Note: Descriptions are shown in the official language in which they were submitted.
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METIiODS AND FORMULATIONS FOR REDUCING
CIRCULATING ANTIBODLES
RELATED APPLICATIONS
This application claims the priority benefit of provisional application U.S.
Serial
No. 60/111,639, filed December 9, 1998, the contents of which are incorporated
by
reference in their entirety.
TECHNICAL FIELD
This invention relates to reducing of circulating antibodies, particularly
disease-
associated antibodies, by binding the antibodies to an epitope-presenting
carrier.
BACKGROUND ART
There are many conditions and disorders which are associated with circulating
1 S antibodies of various classes. Some of these conditions are, for example,
autoimmune
disorders (such as lupus and idiopathic thrombocytopenia purpura) and IgE
associated
disorders.
One approach to treating, or combating, these disorders is to remove andlor
reduce
the circulating antibodies, even on a transient basis. One example of such an
approach is
apheresis, in which blood is removed from an individual and the antibodies are
removed
extracorporeally using an affinity column, such as a protein A column. See,
e.g., U.S. Pat.
Nos. 4,851,126, 4,2SS,627, 4,086,294, 5,147,290, 4,411,792; Badnarenko {1996)
Clinics in
Laboratory Medicine 16:907-929; Snyder et al. (1992) Blood 79:2237-245;
Richter et al.
(1993) Metabol. Clin. ~xp. 42:888-894'; Richter et al. (1997) ASAIOJ. 43(1):S3-
S9;
2S Pascher et al. (1997) Transplantation 6363{6):867-875; Wailukat et al.
{1996) Int'l J. Card.
54:191-195; Nillson et al. (1981) Blood 58:38-44; Watson et al. (1989) Cancer
64:1000;
Suzuki et al. (1994)Autoimmunity l9:lOS-112; Suzuki et al. (1995) Artificial
Organs
20(4):296-302; Bandarenko (1996) Clinics in Laboratory Medicine 16:907-929.
The problem with apheresis as a technique to reduce levels of circulating
antibodies
is that it is cumbersome and expensive. Further, apheresis often requires
frequent and
repetitive administrations.
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Other literature describes inducing tolerance, i.e., reducing levels of
circulating
antibodies by inducing B cell anergy. See, e.g., U.S. Pat. Nos. 5,276,013,
5,391,785,
5,786,512, 5,726,329, 5;552,391, 5,268,454; PCTILTS96/009976; PCTICTS97110075;
PCT/tTS91109176; U.S. 5,268,454; U.S. Ser. No. 081118,055; U.S. Ser. No.
60/088,656;
U.S.Ser. No. 60/103,088.
What is needed are improved methods of reducing circulating antibodies;
particularly antibodies associated with disease.
All publications cited herein are hereby incorporated by reference in their
entirety.
DISCLOSURE OF THE INVENTION
The invention provides methods of reducing levels of circulating antibodies,
particularly disease or disorder-associated antibodies.
Accordingly, in one aspect, the invention provides methods for reducing levels
of
circulating antibodies in an individual, particularly disease-associated
antibodies,
comprising administering to the individual an effective amount of an epitope-
presenting
moiety. The epitope-presenting moiety may be any of a number of embodiments,
and is
preferably a conjugate comprising a valency platform molecule and an
epitope(s). The
epitope may be any moiety, as long as it exhibits the requisite binding
activity.
In another aspect, the invention. provides ex vivo methods of reducing
circulating
antibodies using an eptiope-presenting moiety. The methods provide methods of
reducing
levels of disease-associated antibodies in an individual, comprising treating
the individual's
blood (including any component thereof which contains antibody)
extracoporeally with an
eptiope-presenting carrier under conditions that permit the antibodies to bind
the epitope;
removing antibody-eptiope-presenting carrier complexes, if any; and returning
the blood to
the individual.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a reaction scheme illustrating the enzymatic synthesis of the aGal
epitope, 2-[2-(2-thioethoxy) ethoxy]ethyl 3-O-(a-D-galactopyranosyl)-[3-D-
galactopyranoside.
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Figure 2 is a reaction scheme illustrating the chemical synthesis of the aGal
epitope, 2-[2-(2-thioethoxy) ethoxy]ethyl 3-O-(a-D-galactopyranosyl)-[i-D-
galactopyranoside.
Figure 3 is a reaction scheme illustrating the chemoenzymatic synthesis of the
aGal
epitope, p-aminophenyl 3-O-a-D-galactopyranosyl-a-D-galactopyranoside.
Figure 4 illustrates two general synthetic strategies for conjugation
chemistry.
Figure 5 illustrates a third general synthetic strategy for conjugation
chemistry.
Figure 6 illustrates two dimeric platforms and four tetrameric platforms.
Figure 7 illustrates four octameric platforms.
Figure 8 illustrates a monomeric aGal conjugate and three dimeric aGal
conj ugates.
Figure 9 illustrates two tetrarneric aGal conjugates as described in Example
3.
Figure 10 illustrates two tetrameric aGal conjugates as described in Example
3.
Figure I 1 illustrates two tetrameric conjugates of two aGal-isomers as
described in
Example 3.
Figure 12 illustrates an octameric aGal conjugate as described in Example 3.
Figure 13 illustrates an octameric aGal conjugate as described in Example 3.
Figure 14 illustrates an octameric aGal conjugate as described in Example 3.
Figure 15 illustrates an octameric aGal conjugate as described in Example 3.
Figure 16 is a graph of OD550 versus fraction number and depicts the elution
profile of anti-aGal from an aGal affinity column.
Figures 17A and 17B are graphs depicting affinity purified IgG (17A) and IgM
{17B) anti-aGal binding to PK15 cells. Flow cytometric analysis results are
shown as
mean fluorescence intensity (MFI) versus dose, in p.g/rnl, of affinity-
purified IgG or IgM
(solid circles), or column flow-through IgG or IgM (open squares).
Figure 18 is a graph depicting the effect of aGal valency of toleragens on
inhibition
of anti-aGal binding, measured by ELISA as described in Example 4. Symbols for
inhibitors are as follows: open circles = aGal monomer; open squares, dashed
lines = LJP
724 (trisaccharide monomer) ; open squares, dashed lines = LJP 725
{pentasaccharide
monomer); solid circles, solid lines = aGal dirner; solid squares, solid lines
= LJP 712
tetramer; solid triangles, solid lines = LJP 719 octamer; open triangles,
dashed lines = LJP
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728 {11-mer pentasaccharide-BSA); solid triangles, dashed lines = LJP 726 (11-
mer
trisaccharide [3-caxbon linker]-HSA); solid squares, dashed lines = LJP 727
(11-mer
trisaccharide [14-carbon linker)-HSA}.
Figure I9 is a bar graph of the percent plasma anti-aGal IgG following
treatment
with LJP 712, as described in Example 5.
Figure 20 is a graph depicting activation of the classical complement pathway
by
various substances, as described in Example 5. Symbols are as follows: open
circles, LJP
712; open squares, cobra venom factor (CVF); solid triangles, aggregated human
gamma
globulin (AHG). The dashed Iine represents results obtained with buffer alone.
Figure 21 is a graph depicting activation of the alternative complement
pathway by
various substances, as described in Example S. Symbols are as follows: open
circles, LJP
712; open squares, CVF; solid triangles, AHG.
Figures 22A and 22B axe graphs depicting the decrease in plasma anti-aGal IgG
(22A) and IgM (22B) following treatment with octameric LJP 920 (cpd 46)
(circles),
compared with PBS (squares}, as described in Example 5.
Figure 23 is a graph comparing the effect of tetramer LJP 712 (open circles)
with
octamer LJP 920 (solid circles) on the percent plasma anti-aGal IgM, as
described in
Example 5. PBS (solid squares) was included as a negative control.
Figure 24 depicts a strategy for the synthesis of compound 29.
Figure 25 depicts a strategy for the synthesis of compounds 31 and 32.
Figures 26A and 26B depict the synthesis of compound 30.
MODES FOR CARRYING OUT THE INVENTION
This invention provides effective methods of removing and/or reducing
circulating
levels of antibodies, particularly disease-associated antibodies. This removal
and/or
reduction is generally transient, as it is based on binding to circulating
antibodies as
opposed to causing B cell anergy, although induction of B cell anergy may
accompany
these methods.
General Techniques
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant
techniques),
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microbiology, cell biology, biochemistry and immunology, which are within the
skill of the
art. Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A
Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide
Synthesis"
(M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987);
"Methods in
Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology"
(D.M.
Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells"
(J.M. Miller
& M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M.
Ausubel et al.,
eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds.,
1994); and
"Current Protocols in Immunology" (J.E. CoIigan et al., eds., 1991 ).
Defzhitions
For purposes of this invention, "reducing" and/or "removing" circulating
antibodies
means that the level of free, or unbound, circulating antibodies has been
reduced. In some
embodiments, by binding of epitope-presenting carrier to antibody, antibody is
prevented
from being an effector molecule, i.e., binding other targets, and is thus
"reduced." In some
embodiments, "reducing" circulating antibodies includes clearance of antibody,
e.g.,
physical removal from circulation. One way this way this clearance is effected
is clearance
of a complex comprising an epitope-presenting carrier and antibody by
reticuloendothelial
system.
An "epitope" is a term well-understood in the art and means any chemical
moiety
which exhibits specific binding to an antibody. An "epitope" can also comprise
an antigen,
which is a moiety that contains an epitope, and, as such, also specifically
binds to antibody.
An epitope or antigen that "specifically binds" to an antibody is a term well
understood in the art, and methods to determine such specific binding are also
well known
in the art. A molecule is said to exhibit "specific binding" if it reacts or
associates more
frequently, more rapidly, with greater duration andlor with greater affinity
with a particular
cell or substance than it does with alternative cells or substances. An
antibody "specifically
binds" to a target if it binds with greater affinity, avidity, more readily,
andlor with greater
duration than it binds to other substances.
An "antibody" (interchangeably used in plural form) is an immunoglobulin
molecule capable of specific binding to a target, such as a carbohydrate or
polypeptide,
through at least one antigen recognition site, located in the variable region
of the
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immunoglobulin molecule. As used herein, the term encompasses not only intact
antibodies, but also fragments thereof (such as Fab, Fab'; F(ab')Z, Fv),
single chain (ScFv),
mutants thereof, fusion proteins comprising an antibody portion, humanized
antibodies, and
any other modified configuration of the immunoglobulin molecule that comprises
an
antigen recognition site of the required specificity.
As is well understood by those skilled in the art, a "disease-associated
antibody" is
an antibody whose production occurs during a disease state and/or whose
production is
undesireably, such as in autoimmune diseases and transplantation rejection.
Examples of
disease-associated antibodies are known in the art and include, but are not
limited to, anti-
double-tranded DNA antibodies (lupus) and anti-aGal antibodies
(transplantation
rejection).
"Naturally occurring" refers to an endogenous chemical moiety, such as a
carbohydrate, polynucleotide or polypeptide sequence, i.e., one found in
nature. Processing
of naturally occurring moieties can occur in one or more steps, and these
terms encompass
all stages of processing. Conversely, a "non-naturally occurring" moiety
refers to all other
moieties, i.e., ones which do not occur in nature, such as recombinant
polynucleotide
sequences and non-naturally occurring carbohydrates.
As used herein, the term "mimetic" (also termed an "analog") means a
biological or
chemical compound which specifically binds to an anti-aGal antibody. As such,
for
purposes of this invention, an "aGal epitope" includes mimetics of naturally-
occurring
aGal (such as peptides). A "mimetic" shares an epitope, or binding
specificity, with aGal.
A mimetic may be any chemical substance which exhibits the requisite binding
properties,
and thus may be, fox example, a simple or complex organic or inorganic
molecule; a
polypeptide; a polynucleotide; a carbohydrate; a lipid; a lipopolysaccharide;
a lipoprotein,
or any combination of the above, including, but not limited to, a
polynucleotide-containing
polypeptide; a glycosylated polypeptide; and a gIycolipid. The term "mimetic"
encompasses the term "mimotope", which is a term well known in the art.
An "individual" is a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, farm animals, sport animals, pets,
primates, mice
and rats.
An "effective amount" is an amount sufficient to effect beneficial or desired
results,
including clinical results. An effective amount can be administered in one or
more
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administrations. For purposes of this invention, an effective amount is an
amount sufficient
to reduce circulating levels of free antibodies {i.e., unbound to epitope on
carrier).
A "carrier" is a molecule which contains attachment sites for epitope(s). One
preferred example of a carrier is a valency platform molecule. The terms
"carrier" and
"moiety" are used interchangeably herein.
An "epitope presenting carrier" is a carrier which contains attached, or
bound,
epitopes, at least some of which (at least two of which) are able to bind to
an antibody of
interest.
