Note: Descriptions are shown in the official language in which they were submitted.
CA 02355348 2001-08-23
METHODS OF TREATING SYSTEMIC LUPUS ERYTHEMATOSUS IN
INDIVIDUALS HAVING SIGNIFICANTLY IMPAIRED RENAL FUNCTION
CROSS-REFEREINCE TO RELATED APPLICATIONS
[0001 ] This application claims the priority benefit of provisional patent
application serial number (attorney docket number 25231-3007800),
filed August 13, 2001, entitled, "'Methods of Treating Systemic Lupus
Erythematosus
in Individuals Having Significantly Impaired Renal Function"), which is hereby
incorporated by reference in its entirety.
'TECHNICAL FIELD
[0002] This invention relal:es to the field of antibody-mediated pathologies
such
as lupus. More particularly, the invention relates to methods of treating
individuals
with systemic lupus erythematosis having significantly impaired renal
function.
BACKGROUND ART
(0003] Systemic lupus erythematosis (SLE) is an autoimmune disease
characterized by the production of antibodies to a number of nuclear antigens,
including double-stranded DNA. (dsDNA). Autoantibodies that react with DNA are
believed to play a role in the pathology of SLE and are closely associated
with lupus
nephritis. See, for example, Morimoto et al. (1982) J. Immunol. 139:1960-1965;
Foster et al. (1993) Lab. Invest. 69:494-507; ter Borg et al. (1990) Arthritis
Rheum.
33:634-643; Bootsma et al. (1995) Lancet 345:1595-1599.
[0004] Synthetic double-stranded oligonucleotides (dsON) have been shown to
cross-react with anti-dsDNA antibodies (U.S. Patent No. 5,276,013). The use of
dsON conjugated with non-immunogenic carriers, also referred to as platforms,
has
been proposed for a therapeutic approach for the treatment of SLE. For
example, a
tetrakis conjugate, LJP 249, composed of four dsON attached to a polyethylene
CA 02355348 2001-08-23
glycol) valency platform was used to demonstrate tolerance in an immunized
mouse
model system (Jones et al. ( 19'94) Bioconjugate Chem. 5:390-399).
[0005] Although overall patient prognosis in SLE has improved, treatment
regimens are not ideal and lupus nephritis continues to be associated with
relatively
poor overall survival as compared to individuals without renal involvement in
lupus
(Seleznick et al. (1991) Semin. ,4rthritis Rheum. 21:73-80). Lupus nephritis
is a
primary cause of morbidity and mortality in SLE. Pistiner et al. (1991 )
Semin.
Arthritis Rehum. 21:55-64. Management of patients with lupus nephritis often
requires immunosuppression in the form of high dose systemic corticosteroids,
azathioprine and/or cyclophos~phamide. However, the utility of these agents
can be
limited by significant drug-induced toxicity, and these drugs lack
specificity.
[0006] LJP 394, a tetravalent conjugate composed of four dsON attached to a
platform, was shown to delay ~~progression of renal disease and extend
survival in the
BXSB experimental murine lupus nephritis model (Plunkett et al. (1995) Lupus
4:599; Coutts et al. (1996) I,upzzs 5:158-159). LJP 394 has also been shown to
lower
anti-dsDNA antibodies in hurr~an patients with SLE (Weisman et al. (1997) J.
Rheumatol. 24:314-318). International Patent Application No. WO 01/41813
discloses methods oi~identifying lupus patients, including those with lupus
nephritis,
with high affinity anti-dsDNA antibodies and treatment of such patients with
LJP
394. Other references discuss LJP394 in the context of a potential therapeutic
agent
for lupus. See Strand (2001 ) Lupus 10:216-221; Wallace (2001 ) Expert Opinion
of
Investigational Drugs 10:111-117; Furie et al. (2001) J. Rheumatol. 28:257-
265.
[0007] Other literature describes methods which may be used in the treatment
of SLE, including methods of reducing levels of circulating antibodies by
inducing B
cell tolerance, including, but not limited to, U.S. Pat. Nos. 5,276,013;
5,391,785;
5,786,512; 5,726,329; 5,552,391; 5,268,454; 5,606,047; 5,633,395; 5,162,515;
U.S.
Ser. No. 081118,055 (U.S. Pat. No. 6,060,056); U.S. Ser. Nos. 60/088,656 and
2
CA 02355348 2001-08-23
60/103,088 (U.S. Ser. No. 09/328,199 and PCT App. No. PCT/I1S99/13194). See
also U.S. Pat. No. 6,022,544.
[0008] All references cited herein, including patents, patent applications and
publications, are hereby incorporated by reference in their entirety.
DISCLOSURE OF THE INVENTION
[0009] The invention provides methods for treatment of systemic lupus
erythematosis, particularly symptoms related to renal dysfunction (e.g., lupus
nephritis or LN) in individuals with significant renal impairment, i.e.,
significantly
impaired renal function. Accordingly, in one aspect, the invention provides
methods
of treating LN in an individual with SLE, said method comprising administering
to
the individual a conjugate comprising: (a) a non-immunogenic valency platform
molecule and (b) two or more double stranded DNA epitopes, preferably
polynucleotides, wherein said individual has significantly impaired renal
function. In
another aspect, the invention provides methods of reducing incidence of renal
flares
in an individual with SLE, said method comprising administering to the
individual a
conjugate comprising: (a) a non-immunogenic valency platform molecule and (b)
two
or more double stranded DNA epitopes, preferably polynucleotides, wherein said
individual has significantly impaired renal function. In another aspect, the
invention
provides methods of treating LN in an individual, comprising selecting a SLE
patient
having significantly impaired renal function and administering to the
individual a
conjugate comprising (a) a non-immunogenic valency platform molecule and (b)
two
or more double stranded DNA epitopes, preferably polynucleotides. Preferably,
at
least one of said epitopes is bound at a high initial affinity by antibodies
from the
patient (that is, as described herein, at least one of the epitopes binds with
high
affinity to anti-double stranded DNA antibody in the individual). Preferably,
all
epitopes on the conjugate bindl with high affinity to anti-double stranded DNA
antibody in the individual. Individuals are selected for treatment in
accordance with
the instant methods on the basis of a diagnosis of systemic lupus
erythematosis (SLE)
3
CA 02355348 2001-08-23
and at least one clinical indica~.ion of significantly impaired renal
function. In certain
preferred embodiments, the conjugate comprises (a) a non-immunogenic valency
platform molecule and (b) two or more polynucleotides comprising, consisting
essentially of or consisting of l:he double stranded DNA sequence
5'-GTGTGTGTGTGTGTG'T<~rGT-3'(SEQ ID NO:1). In other embodiments, the
invention provides methods of treating SLE in an individual comprising
administering a conjugate described herein, wherein assessment of renal
function in
the individual, particularly identifying individuals based on significantly
impaired
renal function as a basis for selecting individuals to receive such treatment.
[0010] In another aspect., the invention provides methods of identifying an
individual who may be suitable for treatment for SLE, said treatment
comprising
administration of a conjugate described herein, said method comprising
measuring
the renal function of said individual, wherein an individual is identified by
having at
least one clinical indication of significantly impaired renal function. In
some
embodiments, the conjugate comprises (a) a non-immunogenic valency platform
molecule and (b) two or more polynucleotides which specifically bind to an
antibody
from the individual which specifically binds to double stranded DNA, said
polynucleotide comprising, consisting essentially of, or consisting of the
dsDNA
sequence S'-GTGTGTGTGT(i'CGTGTGTGT-3'(SEQ ID NO:1).
[0011] Preferably, an individual is also selected (and in some embodiments,
treated) based on affinity of anti-double stranded DNA antibodies for an
epitope(s) of
the conjugate. In accordance with the invention, antibody affinity is measured
as the
apparent equilibrium dissociation constant (K~~') (or its functional
equivalent) for a
dsDNA epitope. In certain err~bodiments the individual is selected for
treatment in
accordance with the instant invention if the K,.~' (or its functional
equivalent) is less
than about 1.0 mg IgG per mL. Other, lower Kp' values are described herein
which
could apply to any of the dsDNA epitopes contemplated for use in treatment, as
are
percentile ranking with respect to a given patient population as described
herein.
4
CA 02355348 2001-08-23
[0012] The invention also provides kits for use in the methods of the
invention.
Such kits comprise a conjugate comprising (a) a non-immunogenic valency
platform
molecule and (b) two or more polynucleotides and instructions for use of the
conjugate comprising a description of selecting an SLE patient having
significantly
impaired renal function, and administering said conjugate to the patient. The
instructions may further relate to measurement of affinity of anti-dsDNA
antibodies
from the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figures lA-C are graphs depicting competitive inhibition by LJP 394 of
antibodies from groups of SL>=: patients' sera binding to ~ZSI-labeled dsDNA.
MODES FOR CARRYING OUT THE INVENTION
[0014] We have discovered that administration of a conjugate comprising a
non-immunogenic platform molecule and four double stranded DNA epitopes,
namely, LJP 394 (having four double stranded DNA molecules with the sequence
5'-GTGTGTGTGTGTGTGT(~'TGT-3'(SEQ 1D NO:1 )) to systemic lupus
erythematosis (SLE) patients having significant impaired renal function
effected
significant improvement in terms of fewer renal flares as well as longer time
to flare.
This result was surprising and unexpected. The greatest benefit was observed
in
those patients having high affinity antibodies to the dsDNA epitope(s) of LJP
394.
The instant invention is based upon analysis of data from a clinical trial of
LJP 394
referred to as the 90-OS study, some accounts of which have been published as
Linnik
et al. (2000) Arth. Rheumat. 43(9 supplement):5241 (abstracts 1045 and 1046)
and
Alarcon-Segovia et al. (2000) Arth. Rheumat. 43(9 supplement):5272 (abstract
1231)
and are described herein. Patients with significantly impaired renal function
in the
placebo group appeared more prone to renal flare as 60% of these patients had
a renal
flare, versus 20% of the intent to treat (ITT) placebo population (Example 1
).
CA 02355348 2001-08-23
LJP394 treatment appeared to reduce renal flares, especially in the high
affinity
patients where there was a complete absence of renal flares. These results are
significant, given the intractability of treatment of significant renal
impairment. With
the benefit of applicants' disccmery, such patients are included in treatment,
or at least
selected as suitable as receiving such treatment based on the condition of
having
significant renal impairment. In some embodiments, suitable individuals are
also
selected based on having high affinity antibodies with respect to an
epitope(s) of the
conjugate.
[0015] Accordingly, the invention provides methods of alleviating one or more
symptoms of lupus nephritis (in some embodiments, reducing incidence of renal
flares) in an individual, comprising administering to said individual a
conjugate
comprising a non-immunogenic valency platform molecule and two or more
polynucleotides, at least one of said polynucleotides comprising the dsDNA
epitope,
wherein the individual has significantly impaired renal function. In some
embodiments, the methods comprising selecting an individual suffering from SLE
who has significantly impaired renal function and administering the
conjugates) as
described. Preferably, at least one of said epitopes in the conjugate is bound
at a high
initial affinity by antibodies from the patient. Preferably, all epitopes on
the
conjugate bind with high affinity to anti-double stranded DNA antibody in the
individual. In accordance with the instant invention, SLE patients are
selected and/or
treated on the basis of the presence of significantly impaired renal function,
and
preferably the presence of high affinity antibodies with respect to the double
stranded
DNA epitope(s) of the conjugate to be used. The invention also provides
methods of
selecting an individual suitable for the conjugate-based treatments described
herein
based on assessment of renal function and selecting a patient suitable for
receiving
the treatment based on identif~~ing those patients having significant renal
impairment.
6
CA 02355348 2001-08-23
General Techniques
[0016] The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of molecular biology (including recombinant
techniques), 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)
Cold Spring Harbor Press; Oli,~-onucleotide 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); Current Protocols in
Immunology (J.E. Coligan et al., eds., 1991) and Short Protocols in Molecular
Biology (Whey and Sons, 199!0. Other useful references include Harnson's
Principles of Internal Medicine (McGraw Hill; J. Isseleacher et al., eds.) and
Dubois'
Lupus Erythematosus (5th ed.; D.J. Wallace and B.H. Hahn, eds.; Willaims &
Wilkins, 1997).
