U.S. patent application number 10/044844 was filed with the patent office on 2004-01-15 for apl immunoreactive peptides, conjugates thereof and methods of treatment for apl antibody-mediated pathologies.
Invention is credited to Jones, David S., Marquis, David Matthew, Victoria, Edward Jess, Yu, Lin.
Application Number | 20040009904 10/044844 |
Document ID | / |
Family ID | 27413625 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009904 |
Kind Code |
A1 |
Victoria, Edward Jess ; et
al. |
January 15, 2004 |
APL immunoreactive peptides, conjugates thereof and methods of
treatment for aPL antibody-mediated pathologies
Abstract
aPL analogs that (a) bind specifically to B cells to which an
aPL epitope binds and are disclosed. Optimized analogs lack T cell
epitope(s) are useful as conjugates for treating aPL
antibody-mediated diseases. Conjugates comprising aPL analogs and
nonimmunogenic valency platform molecules are provides as are novel
nonimmunogenic valency platform molecules and linkers. Methods of
preparing and identifying said analogs, methods of treatment using
said analogs, methods and compositions for preparing conjugates of
said analogs and diagnostic immunoassays for aPL antibodies are
disclosed.
Inventors: |
Victoria, Edward Jess; (San
Diego, CA) ; Marquis, David Matthew; (Encinitas,
CA) ; Jones, David S.; (San Diego, CA) ; Yu,
Lin; (San Diego, CA) |
Correspondence
Address: |
Madeline I. Johnston
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
27413625 |
Appl. No.: |
10/044844 |
Filed: |
January 10, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10044844 |
Jan 10, 2002 |
|
|
|
09160513 |
Sep 24, 1998 |
|
|
|
6410775 |
|
|
|
|
09160513 |
Sep 24, 1998 |
|
|
|
08760508 |
Dec 5, 1996 |
|
|
|
08760508 |
Dec 5, 1996 |
|
|
|
08660092 |
Jun 6, 1996 |
|
|
|
6207160 |
|
|
|
|
08660092 |
Jun 6, 1996 |
|
|
|
08482651 |
Jun 7, 1995 |
|
|
|
5874409 |
|
|
|
|
Current U.S.
Class: |
544/388 ; 435/5;
435/7.1; 514/14.9; 514/183; 514/21.1; 514/58; 530/317 |
Current CPC
Class: |
G01N 33/564 20130101;
A61K 2039/645 20130101; C07C 323/12 20130101; G01N 33/92 20130101;
C07K 14/001 20130101; C07K 16/18 20130101; A61K 39/0008 20130101;
C07K 14/4713 20130101; G01N 33/54353 20130101; G01N 33/6878
20130101; C07K 2317/34 20130101; A61K 38/00 20130101; A61K
2039/6093 20130101 |
Class at
Publication: |
514/9 ; 530/317;
514/183; 514/58; 435/7.1; 435/5 |
International
Class: |
A61K 038/12; A61K
031/724; C07K 007/64; C12Q 001/70; G01N 033/53 |
Claims
We claim:
1. An aPL analog that binds specifically to B cells to which an aPL
epitope binds.
2. The analog of claim 1 wherein the analog lacks a T cell
epitope.
3. The analog of claim 1 wherein the analog is a peptide.
4. The analog of claim 3 wherein the peptide comprises the
sequence
15 CLILAPDRC, CLILTPDRC, CLLLAPDRC, CTILTLDRC, CLVLALDRC,
CTILTPDRC, CILLAHDRC, CGNAADARC, CTNWADPRC, CGNIADPRC, CTNLTDSRC,
CGNPTDVRC, GILLNEFA, GILTIDNL, GILNALDYV, LSDPGYVRNIFH or
LTDPRYTRDISNFTD.
5. The analog of claim 3 wherein the peptide comprises the
sequence
16 AGPCLGVLGKLCPG, GPCLGVLGKLCPG, PCLGVLGKLCPG, CLGVLGKLCPG,
AGPCLGVLGKLCG, CLGVLGKLC, GPCILLARDRCG or AGPILLARDRCPG.
6. The analog of claim 3 wherein the peptide contains at least one
proline and further wherein .alpha.-methyl proline is substituted
for at least one said proline.
7. The analog of claim 3 wherein a D-amino acid is substituted for
at least one L-amino acid.
8. The analog of claim 3 wherein the peptide is cyclized by a
disulfide bond.
9. The analog of claim 8 wherein a thioether bond is substituted
for the disulfide bond.
10. The analog of claim 3 wherein the peptide contains at least one
leucine and further wherein isoleucine is substituted for at least
one said leucine.
11. A composition for inducing specific B cell tolerance to an aPL
immunogen comprising a conjugate of a nonimmunogenic valency
platform molecule and an aPL antibody-binding analog that (a) binds
specifically to B cells to which an aPL immunogen binds and (b)
lacks the T cell epitope(s) of the immunogen.
12. The composition of claim 11 wherein the aPL antibody-binding
analog is a peptide comprising the sequence
17 CLILAPDRC, CLILTPDRC, CLLLAPDRC, CTILTLDRC, CLVLALDRC,
CTILTPDRC, CILLAHDRC, CGNAADARC, CTNWADPRC, CGNIADPRC, CTNLTDSRC,
CGNPTDVRC, GILLNEFA, GILTIDNL, GILNALDYV, LSDPGYVRNIFH or
LTDPRYTRDISNFTD.
13. The composition of claim 11 wherein the aPL antibody-binding
analog is a peptide comprising the sequence
18 AGPCLGVLGKLCPG, GPCLGVLGKLCPG, PCLGVLGKLCPG, CLGVLGKLCPG,
AGPCLGVLGKLCG, CLGVLGKLC, GPCILLARDRCG or AGPILLARDRCPG.
14. The composition of claim 11 wherein the aPL antibody-binding
analog is an analog according to claim 6.
15. The composition of claim 11 wherein the aPL antibody-binding
analog is an analog according to claim 7.
16. The composition of claim 11 wherein the aPL antibody-binding
analog is an analog according to claim 8.
17. The composition of claim 11 wherein the aPL antibody-binding
analog is an analog according to claim 9.
18. The composition of claim 11 wherein the aPL antibody-binding
analog is an analog according to claim 10.
19. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises triethylene glycol.
20. The composition of claim 19 wherein the valency platform
molecule comprises AHAB-TEG.
21. The composition of claim 19 wherein the valency platform
molecule comprises compound 46, A-DABA-ATEG.
22. The composition of claim 19 wherein the valency platform
molecule comprises compound 51, A-PABA-DT-TEG.
23. The composition of claim 19 wherein the valency platform
molecule comprises compound 55, MP-TEG.
24. The composition of claim 19 wherein the valency platform
molecule comprises compound 60, A-PIZ-IDA-TEG.
25. The composition of claim 19 wherein the valency platform
molecule comprises compound 68, A-PIZ-IDA-HB-TEG.
26. The composition of claim 19 wherein the valency platform
molecule comprises compound 72, A-PIZ-HIP-TEG.
27. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises polyethylene glycol.
28. The composition of claim 28 wherein the valency platform
molecule comprises DABA-PEG.
29. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises tetraaminobenzene.
30. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises heptaaminobetacyclodextrin.
31. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises tetraaminopentaerythritol.
32. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises 1,4,8,11-tetraazacyclotetradecane
(Cyclam).
33. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises 1,4,7,10-tetraazacyclododecane
(Cyclen).
34. The composition of claim 11 wherein the nonimmuriogenic valency
platform molecule comprises compound 63, tetrakis-A-PIZ-PMA.
35. The composition of claim 11 wherein the nonimmunogenic valency
platform molecule comprises compound 55, MP-TEG.
36. The composition of claim 11 wherein the conjugate is derived
from tetrakis-BMB.
37. A non-immunogenic valency platform molecule comprising
AHAB-TEG.
38. A non-immunogenic valency platform molecule comprising compound
46, IA-DABA-ATEG.
39. A non-immunogenic valency platform molecule comprising compound
51, BA-PABA-DT-TEG.
40. A non-immunogenic valency platform molecule comprising compound
55, BMP-TEG.
41. A non-immunogenic valency platform molecule comprising compound
60, BA-PIZ-IDA-TEG.
42. A non-immunogenic valency platform molecule comprising compound
68, BA-PIZ-IDA-HB-TEG.
43. A non-immunogenic valency platform molecule comprising compound
72, BA-PIZ-HIP-TEG.
44. A non-immunogenic valency platform molecule comprising compound
63, tetrakis-BA-PIZ-PMA.
45. A method of treating an individual suffering from an aPL
antibody-mediated disease comprising administering an effective
amount of the composition of claim 11 to an individual in need
thereof.
46. The method of claim 45 wherein said aPL antibody-mediated
disease is stroke.
47. The method of claim 45 wherein said aPL antibody-mediated
disease is fetal loss.
48. The method of claim 45 wherein said aPL antibody-mediated
disease is antiphospholipid antibody syndrome (APS).
49. The method of claim 45 wherein said aPL antibody-mediated
disease is primary antiphospholipid antibody syndrome (PAPS).
50. The method of claim 45 wherein said aPL antibody-mediated
disease is thrombosis.
51. A method for identifying analogs of epitopes which specifically
bind aPL antibodies isolated from humans suffering from an aPL
antibody-mediated disease comprising: (a) preparing phage random
peptide libraries; (b) screening said libraries with aPL antibodies
to identify aPL mimetic epitopes, wherein said screening comprises
(i) screening said libraries by biopanning; (ii) further screening
phage isolated by biopanning in (i) by micropanning; and (iii)
identifying phage containing aPL antibody high-affinity binding
peptides recovered in (ii) by immunoassay.
52. A method of biopanning phage random peptide libraries to
identify and isolate peptides which bind to aPL antibody
comprising: (a) reacting affinity-purified aPL antibody with phage
bearing random peptide inserts; (b) recovering phage bearing random
peptide inserts which bind to the aPL antibody; (c) infecting a
microorganism with phage recovered in (b); and (d) culturing the
infected microorganism in an antibiotic-containing medium in order
to isolate the phage.
53. A method of micropanning phage random peptide libraries to
identify and isolate peptides having a high binding affinity to aPL
antibodies comprising: (b) isolating phage bearing random peptide
inserts by biopanning; (b) incubating the phage recovered in step
(a) in microplate wells coated with aPL antibody bound to Protein
G; (c) washing the microplate wells to remove unbound phage; (d)
eluting bound phage; and (e) infecting a microorganism with phage
recovered in (d); and (f) culturing the infected microorganism in
an antibiotic-containing medium in order to isolate the phage.
54. The method of claim 51 wherein the immunoassay is a
phage-capture ELISA comprising: (a) incubating phage bearing random
peptide inserts isolated by micropanning in the microplate wells
coated with aPL antibody; (b) washing away unbound phage; (c)
incubating a labeled anti-phage antibody to the wells; (d) washing
away unbound labeled anti-phage antibody; (e) adding a label
substrate; and (f) measuring signal development of the substrate to
identify high affinity-binding phage.
55. The method of claim 54 wherein the label is an enzyme.
56. Then method of claim 54 wherein the substrate is
colorimetric.
57. The method of claim 54 further comprising performing an
additional phage-capture ELISA assay of the high affinity-binding
phage comprising: (a) coating a uniform amount of the phage on
microplate wells; (b) incubating aPL antibody in the wells, (c)
washing away unbound antibody, (e) incubating a labeled anti-aPL
antibody with the bound aPL antibody; (f) washing away unbound
labeled anti-aPL antibody; (g) adding a substrate to the wells; and
(h) measuring signal development of the substrate to measure the
relative binding affinity of the phage.
58. The method of claim 57 wherein the label is an enzyme.
59. The method of claim 57 wherein the substrate is
calorimetric.
60. The method of claim 51 wherein the immunoassay is a colony-blot
immunoassay comprising: (a) culturing a microorganism infected with
phage bearing random peptide inserts on a membrane atop an
agar-containing culture medium; (b) replicate transferring the
microorganism cultured in (a) by blotting the microorganism on a
membrane atop an agar-containing culture medium; (c) incubating the
transferred microorganism; (d) lysing the microorganism; (e)
digesting the microorganism; (f) blocking the membrane; (g)
incubating the membrane with aPL antibody; (h) washing away unbound
aPL antibody; (i) incubating a labeled anti-aPL antibody with the
membrane; (j) washing away unbound labeled anti-aPL antibody; (k)
adding a substrate; and (l) measuring signal development of the
substrate to identify high affinity-binding phage.
61. The method of claim 60 wherein the membrane is
nitrocellulose.
62. The method of claim 60 wherein the microorganism is digested
with lysozyme.
63. The method of claim 60 wherein the blocking solution is
gelatin.
64. The method of claim 60 wherein the label is an enzyme.
65. The method of claim 60 wherein the substrate is
calorimetric.
66. A method for assaying and ranking for affinity-binding
characteristics epitopes which specifically bind aPL antibodies
isolated from humans suffering from an aPL antibody-mediated
disease is also encompassed, the method comprising: (a) coating
wells of a microtitration plate with cardiolipin; (b) adding adult
bovine or human serum as a source of .beta.2-GPI to bind to the
cardiolipin and to prevent non-specific binding to the wells of the
plate; (c) incubating a solution of monomeric analog and a
high-titered aPL antibody for a pre-determined time; (d) adding the
aPL antibody/analog mixture to wells of the microtitration plate
and incubating for a pre-determined time; (e) washing the wells to
wash away unbound aPL antibody; (f) adding anti-human IgG
conjugated with a label to the wells of the plate and incubating
for a pre-determined time; (g) washing the wells to wash away
unbound anti-human IgG conjugate; (h) adding a substrate for the
labeled conjugate and developing the substrate/label reaction for a
pre-determined time; (i) measuring the end-product of the
substrate/label reaction to quantitate the amount of aPL antibody
bound to the well; (j) calculating the percentage inhibition, if
any, of binding of the aPL antibody to determine the affinity of
the analog to the aPL antibody.
67. The method of claim 66 wherein the conjugate is labeled with an
enzyme.
68. The method of claim 66 wherein the substrate is
calorimetric.
69. A diagnostic immunoassay for determining the presence of aPL
antibody in body fluids taken from subjects suspected of suffering
from an aPL antibody-mediated disease comprising (a) contacting a
sample of a body fluid with an analog of an epitope which
specifically binds aPL antibodies (b) detecting aPL antibodies
bound by the analog.
70. The immunoassay of claim 69 wherein the immunoassay comprises:
(a) coating wells of a microtitration plate with an analog of an
epitope which specifically binds aPL antibodies; (b) washing the
wells to wash away unbound analog; (c) adding a test sample of a
body fluid to the wells and incubating for a pre-determined time;
(d) washing the wells to remove unbound test sample; (e) adding
anti-human IgG conjugated with a label to the wells of the plate
and incubating for a pre-determined time; (f) washing the wells to
wash away unbound anti-human IgG conjugate; (g) adding a substrate
for the labeled conjugate and developing the substrate/label
reaction for a pre-determined time; (h) measuring the end-product
of the substrate/label reaction to determine the presence of
anti-aPL antibody in the test sample.
71. The immunoassay of claim 70 wherein the label is an enzyme and
the substrate is calorimetric.
72. Hydrophilic linkers for connecting peptides or other bioactive
molecules to valency platform molecules with the
formulaR.sup.1S(CH.sub.2-
CH.sub.2O).sub.nCH.sub.2CH.sub.2O(CH.sub.2).sub.mCO.sub.2R.sup.2wherein
n=0-200, m=0 to 10, R.sup.1=H or a protecting group such as trityl,
R.sup.2=H or alkyl or aryl, such as 4-nitrophenyl ester.
73. The linkers of claim 72 wherein m=0 to 2.
74. The conjugate of claim 11 wherein the aPL analog is bound to
the nonimmunogenic valency platform molecule by a sulfhydryl
containing moiety.
Description
CROSS-REFERENCE APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. Ser. No. 08/660,092, filed Jun. 6, 1996, which is a
continuation-in-part of U.S. Ser. No. 08/482,651, filed Jun. 7,
1995.
TECHNICAL FIELD
[0002] This invention is in the field of immunology and relates to
compositions and methods for treating and diagnosing
antiphospholipid (aPL) antibody-mediated pathologies. More
specifically, the invention relates to conjugates of
chemically-defined nonimmunogenic valency platform molecules and
immunospecific analogs of aPL-binding epitopes as well as methods
and compositions for producing these conjugates. Optimized analogs
lack T cell epitopes. In addition, the invention relates to
diagnostic assays for detecting the presence of and quantitating
the amount of antiphospholipid antibodies in a biological sample.
The invention also relates to a method of utilizing random peptide
libraries to identify immunospecific analogs of aPL-binding
epitopes.
BACKGROUND OF THE INVENTION
[0003] Antiphospholipid antibodies occur in autoimmune diseases
such as systemic lupus erythematosus (SLE) and antiphospholipid
antibody syndrome (APS) as well as in association with infections
and drug therapy. APS is characterized by one or more clinical
features such as arterial or venous thrombosis, thrombocytopenia
and fetal loss. APS may be primary or it may be associated with
other conditions, primarily SLE. (PHOSPHOLIPID-BINDING ANTIBODIES
(Harris et al., eds., CRC Press, Boca Raton, Fla., 1991); McNeil et
al. ADVANCES IN IMMUNOLOGY, Vol. 49, pp. 193-281 (Austen et al.,
eds., Academic Press, San Diego, Calif., 1991)). Approximately
30-40% of patients with SLE have aPL, however, 50% of patients with
aPL antibodies do not have SLE. This 50% may have other autoimmune
rheumatic diseases, miscellaneous conditions or they may have been
subjected to drug therapy, particularly chlorpromazine. In one
study of 70 patients, 26 males and 44 females, with primary APS
(PAPS) but no evidence of SLE, the following features were
observed: deep venous thrombosis (DVT) in 31; arterial occlusion in
31, particularly stroke or transient ischemia; myocardial
infarctions in 15; recurrent fetal loss in 24; thrombocytopenia
(TCP) in 32; 10 had a positive Coombs' test; Evans' syndrome in 7;
anti-nuclear antibody (ANA) in 32, but less than 1:160 in 29; and
antimitochondrial antibody (AMA) in approximately 24. (McNeil et
al., supra.). Estimates vary but in about 5% of all stroke
patients, aPL antibodies are thought to be an important
contributing factor.
[0004] Transient aPL antibodies, such as those detected in a VDRL
test, occur during many infections. Approximately 30% of patients
possessing persistent aPL antibodies have suffered a thrombic
event. The presence of aPL antibodies defines a group of patients
within SLE who display a syndrome of clinical features consisting
of one or more of thrombosis, TCP, and fetal loss. The risk of this
syndrome in SLE overall is around 25%; this risk increases to 40%
in the presence of aPL antibodies and decreases to 15% in their
absence. Because aPL antibodies were thought to be directed at
phospholipids in plasma membranes, it has been postulated that they
may exert direct pathogenic effects in vivo by interfering with
hemostatic processes that take place on the phospholipid membranes
of cells such as platelets or endothelium. In patients with PAPS,
the fact that aPL antibodies appear to be the only risk factor
present is further evidence that these antibodies have a direct
pathogenic role. Induction of PAPS by immunizing mice with human
anticardiolipin antibodies is the best evidence yet that aPL
antibodies are directly pathogenic (Bakimer et al. 1992 J. Clin.
Invest. 89:1558-1563; Blank et al. 1991 Proc. Natl. Acad. Sci.
88:3069-3073).
[0005] Measurement of aPL antibodies in the clinical environment is
still an imperfect art. A commercially available set of standard
antisera (APL Diagnostics, Inc., Louisville, Ky.) allow generation
of a standard curve for comparison of assays performed in various
laboratories. A great deal of inconsistency exists, however,
between the results obtained at these laboratories regarding the
exact GPL and MPL, the unit of measurement for IgG and IgM
antiphospholipid antibodies, respectively, ratings for given sera
and the levels of GPL and MPL that are categorized as high, medium
or low titer. The available commercial kits vary greatly in the
values assigned to the commercially available standards (Reber et
al. (1995) Thrombosis and Haemostat. 73:444-452). In spite of these
limitations, there is general agreement that the epitopes
recognized by antibodies in APS, PAPS and other aPL
antibody-mediated diseases including recurrent stroke and recurrent
fetal loss are located in the 5th domain of .beta..sub.2-GPI and
are exposed to the antibody following binding of .beta..sub.2-GPI
to cardiolipin.
[0006] It is now generally accepted that aPL antibodies recognize
an antigenic complex comprised of .beta..sub.2-glycoprotein I
(.beta..sub.2-GPI) and negatively-charged phospholipid, e.g.,
cardiolipin (McNeil et al. (1990) Proc. Natl. Acad. Sci.
87:4120-4124; Galli et al. (1990) Lancet 1:1544-1547) (hereinafter
"aPL immunogen"). .beta..sub.2-GPI is a minor plasma glycoprotein
found free and in association with lipoprotein lipids where it is
also known as apolipoprotein H (apo H). It consists of five
independently folding domains referred to as Sushi or short
consensus repeat domains that resemble similar domains in other
proteins. .beta..sub.2-GPI has been reported to undergo antigenic
and conformational changes upon binding phospholipid (Wagenkneckt
et al. (1993) Thromb. Haemostas. 69:361-365; Jones et al. (1992)
Proc. 5th Intl. Symp. Antiphospholipid Antibodies (Abstract S5)).
The fifth domain contains the putative sites of lipid binding and
aPL antibody binding (Hunt J. and S. Krilis, (1994) J. Immunol.
152:653-659; Lauer et al. (1993) Immunol. 80:22-28). The
pathological mechanism for aPL is unknown (McNeil et al., supra).
Most explanations invoke endothelial cell function or platelet
involvement (Haselaar et al. (1990) Thromb. Haemostas. 63:169-173).
These explanations suggest that following blood vessel endothelial
cell injury or platelet activation, the exposure or transbilayer
migration of anionic phospholipid to the plasma-exposed surface may
lead to .beta..sub.2-GPI-binding and trigger aPL antibody
formation.
[0007] aPL antibodies may be directly prothrombotic by reducing
prostacyclin formation (Vermylen, J. and J. Arnout (1992) J. Clin.
Lab. Med. 120:10-12); by direct interference with the action of
coagulation proteins; or by blocking the ability of
.beta..sub.2-GPI to inhibit the intrinsic blood coagulation
pathway, platelet prothrombinase activity, and ADP-mediated
platelet aggregation (Arvieux et al. (1993) Thromb. Haemostas.
60:336-341).
[0008] A major new tool in medicinal chemistry in the search for
lead compounds has been the advent of combinatorial libraries
providing vast molecular diversity. Molecular diversity may arise
from chemical synthesis or from biological systems (Scott., J. K.
RATIONAL DRUG DESIGN (CRC Press, Weiner, D. B. and W. V. Williams,
eds., Boca, Raton, Fla., 1994); Moos et al. (1993) Ann. Reports
Med. Chem. 28:315-324). By displaying random peptides on the
surface of filamentous phage, epitope libraries containing hundreds
of millions of clones for probing by clinically significant
antibodies have been created (Scott, J. K. and G. P. Smith (1990)
Science 249:286-390; Cesareni, G. (1992) FEBS Lett. 307:66-70).
Such phage libraries are prepared by incorporating randomized
oligonucleotide sequences into the phage genome, usually the pIII
gene, which encode unique peptide sequences on the surface of each
phage. Following sequential rounds of affinity purification and
amplification, those phage that bind antibody are propagated in E.
coli and the binding peptides identified by sequencing the
corresponding coding region of viral DNA. In most cases, subsequent
study will involve corresponding synthetic peptides after
establishing their ability to bind antibody. Phage-based libraries
have been used to mimic discontinuous epitopes (Luzzago et al.
(1993) Gene 128:51-57; BaLass et al. (1993) Proc. Natl. Acad. Sci.
90:10638-10642). The potential plasma instability of peptide-based
drugs has been successfully overcome by N-terminal blocking or by
the judicious use of amino acid analogs (Powell, M. F. (1993) Ann.
Reports Med. Chem. 28:285-293).
[0009] At present there is no selective, immunospecific therapy for
patients showing high titers of aPL antibodies. In many cases use
of drugs such as aspirin, steroids, and warfarin has proven to be
largely inadequate (PHOSPHOLIPID-BINDING ANTIBODIES (Harris et al.,
eds., CRC Press, Boca Raton, Fla., 1991); McNeil et al., supra).
Synthetic mimetic peptides, characterized by (i) the inability to
activate T cells while (ii) retaining the ability to bind immune B
cells, are used to tolerize B cells in an antigen-specific manner.
This technology is disclosed in co-owned, co-pending U.S. patent
application, Ser. No. 08/118,055, filed Sep. 8, 1993, and U.S. Pat.
No. 5,268,454, which are incorporated by reference herein in their
entirety. As disclosed in the application and patent cited above, B
cell tolerance entails administering such peptides conjugated to
multivalent, stable, non-immunogenic valency platforms in order to
abrogate antibody production via B cell anergy or clonal deletion
after cross-linking surface immunoglobulin.
[0010] Although the exact molecular nature of the target epitopes
recognized by aPL antibodies is unknown, the use of peptides
derived from epitope libraries will allow for the construction of
successful tolerogens. B cell tolerogens for the treatment of human
systemic lupus erythematosus-related nephritis have also been
disclosed in co-owned U.S. Pat. Nos. 5,276,013 and 5,162,515 which
are incorporated by reference herein in their entirety.
DISCLOSURE OF THE INVENTION
[0011] This invention resides in the discovery of a method for
identifying analogs of key epitopes recognized by aPL antibodies in
patients suffering from PAPS, APS and other aPL antibody-mediated
diseases, such as recurrent stroke and recurrent fetal loss, using
random peptide phage libraries.
[0012] Accordingly, one aspect of the invention is an improved
method for screening random peptide phage libraries in order to
identify the peptide sequences which best mimic the epitopes
recognized by aPL antibodies. This method comprises the steps of:
(a) biopanning the library using methods modified from those known
in the art; (b) eliminating very weakly-binding phage by
micropanning the phage screened from step (a) by (i) incubating the
phage in microplate wells coated with aPL antibody bound to Protein
G, (ii) washing the microplate wells to remove unbound phage, (iii)
eluting the bound phage, and (iv) infecting a microorganism such as
E. coli with the eluted phage and counting the number of infected
microorganisms by plating on agar; (c) determining the
strongest-binding clones recovered in (b) by evaluation via
phage-capture ELISA by (i) coating the wells of a microplate with
aPL antibody, (ii) incubating the strongest-binding clones
identified by micropanning in (b) in the coated wells and washing
away unbound phage, (iii) quantitating the number of phage bound to
the antibody using an enzyme-conjugated goat anti-phage antibody in
a colorimetric ELISA assay and, if several equivalent
strongly-binding clones are identified, an additional round of (d)
phage-ELISA on the strongest-binding phage-capture-ELISA clone.
