Note: Descriptions are shown in the official language in which they were submitted.
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aPL IMMUNOREACTIVE PEPTIDES, CONJUGATES THEREOF AND
METHODS OF TREATMENT FOR APL ANTIBODY-l~IEDIATED
PATHOLOGIES
CROSS-REFERENCE APPLICATIONS
This application is a continuation-in-part application of U.S. Serial
No. 08/660,092, filed June 6, 1996, which is a continuation in-part of U.S. Serial
No. 08/482,651, filed June 7, 1995.
TECHNICAL FIELD
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 m-ethods 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 utili7in~ random
peptide libraries to identify immunospecific analogs of aPL-binding epitopes.
BACKGROUND OF THE INVENTION
Antiphospholipid antibodies occur in autoimmune ~ e~ce~ such as
systemic lupus eryth~m~tosus (SLE) and antiphospholipid antibody syndrome
(APS) as well as in association with infections and dru~ 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, FL, 1991); McNeil
et al. ADVANCES IN IMMUNOLOGY, Vol. 49, FP- 193-2~1 (Austen et al., eds.,
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Academic Press, San Diego, CA, 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 rheurnatic ~ e~es, 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 S~E, the following features were
observed: deep venous thrombosis (DVT) in 31; arterial occlusion in 31,
particularly stroke or transient ischemia; myocardial infarctions in 15; l'eCUllellt
fetal loss in 24; thrombocytopenia (TCP) in 32; 10 had a positive Coombs' test;
l 0 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.
Transient aPL antibodies, such as those detected in a VDRL test, occur
during many infections. Approximately 30% of patients po~cessing 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 di-e~,t ~dlhogenic effects in vivo by
interfering with hemostatic processes that take place on the phospholipid
membranes of cells such as platelets or endothelium. ln patients ~,vith 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).
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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 mea~u~e~ ,nl 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 byantibodies in APS, PAPS and other aPL antibody-me~ ted Ai~e~ces including
recurrent stroke and recurrent fetal loss are located in the 5th domain of ~2-GPI
and are exposed to the antibody following binding Of ,B2-GPI to cardiolipin.
It is now generally accepted that aPL antibodies recognize an antigenic
complex comprised of ~2-glycoprotein I (~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 I:1544-1547) (hereinafter "aPL
imrnunogen"). ~2-GPI is a minor plasma glycoprotein found free and in
''O 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.
~2-GPI has been reported to undergo antigenic and conformational changes upon
-- binding phospholipid (Wag~onkneckt et al. (1991) 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 meç~h~ni~m for aPL is
unknown (McNeil et al., supra). Most explanations invoke endothelial cell
function or platelet involvement (Haselaar et al. (1990) Thromb. Haemostas.
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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 ~2-GPI-binding and
trigger aPL antibody formation.
aPL antibodies may be directly l~lvlhlolnbotic by reducing prostacyclin
forrnation (Vermylen, J. and J. Amout (1992) J. Clin. Lab. Med 120:10-12); by
direct interference with the action of coagulation proteins; or by blocking the
ability of ,~2-GPI to inhibit the intrinsic blood coagulation pathway, platelet
p~.~thlolllbinase activity, and ADP-mediated platelet aggregation (Arvieux et al.
(1993) Thromb. Haemostas. 60:336-341).
A major new tool in medicinal chPrni~try 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~ ~- einer, D.B. and
W.V. Williarns, eds., Boca, Raton, FL., 1994); Moos et al. (1993) Ann. Reports
Med. Chem. 28:315324). By displaying random peptides on the surface of
filarnentous phage, epitope libraries cont~ining 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 arnplification, those phage that bind antibody are
prop~g~t~d in E. coli and the binding peptides identified by sequ~nring the
col,es~onding 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 rnimic discontinuous epitopes
(Luzzago et al. (1993) Gene 128:51-57; BaLass et al. (1993) Proc. Matl. Acad Sci.
90:10638-10642). The potential plasma instability of peptide-based drugs has
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been succes~fully overcome by N-terrninal blocking or by the judicious use of
amino acid analogs (Powell, M.F. (1993) Ann. Reports Med. Chem. 28:285-293).
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,
S steroids, and warfarin has proven to be largely inadequate (PHOSPHOLIPID-
BINDING ANTIBODIES (Harris et al., eds., CRC Press, Boca Raton, FL, 1991);
McNeil et al., supra). Synthetic mimetic peptides, characterized by (i) the
inability to activate T cells while (ii) ret~inine 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, Serial
No. 08/118,055, filed September 8, 1993, and U.S. Patent 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 ~flmini~tçring suchpeptides conjugated to multivalent, stable, non-immunogenic valency platforms inorder to abrogate antibody production via B cell anergy or clonal deletion aftercross-linking surfare irnmunoglobulin.
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 s.lcces~ful tolerogens. B cell tolerogens for the
treatment of human systemic lupus erythem~tosus-related nephritis have also beendisclosed in co-owned U.S. Patent Nos. 5,276,013 and 5,162,515 which are
incorporated by reference herein in their entirety.
DISCLOSURE OF THE ~NVENTION
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-me~ te~ e~ces, such as recurrent stroke and
recurrent fetal loss, using random peptide phage libraries.
Accordingly, one aspect of the invention is an improved method for
screening random peptide phage libraries in order to identify the peptide
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sequences which best mimic the epitopes recognized by aPL antibodies. This
method comprises the steps of. (a) biopanning the library using methods modifiedfrom those known in the art; (b) elimin~ting very weakly-binding phage by
rnicropA~ g 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 microorg~ni.cmc by plating on agar; (c) determining the
strongest-binding clones recovered in (b) by evaluation via phagecapture ELISA
by (i) coating the wells of a microplate with aPL antibody, (ii) incubating the
strongest-binding clones identified by micl~palmillg in (b) in the coated wells and
washing away unbound plialye, III I ing the nurnber of phage bound to the
antibody using an enzyme-conjugated goat anti-phage antibody in a calorimetric
ELISA assay and, if several equivalent strongly-binding clones are identified, an
additional round of (d) phage-ELISA on the strongest-binding phagecapture-
ELISA clone.
In this regard, the invention encomp~csec a method for identifying analogs
of epitopes which specifically bind aPL antibodies isolated from hum~n~ suffering
from an aPL antibody-mediated disease comprising: (a) pr~pa~ g 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
biopal~ning; (ii) further screening phage isolated by biopa~ g in (i) by
mi~;loyal~ling; and (iii) identifying phage cont~ining aPL antibody high-affinity
binding peptides recovered in (ii) by imm-lno~cc~y.
The invention also encomp~cses a method of biop~ ing phage random
peptide libraries to identify and isolate peptides which bind to aPL antibody
comprising: (a) reacting affmity-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
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recovered in (b); and (d) culturing the infected microorganism in an antibiotic-cont~ining medium in order to isolate the phage.
The invention further encomr~ses a method of micrup~ ing 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 biop~nning; (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-cont~ining medium in order to isolate the phage.
The invention also encompacses the above method described wherein the
immunoassay is a phage-capture ELISA comprising: (a) incubating phage bearing
random peptide inserts isolated by mi~lupalming 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 calorimetric substrate; and (f) measuring
the absorbance of the substrate to identify lilgt-t affinity-binding pliage.
Also encomp~sed by the invention is the method described above and
further comprising performing an additional phage-capture ELISA assay of the
high affinitybinding phage comprising: (a) coating a uniform amount of the phageon 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 calorimetric substrate to the wells; and (h) measuring the
absorbance of the substrate to measure the relative binding affinity of the phage.
The invention also encomp~c.ces the method described above wherein the
immunoassay is a colony-blot imm--no~cs~y comprising: (a) culturing a
microorganism infected with phage bearing random peptide inserts on a
nitrocellulose membrane atop an agar-cont~ining culture medium; (b) replicate
transferring the microorganism cultured in (a) by blotting the microorganism on a
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8 ~ -
second nitrocellulose membrane atop an agarcont~ining culture medium;
(c) in~ubatin~ the transferred microorg~ni~m;(d) Iysing the microorg~ni.cm;
(e) digesting the microorganism with Iysozyme; (f) blocking the membrane with a
gelatin solution; (g) incub~ting the membrane with aPL antibody; (h) washing
away unbound aPL antibody; (i) incub~tine 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 (I) measuring the
abso~ ce of the substrate to identify high affmity-binding phage.
A method for assaying and ranking, for affinity-binding characteristics,
epitopes which specifically bind aPL antibodies isolated from humzln~ suffering
from an aPL antibody-me~liated 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 ~2-GPI to bind to the cardiolipin andto 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.
Another aspect of the invention encomp~cses 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.
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The invention also encompasses a diagnostic immunoassay, for
deterrnining the presence of aPL antibody in body fluids taken from subjects
suspected of suffering from an aPL antibody-me~ te~l disease comprising
contacting a sample of a body fluid with an analog of an epitope which
specifically binds aPL antibodies and det~ hling by methods well known in the
art whether aPL antibodies are present in the body fluid and, if present, '
quanlilaLing the amount of aPL antibodies present in the fluid. One such
immunoassay comprises: (a) coating wells of a microlil~Lion plate with an analogof an epitope which specifically binds aPL antibodies; (b) washing the wells to
wash away unbound analog; (cladding a test sarnple of a body fluid to the wells
and incubating for a pre-deterrnined 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 predetermined 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 determinethe presence of anti-aPL antibody in the test sarnple. A diagnostic imrnunoassayas described above wherein the imm~.no~cs~y is quantitative is also encomp~cseclThe phage-ELISA assay consists of (i) coating a uniform arnount of
dirre,c.,~ 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-hurnan 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 synth-oci7ed and ranked for strength of binding using col,~l,elilion
assays.
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
~ cell epitope(s).
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Yet another aspect of the invention is a composition for inducing specific
B cell tolerance to an aPL immunogen comprising a conjugate of a
nonimmlmogenic valency platform molecule and an aPL antibody-binding analog
that (a) binds specifically to B cells to which an aPL inlinllnogen binds and (b)
lacks T cell epitope(s).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows that the substitution of fish gelatin for adult bovine senitn
abolished all anticardiolipin (ACA) activity in an ELISA assay of a commercial
aPL antibody standard. This result supported the fin-1ings of McNeil et al., supra,
and Galli et al., supra, concerning the importance of ~2-glycoprotein I (~2-GPI) in
defining the target epitope(s) of ACA.
Figure 2 shows that resin-bound analog SAI2 immunospecifically binds to
affinity-purified IgG designated ACA-6501.
Figure 3 illustrates that the aPL antibody-binding analogs derived from
screening with ACA-6626 bound aPL antisera but did not bind normal sera.
Figure 4 also illustrates that the ACA-6501/5A12 analog
immunospecifically binds ACA-6501 antiserum and is crossreactive with ACA-
6626.
Figures 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 pre~elel-lially with the s~l~ellillg antibody, a
significant degree of crossreactivity was detecte-l
Figure 6 illustrates method for calcul~ting the GPL value for ACA-6501
aPL antibody.
Figure 7 shows the activity of affinity-isolated ACA-6501 compared to
GPL standard sera.
Figure 8 illustrates the dramatic drop in se~uence diversity of the isolated
clones by the fourth round of biopanning.
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11 '
Figure 9 illustrates that three clones (3AI2, 3B3 and 3A5) exhibited a very
strong imrnunospecific signal in the phage-capture ELISA using ACA-6635
whereas all clones tested were unreactive with normal IgG.
Figure 10 shows the strong signal exhibited by seven clones in a phage-
ELISA using ACA-65 0 1.
Figure 11 shows the results of a co~ e-binding ELISA obtained with
peptides 5A12, CB2 and 3B10 using ACA-6501. 0.16 ~lg of Peptide 5AI2
produced 50% inhibition of binding of ACA-6501 aPL antibody to tetravalent
peptide ACA6501/3B10 bound to polystyrene microplate wells, whereas 0.08 ~lg
of CB2 and 0.54 ~g of 3B10 were required to produce 50% inhibition.
Figure 12 illustrates the comparative activity of modified ACA-6641 /3G3
analogs.
Figure 13 illustrates that the 50% inhibition values for peptides 139, 142
and 143 in a competitive-binding ELISA usi ng ACA-6501 aPL antibody were
6.9, 0.7 and 0.9 llg, respectively.
Figure 14 illustrates the effects of sub~liluling a-Me-Pro at positions 3, 9
and both 3 and 9 in peptide 3B 10. Substitution of a-Me-Pro at position 9
increased activity of the peptide four-fold co~ ~ed to the "native" peptide.
Figure 15 shows that peptide 6641/3G3 (LJP 688) is highly cross-reactive
with nine affmity-purified ACA antibodies.
Figures 16 and 17 show the dose-dependent reduction in anti-685 antibody
ABC at 108M using spleen cells from mice -immnni7Pd with LJP 685-KLH after
the spleen cells were incubated with 100, 20 and 4 ~lM of LJP 685-MTU-DABA-
PEG conjugate (compound 36 and LJP 685-ATU-MTU-AHAB-TEG conjugate
(compound 35), respectively, for 2 hours.
Figure 18 displays the NMR structure closest to the centroid of the nine
- structures elucidated for peptide 925 and is a reasonable re~leselltation of the
shape of the peptide 925 molecule.
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Figure 19 compares the structure of peptide 925 (labeled at the bottom of
the figure as 3G33 with the str~cture of peptide 5A12. Both peptides have turns at
approximately the same positions in the peptide sequence.
Figures 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 92S are tentatively
identified as the ph~ cophore of this peptide as shown in Figure 20A. The
lengths of the hydrocarbon linkers that tether the phan-nacophore groups to somescaffold are specified in Figure 20A as well as the distances s~ g the points
at which these linkers are attached to the scaffold.
Figure 21 illustrates inhibition of ~2GPI by 6501-derived peptides with
diluted 6501 serum.
Figure 22 shows the ACA-6701 titration of CB2*-F;
FITCGPCILLARDRCG .
Figure 23 shows the ACA-6501 titration of CB2*-F:
FITCGPCILLARDRCG .
Figure 24 shows complete ACA-6501 titration of CB2*-F:
FITCGPCILLARDRCG .
Figure 25 shows the displacement of CB2*-F from ACA-6701 using 1.04
equivalents of CB2*.
Figure 26 shows cFP titration using CB2* to displace CB2*-F from
ACA6701.
Figure 27 shows cFP titration using 3B10 to displace CB2* -F from
ACA6701.
Figure 28 shows the dose response of the (LJP685)4/MTU-AHAB-TEG
conjugate for tolerance activity.
Figure 29 shows the dose response of the (LJP685)4/MTU-DABA-TEG
conjugate for tolerance activity.
Figure 30 shows the dose response of tolerance activity of the
(LJP685)4/MTI~-DABA-TEG conjugate tested in the in vitro model.
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13
Figure 31 shows the dose response of tolerance activity of the
(L~P685)4/MTU-DABA-TEG conjugate tested in the in vitro model.
Figure 32 shows the tolerizing effect of the (LJP685)4/MTU-AHAB-TEG
- conjugate co~ )~;ng various ~rlmini~trative routes and dosage ranges.
s
MODES FOR CARRYING OUT THE INVENTION
A. Definitions
As used herein the term "aPL antibody" means any antibody which
specifically binds ~2-GPI that mediates disease.
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 imm~tllre and/or mature B cells and/or the inability
of B cells to produce antibody.
"Unresponsiveness" means a therapeutically effective reduction in the
humoral response to an immunogen. Qu~lli~lively the reduction (as measured by
reduction in antibody production) is at least 50%, preferably at least 75%, and
mostpreferably 100%.
"AntibodY" means those antibodies which are T cell dependent.
As used herein the term "immunogen" means an entity that elicits a
humoral immune response comprising aPL antibodies. Immunogens 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 theld~,~ulic proteins, peptides and antibodies, and the like or
self-imrnunogens (autoi,l.nlullogens) such as those associated with antibody-
mediated hypercoagulability (stroke).
The term "analog" of an immunogen intends a molecule that (a) binds
specifically to an antibody to wl-iich the immunogen binds specifically and (b)
lacks T cell epitopes. Although the analog will normally be a fragment or
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14
derivative of the immunogen and thus be of the sarne 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 inllmmogen (e.g., the imrnunogen 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, earbohydrate,
lipid, lipopolysaccharide, nucleic acid or other biochemical entity. Further, the
chemical structure of neither the inununogen nor the analog need be defined for
the purposes of this invention.
The terrn "analog" of an immunogen also encomp~ses the terrn
"mimotope." The term "mimotope" intends a molecule which co"lpelilively
inhibits the antibody from binding the immunogen. Because it specifically binds
the antibody, the mimotope is considered to mimic the antigenic detennin~ntc of
the immunogen.
As used herein "valency platforrn molecule" means a nonimmnnogenic
molecule cont~ining sites which facilitate the at~chment of a discreet number ofanalogs of imrnunogens.
~'Nonimmuno~enic" is used to describe the valency platform molecule and
means that the valency platform molecule elicits substantially no imrnune
response when it is ~1minictered by itself to an individual.
As used herein "individual" denotes a member of the m~mm~ n species
and includes hllm~nc, primates, mice and dornestic ~nimz~lc such as cattle and
sheep, sports ~nim~lc such as horses, and pets such as dogs and eats.
As used herein "pharmaeophore" means the three ~1imPncional orientation
and chemical plupel Lies of key groups involved in binding of an aPL analog to the
antibody target.
B. Identification of aPL Antibody-binding Analo~s
aPL antibody-binding analogs may be identified by screening candidate
molecules to determine whether or not they (a) bind specifieally to aPL antibodies
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and (b) lack T cell epitopes. Specific binding to aPL antibodies may be
determined using conventional immllno~ ys 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 analo~ which "binds specificàlly" to serum
antibodies to the immunogen exhibits a reasonable affinity thereto, e.g, 107M-I.The presence or absence of T cell epitopes may be determined using a tritiated
thymidine incorporation assay disclosed in Serial No. 08/118,055. The presence
of T cell epitopes can also be determined by measuring secretion of T cell-derived
0 Iymphokines by methods well known in the art. Analogs that fail to induce
statistically significant incorporation of thymidine above background are deemedto lack T cell epitopes. It will be appreciated that the ~uantitative 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 cell1 5 epitopes.
C. Pl~p~.llion of Conju,eates
The aPL antibody-binding analogs are coupled to a nonimmunogemc
valency platfor;n molecule to prepare the conjugates of the invention. Preferredvalency platform molecules are biologically stabilized, i.e., they exhibit an in vivo
excretion half-life oRen 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. P~efe.led
polymers are based on polyethylene glycols (PEGS) having a molecular weight of
about 200 to about 8,000.
Other valency platform molecules suitable for use within the present
invention are the chemically-defined, non-polymeric valency platform molecules
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. 16
disclosed in co-owned, co-pending U.S. patent application Serial No. 08/152,506,filed November 15, 1993, which is incorporated by reference herein in its entirety.
Particularly plcr~ d homogeneous chemically-defined valency platform
molecules suitable for use within the present invention are derivatized
2,2'-ethylenedioxydiethylarnine (EDDA) and triethylene glycol (TEG).
Additional suitable valency platform molecules include
t~ obenzene~ hept~minobetacyclodextrin, tetraaminol)enlaerytbritol~
1,4,8,1 I tetraazacyclotetra(~ec~ne (Cyclam) and 1,4,7,10-tetraazacyclododecane
(Cyclen).
Con}ugation of the aPL antibody-binding analog to the valency platform
molecule may be effected in any number of ways, typically involving one or more
crossliriking agents and functional groups on the analog and valency platform
molecule.
Polypeptide analogs will contain amino acid sid;~..ain 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 functionalgroups 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 orrecombinant 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 ch~ try. For in~t~nce,
primary amino groups may be incorporated by reaction with ethylen~ mine in
the presence of sodium cyanoborohydride and sulffiydryls may be introduced by
reaction of c~ ehn.~ 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
applopl;ate functional groups.
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
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17
linkers with the formula RlS(CH2CH20)n.CH2CH20(CH2)mCO2R2 wherein n = 0-
200? m = 1 or 2, Rl = H or a protecting group such as trityl, R2 = H or alkyl oraryl, e.g., 4-nitrophenyl ester. These linkers are useful in connecting a molecule
cont~ining a thiol reactive group such as haloaceyl, maleiamide, etc., via a
thioether to a second molecule which conlains an amino group via an amide bond.
These linkers are flexible with regard to the order of ~tt~chm~nt i.e., the thioether
can be formed first or last.
