid.
This is further confirmed by the inability of the
peptides S3Pl (amino acids 9-23 of SEQ ID NO: 6) and
S3P2 (SEQ ID No. 6), which possess the strongest
homologies to the sequence found in S2P1 (amino acids
9-23 of SEQ ID NO: 3), to inhibit binding even at high
concentrations of peptide. The homologous sequence
found in the S3 peptides (GAYGRC) (amino acids 18-23 of
SEQ ID NO: 6) lacks the serine amino acid residue,
which was shown to be important for forming an
important hydrogen bond with the carbonyl of the N-
acetyl group in the sialic acid binding site of WGA62.
This indicates that the serine residue found in the
S2P1 (amino acids 9-23 of SEQ ID NO: 3) peptide may
function in an analogous manner in the binding of S2P1
(amino acids 9-23 of SEQ ID NO: 3) to sialic acid.
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-52-
The results of this example are set forth in
Table V below:
Table V
Screening of S2 and S3 Peptides from PT for the
Ability to Inhibit ACS2Pl-biotin (amino acids 9-23
of SEQ ID NO:3) Binding
Pcp~ide SEQ ID NO Co~ dLiu~ Fc~uin' Asialo-
(IlM) fet.uin~
ACS2P1 3 (aa 9-23) 4.1 -50 +lOli:2
S2~ 3 42.5 -50 +6+ 1
0 SPYGRC 3 (aa 18-23) S5~0 -24:t:2 -S4i3
S2P3 4 22.4 +70ill +1Sill
S2P4 S (aa 1~3,) 6.0 -8il6 +2i4
S2P6 S 319 +49:!:5 +18il6
S3P1 6 (aa 9-23) 1142 +24Si2 +4iO
S3P2 6 934 +88i72 +12il6
S3P3 7 (aa 10-31) 16.1 -l3il6 -lOill
S3P4 7 6.4 +55i26 +26i2
S3P5 8 22.5 -19i4 -17i8
The effect of S2 and S3 PT peptides on the
binding of ACS2Pl-biotin (amino acids 9-23 of
SEQ ID NO:3) (O.l~g) to fetuin or
asialofetuin. Negative and positive values
refer to percent inhibition or enhancement
respectively.
aa amino acid
~ Percent Change in Binding Activity--; and
ExamPle 7--Iodination of ACS2Pl-biotin (amino acids
9-23 of SEQ ID NO. 3)
Previous reports have suggested the importance
of tyrosine amino acid residues in the binding of
PT to sialylated glycoprotein receptors~63. These
reports were based on the observation that if PT is
iodinated
- SUeSTlTUTE Si~EET
CA 02211~63 1997-09-19
WO94/07517 PCTICA9310~15
by the conventional IODO-GEN procedure (selectively
modifies tyrosine residues) without first protecting
the binding site for fetuin, the binding activity of PT
was reduced.
To determine if the tyrosine residue found in
ACS2Pl-biotin (amino acids 9-23 of SEQ ID NO: 3) plays
a role in the binding activity, the peptide was
iodinated by the IODO-~EN procedure and its binding
activity compared with the uniodinated peptide.
Specifically, ACS2Pl biotin (amino acids 9-23 of SEQ ID
NO: 3) (l00 ~g 0.052 ~mol) in l00 ~l of PBS was placed
in a 12 x 75-mm glass tube coated with IODO-GEN, and
0.1 mM solution of NaI (50 ~L 5 ~mol) was added and
gently mixed over a period of l0 minutes at room
temperature. The reaction was terminated by removing
the mixture from the IODO-GEN tube and the iodinated
peptide was purified on a Sephadex G-25 column (l x 15
cm equilibrated with PBS). Individual fractions were
analyzed for the presence of peptide by measuring
absorbance at 220 nm, and the concentration was
determined by comparing the absorbance with
underivatized ACS2Pl-biotin (amino acids 9-23 of SEQ ID
NO: 3). A sample of iodinated and underivatized
ACS2Pl-biotin (amino acids 9-23 of SEQ ID NO: 3) were
diluted to a concentration of lO ~g/ml and analyzed for
binding to fetuin coated microtiter wells as described
in Example 6. In addition, binding of ACS2Pl-biotin
(amino acids 9-23 of SEQ ID NO: 3) (l0 ~g/ml) to
fetuin, asialofetuin, and asialoagalactofetuin (each at
3 ~g/ml) was done in a similar manner.