As used herein "valency platform molecule" means a nonimmunogenic molecule
containing sites which allow the attachment of a discrete number of epitopes
and/or
mimetic{s) of epitopes. A "valency" of a conjugate or valency platform
molecule indicates
the number of attachment sites per molecule for an epitope(s). Alternatively,
the valency of
a conjugate is the ratio (whether absolute or average) of epitope to valency
platform
molecule.
"Nonimmunogenic", when used to describe a carrier (including a valency
platform
molecule), means that the carrier (such as a valency platform molecule) fails
to elicit an
immune response (i.e., T cell and/or B cell response), and/or fails to elicit
a sufficient
immune response, when it is administered by itself to an individual. The
degree of
acceptable immune response depends on the context in which the valency
platform
molecule is used, and may be empirically determined.
An epitope that is "conjugated" to a carrier (such as a valency platform
molecule) is
one that is attached to the carrier, either by covalent andlor covalent
interactions.
A "stable complex" is one that sufficiently persists after its formation to
allow
subsequent detection and/or removal.
A "T cell epitope" means a component or portion thereof for which a T cell has
an
antigen-specif c specific binding site, the result of binding to which
activates the T cell.
Where an embodiment of the invention is described as "lacking" a T cell
epitope, this is
taken to mean that a T cell epitope is not detectable using standard assays in
the art. For
purposes of this invention, an epitope that "lacks" a T cell epitope means
that the epitope
lacks a T cell epitope which causes T cell activation in the individuals) to
be treated (i.e.,
who is to receive an epitope-presenting carrier. It is likely that, for
example, an epitope
may lack a T cell epitope(s) with respect to an individual, or a group of
individuals, while
possessing a T cell epitope{s) with respect to other individual(s). Methods
for detecting the
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presence of a T cell epitopc include assays which detect T cell proliferation
(such as
thymidine incorporation). Polypeptides or other antigens that fail to induce
statistically
significant incorporation of thymidine above background (i.e., generally p
less than 0.05
using standard statistical methods) are generally considered to Iack T cell
epitopes,
although it will be appreciated that the quantitative amount of thymidine
incorporation may
vary, depending on the polypeptide (or other antigen) being tested. Generally,
a
stimulation index below about 2-3, more preferably less than about 1,
indicates lack of T
cell epitopes. The presence of T cell epitopes can also be determined by
measuring
secretion of T cell-derived lymphokines according to standard methods.
Location and
content of T cell epitopes, if present, can be determined empirically.
The term "blood" as used herein is bodily fluid including a cellular component
and
plasma. "Blood" means whole blood or a component thereof. Treating an
"individual's
blood" means that any or all of an individual's blood is treated.
A "stable complex" is one that sufficiently persists after its formation to
allow
subsequent detection and/or removal.
Metlaods of the invetztion
The invention provides methods of reducing circulating levels of antibodies,
particularly disease-associated antibodies. These methods generally comprise
administering an effective amount of an epitope-presenting (which includes an
antigen-
presenting) carrier {or a composition comprising an epitope-presenting
carrier) to an
individual. These methods are especially useful for effecting safe (i.e., lack
of
inflammation and/or other undesirable side effects) and rapid clearance in a
more simple
and desirable way than using other methods known in the art, such as
apheresis. Without
wishing to be bound by a particular theory, the inventors note that, by
binding antibody on
multivalent carrier, sufficiently large complexes are likely formed which may
effect rapid
clearance from circulation without formation of excessively large mufti-valent
complexes
which would cause undesired side effects, such as inflammation, although, as
noted below,
"reduction" of circulating antibody levels does not require this clearance for
purposes of
this invention. The initial reduction of circulating antibody is due to
binding the epitope
presenting carrier, and further more effective reduction is obtained by
clearance.
For purposes of this invention, the reduction and/or removal of antibody is
preferably selective, i.e., only that antibody for which reduction or removal
is desired is
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affected. However, it may be acceptable that larger classes of antibodies are
removed
which include the antibody of interest. The extent of cross-reactivity of
binding by the
epitope will generally govern the types of antibody that are removed from
circulation.
Preferably, the reduction (as reflected, for example, by titer) is preferably
at least about
S SO%, preferably at least about 60%, preferably at least about 80%,
preferably at least about
85%, preferably at least about 90%, preferably at least about 95%. It is
understood that, for
purposes of this invention, total reduction (i.e., 100%) need not be effective
in order for
these methods to be efFcacious. Methods of measuring antibody titer, either by
binding or
neutralizing assays, are well known in the art.
It is also understood that reduction and/or removal of particular sub-classes
of
antibodies may be desirable, and that not all classes of epitope binding
antibodies need be
reduced and/or removed. For example, because IgM antibodies mediate acute
rejection in
xenotransplantation, reduction of this class of aGal antibodies may be
indicated. For
example, we have found that, in the case of aGal antibodies, varying the
valency may
affect, and can have a signif cant impact, on adsorbing (binding) particular
classes of
antibody, as described in the Example section.
In some embodiments, methods are provided for reducing levels of circulating
disease-associated antibodies in an individual, comprising administering to
the individual
an effective amount of an epitope presenting carrier (or composition
comprising an epitope-
presenting carrier) comprising a plurality of epitopes conjugated to a carrier
which presents
the epitopes in a manner effective to adsorb (i.e., bind) the antibodies.
The epitope presenting carrier is multivalent, i.e., is capable of presenting
more than
one epitope. Preferably, the valency is at least two. In other embodiments,
the valency is
at least three, at least four, at least six, at least eight, at least 10, at
least 12, at least 16, at
least 20, at least 24, at least 30, at least 32, at least 36, at least 40, at
least 42, at least 46, at
least 50. The upper limit of the valency is not necessarily critical, as long
as the epitope-
presenting carrier effects reduction and/or clearance without undesirable side
effects.
In some embodiments, the valency is two. In other embodiments, the valency is
four. In other embodiments, the valency is six. In other embodiments, the
valency is any
of the following: eight; 10; 12; 16; 20; 22; 24; 26; 28; 30; 32; 34; 36; 38;
40; 42; 44; 46;
48; 50; 52; 54; 56; 58; 60; 62; 64; 66; 68; 70; and increments of two, until
128.
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Epitope presenting carriers
Any of a variety of corners may be used, as long as the carrier does not
elicit an
undesirable or unacceptable immune response. The carrier may be any chemical
moiety,
and have any chemical structure, including, but not limited to, organic and
inorganic
molecules, polypeptides (i.e., polymers of amino acids), nucleic acids,
carbohydrates, other
polymers, artificial structures, and lipid structures (such as liposomes or
micelles) made by
standard techniques, or polymerized as described in U.S. Pat. No. 5,512,294.
A carrier may be proteinaceous or non-proteinaceous (i.e., organic). Examples
of
proteinaceous platforms include, but are not limited to, albumin,
gammaglobulin,
immunoglobulin (IgG) and ovalbumin. Borel et al. (1990) Immunol. Methods
126:159-
168; Dumas et al. (1995) Arch. Dematol. Res. 287:123-128; Bore1 et al. (1995)
Int. Arch.
Allergy Immunol. 107:264-267; Borel et aI. (1996) Ann. N. Y. Acad. Sci. 778:80-
87.
Preferably, the epitope-presenting carriers are conjugates which comprise a
chemically defined valency platform molecule in which a precise valency (as
opposed to an
average) is provided. See, for example, commonly owned U.S. Pat. Nos.
5,162,515;
5,276,013; 5,552,391; 5,391,785; 5,786,512; 5,726,329; 5,268,454; 5,606,047;
and
5,663,395. See also commonly-owned U.S. Serial Nos. 08/482,651; 08/660,092;
08/760,548. Accordingly, a def ned valency platform is a platform with defined
structure,
thus a defined number of attachment points and a defined valency. In contrast
to
previously described, more traditional platforms, these platforms have the
advantage of
having a homogeneous (i.e., uniform) molecular weight (as opposed to
polydisperse
molecular weight), and are thus "chemically defined". Accordingly, it is
understood that a
population of conjugates using these platforms comprise a platform of
homogeneous
molecular weight or are substantially monodisperse (i.e., have a narrow
molecular weight
distribution). A measure of the breadth of distribution of molecular weight of
a sample
(such as a composition and/or population of platform molecules) of a platform
molecule is
the polydispersity of the sample. Polydispersity is used as a measure of the
molecular
weight homogeneity or nonhomogeneity of a polymer sample. Polydispersity is
calculated
by dividing the weight average molecular weight (Mw) by the number average
molecular
weight (Mn). The value of Mw/Mn is unity for a perfectly monodisperse polymer.
Polydispersity (Mw/Mn) is measured by methods available in the art, such as
gel
permeation chromatography. The polydispersity (Mw/Mn) of a sample of platform
molecules is preferably less than 2, more preferably, less than 1.5, or less
than 1.2, less than
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1.07, less than 1.02, or, e.g., about 1.05 to 1.5 or about I .05 to 1.2.
Typical polymers
generally have a polydispersity of 2-5, or in some cases, 20 or more.
Advantages of the
low polydispersity property of the valency platform molecules include improved
biocompatibility and bioavailability since the molecules are substantially
homogeneous in
size, and variations in biological activity due to Wide variations in
molecular weight are
minimized. The low polydispersity molecules thus are pharmaceutically
optimally
formulated and easy to analyze. Further there is controlled valency of the
population of
molecules in the sample.
In some embodiments, the valency platform molecule is a carbamate, i.e., -O-
IO C(=O)-N<). See U.S. Ser. No. 60/I 11,641 ("Valency Platform Molecules
Comprising
Carbamate Linkages"). An example of a carbamate platform is compound 30, Fig.
7.
Preferred valency platform molecules are biologically stabilized, i.e., they
exhibit
an in vivo excretion half life often of hours to days to months to confer
therapeutic efficacy,
and are preferably composed of a synthetic single chain of defined
composition. They
generally have a molecular weight in the range of about 200 to about 200,000,
preferably
about 200 to about 50,000 (or less, such as 30,000). Examples of valency
platform
molecules within the present invention are polymers (or are comprised of
polymers) such
as polyethylene glycol (PEG), poly-D-lysine, polyvinyl alcohol,
polyvinylpyrollidone, D-
glutamic acid and D-lysine (in a ratio of 3:2). Preferred polymers are based
on
polyethylene glycols (PEGS) having a molecular weight of about 200 to about
8,000. Other
suitable platform molecules for use in the conjugates of the invention are
albumin and IgG.
Preferred valency platform molecules suitable for use within the present
invention
include the chemically-defined, non-polymeric valency platform molecules
disclosed in co-
owned U.S. Pat. No. 5,552,391. Particularly preferred homogeneous chemically-
defined
valency platform molecules suitable for use within the present invention are
derivatized
2,2'-ethylenedioxydiethylamine (EDDA) and triethylene glycol (TEG).
Additional suitable valency platform molecules include, but are not limited
to,
tetraaminobenzene, heptaaminobetacyclodextrin, tetraaminopentaerythritol,
1,4,8,11-
tetraazacyclotetradecane (Cyclam) and 1,4,7,10-tetraazacyclododecane (Cyclen).
In other embodiments, the tetra-bromoacetyl platform PIZ/IDAITEG platform is
used. Derivatives of the PIZ/IDAITEG (PITG) platform can be prepared as shown
below.
See PCT/US97/10075 and PCTlUS96/09976 for other examples of suitable
platforms.
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Examples of Compatible Cross-linking Groups on PITG Platform
Platform (bromoacetyl-PITG)
R
R
Platform Epitope Conjugate
R = XCH2C0 D1-SH R' = D1-SCH2C0
By way of example of a conjugate embodiment used in the methods of this
invention, an polypeptide epitope is prepared with a thiol linker at the N
terminus by
chemical or enzymatic synthesis, or by recombinant methods. The linker can be
cysteine or
an SHI containing moiety The modified epitope may then be alkylated by a
suitably
derivatized platform (such as bromoacetyl or iodoacetyl).
In general, the above-described platforms are made by standard chemical
synthesis
techniques. PEG must be derivatized and made multivalent, which is
accomplished using
standard techniques. Some substances suitable for conjugate synthesis, such as
PEG,
albumin, and IgG are available commercially.
In other embodiments, valency platforms may be used which, when conjugated,
provide an average valency (i.e., these platforms are not chemically defined
in terms of
their valency). Examples of such platforms are polymers such as linear PEG;
branched
PEG; star PEG; polyamino acids, such as DEK; polylysine; proteins; amino-
functionalized
soluble polymers.
lz
Conjugate
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Covalent conjugation of epitope(s) with a catTier such as a valency platform
molecule is generally performed using standard chemical techniques. The
following are
examples of standard chemistry which can be used: 1) thiol substitution; 2)
thiol Michael
addition; 3) amino alkylation; 4) disulfide bond formation. Figures 4 and 5
provide
general, exemplary conjugation strategies.
