U.S. patent application number 16/282389 was filed with the patent office on 2019-07-18 for predictive biomarkers of clinical response to anti-lps immunoglobulin treatment.
The applicant listed for this patent is IGNOVA GMBH, JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG. Invention is credited to Gunter SPROTTE, Ana Maria WAAGA-GASSER.
Application Number | 20190219594 16/282389 |
Document ID | / |
Family ID | 52997897 |
Filed Date | 2019-07-18 |
United States Patent
Application |
20190219594 |
Kind Code |
A1 |
SPROTTE; Gunter ; et
al. |
July 18, 2019 |
PREDICTIVE BIOMARKERS OF CLINICAL RESPONSE TO ANTI-LPS
IMMUNOGLOBULIN TREATMENT
Abstract
The present invention relates to the biomarkers for predicting
the clinical response to anti-LPS immunoglobulin treatments in
patients in need thereof. In particular, the invention provides
methods for predicting the clinical response to an anti-LPS
immunoglobulin treatment in a patient in need thereof, said method
comprising the steps of evaluating the expression of a predictive
biomarker selected from the group consisting of CD14, CD68, TLR4,
TLR7, IL6, IL8, IL10, IFN-alpha, IGF1, CXCL1, CXCL9, CXCL10, RAGE,
GDNF, BCHE, and combination thereof, in said patient.
Inventors: |
SPROTTE; Gunter;
(Rottenbuch, DE) ; WAAGA-GASSER; Ana Maria;
(Wurzburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG
IGNOVA GMBH |
Wurzburg
Oberursel |
|
DE
DE |
|
|
Family ID: |
52997897 |
Appl. No.: |
16/282389 |
Filed: |
February 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15562174 |
Sep 27, 2017 |
10261095 |
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PCT/EP2016/058276 |
Apr 14, 2016 |
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16282389 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
G01N 2333/54 20130101; G01N 2333/56 20130101; G01N 2800/52
20130101; A61P 29/00 20180101; G01N 2333/916 20130101; G01N
2800/125 20130101; C12Q 1/6876 20130101; A61P 25/06 20180101; G01N
2333/70596 20130101; G01N 2800/26 20130101; C07K 16/44 20130101;
A61P 19/04 20180101; C07K 2317/23 20130101; C12Q 2600/106 20130101;
A61P 9/10 20180101; A61P 17/00 20180101; A61P 37/02 20180101; C12Q
1/6883 20130101; G01N 2333/5421 20130101; A61P 1/00 20180101; C12Q
2600/158 20130101; G01N 33/56911 20130101; A61P 1/02 20180101; A61P
13/10 20180101; A61P 19/02 20180101; G01N 2333/521 20130101; G01N
2400/50 20130101; G01N 33/6863 20130101; G01N 33/6869 20130101;
A61P 1/04 20180101; G01N 33/6866 20130101; G01N 2333/5412 20130101;
A61P 17/02 20180101; C07K 16/1203 20130101; G01N 2333/5428
20130101; G01N 2333/4756 20130101; G01N 2333/705 20130101; G01N
33/92 20130101; C07K 2317/11 20130101; A61P 25/04 20180101; G01N
2333/475 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 16/44 20060101 C07K016/44; C12Q 1/6876 20060101
C12Q001/6876; C12Q 1/6883 20060101 C12Q001/6883; G01N 33/569
20060101 G01N033/569; G01N 33/92 20060101 G01N033/92; C07K 16/12
20060101 C07K016/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2015 |
EP |
15164087.7 |
Claims
1-15. (canceled)
16. An IgY composition comprising one fraction consisting of
polyclonal IgY directed against Escherichia coli and another
fraction consisting of polyclonal IgY directed against Salmonella
typhimurium.
17. The IgY composition of claim 16, which is obtained from egg
yolk of immunized hens.
18. The IgY composition of claim 16 which is obtained by: a)
immunizing at least 2 distinct groups of hens, one group with E.
coli gram-negative bacteria and another group with S. typhimurium
gram-negative bacteria, b) obtaining IgY-containing fractions of
egg yolks obtained from each of the 2 distinct groups of immunized
hens, and c) mixing said IgY-containing fractions obtained from the
2 distinct groups of immunized hens.
19. The IgY composition of claim 16, which is an egg yolk powder
formulated for oral administration.
20. The IgY composition of claim 19, which is defatted or partially
defatted.
21. A method for treating chronic disease induced by CD14+
activated monocytes in peripheral blood, comprising administering a
therapeutically efficient amount of an IgY composition to a patient
affected with said chronic disease, wherein said chronic disease
induced by CD14+ activated monocytes in peripheral blood is
selected from the group consisting of: idiopathic chronic pain
syndromes, and, migraine, chronic head and neck deceleration
trauma, epicondylitis, impingement syndrome, wherein said IgY
composition comprises one fraction consisting of polyclonal IgY
directed against Escherichia coli and another fraction consisting
of polyclonal IgY directed against Salmonella typhimurium.
22. The method of claim 21, wherein said IgY composition is
obtained from egg yolk of immunized hens.
23. The method of claim 21 wherein said IgY composition is obtained
by: a) immunizing at least 2 distinct groups of hens, one group
with E. coli gram-negative bacteria and another group with S.
typhimurium gram-negative bacteria, b) obtaining IgY-containing
fractions of egg yolks obtained from each of the 2 distinct groups
of immunized hens, and c) mixing said IgY-containing fractions
obtained from the 2 distinct groups of immunized hens.
24. The method of claim 21, wherein said IgY composition is an egg
yolk powder formulated for oral administration.
25. The method of claim 21, wherein said IgY composition is
defatted or partially defatted.
26. The method of claim 21, wherein said IgY composition is
administered orally to the patient.
27. The method of claim 21, wherein said idiopathic chronic pain
syndrome is selected from the group consisting of chronic
widespread pain, fibromyalgia, frozen shoulder or adhesive
capsulitis, oral mucositis, carpal tunnel syndrome, and bladder
syndrome.
28. The method of claim 21, wherein said patient is selected as a
responder patient which is identified by quantifying the expression
level of one or more of said biomarkers in a biological sample
obtained from said patient to obtain an expression value for each
quantified biomarker and comparing each obtained expression value
to an expression value observed in non-responder patients, wherein
a decrease of at least 10% in the obtained expression value of
TLR-4, TLR-7, IL-6, IL-8, IL-10, IFN-alpha, IGF-1, CXCL1, CXCL9,
CXCL10, RAGE, and/or GDNF relative to the expression value observed
in non-responder patients and/or an increase of at least 10% in the
obtained expression value of BCHE relative to the expression value
observed in non-responder patients identifies said patient as a
responder to said anti-LPS immunoglobulin drug.
29. The method of claim 28, wherein the expression level of at
least three of said biomarkers is evaluated in the patient.
30. The method of claim 28, wherein the expression level of at five
of said biomarkers is evaluated in the patient.
Description
[0001] The present invention relates to the biomarkers for
predicting the clinical response to anti-LPS immunoglobulin
treatments in patients in need thereof. In particular, the
invention provides methods for predicting the clinical response to
an anti-LPS immunoglobulin treatment in a patient in need thereof,
said method comprising the steps of evaluating the expression of a
predictive biomarker selected from the group consisting of CD14,
CD68, TLR4, TLR7, IL6, IL8, IL10, IFN-alpha, IGF1, CXCL1, CXCL9,
CXCL10, RAGE, GDNF, BCHE, and combination thereof, in said
patient.
BACKGROUND
[0002] There are growing evidence that a variety of chronic
diseases are triggered, sustained or reinforced by systemic
translocation of intestinal lipopolysaccharides (LPS) [Pinzone et
al. 2012]. Bacterial lipopolysaccharide (LPS) is thought to be
responsible for the multiple organ dysfunction syndrome [Louis et
al. 2013] and the acute respiratory distress syndrome [Bernard et
al., Am J Respir Crit Care Med 1994; 149: 818-824]. Both in vitro
and in vivo studies have shown that administration of LPS causes a
variety of reactions [Windsor et al. 1993; Louis et al. 2013]. In
vitro studies indicate that LPS does not directly induce apoptosis
of endothelial cells. Several investigations have pointed to
LPS-induced tumor necrosis factor alpha (TNF-.alpha.) as the cause
of endothelial cell apoptosis [Bazzoni and Beutler N Engl J Med
1996; 334: 1717-1725; Buchman et al. Am J Physiol 1993, 265:
H165-170; Polunovsky et al. Exp Cell Res 1994, 214: 584-594; Robaye
et al. Am J Pathol 1991, 138: 447-453; Takei et al., J
Gastroenterol Hepatol, 1995, 10: 65-67; Munshi et al. J Immunol.
2002 Jun. 1; 168(11):5860-6].
[0003] LPS is also known to be attracted by CD14 on the surface of
monocytic cells [Devitt et al. 1998; Tapping et al. Nature 1998;
392: 505-509]. Interestingly, it is widely believed that this
reflects the first step in signal transduction via LPS.
[0004] Another question from the clinical perspective concerns the
impact of a therapeutic intervention involving the LPS turnover on
the overall apoptotic response. In this context, in the long-term,
immunoglobulins may exert several beneficial immunological effects
in patients affected with subclinical immune activation. This is
primarily reflected by antibodies against bacterial LPS molecules
and Lactoferrin which also exerts strong inhibitory LPS activity
[Bellamy et al. Biochim Biophys Acta 1992; 1121: 130-136, Appelmelk
et al Infection and Immunity 1994; 62(2): 2628-2632; Inubushi et
al. 2012]. However, the whole spectrum of effects and specificities
of the effector mechanisms of oral anti-LPS immunoglobulins has not
been fully investigated. One key effector mechanism may be related
to apoptosis in monocytic cells.
