NZ716381B2 - Use of a cd6 binding partner and method based thereon - Google Patents
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- NZ716381B2 NZ716381B2 NZ716381A NZ71638114A NZ716381B2 NZ 716381 B2 NZ716381 B2 NZ 716381B2 NZ 716381 A NZ716381 A NZ 716381A NZ 71638114 A NZ71638114 A NZ 71638114A NZ 716381 B2 NZ716381 B2 NZ 716381B2
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Abstract
Multiple Sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system (CNS), characterised by inflammatory infiltrates of T-cells, B cells, macrophages and focal demyelinating plaques within the CNS. Both Th1 and Th17 cell-mediated responses have been shown to play a role in the development of inflammatory demyelination. CD6 is an important cell surface protein predominantly expressed by human T cells and a subset of B cells, as well as by some B cell chronic lymphocytic leukemias and neurons. It has been shown that CD6 can function as an important accessory molecule in T cell activation. The present disclosure relates to a method and a use of a CD6 binding partner. In particular, the present disclosure relates to the use of an Itolizumab antibody specifically binding to Domain 1 (D1) of CD6 in the manufacture of a medicament for use in a subject having a stage of multiple sclerosis (MS) that exhibits increased levels of T helper 17 (Th17) cells and having an increased expression of IL-23R, wherein the use in the subject causes a reduction of Th17 cells and a reduction of TNF-alpha, IFN-gamma, IL-6, and IL-17A, and a reduction of expression of IL-23R on one or more of monocytes, T helper cells and natural killer cells in a body fluid of the subject. the development of inflammatory demyelination. CD6 is an important cell surface protein predominantly expressed by human T cells and a subset of B cells, as well as by some B cell chronic lymphocytic leukemias and neurons. It has been shown that CD6 can function as an important accessory molecule in T cell activation. The present disclosure relates to a method and a use of a CD6 binding partner. In particular, the present disclosure relates to the use of an Itolizumab antibody specifically binding to Domain 1 (D1) of CD6 in the manufacture of a medicament for use in a subject having a stage of multiple sclerosis (MS) that exhibits increased levels of T helper 17 (Th17) cells and having an increased expression of IL-23R, wherein the use in the subject causes a reduction of Th17 cells and a reduction of TNF-alpha, IFN-gamma, IL-6, and IL-17A, and a reduction of expression of IL-23R on one or more of monocytes, T helper cells and natural killer cells in a body fluid of the subject.
Description
USE OFA CD6 BINDING PARTNER AND METHOD BASED THEREON
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of and the ty to provisional Indian patent
application 3264/CHE/2013 filed on 23 July 2013 with the Indian Patent Office. The content of
said application filed on 23 July 2013 is incorporated herein by reference for all purposes in its
ty, including an incorporation of any element or part of the description, claims or drawings
not contained herein and referred to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.
TECHNICAL FIELD
The present disclosure relates to a method and a use of a CD6 binding partner. In particular, the
present disclosure relates to a method for treatment, including prevention, of e conditions
mediated by T-helper l7 (Th17) and/or T-helper l (Th1) T lymphocytes (T . Furthermore,
the present disclosure relates to the use of anti—CD6 binding partner for treatment of disease
conditions ed by eactive Thl7 and Th1 T lymphocytes. The binding partners,
compositions, methods and uses of the present sure further have utility in methods and uses
for modulating an immune response by suppressing the production of the cytokine IL—23R
(interleukin 23 receptor), thereby decreasing inflammation mediated by Th17 cells.
BACKGROUND OF THE DISCLOSURE
The following discussion of the background of the disclosure is merely provided to aid the reader
in understanding the binding rs, itions, methods and uses described in this document,
and is not admitted to be or constitute prior art.
Protective immunity against certain diseases is dependent on the differential induction of specific
pro-inflammatory T-cell (T lymphocyte) populations by antigen ting cells (APCs) of the
innate immune system, such as dendritic cells (DCs) and macrophages. Two such T-cell
populations, responsible for mediating cellular immunity to a wide range of ens, are Th1
and Th1? cells. Both Th1 and more recently Th17 T cell populations have been implicated
ors of autoimmune and chronic inflammatory diseases, and thus serve as relevant cellular
targets for immunosuppressive agents. Furthermore, Dendritic Cells, as initiators of T—cell
responses, are a second cellular target for therapies designed to combat inflammatory disease.
W0 2015/01 1658
Multiple Sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system
(CNS), characterized by inflammatory ates of s, B cells, macrophages and focal
demyelinating plaques within the CNS. Both Th1 and Th17 cell-mediated responses have been
shown to play a role in the development of atory demyelination. Myelin-reactive T-cells
from MS patients produce cytokines consistent with a Th1 -mediated
response, while microarray
studies of MS lesions from patients demonstrate increased expression of TL-23R.
A relevant model for studying the mechanisms of autoimmune inflammatory
responses, and in
particular MS, is the experimental autoimmune encephalomyelitis (EAE) animal model of
inflammatory inating disease that shares clinical and neuropathological changes with
multiple sclerosis (MS). It has been accepted for many years that EAE is y a CD4+ Th1 -
mediated disease, though a pathogenic role for CD8+ T-cells in the induction of EAE has also
been demonstrated. More recently however, it has been demonstrated that an IL-17 producing T
cell subset plays a critical role in the enesis of EAE. While there is still some debate in the
ture, it is likely that Th1 and Thl7 cells cooperate to induce the development of organ-
speciflc autoimmunity.
CD6 is an important cell surface protein predominantly expressed by human T cells and a subset
ofB cells, as well as by some B cell chronic lymphocytic leukemias and neurons [Aruffo et al., J
Exp. Med. 1991, 174:949; Kantoun et al., J. Immunol. 1981, 127:987; Mayer et al., J.
Neuroimmunol. 1990. 29:193]. CD6 is a member of a large family of proteins characterized by
having at least one domain homologous to the scavenger or cysteine-rich domain (SRCR)
of type I macrophages moto, et al., J. Exp. Med. 1991, 173:55 and Resnick et al., Trends
Biochem. Sci. 1994, 19:5]. Other members ofthis family include CD5 [Jones et al., Nature. 1986,
323 2346]; cyclophilin C [Friedman et a1. 1993, PNAS 9016815]; complement factor I, which binds
activated ment proteins C3b and C4b [Goldbergen et al., J. Biol. Chem. 1987, 262: 10065];
bovine WC—l expressed by tau/delta. T cells gaard et al., J. Immunol. 1992, 14923273]
and M130 [Law et al., Eur J. Immunol. 1993, 23 :2320], a macrophage activation marker.
Blocking studies using anti-CD6 monoclonal dies (mAbs) suggest that CD6 plays an
important role in T cell development by ting T cell adhesive interactions with thymic
epithelial (TE) cells (Patel et al., J. Exp. Med. (1995) 181:1563-1568). Additional studies have
shown that CD6 can function as an important accessory molecule in T cell activation. For
example, certain anti—CD6 mAb are directly mitogenic for T cells (Gangemi et al., J. Immunol.
W0 2015/01 1658
(1989) 143 :2439; Bott et al., Int. Immunol. (1993) 7:783), whereas others are able to co-stimulate
T cell proliferation in ction with anti-CD3, anti—CD2 or PMA (Gangemi et al., J. Immunol.
(1989) 143:2439; Morimoto et al., J. Immunol. (1988) 14022165-2170; Osorio et al., Cell.
Immunol. (1994) ). Yet additional ce of the role of CD6 in T cell activation comes
from studies showing that CD6 becomes hyperphosphorylated on Ser and Thr residues (Swack et
al., Mol, Immunol. (1989) 26:1037-1049; Swack et al., J. Biol. Chem. (1991) 266:7137; Cardenas
et al., J. Immunol., 145: 1450—1455 (1990)) and phosphorylated on Tyr residues (Wee et al., J. Exp.
Med. (1993) 177:219-223) following T cell activation. These and other studies implicate CD6 as
an important modulator of both immature and mature T cell function in viva, affecting both T cell
activation and signal transduction (De Wit, J et al., Blood (2011) 118:6107—6114).
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will
now be made to exemplary ments as illustrated with reference to the accompanying
Figures. The Figures together with the detailed description below, are incorporated in and form
part of the specification, and serve to further illustrate the embodiments and n various
principles and advantages, in accordance with the present sure:
Figure 1 depicts inhibition of Th1 and Thl7 cytokines by the humanized monoclonal dy
Itolizumab as compared to an isotype control antibody, namely the humanized monoclonal
antibody Nimotuzumab (“Tlh”), in a mixed cyte reaction assay.
Figure 2: An anti—mouse CD6 domain 1 specific antibody (surrogate dy to Itolizumab)
shows the inhibition of Th1 and Th17 cytokines. Splenocytes from EAE induced animals treated
with anti~mouse CD6 antibody, and a group d with rat antibody
were re-stimulated in culture
with an anti CD3 antibody. The group treated with the rat antibody showed high proliferation
with associated release of high amounts of Th1 and Thl7 nes. On the other hand, the
anti—
mouse CD6 treated group of animals showed decreased proliferation and lower release ofTh1 and
Th17 specific nes in this mouse model of MS‘
Figure 3: CD6 overexpression in pro-inflammatory CD4+T cell subsets:
(A) PBMCS were stimulated in Thnp (nonpolarizing for CD4+ T cells) or Th17pol (Th17-
polarizing) conditions, supernatant was collected from quadruplicate wells, pooled and analyzed
using ELISA for secreted IFN—y (Th1 signature cytokine) and IL-17 (Thl7 signature cytokine).