Conjugation of an epitope to a valency platform molecule may be effected in
any
number of ways, typically involving one or more crosslinking agents and
functional groups
on the epitope and valency platform molecule. Platforms and epitope(s) must
have
appropriate linking groups. Linking groups are added to platforms using
standard synthetic
chemistry techniques. Linking groups may be added to an aGai epitope(s) using
either
standard solid phase synthetic techniques or recombinant techniques (if, for
example, the
aGal epitope is a peptide). Recombinant approaches may require post-
transLational
modification in order to attach a linker, and such methods are known in the
art.
As a further example, if the epitope is a polypeptide, polypeptides contain
amino
acid side chain moieties containing functional groups such as amino, carboxyl,
or
sulfl~ydryl groups that serve as sites for coupling the polypeptide to the
platform. Residues
that have such functional groups may be added to the polypeptide if the
polypeptide does
not already contain these groups. Such residues may be incorporated by solid
phase
synthesis techniques or recombinant techniques, both of which are well known
in the
peptide synthesis arts. When the polypeptide has a carbohydrate side chain(s),
functional
amino, sulfhydryl and/or aldehyde groups may be incorporated therein by
conventional
chemistry. For instance, primary amino groups may be incorporated by reaction
with
ethylenediamine in the presence of sodium cyanoborohydride, sulflrydryls may
be
introduced by reaction of cysteamine dihydrochloride followed by reduction
with a
standard disulfide reducing agent, while aldehyde groups may be generated
following
periodate oxidation. In a similar fashion, the valency platform molecule may
also be
derivatized to contain functional groups if it does not already possess
appropriate functional
groups.
Hydrophilic linkers of variable Lengths are useful for connecting epitopes to
valency
platform molecules. Suitable linkers include linear oligomers or polymers of
ethylene
glycol. Such linkers include linkers with the formula
R' S{CH2CH20)nCH2CH20(CH2),~C02R2 wherein n = 0-200, m = 1 or 2, Rl = H or a
protecting group such as trityl, RZ = H or alkyl or aryl, e.g., 4-nitrophenyl
ester. These
13
CA 02353620 2001-06-05
WO 00/33887 PCTIUS99/29336
linkers are useful in connecting a molecule containing a thiol reactive group
such as
haloaceyl, maleiamide, ete., via a thioether to a second molecule which
contains an amino
group via an amide bond. These linkers are flexible with regard to the order
of attachment,
i. e., the thioether can be formed first or last.
Particular conjugates are described in Example 2 and are depicted in Figures 6-
15;
which accordingly are provided as embodiments of the invention.
While the above discussion has exemplified preferred embodiments, namely those
which employ valency platform molecules, it is clear that the carriers used in
the invention
may be any of a number of other moieties, and that many of the principles
described above
would likewise apply to other types of carriers.
As an example, liposomes may be used. Liposorne technology is known in the art
and need not be described in detail herein. As a brief summary, epitopes are
appropriately
inserted (i.e., inserted so that the binding moiety is available to bind to
antibody, which
may involve attaching "tails" to the epitope(s) for insertion) into liposomes,
which may be
i 5 of varying size. Mahato et al. ( 1997) Pharm. Res. 14:853-859. Liposomal
preparations
include, but are not limited to, cytofectins, multilamellar vesicles and
uniiamellar vesicles.
Polymeric liposomes are described in U.S. Pat. No. 5,512,294.
As another example, multiple antigen peptides {MAPS) may be used. Posnett et
al.
(1988) J. Biol. Chem. 263:1719-1725; Tam (1989) Methods Enz. 168:7-15. MAPS
have a
small immunologically inert core having radially branching lysine dendrites,
onto which
polypeptide epitopes may be anchored. MAPS may be synthesized using methods
known
in the art, for example, a solid-phase methods, such as that described in
Merrifield et al.
(1963) J. Am. Chem. Soc. 85:2149.
Epitopes
Any epitope, or antibody-binding moiety, can be used. An epitope may be a
polypeptide, organic, or inorganic molecule. Such antibody-binding moieties
are known in
the art and/or may be developed using standard methods and assays in the art,
such as
antibody binding assays. Antibodies used for assays to test and/or develop
epitopes may be
also obtained using standard assays in the art, such as affinity purification
(or in some cases
may be commercially available). Assays that may be used to determine whether a
putative
epitope or antigen exhibits requisite binding activity include, but are not
limited to,
14
CA 02353620 2001-06-05
WO 00/33887 PCT/US99/29336
filamentous phage random peptide libraries and screening (by, far example,
biopanning,
micropanning, phage-capture ELISA, phage-ELISA, colony blot, peptide ELISA,
competitive binding peptide ELISA).
Examples of suitable epitopes include, but are not limited to, those that bind
to:
lupus anti-DNA antibodies (see U.S. Pat. Nos. 5,162,515; 5,391,785; 5,276,013;
5,786,512;
5,726,329; 5,552,391, 5,268,454) ; anti- galactose alpha 1,3 galactosyl (aGal)
antibodies;
anti-cardioiipin antibodies; antiphospholipid antibodies; IgE antibodies; anti-
factor VIII
antibodies; anti-factor IX antibodies; anti-(32GPI antibodies, particularly to
domain 1; anti-
platelet antibodies; antibodies associated with idiopathic thrombocytopenia
purpura (ITP);
anti-adenovirus antibodies (which may be problematic when adenovirus is
administered as
a therapeutic agent); anti-adeno-associated virus (AAV) antibodies {which may
be
problematic when AAV is administered as a therapeutic agent); anti-alpha chain
acetyl
choline receptor (myasthenia gravis); anti-RhD antigen antibodies (i.e., Rh
disease); anti-
thyroid antibodies {for example, in auto-immune thyroiditis).
Of particular interest are epitopes that bind any undesired blood group
antibody that
may interfere with alto- or xenotransplantation. For example, antibodies that
bind the
structure Galal-3Ga1[31-4GlcNAc~i1-3Ga1(31-4Glc~i1- can be removed using aGal
epitopes
described elsewhere in this application. In a similar fashion, epitopes can be
designed that
antigenically resemble the antigenic site on polysaccharides and are suitable
for removing
antibodies against other carbohydrate-based blood group antigens, including
but not limited
to those of the ABO system, the MN system, the Lewis system, and the Bombay
phenotype.
Also of interest are epitopes that bind the anti-polynucleotide (particularly
anti-
double stranded DNA) antibodies that occur in systemic lupus erythematosis.
Preferred
platforms for such epitopes are tetrabromoacetyl compounds, and other
tetravalent and
octavalent valency platform molecules: D. Jones et al: (1995) J. Med Chem.
38:2138-2144;
and U.S. Patent references provided above. We have observed that
administration of such
lupus conjugates in humans have resulted in reduction of circulating anti-ds
DNA
antibodies. Jones (1995); Weisman et al. (1997) J. Rheum. 24:314-318; Iverson
et al.
( 1998) Lupus 7 (Suppl. 2): S 166-S 169.
The suitability of particular epitopes for removing antibodies according to
this
invention can be confirmed empirically. For example, to select the optimum
epitope from a
library of small drug molecules believed to mimic the immunogenic epitope for
a particular
CA 02353620 2001-06-05
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autoimmune disease, a family of carriers can be constructed in which each of
the
candidates is alternatively displayed on a similar carrier molecule or
platform. The
composition is then tested far efficacy. For example, for in vivo use, an
animal model is
used in which there are circulating antibodies of the undesired type. The
animals can be
immunized with an appropriate antigen to initiate the antibody response, if
necessary. Test
candidates assembled onto a carrier are then used to treat separate animals,
either by
administration, or by ex vivo use, according to the intended purpose. The
animals are bled
before and after treatment, and the antibody levels in plasma are determined
by standard
immunoassay as appropriate for the specific antibody. Efficacy of the
candidates is then
assessed according to the comparative degree in reduction in the antibody
level.
In some embodiments, the epitope used lacks T cell epitope(s}. Methods for
detecting T cell epitopes are well known in the art. For example, various
assays which
detect T cell proliferation (such as thymidine incorporation) may be used. The
presence of
T cell epitopes can also be determined by measuring secretion of T cell-
derived
lymphokines by methods well known in the art. Antigens that fail to induce
statistical
significant incorporation of thymidine above background (i.e., generally p
less than 0.05
using standard statistical methods) are generally considered to lack T cell
epitopes,
although it will be appreciated that the quantitative amount of thymidine
incorporation may
vary, depending on the polypeptide being tested. Generally, a stimulation
index below
about 2-3, more preferably less than about 1, indicates lack of T cell
epitopes. Location
and content of T cell epitopes are determined empirically.
Removing antibody from biological fluids ex vivo
This invention further includes methods for reducing levels of disease-
associated
antibodies in the biological fluid of an individual, comprising contacting the
fluid with an
epitope-presenting carrier ex vivo under conditions that permit the antibodies
to bind
epitopes on the corner. Suitable bodily fluids include those that can be
returned to the
individual, such as blood, plasma, or lymph.
Affinity adsorption apheresis is~described generally in Nilsson et al. (1981)
Blood
58(1):38-44; Christie et al. (1993) Transfusion 33:234-242; Richter et al.
(1997) ASAIO J.
43(1):53-59; Suzuki et al. (1994) Autoimmunity 19: lOS-112; U.S. Patent No.
5,733,254;
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WO 00/33887 PCT/US99I29336
Richter et al. (1993) Metabol. Clin. Exp. 42:888-894; Richter et al. (1997)
ASAID J.
43(1):53-59; and Wallukat et al. (1996) Int'l J Card. 54:191-195.
Accordingly, the invention includes methods of reducing levels of disease-
associated antibodies in an individual, comprising treating the individual's
blood (including
any component thereof which contains antibody) extracoporeally (i.e., outside
the body or
ex vivo) with an eptiope-presenting carrier under conditions that permit the
antibodies to
bind the epitope; removing antibody-eptiope-presenting carrier complexes, if
any; and
returning the blood to the individual.
In the methods of the invention, the bodily fluid is removed from the
individual for
extracorporeaI binding to an epitope-presenting Garner of this invention. For
example,
apparatuses and methods for removing blood and separating it into its
constituent
components are known in the art (see, e.g., U.S. Patent Nos. 4,086,924;
4,223,672). The
blood or portions thereof are then exposed to the carrier. The carrier
neutralizes (i.e.,
binds) the unwanted antibody, and the blood components are then returned to
the
individual.
In a preferred technique, the antibody-Garner complex is removed before the
fluid is
returned to the individual. This may be done, for example; by using a carrier
attached to a
solid phase, or by using a soluble carrier and selectively removing the
complex from the
treated solution.
To create a solid phase, the carrier is adapted to render it insoluble. For
example,
where the carrier is one of the preferred platforms listed above, then the
platform can be
chemically adapted during synthesis to include an additional reactive group in
the core
structure. For example, an additional linkage can be added to a triethlyene
glycol structure
present in the core. The linkage is then used to attach the platform to an
insoluble
structure, such as a polystyrene or polyethylene bead, a polycellulose
membrane, or other
desirable structure. Commercially available matrices include agarose (a
neutral linear
polysaccharide generally composed of D-galactose and altered 3,6-
anhydrogalactose
residues, for example SepharoseTM, Pharmacia), activated gels, nitrocellulose,
borosilicate,
glass fiber filters, silica, polyvinylchloride, polystyrene, and diazotized
paper. Methods for
preparing peptide-peptide conjugates are described in Hermanson, G.T.,
"Bioconjugate
Techniques", Academic Press: New York, 1996; and in "Chemistry of Protein
Conjugation
and Cross-linking" by S.S. Wong, CRC Press, 1993. The biological fluid to be
treated is
contacted with the solid phase, and antibodies in the fluid complex to the
solid phase. The
17
CA 02353620 2001-06-05
WO 00133887 PCTNS99/29336
supernatant fluid can then be removed from the solid phase fox return to the
individual. In
some instances, the solid phase can also be cleared of antibody for repeat use
by using a
suitable wash, providing both the epitope and the carrier is resistant to the
washing
solution. Suitable washing solutions may include 0.1 M glycine buffer, pH 2.4,
dilute
acetic acid, or 1 M KSCN buffered to ~pH 7.
If the carrier is not part of a solid phase, then the antibody-carrier complex
can be
removed from the fluid by any other appropriate method, including but not
limited to
microfiltration, antibody capture, or precipitation. Solutions suitable to
cause precipitation
of the complex depend on the solubility of the complex, and may include
ammonium
I 0 sulfate or polyethylene glycol. If the fluid is to be returned to the
individual, then the
precipitating solution should be chosen so that any that remains in the fluid
does not cause
an adverse reaction in the individual.
it is understood that the in vivo and ex vivo methods described herein may
used in
conjunction with each other.