[0013] In this regard, the invention encompasses a method for
identifying analogs of epitopes which specifically bind aPL
antibodies isolated from humans suffering from an aPL
antibody-mediated disease comprising: (a) preparing phage random
peptide libraries; (b) screening said libraries with aPL antibodies
to identify aPL mimetic epitopes, wherein said screening comprises
(i) screening said libraries by biopanning; (ii) further screening
phage isolated by biopanning in (i) by micropanning; and (iii)
identifying phage containing aPL antibody high-affinity binding
peptides recovered in (ii) by immunoassay.
[0014] The invention also encompasses a method of biopanning phage
random peptide libraries to identify and isolate peptides which
bind to aPL antibody comprising: (a) reacting affinity-purified aPL
antibody with phage bearing random peptide inserts; (b) recovering
phage bearing random peptide inserts which bind to the aPL
antibody; (c) infecting a microorganism with phage recovered in
(b); and (d) culturing the infected microorganism in an
antibiotic-containing medium in order to isolate the phage.
[0015] The invention further encompasses a method of micropanning
phage random peptide libraries to identify and isolate peptides
having a high binding affinity to aPL antibodies comprising: (a)
isolating phage bearing random peptide inserts by biopanning; (b)
incubating the phage recovered in step (a) in microplate wells
coated with aPL antibody bound to Protein G; (c) washing the
microplate wells to remove unbound phage; (d) eluting bound phage;
and (e) infecting a microorganism with phage recovered in (d); and
(f) culturing the infected microorganism in an
antibiotic-containing medium in order to isolate the phage.
[0016] The invention also encompasses the above method described
wherein the immunoassay is a phage-capture ELISA comprising: (a)
incubating phage bearing random peptide inserts isolated by
micropanning in the microplate wells coated with aPL antibody; (b)
washing away unbound phage;(c) incubating an enzyme-labeled
anti-phage antibody to the wells; (d) washing away unbound
enzyme-labeled anti-phage antibody; (e) adding a colorimetric
substrate; and (f) measuring the absorbance of the substrate to
identify high affinity-binding phage.
[0017] Also encompassed by the invention is the method described
above and further comprising performing an additional phage-capture
ELISA assay of the high affinity-binding phage comprising: (a)
coating a uniform amount of the phage on microplate wells; (b)
incubating aPL antibody in the wells; (c) washing away unbound
antibody; (e) incubating an enzyme-labeled anti-aPL antibody with
the bound aPL antibody; (f) washing away unbound enzyme-labeled
anti-aPL antibody; (g) adding a colorimetric substrate to the
wells; and (h) measuring the absorbance of the substrate to measure
the relative binding affinity of the phage.
[0018] The invention also encompasses the method described above
wherein the immunoassay is a colony-blot immunoassay comprising:
(a) culturing a microorganism infected with phage bearing random
peptide inserts on a nitrocellulose membrane atop an
agar-containing culture medium; (b) replicate transferring the
microorganism cultured in (a) by blotting the microorganism on a
second nitrocellulose membrane atop an agar-containing culture
medium; (c) incubating the transferred microorganism; (d) lysing
the microorganism; (e) digesting the microorganism with lysozyme;
(f) blocking the membrane with a gelatin solution; (g) incubating
the membrane with aPL antibody; (h) washing away unbound aPL
antibody; (i) incubating a enzyme-labeled anti-aPL antibody with
the nitrocellulose membrane; (j) washing away unbound
enzyme-labeled anti-aPL antibody; (k) adding a calorimetric
substrate; and (l) measuring the absorbance of the substrate to
identify high affinity-binding phage.
[0019] A method for assaying and ranking, for affinity-binding
characteristics, epitopes which specifically bind aPL antibodies
isolated from humans suffering from an aPL antibody-mediated
disease is also encompassed, the method comprising: (a) coating
wells of a microtitration plate with cardiolipin; (b) adding adult
bovine or human serum as a source of .beta..sub.2-GPI to bind to
the cardiolipin and to prevent non-specific binding to the wells of
the plate; (c) incubating a solution of monomeric analog and a
high-titered aPL antibody for a pre-determined time; (d) adding the
aPL antibody/analog mixture to wells of the microtitration plate
and incubating for a pre-determined time; (e) washing the wells to
wash away unbound aPL antibody; (f) adding anti-human IgG
conjugated with a label (e.g., an enzyme) to the wells of the plate
and incubating for a pre-determined time; (g) washing the wells to
wash away unbound anti-human IgG conjugate; (h) adding a substrate
for the labeled conjugate and developing the substrate/label
reaction for a pre-determined time; (i) measuring the end-product
of the substrate/label reaction to quantitate the amount of aPL
antibody bound to the well; (j) calculating the percentage
inhibition, if any, of binding of the aPL antibody to determine the
affinity of the analog to the aPL antibody.
[0020] Another aspect of the invention encompasses a fluorescence
polarization peptide binding assay for determining the dissociation
constants for peptides that bind to aPL antibodies. This assay
detects direct binding of peptides to aPL antibodies.
[0021] The invention also encompasses a diagnostic immunoassay for
determining the presence of aPL antibody in body fluids taken from
subjects suspected of suffering from an aPL antibody-mediated
disease comprising contacting a sample of a body fluid with an
analog of an epitope which specifically binds aPL antibodies and
determining by methods well known in the art whether aPL antibodies
are present in the body fluid and, if present, quantitating the
amount of aPL antibodies present in the fluid. One such immunoassay
comprises: (a) coating wells of a microtitration plate with an
analog of an epitope which specifically binds aPL antibodies; (b)
washing the wells to wash away unbound analog; (c) adding a test
sample of a body fluid to the wells and incubating for a
pre-determined time; (d) washing the wells to remove unbound test
sample; (e) adding anti-human IgG conjugated with a label to the
wells of the plate and incubating for a pre-determined time; (f)
washing the wells to wash away unbound anti-human IgG conjugate;
(g) adding a substrate for the labeled conjugate and developing the
substrate/label reaction for a pre-determined time; (h) measuring
the end-product of the substrate/label reaction to determine the
presence of anti-aPL antibody in the test sample. A diagnostic
immunoassay as described above wherein the immunoassay is
quantitative is also encompassed.
[0022] The phage-ELISA assay consists of (i) coating a uniform
amount of different clones on wells of a microtitration plate
followed by (ii) identifying the peptide inserts which most
strongly bind aPL antibody by adding antibody to the wells and
developing the reaction with an enzyme-labeled anti-human IgG
conjugate. The random peptides displayed by the phage which have a
high binding affinity to aPL antibody as measured by phage-ELISA,
colony blot or phage-capture-ELISA represent the analogs of the
aPL-specific epitope. These peptides are then synthesized and
ranked for strength of binding using competition assays.
[0023] Another aspect of the invention is aPL antibody-binding
analogs that bind specifically to B cells to which an aPL epitope
binds. Optimized analogs lack T cell epitope(s).
[0024] Yet another aspect of the invention is a composition for
inducing specific B cell tolerance to an aPL immunogen comprising a
conjugate of a nonimmunogenic valency platform molecule and an aPL
antibody-binding analog that (a) binds specifically to B cells to
which an aPL immunogen binds and (b) lacks T cell epitope(s).
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows that the substitution of fish gelatin for adult
bovine serum abolished all anticardiolipin (ACA) activity in an
ELISA assay of a commercial aPL antibody standard. This result
supported the findings of McNeil et al., supra, and Galli et al.,
supra, concerning the importance of .beta..sub.2-glycoprotein I
(.beta..sub.2-GPI) in defining the target epitope(s) of ACA.
[0026] FIG. 2 shows that resin-bound analog 5A12 immunospecifically
binds to affinity-purified IgG designated ACA-6501.
[0027] FIG. 3 illustrates that the aPL antibody-binding analogs
derived from screening with ACA-6626 bound aPL antisera but did not
bind normal sera.
[0028] FIG. 4 also illustrates that the ACA-6501/5A12 analog
immunospecifically binds ACA-6501 antiserum and is crossreactive
with ACA-6626.
[0029] FIGS. 4 and 5 illustrate that while the ACA-6501/5A12 and
ACA-6626/4D3 aPL antibody-binding analogs derived from screening
with methods within the instant invention bind preferentially with
the screening antibody, a significant degree of crossreactivity was
detected.
[0030] FIG. 6 illustrates method for calculating the GPL value for
ACA-6501 aPL antibody.
[0031] FIG. 7 shows the activity of affinity-isolated ACA-6501
compared to GPL standard sera.
[0032] FIG. 8 illustrates the dramatic drop in sequence diversity
of the isolated clones by the fourth round of biopanning.
[0033] FIG. 9 illustrates that three clones (3A12, 3B3 and 3A5)
exhibited a very strong immunospecific signal in the phage-capture
ELISA using ACA-6635 whereas all clones tested were unreactive with
normal IgG.
[0034] FIG. 10 shows the strong signal exhibited by seven clones in
a phage-ELISA using ACA-6501.
[0035] FIG. 11 shows the results of a competitive-binding ELISA
obtained with peptides 5A12, CB2 and 3B10 using ACA-6501. 0.16
.mu.g of Peptide 5A12 produced 50% inhibition of binding of
ACA-6501 aPL antibody to tetravalent peptide ACA-6501/3B10 bound to
polystyrene microplate wells, whereas 0.08 .mu.g of CB2 and 0.5
.mu.g of 3B10 were required to produce 50% inhibition.
[0036] FIG. 12 illustrates the comparative activity of modified
ACA-6641/3G3 analogs.
[0037] FIG. 13 illustrates that the 50% inhibition values for
peptides 139, 142 and 143 in a competitive-binding ELISA using
ACA-6501 aPL antibody were 6.9, 0.7 and 0.9 .mu.g.
respectively.
[0038] FIG. 14 illustrates the effects of substituting
.alpha.-Me-Pro at positions 3, 9 and both 3 and 9 in peptide 3B10.
Substitution of .alpha.-Me-Pro at position 9 increased activity of
the peptide four-fold compared to the "native" peptide.
[0039] FIG. 15 shows that peptide 6641/3G3 (LJP 688) is highly
cross-reactive with nine affinity-purified ACA antibodies.
[0040] FIGS. 16 and 17 show the dose-dependent reduction in
anti-685 antibody ABC at 10.sup.8M using spleen cells from mice
immunized with LJP 685-KLH after the spleen cells were incubated
with 100, 20 and 4 .mu.M of LJP 685-MTU-DABA-PEG conjugate
(compound 36 and LJP 685-ATU-MTU-AHAB-TEG conjugate (compound 35),
respectively, for 2 hours.
[0041] FIG. 18 displays the NMR structure closest to the centroid
of the nine structures elucidated for peptide 925 and is a
reasonable representation of the shape of the peptide 925
molecule.
[0042] FIG. 19 compares the structure of peptide 925 (labeled at
the bottom of the figure as 3G3) with the structure of peptide
5A12. Both peptides have turns at approximately the same positions
in the peptide sequence.
[0043] FIGS. 20A and 20B illustrate that the pharmacophore of aPL
analogs has been tentatively identified as a small hydrophobic
group and a positively charged group. The gem-dimethyl and amino
groups of peptide 925 are tentatively identified as the
pharmacophore of this peptide as shown in FIG. 20A. The lengths of
the hydrocarbon linkers that tether the pharmacophore groups to
some scaffold are specified in FIG. 20A as well as the distances
separating the points at which these linkers are attached to the
scaffold.
[0044] FIG. 21 illustrates inhibition of .beta..sub.2GPI by
6501-derived peptides with diluted 6501 serum.
[0045] FIG. 22 shows the ACA-6701 titration of CB2*-F;
FITC-GPCILLARDRCG.
[0046] FIG. 23 shows the ACA-6501 titration of CB2*-F:
FITC-GPCILLARDRCG.
[0047] FIG. 24 shows complete ACA-6501 titration of CB2*-F:
FITC-GPCILLARDRCG.
[0048] FIG. 25 shows the displacement of CB2*-F from ACA-6701 using
1.04 equivalents of CB2*.
[0049] FIG. 26 shows cFP titration using CB2* to displace CB2*-F
from ACA-6701.
[0050] FIG. 27 shows cFP titration using 3B10 to displace CB2*-F
from ACA-6701.
[0051] FIG. 28 shows the dose response of the
(LJP685).sub.4/MTU-AHAB-TEG conjugate for tolerance activity.
[0052] FIG. 29 shows the dose response of the
(LJP685).sub.4/MTU-DABA-TEG conjugate for tolerance activity.
[0053] FIG. 30 shows the dose response of tolerance activity of the
(LJP685).sub.4/MTU-AHAB-TEG conjugate tested in the in vitro
model.
[0054] FIG. 31 shows the dose response of tolerance activity of the
(LJP685).sub.4/MTU-DABA-TEG conjugate tested in the in vitro
model.
[0055] FIG. 32 shows the tolerizing effect of the
(LJP685).sub.4/MTU-AHAB-- TEG conjugate comparing various
administrative routes and dosage ranges.
MODES FOR CARRYING OUT THE INVENTION
[0056] A. Definitions
[0057] As used herein the term "aPL antibody" means any antibody
which specifically binds .beta.2-GPI that mediates disease.
[0058] As used herein the term "B cell anergy" intends
unresponsiveness of those B cells requiring T cell help to produce
and secrete antibody and includes, without limitation, clonal
deletion of immature and/or mature B cells and/or the inability of
B cells to produce antibody.
[0059] "Unresponsiveness" means a therapeutically effective
reduction in the humoral response to an immunogen. Quantitatively
the reduction (as measured by reduction in antibody production) is
at least 50%, preferably at least 75%, and most preferably
100%.
[0060] "Antibody" means those antibodies which are T cell
dependent.
[0061] As used herein the term "immunogen" means an entity that
elicits a humoral immune response comprising aPL antibodies.
Immunogen have both B cell epitopes and T cell epitopes. aPL
immunogens that are involved in aPL antibody-mediated pathologies
may be external (foreign to the individual) immunogens such as
drugs, including native biological substances foreign to the
individual such as therapeutic proteins, peptides and antibodies,
and the like or self-immunogens (autoimmunogens) such as those
associated with antibody-mediated hypercoagulability (stroke).
[0062] The term "analog" of an immunogen intends a molecule that
(a) binds specifically to an antibody to which the immunogen binds
specifically and (b) lacks T cell epitopes. Although the analog
will normally be a fragment or derivative of the immunogen and thus
be of the same chemical class as the immunogen (e.g., the immunogen
is a polypeptide and the analog is a polypeptide), chemical
similarity is not essential. Accordingly, the analog may be of a
different chemical class than the immunogen (e.g., the immunogen is
a carbohydrate and the analog is a polypeptide) as long as it has
the functional characteristics (a) and (b) above. The analog may be
a peptide, carbohydrate, lipid, lipopolysaccharide, nucleic acid or
other biochemical entity. Further, the chemical structure of
neither the immunogen nor the analog need be defined for the
purposes of this invention.
[0063] The term "analog" of an immunogen also encompasses the term
"mimotope." The term "mimotope" intends a molecule which
competitively inhibits the antibody from binding the immunogen.
Because it specifically binds the antibody, the mimotope is
considered to mimic the antigenic determinants of the
immunogen.
[0064] As used herein "valency platform molecule" means a
nonimmunogenic molecule containing sites which facilitate the
attachment of a discreet number of analogs of immunogens.
[0065] "Nonimmunogenic" is used to describe the valency platform
molecule and means that the valency platform molecule elicits
substantially no immune response when it is administered by itself
to an individual.
[0066] As used herein "individual" denotes a member of the
mammalian species and includes humans, primates, mice and domestic
animals such as cattle and sheep, sports animals such as horses,
and pets such as dogs and cats.
[0067] As used herein "pharmacophore" means the three dimensional
orientation and chemical properties of key groups involved in
binding of an aPL analog to the antibody target.
[0068] B. Identification of aPL Antibody-Binding Analogs
[0069] aPL antibody-binding analogs may be identified by screening
candidate molecules to determine whether or not they (a) bind
specifically to aPL antibodies and (b) lack T cell epitopes.
Specific binding to aPL antibodies may be determined using
conventional immunoassays such as the ELISA assays described in the
examples below and the presence or absence of T cell epitopes may
be determined by conventional T cell activation assays also
described in the examples. In this regard, an analog which "binds
specifically" to serum antibodies to the immunogen exhibits a
reasonable affinity thereto, e.g., 10.sup.7 M.sup.-1. The presence
or absence of T cell epitopes may be determined using a tritiated
thymidine incorporation assay disclosed in Ser. No. 08/118,055. The
presence of T cell epitopes can also be determined by measuring
secretion of T cell-derived lymphokines by methods well known in
the art. Analogs that fail to induce statistically significant
incorporation of thymidine above background are deemed to lack T
cell epitopes. It will be appreciated that the quantitative amount
of thymidine incorporation may vary with the immunogen. Typically a
stimulation index below about 2-3, more usually about 1-2, is
indicative of a lack of T cell epitopes.
[0070] C. Preparation of Conjugates
[0071] The aPL antibody-binding analogs are coupled to a
nonimmunogenic valency platform molecule to prepare the conjugates
of the invention. Preferred valency platform molecules are
biologically stabilized, i.e., they exhibit an in vivo excretion
half-life often of hours to days to months to confer therapeutic
efficacy, and are preferably composed of a synthetic single chain
of defined composition. They will normally have a molecular weight
in the range of about 200 to about 200,000, usually about 200 to
about 20,000. Examples of valency platform molecules within the
present invention are polymers such as polyethylene glycol (PEG),
poly-D-lysine, polyvinyl alcohol and polyvinylpyrrollidone.
Preferred polymers are based on polyethylene glycols (PEGs) having
a molecular weight of about 200 to about 8,000.
[0072] Other valency platform molecules suitable for use within the
present invention are the chemically-defined, non-polymeric valency
platform molecules disclosed in co-owned, co-pending U.S. patent
application Ser. No. 08/152,506, filed Nov. 15, 1993, which is
incorporated by reference herein in its entirety. Particularly
preferred homogeneous chemically-defined valency platform molecules
suitable for use within the present invention are derivatized
2,2'-ethylenedioxydiethy- lamine (EDDA) and triethylene glycol
(TEG).
[0073] Additional suitable valency platform molecules include
tetraaminobenzene, heptaaminobetacyclodextrin,
tetraaminopentaerythritol, 1,4,8,11-tetraazacyclotetradecane
(Cyclam) and 1,4,7,10-tetraazacyclodode- cane (Cyclen).
[0074] Conjugation of the aPL antibody-binding analog to the
valency platform molecule may be effected in any number of ways,
typically involving one or more crosslinking agents and functional
groups on the analog and valency platform molecule.
[0075] Polypeptide analogs will contain amino acid sidechain
moieties containing functional groups such as amino, carboxyl, or
sulfhydryl groups that will serve as sites for coupling the analog
to the carrier. Residues that have such functional groups may be
added to the analog if the analog does not already contain these
groups. Such residues may be incorporated by solid phase synthesis
techniques or recombinant techniques, both of which are well known
in the peptide synthesis arts. In the case of carbohydrate or lipid
analogs, functional amino and sulfhydryl groups may be incorporated
therein by conventional chemistry. For instance, primary amino
groups may be incorporated by reaction with ethylenediamine in the
presence of sodium cyanoborohydride and sulfhydryls may be
introduced by reaction of cysteamine dihydrochloride followed by
reduction with a standard disulfide reducing agent. In a similar
fashion, the valency platform molecule may also be derivatized to
contain functional groups if it does not already possess
appropriate functional groups.
[0076] Hydrophilic linkers of variable lengths are useful for
connecting peptides or other bioactive molecules to valency
platform molecules. Suitable linkers include linear oligomers or
polymers of ethylene glycol. Such linkers include linkers with the
formula R.sup.1S(CH.sub.2CH.sub.2O)-
.sub.nCH.sub.2CH.sub.2O(CH.sub.2).sub.mCO.sub.2R.sup.2 wherein
n=0-200, m=1 or 2, R.sup.1=H or a protecting group such as trityl,
R.sup.2=H or alkyl or aryl, e.g., 4-nitrophenyl ester. These
linkers are useful in connecting a molecule containing a thiol
reactive group such as haloaceyl, maleiamide, etc., via a thioether
to a second molecule which contains an amino group via an amide
bond. These linkers are flexible with regard to the order of
attachment, i.e., the thioether can be formed first or last.
[0077] The conjugates will normally be formulated for
administration by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). Accordingly,
they will typically be combined with pharmaceutically acceptable
vehicles such as saline, Ringer's solution, dextrose solution, and
the like. The conjugate will normally constitute about 0.01% to 10%
by weight of the formulation. The conjugate is administered to an
individual in a "therapeutically effective amount", i.e., an amount
sufficient to produce B cell anergy to the involved immunogen and
effect prophylaxis, improvement or elimination of the
antibody-mediated condition being addressed. The particular dosage
regimen, i.e., dose, timing and repetition, will depend on the
particular individual and that individual's medical history.
Normally, a dose of about 1 .mu.g to about 100 mg conjugate/kg body
weight, preferably about 100 .mu.g to about 10 mg/kg body weight,
will be given weekly. Other appropriate dosing schedules would be
as frequent as daily or 3 doses per week, or one dose per week, or
one dose every two to four weeks, or one dose on a monthly or less
frequent schedule depending on the individual or the disease state.
Repetitive administrations, normally timed according to B cell
turnover rates, may be required to achieve and/or maintain a state
of humoral anergy. Such repetitive administrations will typically
involve treatments of about 1 .mu.g to about 10 mg/kg body weight
or higher every 30 to 60 days, or sooner, if an increase in
antibody titer is detected. Alternatively, sustained continuous
release formulations of the conjugates may be indicated for some
pathologies. Various formulations and devices for achieving
sustained release are known in the art.
[0078] Anti-helper T cell treatments may be administered together
with the conjugates. Such treatments usually employ agents that
suppress T cells such as steroids or cyclosporin.
[0079] D. Nature of the Antigen for aPL Antibodies
[0080] Initial studies on aPL antibodies by the inventors coincided
with the publication of reports questioning the nature of the
antigenic site recognized by these antibodies. Initially these
antibodies were thought to recognize the cardiolipin molecule much
like those antibodies detected in a VDRL test. As shown in FIG. 1,
substitution of fish gelatin for adult bovine serum in the
anticardiolipin antibody (ACA) solid phase ELISA essentially
abolished all ACA activity of an antibody preparation obtained
commercially as an ACA standard. This finding indicated the ACA
antibodies recognized a determinant on a serum protein as proposed
by McNeil et al., supra and Galli et al., supra, rather than
cardiolipin itself. This protein was shown by these authors to be
.beta..sub.2-GPI and, thus, the terms "ACA" or "anti-cardiolipin
antibodies" are really misnomers but are still used today to refer
to these antibodies.
[0081] The majority of autoimmune, IgG aPL antibodies recognize
determinants on the .beta..sub.2-GPI molecule. These epitopes may
be formed or exposed on .beta..sub.2-GPI only upon its binding to
cardiolipin (a neo epitope). Alternatively, the epitope on
.beta..sub.2-GPI may exist in a single copy per .beta..sub.2-GPI
molecule and may have low affinity for aPL antibodies. Sufficient
avidity to maintain an antibody-antigen interaction might be
reached only with the alignment of two of these sites on adjacent
.beta..sub.2-GPI molecules by their binding to cardiolipin.
[0082] Additional insight into the .beta.2-GPI epitope structure
has been gained by nuclear magnetic resonance (NMR) analysis of aPL
analogs within the present invention which mimic the native
epitope(s) of .beta.2-GPI. For, example, a comparison of the NMR
solution structure of two peptide aPL analogs that are highly
cross-reactive with aPL antibodies showed that both peptides have
turns at approximately the same positions in the peptide sequence
(see FIGS. 18 and 19).
[0083] E. ACA ELISA
[0084] The necessity of large-scale testing in connection with GPL
scoring of clinical samples and chromatographic purifications of
research antibodies and .beta..sub.2-GPI led to the development of
an ELISA assay for aPL antibodies with performance in close
agreement with a commercial kit and similar to the design found to
have the best reproducibility (Reber et al., supra). A modified ACA
ELISA has also been performed wherein .beta..sub.2-GPI bound
directly to certain microplates is used to bind aPL IgG directly in
the absence of any cardiolipin first added to the microplate wells.
(See, Roubey et al. (1996) Arthritis & Rheumatism 39:1606-1607;
R. A. S. Roubey (1996) Arthritis & Rheumatism
39:1444-1454).
[0085] F. Immunoaffinity Purification of aPL Antibodies
[0086] In order to isolate the aPL antibodies, multilamellar,
cardiolipin-containing dispersions (liposomes; also containing
cholesterol and dicetylphosphate) are incubated with aPL plasma (or
serum). These liposomes are pelleted from the serum by
centrifugation. After washing, the liposome mixture is disrupted by
2% octylglucoside detergent and applied to a protein A-agarose
column. Following extensive washings to first remove lipids and
then to remove non-IgG components, IgG aPL antibody is eluted from
protein A with mild acid, neutralized, buffer-exchanged, and tested
in the ACA ELISA. This procedure yields aPL antibody enriched up to
10,000-fold that is devoid of any contaminating .beta..sub.2-GPI as
shown by western blotting with rabbit IgG anti-human
.beta..sub.2-GPI antisera. An additional affinity-purification step
is performed by chromatography of the affinity-purified antibody on
solid phase .beta..sub.2-GPI. This second affinity-purification
step is recommended as a result of the new awareness regarding the
greater clinical relevance of aPL antibodies that directly bind to
.beta..sub.2-GPI. It also serves to further ensure a final
preparation devoid of contaminants, in particular
.beta..sub.2-GPI.
[0087] G. Construction of Filamentous Phage Random Peptide
Libraries
[0088] Eleven different fUSE 5 filamentous phage random peptide
libraries on the p-III protein (five copies of p-III with peptide
per phage) are constructed. These libraries provide a vast array of
shapes and structures for the discovery of mimetic epitopes. Four
libraries, designated "x" libraries, have peptide inserts that are
8, 10, 12, and 15 residues in length, respectively, and are flanked
by proline residues on both the amino and carboxyl ends. The
purpose of these proline residues is to disrupt any contribution to
secondary structure that might arise from the native p-III protein
and to project the insert into the solvent. The "y'" libraries
contain cysteine-bounded inserts that are 6, 7, 8, 9, 11, and 13
amino acids long. The "y" library is the same as the "y'" library
except that it lacks the 6 and 8 amino acid inserts. These peptide
inserts for both "y" and "y'" libraries are flanked by cysteine
residues at both the amino and carboxyl ends to form cyclic, more
rigid structures. Proline residues are incorporated outside these
cysteine residues for reasons similar to those for the "x"
libraries above. The "x," "y'", and "y" libraries are located five
residues from the amino terminus of the native p-III protein. The
"z" library consists of random eight amino acid inserts located at
the amino terminus of the p-III protein and do not contain any
flanking proline or cysteine residues. A combination of the "x,"
"y'" and "z" libraries represents eleven different libraries each
with approximately one hundred million different peptide
inserts.