[0005] The inventors have shown that a new therapeutic approach
based on an oral administration of anti-LPS antibodies
(immunoglobulins) lead to a significant or complete symptom relief
in more than 50% of the patients in need of such treatment, e.g.
with chronic pain syndromes (unpublished data).
[0006] The fit-for-purpose, scientific validation, and the overall
clinical qualification of parameters for diseases triggered by LPS
translocation and inflammation could dramatically change the
current outlook on treatment of such diseases.
[0007] A predictive biomarker is associated with the likelihood of
sensitivity or resistance (response) to a specific therapy (drug).
The concept of a predictive biomarker is usually applied to
individual patients with the goal of tailoring therapy to maximize
efficacy. In more specific and practical terms, a predictive
biomarker as herein demonstrated could assist the clinician in
deciding which patients are responders to the anti-LPS
immunoglobulin treatment.
[0008] Accordingly, one object of the present invention is to
provide a reliable prognosis assay for determining individual
patient clinical benefit of oral immunoglobulin therapy (i.e
responder to oral anti-LPS immunoglobulin treatment) and for
avoiding administering the anti-LPS immunoglobulin treatments to
non-responders. Methods for selecting the responders prior to
treating patients would allow for an individualized therapeutic
decision, which in turn is of great psychological benefit for the
patients, improves health outcomes and provides economic benefit
for the community.
[0009] Another object of the present invention is to provide a
prognosis assay which could be performed from a biological sample
of the patient, preferably a blood sample.
[0010] A further object of the present invention is to provide a
prognosis assay that would be highly specific, i.e. whereas at
least 80%, or 90% or more preferably at least 95% and even more
preferably at least 98% of the patients that are diagnosed as
responder according the assay are indeed true responders to the LPS
immunoglobulin treatments.
[0011] In order to fulfil this need, the inventors performed a
laboratory screen focusing on a broad spectrum of immune parameters
in patients before and after oral anti-LPS immunoglobulin treatment
and comparing the results to those of healthy control
individuals.
[0012] Thus, according to the method of the present invention, a
selection of the patients can take place before these individuals
receive the anti-LPS immunoglobulin treatment. This would relief
mental stress to the patients and save costs.
[0013] The results of the screen performed by the inventors
demonstrated that 15 specific parameter profiles (i.e. the
predictive biomarkers) enable to differentiate between responders
and non-responders to a planned anti-LPS immunoglobulin
treatment.
SUMMARY OF PREFERRED EMBODIMENTS
[0014] In one aspect, the invention relates to an in vitro method
for predicting the response to an anti-LPS immunoglobulin treatment
in a patient in need thereof, said method comprising the step of
evaluating the expression of one or more biomarkers in a biological
sample obtained from said patient, wherein said one or more
biomarkers are selected from the group of predictive biomarkers
consisting of CD14, CD68, TLR-4, TLR-7, IL-6, IL-8, IL-10,
IFN-alpha, IGF-1, CXCL1, CXCL9, CXCL10, RAGE, GDNF and BCHE.
[0015] More specifically, said evaluating step comprises a step of
quantifying expression of one or more of the selected predictive
biomarkers in a biological sample of said patient to obtain
expression values, and comparing each expression value to a
corresponding control value, for example to corresponding
expression values from healthy volunteers. In certain embodiments,
such control value may be normalized mean expression value of
corresponding biomarker that is observed in healthy subjects.
[0016] In a preferred embodiment, said anti-LPS immunoglobulin
treatment is a composition for oral administration, for example, an
IgY composition. In a specific embodiment, said IgY composition
comprises IgY (or their antigen-binding portions), obtained from
hens immunized with gram-negative bacteria or their LPS-containing
portions, preferably obtained from at least two distinct bacterial
species.
[0017] In another specific embodiment, that may be combined with
the preceding embodiments, the method is applied for a human
subject suffering from a chronic disease induced by activated
monocytes (CD14+) in peripheral blood. Such chronic disease induced
by activated monocytes (CD14+) in peripheral blood include, without
limitation,
[0018] idiopathic chronic pain syndromes including without
limitation chronic widespread pain, fibromyalgia, and bladder
syndrome;
[0019] migraine, chronic head and neck deceleration trauma,
epicondylitis, impingement syndrome;
[0020] Graft vs host disease (GVHD), chronic inflammatory gastro
intestinal diseases including without limitation Crohn's disease,
ulcerative colitis, and irritable bowel syndrome;
[0021] crest syndrome, systemic lupus erythematodes, pemphigus
vulgaris, sclerodoma; and,
[0022] atherosclerosis.
[0023] In a specific embodiment of the method of the invention, the
expression of 2, 3, 4, or 5 biomarkers among the 15 predictive
biomarkers of the invention is evaluated.
[0024] In another specific embodiment that may be combined with the
preceding embodiments, the biological sample is a blood sample.
[0025] In another specific embodiment that may be combined with the
preceding embodiments, said expression is gene expression as
quantified by real-time quantitative PCR.
[0026] In another specific embodiment, that may be combined with
the preceding embodiments, said expression is protein biomarker
expression as quantified by specific antibodies.
[0027] In another aspect, the invention relates to a method for
treating a chronic disease induced by activated monocytes (CD14+)
in peripheral blood, comprising administering a therapeutically
efficient amount of an anti-LPS immunoglobulin composition to a
patient affected with said chronic disease, wherein said patient is
a responder to said anti-LPS immunoglobulin composition and wherein
said responder has been selected by evaluating the expression of
one or more predictive biomarkers selected from the group
consisting of CD14, CD68, TLR-4, TLR-7, IL-6, IL-8, IL-10,
IFN-alpha, IGF-1, CXCL1, CXCL9, CXCL10, RAGE, GDNF and BCHE.
Typically, the method may comprises the steps of: (a) providing a
biological sample from a patient; (b) quantifying the expression of
one or more predictive biomarkers, as defined above, in said
biological sample, and, (c) administering a therapeutically
effective amount of said anti-LPS immunoglobulin composition to the
patient only if said patient is predicted to be a responder to said
anti-LPS immunoglobulin composition, based on the expression level
of said one or more biomarkers.
DETAILED DESCRIPTION
[0028] Prognosis methods allowing prediction of a response to
anti-LPS treatment in patients in need of such treatment are
provided by the present invention. Particularly, it is provided
herein methods and kits allowing prediction of a clinical response
to anti-LPS treatment, such as an anti-LPS IgY treatment in
patients suffering from chronic diseases induced by activated
monocytes (CD14+) in peripheral blood.
[0029] According to the present invention, a set of 15 biomarkers
that are, individually or in combination, predictive of high
probability of clinical response to anti-LPS treatment has been
identified. The identification of these predictive biomarkers was
permitted due to the systematic quantification of a number of
cytokines or cytokine receptors expressions in peripheral blood
sample of responder vs non-responder patients.
[0030] Thus, a first object of the present invention consists of a
method for predicting the response to an anti-LPS immunoglobulin
treatment in a patient in need thereof, said method comprising the
step of evaluating the expression of one or more biomarkers in a
biological sample obtained from said patient, wherein said one or
more biomarkers are selected from the group of predictive
biomarkers consisting of CD14, CD68, TLR-4, TLR-7, IL-6, IL-8,
IL-10, IFN-alpha, IGF-1, CXCL1, CXCL9, CXCL10, RAGE, GDNF and
BCHE.
[0031] As it is shown in the examples herein, when comparing the
expression level value of candidate biomarkers between responder
and non-responder patients to anti-LPS treatment, the inventors
have identified biomarkers with statistically different expression
in responder subject when compared to either healthy subject and
non-responder subjects, hereafter called the "predictive
biomarkers" and listed in Table 1 below.
The Patient in Need of Anti-LPS Immunoglobulin Treatment
[0032] The term "patient" and "subject" which are used herein
interchangeably refer to any member of the animal kingdom,
preferably a mammal, or a human being, including for example a
subject that has or is suspected to have a chronic disease induced
by activated monocytes (CD14+) in peripheral blood.
[0033] Anti-LPS immunoglobulin treatment have indeed been shown to
be effective in patients suffering from chronic diseases induced by
activated monocytes (CD14+) in peripheral blood. Said
CD14+monocytes are activated in the gastrointestinal tract by gram
negative bacteria or parts thereof and lead to an overproduction of
monocyte/macrophage-related cytokines.
[0034] Gram negative bacteria are part of the human
gastrointestinal microbiome. In a physiologically healthy
gastrointestinal environment, these gram-negative bacteria do not
pose any risk to human health, as a certain degree of the endotoxin
lipopolysaccharide LPS is tolerated by the human defense system. It
is actually needed as a positive feedback to the host immune
system.
[0035] In a pathologic situation, there can be an overgrowth of
gram-negative bacteria and an increase in gastrointestinal mucosal
permeability, triggered by mucositis. Both lead to an increased
presence of lipopolysaccharide (LPS) and other components of gram
negative bacteria such as flagella, surface proteins etc at the
submucosal level and consequent increased contact with the innate
immune system's pattern recognition receptors.