The results of this experiment indicate that
extensive iodination of ACS2Pl-biotin (amino acids 9-23
of SEQ ID NO: 3) reduced binding of the peptide to
fetuin by 58 + 3% (n=3), which suggests the importance
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-54-
of the tyrosine amino acid in addition to the serine
for the binding activity of ACS2P1-biotin (amino acids
9-23 of SEQ ID N0: 3) to terminally linked ~-sialic
acid(2-6)~Gal- and ~-sialic acid(2~3)~Gal- structures.
S To determine whether the amino acid sequence in
the S2 subunit of PT that corresponds to the peptide
S2Pl (amino acids 9-23 of SEQ ID N0: 3) plays an actual
role in the lectin-like binding activity of PT, the
binding specificity of the biotinylated peptide was
compared with that of PT using fetuin, asialofetuin,
and asialoagalactofetuin. A previous report had
determined that the binding of l25I-PT to asialofetuin
was 53 + 7%, while asialoagalactofetuin was 81 + 8%
relative to control binding to fetuin~. This is in
good agreement with the results obtained with
ACS2Pl-biotin (amino acids 9-23 of SEQ ID N0: 3) which
showed 45 + 9% and 93 + 18% (n=3) binding to asialo-
and asialoagalactofetuin, respectively, relative to
fetuin. These results suggest that this amino acid
sequence may contain a portion of a lectin-like binding
site in the S2 subunit of PT, which is responsible for
the binding results previously observed.
Example 8
Binding assays were carried out essentially as
described above using microtiter wells that were coated
with 50 ~1 of BSA glycoconjugate (50 ~g/ml) in 50 mM
sodium phosphate buffer (pH 6.8) containing 5 mM MgCl2
nd 15 mM NaN3 for 16 hours at 4OC. The solution was
removed by aspiration and replaced with 100 ~1 of 1%
BSA in PBS containing 0.05% Tween 20 (PBST) and
incubated at room temperature for an additional 2-3
hours. The microtiter wells were washed four times
with 300 ~1 of PBST and then replaced with ACS2P1-
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biotin (amino acids 18-23 of SEQ ID NO. 3) (0.5 ~g)
in 50 ~1 of PBS. After incubating for 1 hour, the
binding reaction was stopped by aspirating the
solutions and the plate was washed with PBST (4 X
300~1). Avidin-peroxidase (100 ~1 of a 1/3000
dilution of a 1 mg/ml solution in PBST) was added
and incubated for an additional 1 hour. After
washing the wells as described above, the substrate
solution (1 mM ABTS in 5 mM citrate buffer, pH 4.2,
containing 0.1% hydrogen peroxide, v/v) was added
and the plates were incubated for 30 minutes.