The conjugates will normally be formulated for ~lmini~tration by injection
(e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.).Accordingly, they will typically combined with pharmaceutically acceptable
vehicles such as saline, Ringer's solution, dextrose solution, and the like. Theconjugate will normally constitute about 0.01% to 10% by weight ofthe
formulation. The conjugate is ~imini~tered to an individual in a ''therapeutically
e~fective amount", i.e., an amount sufficient to produce B cell anergy to the
involved irnmunogen and effect prophylaxis, improvement or elimin~tion 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 ,ug to about 100 mg
conjugate/kg body weight, preferably about 100 ~lg to about 10 mg/kg body
weight, will be given weekly. Other a,o,ulo~iate 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 ~mini~trations, norrnally timed
according to B cell turnover rates, may be required to achieve and/or m~int~in astate of humoral anergy. Such re~elili.le ~lmini~trations will typically involvetreatments of about 1 llg to about 10 mg/kg body weight or higher every 3 0 to 60
days, or sooner, if an increase in antibody titer is detected. Alternatively,
sllct~ined continuous release formulations of the conjugates may be indicated for
some pathologies. Various forrnulations and devices for achieving sustained
release are known in the art.
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18
Anti-helper T ceil treatments may be ~rlmini~tered together with the
conjugates. Such treatments usually employ agents that suppress T cells such as
steroids or cyclosporin.
D. Nature of the anti~een for aPL antibodies
S 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 Figure 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 plep~dlion obtained commercially as an ACA standard.
This finding indicated the ACA antibodies recognized a determin~nt on a serum
protein as proposed by McNeil et al., supra and Galli et al., supra, rather thancardiolipin itself This protein was shown by these authors to be ~2-GPI and, thus,
the terms "ACA" or '~anti-cardiolipin antibodies" are really misnomers but are still
used today to refer to these antibodies.
The majority of autoimmune, IgG aPL antibodies recognize determin~nt.
on the ,B2-GPI molecule. These epitopes may be formed or exposed on ~2-GPI
only upon its binding to cardiolipin (a neo epitope). Alternatively, the epitope on
,B2-GPI may exist in a single copy per ,B2-GPI molecule and may have low affinity
for aPL antibodies. Sufficient avidity to m~int~in an antibody-antigen interaction
might be reached only with the alignment of two of these sites on adjacent ~2-GPI
molecules by their binding to cardiolipin.
-- Additional insight into the ~2-GPI epitope structure has been gained by
nuclear magnetic .~sonallce (NMR) analysis of aPL analogs within the present
invention which mimic the native epitope(s) of ~2-GPI. For, example, a
comparison of the NMR solution structure of two peptide aPL analogs that are
highly crossreactive with aPL antibodies showed that both peptides have turns atapproximately the same positions in the peptide sequence (see Figures 18 and 19).
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E. ACA ELISA
The necessity of large-scale testing in connection with GPL scoring of
clinical samples and chromatographic purifications of research antibodies and
~2-GPI led to the development of an ELISA assay for aPL antibodies with
perforrnance in close agreement with a cornmercial kit and similar to the designfound to have the best reproducibility (Reber et al., supra). A modified ACA
ELISA has also been performed wherein ~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 & ~h~llm~ticm
39:1606-1607; R.A.S. Roubey (1996) Arthritis & Rhenm~ti~m 39:1444 1454
F. Imrnnnoaffinity Purification of aPL Antibodies
In order to isolate the aPL antibodies, multilarnellar, cardiolipin-
cont~ining 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
colurrm. 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 bufferexchanged, and tested in the ACA ELISA. This procedure
yields aPL antibody enriched up to I 0,000-fold that is devoid of any
cont~min~ting ~2-GPI as shown by western blotting with rabbit IgG anti-human
~2-GPI antisera. An additional affinitypurification step is performed by
chromatography of the affinity-purified antibody on solid phase ~2-GPI. This
second affinity-purification step is ~co,l~l~.ended as a result of the new awareness
rega~ dhlg the greater clinical relevance of aPL antibodies that directly bind to
~2-GPI. It also serves to further ensure a final p,e~dlion devoid of
cont~min~nt~, in particular ,B2-GPI.
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G. Construction of Filamentous Pha~e Random Peptide Libraries
Eleven different FTJSE 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, .~esign~te.l "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 eysteine-bounded inserts that are 6, 7, 8, 9, 1 1, and 13
amino acids long. The "y" library is the same as the "y"' library except that itlacks the 6 and 8 arnino 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 terrninllc of the
native p-III protein. The "z" library consists of random eight amino acid inserts
located at the amino terrninus of the p-III protein and do not contain any fl~nking
proline or cysteine residues. A cornbination of the "x," "y"' and "z" libraries
represents eleven different libraries each with approximately one hundred million
different peptide inserts.
These libraries are constructed by incorporation of random oligonucleotide
sequences of the length app,opl;ate 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 kin~ecl
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-cont~ining media. The phage from this culture (which contain the
peptide insert) are isolated from the supernatant~ washed and resuspended in
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buffer. Typically libraries are shown to have 7X108 independent clones at 8X10l2transducing units per mL.
~. Pha~e-screenin~ Methodology
The essence of screening phage display peptide libraries lies in the ability
to collapse billions of potential candidate phage to a relative few with 0l1tct~n-1ing
l,lo~e.lies. The original screening protocols reco.. ~n-led 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 sequencescan be thoroughly investig~te~l 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 theother 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 developedwith 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.
(i) Biopanning
"Biopa ~ g" describes the technique wherein affinity-purified aPL
antibody and phage bearing random peptide inserts are allowed to mix, following
which antibody-specific recovery c~ ,s the bound phage. The phage confer
tetracycline re~i~t~nce to E. coli that are prop~gAted in a tetracycline-cont~ining
medium and then isolated. Multiple rounds of biopA~ g 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
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22
nonspecifically bound at low affinities for the selecting antibodies. A method for
further evaluating these phage (miclop~ ing) is required.
(ii) Mi-,~ op~ ~lling
An estim~tion of the relative strength of binding of the phage to the aPL
antibody can be determined by "microl)An~ -g " Miclopal~l~,ng is carried out
following three or more rounds of bio,oalllling and uses the same antibody as
employed in the bio~ g method. The method consists of dilution of the phage
from the !ast round of bi(,p~ g and analyzing fifty or more of these clones by
miclol,a~ g. Miclop~l~"ing 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 cor.centration most likely to reveal
the widest range of miclop~ g scores (from 0 to 4+) and~ thus, permit the
greatest discrimination among the clones being tested. Il lS based on the
'mi~,lvpdl)llmg 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 withmild acid followed by neutralization and infection of E. coli. The nurnber of
infected ~. coli are then quantitated by plating the microorg~ni~m~ on agar plates
cont~ining tetracycline and then dete~ inillg colony densities achieved by each
clone.
(iii) Phage-Capture ELISA
The phage-capture ELISA test was developed to provide an intermediate
level assay to bridge the gap between the relatively low stringency of the
micl~ ing assay and the high stringency of the phage- or peptide-ELISA
assays. Preliminary studies show that some antibody plepaldlions give too many
positive clones by micropanning but none by phage-ELISA or peptide-ELISA.
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The limitation of the phage-ELlSA described below is that only five copies of
p-III are located on each phage and even with a large nurnber 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.
The phage-capture ELISA consists of the following steps. Microtitration
wells are coated with aPL antibody and phage clones are added as in the
mi~;lop~ ing 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 microp~nning-positive phage are phage-captureELISA positive. As a result, peptides synthesized from positive phage-capture
ELISA phage are generally immunoreactive.
(iv) Phage-FLISA
This method of selecting phage requires very tight binding of the insert to
the screening antibody. Phage are directly coated onto wells of a microtitrationplate and incubated with the screening antibody. Following washes to remove
unbound antibody, an anti-human IgG ~ lin~o phosph~t~ce 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.
(v) Colony Blot
This assay allows large-scale colony scleening of ~. coli infected by
biopanned phage. This procedure is an alternative to phage-ELISA for identifyingimmunoreactive clones and exhibits a comparable level of sensitivity without
requiring culturing of individual phage clones prior to testing. In this assay, E.
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coli infected with phage from a round of biop~nning are spread on a large
diameter nitrocellulose (NC) membrane and cultured overnight on the surface of
an agar plate cont~ining tetracycline (Barbas et al. (1991) Proc. Natl. Acad. Sci.
USA 88:7978-7982). Each colony results from infection by phage cont~ining
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 iitm-iunobtotting. Blots that have been blocked may be insub~t~d 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 allowsone to localize the discrete colonies in the master plate which replese
immunospecific phage that may be cloned for further study.
(vi) Peptide-ELISA
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-ELlSA assay, the peptides can be made, for exarnple, as branched
tetravalent molecules, i. e., each molecule has four copies of the insert. Such a
molecule can coat the well of a microlill~tion plate and still have epitopes exposed
to the solution to allow binding by an antibody. The tetravalent peptides are
synthPsi7Pd by incorporating Iysines as branch points at the first two couplingsanalogous to the methods used for Multiple Antigenic Peptides (MAPS) (Posnett
et al. (1988) J. Biol. C~em. 263:1719-1725). A spacer con~i~ting of glycine-
serine-glycine-serine is added on each arm after the Iysines and then the insert,
including the framework amino acids found in the phage, proline-glycine at the
carboxyl ten-ninus and alanine-glycineproline at the amino terminus. All amino
acids in this synthesis are added one at a time using standard Fmoc methods.
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These peptides are then assayed by ELISA which is carried out by coating
the peptides on miclo~ dLion wells and then assaying their reactivity with aPL
antibody in a standard ELISA format. In practice, the peptides usually bind verystrongly to the original screening antibody and show some cross-reactivity with
other aPL antibodies. Controls of non-aPL antibodies are included to elimin~te
nonspecific binding peptides.
(vii) Competitive Binding Peptide-ELlSA
Once ELISA-positive peptides are identified, it is necessary to quantitate
their relative binding affinity to the aPL antibodies and to deterrnine whether or
not two peptides bind the same population of antibodies in a given patient serumvia 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,
i.e., without the Iysine 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 miclotilldlion 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 inrllb~tin~ the antibody and monomeric
peptides for one hour, the antibody/peptide solutions are added to the
miclolilldlion 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 dçtçrrninPrl by plotting the calorimetric
readings obtained for each well. The 50% inhibition point is used as the measureof the relative strength of binding for the monomeric peptides.
A variation of this assay uses micloLil-dlion plates coated with human
-glycoprotein I/cardiolipin (~2.GPI/CL) instead of tetravalent peptide and tests
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the ability of monomeric peptides to block the binding of aPL antibody to the
epitope(s) on ~2 GPI/CL. In this assay, IgG-depleted hurnan serum at an
optimized concentration is used as a source of ~2 GPI. The monomeric peptides atseveral concentrations are incubatecl with an optimized concentration of aPL
antibody in a manner analogous to the assay which employs tetravalent peptide asa plate substrate. Following the incubation of aPL/peptide in (~2 GPI/CL) plates,
antibody binding and the peptide concentration required for 50% inhibition is
deterrnined at half-maxim-al absorbance as in the tetravalent assay.
An additional variation of this assay tests the ability of monomeric
peptides to block the binding of aPL antibody to ,~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.
(viii) Fluorescence Polarization Peptide Binding Assay
This assay detects direct binding of the peptide to aPL antibody. Since
aPL antibodies bind to ,B2 GPI (the antigen~, the ELISA competitive inhibition
assay can show inhibition due to binding to ,B2-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 essenti~l toestablish that a peptide can directly bind to an aPL antibody. This assay is used to
deterrnine the dissociation con~t~nt~ for peptides that bind to two aPL antibodies,
ACA-6501 and ACA-6701. While the ELISA assay is useful for high throughput
s~ g because it requires less antibody than the Fluor~sct~n~e Polarization
assay, however, ELISA-positive peptides should be fflfther evaluated by the
Fluorescence Polarization assay to deterrnine whether they are capable of directly
binding with aPL antibody.
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-
(ix) Evaluation of amino acid contributions to-binding by
substitution and deletion synthesis
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
s ~Innecess~ry residues. In order to .~.duce the minimllm 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 terrnini are deleted, or (ii) inwhich 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 ieucine, or unnatural, e.g., alpha methyl proline for
proline. The effect of these deletions and/or substitutions are then measured via
peptide-competition ELISA.
(x) Grouping of aPL Sera Specificities by Mutagenesis of the
5th domain Of ~2 GPI
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 deterrnine ifseveral antibodies from different patients bind identical residues within the eighty-
four amino acid 5th domain of ~2-GPI which has been suggested by others to
contain the target epitope. If several antibodies bind identical residues, a single
'0 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 ,B2-GPI. The resl.lting mutant
~2-GPIs were assayed for reactivity with several aPL antibodies. The results were
inconclusive. A fusion protein comprising the 5th domain Of ~2 GPI and
glutathionine S transferase (GST) was obtained from A. Steink~c~erer and
expressed in E coli (Steink~serer et al. (1992) FEBS Lett. 313:193-197). This
fusion protein was sllcces~fully substituted for native ~2 GPI in the ACA ELISA.Amino acid substitutions are engineered using standard sitedirected mutagenesis.
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. 28
I. Isolation of SYnthetic aPL Epitopes
Antibody ACA-6501, from a patient with a GPL score of 151 (high titer)
and a historyof recurrent stroke, fetal loss, lupus and three aortic valve
replacements was immunoaffinity purified on dardiolipin liposomes. The
antibody was used in four separate phage library screens, using the xy, the xyz,the xy'z, and a special pro/cysbounded 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 micropalmed (out of 140 tested). These
appear quite homologous and the conserved DR residues at positions 6 and 7 are
l O 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 uniquesequences that were phage-ELISA tested. None were coi~)r 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.
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29
TABLE I
ACA-6501 Pha~e Library Sequences
Clone Sequence Clone Sequence
xy Library
2101 CNILV~LDRC
xyz Library
5A12 CLILAPDRC 3CIOCILLAKNRC
2D7 CLLLAPDRC 3C5 CIVLVPDRC
3B6 CLVLALDRC 2F4 CLVIALDRC
3E4 ' CLFVALDRC SBI CWFRSQSSC
3E7 CILLAHDRC 3El ICSPILRGNC
2HI CIILAPGRC 3E8 CHKFFWLTC
xy'z Library
2AI0 CTILAPDRC 2DI2CLVLAADRC
2G12 CLLITPDRC 3BIOCLLLAPDRC
2GII CLLITHDRC 3F2 CFFHFDHSC
2FlO CNILVLDRC 2D3 CPLHTHHTC
2E3 CPLITHDRC
Custom (X)6R Library
Gll CTILTPDRC IA4 CNLLALDRC
2H5 CTILTPDRC 2H6 CNLLAIDRC
2H2 CTILTLDRC lC3 CLLLAIDRC
2HIO CTLLTPDRC ~ IDIOCTIITQDRC
2EI0 CIQLTPDRC 2H4 CNIITRDRC
SUBSmU~E SHEEr ff~ULE 26)
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ACA-6501 Phage Library Sequences
Clone Sequence CloneSequence
IB7 CHLLTPDRC 2GI2CILHAAHRC
2Hl CLILTPDRC IA9 CSSKSYWRC
2HI2 CSILAPDRC
xy'z Libraly, Colony Blot Screening Assay
CB2 CILLARDRC
SUBSmUlE SHEEr (~UlE 26)
.
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31
TABLE 2
ACA-6501 Colorimetric ELISA-positive Phage
Clone Sequence Clone Sequence
5AI2 CLILAPDRC 3B6 CLVLALDRC
2HI CLILTPDRC 2GI 1 CTILTPDRC
3BlO CLLLAPDRC* 3E7 CILLAHDRC
2H2 CTILTLDRC
* Corresponds to consensus or average sequence
TABLE 3
ACA-6626 xy'z Phage Library Sequences
Clone Sequence Clone Sequence
4B11 CGNAADARC 4G7 CTNLTDSRC
4D3 CTNWADPRC 4A2 CGNPTDVRC
4C7 CGNIADPRC
ACA-6644 is another high-titered aPL antibody that was used to screen
thepooled P-IlI phage libraries according to mPthorl~ described herein. The
following sequences were discovered:
ACA-6644/CBe GILLNEFA
ACA-6644/CBd GILTIDNL
ACA-66644/CBf GILALDYV
SUBSnTUTE SHEEr ~n~ 26)
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32
These sequences all were derived from the component "z" epitope library
that lacks phage framework residues at the N-terrninus. When synthesized as
peptides the sequences were im.munoreactive with several ACA sera including
ACA-6644 and ACA-6501. Analysis revealed unsuspected homologies with the
S sequences previously obtained with ACA-6501 as illustrated in Table 4.
TABLE 4
ACA-6644/CBdGILTIDNL
ACA-6501/2H2CTILTLDRC
ACA-6644/CBf GILALDW
ACA-6501/2FIOCNILVLD~C
ACA-6644/CBcGILLNEFA
ACA-6501/lDIOCTIITODRC
The convergent sequence homology from two very ~ imil~r source
libraries screened by these two aPL antibodies suggests that the sequences may
mimic a major, perhaps imrnunodominant, region in the native target antigen.
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:
ACA-6701/3BI L SD P G yv R NIF H
ACA-6701/3EI L TDPR YTRDISNFTD
As resin-bound peptides, the sequences were strongly immunoreactive
with the parent serum (ACA-670 1) but were minim~lly cross-reactive with other
aPL antibodies.
Continued screening of random pIlI 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 perfonnance of this peptide as we}l as four others is shown in
SUBSlllUI E SHEEr fflULE 26)
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Table 5. Peptide sequences for all five peptides inunediately follow the table. All
were tested using the competitive binding peptide ACA ELISA using CL/~2 GPI
plates as described herein.
SUBSmUrE SHEET ffWlE 26)
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34
TABLE 5
Percent Inhibition of Af~ACA 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
I m ~mL
6501/3BIO 76 87 52 55 5] 87 47 69 60
I m ~mL
6626/4C7 88 96 45 45 69 99 54 77 39
68
I m ~mL
6701/3B1 43 65 43 37 96 72 23 51 41
52
1 m ~mL
6644/CBf 37 60 28 18 22 54 57 43 37
39
200~mL
Peptide Sequence
ACA-6641/3G3: - AGP CLGVLGKLC PG
ACA-6501/3BIO: AGP CLLLAPDRC PG
ACA-6626/4C7: AGPDNIADPRC PG
ACA-6701/3BI: AGP LSDPGYVRNIFH PG
ACA-6644/CBf: GILALDYV GG
-
SUBSrllUlE SHEEr ff~UlE 26)
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Subsequent testing deterrnined 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-di~ulfide cyclization
studies.
The motifs of several peptides selected from the random phage library
screening is set forth below.
TABLE 6
ACA Antibody Phage Insert Sequence
6501 C L L L A P D R C (3BIO)
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 LTDPRYTRDI SNFTD
6707 C A H P D W D R C
J. Immunoreactiviiv of aPL-related Peptides with Affiniiy Purified
IgG aPL Antibody
Phage sequences obtained with affinity-purified ACA-6501 and found to
be phage-ELlSA-posit ive were synth~i7~d on a solid support recently developed
for combinatorial synthetic peptide libraries. This support, a Rapp resin, has ahigh 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 Figure 2, peptide 5AI2
(sequence CLILAPDRC) dramatically outperformed an unrelated control peptide
while not significantly binding normal IgG. Similar results were obtained with
the other phageELlSA-positive peptides tested. In the experiment shown, resin
sussrrru E ~Er ~U~ 26)
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36
peptide-bound affinity-purified ACA-6501 aPL antibody was detected by an
irnmunoconjugate color reaction.
K. aPL Serum Antibody ReactivitY with Synthetic Peptides
The discovery that resin-bound peptides could bind aPL
immunospecifically using serum significantly ~nh~nred the ability to test aPL
antibodies. As shown in Figure 3, the-peptide derived from screening with ACA-
6626 bound aPL antiserum but did not demonstrate significant binding of normal
serum. Figure 3 also illustrates the immunospecific-binding behavior of ACA-
6501 -5A12 peptide towards aPL serum. Binding of norrnal serum to peptide
5A12was nil in data not shown.
L. CrossreactivitY of Synthetic Peptides Towards aPL Antiserum That
Was Not the Source of the Library Screenin~ Antibody
Two peptides were selected for aPL sera testing, one (5A12) Icplesellting
the ACA-6501 screen and another one (4D3) representing the ACA-6626 screen.
As shown in Figures 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 patho!ogic 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 19control sa~nples showed low peptide binding. These results evidence that
synthetic peptides are useful for diagnostic or prognostic assays in stroke patient
care.