[0036] Waaga-Gasser et al. 2009 [International Journal of Clinical
Pharmacology and Therapeutics, Vol 47, No. 7/2009 (421-433)] have
shown that patients showing idiopathic pain syndromes possess such
activated monocytes which fail to go into apoptosis after they have
been activated by LPS.
[0037] Therefore, LPS triggers activation of macrophages followed
by a dysfunctional induction of apoptosis in these cells. The
combination of the two leads to a positive feedback loop and a
translocation of the LPS signal to systemic parts of the body,
leading in essence to a subliminal chronic inflammation.
[0038] This chronic inflammation in turn leads to phenotype
specific to symptomatic diseases.
[0039] Accordingly, in specific embodiments, those patients in need
of such anti-LPS treatment suffer from one or more of the following
chronic diseases, all being characterized by activated (CD14+)
monocytes activated by LPS:
[0040] Diseases related with mechanisms of translocation: [0041]
Pain related diseases such as: idiopathic chronic pain syndromes
(including without limitation chronic widespread pain,
fibromyalgia, bladder syndrome [0042] migraine, head and neck
deacceleration trauma, epicondylitis, impingement syndrome.
[0043] Diseases related with mechanisms of local LPS neutralization
in the gut: [0044] Graft vs host disease (GVHD),chronic
inflammatory gastro intestinal diseases such as Crohn's disease,
ulcerative colitis, irritable bowel syndrome,
[0045] Autoimmune diseases such as: [0046] crest syndrome, systemic
lupus erythematodes, pemphigus vulgaris, sclerodoma,
[0047] Other diseases such as: [0048] Atherosclerosis,
osteoarthritis, dementia, Alzheimer's, and psychiatric diseases
such as depression and schizophrenia.
[0049] In a specific embodiment, the method of the invention is
applied to patients suffering from idiopathic pain syndrome, graft
vs host disease (GVHD) and/or pemphigus vulgaris and epicondylitis,
migraine, osteoarthritis, frozen shoulder or adhesive capsulitis,
oral mucositis, carpal tunnel syndrome.
Anti-LPS Immunoglobulin Treatment
[0050] As used herein, an "anti-LPS immunoglobulin treatment"
relates to any therapeutic treatment comprising, as the active
principle, a substance or composition made of immunoglobulins or
their antigen-binding portions, directed against lipopolysaccharide
(LPS) or micro-organism producing such lipopolysaccharide or their
LPS-containing portions.
[0051] As used herein, and as well understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. Beneficial or desired results
can include but not limited to, alleviation or amelioration of one
or more symptoms or conditions, diminishment of extent of disease,
stabilized (i.e. not worsening) state of disease, preventing spread
of disease, delay or slowing of disease progression, reversal of
disease, amelioration or palliation of the disease state, and
remission (whether partial or total).
[0052] Such anti-LPS immunoglobulin treatment has indeed been shown
to be effective in treating patients suffering from chronic
diseases induced by activated monocytes (CD14+) in peripheral blood
as discussed above.
[0053] Preferred examples of such anti-LPS immunoglobulin treatment
and their beneficial results in patients suffering from chronic
disease induced by activated monocytes are described in
WO2012136522 and WO2012136534.
[0054] In one specific embodiment, such anti-LPS immunoglobulin
treatment comprises polyclonal antibodies or monoclonal antibodies
raised against LPS-expressing microorganism or the LPS-containing
portions, more preferably gram-negative bacteria or their
LPS-containing portions.
[0055] As used herein the term "antibody" or "immunoglobulins"
includes whole antibodies and any antigen binding fragments or
derivatives (i.e., "antigen-binding portion") or single chains
thereof. In specific embodiment, such antibody or immunoglobulin
may include monoclonal or polyclonal antibodies or immunoglobulins,
or immunoglobulins obtained from immunized animals or from
recombinant cells, and their antigen-binding portions.
[0056] In naturally occurring antibodies, two heavy chains are
linked to each other by disulfide bonds and each heavy chain is
linked to a light chain by a disulfide bond. There are two types of
light chain, lambda (l) and kappa (k). The light chain includes two
domains, a variable domain (VL) and a constant domain (CL). The
heavy chain includes four domains, a variable domain (VH) and three
constant domains (CH1, CH2 and CH3, collectively referred to as
CH). The variable regions of both light (VL) and heavy (VH) chains
determine binding recognition and specificity to the antigen. The
constant region domains of the light (CL) and heavy (CH) chains
confer important biological properties such as antibody chain
association, secretion, trans-placental mobility, complement
binding, and binding to Fc receptors (FcR). The Fv fragment is the
N-terminal part of the Fab fragment of an immunoglobulin and
consists of the variable portions of one light chain and one heavy
chain. The specificity of the antibody resides in the structural
complementarity between the antibody combining site and the
antigenic determinant. Antibody combining sites are made up of
residues that are primarily from the hypervariable or
complementarity determining regions (CDRs). Occasionally, residues
from nonhypervariable or framework regions (FR) influence the
overall domain structure and hence the combining site.
Complementarity Determining Regions or CDRs refer to amino acid
sequences which together define the binding affinity and
specificity of the natural Fv region of a native immunoglobulin
binding site. The light and heavy chains of an immunoglobulin each
have each three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1,
HCDR2, HCDR3, respectively. An antigen-binding site, therefore,
typically includes six CDRs, comprising the CDRs set from each of a
heavy and a light chain V region. Framework Regions (FRs) refer to
amino acid sequences interposed between CDRs. Each VH and VL is
composed of three CDRs and four FRs arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4.
[0057] Furthermore, although the two domains of the Fv fragment, VL
and VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single chain protein in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv); see
e.g., Bird et al., 1988 Science 242:423-426; and Huston et al.,
1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments
are obtained using conventional techniques known to those of skill
in the art, and the fragments are screened for utility in the same
manner as are intact antibodies.
[0058] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0059] The term "recombinant antibody", as used herein, includes
all antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies isolated from an animal
(e.g., a mouse) or a hybridoma prepared therefrom, antibodies
isolated from a host cell transformed to express the antibody,
e.g., from a transfectoma, antibodies isolated from a recombinant,
combinatorial antibody library, and antibodies prepared, expressed,
created or isolated by any other means that involve splicing of all
or a portion of a immunoglobulin gene, sequences to other DNA
sequences.
[0060] The term "immunoglobulin" also includes chimeric or
humanized antibody.
[0061] The term "chimeric antibody" refers to an antibody which
comprises a VH domain and a VL domain of a non-human antibody, and
a CH domain and a CL domain of a human antibody.
[0062] According to the invention, the term "humanized antibody"
refers to an antibody having variable region framework and constant
regions from a human antibody but retains substantially the same
CDRs of a non-human antibody.
[0063] In specific embodiments, the term "antigen-binding portions"
refers to a fragment of an antibody or immunoglobulin which
contains the variable domains comprising the CDRs of said antibody.
The basic antibody fragments include Fab, Fab', F(ab')2 Fv, scFv,
dsFv, and the like. For example of antibody fragment see also for
review, Holliger et al Nature Biotechnology 23, issue 9 1126-1136
(2005).
[0064] The term "Fab" denotes an antibody fragment having a
molecular weight of about 50,000 and antigen binding activity, in
which about a half of the N-terminal side of H chain and the entire
L chain, among fragments obtained by treating IgG with a protease,
papaine, are bound together through a disulfide bond.
[0065] The term "F(ab')2" refers to an antibody fragment having a
molecular weight of about 100,000 and antigen binding activity,
which is slightly larger than the Fab bound via a disulfide bond of
the hinge region, among fragments obtained by treating IgG with a
protease, pepsin.
[0066] The term "Fab'" refers to an antibody fragment having a
molecular weight of about 50,000 and antigen binding activity,
which is obtained by cutting a disulfide bond of the hinge region
of the F(ab')2.
[0067] A single chain Fv ("scFv") polypeptide is a covalently
linked VH::VL heterodimer which is usually expressed from a gene
fusion including VH and VL encoding genes linked by a
peptide-encoding linker. "dsFv" is a VH::VL heterodimer stabilised
by a disulfide bond. Divalent and multivalent antibody fragments
can form either spontaneously by association of monovalent scFvs,
or can be generated by coupling monovalent scFvs by a peptide
linker, such as divalent sc(Fv)2.
[0068] Hens' naturally occurring immunoglobulins are like mammals'
in having light (L) and heavy
[0069] (H) chains, bridged by disulphide bonds. The molecule is
made up of a variable part with an antigen binding site and a
constant part. In the cases of hens a distinction is made between
the immunoglobulins M (IgM), Y (IgY) and A (IgA). IgM has the same
function as mammals' IgM. Present in all vertebrates, IgM delivers
the first response with its high molecular weight. Results of
recent genetic research suggest that the IgY molecule is
phylogenetically a progenitor of mammals' IgG and IgE. Structurally
there is a clear difference between IgY and mammals' IgG, as the
heavy chain of hens' IgY has an additional constant domain instead
of the hinge region of mammals' IgG. So the molecular weight of IgY
is higher as compared to IgG. IgY, like mammals' IgG, is the
immunoglobulin delivering the second response with its high
serum-concentration and low molecular weight. In the literature,
the terms IgG and IgY are sometimes used as synonyms as regards
hens, so on the basis of the newest findings it has been decided
within the framework of an international ECVAM workshop that the
term IgY should be used throughout. (Schade et al 2005). As used
herein however, the term IgY also includes any antigen-binding
portions of such IgY.