Binding assays for each BSA conjugate were done in
triplicate, and background binding was measured in
wells coated with BSA only. The extent of
biotinylated peptide ~inding is expressed as a
percentage relative to fetuin as shown in Table VI
below:
Table VI
Binding of ACS2P1-biotin (amino acids 9-23 of SEQ
ID NO. 3) and SPYGRC-biotin (amino acids 18-23 of
SEQ ID NO. 3) to BSA Conjugates*
C~ uL~L~.~ci Structure of BSA % Bindu~g % Bu ding Common
r~ju~, Rclative to Rel~tivc to Nnme for
Fetuin of Fetuin of Carbohydrat
ACS2PI- SPYGRC- c Structure
biotin (n=3) biotin (n=3)
~NeuAc(2-3),~Gal(l 4),BGlcNAc-BSA 117il2 120i7 SLscNAc
25 ~NeuAc(2-3),BGal(1-3),BGlcNAc-BSA 70i4 97il2 SLe'
~YNeuAc(2~),~Gal(l~),~GlcNAc-BSA 94+2 122i5 SLacNAc
cYNeuAc(2-3),BGal(1-4),BGlcNAc-BSA 84i3 120i4 SW
(1-3)
tYFuc
30 ~NeuAc(2-3),BGal(1-3),BGlcNAc-BSA 84il4 103i8 SLe (C19.9)
(1-4)
cYFuc
~Experiments were done by coat_ng 50 ~g/mL BSA-
conjugate or fetuin and probed with 0.1 ~g ACS2Pl-
biotin (amino acids 9-23 of SEQ ID NO. 3) or 0.5 ~g
of SPYGRYC-biotin (amino acids 18-23 of SEQ ID NO.
3) for 1 hour at room temperature.
SlJBSTITlJTE SftEET
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Exam~le 9 -- Formation of a SPYGRC-Streptavidin-
Biotin
Conjugate
Streptavidin (50 ~g) in PBS was combined with
SPYGRC-biotin (amino acids 18-23 of SEQ ID N0. 3)
(50 ~l, 200 ~g) and PBS (150 ~1) and mixed for 1
hour at room temperature. The reaction mixture was
loaded onto a Sephadex G-25 column (1 x 15 cm that
had been equilibrated in PBS) and fractions
collected. Protein cont~;n;ng fractions were
analyzed by SDS PAGE gel electrophoresis, and the
molecular weight was determined. The results were
consistent with the formation of a tetravalent
complex between peptide and streptavidin. Lectin
binding inhibition experiments were carried out as
described previously for determination of IC50's.
The results are set forth in Table VII below:
Table VII
The Effect of the Peptide SPYGRC or SPYGRC-biotin-
Streptavidin Conjugate (amino acids 18-23 of SEQ ID
NO. 3) on the Binding of Biotinylated PT and
Lectins to Fetuin*
Bi~ti.. ~' ' lcctin cnA~ of % Changes C~n~ of %
SPYGRC (a~ 18- in Binding Pc~tidc- Chnngcs
23 of SEQ ID ActivityS1.C~J~.;dh~ in Binding
NO.3) Peptide C~nj~ g/ml) Acti~rity
(mglml)
PT-b 4.4 +34i2 500 +13ill
2~i MAL-b 4.4 -16iO 1.0 -SO+3
SNA-b 4.4 -2i 1 1.2 -50i5
WGA-b 4.4 +7i3 64 -SO+I
*Positive signs pr~c~;ng the numbers in this table
indicate enhancement, and negative signs indicate
inhibition of binding.
aa amino acids
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ExamPle 10 -- Neutralization of Pertussis Toxin
Binding to Chinese Hamster Ovary (CHO)
Cell by Synthetlc Peptides
Confluent monolayers of CHO cells were lifted from
plastic tissue culture flasks with 0.25% trypsin and
suspended at a concentration of 5 x 104 cells/ml in
Ham's F12 media containing 10% fetal bovine serum
(FBS). 100 ~l of the cell suspension was added to 96
well tissue culture plates and was allowed to establish
contact with the plastic for 24 to 48 hours. The
expended media was then removed and filtered-sterilized
peptide 5-fold dilutions ranging in concentration from
300 to 30 femptogram per ml (80 ~l) in Ham's Fl2 media
containing FBS were added to the CHO cells. 20 ~l of a
PT solution (final PT concentration 2.7 ng/ml) was then
added to the tissue culture wells containing peptide.
The incubation mixtures were gently mixed and incubated
at 37~C for 1 hour in a C02 incubator. The incubation
mixtures were then removed and replaced with fresh
media. The tissue culture plates were then incubated
for 24 hours at 37~C, fixed with 100% methanol, and
stained with Geimsa stain. Control experiments were
done in the absence of peptide alone or in the absence
of PT. All determinations were done in triplicate.