Ratio of absolute concentration of IFN—y (empty le) or IL-17A (filled square) in Th17pol
and Thnp conditions ol: Thnp) is plotted across the days of analysis. (B) te levels
ofIFN—y (empty bars) and IL-17 (shaded bars) on day 13 is compared n Thnp and Th17pol
conditions. Data shown is mean :I:SD for triplicate ELISA wells (p<0.001) (C) PBMCs were left
unstimulated or stimulated in Thnp or Th17pol conditions. CD25 expression on CD4+T cells was
analyzed on Day 3. (D) PBMCs were left unstimulated (shaded histogram) or stimulated in Thnp
(solid line) or Th17pol d line) ions. CD6 expression (using biotin Itolizumab as
detection reagent) was analyzed on Day 9 and plotted as CD6 overlay histograms gated on CD4'T
cells. (E) Fold increase in CD6 MFI on gated CD4+T cells was calculated over unstimulated, and
plotted as bar graph for both Thnp (shaded bar) and Th17pol (empty bar) conditions using 3
different antibodies as CD6 detection reagent i.e. MEM98, MT605 and Biotinylated Itolizumab.
Data shown is mean iSD (p<0,05). In panel A-C, data are representative of 2 independent
experiments from different donors and in panel D&E data are entative of 2 independent
experiments from 6 ent donors.
Figure 4: Itolizumab Inhibits T cell activation and proliferation in both Thnp and Th17pol
ions:
(A) PBMCS were stimulated in Thnp or Th17pol conditions in presence of Itolizumab or control
dy. On day 3 cells were analyzed for CD25 expression on gated CD4+T cells. %
CD4+CD25+T cells in ated PBMCs, is plotted as bar graphs. Data shown is meaniSD from
2 independent experiments from different donors. (B) PBMCs labelled with CFSE dye were
stimulated in Thnp or Th17pol conditions in presence of Itolizumab or control antibody. On day
3 cells were analyzed for CFSE dilution on gated CD4+T cells. Data shown is from 1 experiment.
Figure 5: Itolizumab treatment causes substantial reduction in expression of IL-17 and IFN-
7 cytokines in cells stimulated in Th17 polarizing condition:
PBMCs were stimulated in l conditions in presence of Itolizumab or control antibody, On
days 3, 6, 8 and 13, cells stimulated in Th17pol conditions with control or Itolizumab monoclonal
dy, were restimulated with PMA-Ionomycin for 5 hours and ed for expression of
intracellular cytokine IFN—y and IL-l7A. Representative flow cytometry dot plots (on gated
CD3+T cells) on day 6 are shown in panel A. Panel B & C show the % of lFN- T cells and IL-
17A+T cells respectively, in presence of Itolizumab (empty triangle) or control antibody (empty
circle), across days as obtained from flow cytometry analysis. Data is representative of 2
independent experiments from different donors. To analyze the basal level of ed nes,
supernatants were collected from quadruplicate wells of PBMCs stimulated in Thl7pol conditions
in presence of Itolizumab (empty triangle) or control antibody (empty circle). As evaluated by
ELISA, ed (D) IFN-γ and (E) IL-17 levels are plotted across days. In Panel D and E data is
shown as mean + SD (p<0.0001) and entative of 2 independent experiments from ent
donors.
Figure 6: Itolizumab causes ion in ure Thl7 specific markers:
(A) PBMCs were stimulated in Thl7pol conditions in presence of control or Itolizumab antibody
and analyzed on Day 3 for expression of transcription factor . Data shown is ram
for pSTAT3 on gated CD4+T cells. (B) Cells stimulated in Thl7pol conditions in presence of
control or Itolizumab antibody were restimulated with PMA-Ionomycin for 5 hours and analyzed
for expression of intracellular cytokine IL-17A and Thl7 signature transcription factor RORyT.
Day 6 representative dot plots of RORyT and IL-17A on gated CD3+T cells are shown. (C) Data
from panel B was plotted as histogram overlays of RORyT MFI on gated CD3+T cells stimulated
in Thl7pol conditions in presence of control (empty histogram) or Itolizumab (dotted line
histogram) antibodies. Data shown is representative of 2 independent similar experiments from
different donors. (D) For the experiment as explained in panel A, Day 6 representative dot plots
of CCR6 and IL-17A on gated CD3+CCR6+ T cells are shown.
SUMMARY OF THE DISCLOSURE
Disclosed herein are methods, uses and compositions directed at treating, including preventing,
an autoimmune disease in a subject, as well as treating, including preventing, allograft rejection,
and treating, including preventing, versus-host disease. A respective method and use
includes stering to the subject a therapeutically effective amount of a binding partner
specifically binding to CD6.
In a particular aspect, the present invention es the use of an Itolizumab antibody specifically
binding to D1 of CD6 in the manufacture of a ment for use in a subject having a stage of
multiple sclerosis (MS) that ts increased levels of T helper 17 (Th17) cells an increased
expression of IL-23R, wherein the use in the subject causes a reduction of Th17 cells and a
reduction of TNF-alpha, IFN-gamma, IL-6, and IL-17A in a body fluid of the subject.
(followed by page 5a)
The present disclosure relates to methods for treatment, including prevention, of e
conditions mediated by T-helper 17 (Thl7) and/or T-helper 1 (Th1) T lymphocytes (T cells). In
ular, the present disclosure relates to the use of an anti-CD6 antibody for the treatment of
disease conditions mediated by auto—reactive Th1 7 and Th1 T lymphocytes. The methods of the
[FOLLOWED BY PAGE 6]
disclosure r have utility in methods for ting an immune
response by suppressing the
production of the cytokine IL-Z3R, thereby decreasing inflammation ed by Thl7 cells.
In a first aspect there is provided a method of treating a subject ing from (i)
an autoimmune
disease characterized by an increased number of T helper l7 (Thl7) T lymphocytes, (ii) allograft
rejection, or (iii) graft—versus—host disease. Thus the subject is known or suspected to have an
increased number of T helper l7 (Th17) T lymphocytes when compared to a healthy subject. The
method includes administering to the subject a binding partner specifically binding to CD6.
In a second aspect there is provided a binding partner specifically binding to CD6 for
use in the
treatment of (i) an autoimmune disease, (ii) allograft rejection, or (iii) graft-versus~host disease in
a subj ect. The t is known or suspected to have an increased number of T helper l7 (Th17)
T lymphocytes when compared to a healthy subject. These Thl7 T lymphocytes may be auto—
reactive Th17 T lymphocytes.
In some ments of the method according to the first
aspect or of the binding partner
specifically binding to CD6 for use according to the second aspect the subject is furthermore
known or suspected to have an increased number of T helper l (Th1) cells when compared to a
healthy t. These Th1 T lymphocytes may be auto-reactive Th1 T lymphocytes.
In some embodiments of the method according to the first aspect
or of the binding partner
specifically binding to CD6 for use according to the second aspect the subject the autoimmune
disease is rheumatoid arthritis. In some embodiments the autoimmune disease is Inflammatory
Bowel e. The mune disease may for example be s disease. In some
embodiments the autoimmune disease is ulcerative colitis. The autoimmune e is in some
embodiments psoriasis. In some embodiments the autoimmune disease is SjOgren‘s syndrome. In
some embodiments the autoimmune disease is Ankylosing spondylitis. In some embodiments the
autoimmune disease is Type I diabetes.
In some embodiments of the method according to the first
aspect or of the binding partner
specifically binding to CD6 for use according to the second aspect the subject the binding partner
is an antibody such as an globulin. The antibody may for example be a polyclonal
immunoglobulin. In some embodiments the antibody is a monoclonal antibody. In some
embodiments the antibody is a fully non-human antibody. In some embodiments the antibody is
a chimeric antibody, The antibody is in some ments a humanized antibody. In some
embodiments the antibody is a fully human antibody such as a fully human immunoglobulin, An
illustrative example of a zed antibody is Itolizumabi
In some ments of the method according to the first aspect or of the binding partner
specifically binding to CD6 for use according to the second aspect the subject the binding r
is a functional fragment of an immunoglobulin. In some embodiments a respective functional
globulin fragment is a Fab-fragment. In some embodiments the functional
immunoglobulin fragment is a single-chain le fragment (scFv) In some embodiments the
functional immunoglobulin fragment is a nanobody.
In some embodiments the binding partner specifically binding to CD6 is included in a
pharmaceutical composition. The pharmaceutical composition includes the binding partner
specifically binding to CD6 and at least one pharmaceutically acceptable diluent, carrier or
excipient.
In a third aspect there is provided a method of treating a subject suffering from (i) an autoimmune
e characterized by an increased number of T helper 17 (Thl7) T lymphocytes, (ii) allograft
rejection, or (iii) versus-host disease. Thus the t is known or suspected to have an
increased number of T helper 17 (Thl7) T lymphocytes when compared to a healthy subject. The
method includes administering to the subject a binding partner specifically binding to CD6 and
binding partner specifically binding to CD3. In some embodiments the binding partner
specifically binding to CD6 and the binding partner specifically g to CD3 are administered
independent from one another. In some embodiments the binding partner specifically binding to
CD6 and the g partner specifically binding to CD3 are administered itantly.
In a fourth aspect there is provided a combination of a binding partner cally binding
to CD6
and a binding partner specifically binding to CD3 for use in the treatment of (i)
an autoimmune
disease, (ii) allograft rejection, or (iii) graft—versus-host disease in a subject. The subject is known
or suspected to have an increased number of T helper 17 (Th17) T lymphocytes when compared
to a healthy subject. These Thl7 T lymphocytes may be auto-reactive Th17 T lymphocytes.
In a fifth aspect there is provided a pharmaceutical ition for the treatment of an
autoimmune disease in a subject known or suspected to have an increased number of Thl7 cells
when ed to a healthy subject. The pharmaceutical composition includes a binding partner
cally binding to CD6 and at least one pharmaceutically acceptable diluent, carrier or
excipient.
In a sixth aspect there is provided a pharmaceutical composition for the treatment of an
autoimmune disease in a subject known or suspected to have an sed number of T1117 cells
when compared to a y subject. The pharmaceutical composition includes a binding partner
specifically binding to CD6. The pharmaceutical composition also includes a binding partner
specifically binding to CD3. Furthermore the ceutical composition includes at least one
pharmaceutically acceptable diluent, r or excipient.
DETAILED DESCRIPTION OF THE DISCLOSURE
ions
Unless otherwise stated, the following terms used in this document, including the description and
claims, have the definitions given below.