The invention also contemplates devices which can be used for reducing the
level of
antibody in a biological fluid using an epitope-presenting carrier of this
invention.
Typically, the device will be a flow system, comprising the following
elements: a) a port
that permits biological fluid to flow into the device; b) a chamber in which
the fluid is
permitted to contact the epitope-bound carrier (optionally in a solid phase);
c) a port that
permits the treated fluid to flow out of the device. Such devices can be
designed as
continuous flow systems, and as systems that permit the treatment of a single
sample from
an individual for purposes of analysis or readministration at a subsequent
time.
Administration and formulations
Various formulations of epitope-presenting carriers) may be used for
administration. In some embodiments, the epitope-presenting carriers) may be
administered neat. Preferably, the epitope-presenting carriers) is in a
composition
comprising an epitope-presenting carriers) and a pharmaceutically acceptable
excipient,
and may be in various formulations. Pharmaceutically acceptable excipients are
known in
the art, and are relatively inert substances that facilitate administration of
a
pharmacologically effective substance. For example, an excipient can give form
or
consistency, or act as a diluent. Suitable excipients include but are not
limited to
1$
CA 02353620 2001-06-05
WO 00/33887 PCT/US99/29336
stabilizing agents, wetting and emulsifying agents, salts for varying
osmolarity,
encapsulating agents, buffers, and skin penetration enhancers. Excipients as
well as
formulations for parenteral and nonperenteral drug delivery are set forth in
Remington's
Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).
Generally, these compositions are formulated for administration by injection
(e.g.,
intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.).
Accordingly, these
compositions are preferably combined with pharmaceutically acceptable vehicles
such as
saline, Ringer's solution, dextrose solution, and the Like. Generally, the
epitope-presenting
carrier will constitute about 0.01 % to 10% by weight of the formulation due
to practical,
empirical considerations such as solubility and osmolarity. The particular
dosage regimen,
i.e., dose, timing and repetition, will depend on, inter alia, the clinical
indication and the
particular individual and that individual's medical history. Generally, a dose
of about 1 ~Cg
to about 100 mg conjugate/kg body weight, preferably about 100 p.g to about IO
mg/kg
body weight, preferably about 50 ~,g to about 5 mg/kg body weight, preferably
about 1 wg
i 5 to about 1 g conjugate/kg body weight, preferably about 5 p,g to about 500
mg body weight
is administered. Empirical considerations, such as the half life, generally
will contribute to
determination of the dosage. An epitope-presenting carrier may be administered
daily,
followed by less frequent administrations, such as two times per week, once a
week, or
even less frequently. In other embodiments, an epitope-presenting carriers) is
administered less frequently, i.e., bi-weekly, weekly, every ten days, or
every two weeks.
Frequency of administration may be determined and adjusted over the course of
therapy,
and is based on maintaining the desired level of antibody. Other appropriate
dosing
schedules may be as frequent as daily or 3 doses per week, or one dose per
week, or one
dose every two to four weeks, or one dose on a monthly or less frequent
schedule
depending on the individual or the disease state. Repetitive administrations
may be
required to achieve and/or maintain the desired Ievel of antibody.
Alternatively, sustained
continuous release formulations of the compositions may be appropriate.
Various
formulations and devices for achieving sustained release are known in the art.
Other
formulations include those suitable for oral administration, which may be
suitable if the
epitope-presenting carrier is able to cross the mucosa. Similarly, an aerosol
formulation
may be suitable.
In some embodiments, more than one epitope-presenting earner may be present in
a
composition. Such compositions may contain at least one, at least two, at
least three, at
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WO 00/33887 PCT/US99/29336
least four, at least five different conjugates. Such "cocktails", as they are
often denoted in
the art; may be particularly useful in treating a broader range of population
of individuals.
They may also be useful in being more effective than using only one (or fewer
than are
contained in the cocktail} epitape-presenting carrier(s).
S The compositions may be administered alone or in conjunction with other
forms of
agents that serve to enhance and/or complement the effectiveness of an epitope-
presenting
carrier. Additionally, or alternatively, a dosage regimen may begin with one
epitope-
presenting carrier, and then switch to another.
An individual suitable for administration of an epitope-presenting carriers)
(or
I O composition comprising an epitope-presenting carrier(s)) is one who
exhibits undesirable
levels of a disease-associated antibody (such as those described above).
Levels of disease-
associated antibodies rnay be determined using standard assays in the art such
as ELISA.
Preferably, the individual is human. Measurable circulating levels of disease-
associated
antibody need not be detectable, it may be predictable (due to, for example,
risk factors;
15 genetic factors; environmental factors; and other known etiologies) that
such antibodies are
likely to be produced. Thus, the methods of the invention also pertain to
those cases in
which a prophylactic effect is contemplated.
In preferred embodiments, the epitope-presenting carrier {such as a valency
platform conjugate) is administered such that the duration of the effect is
longer than when
20 compared to other epitope-presenting carrier(s). In this vein,
considerations such as (a) the
particular carrier used; (b) valency; (c).type of epitope); (d) dosage
regimen; and (e} means
of administration may enter into producing this duration. Any one or more of
the above
factors may provide a basis for the extent of duration of effect (i.e., length
of time that the
antibodies are reduced). Similarly, any one or more of the above factors may
provide a
25 basis for the rapidity of reduction of antibody.
Kits for use in conjunction with the methods of the invention
30 The invention also includes kits for use in conjunction with the methods
described
herein. In some embodiments, the kits effect in vivo reduction of circulating
antibody
levels (i.e., disease-associated antibody). In other embodiments, the kits
effect
extracorporeal selective formation and/or removal of immunosorbent-anti-viral
antibody
CA 02353620 2001-06-05
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complexes (i.e., ex vivo removal). These kits contain components specific for
whatever
antibody(ies) is targetted for removal. In other embodiments, kits and
compositions are
provided for use in detection of antibody(ies) to be reduced. These kits aid
in assessing (a)
whether an individual is indicated for selective removal of disease-associated
antibody (for
example, if the titer is considered to be above a requisite threshold); (b)
monitoring an
individual after treatment (i.e., selective removal) to determine whether
further su~cient
removal has occurred and/or whether further is indicated (for example, when a
period of
time has elapsed since removal, and the titer of anti-viral antibody has risen
to or past a
requisite threshold, and treatment with a viral therapeutic agent is still
indicated); (c) which
antibody(ies) an individual is producing (this would indicate which antibody
or antibodies
should be selectively removed). These kits contain components specific for
antibodies
targeted for reduction and/or removal, aiding detection and/or monitoring.
It is understood that these kits, especially those used to effect selective
removal of
antibody, may also be denoted as "systems".
Kits of the invention, comprise an epitope-presenting carrier (preferably, the
carrier
is a valency platform molecule) that specifically binds the antibody to be
removed in
suitable packaging. Preferably, the kit also contains instructions for its
use. Appropriate
carriers {including those conjugated to epitope) have been discussed above.
The kits of the invention may further comprise reagents for testing for the
presence
(and/or level) of antibody targetted for reduction and/or removal, which would
be useful for
monitoring purposes.
The following Examples are provided to illustrate but not limit the present
invention.
EXAMPLES
Exam,~le 1: Synthesis of aGal epit0pes
21
CA 02353620 2001-06-05
WO 00133887 PCTIUS99/29336
The general analytical methods and characterization techniques used in the
present
disclosure are identified below. NMR spectra wee recorded on a Broker AC300
spectrometer at 300 MHz for ~H and 75 MHz for ~3C. Chemical shifts were
recorded in
parts per million (8) relative to TMS (i.e,, tetramethylsilane, b = 0.0 ppm)
or to the residual
signal of deuterated solvents: chloroform (8 = 7.27 ppm for iH; 8 = 77.23 ppm
for ~3C),
methanol (8 = 4.87 ppm for IH; 8 = 49.15 ppm for ~3C) and DZO (8 = 4.80 (DSS)
ppm for
~ H). Coupling constants (J) are reported in hertz. Analytical HPLC analyses
were
performed on a Hewlett Packard liquid chromatography HP 1090 instrument fitted
with a
Vydac C 18 column (4.6 x 250 mm, 5 p,rn particle size). Preparative HPLC was
performed
on Dynamax SD 200 system with Vydac C 18 column (22 x 250 mm, 10 pm particle
size).
Mass spectra were recorded on Finnigan LCQ mass spectrometer.
Enzymatic synthesis of the aGal epitape. 2-(2-(2-thioethoxy)ethoxyJethyl 3-Q~a
D-
galactop!vranosyl~~-D- a~ lactopvranoside: A reaction scheme illustrating the
synthesis is
I S shown in Figure 1.
Compound 2
S-2-[2-(2-Hydroxylethoxy)ethoxy]ethyl thiobenzoate
To a mixture of 2-[2-(2-cloroethoxy)ethoxyjethanol, compound 1 (20 g, 0.12
mot)
and thiobenzoic acid (16.4 g, 0.12 mol) was added I2 g of triethylamine at
room
temperature. The mixture was then stirred at 90°C for I h. After cooled
to room
temperature, ethyl acetate (I00 mL) was added to the reaction mixture and
filtered. The
filtrate was concentrated and purified via silica gel chromatography
(hexane/ethyl acetate,
I :1) to give compound 2 (30.6 g, 95%) as an orange syrup: ~H NMR (CDCI3): b
7.97 (dd, J
= 8.3, 1.4, 2 H), 7.56 (d, J= 7.4, 1 H), 7.45 (t, J= 6.9, 2 H), 3.73 (m, 4 H),
3.68 (s, 4 H),
3.62 (m, 2 H), 3.31 (t, 2 H).
Compound 4
2-[2-(2-Benzoylthioethoxy)ethoxy] ethyl 2,3,4,6-tetra-O-acetyl-(3-D-
galactopyranoside
To a solution of compound 2 (7.79 g, 28.9 mmol) and acetobromo-oc~D-gaiactose,
compound 3 ( 17.80 g, 43.28 mmol) in dry CH2C12 ( 100 mL) were added AgZC03
(9.27 g,
22
SU6STlTUTE SHEET (RULE 26j
CA 02353620 2001-06-05
WO 00/33887 PCT/US99/29336
36.1 mmol) and activated 4th molecular sieve (powder, 10 g) at 0 °C.
After stirred at room
temperature for 3 d, the reaction mixture was filtered through Celite and the
ftltrate was
concentrated and purified via silica geI chromatography (hexane/ethyl acetate,
4:1 ) to give
compound 4 (12.29 g, 7I%) as a colorless syrup: ~H NMR (CDCl3): 8 7.99-7.95
(m, 2 H),
7.61-7.55 (m, 1 H), 7.48-7.43 {m, 2 H), 5.39 (dd, J= 1.0, 3.4, 1 H), 5.2I (dd,
J= 7.9, 10.5,
1 H), 5.03 {dd, J= 3.4, 10.5, 1 H), 4.58 (d, J= 7.9, 1 H), 4.14 (dd, J= 2.9,
6.9, 1 H), 3.99
3.89 (m, 2 H), 3.79-3.65 (m, 10 H), 3.30 (t, J= 6.4, 2 H), 2.15-1.98 (4s, 12
H); 13C NMR
(CDC13}: 8 191.4, 170.3, 170.2, 170.0, 169.4, 136.9, 133.4, 128.5, 127.2,
101.3, 97.4, 70.9,
70.6, 70.5, 70.3, 69.8, 69.0, 68.8, 67.0, 61.2, 20.7, 20.6, 20.5; MS (ESI):
m/e (M + Na+)
Calcd. for C2~H36O13SNa: 623.2, obsd.: 623.2.
Compound 6
2-[2-(2-tert-Butyldithioethoxy)ethoxy]ethyl (3-D-galactopyranoside
To a stirred solution of compound 4 (3.47 g, 5.77 mmol) in methanol {15 mL)
was
added NaOCH3 (0.50 g, 9.28 mmoi) at 0 °C. After 3 h, diethyl 1-(tent-
bytylthio)-1,2-
hydrzainedicarboxylate, compound 5 (2.2 g, 8.3 mmol), which was prepared as
described
('V~unsch, E., et al. Hoppe-Seyler's Z. Physial. Chem. (1982) 363: 1461-1464),
was added.
The reaction mixture was stirred at room temperature for another 2 h. Dowex
50X2-404
resin was then added to neutralized the solution and filtered. The filtrate
was concentrated
and purified via silica gel chromatography (CH2C12/MeOH, 9:1) to give compound
6 (1.22
g, 51 %) as a white solid: 1H NMR {CDCl3): b 4.39 (br s, 1 H), 4.35 {br d, 2
H}, 4.11 {br s, 1
H), 4.02 (br s, 2 H), 3.84 (br s, 2 H), 3.78-3.48 (m, 13 H), 2.89 (t, J= 6.7,
2 H), 1.33 (s,
9H); MS (ESI): m/e (M + 1) Calcd. for C~bH33OgSz: 417.2, obsd.: 417.5.