[0089] These libraries are constructed by incorporation of random
oligonucleotide sequences of the length appropriate to give the
desired length insert into the p-III gene of fUSE 5 using standard
molecular biology techniques. Following restriction endonuclease
digestion of the fUSE 5 DNA, an excess of kinased oligonucleotides
provided as gapped duplexes is added and ligated. The DNA is then
electroporated into E. coli and inserts are selected by culturing
in tetracycline-containing media. The phage from this culture
(which contain the peptide insert) are isolated from the
supernatant, washed and resuspended in buffer. Typically libraries
are shown to have 7.times.10.sup.8 independent clones at
8.times.10.sup.12 transducing units per mL.
[0090] H. Phage-Screening Methodology
[0091] The essence of screening phage display peptide libraries
lies in the ability to collapse billions of potential candidate
phage to a relative few with outstanding properties. The original
screening protocols recommended by Scott, J. K. and G. P. Smith,
(1990) Science 249:315-324 are significantly modified to facilitate
the selection of the best epitopes for various aPL antibodies.
These procedures are designed to apply greater stringency of
selection as the screen progressed until a point is reached where a
useful number of clones representing the best sequences can be
thoroughly investigated. With some antibodies, the library does not
appear to have sequences which bind very tightly and if a method
with a high degree of stringency is applied to the screen, no
clones survive that are specific. On the other hand, the library
frequently yields many clones that represent good analogs of the
antigen and it is necessary to employ a method with a high degree
of stringency to identify the best epitopes. For that reason,
assays were developed with varying degrees of stringency in order
to identify the best epitopes from an epitope library screen. The
assays are listed here in order of increasing stringency:
Biopanning<Micropanning<Phage-Capture ELISA<Phage
ELISA=Colony Blot=Peptide ELISA.
[0092] (i) Biopanning
[0093] "Biopanning" describes the technique wherein
affinity-purified aPL antibody and phage bearing random peptide
inserts are allowed to mix, following which antibody-specific
recovery captures the bound phage. The phage confer tetracycline
resistance to E. coli that are propagated in a
tetracycline-containing medium and then isolated. Multiple rounds
of biopanning enrich the number of immunospecific phage in a
sample. Phage are always recovered at the end of three to five
rounds of selection but may represent only sequences that are
nonspecifically bound at low affinities for the selecting
antibodies. A method for further evaluating these phage
(micropanning) is required.
[0094] (ii) Micropanning
[0095] An estimation of the relative strength of binding of the
phage to the aPL antibody can be determined by "micropanning."
Micropanning is carried out following three or more rounds of
biopanning and uses the same antibody as employed in the biopanning
method. The method consists of dilution of the phage from the last
round of biopanning and analyzing fifty or more of these clones by
micropanning. Micropanning is accomplished by growing each clone to
a similar density and then incubating dilute phage at an optimal
single concentration in microtitration wells previously coated with
a constant amount of antibody. The optimal single concentration of
phage is that concentration most likely to reveal the widest range
of micropanning scores (from 0 to 4+) and, thus, permit the
greatest discrimination among the clones being tested. It is based
on the micropanning behavior of six randomly selected clones where
the score is determined at each of several concentrations of phage
obtained by serial dilution. Following the incubation with
antibody, the unbound phage are washed away and the amount of bound
phage is used as an indication of the affinity of the phage insert
for the antibody. The amount of bound phage is determined by
elution with mild acid followed by neutralization and infection of
E. coli. The number of infected E. coli are then quantitated by
plating the microorganisms on agar plates containing tetracycline
and then determining colony densities achieved by each clone.
[0096] (iii) Phage-Capture ELISA
[0097] The phage-capture ELISA test was developed to provide an
intermediate level assay to bridge the gap between the relatively
low stringency of the micropanning assay and the high stringency of
the phage- or peptide-ELISA assays. Preliminary studies show that
some antibody preparations give too many positive clones by
micropanning but none by phage-ELISA or peptide-ELISA. The
limitation of the phage-ELISA described below is that only five
copies of p-III are located on each phage and even with a large
number of phage coated on a well, few copies of the insert are
represented and detection requires that the antibody have a very
high affinity for the insert. With the phage-capture ELISA, the
signal is amplified many times which facilitates the detection of
lower affinity, stable interactions between the antibody and the
insert.
[0098] The phage-capture ELISA consists of the following steps.
Microtitration wells are coated with aPL antibody and phage clones
are added as in the micropanning assay. Unbound phage are washed
away and the amount of bound phage is quantitated using an
enzyme-conjugated goat antiserum which binds the major coat protein
of the phage. Phage screened using phage-capture ELISA react with
many aPL antibodies and provide a strong signal in subsequent ELISA
assays. This intermediate level of sensitivity allows for greater
efficiency in the peptide synthesis effort since few
micropanning-positive phage are phage-capture ELISA positive. As a
result, peptides synthesized from positive phage-capture ELISA
phage are generally immunoreactive.
[0099] (iv) Phage-ELISA
[0100] This method of selecting phage requires very tight binding
of the insert to the screening antibody. Phage are directly coated
onto wells of a microtitration plate and incubated with the
screening antibody. Following washes to remove unbound antibody, an
anti-human IgG alkaline phosphatase conjugate is added to bind any
aPL antibodies bound to the phage. APL antibodies are then detected
by adding a calorimetric substrate to the well which will react
with alkaline phosphatase according to methods well known in the
art.
[0101] (v) Colony Blot
[0102] This assay allows large-scale colony screening of E. coli
infected by biopanned phage. This procedure is an alternative to
phage-ELISA for identifying immunoreactive clones and exhibits a
comparable level of sensitivity without requiring culturing of
individual phage clones prior to testing. In this assay, E. coli
infected with phage from a round of biopanning are spread on a
large diameter nitrocellulose (NC) membrane and cultured overnight
on the surface of an agar plate containing tetracycline (Barbas et
al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982). Each colony
results from infection by phage containing identical sequences.
Several replicate transfer blots on NC are made using this NC
"master" and are allowed to grow on the surface of an agar plate.
Following the chemical and enzymatic disruption of phage-infected
colonies on the blots, the phage may be probed by the techniques
commonly used in Western blotting, i.e., staining or
immunoblotting. Blots that have been blocked may be incubated with
the screening aPL antibody. Following washes to remove unbound
antibody, an anti-human IgG horseradish peroxidase conjugate is
added to bind to any aPL-antibody that is bound to phage. The
addition of a calorimetric substrate allows one to localize the
discrete colonies in the master plate which represent
immunospecific phage that may be cloned for further study.
[0103] (vi) Peptide-ELISA
[0104] Following DNA sequencing to determine the peptide insert
sequences of the best-reacting phage in the assays described above,
the corresponding peptides are made using standard Fmoc peptide
chemistry as is well known in the art. For the peptide-ELISA assay,
the peptides can be made, for example, as branched tetravalent
molecules, i.e., each molecule has four copies of the insert. Such
a molecule can coat the well of a microtitration plate and still
have epitopes exposed to the solution to allow binding by an
antibody. The tetravalent peptides are synthesized by incorporating
lysines as branch points at the first two couplings analogous to
the methods used for Multiple Antigenic Peptides (MAPS) (Posnett et
al. (1988) J. Biol. Chem. 263:1719-1725). A spacer consisting of
glycine-serine-glycine-serine is added on each arm after the
lysines and then the insert, including the framework amino acids
found in the phage, proline-glycine at the carboxyl terminus and
alanine-glycine-proline at the amino terminus. All amino acids in
this synthesis are added one at a time using standard Fmoc
methods.
[0105] These peptides are then assayed by ELISA which is carried
out by coating the peptides on microtitration wells and then
assaying their reactivity with aPL antibody in a standard ELISA
format. In practice, the peptides usually bind very strongly to the
original screening antibody and show some cross-reactivity with
other aPL antibodies. Controls of non-aPL antibodies are included
to eliminate nonspecific binding peptides.
[0106] (vii) Competitive Binding Peptide-ELISA
[0107] Once ELISA-positive peptides are identified, it is necessary
to quantitate their relative binding affinity to the aPL antibodies
and to determine whether or not two peptides bind the same
population of antibodies in a given patient serum via a
peptide-competition ELISA assay. In this assay, various monomeric
peptides compete with tetravalent peptides coated on a
microtitration plate well. To perform the assay, the peptides to be
evaluated are synthesized as monomers, ie., without the lysine
branches employed in the synthesis of the tetravalent peptides,
using standard Fmoc chemistry. The monomeric peptides are then
purified and dissolved at known concentrations. Wells of a
microtitration plate are coated with a tetravalent peptide known to
bind to the aPL antibody. Serial dilutions of the monomeric
peptides are incubated with a constant dilution of the aPL
antibody. The dilution of the aPL antibody was previously
determined by titering the antibody against the tetravalent peptide
and selecting a dilution on the downslope of the titration curve.
After incubating the antibody and monomeric peptides for one hour,
the antibody/peptide solutions are added to the microtitration
wells and a standard calorimetric ELISA is performed. The
concentration of each monomeric peptide that decreases binding of
the aPL antibody with the tetravalent peptide is determined by
plotting the calorimetric readings obtained for each well. The 50%
inhibition point is used as the measure of the relative strength of
binding for the monomeric peptides.
[0108] A variation of this assay uses microtitration plates coated
with human .beta..sub.2-glycoprotein I/cardiolipin
(.beta..sub.2-GPI/CL) instead of tetravalent peptide and tests the
ability of monomeric peptides to block the binding of aPL antibody
to the epitope(s) on .beta..sub.2-GPI/CL. In this assay,
IgG-depleted human serum at an optimized concentration is used as a
source of .beta..sub.2-GPI. The monomeric peptides at several
concentrations are incubated with an optimized concentration of aPL
antibody in a manner analogous to the assay which employs
tetravalent peptide as a plate substrate. Following the incubation
of aPL/peptide in (.beta..sub.2-GPI/CL) plates, antibody binding
and the peptide concentration required for 50% inhibition is
determined at half-maximal absorbance as in the tetravalent
assay.
[0109] An additional variation of this assay tests the ability of
monomeric peptides to block the binding of aPL antibody to
.beta..sub.2-GPI coated directly on the wells of Nunc Maxisorp
microtitration plates. In this variation, the use of cardiolipin is
omitted and instead of fish gelatin, the reagent diluent and
blocker used is nonfat milk/Tween.
[0110] (viii) Fluorescence Polarization Peptide Binding Assay
[0111] This assay detects direct binding of the peptide to aPL
antibody. Since aPL antibodies bind to .beta..sub.2-GPI (the
antigen), the ELISA competitive inhibition assay can show
inhibition due to binding to .beta..sub.2-GPI as well inhibition
due to binding of the peptide to the aPL antibody. Because binding
to antibodies is required in order for the peptide to function as a
Toleragen, it is essential to establish that a peptide can directly
bind to an aPL antibody. This assay is used to determine the
dissociation constants for peptides that bind to two aPL
antibodies, ACA-6501 and ACA-6701. While the ELISA assay is useful
for high throughput screening because it requires less antibody
than the Fluorescence Polarization assay, however, ELISA-positive
peptides should be further evaluated by the Fluorescence
Polarization assay to determine whether they are capable of
directly binding with aPL antibody.
[0112] (ix) Evaluation of Amino Acid Contributions to Binding by
Substitution and Deletion Synthesis
[0113] The desired epitope for tolerance induction should have as
strong an interaction with as many of the aPL antibodies as
possible but not contain any unnecessary residues. In order to
deduce the minimum constitution of an epitope, analogs of each
peptide are made (i) that lack given residues, for example, the
framework residues at the carboxyl and/or amino termini are
deleted, or (ii) in which amino acid substitutions have been made
which differ from sequences found in the epitope library screen.
These amino acid substitutions may be either natural, e.g.,
isoleucine for leucine, or unnatural, e.g., alpha methyl proline
for proline. The effect of these deletions and/or substitutions are
then measured via peptide-competition ELISA.
[0114] (x) Grouping of aPL Sera Specificities by Mutagenesis of the
5th Domain of .beta..sub.2-GPI
[0115] For a Tolerogen to be generally effective, it must bind a
major portion of the aPL antibodies in the majority of patients. It
is important to determine if several antibodies from different
patients bind identical residues within the eighty-four amino acid
5th domain of .beta..sub.2-GPI which has been suggested by others
to contain the target epitope. If several antibodies bind identical
residues, a single mimotope derived from the structural data of the
peptides can be constructed which will react with all the
antibodies. On the other hand, if the antibodies bind to different
residues, a unique tolerogen would be required for each antibody.
Site-directed mutagenesis was performed to identify if key residues
involved in aPL antibody binding reside in the 5th domain of
.beta..sub.2-GPI. The resulting mutant .beta..sub.2-GPIs were
assayed for reactivity with several aPL antibodies. The results
were inconclusive. A fusion protein comprising the 5th domain of
.beta..sub.2-GPI and glutathionine S transferase (GST) was obtained
from A. Steinkasserer and expressed in E. coli (Steinkasserer et
al. (1992) FEBS Lett. 313:193-197). This fusion protein was
successfully substituted for native .beta..sub.2-GPI in the ACA
ELISA. Amino acid substitutions are engineered using standard
site-directed mutagenesis.
[0116] 1. Isolation of Synthetic aPL Epitopes
[0117] Antibody ACA-6501, from a patient with a GPL score of 151
(high titer) and a history of recurrent stroke, fetal loss, lupus
and three aortic valve replacements was immunoaffinity purified on
cardiolipin liposomes. The antibody was used in four separate phage
library screens, using the xy, the xyz, the xy'z, and a special
pro/cys-bounded 7-mer library where, based on the previous screens,
Arg was fixed at the seventh position. As shown in Table 1, 36
sequences were obtained in phage that micropanned (out of 140
tested). These appear quite homologous and the conserved DR
residues at positions 6 and 7 are notably striking. The consensus
sequence is CLLLAPDRC. Despite this homology, only seven phage (see
Table 2) were positive after phage-ELISA calorimetric testing.
Screening the xy'z phage with affinity purified ACA-6626 (from a
patient with a high, but lower than ACA-6501, titer) yielded five
unique sequences that were phage-ELISA tested. None were color
positive but two were positive when the ELISA immunoconjugate was
developed with a chemiluminescent substrate. The sequence motif
associated with ACA-6626 appears related but is different from that
seen with ACA-6501 (see Table 3). Both antibodies preferred the
cys-bounded (probably cyclized and constrained) epitopes over the
open pro-bounded sequences.
1TABLE 1 ACA-6501 Phage Library Sequences Clone Sequence Clone
Sequence xy Library 2101 CNILVLDRC xyz Library 5A12 CLILAPDRC 3C10
CILLAKNRC 2D7 CLLLAPDRC 3C5 CIVLVPDRC 3B6 CLVLALDRC 2F4 CLVIALDRC
3E4 CLFVALDRC 5B1 CWFRSQSSC 3E7 CILLAHDRC 3E11 CSPILRGNC 2H1
CIILAPGRC 3E8 CHKFFWLTC xy'z Library 2A10 CTILAPDRC 2D12 CLVLAADRC
2G12 CLLITPDRC 3B10 CLLLAPDRC 2G11 CLLITHDRC 3F2 CFFHFDHSC 2F10
CNILVLDRC 2D3 CPLHTHHTC 2E3 CPLITHDRC Custom (X).sub.6R Library G11
CTILTPDRC 1A4 CNLLALDRC 2H5 CTILTPDRC 2H6 CNLLAIDRC 2H2 CTILTLDRC
1C3 CLLLAIDRC 2H10 CTLLTPDRC 1D10 CTIITQDRC 2E10 CIQLTPDRC 2H4
CNIITRDRC 1B7 CHLLTPDRC 2G12 CILHAAHRC 2H1 CLILTPDRC 1A9 CSSKSYWRC
2H12 CSILAPDRC xy'z Library, Colony Blot Screening Assay CB2
CILLARDRC
[0118]
2TABLE 2 ACA-6501 Colorimetric ELISA-positive Phage Clone Sequence
Clone Sequence 5A12 CLILAPDRC 3B6 CLVLALDRC 2H1 CLILTPDRC 2G11
CTILTPDRC 3B10 CLLLAPDRC* 3E7 CJLLAHDRC 2H2 CTILTLDRC *Corresponds
to consensus or average sequence
[0119]
3TABLE 3 ACA-6626 xy'z Phage Library Sequences Clone Sequence Clone
Sequence 4B11 CGNAADARC 4G7 CTNLTDSRC 4D3 CTNWADPRC 4A2 CGNPTDVRC
4C7 CGNIADPRC
[0120] ACA-6644 is another high-titered aPL antibody that was used
to screen the pooled p-III phage libraries according to methods
described herein. The following sequences were discovered:
4 ACA-6644/CBc GILLNEFA ACA-6644/CBd GILTIDNL ACA-6644/CBf
GILALDYV
[0121] These sequences all were derived from the component "z"
epitope library that lacks phage framework residues at the
N-terminus. When synthesized as peptides the sequences were
immunoreactive with several ACA sera including ACA-6644 and
ACA-6501. Analysis revealed unsuspected homologies with the
sequences previously obtained with ACA-6501 as illustrated in Table
4.
5 TABLE 4 ACA-6644/CBd G I L T I D N L ACA-6501/2H2 C T I L T L D R
C ACA-6644/CBf G I L A L D Y V ACA-6501/2F10 C N I L V L D R C
ACA-6644/CBc G I L L N E F A ACA-6501/1D10 C T I I T O D R C
[0122] The convergent sequence homology from two very dissimilar
source libraries screened by these two aPL antibodies suggests that
the sequences may mimic a major, perhaps immunodominant, region in
the native target antigen.
[0123] Screening of the p-III libraries with ACA-6701 yielded two
unique sequences with a high degree of internal homology but unlike
others previously obtained with other aPL antibody. The sequences
are as shown:
6 ACA-6701/3B1 L S D P G Y V R N I F H ACA-6701/3E1 L T D P R Y T R
D I S N F T D
[0124] As resin-bound peptides, the sequences were strongly
immunoreactive with the parent serum (ACA-6701) but were minimally
cross-reactive with other aPL antibodies.
[0125] Continued screening of random pIII phage libraries with
affinity purified ACA antibody resulted in the discovery of a
peptide that displayed significant crossreactivity with all nine
affinity purified ACA antibodies against which it was initially
tested. The performance of this peptide as well as four others is
shown in Table 5. Peptide sequences for all five peptides
immediately follow the table. All were tested using the competitive
binding peptide ACA ELISA using CL/.beta.2-GPI plates as described
herein.
7TABLE 5 Percent Inhibition of AffA CA by Peptide Monomers Peptide
6626 6638 6641 6644 6701 7004 7005 6903 6501 Ave. 6641/3G3 100 100
93 100 100 100 95 100 86 97 1 mg/mL 6501/3B10 76 87 52 55 51 87 47
69 60 65 1 mg/mL 6626/4C7 88 96 45 45 69 99 54 77 39 68 1 mg/mL
6701/3B1 43 65 43 37 96 72 23 51 41 52 1 mg/mL 6644/CBf 37 60 28 18
22 54 57 43 37 39 200 .mu.g/mL Peptide Sequence ACA-6641/3G3: AGP
CLGVLGKLC PG ACA-6501/3B10: AGP CLLLAPDRC PG ACA-6626/4C7: AGP
DGNIADPRC PG ACA-6701/3B1: AGP LSDPGYVRNIFH PG ACA-6644/CBf:
GILALDYV GG
[0126] Subsequent testing determined that this peptide,
ACA-6641/3G3 having the sequence AGP-CLGVLGKLC-PG (LJP 688) was
cross-reactive with a number of ACA antisera. Chemical optimization
of this peptide was pursued by truncation, systematic amino acid
substitution, and non-disulfide cyclization studies.
[0127] The motifs of several peptides selected from the random
phage library screening is set forth below.
8TABLE 6 ACA Antibody Phage Insert Sequence 6501 C L L L A P D R C
(3B10) 6626 C T N W A D P R C 6641 C L G V L G K L C (3G3) 6644 G I
L A L D Y V 6701 L T D P R Y T R D I S N F T D 6707 C A H P D W D R
C
[0128] J. Immunoreactivity of aPL-Related Peptides with Affinity
Purified IgG aPL Antibody
[0129] Phage sequences obtained with affinity-purified ACA-6501 and
found to be phage-ELISA-positive were synthesized on a solid
support recently developed for combinatorial synthetic peptide
libraries. This support, a Rapp resin, has a high peptide density
and uses a hydrophilic polyethylene glycol spacer before the first
amino acid is coupled. The synthesis resulted in resin-bound
peptide that was ideally suited for antibody binding studies. As
shown in FIG. 2, peptide 5A12 (sequence CLILAPDRC) dramatically
outperformed an unrelated control peptide while not binding normal
IgG. Similar results were obtained with the other
phage-ELISA-positive peptides tested. In the experiment shown,
resin peptide-bound affinity-purified ACA-6501 aPL antibody was
detected by an immunoconjugate color reaction.
[0130] K. aPL Serum Antibody Reactivity with Synthetic Peptides
[0131] The discovery that resin-bound peptides could bind aPL
immunospecifically using serum significantly enhanced the ability
to test aPL antibodies. As shown in FIG. 3, the peptide derived
from screening with ACA-6626 bound aPL antiserum but did not
demonstrate significant binding of normal serum. FIG. 3 also
illustrates the immunospecific-binding behavior of ACA-6501-5A12
peptide towards aPL serum. Binding of normal serum to peptide 5A12
was nil in data not shown.
[0132] L. Crossreactivity of Synthetic Peptides Towards aPL
Antiserum that Was Not the Source of the Library Screening
Antibody
[0133] Two peptides were selected for aPL sera testing, one (5A12)
representing the ACA-6501 screen and another one (4D3) representing
the ACA-6626 screen. As shown in FIGS. 4 and 5, each of the
peptides reacted preferentially with the parent serum of the
screening antibody. However, a significant degree of
crossreactivity was detectable especially between ACA-6501 and
ACA-6626. A survey of 19 patient sera with low or no GPL score and
of 13 pathologic sera with moderate to high GPL carried out with
the 5A12 resin-bound peptide showed 8 of the 13 pathologic samples
with detectable peptide binding while only 2 out of 19 control
samples showed low peptide binding. These results evidence that
synthetic peptides are useful for diagnostic or prognostic assays
in stroke patient care.
[0134] As noted in Section I above, one synthetic peptide, 6641/3G3
was tested against several high titer ACA antisera. This peptide
appeared to be crossreactive with the vast majority of ACA antisera
tested as illustrated in FIG. 14. 6641/3G3 demonstrated
dose-dependent cross-reactivity with 10 of 10 ACA antibodies with
complete inhibition (>80%) at 1.8 mg/mL and appeared to be
specific for ACA antibodies as demonstrated in Table 7 below.
9TABLE 7 CROSSREACTIVITY DATA FOR AGP-CLGVLGKLC-PG (LJP 688) ACA
SERA WITH CARDIOLIPIN/.beta.2-GPI-C- OATED PLATES IC.sub.50, mg/mL,
for peptide LJP ACA SERUM Nr. 688 [1] 6501 RS, RFL 0.89, 1.1 [2]
6626 RS 1.09 [3] 6635 RS 1 [4] 6638 RS 0.81 [5] 6641 RS 0.89, 0.51
[6] 664 RS 0.76 [7] 6701 RS 1.07 [8] 6903 RS 0.8 [9] 7004 RFL 0.67
[10] 7005 RFL 0.75 mean .+-. sd 0.86 .+-. 0.16 RS = recurrent
stroke RFL = recurrent fetal loss
[0135] M. New Synthetic Peptide Methodologies
[0136] The identification of new candidate sequences by aPL library
screening required testing of the new synthetic peptides for
antibody binding. With Rapp resin of known peptide substitution, it
is possible to carry out quantitative binding studies such as
saturation binding analysis and equilibrium measurements using
radiolabeled aPL IgG.
[0137] Peptide synthesis allows the molecular dissection of the
mimotope by selective synthesis. This includes the modification of
each amino acid along the chain with the goal of enhancing antibody
binding. Selective synthesis reveals the relative importance of
each amino acid in the sequence. If necessary, selective
substitution at particular residue locations can be designed to
maintain B cell reactivity while abolishing any T cell
proliferative reactivity discovered during T cell assays.
[0138] Peptide 6641/3G3 (LJP 688) was subjected to a number of
analyses. The analyses included truncation at both the N-terminus
and the C-terminus, disulfide substitution, substitution of alanine
and glycine for amino acids in positions 2 through 8, substitution
of branched aliphatic amino acids in positions 2, 4, 5 and 8,
substitution of amino acids affecting conformation of the peptide,
including substitution of .alpha.-methyl amino acids, substitution
of basic amino acids in position 7, substitution of D-amino acids
in positions 2 through 9, and the substitution of N-.alpha.-methyl
amino acids in positions 1 through 9. The results are shown below
in Table 9. The structure/activity relationship of these
substitutions and truncations is shown in Table 8.