Definitions
[0017] An individual having "significantly impaired renal function" or
"significant renal impairment'" is an individual exhibiting one or more
clinical signs
of significant renal dysfunction, as described herein. Clinical signs of renal
dysfunction include anuria, oliguria, elevated blood urea nitrogen (BLIN),
elevated
serum creatinine, clinically significant proteinuria, hematuria, reduced
creatinine
clearance, and other clinical indications of renal dysfunction known in the
art. As
described herein, generally, an individual displays significant renal
impairment if any
one of more of these clinical indicia are at least above the upper limit of
"normal"
range, as defined in the clinical arts. In some embodiments, significant renal
impairment is indicated if the value exceeds the upper limit of normal by
about any of
7
CA 02355348 2001-08-23
the following percentages: 10., 20, 25, 30, 50, 60, 75, I 00, 125, 1 S0, 200,
250, 275,
300, 350, 400, 450, 500. As is. known in the art, with respect to at least one
indicia of
kidney function, such as creatinine, an individual can have at least about 2,
3, 5, or 10
fold or greater values compared with the upper limit of normal. Generally, an
individual is determined to have, or in fact has, significant renal impairment
at the
onset (before the individual receives the first administration), or shortly
after the
onset (within about 4 weeks, preferably within about 2 weeks, preferably
within
about 1 week, preferably within about 5 days, preferably within about 2 days,
preferably within about 1 day) upon receiving the first adminsitration), of
the
therapeutic methods described herein.
[0018] When significantly impaired renal function "is used as a basis" for
administration of the treatment methods described herein, or selection for the
treatment methods described herein, renal function is measured before and/or
during
treatment, and the values obtained are used by a clinician in assessing
probable or
likely suitability of an individual to receive treatment(s). As would be well
understood by one in the art, measurement of antibody affinity in a clinical
setting is
a clear indication that this parameter was used as a basis for initiating,
continuing,
adjusting and/or ceasing administration of the treatments described herein.
[0019] "Affinity" of an antibody from an individual for an epitope to be used,
or used, in treatments) described herein is a term well understood in the art
and
means the extent, or strength, of binding of antibody to epitope. Affinity may
be
measured and/or expressed in a number of ways known in the art, including, but
not
limited to, equilibrium dissociation constant (K~ or K~), apparent equilibrium
dissociation constant (Ko' or k:~j'), and ICso (amount needed to effect SO%
inhibition
in a competition assay; used interchangeably herein with "ISO"). It is
understood that,
for purposes of this invention, an affinity is an average affinity for a given
population
of antibodies which bind to an epitope. Values of KD' reported herein in terms
of mg
IgG per mL or mg/mL indicate; mg Ig per mL of serum, although plasma can be
used.
8
CA 02355348 2001-08-23
[0020) When antibody affinity "is used as a basis" for administration of the
treatment methods described herein, or selection for the treatment methods
described
herein, antibody affinity is measured before and/or during treatment, and the
values
obtained are used by a clinician in assessing any of the following: (a)
probable or
likely suitability of an individual to initially receive treatment(s); (b)
probable or
likely unsuitability of an individual to initially receive treatment(s); (c)
responsiveness to treatment; (d} probable or likely suitability of an
individual to
continue to receive treatment(s); (e) probable or likely unsuitability of an
individual
to continue to receive treatment(s); (f) adjusting dosage; (g) predicting
likelihood of
clinical benefits. As would be well understood by one in the art, measurement
of
antibody affinity in a clinical setting is a clear indication that this
parameter was used
as a basis for initiating, continuing, adjusting and/or ceasing administration
of the
treatments described herein.
[0021] An antibody affinity measured "before or upon initiation of treatment"
or an "initial affinity" is antibody affinity measured in an individual before
the
individual receives the first administration of a treatment modality described
herein
and/or within at least about 4 weeks, preferably within at least about 2
weeks,
preferably within at least about 1 week, preferably within at least about 5
days,
preferably within at least about 3 days, preferably within at least about 2
days,
preferably within at least about 1 day upon receiving the first administration
of a
treatment modality described herein.
[0022] A "population" is a group of individuals with lupus. For a given
population (which may vary in terms of number of members, depending on the
context) antibody affinities vary over a range (i.e., maximum and minimum
affinities).
[0023] As used herein, "t:reatment" is an approach for obtaining beneficial or
desired results including and preferably clinical results. For purposes of
this
invention, beneficial or desired clinical results include, but are not limited
to, one or
9
CA 02355348 2001-08-23
more of the following: alleviation of one or more symptoms, diminishment of
extent
of disease, stabilized (i.e., not worsening) state of disease, preventing
spread of
disease, preventing occurrence or recurrence of disease, decreasing, delaying
or
preventing the occurrence of renal "flares," amelioration of the disease
state,
remission (whether partial or total), reduction of incidence of disease and/or
symptoms, stabilizing (i.e., not worsening) of renal function or improvement
of renal
function. During lupus nephritis, which is a chronic inflammatory kidney
disease,
"flares" may occur. "Flares" refer to an increase in activity, generally
inflammatory
activity. If the activity is in the kidneys, then the flare is referred to as
a "renal flare".
"Renal flares" can be identified by evaluating factors including, but not
limited to,
proteinuria levels, hematuria levels, and serum creatinine levels. The
"treatment" of
lupus nephritis may be administered when no symptoms of lupus nephritis are
present, and such treatment (as, the definition of "treatment" indicates)
reduces the
incidence of flares. Also encompassed by "treatment" is a reduction of
pathological
consequences of any aspect of lupus nephritis.
[0024) "SLE flares" are used herein to refer to flares (i.e. acute clinical
events)
which occur in patients with SLE. The SLE flares may be in various major
organs,
including but not limited to, kidney, brain, lung, heart, liver, and skin. SLE
flares
include renal flares.
[0025] "Reducing incidence" of renal flares in an individual with SLE means
any of reducing severity (which can include reducing need and/or amount of
other
drugs generally used for this conditions, including, for example, high dose
corticosteroid and/or cyclophosphamide), duration, and/or frequency
(including, for
example, delaying or increasing time to renal flare as compared to not
receiving
treatment) of renal flares) in an individual. As is understood by those
skilled in the
art, individuals may vary in terms of their response to treatment, and, as
such, for
example, a "method of reducing incidence of renal flares in an individual"
reflects
administering the conjugates) described herein based on a reasonable
expectation
CA 02355348 2001-08-23
that such administration may likely cause such a reduction in incidence in
that
particular individual.
[0026] "High dose cortic:osteroid and/or cyclophosphamide" or "HDCC" as
used herein refers to intervention with an increased dosage of corticosteroid
alone or
with cyclophosphamide. High dose generally refers to corticosteroids. Such
intervention generally occurs upon a flare, or acute episode. Generally, for
example,
the increased dosage is at least. a I S mg/day and can be greater than 20
mg/day.
HDCC may be administered using standard clinical protocols. A clinician may
monitor a patient and determine when HDCC treatment is needed by evaluating
factors including, but not limited to, proteinuria levels, hematuria levels,
and serum
creatinine levels. In general, patients who experience renal flares are given
HDCC
treatment, although this treatment is used for other aspects of lupus.
[0027] An "equivalent" or "functional equivalent" of KD' or a numerical value
for Kp' is a parameter or value; for a parameter which also reflects affinity.
For
example, an equivalent of KD' is ICSO. As another example, an equivalent value
of
KD' of 0.5 could be an ICSO of 200, if they reflect the same, or about the
same,
affinity. Determining such equivalents is well within the skill of the art and
such
equivalents and their determination are encompassed by this invention.
Generally,
reference to K~' includes reference to functional equivalents of KD'.
[0028] As used herein, the singular form "a", "an", and "the" includes plural
references unless indicated otherwise. For example, "an" antibody includes one
or
more antibodies.
[0029] 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, whi<:h is a moiety or molecule that contains an
epitope, and,
as such, also specifically binds to antibody.
[0030] A "double-stranded DNA epitope" or "dsDNA epitope" is any chemical
moiety which exhibits specific: binding to an anti-double-stranded DNA
antibody and
11
CA 02355348 2001-08-23
as such includes molecules which comprise such epitope(s). Further discussion
of
double-stranded DNA epitopes suitable for the conjugates of the invention are
described below. The term "e~pitope" also includes mimetics of double-stranded
DNA itself, which are described below.
[0031) An epitope 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 and/or 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, and/or with greater duration than it binds to
other
substances.
[0032) An "anti-double-stranded DNA antibody" or "anti-dsDNA antibody" or
"double-stranded DNA antibody" or "antibodies to dsDNA", used interchangeably
herein, is any antibody which specifically binds to double-stranded DNA
(dsDNA).
Any antibody includes an antibody of any class, such as IgG, IgA, or IgM, and
the
antibody need not be of any particular class. As clearly indicated in the
definition of
"antibody" provided herein, a "anti-double-stranded DNA antibody" encompasses
any fragments) that exhibits this requisite functional (i.e., specific binding
to
dsDNA) property, such as fragments that contain the variable region, such as
Fab
fragments. As discussed below , it is understood that specific binding to any
anti-
double-stranded DNA antibody (or functional fragment) is sufficient.
[0033) The term "circulating anti-double-stranded DNA antibody", as used
herein, intends an anti-double-stranded DNA antibody which is not bound to a
double-stranded DNA epitope on and/or in a biological sample, i.e., free
antibody.
[0034) An "antibody" (interchangeably used in plural form) is an
immunoglobulin molecule capable of specific binding to a target, such as a
carbohydrate, polynucleotide or polypeptide, through at least one antigen
recognition
12
CA 02355348 2001-08-23
site, located in the variable region of the immunoglobulin molecule. As used
herein,
the term encompasses not only intact antibodies, but also fragments thereof
(such as
Fab, Fab', F(ab')2, 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.
[0035] The terms "polynucleotide" and "nucleic acid", used interchangeably
herein, refer to a polymeric foam of nucleotides of any length, either
ribonucleotides
or deoxyribonucleotides. These terms include a single-, double- or triple-
stranded
DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising
purine and pyrimidine bases, csr other natural, chemically, biochemically
modified,
non-natural or derivatized nctcleotide bases. For purposes of this invention,
unless
otherwise indicated, sequences presented herein denote double stranded
sequences.
For example, the polynucleotide comprising, consisting essentially of, or
consisting
of the double stranded sequence 5'-GTGTGTGTGTGTGTGTGTGT-3'(SEQ ID
NO:1 ) includes the complementary polynucleotide sequence, particularly the
sequence 3'-CACACACACACACACACACA-5'(SEQ ID N0:2). It is understood
that the double stranded polynucleotide sequences described herein also
include the
modifications described herein. The backbone of the polynucleotide can
comprise
sugars and phosphate groups (.as may typically be found in RNA or DNA), or
modified or substituted sugar or phosphate groups. Alternatively, the backbone
of the
polynucleotide can comprise a polymer of synthetic subunits such as
phosphoramidates and thus can be a oligodeoxynucleoside phosphoramidate (P-
NH2)
or a mixed phosphoramidate-phosphodiester oligomer. A phosphorothioate linkage
can be used in place of a phosphodiester linkage. In addition, a double-
stranded
polynucleotide can be obtained from the single stranded polynucleotide product
of
chemical synthesis either by synthesizing the complementary strand and
annealing
13
CA 02355348 2001-08-23
the strands under appropriate conditions, or by synthesizing the complementary
strand de novo using a DNA polymerase with an appropriate primer.
[0036] The following are, non-limiting examples of polynucleotides: a gene or
gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched pol~,mucleotides, plasmids, vectors, isolated DNA of
any
sequence, isolated RNA of any sequence, nucleic acid probes, and primers. For
purposes of this invention, a polynucleotide is generally an isolated
polynucleotide of
less than about 1 kb, preferably less than about 500 base pairs (bp),
preferably less
than about 250 bp, preferably less than about 100 bp, preferably less than
about 50
bp. However, it is understood that a polynucleotide of any size or
configuration
could be used as long as it exhibits the requisite binding to anti dsDNA
antibody from
an individual. It is further understood that a different polynucleotide (for
example, in
terms of size and/or sequence) other than the one that is to be, was, or will
be used in
treatment, as long as both polynucleotides exhibit equivalent (or convertible)
binding
affinities to anti-dsDNA antibodies from an individual. In other words, non-
identical
polynucleotides may be employed with respect to affinity determination and
treatment.
[0037] Preferably, the polynucleotide is DNA. As used herein, "DNA"
includes not only bases A, T, (.', and G, but also includes any of their
analogs or
modified forms of these bases, such as methylated nucleotides, internucleotide
modifications such as uncharged linkages and thioates, use of sugar analogs,
and
modified and/or alternative backbone structures, such as polyamides.
[0038] "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 occurnng 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 moiel:ies, i.e., ones which do not occur in nature,
such as
recombinant polynucleotide sequences and non-naturally occurnng carbohydrates.
14
CA 02355348 2001-08-23
[0039] As used herein, the term "immunogen" means a chemical entity that
elicits a humoral immune response when injected into an animal. Immunogens
have
both B cell epitopes and T cell epitopes.
[0040) As used herein, the term "analog" (also termed an "mimetic") of an
immunogen means a biological or chemical compound which specifically binds to
an
antibody to which the immunogen specifically binds. As such a "double-stranded
DNA epitope" includes mimetics of naturally-occurring double-stranded DNA. An
"analog" or "mimetic" shares an epitope, or binding specificity, with double-
stranded
DNA. An analog may be any chemical substance which exhibits the requisite
binding properties, and thus may be, for 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
glycolipid. The term "analog" encompasses the term "mimotope", which is a term
well known in the ark.
