U.S. patent application number 16/537224 was filed with the patent office on 2019-11-28 for treating respiratory conditions.
The applicant listed for this patent is TLA TARGETED IMMUNOTHERAPIES AB. Invention is credited to Graham Cotton, Ola Winqvist.
Application Number | 20190361020 16/537224 |
Document ID | / |
Family ID | 50973441 |
Filed Date | 2019-11-28 |
View All Diagrams
United States Patent
Application |
20190361020 |
Kind Code |
A1 |
Winqvist; Ola ; et
al. |
November 28, 2019 |
TREATING RESPIRATORY CONDITIONS
Abstract
A method for treating a respiratory conditions comprises
applying peripheral blood from a patient or subject to an apheresis
column loaded with a solid support comprising one or more binding
reagents capable of specifically binding to a chemokine receptor,
optionally the chemokine receptor CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7 immobilized directly or indirectly on the support
thus removing one or more chemokine receptor, optionally CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells from
the peripheral blood of the patient or subject. Various companion
diagnostic methods and useful binding reagents are also
described.
Inventors: |
Winqvist; Ola; (Uppsala,
SE) ; Cotton; Graham; (Edinburgh, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TLA TARGETED IMMUNOTHERAPIES AB |
STOCKHOLM |
|
SE |
|
|
Family ID: |
50973441 |
Appl. No.: |
16/537224 |
Filed: |
August 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15629700 |
Jun 21, 2017 |
10422800 |
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16537224 |
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14105628 |
Dec 13, 2013 |
9726666 |
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15629700 |
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PCT/GB2012/051357 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051349 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051348 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051351 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051350 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051355 |
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14105628 |
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PCT/GB2012/051345 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051352 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051346 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051353 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051356 |
Jun 13, 2012 |
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14105628 |
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PCT/GB2012/051354 |
Jun 13, 2012 |
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14105628 |
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61496442 |
Jun 13, 2011 |
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61496167 |
Jun 13, 2011 |
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61496288 |
Jun 13, 2011 |
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61496242 |
Jun 13, 2011 |
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61496209 |
Jun 13, 2011 |
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61496195 |
Jun 13, 2011 |
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61496228 |
Jun 13, 2011 |
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61496264 |
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61496184 |
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61496329 |
Jun 13, 2011 |
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61496377 |
Jun 13, 2011 |
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61496352 |
Jun 13, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61M 1/3618 20140204;
G01N 33/56972 20130101; G01N 2333/7158 20130101; G01N 2333/70596
20130101; G01N 33/566 20130101; G01N 2800/065 20130101; A61M 1/3679
20130101 |
International
Class: |
G01N 33/566 20060101
G01N033/566; G01N 33/569 20060101 G01N033/569; A61M 1/36 20060101
A61M001/36 |
Claims
1. A method for treating a respiratory condition in a subject in
need thereof, the method comprising applying peripheral blood from
the subject to an apheresis column loaded with a solid support
comprising one or more binding reagents capable of specifically
binding to a chemokine receptor CCR1 immobilized directly or
indirectly on the support, whereby one or more cells expressing
chemokine receptor CCR1 are removed from the peripheral blood of
the subject, wherein the applied blood is returned to the subject,
and whereby the respiratory condition is treated.
2. The method of claim 1, wherein the respiratory condition is
sarcoidosis or Chronic Obstructive Pulmonary Disease (COPD).
3. The method of claim 1, wherein the binding reagent is an agonist
or an antagonist of CCR1.
4. The method of claim 1, wherein the binding reagent is an
antibody or a chemokine.
5. The method of claim 4, wherein: (a) the chemokine is selected
from CCL2 (MCP-1), MCP-2, MCP-3, MCP-4 (CCL12), MCP-5, CCL5
(RANTES), CCL9, MRP-2 (CCL10), CCL14, CCL15, CCL16, CCL23, CCL11
(Eotaxin), CCL28, CCL24 (Eotaxin 2), CCL26, CCL3, CCL4, CCL5, CCL8,
CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8; (b) the
chemokine is RANTES; and/or (c) the chemokine is selected from
CCL9, MRP-2 (CCL10), CCL14, CCL15, CCL16, and CCL23.
6. The method of claim 5, wherein the chemokine is MCP-1 or
MCP-5.
7. The method of claim 1, wherein the one or more cells are
monocytes, eosinophils, or T lymphocytes.
8. The method of claim 1, wherein the subject has increased levels
of expression of CCR1 as compared to a subject that does not have a
respiratory condition.
9. The method of claim 1, wherein 20-50% of the subject's blood is
applied to the column in a single treatment.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/629,700, filed on Jun. 21, 2017, which is a divisional
of U.S. patent application Ser. No. 14/105,628, filed on Dec. 13,
2013, now issued as U.S. Pat. No. 9,726,666, which is a
continuation-in-part of International Patent Application No.
PCT/GB2012/051357, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,442, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051349, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,167, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051348, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,288, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051351, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,242, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051350, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,209, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051355, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,195, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051345, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,228, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051352, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,264, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051346, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,184, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051353, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,329, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051356, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,377, filed on Jun.