Compounds 7 and 8
2-[2-{2-tent-Butyldithioethoxy)ethoxy]ethyl 3-O-(a-D-galactopyranosyl}-[3-D-
galactopyranoside(7)
2-[2-(2-tert-butyldithioethoxy}ethoxy]ethyl 3-O-(a-D-galactopyranosyl)-j3-D-
galactopyranoside (8):
To a solution of compound 6 (2.87 g, 6.89 mmol) and p-nitrophenyl a-D-
galactopyranoside (200 mg) in 10 mL of sodium phosphate buffer (50 mM, pH 6.5)
was
added 20 mg of coffee bean a-galactosidase. The reaction was proceeded at room
23
CA 02353620 2001-06-05
WU 00/33887 PCT/US99/29336
temperature with gradual addition of donor until 300 mMp-nitraphenol had been
formed (3
d). The reaction mixture was lyophilized and the residue was suspended in
methanol,
fzltered and concentrated. The unreacted starting material (2.S g) was
recovered by silica
gel chromatography (CH2Cl2/MeOH, 9S:S to 70:30). The products were first
purified on P2
S Gel filtration column eluted with water and then an reverse phase HPLC
column to yield
compound 7 (80 mg, 2.0%) and compound 8 (106 mg, 2.6%) as white solids. For
compound 7: analytical RF-HPLC: tR 8.49 min with a gradient of 2S to 30% ACN
in H20
at a flow rate of 1 mL/min, purity, 100%;1H NMR (CD30D): 5.04 (d, J= I .9, I
H), 4.32
(dd, J= 1.6, 6.2, 1 H), 4.25 (t, J= S.S, 1 H), 4.05-4.00 (m, 2 H); 3.93 (d, J=
1.2, 1 H),
3.83-3.61 (m, 17 H), 3.52 (t, 1 H), 2.89 {t, J= 6.6, 2 H}, 1.33 (s, 9H); MS
(ESI): m/e {M +
Na+) Calcd. for C22Hq2O13S2Na: 60I .2, obsd.: 601.2. For compound $:
analytical RF-
HPLC: tR 7.14 min with a gradient of 2S to 30% ACN in H20 at a flow rate of 1
mL/min,
purity, I00%;'H NMR (CD30D): 4.27(d, J= 7.5, 1 H), 4.00-3.62 (m, 22 H), 3.50
(rn, 1
H), 2.89 (t, J=6.6, 2 H), 1.36 (s, 9 H); MS (ESI): m/e (M + Na+} Calcd. for
C22H420,3S2Na:
1 S 60I .2, obsd.: 601.1.
These two disaccharides were further characterized after acetylated with Ac20
in
pyridine at room temperature overnight: for acetylated compound 7:'H NMR
(CDCI3): $
5.46 (br d, J= 3.3, 1 H), 5.37 (br d, J= 3.3, I H}, 5.31-5.22 (m, 3 H), 5.14
{dd, J= 3.3,
10.2, 1 H), 4.50 {d, J= 7.8, 1 H), 4.29 {br t, J= 6.5, 7.3, 1 H); 4.2I (dd, J=
7.3, 11.3, 1 H},
4.14 (d, J = 7.3, 2 H), 4.04 {dd, J = 6. S, i 0.8, 1 H), 3 .91 (dd, J = 10.4,
2.9, 1 H), 3 .84 (br t,
J= 7.3, I H), 3.87-3.62 (m, 10 H), 2.9I (t, J= 7.6, 2 H), 2.15-1.95 (7s, 21
H), 1.35 (s, 9 H)
and for acetylated compound 8: 'H NMR (CDC13): 8 5.42 (br d, J= 3.1, 1 H),
5.41 (br d, J
= 3.0, 1 H); 5.28 (dd, J= 3.0, 10.4, 1 H), 5.19 (dd, J= 7.8, 10.4, 1 H), 5.12
{dd, J= 3.3,
10.4, 1 H), 5.02 (dd, J= 3.1, 10.4, I H), 4.93 (d, J= 3.3, 1 H), 4.57 (d, J=
7.8, 1 H), 4.21
2S {br t, J= 6.8, 1 H), 4.09 (m, 2 H), 3.95 (dt, J= 3.9, 10.8, 1 H), 3.86 {br
t, J= 6.6, I H),
3.81-3.62 (m, 10 H), 3.44 (dd, J= 7.4, 10.2, 1 H), 2.89 (t, J = 6.7, 2 H),
2.12-1.98 (7s, 2I
H), 1.3 S (s, 9 H).
Chemical synthesis ofthe aGal epitope 2-~2-~(2 thioethoxv)ethoxy7ethvl 3 O na
D
~alacton rahos~l) a!3 D-galactopyrahoside: A reaction scheme illustrating this
synthesis is
shown in Figure 2.
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Compound 10
2-[2-(2-chloroethoxy)ethoxy]ethyl 2,3,4,6-tetra-O-acetyl-(3-D-
galactopyranoside
To a mixture of gaiactose pentaacetate, compound 9 (70 g, 179 mmol), 2-[2-(2-
chloroethoxy)ethoxy]ethanol (45.4 g, 270 mmol} and activated 41~ molecular
sieve (20 g)
in dry CH2CI2 (500 mL) was added BF3'Et20 (52 g, 370 mmol) dropwise at room
temperature for 3 h. After stirred for 2 d, the suspension was filtered
through CeIite and the
filtrate was poured into 340 mL of saturated aqueous NaHC03 cooled in an ice
bath. The
organic phase was separated and the aqueous phase was extracted with CH2Cl2.
The
combined organic phases were washed with brine, dried, and concentrated. The
residue was
IO purified via silica gel chromatography (hexane%thyl acetate, 1:1) to give
compound 10
(67g, 75%) as a colorless oil: ~H NMR (CDCl3): 8 5.36 (d, J= 3.2, 1 H), 5.19
(dd, J= 10.4,
8.0, 1 H), 5.00 (dd, J= 10.4, 3.6, 1 H), 4.55 (d, J= 8.0, 1 H), 4.18-4.08 (m,
3 H), 391-3.87
(m, 2 H), 3.74 (m, 2H), 3.72-3.61 (m, 8 H), 2.13 (s, 3 H), 2.04 {s, 3 H), 2.03
(s, 3 H), I .96
(s, 3 H}.
Compound 11
2-[2-(2-chloroethoxy)ethoxy]ethyl (3-D-galactopyranoside
A solution of compound 10 (67g, 134 mmol} in 200 mL of methanol and 2S0 mL of
1 M K2C03 aqueous solution was stirred at room temperature overnight. The
reaction
mixture was poured into 700 mL of methanol cooled with ice-water bath. The
precipitate
was filtered through celite and washed with methanol. The filtrate was
combined and
neutralized with Dowex resin (H form} until pH 6. The resin was filtered and
washed with
water. The filtrate was concentrated and Lyophilized to give compound 11 (37
g, 83%) as a
colorless oil: 'H NMR (D20}: 8 4.30 (d, J= 7.2, 1 H), 3.95 (m, 1 H), 3.80 (d,
J= 3.6, I H),
3.80-3.50 (m, 15 H), 3.40 {m, 1 H).
Compound I2
2-[2-(2-chloroethoxy)ethoxy]ethyl 3-O-p-methoxybenzyl-(3-D-galactopyranoside
A mixture of compound 11 (37 g, I 12 mmol) and dibutyltin oxide (46 g, 210
mmol)
in dry MeOH (300 mL) was refluxed under nitrogen until clear (10 hr). The
reaction
mixture was concentrated and the residue was dried under vacuum. The residue
was
CA 02353620 2001-06-05
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dissolved in 800 mL of dioxane and 80 mL of DMF andp-methoxybenzyl chloride
(32 g,
28 rnl, 0.20 mol) was added. The resulting mixture was stirred at 100
°C for 10 h to give a
brownish solution with precipitate. After cooled to room temperature, the
precipitate was
removed by filtration through Celite and washed with dioxane (100 mL) and
chloroform
( I 00 mL). The combined organic phases were concentrated and purified by
silica gel
chromatography (ethyl acetate) to give compound 12 (30 g, 60%) as a colorless
oil.
Compound 13
2-[2-(2-chloroethoxy)ethoxy]ethyl 2,4,6-tri-O-acetyl-3-O-p-methoxybenzyl (3-D
I O galactopyranoside
Compound 12 (30 g, 66.5 mmol) was acetylated with Ac20 {150 mL} in pyridine
(150 mL), catalyzed by DMAP (120 mg). After stirred for 5 h, the reaction
mixture was
concentrated. The residue was dissolved in chIoraform (300 mL} and washed with
HCI
solution (0.5 M), water, saturated aqueous NaHC03 and brine. The organic phase
was dried
over anhydrous Na2S04 and concentrated to give compound I3 (34 g, 89%) as a
colorless
oil:'H NMR {CDCI~): 8 7.19 (d, J= 8.6, 2 H), 6.87 {d, J= 8.6, 2 H), 5.48 (d,
J= 3.3, 1 H),
5 .10 (dd, J = 10.0, 8.1, 1 H), 4.62 (d, J = 11.6, 1 H), 4.45 (d, J = 8.1, 1
H), 4.34 (d, J = 11.6,
1 H), 4.17 {dd, J = 6.7, 1.0, 2 H), 3.94 (m, 1 H), 3 .82-3 .61 (m, 15 H), 3
.49 (dd, J = 10.0,
3.3, 1 H), 2.16 (s, 3 H), 2.08 (s, 3 H), 2.04 (s, 3 H}; MS (ESI}: mle (M + Na~
Calcd. for
2O C26H37CIOi2Na: 599.2, obsd: 599.4.
Compound 14
2-[2-(2-chloroethoxy)ethoxy]ethyl 2,4,6-tri-O-acetyl (3-D-galactopyranoside
To a solution of compound 13 (34 g, 59 mmol) in 300 mL of CH3CN/water (9:1)
was added CAN (64 g, 120 mmol) slowly during 3 h at 0 °C. After
addition, the mixture
was stirred at the same temperature for, 3 h. The reaction mixture was then
concentrated,
diluted with 250 mL of water, and extracted with chloroform. The organic
extracts were
washed with saturated aqueous NaHC03 and brine, dried over anhydrous Na2S04,
and
concentrated. The residue was purified via flash column chromatography
(hexane/ethyl
acetate, 3:1 to 2:3) to give compound 14 (20.4 g, 76%) as a colorless oil: 'H
NMR (CDC13):
8 5 .31 (d, J = 3 .6, 1 H), 4. 9 S (dd, J = 10.0, 7. 8, I H}, 4. 51 (d, J =
8.0, 1 H), 4.13 (m, 2 H},
3.94 (m, 1 H), 4.00-3.61 (m, 14 H}, 2.15 (s, 3 H), 2.11 (s, 3 H), 2.04 {s, 3
H).
26
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Compound 16
2-[2-(2-chloroethoxy)ethoxy]ethyl 3-O-(2,3,4,6-tetra-O-benzyl-a-D
galactopyranosyl)-2,4,6-tri-O-acetyl-(3-D-galactopyranoside
S To a mixture of compound 14 ( 10 g, 22 mmol), compound 15 (25.4 g, 45 mmol),
4-methyl-2,6-di-t-butyl pyridine (6.8 g, 33 mmol), and activated 4A molecular
sieve (10 g)
in dry ether (300 mL) was added a solution of methyl triflate (7.2 mL) in
ether (SO mL)
dropwise via a syringe-pump over a period of 24 h. After stirred at room
temperature for 36
h, the reaction mixture was filtered through Celite. The filtrate was
concentrated and
purified via silica gel chromatography (hexane/ethyl acetate, 4:1 to I:I) to
give compound
1b ( 17.3 g, 81 %) as a yellowish oil: 'H NMR (CDC 13): S 7.35-7.22 (m, 20 H),
5.43 (d, J =
3 .2, 1 H), 5.16 (dd, :l = 10.0, 8.0, 1 H), 5.06 (d, J = 3 .4, 1 H}, 4.90 (d,
J = 11.6, 1 H), 4. 81
(d, J= 12.0, 1 H), 4.68-4.62 (m, 3 H}, 4.46-4.39 {m, 4 H), 4.12-3.59 (m, 20
H), 3.49 (d, J=
6.4, 2 H), 2.03 (s, 3 H), 1.94 (s, 3 H), 1.79(s, 3 H); MS (ESI): m/e (M + Na+)
Calcd. for
CszH63C10,6Na: 1001.4, obsd: 1001.8.