[0041] 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.
[0042] "Inducing tolerance" or "inducing immunotolerance" means a reduction
and/or stabilization of the extent of an immune response to an immunogen, and,
as
such, means immune unresponsiveness (or at least a reduction in the extent of
an
immune response). An "immune response" may be humoral and/or cellular, and may
be measured using standard assays known in the art. For purposes of this
invention,
the immune response is generally reflected by the presence of, and/or the
levels of,
anti-double-stranded DNA antibodies. Quantitatively the reduction (as measured
by
reduction in antibody production and/or levels) is at least about 15%,
preferably at
least about 25%, more preferably at least about 50%, more preferably at least
about
75%, more preferably at least about 90%, even more preferably at least about
95%,
CA 02355348 2001-08-23
and most preferably 100%. It is understood that the tolerance is antigen-
specific, and
applies for purposes of the invention to those individuals having anti-double-
stranded
DNA antibodies. "Inducing tolerance" also includes slowing and/or delaying the
rate
of increase of antibody level.
[0043] As used herein, the term "B cell anergy" intends unresponsiveness of
those B cells requiring T cell help to produce and secrete antibody and
includes,
without limitation, clonal deletion of immature and/or mature B cells and/or
the
inability of B cells to produce .antibody. "Unresponsiveness" means a
therapeutically
effective reduction in the humoral response to an immunogen. Quantitatively
the
reduction (as measured by reduction in antibody production) is at least 50%,
preferably at least 75% and most preferably 100%.
[0044] An "effective amount" (when used in the lupus context, or in the
antibody-mediated pathology context) is an amount sufficient to effect
beneficial or
desired results including clinical results. An effective amount can be
administered in
one or more administrations. hor purposes of this invention, an effective
amount of
conjugate described herein (or a composition comprising a conjugate) an amount
sufficient to reduce circulating levels of anti-double-stranded DNA
antibodies,
preferably by inducing tolerance, particularly with respect to anti-double-
stranded
DNA antibodies. In terms of treatment, an "effective amount" of conjugate
described
herein (or a composition comprising a conjugate) is an amount sufficient to
palliate,
ameliorate, stabilize, reverse, slow or delay progression of or prevent
systemic lupus
erythematosis (SLE), including; the progressive inflammatory degeneration of
the
kidneys that results from SLE (i.e., lupus nephritis).
[0045] A "stable complex"' formed between any two or more components in a
biochemical reaction, refers to a duplex or complex that is sufficiently long-
lasting to
persist between formation of the duplex or complex and subsequent detection,
including any optional washing; steps or other manipulation that may take
place in the
interim.
16
CA 02355348 2001-08-23
[0046] An "isolated" or "purified" polypeptide or polynucleotide is one that
is
substantially free of the materials with which it is associated in nature. By
substantially free is meant at least 50%, preferably at least 70%, more
preferably at
least 80%, even more preferably at least 90% free of the materials with which
it is
associated in nature.
[0047] 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 c;pitopes. A "valency" of a conjugate or valency
platform molecule indicates the number of attachment sites per molecule for a
double-stranded DNA epitope(s). Alternatively, the valency of a conjugate is
the
ratio (whether absolute or average) of double-stranded DNA epitope to valency
platform molecule.
[0048] "Nonimmunogeniic", when used to describe the valency platform
molecule, means that the valency platform molecule fails to elicit an immune
response (i.e., T cell and/or B cell response), andlor 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.
[0049] An epitope which is "conjugated" to a valency platform molecule is one
that is attached to the valency platform molecule by covalent and/or non-
covalent
interactions.
[0050] An "epitope-presenting valency platform molecule" is a valency
platform molecule which contains attached, or bound, epitopes, at least some
of
which (at least two of which) a.re able to bind an antibody of interest.
[0051) A "biological sarr~ple" encompasses a variety of sample types obtained
from an individual and can be used in a diagnostic or monitoring assay. The
definition encompasses blood and other liquid samples of biological origin,
solid
tissue samples such as a biopsy specimen or tissue cultures or cells derived
therefrom,
17
CA 02355348 2001-08-23
and the progeny thereof. The definition also includes samples that have been
manipulated in any way after their procurement, such as by treatment with
reagents,
solubilization, or enrichment for certain components, such as proteins or
polynucleotides. The term "biological sample" encompasses a clinical sample,
and
also includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological
fluid, and tissue samples.
(0052] "In conjunction with" refers to administration of one treatment
modality
in addition to another treatment modality, such as administration of a
conjugate
described herein in addition to administration of corticosteroid
cyclophosphamide
immunosuppressants (or other immunosuppressant therapy) to the same
individual.
As such, "in conjunction with'" refers to administration of one treatment
modality
before, during or after delivery of the other treatment modality to the
individual.
[0053] "Receiving treatment" includes initial treatment and/or continuing
treatment.
[0054] "Comprising" means including.
Methods of treatment
[0055] The invention provides methods for treatment of systemic lupus
erythematosis (SLE), particularly symptoms related to renal dysfunction (e.g.,
lupus
nephritis). Accordingly, in one aspect, the invention provides methods of
treating
lupus nephritis (LN) in an individual, comprising administering to the
individual an
epitope presenting conjugate comprising (a) a non-immunogenic valency platform
molecule and (b) two or more double stranded DNA epitopes, preferably
polynucleotides, wherein the individual has significantly impaired renal
function (as
indicated by measuring one or more clinical indicia of renal function as known
in the
art and/or described herein). Preferably, at least one of said epitopes is
bound at a
high initial affinity by at least one anti dsDNA antibody from the patient
(that is, as
described herein, at least one population or type of antibodies from the
individual
18
CA 02355348 2001-08-23
binds at high affinity to an epit.ope(s) of the conjugate). In some
embodiments, the
methods comprising selecting an SLE patient having significantly impaired
renal
function. Individuals having SI,E, or who are suspected of having SLE, are
selected
for treatment in accordance with the instant methods on the basis of the
presence of at
least one clinical indication of significantly impaired renal function.
Preferably,
selection is also based upon the presence of antibodies which bind to a double-
stranded DNA (dsDNA) epitope at high affinity in the individual. Accordingly,
in
some embodiments of the invention, the methods include an additional step of
assessing the affinity of the individual's antibodies for a dsDNA epitope
present in the
conjugate before or upon initiation of treatment, as described in, for
example,
PCT/LJS00/42307 (W001/41813).
[0056] In certain embodiments, the methods of the invention include an
reassessment step, in which thc: affinity of the individual's antibodies for
at least one
of the dsDNA epitope(s) on the conjugate is remeasured. This remeasurement may
serve as the basis for continuing, or discontinuing, the treatment. In such
embodiments including a reassessment step, treatment is generally, but not
necessarily, continued if the affinity of the individual's antibodies has
decreased, or
generally, but not necessarily, discontinued if the affinity of the
individual's
antibodies has failed to decrease.
[0057) In some embodiments, a conjugate is administered in an amount
sufficient to reduce incidence of, or likelihood of, renal flares.
Accordingly, the
invention provides methods of treating LN in an individual, comprising
administering
to the individual an epitope presenting conjugate comprising (a) a non-
immunogenic
valency platform molecule andl (b) two or more double stranded DNA epitopes,
preferably polynucleotides, wherein the individual has significantly impaired
renal
function. In some embodiments, the methods comprise selecting an individual
with
LN having significantly impaired renal function. Preferably, at least one of
said
epitopes is bound at a high initial affinity by at least one antibody from the
patient. In
19
CA 02355348 2001-08-23
some embodiments, the apparent equilibrium dissociation constant (Ko') for the
polynucleotide in the conjugate with respect to the antibody from the
individual
before or upon initiation of treatment is less than about 1.0 mg IgG per mL.
Preferably, the KD' value is used as a basis for selecting the individual to
receive the
treatment. In other embodiments, the K~' is less than about any of the
following:
0.8; 0.7; 0.6; 0.5; 0.4; 0.3; 0.2; 0.1; 0.09; 0.08; 0.07; 0.06 0.05; 0.025.
[0058) In some embodiments, a conjugate as described herein is administered in
an amount sufficient to reduce the dosage of corticosteroid and/or
cyclophosphamide
immunosuppressive therapy that would otherwise be administered in the absence
of
administering the conjugate. This is significant, as this type of
immunotherapy is
toxic. Accordingly, the invention provides methods of treating lupus nephritis
comprising administering to the individual a conjugate comprising (a) a non-
immunogenic valency platform molecule and (b) two or more double stranded DNA
epitopes, preferably polynucle~otides, wherein the individual has
significantly
impaired renal function. In some embodiments, the methods comprising selecting
an
individual with LN having siyificantly impaired renal function. Preferably, at
least
one of said epitopes is bound apt a high initial affinity by at least one
antibody from
the patient.
[0059) The invention also provides methods of treating SLE, preferably lupus
nephritis, comprising administering a conjugate described herein in
conjunction with
corticosteroid and/or cyclophosphamide, wherein the individual has (or is
suspected
of having) SLE, and wherein tlhe individual has significantly impaired renal
function.
In some embodiments, the methods comprise selecting an individual having
significantly impaired renal function. The conjugate is generally administered
in an
amount effective to reduce antibody affinity for the epitope in the conjugate,
although
any amount that effects a desired result (such as reduction of incidence of
renal flares,
or any other description in the definition of "treatment") in conjunction with
corticosteroid and/or cyclophosphamide is acceptable. Preferably, the
conjugate is
CA 02355348 2001-08-23
LJP 394, which is described hE;rein. Methods of administering corticosteroid
and/or
cyclophosphamide are known in the art. Reducing the dosage of corticosteroid
and/or cyclophosphamide thers~py (which reduces the dependence on
administration
of these drugs and in effect delays administration of these drugs) can be
assessed by,
for example, comparing to known and/or established averages of dosage (in
terms of
amount and/or intervals) generally given over time which are known in the art.
[0060] In certain embodiments, the affinity of the antibodies from the patient
is
quantified as the apparent equilibrium dissociation constant (KD') for the
epitope(s)
in the conjugate. In such embodiments, the individual's antibodies are
considered to
have a high affinity if the K«' with respect to the dsDNA epitope(s) before or
upon
initiation of treatment is less than about 1.0 mg IgG per mL. In other
embodiments,
the Ko' is less than about any of the following: 0.8; 0.7; 0.6; 0.5; 0.4; 0.3;
0.2; 0.1;
0.09; 0.08; 0.07; 0.06; 0.05; 0.X025. It should be noted that measurement of
affinity,
either represented by measuring KD' or by some other method, either before or
during
treatment is strong, if not conclusive, indication that this parameter was a
basis for
selecting the individual to receive treatment.
Selection of individuals for treatment
[0061] The instant method involves treating and/or selecting an individual who
has, or is suspected of having, systemic lupus erythematosus (SLE) who also
has
significantly impaired renal function. The symptoms of SLE are well known in
the
art, and it is well within the knowledge of those of ordinary skill in the art
to identify
individuals having, or who are suspected of having, SLE. Within the group of
individuals having, or being suspected of having, SLE, selecting those having
significantly impaired renal function may be on the basis of any clinical
indication of
significant renal impairment known in the art, including, but not limited to,
anuria,
oliguria, elevated serum creatinine levels, elevated BUN, proteinuria,
hematuria
(occult or gross), reduced creatinine clearance, impaired glomeral filtration,
and the
like. As will be apparent to one of skill in the art, a diagnosis of renal
dysfunction,
21
CA 02355348 2001-08-23
such as a diagnosis of subacute; glomerulonephritis, nephrotic syndrome, or
mild to
severe nephritis, will also identify a significant impairment of renal
function and thus
serve as a basis for treating that individual and/or selection of the
individual for
treatment in accordance with the instant methods.
[0062] As will be apparent, the quantitative level of a particular clinical
parameter that indicates a significant impairment of renal function will
depend on the
particular clinical parameter. Proteinuria is easily detected at a'screening'
level using
colorimetric "dipstick" testing of urine, and can be followed up by more
sensitive and
accurate laboratory testing. Preferably, when the presence of a significant
impairment of renal function is identified by proteinuria, an individual is
considered
to have significantly impaired :renal function when at least about 500 mg of
protein is
excreted in the urine per day, nnore preferably at least about (i.e., greater
than or equal
to about) 1.5, 2, 2.5, 3, 3.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 grams of protein
per day.
When serum creatinine is used as the indicator of significant impairment of
renal
function, an individual will be considered to have significantly impaired
renal
function when serum creatinine levels are at least about (i.e., greater than
or equal to
about) 1.5, 2, 2.5 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 milligrams
per deciliter
(mg/dL).