Stained CHO cells were then examined for inhibition of
characteristic CHO cell clumping which is mediated by
pertussis toxin binding and scored as to whether there
was greater than 50% inhibition of clumping of CHO
cells relative to control wells in the presence of PT
only. The results reported in Table VIII show the
maximum peptide concentration required to cause a 50%
reduction in CHO cell clumping.
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Table VIII
CHO Cell Neutralization Experiments
Using PT S2 and S3 Peptides*
PT SEQ ID N0 M~Y; -1 Inhibitory
P~ptide Concentration for Greater
than 50~ Inhibition
AC52P1 3 (aa 9-23) 96 pg/ml
SPYGRC 3 (aa 18-23) 480 pg/ml
S2P2 3 NI
S3P1 6 (aa 9-23) NI
0 S3P2 6 NI
S3P9a 9 NI
* CHO cell neutralization experiments were
carried out using a PT concentration of 2.4
ng/ml. CHO cell (5.4 x 104 cells/ml) were
exposed to incubation mixtures containing
peptide and PT for l hour at room temperature.
Control experiments were done in the absence
of peptide.
aa amino acid
~0 NI Not inhibitory at a peptide concentration of
300 ng/ml.
pg picogram--.
The above data demonstrates that at least
certain of the subject peptides would be effective
in inhibiting the attachment of bacterial/viral
agents and/or their toxins which utilize ~-sialic
acid(2-6)~Gal- and/or ~-sialic acid(2-3)~Gal-
structures as the attachment point on the surface
of mammalian cells. Such agents/toxins include
known toxins such as pertussis toxin, cholera
toxin, etc., and, accordingly, administration of an
effective amount of at least one of the subject
peptides to a mammal would be effective in
inhibiting such attachment.
Examples ll and 12 below illustrate in vivo
results for the subject peptides.
SUBSTITUTE SHEtT
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59
ExamDle 11 -- Inhibition of DTH Inflammatory Response
DTH inflammatory responses were measured using the
mouse footpad swelling assay as described by Smith and
Ziola~. Briefly, groups of Balb/c mice (about 19-20
grams each) were immunized with 100 ~g of the OVA
antigen containing 20~g of the adjuvant (DDA --
dimethyl-dioctadecylammonium bromide) which also
induces a strong inflammatory DTH response. Seven days
later, each group of mice was footpad-challenged with
20 ~g of the OVA antigen (without adjuvant). The
resulting inflammatory footpad swelling was measured
with a Mitutoyo Engineering micrometer 24 hours after
challenge.
To assess the effect of different peptides on the
inflamma~ory DTH response, groups of mice received 100
~g of the following peptides: ACS2P1 (2275) (amino
acids 9-23 of SEQ ID NO: 3), SPYG~C (2283) (amino acids
18-23 of SEQ ID NO: 3), and S3P9a (SEQ ID NO: 9).
These peptides were injected as a solution into the
tail vein 5 hours after challenge. Control groups were
left untreated or received 100 ~L of phosphate-buffered
saline (PBS). The results of this experiment are shown
in Figure 2 which illustrates that the peptides
employed were effective in reducing a DTH response in
mice.
ExamPle 12 -- Persistence of Suppression of the DTH
Inflammatory Response at 2 Weeks After
Challenge
Identical groups of mice treated with the peptides
in Example 11 above were re-challenged with OVA antigen
2 weeks after primary immunization. Untreated controls
responded with the usual degree of footpad swelling,
whereas all other groups showed reduced footpad
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swelling. Specifically, these results are set forth in
Figure 3 which illustrates reduction in the degree of
footpad swelling in mice previously treated with the
subject peptides.
In addition to providing suppression of antigen
induced inflammation in a sensitized mouse, the above
data demonstrate that treatment with the subject
peptides as per this invention also imparts tolerance
to still later challenges from the same antigen.