Compound 17
2-[2-(2-chloroethoxy)ethoxyJethyl 3-O-a-D-galactopyranosyi-2,4,6-tri-O-acetyl-
~3
D-galactopyranoside
A mixture of compound I6 (I8. I g, 18.5 mmol) and Pd/C (20%, 3 g) in 300 mL of
methanol and 1.5 mL of acetic acid was shaken under compressed hydrogen (50
psi) at
room temperature for 9 h. The reaction mixture was f ltered through Celite and
concentrated. Water {10 mL) was added to the residue to assist the removal of
acetic acid
and the crude product was used directly in next reaction without purification.
IH NMR
(CD30D): 8 5.52 (dd, J= 10.0, 8.4, I H), 4.98 {d, J= 3.6, 1 H), 4.62 (d, J=
8.0, 1 H}, 4.14
{m, 2 H), 4.06 (m, 1 H), 3.98 (t, J= 8.4, 1 H), 3.92 (m, 1 H), 3.86 (d, J=
2.7, 1 H}, 3.77-
3.61 (m, 16 H), 3.SS (dd, J= 10.0, 3.2, 1 H), 2.13 (s, 3 H), 2.11 (s, 3 H),
2.03 (s, 3 H). MS
(ESI): m/e (M + Na*) Calcd. for C2~H39CIO16Na: 641.2; obsd: 641.3.
Compound I8
2-[2-(2-chloroethoxy)ethoxy)ethyl 3-O-a-D-galactopyranosyl-/3-D-
galactopyranoside
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A solution of compound 17 in 40 mL of 1 M NaOH and 300 mL of methanol was
stirred at room temperature for 6 h. The reaction mixture was neutralized with
Dowex resin
(H form) to pH 6, f ltered and lyophilized to give compound 18 (8.9 g, 97%) as
a white
solid. 'H NMR (D20): 8 5.18 {d, J= 3.7, 1 H), 4.53 (d, J= 7.7, 1 H), 4.25-3.68
(m, 24 H).
MS (ESI): mle (M +Na+) Calcd. for C1gH33C1OI3Na: 515.2, obsd: 515.5.
Compound 19
2-[2-(2-acetylthioethoxy)ethoxyJethyl 3-O-(a-D-galactopyranosyl)-(3-D
galactopyranoside
A solution of compound 18 (8.9 g, 18.1 mmol) in 100 mL of I M potansium
thioacetate was stirred at 95 °C under nitrogen for 36 h. The reaction
mixture was cooled
and loaded on a column packed with Dowex ion exchange resin (H form, 100 g)
and eluted
with water. The aqueous solution was neutralized with Dowex Marathon WBA anion
exchange resin (from pH 2 to 5), filtered, and lyophilized to give compound 19
(7.5 g,
78%) as an off white solid. 'H NMR (D20): 8 5.17 (d, J= 3.8, 1 H), 4.52 (d, J=
7.7 Hz, 1
H), 4.24-3.67 (m, 25 H), 3.15 (t, J= 6.2, 2 H); MS (ESI): mle (M + Na+) Calcd.
for
C20H36~14SNa: 555.2, obsd: 555.3.
Compound 20
2-[2-(2-thioethoxy)ethoxy]ethyl 3-O-(a-D-galactopyranosyl)-(3-D-
galactopyranoside
A mixture of compound 19 (650, 1.22 mmol) and 4 g of DOWER S50A OH anion-
exchange resin, pre-washed with methanol, in 50 mL of methanol was stirred at
room
temperature overnight. The reaction mixture was filtered and the resin was
washed with 5%
acetic acid in methanol. The filtrate was concentrated to give compound 20
(578 mg, 97%)
as a white solid: 'H NMR (CD30D): 8 5.00 (d, J= 3.6, 1 H), 4.35 (d, J= 7.6 Hz,
1 H), 4.04
(m, 2H), 3.93 (m, 1 H), 3.84 (m, 1 H), 3.82 (dd, J= 10.4, 3.2, 1 H), 3.73-3.51
(m, 18 H),
2.83 (t, J= 6.0, 2 H);'3C NMR (D20): S 103.2, 95.8, 77.8, 75.4, 72.7, 71.4,
70.2, 70.1,
69.9, 69.7, 69.2, 68.9, 68.7, 65.4, 61.5, 37.9, 23.5; MS (ESI): m/e (M + Na+)
Calcd. for
C~gH340~3SNa: 513.5, obsd: 513.3.
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The synthetically prepared aGal epitope on resin was antigenically active as
demonstrated by its ability to remove >95% of anti-aGa1 Ig from normal rhesus
monkey or
human serum, as measured by FRCS.
Compound 22
p-aminophenyl 3-O-a-D-galactopyranosyl-a-D-galactopyranoside
A reaction scheme illustrating the chemoenzymatic synthesis of the aGal
epitope,
p-aminophenyl 3-O-a-D-galactopyranosyl-a-D-galactopyranoside (22) is shown in
Figure
3. p-Nitrophenyl 3-O-a-D-galactopyranosyl-a-D-galactopyranoside (compound 21)
was
prepared enzymatically as described by Nilsson (Tetrahedron Lett. (1997)
38:133-136). A
mixture ofp-nitrophenyl 3-O-a-galactopyranosyl-a-D-galactopyranoside (70 mg,
0.15
mmol) and IO mg Pd/C (10%) in methanol (4 mL) was stirred under hydrogen of
room
temperature overnight. The reaction mixture was then filtered through Celite
and the filtrate
was concentrated in vacuo to give (50 mg) as a yellowish solid: ' H NMR
(CD3OD): 8 6.87
(t, 2 H), 6.57 (d, 2 H), 5.21 (d, 1 H), 4.98 (d, 1 H), 4.2-4.1 (m, 2 H), ~.1-
3.8 (m, 3 H), 3.8-
3.7 (m, 3 H), 3.7-3.5 (m, 4 H); 13C NMR (CD30D): 8 143.8, 120.1, 117.9, 1
I5.4, 101.2,
97.6, 78.3, 77.0, 72.6, 71.5, 71.4, 70.3, 68.7, 67.4, 63.2, 62.6; MS (ESI):
xn/e (M + Nay)
Calcd. for CrgH2~NO~~Na: 456.2, obsd: 456.2.
Example 2: Synthesis of valency platforms
Synthesis of compound 23
A solution of 1,4-diaminobutane and NaHC03 in water/dioxane I/1 is treated
with
bromoacetic anhydride. The mixture is extracted with CH2C12, and the CH2CI2
layer is
dried and concentrated to give crude product which is purified by silica gel
chromatography to give compound 23..
Synthesis of compound 24
A solution of 4,7,10-trioxa-1,3-tridecanediamine and NaHC03 in water/dioxane
1/I
is treated with bromoacetic anhydride. The mixture is extracted with CH2C12,
and the
CH2Cl2 layer is dried and concentrated to give crude product which is purified
by silica geI
chromatography to give compound 24.
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Synthesis of Compound 29
A strategy for synthesis of compound 29 is shown in Figure 24.
Compound A: A solution of 1,3-diamino-2-hydroxypropane in aqueous dioxane
was treated with di-t-butyldicarbonate and NazCO3. The mixture was extracted
with
CH2C12, and the CHZC12 layer was dried and concentrated to give crude product
which was
purified by silica gel chromatography to give compound B.
Compound B was treated with p-toluensulfonyl chloride in pyridine. The mixture
was acidified with aqueous HCl and extracted with CH2CIz. The CH2C12 layer was
dried
and concentrated to give crude product which was purified by silica gel
chromatography to
give compound C.
A solution of compound C in a suitable solvent is treated with thiobenzoic
acid and
a suitable base. The mixture is extracted with CH2G12, and the CHZCI2 Layer is
dried and
concentrated to give crude product which is purified by silica gel
chromatography to give
compound D.
A solution of compound D in MeOH is treated with one equivalent of NaOH until
the thiobenzoate ester is hydrolyzed as evidenced by TLC. To the resulting
mixture is
added 0.25 equivalents of compound 26. The mixture is stirred until complete
as evidenced
by TLC. The mixture is acidified with aqueous H2SO4 and extracted with CHzCl2.
The
CH2CI2 layer is dried and concentrated to give crude product which is purified
by silica gel
chromatography to give compound E.
Compound E is treated with trifluoroacetic acid to remove the BOC protecting
groups. The mixture is concentrated, and the residue is dissolved in a
solution of NaHC03
in 1 /1 dioxane/water. To the resulting solution is added eight equivalents of
bromoacetic
anhydride. The mixture is stirred until complete as evidenced by TLC. The
mixture is
acidif ed with aqueous H2S04 and extracted with CH2CI2. The CH2Cl2 layer is
dried and
concentrated to give crude product which is purified by silica gel
chromatography to give
compound 29.
Synthesis of Compound 30
A chemical scheme for the preparation of an octamer of HEGA/TEG is shown in
Figures 26A and 26B. Compound 30. .The bis-hexaethyleneglycolamine (compound
_4')
was reacted with di-tert-butyldicarbonate to yield the N-BOC compound
(compound 8'),
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which was then reacted with para-nitrophenylchloroformate to yield the para-
nitrophenylcarbonate compound (compound 9~. Thepara-nitrophenylcarbonate (PNP)
group was then converted to a carbarnate group by reaction with mono-CBZ-
protected
piperazine, yielding compound 10'. The BOC group was removed using
trifluoroacetic
acid to yield compound 11'. Compounds 9' and 11' were then reacted together to
form a
"one-sided" dendritic compound (compound I2'). Again, the BOC group was
removed
using trifluoroacetic acid to yield compound I 3' . Compound I 3' was then
reacted with
triethyleneglycol bis chloroformate (from which the "core" is derived) to
yield the "two-
sided" dendritic compound (compound 14'). The terminal CBZ-protected amino
groups
were then converted to the hydrobromide salt of amino group, and further
reacted with
bromoacetic anhydride to yield reactive bromoacetyl groups at each of the
termini in
compound 30.
Synthesis of Compounds 31 and 32
I 5 A strategy for synthesis of compound 31 and 32 is shown in Figure 25.
Tetraamino platform, compound F, was reacted with the N-hydroxysuccinimidyl
ester of N«,NE-bis-CBZ-lysine in a solution of water/acetonitrile which
contained Na2C03.
The acetonitrile was removed under vacuum, and the product precipitated. The
precipitate
was washed with water and recrystallized from acetonitrile to give G.
The CBZ groups were removed from compound G by catalytic hydrogenation using
I 0% Pd on carbon in ethanol. The mixture was filtered, and the filtrate was
concentrated to
give the octa-amine, compound H, as a brown oil.
Compound H was reacted with chloroaceticanhydride in methanollacetonitrile at
room temperature overnight. The crude product was purified by silica gel
chromatography
to give compound 31.
Compound 32:
A solution of compound H is reacted with acryloyl chloride or acrylic
anhydride in
the presence of suitable base. The solvent is removed, and the crude product
is purified by
silica gel chromatography to give compound 32.
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Example 3: Synthesis of aGal conjugates
Compound 33
Synthesis of monomeric aGal Conjugate
A mixture of aGal 20 (100 mg, 0.204 mmol), chloroacetamide (38 mg, 0.40$
mmol), and tributylphosphine ( 10 p,L) in 1 mL of Na2C03 ( i 0 mg/mL) solution
in
water/ACN (1:I) was stirred at room temperature overnight. After removing the
organic
solvent, the remaining aqueous solution was purified on reversed phase HPLC
column
I O eluted at 10 mL/min with a gradient of acetonitrile-water (5 to 15%) over
40 minutes to
give 33 (96.3 mg, 86%) as a white solid: analytical RF-HPLC: tR 4.78 min with
a gradient
of 5 to 20% ACN in H20 at a flow rate of 1 mL/min. purity, 100%; MS (ESI):
rn/e (M +
Na+) Calcd. for C2oHs~N0,4SNa: 570.2, obsd: 570.3.
I 5 Compound 34
Synthesis of dimeric aGal Conjugate
A mixture of aGal 20 (65 mg, O.I33 mmol) in 1 mL of Na2C03 (20 mg/mL)
aqueous solution was stirred at roam temperature overnight. The solution was
purified on
reversed phase HPLC column eluted at 10 mL/min with a gradient of acetonitrile-
water (10
20 to 25%) over 40 minutes to give 34 (3I .3 mg, 48%) as a white solid:
analytical RF-HPLC:
tR 9.96 min with a gradient of 5 to 30% ACN in H20 at a flow rate of 1 mL/min.
purity,
100%; MS (ESi): m/e (M + Na'~ Calcd. for C36H66NO26S2Na: 1001 .3, obsd:
1001.3.