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 Figure 14.
66~1/3G3 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.
SUBSmUrE SHEEr ff~ULE 26) -
. .
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TABLE 7
CROSSREACTIVITY DATA FOR
AGP-CLGVLGKLC-PG (LJP 688)
ACA SERA WITH CARDIOLlPfN/~2.GPI-COATED PLATES
ACA SERUM Nr. ICso~ mg/mL, for peptide LJP
688
[1~ 6501 RS, RFI, 0.8991.1
[2] 6626 RS 1.09
[3] 6635 RS
[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 i sd 0.86 +0.16
RS = recurrent stroke
RFL = recurrent fetal loss
M. New SYnthetic Peptide Methodolo,eies
The identification of new candidate sequences by aPL library screening
required testing of the new ~ylllll~lic peptides for antibody binding. With Rappresin of known peptide substitution, it is possible to carry out qu~llilali~re binding
studies such as saturation binding analysis and e4uilibrium measurements using
radiolabeled aPL IgG.
Peptide synthesis allows the molecular dissection of the mimotope by
selective synthesis. This includes the modification of each arnino acid along the
SUBSTllUlE SHEr ff~ULE 26)
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chain with the goal of enhancing antibody binding. Se}ective synthesis reveals the
relative importance of each arnino acid in the sequence. If necessary, selectivesubstitution at particular residue locations can be designed to m~int~in B cell
reactivity while abolishing any T cell proliferative reactivity discovered during
S T cell assays.
Peptide 6641/3G3 (LJP 688) was subjected to a number of analyses. The
analyses included truncation at both the N-terrninus and the C-terrninllc, disulfide
substitution, substitution of alanine and glycine for arnino acids in positions 2
through 8, substitution of branched aliphatic arnino acids in positions 2, 4, 5 and
8, substitution of amino acids affecting conformation of the peptide, including
substitution of a-methyl arnino acids, substitution of basic arnino acids in position
7, substitution of D-amino acids in positions 2 through 9, and the substitution of
N-a-methyl arnino acids in positions 1 through 9. The results are shown below inTable 9. The structure/activity relationship of these substitutions and truncations is
shown in Table 8.
SUBSTITUlE SHEEr ffUllE 26)
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TABLE 8
Optimization Of Peptide 3G3
Total Peplides 144 ¦
3n8t96 , with ACA 6501
Position Numbtr ¦ -2 - I O 1 2 3 4 5 6 7 8 9 A B ¦ I S0 ~I~JmL std
TruDcntion ~n~lysis
A G P C L G V L G }; L C P G 850
2 G P C L G V L G }; L C P G 180
3 P C L G V L G ~; L C P G 420
4 C L G V L G ~ L C P G 460
S A G P C L G ~ L G li L C G 450
6 C L G V L G ~; L C 10 #906 (LJP 690)
Disuladt surrogst~s
C L G V L G li L C 110 #952=100
2 Hc L G V L G ~; L C _ 46
3 C L G V L G ~; L tL~ 9~
4 H~ L G V L G ~ L Hc 80
Al~ c~n
C A G V L G ~ L C 200 #952 - 80
2 C L A V L G ~; L C 120
3 C L G A L G ~; L C 260
4 C L G V A G l; L C inf
C L G V L A l; L C 40
6 C L G V L G A L C inf
7 C L G V L G ~; A C 360
Gly cnn
C G G V L G ~; L C 310 #952 = 120
2 C L G G L G }; L C inf
3 C L G V G G }; L C inf
4 C L G V L G G L C inf
C L G V L G 1: G C SoO
SUBS~llUlE SHEET ffWlE 26)
' CA 02256449 1998-11-23
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. 40
Sequence optim. Branched ~liph-tic Nmino ~cids
I CIGVLG~LC 60 ~910=40
2 CVGVLG~ L C 130
3 CMGVLG~LC
4 CCyGV L G~ L C
S C tL G V LG}; L C
6 C mL G V LG~LC
7 ClvGVLG~LC
8 CLGL L G~ L C
g C L Gl L G~LC
CLGM L G~ L C
Il C L GC~LG~LC
12 CLG tL L G ~ L C
13 C L GmL L G 1~ L C
14 C L G IY L Gl~ L C
C L GVI G }~ L C
16 C L GVVG~ L C
17 C L GV M G ~ L ~.
18 CLGVC~ G ~ L C
19 C L GV tL G~LC
CLGV mL G~ L C
21 CLGVlvG~ L C
22 C L GVLG~IC
23 CLGVLG~VC
24 C L GVL G }; M C
2S C L GV L G~C~C
26 C L GV L G ~ tL C
27 CL G V LG~ mL C
28 CLGVLG~lvC
SUBS'rlTUlE SHEEr (~ULE 26)
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41
C~ scanaminoNcids
C L P V L G ~; L C
2 C LmP V L G ~ L C
- 3 C LmA V L G l;LC
4 C LcG V L G }~ L C
S C L G V L P }~ L C
6 C L G V L mP }~LC
7 CL G V L mA ~; L C
8 CLGV L cG li L C
g CL dA V L G 1: L C
C L GVL dA ~ L C
I I C P G V L G l;LC 160 #952 = 100
12 C L P V L G l;LC 340 (Pro Scan)
13 C L G P L G ~; L C 180
14 C L G V PG};LC inf
CLGVLP~LC 350
16 C L GV L G P LC 160
17 C L G V L G }; P C 550
18 C pG GV L GTi L C 130 #910=40
19 C L pGV L G~; L C 70
C LG pG L G~; L C 70
21 C L G V pG G l; L C 35
22 C L G V L pG ~; L C 30 #910 = 5
23 C L G V L G pG L C 230
24 C L G V LGI; pG C 80
slpha Mc AA 2~ C aiB G V L G l; L C 370 #910 = 40
26 C L aiB V L G l; L C 80
27 C LG aiB L G}; L C 110
28 C L G V aiB G~; L C inf
29 C L GV L aiB }~LC 110
C L G V L G aiB L C 460
31 C L G V L G }~aiB C >470
Basic amino ~cid
C L GV L GR L C 160 #952= 120
C L GVL G OrLC 40 910 = 60
3 C LGV L Gm~;LC
SUBSmUlE SHEEl ~WIE 26)
. . .
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42
D ~Imino ~cids (lowrr crsr d)
dPC L G V L G K L C 120 #952 = 100
2 C dL G V L G K L C 180 #952 = 30
3 C L G dV L G ~; L C 260 #952 = 30
4 C L G V dL G }~ L C 230 #952 = 30
C L G V L G dK L C 170 #9S2 = 50
6 C L G V L G ~; dL C 140 #952 = 30
7 C dL G dV L G J; L C
8 C dV G dV L G l; L C
9 C dL G dL L G li L C
C L G dV dL G K L C 50 #952 = 30
Il C L G dL dV G K L C
11 C L G dV dV G l; L C
13 C L G dL dL G K L C
14 C L G V dL G dl; L C
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 dl; dL C 150 #952=S0
19 C L G V L G dL dK C
C L G V L G dK d~; C
21 C L G V L G dL dL C 140 #952=30
2' C L G V L dA }~ L C 140 #952 = S0
C L G V L G li L dC
I~'-,~lc smino ~cids ~nAA)
nC L G V L G l; L C
2 ~' C L ~G ~ ':V .L ~ -~ G ~ .~; .i C
. ~ . , .. ... ,~ , ..... ...
3 C L nG V L G }~ L C 70 #952= S0
4 C L G nV L G K L C 220
C L G V nL G ~; L C 180
6 C L G V L nG ~ 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 = S0
9 C L G V L G ~ L nC
SU~STllUrE SHEEr ~WLE 26)
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43
Others
C L G V L G 1;. L C ATU Y 300 ~952 z 50
2 Y ATU C L G V L G ~; L C 170
3 C L G V L G l; L C TGR resin 500
4 C L G V L G l; L C dC TGRresin 620
dC L G V L G 1~ L C C
6 cC L G V L G ~ L C dC TGR resin
Thioelhtrs: sin~le delection
Hc L G V L A }~ L C
2 Hc G V L A li L C
3 Hc L V L A li L C
4 Hc L G L A ~ L C
Hc L G V A 1~ L C
6 Hc L G V L ~; L C
7 Hc L G V L A L C
8 Hc L G V L A l; C
shrink I
Hc V L A }; L C
2 Hc L A l; L C
3 Hc L A li C
br~lnch~d
Hc L G V L G l; L Pe
2 Hc L G V L G li L dPe
3 Pe L G V L G ~; L Hc
4 dPe L G V L G }~ L Hc
dPe L G V L G l; L dPe
D ~mino ~cids
dHc L G V L G ~; L C
2 dHc L G V L G }; L dC
3 Hc L G V L G l; L dC
SUBSrllUrE SHEEr ~IE 26)
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44
Des ~mino
H~ L G V L G }; L Hc desamino
2 B~ L G V L G l; L C desamino
HCTE
3 Pp L G V L G }~ L ~Ic desamino
CHTE
4 Pp L G V L G ~; L C des~mino
CHTE
SUBSmUrE SHEEr (RlJLE 26)
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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 Iysine
mK......... N epsilon-methyl lys ine
Pe......... penicillamine
SUBSlllUrE SHEEl' ffWI~ 26)
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46
Abbreviations
By......... butyroyl
Pp......... proprionyl
Or......... omithine
TABLE 9
Structure Activily Relationship of the Crossreactive ACA Epitope
Relative
Structure Activity
AGPCLGVLGKLCPG (3G3) (LJP 688) 100
CLGVLGKLC (LJP 690) 3.5
CLGVLAKLC 1.8
C~GVLpGKLC 1.4
CLGdVdLGKLC 5.9
HCLGVLGKLC (thioether) 1.6
SLGV-LGKLS 00
CLGVAGKLC 00
CLGVLGALC 00
N. Use of a-methvl Amino Acids Substitutions and Unnatural Amino
Acids to Enhance Peptide ImmunoreactivitY and Confer Resistance
to Protease Attack
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 ,B-turn.
~-turn mimetics can be used to enhance the stability of reverse tD confon-
SUBSmUI E SHEEr fflULE 26)
- CA 022~6449 1998-ll-23
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nations in small peptides. Such a mimetic is (S~ methyl proline (a-MePro), a
proline analog that, in addition to stabilizing turn confo~nations, confers
resi~t~nce to protease degradation. Protease resi~t~nre is a desirable prop~lly for a
potential dlug designed to act in the plasma. Peptide ACA-6501/3B10
S AGPCLLLAPDRCPG (insert highlighted) is a consensus peptide. It has a
sequence featuring the most prevalent residue at each position based on a
con~ u;son 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 hlll~ol ~ fintlin~ 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 a-MePro for proline at the 9-position. This
substitution led to a six-fold enhancement in immunoreactivity.
The results of a-methyl and a-methyl amino acid substitution studies
carried out on peptide 6641/3G3 are shown above in Table 8.
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"-alkyl amino acids, alpha-alkyl amino acids, cyclic amino acids,
chirrieric 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 consllai
to improve biological activity (Hruby et al. (1990) Biochem. J268:249-262;
Hruby and Bonner (1995) Me~hods in Molecular Biology 35:201-240).
The most common Na~alkyl amino acids are the NB-methyl amino acids,
such as Na-methyl cysteine (nC), Na~methyl glycine (nG), Na methyl leucine (nL),N~~methyl Iysine (nK), and Na methyl valine (nV). Examples of alpha-alkyl amino
acids include alpha-methyl alanine (niA), alpha-aminoisobutyric acid (aiB), alpha-
methyl proline (mP), alpha-methyl leucine (mL), alpha-methyl valine (mV),
SUBSmU~E SHEEr (RULE 28)
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48
alpha-methylalpha-aminobutyric acid (tv), diethylglycine (deG), diphenylglycine
(dpG), and dicyclohexyl glycine (dcG) (Balararn (1992) Pure & Appl. Chem.
64:1061-1066; Toniolo et al. (1993) Biopolymers 33:1061-1072; Hinds et al.
(1991) Med Chem. 34: 1777-1789).
Exarnples of cyclic amino acids include 1-annino-1-cycloplopane
carboxylic acid (cG), I-amino-1-cyclopentane carboxylic acid (Ac5 c),
I-arnino-l-cyclohexane carboxylic acid (Ac6c), arninoin.dane carboxylic acid
(ind), tetrahydroisoquinoline carboxylic acid (Tic), and pipecolinic acid (Pip) (C.
Toniolo (1990) Int'l. ~. Peptide Protein Res. 35:287-300; Burgess et al. (1995)
J. Am. Chem. Soc. 117:3808-3819). Exarnples of chimeric arnino 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.
Exarnples of miscellaneous alpha arnino acids include basic amino acid analogs
such as omithine (Or), N~-methyl Iysine (mK), 4-pyridyl ~l~nine(pyA), 4
piperidino alanine (piA), and 4-aminophenylalanine; acidic amino acid analogs
such as citrulline (Cit), and 3-hydroxyvaline; aromatic arnino acid analogs such as
I-naphthy~ nine (l-Nal), 2-naphthyl~ nine (2-Nal), phenylglycine (pG), 3,3-
diphenyl~l~nine (dpA), 3-(2-thienyl)alanine (TE). and halophenylalanines (e.g., 2-
fluorophenyl~l~nine and 4-chlorophenylalanine); hydrophobic arnino acid analogs
such as t-butylglycine (i.e., tertiary leucine (tL)), 2-arninobutyric acid (Abu),
cyclohexyl~l~nine (Cy), 4-tetrahydropyranyl alanine (tpA), 3,3-dicyclohexyl
alanine (dcA), and 3,4-dehydroproline.
In addition tg alpha-arnino acids, others such as beta amino acids can also
be used in the present invention. Exarnples of these other amino acids include 2-
arninobenzoic acid (Abz), ~-aminoplopalloic acid (~-Apr),7~-aminobutyric acid
(~-Abu), and 6-arninohexanoic acid (~-Ahx). Carboxylic acids such as 4-
chlorobutyric acid (By) and 3-chloro,t,ro~ionic acid (Pp) have also been used asthe first residue on the N-terîninal in the synthesis of cyclic thioether peptides.
SUBSlTrU~E SHEEI ffWLE 26)
,
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49
O. Preparation of CYclic Thioether Analogs
T'he 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 elimin~te 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 I ~ 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 platforTn molecule
conjugates.
Four cyclic thioether analogs were ~re~ed 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 RilllCTM
arnide 4-methyl benzhydrylarnino resin or MBHA resin, fall 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.
CCTE HCTE
S I . ~ S~
AFLGVLG'KLAF-NH2 AFLGVLGKLAF-NH2
CHTE HHTE
~S ~ ~S ~
Af LGVLGKLAF-NH2 AFLGVLGKLAF-NH2
AF =--HN CO--
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CYCLIC THIOETHER REACTION SCHEME I
JBDMSO fBIlSS
(BOC)AFLGVLG(BOCJKLA
Ph3PCI2
Cl tBuSS
(BocJAFLGvLG(Boc)KLA
HFJDMSIEDT
(64%)
~.
C/~ HS~
AFLGVLGKl AF-NH2
Na2CO3 in H~O/ACN (1:1)
(49%)
CCTE
S
AFLGVLGKl AF-NH2
H
AF = H N C O
SUBSmU~E SHEET ~lUlE ~6)
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51
Alternatively, thioether analogs are prepared according to the reaction
scheme below.
- CYCLIC THIOETHER REACTION SCHEME 2
t) isobutylchloro~orrnate
N-m~ ",o pholin~ Hf
CO2~1 2) N-hydr~xy-2-m~rc~p~opyndine
BOCHN~ .3N E30CllN~f C~2 ~r
Co2c~cH3)3 co2c~CH3)3
~nitrophenol ~
80C~ly-pr~C02H ~BOC~ly-pr~COz~NO2
DCC,THF
(ctt~)3SS ~CO2H ~ (H~OCC (CH3)~SS~CONH2
NHFMOC 2) I~ HCO3 NHFMOC
~MO~t~utylthio~r~t ~e 1) PBu3t~aHCO3
dio~ne/l 120
2) compolJnd 1
KzCO~ ~
2~
BOCNH-Gly Pro-COHNrC~S~CONH~ BOCHNrCH~S ~CONH2
CO2H NHFMOC ~ Co2c(cHl~3 NHFMOC
l) TFA/H20/HOCH2CH2SH
2) cDmpound ~
- NaHCO~ o~ eJH20
BOCN~ P CONH~CH2--S ~CONH2
NH-I-L-CO2H NHCO-R(Prnc)-C~(Ot~3u) P~b-A-L~NHFMOC
1) OBU/CH3CN H2N~-P-CO~N --CH~S /CONH2
2) C)PPAlCH3Chl/pyrldin~ l l
3) TFA/H20/lllioanisole/EDT/phenol CO NH
HN-I-B-L-A-P~-D-R- CO
SUBSmUrE SHEEI fftllLE 28)
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52
The following analogs are representative as analogs made according to
cyclic thioether reaction scheme 2.
112N~;p CONIlrCH~S~
~tl2-GP-CONlt~ 5~2 C~LIlAPDR-NH
CTE c O
CO-lLlJ~bOR-NH
cl~ A ~2~GP~ONH
c~LLw
HzN GP~ONH C~ a:lNH2 CTE D
r ~ Ad~-P CoNl l~c~ r CON~2
C~LLtAPDR~ : ; l I
CTE B CTE E CO~llAPDR~NH
E~emplary cyclic thioether analogs were tested for activity against ACA
antibodies ACA-6501 and ACA-6701. The results are shown in Table 10 below.
TABLE 10
IC50 Values (llg/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
The HCTE analog, LJP 699, outperforrned the reference peptide, LJP 690,
which is the truncated version, CLGVLGECLC, of LJP 688,
AGPCLGVLGKLCPG.
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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.
EX~ PT F~
Example 1: Measurement of Anticardiolipin Antibodies (~CA) in serum 6501
Even numbered wells of an Inunulon I microtitration plate (Dynatech
Laboratories, Inc., Chantilly~ VA) were coated with 50 ,ug cardiolipin (Sigma
Chemical, St. Louis, MO) in 30 ~L ethanol per well. The plate was dried
overnight at 4~C and blocked with 200 ~lL of 10% adult bovine senun (ABS)
(Irvine Scientific Co., Santa Ana, CA) in phosphate-buffered saline (ABS/PBS)
for 2 hours at room t~ll,pe~ re (RT). The plates were washed S times with Tris-
buffered saline (TBS) prior to the addition of 10 ~lLof 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 I :50
with 10% ABS-PBS. The test serum. ACA-6501 ~ diluted 1 :50 to 1 :2,000 in serialdilutions 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 ~L of 1: 1,000 goat-anti-human-lgG/alkaline phosphatase conjugate (Zymed,
South San Francisco, CA, Cat No. 62,8422), in 10% ABS-PBS was added and
incubated for I hour at RT. Again, the plate was washed five times with TBS and
the assay was developed by adding 100 ~lL phenolphthalein monophosphate
(PPNM) substrate solution (Sigma, Cat. No. P-5758), ~ ,d from a stock
solution of 0.13 M PPMP and 7.8 M2-amino-2-methyl- 1 -propanol, adjusted to
- pH 10.15 with HCI, after a dilution of 1 :26 with deionized water. After
approximately 30 minutes, the reaction was stopped with 50 ~lL 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,
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54
Winooski, VT, Model EL3 1 1). The optical density of the odd-numbered control
wells (blank, without cardiolipin (CL)) was subtracted from the optical density of
the evennl-mhered wells. The absorbance readings of the aPL standards were
plotted using Graph Pad Prizm (Graph Pad Software, Inc., San Diego, CA) to
generate the GPL (IgG phospholipid) standard cunre. The diluted 6501 test serum
absorbance readings were used to calculate GPL scores based on the GPL
standard curve.
In the modified ACA ELISA, Nunc Maxisorp microtitration plates were
coated with 100 ~L /mL ~2-GPI made up in PBS following incubation at I hour at
room temperature. Following this step, microplates were blocked with PBS
cont~3ining 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 ~2-GPI directly were used for
ACA IgG binding studies as previous described for the Dynatech microplates
coated first with cardiolipin before the ,B2-GPI was added.
Example 2: Anticardiolipin Antibod~ (ACA) Purification from Serum 650l
In a 25 mL round-bottom flask (Kontes Scientific Co.~ Vinelands N.J.) a
mixture of 1.2 mL cardiolipin (Sigma Chemical, St. Louis, MO, #C- l 649),
0.464 mL cholesterol (Sigma Diag., St. Louis, MO., #965-25), 0.088 mL of 5 mg
dicetylphosphate (Sigma Chemical, St. Louis, MO, D-263 1 ) per mL chloroform
was dried for approximately S minutes in a Rotavap (Buchi, Switzerland).