In view of the fact that the immune system of mice
serves as a good model for the immune system of humans,
the above data demonstrates that the subject peptides
would be effective in suppressing cell-mediated immune
responses in humans, and, when the cell-mediated immune
response is to an antigen, this data also shows that
the subject peptides would also impart tolerance
to later challenges to the human of that antigen.
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TABLE IX
Initial screening of synthetie pcptidcs for inhibitory aeti~ity
Peptid~ C ~ ;.. , Changes in binding activity
(mglml)
Pl'/fcluin- MAL/fctuisl SNA/fe~uLn WGA/fauin
ACS2PI~ 1.0 1.89 1:0.03 1.16iO.09 0.97:t:0.01 0.78iO.06
5 NSS2P1 2.4 1.18 t0.03 1.03iO.02 I.lOiO.02 1.03iO.02
SPYGRC 4.4 1.34:~0.0'7 0.84+0.00 0.98 t0.02 1.07iO.03
GSYRPC 2.2 1.48+0.21 0.95iO.16 0.96+0.02 l.OgiO.OI
SPYGYC 1.0 2.85~0.16 1.21~0.08 1.07:~0.15 1.09t0.07
SPWGRC 1.0 1.75iO.53 1.39iO.13 1.00:t0.02 1.11+0.01
10 SPFGRC 1.0 1.32 1 0.08 1.06i0.12 1.0OiO.01 1.01 +0.01
2420 1.0 1.79' 1.43' 1.11' 2.1'
' Biotinylatcd leclinl~ly~o~J~ut~ bound to mierotiter wells. Values greater than I in the table
indicatc r-~ nr- ---- -~1 while numbers less than 1 indicate inhibition of binding.
,v;ou~l~ rcport~d valucs.
15 ' One J~.,.. ~.tion only.
~amPle 13 -- Binding Inhibition Assays (Initial
Peptide Screening).
Microtiter wells were coated with lO0 ~l of
fetuin or asialofetuin (50 ~g/ml) in ~0 Mm sodium
phosphate buffer (pH 6.8) containing 5 mM MgCl2 and
15 mM NaN3, for 16 hours at 4~C. The solution was
removed by aspiration and replaced with lO0 ~l of
1% BSA in PBS containing 0.05% Tween 20 (PBST).
After incubation for 2-4 hours at room temperature,
the microtiter wells were washed four times with
300 ~l of PBST. Peptides ranging in concentration
from l.0 to 4.4 mg/ml in PBS (40 ~l) were added to
each well, and PT-biotin (lO ~l containing lO ng in
PBS) was then added to the microtiter wells. After
incubating for l hour, the
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binding reaction was stopped by aspirating the
solutions, and the plate was washed with PBST (300 ~l).
Horseradish peroxidase-conjugated avidin (lO0 ~l,
l/3000 dilution in PBST to a concentration of 0.3
~g/ml) was then added to the wells, and the plates were
incubated at ambient temperature for l hour. After
washing the wells as described above, the substrate
solution (l mM ABTS in 5 mM citrate buffer, pH 4.2,
containing 0.1% hydrogen peroxide, v/v) was added, and
the plates were incubated for 30 minutes. Color
development was recorded at 405 nm using a Titertek
Multiskan MC plate reader. Maximum binding was
determined in the absence of peptide, and background
binding was measured in wells coated with BSA only.
Binding assays for each peptide were done in duplicate.
Binding inhibition experiments utilizing SNA-, WGA-,
and MAL-biotin were performed as described above using
lO ng of each biotinylated lectin in PBS.
The panel of 8 peptides was assayed for the
ability to inhibit PT-, WGA-, MAL-, and SNA biotin
binding to fetuin or asialofetuin. The biotinylated
plant lectins have proved to be useful controls, since
we have previously showed that these lectins possess
similar binding specificities as PT2546.
The results from this example are set forth in
Table IX above. These results indicate that peptides
based on the SPX~GX2C (SEQ ID NO:l) motif inhibited PT
biotin binding.