10TABLE 8 Optimization of Peptide 3G3 Total Peptides 144 with ACA
Mar. 28, 1996 6501 Position Number -2 -1 0 1 2 3 4 5 6 7 8 9 A B
150 ug/mL std Truncation analysis 1 A G P C L G V L G K L C P G 850
2 G P C L G V L G K L C P G 180 3 P C L G V L G K L C P G 420 4 C L
G V L G K L C P G 460 5 A G P C L G V L G K L C G 450 6 C L G V L G
K L C 10 #906(LJP 69O) Disulfide surrogates 1 C L G V L G K L C 110
#952 = 100 2 HcL G V L G K L C 46 3 C L G V L G K L Hc 92 4 HcL G V
L G K L Hc 80 Ala scan 1 C A G V L G K L C 200 #952 = 80 2 C L A V
L G K L C 170 3 C L G A L G K L C 260 4 C L G V A G K L C inf 5 C L
G V L A K L C 40 6 C L G V L G A L C inf 7 C L G V L G K A C 360
Gly scan 1 C G G V L G K L C 310 #952 = 120 2 C L G G L G K L C
inf. 3 C L G V G G K L C inf 4 C L G V L G G L C inf 5 C L G V L G
K G C 500 Sequence optim. Branched aliphatic amino acids 1 C I G V
L G K L C 60 #910 = 40 2 C V G V L G K L C 130 3 C M G V L G K L C
4 C Cy G V L G K L C 5 C tL G V L G K L C 6 C mL G V L G K L C 7 C
Iv G V L G K L C 8 C L G L L G K L C 9 C L G I L G K L C 10 C L G M
L G K L C 11 C L G Cy L G K L C 12 C L G tL L G K L C 13 C L G mL L
G K L C 14 C L G Iv L G K L C 15 C L G V I G K L C 16 C L G V V G K
L C 17 C L G V M G K L C 18 C L G V Cy G K L C 19 C L G V tL G K L
C 20 C L G V mL G K L C 21 C L G V Iv G K L C 22 C L G V L G K I C
23 C L G V L G K V C 24 C L G V L G K M C 25 C L G V L G K Cy C 26
C L G V L G K tL C 27 C L G V L G K mL C 28 C L G V L G K Iv C
Conformational scan amino acids 1 C L P V L G K L C 2 C L mP V L G
K L C 3 C L mA V L G K L C 4 C L cG V L G K L C 5 C L G V L P K L C
6 C L G V Lm P K L C 7 C L G V Lm A K L C 8 C L G V Lc G K L C 9 C
L dA V L G K L C 10 C L G V Ld A K L C 11 C P G V L G K L C 160
#952 = 100 12 C L P V L G K L C 340 (ProScan) 13 C L G P L G K L C
180 14 C L G V P G K L C Inf. 15 C L G V L P K L C 350 16 C L G V L
G P L C 160 17 C L G V L G K P C 550 18 C pG G V L G K L C 130 #910
= 40 19 C L pG V L G K L C 70 20 C L G pG L G K L C 70 21 C L G Vp
G G K L C 35 22 C L G V Lp G K L C 30 #910 = 5 23 C L G V L Gp G L
C 230 24 C L G V L G K pG C 80 alpha Me AA 25 C aiB G V L G K L C
370 #910=40 26 C L aiB V L G K L C 80 27 C L G aiB L G K L C 110 28
C L G V aiB G K L C inf. 29 C L G V L aiB K L C 110 30 C L G V L G
aiB L C 460 31 C L G V L G K aiB C >470 Basic amino acid 1 C L G
V L G R L C 160 #952 = 120 2 C L G V L G Or L C 40 910 = 60 3 C L G
V L G mK L C D amino acids (lower case d) 1 dP C L G V L G K L C
120 #952 = 100 2 CdL G V L G K L C 180 #952 = 30 3 C L G dV L G K L
C 260 #952 = 30 4 C L G V dL G K L C 230 #952 = 30 5 C L G V L G dK
L C 170 #952 = 50 6 C L G V L G K dL C 140 #952 = 30 7 C dL G dV L
G K L C 8 C dV G dV L G K L C 9 C dL G dL L G K L C 10 C L G dV dL
G K L C 50 #952 = 30 11 C L G dL dV G K L C 12 C L G dV dV G K L C
13 C L G dL dL G K L C 14 C L G V dL G dK L C 15 C L G V dK G dL L
C 16 C L G V cL G dL L C 17 C L G V dK G dK L C 18 C L G V L G dK
dL C 150 #952 = 50 19 C L G V L G dL dK C 20 C L G V L G dK dK C 21
C L G V L G dL dL C 140 #952 = 30 22 C L G V L dA K L C 140 #952 =
50 C L G V L G K L dC N-Me amino acids (nAA) 1 nC L G V L G K L C 2
C L G V L G K L C 3 C L nG V L G K L C 70 #952 = 50 4 C L G nV L G
K L C 220 5 C L G V nL G K L C 180 6 C L G V L nG K L C 550 7 C L G
V L G nK L C 8 C L G V L G K nL C 240 #952 = 50 9 C L G V L G K L
nC Others 1 C L G V L G K L C ATU-Y 300 #952 = 50 2 Y ATU C L G V L
G K L C 170 3 C L G V L C K L C TGR resin 500 4 C L G V L G K L C
dC TGR resin 620 5 dC L G V L G K L C C 6 cC L G V L G K L C dC TGR
resin Thioethers: single deletion 1 Hc L G V L A K L C 2 Hc G V L A
K L C 3 Hc L V L A K L C 4 Hc L G L A K L C 5 Hc L G V A K L C 6 Hc
L G V L K L C 7 Hc L G V L A L C 8 Hc L G V L A K C shrink
minimization 1 Hc V L A K L C 2 Hc L A K L C 3 Hc L A K C branched
1 Hc L G V L G K L Pe 2 Hc L G V L G K L dPe 3 Pe L G V L G K L Hc
4 dPe L G V L G K L Hc 5 dPe L G V L G K L dPe D amino acids 1 dHc
L G V L G K L C 2 dHc L G V L G K L dC 3 Hc L G V L G K LdC Des
amino 1 By L G V L G K L Hc desamino HHTE 2 By L G V L G K L C
desamino HCTE 3 Pp L G V L G K L Hc desamino CHTE 4 Pp L G V L G K
L C desamino CHTE Abbreviations Hc homocysteine Cy cyclohexyl
alanine tL tertiary leucine mL alpha methyl leucine Iv alpha
methyl, alpha amino butyric acid mP alpha methyl proline mA alpha
methyl alanine aiB aminoisobutryic acid pG phenylglycine cG
cyclopropyl glycine dA D alanine dP D proline dL D leucine dV D
valine dK D lysine nC N alpha-methyl cysteine nL N alpha-methyl
leucine nG N methyl glycine nV N alpha-methyl valine nK N
alpha-methyl lysine mK N epsilon-methyl lysine Pe penicillamine By
butyroyl Pp proprionyl Or ornithine
[0139]
11TABLE 9 Structure Activity Relationship of the Crossreactive ACA
Epitope Structure Relative Activity AGPCLGVLGKLCPG (3G3) (LJP 688)
100 CLGVLGKLC (LJP 690) 3.5 CLGVLAKLC 1.8 CLGVL.sub.PGKLC 1.4
CLG.sub.dV.sub.dLGKLC 5.9 HcLGVLGKLC (thioether) 1.6 SLGVLGKLS
.infin. CLGVAGKLC .infin. CLGVLGALC .infin.
[0140] N. Use of .alpha.-Methyl Amino Acids Substitutions and
Unnatural Amino Acids to Enhance Peptide Immunoreactivity and
Confer Resistance to Protease Attack
[0141] Proline residues have a special significance due to their
influence on the chain conformation of polypeptides. They often
occur in reverse turns on the surface of globular proteins. In the
phage epitope libraries of the present invention, all random
peptide inserts are flanked by boundary prolines. In addition, most
of the mimotopes discovered with ACA-6501 have a third proline
which, based on computer-based predictions, likely exists as part
of a .beta.-turn. .beta.-turn mimetics can be used to enhance the
stability of reverse turn conformations in small peptides. Such a
mimetic is (S)-.alpha.-methyl proline (.alpha.-MePro), a proline
analog that, in addition to stabilizing turn conformations, confers
resistance to protease degradation. Protease resistance is a
desirable property for a potential drug designed to act in the
plasma. Peptide ACA-6501/3B10 AGPCLLLAPDRCPG (insert highlighted)
is a consensus peptide. It has a sequence featuring the most
prevalent residue at each position based on a comparison with 35
other homologous sequences. Due to its representative character,
the sequence was subjected to a number of systematic modifications
and deletions and its activity subsequently evaluated by aPL
antibody binding. Among the most important findings was the
discovery that the prolines at the 3 and 9 positions are important
for activity. Proline-3 is derived from the phage framework and is
not part of the random insert. The most dramatic effect was
obtained by the substitution of .alpha.-MePro for proline at the
9-position. This substitution led to a six-fold enhancement in
immunoreactivity.
[0142] The results of .alpha.-methyl and N-.alpha.-methyl amino
acid substitution studies carried out on peptide 6641/3G3 are shown
above in Table 8.
[0143] In addition to the twenty naturally-occurring amino acids
and their homoanalogs and noranalogs, several other classes of
alpha amino acids can be employed in the present invention.
Examples of these other classes include d-amino acids,
N.sup..alpha.-alkyl amino acids, alpha-alkyl amino acids, cyclic
amino acids, chimeric amino acids, and miscellaneous amino acids.
These non-natural amino acids have been widely used to modify
bioactive polypeptides to enhance resistance to proteolytic
degradation and/or to impart conformational constraints to improve
biological activity (Hruby et al. (1990) Biochem. J. 268:249-262;
Hruby and Bonner (1995) Methods in Molecular Biology
35:201-240).
[0144] The most common N.sup..alpha.-alkyl amino acids are the
N.sup..alpha.-methyl amino acids, such as N.sup..alpha.-methyl
cysteine (nC), N.sup..alpha.-methyl glycine (nG),
N.sup..alpha.-methyl leucine (nL), N.sup..alpha.-methyl lysine
(nK), and N.sup..alpha.-methyl valine (nV). Examples of alpha-alkyl
amino acids include alpha-methyl alanine (mA),
alpha-aminoisobutyric acid (aiB), alpha-methyl proline (mP),
alpha-methyl leucine (mL), alpha-methyl valine (mV),
alpha-methyl-alpha-aminobutyric acid (tv), diethylglycine (deG),
diphenylglycine (dpG), and dicyclohexyl glycine (dcG) (Balaram
(1992) Pure & Appl. Chem. 64:1061-1066; Toniolo et al. (1993)
Biopolymers 33:1061-1072; Hinds et al. (1991) Med. Chem.
34:1777-1789).
[0145] Examples of cyclic amino acids include
1-amino-1-cyclopropane carboxylic acid (cG), 1-amino-1-cyclopentane
carboxylic acid (Ac5c), 1-amino-1-cyclohexane carboxylic acid
(Ac6c), aminoindane carboxylic acid (ind), tetrahydroisoquinoline
carboxylic acid (Tic), and pipecolinic acid (Pip) (C. Toniolo
(1990) Int'l. J. Peptide Protein Res. 35:287-300; Burgess et al.
(1995) J. Am. Chem. Soc. 117:3808-3819). Examples of chimeric amino
acids include penicillamine (Pe), combinations of cysteine with
valine, 4R- and 4S-mercaptoprolines (Mpt), combinations of
homocysteine and proline and 4R- and 4S-hydroxyprolines (hyP) and a
combination of homoserine and proline. Examples of miscellaneous
alpha amino acids include basic amino acid analogs such as
ornithine (Or), N.sup..epsilon.-methyl lysine (mK), 4-pyridyl
alanine(pyA), 4-piperidino alanine (piA), and 4-aminophenylalanine;
acidic amino acid analogs such as citrulline (Cit), and
3-hydroxyvaline; aromatic amino acid analogs such as
1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), phenylglycine
(pG), 3,3-diphenylalanine (dpA), 3-(2-thienyl)alanine (Thi), and
halophenylalanines (e.g., 2-fluorophenylalanine and
4-chlorophenylalanine); hydrophobic amino acid analogs such as
t-butylglycine (i.e., tertiary leucine (tL)), 2-aminobutyric acid
(Abu), cyclohexylalanine (Cy), 4-tetrahydropyranyl alanine (tpA),
3,3-dicyclohexyl alanine (dcA), and 3,4-delhydroproline.
[0146] In addition to alpha-amino acids, others such as beta amino
acids can also be used in the present invention. Examples of these
other amino acids include 2-aminobenzoic acid (Abz),
.beta.-aminopropanoic acid (.beta.-Apr), .gamma.-aminobutyric acid
(.gamma.-Abu), and 6-aminohexanoic acid (.epsilon.-Ahx). Carboxylic
acids such as 4-chlorobutyric acid (By) and 3-chloropropionic acid
(Pp) haved also been used as the first residue on the N-terminal in
the synthesis of cyclic thioether peptides.
[0147] O. Preparation of Cyclic Thioether Analogs
[0148] The mimetic peptides identified by the methods of the
instant invention can be further modified to contain thioether
substitutions. Modification of cyclic disulfide analogs to cyclic
thioether analogs will extend the plasma half-life of the analog
conjugates and, therefore, require a lower dosage. Cyclic thioether
analogs also eliminate the problem of disulfide bond exchange which
often occurs with cyclic disulfide polypeptides. In addition, the
cyclic thioether analogs may also interfere with MHC Class II
presentation to T cells and, thus, facilitate induction of anergy.
Finally, the cyclic thioether analogs are useful in the
thiol-dependent conjugation reactions used in the production of
valency platform molecule conjugates.
[0149] Four cyclic thioether analogs were prepared according to the
methodology described in co-owned, co-pending patent application,
attorney docket number 252312006600, which is incorporated herein
in its entirety. Using either a Rink.TM. amide 4-methyl
benzhydrylamino resin or MBHA resin, full length peptide analogs of
6641/3G3 were prepared, converted into chloro-peptides, cleaved
from a solid support and then cyclized. See Thioether Reaction
Scheme below. Suitable cyclic thioether analogs include the analogs
shown below. 1 2
[0150] Alternatively, thioether analogs are prepared according to
the reaction scheme below. 3
[0151] The following analogs are representative as analogs made
according to cyclic thioether reaction scheme 2. 4
[0152] Exemplary cyclic thioether analogs were tested for activity
against ACA antibodies ACA-6501 and ACA-6701. The results are shown
in Table 10 below.
12TABLE 10 IC.sub.50 Values (.mu.g/0.1 mL) Thioether ACA-6501
ACA-6701 CCTE, LJP 698 11.0 5.0 HCTE, LJP 699 4.6 3.0 CHTE, LJP 702
9.2 3.2 HHTE, LJP 703 8.0 3.6 LJP 690 10.0 4.0 CTE A 0.08 CTE B 0.5
CTE C 0.9 CTE D 3.2 CTE E 6.3
[0153] The HCTE analog, LJP 699, outperformed the reference
peptide, LJP 690, which is the truncated version, CLGVLGKLC, of LJP
688, AGPCLGVLGKLCPG.
[0154] All articles, documents, patents and patent applications
cited herein are incorporated by reference herein in their
entirety. The following examples are intended to further illustrate
the invention and its uniqueness. These examples are not intended
to limit the scope of the invention in any manner.
EXAMPLES
Example 1
[0155] Measurement of Anticardiolipin Antibodies (ACA) in Serum
6501
[0156] Even numbered wells of an Immulon I microtitration plate
(Dynatech Laboratories, Inc., Chantilly, Va.) were coated with 50
.mu.g cardiolipin (Sigma Chemical, St. Louis, Mo.) in 30 .mu.L
ethanol per well. The plate was dried overnight at 4.degree. C. and
blocked with 200 .mu.L of 10% adult bovine serum (ABS) (Irvine
Scientific Co., Santa Ana, Calif.) in phosphate-buffered saline
(ABS/PBS) for 2 hours at room temperature (RT). The plates were
washed 5 times with Tris-buffered saline (TBS) prior to the
addition of 10 .mu.L of aPL standards and the test serum, ACA-6501.
The aPL standards (APL Diagnostics, Inc., Louisville, Ky.) were
reconstituted according to the manufacturer's instructions and
diluted 1:50 with 10% ABS-PBS. The test serum, ACA-6501, diluted
1:50 to 1:2,000 in serial dilutions with 10% ABS-PBS, was added to
selected duplicate wells and incubated for 2 hours at RT. The plate
was washed five times with TBS and 100 .mu.L of 1:1,000
goat-anti-human-IgG/alkaline phosphatase conjugate (Zymed, South
San Francisco, Calif., Cat. No. 62,8422), in 10% ABS-PBS was added
and incubated for 1 hour at RT. Again, the plate was washed five
times with TBS and the assay was developed by adding 100 .mu.L
phenolphthalein monophosphate (PPMP) substrate solution (Sigma,
Cat. No. P-5758), prepared from a stock solution of 0.13 M PPMP and
7.8 M 2-amino-2-methyl-1-propanol, adjusted to pH 10.15 with HCl,
after a dilution of 1:26 with deionized water. After approximately
30 minutes, the reaction was stopped with 50 .mu.L of 0.2 M dibasic
sodium phosphate (Mallinckrodt, Analytical Reagent) added per well.
The optical density was read at 550 nm in a microplate autoreader
(Bio-Tek Instruments, Winooski, Vt., Model EL311). The optical
density of the odd-numbered control wells (blank, without
cardiolipin (CL)) was subtracted from the optical density of the
even-numbered wells. The absorbance readings of the aPL standards
were plotted using Graph Pad Prizm (Graph Pad Software, Inc., San
Diego, Calif.) to generate the GPL (IgG phospholipid) standard
curve. The diluted 6501 test serum absorbance readings were used to
calculate GPL scores based on the GPL standard curve.
[0157] In the modified ACA ELISA, Nunc Maxisorp microtitration
plates were coated with 100 .mu.L /mL .beta..sub.2-GPI made up in
PBS following incubation at 1 hour at room temperature. Following
this step, microplates were blocked with PBS containing 2% nonfat
milk and 0.4% (w/v) Tween 80 for 2 hours at room temperature.
Except for the use of nonfat milk/Tween as reagent diluent and
blocking agent, the Nunc microplates coated with .beta..sub.2-GPI
directly were used for ACA IgG binding studies as previous
described for the Dynatech microplates coated first with
cardiolipin before the .beta..sub.2-GPI was added.
Example 2
[0158] Anticardiolipin Antibody (ACA) Purification from Serum
6501
[0159] In a 25 mL round-bottom flask (Kontes Scientific Co.,
Vineland, N.J.) a mixture of 1.2 mL cardiolipin (Sigma Chemical,
St. Louis, Mo., #C-1649), 0.464 mL cholesterol (Sigma Diag., St.
Louis, Mo., #965-25), 0.088 mL of 5 mg dicetylphosphate (Sigma
Chemical, St. Louis, Mo., D-2631) per mL chloroform was dried for
approximately 5 minutes in a Rotavap (Buchi, Switzerland).
Following the removal of solvent, 2 mL of 0.96% (wt./vol.) NaCl (J.
T. Baker, Inc., Phillipsburg, N.J.) Baker analyzed reagent) was
added and mixed in a Vortex Genie Mixer (Scientific Industries,
Inc., Bohemia, N.Y.) for 1 minute. The liposome suspension was
incubated for 1 hour at 37.degree. C. Meanwhile, serum 6501 was
spun at 600.times.g in a Sorvall RT 6000 centrifuge (Dupont Co.
Wilmington, Del.) for 10 minutes at 8.degree. C. Four mL of the
supernatant was placed in a 25 mL round-bottom flask with 1 mL of
the prepared liposome suspension and the mixture was incubated with
agitation at medium speed in an orbital shaker, Tektator V
(Scientific Products, McGraw Park, Ill.) for 48 hours at 4.degree.
C., and an additional 2 hours at 37.degree. C. Twenty mL of cold
TBS was added and the mixture was transferred into a 50 mL
polycarbonate centrifuge tube (Nalge Co., Rochester, N.Y.) and
centrifuged at 27,000.times.g for 15 minutes at 4.degree. C. in an
RC3 centrifuge in a SS-34 rotor (Sorvall-Dupont, Wilmington, Del.).
The precipitate was washed 3 times with 25 mL of cold 0.96% NaCl
using the RC3 centrifuge. The pellet was dissolved in 1 mL of 2%
(wt/vol) solution of n-octyl-.beta.-D-glucopyranoside (Calbiochem,
La Jolla, Calif.) in TBS and applied to a 0.6 mL protein
A/cross-linked agarose (Repligen Corporation, Cambridge, Mass.)
column which had been pre-washed with 15 times bed volume of 1 M
acetic acid and equilibrated with 15 times bed volumes of TBS. The
antibody-protein A/agarose column was washed with 40 times bed
volume of 2% octylglucopyranoside to remove lipids, followed by
extensive washings with TBS until the optical density of the eluate
at 280 nm approached the baseline. The bound antibody was eluted
with 1 M acetic acid. One mL fractions were collected, neutralized
immediately with 0.34 mL 3 M Tris (Bio-Rad, electrophoresis grade
reagent) per fraction and kept in an ice bath. The optical density
of each fraction was determined at 280 nm in a spectrophotometer
(Hewlett-Packard, 8452A Diode Array Spectrophotometer, Palo Alto,
Calif.). Fractions containing antibody were pooled, concentrated
and washed 4 times with TBS in Centricon-30 concentrators (Amicon
Division, W. R. Grace & Co., Beverly, Mass.) per manufacturer's
protocol. The final yield of purified antibody from 4 mL of serum
6501 was determined by reading the optical density at 280 nm of an
aliquot from the concentration, where 1 mg=1.34 OD.sub.280. The
average yield obtained was 750 .mu.g antibody from 4 mL of serum
6501. The purified antibody was tested for ACA activity and checked
for purity with Laemmli SDS-PAGE. An affinity adsorbent containing
.beta..sub.2-GPI was prepared using CNBr-activated
agarose)Pharmacia, Inc., Piscataway, N.J.) or Affi-Gel 10 (BioRad,
Richmond, Calif.) in accordance with the manufacturer's
instructions using purified .beta..sub.2-GPI obtained commercially
(PerImmune, Rockville, Md.). To a 1 mL gel column containing the
.beta..sub.2-GPI affinity adsorbent, up to 100 .mu.g of
liposome-purified aPL IgG in 1 mL TBS was added. After 1 hour, the
column was extensively washed with buffer to displace contaminants
and any IgG that did not bind .beta..sub.2-GPI. The
.beta..sub.2-GPI-binding IgG was displaced with 1 M HOAc and the
fractions neutralized with Tris as described above. Fractions
containing IgG were pooled, concentrated and subjected to buffer
exchange as previously described.
Example 3
[0160] Construction of a p-III Library Vector Preparation
[0161] fUSE 5 (Scott, J. K. and G. Smith, supra) was used as the
vector for the construction of p-III libraries, and a variation of
the method of Holmes, D. S. and M. Quigley (1981), Anal. Biochem.
144:193) was employed to generate the double-stranded replicative
form (RF). Briefly, an 800 mL culture of E. coli K802, harboring
fUSE 5, was grown in 2YT medium (Difco Labs, Ann Arbor, Mich.)
containing 20 micrograms/mL tetracycline for 18 hours at 37 degrees
with vigorous shaking. Cells were collected by centrifugation and
resuspended in 75 mL STET. STET consists of 8% sucrose in 50 mM
Tris/HCl pH 8.0, 50 mM EDTA and contains 0.5% Triton X-100.
Lysozyme, 10 mg/mL in STET, was added to a final concentration of 1
mg/mL. After 5 minutes at RT, three equal aliquots were placed in a
boiling water bath with occasional shaking for 3.5 minutes. The
viscous slurry was centrifuged for 30 minutes at 18000.times.G and
an equal volume of isopropanol was added to the supernatant. The
solution was cooled to -20.degree. C. and the nucleic acids were
collected by centrifugation. The RF was isolated from a CsCl
gradient as described by Sambrook et al. MOLECULAR CLONING: A
LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2d ed., 1989).
[0162] Preparation of the Random Insert
[0163] The DNA for insertion was generated by the "gapped duplex"
method described by Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA
87:6378-6382. In this method, an oligonucleotide containing a
degenerate region in the middle is surrounded by short constant
regions on each end of the oligonucleotide. Two shorter,
complementary oligonucleotides are annealed to form a "gapped
duplex" possessing overhangs that are complementary to the sticky
ends produced by the restriction endonuclease used to digest the
vector. In this case, the longer degenerate oligonucleotide has the
sequence:
[0164] 5' GGGCTGGACCC(NNK).sub.xCCGGGGGCTGCTG 3' where N=A or C or
G or T, K=G or T, and x is the number of codons in the random
regions. EpiGS2 was designed to base pair to the 5' end of the
degenerate oligonucleotide and has the sequence 5' GGGTCCAGCCCCGT
3'. Similarly EpiGS3 was designed to anneal to the 3' end of the
degenerate oligonucleotide and has the sequence 5' CAGCCCCCGG 3'.
When correctly annealed, the three oligonucleotides form a "gapped
duplex" which, when inserted into fUSE 5 digested with Sfi1,
restores the reading frame of p-III with the random insert near the
5' end.
[0165] Oligonucleotides described above were prepared by excision
from polyacrylamide gels. One nanomole of EpiGS2, EpiGS3, and 50
picomoles of the degenerate oligonucleotide were kinased separately
in 66 microliter volumes. The three oligonucleotides were then
pooled, NaCl was added to 50 mM and the mixture was heated to
65.degree. C. for 5 minutes followed by slow cooling to RT. The
annealed oligonucleotides were then cooled on ice and used
immediately in the ligation reaction with fUSE 5. The ligation
reaction consisted of 10 micrograms of fUSE 5 DNA digested to
completion with Sfil, 30 .mu.L of the "gapped duplex" solution and
1000 U of T4 ligase in a total volume of 450 microliters. The
ligation was incubated for 18 hours at 16.degree. C. The mixture
was then phenol-chloroform extracted, precipitated with ethanol and
the precipitate dissolved in 20 .mu.L of water.
[0166] Generating and Amplifying the Library
[0167] The ligated DNA was introduced into E. coli by
electroporation (Dower et al. (1988) Nucleic Acid Res.
16:6127-6145). Frozen electrocompetent MC1061 cells (0.1 mL) were
mixed with 4 .mu.L of ligated DNA in a cold 2 mm cuvette and
subjected to 2.5 kV, 5.2 mS pulse by a BTX electroporation device
(BTX Corp., San Diego, Calif.). Immediately after the pulse, 1 mL
of SOC, a cell growth medium (see Dower et al., supra) was added.
Five separate electroporations were carried out, pooled, and
incubated at 37.degree. C. for 1 hour. At that time, samples were
removed and diluted to determine the total number of clones
generated. The balance of the mixture was diluted in 1 L of 2YT
(1.6% Peptone, 140, 1% Yeast Extract, and 0.5% NaCl, Difco Labs,
Ann Arbor, Mich.) containing 20 micrograms per mL of tetracycline
and grown for 18 hours at 37.degree. C. while shaken at 275 rpm.
Phage were purified by 2 rounds of PEG/NaCl precipitation and
resuspension in 1.2 mL TBS containing 0.02% sodium azide. Particle
number was estimated by absorbance at 269 nm. The titer of the
phage was determined by mixing 10 .mu.L of dilutions of phage with
10 microliters of starved E. coli.
Example 4
[0168] Screening a p-III Library with aPL Antibody
[0169] Affinity-purified ACA-6501 (affACA-6501, 10 .mu.g; 7 .mu.L
of 1.44 mg/mL stock solution) was incubated for 2 hours at RT in
siliconized 1.4 mL microfuge polypropylene tubes with pooled phage
from the x, y and z libraries (epi.sup.xyz) [11 .mu.L+8.5 .mu.L+2
.mu.L, respectively; total volume=21.5 .mu.L.about.10.sup.10
clones] in a final volume of 100 .mu.L TBS, pH 7.4, with 0.5%
bovine serum albumin (BSA). During this incubation, the final steps
in preparing freshly starved E. coli, strain K-91, were carried
out. A suspension of E. coli freshly grown in 2YT medium for about
5 hours with 250 rpm shaking at 37.degree. C. was spun at
1000.times.g for 10 minutes at RT in 50 mL polypropylene tubes.
Twenty mL of 80 mM NaCl was added to the packed E. coli pellet and
then incubated for 45 minutes at 37.degree. C. at 100 rpm.
Following centrifugation as above, the starved E. coli pellet was
suspended in 1 mL of 50 mM ammonium phosphate/80 mM NaCl and used
later for phage amplification. Protein G-agarose beads were washed
2.times. in TBS/BSA and 2 times in TBS/0.5% Tween-20 and stored at
4.degree. C. as a 50% suspension in TBS/Tween.
[0170] Two hundred .mu.L of the protein G-agarose bead suspension
was then added to the ACA-6501/phage mixture and incubation
continued for an additional 1 hour at RT. At this point, the
mixture was chilled and washed 3 times with cold TBS/Tween and the
precipitate was collected in a microfuge in a cold room. The washed
beads were transferred to new microfuge tubes prewashed with
TBS/BSA and TBS/Tween to prevent non-specific adherence. After
three additional washes with TBS/Tween, the beads with bound
ACA-6501-bearing phage were eluted with 300 .mu.L 0.2 N
HCl/glycine, pH 2.1 by tumbling for 10 minutes at RT. Following
centrifugation at 16,000.times.g, the acidic eluate supernatant was
collected and an additional 100 .mu.L of elution solution was added
to the bead pellet and the procedure repeated. After 10 minutes,
the phage-containing eluates (representing the unamplified first
round phage) were pooled (.about.400 .mu.L) and placed in a sterile
17.times.100 mm polypropylene cell culture tube to which was added
50 .mu.L 0.5 M NaCl followed by pH neutralization with 2.5 M Tris
base (usually .about.25-35 .mu.L). An equal volume of the starved
E. coli suspension was added inmmediately and then incubated for 10
minutes at 37.degree. C. at 100 rpm. The mixture was then
transferred to a 250 mL sterile culture flask containing 25 mL 2YT
with 20 .mu.g/mL tetracycline (Tet) and incubated overnight at
37.degree. C. at 250 rpm.
