NZ615912B2 - Means and methods for diagnosing and treating multiple sclerosis - Google Patents
Means and methods for diagnosing and treating multiple sclerosis Download PDFInfo
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- NZ615912B2 NZ615912B2 NZ615912A NZ61591212A NZ615912B2 NZ 615912 B2 NZ615912 B2 NZ 615912B2 NZ 615912 A NZ615912 A NZ 615912A NZ 61591212 A NZ61591212 A NZ 61591212A NZ 615912 B2 NZ615912 B2 NZ 615912B2
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Abstract
Discloses a peptide comprising or consisting of at least 8 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 3, the upper limit for the length of said peptide being 100 amino acid residues, provided that said peptide does not consist of the sequence set forth in SEQ ID NO: 3; or a corresponding peptidomimetic, wherein said peptide or peptidomimetic is capable of binding to an anti-KIR4.1 antibody comprised in a sample from a patient having multiple sclerosis or a predisposition thereto wherein: (i) said at least 8 consecutive amino acid residues are a subsequence of an extracellular domain of KIR4.1, said extracellular domain consisting of the sequence set forth in SEQ ID NO: 1 or 2; or (ii) said peptide comprises or consists of the sequence of SEQ ID NO: 1 or 2; wherein the sequences are as defined in the complete specification. Further discloses methods involving the use of said peptide or peptidomimetic for diagnosing multiple sclerosis, screening drugs for multiple sclerosis, or to treat multiple sclerosis. or a corresponding peptidomimetic, wherein said peptide or peptidomimetic is capable of binding to an anti-KIR4.1 antibody comprised in a sample from a patient having multiple sclerosis or a predisposition thereto wherein: (i) said at least 8 consecutive amino acid residues are a subsequence of an extracellular domain of KIR4.1, said extracellular domain consisting of the sequence set forth in SEQ ID NO: 1 or 2; or (ii) said peptide comprises or consists of the sequence of SEQ ID NO: 1 or 2; wherein the sequences are as defined in the complete specification. Further discloses methods involving the use of said peptide or peptidomimetic for diagnosing multiple sclerosis, screening drugs for multiple sclerosis, or to treat multiple sclerosis.
Description
Means and methods for diagnosing and treating multiple sclerosis
This invention relates to a peptide comprising or consisting of at least 8 consecutive amino
acid residues of the sequence set forth in SEQ ID NO: 3, provided that said peptide does
not consist of the sequence set forth in SEQ ID NO: 3, or a corresponding peptidomimetic,
wherein said peptide or peptidomimetic binds to an anti-KIR4.1 antibody comprised in a
sample from a patient, said patient having multiple sclerosis or a predisposition therefor,
wherein preferably (i) said at least 8 consecutive amino acid residues are a subsequence of
an extracellular domain of KIR4.1 , said extracellular domain consisting of the sequence set
forth in SEQ D NO: 1 or 2 ; or (ii) said peptide comprises or consists of the sequence of
SEQ ID NO: 1 or 2. The present invention furthermore relates to a method for diagnosing
multiple sclerosis or a predisposition for multiple sclerosis in a subject, the method
comprising determining the presence of an anti-KIR4.1 antibody in a sample obtained from
said subject, wherein the presence of an anti-KIR4.1 antibody in said sample is indicative of
multiple sclerosis or a predisposition for multiple sclerosis.
In this specification, a number of documents including patent applications and
manufacturer's manuals is cited. The disclosure of these documents, while not considered
relevant for the patentability of this invention, is herewith incorporated by reference in its
entirety. More specifically, all referenced documents are incorporated by reference to the
same extent as if each individual document was specifically and individually indicated to be
incorporated by reference.
Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central
nervous system (CNS) leading to disability in the majority of affected patients (1). The
etiology of MS is unknown but epidemiological evidence suggests a complex interplay
between genetic and environmental factors (2-4). An uncertain pathogenic mechanism,
clinical heterogeneity and unpredictability of the outcome of individual patients add to the
complexity of the disease (5).
The current working hypothesis for MS pathogenesis suggests that autoreactive T cells play
a central role (6). However, histopathological studies have revealed a subset of MS patients
exhibiting prominent deposition of immunoglobulins and complement activation in acute
demyelinating lesions (7, 8). These patients respond particularly well to therapeutic plasma
exchange (9). Moreover, B cell depletion by a therapeutic monoclonal antibody has a
profound impact on inflammatory activity in MS (10). All these findings support the
contention that at least in a subset of MS patients B cells and antibodies substantially
contribute to the development and progression of the disease (11, 12). Despite this
circumstantial evidence, a direct proof of clinically relevant antibodies in MS has not
been established owing to the fact that specific molecular targets for humoral responses
in MS remain undiscovered.
The technical problem can be seen in the provision of alternative or improved means
and methods for diagnosing and/or treating multiple sclerosis.
Any discussion of the prior art throughout the specification should in no way be
considered as an admission that such prior art is widely known or forms part of common
general knowledge in the field.
According to a first aspect, the present invention relates to a peptide comprising or
consisting of at least 8 consecutive amino acid residues of the sequence set forth in
SEQ ID NO: 3, the upper limit for the length of said peptide being 100 amino acid
residues, provided that said peptide does not consist of the sequence set forth in SEQ
ID NO: 3; or a corresponding peptidomimetic,
wherein said peptide or peptidomimetic is capable of binding to an anti-KIR4.1 antibody
comprised in a sample from a patient having multiple sclerosis or a predisposition
thereto wherein:
(i) said at least 8 consecutive amino acid residues are a subsequence of an
extracellular domain of KIR4.1, said extracellular domain consisting of the sequence set
forth in SEQ ID NO: 1 or 2; or
(ii) said peptide comprises or consists of the sequence of SEQ ID NO: 1 or 2.
According to a second aspect, the present invention relates to a method for diagnosing
multiple sclerosis or a predisposition to multiple sclerosis in a subject, the method
comprising determining, in a sample obtained from said subject, the presence of an
anti-KIR4.1 antibody, wherein the presence of the anti-KIR4.1 antibody is determined
by:
- 2a -
(i) contacting a peptide or peptidomimetic of the first aspect; and
(ii) detecting the formation of a receptor-anti-KIR4.1 antibody complex,
wherein the presence of the anti-KIR4.1 antibody in said sample is indicative of multiple
sclerosis or a predisposition thereto.
According to a third aspect, the present invention relates to a peptide or peptidomimetic
according to the first aspect when used in determining, in a sample, previously obtained
from a patient having multiple sclerosis or a predisposition thereto, the presence of the
anti-KIR4.1 antibody.
According to a fourth aspect, the present invention relates to use of a peptide or
peptidomimetic of the first aspect in the manufacture of a medicament for the treatment
or prevention of multiple sclerosis.
According to a fifth aspect, the present invention relates to a composition comprising a
peptide or peptidomimetic of the first aspect.
According to a sixth aspect, the present invention relates to a method of screening for a
drug or lead compound, said method comprising bringing into contact a complex
comprising or consisting of:
(a) an anti-KIR4.1 antibody comprised in a sample from a patient having
multiple sclerosis or a predisposition thereto; and
(b) a peptide or peptidomimetic of the first aspect,
with a test compound,
wherein a reduction of the amount of said complex is indicative of the test compound
being a drug or lead compound suitable for the treatment or prevention of, or for the
development of drugs suitable for the treatment or prevention of, multiple sclerosis.
According to a seventh aspect, the present invention relates to use of a peptide or
peptidomimetic of the first aspect for removing anti-KIR4.1 antibodies from, or reducing
the amount thereof in, blood or serum of a multiple sclerosis patient or of a subject
carrying a predisposition to develop multiple sclerosis, wherein said use is to be
effected ex vivo.
According to an eighth aspect, the present invention relates to an ex vivo method of
removing anti-KIR4.1 antibodies from, or reducing the amount thereof in, blood or
serum of a multiple sclerosis patient or a subject with a predisposition to develop
- 2b -
multiple sclerosis, said method comprising bringing blood previously obtained from said
patient or said subject into contact with a peptide or peptidomimetic of the first aspect.
According to a ninth aspect, the present invention relates to a carrier with a receptor
being immobilized thereon, wherein said receptor is a peptide or peptidomimetic of the
first aspect.
According to a tenth aspect, the present invention relates to a device for removing antiKIR4.1 antibodies from blood, said device comprising the carrier of the ninth aspect.
According to an eleventh aspect, the present invention relates to a use of a peptide or
peptidomimetic of claim 1 in the preparation of a medicament for removing anti10 KIR4.1antibodies from, or reducing the amount thereof in, blood or serum of a multiple
sclerosis patient or of a subject carrying a predisposition to develop multiple sclerosis
Unless the context clearly requires otherwise, throughout the description and the
claims, the words “comprise”, “comprising”, and the like are to be construed in an
inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the
sense of “including, but not limited to”.
The present invention provides in another aspect a peptide comprising or consisting of
at least 8 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 3,
provided that said peptide does not consist of the sequence set forth in SEQ ID NO: 3,
or a corresponding peptidomimetic, wherein said peptide or peptidomimetic binds to an
anti-KIR4.1 antibody comprised in a sample from a patient, said patient having multiple
sclerosis or a predisposition therefor, wherein preferably (i) said at least 8 consecutive
amino acid residues are a subsequence of an extracellular domain of KIR4.1, said
extracellular domain consisting of the sequence set forth in SEQ ID NO: 1 or 2; or (ii)
said peptide comprises or consists of the sequence of SEQ ID NO: 1 or 2. Accordingly,
preferred are also peptidomimetics corresponding to the peptides according to options
(i) and (ii). It is also preferred that said peptide does not comprise the sequence of SEQ
ID NO: 3.
“KIR4.1” is a shorthand designation of a specific inward rectifying potassium channel.
Preferably, said KIR4.1 is of human origin. The sequence of human KIR4.1 is provided
in SEQ ID NO: 3. The terms “the sequence of human KIR4.1 protein” and “the
sequence of SEQ ID NO: 3” are used herein to characterize the same entity.
- 2c -
The recited anti-KIR4.1 antibody is also referred to as “autoantibody” according to the
invention. The autoantibody is a naturally occurring antibody and is preferably an IgG
antibody. In particular, the autoantibody is the anti-KIR4.1 antibody occurring in MS
patients and subjects having a predisposition for developing MS and is to be
distinguished from any other anti-KIR4.1 antibodies which do not occur in said patients
or subjects as well as further antibodies which may be used for therapeutic purposes as
disclosed herein below. Said latter types of anti-KIR4.1 antibodies are not naturally
occurring and not indicative of the disease. The autoantibody preferably binds to an
extracellular domain of KIR4.1, said extracellular domain consisting of the sequence set
forth in
SEQ D NO: 1.
Preferred subsequences of the sequences of SEQ ID NOs: 1 and 2 are the sequences of
SEQ ID NOs: 4 and 5 , respectively. The sequences of SEQ ID NOs: 4 and 5 are expected
to be extracellular in their entirety. Therefore, it is also preferred that said peptide comprises
or consists of the sequence of SEQ ID NO: 4 or 5. Also, a peptidomimetic corresponding to
the latter peptide is deliberately envisaged.
While peptides having sequences comprising or consisting of the sequence of any one of
SEQ ID NOs: 1, 2, 4 or 5 are preferred, further peptide sequences are envisaged, wherein
said peptide sequences may only partially or not at all overlap with the sequences of any
one of SEQ ID NOS: 1, 2, 4 and 5. To explain further, and as is known in the art, T-cells and
B-cells may have different epitope preferences within a given antigen.