The word “about” as used herein refers to a value being within an acceptable
error range for the
particular value as determined by one of ordinary skill in the art, which will depend in part on how
the value is measured or determined, i.e., the limitations of the measurement
system. For example,
“about” can mean within '1 or more than 1 standard deviation, per the ce in the art. The term
“about” is also used to indicate that the amount or value in question
may be the value designated
or some other value that is approximately the same. The phrase is intended to
convey that similar
values promote equivalent results or effects according to the binding partners, compositions,
s and uses described herein. In this context “about” may refer to a
range above and/or
below of up to 10%. The word “about” refers in some embodiments to a
range above and below
a certain value that is up to 5%, such as up to up to 2%, up to 1%, or
up to 0,5 % above or below
that value. In one embodiment “about” refers to a
range up to 0.1 % above and below a given
value.
The term istering”, as used herein, refers to any mode of transferring, delivering,
introducing, or transporting matter such as a compound, e.g. a pharmaceutical compound, or other
agent such as an antigen, to a t. Modes of administration include oral administration, topical
contact, intravenous, intraperitoneal, intramuscular, intranasal, or subcutaneous administration (cf.
below). stration “in combination with” r matter such as one or more therapeutic
agents includes simultaneous (concurrent) and consecutive administration in any order.
The term “antibody” generally refers to an immunoglobulin, a fragment thereof or a naceous
binding molecule with globulin-like functions (cf. .
The term “binding partner” as used herein refers to matter, such as a molecule, in particular
polymeric molecule, that can bind a nucleic acid le such as a DNA or an RNA molecule,
ing an mRNA molecule, as well as a peptide, a protein, a saccharide, a polysacchande or a
lipid through an interaction that is sufficient to permit the agent to form a complex with the nucleic
acid molecule, peptide, n or saccharide, a polysaccharide or a lipid, generally via non—
nt bonding. In some embodiments the binding partner is a PNA molecule. In some
embodiments the binding partner is an immunoglobulin or a proteinaceous binding molecule with
immunoglobulin—like functions as defined below. In some embodiments the binding partner is an
aptamer. In some embodiments a binding partner is specific for a particular target. In some
ments a binding partner includes a plurality of binding sites, each binding site being
specific for a particular target. As an illustrative example, a binding partner may be a
proteinaceous agent with immunoglobulin-like functions with two binding sites. It may for
instance be a bispecific diabody, such as a bispecific single chain diabody.
As used , the term ric antibody“ refers to an immunoglobulin polypeptide
or domain
antibody that includes sequences from more than one species. In a ic antibody a heavy chain
or a light chain may contain a variable region sequence from one species such
as human and a
constant region sequence from another species such as mouse. As an example,
a “chimeric
antibody” may be an immunoglobulin that has variable regions derived from an animal antibody,
such as a rat or mouse antibody, fused to another molecule, for example, the
constant domains
derived from a human antibody. The term "chimeric antibody" is intended to encompass
antibodies in which: (i) the heavy chain is chimeric but the light chain comprises V and C regions
from only one species; (ii) the light chain is chimeric but the heavy chain comprises V and C
regions from only one species; and (iii) both the heavy chain and the light chain are chimeric.
In this regard, a “humanized antibody” as used herein is an immunoglobulin ptide or
domain antibody containing ural elements of a human antibody and the antigen binding
site
of a non-human antibody. “Humanized dies” contain
a minimal number of residues from
the non-human antibody. For instance, they may contain only the CDR regions of the non-human
antibody, or only those residues that make up the hypervariable regions of the non-human
antibody. They may also contain n residues from outside the variable regions of the non-
human polypeptide, such as residues that are necessary to mimic the structure of the man
antibody or to minimize steric interference. Typically a humanized dy contains a human
framework, at least one CDR from a non-human antibody, with any constant region present being
substantially identical to a human immunoglobuiin constant region, i.e., at least about 85—90%,
such as at least 95% identical. Hence, all parts of a humanized globulin, except possibly
the CDRs, are substantially cal to corresponding parts of one or more native human
immunoglobulin sequences. In addition, humanized antibodies may contain es that do not
pond to either the human or the non-human antibodies.
The term “detect” or “detecting”, as well as the term “determine” or “determining” when used in
the context of a biomarker, refers to any method that can be used to detect the
presence of a nucleic
acid (DNA and RNA) or a protein/polypeptide. When used herein in combination with the words
“level”, “amount” or “value”, the words “detect”, “detecting”, “determine” or “determining” are
understood to lly refer to a quantitative or a qualitative level. Accordingly, a method or use
as disclosed herein may include a quantification of Th'l7 cells in absolute numbers, A method or
use as disclosed herein may also include a comparison by measuring a relative amount of Thl7
cells, for example compared to a nce sample from one or more healthy ts. As a further
example, absolute amounts of IL-17A or TNFoc may in some embodiments be measured. In some
embodiments it may be analysed whether a first sample ns a higher or a lower or the same
amount of IL-17A or TNFoc than a second sample. The terms “value,” “amount” and ” also
refer to the rate of synthesis of for example IL~17A, TNFOL, IL-22, IL—l7F, lL-Zl, or IL—6. The
exact nature of the “level”, “amount” or “value” s on the specific design and components
of the particular analytical method employed to detect T cells or
erg. IL-17A, TNFoc, IL-22, IL—
17F,IL-21, or IL-6.
An “effective amount” or a “therapeutically effective amount” of an agent such as a binding
partner, is an amount — either as a single dose or as part of a series of doses — sufficient to provide
a therapeutic benefit in the treatment or management of the relevant pathological condition,
or to
delay or minimize one or more symptoms associated with the presence of the condition. Such a
condition may be associated with immunosuppression,
e.g. an autoimmune disease.
W0 2015/01 1658
The terms “expressing” and “expression” in nce to a biomarker are intended to be understood
in the ordinary meaning as used in the art. A biomarker is expressed by a cell via transcription of
a nucleic acid into mRNA, followed by translation into a polypeptide, which is folded and possibly
further processed. The biomarkers sed in this disclosure are in addition being transported
to the surface ofthe respective cell. Hence, the statement that a cell is expressing such a biomarker
indicates that the biomarker is found on the surface of the cell and s that the ker has
been synthesized by the expression machinery of the respective cell. Accordingly, the term
“expression level” in the t of a cell population such as T cells refers to the number or
percentage of cells that have the biomarker of interest on their cell surface. The determination of
expression may be based on the normalized sion level of the biomarkers. Expression levels
are normalized by correcting the te expression level of a biomarker by comparing its
expression to the sion of a gene that is not a biomarker in the context of the invention. The
expression level may also be provided as a relative expression level.
With regard to the respective ical process itself, the terms ssion”, “gene expression”
or “expressing” refer to the ty of regulatory ys converting the information encoded in
the nucleic acid sequence of a gene first into messenger RNA (mRNA) and then to a protein.
Accordingly, the expression of a gene includes its transcription into a primary hnRNA, the
processing of this hnRNA into a mature RNA and the translation of the mRNA sequence into the
corresponding amino acid sequence of the protein. In this context, it is also noted that the term
“gene produc " refers not only to a protein, including e.g. a final protein (including a splice variant
thereof) encoded by that gene and a respective precursor protein where applicable, but also to the
respective mRNA, which may be regarded as the “first gene product” dun'ng the course of gene
expression.
By ent” in reference to a polypeptide such as an immunoglobulin or a proteinaceous
binding molecule is meant any amino acid sequence t in a corresponding polypeptide, as
long as it is shorter than the full length sequence and as long as it is capable of performing the
function of interest of the protein — in the case of an immunoglobulin specifically binding to the
desired target, e. g. antigen (CD6, for example). The term “immunoglobulin fragment” refers
to a
portion of an immunoglobulin, often the hypervariable region and portions of the surrounding
heavy and light chains that displays specific binding affinity for a particular molecule. A
ariable region is a portion of an immunoglobulin that physically binds to the polypeptide
W0 2015/01 1658
target. Due to the usage in the art, the terms “antibody fragment” and “immunoglobulin fragment”
are used interchangeably herein.
A “functional fragment” as used herein, refers to a fragment of a molecule such as a peptide or a
nucleic acid molecule, which retains at least one biological activity of the full length molecule. In
the context of an immunoglobulin a functional nt is an immunologically functional
fragment. Typically a functional fragment of a peptide is capable of performing ntially the
same ons as those of the intact polypeptide.
As used in this document, the expression “pharrnaceutically acceptable” refers to those active
nds, als, compositions, rs, and/or dosage forms which are, within the scope of
sound medical judgment, le for use in contact with the tissues of human beings and animals
without excessive toxicity, irritation, allergic response, or other problems or complications,
surate with a reasonable benefit/risk ratio.
The terms “polypeptide” and “protein” refer to a polymer of amino acid residues and
are not
limited to a n minimum length of the product. Where both terms are used concurrently, this
twofold naming accounts for the use of both terms side by side in the art.
The term “preventing” in the medical/physiological context, i.e. in the context of a physiological
state, refers to decreasing the probability that an organism contracts or develops an abnormal
condition.
The term “specific” as used in this document is tood to indicate that
a binding partner is
directed against, binds to, or reacts with a biomarker disclosed in the
present application, such as
CD6. Thus, being directed to, binding to or reacting with includes that the binding partner
specifically binds to e.g. CD6. The term “specifically” in this context means that the binding
partner reacts with CD6, as able, or/and a portion thereof, but at least essentially not with
r protein. The term “another protein” includes any protein, including proteins closely
d to or being homologous to e.g. CD6 t which the binding
partner is directed to. The
term “does not essentially bind” means that the binding partner does not have particular affinity
to another protein, i.e., shows a cross-reactivity of less than about 30%, when compared to the
affinity to CD6. In some embodiments the binding partner shows a cross—reactivity of less than
about 20%, such as less than about 10%. In some embodiments the binding partner shows a cross-
1658
reactivity of less than about 9, 8, or 7%, when compared to the affinity to CD6. In some
embodiments the binding partner shows a cross-reactivity of less than about 6%, such as less than
about 5%, when compared to the affinity to e.g. CD6. Whether the binding partner specifically
reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of a
respective binding partner with e.g. CD6, as applicable, and the reaction of the binding partner
with (an) other protein(s). The term “specifically recognizing”, which can be used interchangeably
with the terms “directed to” or “reacting with” means in the context of the present sure that
a particular molecule, generally an globulin, an immunoglobulin fragment or a
naceous binding molecule with immunoglobulin-like functions is capable of specifically
interacting with and/or binding to at least two, including at least three, such as at least four or even
more amino acids of an epitope as defined herein. Generally the immunoglobulin or proteinaceous
binding molecule can thereby form a complex with the respective epitope of eg. CD6. Such
binding may be exemplified by the specificity of a “lock—and—key—principle”. “Specific binding”
can also be determined, for e, in ance with a Western blot, ELISA-, RIA—, ECL—,
est, FACS, IHC and a peptide scan.