[0171] To isolate amplified phage from overnight cultures, the
suspension was centrifuged at 12,000.times.g for 10 minutes in
polycarbonate tubes and the pellet discarded. After heating the
supernatant at 70.degree. C. for 30 minutes in polypropylene tubes,
the material was spun again in polycarbonate tubes and the
supernatant saved. To the supernatant, 1/4 volume of 20% (w/v)
polyethylene glycol, molecular weight 8000 (PEG 8000) was added to
precipitate phage. The solution was mixed by inversion 100 times
and then incubated at 4.degree. C. for 2 hours. After
centrifugation at 35,000.times.g for 30 minutes at 4.degree. C.,
the phage-containing pellet was resuspended in .about.0.5 mL of
TBS/BSA and transferred to a 1.4 mL microfuge tube. After a 1
minute spin in a microfuge at 16,000.times.g, the supernatant was
transferred to a clean tube and labeled first round amplified
phage.
[0172] During second, third and fourth rounds of biopanning, 75
.mu.L of amplified phage from the preceding round was incubated
with 7 .mu.L affACA-6501 in a final volume of 100 .mu.L. For fifth
round phage, affACA-6501 was diluted first at 1:1000, then treated
as described for the other rounds. All subsequent steps were
carried out as described for the first round. Phage from five
rounds of biopanning were spot-titered on 2YT/Tet plates to
determine phage concentration. The spot titer of amplified phage
requires an initial phage dilution of 1.times.10.sup.6 in TBS/BSA
or 2YT media. For each round, 10 .mu.L of the dilute phage was
incubated with 40 .mu.L of starved E. coli for 10 minutes at
37.degree. C. with no shaking. Following the addition of 950 .mu.L
of 2YT/dilute Tet (0.2 .mu.g/mL), the mixture was incubated for
30-45 minutes at 37.degree. C. with 250 rpm shaking. Ten .mu.L
aliquots of neat and diluted phage solutions, 1:10, 1:100, and
1:1000, were spotted in 2YT/dilute Tet using agar plates containing
20 .mu.g/mL Tet.
[0173] Micropanning
[0174] Immulon type 2 plates were coated with protein G. Protein G
was prepared at 10 .mu.g/mL in 0.1 M NaHCO.sub.3 and 100 .mu.L per
well was added to the wells of microtitration plates and incubated
overnight at 4.degree. C. After discarding excess protein G
solution from plates, each well was blocked with 250-300 .mu.L 2YT
for 1 hour at RT with agitation on an oscillating platform.
Tris-buffered saline, pH 7.4/0.5% Tween 20 (TBS/Tween), was used
with an automatic plate washer to wash the wells 4 times with 200
.mu.L. One hundred .mu.L affACA-6501 (or control normal IgG),
diluted to 2.5 .mu.g/mL with 2YT, was added to washed wells. The
plate was transferred to a cold room rotator near the end of a 1
hour incubation at RT on a rotating platform.
[0175] Phage to be tested by micropanning were obtained from the
agar plates generated by biopanning. Each clone to be tested was
transferred using sterile toothpicks to a separate well of a
round-bottom 96-well microtitration plate (Corning, Corning, N.Y.)
containing 250 .mu.L 2YT/Tet per well and cultured overnight at
37.degree. C. Clone designations are based on the screening
antibody, the biopanning round of origin, and the location of the
clone in the overnight culture plate, e.g., ACA-6501/3B10 refers to
the clone isolated by ACA-6501 in the third round located in the
well designated B10 on the microtitration plate. Following
overnight incubation, phage cultures were centrifuged using a
microtitration plate holder at 1300.times.g for 10 minutes at RT.
Supernatants constituted the source of "neat" phage.
[0176] Initial micropanning was restricted to six clones which were
tested at dilutions expanded by factors from 1:10 to 1:10.sup.6.
The results from these pilot clones were used to suggest the
appropriate single dilution that would yield gradable results for
clones in the source plate for each round. Plates representing
cultured, randomly chosen clones from the 3rd, 4th, and 5th rounds
of biopanning were diluted to 1:100,000 from the "neat" solution
using 2YT in microtitration plates with a final volume per well of
250 .mu.L. From the last plate representing the desired dilution,
100 .mu.L was added to the plate containing protein G-bound
ACA-6501 and normal IgG prepared as described above. The incubation
of dilute phage with aPL antibody or control IgG was carried out
for 2 hours at 4.degree. C. on a flat rotator. After 9 washes with
TBS/Tween in an automated plate washer, the IgG-bound phage was
eluted with 20 .mu.L of 0.2 N HCl-glycine/0.1% BSA, pH 2.2. The
elution incubation continued for 10 minutes at RT, during which
time a new Corning microtitration plate was prepared containing 20
.mu.L of freshly starved E. coli per well and kept chilled. One
hundred forty .mu.L of 29 mM Tris was added to the plate containing
the phage eluates in order to neutralize the pH, following which 20
.mu.L of phage suspension was transferred from each well to the
corresponding well in the plate containing starved E. coli. After a
10 minute incubation at 37.degree. C., 200 .mu.L 2YT/dil Tet was
added and incubation carried out an additional 30 minutes at
37.degree. C. Using multi channel pipettors, 10 .mu.L from each
well was spotted on a large 2YT/Tet agar plate while retaining the
original 8.times.12 well pattern and orientation from the last
microtitration plate. After allowing the spots to dry for 30
minutes, the plate was incubated overnight at 37.degree. C. The
following day, colonies were semiquantitatively scored from 0 to
4+, with 0 symbolizing <10 colonies; +/-, 10-20; 1+, 20-50; 2+,
50-70% confluent; 3+, 70-90% confluent; and 4+ representing >90%
confluent colonies. Of the 94 clones that were examined at a
dilution of 1:10.sup.5 [representing 81 third, 6 fourth, and 7
fifth round clones], six clones had micropanning scores of zero,
three scored 1+, 14 scored 2+, 62 scored 3+, and 9 scored 4+. A
survey of random clones from the plates representing the second
through fifth rounds of biopanning was carried out by G-track DNA
sequencing as described below. The results showed a dramatic drop
in sequence diversity by the fourth round of biopanning (see FIG.
8), therefore, a second plate was micropanned using phage at a
dilution of 1:3.times.10.sup.5, this time including clones from an
earlier (second) round. A total of 94 clones were tested, including
29 which had scored high at 1.times.10.sup.5 dilution [26 from the
3rd round, 1 from the 4th found, and 2 from the 5th round] plus 65
clones from the 2nd round that had not been previously tested. Of
the 94 clones tested at a dilution of 1:3.times.10.sup.5, 26 scored
zero, 11 scored +/-, 10 scored 1+, 13 scored 2+, 34 scored 3+, and
zero scored 4+.
[0177] G-Track DNA Sequencing
[0178] Single-stranded viral DNA was isolated from cultures
incubated overnight at 37.degree. C. To prepare cultures, 2YT/Tet,
either as 2 mL in tubes or 250 .mu.L in microtitration plate
round-bottom wells, was inoculated with individual phage from
spread plates of previously grown cultures in microtitration
plates. The purification of phage by 20% PEG/2.5 M NaCl
precipitation of culture supernatants as well as the isolation or
release of virion DNA by phenol-chloroform extraction or by alkali
denaturation was performed as described in Smith, G. P. and J. K.
Scott, "Libraries of peptides and proteins displayed on filamentous
phage" (1993) Meth. Enzymol. 217:228-257 for cultures in tubes and
as described in Haas, S. J. and G. P. Smith G. P., "Rapid
sequencing of viral DNA from filamentous phage" (1993)
BioTechniques 15:422-431 for phage in microtitration plates. The
dideoxy nucleotide chain termination DNA sequencing technique of
Sanger et al. (Sanger et al., "DNA sequencing with chain
terminating inhibitors" (1970) Proc. Natl. Acad. Sci. 74:5463-5467)
was carried out using a commercial Sequenase kit (U.S.
Biochemical/Amersham, Arlington Heights, Ill.) as described by
Smith and Scott, supra, for tube culture phage DNA and as in Haas
and Smith, supra, for phage DNA from microtitration plates. By
using only the ddCTP termination mixture, G tracking or the
sequence pattern suggested by a single base (G) was obtained
following electrophoresis on 7% polyacrylamide/urea sequencing gels
and exposure by autoradiography. Standard gel electrophoresis and
autoradiography procedures were followed (Sambrook et al,
supra).
[0179] G-tracking of 76 clones from the micropanning plate tested
at a phage dilution of 1:1.times.10.sup.5 from the ACA-6501 library
screen revealed 11 unique sequences, while 64 clones from the
second micropanning plate tested at a phage dilution of
1:3.times.10.sup.5 showed 30 unique sequences. Conventional DNA
sequencing using all four dideoxynucleotide triphosphates was
applied to the phage clones with the highest micropanning scores
and unique sequences and resulted in the 12 sequences shown in
Table 1 for the xyz library.
[0180] Phage-Capture ELISA
[0181] Clones ACA-6635/3A12, 3B3, 3C8, 3A5, 3C9, and 3B7 were grown
as 3 mL cultures. Affinity purified ACA-6635 was diluted to 2.5
.mu.g/mL in phosphate-buffered saline, pH 7.2, and 100 .mu.L added
to Immulon-2 microtitration plate wells. After 2 hours, the plates
were washed 3 times with TBS/Tween in an automated plate washer
with no shaking. The plate was then blocked with 150 .mu.L 0.1% BSA
(globulin-free) in PBS per well. After 1 hour at 4.degree. C., the
plate was washed 3 times as previously described. After
centrifuging each phage culture 3 minutes at 17,000.times.g, each
supernatant was diluted 1:10 in 0.1% BSA/PBS and 100 .mu.L added to
each of the wells coated with affinity purified ACA-6635 and then
incubated for 2 hours at 4.degree. C. Plates were then washed with
TBS/Tween as before. Horseradish peroxidase-conjugated sheep IgG
anti-M13 phage antibody (Pharmacia, Inc., Piscataway, N.J.) was
diluted 1:5,000 in 0.1% BSA/PBS and 100 .mu.L applied to each well.
Following incubation for 1 hour at 4.degree. C., the plate was
washed 4 times as before. One hundred .mu.L of substrate prepared
according to the conjugate manufacturer's instructions was added to
each well. After 19 minutes, the absorbance at 405 nm of each well
was read in the automated microplate absorbance reader (Biotek,
Winooski, Vt.) The seven clones tested from the ACA-6635 phage
library screen were selected because of their high micropanning
scores and negative phage-ELISA scores (see below). As shown in
FIG. 9, three clones (3A12, 3B3, and 3A5) out of the seven tested
exhibited a very strong immunospecific signal in the phage-capture
ELISA.
[0182] Phage-ELISA
[0183] Three mL cultures were prepared from 35 clones previously
isolated with affinity purified ACA-6501 in several phage library
screens as well as one fUSE 2 phage clone lacking a peptide insert
which was used as a control. After centrifugation for 3 minutes at
17,000.times.g, 100 .mu.L from each phage supernatant (adjusted to
-2.times.10.sup.11 particles/mL, based on absorbance) was added to
microtitration plate wells (Falcon, Becton-Dickinson Labware,
Lincoln Park, N.Y.) and allowed to incubate overnight at 4.degree.
C. Following four washes with TBS 7.4, the plate was blocked with
125 .mu.L of 0.5% BSA/TBS for 1 hour at RT. After another four TBS
washes, 100 .mu.L of affACA-6501 previously diluted to 2.5 .mu.g/mL
in TBS/BSA was added to each well and allowed to incubate 1 hour at
37.degree. C. Following an additional four washes, 100 .mu.L
anti-human IgG diluted 1:1000 in TBS/0.5% Tween was added to each
well. After 1 hour at RT, enzyme substrate was added and the
incubation allowed to proceed for 2 hours. Following the addition
of 50 .mu.L 0.2 M Na.sub.2HPO.sub.4 to stop the reaction,
absorbance was measured at 550 nm in the automated plate absorbance
reader. As shown in FIG. 10, seven of the clones had significant
signals in the phage-ELISA with ACA-6501: 5A12, 3B6, 3E7, 3B10
(same sequence as 2D7), 2G11, 2H1, and 2H2. The sequences for these
clones are showvn in Table 2.
[0184] Peptide-ELISA
[0185] In the standard protocol, stock solutions of tetrameric
peptide in dimethylformamide were diluted 1000 times to 10 .mu.g/mL
in pH 9.5 carbonate buffer. Each microtitration plate well was
coated overnight at 4.degree. C. with 100 .mu.L of dilute peptide
followed by blocking with buffered albumin. Peptide-coated
microtitration plates were incubated 1-2 hours at room temperature
with aPL sera at several dilutions starting at 1:50. Following
washes, the presence of peptide-bound human IgG was determined with
enzyme-conjugated anti-human IgG according to standard ELISA
procedures.
[0186] Competitive Binding Peptide-ELISA
[0187] (A) Each well of an Immulon II plate (Dynatech Laboratories,
Inc., Chantilly Va.) was coated with 100 .mu.L of a solution
containing 10 .mu.g tetravalent peptide ACA 6501/3B10 in 50 mM
sodium carbonate, pH 9.5, containing 35 mM sodium bicarbonate
(Fisher Scientific, Pittsburgh, Pa., reagent grade) for at least 1
hour at RT, except for three wells used as blank controls. The
liquid was then removed from the wells and 200 .mu.L of 0.5%
(wt/vol) BSA (Sigma Chemical, St. Louis, Mo., #A7638) in TBS was
added per well including the blank wells for blocking and incubated
for at least 1 hour at RT. Four 1.5 mL microfuge tubes were
numbered 1 to 4. The following reagents were mixed in the first
microfuge tube (Brinkman Instruments, Westbury, N.Y.): 30 .mu.L of
5% BSA; 284 .mu.L TBS; 8 .mu.L of a stock solution of approximately
400-500 .mu.g/mL of monomeric peptide (ACA-5A12 or -CB2 or -3B10 or
scrambled -3B10 as negative control) in TBS; and 8.2 gL of 1:10
diluted serum 6501 in 0.5% BSA-TBS. The following reagents were
mixed in the second microfuge tube: 30 .mu.L of 5% BSA; 290 .mu.L
TBS; 2 .mu.L of a stock solution of approximately 400-500 .mu.g/mL
of monomeric peptide (ACA-5A12 or -CB2 or -3B10or scrambled -3B10
as negative control) in TBS; and 8.2 .mu.L of 1:10 diluted serum
6501 in 0.5% BSA-TBS. The following reagents were mixed in the
third microfuge tube: 30 .mu.L of 5% BSA, 287 .mu.L of a 1:10
dilution of approximately 400-500 .mu.g/mL of monomeric peptides
(5A12, CB2, 3B10, or scrambled sequence 3B10 control), and 8.2
.mu.L of ACA-6501 serum previously diluted 1:10 in 0.5% BSA-TBS.
The following reagents were mixed in the fourth Eppendorf microfuge
tube: 60 .mu.L 5% BSA; 584 .mu.L TBS and 16.5 .mu.L of 1:10 diluted
serum 6501 in 0.5% BSA-TBS. The blocked plate was washed 5 times
with TBS. The solution in the first microfuge tube was added to
triplicate wells, 100 .mu.L per well. Identical amounts of the
solutions in the second, third and fourth microfuge tubes were also
added to triplicate wells. An aliquot of 100 .mu.L of the solution
in the fourth microfuge tube was added to each of the three blocked
blank wells of the microtitration plate. The plate was incubated
for 1 hour at RT with agitation at 40 rpm in an orbital shaker
(American Dade, Miami, Fla., Rotator V) and then washed 5 times
with TBS. An aliquot of 100 .mu.L of 1:1000 diluted
goat-anti-human-IgG/alkaline phosphatase conjugate (Zymed, South
San Francisco, Calif., Cat. no. 62-8422) in 0.5% BSA-TBS was added
and incubated for 1 hour at RT. The plate was then washed 5 times
with TBS and the assay was developed by adding 100 .mu.L PPMP
diluted substrate solution as described in Example 1. After 20
minutes, the reaction was stopped by adding 50 .mu.L of 0.2 M
Na.sub.2HPO.sub.4 (Mallinckrodt, St. Louis, Mo., reagent grade) per
well. The optical density was read at 550 nm in a microplate reader
(Bio-Tek Instruments, Winooski, Vt., Model EL 311). The optical
density at 550 nm versus the amount of the peptide per well was
plotted in Graph Pad Prizm (Graph Pad Software, Inc., San Diego,
Calif.). The amount of peptide required for 50% inhibition of
binding of serum 6501 to tetravalent 3B10 was calculated from the
graph.
[0188] (B) Immulon I.RTM. 96-well, flat-bottom, polystyrene
microtitration plates (Dynatech Laboratories, Inc., Chantilly, Va.)
were coated with 30 .mu.L/well of cardiolipin (CL, 50 .mu.g; Sigma
Chemical Co., St. Louis, Mo.) in ethanol. Two control wells
received 30 .mu.L ethanol only. After overnight evaporation at
4.degree. C., each well was blocked with 200 .mu.L of 5% (w/v) fish
gelatin in PBS for 2 hours at RT. The CL-coated, blocked plate was
washed 5 times with TBS and then to each well was added .beta.2-GPI
as 100 .mu.L of 2.3% (v/v in PBS) IgG-depleted human serum (Sigma
Chemical Co., St. Louis, Mo.) and incubated 2 hours at RT.
[0189] During this incubation, variable amounts of each of six
peptides were mixed with 22 .mu.L ACA-6501 serum diluted with 3%
fish gelatin in 1:1 TBS/PBS (final dilution of 1:400) in a final
volume of 220 .mu.L using Eppendorf microcentrifuge tubes.
Specifically, in tube #1 were mixed 181.3 .mu.L of 3% fish gelatin
in TBS-PBS, 16.7 .mu.L of peptide stock solution plus 22 .mu.L of
ACA-6501 serum diluted 40 times in 3% fish gelatin/TBS-PBS. Stock
solutions ranged from 450-800 .mu.g/mL for peptides #951 (diserine
non-cyclized negative control), #952 (a lot of LJP 690), and
thioethers CCTE-3G3, CHTE-3G3, HCTE-3G3 and HHTE-3G3. To tube #2
the following were added: 148 .mu.L fish gelatin/TBS-PBS, 50 .mu.L
of peptide stock solution, plus 22 .mu.L of 40 times diluted
ACA-6501 serum. Tube #3 contained 48 .mu.L fish gelatin/TBS-PBS, 50
.mu.L of peptide stock solution, plus 22 .mu.L of 40 times diluted
ACA-6501 serum. The control tube #4 received 396 .mu.L fish
gelatin/TBS-PBS and 44 .mu.L of 40 times diluted ACA-6501 serum (no
peptide). Each of the tubes incubated for approximately 1 hour at
RT.
[0190] The CL/.beta.2-GPI microtitration plate was washed 5 times
with TBS and 100 .mu.L aliquots in duplicate from Eppendorf tubes
#1, 2, 3, and 4 containing the antibody-peptide (or no peptide
mixtures) were added to the wells. A volume of 100 .mu.L from tube
#4 was added to the duplicate control wells containing no
cardiolipin. The microtitration plate was incubated for 1 hour at
RT with agitation at 40 rpm in an orbital shaker (American
Scientific, Rotator V), washed 5 times with TBS and then 100 .mu.L
of 1:1000 goat anti-human IgG alkaline phosphatase conjugate
(Zymed, Cat No. 62-8422) in 0.5% (w/v) BSA-TBS was added. Following
incubation for 1 hour at RT, the microtitration plate was again
washed 5 times with TBS and the colorimetric enzyme detection
developed by adding 100 .mu.L of PMPP solution (7.8 g
phenolphthalein monophosphate plus 69.5 g of
2-amino-2-methyl-1-propanol in 100 mL water stock solution diluted
1:26 with water). After 21 minutes, the reaction was stopped by
adding 50 .mu.L of 0.2 M Na.sub.2HPO.sub.4 (Mallinckrodt) to each
well. Absorbance at 550 nm was read in a microplate reader (Bio-Tek
Instruments, Model EL 311). Absorbance vs. peptide added was
plotted on Graph Pad Prism (Graph Pad Software, Inc.) as shown in
FIG. 12. The amount of peptide that inhibited ACA-6501 binding by
50%, the IC.sub.50, was calculated from the graph at the
intersection of half-maximal absorbance with amount of peptide
added.
[0191] (C) Nunc Maxisorp.RTM. 96-well, flat-bottom microtitration
plates were coated with 100 .mu.L/well of purified human
.beta..sub.2-glycoprote- in I (PerImmune, Rockville, Md.) at 10
.mu.g/well in PBS. Two control wells received 100 .mu.L PBS only.
After two hours incubation at room temperature, the liquid was
removed. The coated plate was blocked for 2 hours at room
temperature with 200 .mu.L/well of PBS containing 2% (w/v) nonfat
dry milk (Carnation, Glendale, Calif.) and 0.4% (w/v) Tween 80
(Calbiochem, San Diego, Calif.). The blocking solution was also
used as a reagent diluent.
[0192] During the second hour of incubation, variable amounts of
each of four test peptides were mixed with 22 .mu.L of ACA-6501
serum diluted with sample diluent in a final volume of 220 .mu.L
using Eppendorf microcentrifuge tubes. Specifically, in tube #1,
188 .mu.L of sample diluent, 10 .mu.L of peptide stock solution (2
mg-4 mg/mL diluent), and 22 .mu.L of AC-6501 serum diluted 1:35 in
sample diluent were added. To tube #2, 158 .mu.L sample diluent, 40
.mu.L peptide stock solution and 22 .mu.L of 1:35 diluted ACA-6501
serum were added. Tube #3 contained 38 .mu.L sample diluent, 160
.mu.L peptide stock solution and 22 .mu.L of 1:35 diluted ACA-6501
serum. The control tube, tube #4, contained 396 .mu.L sample
diluent and 44 .mu.L of 1:35 diluted ACA-6501 serum (no peptide).
Each of the tubes was incubated for approximately 1 hours at room
temperature.
[0193] The .beta..sub.2-GPI microplate was washed 5 times with TBS
and 100 .mu.L of the mixtures contained in Eppendorf tubes #1, 2, 3
and 4 was added to duplicate wells of the microplate. 100 .mu.L of
the contents of tube #4 was added to each of the duplicate control
wells that were not coated with .beta..sub.2-GPI. The plate was
incubated for 1 hour at room temperature, washed 5 times with TBS
and 100 .mu.L of 1:1000 goat anti-human IgG/AP conjugate (Zymed,
Cat. No. 62-8422) diluted in sample diluent buffer was added.
Following incubation for 1 hour at room temperature, the plate was
again washed 5 times with TBS and the calorimetric enzyme detection
developed by adding 100 .mu.L PPMP solution (7.8 g phenolphthalein
monophosphate and 69.5 g 2-amino-2-methyl-1-propan- ol in 100 mL
water stock solution diluted 1:26 with water). After 22 minutes,
the reaction was stopped with 50 .mu.L of 0.2 M Na.sub.2HPO.sub.4
(Mallinckrodt) added per well. A.sub.550 nm was read in a
microplate reader (Bio-Tek Instruments, Model EL 311). Absorbance
vs. peptide added was plotted on Graph Pad Prism (Graph Pad
Software, Inc.) as shown in FIG. 21. The amount of peptide that
inhibited ACA-6501 binding by 50%, the IC-50, was calculated from
the graph at the intersection of half-maximal absorbance with the
amount of peptide added.
Example 5
[0194] Truncation Experiments of Peptide 3B10 and the Resulting
Peptide Competition ELISA Results
[0195] Microtitration plates (96-well, flat bottom polystyrene,
Immulon-2, Dynatech Laboratories, Inc., Chantilly, Va.) were coated
with 100 .mu.L/well for 1 hour at RT with tetrameric ACA-6501/3B10
peptide at 10 .mu.g/mL in carbonate buffer, pH 9.6 (15 mM
Na.sub.2CO.sub.3/35 mM NaHCO.sub.3). After the liquid from the
wells was removed, each well was blocked for 1 hour at RT with 200
.mu.L 0.5% (wt/vol) BSA (globulin-free, cat. no. A7638, Sigma
Chemical Co., St. Louis, Mo.) in TBS. Three wells on the plate were
left uncoated by tetravalent peptide to serve as blank control
wells.
[0196] During the blocking step, each soluble, monomer peptide to
be tested was set up in three test tubes (Eppendorf micro test
tubes, Brinkmann Instruments, Westbury, N.Y.) each containing 30
.mu.L 5% BSA/TBS, 8.2 .mu.L ACA-6501 serum (at 1:10 dilution with
0.5% BSA/TBS), plus a variable volume of the peptide/TBS stock and
the necessary volume of TBS buffer to yield a final volume of 330
.mu.L. The 330 .mu.L volume was sufficient to generate triplicate
100 .mu.L samples for each peptide concentration that was tested
for its ability to block ACA-6501 binding to the tetravalent
peptide-coated plate. For peptide 139, which was truncated at the
amino terminus and lacks the framework ala-gly contribution
normally tested, the concentration of the stock solution was
approximately 340-400 .mu.g/mL TBS and aliquots of 19 .mu.L, 75
.mu.L, and 292 .mu.L were removed to prepare the three peptide
concentration tubes. For peptide 142 (lacking only the N-terminal
ala) and peptide 143 (not truncated), stock solution concentrations
were 400-500 .mu.g/mL in TBS. Aliquots from each stock solution of
1 .mu.L, 4 .mu.L, and 16 .mu.L were removed to set up the three
concentration tubes for each peptide. For the peptide monomer
control tube (lacking peptide), a tube with a final volume of 660
.mu.L was prepared containing 60 .mu.L 5% BSA, 16.5 .mu.L of a 1:10
dilution of ACA-6501 and 583.5 .mu.L TBS, i.e., the same final
concentrations (0.5% BSA and ACA-6501 serum at 1:400) as the 330
.mu.L tubes but with twice the volume and without peptide.
[0197] Following the blocking incubation, the plate coated with
tetravalent peptide was washed 5 times with TBS. From each 330
.mu.L tube containing peptides 139, 142 and 143 at different
concentrations, 100 .mu.L was added to coated triplicate wells.