Following the removal of solvent, 2 mL of0.96% (wt./vol.) NaCl (J.T. Baker,
Inc., Phillipsburg, NJ) Baker analyzed reagent) was added and mixed in a Vortex
Genie Mixer (Scientific Industries, lnc., Bohemia, NY) for 1 minute. The
liposome suspension was incubated for 1 hour at 37~C. Meanwhile, serum 6501
was spun at 600 X g in a Sorvall RT 6000 centrifuge (Dupont Co. Wilmington,
DE) for 10 minutes at 8~C. Four mL of the supernatant was placed in a 25 mL
round-bottom flask with I mL of the prepared liposome suspension and the
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mixture was incubated with agitation at medium speed in an orbital shaker,
Tektator V (Scientific Products, ~IcGraw Park, IL) for 48 hours at 4~C, and an
additional 2 hours at 37~C. Twenty mL of cold TBS was added and the mixture
was transferred into a 50 mL polyc~rbonate centrifuge tube (Nalge Co., Rochester,
NY) and centrifuged at 27,000 X g for 15 minutes at 4~C. in an RC3 centrifuge ina SS-34 rotor (Sorvall-Dupont Washington, DE). The precipitate was washed 3
times with 25 mL of cold 0.96% NaCl using the RC3 centrifuge. The pellet was
dissolved in l mL of 2% (wt/vol) solution of n-octyl-p-D-glucopyranoside
(Calbiochem, La Jolla, CA) in TBS and applied to a 0.6 mL protein A/cross-
linked agarose (Repligen Corporation, Cambridge, MA) column which had been
pre-washed with 15 times bed volume of I 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 o tical density of the eluate at 280 nmapproached the baseline. The bound antibody was eluted with I M acetic acid.
One mL fractions were collected7 neutralized immediately with 0.34 mL 3 M Tris
(Bio-Rad. electrophoresis grade reagent) per fraction and lcept in an ice bath. The
optical density of each fraction was deterrnined at 280 mn in a spectrophotometer
(Hewlett-Packard~ 8452A Diode Array Spectrophotometer, Palo Alto, CA).
Fractions cont~inin~ antibody were pooled, concentrated and washed 4 times with
TB S in Centricon-3 0 concentrators (Amicon Division, W.R. Grace & Co.,
Beverly, MA) per m~nnf~cturer's protocol. The final yield of purified antibody
from 4 mL of serum 6501 was deterrnin~d by reading the optical density at
280 nm of an aliquot from the concentration, where I mg = 1.34 OD280. The
average yield obtained was ~50 ,ug 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 cont~inine ~2-GPI was prepared
using CNBr-activated agarose ) Pharmacia, Inc., Piscataway, NJ) or Affi-Gel 10
(BioRad. Richmond. CA) in accordance with the manufacturer s instructions
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using purified ,B2-GPI obtained commercially (Perlmrnune, Rockville, MD). To a
1 mL gel column contS~ining the ~2-GPI affinity adsorbent, up to 100 ~g of
liposome-purified aPL IgG in 1 mL TBS was added. After 1 hour, the colurnn
was extensively washed with buffer to displace co~ nt.~ and any IgG that did
not bind ~2-GPI. The ,B2-GPI-binding IgG was displaced with I M HOAc and the
fractions neutralized with Tris as described above. Fractions cont~ining IgG were
pooled, concentrated and subjected to buffer exchange as previously described.
Example 3: Construction of a p-I~T T ibrary Vector P~ lion
fUSE 5 (Scott, J.K. and G. Smith. supra) was used as the vector ~or 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
doublestranded replicative forrn (RF). Briefly, an 800 m' ~ulture of ~. coli K802,
harboring fUSE 5, was grown in 2YT medium (Difco Labs, Ann Arbor, NU)
con~ining 20 micrograms/mL tetracycline for 18 hours at 37 degrees with
vigorous sh~kine. Cells were collected by centrifugation and resuspended in 75
mL STET. STET consists of 8% sucrose in 50 mM Tris/HCI 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 l mg/mL. After 5 minutes at RT, three equal
aliquots were placed in a boiling water bath with occasional .ch~king for 3.5
minutes. The viscous slurry was centrifuged for 30 minutes at 18000 x G and an
equal volume of isopropanol was added to the sUpçrn~t~nt. The solution was
cooled to -20~C and the nucleic acids were collected by .centrifugation. The RF
was isolated from a CsCI gradient as described by Sarnbrook et al. MOLECULAR
CLON~G:ALABORATORY MANUAL (Cold Spring Harbor Laboratory Press,
- Cold Spring Harbor, NY, 2d ed., 1989).
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Proration of the random in~rt
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 cont~ining 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:
5' GGGCTGGACCC(NNK)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'. Simi}arly 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 forrn
a "gapped duplex" which, when inserted into fUSE 5 digested with Sfil, restores
the reading ftme of P-IIl with the random insert near the 5' end.
Oligonucleotides described above were prepared by excision from
polyacrylainide gels. One nanomole of EpiGS2, EpiGS3, and 50 picomoles of the
degenerate oligonucleotide were kinased separately in 66 microliter volumes. Thethree oligonucleotides were then pooled, NaCI was added to 50 mM and the
mixture was heated to 65~C for 5 minnte~ 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 S DNA digested to completion with Sfil, 30 ~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~C. The mixture was then phenol-
chloroform extracted, precipitated with ethanol and the precipitate dissolved in20 ~lL of water.
SUBSrlTUrE SHEEr ffWLE 26) -~
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58 ~ -
Generatin~ and Am~ in~ the T ibrary
The ligated DNA was introduced into E. coli by electroporation (Dower et
al. (1988) NucleicAcid Res. 16:6127-6145). Frozen electrocompetent MC 1061
cells (0.1 mL) were mixed with 4 ~L of ligated DNA in a cold 2 mm cuvette and
subjected to 2.5 kV, 5.2 mS pulse by a BTX eleclro~olalion device (BTX Corp.,
San Diego, CA). Immediately after the pulse, I mL of SOC, a cell growth
medium (see Dower et al., s2~pra) was added. Five separate electroporations werecarried out pooled, and incubated at 37~C for 1 hour. At that time, samples wereremoved 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% NaCI? Difco Labs, Ann Arbor, MI) containing 20 micrograms.
per mL of tetracycline and grown for 18 hours at 37~C while shaken at 275 rpm.
Phage were purified by 2 rounds of PEGlNaCI precipitation and resuspension in
1.2 mL TBS cont~ining 0.02% sodium azide. Particle number was estimated by
absorbance at 269 mn. The titer of the phage was deter-mined by mixing 10 ~lL ofdilutions of phage with 10 microliters of starved E. coli.
Example 4: Screenin.~ a p-JlT T ibrary with aPL Antibody
Affinity-purified, ACA-6501 (affACA-6501, 10 ~lg, 7 ~lL 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 (epiXYZ) [11
IlL + 8.5 ~lL + 2 ~LL, respectively; total volume = 21.5 ~lL, ~101~ clones] in a final
volume of 100 IlL TBS, pH 7.4, with 0.5% bovine serum albumin (BSA). During
2S 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 ~h~king at 370~C was spun at 1000 x g for 10 minutes at RT
in 50 mL polypropylene tubes. Twenty mL of 80 mM NaCI was added to the
packed E. coli pellet and then incubated for 45 minutes at 37~C at 100 rpm.
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Following centrifugation as above, the starved E. coli pellet was suspended in
I mL of 50 mM ammonium phosphate/80 rnm NaCI and used later for phage
amplification. Protein G-agarose beads were washed 2x in TBS/BSA and 2 times
in TBS/0.5% Tween-20 and stored at 4~C as a 50% suspension in TBS/Tween.
Two hundred gL of the protein G-agarose bead suspension was then added
to the ACA-6501/phage mixture and incllh~tion continued for an additional 1 hourat 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/E~SA
and TBS/Tween to prevent non-s ecific adherence. After three additional washes
with TBS/Tween, the beads with bound ACA-6501-bearing phage were eluted
with 300 ,uL 0.2 N HCI/glycine, pH 2.1 by tumbling for 10 minutes at RT.
Following centrifugation at 16,000 X g, the acidic eluate supernatant was
collected and an additional 100 !lL of elution solution was added to the bead pellet
and the procedure repeated. After 10 minutes, the phage-cont~ining eluates
(representing the unamplified first round phage) were pooled (~400 ~L) and
placed in a sterile 17xlO0 mm polypropylene cell culture tube to which was added50 !lL 0.5 M NaCI followed by pH neutralization with 2.5 M Tris base (usually
~25-35 ~L). An equal volume of the starved ~. coli suspension was added
immediately and then incubated for 10 minutes at 37~C at 100 rpm. The mixture
was then transferred to a 250 mL sterile culture flask cont~ining 25 mL 2YT with20 ~g/mL tetracycline (Tet) and incubated overnight at 37~C at 250 rpm.
To isolate amplified phage from overnight cultures, the suspension was
centrifuged at 12,000 x g for 10 minutes in polycarbonate tubes and the pellet
discarded. After heating the sUp~ t~nt at 70~C for 30 minutes in polypropylene
tubes, the material was spun again in polymbonate 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~C for 2 hours. After
centrifugation at 35,000 x g for 30 minutes at 40~C, the phage-containing pellet
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was resuspended in ~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 x g, the supematant was
transferred to a clean tube and labeled first round amplified phage.
During second, third and fourth rounds of biop~nnin~, 75 ~L of amplified
phage from the prece~ine round was incubated with 7 ~LL affACA-6501 in a final
volume of 100 ~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 biop~nning
were spottitered on 2YT/Tet plates to detemmine phage concentration. The spot
titer of amplified phage requires an initial phage dilution of lx I o6 in TBS/BSA or
2YT media. For each round. 10 ~lL of the dilute phage was incubated with 40 ~lL
of starved E. coli for 10 minutes at 37~C with no ~h~kin~. Following the addition
of 9S0 ,uL of 2YT/dilute Tet (0.2 llg/mL), the mixture was incubated for 30-45
minutes at ,37~C with 250 rpm shaking. Ten ~lL aliquots of neat and diluted phage
solutions, 1: 10, 1: 100, and 1: 1000, were spotted in 2YT/dilute Tet using agarplates cont~ininE 20 llg/mL Tet.
Micropanning
Immulon type 2 plates were coated with protein G. Protein G was prepared
at 10 ~g/mL in 0. I M NaHCO3 and 100 ~lL per well was added to the wells of
microtitration plates and incubated ovemight at 4~C. After discarding excess
protein G solution from plates, each well was blocked with 250-300 ~LL 2YT for Ihour at RT with agitation on an oscillating platfomm. 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 ~L. One hundred ~L affACA-6501 (or control
nomlal IgG), diluted to 2.5 ~lg/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.
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Phage to be tested by mi~;lopa~ ing were obtained from the agar plates
generated by biopa~ lg. Each clone to be tested was transferred using sterile
toothpicks to a separate well of a round-bottom 96-well miclotillalion plate
(Corning, NY) cont~ining 250 ~L '~,YT/Tet per well and cultured overnight at
37~C. Clone desi~n~tions are based on the screening antibody, the bio~ g
round of origin, and the location of the clone in the overnight culture plate, e.g.,
ACA6501/3B10 refers to the clone isolated by ACA-6501 in the third round
located in the well designated B10 on the mi-;lotiLlation plate. Following
overnight incubation, phage cultures were centrifuged using a microtitration plate
holder at 1300 x g for 10 minutes at RT. Supernatants constituted the source of
' neat" phage.
Initial mi~;l. palming was restricted to six clones which were tested at
dilutions expanded by factors from 1: 10 to 1: 1 o6 ~ The results from these pilot
clones were used to suggest the ~plol.l.ate 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 bioparmingwere diluted to 1: 100,000 from the "neat'' solution using 2YT in miclo~ ion
plates with a final volume per well of 250 ~LL. From the last plate representing the
desired dilution, 100 ~LL was added to the plate cont~ining protein G-bound ACA-6501 and norrnal IgG prepared as described above. The incubation of dilute
phage with aPL antibody or control IgG was carried out for 2 hours at 4~C on a
flat rotator. After 9 washes with TBS/Tween in an automated plate washer, the
IgG-bound phage was eluted with 20 ~L of 0.2 N HCIglycine/0.1% BSA, pH 2.2.
The elution incubation continued for 10 minutes at RT, during which time a new
Corning microtitration plate was l~le~)ared cont~ining 20 ~lL of freshly starvedE. coli per well and kept chilled. One hundred forty ~lL of 29 mM Tris was addedto the plate cont~ining the phage eluates in order to neutralize the pH, following
which 20 ~L of phage suspension was transferred from each well to the
corresponding well in the plate cont~inin& starved E. coli. After a 10 minute
incubation at 37~C, 200 ~L 2YT/dil Tet was added and incubation carried out an
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additional 30 minutes at 37~C. Using multichannel pipettors, 10 ~L from each
well was spotted on a large 2YT/Tet agar plate while retaining the original 8x12well pattern and orientation from the last microtitration plate. After allowing the
spots to dry for 30 min~ltes, the plate was inr.llbated overnight at 37~C. The
following day, eolonies were semi4~ tiL;~ ely seored 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% confllient colonies. Of the 94 clones
that were examined at a dilution of 1:105 [representing 8 I third, 6 fourth, and 7
fifth round clones~. six clones had mic~u~ in~ scores of zero, three seored 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 biop~nning was earried 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 Figure 8),
therefore, a seeond plate was micropanned using phage al a dilution of 1:3 X 10
this time including clones from an earlier (second) round. A total of 94 clones
were tested, including 29 whieh had scored high at I x l Os dilution ~26 from the 3rd
round, I 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 elones tested at a
dilution of I :3xl 05, 26 scored zero, I I seored +/, 10 seored I +, 13 scored 2+, 34
scored 3+, and zero scored 4+.
G-track DNA Sequencin~
Single-stranded viral DNA was isolated from cultures incubated overnight
at 37~C. To prepare cultures, 2YT/Tet, either as 2 mL in tubes or 250 ~IL in
mier~ ion plate round-bottom wells, was inoeulated with individual phage
from spread plates of previously grown cultures in microtitration plates. The
purifieation of phage by 20% PEG/2.5 M NaCI plGcipitalion of eulture
supernatants as well as the isolation or release of virion DNA by phenol-
chloroform extraction or by alkali denaturation was performed as described in
SUBSmUlE SffEEr ff'(ULE 26)
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63
Smith, G.P. and J.K. Scott, "Libraries of peptides and proteins displayed on
fil~mentous 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 seql~onr,ing of viral DNA
from filamentous phage" (1993) BioTechniques 15:422-431 for phage in
microtitration plates. The dideoxy nucleotide chain t~rmin~tion DNA sequencing
technique of Sanger et al. (Sanger et al., "DNA sequencing with chain terrnin~ting
inhibitors" (1970) Proc. Natl. Acad. Sci., 74:5463-5467) was carried out using acommercial Sequenase kit (U.S. Biochemical/ Amersharn, Arlington Heights, IL)
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 asingle base (G~ was obtained following electrophoresis on 7%
polyacrylamide/urea sequencing gels and exposure by autoradiography. Standard
gel electrophoresis and autoradiography procedures were followed (Sarnbrook et
al., supJ~a)
G-tracking of 76 clones from the mi~;lop~ g plate tested at a phage
dilution of l:I X I05 from the ACA-6501 library screen reve~led l I unique
sequences, while 64 clones from the second microp~nning plate tested at a pha~e
dilution of I :3 X 105 showed 30 unique sequences. Conventional DNA
sequencing using all four dideoxynucleotide triphosphates was applied to the
phage clones with the highest miclop~-t-;l-g scores and unique sequences and
resulted in the 12 sequences shown in Table I for the xyz library.
Phage-Capture F.T TSA
Clones ACA-663S/3A12, 3B3, 3C8, 3A5, 3C9, and 3B7 were grown as 3
mL cultures. Affinity purified ACA-6635 was diluted to 2.5 ~lg/mL in phosphate-
buffered saline, pH 7.2, and 100 ~lL 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 ~h~kin~. The plate was then blocked with 150 ~lL
SUBS T U E S~Er ~LE 26)
.
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64
0.1 % BSA (globulin-free) in PBS per well. After 1 hour at 4~C, the plate was
washed 3 times as previously described. After centrifuging each phage culture 3
minutes at 17,000 x g, each supçrn~t~nt was diluted 1:10 in 0.1% BSA/PBS and
100 ~lL added to each of the wells coated with affinity purified ACA-6635 and
then incubated for 2 hours at 4~C. Plates were then washed with TBS/Tween as
before. Horseradish peroxidase-conjugated sheep IgG anti-M13 phage antibody
(Pharmacia, Inc., Piscataway, NJ) was diluted 1 :5,000 in 0.1 % BSA/PBS and 100
~L applied to each well. Following incubation for 1 hour at 4~C, the plate was
washed 4 times as before. One hundred ~L of substrate prepared according to the
conjugate manufacturer's instructions was added to each well. After 19 minutes~
the absorbance at 405 nrn 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 Figure 9, three
clones (3A12, 3B3, and 3AS) out ofthe seven tested exhibited a very strong
immunospecific signal in the phage-capture ELISA.
Phage-F~ ISA
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 minl-tes at 17,000 X g, 100 ~L from each phage supernatant
(adjusted to -2 X 1011 particles/mL, based on absorbance) was added to
microtitration plate wells ~Falcon, Becton-Dickinson Labware, Lincoln Park, NY)
and allowed to incubate overnight at 40~C. Following four washes with TBS 7.4,
the plate was blocked with 125 ~lL of 0.5% BSA/TBS for I hour at RT. After
another four TBS washes ~00 ~lL of affACA-6501 previously diluted to 2.5
,u~/mL in TBS/BSA was added to each well and allowed to incubate 1 hour at
37~C. Following an additional four washes, 100 ~L anti-human IgG diluted
SUBSTllUrE SHEEr ~IE 26)
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1:1000 in TBS/0.5% Tween was added to each well. After I hour at RT, enzyme
substrate was added and the incubation allowed to proceed for 2 hours. Followingthe addition of 50 ~lL 0.2 M Na2HPO4 to stop the reaction, absorbance was
measured at 550mn in the automated plate absorbance reader. As shown in Figure
10, seven of the clones had significant signals inthephage-ELISA with ACA-
6501: 5A12, 3B6, 3E7, 3B10 (same sequence as 2D7), 2G11, 2H1, and 2H2. The
sequences for these clones are shown in Table 2.
Peptide-ELlSA
In the standard protocol, stock solutions of tetrameric peptide in
dimethylformamide were diluted 1000 times to 10 llg/mL in pH 9.5 carbonate
buffer. Each microtitration plate well was coated overnight at 40~C with 100 ~lLof dilute peptide followed by blocking with buffered albumin. Peptide-coated
microtitration plates were incubated 1-2 hours at room temperature with aPL seraat several dilutions starting at 1:50. Following washes, the presence of peptide-
bound human IgG was determined with enzyme-conjugated anti-human lgG
according to standard ELISA procedures.