The term “Surrogate antibody” as used herein is understood to indicate the surrogate antibody to
Itolizumab that was ped in-house to study the effects of an Anti CD6 domain 1 binding
antibody in mice and is identified as a rat anti mouse CD6 IgG 2c. It has the equivalent ties
to Itolizumab as 1. Binds to domain I of mouse CD6, 2. Does not compete with ALCAM
binding. 3. ts the proliferation of naive T cells from splenocytes stimulated with anti CD3.
4. Is not ically depleting in mice. 5. Has a comparable y to that of Tlh.
The term “subj ect” as used herein, also addressed as an individual, refers to a human
or non—human
animal, lly a mammal. A subject may be a mammalian species such as a , a mouse, a
rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an
or a human. Thus, the methods, uses and compositions described in this document are applicable
to both human and veterinary disease. The sample has been obtained from the subject. Where the
same is a body fluid sample or a biopsy sample, it may be obtained using conventional techniques
routinely employed in the art. It is thus understood that conclusions drawn from expression levels
in the sample and decisions based thereon concern the subject from whom/which the sample has
been taken. Where the subject is a living human who is receiving medical care for
a disease or
condition, it is also addressed as a “patient”.
PCT/132014/063345
The terms “treatment” and “treating” as used herein, refer to a prophylactic or preventative
measure having a therapeutic effect and preventing, slowing down (lessen), or at least partially
alleviating or abrogating an abnormal, including pathologic, condition in the organism of a t.
Those in need of treatment include those already with the er as well as those prone to having
the disorder or those in whom the disorder is to be prevented (prophylaxis). Generally a treatment
reduces, izes, or ts progression of a symptom that is associated with the presence and/or
progression of a disease or pathological condition. The term “administering” relates to a method
of incorporating a nd into cells or tissues of a subject. The term “therapeutic effect” refers
to the inhibition or activation of factors causing or contributing to the abnormal condition. A
therapeutic effect relieves to some extent one or more of the ms of an abnormal condition
or disease. The term “abnormal condition” refers to a function in the cells or tissues ofan organism
that deviates from their normal functions in that sm. An abnormal condition can inter alia
relate to cell proliferation, cell differentiation, or cell al.
The terms “comprising”, “including,” ning”, “having” etc. shall be read expansively or
open—ended and without tion. Singular forms such as “a“, “an” or “the” include plural
references unless the context clearly indicates ise. Thus, for example, reference to a
“vector” includes a single vector as well as a ity of vectors, either the same - e.g. the same
operon — or different, Likewise reference to “cell” includes a single cell as well as a plurality of
cells. Unless otherwise indicated, the term “at least” preceding a series of elements is to be
understood to refer to every element in the series. The terms “at least one” and “at least one of”
include for example, one, two, three, four, or five or more elements. It is furthermore understood
that slight variations above and below a stated range can be used to achieve substantially the same
results as a value within the range. Also, unless indicated otherwise, the disclosure of ranges is
intended as a continuous range including every value between the minimum and maximum values.
The scope and meaning of any use of a term will be apparent from the specific context in which
the term is used. Certain further definitions for selected terms used throughout this document are
given in the riate context of the detailed description, as applicable. Unless otherwise
defined, all other scientific and technical terms used in the description, figures and claims have
their ordinary meaning as ly understood by one of ordinary skill in the art.
Methods and uses for treating a disease
The present inventors made the surprising finding that anti CD6 dy ed co-stimulation
with an anti-CD3 antibody is of high significance, and apparently more significant as compared
to the co-stimulation induced by anti CD28/CD3, which primes naive T cells for stable Th17
development by promoting the expression of IL-Z3R. Thus, the anti-CD6 dy has
suppressive activity and acts to selectively suppress Th1 and Th17 (IL—23R producing T
cell) mediated inflammatory autoimmune disease.
Without wishing to be bound by theory, the inventors predict that IL-23R is expressed by antigen
presenting cells, such as dendritic cells and macrophages. The expression of]L-23R by the antigen
ting cells skews the resulting T cell response away from the expansion of T cells with a Thl
and T1117 ype which can in some instances become auto-reactive T cells which e
autoimmune and chronic inflammatory conditions.
The current therapies for the treatment of autoimmune and chronic inflammatory diseases, such
as multiple sclerosis, are mainly focused on the use of steroids and other NSAIDs, which
are non-
specific and have serious side effects. In particular, n such treatments primarily act to
suppress the expression or functional activity of Tumor necrosis factor alpha (TNT-alpha). For
example, the chimeric monoclonal antibody INFLIXIMAB (Remicade®) targets TNF-alpha
function. Although effective'in certain patients, such anti—TNF-alpha treatments
can be ctive
when treating certain patients, or certain autoimmune conditions, or r, could also result in
the occurrence of undesirable side effects
The inventors have fied the utility of a binding partner or composition of the
present
sure in the treatment of Th1 and/or Th 17—mediated diseases and conditions, in particular
autoimmune or immune-mediated conditions, which occur where aberrant Th1 and/or Th17
responses occur due to the occurrence of auto-reactive Th1 and/or Th17 T cells.
A t to be treated according to the present disclosure is known or suspected to have an
increased number of Thl7 T cells when compared to a healthy subject. Th1? T cells
were named
after their production ofthe signature cytokine IL-17A. In addition Th17 T cells also
produce IL-
17F, lL-Zl, 1L-22, GM-CSF, TNFoc and IL—6.
The presence of an increased number of Thl7 T cells in a subject
may be detected by analyzing
the T cells present in body fluid in a subject or body fluid obtained from
a subject.
2014/063345
The presence of an sed number of Th17 T cells in a subject may be detected by analyzing
the level of cytokines known to be produced by Th17 T cells. Accordingly in some embodiments
an increased number of Th17 T cells in a subject may be detected by detecting the level of IL—
17A in the subject and comparing the level to the level of 1L~17A in a healthy subject. In some
embodiments an increased number of Thl7 T cells in a subject may be detected by detecting the
level of TNFoc in the subject and comparing the level to the level of TNFoc in a healthy subject.
In some embodiments an increased number of Th17 T cells in a subject
may be detected by
detecting the level of IL—6 in the t and comparing the level to the level ofTNFoc in a healthy
subject. In some embodiments an sed number of Thl7 T cells in a subject
may be detected
by detecting the level of Interferon gamma (IFN—y) in the subject and comparing the level to the
level of Interferon gamma in a healthy subject.
A binding partner sed herein cally binds to CD6. “CD6” is an abbreviation of “Cluster
ofDifferentiation 6”. The protein is also called T—cell differentiation antigen CD6, as well as T12
or TP120. CD6 is in some embodiments the mouse n of SwissProt/UniProt accession
Q61003 (version 117 of 9 July 2014). In some embodiments CD6 is the human protein with the
SwissProt/UniProt accession no. P30203 (version 125 of 9 July 2014). In some embodiments
CD6 is isoform d of the human protein CD6, having rot/UniProt accession
no. Q8WWJ4
(version 59 of 16 April 2014). In some embodiments CD6 is isoform c ofthe human protein CD6,
having SwissProt/UniProt accession no. QSWWJ6 (version 59 of 16 April 2014). In some
embodiments CD6 is isoform b of the human n CD6, having SwissProt/UniProt accession
no. QSWWJ3 (version 59 of 16 April 2014). In some embodiments CD6 is isoform e of the human
protein CD6, having SwissProthniProt accession no. Q8WWI7 (version 60 of 16 April 2014). In
some embodiments CD6 is the human protein of SwissProt/UniProt accession
no. Q8N4Q7
(version 66 of 16 April 2014). In some ments CD6 is isoform CRA_d ofthe human protein
CD6, having SwissProt/UniProt ion no. G5E973 (version 26 of 9 July 2014). In some
embodiments CD6 is the rat protein with the SwissProt/UniProt accession
no. Q5FVU4 (version
69 of 9 July 2014). In some embodiments CD6 is the Rhesus
e (Macaca mulatta) protein
with the SwissProt/UniProt accession no. H9ZFC2 (version 7 of 16 April 2014).
In some embodiments of a method or use disclosed herein, IL-23R expression
on blood cells
and/or dendritic cells of the subject is being analysed. In some embodiments the level of one or
more of INF—alpha, IFN—gamma, IL—17, and IL-17A in a body fluid of the patient is being
WO 11658
analysed. In a mixed lymphocyte reaction, it is observed that the Th1 and Thl7 ne IL17A
is reduced in presence of Tlh as illustrated in Fig. 1, In animal models using surrogate antibody
to domain 1 of CD6, reduction in Th1 and Thl7 (IL17A) cytokines is observed as illustrated in
Fig. 2.
Any available method can be used to detect the presence of a nucleic acid or a protein in the
context of the present invention. Such a method may include established standard procedures well
known in the art. Examples of such techniques include, but are not limited to, RT—PCR, RNAse
protection assay, rn analysis, Western analysis, ELISA, radioimmunoassay or fluorescence
ion assay. Assessing the amount of a biomarker such as IL-l7, IFN—gamma or TNF—alpha
in/on a cell may include assessing the amount of a nucleic acid, e. g. RNA, in a cell encoding the
respective biomarker. A nucleic acid probe may be used to probe 21 sample by any common
hybridization method to detect the amount of nucleic acid molecules of the biomarker. In order
to obtain nucleic acid probes chemical sis can be d out. The synthesized nucleic acid
probes may be first used as primers in a polymerase chain reaction (PCR) carried out in accordance
with recognized PCR techniques, essentially according to standard PCR protocols utilizing the
appropriate template, in order to obtain the respective probe, One skilled in the art will readily be
able to design such a probe based on the ces ble for the biomarker. The hybridization
probe can be labeled by standard labeling ques such as with a radiolabel, enzyme label,
fluorescent label, -avidin label, chemiluminescence or a nanoparticle. After hybridization,
the probes may be visualized using a standard technique.