The term "peptide" refers to a polycondensate of amino acids. Preferably, said amino acids
are selected from the 20 naturally occurring amino acids. The peptide according to the
invention has a length of at least 8 amino acid residues. Preferred upper limits for the length
of said peptide are 100, 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10 or 9 amino acid residues
and, in its broadest form, no upper length limit and accordingly includes polypeptides of any
length. Preferably, the length of said peptide is chosen such that it is unique. As detailed
further below, the length is preferably chosen such that the peptide is capable of binding to
an MHC molecule. In particular, MHC I molecules are known to generally impose certain
size limits on peptides being capable of binding thereto. Accordingly, preferred lengths and
length ranges are from 8 to 12, from 8 to 10, and most preferred 9 amino acids. MHC II
molecules on the other hand are generally capable of binding peptides of larger lengths as
well and accordingly do not impose upper limits on the length of peptide according to the
invention.
The term "peptidomimetics" is well-known in the art. It refers to derivatives of peptides, said
derivatives being defined in structural terms further below. A "corresponding
peptidomimetic" is a peptidomimetic which binds to the recited antibody. Such binding may
be achieved by retaining structural features of each constituent amino acid of the peptide it
is derived from, such parent peptide binding to the recited antibody as well. In a preferred
embodiment, each of the side chains of said at least eight consecutive amino acid residues
is retained in said corresponding peptidomimetic in modified or unmodified form. Side chain
modifications include the replacement of one or more hydrogen atoms with halogen atoms,
preferably F atoms. Further preferred side chain modifications include cyclisations.
Independent thereof, one or more main chain peptide bonds may independently be
replaced with functional groups which are isosteric or, in other words, mimic the peptide
bond. Preferably, a peptide bond -CO-NH- may be replaced with any one of -NH-CO-, -CH-
(OH)-CH2
-, -CO-CH2
-, -CH2
-NH-, -CH2
, -CH2
-CH2
-, -CH=CH-, -CO-N(CH 3
)-, and
Ρ0 2
-Χ-, X preferably being selected from NH, O and CH2
. As an example, in a
corresponding peptidomimetic, all peptide bonds of the parent peptide may be replaced with
retro-inverso bonds (-NH-CO-).
It is understood that substantially unaltered functional properties of the parent peptide are
inherent to a peptidomimentic of the invention. In particular, a corresponding peptidomimetic
binds to an anti-KIR4.1 antibody comprised in the sample of a patient with multiple
sclerosis. Such binding can be assessed without further ado using means and methods
described herein.
It is understood that the first aspect of the present invention relates to a peptide on the one
hand, and, in the alternative, to a peptidomimetic. The disclaimer removes the amino acid
sequence consisting of the sequence set forth in SEQ ID NO: 3 from the definition of the
peptide. In other words, the first aspect relates to (a) a peptide comprising or consisting of
at least 8 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 3,
provided that said peptide does not consist of the sequence set forth in SEQ ID NO: 3 , or
(b) a corresponding peptidomimetic, wherein said peptide or peptidomimetic binds to an
anti-KIR4.1 antibody comprised in a sample from a patient, said patient having multiple
sclerosis or a predisposition therefor, wherein preferably (i) said at least 8 consecutive
amino acid residues are a subsequence of an extracellular domain of KIR4.1 , said
extracellular domain consisting of the sequence set forth in SEQ ID NO: 1 or 2; or (ii) said
peptide comprises or consists of the sequence of SEQ ID NO: 1 or 2 .
The peptide or peptidomimetic according to the invention bind to an anti-KIR4.1 antibody,
wherein said anti-KIR4.1 antibody is comprised in a sample of a patient with multiple
sclerosis (MS) or a subject having a predisposition to develop MS. As further detailed
below, the present invention provides various means and methods for determining whether
an MS patient carries anti-KIR4.1 autoantibodies as well as for isolating such antibodies
from an MS patient. Said means include the agents generally referred to as "receptors"
herein. Methods for isolating autoantibodies according to the invention include the step of
bringing into contact said receptors with a sample obtained from a subject, the sample
being suspected of containing anti-KIR4.1 antibodies. Using such anti-KIR4.1 antibodies
obtained from an MS patient, the skilled person can determine without further ado whether
a peptide comprising or consisting of at least 8 consecutive amino acid residues of KIR4.1
or a corresponding peptidomimetic is capable of binding to the antibody or not. A preferred
means the skilled person can use is an ELISA assay. In such an assay, said peptide or
peptidomimetic according to the main embodiment is immobilized on a carrier, the
autoantibody from an MS patient or a subject having a predisposition for MS is allowed to
bind the peptide or peptidomimetic, and said binding is detected by means of a secondary
antibody which in turn is enzyme-linked. Said secondary antibody may, for example, be an
antibody capable of binding F fragments and accordingly would bind the F part of the
autoantibody.
Related to the above, the present invention furthermore provides an anti-KIR4.1 antibody
obtainable from a multiple sclerosis patient or a subject having a predisposition to develop
MS. This is the autoantibody defined herein above.
As described in more detail in the examples enclosed herewith, at least two extracellular
domains are present in KIR4.1 . The two extracellular domains are also referred to as large
and small extracellular domain. An anti-KIR4.1 antibody indicative of multiple sclerosis
preferably binds to the large extracellular domain, the small extracellular domain or both
extracellular domains. The sequences of large and small extracellular domain of KIR4.1 ,
respectively, are provided in SEQ ID NOs: 1 and 2. The strictly extracellular parts thereof
are provided in SEQ ID NOs: 4 and 5 , respectively.
The term "multiple sclerosis" refers to an inflammatory disease affecting the nervous
system; see also the literature quoted in the background section above. Whether or not a
subject or patient has multiple sclerosis can be determined with the method of diagnosing
according to the invention which is subject of the second aspect of the invention and
described further below. Alternatively or in addition, a diagnosis of multiple sclerosis can be
established on the basis of established clinical symptoms, said clinical symptoms being
known to the skilled person. The clinical symptoms of multiple sclerosis include vision
problems, dizziness, vertigo, sensory dysfunction, weakness, problems with coordination,
loss of balance, fatigue, pain, neurocognitive deficits, mental health deficits, bladder
dysfunction, bowel dysfunction, sexual dysfunction, heat sensitivity.
While a detection of anti-KIR4.1 autoantibodies in a sample taken from a patient or a
subject indicates multiple sclerosis or a predisposition therefor, it has to be understood that
multiple sclerosis or a predisposition therefor is not necessarily characterized in that said
autoantibodies are present in said subject or patient or a sample taken therefrom.
Accordingly, the presence of anti-KIR4.1 autoantibodies defines a subgroup of individuals
having a predisposition to develop MS, said subgroup being characterized in that they have
said autoantibodies. Similarly, a subgroup of MS patients is disclosed herein, said subgroup
being characterized in that they have said autoantibodies. In other terms, presence of the
autoantibody defines a sub-indication within the indication which is multiple sclerosis. It is
expected that patients exhibiting this sub-indication of MS respond differently to treatment
when compared to MS patients which do not have said autoantibodies. Similarly, it is
expected that the risk profile of subjects having said autoantibodies differs from the risk
profile of subjects which do not have said autoantibodies. As a consequence, different
curative treatments as well as different preventive treatments may be chosen in
dependence of whether an MS patient has autoantibodies or not, and whether a subject at
risk of developing MS has said autoantibodies or not, respectively.
The term "subsequence" refers to a stretch of contiguous amino acid residues taken from a
larger sequence. In other words, if said larger sequence consists of n residues, the maximal
length of a subsequence is (n - ) residues.
The present invention furthermore provides a nucleic acid encoding the above defined
peptide according to the invention. The nucleic acid may be DNA, such as cDNA or
genomic DNA, or RNA. Furthermore provided is a vector comprising said nucleic acid.
Moreover, the present invention relates to a host cell comprising a nucleic acid and/or
vector according to the invention. The host cell may be of any origin and is preferably in
vitro such as isolated or in culture. While it is noted that human embryonic stem cell lines
are at the skilled person's disposal, it is preferred that the host cell is not obtained by using
or destroying human embryos. Also, it is preferred that the host cell, to the extent it is an
embryonic cell or an embryonic stem cell, is non-human.
In a second aspect, the present invention provides a method for diagnosing multiple
sclerosis or a predisposition for multiple sclerosis in a subject, the method comprising
determining the presence of an anti-KIR4.1 antibody in a sample obtained from said
subject, wherein the presence of an anti-KIR4.1 antibody in said sample is indicative of
multiple sclerosis or a predisposition for multiple sclerosis.
This method permits to diagnose multiple sclerosis, or, to the extent multiple sclerosis is not
apparent in said subject, for diagnosing a predisposition therefor. The term "predisposition"
has the meaning as established in the art and prefers a likelihood to develop a disease. In
particular, said likelihood is higher than in a normal control subject. Said likelihood in a
normal control subject may be represented as the average likelihood to develop MS in a
random sample from the population.
Suitable agents for determining said presence of an anti-KIR4.1 antibody are described
further below, in particular as active agents in relation to the disclosed diagnostic
compositions and diagnostic uses.
A preferred group of individuals to be tested for said predisposition are individuals with a
history of MS in the family.
The present inventors are the first ones to identify a molecular target of the previously
suspected autoimmune response in MS. It is noteworthy that conventional strategies to
uncover autoantibodies in MS have largely focused on serological screening for
immunoglobulins to preselect candidate target molecules based on their functional
relevance to myelin biology and encephalitogenic potential in animal models ( 13). Also, E.
coli expression, phage display and peptide libraries were screened to identify linear targets
of MS specific autoantibodies (14-1 7). Neither strategy has yet yielded any potential targets
that could either be MS-specific or prognostic (18, 19).
The present inventors detected high titers of anti-KIR4.1 antibodies in patient sera of 50.8%
of two independent cohorts. Accordingly, the means and methods described herein allow
diagnosis of MS or a predisposition therefor in about half of the MS cases or subjects being
at risk to develop the disease, respectively. In particular, the methods of the invention
permit early diagnosis of MS or a predisposition therefor or a confirmation of an uncertain
diagnosis. The antibody test may allow to diagnose CIS or MS without invasive procedures
(such as cerebrospinal fluid analysis) and to diagnose MS, CIS or predisposition to MS
earlier than this would be possible by diagnostic procedures known in the art. "CIS" refers to
"clinically isolated syndrome" and is discussed further below. It is well known that MS
therapy works best when started as early as possible during the course of disease.
Therefore early diagnosis may allow to implement early treatment of patients with CIS, MS
or at risk to develop these diseases. In some individuals at risk treatment may even prevent
the (further) development of disease.
As shown in the Examples enclosed herewith, the autoantibodies may deplete KIR4.1
expressing glial cells via antibody dependent cell-mediated cytoxicity (ADCC) or
complement activation (Figure 6). In addition, the antibody may interfere with the function of
the potassium channel resulting in functional consequences for ion buffering and
neurotransmitter homeostasis (20, 2 1, 22). This may result in tissue injury or impaired
remyelination.
In a preferred embodiment of the methods according to the invention, and in case an antiKIR4.1 antibody is present in said sample, (i) presence of at least one clinical symptom of
multiple sclerosis in said subject is indicative of multiple sclerosis; and (ii) absence of any
clinical symptom of multiple sclerosis is indicative of said predisposition for multiple
sclerosis.
As disclosed above, the methods according to the invention provide for diagnosing multiple
sclerosis as well as for diagnosing a predisposition therefor. The present preferred
embodiment provides for further information to be acquired for said subject, said further
information aiding in distinguishing between diagnosis of the disease and diagnosis of a
predisposition therefor. In particular, said further information consists of or comprises at
least one clinical symptom of multiple sclerosis. Multiple sclerosis is a well-known disease
with established clinical symptoms. The skilled person is well aware of clinical symptoms
being characteristic or indicative of multiple sclerosis (see also further below) and can
determine the presence or absence thereof without further ado.