[0063] As will be understood by one of skill in the art, administration of a
conjugate comprising a non-irr~munogenic valency platform molecule and two or
more double stranded DNA epitopes, preferably polynucleotides, and preferably
wherein at least one of said epitopes is bound at a high initial affinity by
antibodies
from the patient entails assessing antibody affinity from an individual in
those
embodiments in which selection is based on antibody affinity, wherein said
individual
has, or is suspected of having, SLE. For purposes of this invention; (a) the
affinity in
question is with respect to an individual's antibodies, that is, antibodies
obtained from
that individual; (b) the antibody for which affinity is measured is an
antibody
associated with, and/or implicated in SLE; and (c) the binding of interest is
binding of
22
CA 02355348 2001-08-23
antibody to an epitope which binds to the antibody(ies), with the epitope to
be used in
the proposed treatment, as described herein (i.e., a dsDNA epitope).
[0064] For all embodiments of the invention which use or are directed to KD',
whether screening, treatment, monitoring, or any other methods directed to
assessing
affinity, it is understood that other, equivalent values can be measured and
used, and
are encompassed by this invention. For example, as discussed below, there are
a
number of methods known in the art which can measure (and express) affinity of
antibodies from an individual for an epitope to be used for treatment (in the
context
of this invention, a double stranded DNA epitope). Kp' is one of these
parameters,
and equivalent parameters can be measured and used in this invention. Further,
with
respect to KD' cut-off values reported herein, the basis of this finding was
administering about 100 mg of LJP 394 conjugate about once a week.
[0065] Measurement of affinity, either represented by measuring Ko' or by
some other method, either before or during treatment is strong, if not
conclusive,
indication that this parameter vvas a basis for selecting the individual to
receive
(and/or continue to receive) treatment. Accordingly, with respect to all
treatment
methods described herein, and as the definition for "is used as a basis"
states, other
embodiments include ( 1 ) assessing, or measuring, the affinity as described
herein
(and preferably selecting an individual suitable for receiving (including
continuing to
receive) treatment); and (2) administering the treatments) as described
herein. As
described herein, in some embodiments, more than one measurement is made, when
change (if any) in affinity is assessed.
[0066] Antibody affinity may be measured using methods known in the art
which assess degree of binding; of DNA epitope to antibody. Generally, these
methods comprise competition assays and non-competition assays. With respect
to
polynucleotide epitopes (which will be used in a conjugate to be
administered),
affinity may be measured using polynucleotide alone or polynucleotide-
containing
conjugates (as long as the polynucleotide and conjugate give equivalent, or at
least
23
CA 02355348 2001-08-23
convertible, values). Affinity may be measured using the epitope (or a
molecule or
moiety comprising the epitope) used in the conjugate; alternatively, a
similar, non-
identical epitope may be used, as long as its affinity may be at least
correlated to the
affinity of the epitope used in the conjugate, so that a meaningful
measurement of
affinity may be obtained.
[0067] In a competition assay, varying concentrations of antibody or epitope
are
reacted with epitope or antibody, and results may be expressed in terms of
amount of
antibody (generally in terms of concentration) required to reach half maximal
binding, generally designated as ICS.
[0068] Another conveniewt way to express affinity is apparent equilibrium
dissociation constant, or Kn', which reflects the titer-weighted average
affinity of the
antibody for the antibody-binding epitope on the conjugate. Antibody is
generally
obtained from whole blood anti measured, by plasma, serum, or as an IgG
fraction,
and the affinity of this fraction for the conjugate is measured. Methods of
obtaining
IgG fractions are known in the art and are described herein. One preferred way
to
measure affinity is to measure Kp' based on a surface plasmon resonance assay
as
described in Example 2.
[0069] Another way to measure affinity is by kinetic (i.e., non-equilibrium)
analysis, methods of which are known in the art. Preferably, rate of
dissociation (i.e.,
off rate) of antibody from epitope is measured.
[0070] In preferred embodiments, the affinity of the individual's antibodies
for
the dsDNA epitope(s) is measured as the apparent equilibrium dissociation
constant
(Kp') for the dsDNA epitope(s) in the conjugate before or upon initiation of
treatment
is less than about (in some embodiments, less than or equal to about) 1.0 mg
IgG per
mL. In other embodiments, the KD' is less than about (in some embodiments,
less
than or equal to about) any of l;he following: 0.8; 0.7; 0.6; 0.5; 0.4; 0.3;
0.2; 0.1;
0.09; 0.08; 0.07; 0.06; 0.05; 0.025. In some embodiments, KD' is less than
about (in
some embodiments, less than or equal to about) 0.8 mg IgG per mL. In some
24
CA 02355348 2001-08-23
embodiments, Kp' is less than or equal to about (in some embodiments, less
than or
equal to about) 0.5 mg IgG per mL. In some embodiments, Kp' is less than about
(in
some embodiments, less than or equal to about) 0.1 mg IgG per mL.
[0071 ] In some embodiments, an individual is considered to have high affinity
for a dsDNA epitope if the antibody affinity of the individual is in a
relatively high
percentile ranking of affinity compared to a population. For example, there is
a range
of antibody affinities over a given patient population, and individuals
considered to
have high affinity for a dsDNA epitope can be identified based on a percentile
ranking of antibody affinity with respect to this population. Accordingly, in
some
embodiments, an individual is considered to have high affinity antibodies if
the
antibody affinity relative to thc: dsDNA epitope(s) for that individual is
greater than
about the 20th percentile (i.e., in about the top 80% of affinities for that
population),
and considered to not have high affinity antibodies (i.e., is not selected for
treatment
in accordance with the invention) if the individual's antibody affinity is in
or below
the 20th percentile. In other embodiments, an individual is included in
treatment, or
identified as suitable to receive treatment, if the antibody for that
individual is greater
than about the 50th percentile for that population. In some embodiments, the
individual is considered to have high affinity antibody if the affinity is
greater than
the 70th, 75th, 80th, 85th, 90th, or 95th percentile. A population may be
about, or
alternatively at least about any of the following, in terms of number of
individuals
measured: 10, 15, 20, 25, 30, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250,
300, 400,
500. Preferably, a sufficient number of individuals are measured to provide a
statistically significant population, which can be determined by methods known
in the
art. An upper limit of a population may be any number, including those listed.
[0072] In those embodiments which include a step wherein the individual's
antibody affinity for the dsDNA epitope(s) is remeasured after initiation of
the
treatment, the treatment is continued if the average affinity of the
individual's
antibodies for the dsDNA epitope(s) is decreased by at least about 15%,
preferably at
CA 02355348 2001-08-23
least about 20%, more preferably at least about 25%, more preferably at least
about
40%, more preferably at least about 50%, compared to the affinity measured
before
or at initiation of treatment, or discontinued if the antibody affinity has
not decreased
by at least about 15% (preferably at least about 20%, more preferably at least
about
25%, more preferably at least about 40%, more preferably at least about 50%).
For
these embodiments, antibody a~ffmity is measured after initiation of treatment
(for
comparison to antibody affmit;y before or upon initiation of treatment) at
least about 4
weeks, preferably at least about 6 weeks, more preferably at least about 10
weeks,
more preferably at least about 12 weeks, after initiation of treatment. In
other
embodiments, treatment is continued if antibody affinity is decreased at least
about
any of the following (as compared to antibody affinity before or upon
initiation of
treatment): 40%, 50%, 75%, 100%, 200%, S00%. Preferably, antibody affinity is
measured as the Ko'. As is understood by those of skill in the art, Kp values
are
inversely proportional to the affinity of the antibodies measured.
Accordingly, when
KD' values are used to measure; antibody affinity, treatment is continued if
the Kn'
increases by at least about 15°ro, and may be continued if Kp' is
increased at least
about any of the following (as compared to antibody affinity before or upon
initiation
of treatment): 40%, SO%, 75°~0, 100%, 200%, 500%.
[0073] When antibody affinity is assayed using surface plasmon resonance, a
reduction in affinity of at least about 15%, preferably at least about 20%,
more
preferably at least about 25%, more preferably at least about 40%, more
preferably at
least about 50% indicates responsiveness and that continuation of the
treatment is
indicated. For a competitive Farr assay, the same reductions in affinity
generally
apply. For other assays, the change can be at least about any of the above
percentages, and further can be at least about any of the following
percentages: 75%,
100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%.
[0074] The invention also provides methods of identifying an individual
suitable for receiving the treatments) described herein based on significant
26
CA 02355348 2001-08-23
impairment of renal function. These methods may be practiced independently of
the
treatment methods, and may bc: practiced by a skilled technician other than a
medical
doctor, using equipment and/or techniques of the art.
[0075] Accordingly, in some embodiments, the invention provides methods of
identifying an individual who may be suitable for treatment for SLE,
especially lupus
nephritis, said treatment comprising administration of a conjugate comprising
(a) a
non-immunogenic valency platform molecule and (b) two or more dsDNA epitopes,
preferably polynucleotides which specifically bind to an antibody from the
individual
which specifically binds to double stranded DNA, said method comprising
assessing
renal function in the individual, wherein an individual is identified by
having
significant renal impairment b;y any of the criteria described herein and/or
known in
the art. In some embodiments, the screening (selection) also involves
measuring
initial antibody affinity, as described herein. Generally, a higher affinity
"cut-off
(for example, as indicated by a lower KD' value) would provide a higher degree
of
certainty with respect to likely success of treatment.
Administration of conjugates
[0076] Various formulations of epitope-presenting conjugates) may be used for
administration, and, as such, the methods of this invention include
administering a
composition comprising any conjugates) described herein. In some embodiments,
the epitope-presenting conjugates) may be administered "neat" (e.g., dissolved
in
pure water, such as j.lSP water for injection). In some embodiments, the
compositions comprise a conjugates) 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
stabilizing agents, wetting anf~ emulsifying agents, salts for varying
osmolarity,
encapsulating agents, buffers, and skin penetration enhancers. Excipients as
well as
27
CA 02355348 2001-08-23
formulations for parenteral and nonparenteral drug delivery are set forth in
Remingtora's Pharmaceutical ~~'ciences 19th Ed. Mack Publishing (1995).
[0077] Generally, these compositions are formulated for administration by
injection (e.g., intraperitoneall:y, 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, and, as is understood in the art, are usually sterile
to be suitable
for injection, especially in humans. Generally, the conjugate will normally
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 the particular individual and that
individual's medical history. Generally, a dose of about 1 ~g to about 100 mg
conjugate/kg body weight, preferably about 100 ~g to about 10 mg/kg body
weight,
preferably about 150 ~,g to about 5 mg/kg body weight, preferably about 250 pg
to
about 1 mg conjugate/kg body weight. Empirical considerations, such as the
half life,
generally will contribute to deuermination of the dosage. Other dosages, such
as
about 50 to 100 mg per week, 50 to 250 mg per week, and 50 to 500 mg per week
(with any value inbetween the lower and upper limit of these ranges) are also
contemplated. Example 1 provides an example of a dosing regimen. If used as a
toleragen, conjugate may be administered daily, for example, in order to
effect
antibody clearance (pheresis), followed by less frequent administrations, such
as two
times per week, once a week, or even less frequently. Frequency of
administration
may be determined and adjusted over the course of therapy, and is based on
maintaining tolerance (i.e., reduced or lack of immune response to dsDNA).
Other
appropriate dosing schedules may be as frequent as continuous infusion to
daily or 3
doses per week, or one dose per week, or one dose every two to four weeks, or
one
dose on a monthly or less frequent schedule depending on the individual or the
disease state. Repetitive administrations, normally timed according to B cell
turnover
28
CA 02355348 2001-08-23
rates, may be required to achieve and/or maintain a state of humoral anergy.
Such
repetitive administrations generally involve treatments of about 1 pg to about
10
mg/kg body weight or higher every 30 to 60 days, or sooner, if an increase in
anti-
dsDNA antibody level is detected. Alternatively, sustained continuous release
formulations of the compositie~ns may be appropriate. Various formulations and
devices for achieving sustained release are known in the art.
[0078] Other formulations include those suitable for oral administration,
which
may be suitable if the conjugate is able to cross the mucosa. Similarly, an
aerosol
formulation may be suitable.
[0079] Other formulations include suitable delivery forms known in the art
including, but not limited to, carriers such as liposomes. Mahato et al. (
1997) Pharm.
Res. 14:853-859. Liposomal preparations include, but are not limited to,
cytofectins,
multilamellar vesicles and unilamellar vesicles.
[0080] In some embodiments, more than one conjugate may be present in a
composition. Such compositicms may contain at least one, at least two, at
least three,
at 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) conjugate(s).
[0081] The compositions may be administered alone or in conjunction with
other forms of agents that serve to enhance and/or complement the
effectiveness of a
conjugate of the invention, including, but not limited to, anti- T cell
treatments. Such
treatments usually employ agents that suppress T cells such as steroids or
cyclosporin. Another agents a.re corticosteroid and/or cyclophosphamide
immunosuppressive therapy.