Competiti~e Bindin~ Peptide-ELISA
(A) Each well of an Imrnulon II plate (Dynatech Laboratories, Inc.,
Chantilly VA) was coated with 100 ~lL of a solution cont~ining 10 ~lg tetravalent
peptide ACA 6501/3B10 in 50 mM sodium carbonate, p3I 9.5, containing 35 mM
sodium bicarbonate (Fisher Scientific, Pittsburgh, PA, reagent grade) for at least I
hour at ~T, except for three wells used as blank controls. The liquid was then
removed from the wells and 200 ~lL 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 I hour at RT. Four 1.5 mL microfuge tubes
were numbered I to 4. The following reagents were mixed in the first microfuge
tube (Brinkman Instruments, Westbury, NY): 30 ~L of 5% BSA; 284 ~L TBS; 8
SUBSTllUI E SHEEr ~RlJlE 26)
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66
~LL of a stock solution of approximately 400-500 ~lg/mL of monomeric peptide
(ACA-5A12 or -CB2 or -3B l O or scrambled -3B 10 as negative control) in TBS,
- and 8.2 ~L of 1:10 diluted serum 6501 in 0.5% BSA-TBS. The following
- reagents were mixed in the second microfuge tube: 30 ~lL of 5% BSA; 290 ,uL
TBS; 2 ',IL of a stock solution of approximately 400-500 ~g/mL of monomeric
peptide (ACA-5A12 or -CB2 or -3B10 or scrarnbled -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 third microfuge tube: 30 ~,IL of 5% BSA, 287 ~L of a
1:10 dilution of approximately 400-500 ~lg/mL of monomeric peptides (5A12,
CB2. 3BlO. or scrambled sequence 3B10 control), and 8.2 ,uL of ACA-6501 serum
previously diluted 1:10 in 0.5% BSA-TBS. The following reagents were mixed in
the fourth Eppendorfmicrofuge tube: 60 ~L 5% BSA; 584 ~L TBS and 16.5 gL 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 triplicatewells, 100 ~LL 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 ,uL
of the solution in the fourth microfuge tube was added to each of the three blocked
blanli wells of the microtitration plate. The plate was incubated for I hour at RT
with agitation at 40 rpm in an orbital shaker (American Dade, Miami, FL, RotatorV) and then washed 5 times with TBS. An aliquot of 100 ~L of 1:1000 diluted
goat-anti-human-IgG/~lk~line phosphatase conjugate (Zymed, South San
Francisco, CA.Cat. no. 62-8422) in 0.5% BSA-TBS was added and incubated for
I hour at RT. The plate was then washed 5 times with TBS and the assay was
developed by adding 100 ,uL PPMP diluted substrate solution as described in
Example 1. After 20 minllte~, the reaction was stopped by adding 50 ~L of 0.2 M
Na2HPO4 (Mallinckrodt, St. Louis, MO., reagent grade) per well. The optical
density was read at 550 mn in a microplate reader (Bio-Tek Instrurnents,
Winooski, VT, Model EL 311). The optical density at 550 mn versus the amount
of the peptide per well was plotted in Graph Pad Prizm (Graph Pad Software, Inc
SUBSmU~E SHEEr ~lllLE 26)
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67
San Diego, CA). The amount of peptide required for 50~/u inhibition of binding of
serum 6501 to tetravalent 3B10 was calculated from the graph.
(B) Immulon I(~) 96-well, flat-bottom, polystyrene microtitration plates
(Dynatech Laboratories, Inc., Chantilly, VA) were coated with 30 ~L/well of
cardiolipin (CL, 50 gg; Sigma Chemical Co., St. Louis, MO) in ethanol. Two
control wells received 30 ~lL ethanol only. After overnight evaporation at 4~C,
each well was blocked with 200 ~lL 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 ~2-GPI as 100 ,uL of 2.3% (v/v in PBS) IgG-depleted human
serum (Sigma Chemical Co., St. Louis, MO) and incubated 2 hours at RT.
During this incubation, variable amounts of each of six peptides were
mixed with 22 ~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 ~lL using Eppendorf
microcentrifugetubes. Specifically,intube#l weremixed 181.3 ~Lof3%fish
gelatin in TBS-PBS, 16.7 ~lL of peptide stock solution plus 22 ~L of ACA-6501
serum diluted 40 times in 3% fish gelatin/TBS-PBS. Stock solutions ranged from
450-800 ~lg/mL for peptides #951 (diserine non-cyclized negative control), #952
(a lot of LJP 69()), and thioethers CCTE-3G3, CHTE-3G3, HCTE-3G3 and
HHTE-3G3. To tube #2 the following were added: 148 ~L fish gelatin/TBS-PBS~
50 ~L of peptide stock solution, plus 22 ~L of 40 times diluted ACA6501 serum.
Tube #3 contained 48 ~lL fish gelatin/TBS-PBS, 50 ~lL of peptide stock solution,plus 22 ~L of 40 times diluted ACA-6501 serum. The control tube #4 received
396 IlL fish gelatin/TBS-PBS and 44 IlL of 40 times diluted ACA-6501 serum (no
peptide). Each of the tubes incubatçd for approximately 1 hour at RT.
The CL/~2-GPI microtitration plate was washed 5 times with TBS and 100
-- ,uL aliquots in duplicate from Eppendorf tubes # 1, 2, 3, and 4 cont~ining the
antibody-peptide (or no peptide mixtures) were added to the wells. A volume of
100 ~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
SUBSmUlE SHEEr ~UlE 26)
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68
at 40 rpm in an orbital shaker (American Scientific, Rotator V), washed 5 times
with TBS and then 100 ~LL of 1:1000 goat anti-human IgG :~lk~line phosphatase
conjugate (Zymed, Cat No. 62-8422) in 0.5% (wlv) BSA-TBS was added.
Following incubation for I hour at RT, the microtitration plate was again washed5 times with TBS and the calorimetric eszyme detection developed by adding 100
~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 ~L of 0.2 M
Na2HP04 (Mallinckrodt) to each well. Absorbance at 550 mn was read in a
microplate reader (13ioTek InstnllnPnt~, Model EL 31 1). Absorbance vs. peptide
added was plotted on Graph Pad Prism (Graph Pad Software, Inc.) as shown in
Figure 12. The amount of peptide that inhibited ACA-6501 binding by 50%, the
IC50, was calculated from the graph at the intersection of l~alf-maximal
absorbance with amount of peptide added.
(C) Nunc Maxisorp~) 96-well, flat-bottom microtitration plates were
coated with 100 ~IL/well of purified human P2-glycoprotein I (Perlmrnune,
Rockville, MD) at 10 ~lg/well in PBS. Two control wells received 100 ~L PBS
onl!~. After two hours incubation at room temperature, the liquid was removed.
Thc coated plate was blocked for 2 hours at room temperature with 200~LL/well of2() PBS cont~ining 2% (w/v) nonfat dry milk (Carnation, Glendale, CA) and 0.4%
(w/v) Tween 80 (Calbiochem, San Diego, CA). The blocking solution was also
used as a reagent diluent.
During the second hour of incubation, variable amounts of each of four
. _ .
test peptides were mixed with 2211L of ACA-6501 serum diluted with sample
2~ diluent in a fmal volume of 220,uL using Eppendorf microcentrifuge tubes.
Specifically, in tube #I, 188 ~L of sample diluent, 10 ~L of peptide stock solution
(2 mg - 4 mg/mL diluent), and 22 ~L of AC-6501 serum diluted 1 :35 in sample
diluent were added. To tube #2, 158 ~L sample diluent, 40 ,uL peptide stock
solution and 22 ~L of I :35 diluted ACA-6501 serum were added. Tube #3
SUBSllTU~ SHEl' ff~(ULE 26)
CA 022~6449 1998-11-23
WO 97146251 PCT/US97/10075
69
contained 38 liL sample dituent, 160 IlL peptide stock solution and 22 ~lL of 1 :35
diluted ACA-6501 sertim. The control tube, tube 94, contained 396 IlL sample
diluent and 44 ~lL of 1:35 diluted ACA-6501 serum (no peptide). Each ofthe
tubes was incubated for approximately I hours at room temperature
S The ~2-GPI microplate was washed S times with TBS and 100 ~lL of themixtures contained in Eppendorf tubes 91, 2, 3 and 4 was added to duplicate wells
of the microplate. 100 ~LL of the contents of tube #4 was added to each of the
duplicate control wells that were not coated with ,B2-GPI. The plate was incubated
for I hour at room temperature, washed 5 times with TBS and 100 ~lL of 1:1000
goat anti-human IgG/A-P conju~ate (Zymed, Cat. No. 62-8422) diluted in sample
diluent buffer was added. Following incubation for I hour at room temperature,
the plate was again washed 5 times with TBS and the calorimetric enzyme
detection developed by adding 100 ',lL PPMP solution (7.8 g phenolphthalein
monophosphate and 69.5 g 2-amino-2-methyl-1-propanol in 100 mL water stock
solution diluted I :26 with water). After 22 minutes, the reaction was stopped with
50 IlL of 0.2 M Na2HPO4 (Mallinckrodt) added per well. A55Onm 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.) às shown in
Figurc 21. The amount of peptide that inhibited ACA-6501 binding by 50~/0, the
IC-50~ was calculated from the graph at the intersection of half-maximal
absorbance with the amount of peptide added.
Example 5: Truncation F~periments of Peptide 3B 10 and the Resultin~ Peptide
Coml-etition F.T lSA Results
Microtitration plates (96-well, flat bottom polystyrene, Irnmulon-2,
Dynatech Laboratories~ Inc., Chantilly, VA) were coated with 100 ~L/well for I
hour at RT with tetrameric ACA-6501/3B10 peptide at 10 ~g/mL in carbonate
buffer, pH 9.6 (15 mM Na2CO3/35 mM NaHCO3). After the liquid from the wells
was removed. each well was blocked for I hour at RT with 200 ,uL 0.5% (wt/vol)
SUBSrllUrE SHEEr (I'~UlE 26) --
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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.
During the blocking step, each soluble, monomer peptide to be tested was
set up in three test tubes (Eppendorf micro test tubes, Brinkm~nn Instruments,
Westbury, NY) each cont~inin~; 30 ~lL 5% BSA/TBS, 8.2 ~IL ACA-6501 serum (at
1: 10 dilution with 0.5% BSA/TBS), plus a variable volume of the peptide/-FBS
stock and the necessary volume of TBS buffer to yield a final volume of 330 ~L.
The 330 ~L volume was sufficient to generate triplicate 100 ',lL sarnples for each
peptide concentration that was tested for its ability to block ACA-6501 bindin~ 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. theconcentration of the stock solution was approximately 340-400 ~g/mL TBS and
aliquots of 19 !lL, 75 ~L. and 292 ~L were removed to prepare the three peptide
concentration tubes. For peptide 142 (lacking only the N-terminal ala) and
peptide ] 4~ (not truncated). stock solution concentrations were 400-500 ~g/mL in
TBS. Aliquots from each stock solution of 1 ~,lL, 4 IlL, and 16 ~lL were removedto 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 ~lL was prepared
cont~inin, 60 IlL 5% BSA, I 6.5 ~L of a I: 10 dilution of ACA-6501 and 583.5 IlLTBS, i.e., the same final concentrations (0.5% BSA and ACA-6501 serum at
1 :400) as the 330 ilL tubes but with twice the volume and without peptide.
Following the blocking incubation, the plate coated with tetravalent
peptide was washed S times with TBS. From each 330 IlL tube cont~inin~
peptides 139, 142 and 143 at different concentrations, 100 IlL was added to coated
triplicate wells. Three 100 IlL aliquots from the 660 ~L control tube without
peptide were each added to coated wells and three additional I 00 ~lL ali~uots were
each added to uncoated blank wells. The plate was incubated at 40 rev/min on a
rotar~ orbital shaker (Rotator V, American Dade. Miami, FL) for I hour at RT andthen washed 5 times with TBS. One hundred ~L/well of goat antihuman
SUBS~lllUI~ SffEEr ~(ULE 26)
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IgG/alkaline phosphatase conjugate (Cat. no. 62-8422, Zymed, South San
Francisco, CA) diluted l :1000 in BSA/TBS was added to the microplate. After
incubation for I hour at RT, the plate again was washed S times with TBS. Color
development followed the addition of 100 ~lL/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 l O.15 with HCI).
After 30 minutes, the reaction was stopped by adding 50 ~lL/well of 0.2M
l 0 Na2HPO3 (reagent grade, Mallinckrodt, St. Louis, MO). Absorbance
measurements at 550 nrn were carried out on a microplate reader (Bio-Tek
Instruments, Winooski, VT) and the As50nm vs. peptide added per well results
plotted using Graph Pad Prizrn (Graph Pad Software, Inc., San Diego, CA). As
shown on Figure l l, 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 Figure 12. The results indicate that while the
loss of the N-terminal Ala had no negative conse~uences, the additional loss of
Gly increased by about 8-fold the concentration necessary to achieve 50%
inhibition.
F.Y~rle 6: Substitution of ~l~ha n ethyl proline into 3B l O ~nd the resultin~
~.r,lSAs
Testing of peptides ACA-6501/3BlO and analogs in which prolines at the
3 and 9 position were replaced by a-Me-Pro was carried out using the
methodology described in Exarnple 5. Peptides 3B10, 726 (aMe-Pro substituted
at the 3 position), 727 (aMe-Pro substituted at the 9 position), and 72B (aMe-Pro
substituted at both the 3 and 9 positions) were tested as soluble monomer peptides
SUBSTllU~E SHEET ~UlE 26)
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W O 97/46251 PCTrUS97/10075
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in a competitive- binding ELISA using tetramer peptide 3B 10-coated
microtitration plates and ACA-6501 serum.
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 ~L. All peptide stock solutions were at
400-500 ~g/mL TBS. To tubes cont~inin~ 30 ~lL of 5% BSA/TBS and 8.2 ~L of
ACA-6501 serum (diluted 1:10 with 0.5% BSA/TBS), aliquots of I ~lL, 4 ~L, or
16 ~lL of each of the four peptide stock solutions were added in addition to an
applu~liate volume of TBS to achieve a final volume of 330 ~lL. A control tube
with no competitor peptide present was prepared with a final volume of 660 ~lL as
described in Example 5.
Following the blocking incubation of the tetravalent peptide-coated plate,
three 100 ,~LL aliquots from each of the peptide concentration tubes for each of the
four peptides as well as the control tube cont~ining no peptide and blank controls
were tested as described in Example 5. The microtitration plate ELISA proceduresas well as the data handling were perfonned as described in Example 5. As shown
in Figure 13, peptide 727, where a-Me-Pro was substituted at the 9 position, wassignificantly more active than unmodified peptide 3B10 or the analog with ~oth
prolines changed (peptide728). Peptide726, which was substituted at position 3,
lost activity as a result of the substitution.
Example 7: Abbréviated Description of Screen with 6626 Antibody and the
Corresponding Sequences
Affinity purified ACA-6626 (AffACA-6626) was isolated by affinity
purification from 8 mL of ACA-6626 plasma as previously described. AffACA-
6626 (10 ,ug) was incubated with the epitope xy'z phage library consisting of a
pool of all p-III component libraries in a final volume of l O0 ~lL as previously
SUBSIl~ SHE~ ~ULE 26) --
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73
described for ACA-6501 biop~nning. Following three rounds of biop~nning,
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 biop:~nning was carried out. Miclop~
of 94 fourth round clones revealed 43 irnmunopositives, 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: Identification of Sequences Specific for the ACA from Patient
6644
The epiX~ 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 ~lg/mL. Of the 150 colonies platedon 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 (epiZ):
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)
SUBSlllUrE SHEEl ff'(VLE 26)
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74
Example 9: Sllmm~y of Ph~e T ibrary Screen with ACA-6641.
AffACA-6641 was isolated from 4 mL of plasma taken from patient
number 6641. AffACA-6641 (10 llg) was incubate~ with the pooled p-III phage
libraries in a final volume of 100,uL as described previously. Following four
rounds of biop~nning, 45 clones from the 3rd and 4th rounds were tested by
microp~nning 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: _
CLGVLGKLC.
~xample 10: Peptide conjugation to non-~mmunogenic. multivalent carriers
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, Serial Nos., 08/118, 055, filed September 8, 1993, 08/152,506, filed
November 15, 1993, and U.S. Patents Nos. 5,268,454, 5,276,013 and 5.162,515
which are incorporated by reference herein in their entirety. ~andidate peptidesselected by aPL antibody screens of epitope libraries are conjugated and tested for
modifications of immunochemical behavior such as antibody binding.
Non-inununogenic multivalent platforms with amine groups are
synthesized as shown in the following scheme.
SUBSTllUrE SHEr ~WlE 26)
, .
CA 02256449 1998-11-23
WO 97/46251 PCT/US97/10075
Amine on Platfolm - Carbox~rl on Pe~tide
C8atN(C~2) sCONH ~ r NHCC)(C~2)6NHCaZ
o~o~lL~N
C8Z11N(Clt~sCONH~ 12 ~ NffOO(C~2)sNHC8
PdlC
c~clohexane/EtOH
~2N(C~23scoNH~ ~ ~ NHCO(CH~)sNH~
N--II~o~,O~ o~N
H2N(C~sCONH~ 2 2-~oo(c~6NH2
PNH~(P = I IF ~3b;10 p~otec~n~ ~a~
f3 ~~ amlno t~rmi~s and sldo ~;)
H08T
OMF
PHN p~OHN(~)sCO~ ~ N~tCO(C~)6NI IOO~HP
N~l ~ o ~ o,lL N
5 PHN-p~de~OHN(C~OONH ~ ~ NHCO~CH~NHCC~pep~NHP
H,2N ~COHN((~)6CO~I~~HCC)~ sNHCO~de Nt~2
~ o
~2N1~XK~IOOHN(C~ C3~ NHC~
Compo--nd 2: ~ solution of 8.0 g (5 .7 mmol) of 1 in 50 mL of absoiute 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. IH NMR (50/50 CDCI3/CD30D) 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).
Protected pepti~ with free ~rboxyl (PH~-peptide-CO2~
A peptide is synth~si7~1 with standard solid phase methods using FMOC
ch-?mi~try on a Wang (p-alkoxybenzyl) resin, using trifluoroacetic acid (TFA)
stable pr~ lillg groups (benzyl ester or cyclohexyl ester on carboxyl groups and
SUBSlTrl~ SHEr ~E 26)
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76
carbobenzyloxy (CBZ) OR 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.
Peptide - Platform Conju~ate, 4
The protected peptide (0.3 mmol) is dissolved in 1 mL of
dimethylformamide (DM'F), and to the solution is added 0.3 mmol of
diisopropylcarbodiimide and 0.3 mmol of l-hydroxybenzotriazole hydrate
(HOBT). The solution is added to a solution of 0.025 mmol tetraamino platforrn,
2 in 1 mL of DMF. W'hen 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~ to give conjugate 4.
Purification is accomplished by plcl~a dlive reverse phase HPLC.
The following scheme shows the ~tt~ ment of an amino group of a
peptide to a carboxy group on a platform.
- Carboxyl on Platform - Amine on L i~and
~2N(cH2)6coNH~ ~ NHCO(CH~sNH2
) 1~ ~ o ~ N
H2N(CH2)sCONH ,, NHCO(CH2)tN~2
2 succmlc anhydride
H(~2CCH2~H2COHN(C~sCONH ~ NHco~cl~sNHcocl~t2co2
N~l~ ~ O ~
H~2coHNta~scoNHJ 5 OCC~NHS (--NHCO(CH2)sNHCO~CO~
H2N-p~ptide CONH2 (~de cnains ~otedea~wi h HF
1 , labUe protectirlg ~roupS)
pep NHOCa~2GH2COHN(C~sCONH~ r t~cO~C~2)d~CON
N~~ ~o~o~lLN
p3~NHOCCH2a~zCOHN(C~2)sCONH ~ ,r f~ 2 --NHOO(C~sNHCOa kC112CONH~
pep = protect~3d pepti~e
' wNHocc~l2(~2coHN(c~coNH~ ~ NHCO(C~ sNHOI~2C~Hpe
N~l' a~ ~0--1~ N
pcpNHOC~l COllN(C~ 1 2 --N~O~
SU~mU~E SHEEr ~ULE 2S)
. .
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77
Compound 5 - Platform with Four Carboxylic Acid Groups
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 mrnol) of NaHCO3 in 20 mL of 1/1
dioxane/H2O, and the mixture is stirred for 16 hours at RT. The mixture is
acidified with lN HCI and concentrated. The concentrate is purified by silica gel
chromatography to provide 5.
Protected peptide with free ~mine (H2N-peptide-CONH2~
A peptide is synthesized with standard solid phase methods on an amide
resin~ which resulted in a carboxy termin~l arnide after cleavage frQm the resin,
using TFA stable protecting groups (benzyl ester or cyclohexyl ester on carboxylgroups 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.
Peptide -Platforrn ConJI~ate. 7
A solution of 0.05 mmol of protected peptide with free amine, (H2N-
peptide-CONH2), 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 analyticalHPLC. The peptide protecting groups are removed by tre~tment with HF in the
presence of anisole at 0~ to give conjugate with protecting groups removed, 1
Compound 1 is purified by plel)alalive reverse phase HPLC.
The following scheme shows how to attach a sulfhydryl lir~er to the
amino terminus of a peptide and, in turn, attach the peptide/linker to a
tetrabromoacetylated platform to give compound 13.