[0070] "LPS-producing microorganisms" may typically be selected
from gram-negative bacteria, most preferably selected from the
group consisting of Streptobacillus moniliformis, meningococcus,
Chlamydophila, chlamydia, spirochetes, cyanobacteria, species of
the Proteobacteria phylum, in particular Enterobacteriaceae
(Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus,
Enterobacter), Pseudomonas bacteria, Legionella bacteria, Neisseria
bacteria, Rickettsia bacteria, Pasteurella multocida bacteria and
species of the Bacteroidetes strain.
[0071] Said LPS-containing portions of LPS-producing microorganism
may be any antigens capable of raising an immunological response
against lipopolysaccharides produced by LPS-producing microorganism
(also called LPS antigenic determinants).
[0072] In a specific embodiment, said anti-LPS immunoglobulin
treatment comprises or essentially consists of immunoglobulin A,
immunoglobulin D, immunoglobulin E, immunoglobulin M,
immunoglobulin G and/or immunoglobulin Y, or their antigen-binding
portions.
[0073] In a specific embodiment, said anti-LPS immunoglobulin
treatment comprises bovine IgG, more specifically colostrum-derived
bovine IgG, or their antigen-binding portions.
[0074] In a preferred embodiment, said anti-LPS immunoglobulin
treatment comprises anti-LPS IgY composition, most preferably,
polyclonal IgY antibodies raised against gram-negative bacteria, or
their antigen-binding portions.
[0075] In a preferred embodiment which may be combined with the
previous embodiment, IgY polyclonal antibodies have been obtained
at least partially from egg yolk powder, preferably from defatted
or partially defatted egg yolk powder.
[0076] Defatted or partially defatted egg yolk powder is obtained
by standard processes (removal of fat from liquid egg yolk or dried
egg yolk powder), preferably by using ultrafiltration, water
dilution, filtration, gel electrophoresis, chromatography, hexane
or supercritical CO.sub.2. After the removal of fat, the defatted
liquid egg yolk or egg yolk powder is dried via lyophilization or
spray drying.
[0077] The IgY composition obtained from egg yolk typically
comprise, for example, lipoproteins, such as HDL and LDL, and the
water-soluble proteins of the egg yolk, a-livetin (80 kDa),
.beta.-livetin (45 kDa) and/or y-livetin (150 kDa), which also
comprise most of the enzymes found in the egg (Ternes, Acker and
Scholtyssek, Ei and Eiprodukte, 1994).
[0078] In order to obtain anti-LPS IgY from hens (or their eggs),
hens may advantageously be immunized by LPS-producing
microorganisms.
[0079] In a specific embodiment, said anti-LPS immunoglobulin
treatment comprises anti-LPS IgY composition, or their
antigen-binding portions, obtained from egg yolk of hens immunized
with gram-negative bacteria or their LPS-containing portions,
preferably from at least two distinct gram-negative bacterial
species.
[0080] For example, said IgY composition suitable for the
preparation of an anti-LPS treatment may be obtained by the
following method: [0081] a) immunizing at least 2 distinct groups
of hens, each group with LPS-producing gram-negative bacteria,
wherein each group is given a different bacterial species, [0082]
b) obtaining the antibody-containing fraction from each of the at
least 2 distinct groups, [0083] c) mixing the at least 2
antibody-containing fractions so that the resulting antibody
preparation comprises at least 3% of each antibody fraction
directed against each bacterial species or their LPS-containing
portions thereof by weight of the total antibody content, and,
preferably, the total amount of each specific antibodies against
each microorganism species is >=7% by weight of the total
antibody content.
[0084] According to the above specific production method, an
anti-LPS immunoglobulin treatment may be an IgY composition which
comprises at least 2 specific antibody fractions which target
distinct lipopolysaccharide-expressing gram-negative bacteria, for
example between 2 and 10 specific antibody fractions; each specific
antibody fraction in each case have an antibody content of at least
3% by weight of the total antibody content of the antibody
preparation; and, preferably, the total amount of such specific
antibody fractions against lipopolysaccharide-expressing
microorganisms is >=7% by weight of the total antibody content
of the antibody preparation.
[0085] In specific embodiments, said IgY composition comprising
antibody fractions directed against gram-negative bacteria selected
from the group consisting of E. coli, Salmonella, Shigella,
Klebsiella, Proteus and Enterobacter. More specifically, in one
preferred embodiment, said IgY composition comprises one fraction
consisting of polyclonal IgY directed against Escherichia coli and
another fraction consisting of polyclonal IgY directed against
Salmonella typhimurium.
[0086] More preferably, each specific antibody fraction account for
at least 4% by weight based on the total antibody content of the
IgY composition, and even more preferably, the total amount of said
specific antibodies is >=10% by weight respectively based on the
total antibody content of the antibody preparation.
[0087] In one specific embodiment, said anti-LPS treatment is
formulated for oral administration.
[0088] Preferably, said anti-LPS treatment is an IgY composition as
described above for oral administration.
The Prognosis Method
[0089] The methods of the invention enable to predict the response
of a patient to an anti-LPS immunoglobulin treatment.
[0090] As used herein, the term "predict" refers to a method that
allows determining with a high level of probability (statistically
significant), prior to treatment, if a patient will respond to said
treatment. Accordingly, the term "predict" does not necessarily
consist of an absolute response. Rather, it may consist of a
response allowing to determine a higher probability of the patient
to be a good responder, as compared to the average probability in a
population.
[0091] As used herein, a "response to anti-LPS immunoglobulin
treatment" or equally a "clinical response to anti-LPS
immunoglobulin treatment" is observed when at least one of the
symptoms of a disease to be treated by said anti-LPS immunoglobulin
is decreased after treatment as compared to said symptom prior to
the treatment. In specific embodiments, said symptom of the chronic
disease is pain, for example specific pain symptom associated to
the chronic disease as measured by a daily summed up score (NRS) by
said patient (NRS-pain score values). In such specific embodiment,
a response to anti-LPS immunoglobulin treatment is a significant
decrease of the mean value of NRS-pain score values after treatment
(for example during a 5-day period) as compared to the mean value
of corresponding NRS-pain score values prior to treatment.
[0092] As used herein, the term "decrease" or "increase" means a
statistically significant decrease or increase of a control value,
preferably, at least 10%, at least 20%, at least 30%, at least 40%,
at least 50%, at least 90%, or at least 99% decrease or increase of
the control value.
[0093] According to the methods of the invention, the patient is
predicted to be responder or non-responder based on evaluation of
the expression of one or more of the predictive biomarkers in a
biological sample, prior to the treatment.
[0094] The term "responder" as used herein means a patient that
demonstrates or is likely to demonstrate a positive treatment
response to anti-LPS immunoglobulin treatment. In an embodiment, a
responder is a patient suffering from idiopathic pain syndromes who
demonstrates or is likely to demonstrate significant pain relief or
even total relief of at least one pain symptom after treatment.
[0095] The term "non-responder" as used herein means a patient that
does not demonstrate or is not likely to demonstrate a positive
treatment or a response to anti-LPS immunoglobulin treatment. In an
embodiment, a non-responder is a patient suffering from idiopathic
pain syndromes and that does not demonstrate or is not likely to
demonstrate any significant pain relief of any of the pain symptoms
after treatment.
[0096] The methods of the invention thus comprises the step of (a)
quantifying the expression of one or more predictive biomarkers in
a biological sample obtained from said patient and (b) comparing
the obtained expression values to corresponding control values.
[0097] As used herein, the term "biological sample" is intended to
include tissues, cells, biological fluids and isolates thereof,
isolated from a subject, as well as tissues, cells and fluids
present within a subject, provided that said biological sample is
susceptible to contain (i) peripheral blood cells or (ii) nucleic
acids or proteins that are produced by the peripheral blood cells
from the patient. In a specific embodiment, said biological sample
for use in the methods of the invention is a blood sample,
including, peripheral blood cell sample and proteins.
Quantifying the Expression of a Predictive Biomarker
[0098] The prediction method of the invention comprises a step of
evaluating the expression of one or more of the predictive
biomarkers in a biological sample.
[0099] As used herein, the term "evaluating" typically include the
steps of (a) quantifying the expression of one or more of the
selected predictive biomarkers in a biological sample obtained from
said subject to obtain expression values, and (b) comparing the
obtained each expression value of said predictive biomarkers to
corresponding control values, wherein differences in the expression
values compared to the respective control values is indicative that
the subject is a responder to anti-LPS immunoglobulin
treatment.
[0100] Expression of the biomarkers can be quantified by
determining gene or protein expression of the predictive biomarkers
in the biological sample of a subject, for example a blood sample.
The quantification may be relative (by comparing the amount of a
biomarker to a control with known amount of biomarker for example
and detecting "higher" or "lower" amount compared to that control)
or more precise, at least to determine the specific amount relative
to a known control amount.
[0101] The terms "nucleic acid" and "polynucleotide" are used
interchangeably and refer to a polymeric form of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides or analogs
thereof. Polynucleotides can have any three-dimensional structure
and may perform any function. The following are non-limiting
examples of polynucleotides: a gene or gene fragment, exons,
messenger RNA (mRNA), cDNA, isolated DNA of any sequence, isolated
RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide can comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure can be imparted before or
after assembly of the polymer. The sequence of nucleotides can be
interrupted by non-nucleotide components. A polynucleotide can be
further modified after polymerization, such as by conjugation with
a labeling component. The term also refers to both double- and
single-stranded molecules. Unless otherwise specified or required,
any embodiment of this invention that is a polynucleotide
encompasses both the double-stranded form and each of two
complementary single-stranded forms known or predicted to make up
the double-stranded form.