[0082] Detection and mc;asurement of indicators of efficacy are generally
based
on measurement of anti-double-stranded DNA antibody and/or clinical symptoms
associated with SLE, especially lupus nephritis, which are known in the art.
29
CA 02355348 2001-08-23
(0083] Lupus nephritis (kidney glomerulonephritis or kidney inflammation) is
characterized by a progressive loss of kidney function culminating in renal
failure.
Lupus nephritis is characterized by hematuria, decreased urine output,
elevated blood
urea nitrogen levels, elevated ~~erum creatinine levels, hypertension, and
proteinuria.
Accordingly, these parameters can be monitored as a means of monitoring kidney
degeneration. In preferred emlbodiments, a conjugates) is administered such
that one
or more symptoms associated with lupus nephritis is alleviated (such as
reduction of
incidence), as described herein..
Treatment modalities: conjugates
[0084] The instant invention relates to a conjugate comprising an non-
immunogenic valency platform molecule and at least two (i.e., two or more)
dsDNA
epitopes, preferably polynucle~otides which bind to anti-dsDNA antibody from
the
individual. Preferably, the polynucleotide is double stranded DNA, preferably
the
sequence 5'-GTGTGTGTGTCiTGTGTGTGT-3'(SEQ ID NO:I). In some
embodiments, the polynucleotide comprises this sequence ((GT)lo), or consists
essentially of this sequence.
dsDNA epitope
[0085] Double-stranded DNA (dsDNA) epitopes for use in the conjugates of
the present invention may be amy chemical moiety which specifically binds to a
dsDNA antibody. In particular, epitopes of interest include those that bind
the anti-
polynucleotide (particularly anti-double stranded DNA) antibodies that occur
in
systemic lupus erythematosis. Generally, but not necessarily, the dsDNA
epitopes
used are polynucleotides, preferably DNA (including DNA analogs).
[0086] Examples of suitable epitopes include, but are not limited to, those
that
bind to lupus anti-DNA antibodies (see U.S. Patent Nos. 5,162,515; 5,391,785;
5,276,013; 5,786,512; 5,726,329; 5,552,391; 5,268,454; 5,633,395; 5,606,047).
CA 02355348 2001-08-23
(0087] The suitability of particular epitopes for binding antibodies according
to
this invention can be identified and/or confirmed using techniques known in
the art
and described herein. For example, to select the optimum epitope from a
library of
small drug molecules believed to mimic the dsDNA epitope for SLE, a family of
platforms can be constructed in which each of the candidates is alternatively
displayed on a similar platform molecule. The composition is then tested for
efficacy. For example, for ira vivo use, an animal model is used in which
there are
circulating antibodies of the undesired type, such as, for example, the BXSB
mouse
model system. The animals ca.n be immunized with an appropriate epitope to
initiate
the antibody response, if necessary. Test candidates assembled onto a platform
are
then used to treat separate aninnals, 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 plasvma are determined by standard immunoassay as
appropriate for the specific antibody. Efficacy of the candidates is then
assessed
according to antibody affinity assays designed to indicate antibodies specific
for the
epitope being tested. Appropriate affinity assays are described herein.
[0088] Polynucleotides rnay be screened for binding activity with antisera
containing the antibodies of interest, for example, SLE antisera, by the
assays
described in the examples and known in the art. Examples of such assays
include
competitive affinity assays, fo:r example, a competitive Farr assay and/or a
competitive ELISA assay, and/or non-competitive, equilibrium affinity assay,
such as
the surface plasmon resonance (for example, using BIACORE~) based assay
described herein.
[0089] A competitive Fa.rr assay in which binding activity may be expressed as
ICSO (the polynucleotide concentration in molar nucleotides resulting in half
maximal
inhibition) is an exemplary assay. Polynucleotide duplexes having an ICSO of
less
than about 500 nM, preferably less than 50 nM, are deemed to have significant
binding activity and are, therefore, useful for making the conjugates of this
invention.
31
CA 02355348 2001-08-23
[0090] Another appropriate assay is the non-competitive, equilibrium affinity
assay described herein, in which a titer-weighted affinity is determined.
[0091] It is understood that, for purposes of this invention, more than one
type
of dsDNA epitope(s) may be used in preparing a conjugate. Alternatively, one
type
(i.e., one chemical species) of an dsDNA epitope may be used. If a
polynucleotide
(such as dsDNA) is used, generally the length is greater than about 10 base
pairs (bp),
more preferably greater than about 15 bp, more preferably greater than or
equal to
about 20 bp. Generally, but not necessarily, the length is less than about 1
kb,
preferably less than about 500 bp, preferably less than about 100 bp.
Valenc~platform molecules
[0092] Any of a variety of non-immunogenic valency platform molecules (also
called "platforms") may be usc;d in the conjugates of the invention. Many have
been
described in the art, such as polymers, and need not be described herein. Any
non-
immunogenic, acceptably low to non-toxic molecule which provides requisite
attachment sites such that the <:onjugate may act to bind circulating anti-ds
DNA
antibody and/or induce B cell ~anergy and/or apoptosis in cells producing
these
antibodies may be used. Preferably, the conjugates comprise a chemically
defined
valency platform molecule in which a precise valency (as opposed to an
average) is
provided. Accordingly, a defined valency platform is a platform with defined
structure, thus a defined number of attachment points and a defined valency.
Certain
classes of chemically defined valency platforms, methods for their
preparation,
conjugates comprising them and methods for the preparation of such conjugates
suitable for use within the pre:~ent invention include, but are not limited
to, those
described in the U.S. Patents Nos. 5,162,515; 5,391,785; 5,276,013; 5,786,512;
5,726,329; 5,268,454; 5,552,391; 5,606,047; 5,663,395; and 6,060,056; and in
commonly-owned U.S. Serial Nos. 60/111,641 (U.5. Ser. No. 09/457,607 and PCT
App. No. PCT/US99/29339); 60/138,260 (U.5. Ser. No. 09/590,592 and PCT App.
No. PCT/US00/15968), U.S. 09/457,913 (PCT App. No. PCT/US99/29338), U.S.
32
CA 02355348 2001-08-23
09/457,607 (PCT/US99/29339) and U.S. 09/877,387 (PCT/LJSO1/18446), all of
which are hereby incorporated by reference.
[0093] A platform 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;
Borel
et al. (1995) Int. Arch. Allergy Immunol. 107:264-267; Borel et al. (1996)
Ann. N. Y.
Acad. Sci. 778:80-87.
(0094] The valency of a chemically-defined valency platform molecule within
the present invention can be predetermined by the number of branching groups
added
to the platform molecule. Suitaible branching groups are typically derived
from
diamino acids, triamines, and amino diacids.
(0095] 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, polyvinylpyrrollidone, D-glutamic acid and D-lysine (in a
ratio of
3:2). Preferred polymers are teased on polyethylene glycols (PEGs) having a
molecular weight of about 200 to about 8,000, or, in some embodiments, about
200 to
about 10,000. In other embodiments, the molecular weight can range between
about
40,000 to about 100,000; with a range of about 10,000 to about 20,000 as
preferable.
Other suitable platform molecules for use in the conjugates of the invention
are
albumin and IgG. Valency platform molecules should be of a size such that a
conjugate made with the valency platform does not become a T cell independent
immunogen.
33
CA 02355348 2001-08-23
[0096] Preferred valency platform molecules suitable for use within the
present
invention are the chemically-de:lined valency platform molecules disclosed,
for
example, in co-owned U.S. Patent No. 5,552,391, hereby incorporated by
reference.
These platforms generally have low polydispersity. 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). The ARAB-TEG platform used for LJP 394 (a
monodisperse platform) is described below.
[0097] In some embodirrrents, the valency platform molecules have the
advantage of having a substantially homogeneous (i.e., uniform) molecular
weight (as
opposed to polydisperse molecular weight). Accordingly, a population of these
molecules (or conjugates thereof) are substantially monodisperse, i.e., have a
narrow
molecular weight distribution. A measure of the breadth of distribution of
molecular
weight of a sample of a platform molecule (such as a composition and/or
population
of platform molecules) 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 valency molecules is preferably less
than
about 2, more preferably, less than about 1.5, or less than about 1.2, less
than about
1.1, less than about 1.07, less than about 1.02, or, e.g., about 1.05 to 1.5
or about 1.05
to 1.2. Typical polymers generally have a polydispersity of about 2-5, or in
some
cases, 20 or more. Advantages of the low polydispersity property of these
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
34
CA 02355348 2001-08-23
molecules thus are pharmaceutically optimally formulated and easy to analyze.
Accordingly, in some embodiments, the valency platform molecules have very low
polydispersity, and, in some embodiments are monodisperse.
[0098] Preferred platfornns for dsDNA epitopes are tetrabromoacetyl
compounds, and other tetravalent and octavalent valency platform molecules,
such as
those described in Jones et al. ( 1995) J. Med C'hem. 38:2138-2144; and U.S.
Patent
references provided above.
[0099] 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).
[0100] In some embodiments, a platform having a defined number of
attachment sites also comprises a (one or more) polyethylene oxide group, as
described, for example, in U.S. patents and patent applications described
above as
well as U.S. Serial No. 09/877,387, filed June 7, 2001 (PCT/USO1/18446). The
molecular weight of PEG can lbe any molecular weight, including, but not
limited to,
greater than about 200, 500, 1000, 2000, 5000, 10,000, 15,000, 18,000, 22,000,
40,000, 50,000, 80,000, 100,000 Daltons. In one embodiment, in the valency
platform molecule, the high molecular weight polyethylene oxide group has the
formula:
-(CHzCI-I~C))n-
wherein n is greater than 500; n is greater than 400; n is greater than 500;
n is greater than 600; n is greater than 700; or n is greater than 800. In
another
embodiment, the valency platform molecule comprises a core group and at least
three
arms wherein each arm comprises a terminus. The core group and/or the arms may
comprise a high molecular weight polyethylene oxide group. The high molecular
weight polyethylene oxide group also may be attached to the core or arm. In
some
embodiments, a composition comprising the valency platform molecules is
provided,
CA 02355348 2001-08-23
wherein the molecules have a polydispersity less than 1.2. In another
embodiment,
the valency platform molecule may comprise at least three reactive conjugating
groups such as hydroxyl, thiol, isocyanate, isothiocyanate, amine, alkyl
halide,
alkylmercurial halide, aldehyd~e, ketone, carboxylic acid halide, a-
halocarbonyl, a,(3-
unsaturated carbonyl, haloforniate ester, carboxylic acid, carboxylic ester,
carboxylic
anhydride, O-acyl isourea, hydirazide, maleimide, imidate ester, sulfonate
ester,
sulfonyl halide, a,(3-unsaturated sulfone, aminooxy, semicarbazide, or [i-
aminothiol.
In another embodiment, the valency platform molecule comprises at least 3
aminooxy
groups and/or at least 3 carbamate groups.
[0101] In general, these 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.
[0102] For purposes of tlhis invention, the valency platform molecules have a
minimum valency of° at least two, preferably at least four, preferably
at least six, more
preferably at least eight, preferably at least 10, preferably at least 12. As
an upper
limit, valency is generally less than 128, preferably less than 64, preferably
less than
35, preferably less than 30, prc;ferably less than 25, preferably less than
24, preferably
less than 20, although the upper limit may exceed 128. Conjugates may also
have
valency of ranges of any of the lower limits of 2, 4, 6, 8, 10, 12, 16, with
any of the
upper limits of 128, 64, 35, 30, 25, 24, 20.
[0103] In some embodinnents, the valency platform molecule comprises a
carbamate linkage, i.e., -O-C.(-=O)-N<). Such platforms are described in a co-
owned
patent application entitled "Valency Platform Molecules Comprising Carbamate
Linkages" U.S. Serial No. 60/I 11,641 (U.S. Ser. No. 09/457,607 and PCT App.
No.
PCT/L1S99/29339), hereby incorporated by reference.
[0104] In other embodinnents, valency platforms may be used which, when
conjugated, provide an average valency (i.e., these platforms are not
precisely
36
CA 02355348 2001-08-23
chemically defined in terms of their valency). Examples of such platforms are
polymers such as linear PEG; branched PEG; star PEG; polyamino acids;
polylysine;
proteins; amino-functionalized soluble polymers.
[0105] In some embodiments, the conjugates include branched, linear, block,
and star polymers and copolymers, for example those comprising polyoxyalkylene
moieties, such as polyoxyethylene molecules, and in particular polyethylene
glycols.
The polyethylene glycols preferably have a molecular weight less than about
10,000
daltons. In one embodiment, polymers with low polydispersity may be used. For
example, polyoxypropylene arud polyoxyethylene polymers and copolymers,
including polyethylene glycols may be modified to include aminooxy groups,
wherein the polymers have a low polydispersity, for example, less than 1.5, or
less
than 1.2 or optionally less than 1.1 or 1.07. Preferably, the polymers
comprise at
least 3 aminooxy groups, or at least 4, 5, 6, 7, 8, or more.