Three 100 .mu.L aliquots from the 660 .mu.L control tube without
peptide were each added to coated wells and the additional 100
.mu.L aliquots were each added to uncoated blank wells. The plate
was incubated at 40 rev/min on a rotary orbital shaker (Rotator V,
American Dade, Miami, Fla.) for 1 hour at RT and then washed 5
times with TBS. One hundred .mu.L/well of goat antihuman
IgG/alkaline phosphatase conjugate (Cat. no. 62-8422, Zymed, South
San Francisco, Calif.) diluted 1:1000 in BSA/TBS was added to the
microplate. After incubation for 1 hour at RT, the plate again was
washed 5 times with TBS. Color development followed the addition of
100 .mu.L/well of freshly prepared dilute PPMP substrate solution.
A dilute solution of PPMP (phenolphthalein monophosphate, cat. no.
P-5758, Sigma Chemical Co., St. Louis, Mo.) was prepared by making
a 1:26 dilution with water of the PPMP stock solution (0.13 M PPMP,
7.8 M amino-2-methyl-1-propanol adjusted to pH 10.15 with HCl).
After 30 minutes, the reaction was stopped by adding 50 .mu.L/well
of 0.2M Na.sub.2HPO.sub.3 (reagent grade, Mallinckrodt, St. Louis,
Mo.). Absorbance measurements at 550 nm were carried out on a
microplate reader (Bio-Tek Instruments, Winooski, Vt.) and the
A.sub.550 nm vs. peptide added per well results plotted using Graph
Pad Prizm (Graph Pad Software, Inc., San Diego, Calif.). As shown
on FIG. 11, a horizontal line has been drawn corresponding to
half-maximal the binding reaction obtained in the absence of
monomer peptide competitive inhibition. The amount of peptide
necessary for 50% inhibition can be read at the intersection of
this line with the plot for each peptide tested. The 50% inhibition
values are shown on FIG. 12. The results indicate that while the
loss of the N-terminal Ala had no negative consequences, the
additional loss of Gly increased by about 8-fold the concentration
necessary to achieve 50% inhibition.
Example 6
[0198] Substitution of Alpha Methyl Proline into 3B10 and the
Resulting ELISAs
[0199] Testing of peptides ACA-6501/3B10 and analogs in which
prolines at the 3 and 9 position were replaced by .alpha.-Me-Pro
was carried out using the methodology described in Example 5.
Peptides 3B10, 726 (.alpha.Me-Pro substituted at the 3 position),
727 (.alpha.Me-Pro substituted at the 9 position), and 728
(.alpha.Me-Pro substituted at both the 3 and 9 positions) were
tested as soluble monomer peptides in a competitive-binding ELISA
using tetramer peptide 3B10-coated microtitration plates and
ACA-6501 serum.
[0200] Microtitration plates were coated with tetramer 3B10 peptide
as described for Example 5. For each of the four peptides tested,
three peptide concentrations were prepared in tubes. As in Example
5, these twelve tubes had final concentrations of 0.5% BSA/TBS and
ACA-6501 serum at a final dilution of 1:400 in a final volume of
330 .mu.L. All peptide stock solutions were at 400-500 .mu.g/mL
TBS. To tubes containing 30 .mu.L of 5% BSA/TBS and 8.2 .mu.L of
ACA-6501 serum (diluted 1:10 with 0.5% BSA/TBS), aliquots of 1
.mu.L, 4 .mu.L, or 16 .mu.L of each of the four peptide stock
solutions were added in addition to an appropriate volume of TBS to
achieve a final volume of 330 .mu.L. A control tube with no
competitor peptide present was prepared with a final volume of 660
.mu.L as described in Example 5.
[0201] Following the blocking incubation of the tetravalent
peptide-coated plate, three 100 .mu.L aliquots from each of the
peptide concentration tubes for each of the four peptides as well
as the control tube containing no peptide and blank controls were
tested as described in Example 5. The microtitration plate ELISA
procedures as well as the data handling were performed as described
in Example 5. As shown in FIG. 13, peptide 727, where
.alpha.-Me-Pro was substituted at the 9 position, was significantly
more active than unmodified peptide 3B10 or the analog with both
prolines changed (peptide 728). Peptide 726, which was substituted
at position 3, lost activity as a result of the substitution.
Example 7
[0202] Abbreviated Description of Screen with 6626 Antibody and the
Corresponding Sequences
[0203] Affinity purified ACA-6626 (AffACA-6626) was isolated by
affinity purification from 8 mL of ACA-6626 plasma as previously
described. AffACA-6626 (10 .mu.g) was incubated with the epitope
xy'z phage library consisting of a pool of all p-III component
libraries in a final volume of 100 .mu.L as previously described
for ACA-6501 biopanning. Following three rounds of biopanning,
randomly selected phage from the second and third rounds were
tested by micropanning. Only a few clones were weakly
immunopositive at a 1:1000 dilution. An additional 4th round of
biopanning was carried out. Micropanning of 94 fourth round clones
revealed 43 immunopositives, some at phage dilutions as high as
1:100,000. G-Tracking DNA sequencing of the 43 immunopositive
clones carried out as previously described for ACA-6501 revealed 5
unique sequences. After conventional four base DNA sequencing, the
translated amino acid sequences of Table 3 were obtained.
Example 8
[0204] Identification of Sequences Specific for the ACA from
Patient 6644
[0205] The epi.sup.xy'z phage display library was screened using
methods similar to those in Example 4 with ACA affinity purified
antibody from patient number 6644. A colony blot assay as described
previously was employed as the final identification step prior to
peptide synthesis. Approximately 150 colonies were plated on the
original nitrocellulose membrane and assayed. Antibody from patient
6644 was used at a concentration of 1 .mu.g/mL. Of the 150 colonies
plated on the nitrocellular membrane and assayed, only 4 were
strongly positive and 2 weakly positive in this screen. Sequencing
of the inserts of the six positive phage selected by this screen
revealed that the inserts were all derived from the 8-mer library
with a free amino-terminus (epi.sup.z):
13 Gly-Ile-Leu-Ala-Leu-Asp-Tyr-Val-Gly-Gly (3 inserts) Gly-Ile
Leu-Thr-Ile-Asp-Asn-Leu-Gly-Gly (1 insert)
Gly-Ile-Leu-Leu-Asn-Glu-Phe-Ala-Gly-Gly (2 inserts)
Example 9
[0206] Summary of Phage Library Screen with ACA-6641.
[0207] AffACA-6641 was isolated from 4 mL of plasma taken from
patient number 6641. AffACA-6641 (10 .mu.g) was incubated with the
pooled p-III phage libraries in a final volume of 100 .mu.L as
described previously. Following four rounds of biopanning, 45
clones from the 3rd and 4th rounds were tested by micropanning. Of
the 45, 23 scored negative. The 3rd round phage yielded two clones
that scored 4+, two that scored 3+ and two that scored 2+. From the
4th round, one clone scored 4+, one scored 3+ and three scored 2+.
G-tracking DNA sequencing revealed six unique sequences. Only one,
clone 3G3, was strongly positive in the phage-capture ELISA. Four
base DNA sequencing gave the following translated peptide
sequence:
C L G V L G K L C.
Example 10
[0208] Peptide Conjugation to Non-Immunogenic, Multivalent
Carriers
[0209] Several tetravalent platforms for the development of B cell
tolerogens have been developed as described in co-owned and
co-pending U.S. patent applications, Ser. Nos., 08/118,055, filed
Sep. 8, 1993, 08/152,506, filed Nov. 15, 1993, and U.S. Pat. Nos.
5,268,454, 5,276,013 and 5,162,515 which are incorporated by
reference herein in their entirety. Candidate peptides selected by
aPL antibody screens of epitope libraries are conjugated and tested
for modifications of immunochemical behavior such as antibody
binding.
[0210] Non-immunogenic multivalent platforms with amine groups are
synthesized as shown in the following scheme. 5
[0211] Compound 2: A solution of 8.0 g (5.7 mmol) of 1 in 50 mL of
absolute EtOH and 35 mL of cyclohexene was placed under nitrogen,
and 500 mg of 10% Pd on carbon was added. The mixture was refluxed
with stirring for two hours. When cool, the mixture was filtered
through Celite and concentrated to give 5.0 g of 2 as an oil.
.sup.1H NMR (50/50 CDCl.sub.3/CD.sub.3OD) d 1.21 (m, 8H), 1.49 (m,
8H), 1.62 (m, 8H), 2.19 (t, J=7.4 Hz, 8H), 2.67 (t, J=7.4 Hz, 8H),
3.36 (bd s, 16H), 3.67 (s, 4H), 3.71 (m, 4H), 4.21 (m, 4H).
[0212] Protected Peptide with Free Carboxyl
(PHN-Peptide-CO.sub.2H
[0213] A peptide is synthesized with standard solid phase methods
using FMOC chemistry on a Wang (p-alkoxybenzyl) resin, using
trifluoroacetic acid (TFA) stable protecting groups (benzyl ester
or cyclohexyl ester on carboxyl groups and carbobenzyloxy (CBZ) on
amino groups). Amino acid residues are added sequentially to the
amino terminus. The peptide is removed from the resin with TFA to
provide a peptide with one free carboxyl group at the carboxy
terminus and all the other carboxyls and amines blocked. The
protected peptide is purified by reverse phase HPLC.
[0214] Peptide--Platform Conjugate, 4
[0215] The protected peptide (0.3 mmol) is dissolved in 1 mL of
dimethylformamide (DMF), and to the solution is added 0.3 mmol of
diisopropylcarbodiimide and 0.3 mmol of 1-hydroxybenzotriazole
hydrate (HOBT). The solution is added to a solution of 0.025 mmol
tetraamino platform, 2 in 1 mL of DMF. When complete, the DMF is
removed under vacuum to yield a crude fully protected conjugate 3.
The conjugate, 3, is treated with hydrofluoric acid (HF) in the
presence of anisole for 1 hour at 0.degree. to give conjugate 4.
Purification is accomplished by preparative reverse phase HPLC.
[0216] The following scheme shows the attachment of an amino group
of a peptide to a carboxy group on a platform. 6
[0217] Compound 5--Platform with Four Carboxylic Acid Groups
[0218] Succinic anhydride (1.0 g, 10 mmol) is added to a solution
of 861 mg (1.0 mmol) of 2 and 252 mg (3.0 mmol) of NaHCO.sub.3 in
20 mL of 1/1 dioxane/H.sub.2O, and the mixture is stirred for 16
hours at RT. The mixture is acidified with 1N HCl and concentrated.
The concentrate is purified by silica gel chromatography to provide
5.
[0219] Protected Peptide with Free Amine
(H.sub.2N-Peptide-CONH.sub.2)
[0220] A peptide is synthesized with standard solid phase methods
on an amide resin, which resulted in a carboxy terminal amide after
cleavage from the resin, using TFA stable protecting groups (benzyl
ester or cyclohexyl ester on carboxyl groups and CBZ on amino
groups). Amino acid residues are added sequentially to the amino
terminus using standard FMOC chemistry. The peptide is removed from
the resin with trifluoroacetic acid to provide a protected peptide
with a free amine linker. The protected peptide is purified by
reverse phase HPLC.
[0221] Peptide--Platform Conjugate, 7
[0222] A solution of 0.05 mmol of protected peptide with free
amine, (H.sub.2N-peptide-CONH.sub.2), 0.1 mmol of diisopropylethyl
amine, and 0.01 mmol of 5 in 1 mL of DMF is prepared. BOP reagent
(benzotriazol-1-yloxy-tris(dimethylamino)phosphonium
hexafluorophosphate) (0.1 mmol) is added, and the mixture is
stirred until the reaction is complete as evidenced by analytical
HPLC. The peptide protecting groups are removed by treatment with
HF in the presence of anisole at 0.degree. to give conjugate with
protecting groups removed, 7. Compound 7 is purified by preparative
reverse phase HPLC.
[0223] The following scheme shows how to attach a sulfhydryl linker
to the amino terminus of a peptide and, in turn, attach the
peptide/linker to a tetrabromoacetylated platform to give compound
13. 7
[0224] Compound 9
[0225] Concentrated sulfuric acid (100 uL) was added to a
60.degree. C. solution of 4.48 g (17.2 mmol) of triphenyl methanol
and 1.62 g (15.3 mmol, 1.3 mL) of 3-mercaptopropionic acid in 35 mL
of EtOAc. The mixture was stirred at 60.degree. C. for 10 minutes,
allowed to cool to room temperature (RT), and placed on ice for 1
hour. The resulting white solid was collected by filtration to give
4.52 g (75%) of 9.
[0226] Compound 10
[0227] Dicyclohexyl carbodiimide (DCC) (2.41 g, 11.7 mmol) was
added to a 0.degree. C. solution of 2.72 g (7.8 mmol) of 9 and 1.08
g (7.8 mmol) of p-nitrophenol in 41 mL of CH.sub.2Cl.sub.2. The
mixture was stirred for 16 hours allowing it to come to RT. The
mixture was filtered to remove N,N-dicyclohexylurea (DCU), and the
filtrate was concentrated. The residue was crystallized from
hexane/CH.sub.2Cl.sub.2 to give 3.17 g (86%) of 10 as pale yellow
crystals.
[0228] Compound 11--Cyclic Thioether Peptide with Mercaptopropionyl
Linker Attached
[0229] A solution of a cyclic thioether peptide (an analogue of a
disulfide cyclized peptide in which one sulfur was replaced with a
CH.sub.2) and sodium bicarbonate in water and dioxane was treated
with p-nitrophenyl ester 10. If the peptide contains lysine, it
must be appropriately blocked. The resulting modified peptide was
treated with trifluoroacetic acid to provide thiol linker modified
peptide 11.
[0230] Compound 13--Conjugate of Cyclic Thioether Peptide and
Bromoacetylated Platform
[0231] To a He sparged solution of 0.10 mmol of thiol modified
peptide 11 in 100 mM sodium borate pH 9, was added 0.025 of
bromoacetylated platform 12 as a 40 mg/mL solution in 9/1
MeOH/H.sub.2O). The solution was allowed to stir under N.sub.2
atmosphere until conjugation was complete as evidenced by HPLC. The
conjugate was purified by reverse phase HPLC.
Example 12
[0232] Synthesis of LJP 685 8
[0233] .gamma.-bromo-N-BOC-.alpha.-aminobutryic acid t-butyl ester,
compound 14: A solution of 4.03 g (13.3 mmol) of N-BOC-glutamic
acid-.alpha.-t-butyl ester and 1.61 mL (1.48 g, 14.6 mmol) of
N-methylmorpholine in 40 mL of dry THF under N.sub.2 atmosphere was
cooled to 15.degree.. Isobutylchloroformate (1.73 mL, 1.82 g, 13.3
mmol) was added to the mixture dropwise. The mixture was stirred
for 10 minutes and a solution of 2.03 g (16.0 mmol) of
N-hydroxy-2-mercaptopyridine in 8 mL of THF was added followed by
2.23 mL (1.62 g, 16.0 mmol) of Et.sub.3N. The mixture was covered
with foil to keep out light and allowed to stir at room temperature
for 1 hour. The mixture was filtered and the filtrate was
concentrated on the rotary evaporator taking care to minimize
exposure to light. The concentrate was dissolved in 20 mL of
BrCCl.sub.3 and the solution was cooled to -70.degree. C. The solid
was placed under vacuum, then purged with N.sub.2, allowed to come
to room temperature, placed in a 20.degree. water bath, and
irradiated from above at close range with a 500 W sunlamp for 5
minutes. The mixture was concentrated on the rotary evaporator and
purified by silica gel chromatography (40 mm.times.150 mm, toluene
was used as eluent until UV active material finished eluting, 2%
EtOAc/toluene (500 mL), 5% EtOAc/toluene (500 mL). Impure fractions
were repurified. Pure fractions by TLC (R.sub.f 0.23, 5%
EtOAc/toluene) were combined and concentrated to give 3.23 g (72%)
of compound 14 as a waxy solid.
[0234] N-BOC-glycylproline-4-nitrophenyl ester, compound 15: A
solution of 3.0 g (11.6 mmol) of N-BOC-glycylproline and 1.93 g
(13.9 mmol) of 4-nitrophenol in 82 mL of dry THF was cooled to
0.degree. C. and 3.34 g (16.2 mmol) of DCC was added. The mixture
was stirred at 0.degree. C. for 1 hour, the ice bath was removed,
and the mixture was stirred for 16 hours at room temperature. HOAc
(579 .mu.L) was added to the mixture and it was allowed to stir for
30 minutes. The mixture was kept in the freezer for 30 minutes and
filtered under vacuum. The filtrate was concentrated and purified
by silica gel chromatography (18.times.150 mm bed, 2.5% EtOAc/97.5%
CH.sub.2Cl.sub.2/1% HOAc). Traces of acetic acid were removed by
concentrating from dioxane several times on the rotary evaporator.
The concentrate was triturated with 3/1 hexane Et.sub.2O and the
resulting white solid was collected by filtration to give 4.1 g
(90%) of compound 15 as a white solid; TLC R.sub.f 0.09, 40/60/1
EtOAc/hexane/HOAc; .sup.1H NMR(CDCl.sub.3) .delta.1.09-2.55 (m,
4H), 1.48 (s, 9H), 3.47-3.77 (m, 2H), 4.05 (m, 2H), 4.74 (m, 1H),
5.45 (bd s, 1H), 7.35 (d, 2H), 8.30 (d, 2H).
[0235] N-FMOC-S-t-butylthiocysteineamide, compound 16: A solution
of 5.0 g (11.6 mmol) of FMOC-S-t-butylthiocysteine and 1.33 g (11.6
mmol) of N-hydroxysuccinimide in 115 mL of THF was cooled to
0.degree. C. To the solution was added 3.58 g (17.37 mmol) of DCC.
The mixture was stirred at 0.degree. C. for 1 hour and 42.9 mL of a
solution of 1.6 g of (NH.sub.4)HCO.sub.3 in 50 mL of water was
added. The mixture was stirred for 4.5 hours, allowing the ice bath
to gradually warm to room temperature and concentrated on a rotary
evaporator to remove THF and give an aqueous phase with white
solid. The mixture was stirred with 200 mL of CH.sub.2Cl.sub.2
until most of the solid dissolved, then was shaken with 100 mL of
1N HCl solution. The CH.sub.2Cl.sub.2 layer was washed with 100 mL
of saturated NaHCO.sub.3 solution, dried (Na.sub.2SO.sub.4), and
filtered. The filtrate was brought to a boil on a hot plate and
crystallized from 300 mL of CH.sub.2Cl.sub.2/hexane to give 4.36 g
(87%) of compound 16 as a white solid: mp 127.degree.-129.degree.
C.; TLC R.sub.f 0.29, 95/5/1 CH.sub.2Cl.sub.2/CH.sub.3CN/MeOH,
.sup.1H NMR (CDCl.sub.3) .delta.1.36 (s, 9H), 3.06-3.24 (m, 2H),
4.25 (t, 1H), 4.55 (m,3H),5.56 (bd s, 1H), 5.70 (bd s, 1H), 6.23
(bd s, 1H); .sup.13C NMR (CDCl.sub.3) 29.8, 41.9, 47.1, 48.5, 54.2,
67.2, 120.0, 125.0, 127.1, 127.8, 141.3, 143.6, 156.1, 172.2.
Analysis, Calculated: C, 61.4%; H, 6.1%; N, 6.5%. Found: C, 61.5%;
H, 6.0%; N, 6.5%.
[0236] Compound 17: Water (16.9 mL) was added to 2.91 g (6.75 mmol)
of compound 16 in 33.8 mL of dioxane and the solution was sparged
with nitrogen for 5-10 minutes. The mixture was kept under a
nitrogen atmosphere and 1.13 g (13.5 mmol) of NaHCO.sub.3 was added
followed by 1.77 mL (1.44 g, 7.09 mmol) of PBu.sub.3. The mixture
was stirred at room temperature for 1 hour and partitioned between
1.times.100 mL of 1N HCl and 2.times.100 mnL of CH.sub.2Cl.sub.2.
The CH.sub.2Cl.sub.2 layers were combined and concentrated and the
resulting white solid was partially dissolved in 33.8 mL of
dioxane. Water (9 mL) was added and the mixture was purged with
nitrogen for 5-10 minutes. The mixture was kept under nitrogen
atmosphere and 2.17 g (16.9 mmol) of K.sub.2CO.sub.3 was added
followed by 2.63 g (8.1 mmol) of compound 14. The mixture was
stirred for 16 hours and partitioned between 1.times.100 mL of 1N
HCl and 2.times.100 mL of 10% MeOH/CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layers were combined, dried with NaSO.sub.4,
filtered and concentrated to a semisolid residue. Purification by
silica gel chromatography (1/3 CH.sub.3CN/CH.sub.2Cl.sub.2) gave
3.2 g (80%) of compound 17 as a white solid; TLC R.sub.f 0.27, 1/3
CH.sub.3CN/CH.sub.2Cl.sub.2. Further purification of an analytical
sample was done by recrystallizing from hexane/EtOAc; mp
104-106.5.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta.1.47 (s, 9H),
1.49 (s, 9H), 1.96 (m, 1H), 2.11 (m, 1H), 2.69 (m, 2H), 2.88 (m,
1H), 3.03 (m, 1H), 4.29 (t, 1H), 4.36 (m, 2H), 4.50 (m, 2H), 5.21
(m, 1H), 5.49 (m, 1H), 5.81 (m, 1H), 6.54 (m, 1H), 7.34 (t, 2H),
7.42 (t, 2H), 7.61 (d, 2H), 7.80 (d, 2H); .sup.13C NMR (MeOH)
.delta.26.1, 26.7, 28.2, 28.7, 34.8, 54.8, 55.7, 68.1, 80.5, 82.7,
120.9, 126.3, 128.2, 128.8, 142.6, 145.2, 160.0, 162.7, 173.4,
175.8. Analysis, Calculated for C.sub.31H.sub.41N.sub.3O.sub.7S: C,
62.08; H, 6.89; N, 7.01. Found: C, 62.23; H, 7.12; N, 7.39.
[0237] Compound 18: A solution of 20/1/1
TFA/H.sub.2O/mercaptoethanol (23.2 mL) was added to 1.27 g (2.11
mmol) of compound 17. The mixture was stirred at room temperature
for 1 hour and concentrated to a volume of about 3 mL. 50 mL of
ether was added to precipitate the product. The resulting solid was
washed with two more portions of ether and dried under vacuum to
give 812 mg of solid. The solid was suspended in 10.6 mL of dioxane
and 10.6 mL of H.sub.2O. NaHCO.sub.3 (355 mg, 4.22 mmol) was added
to the mixture followed by a solution of 1.33 g (3.38 mmol) of
compound 15 in 10.5 mL of dioxane. The mixture was allowed to stir
at room temperature for 20 hours and partitioned between 100 mL of
1N HCl and 3.times.100 mL of CH.sub.2Cl.sub.2. The combined
CH.sub.2Cl.sub.2 layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated. Purification by silica gel chromatography
(35.times.150 mm; step gradient, 5/95/1 MeOH/CH.sub.2CL.sub.2/HOAc
(1 L) to 10/95/1). Pure fractions were concentrated and the residue
was triturated with ether to give 881 mg (60%) of compound 18; TLC
R.sub.f 0.59, 10/90/1 MeOH/CH.sub.2Cl.sub.2/HOAc; mp
116-117.5.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta.1.41 (s, 9H),
2.00 (m, 2H), 2.18 (m, 2H), 2.56 (m, 1H), 2.69 (m, 1H), 2.85 (m,
2H), 3.49 (m, 2H), 3.62 (m, 2H), 3.85 (m, 2H), 4.08 (m, 1H), 4.12
(m, 1H), 4.22 (t, 1H), 4.38 (m, 1H), 4.49 (m, 2H), 4.61 (m, 2H),
7.78 (d, bd s, 1H), 6.12 (bd s, 1H), 6.43 (bd s, 1H), 7.35 (d, 2H),
7.40 (d, 2H), 7.61 (d, 2H), 7.78 (d, 2h); .sup.13C NMR (CDCL.sub.3)
.delta.25.3, 27.8, 28.6, 29.4, 32.6, 35.1, 44.1, 46.9, 47.3, 52.2,
53.9, 61.2, 67.8, 80.8, 120.8, 125.7, 128.2, 129.0, 142.0, 144.5,
157.6, 157.8, 170.6, 181.2, 182.9, 183.1. Analytical, Calculated
for C.sub.34H.sub.43N.sub.5O.sub.9S: C,58.52; H, 6.21; N, 10.03.
Found: C, 58.38, H, 6.17; N, 10.20. 9
[0238] N-FMOC-L-Alanyl-L-2-methylproline, compound 19: A solution
of 2-methylproline (Seebach et al. (1983) J. Am. Chem. Soc.
105:5390-5398) (1.00 g, 4.76 mmol), 4.00 g (47.6 mmol) of
NaHCO.sub.3, and 31 mg (0.23 mmol) of HOBT in 6.9 mL of DMF was
cooled to 0.degree. C. and 3.18 g (6.66 mmol) of N-FMOC-L-alanine
was added. The reaction was stirred for 1 hour at 0.degree. C.,
then at room temperature for 18 hours. The mixture was partitioned
between 50 mL of EtOAc and 3.times.50 mL of 1N HCl. The EtOAc layer
was dried (MgSO.sub.4), filtered and concentrated. Purification by
silica gel chromatography (step gradient 45/55/1 EtOAc/Hexane/HOAc
to 47/53/1 EtOAc/Hexane/HOAc to 50/50/1 EtOAc/Hexane/HOAc) gave
1.72 g (86%) of compound 19 as a white solid. The product was
concentrated several times from dioxane to remove traces of acetic
acid: mp 59-60.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta.1.39(d,
3H), 1.93 (m, 2H), 2.06 (m, 2H), 3.78 (m, 2H), 4.22 (m, 1H), 4.40
(d, 2H), 4.56 (m, 1H), 5.09 (t, 1H), 5.69 (d, 1H), 7.32 (t, 2H),
7.42 (t, 2H), 7.62 (d, 2H), 77.8 (d, 2H); .sup.13C NMR (CDCl.sub.3)
.delta.17.8, 21.8, 23.8, 37.9, 47.1, 48.5, 65.9, 67.0, 120.0,
125.1, 127.1, 127.7, 141.3, 144.1, 155.6, 172.9, 175.1.
[0239] N-FMOC-L-Leucinyl-HMPB-MBHA resin: A solution of
N-FMOC-L-leucine in 22.5 mL of CH.sub.2Cl.sub.2 and a few drops of
DMF was prepared and cooled to 0.degree. C. To the solution was
added 1.71 mL (1.38 g, 10.9 mmol) of diisopropylcarbodiimide (DIC)
and the mixture was stirred for 20 minutes at 0.degree. C. The
mixture was concentrated to an oil; meanwhile, enough DMF
(approximately 3 mL) was added to 2.5 g (0.87 mmol/g, 2.18 mmol)
HMPB-MBHA resin (Nova Biochem) to swell the resin. The concentrated
oil was dissolved in a minimal amount of DMF (approximately 1 mL)
and added to the swelled resin followed by a solution of 266 mg
(2.18 mmol) of DMAP dissolved in approximately 1 mL of DMF. The
mixture was gently rocked for 1 hour and washed (2.times.DMF,
2.times.MeOH, 2.times.DMF, 2.times.MeOH). The resin was dried under
vacuum to give 2.77 g (85%) and the substitution was determined by
the Geisen test to be 0.540 mmol/g.