[0102] A "gene" refers to a polynucleotide containing at least one
open reading frame (ORF) that is capable of encoding a particular
polypeptide or protein after being transcribed and translated. A
polynucleotide sequence can be used to identify larger fragments or
full-length coding sequences of the gene with which they are
associated. Methods of isolating larger fragment sequences are
known to those of skill in the art. "Gene expression", "gene
product" or "expression" are all used herein interchangeably and
refer to the nucleic acids or amino acids (e.g., peptide or
polypeptide) generated when a gene is transcribed and translated,
cDNA or RNA sequence of the biomarker; biomarker gene expression,
biomarker protein expression, biomarker mRNA expression; functional
effect of the biomarker protein, functional effect of the biomarker
gene, cDNA or mRNA, protein, cDNA, gene or mRNA activity.
[0103] In a particular embodiment "gene expression", "gene product"
or "expression" denotes mRNA expression, cDNA expression, protein
transcription and protein expression.
[0104] The term "polypeptide" is used interchangeably with the term
"protein" and in its broadest sense refers to a compound of two or
more subunit amino acids. The subunits can be linked by peptide
bonds.
[0105] Such quantification methods may alternatively include
detection and quantification of the corresponding gene expression
level of said predictive biomarker which encompasses the
quantification of corresponding mRNA of said predictive biomarker,
for example by performing Real-Time quantitative PCR, as well as by
using DNA microarrays, i.e. substrate onto which are bound nucleic
acids, at defined position, that specifically hybridize with the
cDNA corresponding to amplified mRNA of said predictive
biomarker.
[0106] Typically, in specific embodiments, a mixture of transcribed
polynucleotides (mRNA) obtained from the biological sample of the
patient is subjected to reverse transcription and quantitative
amplification. Said cDNA or mRNA may be detected by in vitro
techniques either by stringent hybridization to DNA microarrays or
Northern blots.
[0107] In any cases, a general principle of such detection and
quantification assays involve preparing a sample or reaction
mixture that may contain a predictive biomarker and a probe under
appropriate conditions and for a time sufficient to allow the
predictive biomarker and probe to interact and bind, thus forming a
complex that can be detected (and quantified) in the reaction
mixture.
[0108] These detection and/or quantification assays of a biomarker
can be conducted in a variety of ways. Appropriate conditions to
the particular assay and components thereof will be well known to
one skilled in the art.
[0109] In a particular embodiment, the level of predictive
biomarker mRNA can be determined both by in vitro formats in a
biological sample using methods known in the art.
[0110] Specific methods for quantifying a biological marker for the
purpose of carrying out the prediction methods of the invention are
described hereunder. Those methods include
[0111] Luminex and ELISA quantification methods as described in the
Examples.
Quantifying Predictive Biomarkers by cDNA Microarrays
[0112] According to this embodiment, a microarray may be
constructed based on one or a combination of 2, 3, 4, 5 or 6 of the
15 predictive biomarkers that are disclosed throughout the present
specification. Probes for these biomarkers may be placed on the
microarray.
[0113] These probes may be different than those used in PCR
methods. However, they should be designed and used in conditions
such that only nucleic acids (mRNA or cDNA or PCR amplificates of
cDNA material) having the predictive biomarkers may hybridize and
give a positive result.
[0114] In a preferred embodiment, the array will further include
one or more control probes.
[0115] In specific embodiments, said probes may be oligonucleotides
that range from about 5 to about 500 or about 5 to about 200
nucleotides, more preferably from about 10 to about 100 nucleotides
and most preferably from about 15 to about 70 nucleotides in
length. In other particularly preferred embodiments, the probes are
about 20 or 25 nucleotides in length. In another preferred
embodiment, test probes are double or single strand DNA
sequences.
[0116] DNA sequences may be isolated or cloned from natural sources
or amplified from natural sources using natural nucleic acid as
templates. These probes have sequences complementary to particular
subsequences of the genes of the predictive biomarkers whose
expression they are designed to detect.
[0117] In addition to test probes that bind the target nucleic
acid(s) of interest (corresponding gene expression of one or more
of the predictive biomarkers), the microarray can contain a number
of control probes. The control probes may fall into three
categories referred to herein as normalization controls; expression
level controls; and mismatch controls. Normalization controls are
oligonucleotide or other nucleic acid probes that are complementary
to labeled reference oligonucleotides or other nucleic acid
sequences that are added to the nucleic acid sample. The signals
obtained from the normalization controls after hybridization
provide a control for variations in hybridization conditions, label
intensity, "reading" efficiency and other factors that may cause
the signal of a perfect hybridization to vary between arrays. In a
preferred embodiment, signals (e.g. fluorescence intensity) read
from all other probes in the array are divided by the signal (,
fluorescence intensity) from the control probes thereby normalizing
the measurements. Virtually any probe may serve as a normalization
control. However, it is recognized that hybridization efficiency
varies with base composition and probe length. Preferred
normalization probes are selected to reflect the average length of
the other probes present in the array; however, they can be
selected to cover a range of lengths. The normalization control(s)
can also be selected to reflect the (average) base composition of
the other probes in the array, however in a preferred embodiment,
only one or a few probes are used and they are selected such that
they hybridize well (i.e., no secondary structure) and do not match
any target-specific probes.
[0118] Expression level controls are probes that hybridize
specifically with constitutively expressed genes in the biological
sample. Virtually any constitutively expressed gene provides a
suitable target for expression level controls. Typical expression
level control probes have sequences complementary to subsequences
of constitutively expressed "housekeeping genes" including the
beta-actin gene, the RNA18S, the transferrin receptor gene, thGAPDH
gene, Ubiquitin C (UBC) gene, ribosomal protein large PO (RPLPO)
gene, beta-2-microglobulin (B2M), hypoxanthine
phosporibosyltransferase 1 (HPRT1) gene, TATA box binding protein
(TBP) gene, peptidylprolyl isomerase A (PPIA) gene, glucuronidase
beta (GUSB) gene, and phosphoglycerate kinase 1 (PGK1) gene.
[0119] Solid supports containing oligonucleotide probes for
differentially expressed genes can be any solid or semisolid
support material known to those skilled in the art. Suitable
examples include, but are not limited to, membranes, filters,
tissue culture dishes, polyvinyl chloride dishes, beads, test
strips, silicon or glass based chips and the like. Suitable glass
wafers and hybridization methods are widely available. Any solid
surface to which oligonucleotides can be bound, either directly or
indirectly, either covalently or non-covalently, can be used. In
some embodiments, it may be desirable to attach some
oligonucleotides covalently and others non-covalently to the same
solid support. A preferred solid support is a high density array or
DNA chip. These contain a particular oligonucleotide probe in a
predetermined location on the array. Each predetermined location
may contain more than one molecule of the probe, but each molecule
within the predetermined location has an identical sequence. Such
predetermined locations are termed features.
Quantifying Predictive Biomarkers by Real-Time Quantitative PCR
[0120] Methods to quantify nucleic acids (for example mRNAs of the
predictive biomarkers) may include real time quantitative PCR
methods (RT-qPCR). RT-qPCR is based on the detection of a
fluorescent report molecule that increases as PCR product
accumulates with each cycle of amplification. Fluorescent reporter
molecules include dyes that bind double-stranded DNA (e.g. SYBR
Green I) or sequence-specific probes (e.g. Molecular Beacons or
TaqMan.RTM. Probes). Such methods include multiplex quantitative
methods which allow to quantify in parallel expression of a
plurality of predictive biomarkers.
[0121] For example, for quantifying the predictive biomarkers of
the invention by use of RT-qPCR methods, a blood cell sample of a
patient is collected. Said blood cells are lysed and total RNA is
extracted according to standard methods.
[0122] Total RNA extract is then subjected to reverse transcription
followed by real-time quantitative PCR (for example, as described
in the examples).
Detecting and Quantifying Biomarker Polypeptides
[0123] Expression level of the biomarker can also be determined by
examining protein expression or the protein product of at least one
of the predictive biomarkers. Determining the protein level
involves measuring the amount of any immunospecific binding that
occurs between an antibody that selectively recognizes and binds to
the polypeptide of the biomarker in a sample obtained from a
patient and comparing this to the amount of immunospecific binding
of at least one biomarker in a control sample. The amount of
protein expression of the biomarker can be increased or reduced
when compared with control expression. Alternatively, a combination
of more than one of the predictive biomarkers can be assayed.
[0124] Various methods are known in the art for detecting protein
expression levels in such biological samples, including various
immunoassays methods. They include but are not limited to
radioimmunoassays, ELISA (enzyme linked immunosorbent assays),
"sandwich" immunoassays, immunoradiometric assays, in situ
immunoassays (using e.g., colloidal gold, enzyme or radioisotope
labels), western blot analysis, immunoprecipitation assays,
immunofluorescent assays, flow cytometry, immunohistochemistry,
confocal microscopy, enzymatic assays, surface plasmon resonance
and PAGE-SDS.