Conjugation of dsDNA epitope(s) with valency platform molecules
[0106] Conjugation of a biological or synthetic molecule to the chemically-
defined platform molecule may be effected in any number of ways, typically
involving one or more crosslin~king agents and functional groups on the
biological or
synthetic molecule and valency platform molecule. Examples of standard
chemistry
which may be used for conjugation include, but are not limited to: 1) thiol
substitution; 2) thiol Michael addition; 3) amino alkyation (reductive
alkylation of
amino groups); 4) disulfide bond formation; 5) acylation of amines.
[0107] The synthetic polynucleotide duplexes that are coupled to the valency
platform molecule are composed of at least about 20 by and preferably 20-50
bp.
Polynucleotides described herein are deoxyribonucleotides unless otherwise
indicated
and are set forth in 5' to 3' orientation. Preferably the duplexes are
substantially
homogeneous in length; that is, the variation in length in the population will
not
normally exceed about ~20'%, preferably ~10%, of the average duplex length in
base
pairs. They are also preferably substantially homogeneous in nucleotide
37
CA 02355348 2001-08-23
composition; that is, their base composition and sequence will not vary from
duplex
to duplex more than about 10°io. Most preferably they are entirely
homogeneous in
nucleotide composition from duplex to duplex.
[0108] Based on circular dichroic (CD) spectra interpretation, duplexes that
are
useful in the invention assume a B-DNA type helical structure. It should be
understood that it is not intended that the invention be limited by this
belief and that
the duplexes may, upon more conclusive analysis assume Z-DNA and/or A-DNA
type helical structures.
[0109] These polynucleotide duplexes may be synthesized from native DNA or
synthesized by chemical or recombinant techniques. Naturally occurnng or
recombinantly produced dsDNA of longer length may be digested (e.g.,
enzymatically, chemically and/or by mechanical shearing) and fractionated
(e.g., by
agarose gel or SephadexTM column) to obtain polynucleotides of the desired
length.
[0110] Alternatively, pairs of complementary single-stranded polynucleotide
chains up to about 70 bases in length are readily prepared using commercially
available DNA synthesizers and then annealed to form duplexes by conventional
procedures. Synthetic dsDNA of longer length may be obtained by enzymatic
extension (5'-phosphorylation followed by ligation) of the chemically produced
shorter chains.
[0111] The polynucleoti~des may also be made by molecular cloning. For
instance, polynucleotides of de sired length and sequence are synthesized as
above.
These polynucleotides may be designed to have appropriate termini for ligation
into
specific restriction sites. Multiple iterations of these oligomers may be
ligated in
tandem to provide for multicovpy replication. The resulting construct is
inserted into a
standard cloning vector and the vector is introduced into a suitable
microorganism/cell by transformation. Transformants are identified by standard
markers and are grown under .conditions that favor DNA replication. The
polynucleotides may be isolated from the other DNA of the cell/microorganism
by
38
CA 02355348 2001-08-23
treatment with restriction enzymes and conventional size fractionation (e.g.,
agarose
gel, SephadexTM column).
[0112] Alternatively, the polynucleotides may be replicated by the polymerase
chain reaction (PCR) technology. Saiki et al ( 1985) .Science 230:1350-1354;
Saiki et
al. (1988) Science 239:487-491; Sambrook et al. (1989) p 14.1-14.35.
[0113] The polynucleotides are conjugated to the chemically-defined valency
platform molecule in a manner that preserves their antibody binding activity.
This is
done, for example, by conjugating the polynucleotide to the valency platform
molecule at a predetermined site on the polynucleotide chain such that the
polynucleotide fortes a pendant chain of at least about 20 base pairs measured
from
the conjugating site to the free (unattached) end of the chain.
[0114] In one embodiment, the polynucleotide duplexes are substantially
homogenous in length and one strand of the duplex is conjugated to the valency
platform molecule either directly or via a linker molecule. Synthetic
polynucleotides
may be coupled to a linker molecule before being conjugated to a valency
platform
molecule. Usually the linker containing strand of the duplex is coupled at or
proximate (i.e., within about 5 base pairs) to one of its ends such that each
strand
forms a pendant chain of at least about 20 base pairs measured from the site
of
attachment of the strand to the linker molecule. The second strand is then
annealed to
the first strand to form a duplex. Thus, a conj ugate within the present
invention may
be generally described by the following formula: [(PN)"-linker]m valency
platform
molecule wherein PN=a double-stranded polynucleotide with "n" nucleotides,
wherein n = at least about 20 zmd m = 2-8.
[0115] In one embodiment, the polynucleotides of the conjugates are coupled to
a linker molecule at or proximate one of their ends. The linker molecule is
then
coupled to the chemically-defined valency platform molecule. As described in
U.S.
Patent 5,552,391 and incorporated herein by reference, exemplary of suitable
linker
molecules within the present invention are 6 carbon thiols such as HAD, a thio-
6
39
CA 02355348 2001-08-23
carbon chain phosphate, and HADP S, a thio-6 carbon chain phosphorothioate.
Chemically-defined valency platform molecules within the present invention are
formed, for example, by reacting amino modified-PEG with 3,5-bis-
(iodoacetamido)
benzoyl chloride (hereinafter "lA-DABA"); 3-carboxypropionamide-N,N-bis-[(6'-
N'-carbobenzyloxyaminohexyll)acetamide] 4"-nitrophenyl ester (hereinafter
"BAHA"); 3-carboxypropionamide-N,N-bis-[(8'-N'-carbobenzyloxyamino-3',6'-
dioxaoctyl )acetamide] 4"-nitrophenyl ester (hereinafter "BAHAoX"); or by
reacting
PEG-bis-chloroformate with N,N-di(2-[6'-N'-
carbobenzyloxyaminohexanoa~mido]ethyl)amine (hereinafter "AHAB") to form
chemically-defined valency platform molecules.
[0116] For example, a de:fmed double-stranded polynucleotide (PN) can be
conjugated to a valency platfoml molecule by first providing a single chain
consisting
of approximately 20 alternating cytosine (C) and adenosine (A) nucleotides.
Four
CA chains may then be covalently conjugated through linkers such as HAD to
four
reactive sites on a derivatized platform molecule such as triethylene glycol.
The
valency platform molecule is synthesized to include groups such as
bromoacetyl.
During the conjugation, a leaving group is displaced by sulfur. A second
single
nucleotide chain consisting of approximately 20 alternating thymidine (T) and
guanosine (G) nucleotides can then be annealed to the CA strand to form a
double-
stranded PN conjugate of the formula, [(PN)ZO -linker]4 -valency platform
molecule.
[0117] Alternatively, in another embodiment, the polynucleotide may be
coupled to the derivatized vale:ncy platform molecule at the 3' end of the
polynucleotide via a morpholino bridge formed by condensing an oxidized 3'
terminal
ribose on one of the strands of the polynucleotide with a free amino group on
the
derivatized platform molecule and then subjecting the adduct to reducing
conditions
to form the morpholino linkage, as described in U.S. Patent 5,553,391. Such
coupling requires the derivatiz;ed platform molecule to have at least an equal
number
of amino groups as the number of polynucleotide duplexes to be bound to the
CA 02355348 2001-08-23
platform molecule. The synthesis of such a conjugate is carried out in two
steps. The
first step is coupling one strand of the polynucleotide duplex to the
derivatized
platform molecule via a condemsation/reduction reaction. The oxidized 3'
terminal
ribose is formed on the single polynucleotide strand by treating the strand
with
periodate to convert the 3' terminal ribose group to an oxidized ribose group.
The
single-stranded polynucleotide is then added slowly to an aqueous solution of
the
derivatized platform molecule with a pH of about 6.0 to 8.0 at 2-8°C,
generally with a
reducing agent (such as sodium borohydride).
[0118] The molar ratio of polynucleotide to platform molecule in all the
conjugation strategies will normally be in the range of about 2:1 to about
30:1,
usually about 2:1 to about 8:1 and preferably about 4:1 to 6:1. In this
regard, it is
preferable that the conjugate not have an excessively large molecular weight
as large
molecules, particularly those v~ith repeating units, of m.w. >200,000 may be T-
independent immunogens. See Dintzis et al. ( 1983) J Immunol. 131:2196 and
Dintzis et al. ( 1989) ,7. Immunol. 143:1239. During or after the condensation
reaction
(normally a reaction time of 24 to 48 hr), a strong reducing agent, such as
sodium
cyanoborohydride, is added to form the morpholino group. The complementary
strand of the duplex is then added to the conjugate and the mixture is heated
and
slowly cooled to cause the strands to anneal. The conjugate may be purified by
gel
permeation chromatography.
[0119] An alternative to the ribose strategy is forming aldehyde
functionalities
on the polynucleotides and using those functionalities to couple the
polynucleotide to
the platform molecule via reacaive functional groups thereon. Advantage may be
taken of the fact that gem vicinal diols, attached to the 3' or S' end of the
polynucleotide, may be oxidized with sodium periodate to yield aldehydes which
can
condense with functional amino groups of the platform molecule. When the diols
are
in a ring system, e.g., a five-membered ring, the resulting condensation
product is a
heterocyclic ring containing nitrogen, e.g., a six-membered morpholino or
piperidino
41
CA 02355348 2001-08-23
ring. The imino-condensation product is stabilized by reduction with a
suitable
reducing agent; e.g., sodium borohydride or sodium cyanoborohydride. When the
diol is acyclic, the resulting oxidation product contains just one aldehyde
and the
condensation product is a secondary amine.
[0120] Another procedure involves introducing alkylamino or alkylsulfhydryl
moieties into either the 3' or 5' ends of the polyrmcleotide by appropriate
nucleotide
chemistry, e.g., phosphoramidite chemistry. The nucleophilic groups may then
be
used to react with a large excess of homobifunctional cross-linking reagent,
e.g.,
dimethyl suberimidate, in the ease of alkylamine derivatives, or an excess of
heterobifunctional cross-linking reagent, e.g., m-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) or succinimidyl (4-iodoacetyl) aminobenzoate
(SIAB), for the alkylsulfhydryl derivatives. Once excess cross-linker is
removed, the
polynucleotide derivatives are reacted with amino groups on the platform
molecule.
Alternatively, the sullhydryl group may be reacted with an electrophilic
center on the
platform, such as a maleimide or a-haloacetyl group or other appropriate
Michael
acceptor.
[0121] Still another strategy employs modified nucleosides. Suitable
deoxynucleoside derivatives can be incorporated, by standard DNA synthetic
chemistry, at desired positions in the polynucleotide, preferably on the 5' or
3' ends.
These nucleoside derivatives rnay then react specifically and directly with
alkylamino
groups on the platform molecule. Alternatively, side reactions seen with the
above-
described dialdehyde chemistry, such as amine catalyzed beta-elimination, can
be
circumvented by employing appropriate nucleoside derivatives as the 3'
terminus of
the chain to be attached. An example of this is S' methylene extension of
ribose; i.e.,
a 5' (2-hydroxyethyl)-group instead of a 5' hydroxymethyl group. An
alternative
would be to use a phosphonatc: or phosphinate linkage for the 3' terminal
dinucleotide
of the polynucleotide to be attached to the platform molecule.
42
CA 02355348 2001-08-23
[0122] A description of the synthesis of the conjugate LJP 394, a tetravalent
conjugate, is described in Jones et al. (1995) and in U.S. Patent 5,552,391,
which are
hereby incorporated by reference. LJP 394 comprises four 20-mer
oligonucleotides
consisting of alternating C and A nucleotides, (CA),o, attached to a platform
and
annealed with complementary 20-mer oligonucleotides consisting of alternating
G
and T nucleotides, (CiT),o, olig;onucleotide. The valency platform molecule
used in
LJP 394 is shown immediately below.
O
I I
NHCO(CH2)SNHCOCH2S(CH2)60-P-O-PN
O ~ O
CH20CH2CH20~-N
O
AHAB-TEG NHCO(CH2)SNHCOCH2S(CH2)60-IP-O-PN
O 2
PN = (CA)lo~(TG)~o
Kits
[0123] The invention also provides kits for use in the instant methods. Kits
of
the invention include one or more containers comprising an epitope presenting
conjugate (i.e., a conjugate comprising a non-immunogenic valency platform
molecule and two or more double stranded DNA epitopes, preferably
polynucleotides) and instructions for use in accordance with the methods of
the
invention. Accordingly, these instructions comprise a description of selecting
an
individual suitable for treatment based on identifying whether that individual
has SLE
and significant renal impairment (as indicated by any clinical indicia
described herein
and/or known in the art), and preferably also further describe administration
of the
conjugate for treatment of SLE and/or lupus nephritis. In some embodiments,
the
instructions comprise description of administering a conjugate to an
individual having
lupus nephritis who has significantly impaired renal function (which may also
describe one or more criteria for determining whether an individual having, or
43
CA 02355348 2001-08-23
suspected of having lupus nephritis has significant renal impairment). In some
embodiments, the kits further comprise one or more compositions for measuring
level
of renal function in an individual.