13, 2011. U.S. patent application Ser. No. 14/105,628 is also a
continuation-in-part of International Patent Application No.
PCT/GB2012/051354, filed on Jun. 13, 2012, which claims priority to
U.S. Provisional Patent Application No. 61/496,352, filed on Jun.
13, 2011. The entire contents of each of these applications are
fully incorporated herein by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 31, 2019, is named
"P81602729US01-211226-9003-US02-SEQ-LIST-08-09-19.txt", and is
26,527 bytes in size.
FIELD OF THE INVENTION
[0003] The various embodiments of the present invention relate to
products for and methods of treating inflammatory conditions, such
as respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). Companion diagnostics are
also described.
BACKGROUND OF THE INVENTION
[0004] Respiratory tract disorders such as asthma and chronic
obstructive pulmonary disease (COPD) are typically characterised by
breathing difficulties as a result of reduced airflow to the lungs.
These symptoms are often caused by inflammation of the airways; for
example, chronic bronchitis is a form of COPD associated with
excessive inflammation of the bronchi.
[0005] In addition to breathing difficulties, airway obstruction
and associated inflammation can cause progressive lung damage. In
the case of patients with COPD, permanent narrowing of the airways
can lead to complications such as chest infections, heart failure
and ultimately pulmonary failure.
[0006] COPD is a very common respiratory disease and in the
majority of cases, smoking is the cause.
[0007] Patients are typically treated using inhalers containing
"bronchodilator" drugs. However, these are of limited use for
patients with permanently-constricted airways and/or end-stage
disease characterised by extensive damage to the lungs.
[0008] Apheresis is a treatment used for depletion of blood
components, such as antibodies, low-density lipoproteins (LDL) and
blood cells. Leukapheresis is the apheresis treatment used for
removal of white blood cells, leukocytes. The patient is connected
to an extracorporeal blood circulating system; the blood is drawn
from a vein in one arm, passed through a column device and returned
into the other arm of the patient. WO2010/029317 describes
apheresis columns useful for treating inflammatory conditions
including a chemokine immobilised on a solid support.
SUMMARY OF THE INVENTION
[0009] Chemokines are a class of cytokine molecules involved in
cell recruitment and activation in inflammation. Chemokines cause
chemotaxis and activation of various subpopulations of cells in the
immune system. The activity of chemokines is mediated primarily
through tight binding to their receptors on the surface of
leukocytes. In certain embodiments the present invention is based
on the realisation that the interaction between chemokines and
cells expressing their receptors may be exploited for the treatment
of respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). In particular, various
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD) include an inflammatory
component. Whilst sarcoidosis is predominantly considered a
respiratory condition, general treatment of sarcoidosis affecting
any organ of the body may be possible within the scope of the
various embodiments of the present invention. The inventors have
determined that targeting increased recruitment of specific
chemokine receptor-expressing cells to the site of inflammation
presents a new therapeutic approach to treat such conditions.
Moreover, in such conditions, chemokine receptor expression on each
cell may be increased again providing a therapeutic approach to
treat such conditions. It is shown herein that subjects suffering
from respiratory conditions such as sarcoidosis exhibit increased
frequency of chemokine receptor expressing cells in the peripheral
blood. Subjects with sarcoidosis exhibit increased frequency of
CCR1 expressing cells such as CCR1 expressing monocytes, compared
to healthy controls. It is also shown herein that the CCR1
expressing cells can be removed using a suitable binding reagent,
in particular RANTES (in biotinylated form) immobilized on a
suitable matrix. Similarly, it is shown herein that the monocytes
also express CCR2. The CCR2 expressing monocytes can be depleted in
sarcoidosis patients using a suitable binding reagent, in
particular MCP-1, in biotinylated form, immobilized on a suitable
matrix. It is also shown herein that subjects suffering from
respiratory conditions such as sarcoidosis exhibit increased
frequency of CCR7 expressing cells such as CCR7 expressing
lymphocytes, and also central memory T cells,compared to healthy
controls. It is also shown herein that the CCR7 expressing cells
can be removed using a suitable binding reagent, in particular
MIP3b (in biotinylated form) immobilized on a suitable matrix.
[0010] Thus, in certain embodiments the invention serves to reduce
the recruitment of inflammatory leukocytes, which express
characteristic chemokine receptors, and possibly express
characteristic chemokine receptors at increased levels, to sites of
inflammation linked to respiratory conditions, in particular
sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD). This
is achieved using specific binding reagents to capture specific
chemokine receptor-expressing inflammatory leukocytes from the
patient. Accordingly, in certain embodiments the invention provides
in a first aspect a method for treating respiratory conditions, in
particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD) comprising applying peripheral blood from a patient to an
apheresis column loaded with a solid support comprising one or more
binding reagents capable of specifically binding to one or more
chemokine receptors, in particular the chemokine receptors CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7, immobilized directly or
indirectly on the support thus removing one or more chemokine
receptor, in particular one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7, expressing cells from the peripheral
blood of the patient. The peripheral blood from which the chemokine
receptor expressing cells have been removed may then be returned to
the patient in order to complete the treatment. The invention may
thus rely on a continuous extracorporeal circuit in some
embodiments. Alternatively, in certain embodiments the invention
may comprise steps of obtaining peripheral blood from the patient,
applying the peripheral blood to the column and subsequently
returning the peripheral blood from which the chemokine receptor
expressing cells have been removed to the patient.