[0125] Determining the protein level involves for example measuring
the amount of any immunospecific binding that occurs between an
antibody that selectively recognizes and binds to the polypeptide
of the biomarker in a sample obtained from a patient. These assays
may also include direct binding of labelled antibody to a target
biomarker.
[0126] Sandwich assays are among the most useful and commonly used
assays. A number of variations of the sandwich assay technique
exist, and all are intended to be encompassed by the present
invention. Briefly, in a typical forward assay, an unlabeled
antibody is immobilized on a solid substrate, and the sample to be
tested brought into contact with the bound molecule. After a
suitable period of incubation, for a period of time sufficient to
allow formation of an antibody-antigen complex, a second antibody
specific to the antigen, but labeled with a reporter molecule
capable of producing a detectable signal is then added and
incubated, allowing time sufficient for the formation of another
complex of antibody-antigen-labeled antibody. Any unreacted
material is washed away, and the presence of the antigen is
determined by observation of a signal produced by the reporter
molecule. The results may either be qualitative, by simple
observation of the visible signal, or may be quantitated by
comparing with a control sample containing known amounts of
biomarker.
[0127] Variations on the forward assay include a simultaneous
assay, in which both sample and labeled antibody are added
simultaneously to the bound antibody. These techniques are well
known to those skilled in the art, including any minor variations
as will be readily apparent. In a typical forward sandwich assay, a
first antibody having specificity for the biomarker is either
covalently or passively bound to a solid surface.
[0128] The binding processes are well-known in the art and
generally consist of cross-linking covalently binding or physically
adsorbing, the polymer-antibody complex is washed in preparation
for the test sample. An aliquot of the sample to be tested is then
added to the solid phase complex and incubated for a period of time
sufficient (e.g. 2-40 minutes or overnight if more convenient) and
under suitable conditions (e.g. from room temperature to 40.degree.
C. such as between 25.degree. C. and 32.degree. C. inclusive) to
allow binding of any subunit present in the antibody. Following the
incubation period, the antibody subunit solid phase is washed and
dried and incubated with a second antibody specific for a portion
of the biomarker. The second antibody is linked to a reporter
molecule which is used to indicate the binding of the second
antibody to the molecular marker.
[0129] An alternative method involves immobilizing the predictive
biomarkers in the sample and then exposing the immobilized
biomarkers to specific antibody which may or may not be labeled
with a reporter molecule. Depending on the amount of biomarker
target and the strength of the reporter molecule signal, a bound
biomarker target may be detectable by direct labelling with the
antibody. Alternatively, a second labeled antibody, specific to the
first antibody is exposed to the target- first antibody complex to
form a target-first antibody-second antibody tertiary complex. The
complex is detected by the signal emitted by the reporter
molecule.
[0130] By "reporter molecule", as used in the present
specification, is meant a molecule which, by its chemical nature,
provides an analytically identifiable signal which allows the
detection of antigen-bound antibody. The most commonly used
reporter molecules in this type of assay are either enzymes,
fluorophores or radionuclide containing molecules (i.e.
radioisotopes) and chemiluminescent molecules.
[0131] In the case of an enzyme immunoassay or ELISA assay, an
enzyme may typically be conjugated to the second antibody,
generally by means of glutaraldehyde or periodate. As will be
readily recognized, however, a wide variety of different
conjugation techniques exist, which are readily available to the
skilled artisan. Commonly used enzymes include horseradish
peroxidase, glucose oxidase, -galactosidase and alkaline
phosphatase, amongst others. The substrates to be used with the
specific enzymes are generally chosen for the production, upon
hydrolysis by the corresponding enzyme, of a detectable colour
change. Examples of suitable enzymes include alkaline phosphatase
and peroxidase. It is also possible to employ fluorogenic
substrates, which yield a fluorescent product rather than the
chromogenic substrates noted above. In all cases, the
enzyme-labeled antibody is added to the first antibody-molecular
marker complex, allowed to bind, and then the excess reagent is
washed away. A solution containing the appropriate substrate is
then added to the complex of antibody-antigen-antibody. The
substrate will react with the enzyme linked to the second antibody,
giving a qualitative visual signal, which may be further
quantitated, usually spectrophotometrically, to give an indication
of the amount of biomarker which was present in the sample.
[0132] Alternatively, fluorescent compounds, such as fluorescein
and rhodamine, may be chemically coupled to antibodies without
altering their binding capacity. When activated by illumination
with light of a particular wavelength, the fluorochrome-labeled
antibody adsorbs the light energy, inducing a state to excitability
in the molecule, followed by emission of the light at a
characteristic color visually detectable with a light microscope.
As in the EIA, the fluorescent labeled antibody is allowed to bind
to the first antibody-molecular marker complex. After washing off
the unbound reagent, the remaining tertiary complex is then exposed
to the light of the appropriate wavelength, the fluorescence
observed indicates the presence of the molecular marker of
interest. Immunofluorescence and EIA techniques are both very well
established in the art. However, other reporter molecules, such as
radioisotope, chemiluminescent or bioluminescent molecules, may
also be employed.
Comparing Expression Level of Predictive Biomarkers to a Control
Value
[0133] In specific embodiment of the prediction method of the
invention, the quantifying step thus allows to obtain an
"expression value" for each biomarker tested in the biological
sample, for use in the comparing step.
[0134] For ease of use in the comparing step, said expression value
may consist of a normalized (relative) value which is obtained
after comparison of the absolute expression level value with a
reference value, said reference value consisting for example of the
expression level value of reference proteins in the biological
sample.
[0135] Each expression level value obtained after quantification of
the expression of one or more of the predictive biomarkers in a
patient prior to treatment, is compared with a corresponding
control value, allowing to determine whether the patient is a
responder or a non-responder.
[0136] Preferably, said expression level value consists of a
normalized relative value which is obtained after comparison of the
absolute expression level value with a normal value, said normal
value consisting of the expression level value of constitutive (or
reference) genes, such as housekeeping genes .beta.-actin, 18S RNA
or peptidylprolyl isomerase A (PPIA).
[0137] Said control value may be for example, the mean value of
normalized (relative) mean value of healthy subjects, responder
patients and/or non-responder patients. Examples of such normalized
mean value are given in the examples below for each of the 15
predictive biomarkers.
[0138] Said control value can also be determined by routine
experimentation depending on the quantification methods and the
predictive biomarkers that will be used for the methods of the
invention.
[0139] For example, said control value corresponds to the
expression level value observed for non-responder patients, and a
patient is predicted to be a responder when the expression level
value is statistically different from the control value, for
example increased as compared to a control value, or decreased as
compared to a control value.
[0140] Alternatively, said control value corresponds to the
expression level value observed for responder patients, and a
patient is predicted to be a responder when the expression level
value is statistically not different from the control value.
[0141] The comparison referred to in step (b) of the methods of the
invention may be carried out manually or computer assisted.
[0142] For a computer-assisted comparison, the expression values
may be compared to control values which are stored in a database by
a computer program. The computer program may further evaluate the
result of the comparison, i.e. automatically provide the desired
assessment in a suitable output format.
[0143] As it is shown in the examples, each of the predictive
biomarkers according to the invention listed herein is relevant for
predicting a response to an anti-LPS immunoglobulin treatment,
since all the biomarkers have a P value equal or inferior to 0.0001
except for IL6 and BCHE predictive biomarker.
[0144] Statistical relevance may be improved by combining the
predictive biomarkers in the assays. In specific embodiments, the
expression level of 2 predictive biomarkers, or 3, 4, 5, 6, 7 or
even 8, out of the 15 predictive biomarkers of the invention, is
evaluated.
[0145] Any combination of two, three, four or more of predictive
biomarkers is encompassed by the methods of the invention: Specific
combinations of predictive biomarkers for use in the methods of the
invention are listed hereafter: [0146] (i)
CD14+TLR4+IGF-1+IL-8+IFN-alpha+CXCL10+GDNF [0147] (ii)
CD14+TLR4+IGF-1+IL-8+IFN-alpha+RAGE [0148] (iii)
CD14+CD68+TLR4+IGF-1+IL-8+IFN-alpha [0149] (iv)
CD14+TLR4+IGF-1+IL-8+IFN-alpha [0150] (v) CD14+TLR4+IGF-1+IL-8
[0151] (vi) CD14+TLR4+IGF-1+IFN-alpha [0152] (vii)
CD14+TLR4+IGF-1+RAGE
[0153] or other combinations with the mentioned markers.
[0154] The comparing step may not necessarily include a separate
comparison of the expression values of each biomarker with their
corresponding control values. In specific embodiments, a
multi-biomarker score value can be obtained by combining together
the expression values or their normalized values and compared to a
corresponding multibiomarker score control value.
The Predictive Biomarkers
[0155] The 15 predictive biomarkers of the invention of the present
invention are described hereafter by their acronym names, also
termed herein "gene symbols", according to Genbank
nomenclature:
TABLE-US-00001 TABLE 1 List of predictive biomarkers Biomarker
UniprotKB/Swiss-Prot1 CD14 P08571 C68 P34810 TLR4 O00206 IL6 P05231
IL8 P10145 IL10 P22301 IFN.alpha. P01562 IGF1 P05019 CXCL1 P09341
CXCL9 Q07325 CXCL10 P02778 RAGE(=AGER) Q15109 GDNF P39905 BCHE
P06276 .sup.1http://www.uniprot.org/
[0156] In the present invention, when referring to the biomarkers,
as already explained above, it may alternatively refer either to
the gene (polynucleotide) encoding said biomarker, any of its
expression product, including transcript RNA molecules or
corresponding cDNAs or the protein and/or its post-translational
modifications.