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TABLE X
Inhibition of biotinylated peptide binding to
fetuin by simple saccharides'
S~ccl.. ;dc Chang~ in binding activity
SPWGRC-biotin SPFGRCbio~ SPYGYCGFGAEY-
(n = 3) (n = 3) biotin
(n = 3)
Sialic acid 0.71+0.11 1.09~0.03 1.21iO.09
Sialylsctose 0.65+0.05 0.92~0.04 1.08iO.03
Lactose 0.96+0.12 0.78 t0.05 1.06~0.00
N-Acctyl~ .... r 0.82~0.10 1.07iO.14 1.10iO.25
~ Inhibition experiments were carried out as
outlined in methods using 0.5 ~g of biotinylated
peptides respectively. Binding values greater than
l in this table indicate enhancement and values
less than l indicate inhibition of binding to
fetuin. Concentration of saccharides used were 8
15 mM.
Exam~le 14 -- Binding Inhibition Assays Using
Oligosaccharides
In order to assess the binding to ~-sialic
acid (2-6)~Gal- and ~-sialic acid (2-3)~Gal-
structures by certain peptides, inhibitionexperiments with simple saccharides were conducted
to determine if the interaction was carbohydrate-
dependent. The simple sugars chosen were sialic
acid, sialyllactose from bovine colostrum (contains
a mixture of ~(2-6) and ~(2-3) linked sialic acid
structures), lactose and N-acetyl-glucosamine at a
concentration of 8mM in PBS. Inhibition assays
were done for l hour as described above (e.g.,
Example
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2) at room temperature, and the amount of biotinylated
peptide bound to fetuin (3~g/ml) quantitated by using
avidin-peroxidase. The pH of free sialic acid was
carefully monitored and adjusted to physiological pH
values with dilute sodium hydroxide. The results of
this example are set forth in Table X above.
The above results indicate that some of the
peptides tested were able to bind to terminally linked
a-sialic acid(2-6)~Gal- and ~-sialic acid(2-~3)~Gal-
structures.
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,A3L, XI
Bin~ing of biotirryfa~c~ pcpn~ o si~ d BSA conjs ~atcs~
CarbohYdrate St~ucmrc of Binding ~cl~siYe Bhti~ ~clati~c Bmding ~da~Ye
BSA t-onJu~sttr to }:ctuin of to Fe~n of to Fen~in of
SPWGRC-bio~n SPFGRC-biot~n SPYGYCGFC;-
AEY-~io~n
(n~3~ (n-3) (n.3)
aNcuAc(2~ 3Gal(1-4)~GIcNAc BSA 0.49 i 0.070.63 + 0.270.27 + 0.12
ah~euAc(2-3),8GaI(1-3)~G1cNAc-BSA 0.43 i 0.100.36 i O.1?0.2S ~ 0.04
~euAc(2-6)~G~ 4)~ Ae-B~A 0.07 ~ 0..030.29 + O.OS 0.28 + 0.11
~NeuAc(2-3)~Gal(1~),~GlcNAc-BSA 0.67 i 0.140.42 i 0.1~ 0.33 + 0.04
(1-3)
~Fuc
~euAc(2-3)~Ga~ 3)~GlcNAG-BsA 0.77 t 0.19 0.52+0.18 0.51iO12
(1-4)
oFuc
a F ~ lr ; ~ were~ncbycoa~g~S.O~ BSA~jn~u~orfe~a~p~ ~0.5
~gbioDnyla~d~f~lhlhe b~dng of b;o~la~l ~p~ ~ fc~ is set at a ~ of 1Ø
Exam~le 15
Binding assays were carried out essentially as
described above using microtiter wells that were coated
with 50 ~l of BSA glycoconjugate (50 ~g/ml) in 50 mM
sodium phosphate buffer (pH 6.8) containing 5 mM MgCl2
nd 15 mM NaN3 for 16 hours at 4~C. The solution was
removed by aspiration and replaced with 100 ~l of 1%
BSA in PBS containing 0.05% Tween 20 (PBST) and
incubated at room temperature for an additional 2-3
hours. The microtiter wells were washed four times
with 300 ~l of PBST and then replaced with 0.5
microgram of biotinylated peptide. After incubating
for 1 hour, the binding reaction was stopped by
aspirating the solutions and the plate was washed with
PBST (4 X 300~1). Avidin-peroxidase (100 ~l of a 1/3000
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-66-
dilution of a l mg/ml solution in PBST) was added and
incubated for an additional l hour. After washing the
wells as described above, the substrate solution (l mM
ABTS in 5 mM citrate buffer, pH 4.2, containing 0.1%
hydrogen peroxide, v/v) was added and the plates were
incubated for 30 minutes. Binding assays for each BSA
conjugate were done in triplicate, and background
binding was measured in wells coated with BSA only.