A detection method used in the context of the t ion
may include an amplification of
the signal caused by the nucleic acid or protein, such as a polymerase chain reaction (PCR)
or the
use of the biotin—streptavidin system, for example in form of a ation to
an immunoglobulin,
as also explained in more detail below. The ion method may for example include the
use of
an antibody, e, g. an immunoglobulin, which may be linked to an attached label, such
as for
instance in Western analysis or ELISA. Where desired, an intracellular immunoglobulin
may be
used for detection. Some or all of the steps of detection may be part of an automated detection
system. rative examples of such systems are automated real-time PCR platforms, automated
nucleic acid isolation platforms, PCR product analysers and real-time detection
systems. As
indicated above, the term “antibody” as used herein, is understood to include
an immunoglobulin
and an immunoglobulin fragment that is capable of specifically binding
a selected protein, e, g. L-
selectin or a protein c for T cells, as well as a respective proteinaceous binding
molecule
W0 2015/01 1658
with immunoglobulin-like functions. An antibody may for instance be an EGF-like domain, a
Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III
domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin
Inhibitor domain, tendamistat, a Kazal—type serine protease tor domain, a Trefoil (P-type)
domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB ,
a thyroglobulin type I repeat, an LDL—receptor class A domain, a Sushi domain, a Link domain, a
Thrombospondin type I domain, an immunoglobulin domain or a an immunoglobulin—like domain
(for example a domain antibody or a camel heavy chain antibody), a C-type lectin domain, a MAM
domain, a von Willebrand factor type A , a Somatomedin B , a WAP-type four
disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3
domain, a Laminin-type EGF-like domain, a C2 , a "Kappabody" (111. et al., Protein Eng
(1997) 10, 949-957), a ody" (Martin et al., ElVIBO I (1994) 13, 5303-5309), a "Diabody"
(Holliger et al., PNAS USA. 90, 6444-6448 (1993)), a in" ecker et al., EMBO J
(1991) 10, 659 or Traunecker et al., Int J Cancer (1992) Suppl 7, 51—52), a nanobody, an
adnectin, a tetranectin, a microbody, an affilin, an y or an ankyrin, a crystallin, a knottin,
ubiquitin, a zinc~finger n, an autofluorescent protein, an ankyrin or ankyrin repeat protein or
a leucine-rich repeat protein (cf. also below).
A ement of a level or amount may for instance rely on spectroscopic, photochemical,
photometric, fluorometric, radiological, enzymatic or thermodynamic means. An example of a
oscopical detection method is fluorescence correlation oscopy. A photochemical
method is for ce photochemical cross-linking. The use of photoactive, fluorescent,
radioactive or enzymatic labels respectively are examples for photometric, fluorometric,
radiological and enzymatic ion methods. An example of a thermodynamic detection method
is isothermal ion calorimetry. As an illustrative example of a label,
a detailed protocol on the
use of water—soluble, bio—functionalized semiconductor quantum dots has been given by Lidke
a1. (Current Protocols in Cell Biology, [2007] Suppl. 36, 2511-25118). Such
quantum dots have
a particularly high photostability, allowing monitoring their localization for minutes to hours
days. They are typically fluorescent nanoparticles. Since different types of quantum dots can be
excited by a single laser line multi-colour labelling can be performed. Detection can for example
iently be carried out in different fluorescence channels of a flow cytometer. A quantum
dot can be coupled to a binding partner of IL-17, IFN-gamma or TNF-alpha.
2014/063345
The measurement used is generally selected to be of a ivity that allows detection ofthe cells
expressing the biomarker of interest, e.g IL-17, mma or TNF-alpha, in the range of a
selected old value, in particular of a sensitivity that allows determining whether IL-1 7, IFN-
gamma or TNF-alpha expressing cells are below the threshold value. Typically a g partner
of IL—l7, IFN-gamma or TNF-alpha, respectively, may be used in combination with a detectable
marker. Such a binding partner of lL—l 7, lFN-gamma or TNF-alpha has a able affinity and
specificity for lL—l7, EN—gamma or TNF—alpha, respectively. Typically, g is considered
specific when the binding y is higher than 10-6 M. A binding partner of eg. lL-l7, IFN—
gamma, "INF-alpha, or IL-23R, respectively, has in some embodiments an affinity of about 10'8
M or higher, or of about 10'9 M or higher.
A respective binding partner of eg. IL-l7, IFN—gamma, TNF-alpha, or IL-23R, as well
as a
binding partner for another selected cell—characteristic protein, may be an immunoglobulin, a
fragment thereof or a naceous binding molecule with immunoglobulin—like ons. An
antibody fragment generally contains an antigen g or variable region. Examples of
(recombinant) antibody fragments are immunoglobulin fragments such as Fab nts, Fab’
fragments, Fv fragments, single-chain Fv fragments (scFv), ies or domain antibodies (Holt,
L.J., et al., Trends Biotechnol. , 21, 11, 0). An example of a proteinaceous binding
molecule with immunoglobulin—like functions is a mutein based on a polypeptide of the lipocalin
family (W0 03/029462, Beste et al., Proc. Natl. Acad. Sci. USA (1999) 96, 1898-1903).
Lipocalins, such as the bilin binding protein, the human neutrophil gelatinase—associated lipocalin,
human Apolipoprotein D or glycodelin, posses natural ligand-binding sites that
can be modified
so that they bind to selected small protein regions known as haptens. Examples of other
proteinaceous binding molecules are the so—called glubodies (see e.g. international patent
application W0 96/23 879 or Napolitano, E.W., et a1., Chemistry & y (1996) 3, 5, 359—3 67),
proteins based on the ankyrin scaffold (Mosavi, L.K., et a1., Protein Science (2004) 13, 6, 1435-
1448) or crystalline scaffold (eg. internation patent application WO 01/04144), the proteins
described in Skerra, J. Mol. Recognit. (2000) 13, 167-187, ins, tetranectins and avimers.
Avimers contain so called A—domains that occur as strings of multiple s in several cell
surface receptors (Silverman, 1., et al., Nature Biotechnology (2005) 23, 1556-1561). Adnectins,
derived from a domain of human fibronectin, contain three loops that
can be engineered for
immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current Opinion in
Biotechnology (2006) 17, 653-658). Tetranectins, derived from the respective human
homotrimeric protein, likewise contain loop regions in a C~type lectin domain that can be
W0 2015/0 1 1658
engineered for desired binding (ibid). Peptoids, which can act as protein ligands, are oligo(N—
alkyl) glycines that differ from peptides in that the side chain is ted to the amide nitrogen
rather than the or carbon atom. Peptoids are typically resistant to proteases and other modifying
enzymes and can have a much higher cell permeability than peptides (see e.g. Kwon, Y.-U., and
Kodadek, T., J. Am. Chem. Soc. (2007) 129, 1508-1509). A le dy may in some
embodiments also be a multispecific antibody that includes several immunoglobulin fragments.
An immunoglobulin or a naceous binding molecule with globulin-like
functions may be PEGylated or hyperglycosylated if desired. In some embodiments a
proteinaceous binding molecule with immunoglobulin—like functions is a fusion protein of one of
the exemplary proteinaceous binding les above and an albumin-binding domain, for
instance an n-binding domain of streptococcal protein G. In some embodiments a
proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of an
immunoglobulin fragment, such as a single—chain y, and an immunoglobulin binding
domain, for instance a bacterial globulin binding domain. As an illustrative example, a
single—chain diabody may be fused to domain B of staphylococcal protein A as described by
Unverdorben et a]. (Protein ering, Design & Selection [2012] 25, 81—88).
According to one aspect of the t disclosure, there is provided a method of treating or
preventing an autoimmune disease which is caused by auto-reactive Thl and/or Thl 7 T cells, the
method including the steps of:
— providing a therapeutically effective amount of a composition including anti—CD6 antibody; and
- administering said antibody to a subject in need of such treatment in an amount sufficient to
suppress the activation of er 17 lymphocytes (Th17 T cells) and/or a T-helper l
lymphocytes (Th1 T cells).
In some embodiments determining the level of expression of the gene of interest includes
determining the level of transcription into mRNA. RNA encoding the protein of interest in the
sample, such as lL-l7, IFN—gamma, TNT—alpha, or IL—23R may be amplified using any available
amplification technique, such as polymerase chain reaction (PCR), including multiplex PCR,
nested PCR and amplification refractory mutation c (ARMS) PCR (also called -
specific PCR (AS-PCR), rolling circle amplification (RCA), nucleic acid sequence based
amplification ), ligase chain reaction (LCR), QB replicase chain reaction, loop-mediated
isothermal amplification (LAMP), transcription ed amplification (TMA) and strand
displacement amplification (SDA), including genome strand displacement amplification
(WGSDA), multiple strand displacement cation (MSDA), and gene specific strand
displacement amplification (GS-MSDA). Detection of the obtained amplification products may
be performed in numerous ways known in the art. Examples e, but are not limited to,
electrophoretic methods such as agarose gel electrophoresis in combination with a staining such
as ethidium bromide staining. In other embodiments the method of the invention is accompanied
by real time detection, such as real time PCR. In these embodiments the time course of the
amplification process is monitored. A means of real time detection commonly used in the art
involves the addition of a dye before the amplification process. An example of such a dye is the
fluorescence dye SYBR Green, which emits a fluorescence signal only when bound to double-
stranded nucleic acids.
lly a detectable label or marker is used. Such a marker or label may be included in a nucleic
acid that includes the ce to be ed A marker
may also be included in a primer or a
probe. It may also be incorporated into the amplification product in the course of the reaction. In
some embodiments such a marker compound, e. g. included in a nucleic acid, is an optically
able label, a fluorophore, or a chromophore. An illustrative example of a marker nd
is 6-carboxyfluorescein (FAM).