[0124] In some embodirrlents, the kits may also contain supplies and
instructions for measuring antibody affinities for use in the methods
described herein,
particularly affinity for an epitope which binds to anti-dsDNA antibodies.
Accordingly, the kits of such embodiments contain (i.e., comprise) one or more
dsDNA epitopes, preferably polynucleotides (preferably, double stranded (ds)
DNA
molecules) comprising an epitope which binds to an anti-dsDNA antibody from an
individual (and the epitope-containing polynucleotide binds to an anti-dsDNA
antibody from an individual). Accordingly, the kits comprise a molecule or
moiety
comprising a dsDNA epitope, such as any described herein. In one embodiment,
the
kit comprises a polynucleotide with (comprising) the sequence (or,
alternatively,
consisting essentially of or consisting of the sequence) 5'-
GTGTGTGTGTGTGTGTGTcsT-3'(SEQ ID NO:I). In certain embodiments the
dsDNA epitopes are not part of a conjugate with a non-immunogenic valency
platform molecule. In other embodiments, the kits comprise the conjugates
described
herein, with instructions for using the conjugate to detect affinity of an
individual's
anti-dsDNA antibodies for the conjugate. Preferably, the conjugate is LJP 394.
[0125] In those embodiments containing materials and instructions for
measurement of antibody affinity, such materials may be used, for example, to
test an
individual to determine if the individual is suitable or unsuitable for
treatment with
the conjugate(s), as well as for monitoring purposes. The affinity testing
materials
may also be used in determining affinity cut-off values (i.e., affinity values
which
correlate with clinical results).
[0126] The kits of this invention are in suitable packaging. Suitable
packaging
for epitope presenting conjugates includes, but is not limited to, vials,
bottles, jars,
flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
44
CA 02355348 2001-08-23
[0127] Kits may optionally provide additional components such as, buffers and
instructions for determining affinity or binding to anti-dsDNA antibody, such
as
capture reagents, developing rc;agents, labels, reacting surfaces, means for
detection,
control samples, and interpretive information. The instructions relating to
measurement of antibody affinity may be for any measurement of antibody
affinity,
including, but not limited to, those assays described herein. Accordingly, in
some
embodiments, the instructions are for determining affinity using surface
plasmon
resonance. In other embodiments, the instruction are for determining affinity
using
direct binding assays and/or Farr assays. In some embodiments, reagents
described
above are supplied such that mmltiple measurements may be made, such as
allowing
for measurements in the same individual over time or multiple individuals.
[0128] In those embodiments comprising materials for testing antibody
affinity,
the dsDNA epitope(s) of the kit, preferably a polynucleotide(s) of the kit
(whether in
free form or attached to a conj ugate or other matrix), generally contains, or
alternatively consists of, the epitope that will be or is used in treatment,
or has been
demonstrated to have about the same affinity for an individual's anti-dsDNA
antibodies as the epitope(s) that will be used in treatment. In other
embodiments, the
kits comprising a dsDNA epitope whose affinity for anti-dsDNA antibodies
mimics
or alternatively can be correlated to that of the dsDNA epitope to be used in
treatment, such as 5'-GTGTG'TGTGTGTGTCTTGTGT-3'(SEQ ID NO:1). These
dsDNA epitopes can be used ass "proxies" for the dsDNA epitope to be used in
treatment, such as LJP 394, in assessing antibody affinity for the methods
described
herein.
[0129] Embodiments including materials for testing antibody affinity may
comprise any appropriate means for detecting binding of the antibodies, such
as a
labeled anti-human antibody, 'when the presence of human anti-dsDNA antibodies
is
tested, wherein the label may loe an enzyme, fluorophore, chemiluminescent
material
radioisotope or coenzyme. Gc;nerally, the label used will be an enzyme.
CA 02355348 2001-08-23
Accordingly, in some embodiments, the kits) of the invention further comprises
a
label. In some embodiments, the polynucleotide in the kits) is conjugated to
biotin.
In a preferred embodiment, the dsDNA epitope (such as a polynucleotide, for
example, double stranded DNA) is biotinylated. Biotinylation may also be
accomplished using commerci;~lly available reagents (i.e., Pharmacia; Uppsala,
Sweden). In another preferred embodiment, the biotinylated dsDNA epitope
comprises, consists essentially or, or consists of is 5'-
GTGTGTGTGTGTGTGTGT(JT-3'(SEQ ID NO:l).
[0130] In other embodiments, the invention provides a kit comprising (a) an
epitope presenting conjugate a;s described herein, such as LJP 394; and (b) a
polynucleotide (or other dsDNA epitope) used in the conjugate, or,
alternatively, a
polynucleotide comprising the polynucleotide used in the conjugate (or a
molecule or
moiety comprising the epitope to be used in the conjugate). These kits also
contain
the instructions for practicing a methods) of the invention, as described
above.
When used for affinity measurements, the conjugate and/or polynucleotide may
be
biotinylated. In some embodiments, the kit contains instructions for
administering
the conjugate to an individual as well as instructions for using the conjugate
and/or
the polynucleotide (including a polynucleotide comprising the polynucleotide
used in
the conjugate) for detecting affinity for an antibody in an individual which
binds to
dsDNA as described herein. As discussed herein, a combination of a conjugate
to be
used for treatment and a molecule comprising a dsDNA epitope, the binding
activity
or affinity of which mimics, or can be correlated with, the epitope of the
conjugates is
used in the kits.
[0131] The following E~,:amples are provided to illustrate but not limit the
invention.
46
CA 02355348 2001-08-23
F'.XAMP1,FS
Examine 1: Treatment of SLE patients having significantly impaired renal
function with LJP 394
[0132] 230 SLI: patients were enrolled in a double-blind, placebo-controlled
trial of LJP 394 (abetimus sodium). The study was a multicenter, double-blind,
randomized, parallel group trial comparing intravenously administered LJP 394
to
placebo during induction (100 mg weekly) and maintenance periods (50 mg
weekly)
in patients with a history of lupus renal disease. LJP 394 is formulated as a
sterile,
colorless liquid in an isotonic phosphate-buffered saline solution for
intravenous (IV)
administration. Each 1 mL of solution contains 50 mg of LJP 394, 1.9 mg
Na2HP04*7H20, 0.30 mg NHzP04*H20, and 5.8 mg NaCI in water for Injection,
USP (pH 6.8 -8.0). The formulation contains no preservatives.
(0133] Prospective patients were observed for four to six weeks prior to
randomization to ensure that entrance criteria were met prior to
randomization. The
initial protocol provided LJP 394 or placebo at 100 mg/week for 52 weeks with
a six
month follow up period. The protocol was later amended to a 75 week treatment
period consisting of a 15 week induction period and a 60 week maintenance
period
consisting of 15 weekly doses of LJP 394 or placebo, followed by alternating
eight
week drug holidays and 12 weekly treatments with 50 mg LJP 394 or placebo for
a
total of 75 weeks. All patients in the original protocol were transferred into
the
amended protocol prior to the end of the first dosing cycle of the maintenance
period.
[0134] A protocol-defined renal flare was reached if one or more of the
following endpoints was met and the flare was attributed to SLE by the
treating
physician and the study's medical monitor: (a) reproducible increase in 24
hour urine
protein to greater than 1000 mg per 24 hours if the screening value was less
than 200
mg per 24 hours, to greater than 2000 mg per 24 hours if the screening value
was
200-1000 mg per 24 hours, or to more than two fold the screening value if it
was
greater than 1000 mg per 24 hours; (b) a reproducible increase in serum
creatinine of
47
CA 02355348 2001-08-23
> 20% or at least 0.3 mg/dL, which ever was greater, accompanied by
proteinuria
(>1000 mg per 24 hours), hematuria (> four red blood cells per high power
field
(RBCs/HPF)) and/or red cell casts; or (c) new reproducible hematuria (>I 1-20
RBCs/HPF) or a reproducible two grade increase in hematuria compared to
baseline
with >25% dysmorphic blood cells (glomerular in origin), exclusive of menses,
and
either an 800 mg increase in 24 hour protein or new red cell casts.
[0135] Baseline for dsDl~lA was calculated as the mean of the last two
screening measurements. Baseline for all other laboratory values was the
measurement taken immediately prior to the first administration of study drug.
[0136] Therapeutic intervention with high doses of corticosteroids and/or
cyclophosphamide (HDCC) was left to the investigator's discretion and
summarized
at study closure using the following criteria: any exposure to
cyclophosphamide;
systemic prednisone (or prednisone equivalent) increase >15 mg per day over
baseline dose to greater than 20 mg per day for more than two days or a dose
of
prednisone (or prednisone equivalent) that exceeded 200 mg in a single day.
Topical,
intra-articular, or intra-ocular usage was excluded. If a patient received
both high
dose corticosteroids and cyclophosphamide, HDCC analysis considered only the
first
exposure.
[0137] The comparison of continuous variables was performed using analysis
of variance. The Fisher's exact test was used for the comparison of incidence
rates
and all other categorical comparisons. No adjustments were made to p-values
for
multiple comparisons.
[0138] All time-to-event comparisons were performed using the Kaplan-Meier
product limit method and the log rank test. When comparing time-to-event
variables
for the entire study period, all patients were included in the analysis until
time of
event (renal flare or HDCC) or their last exposure to drug. Only the first
renal flare
and the first exposure to HDCC' were used in the time to event and incidence
analysis.
48
CA 02355348 2001-08-23
[0139] Two hundred and thirty patients were randomized to receive study drug
( 116 received LJP 394, 114 received placebo). Pretreatment samples for
determination of affinity were available for 213 patients. The trial was
prematurely
terminated after an interim analysis established that the trial was unlikely
to reach
statistical significance for time to renal flare in the ITT (intent to treat)
population.
[0140] Twenty seven patients ( 17 receiving LJP 394, 10 receiving placebo)
were enrolled exhibiting significantly impaired renal function (i.e., with
baseline
serum creatinine levels between 1.5 mg/dL and 2.5 mg/dL (the upper limit for
inclusion)). In these patients, renal flare was observed in 3/17 (18%) of LJP
394-
treated patients and 6/10 (60%) placebo patients. LJP 394-treated patients had
a
significantly longer time to renal flare than patients treated with placebo
(p=0.003).
Eleven of 17 (65%) of LJP 394 patients and all of the placebo patients had
high
affinity antibodies to LJP 394 prior to treatment. Of the patients with high
affinity
antibodies (Kd' less than or equal to 0.8 mg IgG/ml), there were no renal
flares in the
LJP 394-treated group, compared to 6/10 (60%) of the placebo group.
[0141] Patients with sigruficantly impaired renal function at baseline
appeared
to fare worse than the overall study population. Six of ten (60%) patients in
the
placebo group developed renall flares versus 23/116 (20%) in the ITT placebo
group.
LJP 394-treated patients that did not have high affinity antibodies to LJP 394
fared
very poorly (same as the placebo population, in which 6 of 10 developed renal
flares),
with three of six (50%) developing renal flares, as compared to 19/116 (16%)
of the
LJP 394 ITT population, and 7/92 (8%) of the LJP 394 treated patients having
high
affinity antibodies. However, there were no renal flares amongst those
patients
having significantly impaired renal function and high affinity antibodies to
LJP 394.
Example 2: Inhibition of binding of anti-dsDNA antibodies to DNA by LJP 394
[0142] After determining the presence of anti-dsDNA antibodies in patients
using a Farr assay, a competitive Farr assay was used to measure the affinity
of anti-
dsDNA antibodies found in sera from patients with SLE to LJP 394. In addition,
the
49
CA 02355348 2001-08-23
assay was used to measure the affinity of anti-dsDNA antibodies found in sera
from
three animals models of SLE (BXSB mice, NZB x NZW F, mice, and MRL/Ipr
mice).
[0143] The Farr assay used ~zSI-labeled recombinant dsDNA (Diagnostic
Products Corporation, Los An~;eles, CA) that was combined with the anti-dsDNA
antibodies found in sera from patients with SLF or from the mouse models of
SLE.