In accordance with the present preferred embodiment, the absence of any clinical symptom
of multiple sclerosis, when concomitantly occurring together with the presence of antiKIR4.1 antibodies, is indicative of predisposition for multiple sclerosis. In other words, where
established methods of diagnosis or prognosis fail, the present invention allows to identify
those subjects which exhibit an elevated risk of developing multiple sclerosis at some point
in the future.
On the other hand, in subjects where at least one clinical symptom of multiple sclerosis is
present, the determination of anti-KIR4.1 antibodies further corroborates the diagnosis of
multiple sclerosis. In those cases where the clinical parameters alone do not permit a clear
diagnosis, the present invention aids in performing and substantiating said diagnosis. This
applies in particular to early forms of multiple sclerosis. As is well-known in the art, an early
diagnosis of multiple sclerosis is highly desirable, given that early stages are generally more
amenable to treatment.
According to a further preferred embodiment, said clinical symptom is at least one selected
from vision problems, dizziness, vertigo, sensory dysfunction, weakness, problems with
coordination, loss of balance, fatigue, pain, neurocognitive deficits, mental health deficits,
bladder dysfunction, bowel dysfunction, sexual dysfunction, heat sensitivity, the presence of
(an) inflammation marker(s) in cerebrospinal fluid (CSF), the presence of lesions of the
brain and/or the spinal cord. The mentioned lesions may be detected in an MRT image.
Typically, such lesions occur in the periventricular, juxtacortical and/or infratentorial region
of the brain. Inflammation markers indicative of MS are well-known in the art and are
preferably to be selected from pleocytosis (abnormally increased number of cells in the
CSF, wherein typical values of increased cell numbers are between 5 and 50 cells/ µ Ι or
above), intrathecal IgG synthesis and the occurrence of oligoclonal IgG bends in the CSF.
According to a further preferred embodiment, said subject has clinically isolated syndrome
(CIS), or said at least one clinical symptom is CIS. CIS is generally perceived in the art as
being an early stage of MS, wherein the clinical parameters characteristic of the latter are
not yet fully developed. For a discussion of CIS, see, for example, Thrower, Neurology 68,
S12-S15 (2007). The means and methods according to the present invention are
advantageous in that they permit collection of further evidence for those patients which
have CIS.
According to a further preferred embodiment, said anti-KIR4.1 antibody, i.e., the anti-KIR4.1
antibody which may occur in MS patients as well as subjects being at risk to develop MS,
binds to KIR4.1 (SEQ ID NO: 3) or an extracellular domain of KIR4.1 consisting of the
sequence set forth in any one of SEQ ID NOs: 1, 2, 4 or 5. The structure of KIR4.1 is further
described in the examples enclosed herewith. In particular, it comprises (at least) two
extracellular domains which are presumably separated by one transmembrane spanning
segment; see Figure 4c. The two extracellular domains are herein also referred to as large
extracellular domain and small extracellular domain and are set forth in SEQ ID NOs: 1 and
2 . The residue ranges indicated in Figure 4c are those of SEQ ID NOs: 4 and 5 ,
respectively.
In a further preferred embodiment, the detection of the anti-KIR4.1 antibody in said sample
is effected by a method selected from the group consisting of ELISA, immunoprecipitation,
Western blotting, immunofluorescence, immunohistochemistry, flow cytometry,
metalloimmunoassay (such as GLORIA), fluorescence resonance energy transfer (FRET)
assay and mass spectroscopy. These methods are well-established and at the skilled
person's disposal. For example, in an ELISA assay, an antibody binding to said anti-KIR4.1
antibody may be used. Similar considerations apply to immunoprecipitation, Western
blotting, immunofluorescence and immunohistochemistry. As noted above, the skilled
person, when provided with the teaching of the present invention, can isolate and
characterize the anti-KIR4.1 antibody without further ado. Such characterization preferably
uses mass spectrometry. Once being characterized, mass spectrometry may be used for
determining presence or absence of anti-KIR4.1 antibodies in any given sample. FRET
assays may be used, for example, in the context of a binding assay, said binding assay
preferably making use of a receptor, said receptor being defined further below. Such FRET
assay may be designed such that a detectable transfer between donor and acceptor of the
FRET pair only occurs in case receptor and anti-KIR4.1 antibody are in close special
proximity, said close special proximity being indicative of the presence of the anti-KIR4.1
antibody.
In further preferred embodiments, the presence of said anti-KIR4.1 antibody is determined
by (a) contacting the sample with a receptor binding to said anti-KIR4.1 antibody; and (b)
detecting the formation of a receptor-anti-KIR4.1 antibody complex, wherein said receptor is
preferably selected from the group consisting of a peptide or peptidomimetic according to
the invention, KIR4.1 protein (SEQ ID NO: 3) and an antibody binding to said anti-KIR4.1
antibody. As described further below, means and methods for preparing an antibody against
a given antigen (including an antibody) are at the skilled person's disposal.
Whereas the previous preferred embodiment provides various readout schemes, the
present preferred embodiment provides specific means for effecting detection of anti-KIR4.1
antibodies, said specific means being characterized in structural terms. Accordingly, these
preferred embodiments - as well as any other embodiments disclosed herein - are
amenable to combination if not indicated otherwise, any of said combinations being the
subject of further preferred embodiments according to the present invention. In a preferred
assay, KIR4.1 protein is expressed in cells, said cells are incubated with serum, and binding
of the autoantibody to KIR4.1 protein is determined by means of flow cytometry or
immunohistochemistry using a secondary antibody. As stated above, said secondary
antibody preferably binds to said autoantibody, for example by binding to the F part
thereof. Further preferred assays are described in the examples enclosed herewith.
Preferred embodiments of the recited receptor are a peptide or peptidomimetic according to
the invention and an antibody binding to said anti-KIR4.1 antibody. In either case, it is
furthermore preferred that said receptor is specific for said anti-KIR4.1 antibody. Specificity
can be determined in comparative or competition assays, wherein binding of the receptor to
said anti-KIR4.1 antibody on the one hand and to other proteins, binding proteins or
antibodies is determined. Preferably, the binding constant (K^) of the receptor for the
autoantibody is at least one order of magnitude, preferably at least two, three, four, five or
six orders of magnitude lower than for the other proteins tested. An "order of magnitude" is
a factor of 10.
Therapeutic or diagnostic antibodies as disclosed herein may be monoclonal or polyclonal
antibodies. Furthermore, and in particular in the context of diagnostic and therapeutic
antibodies as disclosed herein, the term "antibody" furthermore includes single chain
antibodies or fragments thereof that specifically bind to their respective target as well as
bispecific antibodies, synthetic antibodies, antibody fragments such as Fab, F(ab2
) Fv and
scFv fragments and the like as well as chemically modified derivatives thereof.
Monoclonal antibodies can be prepared, for example, by the techniques as originally
described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73
(1981 ), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from
immunized mammals with modifications developed by the art. Furthermore, antibodies or
fragments thereof to the aforementioned peptides can be obtained by using methods which
are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press,
Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage
display technique, surface plasmon resonance as employed in the BIAcore system can be
used to increase the efficiency of phage antibodies which bind to an epitope of the peptide
or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105;
Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is
described, for example, in WO89/09622. A further source of antibodies to be utilized in
accordance with the present invention are so-called xenogenic antibodies. The general
principle for the production of xenogenic antibodies such as human antibodies in mice is
described in, e.g., WO 91/10741 , WO 94/02602, WO 96/34096 and WO 96/33735.
Antibodies to be employed in accordance with the invention or their corresponding
immunoglobulin chain(s) can be further modified using conventional techniques known in
the art, for example, by using amino acid deletion(s), insertion(s), substitution(s),
addition(s), and/or recombination(s) and/or any other modification(s) known in the art either
alone or in combination. Methods for introducing such modifications in the DNA sequence
underlying the amino acid sequence of an immunoglobulin chain are well known to the
person skilled in the art; see, e.g., Sambrook, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
The term "monoclonal" or "polyclonal antibody" (see Harlow and Lane, (1988), loc. cit.) also
relates to derivatives of said antibodies which retain or essentially retain their binding
specificity. Whereas particularly preferred embodiments of said derivatives are specified
further herein below, other preferred derivatives of such antibodies are chimeric antibodies
comprising, for example, a mouse or rat variable region and a human constant region.
The term "scFv fragment" (single-chain Fv fragment) is well understood in the art and
preferred due to its small size and the possibility to recombinantly produce such fragments.
In a particularly preferred embodiment of the method of the invention, said antibody or
antibody binding portion is or is derived from a human antibody or a humanized antibody.
The term "humanized antibody" means, in accordance with the present invention, an
antibody of non-human origin, where at least one complementarity determining region
(CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been
replaced by CDRs of an antibody of human origin having a desired specificity. Optionally,
the non-human constant region(s) of the antibody has/have been replaced by (a) constant
region(s) of a human antibody. Methods for the production of humanized antibodies are
described in, e.g., EP-A1 0 239 400 and WO90/07861 .
In a further preferred embodiment, said sample is selected from blood, serum, plasma,
lymph nodes, CSF, lacrimal fluid, urine, sputum and brain biopsy.
In a third aspect, the present invention provides a receptor as defined above for use in the
treatment of multiple sclerosis or in the diagnosis of multiple sclerosis or a predisposition
therefor. The receptor as defined herein above as well as the entire KIR4.1 protein (i.e. the
protein consisting of the sequence set forth in SEQ ID NO: 3) are envisaged for these
medical uses. The administration of these agents serves to reduce the number of circulating
anti-KIR4.1 antibodies. As will be described in more detail below, these agents may not only
be administered to a patient suffering from MS or to a subject having or being suspected of
having a predisposition therefor, but may also be used in ex vivo methods, said ex vivo
methods providing for the removal of the autoantibodies from an MS patient or, more
specifically, from a bodily fluid of said patient or a subject at risk to develop MS.
The term "treatment" refers to treatment by therapy and embraces amelioration of the
disease and/or its symptoms as well as complete remission. Furthermore, the term
"treatment" extends to prevention.
In preferred embodiments of the medical uses according to the present invention, one or
more of the recited agents are the only active agents to be used. In alternative preferred
embodiments, one or more of said explicitly recited agents may be used in conjunction with
one or more of agents known to be beneficial to MS patients or known to aid in diagnosis of
MS. Until now, progression of MS is prevented or mitigated and relapses are prevented by
administration of one or more of the following: Interferon-beta, Glatirameracetate,
Natalizumab, Mitoxantrone, Fingolimod, Azathiophne. Relapses are treated with high doses
of Methlprednisolone and/or plasma exchange treatment.
In preferred embodiments, said receptor is to be contacted with blood, serum, plasma,
lymph nodes, CSF, lacrimal fluid, urine, sputum and/or brain biopsy obtained from a subject.
This embodiment refers to preferred samples which are to be used in diagnosis.
In a further preferred embodiment of the third aspect of the invention, to the extent said third
aspect relates to therapy, said receptor is to be administered to the patient at least two
times. Multiple administration provides for high degrees of removal, preferably complete
removal of autoantibodies and eventually for amelioration or remission of the disease.
In a further preferred embodiment, said peptide may be chosen such that it binds to an
MHC allele of the patient to be treated. Said MHC molecule may be an MHC class I or class
II molecule. In order to ensure binding to an MHC class I molecule, it is preferred that said
peptide consists of 8 to 12, preferably of 8 to 10 and most preferred of 9 amino acids.
Furthermore, it is preferred that anchor amino acid residues, said anchor amino acid
residues being residues known to be involved in MHC binding, are present. Selection of
suitable peptide sequences within the SEQ ID NOs: 1 and 2 are well within the skills of the
skilled person. For example, Rammensee et al. (Immunogenetics, 4 1: 178-228, 1995)
describes features including anchor amino acids of MHC binding peptides. Presenting of
said peptide in an MHC context allows to target an additional or alternative mechanism in
the treatment or prevention of MS. More specifically, it permits the drive T-cells specific for
KIR4.1 into apoptosis, thereby reducing or abolishing the autoimmune reaction to
endogenous KIR4.1 protein. In a yet further preferred embodiment, MHC-peptide
complexes for use in the treatment of multiple sclerosis are provided. Particularly preferred
is the use of MHC multimers such as MHC tetramers, wherein preferably each MHC
molecule has a peptide or peptidomimetic according to the invention bound. Preferably, said
peptide or peptidomimetic is the same for all MHC molecules of said multimer or tetramer.