SUBSIllUrE SHEEr ~(IJLE 26)
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. 78
Haloacetyl on Platforrn and Sulfhydryl on Peptide
tri~ohor~ylmethanol
H2S0~
HS(CH2)2C02H ~rS(CH2)2CO2H
E~OAc 2
R f~rut~op~7en~
DCC
1) H~N-p~p ~ r C~2CH2
- . N~
Jiu~
HS(CH2)2CONH~ r-S(CH2)2C0~ No2
1 1 2~ tr~luoro2c~ic ocid ~Y
B-C~12COHN(C~)s;CONH~ ~ NHco(cH2)sNH
N~l~c~ O~t~N
BrC1 12COHN(Cf.~2)sCONH 1~ t'll lCC~(Cf~2)sNHCOC~2f3<
F~NHCo(ct 12)2SCH2COHN(CI~2)sCONH~ ~ N~CC(Cl~12)si~1COC~2S(C~2)2
N~~
pel~NHCO(aO25C~12COHN(a~s~NHJ 13 2 (~ N~co~ 2)sNHcocH2s(c~)2
Con~3ound 9
Concentrated sulfuric acid ( 100 ~L) was added to a 60~C solution of
4.48 g (17.2 mmol) oftriphenyl 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~C
for 10 minutes, allowed to cool to room temperature (RT), and placed on ice for
I hour. The resulting white solid was collected by filtration to give 4.52 g (75%)
of 9.
Compolmd 10
Dicyclohexyl carbodiimide (DCC) (2.41 g, 1 1.7 mmol) was added to a
0~C solution of 2.72 g (7.8 mmol) of 2 and 1.08 g (7.8 mmol) of p-nitrophenol in41 mL of CH2C I2 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
SUBSmUrE SHEEr ffUllE 26)
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79
filtrate was concentrated. The residue was cryst~lli7~d from hexane/CH2CI2 to
give 3.17 g (86%) of 10 as pale yellow crystals.
Con~ound 11 - cyclic thioether peptide with rner~topropionyl linker
S attached
A solution of a cyclic thioether peptide (an analogue of a disulfide cyclized
peptide in which one sulfur was replaced with a CH2) and sodium bicarbonate in
water and dioxane was treated with p-nitrophenyl ester 10. If the peptide contains
Iysine, it must be al.proJ~I;ately blocked. The resulting modified peptide was
treated with trifluoroacetic acid to provide thiol linker modified peptide 1 1
Compound 13 - coniu~ate of cyclic thioether peptide ~nd bromoacetylated
platform
To a He sparged solution of 0.10 mmol of thiol modified peptide 1 1 in
100 mM sodiurn borate pH 9, was added 0.025 of bromoacetylated platform L2 as
a 40 mg/mL solution in 9/1 MeOH/H2O). The solution was allowed to stir under
N2 atmosphere until conjugation was complete as evidenced by ~IPLC. The
conjugate was purified by reverse phase HPLC.
SUBSllIU~ SHEEI ~IULE 26)
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wO 97/46251 PCT/uSs7/l00
Example 12: Synthesis of LJP 685
1) is~butyldllotofotms~e
N-., . ~,ll .~1, . IVI pl " ~e, 'THf
C02~1 2) N-hydroxy-2-mcr~plopyridin~3
BOCHN~-- Et~N BOCHN~/ CH2-Br
Co2c~cH~)3 co2C(CH?)3
4 <litlvl.llcn~,t f=\
80C-~ly-pro-CO2H DCC, THf80C~ly-pto-CO~c>--NO2
~CH~)3SS--~CO2H ~) NHS/DCC(CH3),SS~cONl 12
NHFMOC 2) ~IH~HCO~ NHF~10C
N-~MOC-t~utyl~hiocysteine 1) P8u~ aHCO3
d:o~n~ l2~
2) ~)~npound 1
K~'COI ~
" x~2O
gocNH-Gly~ro-coH~clt~s~coNH2BOCHNrCH7 S~CONH2
18 C02H NHFMOC ~ 1 7 CO2C(CH3)3 NI~FMOC
~) rFA~H20tHOCH2CH2SH
~) compound 15
solid phase NaHCO~dio~ne~H2O
peptide synthesis
E3ocNH~-p-coNH~,rcH2--S CONH2
l l
20 ~H-I-L~02H NHCO-R(Pn~c)-D(Ot~u)-P~-A-L-NI IFMOC
1) OBU/CH3CN H2N~-P-COHN~, CH~S ~CONH2
2) C~PPAlCH3Crllpyridine l l
3) TFA/H2Orll, -i5 le~~DTlpheno/ CO NH
(90%) ~ HN-I~-~-A-P~'-D-R-CO
21 (LJP 6~5)
y-bromo-N-BOC-a-~minobutryic acid t-butyl ester. compolmtl 14: A solution of
4.û3 g (13.3 mmol) of N-BOC-glutamic acid-a-t-butyl ester and 1.61 mL (1.48 g,
14.6 mmol) of N-methylmorpholine in 40 mL of dry THF under N2 atmosphere
was cooled to 15~. Isobutylchloroformate (1.73 mL, 1.82 g, 13.3 mmol) was
added to the mixture dropwise. The mixture was stirred for 10 minllte~ and a
solution of 2.03 g (16.û 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 Et3N. The mixture was
covered with foil to keep out light and allowed to stir at room temperature for
SUBSrllllFE SHEEr ~UIE 26)
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81
I hour. The mixture was filtered and the filtrate was concentrated on the rotaryevaporator taking care to minimi7~ exposure to light. The concentrate was
dissolved in 20 mL of BrCC 13 and the solution was cooled to -70~C. The solid
was placed under vacuum, then purged with N2, allowed to come to room
S temperature, placed in a 20~ water bath, and ilTadiated from above at close range
with a 500 W s.ml~mp for 5 minlltes The mixture was concentrated on the rotary
evaporator and purified by silica gel chromatography (40 mm X 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
l 0 fractions by TLC (Rf 0.23, 5% EtOAc/toluene) were combined and concentrated
to give 3.23 g (72%) of compound 14 as a waxy solid.
N-P~OC-Elycylproline-4-nitrophenyl ester. con~ound 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~C and 3.34 g (16.2 mmol) of DCC was added.
The mixture was stilTed at 0~C for 1 hour, the ice bath was removed, and the
mixture was stirred for 16 hours at room temperature. HOAC (579 ~lL) 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 X 150 mm bed, 2.5%
EtOAc/97.5% CH2C 12/l %HOAc). Traces of acetic acid were removed by
concentrating from dioxane several times on the rotary evaporator. The
concentrate was lrilu-dl~d with 3/l hexane Et2O and the reslllting white solid was
collected by filtration to give 4.1 g (90%) of compound 15 as a white solid; TLCRf 0.09, 40/60/l EtOAc/hexanelHOAc; lH NMR(CDC13) o 1.09-2.55 (m, 4H),
1.48 (s, 9H), 3.47-3.77 (m, 2H), 4.05 (m, 2H), 4.74 ~m, lH), 5.45 (bd s, lH), 7.35
(d, 2H), 8.30 (d, 2H).
N-FMOC-S-t-butylthiocysteine~mide. com~ound 16 A solution of 5.0 g
(11.6 mmol) of FMOC-S-t-butylthiocysteine and 1.33 g (11.6 mmol) of N-
SUBSlllUlE SHEEr ~UIlE 2B)
CA 022~6449 1998-11-23
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82
hydroxysuccinimide in 115 mL of THF was cooled to 0~C. To the solution was
added 3.58 g (17.37 rnmol) of DCC. The mixture was stirred at 0~C for I hour
and 42.9 mL of a solution of 1.6 g of (NH4)HCO3 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
CH2C 12 until most of the solid dissolved, then was shaken with 100 mL of IN
HCI solution. The CH2C 12 layer was washed with 100 mL of saturated NaHCO3
solution~ dried (Na2SO4), and filtered. The filtrate was brought to a boil on a hot
plate and crystallized from 300 mL of CH2CI2/hexane to give 4.36 g (87%) of
compound 16 as a white solid: mp 127~-129~C; TLC Rf 0.29, 95/5/1
CH2CI./CH3CN/MeOH, IH NMR (CDC13) ~ 1.36 (s, 9H), 3.06-3.24 (m, 2H),
4.25 (t, lH)~ 4.55 (m. 3H). 5.56 (bd s, lH), 5.70 (bd s, lH), 6.23 (bd s, lH); 13c
NMR (CD,CI3) 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%.
~ound: C, 61.5%; H, 6.0%; N, 6.5%.
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 I .13 g
(13.5 rnrnol) of NaHCO3 was added followed by 1.77 mL (1.44 g, 7.09 mmol) of
PBu3. The mixture was stirred at room te~ Idlure for 1 hour and partitioned
between 1 X 100 mL of IN HCI and 2 X 100 mL of CH2C 12. The CH2C 12 layers
were combined and concentrated and the resulting white solid was partlally
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 K2CO3 was added followed by 2.63 g (8.1
mmol) of compound 14. The mixture was stirred for 16 hours and partitioned
between 1 X 100 mL of lN HCI and 2 X 100 mL of 10% MeOH/CH2Cl2. The
CH2Cl2 layers were combined, dried with NaSO4, filtered and concentrated to a
SUBSmUrE SHEEr ~lULE 28)
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83
semisolid residue. Purification by silica gel chromatography (1/3
CH3CN/CH2Cl2) gave 3.2 g (80%) of compound 17 as a white solid; TLC Rf
0.27, 1/3 CH3CN/CH2Cl2. Further purification of an analytical sample was done
by recryst~lli7ing from hexane/EtOr',c; mp 104-106.5~C; IH NMR (CDCI3) o
1.47 (s, 9H), 1.49 (s, 9H), 1.96 (m, lH), 2.11 (m, lH), 2.69 (m, 2H), 2.88 (m, lH),
3.03 (m, lH), 4.29 (t, lH), 4.36 (m, 2H), 4.S0 (m, 2H), 5.21 (m, lH), 5.49 (m,
lH), 5.81 (m, lH), 6.54 (m, lH), 7.34 (t, 2H), 7.42 (t, 2H), 7.61 (d, 2H), 7.80 (d,
2H); 13C-NMR (MeOH) ~ 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 C3~H4~N307S: C, 62.08; H, 6.89; N, 7.01. Found: C, 62.23; H,
7.12; N, 7.39.
Compound 18: A solution of 20/1/1 TFA/H2O/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 H2O. NaHCO3 (355
mg, 4.22 mmol) was added to the mixture followed by a solution of 1.33 g (3.38
mmol)ofcompound 15in 10.5mLofdioxane. Themixturewasallowedtostirat
room temperature for 20 hours and partitioned between 100 mL of lN HCI and 3
X 100 mL of CH2Cl2. The combined CH2Cl2 layers were dried (Na2SO4),
filtered and concentrated. Purification by silica gel chromatography (35 X
150 mrn; step gradient, 5195/1 MeOH/CH2CL2/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 1~ TLC Rf 0.59, 10/90/1 MeOH/CH2Cl2/HOAc; mp
116-117.5~C; IH NMR (CDC13) o 1.41 (s, 9H), 2.00 (m, 2H), 2.18 (m. 2H), 2.56
(m, lH), 2.69 (m, lH), 2.85 (m, 2H), 3.49 (m, 2H), 3.62 (m, 2H), 3.85 (m, 2H),
4.08 (m, lH), 4.12 (m, lH), 4.22 (t, lH), 4.38 (m, lH), 4.49 (m, 2H), 4.61 (m,
2H), 7.78 (d, bd s, lH), 6.12 (bd s, lH), 6.43 (bd s, lH3, 7.35 (d, 2H), 7.40 (d,
SUBSTllUrE SHEEr ff'(ULE 26)
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84
2H), 7.61 (d, 2H), 7.78 (d, 2h); 13C NMR (CDCL3) o 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
C34H43NsO9S: C, 58.52; H, 6.21; N, 10.03. Found: C, 58.38, H, 6.17; N, 10.20.
S F f
FmocHN ~~ ~ F
CH3 ~ F F
c~O Fmoc~N ~ N~
H HBr N~Ht~O~ CO2H
HOBtll~MF (66%)
10a-rnethyl proline 19
N-FMOC-L-Alanyl-L-2-metllylprolin~? co~o--n~ 19: A solution of 2-
methylproline (Seebach et al. (1983) JAm. Chem. Soc. 105:5390-5398) (1.00 g5
4.76 mmol), 4.00 g (47.6 mmol) of NaHCO3, and 31 mg (0.23 mmol) of HOBT in
6.9 mL of DMF was cooled to 0~C and 3.18 g (6.66 mmol) of N-FMOC-L-alanine
was added. The ~ ~ction was stirred for 1 hour at 0~C, then at room temperature
for 18 hours. The mixture was partitioned between 50 mL of EtOAc and 3 X
50 mL of lN HCI. The EtOAc layer was dried (MgSO4), 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~C; IH NMR (CDC13) ~ 1.39(d, 3H), 1.93 (m, 2H), 2.06 (m, 2H),
3.78 (m, 2H), 4.22 (m, lH), 4.40 (d, 2H), 4.56 (m, lH), 5.09 (t, lH)5 5.69 (d, lH),
7.32 (t, 2H), 7.42 (t, 2H), 7.62 (d, 2H), 7.78 (d, 2H); 13C NMR (CDCl3) ~ 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.
N-FMOC-L-T eucinyl-HMPF~-~RHA resin: A solution of N-FMOC-L-leucine in
22.5 mL of CH2C12 and a few drops of DMF was prepared and cooled to 0~C. To
SUBS~lIUlE SHEEI ffWLE 26)
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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~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 minim~l 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 X DMF, 2 X MeOH, 2 X
DMF, 2 X 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.
N-FMOC-linear peptide with t-butyl ester on aspartic acid ;~n~l Pmc ~roup on
ar~inine and with thioether in~ert~ compound~Q: This peptide was prepared by
standard FMOC synthesis on N-FMOC-L-leucinyl-HMPb-MBRA 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
diisopropy}carbodiimide. Each step was monitored by using 10 ~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 at1 00~C for 2 minutes with one drop of pyridine and one drop of 5% ninhydrin in
EtOH and one drop of 80% phenolin EtOH). Thus, 1.13 mg (0.613 mmol) of resin
was used to prepare the peptide. After the final coupling step, cleava~e from the
resin was accomplished by treatment with 15 mL of a solution of 1%
trifluoroacetic acid in CH2C12 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,
~radient, 60/40/0.1 CH3CH/H20/TFA to 90/10/0.1 CH3CN/H201TFA, 210 mn,
1 mL/min, 4.6 mm X 250 mm column) were combined and concentrated. The
SUBSrllUTE SHEEr (I'~ULE 26)
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86
concentrate was dissolved in 40 mL of 10% HOAc solution and purified by HPLC
to give 0.528 g (49%) of peptide ~.
Conversion of peptide 20 to cyclic peptide 21 (removal of FMOC ~roup
cyclization and removal of protecti~ grou~s: A solution (5 mL) of 99 ~lL of
DBU in 10 mL of CH3CN was added to 96 mg (0.052 mmol) of peptide ~Q. The
solution was stirred for I hour and concentrated to a residue. The residue was
triturated with 2 X 10 mL of Et2O to give a white solid. The solid was dissolvedin 100 mL of CH3CN and 312 IlL (0-312 mmol) of 0. I M solution of
diphenylphosphorylazide (DPPA) in CH3CN. The mixture was stirred for
20 hours and concentrated on a rotary evaporator. The residue was triturated with
2 X 50 ml of Et20 and the white opaque residue was treated with 5 mL of
9213I213 TFA/anisole/EDT/Me~S for I hour. The product was precipitated by
adding the mixture to 40 mL of Et2O in a 50 mL polypropylene centrifuge tube.
The precipitate was cooled to 0~C and centrifuged for S minutes at 2000 rpm. Thesupernatant was decanted and the pellet was washed with Et2O and recentrifuged.
The pellet was dried and dissolved in 4 mL of 50/50 CH3CN/H2O. The mixture
was diluted with 36 mL of H2O/0.1 % TFA, filtered and purified by HPLC (1 " C 18column, gradient, 10/90/0/l CH3CN/H20/TFA to 35/65/0.1 CH3CN/H20/TFA.
230 nm). The pure fractions, as evidenced by HPLC, were Iyophilized to give
52 mg (90%) of compound 21.
S~BSmUrE SffEEr ~nE 26)
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. 87
Example 13: Synthesis of conju~ates of LJP 685
H2N~-P-COHN ~ CH~S ~ CONH2
CO NH
I~N-l-L-L-A-p~ D-R-co 21 (LJP 6~5)
O
Hs~)~HN~-p-coHN~--CH~S CONH2 ~ ~) TrscH2cH2co2pNp
--1~ EtJNlDMF 2~TFA~cH2c~ aphen
2 2 CO'NH 2)TFA/CH2CI21thiophenol
H~-L-L-A-p~-D-R- CO
HS O O O ~
HN-~P-COHNf CH2-S ~CONH2
24 CO NH
HN-I-L~-P~b~R-co
O H
t\o O t'l H ~S~ff2CH2CONH-peptide
\ 2 ~ N~~~N~S CH2CH2CON~peptid~ -
O H
2 5 ~ p~ptid~ = a JP 6~5~
O O 11 N HH N~\o NH~epcde
2 \ N~f~~'' ~S O O~O NH-peptide
O H \~
26 pepLide = (I JP 6a5)
SUBS~llUlE SHEr ~ULE 26)
CA 022~6449 1998-11-23
WO 97/46251 PCT~US97110075
88
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 ~, the peptide with the free thiol linker. Reaction of an excess of
compound ~ with valency platform molecule 12 produced tetravalent conjugate
~. Treatment of compound 21 with the longer linker, compound 33 (see reaction
scheme below), followed by detritylation, gave compound ~. Compound 24
reacted with valency platform molecule 12 to give the tetraconjugate 2~. Both
conjugation reactions appeared very clean by HPLC.
Attachment of MTU linker to LJP 685. synthesis of compound 24: To
15 mg.(0.013 mmol) of compound 21 was added 160 IlL of a solution of 29.5 mg
of compound 23 and 17.5 ~L of diisopropylethylamine in 0.5 mI, of DMF. The
mixture was stirred for 2 hours and precipitated from Et2O. The precipitate was
dried and dissolved in 650 ~L of a solution of 1/1/0.056/0.040
TFA/CH2CI2/thiophenol/Me2S and the solution was allowed to stand for I hour.
Precipitation from Et20 gave crude compound ~L which was purified by HPLC
(Cl8,15-45% CH3CN/H2O, 0.1% TFA). Fractions Cont~ining pure product were
Iyophilized to give 8.6 mg of compound 24 as a white solid.
LJP 685-MTU-AHAB-TEG~ compound 26: To a solution of 8.6 mg (6.3 X 10 6
mol) of compound 24 in 630 ~L of He sparged pH 8.5 200 mM borate buffer was
40 ~lL of a 40 mg/mL solution of compound 12 in 9/1 MeOH/H20. The mixture
was stirred for 24 hours and I mL of 10% HOAc/H2O solution was added. The
mixture was purified ~y HPLC (C~8, gradient 25-55% CH3CN/H20, 0.1% TFA) to
give 8.3 mg of compound 26 (LJP 685-MTU-AHAB-TEG).
Example 14: Development of extended SH linkers
The thiobenzoate ester, compound 28, was prepared from compound 27.
Compound 28 was converted to compound ~ in portions. The thiobenzoate was
SUBSTllUrE SHEET ~WLE 26)
.
CA 02256449 1998-11-23
WO 97/46251 PCT/US97/10075
. . .
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 ~ p~d from compound 29. Aminotrioxoudecanoicacid
ethylester, compound 3 1, was ~Ic~ud~ed by treatment of compound ~ Z with
sodium azide and reduction to the amine. Amine 3-1 was acylated with
cornpound 3-0 to prbvide compound 32. Hydrolysis of compound 32 was
achieved by tre~tmPnt with sodium hydroxide to give a free carboxylic acid. The
intermediate carboxylic acid was con-l~Pn~e~l with p-nitrophenol 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.
a o o o-cf1,ca ~ D9U ~8 O O O-CH2CO2R
27 2~ 1) N-OEUEtOH
2t ~
~ , 3)~4ROt~,
TrS O O O-CH~CO2PNP ~ ./~
TrS O O O--a~2Co~H
1) N,N~Mfl~2~~
a o o o-~2co2E~ 2) Il~ C ~ C/ 0 3-a~ ,B
~0
, r ~4
TrS o O o HN O O O-c1~2co2E
I) N~OH 2
EIOH
__ ~ ; 2) f . ~L ~
TrS O O O ~ O O O-CH2CO2PNP
H~ O O O ~N O O o-CH2CONH-G-P.COHN --CH2-5 ~CONH2
3 4 CO NH
tl~l~-~-/~-D-R CO
SUBST TU~E SHEEr ~n~ 26)
CA 02256449 199~ -11- 23
rCT/11S97/tO075
WO g7146251
Example 15: Synthesi~ of a (I lp68s~4/~Tu-ATl~-AHAB-TEG CoF~ t~
co~ollnd 35
Tetravalent conjugate 3~ was prepared as shown below. The peptide with
- ~ linkers attached, compound 34 was dissolved in ~e sparged, p~ 8.~, 200 ni~ borate
buffer To the mixture was added 0.3 mo~ e~uivalents of platform compound 1-2.