As an illustrative example, real-time PCR may be used to determine the level of RNA ng
the protein of st in the sample, such as IL~17, IFN-gamma, TNF-alpha, or lL-23R. Such a
PCR procedure is carried out under real time detection, so that the time course ofthe amplifi cation
process is monitored. PCR is characterized by a logarithmic cation ofthe target sequences.
For the amplification of RNA, a reverse transcriptase-PCR is used. Design of the primers and
probes required to detect expression of a biomarker of the invention is within the skill of a
tioner of ordinary skill in the art. In some embodiments RNA from the sample is isolated
under RNAse free conditions and then converted to DNA via the use of a
reverse transcriptase.
Reverse transcription may be performed prior to RT-PCR analysis or simultaneously, within
single reaction vessel. RT-PCR probes are ucleotides that have a fluorescent moiety, also
called reporter dye, attached to the 5' end and a quencher moiety coupled to the 3' end (or vice
versa). These probes are typically designed to hybridize to an internal region of
a PCR product.
In the non-hybridized state, the proximity of the fluor and the quench molecules prevents the
detection of fluorescent signal from the probe. During PCR amplification, when the polymerase
replicates a template on which an RT-PCR probe is bound, the 5'—3' nuclease activity of the
polymerase cleaves the probe. Thereby the fluorescent and quenching moieties are decoupled.
W0 2015/01 1658
Fluorescence increases then in each cycle, in a manner proportional to the amount of probe
cleavage. Fluorescence signal emitted from the on can be measured or followed overtime
using ent which is commercially available using e and conventional techniques.
Quantitation of biomarker RNA in a sample being evaluated may be performed by comparison of
the amplification signal to that of one or more standard curves Where known quantities of RNA
were ted in a similar manner. In some ments, the difference in biomarker expression
is measured as the difference in PCR cycle time to reach a threshold fluorescence, or “dCT.”
In some embodiments of a method or use disclosed herein an autoimmune disease is being treated.
In certain embodiments of the present disclosure, the composition disclosed herein
suppresses
both a T—helper 17 lymphocyte (Thl7) mediated immune se and a T-helper 1 lymphocyte
(Th1) mediated immune response. In certain embodiments, the disease which is mediated by the
auto—reactive T cells is an autoimmune disease or chronic inflammatory disease.
In certain embodiments of the present sure, the autoimmune disease is Multiple Sclerosis
(MS). In some embodiments the autoimmune disease is Rheumatoid Arthritis (RA). In some
embodiments the autoimmune disease is Inflammatory Bowel Disease (IBD) such as Crohn's
disease. The autoimmune disease may in some ments be Ulcerative Colitis. In some
embodiments the autoimmune disease is Type 1 Diabetes. In some embodiments the autoimmune
disease is Psoriasis.
The listing or discussion of a previously published document in this specification should not
necessarily be taken as an acknowledgement that the document is part of the state of the art or is
common general knowledge.
The binding partners, compositions, methods and uses ratively described herein
may suitably
be practiced in the absence of any element or elements, limitation or tions, not specifically
disclosed herein. Additionally, the terms and expressions employed herein have been used as
terms of ption and not of tion, and there is no intention in the use of such terms and
expressions of excluding any equivalents of the features shown and described or portions thereof,
but it is recognized that various modifications are possible. Thus, it should be understood that
although the t binding partners, compositions, methods and uses has been specifically
disclosed by exemplary ments and al features, modification and variation of
binding rs, compositions, methods and uses embodied therein herein disclosed may be
resorted to by those skilled in the art, and that such modifications and variations are considered to
be within the scope of the g partners, compositions, methods and uses disclosed.
The binding partners, itions, s and uses have been described broadly and
generically herein. Each of the narrower species and subgeneric groupings falling within the
generic sure also form part of the binding partners, compositions, methods and uses. This
includes the c description of the binding partners, compositions, methods and uses with a
proviso or negative limitation removing any t matter from the genus, regardless ofwhether
or not the excised al is specifically recited herein.
Other embodiments are within the appending claims. In addition, where features or aspects of the
binding partners, compositions, methods and uses are described in terms ofMarkush groups, those
skilled in the art will ize that the binding rs, compositions, methods and uses is also
y described in terms of any individual member or subgroup of s of the Markush
group.
In order that the binding partners, compositions, methods and uses may be readily understood and
put into practical effect, particular embodiments will now be described by way of the following
non-limiting examples.
Example 1:
Mixed lymphocyte reaction and cytokine anlaysis:
Preparation of PBMCs:
ml ofblood is collected from a healthy donor. PBMCs are isolated by standard FICOLL density
gradient centrifugation Monocyte Depletion & Setting up Dendritic Cell Derivation Assay: These
cells are incubated in a C02 incubator for two hours. Monocytes are allowed to adhere onto the
plastic surface. The non-adhered cells (PBLs) are subsequently removed from the flasks. All the
flasks are washed with lXPBS once. 20 ml of DC media (made 50 ml stock, 10 [.ll of GMCSF
and 5 til of IL—4 in 50 ml of assay media) is added to each flask. The flasks are kept in C02
incubator for 6 days.
LPS Treatment to on-growing Dendritic Cells:
At day 6, DC media with LPS is added to each flask (final concentration of LPS in the flask is 4
ug/ml) and kept back in C02 incubator for 40 —— 48 hrs.
Preparation of Cells for Mixed Lymphocyte Assay:
Preparation of DCs:
After LPS treatment the cell suspension (DC) are collected from the two flasks. Each flask is
washed with IXPBS once. The cell suspension is spun down @ 1500 rpm for 5 minutes and
reconstituted in 3 ml media. LPS treated DCs are counted and tituted in media as per assay
requirement.
ation of PBLs:
Following the same protocol as mentioned before, Ficoll separation is performed after collecting
blood from another healthy individual. After monocyte ion the non adhered cells (PBLS)
are collected and spun down @ 1500 rpm for 5 minutes and reconstituted in 5 ml media. PBLs
are counted and reconstituted to 1.0 X 106 cells/ml.
SEB treatment to tic Cells:
Preparation of SEB: SEB stock concentration is 1mg/ml. From the stock 3 ul of SEB is ved
in 3 ml of media to get 1 ug/ml working solution of SEB. Treatment: As per the standardized
protocol 0.06 X 106 DCs are treated with 0.6 pg of SEE. A stock 0.1 X 106 cells/ml (LPS treated
matured DCs) is made. From this, 600 pl of cell suspension is dissolved in 2.4 ml of assay media
(total volume of cell suspension is 3 m1 that contains 0.02 X 106 m1). This is spun down @
1500 rpm for 5 min and 600 pl of SEB (lug/ml) is added to the pellet. This is incubated inside
C02 incubator @ 37°C for 20 minutes. Excess media (2 ml) is added to the tube after incubation
and washed @ 1500 rpm for 5 min. Supernatant is discarded and the cells are washed again with
3 ml of media. Finally the pellet is dissolved in 3 ml of assay media.
Mytomycin C treatment to PBLs:
rig/ml cin solution is made from the mytomycin stock of lmg/ml. 0.5 X 106 PBLs are
treated with 500 pl of 25 ug/ml cin for 30 min inside CO2 incubator @ 37°C. Excess
media (2 ml) is added to it after the incubation and the cells are washed @ 1500
rpm for 5 media.
Supernatant is discarded and the cells are washed again with 3 m1 of media.
MLR Assay - Inhibition of Proliferation:
MLR assay is performed at DC:PBL = 1:50 ratio. Negative control used is Nimotuzumab. After
6 days the plate is read with alamar blue. Supernatant from a parallel plate is taken and assayed
for cytokines using a Th1, Th2 and Th17 CBA human kit (BD ences, Pharmingen, USA) is
used as per the manufacturer’s instruction. Concentration of cytokines is ined from a
standard supplied along with the kit. All samples were analysed with a Cyan —ADP, Beckman
Coulter, Flow cytometer with the Summit 4.3 Software.
W0 1 1658 PCT/IB201M063345
s: icant difference in the key cytokines evaluated is observed in the presence of
Itolizumab as compared to control (Nimotuzumab) treated group. The mean Th17 determining
cytokine, IL17 is reduced almost 50%.
Example 2:
Induction of EAE and treatment:
Mice were immunized subcutaneously on day 0 with 200ul of emulsion consisting of ZOug of
MOG 35-65 in PBS combined with an equal volume of CFA containing 500ug heat-killed M.
tuberculosis H37Ra. The emulsion was injected in one of lower dorsum and followed by an
intravenous ion of IOOpl of B. pertussis toxin (0.2ug/100ul) in 0.01M PBS through lateral
tail vein. The booster dose of 100 pl of B. pertussis toxin was given on day 3. Mice were re-
immunized on day 7 with 200ul of MOG/CPA emulsion injected subcutaneously on the other
flank. On day 14 mice were randomized and grouped as control and anti CD6 treated mice.
Treated group of mice were injected with anti-mouse CD6 MAb (R&DSystem)/10D12
60ug/100ul/dose, intraperitoneally on every alternate day. Control mice were injected with same
dose of anti-rat IgG MAb. Mice were observed for symptoms ofEAE. Disease severity and onset
in control and treated group was scored as clinical score from day 14 to 32 on
a 5 point scale as-
0, normal; 05, stiff tail; 1; limp tail; 1.5, limp tail with ity to right; 2; paralysis of one limb;
2.5 paralysis of one limb and ss of one other limb; 3, complete paralysis ofboth hind limbs;
4 moribund; 5, death. Mean clinical score was calculated by adding the clinical scores for one
group and dividing by the total number of mice in that group. The induction of disease in mice
was more than 95%.