As is known in the art, tetramer formation may be achieved by using biotinylated MHC
molecules. The carboxy terminus of an MHC molecule is a preferred target of biotenylation.
When incubated with a streptavidin, tetramers are formed because streptavidin has four
biotin binding sites. Such multimers or tetramers bind antigen-specific T-cell receptors with
particularly high affinity. Therefore, use of multimers, preferably tetramers of peptide bound
MHC molecules permits identification and furthermore inactivation of KIR4.1 -specific Tcells.
Furthermore, and without being bound by specific theory, it is envisaged that the
administration of peptides or peptidomimetics according to the invention permits
desensitisation of T-cells and B-cells responsible for the disease. In this regard, one or
more positions of said peptide or peptidomimetic, said positions interacting with the T-cell
receptor, may be modified for the purpose of fine-tuning T-cell receptor interaction of said
peptide or peptidomimetic. Such positions and approaches for their modification are known
in the art; see, for example, Kappos et al., Nature Med. 6, 1176-1 182 (2000).
In other words, the invention provides means and methods for induction of tolerance of
KIR4.1. The term "tolerance" has the meaning as established in the art and refers to a nonreactivity of the immune system to a given antigen. Typically, there is tolerance with regard
to self antigens. In the absence of tolerance to a self antigen, autoantibodies may be
generated and an autoimmune disease may arise. As is apparent from the disclosure of this
invention, multiple sclerosis has characteristics of an autoimmune disease. In order to
reduce or abolish the generation of autoantibodies against the KIR4.1 protein, induction of
tolerance or desensitisation is one of the preferred approaches. The envisaged effect is the
establishment of self-tolerance with regard to KIR4.1. Since tolerance is an antigendependent effect, it can exist in B-cells, T-cells or both B-cells and T-cells. The
phenomenon of tolerance as such as well as the mechanisms underlying B-cell and T-cell
tolerance are known in the art.
In a fourth aspect, the present invention provides an antibody binding to KIR4.1 (SEQ ID
NO: 3) or an extracellular domain of KIR4.1 for use in the treatment of multiple sclerosis,
said domain consisting of the sequence set forth in SEQ ID NO: 1 or 2, and said antibody
interfering with the binding to KIR4.1 of an anti-KIR4.1 antibody comprised in a sample from
a patient, said patient having multiple sclerosis or a predisposition therefor.
Preferably, said antibody binding to an extracellular domain of KIR4.1 is specific therefor.
Means and methods for determining specificity of antibodies are at the skilled person's
disposal and described herein above. It is particularly preferred that the above antibody to
be used in therapy is capable of binding to an epitope which is not recognized by any
autoantibody. In this regard, it is noted that means and methods for epitope mapping are at
the skilled person's disposal. By choosing an appropriate epitope, the above mentioned
functional requirements, i.e., interference with binding of the autoantibodies, can be
ensured. It is furthermore preferred that binding of the therapeutic antibody does not or not
significantly interfere with the biochemical or cellular function of KIR4.1 . KIR4.1 is known to
be involved in homeostasis of water and potassium ions in the central nervous system.
Maintaining or re-establishing the function of KIR4.1 is therefore envisaged to exhibit a
protective effect for neurons and clear cells.
These embodiments provide means of interfering with binding of the autoantibodies to their
cognate target and thereby alleviating the disease.
In a fifth aspect, the present invention provides a composition comprising (i) a peptide of the
invention, (ii) an antibody binding to an anti-KIR4.1 antibody as defined above (i.e., an
antibody binding to an autoantibody), and/or (iii) an antibody of the preceding embodiments,
i.e., an antibody interfering with the binding of the autoantibody to its target.
Accordingly, the present disclosure refers to three distinct types of antibodies. First, there is
disclosure of the autoantibody binding to an extracellular loop of KIR4.1 . This is the
antibody considered causative and/or indicative of MS in about half of the MS patients.
Secondly, the invention provides an antibody capable of binding to the autoantibody. This
second type of antibody is suitable for both therapeutic and diagnostic purposes detailed
herein above. Finally, the invention provides an antibody which binds to KIR4.1 and at the
same time interferes with the binding of the autoantibody to its target. This latter antibody is
suitable for therapeutic purposes described herein above.
Preferred embodiments of the composition according to the invention relate to a
pharmaceutical composition optionally further comprising a pharmaceutically acceptable
carrier and/or diluent, and, to the extent said composition relates to a peptide or antibody
binding to an anti-KIR4.1 antibody, to a diagnostic composition. Suitable pharmaceutically
acceptable carriers, excipients, and/or diluents can be chosen by the skilled person without
further ado. For example, the antibody may be provided in solution such as buffered
solution.
Buffers are well known in the art and the skilled person is aware of appropriate buffers in
dependency of the substances being assayed. Common buffers comprise (pKa
values in
brackets) H3
P0 / NaH2
P0 4
(pKa
. = 2.12), Glycine (pK = 2.34), Acetic acid (4.75), Citric
acid (4.76), MES (6.15), Cacodylic acid (6.27), H2
C0 3
/ NaHC0 3
(pK = 6.37), Bis-Tris
(6.50), ADA (6.60), Bis-Tris Propane (pK3
= 6.80), PIPES (6.80), ACES (6.90), Imidazole
(7.00), BES (7.15), MOPS (7.20), NaH2
P0 4
/ Na2
HP0 4
(pKa,2 = 7.21), TES (7.50), HEPES
(7.55), HEPPSO (7.80), Triethanolamine (7.80), Tricine (8.10), Tris (8.10), Glycine amide
(8.20), Bicine (8.35), Glycylglycine (pKa,2 = 8.40), TAPS (8.40), Bis-Tris Propane (pKa
,
2
=
9.00), Boric acid (H3
B0 3
/ Na2
B4
0 7
) (9.24), CHES (9.50), Glycine (pKa,2 = 9.60), NaHC0 3
/
Na2
C0 3
(pKa,2 = 10.25), CAPS (10.40) and Na2
HP0 4
/ Na3
P0 4
(pKa,3 = 12.67).
Furthermore, ionic strength of said buffer may be adjusted, e.g., by the addition of sodium
chloride and/or potassium chloride. Preferred concentrations of sodium chloride are
between 0 and 2 M , preferably between 100 and 200 mM. Examples of buffers comprising
sodium chloride include PBS (phosphate buffered saline) containing 1.37 M NaCI, 27 mM
KCI, 43 mM Na2
HP0 4
-7H2
0 and 14 mM KH2
P0 4
in the 10-fold aqueous stock solution,
which is adjusted to pH 7.3; SSC containing 3 M NaCI and 0.3 M sodium citrate in 20-fold
aqueous stock solution, which is adjusted to pH 7.0; and STE (Saline Tris EDTA) containing
mM Tris base, 10 mM NaCI and 1mM ETA (acid). Alternatively, sodium chloride is
absent from the buffer preparation. Examples for common buffer preparations without
sodium or potassium chloride are TAE (Tris acetate EDTA) containing 2 M Tris acetate and
0.1 M EDTA in the 50-fold aqueous stock solution at pH 8.5; TBE (Tris borate EDTA)
containing 0.89 M Tris base, 0.89 M Boric acid and 0.02 M EDTA in the 10-fold aqueous
stock solution at pH 8.0; and TE (Tris EDTA) containing 10 mM Tris base and 1 mM EDTA
(acid) at pH 7.5.
The pharmaceutical compositions described herein can be administered to the subject at a
suitable dose. Administration of the suitable compositions may be effected by different
ways, e.g., by intravenous, intraperitoneal, subcutaneous, as well as transdermal
administration.
More specifically, the pharmaceutical compositions may be administered orally,
parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally,
intrathecally, transdermally, transmucosally, subdurally, nasal, locally or topically via
iontopheresis, sublingually, by inhalation spray, aerosol or rectally and the like in dosage
unit formulations optionally comprising conventional pharmaceutically acceptable
excipients.
The dosage regimen will be determined by the attending physician and clinical factors. As is
well known in the medical arts, dosages for any one patient depends upon many factors,
including the patient's size, body surface area, age, the particular compound to be
administered, sex, time and route of administration, general health, and other drugs being
administered concurrently.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as
ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or
fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like. Furthermore, the pharmaceutical
composition described herein may comprise further agents depending on the intended use
of the pharmaceutical composition.
Pharmaceutically useful excipients that may be used in the formulation may comprise
carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol,
isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil,
coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl
myristate; binders, adjuvants, solubilizers, thickening agents, stabilizers, disintergrants,
glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending
agents, sweetening agents, colourants, flavours, coating agents, preservatives,
antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium
phosphate, magnesium state, talc, monosaccharides, disaccharides, starch, gelatine,
cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl- βcyclodextrin, polyvinylpyrrolidone, low melting waxes, and/or ion exchange resins.
Other suitable pharmaceutically acceptable excipients are described in Remington ' s
Pharmaceutical Sciences, 15
th Ed., Mack Publishing Co., New Jersey (1991 ).
Dosage forms for oral administration include tablets, capsules, lozenges, pills, wafers,
granules, oral liquids such as syrups, suspensions, solutions, emulsions, powder for
reconstitution.
Dosage forms for local/topical administration comprise insufflations, aerosols, metered
aerosols, transdermal therapeutic systems, medicated patches, rectal suppositories, and/or
ovula.
For the purpose of the present invention, a therapeutically effective dosage of the recited
agents may generally be from about 2.5 to 100 mg/day, preferably from about 5 to about 50
mg/day, and most preferably from about 10 to about 30 mg/day, which may be administered
in one or multiple doses.
It will be appreciated, however, that specific dose level of the compounds of the invention
for any particular patient will depend on a variety of factors such as age, sex, body weight,
general health condition, diet, individual response of the patient to be treated time of
administration, severity of the disease to be treated, the activity of particular compound
applied, dosage form, mode of application and concomitant medication. The therapeutically
effective amount for a given situation will readily be determined by routine experimentation
and is within the skills and judgement of the ordinary clinician or physician.
In a sixth aspect, the present invention provides an agent selected from Rituximab,
Ocrelizumab, Ofatumumab and an Fc
-binding agent for use in the treatment of multiple
sclerosis in a patient, said patient being characterized by the presence of anti-KIR4.1
antibodies in any one of blood, serum, plasma, lymph nodes, cerebrospinal fluid (CSF),
lacrimal fluid, urine, sputum and brain biopsy. As explained herein above, it is expected that
MS patients with autoantibodies on the one hand and MS patients without autoantibodies
on the other hand respond differently to these agents.
Fc-binding agents are known agents suitable for the removal of antibodies. Such agents
bind to the F part of antibodies and are suitable for the removal or inactivation of
antibodies. Such process of removal is also referred to as immunoadsorption.
In a seventh aspect, the present invention furthermore provides a method of screening for a
drug or lead compound, said method comprising: bringing into contact a complex
comprising or consisting of (i) an anti-KIR4.1 antibody as defined above (i.e., an
autoantibody); and (ii) KIR4.1 protein or a peptide or peptidomimetic as defined above with
a test compound, wherein a reduction of the amount of said complex is indicative of the test
compound being a drug or lead compound.
In view of the surprising discovery that anti-KIR4.1 antibodies are involved in multiple
sclerosis, the present invention furthermore provides for means and methods of identifying
drugs and lead compounds, said drugs and lead compounds being suitable for the
treatment or for the development of drugs suitable for the treatment of multiple sclerosis. It
is understood that the recited bringing into contact is effected under conditions which
maintain said complex. The skilled person is aware of such suitable conditions which
include, for example, buffered solutions comprising the test compound, an anti-KIR4.1
antibody, and KIR4.1 protein or a peptide or peptidomimetic according to the invention.