~he mixture was stirred for I hour and the product was purified by HPLC.
~~~o~~or~4~N~~ ~ o~C~2C~NIt G P --r CONff2
HN-l~-L A-P~e-D R CO
34 ~ 12
R~S O O O ~l~l O O O-CH2CONH-G P COHN--~-- 2 S~CQN~)
3 5 ffN-t~ L~ P~e~l}R~o
Fc = O , ~N~J
H O
Example 16: Synthesi~ of a (l~lP685~lll)ARA-PEG conju~ate compollnd 36
Treatment of IA-DABA-PEG with compound ~ in ~.5 borate buffer gave
conjugate 36 O
t~S 24
t\of\oio~N--N~ ~ Pep=WP685
IA-D~BA-P~G HN~I O
~0 0~~--~ Pe,c
~\~ --N~
HN~S o~0 0--
SUBS~T~ SHEr ~E26)
. , . , . _ .. .. . .
CA 022~6449 1998-11-23
wo 97/46251 PCT/US97/10075
91 ''
Example 17: T cell assay for activation
Tritiated thymidine uptal~e by peptide-stimulated T cells was monitored in
96well round bottom plates. A single-cell suspension of draining Iymph node
cells (mice) or isolated peripheral blood Iymphocytes (human), 5 x 105 were
mixed with between I and 30 jig of peptide in a final volume of 150 ,uL per welland incubated for 5 days at 37~C in 5% C02. At that point, I 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: In vitro induction of tolerance
Eight groups~ each cont~ining five C57Bl/6 mice, were primed with
10 ~lg/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~C with (LJP685)4-DABA-PEG,
compound 36, and (LJP685)4-TEG~ compound 35, in concentrations of 100 ~M,
20 !lM and 4 IlM. 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 b~l~nced salt solution. The
cells were then injected into 650R irradiated syngeneic recipient mice in such amanner 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 imrnunization of 10 ~lg of LJP 685-KLH in saline,
intraperitoneally. Seven days after the booster immunization, the mice were bledand 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
SUBSll~ SffEEr (P(llL~ 26)
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92
antibodies as shown in Figures 16 and 17, which is measured by Antigen Binding
Capacity (ABC) as described in Iverson, G.M., "Assay for in vivo adoptive
imrnune responses," in HANDBOOK OF EXPERIMENTAL IMMUNOLOGY,
Volume 2, Cellular Immunology ~Weir, D.M., ed., Blackwell Scientific
S Publications, Palo Alto, CA, 1986).
Example 19: NMR solution structure ~n~lysis of the SA12. CB2 ~nll 3G3
peptides
Two peptides isolated from pliage library screens using the methodology
0 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 Ci5' and trans isomers at this position. Removal of the proline
gave peptides with KD'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 SA12 (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
2() dispersion in the ID IH NMR spectnun of CB2 which was consistent with SA12
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 SA12
peptide. Although deuterium exchange and tempelaLure 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 2.1 + 0.2 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
SUBSTI~Ul E SHEEr (RlllE 26)
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wO 97/46251 PCT/US97/10075
93 ~ -
the backbone LIL region. Finally, the carboxy terminal glycine is very mobile asit is the only residue with a positive intraresidue NOE. This represents the first
structural infon-nation determined about a peptide that mimics the ACA epitope
on ,B2-GPI.
NMR data coupled with distance geometry calculations were used to
determine the three dimensional structure of peptide 925 (CLGVLAKLC), a
truncated version of peptide 3 G3 (AGP CLGVLGKLCPG) with alanine
substituted for glycine in position 6 of the 925 peptide. The structure of peptide
925 was detenriined in water at pH 3.8 and at 25~C. An ensemble of nine
l 0 structures were calculated all of which were consistent with the NMR data. The
RMSD for all non-hydrogen atoms was 2.45 + 0.36 angstroms when each
structure was compared to the centroid. Figure 18 displays the structure closest to
the centrold of the ensemble and, therefore, is a reasonable representation of the
shape of the peptide 925 molecule. Figure 19 compares the structure of peptide
925 (labeled at the bottom of the figure as 3G3) with the structure of peptide 5Al2.
Both peptides have turns at approximately the same positions in the peptide
sequence.
The pharmaeophore 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 pharmaeophore of
this peptide as shown in Figure 20. The hydrocarbon linkers that tether the
pharmacophore groups to some scaffold have the lengths specified in Figure 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~.
SUBSlTlUlE SffEEr ~lJLE 26)
, ..
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.
Example 20: Synthesis of TFG carbamate linker
Cl~/~o~O~O~
37
fBuSH, DBU
~ (99% )
t~US~o~~~ OH
38
CICOO ~NO2, Pyr.
O
BuS O ~~ o~o~NO2
2-[2-(2-tert-Butylthioethoxj~)ethoxy]ethanol. com~ound 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 mrnol) cooled in an ice bath was slowly added 1,8-
diazabicyclo[5.40]undec-7-ene ~DBU, 9.75 mL, 65.23 mmol). The reaction
mixture was allowed to warm to room termperature and stirred overnight. This
mixture was then diluted with ethyl acetate and filtered. The crude product in the
- filtrate was purified on a filter colurnn eluted with ethyl acetate to give yellowish
oil (14.4 g, 64.6 mmol, 99%): lH NMR (300 Mhz, CDCI3): d 3.73 (br s, 2H),
3.69-3.60 (m, 8H, 2.75 (t, J= 7.4, 2H), 2.62 (br s, lH), 1.32 (s, 9H); 13C NMR
(75 Mhz, CDC13): d 72.47, 71.08, 70.33, 70.27, 61.71, 42.09, 30.95, 27.87; MS
(ESI): m/e (M+l) Calcd. for CloH23O3S: 223, obsd.: 223.
SVBSllTUrE SHEEr ~WE 26)
,
CA 022~6449 1998-11-23
wo 97/46251 PCT/US97/10075
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
chloroforrnate (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
S precipitate came out imrnediately. 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
purlficatlon.
Example 21: Synthesis of the Tetravalent Platform IA/DAB~/ATEG. 46.
Bis-N-(t-butoxycarbonyl)-diaminoben70ic acid. corr~ound 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 :~nd 2.76 g (32.9 mmol)of NaHCO3 in 44.5 mL of H20 and 22.5 mL of MeOH, and the mixture was
stirred at room temperature for 24 h. The mixture was cooled to 0~, and 6.53 g of
citric acid was added, and the mixture was extracted with EtOAc. The combined
EtOAc layers were dried (MgS04), filtered, and concentrated. The residue was
dissolved in 40 mL of Et,O and the solution was filtered through Celite. The Et20
layer was extracted with two 40 mL portions of HCl. The Et20 layer was dried
(MgSO4), filtered, and concentrated to give 3.81 g (66%) of 40 as a foamy pink
solid.
N-hydroxysuccinimidyl ester of co~r~ound 40. comround 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 mrnol) of N-hydroxysuccinimide
in 55 mL of EtOAc which had been cooled to 0~, and the resulting mixture was
stirred for 18 h allowing to come to room t~ p~ldL~re. 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
SUBSllTUlE SHEr ff~ E 26) ~-
CA 022~6449 1998-11-23
W O 97/46251 PCTAUS97/10075
96
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.
Mono-N-(t-butoxycarbonyl)-ethylene~liAmine. compound 42: A solution of 1.5 g
(25.0 mmol) of ethylene~ mine in 15 mL of CH2CI2 was cooled to 0~, 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
CH2CI2/MeOH/HOAc) gave 0.98 g (67%) of compound 42 as an oil.
Bis- N-(t-butoxycarbonvl)-~mino-TFG. compound 43: To a solution of 750 mg
(4.25 mmol) compound 42 and 345 uL (337 mg, 4.25 mmol) of pyridine in 6 mL
of CH2CI was added 445 uL (559 mg, 2.02 mmol) of triethyleneglycol bis-
chloroforrnate. The mixture was stirred for 3.5 h, and the mixture was partitioned
between 35 mL of CH2CI2 and 35 mL of I N HCI. The CH2CI2 layer was washed
with H2O? dried (Na,SO4), filtered and concentrated to give I .14 g of crude
compound 43 which was used directly in the next step.
Diamino-TEG bis-tr;fluoroacetate salt~ compound 44: 300 mg (0.57 mmol) of
compound 43 was dissolved in 3.5 g of CH2Cl2, and 3.5 mL of trifluoroacetic acidwas added. The mixture was stirred for 3 h at room temperature, and the solutionwas concentrated to give 398 mg of crude compound 44 which was used directly
in the next step.
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 NaHCO3. The mixture was stirred for 3 h and
acidified with I N HCI and partitioned between 20 mL of I N HCI and 30 mL of
EtOAc. The combined EtOAc layers are washed with sat NAHCO3 solution,
SUBSllIUI E SHEEr ffWlE 26)
- CA 022~6449 1998-11-23
W O 97/462~1 PCT~US97/10075
97
dried (MgSO4), 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 ~ as a white foamy solid.
IA/DABAIATFG. Con~l?o~lnd 46: A solution of 100 mg (0.1 mmol) of compound
45 was prepared, and I mL of trifluoroacetic acid was added, and the mixture wasstirred for I h at room temperature. The mixture was concentrated under vacuum.
The residue was triturated with Et2O and dried under vacuum to give a white
crystalline solid. The solid was dissolved in I mL of DMF, 104 uL (77 mg,
0.6 mmol) of diisopropylethylamine was added, the mixture was cooled to 0~, 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~, acidified with I N H2SO4, and partitioned between 10 mL of
I N H2SO4 and 6 X 20 mL portions of 8/2 CH2CI2/MeOH. The combined organic
layers were dried (MgS04), filtered, and concentrated to give 330 mg of orange
oil. Purification by pl~.ai~tive HPLC (25 cm X 22.4 mm C~g, gradient: 30%B to
40%B 0-40 min, A = H2O/0. 1% TFA, B = CH3CN/0. 1% TFA, 12 mL/min) gave
I g mg of compound 46 as a white solid.
SUBSmUrE SHEEr ~UlE 2B)
CA 02256449 1998-11-23
W O97146251 PCTAUS9711007S
98
NH2 (BOC)20 NHBOC NHS NHBOC O
40 ~ 41 O O
3,5-diaminobenzoic acid
NH2~ NH2 (BOC)20 NH2~ --NHBOC
ethylenediamineCH2CI2 42
¦ triethyleneglycol bis-chloroformate
~ pyridine/CH2CI2
NH o ~ i 2 CH2CI2 BOCNH N ~ r~
Compound 2
NaHC03
dioxane/H20
1)TFA
CH2C12
B OC N H ~ N ~ N ~ o~L2 I C H2 CO H N ~[~ ~ N ' ' ~ ~1L2
2) iodoacelic-
NHBOC -- anhydride ICH2COHN 46 (IA/DABA/ATEG)
DIPE~DMF
Example 22: Synthesi~ ofthe Tetravalent Platfonn BA/PABA/DTITFG. 51.
N-(t-butoxycarbonyl)PARA~ compound 47: A solution was prepared of 3.0 g(21.9 mmol) of p-aminobenzoic acid in 60 mL of H2O. Na2CO3 (2.16 g, 25.7
SUBSrllUrE SHET (RUlE 26)
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99
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 ofMeOH was added and the mixture was stirred at room telllpeld~ure for 18 h. To
the mixture was added 4.92 g (25.6 mmol) of citric acid and the resulting cloudyS mixture was partitioned between 200 mL of H2O and 200 mL of EtOAc. The
EtOAc layer was washed successively with 200 mL of 0.1 N HCI and 200 mL of
H20, dried (Na2SO4), filtered, and concentrated to yield 3.0 g (58%) of compound47 as a white solid.
N-(t-butoxycarbonyl)P~BA N-hydroxysuccinimidyl ester~ compound 48: DCC
(2.61 g, 12.6 mrnol) was added to a 0~ solution of 2.0 g (8.43 mmol) of compound47 and 0.97 g (8.43 mrnol) of N-hydroxysuccinimide in 5(~ 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
l S min, placed in the freezer for l .S 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.
Compound 49: A solution of 3.00 g (8.97 mmol) of compound 48 in 50 mL Of
CH2CI2 was added dropwise over 30 min to a solution of 485 uL (4.49 mmol) of
diethylenetriamine in 30 uL of CH2CI2 which had been cooled in an ice bath. The
mixture was stirred at 5-7~ for 30 min then at-room temperature for 16 h. The
milky mixture was placed in a separatory funnel with 200 mL of H20, the pH of
the H2O layer was adjusted to 10 with 3 M NaOH solution, and the mixture was
extracted with 200 mL of 4/1 CH3Cl/MeOH. The organic phase was dried
(Na2SO4) 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 CH3CI/MeOH. The organic layers were
combined, dried (Na,SO4), and concentrated to give another 1.08 g (44%) of
compound 49 which required further purification. Further purification can be
SUBSrllUrE SHEEr ff'~ULE 26)
CA 022~6449 1998-ll-23
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100
accomplished by silica gel chromatography (gradient~ 80/20 to 70/30
CH3CI/MeOH).
Compollnd 50: A solution of 135 uL (0.66 mmol) of triethyleneglycol bis-
chloroformate in 0.3 mL of THF was added to a 0~ solution of 855 mg ~1.58
mmol) of compound 49 and 275 uL (1. mmol) of diisopropylethylamine in 13 mL
of THF. The cloudy mixture cleared when the ice bath was removed. An
additional 70 uL of dilsopropyethylamine was added to m~int~in a basic pH. The
mixture was stirred at room temperature for a total of 3 h and partitioned between
25 mL of H2O 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
(Na2CO3), filtered, and concentrated to give 0.986 g of crude 50. Purification by
silica gel chromatography (80/4/16 CH3CI/dioxane/isopropanol) gave 516 mg
(61%) of compound 50 as a white solid.
P,e~a.d~ion of BA/PABA/DT/TEG. compound 51: Compound 50 (487 mg,
8.79 mmol) was dissolved in 9 mL of CH2Cl2 and 5 mL of trifluoroacetic acid.
The mixture was stirred for 1.5 h and concentrated. The residue was triturated
with 7 mL of Et2O, 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 H2O, and 191 mg (2.27 mmol)
of NaHCO3 was added. A solution of 591 mg (2.27 mmol) of bromoacetic
anhydride in 10 mL dioxane was added to the mixture. An additional 2 mL of
dioxane was used to rinse. More NaHCO3 was added as needed to m~int~in a
basic pH. The mixture was stirred for 2 h at room temperature, and acidifiéd with
I N H2SO4. The mixture was extracted with 3 X 25 mL of ~tOAc. The combined
EtOAc layers were dried (Na2SO4), filtered, and concentrated to give 773 mg of
an oil. Purification was accomplished by silica gel chromatography (gradient~
90/10 to 85/15 CH3CI/MeOH) to give 401 mg (77%) of 51 as a white solid.
SUBSTlTUlE SHEEI- ~WI~ 26)
CA 02256449 1998-11-23
W O 97/46251 PCT~US97/10075
101
(BOC)20
O MeOH/H2o ~ 0
H2N ~ OH Na2CO3 BOCH N ~~' OH
4-aminobenzoic acid 47
NHS
DCC
EtOAc
/=\ 11 diethylenetriamine
BOCHN~ ~ ~ CH2C12 0 ~_
H N - H ~ BOCHN~O-N~
BOCHN~ N J
49 O
triethyleneglycol bis-
chloroformate
~ Et3N/THF
BOCHN~ N ~10 1) TFAJCH2CI2 BrCH2COHN~ H ~\ ~ '~
H ~7/2 H NuO o7'2
BOCHN~, H J BrCH2COHN~ N J
0 50 2) bromoacetic anhydride ~
NaHCO3 51 (BAIPABA/DT/TEG)
dloxane/H2~
Example 23: Synthesis of the Tetravalent Platform BMP/TEG. 55.
Dimethyl-5-bydroxyisophth~l~t~. compound 52: A solution of 2.00 g (11 mmol)
of 5-hydroxylsophthatic acid and 0.5 mL of con HCI in 30 mL of MeOH was
refluxed for 5 h. The mixture was concentrated and the resulting residue was
disso~ved in 100 mL of EtOAc. The EtOAc solution was washed successivel~
with two 50 mL portions of 5% NaHCO3 solution two 50 mL portions of sat NaCI
solution, dried (Na2CO3), 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
K2CO3 in 11 mL of CH3CN and the mixture was refluxed under N2 for 16 h. The
mixture was concentrated, alld the residue was dissolved in 25 mL of CHCI3 and
washed with 25 mL of H2O and 25 mL of sat NaCI solution. The CHCl3 layer
was dried (Na2SO4), filtered, and concentrated. Purification by silica gel
chromatography (gradient, 50/50 hexane/EtOAc to 100% EtOAc) gave 93 mg
(41 %) of compound 53.
SUBSlllUrE SHEE~ ff'lUlE 26)
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Compound 54: A suspension of 93 mg (0.18 mmol) of compound 53 in 4 mL of
THF was stirred under a N2 atmosphere while 1.82 mL(1.82 mmol) of a I M
solution of LiBHEt3 was added to the mixture dropwise. A clear solution was
obtained which was stirred for 18 I~, ~t which time 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 witl
3 X 10 mL portions of EtOAc, and the combined EtOAc layers were dried
(Na2SO4), filtered and concentrated. Purification was accomplished by silica gelchromatography (gradient, 90/10 to 85/15 CH3CI/MeOH) to give 20 mg (27%) of
54 as a white solid.
Cornpound 55: To a suspension of 20 mg (0.048 mmol) of 54 in 5 mL of Et,O
and I mL of THF is added 9.6 uL (0.1 mmol) of PBr3. The mixture is stirred for
3 h and partitioned between and EtOAc. The EtOAc layer is dried (Na2SO4),
filtered and concentrated, and the residue is purified by silica gel chromatograph~
(CH3CI/MeOH) to pro~ide compound 55.
HO2C HCOH MeO2C
~ OH > ~OH
HO2C MeO2C 52
5-hydroxyisophthalic acid K2Co3/CH3CN
r\~
TosO O/ 2
HO~_ LiBHFt3 M c~ 53
HO 54
PBr
TH~/Et20
Br~
~O/ \Or/2
Br
55. fBMP/TEG)
SUBSllTUl E SHEEr (P((ILE 26)
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Example 24: Synthesis of the Telravalent Platfo~n BA/P17/~DA/TF.G. 60.
Compound 56 To a solution of 1.02 g (4.37 mmol) of N-(t-butoxycarbonyl)-
iminodiacetic acid (compound 5 in U.S. 5,552,391, Chemically-Defined Non-
Polymeric Valency Platform Molecu!es and Conjugates Thereof) and 1.01 g
(8.75 mmol) of N-hydroxysuccinimide in 50 mL of dry THF. cooled to 0~, was
added 2.26 g (10.94 mmol) of dicyclohexylcarbodiimide. The mixture was stirred
for 16 h allowing to slowly warrn 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 rnmol) of Et3N. The mixture was stirred for
7 h at room temperature, filtered. The filtrate was concentrated and the residuewas dissolved in 125 mL of EtOAc and shaken with 2 X 125 mL portions of I N
HCI, 125 mL of sat NaHCO3 solution, dried (MgSO4 filtered, and concentrated to
give 2.39 g of a sticky solid. Purification by silica gel chromatography (95/J
CH2CI~/MeOH) gave 1.85 g (66%) of 56.
Compound 57: To a solution of 1.74 g (2.74 mmol) of compound 56 in 10 mL of
CH2CI2 was added 10 mL of trifluoroacetic acid, and the mixture was stirred for
3 h at room te,llpe,~ re. mixture was concentrated, and the residue was dissolved
in 5 mL of CH2CI2. The mixture cooled to 0~ and 100 mL of sat NaHCO3 was
added. The mixture was then extracted with four 100 mL portions of CH2CI2.
The CH2CI2 layers were combined, dried (MgS04), filtered, and concentrated to
give 1.46 g (99%~ of 57 as a sticky hygroscopic solid which was used directly the
next step.