[0240] N-FMOC-linear peptide with t-butyl ester on aspartic acid
and Pmc group on arginine and with thioether insert. compound 20:
This peptide was prepared by standard FMOC synthesis on
N-FMOC-L-leucinyl-HMPB-MBHA resin. Three equivalents of amino acid,
HOBT and (DIC) were used for each coupling step with the exception
of the coupling step of compound 18. Two equivalents of compound 18
were used with three equivalents of HOBT and
diisopropylcarbodiimide. Each step was monitored by using 10 .mu.L
of bromophenol blue indicator. Completeness of the reaction was
also assessed with a ninhydrin test (beads turn blue for incomplete
reaction with 1 mg is heated at 100.degree. C. for 2 minutes with
one drop of pyridine and one drop of 5% ninhydrin in EtOH and one
drop of 80% phenol in EtOH). Thus, 1.13 mg (0.613 mmol) of resin
was used to prepare the peptide. After the final coupling step,
cleavage from the resin was accomplished by treatment with 15 mL of
a solution of 1% trifluoroacetic acid in CH.sub.2Cl.sub.2 for 2
minutes. After 2 minutes, the solution was filtered under pressure
into 30 mL of 10% pyridine in MeOH. This was repeated ten times and
the filtrates which contained peptide as evidenced by HPLC (C18,
gradient, 60/40/0.1 CH.sub.3CH/H.sub.2O/TFA to 90/10/0.1
CH.sub.3CN/H.sub.2O/TFA, 210 mn, 1 mL/min, 4.6 mm.times.250 mm
column) were combined and concentrated. The concentrate was
dissolved in 40 mL of 10% HOAc solution and purified by HPLC to
give 0.528 g (49%) of peptide 20.
[0241] Conversion of peptide 20 to cyclic peptide 21 (removal of
FMOC group, cyclization and removal of protecting groups: A
solution (5 mL) of 99 .mu.L of DBU in 10 mL of CH.sub.3CN was added
to 96 mg (0.052 mmol) of peptide 20. The solution was stirred for 1
hour and concentrated to a residue. The residue was triturated with
2.times.10 mL of Et.sub.2O to give a white solid. The solid was
dissolved in 100 mL of CH.sub.3CN and 312 .mu.L (0.312 mmol) of 0.1
M solution of diphenylphosphorylazide (DPPA) in CH.sub.3CN. The
mixture was stirred for 20 hours and concentrated on a rotary
evaporator. The residue was triturated with 2.times.50 mL of
Et.sub.2O and the white opaque residue was treated with 5 mL of
92/3/2/3 TFA/anisole/EDT/Me.sub.2S for 1 hour. The product was
precipitated by adding the mixture to 40 mL of Et.sub.2O in a 50 mL
polypropylene centrifuge tube. The precipitate was cooled to
0.degree. C. and centrifuged for 5 minutes at 2000 rpm. The
supernatant was decanted and the pellet was washed with Et.sub.2O
and recentrifuged. The pellet was dried and dissolved in 4 mL of
50/50 CH.sub.3CN/H.sub.2O. The mixture was diluted with 36 mL of
H.sub.2O/0.1% TFA, filtered and purified by HPLC (1" C.sub.18
column, gradient, 10/90/0/1 CH.sub.3CN/H.sub.2O/TFA to 35/65/0.1
CH.sub.3CN/H.sub.2O/TFA, 230 nm). The pure fractions, as evidenced
by HPLC, were lyophilized to give 52 mg (90%) of compound 21.
Example 13
[0242] Synthesis of Conjugates of LJP 685 10
[0243] LJP 685, also referred to as compound 21, was treated with
the PNP ester of 3-tritylmercaptopropionic acid and the resulting
product was detritylated to give compound 22, the peptide with the
free thiol linker. Reaction of an excess of compound 22 with
valency platform molecule 12 produced tetravalent conjugate 25.
Treatment of compound 21 with the longer linker, compound 33 (see
reaction scheme below), followed by detritylation, gave compound
24. Compound 24 reacted with valency platform molecule 12 to give
the tetraconjugate 26. Both conjugation reactions appeared very
clean by HPLC.
[0244] Attachment of MTU linker to LJP 685, synthesis of compound
24: To 15 mg (0.013 mmol) of compound 21 was added 160 .mu.L of a
solution of 29.5 mg of compound 23 and 17.5 .mu.L of
diisopropylethylamine in 0.5 mL of DMF. the mixture was stirred for
2 hours and precipitated from Et.sub.2O. The precipitate was dried
and dissolved in 650 .mu.L of a solution of 1/1/0.056/0.040
TFA/CH2Cl2/thiophenol/Me.sub.2S and the solution was allowed to
stand for 1 hour. Precipitation from Et.sub.2O gave crude compound
24 which was purified by HPLC (C.sub.18, 15-45%
CH.sub.3CN/H.sub.2O, 0.1% TFA). Fractions containing pure product
were lyophilized to give 8.6 mg of compound 24 as a white
solid.
[0245] LJP 685-MTU-AHAB-TEG, compound 26: To a solution of 8.6 mg
(6.3.times.10.sup.-6 mol) of compound 24 in 630 .mu.L of He sparged
pH 8.5 200 mM borate buffer was 40 .mu.L of a 40 mg/mL solution of
compound 12 in 9/1 MeOH/H.sub.2O. The mixture was stirred for 24
hours and 1 mL of 10% HOAc/H.sub.2O solution was added. The mixture
was purified by HPLC (C.sub.18, gradient 25-55%
CH.sub.3CN/H.sub.2O, 0.1% TFA) to give 8.3 mg of compound 26 (LJP
685-MTU-AHAB-TEG).
Example 14
[0246] Development of Extended SH Linkers
[0247] The thiobenzoate ester, compound 28, was prepared from
compound 27. Compound 28 was converted to compound 29 in portions.
The thiobenzoate was removed by ethanolysis and the resulting thiol
was tritylated. The ethyl ester was then hydrolyzed to give
compound 29. The nitrophenyl phosphate (PNP) ester, compound 30,
was prepared from compound 29. Aminotrioxoudecanoicacid ethylester,
compound 31, was prepared by treatment of compound 27 with sodium
azide and reduction to the amine. Amine 31 was acylated with
compound 30 to provide compound 32. Hydrolysis of compound 32 was
achieved by treatment with sodium hydroxide to give a free
carboxylic acid. The intermediate carboxylic acid was condensed
with p-nitropheniol to give para-nitrophenyl (PNP) ester, compound
33. Linker 33 was attached to the peptide and the trityl group
removed to give compound 34 which was used to produce an
MTU-ATU-AHAB-TEG conjugate. 11
Example 15
[0248] Synthesis of a (LJP685).sub.4/MTU-ATU-AHAB-TEG Conjugate,
Compound 35
[0249] Tetravalent conjugate 35 was prepared as shown below. The
peptide with linkers attached, compound 34 was dissolved in He
sparged, pH 8.5, 200 mM borate buffer. To the mixture was added 0.3
mol equivalents of platform compound 12. The mixture was stirred
for 1 hour and the product was purified by HPLC. 12
Example 16
[0250] Synthesis of a (LJP685).sub.4/DABA-PEG Conjugate, Compound
36
[0251] Treatment of IA-DABA-PEG with compound 24 in 8.5 borate
buffer gave conjugate 36. 13
Example 17
[0252] T Cell Assay for Activation
[0253] Tritiated thymidine uptake by peptide-stimulated T cells was
monitored in 96-well round bottom plates. A single-cell suspension
of draining lymph node cells (mice) or isolated peripheral blood
lymphocytes (human), 5.times.10.sup.5 were mixed with between 1 and
30 .mu.g of peptide in a final volume of 150 .mu.L per well and
incubated for 5 days at 37.degree. C. in 5% CO.sub.2. At that
point, 1 micro curie of labeled thymidine was added and incubated
for an additional 15-24 hours. The harvested cells were collected
on filters and counted by liquid scintillation spectrometry.
Example 18
[0254] In vitro Induction of Tolerance
[0255] Eight groups, each containing five C57B1/6 mice, were primed
with 10 .mu.g/mouse of a conjugate of LJP685-KLH on alum plus B.
pertussis vaccine as an adjuvant. After three weeks, spleen were
harvested and single cells suspensions were prepared, washed three
times with balanced salt solution and resuspended in complete
RPMI-1640 medium at a concentration equivalent to one spleen/1.5 mL
of medium. The cell suspension was divided into aliquots of 2.5
mL/petri dish and incubated for 2 hours at 37.degree. C. with
(LJP685).sub.4-DABA-PEG, compound 36, and (LJP685).sub.4-TEG,
compound 35, in concentrations of 100 .mu.M, 20 .mu.M and 4 .mu.M.
One group of cells was incubated without toleragen and acted as the
positive control. The cells were then washed with large volumes of
balanced salt solution and resuspended in 2.5 mL of balanced salt
solution. The cells were then injected into 650R irradiated
syngeneic recipient mice in such a manner that all of the cells
from a given treatment group were divided evenly into five
recipients. All of the recipient mice, including the positive
controls, were then given a booster immunization of 10 .mu.g of LJP
685-KLH in saline, intraperitoneally. Seven days after the booster
immunization, the mice were bled and their sera tested for the
presence of anti-LJP 685 antibody. Treatment with either conjugate
produced a significant, dose-dependent reduction of anti-LJP 685
antibodies as shown in FIGS. 16 and 17, which is measured by
Antigen Binding Capacity (ABC) as described in Iverson, G. M.,
"Assay for in vivo adoptive immune responses," in HANDBOOK OF
EXPERIMENTAL IMMUNOLOGY, Volume 2, Cellular Immunology (Weir, D.
M., ed., Blackwell Scientific Publications, Palo Alto, Calif.,
1986).
Example 19
[0256] NMR Solution Structure Analysis of the 5A12, CB2 and 3G3
Peptides
[0257] Two peptides isolated from phage library screens using the
methodology described in the examples above were subjected to NMR
analysis. The original peptide has a proline in the second to last
position, however, this amino acid was removed since two
dimensional (2D) double quantum filtered correlated spectroscopy
(DQF-COSY) NMR data suggested the presence of two different
structures from the cis and trans isomers at this position. Removal
of the proline gave peptides with K.sub.D'S in the 50-100 nM range
for binding to ACA antibodies, and the expected number of peaks in
the fingerprint region of the DQF-COSY spectrum. The resulting
cyclic peptides are 5A12 (GPCLILAPDRCG) AND CB2 (GPCILLARDRCG). The
major difference between the peptides was the substitution of
arginine for proline at position 8 which resulted in much less
dispersion in the 1D 1H NMR spectrum of CB2 which was consistent
with 5A12 being a more rigid peptide. The arginine substitution
also produces a 0.55 kcal/mol stabilization of the ionized aspartyl
carboxy group as reflected in pKa values. A structural analysis was
carried out on the more ordered 5A12 peptide. Although deuterium
exchange and temperature coefficient data show no evidence of
hydrogen bonding, the Nuclear Overhauser Effect (NOE), Rotating
Overhauser Effect (ROE) and coupling data are consistent with one
structure. Distance geometry calculations yielded a family of 50
structures. The 15 best had a mean root mean square deviation
(RMSD) for all atoms of 2.10.2 angstroms. We determined that the
peptide has an oval shape with turns at opposite ends of the
molecule, at the disulfide and at Proline 8 which is cis. There is
also a kink in the backbone LIL region. Finally, the carboxy
terminal glycine is very mobile as it is the only residue with a
positive intraresidue NOE. This represents the first structural
information determined about a peptide that mimics the ACA epitope
on .beta.2-GPI.
[0258] NMR data coupled with distance geometry calculations were
used to determine the three dimensional structure of peptide 925
(CLGVLAKLC), a truncated version of peptide 3G3 (AGPCLGVLGKLCPG)
with alanine substituted for glycine in position 6 of the 925
peptide. The structure of peptide 925 was determined in water at pH
3.8 and at 25.degree. C. An ensemble of nine structures were
calculated all of which were consistent with the NMR data. The RMSD
for all non-hydrogen atoms was 2.450.36 angstroms when each
structure was compared to the centroid. FIG. 18 displays the
structure closest to the centroid of the ensemble and, therefore,
is a reasonable representation of the shape of the peptide 925
molecule. FIG. 19 compares the structure of peptide 925 (labeled at
the bottom of the figure as 3G3) with the structure of peptide
5A12. Both peptides have turns at approximately the same positions
in the peptide sequence.
[0259] The pharmacophore of the peptides has been tentatively
identified as a small hydrophobic group and a positively charged
group. The gem-dimethyl and amino groups of peptide 925 are
tentatively identified as the pharmacophore of this peptide as
shown in FIG. 20. The hydrocarbon linkers that tether the
pharmacophore groups to some scaffold have the lengths specified in
FIG. 20 and the points at which these linkers are attached to the
scaffold are separated by the distance specified. Finally, the
dihedral angle defining the relative orientation of the two linkers
was determined to be 22.degree..
Example 20
[0260] Synthesis of TEG Carbamate Linker 14
[0261] 2-[2-(2-tert-Butylthioethoxy)ethoxy]ethaniol. compound 38:
To a mixture of 2-[2-(2-chloroethoxy)ethoxy]ethanol (11.0 g, 65.24
mmol) and tert-butylthiol (7.35 mL, 65.23 mmol) cooled in an ice
bath was slowly added 1,8-diazahicyclo[5.4.0]undec-7-ene (DBU, 9.75
mL, 65.23 mmol). The reaction mixture was allowed to warm to room
temperature and stirred overnight. This mixture was then diluted
with ethyl acetate and filtered. The crude product in the filtrate
was purified on a filter column eluted with ethyl acetate to give
yellowish oil (14.4 g, 64.6 mmol, 99%/): .sup.1H NMR (300 MHz,
CDCl.sub.3): d 3.73 (br s, 2H), 3.69-3.60 (m, 8H), 2.75 (t, J=7.4,
2H), 2.62 (br s, 1H), 1.32 (s, 9H); .sup.13C NMR (75 MHz,
CDCl.sub.3): d 72.47, 71.08, 70.33, 70.27, 61.71, 42.09, 30.95,
27.87; MS (ESI): m/e (M+1) Calcd. for C.sub.10H.sub.23O.sub.3S:
223, obsd.: 223.
[0262] 2-[2-(2-tert-Butylthioethoxy)ethoxy]ethyl p-nitrophenyl
carbonate, compound 39: To a solution of compound 38 (2.0 g, 8.98
mmol) and p-nitrophenol chloroformate (1.81 g, 8.98 mmol) in 5 mL
of dry THF cooled in an ice bath was slowly added dry pyridine
(0.73 mL, 8.98 mmol) in 1 mL of dry THF. White precipitate came out
immediately. After stirred at room temperature for 15 min, the
reaction mixture was diluted with 10 mL of ether and filtered. The
filtrate was concentrated and used directly in the peptide
synthesis without further purification.
Example 21
[0263] Synthesis of the Tetravalent Platform IA/DABA/ATEG, 46.
[0264] Bis-N-(t-butoxycarbonyl)-diaminobenzoic acid. compound 40: A
solution of 7.18 g (32.9 mmol of di-t-butyldicarbonate in 5.5 mL of
MeOH was slowly added to a solution of 2.5 g (16.4 mmol) of
3,5-diaminobenzoic acid and 2.76 g (32.9 mmol) of NaHCO.sub.3 in
44.5 mL of H.sub.2O and 22.5 mL of MeOH, and the mixture was
stirred at room temperature for 24 h. The mixture was cooled to
0.degree., and 6.53 g of citric acid was added, and the mixture was
extracted with EtOAc. The combined EtOAc layers were dried
(MgSO.sub.4), filtered, and concentrated. The residue was dissolved
in 40 mL of Et.sub.2O and the solution was filtered through Celite.
The Et.sub.2O layer was extracted with two 40 mL portions of HCl.
The Et.sub.2O layer was dried (MgSO.sub.4), filtered, and
concentrated to give 3.81 g (66%) of 40 as a foamy pink solid.
[0265] N-hydroxysuccinimidyl ester of compound 40, compound 41:
Dicyclohexylcarbodiimide (3.34 g, 16.2 mmol) was added to a
solution of 3.8 g (10.8 mmol) of compound 40 and 1.24 g (10.8 mmol)
of N-hydroxysuccinimide in 55 mL of EtOAc which had been cooled to
0.degree., and the resulting mixture was stirred for 18 h allowing
to come to room temperature. To the mixture was added 0.55 mL of
acetic acid. The mixture was stirred for 30 min and placed in the
freezer for 2 h. The mixture was filtered to remove solids, and the
filtrate was concentrated to give 5.80 g of pink foamy solid.
Purification by silica gel chromatography (60/40/1
hexane/EtOAc/HOAc) gave 4.30 g (89%) of compound 41 as a slightly
pink solid.
[0266] Mono-N-(t-butoxycarbonyl)-ethylenediamine, compound 42: A
solution of 1.5 g (25.0 mmol) of ethylenediamine in 15 mL of
CH.sub.2Cl.sub.2 was cooled to 0.degree., and a solution of 1.82 g
(8.33 mmol) of di-t-butyldicarbonate was added slowly to the
mixture. The mixture was stirred at room temperature for 18 h and
filtered, and the filtrate was concentrated. Purification by silica
gel chromatography (90/10/1 CH.sub.2Cl.sub.2/MeOH/HOAc) gave 0.98 g
(67%) of compound 42 as an oil.
[0267] Bis-N-(t-butoxycarbonyl)-amino-TEG, compound 43: To a
solution of 750 mg (4.25 mmol) of compound 42 and 345 .mu.L (337
mg, 4.25 mmol) of pyridine in 6 mL of CH.sub.2Cl.sub.2 was added
445 uL (559 mg, 2.02 mmol) of triethyleneglycol bis-chloroformate.
The mixture was stirred for 3.5 h, and the mixture was partitioned
between 35 mL of CH.sub.2Cl.sub.2 and 35 mL of 1 N HCl. The
CH.sub.2Cl.sub.2 layer was washed with H.sub.2O, dried
(Na.sub.2SO.sub.4), filtered and concentrated to give 1.14 g of
crude compound 43 which was used directly in the next step.
[0268] Diamino-TEG bis-trifluoroacetate salt, compound 44: 300 mg
(0.57 mmol) of compound 43 was dissolved in 3.5 g of
CH.sub.2Cl.sub.2, and 3.5 mL of trifluoroacetic acid was added. The
mixture was stirred for 3 h at room temperature, and the solution
was concentrated to give 398 mg of crude compound 44 which was used
directly in the next step.
[0269] Compound 45: A solution of 567 mg of compound 41 in 6 mL of
dioxane was added to a solution of 398 mg of crude compound 44
(est. 316 mg, 0.57 mmol) and 193 mg (2.30 mmol) of NaHCO.sub.3. The
mixture was stirred for 3 h and acidified with 1 N HCl and
partitioned between 20 mL of 1 N HCl and 30 mL of EtOAc. The
combined EtOAc layers are washed with sat NaHCO.sub.3 solution,
dried (MgSO.sub.4), filtered and concentrated to give 490 mg (86%)
of crude compound 45 as a white foamy solid. Purification by silica
gel chromatography (EtOAc) gave 276 mg (48%) of compound 45 as a
white foamy solid.
[0270] IA/DABA/ATEG, Compound 46: A solution of 100 mg (0.1 mmol)
of compound 45 was prepared, and 1 mL of trifluoroacetic acid was
added, and the mixture was stirred for 1 h at room temperature. The
mixture was concentrated under vacuum. The residue was triturated
with Et.sub.2O and dried under vacuum to give a white crystalline
solid. The solid was dissolved in 1 mL of DMF, 104 uL (77 mg, 0.6
mmol) of diisopropylethylamine was added, the mixture was cooled to
0.degree., and 212 mg (0.6 mmol) of iodoacetic anhydride was added.
The ice bath was removed, and the mixture was stirred at room
temperature for 2 h. The mixture was cooled to 0.degree., acidified
with 1 N H.sub.2SO.sub.4, and partitioned between 10 mL of 1 N
H.sub.2SO.sub.4 and 6.times.20 mL portions of 8/2
CH.sub.2Cl.sub.2/MeOH. The combined organic layers were dried
(MgSO.sub.4), filtered, and concentrated to give 330 mg of orange
oil. Purification by preparative HPLC (25 cm.times.22.4 mm
C.sub.18, gradient: 30% B to 40% B 0-40 min, A=H.sub.2O/0.1% TFA,
B=CH.sub.3CN/0.1% TFA, 12 mL/min) gave 19 mg of compound 46 as a
white solid. 15
Example 22
[0271] Synthesis of the Tetravalent Platform BA/PABA/DT/TEG,
51.
[0272] N-(t-butoxycarbonyl)PABA, compound 47: A solution was
prepared of 3.0 g (21.9 mmol) of p-aminobenzoic acid in 60 mL of
H.sub.2O. Na.sub.2CO.sub.3 (2.16 g, 25.7 mmol) was added slowly
followed by 30 mL of MeOH. When all solids were dissolved, a
solution of 4.77 g (21.9 mmol) of di-t-butyldicarbonate in 10 mL of
MeOH was added and the mixture was stirred at room temperature for
18 h. To the mixture was added 4.92 g (25.6 mmol) of citric acid
and the resulting cloudy mixture was partitioned between 200 mL of
H.sub.2O and 200 mL of EtOAc. The EtOAc layer was washed
successively with 200 mL of 0.1 N HCl and 200 mL of H.sub.2O, dried
(Na.sub.2SO.sub.4), filtered, and concentrated to yield 3.0 g (58%)
of compound 47 as a white solid.
[0273] N-(t-butoxycarbonyl)PABA N-hydroxysuccinimidyl ester,
compound 48: DCC (2.61 g, 12.6 mmol) was added to a 0.degree.
solution of 2.0 g (8.43 mmol) of compound 47 and 0.97 g (8.43 mmol)
of N-hydroxysuccinimide in 50 mL of EtOAc. The ice bath was
removed, the mixture was stirred for 16 h at room temperature, and
0.5 mL of acetic acid was added. The mixture was stirred for an
additional 30 min, placed in the freezer for 1.5 h, filtered, and
concentrated to give 3.75 g of crude 48. Purification by silica gel
chromatography (50/50 hexane/EtOAc) gave 2.53 g (90%) of compound
48 as a white solid.
[0274] Compound 49: A solution of 3.00 g (8.97 mmol) of compound 48
in 50 mL of CH.sub.2Cl.sub.2 was added dropwise over 30 min to a
solution of 485 uL (4.49 mmol) of diethylenetriamine in 30 uL of
CH.sub.2Cl.sub.2 which had been cooled in an ice bath. The mixture
was stirred at 5-7.degree. for 30 min then at room temperature for
16 h. The milky mixture was placed in a separatory funnel with 200
mL of H.sub.2O, the pH of the H.sub.2O layer was adjusted to 10
with 3 M NaOH solution, and the mixture was extracted with 200 mL
of 4/1 CH.sub.3Cl/MeOH. The organic phase was dried
(Na.sub.2SO.sub.4) and concentrated to give 0.971 g (40%) of
compound 49 which was pure enough to be used in the following step.
The aqueous layer was extracted with six 100 mL portions of 4/1
CH.sub.3Cl/MeOH. The organic layers were combined, dried
(Na.sub.2SO.sub.4), and concentrated to give another 1.08 g (44%)
of compound 49 which required further purification. Further
purification can be accomplished by silica gel chromatography
(gradient, 80/20 to 70/30 CH.sub.3Cl/MeOH).
[0275] Compound 50: A solution of 135 uL (0.66 mmol) of
triethyleneglycol bis-chloroformate in 0.3 mL of THF was added to a
0.degree. solution of 855 mg (1.58 mmol) of compound 49 and 275 uL
(1.58 mmol) of diisopropylethylamine in 13 mL of THF. The cloudy
mixture cleared when the ice bath was removed. An additional 70 uL
of diisopropyethylamine was added to maintain a basic pH. The
mixture was stirred at room temperature for a total of 3 h and
partitioned between 25 mL of H.sub.2O and 25 mL of EtOAc. The
aqueous layer was extracted with a second 25 mL portion of EtOAc,
and the combined EtOAc layers were dried (Na.sub.2CO.sub.3),
filtered, and concentrated to give 0.986 g of crude 50.
Purification by silica gel chromatography (80/4/16
CH.sub.3Cl/dioxane/isopropanol) gave 516 mg (61%) of compound 50 as
a white solid.
[0276] Preparation of BA/PABA/DT/TEG, compound 51: Compound 50 (487
mg, 8.79 mmol) was dissolved in 9 mL of CH.sub.2Cl.sub.2 and 5 mL
of trifluoroacetic acid. The mixture was stirred for 1.5 h and
concentrated. The residue was triturated with 7 mL of Et.sub.2O,
and dissolved in 5 mL of MeOH and 1 mL of 48% HBr solution. The
mixture was concentrated and placed under vacuum until a dry. The
resulting HBr salt was dissolved in 10 mL of H.sub.2O, and 191 mg
(2.27 mmol) of NaHCO.sub.3 was added. A solution of 591 mg (2.27
mmol) of bromoacetic anhydride in 10 mL of dioxane was added to the
mixture. An additional 2 mL of dioxane was used to rinse. More
NaHCO.sub.3 was added as needed to maintain a basic pH. The mixture
was stirred for 2 h at room temperature, and acidified with 1 N
H.sub.2SO.sub.4. The mixture was extracted with 3.times.25 mL of
EtOAc. The combined EtOAc layers were dried (Na.sub.2SO.sub.4),
filtered, and concentrated to give 773 mg of an oil. Purification
was accomplished by silica gel chromatography (gradient, 90/10 to
85/15 CH.sub.3Cl/MeOH) to give 401 mg (77%) of 51 as a white solid.
16
Example 23
[0277] Synthesis of the Tetravalent Platform BMP/TEG, 55.
[0278] Dimethyl-5-hydroxyisophthlalate. compound 52: A solution of
2.00 g (11 mmol) of 5-hydroxyisophthalic acid and 0.5 mL of con HCl
in 30 mL of MeOH was refluxed for 5 h. The mixture was concentrated
and the resulting residue was dissolved in 100 mL of EtOAc. The
EtOAc solution was washed successively with two 50 mL portions of
5% NaHCO.sub.3 solution and two 50 mL portions of sat NaCl
solution, dried (Na.sub.2CO.sub.3), filtered, and concentrated to
give 2.09 g (90%) of 52 as a white crystalline solid. Compound 53:
Triethyleneglycol ditosylate 546 mg (1.19 mmol) was added to a
suspension of 500 mg (2.38 mmol) of compound 52 and 395 mg (2.86
mmol) of K.sub.2CO.sub.3 in 11 mL of CH.sub.3CN and the mixture was
refluxed under N.sub.2 for 16 h. The mixture was concentrated, and
the residue was dissolved in 25 mL of CHCl.sub.3 and washed with 25
mL of H.sub.2O and 25 mL of sat NaCl solution. The CHCl.sub.3 layer
was dried (Na.sub.2SO.sub.4), filtered, and concentrated.