Preferably, said method is effected in high-throughput format. High-throughput assays,
independently of being biochemical, cellular or other assays, generally may be performed in
wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of
the plates, including incubation at temperatures other than ambient temperature, and
bringing into contact of test compounds with the assay mixture is preferably effected by one
or more computer-controlled robotic systems including pipetting devices. In case large
libraries of test compounds are to be screened and/or screening is to be effected within
short time, mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be
added to each well. In case a well exhibits biological activity, said mixture of test
compounds may be de-convoluted to identify the one or more test compounds in said
mixture giving rise to said activity
Test compounds, lead compounds and/or drugs are preferably small molecules, more
preferred small organic molecules. The molecular weight is preferably below 2000, more
preferred below 1500, 1000, 900, 800, 700, 600, 500 or 400 Daltons. Any hit identified in
the screen may be subjected to an optimization of its pharmacological properties (including
absorption, distribution, metabolism and excretion), thereby developing the lead compound
into a drug.
Methods for the optimization of the pharmacological properties of compounds identified in
screens, generally referred to as lead compounds, are known in the art and comprise a
method of modifying a compound identified as a lead compound to achieve: (i) modified site
of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii)
decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or
(v) modified onset of therapeutic action, duration of effect, and/or (vi) modified
pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii)
modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability,
state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix)
optimized application form and route by (i) esterification of carboxyl groups, or (ii)
esterification of hydroxyl groups with carboxylic acids, or (iii) esterification of hydroxyl
groups to, e.g. phosphates, pyrophosphates or sulfates or hemi-succinates, or (iv) formation
of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable
complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of
hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side
chains, change of substituent pattern, or (ix) modification by introduction of isosteric or
bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of
branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii)
derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides,
phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of
ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines,
thiazolidines or combinations thereof.
The various steps recited above are generally known in the art. They include or rely on
quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and
Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical
chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker
Zeitung 140(8), 813-823, 2000).
The present invention, in an eighth aspect, provides the use of a receptor as defined above
for removing anti-KIR4.1 antibodies from blood or serum or reducing the amount thereof,
wherein said use is to be effected ex vivo.
Related thereto, the present invention provides an ex vivo method of removing anti-KIR4.1
antibodies from a bodily fluid such as blood or serum or reducing the amount thereof, said
method comprising
(a) bringing blood removed from a subject into contact with a receptor as defined above
and/or an Fc-binding agent; and/or
(b) performing plasmapheresis.
These aspects relate to ex vivo applications, said ex vivo applications aiming at a reduction
of a number of autoantibodies or a complete depletion thereof. Preferably, blood or serum
of an MS patient or of a subject carrying a predisposition to develop MS are subjected to the
ex vivo treatment. It is understood that said bringing into contact is effected under
conditions which allow binding of autoantibodies, if present, to said receptor. In one
embodiment, said conditions may be established by bringing into contact blood or serum
with a carrier or device according to the invention, said carrier or device being further
defined below. Plasmapheresis as such is known in the art and may be used, in accordance
with the invention, to reduce the number of circulating autoantibodies.
In a preferred embodiment of the ex vivo method according to the invention, the blood or
serum, after said bringing into contact, is to be returned to the same subject.
In further preferred embodiments of the ex vivo use or the ex vivo method of the invention,
said protein, peptide and/or antibody is bound to a carrier. Any carrier, including a solid
carrier is envisaged. Support or carrier materials commonly used in the art and comprising
glass, plastic, gold and silicon are envisaged for the purpose of the present invention.
Suitable coatings of the carrier or support, if present, include poly-L-lysine- and aminosilane-coatings as well as epoxy- and aldehyde-activated surfaces. In a preferred
embodiment, said carrier is the matrix of a column. Suitable matrices are known in the art
and may be derivatized by the attachment of said receptor.
The present invention furthermore relates to a carrier with a receptor as defined above
being immobilized thereon.
Related thereto, provided is also a device for removing anti-KIR4.1 antibodies from blood,
said device comprising the carrier as defined above.
In a preferred embodiment of the device, said device further comprises an inlet and/or an
outlet permitting to let blood or serum of the subject flow across the filter and/or the blood or
serum being returned to the same subject.
As regards the embodiments characterized in this specification, in particular in the claims, it
is intended that each embodiment mentioned in a dependent claim is combined with each
embodiment of each claim (independent or dependent) said dependent claim depends from.
For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a
dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1
and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification
unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A,
D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H ; B, D, I; B, E, G; B, E,
H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F,
G; C, F, H; C, F, I, unless specifically mentioned otherwise.
Similarly, and also in those cases where independent and/or dependent claims do not recite
alternatives, it is understood that if dependent claims refer back to a plurality of preceding
claims, any combination of subject-matter covered thereby is considered to be explicitly
disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring
back to claim , and a dependent claim 3 referring back to both claims 2 and , it follows
that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously
disclosed as is the combination of the subject-matter of claims 3 , 2 and 1. In case a further
dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the
combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims 4 , 3 and
1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.
The above considerations apply mutatis mutandis to all attached claims. To give a few
examples, the combination of claims 6, 5 , 4(b), 3 and 2 is clearly and unambiguously
envisaged in view of the claim structure. The same applies for the combinations of claims 6,
, 4(a), 3 and 2, and, to give a few further examples which are not limiting, the combination
of claim 4(a) and 2 and the combination of claim 5, 4(a) and 2.
The figures show:
Figure 1: MS Serum IgG reactivity with CNS membrane antigens
(a) Representative photomicrographs of immunofluorescence labeling performed on rat
cerebellar (upper panels) and human brain sections (lower panels) with serum IgG from
patients with MS or OND patients as indicated. Scale bars 100 pm (upper panels) and 20
pm (lower panels).
(b) Capture ELISA assay with membrane protein fractions prepared from rat brain tissue.
Serum reactivities in MS and OND patients are shown (OD, optical density).
Figure 2: Identification of KIR4.1 as target of serum IgG in MS
(a) One dimensional SDS gel electrophoresis (left) of human brain lysate precipitated with
pooled IgG from OND or MS patients. Note that unique bands (third lane) above and below
the IgG heavy chain band (arrow) were obtained after immunoprecipitation with pooled
serum IgG purified from MS patients. Two dimensional electrophoresis (right) of brain
antigens obtained after immunoprecipitation with serum IgG from MS patients. The spot
containing the KIR4.1 protein identified by MALDI-MS/MS analysis is marked with a frame.
The arrow marks the IgG heavy chain spot.
(b) KIR4.1 detection by Western blot analysis in various immunoprecipitates as indicated.
Immunoprecipitations were performed with serum IgG from OND or MS patients on
enriched membrane protein fractions of rat kidney and human brain lysates, respectively.
(c) Western blot analysis of KIR4.1 in immunoprecipitates of in vitro translated KIR4.1
protein with serum IgG from OND or MS patients.
Figure 3: Validation ofKIR4. 1 as the target of the serum IgG reactivity in MS patients
(a) Double immunofluorescence labeling showing co-localization of serum IgG from an MS
patient with monoclonal anti-KIR4.1 in rat brain cerebellum sections. Staining with serum of
an OND patient is shown as control. Scale bar 200 pm.
(b) Immunofluorescence labeling of cerebellar sections of wild type (left panels) and Kir4.
mice (right panels) with purified serum IgG from an MS patient. Scale bars 100 m (upper
panels) and 50 pm (lower panels).
(c) Double immunofluorescence staining of mouse primary astroglial cell cultures. Staining
with OND serum IgG (upper panels) and MS serum IgG (lower panels) was detected in the
green channel. Additional GFAP staining (right panels) was detected in the red channel.
(d) Staining and flow cytometric analysis of mouse primary astrocytes with serum from MS
and OND patients.
Figure 4: High titer serum reactivity to KIR4. 1 in a subset of MS patients.
(a) Protein based ELISA screening approach for anti-KIR4.1 serum reactivity. Purified
recombinant KIR4.1 from HEK293 cells was covalently coupled to the solid phase of ELISA
plates. Serum antibody binding to KIR4.1 was determined in HD, OND, and MS patients.
The frequency of antibody positive and negative sera were compared between HD (n=14),
OND (n=71) and MS patients (n=122) by Kruskal-Wallis test. The threshold for anti-KIR4.1
antibody positivity (cut off OD 0.866, 5 SD above median OD of HD subjects) is indicated by
a dashed horizontal line.
(b) ROC curves depicting the diagnostic performance of the anti-KIR4.1 antibody ELISA test
in two independent MS and OND patient groups. The discovery cohort (solid line)
corresponds to the cohort shown in (a). The validation group (broken line) consisted of 132
OND and 147 MS patients. Area under the ROC curve (AUC), discovery cohort: 0.76 (95%
CI: 0.69-0.81), validation cohort: 0.82 (95% CI: 0.76-0.87).
(c) Two dimensional graphical illustration of KIR4.1 protein based on the sequence
annotation from uniprot database (http://www.uniprot.org/uniprot/P78508). The large and
small extracellular loops are highlighted in red and yellow, respectively.
(d) ELISA assay with plate bound peptide KIR4 .18 3
. 12 o which contains the first extracellular
loop of KIR4.1 . Serum reactivity against KIR4 .1 3
. 12 was determined in HD, OND patients,
and MS patients of the discovery cohort (see (a)). Antibody positive and negative sera were
compared between HD, OND and MS patients by Kruskal-Wallis test. The threshold for antiKIR4.1 positivity (cut-off OD 0.7558, 5 SD above median OD of HD subjects) is indicated by
a broken horizontal line.
Figure 5: KIR4.1-specific MS serum IgG antibodies are specific to the extracellular
loop or KIR4. 1 (KIR4. 183
.
12
o).
(a) Competitive binding of affinity purified anti-KIR4.1 serum IgG by KIR4.183-12 o (first
extracellular loop) or KIR4.1 356 375 (c-terminal domain) against full length purified
recombinant His-tagged KIR4.1. Anti-KIR4.1 serum IgG was immobilized on ELISA plates
and incubated with increasing concentrations of peptides (concentration range 0.045-150
nM) in the presence of a fixed concentration (150 nM) of His-tagged recombinant KIR4.1
protein. Binding of KIR4.1 protein was determined by anti-His tag detection antibodies.
(b) Correlation of ELISA assays based on plate bound KIR4.1 protein or KIR4.1e 3
- 2 peptide
for the quantification of anti-KIR4.1 serum reactivity in samples from MS patients (n = 122).
(c) Cell based competitive binding assay. HEK293 cells expressing KIR4.1 were
immunolabelled with MS serum gG either without competition (left) or in the presence of
KIR4.1 83-120 (middle) or KIR4.1 356.
3
(right). Representative microphotographs.
Figure 6:
(a) Immunofluorescence labeling performed on P10 mouse cerebellar sections. Stainings
with MS serum IgG (left panels) and anti-GFAP antibodies (right panels). Scale bar 50 pm.
(b) Perivascular staining pattern obtained with MS serum IgG (left panels) and anti-GFAP
antibody immunolabeling (right panels) on human cortical sections. Frames indicate areas
of higher power magnification presented in the lower panels. Scale bars 100 pm (upper
panels) and 20 pm (lower panels).
Figure 7: KIR4. 1-specific MS serum IgG antibodies induce loss of KIR4. 1 staining,
disruption of GFAP filament structures and activation of complement in vivo.