- Con~ound 58: To a solution of 0.7 g (1.3 mmol) of compound 57 and 226 uL
( 168 mg~ 1.30 mmol) of diisopropylethylamine at 0~ was added a solution of 127
SUBSmUlE SHEEr ffWl E 2S)
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uL of triethyleneglycol biscmoroforinate in 4 mL of CH2CI2, and the ~ e was
stirred for 3 h at room temperature. mixture was partitioned between 80 mL of
CH2CI2 and 80 mL of I N HCI. The ~ 2 ~ layer was washed with two 80 mL
portions of water, dried (MgSO4), filtered, and concentrated to give 736 mg (93%)
of compound 58 as a crystalline solid.
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 I h at
which time 5 mL of Et2O added. The mixture was placed in the freezer for 1 h
and centrifuged. The resulting pellet was washed with Et2O and dried to give thetetralaydrobromide salt 59 which was dissolved in 1 mL of H2O. To the mixture
is added 49 mg (0.58 mmol) of NaHCO3 and 3 mL of dioxane. More NaHCO3 is
added, if needed, to make the mixture basic. The mixture - i cooled to 0~, and
748 mg (2.89 mmol) of bromoacetic anhydride is added. The mixture is stirred
for 2 h and partitioned between 20 mL of I N H2SO4 and 20 mL of 80/20
CH2CI2/MeOH. The organic layer is dried (Na2SO4), filtered and concentrated to
give crude 60 which is purified by silica gel chromatography (CH2CI2/MeOH) to
,~ive 60.
SUBSllTUrE SHEEr (RUI~ 26)
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NCBZ N-BOC iminodiacetir~acid A ~
HN THF CBZN N~
mono-CBZ-piperazine ~ A NBOC
CBZN N~
\_/ 0 56
TFAICH2Cl2
~rielhyleneglycol bis-
o chloroformate/DlPEA O
CBZN N~ CH2CI2 CBZN N
I O l--r/
/_\ N ~ ~/ 2 ~ / \ îNH
CBZNJN~ CBZN N~
HBr
acetic acid
bromoacetic anhydride
NaHC03
~_~ o dloxane/H20 A ~
~/ ~N ~ Ol~Or/ 2 ~J ~ 0 1~ r/'
HBr.HN N~ BrCH2CON N~
~ 59 60 (sAlplzllDAlTEG)
Example 25: Synthesis of the Tetravalent Platform tetrakis-BATPI7/PMA. 63.
Compound 61: To a 0~ 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 telllp~,dl-lre 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 Et3N. The mixture-was filtered, and the filtrate
was concentrated. The residue was partitioned between 100 mL of EtOAc and
2 X 100 mL of 1 N HCI, and the EtOAc layer was washed with 100 mL of sat
NaHCO3, 100 mL of H2O, 100 mL of sat NaHCO3, dried (MgSO4;, filtered, and
- 25 concentrated. Purification was accomplished by silica gel chromatography
(97.5/2.5 CH2C1~/MeOH) to give 1.78 g (66%) of 63 as a white crystalline solid.
SUBSmUI E SHEr ~RUlE 26)
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106
Compound 63: Compound 6 l 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.
HO2C,~ 02H DCC CBZHN N~N NCBZ
HO2C CO2H HN NCBZ CBZHN N N NCBZ
1 ,2,4,5-benzenetetracarboxylic acid 6
(pyrometlitic acid)
HBr/HOAc
bromoacetic anhydride HBr.HN N~ ~N N11.HBr
NaHC03/diOxanelH2o A ~
HBr.HN N~N NH.HBr
BrCH2CON N~N NCOCH2Br 62
BrCH2CON N N NCOCH2Br
63 (tetrakis-BA/PlZ/PMA)
~O
Example 26: Synthesis of the Tetravalent Platform 3A/PIZ/II)A/E~B/TEG 68.
Compound 64: Triethyleneglycol ditosylate (1.0 g, 2.18 mmol) was added to a
solution o 725 mg (4.36 mmol) of ethyl 4-hydroxybenzoate and 723 mg (5.23
mmol) of K2CO3, and the mixture was refluxed for 16 h. The mixture was
concentrated, and the residue was partitioned between 20 mL of water and 3 X
20 mL of Et2O. The combined organic layers were washed with 2 X 40 of sat
NaHCO3 solution, 40 inL of sat NaCI solution. The aqueous layers were washed
with Et2O, and the combined Et2O layers were dried (MgSO4), filtered, and
concentrated. Purification by silica gel chromatography (70/30 hexanes/EtOAc)
have 902 mg (93%) of 64 as a crystalline solid.
SUBSllTUlE SHEET ~UI,E 26)
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Compound 65: Compound 64 is dissolved in acetone cont~inine 2.2 equivalents
of LiOH and mixture is stirred for 3 h (until complete as evidenced by TLC). Themixture is acidified with acetic acid and concentrated, and the residue is purified
by silica gel chromatography to give 65.
Compound 66: Compound Ç~ 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
Compound 68: Compound 66 is converted to compound 68 in essentially the
same marmer as described for the conversion of 58 to 60 in Example 23.
Purification is accomplished usin~ silica gel chromatography.
Ho2c acetone MeO2C
~_ A r/ ~ ~~ ~ ~/2
HO2C 69 MeO2C 53
NHS
DCC
THF
HN NCBZ
CBZN N ~ ~ HBr/HOAc HBr.HN N ~=~
~ <~0 2 ~ ~ ~~ ~ 2
CBZN N ~; 7Q HBr.HN N ~
bromoacetic anhydride
~" NaHCO3/dioxane/H20
~ o
BrCH2CON N ~=N A r/
~ ~o o/2
72 (BA/PIZIHIP/TE(~) BrCH2CON N~
SUBSllTUrE SHER ffWI~ 28) ---
,
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Example 27: Synthesis of the Tetravalent Platfonn BA/PT7./HIP/TFG. 72.
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.
Comr~o~nd 70: The tetra-acid, compound 69, is converted to compound 70 in
- essenti~lly 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.
Compound 72: Compound 70 is converted to compound 72 in essentially the
same manner described for the conversion of 58 to 60 in Example 23. Purificationis accomplished using silica gel chromatography.
K2C03/CH3CN
t~ r/
TosO O/2 ~ ~/.
EtO2C ~ OH ~ ESO2C ~ o O~/2
ethyi4-hydroxybenzoate 64
A ~acetone
C BZN N
CBZN N~CBZN N~
N~O O/2 ~ O HOzC~O (~
CBZN N ~ DCC
\~ 11 66 NHS 65
o -- THF
HBr
acetic aci;l
bromoacetic anhydride
NaHCO3
~_~ O dioxanetH20 ~ ~
HBr.HN N 1l . Bt-CH2CON N~
\~ ~ O r=~ r~ r/ ~ \J I O ~ r~ r/
N~O O/2 ~_~ N~o O/2
HBr.HN N~ BrCH2CON N~
0 6, 6~3 (BtVPlZ/lDA/HBtTEG)
SUB~ SHEEr ~RlJlE 26)
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Example 28: Sy~thesis of Coruu~t~s of Haloacetylated Tetravalent Con~ounds
Synthesis of (~ rP685)1/MTU/A/DABA/AT~G corU-~eAt~
compound 73:
A solution was prepared of 21 mg ( 15.5 umol) of compound 24 in Helium
S sparged pH 8.5 200 mM borate buffer To the solution was added a second
solution consisting of 3.25 mg'(2.6 urnol) of IA/DABA/ATEG, compound 46,
dissolved in 396 uL of MeOH. A precipitate fo~ned, and I 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 H2O/HOAc was added. The mixture was diluted
] 0 with 50 ML of H2O/CH3CN and loaded onto an HPLC p~ e~aldlive column.
Purification was accomplished by p.~ d~ive HPLC (25 cm X 22.4 mm C~8.
gradient: 35%B to 45%B 0-40 min, A = H2O/0. 1% TFA, B = CH3CN/0. 1% TFA,
12 mL/min) to provide 10.8 mg (67%) of compound 73 as a solid after
Iyophilization.
Pep~O~O~O~SH Pep = LJP 685
0 24
ICH2COHN~ ~NILO ~~2
~r ICH2COHN 46 (IA/DABA/ATEG)
P ~~ ~ ~ ~S ~~~
HN~ ~N 'l
HN
~0~~ O~S~
73 (LJI'68~)4/MTU/A/DA8A/ATEG
Syntllesis of (T TP685)~/MTU cor~lu~tP~ co~oIm~lc 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 ay~u-oyl;ate platform compound for platform
compound 46. Pep can be LJP685 or other relevant peptide.
SU~SmUrE SHEEr ~RULE 26)
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~0 ~0 o~ S ~l~ a~ H--l O ~
P ~O O~O 5~ ~H N"O ~/2
~~--o~S~t~,N/--\N~
N 0 0/
Jl~, o ~ O s ~ N
O O
~ ~ o ~ S J~ NN 4~ N N ~ S O ~ O ~O ~-
P~P~ --o~~ S~N N~NN~1~5~o 0~ ~~Pep
~~~ ~O~ ~ N ~
~ ~1 or~r/
N ~ O 0/ ?
c~~~ 0~~ - s~NN~
~O~O o~S~,,U~ /~~ O
Pop~ --o~O S~N\ N
Example 29: Synthesis of Con~in~t~s Tetravalent Platforrn 55~ Co~ ~te
Con~ol-nd 79. a TP685)JMP/TEG:
A solution is prepared of four equivalents of compound 2~L and five
equivalents of Cs2C3 in DMF. One equivalent of BMP/TEG, platform compound
55, is added to the mixture is then stirred for I h. The mixture is diluted with80/20/10 H20/CH3CN/HOAc and loaded onto a plep~d~ e HPLC column.
Purification is accomplished by plc~a~live HPLC (C~x. A = H20/0.1% TFA. B =
25 CH3CN/0.1% TFA) to give compound 79 after Iyophilization. Pep can be LJP685
or other relevant peptide.
SUBS~TUlE SHEEl ~ULE 26) -'-
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Pep~O~O~ O~sH Pep = LJP 685
o 24
Br
cs2co3 ~\ / \ ,7l
DMF _~~ ~/ 2
Br
- 55 (BMP/~EG)
PepJ~~ 'O~ 0
~~/ '~7L2
Pep~f~O~\/O~ - O~/S
o 79 ((LJP685)4/MTlJ/MP/TEG)
l~xample 30: Synthesis of Cor~u~ates Tetravalent Platforn- tetrakis-BMB~
Conjug~t~ Compound B0, (L~P685)~/MTU/tetrakis-MR:
A solution is ~le~a~ed of four equivalents of compound 24 and five
equivalents of Cs2C3 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 H2O/CH3CN/HOAc and loaded onto a p,e~ dli~e HPLC column.
Purification is accomplished by pIepdldlive HPLC (Cl8 A = H2O/0.1% TFA, B =
CH3CN/0.1% TFA) to give compound 80 after Iyophilization. Pep can be LJP685
or other relevant peptide.
SUBSrlTU~ SHEEr (~ULE 26)
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Pep ~O ~~~O~ SH Pep = LJP 685
O 24
Br ~=~Br
Br ~Br
tetrakis-BMB
5~s~ ~o~ ~Pep
--' o ~ O ~~ ~'~ ~T' Pep
~ 80 ((LJP685)4/MTU/MB) ~
Example 30: ~luorescence po~ri7~tion Peptide Bin~lin.~ Assa~s Synthesis of
FITC-GPCILLARnRCG (CR2*)
A solution of the cyclic disulfide peptide GPCILLARDRCG (20.0 m~.
14.4 !lmol) and fluorescein isothiocyanate (FITC) (5.6 mg. 14.4 ~umol) in 20 mL
of ACNlwater (1:1), co~ 20 mg sodium carbonate (Na2CO3, pH ~ 10.5),
was stirred at room telll?~ u~ e. The reaction was monitored by analytical HPLC.After consuming the fluolescent labeling reagent, the crude material was purified
on a ~l~;par~live HPLC eluted at 10 mL/min with a linear gradient from 30 to 55%B over 40 minutes where A was 0.1% (vlv) TFA in H2O and B was 0.085% (vlv)
TFA in CAN. The FITC peptide was obtained as a bri~ht yellow powder after
lyophi~i7~tion (3.7 mg, 15% yield): MS (ESI): rnle (M+l) Calcd. for
C73H,02N,902oS3: 1661, obsd.: 1661.
SUBSmUlE St~EEr ~WLE 26
. .
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Direct Binclir~ Fluorescence Polarization Rintli~ A~y (dbFP)
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.
Equation I KD/ R + 1 0
where Y is the Y-axis value (milli-polarization units, mP), R is the total
concentration of antibody receptor, PL is the polarization for free F}TC-labeledpeptide (F), and PH is the polarization for F complexed with R (FR). KD is the
dissociation constant (reciprocal binding constant) for F from the FR complex.
l S For these equations to be valid, it must be true that F <~ R. This titration is
shown in Figures 22 and 23 for CB2*-F binding to ACA-6701 and ACA-6501
antibodies. respectively. A complete titration was not obtained with ACA-6501 asshown in Figure 23, but a previous titration is shown in Figure 24 gave a KD of
241 nM. By adding CB2* (GPCILLARDRCG) in slight excess over antibody
6701, to displace CB2*-F from 6701 (see Figure 25), a dissociation rate constantof Kofl~ = 0.0184 sec~', which corresponds to t~2 = 38 seconds, was determined for
CB2*-F. Given the KD of 256 nM, this corresponds to an association rate constantof Kon = 3.6 X 104 M-l sec~l (after correcting for antibody bivalency). Thus,
CB2*-F binding to ACA-6701 is limited only by diffusion ofthese two molecules
together.
SUBSITnUTE SHEEr ffUULE 26)
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Competitive Fluorescence Pol~ri7~tion A~ y (cFP)
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 llg. 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 I hour for ACA 6701 and CB2*-F.
Increasing concentrations ofthe 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 (~) and CB2*-F (F) such
that F << R, the concentration of R need only be high enough such that the
measured polarization (PH) is significantly higher than PL (see Figure 22). This A
(mP) value should be 20 or more mP units to insure reliable results.
Equation 2 ~(mP) = Pl~ - Pl
As unlabeled (no FITC) peptide (I) is added to inhibit F from binding to R, Y
decreases from its maximum value of PH to a plateau of PL which should agree
with that in Equation 1. These titrations are shown in Figures 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:
Equation 3 y = PH +PL * (I / Kl ' )
I/KI'+l.O
SUBSTllUrE SHEEr ~UILE 26)
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where PLjS the same as in Equation 1, I is the concentration of unlabeled peptide
competitor, and K~' is the apparent dissociation constant for that peptide. Values
for these parameters were obtained by fitting cFP titration data to the above
equation.
S The true dissociation constant for I is obtained from Equation 4.
Equation 4 Kl= Kl'/~1.0 + RUKD)
where R and Kl, are defined as in Equation 1. The R/KD ratio is obtained from the
values Of PH' (from Equation 3) and PH and PL (from Equation I ) and using
Equation 5. .
Equation S RJKD =(PH _PL)I(PH_PH )
In general, Equation 5 can be used to determine aPL antibody concentrations oncethe titration defined by Equation I is performed as a ''standard curve." Thus, in
addition to providing a means for detçnnining Kl, this method provides a means
of standardizing all aPL antibody stock solution concentrations and of analyzingtheir binding activity/stability over time using only 5-10 ~g of antibody per cFP
assay.
SUBSTrrUlE SHEEl- ~llllE 26)
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DISSOCIATION CONSTANTS DFTERMRNED BY dbFP AND cFP
Peptide Antibody Sequence KDa or K
CB2*-F 6501FITC-GPCILLARDRCG 482 nMa
CB2*-F 6701FITC-GPCILLARDRCG 512 nMa
CB2* 6701 GPCILLARDRCG 35 nM
3B10 6701 AGPCLLLAPDRCPG 313 nM
a The KD values have been multiplied by 2 to correct for KD values determined
from Equation I that do not factor in the bivalency of the antibody.
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, 3B 10, 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 SA12, a peptide similar to 3B 10, 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 er al., p. 251,
GUIDEBOOK ON MOLECULAR MODELING IN DRUG DESIGN (Academic
Press, N. Cohen, ed., 1996).
Example 31: Tolerance Activity of Peptide Conjn~t~s
Two different conjugates co~ g 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
l~emocyanin (KLH) to generate peptide-specific memory B cells. Three weeks
SUBSTIlll~E SffEET ffUlLE 26)
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117
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.,
l 0 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 ind~lced tolerance, the other one did not
inhibit antipeptide 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 ~LH were harvested and incubated in complete RPMI- 1640
medium for 2 hours at 37~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 thedifference between conjugates is due to a short half-life of the conjugate. To
directly test this hypothesis, the conjugate was ~mini~tered continuously by an
implanted osmotic pump over a prolonged period of time. The results clearly
show that this conjugate induced tolerance when a~1ministered by sustained release
but not when ~-lmini~tered as a bolus as shown in Figure 32.
SUBSmU E S~EEr ~n~ 26)
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Tes~in~ of (LJP685)~/MTU-AHAB-TEG for Tolerance Activity in the in vivo
Model
Mice were primed with LJP-685-KLH on alum plus per~ussis as an
S adjuvant. Three weeks later, the mice were treated with a range of doses of the
(LJP685)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 l 0 ~lg 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 Figure 28 demonstrate that the treatment
with the (LJP685)4/MTU-AHAB-TEG conjugate, over a dose range of I to 50
nmoles, had no detectable effect on the anti-LJP685 response.
Testin~ the (LJP685),/MTU-DAP~A-TEG Conju~ate for ~ ~,lerance Induction in
the in vivo Model
Mice were primed with LJP685-KLH on alum plus pertussis. Three
weeks later, the mice were treated with 5, 10 or 50 nrnoles of the (LJP685)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 with10 ~g 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 Figure 29 demonstrate that (LJP685)4/MTU-DABA-TEG conjugate
induces tolerance in the in vivo model with an EDso of 5 nmoles.
Testing the (T TP685)~/MTU-AHAB-TF~G Coruu~tP for Toler;lnce Induction in
the in vitro Model
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~C
with 4, 20 or 100 ~lM of (LJP685)4/MTU-AHAB-TEG conjugate. One group of
cells was incubated without toleragen and acted as a positive control group. The
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cells were washed, transferred to irradiated recipients and boosted with 10 ~lg of
LJP685-K-LH. 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
Figure 30 clearly illustrate that the (LJP685)4/MTU-AHAB-TEG conjugate can
induce tolerance when tested in the in vitro model achieving an ICso of ~ 4 ~lM.
Testin~ the (LJP6851~/MTU-DAP~A-TFG conJugate for Toler:~nre Tnduction in the
in vitro Model
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~C
with 0.4. 1.3 and 4 IlM of (LJP685)4/MTU-DABA-TEG conjugate. One group of
cells was incubated without toleragen and acted as a positive control group. Thecells were washed, transferred to irradiated recipients and boosted with l 0 ~lg 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
Figure 31 demonstrate that (LJP685)4/MTU-DABA-TEG conjugate can induce
tolerance when tested in the in vi~ro model, achieving an IC50 of < 4 ~lM.
Testing the (LJP685)~/MTU-AHAB-TEG Con~ugate for Tolerance Induction in
vi~o U~ a Continuous l )elivery Pump
Mice were primed 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 cont~ining 50 nmoles of the (LJP685)4/MTU-AHAB-TEG conjugate. The
three rem~inin~ groups were implanted with osmotic purnps. In one group, the
pumps were filled with saline and delivered at 1 IlL/hour for 3 days. The two
rem~ining groups received pumps filled with the (LJP685)4/MTU-AHAB-TEG
conjugate (50 nmoles). One group received pumps that deliver at 1 IlL/hour for
three days and the other received pumps that deliver at 0.5 ~L/hour for seven days.
On day 5, the pumps that deliver for three days were surgically removed. On day
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7, all of the mice, including the control group, were boosted with 10 ~g of
LJP685-KLH. On day 10, the purnps 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 Figure 32.
Teslin~ the U TP-Peptide)~/MTU-BMP-TEG Coruu~ate for Toler~nce Induction in
the in vivo Model
Mice are primed with peptide-KLH on alum plus pertussis. Three weeks
later, the mice are treated with the (LJP-peptide)4/MTU-BMP-TEG conjugate, one
group is not treated and acts as the control group. Five days later. all of the mice7
including the control group, are boosted with 10 ',Ig 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)4/MTU-BMP-TEG
conjugate induces tolerance in the in vivo model at a potency equal to or greater
than that of the (LJP685)4/MTU-AHAB-TEG conjugate.
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