T cell receptor (CD3) mediated Proliferation
assay
Mice were sacrificed on day 30. Spleen and blood was assessed from each mouse. Spleens
obtained from same group of mice were pooled for in vitm experiments. Splenocytes (0.2xl 06
cells/well) obtained from MOG immunized; control and antiCD6 MAb treated mice were
stimulated with ted antiCD3 MAb (2.5 ug/ml) in 96 well plates (Nunc). Splenocytes in
ed wells were used as unstimulated l cells. Cells were incubated for 3 days. On day
3 alamar blue (30 pl/well; Invitrogen) was added. After overnight incubation plates were read at
530/590 nm using Biotek Synergy plate reader next morning. Data was represented as mean
Relative fluorescence unit (RFU) values from ulated and stimulated cytes of both
groups
Cytokine Analysis
ZOI4/063345
For cytokine analysis same set up of eration assay was used for stimulation of splenocytes.
Briefly, splenocytes (0.2 x 106 cells/well) obtained from MOG immunized; control and antiCD6
MAb treated mice were stimulated with precoated antiCD3 MAb (25 ug/ml) in 96 well plates
(Nunc). Splenocytes in uncoated wells were used as unstimulated control cells. On day 3
supernatants were collected and preserved at —80°C until cytokine measurement was performed
using Cytometeric bead array (CBA) inflammatory cytokine kit (Interleukin—6, Interleukin -l0,
Interleukin—12p70, Interferon-y, Tumor Necrosis Factor~oc) (BD Biosciences, USA) as per
manufacturer's instructions. Briefly, 50 ul of mixed bead tion with discrete fluorescence
intensities and coated with cytokine specific capture antibodies was added to 50 ul of supernatant,
and 50 ul of phycoerythrin detection reagent for inflammatory cytokine antibodies.
Simultaneously, standards for each ne (0-5000 pg/ml) were likewise mixed with cytokine
capture beads and phycoerythrin detection n t. The vortexed mixtures were allowed to
incubate for 2 hrs at room temperature in dark. Beads were washed and acquired on flow
ter (CyAnDMADP, Dako). Standard curves were derived from the cytokine standards
supplied with the kit and quantity ) of respective cytokine was calculated using standard
graphs.
For detection oflnterleukin-17A flex set assay kit (BD Biosciences, USA) was used
as per
manufacturer's instructions. Briefly, 50 ul of capture bead solution coated with IL—17A c
capture dies was added to 50 ul of supernatant or standard (0—5000 pg/ml) and incubated for
1hr at room ature. Beads were washed and ted with 50 pl of phycoerythtin detection
reagent for 1hr at room temperature Beads were washed and acquired on flow cytometer
(CyAnDMADP, Dako). Standard curve was derived from the cytokine standards supplied with
the kit and quantity (pg/ml) of IL—17A was calculated using standard graph.
Results: Mice treated with anti—mouse CD6 antibody showed significant clinical
improvement over the Rat antibody d group. Statistically significant ence in the anti
mouse CD6 treated group as compared to the control Rat IgG treated
group was observed for the
following cytokines TNFoc, IL6, IFNy and 11,17.
Example 3:
Method:
1. Human PBMC culture for Thl7 polarization
Human blood was collected from y volunteers after signing
an ed consent form and
PBMCS (peripheral blood mononuclear cells) were separated by standard density gradient
PCT/[82014/063345
centrifugation over Ficoll Paque (GE Healthcare Bio-sciences AB, Uppsala, Sweden). PBMCs
were counted and plated in 96 well flat bottom plates, at a density of 0.1 million cells per well and
stimulated with anti-CD3/anti-CD28 coated beads (each antibody coated on beads at a final
concentration of Sng for 0,1 million cells per well) under non-polarizing (Thnp) and Thl7
zing conditions (Thl7pol). In Thl7pol conditions, polarizing cytokines and growth factors
were added at a final concentration of: lL-lB lOng/mL, lL-6 L, TGF-B 15 ng/mL, lL-23
lOng/mL, anti—IL-4 lOug/mL and anti-IFN—y 2.5ug/mL. Itolizumab or irrelevant isotype control
antibody (control) were added to cells in both Thnp and Thpol ions at a final concentration
of 40ug/mL. Cells were cultured in a 37 °C C02 incubator for 13 days and sampled at various
time points (day 3, day 6, day 8 and day 13) for nes, cell-surface and intracellular marker
analysis. For cells that were continuously cultured, SOuL medium was replaced with RPMI
complete medium containing an/mL IL-2 for Thnp condition and 2ng/mL IL-2 along
g/ml IL-23 for Thl7pol condition on days 6 and 10.
2. Cytokine analysis
At each time point, lOOuL of cell supernatant was collected and pooled from quadruplicate wells
and frozen at -80°C. Cytokine analysis was performed as per manufacturer instructions using
Human IFN—y Quantikine SixPak (SIFSO) and Human IL—l7 (IL-17A) (Quantikine SixPak
(Sl700) kits from R&D Systems. Absorbance was read at 450/630nm using SpectraMax M5e
reader, lar Devices, Sunnyvale, CA, USA.
3. Flow cytometry analysis
Cell surface marker staining: At each time point, cells from plicate wells were
harvested and d with Human Fc receptor binding inhibitor and incubated at 40C for 30
minutes. Cells were centrifuged and re~suspended in staining buffer (2% PBS in DC PBS)
containing the antibodies for cell surface markers (6. g. CD4, CD8, CD25, CD6). Acquisition and
analysis of samples were performed using a Cyan-ADP flow cytometer with the Summit version
4.3 re (Beckman r, Fullerton, CA, USA). Lymphocytes were gated by forward and
side scatter and further gated on CD4+T cells.
4. tical analyses
GraphPad Prism 6.0 software (GraphPad software, San Diego, CA, USA) was used for all
tical analysis. Statistical cance was determined using unpaired t test followed by
Holm-Sidak method, with alpha=5.000% (Figures 33).
Results:
W0 2015/01 1658
T cell activation and CD6 pression in Th17 polarizing conditions.
As shown in Figure 3A, by day 13 the increase in ed IL-l7 levels was 3-4 fold higher than
the rise in IFN-y release in cells stimulated in Th17pol conditions (PBMCs stimulated with anti-
CD3/anti-CD28 in presence of Thl7 polarizing, Th17pol, cytokines) as compared to Thnp cells
(PBMCS ated with anti-CD3/anti-CD28, Non—Polarizing condition; Thnp). Absolute
concentrations of lL—17 and IFN-y on day 13 are shown in Figure 3B. IFN—y released by cells
ated in Th17pol condition was significantly low as compared to the Thnp cells. Similarly
these Th17pol cells showed a 3-4 fold increase in secreted IL—l7 levels as compared to Thnp cells.
These results indicate that under Th17pol conditions a shift towards Thl7 cells was initiated
early as day 3 with full establishment by day 13. There was similar activation of CD4+ T cells on
day 3, as indicated by CD25 (IL-2R0t) over expression in Thnp as well as Th17pol conditions
e 3C).
The surface expression of CD6 receptors on CD4+ T cells increased after 48 hours of stimulation
and was sustained till day 12/13, the end of the polarizing experiment. Representative analysis on
day 9, as histogram overlay for CD6 expression on CD4+T cells for one donor is shown in Figure
3D. Biotinylated Itolizumab (anti-human CD6) as a detection reagent, identified CD6
overexpression in 15-30% and 25-3 5% of CD4+T cells in Thnp and Th17pol conditions
respectively e 3D). As shown in Figure 3E (data from 6 different donors), Thnp and
Th17pol CD4+T cells showed consistent increase in CD6 MFI over the unstimulated cells.
se in CD6 expression was also confirmed with the use of commercially available domainl
binding anti—CD6 dies (MEM98 and MT605 clones Figure 3E). The increase in CD6 MFI
in Th17pol over Thnp cells (Figure 3D and E) are in line with the
reports from clinical sample
d clones of Th17 cells overexpressing CD6 as compared to Th1 cells (Brucklacher—Waldert,
Stuerner et a1. 2009). This overexpression was not only limited to CD4+ T cells but
was also
ed in activated CD8+ T cells.
Example 4:
Method:
1. Human PBMC culture for Th17 polarization: Same as described in Example
3. The
experiment was carried out only till day 3 and day 6 in this ular case.
2. CFSE labelling and T cell proliferation
assay
PBMCs were incubated with CFSE dye at a final concentration of SuM, incubated
at 37 °C, C02
tor for 15 minutes (with intermittent shaking), centrifuged and resuspended in
complete
W0 2015/01 1658
medium and again incubated at 37 °C, CO2 incubator for 30 minutes for the stabilization. Cells
were washed and stimulated in Thnp and Th17pol conditions with control or Itolizumab antibody.
On day 3 post stimulations cells were analysed for CFSE dilution on CD4+T cells using flow
cytometry.
3. Flow cytometry analysis
Cell surface marker staining: At each time point, cells from quadruplicate wells were harvested
and blocked with Human Fc or binding inhibitor and incubated at 4°C for 30 minutes. Cells
were centrifuged and re—suspended in staining buffer (2% FBS in 1X PBS) containing the
antibodies for cell surface s (e.g. CD4, CD8, CD25). Acquisition and analysis of samples
were performed using a Cyan—ADP flow cytometer with the Summit version 4.3 re
(Beckman r, Fullerton, CA, USA). Lymphocytes were gated by forward and side scatter
and further gated on CD4+T cells.
Results:
Itolizumab is associated with inhibition of T cell activation and proliferation in Thnp and
Th17pol conditions.
We had previously reported a 50-55% decrease in CD25 expression on D3
as well as anti-
CD3 and ALCAM co-stimulated cells in the presence ofltolizumab (Nair, Melarkode et a1. 2010).
Here, we show that, there is significant reduction in CD25 expression on stimulated CD4+T cells
(~50% reduction) in Thnp and Thl7pol conditions in presence of umab (Figure 4A). This
would imply that the antibody in various contexts of stimulation is able to consistently and
similarly t the activation of T cells.
Similar to inhibition of activation of T cells, we observed that Itolizumab also ted the
proliferation of T cells. In presence of Itolizumab, a significant reduction in CD4+T cell
proliferation (~38% in Thnp and ~62 % reduction in Thl7pol condition) was observed (indicated
by reduced number of CFSE lo cells) confirming the inhibitory role of Itolizumab in T cell
activation and proliferation (Figure 4B).