PBS (first row), MS patient serum IgG depleted KIR4. 1-specific antibody reactivity (second
row), or serum IgG with preserved anti-KIR4.1 reactivity (third and forth row) were injected
into the cisterna magna of C57BL/6 mice together with human complement. 24 hrs after
injection mice were sacrificed and brain sections were assessed for GFAP (left), KIR4.1
(middle) and C9neo reactivity (right) by immunohistochemistry. Scale bars 50 m and 20
pm (bottom panels).
Figure 8:
(a) Purification of His-tagged KIR4.1 protein from HEK293 cells transfected with KIR4.1
expression construct [PcDNA3.1 (+)/KIR4.1]. Lane 1: HEK293 cleared lysate; Lane 2: flowthrough; Lane 3-5: wash fractions; lane 6-8: elution fractions.
(b) Depletion of anti-KIR4.1 reactivity from the serum IgG of MS patients. Lane 1: beads
mixed with mock transfected HEK293 cell lysate and Lane 2: beads mixed with PcDNA3.1
(+)/KIR4.1 transfected HEK293 cell lysate. These beads were used to generate mock
preabsorption and preabsorption columns, respectively. Immunoblot on the right shows
serum IgG captured by preabsorption column based on bead-bound KIR4.1 .
(c) The non-concentrated flow through from mock preabsorption and preabsorption columns
was tested for KIR4.1 reactivity by recombinantly purified KIR4.1 ELISA.
The examples illustrate the invention:
Example 1:
Materials and Methods
Patients and controls
Patients and controls were recruited at the Department of Neurology, Klinikum rechts der
Isar of the Technische Universitat in Munich. Two independent cohorts of MS patients,
patients with high-risk clinically isolated syndrome were included in the study. Control
groups consisted of age matched healthy donors (HD) or patients with other neurological
diseases (OND). The characteristics of patients and controls are given in table 1. The ethics
committees of the University approved the study.
Abbreviations: HD = healthy donors, OND = other neurological diseases, MS = multiple
sclerosis. † OND include patients with bacterial or viral meningitis, viral encephalitis,
neurosyphilis and HIV infection.
Antibodies
Rabbit polyclonal anti-human/mouse/rat KIR4.1 (obtained from Millipore, Billerica, MA, USA,
and Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-human/rat KIR4.1 (SigmaAldrich), monoclonal anti-rat/mouse GFAP (Invitrogen), rabbit anti-human C9 neo or purified
serum IgG were used as primary antibodies and biotin-, AlexaFluor 488-, or AlexaFluor 555-
tagged rabbit polyclonal anti-human, anti-rat (Invitrogen, Carlsbad, CA, USA), or anti-mouse
IgG (Vector Laboratories Inc., Burlingame, CA, USA) were used as secondary antibodies in
all immunolabeling experiments.
Immunofluorescence and Immunohistochemistry
For immunofluorescence staining freshly dissected CNS tissue of mouse, rat, or human
origin was snap frozen and embedded in tissue-tek O.C.T (VWR Int., LLC, Radnor, PA,
USA). Cryo-sectioning was performed at -20°C to obtain 10 m sections. After fixation with
100% ice cold methanol for 10 min, blocking steps were performed with peroxidase, avidin
and biotin blocking reagents (Vector Laboratories Inc.) for 15 min each and with 10% goat,
mouse or rat serum in PBS-T (0.05% tween-20 in phosphate buffer saline pH 7.0) for 30
min. Sections were then incubated with diluted purified serum IgG ( 0 g/ml in PBS-T) or
with a commercial antibody solution overnight at 4
°C. After multiple washing steps, sections
were incubated with biotin-tagged secondary antibodies for 1 hr at room temperature.
Section were further incubated with Avidin-biotin complex (Vector) for 1 hr, with 1 ul of
biotinylated tyramide in PBS with 8.8 m of H2
0 2
. All washing steps were performed with
PBS-T. Antibody binding was detected with AlexaFluor 488- or AlexaFluor 555-labeled
avidin. Nuclear staining was performed using Gold antifade with DAPI (Invitrogen). After
incubuation with Avidin-biotin complex, secetion were developed either with DAB
chromogen (Dako) or AEC chromogen (Sigma). Counterstaining was done with hemalum
solution. In case of DAB chromogen they were dehydrated and mounted with xylene
compatible roto-histo kit mounting medium and for AEC with water soluble mounting
medium (Vector). Images were taken using a Zeiss Cell Observer microscope with an
AxioCam MRm camera (Carl Zeiss Microimaging, Ltd., Gottingen, Germany).
Preparation of membrane protein enriched CNS tissue fraction
CNS tissue from 8 rat brains or human brain (2.4 g) was homogenized using a glass tissue
homogenizer in ice cold homogenization buffer (0.32M sucrose, 10mM HEPES pH 7.4, 2mM
EDTA) and protease inhibitor cocktail (Sigma-Aldrich). The suspension was centrifuged at
1000 g to pellet down the nuclear fraction. High speed centrifugation and sucrose gradient
method was used for the enrichment of the membrane fraction. The enriched membrane
pellet was resuspended in HEPES lysis buffer (50 m HEPES pH 7.4, 2mM EDTA, and
protease inhibitor cocktail). The enriched membrane fraction from CNS tissue was used to
prepare a cyanogen bromide (CNBr) activated sepharose bead-based enrichment column
(GE Healthcare Life Sciences, Pittsburgh, PA, USA) according to the manufacturer's
protocol.
Immunoprecipitation, 2-D electrophoresis and western blotting
CNS membrane reactive serum IgG antibodies from 12 MS patients were enriched using
CNBr activated sepharose enrichment column (see above) and were purified using a
protein G bead-based approach (GE Healthcare Life Sciences). The purified MS serum IgG
antibodies were pooled together and used for immunoprecipitation of reactive antigens with
magnetic protein G beads (Invitrogen) based purification columns according to the
manufacturer's protocol. The eluted antigen fractions were precipitated with chloroformmethanol and were solublized with a 2-D protein solubilizer (Invitrogen). The solublized
fractions were loaded on iso-electric focusing strips (Invitrogen) and run at pH 3-10 or pH 4-
6. To identify the immunoprecipitated CNS antigens, 2-D-electrophoresis was performed
with small 2-D benchtop technology (Invitrogen). Spots were removed and subjected to
matrix-assisted laser desorption/ tandem mass spectrometry (MALDI-MS/MS; Alphalyse,
Inc., CA, USA) for identification. As control, we ran parallel samples involving pooled serum
IgG antibodies purified from 24 OND patients.
For validation, rat kidney lysate ( KL)
4 1
, human brain lysate (HBL) and in vitro translated
KIR4.1 protein were subjected to immunoprecipitation with serum IgG from MS patients and
controls using a protein G sepharose beads (Invitrogen). A total of 4 g purified serum IgG
diluted in 5 ml PBS was captured on 400 µ Ι bead suspension and cross linked by dimethyl
pimelimidate (DMP) - 2HCI in 50 mM borate buffer at room temperature (RT). After cross
linking, excess DMP was quenched with 50 mM borate buffer and blocking was performed
with ethanolamine buffer (200 mM, pH 8.0). Prepared beads were used to
immunoprecipitate KIR4.1 from RKL HBL and KIR4.1 in vitro translation reaction mix. All
western blotting experiments were performed on 4-12% SDS gels (Invitrogen) with rabbit
polyclonal anti-human KIR4.1 antibody using ECL detection system (GE Healthcare Life
Sciences).
In vitro-translation of KIR4. 1 protein
Human brain total RNA was used to synthesize full-length cDNA encoding KIR4.1 . The
primers 5
' GGA TCC ATG ACG TCA GTT GCC AAG GTG 3 and 3
" CTC GAG TCA GAC
ATT GCT GAT GCG CAC 5
' were used to add the restriction sites BamHI and Xho1 at the
' and 3
' ends, respectively. The PCR product was cloned into the plasmid pT7CFE1-CHIS
(Pierce, Thermo Fisher Scientific, Rockford, IL, USA). In-vitro translation was performed
with human protein expression kit (Pierce, Thermo Fisher Scientific) according to the
manufacturer's protocol. A pT7CFE1-CHIS construct encoding green fluorescence protein
(GFP) was used as control in all in-vitro translation experiments. Western blotting was
performed on 4-12% SDS gel (Invitrogen) to confirm the KIR4.1 expression using a rabbit
polyclonal anti-human KIR4.1 antibody with ECL detection.
Preparation of murine primary cortical astroglial culture and flow cytometry
For the isolation of primary cortical astroglial cells mouse pups were sacrificed, and
cerebellum and optic nerve were dissected and placed in ice cold buffer [ 1 .47 M Nacl, 5 mM
Kcl, 0.2mM NaHP04(2H20), 0.2mMKH2PO4,5.5mM glucose,0.058 M sucrose in 1
Iiter,ph6.5]. The tissue was minced and digested with 0.5% trypsin at 37°C for 10 min,
subsequently. After washing with MG medium [MEM medium (Sigma-Aldrich)
supplemented with 10% FCS (low endotoxin), 1% L-glutamine, and 0.5% Pen/Strep], a
pasteur pipette with a melted tip was used to generate tissue suspensions. For astroglial
culture, the tissue suspension was seeded in MG medium. Fresh medium was provided
after every two to three days. After two weeks, the mixed glial cell culture obtained was
subjected to gentle shaking at 37
°C for 6 hrs to remove microglia. The astroglial culture was
used in double immuoflourescence staining experiments and flow cytometric analyses
(CyAn ADP, Beckmann Coulter Inc., FL) using serum IgG antibodies from MS and OND
patients and anti-mouse GFAP as primary antibodies.
Cloning, expression and purification
For recombinant KIR4.1 expression in HEK293 cells, a full length cDNA encoding human
KIR4.1 with C-terminal hexa-histidine tag (his-tag) was synthesized from total human brain
mRNA (BD Biosciences. San Jose, California) using 5
'
-GCG GCC GCA CCA TGA CGT
CAG TTG CCA AGG TGT ATT ACA GTC AG-3 ' and 5
'
-CTC GAG TCA GTG GTG GTG
GTG GTG GTG GAC ATT GCT GAT GCG CAC-3 ' as forward and reverse primers (his-tag
encoding sequence is underlined) . Cloning into pcDNA 3.1(+) (invitrogen) was carried out
using Notl and Xhol restriction sites inserted via forward and reverse primers respectively
to obtain pcDNA 3.1 (+)/KIR4.1 expression construct. HEK 293 cells were transiently
transfected with pcDNA 3.1(+)/KIR4.1 using lipofectamine 2000 transfection reagent
(Invitrogen) according to the manufacturer's instructions. At 6 hr post-transfection medium
was supplemented with 10 % FCS and 300 mM barium chloride. At 36 hours posttransfection cells were harvested and washed twice with ice cold PBS. After counting 30
million cells were subjected to lysis in 10 ml of 50 mM sodium phosphate buffer pH 7.4
containing 550mM sodium chloride, 5 mM Tris-HCI, 1.0 % Fos-Choline, 500 unit of
Benzonase® nuclease (Sigma) and 1X EDTA free protease inhibitor cocktail (Sigma). Cell
lysate was centrifuged at 20,000 rpm, using SS34 rotor on Sorvall RC6 plus centrifuge for
minutes at 4 °C. After centrifugation supernatant (cleared lysate) was collected and a
total of 40 mg protein was loaded onto a purification column containing 1 ml of HisPure™
cobalt resin (Pierce) pre-equilibrated with 5 ml of binding buffer (same as lysis buffer).
Washing was carried out with 6 ml of washing buffer (same as lysis buffer). Elution of histagged protein fraction was carried out with 3 ml elution buffer (50mM sodium phosphate,
300 mM sodium chloride, 150 mM imidazole; pH 6.0). Finally, the elution fraction was
dialyzed against PBS and tested for the presence of purified KIR4.1 by western blot
analysis probing with rabbit anti human 4.1 antibody (Millipore).