[0157] As shown in the Table 2 in the examples, the relative
expression value dCt of the predictive biomarkers CD14, C68, TLR4,
TLR7, IL2, IL6, IL8, IFN.alpha., CXCL1, CXCL9, CXCL10, GDNF, RAGE
and IGF1 has been shown to be significantly lower in responder
patients as compared to the corresponding relative expression value
in non-responder patients.
[0158] The relative expression value of the predictive biomarker
BCHE has been shown to be significantly higher in responder
patients as compared to the corresponding relative expression value
in non-responder patients.
Assaying for Biomarker Expression and the Treatment with Anti-LPS
Immunoglobulin
[0159] Once a patient has been predicted to be a responder to
anti-LPS immunoglobulin treatment, administration of a suitable
anti-LPS immunoglobulin treatment only to said responder patient
can be effected in one dose, continuously or intermittently
throughout the course of treatment.
[0160] Methods of determining the most effective means and dosage
of administration are well known to those of skill in the art and
will vary with the composition used for therapy, the purpose of the
therapy, and the subject being treated. Single or multiple
administrations can be carried out with the dose level and pattern
being selected by the treating physician. Suitable dosage
formulations and methods of administering the agents may be
empirically adjusted. Preferably, oral formulations are used.
[0161] If a patient is predicted to be a non-responder to anti-LPS
immunoglobulin treatment, alternative therapies may be
preferred.
[0162] At least one of the biomarkers selected from Table 1 can be
assayed, prior to administration of anti-LPS treatment.
Alternatively, more than one, for example 2, 3, 4, 5, 6, 7, or 8,
or all of the 15 biomarkers selected from Table 1 can be assayed
together.
Kits of the Invention
[0163] The invention also relates to a kit for carrying out the
prediction methods of the invention as disclosed above. The kit may
comprise a plurality of reagents, each of which is capable of
binding specifically with one or more of the predictive biomarkers
(either its nucleic acid or protein). Suitable reagents for such
kit include antibodies or nucleic acids. For example, such kits
include a DNA microarray as described above. Such kit may
alternatively comprise, primers and probes for carrying out RT-qPCR
on one or more of the predictive biomarkers as listed in Table
1.
[0164] The monitoring or prediction kit of the invention may thus
include a plurality of reagents, each of which is capable of
binding specifically with a gene or protein specific of one of the
predictive biomarkers. Suitable reagents for binding specifically
with a protein biomarker include, without limitation,
antibodies.
[0165] In specific embodiments, the kit comprises specific reagents
for quantifying the following group of biomarkers: [0166] (i)
CD14+TLR4+IGF-1+IL-8+IFN-alpha+CXCL10+GDNF; [0167] (ii)
CD14+TLR4+IGF-1+IL-8+IFN-alpha+RAGE; [0168] (iii)
CD14+CD68+TLR4+IGF-1+I L-8+I FN-alpha; [0169] (iv)
CD14+TLR4+IGF-1+IL-8+IFN-alpha; [0170] (v) CD14+TLR4+IGF-1+IL-8;
[0171] (vi) CD14+TLR4+IGF-1+IFN-alpha; or, [0172] (vii)
CD14+TLR4+IGF-1+RAGE,
[0173] or other combinations with the above mentioned specific
biomarkers.
EXAMPLES
Preparation of Anti-LPS IgY Treatment
[0174] The manufacturing process of the drug substance for the
below samples comprised four main steps. The first step was the IgY
egg production via vaccination of six herds of hens (Gallus gallus
domesticus) with antigen preparations of inactivated whole cell
bacteria of Escherichia coli F18 and Salmonella typhimurium,
Porphyromonas gingivalis, Clostridium perfringens C, Streptococcus
Mutans, and the fungal cells of Candida albicans respectively.
[0175] Six distinct hen herds were held. Each herd was immunized
with one of the above mentioned antigens. The second step was the
egg processing including egg yolk separation, pasteurization and
egg yolk spray drying. The third step was the delipidation with
hexane and the stabilization with oligosaccharides, which was
followed by the fourth step, the preparation of the drug substance
by mixing equal amounts of the delipidated egg yolk powders from
the six vaccinated herds. The mixed delipidated egg yolk powder
(drug substance) was not further formulated. It was given to the
patients and swallowed with water. Alternatively it could be mixed
into plain yoghurt and then eaten. Specific IgY activity was
measured by competitive ELISA using respective in house
standards.
Example 1: Predictive Biomarkers in Patients Suffering from
Idiopathic Chronic Pain Syndrome
Studied Patient Cohort
[0176] Patients (n=40) were included if they had chronic idiopathic
pain irrespective of classification and no acceptable response to
any symptomatic treatment (chronic intractable pain syndromes).
Those with allergy against egg components were excluded. The
patients were treated over a period of 4 weeks. The dosage during
the first 2 weeks of IgY treatment was 1.25 g twice daily followed
by 2.5 g twice daily during the last two weeks. The therapeutic
effects were assessed via both clinical and laboratory parameters.
Thereby, the laboratory parameters underwent a blinded assessment.
The protocol used was approved by the local medical ethics
committee and informed consent was given by the patient prior to
sample acquisition.
Clinical Parameters
[0177] Patient pain diaries containing a daily summed up score
(numeric rating scale NRS) of pain and 5 quality of life
parameters. The pain diary had the option of daily documentation of
three differently classified pain symptoms (patients suffering from
long standing idiopathic pain present in the majority of cases more
than one chronic pain syndrome). Patients with more than one
chronic pain entity graded them in their personal perception of
pain intensity, once before the start of the trial (pain 1=highest
grade, pain 3=lowest grade).
[0178] The primary clinical end point of the study was defined as
the change of the two mean NRS-pain score values of at least one of
the three pain symptoms.
Primary Endpoint
[0179] The change in pain diary VAS-pain score between the mean
value of the 5-day period prior to the start and the 5-day period
prior to the end of the study. A reduction of at least 2 points on
the numeric rating scale (NRS) of at least one of the three
constant pain syndromes was defined as a positive result. Efficacy
of the delipidated egg yolk powder containing the target IgY could
be shown. The respective patients were named "Responders".
Secondary Endpoints
[0180] (i) Change in "quality of life parameters" (pain diary data)
between the mean value of the 5-day period prior to the start and
the 5-day period prior to the end of the study. A reduction of at
least 2 points on the numeric rating scale (NRS) was defined as a
positive result. [0181] (ii) Significant changes of biomarker
values between the pre and end of treatment values (represent
responder patients). [0182] (iii) Significant laboratory
differences between the mean pre-therapeutic laboratory values of
responders vs. non-responders (for the identification of
response-predictive biomarker). [0183] (iv) Incidence and severity
of unexpected adverse reactions
[0184] From a total of 40 patients, peripheral blood from 38
individuals was analyzed and compared with peripheral blood of
untreated healthy volunteers (n=30).
Real Time PCR
[0185] The qRT-PCR was carried out as followed: Peripheral blood
was taken from the patients with chronic diseases (chronic pain
syndromes) treated with IgY at the University of Wurzburg, Germany,
at two time points: (i) Before the beginning of the treatment with
IgY (T1=week 0) and (ii) after four weeks of treatment with IgY
(T2=week 4).
[0186] For this purpose 40 ml EDTA peripheral blood was taken from
the patients at both time points. To obtain the blood cells, one
part of EDTA-blood plus five parts Lysis Buffer (Fa. Qiagen) was
mixed and kept for 15 min at room temperature (RT). Then the tubes
were centrifuged by 311 g for 10 Min and the supernatant discarded.
The pellet of peripheral blood mononuclear cells (PBMCs) was washed
with RPMI 1640 Medium (Fa.Gibco). This procedure was performed
three times. The cells were counted and 5.times.10.sup.6 cells/ml
were diluted using "freeze medium", (i.e. inactivated fetal calf
serum (FCS, Fa. Gibco)+10% Dimethylsulfoxid (DMSO, Fa. Sigma) and
stored at -80.degree. C.).
[0187] Gene expression was analyzed using reverse transcription
following quantitative real-time PCR (RT-qPCR). Reverse
transcription from total RNA to cDNA was carried out by using High
Capacity cDNA Reverse Transcription Kit (Life Technologies,
Carlsbad, Calif.) according to the manufacturer's instructions.
[0188] Gene quantification was performed using (i) Taqman Gene
Expression Master Mix (Life
[0189] Technologies) and Taqman Gene Expression Assays (Life
Technologies) according to the manufacturer's instructions.
Analysis was performed on a Biorad CFX96 Touch Real-Time PCR
Detection System (Biorad, Hercules, Calif.). Quantification data
were analyzed with the Biorad CFX Manager Analysis software
(Biorad) and Microsoft Excel 2010 (Microsoft Corporation, Redmond,
Wash.) and (ii) MESA GREEN qPCR MasterMix Plus for SYBR.RTM.
[0190] Assay (Eurogentec, Seraing, Belgium) and RT2 qPCR Primer
Assays (Qiagen, Hilden, Germany). For preparation of the reaction
mix 12.5 .mu.l MESA GREEN qPCR MasterMix Plus for SYBR.RTM. Assay,
9.5 .mu.l dH2O and 1.0 .mu.l RT2 qPCR Primer Assay were mixed and
2.0 .mu.l cDNA dilution (containing 100 ng cDNA) was added.