Compound 35
25 Synthesis of Dimeric aGal Conjugate
A mixture of aGal 19 (30 mg, 0.056 mmol), dimeric platform 23 (9.3 mg, 0.028
mmol), and tributylphosphine (0.030 mL) in 3 mL of Na2C03 solution (20 mg/mL)
and 2
mL of ACN was stirred under N2 at room temperature overnight. After removing
the
organic solvent, the remaining aqueous solution was purified an reversed phase
HPLC
30 column eluted at I O mL/min with a gradient of acetonitrile-water (5 to
15%) over 40
minutes to give 35 ( I 0 mg, 31 %) as a white solid: analytical RF-HPLC: t~
8.69 min with a
gradient of 5 to 30% ACN in H20 at a flow rate of I mL/min. purity, 97.9%;'H
NMR
32
CA 02353620 2001-06-05
WD 00133887 PCT/US99129336
(D20): & 5.09 (d, J= 3.5, 2 H), 4.39 (d, J= 7.3, 2 H), 4.21 (t, J= 6.5, 2 H),
4.10 (d, J= 2.5,
2 H}, 4.05 (m, 2 H}, 3.96 (d, J= 2.6, 2 H}, 3.88 (d, J= 2.5, 2 H}, 3.85 (d, J=
3.0, 2 H),
3.82-3.57 (m, 32 H), 3.27 (s, 4 H), 3.23 (s, 4 H}, 2.79 (t, J= 6.5, 4 H), 1.56
{br s, 4 H); ~3C
NMR (D20): 8 173.1, 105.6, 103.4, 98.2, 96.0, 80.2, 78.3, 77.2, 75.3, 73.16,
7I.8, 71.3,
70.9, 69.8, 69.4, 69.1, 65.4, 32.4, 40.5, 36.5, 34.6, 32.8; MS (ESI): m/e {M +
Nay') Calcd.
for C~,H8oN202xS2Na: 1171.4, obsd: 1172.5.
Compound 36
Synthesis of Dimeric aGal Conjugate
I 0 This compound was prepared following the procedure described above for
compound 35. Compound 19 {30 mg, 0.056 mmol) was conjugated with dimeric
platform
24 (13.0 mg, 0.028 mmol) to give 36 as a white solid: analytical RF-HPLC: tR I
1.3 min
with a gradient of 5 to 30% ACN in H20 at a flow rate of 1 mL/min. purity,
97.4%; 'H
NMR (D20): 8 5.19 (d, J = 3.5, 2 H), 4.53 (d, J = 8.8, 2 H), 4.22 (t, J = 3.9,
4 H), 4. I 3 (m,
2 H}, 4.05-3.98 (m, 2 H), 3.91 {d, J= 2.7, 2 H), 3.88-3.66 (m, 42 H), 3.63 (t,
J= 6.5, 4 H),
3.35 (t, J= 7.2, 8 H), 2.84 (t, J = 6.4, 4 H), i .81 (m, 4 H); MS (ESI): m/e
(M + Nay} Calcd.
for CSflH92NZO3~S2Na: 1303.5, obsd: 1303.4.
Compound 37
Synthesis of Tetrameric aGal Conjugate
The tent-butylthio protecting group of a.Gal 7 (30 mg, 0.052 mmol) was removed
by
reducing with tributylphosphine (25 ~L) in 5 mL of water at room temperature
overnight.
The reaction mixture was concentrated and dried under high vacuum overnight to
remove
any residual tent-butylthiol. The residue was dissolved in 5 mL of Na2C03 { I
0 mg/mL)
solution in water/ACN (1:1). Tetrarneric platform 25 (5.0 mg, 0.0074 mmol) was
added
and the resulted solution was stirred at room temperature overnight. After
removing the
organic solvent, the remaining aqueous solution was purified on a reverse
phase HPLC
column eluted at 10 mL/min with a gradient of acetonitrile-water (20 to 25%)
over 40
minutes to give 37 (9.6 mg, 56%) as a white solid after lyophilization: MS
(ESI): mle (MJ2
+ Na+) Calcd. for C4~H~9028S2Na: 1178.4, obsd.: 1178.4.
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Compound 38
Synthesis of Tetrameric aGal Conjugate
This compound was prepared following the procedure described above for
compound 37. Compound 7 (22 mg, 0.038 mmol) was conjugated with platform 26
(7.0
S mg, O.OOS7 mmol). The product was purified on a reverse phase HPLC column
with a
gradient of acetonitrile-water (1 S to 20%) over 40 minutes to yield 3$ (13
mg, 80%) as a
white solid: analytical RF-HPLC: tR 5.24 min with a gradient of 1 S to 20% ACN
in H20 at
a flow rate of 1 mL/min, purity, 100%; MS (ESI): m/e (M/2 + Na~ Calcd. for
CssH9sNs03aSzNa: 1454.6, obsd.: 1454.5.
Compound 39
Synthesis of Tetrameric aGa1 Conjugate
This compound was prepared following the procedure described above for
compound 38. The conjugation of compound 7 (22 mg, 0.038 mmol) with platform
27 (6.0
mg, 0.0048 mmol) yielded 39 (12 mg, 93%) as a white solid: analytical RF-HPLC:
tR 11.32
min with a gradient of 1 S to 20% ACN in H20 at a flow rate of 1 mL/min,
purity, 100%;
MS (ESI): m/e (M/2 + Na+) Calcd. for Cs3Hg7N4O32S2Na: 1379.1, obsd.: 1379.1.
Compound 40
Synthesis of Tetrameric aGal Conjugate
This conjugate is prepared by following the procedure described above for
compound 38 using aGal 7 and platform 28.
Compound 41
Synthesis of Tetrameric aGal Conjugate 41
A mixture ofp-aminophenyl 3-O-a-D-galactopyranosyl-a-D-galactopyranoside 22
(11 mg, 0.025 mmol), tetrameric platform 28, and NaHC03 (3 mg, 0.030 mmol) in
0.15 mL
of H20/CH3CN (1:1) was slightly shaken for 1 h. After addition of 0.15 mL of
H20, the
reaction mixture was set at room temperature for 2 d and purified on reversed
phase HPLC
column eluted at 1 mL/min with a gradient of acetonitrile-water (0 to 30%)
over 1 S minutes
to yiled 41(4.6 mg, 40%) as a white solid: MS (ESI): m/e (Ml2 + 1) Calcd. for
C60H95N7~29~ 1377.6 obsd: 1377.9.
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Compound 42
Synthesis of Tetrameric aGal Conjugate
A solution of the beta isomer of aGal 7, 2-[2-(2-thioethoxy)ethoxy]ethyl 3-O-
([3-D-
galactopyranosyl)-[3-D-galactopyranoside (30 mg, 0.061 mmol), platform 25 (5
mg, 0.0074
mmol), and tributylphosphine {0.10 mL) in 5 mL of Na2C03 ( 10 mg/mL) solution
in
water/ACN (1:1) was stirred at room temperature overnight. After removing the
organic
solvent, the remaining aqueous solution was purified on a reverse phase HPLC
column
eluted at 10 mL/min with a gradient of acetonitrile-water (20 to 25%) over 40
minutes to
give 42 (2.4 mg, 14%) as a white solid: MS (ESi): m/e (M/2 + Na+) Calcd. for
C4~H~9O2gS2Na: 1178.4, obsd.: 1179Ø
Compound 43
Synthesis of Tetrameric aGal Conjugate
This conjugate was prepared by following the procedure described above for
compound 38. The conjugation of compound 8 (25 mg, 0.043 mmol) with platform
27 (6.0
mg, 0.0048 mmol) yielded 43 (8.8 mg, 68%) as a white solid: analytical RF-
HPLC: tR 6.84
min with a gradient of 15 to 20% ACN in H20 at a flow rate of 1 mL/min,
purity, 100%;
MS (ESI): m/e (M/2 + Nay) Calcd. for C53H87Na032S2Na: 1379.4, obsd.: 1379.1.
Compound 44
Synthesis of Octameric aGal Conjugate
A solution of aGal 19 (23 mg, 0.429 mrnol) and platform 29 (10 mg, 0:0043
mmol)
in 2 mL of Na2C03 (10 mg/mL) solution in water/ACN (1:1) was stirred under N2
at room
temperature overnight. The reaction mixture was concentrated and purified on a
reverse
phase HPLC column eluted at 10 mL/min with a gradient of acetonitrile-water
(10 to 30%)
over 40 minutes to give 44 (8.5 mg, 37%) as a white solid.
Compound 45
Synthesis of Octameric aGal Conjugate
This compound was prepared following the procedure described above for
compound 44. Compound 19 (50 mg, 0.094 mmol) was conjugated with platform 30
(I O
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mg, 0.0018 mmoi}. The product was purified on a reverse phase HPLC column with
a
gradient of acetonitrile-water (10 to 50%) over 40 minutes to yield 45 (10.8
mg, 67%) as a
white solid: analytical RF-HPLC: tR I I .16 min with a gradient of 5 to 70%
ACN in H20 at
a flow rate of 1 mL/min, purity, I00%.
Compound 46
Synthesis of Octameric aGal Conjugate
This compound was prepared following the procedure described above for
compound 44. Compound 19 (691 mg, 1.41 rnmol) was conjugated with platform 31
(280
mg, 0.141 mmol). The product was purified on a reverse phase HPLC column with
acetonitrile-water (I9.5%) over 40 minutes to yield 46 (604 mg, 76%) as a
white solid:
analytical RF-HPLC: tR 9.16 min with a gradient of I S-25% ACN in H20 at a
flow rate of
1 mL/min, purity, 100%; jH NMR (DZO): S 5.13 (d, J= 3.8, 8 H), 4.47 (d, J=
7.8, 8 H),
4.I2-4.16 (m, 24 H}, 4.12-4.05 (m, 8 H), 4.00 (d, J= 0.8, 8 H), 3.99-3.92 (m,
8 H}, 3.86-
3.64 (m, 144 H), 3.39-3.34 (m, 24 H), 3.28 (S, 8 H), 3.22-3.11 (m, 16 H), 2.79
(dd, J= 6.1,
10.6, 16 H), 2.20 {t, J= 7.1; 8 H), 1.82-1.68 (m, 8 H), 1.59-1.20 (m, 40 H);
f3C NMR
(Dz0): b 178.5, 178.4, i 75.1, 174.2, 174.0, 159.3, 104.4, 97.0, 78.9, 76.6,
72.5, 71.4, 71.3,
71.1, 71.0, 70.9, 70.8, 70.3, 69.9, 66.5, 62.6, 55.9, 48.5, 48.I, 41.I, 40.8,
39.1, 38.9, 37.4,
37.0, 36.5, 33.0, 32.9, 32.6, 29.8, 29.6, 27.4, 26.7, 24.3; MS (ESI): m/e (M/3
+ Na+) Calcd.
for (C22aHaooN~sOi26Sa)/3 + Na: 1895.7, obsd.: 1895.3.
Compound 47
Synthesis of Octameric aGal Conjugate
This compound was prepared following the procedure described above for
compound 44. Compound 19 (29 mg, 0.055 mmol) was conjugated with platform 32
(10
mg, 0.0055 mmol). The product was purified on reverse phase HPLC column with a
gradient of acetonitriIe-water (15 to 20%) over 40 minutes to yield 47 (20 mg,
65%) as a
white solid: analytical RF-HPLC: tR I 1.35 min with a gradient of 15 to 25%
ACN in H20
at a flow rate of 1 mL/min. purity, 82%; MS (ESI): m/e (M/3 + Na~) Calcd. for
(C232H416N18~126s8)/3 +Na: 1933.1, obsd.: 1932.1.
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Example 4: In vitro characterization of aGal conjugates
Materials and Methods
Antibodies. Blood was drawn from healthy normal volunteers. Plasma was
separated by centrifugation and allowed to clot. Fibrin was removed and plasma
was used
immediately or stored in aliquots at -70°C. Rhesus monkey serum
(California Regional
Primate Research Center,' Davis, CA) was obtained from blood drawn into
vacutainer tubes
and allowed to clot. After serum was separated by centrifugation, it was
pooled, aliquoted
and stored at -20°C or -70°C. In some experiments, sera was heat-
inactivated at 56°C for
30 minutes to destroy complement hemolytic activity. Antibodies to the aGal
epitope were
affinity purified on an aGal-Sepharose column, which was prepared by coupling
aGal-SH
to maleimide-Sepharose (Pierce) through Michael addition chemistry at 10 mg/mL
resin.
Up to 20 mL pooled NHS (or NMS) normal monkey serum was applied to a 2 mL
volume
of packed aGal-Sepharose. After the flow through was collected, the column was
washed
with 10-20 column volumes or until A28o reached baseline values and eluted
with 0.1 M
triethanolamine, pH 11.5 into tubes containing 1 M Tris, pH 8Ø The column
was
immediately washed with 10-20 volumes of phosphate-buffered saline (PBS).