Enzyme linked immunosorbent assays (ELISA)
For the detection of serum reactivity in MS and control patients with CNS membrane
proteins, rat cerebellum (400 mg snap frozen) was used to prepare protein fractions
enriched for membrane and cytoplasmic antigens. Protein fractions were surface
biotinylated with Suifo-NHS-SS-Biotin (Pierce) and were diluted in PBS to final
concentration of 80 g/ l . For coating 00 µ Ι of diluted protein fraction was added to each
well of Nunc immobilizer™ streptavidin pre-coated and pre-blocked ELISA plates (Pierce).
Plates were left overnight 4
°C on a rotary shaker with slight shaking. After coating plates
were washed twice with PBS-T.
For screening of anti-KIR4.1 reactivity in serum samples solid phase bound purified
recombinant KIR4.1 was used. Purified KIR4.1 protein was diluted in PBS to a final
concentration of 6 g/ml and 100 µ Ι were added to each well of Nunc immobilizer™ amino
plates (Pierce). Plates were left overnight 4
°C on a rotary shaker with slight shaking.
Coated plates were washed twice with PBS-T and blocked for 1 hr using 10mM
ethanolamine in 100mM Na-Carbonate pH 9.6.
For screening of anti-KIR4.1 extracellular peptide reactivity in serum samples, the amino
acid sequence representing the first and second extracellular loops of KIR4.1 protein
rGVVWYLVAVAHGDLLELDPPANHTPCWQVHTLTGAFL (large extracellular domain;
KIR4.183
-i2o; SEQ ID: NO: 1; underlined sequence: KIR4go-n4
; SEQ ID NO: 4) and
TIGYGF RYISEECPL AIVLLI (small extracellular domain; KIR4.1 12S -148
; SEQ ID NO: 2 ;
underlined sequence: SEQ ID NO: 5) respectively] with N-terminal biotin
modification were purchased from JPT peptide Technologies Ltd. (Berlin, Germany). The
peptides were diluted at 16 g/ l in sodium phosphate buffer pH 8.0. Coating was
performed on Nunc immobilizer™ streptavidin pre-coated and pre-blocked ELISA plates
(Pierce) as described above. Control ELISA plates were coated with bovine serum albumin
(Sigma) in all screening experiments. Serum samples were diluted in 3 % skimmed milk
(Biorad Inc.) to obtain IgG concentration of 10 g/ml. An HRP-conjugated anti-human IgG
antibody (Dako) was used for detection. The optical density (OD) measurements were
carried out at 450 nm on a Tecan microplate reader (Tecan Group Ltd., Switzerland).
Competitive-binding assay
Total serum IgG was purified by protein G sepharose beads (GE biosciences) according to
manufacturer's protocol. For the isolation of KIR4.1 reactive IgG fraction from total serum
IgG KIR4.1-bound CNBr activated sepharose affinity beads (GE biosciences) were used.
The binding capacity of the isolated KIR4.1 reactive serum IgG fraction was estimated by
direct ELISA with purified recombinant KIR4.1 . For competitive-binding assays, the KIR4.1
reactive serum IgG was diluted to 5 Mg/ml in PBS and added to each well of Nunc
immoblizer™ amino strips (Pierce). Coating and blocking was performed as described.
Increasing concentration (12 nM to 144 nM) of extracellular KIR4.1 peptide (KIR4.1 128-148)
KIR4.1 intracellular C-terminal peptide (KIR4.1 356-375) was then added to the wells. After 1 hr
incubation plates were washed 3 times with PBS-T and 145 nM purified recombinant histagged KIR4.1 protein was added to each well for 1 hr. After washing (3 times with PBS-T,
an HRP conjugated anti-his tag antibody was used for detection. The competitive-binding
assay was performed in duplicates and the performance of the assay was validated with
binding of a commercially available anti-KIR4.1 monoclonal antibody (Millipore) to the Cterminal KIR4.1 peptide (KIR4.1 356-375)· The cell based competitive-binding assay was
performed on KIR4.1 transfected HEK293 cells using KIR4.1 reactive serum IgG with and
without pre-incubation with the extracellular (KIR s- s) and intracellular C-terminal (KIR 356.
375) peptides, respectively.
Intrathecal injection of MS serum IgG in mice
MS serum total IgG, MS serum IgG depleted of KIR4.1 reactivity and PBS (control) injection
aliquots were prepared. For depletion of KIR4.1 reactivity in MS serum IgG KIR4.1 -bound
Ni-NTA agarose beads (Pierce) were used following the manufacturer's instructions. To
prepare injection aliquots all serum IgG preparations were concentrated to 30 mg/ml using a
kD cutoff spin concentrator (Pierce). Twenty microlitre injection aliquot containing equal
volume of concentrated serum IgG (or PBS) and human total complement (30 units / ml)
were prepared. Six to eight-week-old C57BL/6 mice were divided into 3 groups (n = 3-6
mice) each receiving either MS serum total IgG with or without depletion of KIR4.1 reactivity
or PBS. Mice were anaesthetized by isoflurane inhalation. A transcutaneous intracisternal
injection protocol was adapted as previously described (Klein M, Ann Neurol. 2003,
Oct;54(4):451-8.) . After 24 hrs the mice were sacrificed followed by perfusion with ice-cold
PBS and paraformaldehyde (4%, pH 7.4) through the left cardiac ventricle. Brainstem and
cerebellum were dissected and placed in 20% sucrose at 4°C overnight. Sagittal pieces of
brainstem and cerebellum were embedded in Tissue Tek (Sakura), frozen in liquid nitrogen
and cryotomized at 10 µιτι (Leica CM3050S). Immunohistochemistry was performed as
described.
Statistical analysis
Sera were considered antibody positive when the OD exceeded the cut-off value
determined by titers observed in HD [median OD plus 5 times standard deviation]. The
Kruskal-Wallis test was used to compare the number of antibody positive and negative
patients in the OND and MS group. A p-value below 0.05 was considered significant.
Receiver operating characteristic (ROC) analysis was performed and the areas under ROC
curves (AUC) were computed for two independent sets of samples using MedCalc (or
Analyse-it) software.
Example 2:
MS serum IgG antibodies specifically bind membrane antigens in the CNS
IgG antibodies were purified from serum samples of 19 MS patients and 24 patients with
other neurological diseases (OND) and tested for their reactivity with rat and human brain
tissue sections by immunofluorescence. Using MS serum IgG, we observed a membrane
immunoreactivity in 37 % (7/19) on rat cerebellar and in 58 % ( 1 1/19) on human brain
sections (Figure 1a). In contrast, we could not find this particular staining pattern using
serum IgG from any of the OND patients (Figure 1a). To confirm the specific membrane
reactivity, we established a capture ELISA based on rat cerebellar protein fractions enriched
for membrane and cytoplasmic antigens. An elevated reactivity with membrane protein
fraction was only observed in sera from MS patients (n = 56) but not in sera from OND
patients (n = 29) suggesting the presence of a specific serum IgG antibody against a CNS
membrane protein in MS patients (Figure 1b). In comparison, reactivity to the cytoplasmic
protein fraction was similar in both sera from MS and OND patients (data not shown). Thus,
in the subsequent immunoprecipitation studies we proceeded with the CNS tissue fraction
enriched for membrane proteins for the identification of target antigens in MS.
Example 3:
Identification of KIR4. 1 as the target of serum IgG in MS
The CNS reactive serum IgG from 12 MS patients were pooled and enriched using CNBr
activated beads coated with membrane protein fraction prepared from human brain tissue.
The MS serum IgG eluted from the enrichment column was used for subsequent antigen
immunoprecipitation. The antigen-lgG complexes eluted from the precipitation column were
then analyzed on SDS-PAGE and separated by 2-D gel electrophoresis (Figure 2a). 7
protein spots were excised and analyzed by MALDI-MS/MS, the abbreviation "MS" referring
to mass spectrometry in this specific context. In one of the spots the inward rectifying
potassium channel KIR4.1 was identified. The identity of KIR4.1 as MS serum IgG antibody
target was subsequently confirmed by immunoprecipitation and Western blotting using
extracts from rat kidney lysate, human brain lysate (Figure 2b), and in vitro translated
KIR4.1 reaction mix (Figure 2c).
Example 4:
KIR4. 1 reactivity with MS serum IgG localizes to hippocampal and cerebellar astroglia
Double immunofluorescence labeling was performed on rat brain sections with both purified
IgG antibodies from MS sera and the anti-KIR4.1 monoclonal antibody (Figure 3a). As
control, a similar staining was performed with purified IgG from sera of OND patients.
Specific co-localization of the monoclonal anti-KIR4.1 and the serum gG antibody on rat
cerebellar sections was only observed for MS-IgG but not for OND-IgG (Figure 3a). To
further validate this observation we performed immunolabeling of cerebellar sections from
day-old wildtype and Kir4. 1 null mice {Kir4. 1^) mice with MS serum IgG (Figure 3b). On
day 10 after birth (P10), KIR4.1 is known to be expressed in high amounts (20). KIR4.1
antibody positive MS sera stained astroglial cells in cerebellar and hippocampal sections of
wild type mice but failed to react with sections from Kir4. V mice (Figures 3b and Figure 6).
KIR4.1 antibody negative sera did not stain CNS tissue from either wildtype mice or Kir4.
mice (data not shown). To confirm the astroglial localization of the anti-KIR4.1 reactivity in
MS sera, we prepared murine mixed glial primary cultures. A highly MS serum-specific
membrane staining was observed in GFAP-positive cells (Figure 3c). A similar MS serumspecific surface staining of glial cells was also observed by flow cytometry (Figure 3d).
Example 5:
High titer serum reactivity to the extracellular loop of KIR4. 1 protein is restricted to
MS
For the quantification of anti-KIR4.1 reactivity we used a capture ELISA assay based on
KIR4.1 protein isolated from the human PC3 cell line. Sera from 122 MS/CIS, 70 OND
patients and 14 healthy donors (HD) were analysed (Figure 4a). Significant KIR4.1 serum
autoantibody concentrations (> 5 SD from median of healthy controls) were detected in
16.9% of OND patients (12/71) and 50.8% of MS patients (62/122) (pO.0001 ). All positive
MS sera contained higher antibody concentrations than any serum of the OND group.
These findings were independently confirmed in second case-control cohort involving 130
OND and 149 MS patients (Figure 4d).
Similar results were obtained in a smaller, group of patients and controls by an ELISA assay
in which in vitro translated KIR4.1 protein was used as capture substrate (data not shown).
None of the sera from OND patients contained significantly elevated antibody titers,
whereas 22.5% (10/44) of MS patient sera were antibody positive (p=0.0108) in this assay.
Membrane topology analysis (Uniprot database version 107, entry 78508 (last modified
April 5, 201 1); http://www.uniprot.org/uniprot/P78508) predicts two extracellular loops for the
KIR4.1 protein; a larger loop spanning 25 amino acids (KIR4.19
-
4
; SEQ ID NO: 4) and a
smaller loop spanning 9 amino acids (KIR4.113
4 _
1
2; SEQ ID NO: 5); see Figure 4c.
To mimic the external loop topology of KIR4.1 , peptides comprising the amino acid
sequence of the extracellular regions of KIR4.1 and the adjacent intramembrane domains
were synthesized with biotin tags and immobilized on avidin coated plates. Sera from MS
patients and controls were tested for antibody binding to these peptides. Antibody reactivity
to the peptide representing the smaller extracellular domain of KIR4.1 (K 4.1 2 -i4s SEQ ID
NO: 2) was observed in only 4% of the MS patients and in no HD or OND patients (data not
shown). However, when MS sera were assayed for their binding capability to the first
extracellular loop of KIR4.1 (KIR4.
83- 2
o; SEQ ID NO: 1), significantly elevated antibody
concentrations were observed in MS patients (37/122, 30.3 %) versus OND patients (1/70,
1.4 %) (p<0.0001 ). This observation was independently replicated in a second case-control
cohort (data not shown).