[0191] Analysis was performed on a Biorad CFX96 Touch Real-Time PCR
Detection System (Biorad, Hercules, Calif.) according to the
manufacturer's instructions. Quantification data were analyzed with
the Biorad CFX Manager Analysis software (Biorad) and Microsoft
Excel 2010 (Microsoft Corporation, Redmond, Wash.).
[0192] Housekeeping genes .beta.-actin, 18S RNA and PPIA were used
for relative quantification. Reproducibility was confirmed by
duplicates of each sample. The average threshold cycle (Ct) value
was calculated as the cycle number at which the fluorescence of the
reporter reaches a fixed threshold. The difference (dCt) between
the average Ct values of the samples in the target wells and those
of the housekeeping genes was assessed.
Identification of Predictive Biomarkers of Therapeutic Response
[0193] Blood samples were taken before study start (prior to first
dose of DYP IgY product intake) and at the last day of the
study.
[0194] The following biomarkers were analyzed: Interleukin
(IL)-1.alpha., IL-113, IL-1R.alpha., IL-2, IL-2R, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, Interferon
(IFN)-.alpha., IFN-.gamma., Tumornekrosefaktor (TNF)-.alpha.,
TNF-RI, TNF-RII, chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL4,
CCLS, CCL7, CCL8, CCL11, Chemokine receptor (CXCR) 3, chemokine
(C-X-C motif) ligand 1 (CXCL1), CXCL9, CXCL10, CXCL12, CXCL13,
Granulocyte-macrophage colony-stimulating factor (GM-CSF),
Insulin-like growth factor 1 (IGF-1), nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-.kappa.B),
Cyclooxygenase (COX)-2, Receptor for Advanced Glycation Endproducts
(RAGE), High-mobility group protein B1 (HMGB1), heat shock protein
(HSP)70, HSP90, CD14, CD19, CD20, CD21, CD45, CD68, toll like
receptor (TLR)2, TLR3, TLR4, TLR7, TLR8, TLR9, Substance P,
Leukotriene B4 (LTB4), Fractalkin,
[0195] Epidermal growth factor (EGF), Vascular endothelial growth
factor (VEGF), Basic fibroblast growth factor (FGF basic), glial
cell line-derived neurotrophic factor (GDNF), Butyrylthiocholine
(BChE).
[0196] Protein and gene expression levels were determined. Protein
levels of IL-1.alpha., IL-1.beta., IL-1R.alpha.,
[0197] IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,
IL-13, IL-15, IL-17, IL-18, IFN-.alpha., IFN-.gamma., TNF-.alpha.,
TNF-RI, TNF-RII, CCL2, CCL3, CCL4, CCLS, CCL7, CCL8, CCL11, CXCR3,
CXCL1, CXCL9, CXCL10, CXCL12, CXCL13, GM-CSF, EGF, VEGF, and FGF
basic were measured in duplicate in the serum samples using
immunobead-based multiplex assays (Luminex analysis), Panels of
capture antibody-coated beads and labeled detection antibodies were
purchased from Biosource, (Camarillo, Germany). The reagents were
pre-tested and qualified by the manufacturer to ensure the absence
of cross-reactivity among antibody-coated beads. The assays were
performed using the Bio-Plex System (Biosource). Immunoassays
(ELISA) were used according the manufacturer's instructions to
measure human IGF-1, Substance P, LTB4, RAGE, and BChE in patient
serum (R&D Systems, Wiesbaden, Germany).
[0198] To determine the amount of the cells flow cytometry (FACS)
analysis were used at each of the two time points were analyzed in
peripheral blood samples obtained from the patients and separated
on Lymphoprep.RTM. according to the manufacturer's instructions
(Nycomed Pharma, Oslo, Norway). Cells (5.times.10.sup.5) were
stained with PE-conjugated anti-CD14, anti-CD68, FITC-conjugated
anti-CD45 and 7 AAD, PE-conjugated anti-CD25, FITC-conjugated
anti-CD3 and 7 AAD and PE-conjugated anti-mouse IgG2a. All
antibodies were purchased from Beckman Coulter (Krefeld, Germany).
Four-color flow cytometry was performed on a FACS-Epics XL-MCL
(Beckman Coulter) and cells were analyzed using Expo 32 acquisition
software (Beckman Coulter). Viable lymphocytes were gated and
10.sup.5 events were collected.
Results
[0199] 38 patients completed the study, 24 women and 14 men; their
mean age was 54 years and the mean duration of pain history 12.6
years. [0200] All 38 patients documented the course of at least one
pain, 30 patients documented the course of at least two and 26, the
course of three differently classified pain syndromes. [0201]
Analysis of variance (ANOVA) showed significant pain relief for the
responder study patient group to the IgY treatment, and in
addition, pain relief was significant for pain syndrome 1, 2 and 3
when responders were grouped together. That means that as a group
the responders patients on average showed significant relief in one
pain syndrome as well as the same relief in their second and third
pain entity. [0202] 24 out of 38 subjects completing the study were
identified as IgY responders in accordance to the definition of the
primary endpoint. [0203] All 24 IgY responders fulfilled both the
clinical and laboratory response criteria. [0204] 14 Subjects were
identified as IgY non-responders missing both clinical and
laboratory response criteria [0205] Relevant laboratory parameters
(gene expression) could be identified as potentially predictive
biomarkers for therapeutically relevant IgY efficacy. [0206] The
two patients dropped out of the study due to violation of the
protocol and one from persistent cough, which happened some days
before the end of the study.
[0207] The major therapeutic effects resulted during the second
part of the study under an IgY dosage of 2.times.2.5 g.
[0208] Statistical analysis and identification of predictive
biomarkers chosen as a potentially predictive marker for the
IgY-treatment.
[0209] Each used parameter was compared between healthy volunteers
and non-responders or responders as well as non-responders and
responders using a T-test. Furthermore, each of the parameters was
also compared between healthy volunteers together with
non-responders and responders using an one way ANOVA. The
statistical programme used was the GraphPad Prism5. The parameters
which were significantly different, p<0.05 between healthy
volunteers and non-responders and responders were chosen as a
potentially predictive marker for the IgY-treatment.
[0210] The table 2 below shows the relative expression data for
each predictive biomarker between responder and non-responder
groups with P values showing statistical significance:
TABLE-US-00002 TABLE 2 Comparison of expression values of
predictive biomarkers in responder, and non-responder Predictive
Relative expression (dCt) Relative expression (dCt) biomarker in
Responder group in Non-responder group P value CD14 5.627 .+-.
0.2718 N = 21 9.959 .+-. 0.3561 N = 11 <0.0001 CD68 4.718 .+-.
0.1754 N = 21 8.449 .+-. 0.2215 N = 11 <0.0001 TLR4 10.58 .+-.
0.2286 N = 21 13.48 .+-. 0.2675 N = 10 <0.0001 TLR7 9.885 .+-.
0.1858 N = 21 11.87 .+-. 0.2018 N = 10 <0.0001 IL2 13.10 .+-.
0.5763 N = 10 16.82 .+-. 0.1823 N = 7 0.0001 IL6 11.72 .+-. 0.6477
N = 16 15.96 .+-. 0.2643 N = 8 0.0002 IL8 6.323 .+-. 0.3445 N = 21
8.775 .+-. 0.3917 N = 11 0.0001 IFN.alpha. 12.80 .+-. 0.4354 N = 13
15.38 .+-. 0.3370 N = 12 0.0001 CXCL1 8.510 .+-. 0.3240 N = 21
11.43 .+-. 0.2247 N = 10 <0.0001 CXCL9 11.45 .+-. 0.2191 N = 16
14.37 .+-. 0.1162 N = 9 <0.0001 CXCL10 11.94 .+-. 0.2639 N = 20
14.20 .+-. 0.2041 N = 9 <0.0001 GDNF 6.403 .+-. 0.2769 N = 20
9.058 .+-. 0.3319 N = 11 <0.0001 RAGE 5.935 .+-. 0.2236 N = 21
10.59 .+-. 0.5092 N = 10 <0.0001 BCHE 12.38 .+-. 0.3535 N = 5
9.952 .+-. 0.6549 N = 9 0.0227 IGF1 11.68 .+-. 0.2025 N = 21 13.83
.+-. 0.1611 N = 11 <0.0001
Conclusions
[0211] The polyvalent anti gram-negative whole bacteria
Immunoglobulin Y offers a completely new approach to treat
idiopathic pain syndromes even in patients with very late stages of
chronification and highlights the first time a new common
pathogenic mechanism as one major component in a very complex
aetiology of a broad spectrum of phenotypes of chronic pain.
[0212] The oral application of polyvalent anti-gram negative whole
bacteria Immunoglobulin Y resulted in a sustainable pain relief in
more than 60% of patients (therapeutic responders).
[0213] Predictive biomarkers were identified, distinguishing not
only between healthy subjects and pain patients but also between
patients responding to IgY treatment and those not responding. This
observation offers the availability of a prognostic laboratory
screening test for pre-application oral polyvalent anti-gram
negative whole bacteria Immunoglobulin Y therapy response
testing.
[0214] Through this observation a representative example of
potential predictive markers allowing to identify the patient
population with such markers in their blood as a treatment response
criteria for the anti-LPS immunoglobulin therapy.
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