Fractions
Were assessed for protein concentration by Bradford assay. Peak fractions were
pooled and
dialyzed against PBS. Affinity-purified anti-aGa1 Ig was negatively selected
for IgG by
purification over an MBP column (Pierce, Rockford, IL) which removed IgM anti-
aGal
antibodies. IgM anti-aGal was negatively selected by purification over a
protein G-
Sepharose column (Boehringer Mannheim, Indianapolis, IN) which removed IgG
anti-aGal
antibodies. An elution profile of anti-octal Ig from an aGal affinity column
is shown in
Figure l d.
SDS PAGE and fmmunoblotAnalysis. Antibody-containing fractions and pools
were resolved by 4-12% SDS-PAGE (Novex, San Diego, CA). Proteins were
electrophoretically transferred to PROTRANTM pure nitrocellulose membranes
(Schleicher
and Schuell, Keene, NH) using XCELL IITM Biot Module blot system (Novex).
Membranes were blocked with 2% non-fat dry milk (NFDM) in PBS and probed with
anti-
human IgG or IgM coupled to alkaline phosphatase (3ackson immunoResearch, West
Grove, PA) or anti-monkey igG or IgM coupled to alkaline phosphatase (Advanced
Chem
37
CA 02353620 2001-06-05
WO 00/33887 PCTIUS99/29336
Tech, Louisville, KY). Second step antibody was developed with Western Blue
Stabilized
Substrate for alkaline phosphatase (Promega Corporation, Madison, WI).
ELISA for anti-aGal antibodies. aGal-SH was coupled to maleimide-BSA {bovine
serum albumin) (Pierce) at a 2:1 ratio {wlw) according to manufacturer's
protocol. The
ratio of aGal molecules coupled per BSA molecule was 10-12:1 or 25:1.
Alternatively,
aGal-BSA or aGal-HAS (human serum albumin) was purchased with C3 or C 14
linker
groups (Dextra, Redding, England). aGal-BSA (100 ~.1 at 5 ~Cg/mL in PBS) was
adsorbed
onto 96 well plates for 18 hours at 4°C. Plates were blocked with 2%
NFDM in PBS at
4°C fox at least 48 hours prior to use. Plates were stable for at least
3 months. New lots of
plates were compared with binding efficacy of the original lot using pooled
standard serum.
Pooled standard sera, individual sera or affinity purified anti-aGal Ig were
titered. Serum
(100 ~.L neat - 1/256 diluted in HBSA) or affinity-purifzed anti-aGal IgG (100
~L of serial
two-fold dilutions from 2 mg/mL -1 ~.g/mL in Hank's balanced salt solution
without Ca+2
or Mg+2 (HBSA)) were incubated in aGal-BSA coated wells for 60 minutes at
20°C. After
washing, anti-aGal Ig was developed with predetermined saturating
concentrations
{100 ~.L, usually 1:1000 dilution) of anti-monkey or anti-human IgG or IgM
coupled to
alkaline phosphatase for 60 minutes at 20°C. After washing the wells 5
times with wash
buffer (1% Tween 20 in PBS), plates were developed with 100 pL PPMP
(phenolphthalein
monophosphate) (Sigma) for 5-20 minutes at 20°C. Reactions were stopped
by addition of
100 pL 0.2M Na2HP04 and plates read at Asso (PowerWave 340 Microplate
Spectrophotometer, Bio-Tek, Winooski, Vermont).
aGal Conjugates. aGal {galactose (al, 3, galactose) epitopes were synthesized
at a
multigram scale as described in Example 1 and were coupled to a well-defined
organic
platform as described in Example 2.
Competition ELISA. Serum or affinity purified anti-aGal Ig preparations were
titered by ELISA and the 50% binding concentration was determined. For serum,
the 50%
binding point was reached at a serum dilution of ~1:5 while for affinity-
purified Ig, the
50% dilution binding concentration was 12.5 ~.g/mL. Serum or Ig (50 ~.L) was
incubated
with an equal volume of HBSA containing inhibitor which was serially diluted
in a two-
38
CA 02353620 2001-06-05
WO 00/33857 PCT/US99/29336
fold manner from 4 mg/mL to 10 p,g/mL or buffer alone for 60 minutes at
20°C. Anti-aGal
Ig or serum + inhibitor was then added to aGal-BSA-coated plates and Ig
binding was
assessed as described. Percent inhibition of anti-diGal binding was calculated
as follows:
[(ODsso Ig source + TNH) - ODsso blank / (ODsso Ig source - INH) - ODsso
blank] x 100.
S (INH = inhibitor)
ELISpot assay. Spleens from normal rhesus monkeys were minced and prepared as
single cell suspension using debarred frosted glass slides. Contaminating
erythrocytes
were hypotonically lysed and mononuclear cells (MNC) isolated by Ficoll-
hypaque density
gradient centrifugation. MNC (100 p,L) were added in serial two-fold dilutions
from
104/cells/mL to 5 x 102 cells/mL in quadruplicate to ELISA plates bearing aGai-
BSA or
anti-monkey IgG or IgM. Plates were incubated overnight at 37°C in a
humidifed
atmosphere with 5% C02 and then washed. The footprint of secreted anti-aGal Ig
bound
to aGal-BSA was developed by incubation with goat anti-monkey IgG or IgM
coupled to
biotin (Advanced ChemTech) for 60 minutes at 37°C followed by the
addition of
ExtrAvidin-alkaline phosphatase (Sigma). Alkaline phosphatase substrate B (100
p,L at
1:100 dilution, Bio-Rad, Hercules, CA) was added and incubation continued
overnight at
20°C. Total Ig-producing cells were similarly determined. The
footprints were quantified
using a Microtek ScanMaker III flat-bed scanner and personal computer
utilizing the
Image-Pro imaging software {Media Cybernetics, Univ. Rochester Medical School,
Rochester, NY). The ratio of anti-aGal IgG- or IgM-producing cells/total IgG-
or IgM-
producing cells were calculated.
Cytoxicity assays. The porcine kidney epithelial cell line PK-15 and porcine
aortic
endothelial cells (PAEC) {ATCC, 10801 University Blvd., Manassas, VA 20110-
2209)
were cultured as directed. For the assay, cells were removed with trypsin -
EDTA and
replated subconfluently in 96 well plates ar on coverslips in 24-well plates.
While still
subconfluent (within 2 days of replating), cells were used in cytotoxicity
assays. Neat,
complement-suffcient serum was incubated with inhibitor as described for 60
minutes at
4°C. Serum was then added to wells containing subconfluent cells from
which medium
had been aspirated immediately prior to serum addition. Wells were incubated
with serum
~ inhibitor for 60-90 minutes at 37°C. Wells were rinsed and cell death
was visualized
39
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WO 00/33887 PCT/US99/29336
using a Live/Dead kit (Molecular Probes, Eugene, OR) and quantif ed
microscopically in
high powered fields or by counting 250 cells. For some experiments, cells were
non-
enzymatically removed from flasks with cell dissociation solution (Sigma) and
single cell
suspensions prepared. Assays were performed as for adherent cells except that
cytoxicity
5 was quantified by flow cytometry on a Becton-Dickinson FACScalibur.
Results
Antigenic activity of aGal epitope
The synthetically prepared aGal epitope (Figure 2) was antigenically active,
as
10 demonstrated by its ability (when coupled to Sepharose) to remove >95% of
anti-aGal Ig
from normal rhesus monkey or human serum as measured by FACS.
Activity of tetrameric conjugates. We tested the aGal tetrameric platform
constructs which included the PITG platform {compound (cpd} 38) and BMTG {cpd
37)
platforms as described in Example 2 and found them to be equivalent in their
ability to
I S inhibit Ab from binding to the aGal epitope in the ELISA. Cpd 38 (LJP712)
bound anti-
aGal Ig and inhibited the binding of IgG anti-aGal to the aGal-expressing
porcine kidney
epithelial cell line PK-15 and to BSA-aGal adsorbed onto ELISA wells at ~1 mM.
The
binding of affinity purified IgM anti-aGal was inhibited 1000-fold less well
by cpd 38 than
was the IgG anti-aGal.
Activity of octameric conjugates. Octameric conjugates {as described in
Example 2)
were tested. Octameric conjugate cpd 44 (also referred to as LJP 719) was
tested in vitro
for its ability to inhibit in serum the anti-aGal IgM binding to BSA-aGal.
ELISA analysis
showed that the aGal epitope when presented on a platform as an octamer
inhibited both
IgG and IgM anti-aGal in serum from binding to BSA-aGal or to the aGal-
expressing
porcine kidney epithelial cell line PK-15 and was 5-IO fold more efficient at
inhibiting the
IgM anti-diGal binding to the diGal epitope, as shown in Figure 18. This
supported our
hypothesis that valency of the toleragen was very important in binding the
lower affinity
IgM molecules.
Example 5: In vivo evaluation of conjugates
The in vivo efficacy of the toleragens was tested in a dose-escalation study
in rhesus
monkeys which were treated IV with tetrameric or octameric toleragen or
buffer.
CA 02353620 2001-06-05
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Six male rhesus monkeys (3.5-4 kg) were housed at the California Regional
Primate
Research Center (CRPRC), Davis, CA. All experimental protocols met CRPRC IACUC
(Institutional Animal Care and Use Committee) standards. Monkeys were bled for
baseline
clinical values and anti-aGal antibody levels. Monkeys were bled for baseline
clinical
values. Four monkeys were treated IV daily with 2-20 mglkg of tetravalent
platform LJP
712 (cpd 38). Two monkeys received PBS alone intravenously (IV) as a control.
Monkeys
were bled weekly (S mL) immediately prior to the IV injection in those animals
treated for
60 days with LJP 712 at 2 mglkg. When monkeys received 10 mglkg or 20 mglkg of
tetrameric LJP 712 or 20 mg/kg octameric LJP 920 (cpd 46), treatment was for 5-
7 days
and animals were bled every 2-3 days for 3 mL. Serum samples were analyzed by
ELISA
far anti-aGal IgG and IgM. In one experiment, two monkeys were treated IV
daily fox 10
days with 20 mglkg octameric platform LJP 719. In another experiment, monkeys
were
treated daily IV with octameric LJP 920 at 20 mglkg.
Tetrameric conjugates. To test the in vivo efficacy of LJP 712, the
tetravalent
toleragen at doses high enough to create a molar excess of toleragen to anti-
aGal antibody
in the plasma based on 1% of circulating antibody being specific for aGal was
used to treat
monkeys. Monkeys were treated IV daily with 2 mg/kg of LJP 712 (n=4) or buffer
(PBS)
(n=2) for 60 days as described above. Blood was drawn weekly and serum tested
by
ELISA for IgG and IgM anti-aGal. LJP 712 was well-tolerated and did not
activate either
the classical or alternative complement pathways in vitro, as shown in Figures
20 and 21,
respectively. There was no statistically significant diminution of anti-aGal
Ig responses.
We next sought to determine whether higher doses of LJP 712 were able to
effect
clearance of anti-aGal Ab from the circulation using shorter term IV dosing
modalities.
When the dose was increased to 10 mglkg LJP 712, little diminution of either
IgG or IgM
anti-aGal was observed. By contrast, daily IV treatment with 20 mglkg LJP 712
resulted
in the diminution of the anti-aGal IgG response by up to 24% (p<0.05) by day 8
of
treatment and anti-aGal IgM levels by up to 12% (p=NS), as shown in Figure 19.
Octameric conjugates
We next determined whether treatment of rhesus monkeys for 7 days with the
octameric toleragen LJP 920 led to a diminution in serum levels of anti-aGal
Ab. Two
monkeys were treated IV daily with PBS and two were treated IV daily with LJP
920 (cpd
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WO 00/33887 PCTNS99/29336
46) at 20 mg/kg, a dose which for tetrameric toleragen had shown a diminution
in
circulating IgG anti-aGal but not IgM anti-aGal. Serum samples were prepared
from
blood drawn immediately prior to drug or control administration on day 0
(prebleed) and on
days 3 and 6 (24 hours post-drug administration). Serum was also prepared on
day 8, 24
hours after the Iast dose with no subsequent dosing administered. LJP 920 (cpd
46) was
well-tolerated in the treated animals with no untoward effects as observed by
veterinary
staff. At day 8, IgG anti-aGal levels were decreased by 11%, similar to the
levels seen
with tetramer. Control animals showed little change (Fig. 22A). Similarly,
there was a
diminution of 18% in IgM anti-aGal levels in one monkey and 5% in the
replicate animal.
By contrast, IgM anti-aGal levels in the control animals did not change in one
animal and
increased in the replicate animal, as shown in Figure 22B. That the octamer is
more
efficient than tetramer at clearing IgM anti-aGal is shown in Fig. 23. These
data show that
increased valency results not only in a more efficacious molecule in vitro but
also i~a vivo.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
apparent to
those skilled in the art that certain changes and modifications will be
practiced. Therefore,
the description and examples should not be construed as limiting the scope of
the invention,
which is delineated by the appended claims.
42