Binding of human KIR4.1 -specific antibodies to the large extracellular domain was further
confirmed in a competition assay; see Figure 5.
Overall, we observed a strong correlation between the antibody reactivity measured by the
KIR4.1 protein based ELISA from PC3 cells and the KIR4.183
.
2
o peptide (SEQ ID NO: 1)
based ELISA assays suggesting that the MS serum antibodies against KIR4.1 recognize an
epitope in the first extracellular loop of KIR4.1.
Example 6:
Serum KIR4.1-specific antibodies induce loss of KIR4.1 expression, disruption of
GFAP filament structures and activation of complement in vivo.
Mice injected with serum IgG containing KIR4.1 -specific antibodies showed disruption of the
GFAP filament structures in astrocytes, loss of KIR4.1 expression and activation of
complement in areas where KIR4.1 loss was observed. These changes were not observed
in mice which received PBS or the serum IgG from the same patient, which was depleted
from KIR4.1 -specific antibodies. Corresponding data are displayed in Figures 7 and 8 .
Further references
1 J. H. Noseworthy, C. Lucchinetti, M. Rodriguez, B. G. Weinshenker, Multiple
sclerosis. N. Engl. J. Med. 343, 938-952 (2000).
2 . A. Ascherio, K. L . Munger, Environmental risk factors for multiple sclerosis. Part II:
Noninfectious factors. Ann. Neurol. 61, 504-513 (2007).
3. A. Ascherio, K. L . Munger, Environmental risk factors for multiple sclerosis. Part I:
the role of infection. Ann. Neurol. 61, 288-299 (2007).
4 . D. A. Hafler, A. Compston, S. Sawcer, E. S. Lander, M. J. Daly, P. L. de Jager, P. I.
de Bakker, S. B. Gabriel, D. B. Mirel, A. J. Ivinson, M . A. Pericak-Vance, S. G.
Gregory, J. D. Rioux, J. L . McCauley, J. L . Haines, L. F. Barcellos, B. Cree, J. R.
Oksenberg, S. L . Hauser, Risk alleles for multiple sclerosis identified by a
genomewide study. N. Engl. J. Med. 357, 851-862 (2007).
. H . F. McFarland, R . Martin, Multiple sclerosis: a complicated picture of
autoimmunity. Nat. Immunol. 8, 913-919 (2007).
6. B. Hemmer, J. J. Archelos, H. P. Hartung, New concepts in the
immunopathogenesis of multiple sclerosis. Nat. Rev. Neurosci. 3, 291-301 (2002).
7. M. K. Storch, S. Piddlesden, M . Haltia, M . livanainen, P. Morgan, H. Lassmann,
Multiple sclerosis: in situ evidence for antibody- and complement-mediated
demyelination. Ann. Neurol. 43, 465-471 (1998).
8. C. Lucchinetti, W. Bruck, J. Parisi, B. Scheithauer, M . Rodriguez, H. Lassmann,
Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of
demyelination. Ann. Neurol. 47, 707-717 (2000).
9. M. Keegan, F. Konig, R. McClelland, W. Bruck, Y. Morales, A. Bitsch, H. Panitch, H.
Lassmann, B. Weinshenker, M. Rodriguez, J . Parisi, C. F. Lucchinetti, Relation
between humoral pathological changes in multiple sclerosis and response to
therapeutic plasma exchange. Lancet 366, 579-582 (2005).
. S. L . Hauser, E. Waubant, D. L. Arnold, T. Vollmer, J . Antel, R. J. Fox, A. Bar-Or, M.
Panzara, N. Sarkar, S. Agarwal, A. Langer-Gould, C. H . Smith, B-cell depletion with
rituximab in relapsing-remitting multiple sclerosis. N. Engl. J. Med. 358, 676-688
(2008).
11. A. Uccelli, F. Aloisi, V. Pistoia, Unveiling the enigma of the CNS as a B-cell fostering
environment. Trends Immunol. 26, 254-259 (2005).
12. E. Meinl, M . Krumbholz, R. Hohlfeld, B lineage cells in the inflammatory central
nervous system environment: migration, maintenance, local antibody production,
and therapeutic modulation. Ann. Neurol. 59, 880-892 (2006).
13. F. J. Quintana, M. F. Farez, V. Viglietta, A. H. Iglesias, Y. Merbl, G. Izquierdo, M.
Lucas, A. S. Basso, S. J. Khoury, C. F. Lucchinetti, I. R. Cohen, H. L. Weiner,
Antigen microarrays identify unique serum autoantibody signatures in clinical and
pathologic subtypes of multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A 105, 18889-
18894 (2008).
I. Cortese, R. Tafi, L. M. Grimaldi, G. Martino, A. Nicosia, R. Cortese, Identification
of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using
phage libraries. Proc. Natl. Acad. Sci. U. S. A 93, 11063-1 1067 (1996).
J. J. Archelos, J. Trotter, S. Previtali, B. Weissbrich, K. V. Toyka, H. P. Hartung,
Isolation and characterization of an oligodendrocyte precursor-derived B-cell epitope
in multiple sclerosis. Ann. Neurol. 43, -24 (1998).
S. Cepok, D. Zhou, R . Srivastava, S. Nessler, S. Stei, K. Bussow, N. Sommer, B.
Hemmer, Identification of Epstein-Barr virus proteins as putative targets of the
immune response in multiple sclerosis. J. Clin. Invest 115, 1352-1360 (2005).
V. Somers, C. Govarts, K. Somers, R. Hupperts, R. Medaer, P. Stinissen,
Autoantibody profiling in multiple sclerosis reveals novel antigenic candidates. J.
Immunol. 180, 3957-3963 (2008).
T. Berger, M. Reindl, Multiple sclerosis: disease biomarkers as indicated by
pathophysiology. J. Neurol. Sci. 259, 21-26 (2007).
T. Derfuss, C. Linington, R. Hohlfeld, E. Meinl, Axo-glial antigens as targets in
multiple sclerosis: implications for axonal and grey matter injury. J. Mol. Med. 88,
753-761 (2010).
Y. V. Kucheryavykh, L . Y. Kucheryavykh, C. G. Nichols, H. M. Maldonado, K. Baksi,
A. Reichenbach, S. N. Skatchkov, M. J. Eaton, Downregulation of Kir4.1 inward
rectifying potassium channel subunits by RNAi impairs potassium transfer and
glutamate uptake by cultured cortical astrocytes. Glia 55, 274-281 (2007).
E. A. Nagelhus, Y. Horio, A. Inanobe, A. Fujita, F. M. Haug, S. Nielsen, Y. Kurachi,
O. P. Ottersen, Immunogold evidence suggests that coupling of K+ siphoning and
water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and
AQP4 in specific membrane domains. Glia 26, 47-54 (1999).
M. Amiry-Moghaddam, A. Williamson, M . Palomba, T. Eid, N. C. de Lanerolle, E. A.
Nagelhus, M . E. Adams, S. C. Froehner, P. Agre, O. P. Ottersen, Delayed K+
clearance associated with aquaporin-4 mislocalization: phenotypic defects in brains
of alpha-syntrophin-null mice. Proc. Natl. Acad. Sci. U. S. A 100, 13615-13620
(2003).
Claims (22)
1. A peptide comprising or consisting of at least 8 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 3, the upper limit for the length of said peptide being 100 amino acid residues, provided that said peptide does not consist of the 5 sequence set forth in SEQ ID NO: 3; or a corresponding peptidomimetic, wherein said peptide or peptidomimetic is capable of binding to an anti-KIR4.1 antibody comprised in a sample from a patient having multiple sclerosis or a predisposition thereto wherein: (i) said at least 8 consecutive amino acid residues are a subsequence of an 10 extracellular domain of KIR4.1, said extracellular domain consisting of the sequence set forth in SEQ ID NO: 1 or 2; or (ii) said peptide comprises or consists of the sequence of SEQ ID NO: 1 or 2.
2. A method for diagnosing multiple sclerosis or a predisposition to multiple 15 sclerosis in a subject, the method comprising determining, in a sample obtained from said subject, the presence of an anti-KIR4.1 antibody, wherein the presence of the antiKIR4.1 antibody is determined by: (i) contacting the sample with a peptide or peptidomimetic of claim 1; and (ii) detecting the formation of a receptor-anti-KIR4.1 antibody complex, 20 wherein the presence of the anti-KIR4.1 antibody in said sample is indicative of multiple sclerosis or a predisposition thereto.
3. The method of claim 2, wherein, when the anti-KIR4.1 antibody is present in said sample: 25 (i) the presence of at least one clinical symptom of multiple sclerosis in said subject is indicative of multiple sclerosis; and (ii) the absence of any clinical symptom of multiple sclerosis is indicative of said predisposition to multiple sclerosis.
4. The method of claim 2, wherein said subject has clinically isolated syndrome 30 (CIS).
5. The method of claim 3, wherein said at least one clinical symptom is CIS.
6. The method of any one of claims 2 to 5, wherein the anti-KIR4.1 antibody is capable of binding to KIR4.1 (SEQ ID NO: 3) or an extracellular domain thereof, said 5 extracellular domain consisting of the sequence set forth in any one of SEQ ID NOs: 1, 2, 4 or 5.
7. A peptide or peptidomimetic of claim 1, when used in determining, in a sample previously obtained from a patient having multiple sclerosis or a predisposition 10 thereto, the presence of the anti-KIR4.1 antibody.
8. Use of a peptide or peptidomimetic of claim 1 in the manufacture of a medicament for the treatment or prevention of multiple sclerosis. 15
9. A composition comprising a peptide or peptidomimetic of claim 1.
10. A method of screening for a drug or lead compound, said method comprising bringing into contact a complex comprising or consisting of: (a) an anti-KIR4.1 antibody comprised in a sample from a patient having 20 multiple sclerosis or a predisposition thereto; and (b) a peptide or peptidomimetic as defined in claim 1, with a test compound, wherein a reduction of the amount of said complex is indicative of the test compound being a drug or lead compound suitable for the treatment or prevention of, or for the 25 development of drugs suitable for the treatment or prevention of, multiple sclerosis.
11. Use of a peptide or peptidomimetic of claim 1 in the preparation of a medicament for removing anti-KIR4.1antibodies from, or reducing the amount thereof in, blood or serum of a multiple sclerosis patient or of a subject carrying a predisposition 30 to develop multiple sclerosis.
12. A carrier with a receptor being immobilized thereon, wherein said receptor is a peptide or peptidomimetic of claim 1.
13. A device for removing anti-KIR4.1 antibodies from blood, said device comprising 5 the carrier of claim 12.
14. A peptide or peptidomimetic according to claim 1, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 10
15. A method according to claim 2, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
16. A peptide or peptidomimetic according to claim 7, substantially as herein 15 described with reference to any one or more of the examples but excluding comparative examples.
17. Use according to claim 8, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 20
18. A composition according to claim 9, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
19. A method according to claim 10, substantially as herein described with reference 25 to any one or more of the examples but excluding comparative examples.
20. Use according to claim 11, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 30
21. A carrier according to claim 12, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
22. A device according to claim 13, substantially as herein described with reference 5 to any one or more of the examples but excluding comparative examples.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ717188A NZ717188B2 (en) | 2011-05-30 | 2012-05-23 | Means and methods for diagnosing and treating multiple sclerosis |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11004423A EP2530088A1 (en) | 2011-05-30 | 2011-05-30 | Means and methods for diagnosing and treating multiple sclerosis |
EP11004423.7 | 2011-05-30 | ||
PCT/EP2012/059622 WO2012163765A1 (en) | 2011-05-30 | 2012-05-23 | Means and methods for diagnosing and treating multiple sclerosis |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ615912A NZ615912A (en) | 2016-05-27 |
NZ615912B2 true NZ615912B2 (en) | 2016-08-30 |
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