Example 5:
Method:
1. Human PBMC culture for Thl7 polarization: Same as bed in e 3
2. Flow cytometry analysis
W0 2015/01 1658
Intracellular ne staining for IL—17A and IFN—y was performed as s. At the time point
of analysis, cells were left unstimulated or stimulated with 50 ng/mL of PMA and lug/mL of
ionomycin in presence of BD Golgi Stop PlugTM and incubated for 5 hours at 37 °C. Cells were
harvested and washed in staining buffer and d with human Fc receptor binding inhibitor.
After surface marker staining, cells were re-suspended in BD Cytofix/CytopermTM
fixation/penneabilization buffer, and intracellular staining was performed as per manufacturer
instructions. ition and analysis of samples were performed using a Cyan-ADP flow
cytometer with the Summit version 4.3 software (Beckman Coulter, Fullerton, CA, USA).
Lymphocytes were gated by forward and side scatter and further gated on CD3'T cells as
mentioned in the respective experiments.
3. Cytokine analysis
At each time point, lOOuL of cell supernatant was collected and pooled from quadruplicate wells
and frozen at-8OOC. Cytokine analysis was performed as per manufacturer instructions using
Human IFN—y Quantikine SixPak (SIFSO) and Human 11-17 A) (Quantikine SixPak
($1700) kits from R&D Systems. Absorbance was read at 450/630nm using SpectraMax M5e
, Molecular Devices, Sunnyvale, CA, USA.
4. Statistical analyses
GraphPad Prism 60 re (GraphPad re, San Diego, CA, USA) was used for all
statistical is. Statistical significance was determined using unpaired t test followed by
Holm—Sidak method, with alpha=5.000% (Figures 5 D and E).
Results:
Itolizumab is ated with reduction in Th17 hallmark cytokines
Under Thl7pol conditions intracellular expression of Thl (IFN—y) and Th17 (IL-17A) ure
cytokines were ed between control antibody and Itolizumab treated cells, Representative
dot plot from day 6, show only minimal expression of both IFN—y and 1L-17A
upon umab
treatment, as compared to controls (Figure 5A). Similar reductions, with Itolizumab treatment
were also ed in Thnp conditions.
The time course study across days 3, 6, 8 and 13 demonstrated maximum decrease in intracellular
IFN—y on days 6 and 8 (Figure 5B). Similarly, maximum inhibition (80-90%) of intracellular IL-
l7A by Itolizumab was also observed on these days (Figure 5C). A corresponding correlation
decrease in lFN—y and 1L—17 release, upon Itolizumab treatment (as evaluated by measuring
secreted cytokines) was also observed es 5D and E). The similar pattern ofinhibition for
both the intracellular cytokines measured as well as the ed cytokines confirmed the effect of
Itolizumab in these two major effector Th subsets.
Example 6:
Method:
1. Human PBMC culture for Th17 polarization: Same as described in Example 3
2. Flow cytometry analysis
ellular staining for pSTAT3 was performed as follows. At the time point of analysis cells
were harvested and washed in staining buffer and blocked with human Fc receptor g
inhibitor. After surface marker (CD4) staining, cells were fixed with 2% PFA (100 pl for each
staining well) and incubated at 4°C for 15—20 mins. Cells were washed with PBS, twice and
permeabilised with 100% chilled ol. SOOul of chilled methanol was added while gently
vortexing the cells and were incubated at 4°C for 20 mins. After centrifugation, at 4 °C at ~250 x g
for 5 mins methanol was decanted and cells were resuspended in ng buffer. Antibodies for
intracellular staining were diluted in staining buffer and added at appropriate dilutions and plates
were incubated at 4°C for 30 minsCells were washed thrice in staining buffer and resuspended in
1x PBS and acquired (all washes done at 4°C).
Intracellular cytokine staining for IL-17A and transcription factor RORyT was performed
follows. At the time point of is, cells were left unstimulated or stimulated with 50 ng/mL
ofPMA and lug/mL ofionomycin in presence ofBD Golgi Stop PlugTM and ted for 5 hours
at 37 °C. Cells were harvested and washed in staining buffer and blocked with human Fc
receptor
g inhibitor. After surface marker (CD3) staining, cells were re-suspended in BD
Cytofix/CytopermTM fixation/penneabilization buffer, and intracellular staining was performed as
per manufacturer instructions.
Intracellular cytokine staining for lL-l 7A and surface marker CCR6 and CD3
was performed as
follows At the time point of analysis, cells were left unstimulated or stimulated with 50 ng/mL
of PMA and 1 ug/mL of ionomycin in presence of BD Golgi Stop PlugTM and incubated for 5
hours at 37 °C. Cells were harvested and washed in ng buffer and blocked with human Fc
receptor binding inhibitor. After surface marker (CD3 and CCR6) staining, cells were re-
suspended in BD Cytofix/CytoperrnTM fixation/penneabilization , and intracellular staining
was performed as per manufacturer instructions
Acquisition and is of samples were performed using a DP flow cytometer with the
Summit version 4.3 software (Beckman Coulter, Fullerton, CA, USA). Lymphocytes were gated
by forward and side scatter and further gated on CD4+ or CD3+T cells as mentioned in the
respective panels.
Results:
Reduction in signature Th17 specific markers in ce of Itolizumab
Th17 differentiation involves the synergistic combination of pSTAT3 and RORyT transcription
factors (Annunziato, Cosmi et al. 2007; li, Korn et a1. 2008; de Wit, Souwer et al. 2011).
Previously we have reported on Itolizumab mediated reduction in pSTAT3 expression on activated
T cells (Nair, Melarkode et a1. 2010) which was further confirmed in the present study (Figure
6A). We also show, in Th17pol conditions there is a substantial reduction in IL-17A and RORyT
double positive T cells in the presence of Itolizumab as compared to control antibody treated cells.
Correlating with the peak inhibition of or cytokines (Figure SB—E), the inventors observe a
substantial reduction in these double positive cells (70—80% ion) and total RORyT MFI
day 6 (Figure 6B and 6C respectively). Similar trend was also observed on day 8.
CCR6 is the signature surface marker for Th17 cells (Liu and Rohowsky-Kochan 2008; Singh,
Zhang et al. 2008). Therefore it was of interest to evaluate CCR6 expression in lL-17 producing
T cells and effect of Itolizumab on them. As analysed on day 6, in Th17pol conditions, 50-60%
reduction in the expression of CCR6+IL-17A+ (dual positive) CD3+T cells
was observed in the
presence of Itolizumab as compared to the control (Figure 6D). Similar reductions were also
observed on day 10.
While CCR6+T cells are either memory or Treg cells aki, Yang et al. 2008) all these
cells
are not IL-17 producing Th17 cells (Sallusto, Lenig et al. 1998; Liao, Rabin et al. 1999;
Kleinewietfeld, Puentes et al. 2005). Itolizumab had little impact on the I‘L—17'CCR6+ (non-Th17
cells). This observation is distinct from the decrease ed in total RORyT MFI in the
presence
of umab as compared to the control (Figure 6C)
, suggesting that Itolizumab could down
regulate the expression of transcription factor RORyT but affects only the IL17+ subset of CCR6
expression T cells. These experiments prove that umab inhibits the activation and
differentiation of Th17 cells by inhibiting key transcription factors , RORyT
along with
RORyT + 1L-17A+ and CCR64'lL-17+ T cells e 6) across days.
References:
W0 2015/01 1658 PCT/lB2014/063345
Annunziato, F, L Cosmi, et a1. (2007). "Phenotypic and functional features of human Th17 cells." 1
Exp Med 204(3): 1849-61 .
Bettelli, E., T. Korn, et a1. . "Induction and effector ons of T(H)l7 cells." m
453(7198): 1051-7.
Brucklacher—Waldert, V., K. Stuerner, et al. (2009). "Phenotypical and functional terization of
T helper 17 cells in multiple sclerosis." Lain. 132(Pt 12): 3329—41.
De Wit, 1., Y. Souwer, et a1. (2011). "CD5 costimulation induces stable Thl7 development by
promoting IL—23R expression and sustained STAT3 activation." 131L094 118(23): 6107—14.
Kleinewietfeld, M., F. Puentes, et a1. . "CCR6 expression defines regulatory or/memory—
like cells within the CD25(+)CD4+ T—cell subset." 13M 105(7): 2877-86.
Liao, F., R. L. Rabin, et al. (1999). "CC-chemokine receptor 6 is expressed on diverse memory subsets
of T cells and ines responsiveness to macrophage inflammatory protein 3 alpha." J
l 162(1): 186—94.
Liu, H. and C. Rohowsky-Kochan (2008). "Regulation ofIL-17 in human CCR6+ effector
memory T
cells." J Immunol 180(12): 7948-57.
Nair, P, R. Melarkode, et a1. . ”CD6 synergistic co-stimulation promoting proinflammatory
response is modulated without interfering with the activated leucocyte cell adhesion molecule
interaction. ” Clin Exp Immunol 162(1): 116-30.
Sallusto, F., D. Lenig, et al. (1998). "Flexible programs of chemokine receptor expression on human
polarized T helper l and 2 cytes." J Exp Med 187(6): 875-83.
Singh, S. P., H. H. Zhang, et a1. (2008). ”Human T cells that are able to produce IL-l7
express the
chemokine receptor CCR6." J Immunol : 214—21.
ki, T., X. 0. Yang, et a1. (2008). "CCR6 regulates the migration of inflammatory and
regulatory T cells." J l 181(12): 8391-401.
Claims (2)
1. Use of an Itolizumab dy specifically binding to Domain 1 (D1) of CD6 in the manufacture of a medicament for use in a subject having a stage of multiple sclerosis (MS) that exhibits increased levels of T helper 17 (Th17) cells and having an increased expression of IL- 23R, wherein the use in the subject causes a reduction of Th17 cells and a reduction of TNF- alpha, IFN-gamma, IL-6, and IL-17A in a body fluid of the subject.
2. The use according to claim 1, wherein the use includes ring IL-23R expression on blood cells and/or dendritic cells of the subject.
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