Anti-dsDNA antibodies were obtained from serum samples of donors with SLE
collected through a volunteer donor program. Blood samples were drawn, serum
harvested, aliquots made, labeled, and stored frozen at -70°C until
used. In this assay,
25 pL of patient's serum was added to 75 ~L of Tris buffer (50 mM Tris, 150 mM
NaCI pH 7.5, 10% normal rabt»t serum), then L00 pL of ~ZSI-labeled recombinant
dsDNA was added, mixed and incubated at 37"C for one hour. Similar samples
containing known amounts of anti-dsDNA antibodies (calibrators) were prepared
and
incubated at the same time. 500 pL of 70% saturated ammonium sulfate was added
to each tube, mixed, and then centrifuged at 800 x g for 15 minutes to
precipitate the
antibodies in solution. The supernatant was decanted and the amount of
radioactivity
in the precipitated product was determined by counting the radioactivity in a
gamma
counter. The amount of radioactivity in the precipitant is proportional to the
amount
of anti-dsDNA antibodies that bound to ~ZSI-labeled recombinant dsDNA.
Calibrators
with known amounts of anti-dsDNA antibodies were used to generate a standard
curve from which the amount of dsDNA binding by anti-dsDNA antibodies could be
calculated.
[0144] Serum samples from 58 patients were assayed for the presence of
antibodies to dsDNA using the Farr assay described above. Forty-two of these
samples had sufficient levels o~f antibody (>_ 20% binding) to use in the LJP
394
inhibition assay.
[0145] LJP 394 was tested for its ability to inhibit binding of anti-dsDNA
antibodies to'zsI_labeled recombinant dsDNA by a competitive Farr assay. Calf
CA 02355348 2001-08-23
thymus DNA (ctDNA) was also used in the inhibition assay as another source of
dsDNA. Calf thymus dsDNA was prepared by dissolving calf thymus DNA in
nuclease-S 1 buffer (0.2 M Na<'.l, 50 mM sodium acetate pH 4.5, 1 mM ZnS04 and
0.5% glycerol) and 100,000 units of S-1 nuclease and incubating for one hour
at
37°C. The dsDNA was extracted from this mixture by adding an equal
volume of
phenol-chloroform, mixing, centrifuging, and harvesting the aqueous layer. The
dsDNA was then precipitated by adding 2 volumes of EtOH, mixing, and
centrifuging. The pellet was harvested, dried under vacuum and dissolved in
water to
approximately 10 mg/mL. The final concentration of the ctDNA preparation was
determined spectrophotometrically assuming an extinction coefficient of 33 ~g
per 1
OD unit at 260 nM.
[0146] Each serum sample that gave >_ 20% binding was tested in the inhibition
assay. Briefly, 25 ~L of patient's serum was added to 75 pL of Tris buffer (50
mM
Tris, 150 mM NaCI pH 7.0, 10°,% normal rabbit serum) containing
various
concentrations of inhibitor (either calf thymus dsDNA or LJP 394), then 100 ~L
of
izsl-labeled recombinant dsDNA was added, mixed and incubated at 37°C
for one
hour. 500 ~L of 70% saturated ammonium sulfate was added to each tube, mixed
and then centrifuged at 800 x for 15 minutes. The supernatant was decanted and
the amount of radioactivity in the precipitated product was determined by
counting
the radioactivity in a gamma counter. Extent of inhibition was calculated by
the
following formula: { [(cpm patient's serum without inhibitor - cpm without
patient's
serum, no inhibitor) - (cpm patient's serum with inhibitor - cpm without
patient's
serum, no inhibitor)] divided by (cpm patient's serum without inhibitor - cpm
without
patient's serum, no inhibitor)} all times 100.
[0147] Figures lA-C illustrate the ability of LJP 394 to inhibit the binding
of
autoantibodies from a representative populations of patients with SLE.
Overall, LJP
394 was capable of inhibiting binding of the autoantibodies to dsDNA in 42 out
of 42
patients with SLE. The inhibition curves for LJP 394 and calf thymus dsDNA
were
51
CA 02355348 2001-08-23
parallel, suggesting that the antigenic determinants being recognized by the
SLE sera
were identical on both the calf thymus dsDNA and LJP 394.
[0148] The ability of LJf 394 to inhibit the binding of anti-DNA antibodies in
a
mouse models of SLE was also tested. Competitive inhibition assays with calf
thymus dsDNA and L~JP 394 were performed as described above and the results
are
shown in Table 1. The 50% inhibition ratios (ICSO LJP 394/ICSO ctDNA) were
lowest
for human anti-dsDNA antibodies (from SLE sera), compared to the mouse
antibodies. LJP 394 showed high affinity for human antibodies and the NZBxNZW
F 1 mouse strain.
TABLE 1
Competitive
Inhibition
of Binding
of Anti-dsDNA
Antibodies
by ctDNA
and
LJP 394
- -
__ICSO~ Ni~mL ICSo
(mean 5D)
LJP 394/ctDNA
No. of sera -
Source of ctDNA LJP 394 ratio
sera
MRL
3 0.356 0.455200 42 562
(Ipr/Ipr)(mouse)
NZBxNZWF
~
3 0.021 0.011 S.5 0.7 258
(mouse)
S BXSB (mouse)0.028 0.000215 144 7679
42 Human SL.E 1.88 0.920 24
. 46 16
Example 3: Determination of titer-weighted average affinity of antibodies for
coniuEate by a surface ulasmon resonance assay
[0149] An assay using surface plasmon resonance is used to directly measure a
titer-weighted average affinity of antibodies from SLE patients for the
conjugate LJP
52
CA 02355348 2001-08-23
394. Surface plasmon resonance is used to quantify the fractional saturation
of
antigen with antibody. This assay was adapted so that it measures the average
affinity of the IgG population o1~ LJP 394.
Materials and Methods
[0150] Reage, nts. Streptavidin CMS chips, HBS buffer (0.01 M HEPES pH 7.4,
0.15 M NaCI, 3 mM EDTA and 0.005% (v/v) surfactant P20) are obtained from
BIACORE AB (Piscataway, NJ).
(0151 ) LJP 394 is composed of four 20-mer dsDNA epitopes that are covalently
attached to a triethyleneglycol--based platform by a thiol linkage. The DNA
epitope is
composed of 5'-(CA)lo-3' strands annealed to complementary GT strands, with
biotin
attached at the free 5' ends of the GT strand. Biotin is incorporated by using
Biodite
biotin amidite (Pharmacia; Llppsala, Sweden) in the final coupling of the
(GT)1o
strand. LJP 394 is prepared essentially as described in Jones et al. (1995)
except that
this biotin-modified (GT)lo strand is used in the annealing step. In some
experiments,
only the dsDNA epitope is immobilized on the streptavidin chip. The epitope is
prepared by annealing 5'-(CA)~o-3' to 5'-biotin-(TG)~o-3' and purifying the
dsDNA
by HPLC.
[0152] Plasma samples acre collected from SLE patients, and total IgG fraction
is isolated from the plasma by combining 100 ~L of plasma with 100 p.Lof IgG
binding buffer (Pierce Chemical Co.; Rockford, IL) and mixing with Immunopure
Plus~ protein G agarose beads (Pierce Chemical Co.) according to manufacturer
recommendations. Elution of IgG from the beads is accomplished by following
the
acid elution/neutralization protocol of Pierce Chemical Co., and 300 ~.L of
acid eluted
IgG is neutralized with 100 ~.L. of 1 M NaP04, pH 7.5. These purified IgG
samples
are then used in the titration e:~cperiments. Total IgG concentrations are
determined
with the Bradford assay (Bior;~d; Hercules, CA).
53
CA 02355348 2001-08-23
[0153] Surface Plasmon Resonance. All measurements are performed using a
BIACORE~ 2000 instrument at 25°C with a flow rate of 10 pl/minute. LJP
394 is
attached to the streptavidin CMS chip through its 5' biotin group by flowing a
50
~,g/mL solution of LJP 394 in JHBS + 0.3 M NaCI over the chip for 20 minutes
at S
~,L/minute. The chip is preconditioned prior to titration with 3 x 1 minute
pulses of
regeneration buffer ( 1 M NaCI and SO mM NaOH). When the dsDNA epitope of LJP
394 is used for immobilization, the biotinylated epitope is flowed over the
chip at a
concentration of 10 ELg/mL using similar conditions as employed for the
biotinylated
LJP 394 epitope.
[0154] Antibody titrations of the dsDNA (LJP 394) chip are performed with
serial 1:2 dilutions of purified IgG in HBS. Sample is injected for 5 minutes,
which
is adequate association time for a significant approach to the response
plateau, and is
followed by a 4 minute dissociiation period where HBS is flowed over the chip,
then a
30 second regeneration is pert~~rmed with 1 M NaCI, 50 mM NaOH.
[0155) Analysis. Response plateau values (R.eq) are obtained by a nonlinear
least squares fit of the association curves to equation 1, after subtraction
of a
background curve for an empty flow cell, to account for bulk response/buffer
effect,
and using the manufacturers software (BiaEvaluation version 2.2, Uppsala,
Sweden)
Rt= Rey(1-e-ksy-z°') + R~ (equation 1)
where R~ is the measured response at time t, Reu is the equilibrium plateau
response, t
is time, to is initial time, ks is am apparent association constant (kS = kaC
+ ka;s, where
ka is the association constant, C is the analyte concentration and ka;s is the
dissociation constant), and Ra is a response offset. These response plateaus
are
plotted versus the concentration of total IgG, and fitted to equation 2 to
obtain values
for Rmax and Ka*.
Rmax AT
Req = ------------ (equation 2)
Ka * + A.r
54
CA 02355348 2001-08-23
where AT is the total antibody (IgG) concentration, RmaX is the maximum
response
plateau and Ka* is an apparent dissociation constant. Kd* is the same as <Kd'>
in
equation 3 (below), the titer-weighted-average (TWA) dissociation constant.
The
derivation of Kd' was performed as described in Sem et al. (( 1999) Arch.
Biochem.
Biophys. 372:62-68) and provides insight into the physical meaning of the Kd*
constant in equation 2. This analysis pertains to the case of a polyclonal
pool of n
different antibody subpopulations, where B = L:fP 394 and A; = antibody
subpopulation i.
AT 1
<y> _ ____________ __ ________________ (equation 3)
n n
(A; ~;) ~ (r. /K;)
i=1 i=1
where r; (relative titer) is the fraction of total antibody present as form i,
defined as
r; = A;/AT. Thus, equation 3 is the general equation describing the observed
dissociation constant for a polyclonal population of n different antibody
subpopulations of relative titer (fractional presence) r; and dissociation
constant K;.
This <Ka'> is the apparent K~ of equation 2, K~*.
[0156] The measured apparent dissociation constant K~' reflects both inherent
affinity of antibody subpopulation i for antigen, and relative titer of
antibody
subpopulation i (r;). In general, 0 < r; < 1, so K~' > K;. That is, the
factors that can
cause Kd' to decrease are an increase in affinity (K; decreases) and/or an
increase in
relative titer of antibody subpopulation i (r; increases). In practice, in a
polyclonal
population of antibodies, there will be many different antibody subpopulations
that
bind, each with slightly different affinity.
[0157] The above analysis, and that further described in Sem et al. (1999),
produces an apparent dissocial:ion constant that is a reflection of the
various affinities
CA 02355348 2001-08-23
and titers of clonally related subpopulations of antibodies within a
polyclonal pool.
The apparent dissociation constant obtained as described is the titer-weighted-
average
(TWA) dissociation constant derived in equation 3, <Kd'>. The value of <Kd'>
is
dominated by antibody subpopulations that have the largest r; (highest
relative titer)
and smallest K; (highest affinity) in combination. Any change in relative
titers of
subpopulations with a given affinity will change the apparent dissociation
constant
according to equation 3.
56
CA 02355348 2002-05-06
SEQUENCE LISTING
GENERAL INFORMATION;
APPLICANT: La Jolla Pharmaceutical Company
TITLE OF INVENTION: METHODS OF TREATING SYSTEMIC LUPUS
ERYTHEMATOSU5 IN INDIVIDUALS HAVING SIGNIFICANTLY IMPAIRED RENAL
FUNCTION
REFERENCE NUMBER: 252312007840
CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2,355,348
APPLICATION DATE: 2001-08-23
CLASSIFICATION:
PRIOR APPLICATION DATA:
APPLICATION NUMBER: US 60/311,858
FILING DATE: 2001-OS-13
CLASSIFICATION
NUMBER OF SEQUENCES: 2
SOFTWARE: PatentIn Ver. 2.1
INFORMATION FOR SEQ ID NO.: 1
SEQUENCE CHARACTERISTICS:
LENGTH: 20
TYPE: DNA
MOLECULE TYPE: Artificial Sequence
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence: synthetic
construct
SEQUENCE DESCRIPTION: SEQ ID NO.: 1
gtgtgtgtgt gtgtgtgtgt 20
INFORMATION FOR SEQ ID NO.: 2
SEQUENCE CHARACTERISTICS:
LENGTH: 20
TYPE: DNA
MOLECULE TYPE: Artificial Sequence
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence: synthetic
construct
SEQUENCE DESCRIPTION: SEQ ID NO.: 2
cacacacaca cacacacaca 20