Purification by silica gel chromatography (gradient, 50/50
hexane/EtOAc to 100% EtOAc) gave 93 mg (41%) of compound 53.
[0279] Compound 54: A suspension of 93 mg (0.18 mmol) of compound
53 in 4 mL of THF was stirred under a N.sub.2 atmosphere while 1.82
mL (1.82 mmol) of a 1 M solution of LiBHEt.sub.3 was added to the
mixture dropwise. A clear solution was obtained which was stirred
for 18 h at which time a 10% solution of HOAc in water was added
until the pH was acidic. The mixture was concentrated under vacuum
and the residue was dissolved in 10 mL of water. The mixture was
extracted with 3.times.10 mL portions of EtOAc, and the combined
EtOAc layers were dried (Na.sub.2SO.sub.4), filtered and
concentrated. Purification was accomplished by silica gel
chromatography (gradient, 90/10 to 85/15 CH.sub.3Cl/MeOH) to give
20 mg (27%) of 54 as a white solid.
[0280] Compound 55: To a suspension of 20 mg (0.048 mmol) of 54 in
5 mL of Et.sub.2O and 1 mL of THF is added 9.6 uL (0.1 mmol) of
PBr.sub.3. The mixture is stirred for 3 h and partitioned between
water and EtOAc. The EtOAc layer is dried (Na.sub.2SO.sub.4),
filtered and concentrated, and the residue is purified by silica
gel chromatography (CH.sub.3Cl/MeOH) to provide compound 55. 17
Example 24
[0281] Synthesis of the Tetravalent Platform BA/PIZ/IDA/TEG,
60.
[0282] Compound 56: To a solution of 1.02 g (4.37 mmol) of
N-(t-butoxycarbonyl)-iminodiacetic acid (compound 5 in U.S. Pat.
No. 5,552,391, Chemically-Defined Non-Polymeric Valency Platform
Molecules and Conjugates Thereof) and 1.01 g (8.75 mmol) of
N-hydroxysuccinimide in 50 mL of dry THF, cooled to 0.degree.. was
added 2.26 g (10.94 mmol) of dicyclohexylcarbodiimide. The mixture
was stirred for 16 h allowing to slowly warm to room temperature,
and a solution of 2.22 g (10.1 mmol) of mono-CBZ-piperazine in 25
mL of THF was added to the mixture followed by 1.22 mL (887 mg,
8.75 mmol) of Et.sub.3N. The mixture was stirred for 7 h at room
temperature, and filtered. The filtrate was concentrated and the
residue was dissolved in 125 mL of EtOAc and shaken with
2.times.125 mL portions of 1 N HCl, 125 mL of sat NaHCO.sub.3
solution, dried (MgSO.sub.4), filtered, and concentrated to give
2.39 g of a sticky solid. Purification by silica gel chromatography
(95/5 CH.sub.2Cl.sub.2/MeOH) gave 1.85 g (66%) of 56.
[0283] Compound 57: To a solution of 1.74 g (2.74 mmol) of compound
56 in 10 mL of CH.sub.2Cl.sub.2 was added 10 mL of trifluoroacetic
acid, and the mixture was stirred for 3 h at room temperature. The
mixture was concentrated, and the residue was dissolved in 5 mL of
CH.sub.2Cl.sub.2. The mixture was cooled to 0.degree. and 100 mL of
sat NaHCO.sub.3 was added. The mixture was then extracted with four
100 mL portions of CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layers
were combined, dried (MgSO.sub.4), filtered, and concentrated to
give 1.46 g (99%) of 57 as a sticky hygroscopic solid which was
used directly in the next step.
[0284] Compound 58: To a solution of 0.7 g (1.3 mmol) of compound
57 and 226 uL (168 mg, 1.30 mmol) of diisopropylethylaminie at
0.degree. was added a solution of 127 uL of triethyleneglycol
bis-chloroformate in 4 mL of CH.sub.2Cl.sub.2, and the mixture was
stirred for 3 h at room temperature. The mixture was partitioned
between 80 mL of CH.sub.2Cl.sub.2 and 80 mL of 1 N HCl. The
CH.sub.2Cl.sub.2 layer was washed with two 80 mL portions of water,
dried (MgSO.sub.4), filtered, and concentrated to give 736 mg (93%)
of compound 58 as a crystalline solid.
[0285] Compound 60: Compound 58 (61 mg, 0.48 mmol) was dissolved in
3 mL of 30% HBr/HOAc and the resulting mixture was stirred at room
temperature for 1 h at which time 5 mL of Et.sub.2O was added. The
mixture was placed in the freezer for 1 h and centrifuged. The
resulting pellet was washed with Et.sub.2O and dried to give the
tetrahydrobromide salt 59 which was dissolved in 1 mL of H.sub.2O.
To the mixture is added 49 mg (0.58 mmol) of NaHCO.sub.3 and 3 mL
of dioxane. More NaHCO.sub.3 is added, if needed, to make the
mixture basic. The mixture is cooled to 0.degree., and 748 mg (2.89
mmol) of bromoacetic anhydride is added. The mixture is stirred for
2 h and partitioned between 20 mL of 1 N H.sub.2SO.sub.4 and 20 mL
of 80/20 CH.sub.2Cl.sub.2/MeOH. The organic layer is dried
(Na.sub.2SO.sub.4), filtered and concentrated to give crude 60
which is purified by silica gel chromatography
(CH.sub.2Cl.sub.2/MeOH) to give 60. 18
Example 25
[0286] Synthesis of the Tetravalent Platform Tetrakis-BA/PIZ/PMA,
63.
[0287] Compound 61: To a 0.degree. solution of 640 mg (2.52 mmol)
of pyromellitic acid and 1.16 g (10.1 mmol) of N-hydroxysuccinimide
in 50 mL of THF was added 2.6 g (12.6 mmol) of
dicyclohexylcarbodiimide, and the mixture was allowed to come to
room temperature while stirring for 16 h. A solution of 2.5 g (11.3
mmol) of mono-CBZ-piperazine in 25 mL of THF was added to the
mixture followed by 1.4 mL (1.02 g, 10.1 mmol) of Et.sub.3N. The
mixture was filtered, and the filtrate was concentrated. The
residue was partitioned between 100 mL of EtOAc and 2.times.100 mL
of 1 N HCl, and the EtOAc layer was washed with 100 mL of sat
NaHCO.sub.3, 100 mL of H.sub.2O, 100 mL of sat NaHCO.sub.3, dried
(MgSO.sub.4), filtered, and concentrated. Purification was
accomplished by silica gel chromatography (97.5/2.5
CH.sub.2Cl/MeOH) to give 1.78 g (66%) of 63 as a white crystalline
solid.
[0288] Compound 63: Compound 61 is converted to compound 63 in
essentially the same manner as described for the conversion of 58
to 60 in Example 23. Purification is accomplished using silica gel
chromatography. 19
Example 26
[0289] Synthesis of the Tetravalent Platform BA/PIZ/IDA/HB/TEG,
68.
[0290] Compound 64: Triethyleneglycol ditosylate (1.0 g, 2.18 mmol)
was added to a solution of 725 mg (4.36 mmol) of ethyl
4-hydroxybenzoate and 723 mg (5.23 mmol) of K.sub.2CO.sub.3, and
the mixture was refluxed for 16 h. The mixture was concentrated,
and the residue was partitioned between 20 mL of water and
3.times.20 mL of Et.sub.2O. The combined organic layers were washed
with 2.times.40 mL of sat NaHCO.sub.3 solution, 40 mL of sat NaCl
solution. The aqueous layers were washed with Et.sub.2O, and the
combined Et.sub.2O layers were dried (MgSO.sub.4), filtered, and
concentrated. Purification by silica gel chromatography, (70/30
hexaties/EtOAc) have 902 mg (93%) of 64 as a crystalline solid.
[0291] Compound 65: Compound 64 is dissolved in acetone containing
2.2 equivalents of LiOH and the mixture is stirred for 3 h (until
complete as evidenced by TLC). The mixture is acidified with acetic
acid and concentrated, and the residue is purified by silica gel
chromatography to give 65.
[0292] Compound 66: Compound 66 is prepared similarly to the method
of preparing compound 56 in example 23. Compound 65 is used instead
of N-BOC-iminodiacetic acid, and compound 57 is used instead of
mono-CBZ-piperazine. Purification is accomplished using silica gel
chromatography.
[0293] Compound 68: Compound 66 is converted to compound 68 in
essentially the same manner as described for the conversion of 58
to 60 in Example 23. Purification is accomplished using silica gel
chromatography. 20
Example 27
[0294] Synthesis of the Tetravalent Platform BA/PIZ/HIP/TEG,
72.
[0295] Compound 69: Compound 53 is hydrolyzed with LiOH in
essentially the same manner as described for the hydrolysis of 64
in example 25 with the exception that 4.4 equivalents of LiOH is
used.
[0296] Compound 70: The tetra-acid, compound 69, is converted to
compound 70 in essentially the same manner as described for the
conversion of pyromellitic acid to 61 in example 24 with the
exception that 69 is used instead of pyromellitic acid.
[0297] Compound 72:: Compound 70 is converted to compound 72 in
essentially the same manner as described for the conversion of 58
to 60 in Example 23. Purification is accomplished using silica gel
chromatography. 21
Example 28
[0298] Synthesis of Conjugates of Haloacetylated Tetravalent
Compounds
[0299] Synthesis of (LJP685).sub.4/MTU/A/DABA/ATEG conjugate
compound 73: A solution was prepared of 21 mg (15.5 umol) of
compound 24 in Helium sparged pH 8.5 200 mM borate buffer. To the
solution was added a second solution consisting of 3.25 mg (2.6
umol) of IA/DABA/ATEG, compound 46, dissolved in 396 uL of MeOH. A
precipitate formed, and 1 mL of MeOH was added to dissolve all the
solids. The mixture was stirred for 18 h at room temperature, and 6
mL of 9/1 H.sub.2O/HOAc was added. The mixture was diluted with 50
mL of 9/1 H.sub.2O/CH.sub.3CN and loaded onto an HPLC preparative
column. Purification was accomplished by preparative HPLC (25
cm.times.22.4 mm C.sub.18, gradient: 35% B to 45% B 0-40 min,
A=H.sub.2O/0.1% TFA, B=CH.sub.3CN/0.1% TFA. 12 mL/min) to provide
10.8 mg (67%) of compound 73 as a white solid after lyophilization.
22
[0300] Synthesis of (LJP685).sub.4/MTU conjugates, compounds 74,
75, 76, 77, and 78: These conjugates are prepared from platform
compounds 51, 60, 63, 68, and 72, respectively, by the same
reaction conditions as described above for the synthesis of
conjugate 73 by substituting the appropriate platform compound for
platform compound 46. Pep can be LJP685 or other relevant peptide.
23
Example 29
[0301] Synthesis of Conjugates Tetravalent Platform 55, Conjugate
Compound 79, (LJP685).sub.4/MP/TEG:
[0302] A solution is prepared of four equivalents of compound 24
and five equivalents of Cs.sub.2CO.sub.3 in DMF. One equivalent of
BMP/TEG, platform compound 55, is added to the mixture which is
then stirred for 1 h. The mixture is diluted with 80/20/10
H.sub.2O/CH.sub.3CN/HOAc and loaded onto a preparative HPLC column.
Purification is accomplished by preparative HPLC (C.sub.18,
A=H.sub.2O/0.1% TFA, B=CH.sub.3CN/0.1% TFA) to give compound 79
after lyophilization. Pep can be LJP685 or other relevant peptide.
24
Example 30
[0303] Synthesis of Conjugates Tetravalent Platform Tetrakis-BMB,
Conjugate Compound 80. (LJP685)/.sub.4/MTU/Tetrakis-MB:
[0304] A solution is prepared of four equivalents of compound 24
and five equivalents of Cs.sub.2CO.sub.3 in DMF. One equivalent of
tetrakis-bromomethylbenzene is added to the mixture which is then
stirred for 1 h. The mixture is diluted with 80/20/10
H.sub.2O/CH.sub.3CN/HOAc and loaded onto a preparative HPLC column.
Purification is accomplished by preparative HPLC (C.sub.18,
A=H.sub.2O/0.1% TFA, B=CH.sub.3CN/0.1% TFA) to give compound 80
after lyophilization. Pep can be LJP685 or other relevant peptide.
25
Example 30
[0305] Fluorescence Polarization Peptide Binding Assays Synthesis
of FITC-GPCILLARDRCG (CB2*)
[0306] A solution of the cyclic disulfide peptide GPCILLARDRCG
(20.0 mg, 14.4 .mu.mol) and fluorescein isothiocyanate (FITC) (5.6
mg, 14.4 .mu.mol) in 20 mL of ACN/water (1:1), containing 20 mg
sodium carbonate (Na.sub.2CO.sub.3, pH.about.10.5), was stirred at
room temperature. The reaction was monitored by analytical HPLC.
After consuming the fluorescent labeling reagent, the crude
material was purified on a preparative HPLC eluted at 10 mL/min
with a linear gradient from 30 to 55% B over 40 minutes where A was
0.1% (v/v) TFA in H.sub.2O and B was 0.085% (v/v) TFA in CAN. The
FITC peptide was obtained as a bright yellow powder after
lyophilization (3.7 mg, 15% yield): MS (ESI): m/e (M+1) Calcd. for
C.sub.73H.sub.102N.sub.19O.sub.20S.sub.3: 1661, obsd.: 1661.
[0307] Direct Binding Fluorescence Polarization Binding Assay
(dbFP)
[0308] The methodology is described in PanVera Application Guide
(1994), PanVera Corporation. Briefly, a trace amount of fluorescein
isothiocyanate (FITC) labeled peptide (CB2*-F) is titrated with
antibody (ACA 6501 or 6701) and the polarization for the sample is
plotted versus the antibody concentration. Polarization was
measured with the PanVera Beacon instrument. Data were fitted to
Equation 1. 1 Y = [ P L * ( K D / R ) + P H ] K D / R + 1.0
Equation 1
[0309] where Y is the Y-axis value (milli-polarization units, mP),
R is the total concentration of antibody receptor, P.sub.L is the
polarization for free FITC-labeled peptide (F), and P.sub.H is the
polarization for F complexed with R (FR). K.sub.D is the
dissociation constant (reciprocal binding, constant) for F from the
FR complex. For these equations to be valid, it must be true that
F<<R. This titration is shown in FIGS. 22 and 23 for CB2*-F
binding to ACA-6701 and ACA-6501 antibodies, respectively. A
complete titration was not obtained with ACA-6501 as shown in FIG.
23, but a previous titration shown in FIG. 24 gave a K.sub.D of 241
nM. By adding CB2* (GPCILLARDRCG) in slight excess over antibody
6701, to displace CB2*-F from 6701 (see FIG. 25), a dissociation
rate constant of K.sub.off=0.0184 sec.sup.-1, which corresponds to
t.sub.1/2=38 seconds, was determined for CB2*-F. Given the K.sub.D
of 256 nM, this corresponds to an association rate constant of
K.sub.on=3.6.times.10.sup.4 M.sup.-1sec.sup.-1 (after correcting
for antibody bivalency). Thus, CB2*-F binding to ACA-6701 is
limited only by diffusion of these two molecules together.
[0310] Competitive Fluorescence Polarization Assay (cFP)
[0311] The above described dbFP assay provides binding constants
for FITC-labeled peptides and requires on the order of 0.5 mg of
purified antibody. The cFP assay provides binding constants for
peptides that lack the FITC group and it consumes less antibody, on
the order of 10 .mu.g. The cFP assay is modified from that reported
in PanVera Applications Guide (1994) PanVera Corporation such that
it consumes 50-fold less antibody. Briefly, antibody (ACA 6701) is
mixed with trace FITC labeled peptide (CB2*-F) and enough time is
allowed for equilibrium to be reached. This was 1 hour for ACA 6701
and CB2*-F. Increasing concentrations of the unlabeled peptide
being tested (CB2* or 3B10) are then added to the tube. After each
addition, sufficient time, approximately 15 minutes, is allowed for
equilibrium to be reached and the mP value was read. Although it is
necessary to choose concentrations of 6701.RTM. and CB2*-F (F) such
that F<<R, the concentration of R need only be high enough
such that the measured polarization (P.sub.H) is significantly
higher than P.sub.L (see FIG. 22). This .DELTA.(mP) value should be
20 or more mP units to insure reliable results.
.DELTA.(mP)=P.sub.H-P.sub.L Equation 2
[0312] As unlabeled (no FITC) peptide (I) is added to inhibit F
from binding to R, Y decreases from its maximum value of P.sub.H'
to a plateau of P.sub.L which should agree with that in Equation 1.
These titrations are shown in FIGS. 26 and 27 for displacement of
CB2*-F from ACA-6701 by CB2* and 3B10, respectively. The equation
describing this titration was derived and is: 2 Y = P H ' + P L * (
I / K I ' ) I / K I ' + 1.0 Equation 3
[0313] where P.sub.L is the same as in Equation 1. I is the
concentration of unlabeled peptide competitor, and K.sub.I' is the
apparent dissociation constant for that peptide. Values for these
parameters were obtained by fitting cFP titration data to the above
equation.
[0314] The true dissociation constant for I is obtained from
Equation 4.
K.sub.I=K.sub.I'/(1.0+R/K.sub.D) Equation 4
[0315] where R and K.sub.D are defined as in Equation 1. The
R/K.sub.D ratio is obtained from the values of P.sub.H' (from
Equation 3) and P.sub.H and P.sub.L (from Equation 1) and using
Equation 5.
R/K.sub.D=(P.sub.H'-P.sub.L)/(P.sub.H-P.sub.H') Equation 5
[0316] In general, Equation 5 can be used to determine aPL antibody
concentrations once the titration defined by Equation 1 is
performed as a "standard curve." Thus, in addition to providing a
means for determining K.sub.I, this method provides a means of
standardizing all aPL antibody stock solution concentrations and of
analyzing their binding activity/stability over time using only
5-10 .mu.g of antibody per cFP assay.
14 DISSOCIATION CONSTANTS DETERMINED BY dbFP AND cFP Peptide
Antibody Sequence K.sub.D.sup.a or K.sub.I CB2*-F 6501
FITC-GPCILLARDRCG 482 nM.sup.a CB2*-F 6701 FITC-GPCILLARDRCG 512
nM.sup.a CB2* 6701 GPCILLARDRCG 35 nM 3B10 6701 AGPCLLLAPDRCPG 313
nM .sup.aThe K.sub.D values have been multiplied by 2 to correct
for K.sub.D values determined from Equation 1 that do not factor in
the bivalency of the antibody.
[0317] The results demonstrate that CB2*-F cross reacts with two
very different aPL antibodies, ACA-6501 and ACA-6701, binding to
both with equal high affinity. Removal of the FITC group improved
binding of CB2* to ACA-6701 by 14-fold. Binding of a related
peptide, 3B10, was 9-fold less than binding of CB2* to ACA-6701.
While this result may be due to additional framework residues on
3B10, it may also be due to the substitution of a proline for
arginine at position 8 in CB2*. Previous NMR structure studies of
5A12, a peptide similar to 3B10, showed that this proline that is
in a turn position gives the structure rigidity. CB2 is a much more
flexible peptide and it has an arginine in this position. A more
flexible peptide like CB2 may be more cross-reactive because it may
more readily adjust its shape to fit a given antibody binding site.
The implications of drug rigidification on binding affinity are
discussed in Koehler et al. p. 251, GUIDEBOOK ON MOLECULAR MODELING
IN DRUG DESIGN (Academic Press, N. Cohen, ed., 1996).
Example 31
[0318] Tolerance Activity of Peptide Conjugates
[0319] Two different conjugates containing the same peptide were
tested for their ability to induce antigen specific tolerance in
vivo. Briefly, mice were immunized with the peptide conjugated to
the immunogenic carrier Keyhole Limpet Hemocyanin (KLH) to generate
peptide-specific memory B cells. Three weeks later, groups of 5
mice per group were treated with various doses of the test
conjugates, one group of mice was not treated and acted as the
control. Five days later, all of the mice, including the control
group, were boosted with the peptide conjugated to KLH and seven
days later all of the mice were bled and their sera assayed for
anti-peptide antibodies using a modified Farr assay. The Antigen
Binding Capacity (ABC) was calculated for each individual serum
sample according to the method described in G. M. Iverson, "Assay
for in vivo adoptive immune response," Volume II, Chapter 67,
HANDBOOK OF EXPERIMENTAL IMMUNOLOGY (Blackwell Scientific
Publications, Weir et al., eds., 4th ed., Oxford, 1986). These
values were then used to determine a mean and standard deviation
for all of the individuals of a group. While one of the conjugates
induced tolerance, the other one did not inhibit anti-peptide
antibodies over the dose range tested. The most likely explanation
for this difference is that the latter conjugate has a short in
vivo half-life. To address this problem, a system was employed that
induced tolerance in vitro, thereby negating half-life
consideration, and then the cells were transferred to irradiated
recipients. Briefly, spleen cells from mice primed with the peptide
conjugated to KLH were harvested and incubated in complete
RPMI-1640 medium for 2 hours at 37.degree. C. with various doses of
the test conjugates. One group of cells was incubated without
toleragen and acted as the positive control. The cells were washed,
transferred to irradiated syngeneic recipients and boosted with the
peptide conjugated to KLH. Seven days later all of the mice were
bled and their sera assayed for anti-peptide antibodies. The
conjugate that did not induce any detectable tolerance when tested
in the in vivo model did induce tolerance when tested in this in
vitro model. This result supports the assumption that the
difference between conjugates is due to a short half-life of the
conjugate. To directly test this hypothesis, the conjugate was
administered continuously by an implanted osmotic pump over a
prolonged period of time. The results clearly show that this
conjugate induced tolerance when administered by sustained release
but not when administered as a bolus as shown in FIG. 32.
[0320] Testing of (LJP685).sub.4/MTU-AHAB-TEG for Tolerance
Activity in the in vivo Model
[0321] Mice were primed with LJP-685-KLH on alum plus pertussis as
an adjuvant. Three weeks later, the mice were treated with a range
of doses of the (LJP685).sub.4/MTU-AHAB-TEG conjugate. One group
was not treated and acted as a control group. Five days later, all
of the mice, including the control group, were boosted with 10
.mu.g LJP685-KLH and seven days later the mice were bled. Their
sera were analyzed for anti-LJP685 antibodies by a modified Farr
assay as described above. The results as shown in FIG. 28
demonstrate that the treatment with the (LJP685).sub.4/MTU-AHAB-TEG
conjugate, over a dose range of 1 to 50 nmoles, had no detectable
effect on the anti-LJP685 response.
[0322] Testing the (LJP685).sub.4/MTU-DABA-TEG Conjugate for
Tolerance Induction in the in vivo Model
[0323] Mice were primed with LJP685-KLH on alum plus pertussis.
Three weeks later, the mice were treated with 5, 10 or 50 nmoles of
the (LJP685).sub.4/MTU-DABA-TEG conjugate. One group was not
treated and acted as a control group. Five days later, all of the
mice, including the control group, were boosted with 10 .mu.g of
LJP685-KLH and seven days later the mice were bled. Their sera were
analyzed for anti-LJP685 antibodies by a modified Farr assay. The
results as shown in FIG. 29 demonstrate that
(LJP685).sub.4/MTU-DABA-TEG conjugate induces tolerance in the in
vivo model with an ED.sub.50 of 5 nmoles.
[0324] Testing the (LJP685).sub.4/MTU-AHAB-TEG Conjugate for
Tolerance Induction in the in vitro Model
[0325] Spleen cells from mice primed 3 weeks earlier with
LJP685-KLH were harvested and incubated in complete RPMI-1640
medium for 2 hours at 37.degree. C. with 4, 20 or 100 .mu.M of
(LJP685).sub.4/MTU-AHAB-TEG conjugate. One group of cells was
incubated without toleragen and acted as a positive control group.
The cells were washed, transferred to irradiated recipients and
boosted with 10 .mu.g of LJP685-KLH. Seven days later, the mice
were bled and their sera were analyzed for anti-LJP685 antibodies
by a modified Farr assay. The results as shown in FIG. 30 clearly
illustrate that the (LJP685).sub.4/MTU-AHAB-TEG conjugate can
induce tolerance when tested in the in vitro model achieving an
IC.sub.50 of <4 .mu.M.
[0326] Testing the (LJP685).sub.4/MTU-DABA-TEG Conjugate for
Tolerance Induction in the in vitro Model
[0327] Spleen cells from mice primed 3 weeks earlier with
LJP685-KLH were harvested and incubated in complete RPMI-1640
medium for 2 hours at 37.degree. C. with 0.4, 1.3 and 4 .mu.M of
(LJP685).sub.4/MTU-DABA-TEG conjugate. One group of cells was
incubated without toleragen and acted as a positive control group.
The cells were washed, transferred to irradiated recipients and
boosted with 10 .mu.g of LJP685-KLH. Seven days later, the mice
were bled and their sera were analyzed for anti-LJP685 antibodies
by a modified Farr assay. The results as shown in FIG. 31
demonstrate that (LJP685).sub.4/MTU-DABA-TEG conjugate can induce
tolerance when tested in the in vitro model, achieving an lC.sub.50
of <4 .mu.M.
[0328] Testing the (LJP685).sub.4/MTU-AHAB-TEG Conjugate for
Tolerance Induction in vivo Using a Continuous Delivery Pump
[0329] Mice were printed with LJP685-KLH on alum plus pertussis.
Three weeks later, the mice were divided into 5 groups of five mice
per group. On day 1, one group was treated with a bolus of saline
and another group was treated with a bolus containing 50 nMoles of
the (LJP685).sub.4/MTU-AHAB-TEG conjugate. The three remaining
groups were implanted with osmotic pumps. In one group, the pumps
were filled with saline and delivered at 1 .mu.L/hour for 3 days.
The two remaining groups received pumps filled with the
(LJP685).sub.4/MTU-AHAB-TEG conjugate (50 nMoles). One group
received pumps that deliver at 1 .mu.L/hour for three days and the
other received pumps that deliver at 0.5 .mu.L/hour for seven days.
On day 5, the pumps that deliver for three days were surgically
removed. On day 7, all of the mice, including the control group,
were boosted with 10 .mu.g of LJP685-KLH. On day 10, the pumps that
deliver for seven days were surgically removed. On day 14, all of
the mice were bled. Their sera were analyzed for anti-LJP685
antibodies by a modified Farr assay. The results are shown in FIG.
32.
[0330] Testing the (LJP-Peptide).sub.4/MTU-BMP-TEG Conjugate for
Tolerance Induction in the in vivo Model
[0331] Mice are primed with peptide-KLH on alum plus pertussis.
Three weeks later, the mice are treated with the
(LJP-peptide).sub.4/MTU-BMP-TE- G conjugate, one group is not
treated and acts as the control group. Five days later, all of the
mice, including the control group, are boosted with 10 .mu.g of
peptide-KLH and seven days later the mice are bled. Their sera are
analyzed for anti-peptide antibodies by a modified Farr assay. The
results show that the (LJP-peptide).sub.4/MTU-BMP-TEG conjugate
induces tolerance in the in vivo model at a potency equal to or
greater than that of the (LJP685).sub.4/MTU-AHAB-TEG conjugate.
* * * * *