The extent of biotinylated peptide binding is expressed
as a ratio relative to fetuin as shown in Table XI
above:
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Example 16 -- Inhibition of DTH Inflammatory Response
DTH inflammatory responses were measured using the
mouse footpad swelling assay as described by Smith and
Ziola~. Briefly, groups of Balb/c mice (about 19-20
grams each) were immunized with 100 ~g of the OVA
antigen containing 20~g of the adjuvant (DDA --
dimethyl-dioctadecylammonium bromide) which also
induces a strong inflammatory DTH response. Seven days
later, each group of mice was footpad-challenged with
20 ~g of the OVA antigen (without adjuvant). The
resulting inflammatory footpad swelling was measured
with a Mitutoyo Engineering micrometer 24 hours after
challenge.
To assess the effect of different peptides on the
inflammatory DTH response, groups of mice received 100
~g of the following peptides: ACS2Pl (2275) (amino
acids 9-23 of SEQ ID NO. 3), SPFGRC (2418), SPWGRC
(2416), AcSPYGYCGFGAEY-CONH2 (2420) SPYGYC(2365), SPYGRC
(amino acids 18-23 of SEQ ID. NO: 3) (2283),
NSS2P1(2295), and GSYRPC(2294). These peptides were
injected as a solution into the tail vein 5 hours after
challenge. Control groups were left untreated or
received 100 ~L of phosphate-buffered saline (PBS).
The results of this experiment are shown in Figures 4
and 6 which illustrate that the peptides employed were
effective in reducing a DTH response in mice.
Exam~le 17 -- Persistence of Suppression of the DTH
Inflammatory Response at 2 Weeks After
Challenge
Identical groups of mice treated with the peptides
in Example 16 above were re-challenged with OVA antigen
two weeks or 15 days after primary immunization.
Untreated controls responded with the usual degree of
footpad swelling, whereas all other groups showed
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-68-
reduced footpad swelling. Specifically, these results
are set forth in Figures 5 and 7 which illustrate
reduction in the degree of footpad swelling in mice
previously treated with the subject peptides.
In addition to providing suppression of antigen
induced inflammation in a sensitized mouse, the above
data demonstrate that treatment with the subject
peptides as per this invention also imparts tolerance
to still later challenges from the same antigen.
In view of the fact that the immune system of mice
serves as a good model for the immune system of humans,
the above data demonstrates that the subject peptides
would be effective in suppressing cell-mediated immune
responses in humans, and, when the cell-mediated immune
response is to an antigen, this data also shows that
the subject peptides would also impart tolerance
to later challenges to the human of that antigen.
CA 022ll563 l997-09-l9
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_og -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: ALBERTA RESEARCH COUNCIL
l'B'I STREET: 250 KARL CLARK ROAD
,C CITY: EDMONTON
,DI STATE: ~T.R~RTA
~E COUNTRY: CANADA
Fl ZIP: T6H SX2
,G TE~EPHONE: 403-450-Slll
,,H, TELEFAX: 403-461-2651
(ii) TITLE OF lhv~L-ION: ANTI-INFLAMMATORY, TOL~O~NlC ~ND
IMMUNOINHIYITING PROPERTIES OF ~PRR~YDRATE
BINDING-~-rll~5
(iii~ NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BLAKE, CASSELS ~ GRAYDON
(B) STREET: BOX 25, COMMERCE COURT WEST
(C) CITY: TORONTO
(D) STATE: ONT
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(B COMPUTER: IBM PC compatible
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(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B~ FI~ING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/995,503
(B) FILING DATE: 21-DEC-1992
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/956,043
~B) FILING DATE: 02-OCT-1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GRAY, BRIAN W.
(B) REGISTP~ATION NUMBER: 3752
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(ix) TELECOMMUNICATION INFORMATION:
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- SUBST~TUTE ~HEET
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-70-
~ (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "X is selected from the
group consisting of amino acids Tyr, Phe, Trp and
His or peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(8) LOCATION: S
(D) OTHER INFORMATION: /note= "X is selected from the
group conqisting of amino acids Tyr, Phe, Arg, Trp
and His or peptide mimetics thereof"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ser Pro Xaa Gly Xaa Cys
~2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/REY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "X is selected from the
group consisting of amino acids Tyr, Phe, Trp and
His of peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: S
(D) OTHER INFORMATION: /note= "X is selected from the
group consisting of amino acids Tyr, Phe, Arg,
Trp, and Hi~ or peptide mimetics thereof"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: ?
(D) OTHER INFORMATION: /note= "4-6 amino acids"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "X is selected from the
group consisting of amino acids Tyr, Phe, Trp and
His or peptide mimetics thereof"
CA 02211563 1997-09-19
WO 94/07517 PCI~/CA93/00415
-71-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ser Pro Xaa Gly Xaa Cys Xaa Xaa
1 5
(2) INFORMATION FOR SEQ ID NO:3:
( i ) ~QU~ N~: CHARACTERlSTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Thr Pro Gly Ile Val Ile Pro Pro Gln Glu Gln Ile Thr Gln Hi~
1 5 10 15
Gly Ser Pro Tyr Gly Arg Cys
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) s~u~ DESCRIPTION: SEQ ID NO:4:
Gly Ala Phe Asp Leu Lys Thr Thr Phe Cy9 Ile Met Thr Thr Arg Asn
1 5 10 15
Thr Gly Gln Pro Ala
(2) INFORMATION FOR SEQ ID NO:5:
(il SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Phe Val Arg Ser Gly Gln Pro Val Ile Gly Ala Cys Thr Ser Pro Tyr
1 5 10 lS
Asp Gly Lys Tyr Trp Ser Met Tyr Ser Arg Leu Arg Lys Met Leu Tyr
CA 02211~63 1997-09-19
WO 94/07517 PCI/CA93/00415
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHAP~ACTERISTIC5:
(A) LENGTH: 2~ amLno acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Val Ala Pro Gly Ile Val Ile Pro Pro Ly~ Ala Leu Phe Thr Gln Gln
1 5 10 15
Gly Gly Ala Tyr Gly Arg Cys
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Gly Phe Ile Tyr Arg Glu Thr Phe Cys Ile Thr Thr Ile Tyr Ly~
1 5 10 15
Thr Gly Gln Pro Ala Ala Asp His Tyr Tyr Ser Lys Val Thr Ala
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Cy5 Ala Ser Pro Tyr Glu Gly Arg Tyr Arg Asp Met Tyr Asp Ala Leu
1 0 1 ~;
Ary Arg Leu Leu Tyr
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
SUBSTil~UTE S~ET
CA 02211563 1997-09-19
WO 94J07~il7 PCI/CA93/00415
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Arg Leu Leu Ala Ser Thr Asn Ser Arg Leu Cys Ala Val Phe Val Arg
A~p Gly
(Z) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Pro Gln Glu Gln Ile Thr Gln His Gly Ser Gln Tyr Gly Tyr Cy~
1 5 10 lS
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amlno acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Ser Gln Tyr Gly Tyr Cys Gly Phe Gly ALa Glu Tyr
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ser Gln Tyr Gly His Cys