[0011] As shown herein, suitable binding reagents can be
immobilized onto a solid support, either directly or indirectly, to
generate an apheresis column suitable for capturing relevant
chemokine receptor-expressing cells. Where increased levels of
chemokine receptor expression are observed, such cells may be
preferably removed from the peripheral blood using the columns of
the various embodiments of the invention. Thus, the methods of the
various embodiments of the invention may preferably target one or
more of CCR2hi, CCR1hi, CCR3hi, CCR5hi, CXCR1hi, CXCR2hi and/or
CCR7hi cells as defined herein for removal from the peripheral
blood. "High" expression may be determined according to standard
flow cytometry techniques. The level is measured relative to levels
of expression of the chemokine receptor in cells taken from a
healthy subject. The attached FIG. 17 provides an example of a
gating strategy.
[0012] Herein, reference to CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 is intended to encompass selection of any one or more,
up to all, of the chemokine receptors listed. In addition, the
combination of CCR2, CCR1, CCR3, CCR5, CXCR1 and/or CXCR2 is
explicitly contemplated as a separate grouping, to include any one
or more of CCR2, CCR1, CCR3, CCR5, CXCR1 and CXCR2.
[0013] In other embodiments the invention further provides a
binding reagent capable of specifically binding to one or more
chemokine receptors, in particular to a chemokine receptor/the
chemokine receptor CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7, for use in the treatment of respiratory conditions, in
particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD), wherein the one or more binding reagents is immobilized,
directly or indirectly, on a solid support contained within an
apheresis column, to which is applied peripheral blood from a
patient thus removing one or more chemokine receptor/CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells from the
peripheral blood of the patient. In certain embodiments the
invention also provides for use of one or more binding reagents
capable of specifically binding to a chemokine receptor/the
chemokine receptor CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
for use in the manufacture of an apheresis column for treatment of
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD), wherein the one or more
binding reagents is immobilized on a solid support contained within
the apheresis column, to which is applied peripheral blood from a
patient thus removing one or more of chemokine receptor/CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells from the
peripheral blood of the patient.
[0014] All embodiments described in respect of the methods of
treatment of the various embodiments of the invention apply to
these aspects mutatis mutandis and are not repeated for reasons of
conciseness. Thus, the following discussion made with reference to
the various embodiments of the methods of treatment is also
applicable to the medical use aspects of the invention.
[0015] In certain embodiments the invention aims to treat a range
of respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). By treatment is meant a
reduction in the specific chemokine receptor expressing cells in
the peripheral blood of the patient. The reduction may comprise a
reduction in cells that express chemokine receptors, in particular
one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7, at
increased levels in diseased patients. The patient is typically a
human patient but the term patient may include both human and
non-human animal subjects in some embodiments. In the context of
the various embodiments of the present invention, this typically
involves a reduction in one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells, such as one or more of
CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi, CXCR1.sup.hi,
CXCR2.sup.hi and/or CCR7.sup.hi expressing cells, in the peripheral
blood of the patient. The CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 expressing cells comprise, consist essentially of or
consist of monocytes, macrophages, lymphocytes, in particular
T-lymphocytes, and/or eosinophils in certain embodiments. In
specific embodiments, the cells removed in order to treat
respiratory conditions such as sarcoidosis comprise monocytes, in
particular CCR1 and/or CCR2 expressing monocytes. In other
embodiments cells removed in order to treat respiratory conditions
such as sarcoidosis comprise lymphocytes, specifically T
lymphocytes such as central memory T cells, in particular CCR7
expressing T cells.
[0016] Monocytes are produced by the bone marrow from
haematopoietic stem cell precursors called monoblasts. Monocytes
may differentiate into macrophages or dendritic cells. Monocytes
and their macrophage and dendritic cell progeny serve a number of
functions in the immune system including phagocytosis, antigen
presentation and cytokine production. Monocytes may be
characterized with reference to expression of the cell surface
marker CD14, optionally together with CD16. Classical monocytes may
be characterized by high level expression of the CD14 cell surface
receptor (CD14++ CD16- monocyte). Non-classical monocytes may be
characterized by low level expression of CD14 and with additional
co-expression of the CD16 receptor (CD14+CD16++ monocyte).
Intermediate monocytes may be characterized by high level
expression of CD14 and low level expression of CD16 (CD14++CD16+
monocytes). Macrophages are derived from monocytes and are
responsible for protecting tissues from foreign substances. They
are cells that possess a large smooth nucleus, a large area of
cytoplasm and internal vesicles for processing foreign material.
The term "macrophage" may refer to a monocyte-derived cell
expressing one or more of the following cell surface markers CD14,
CD11b, Lysozyme M, MAC-1/MAC-3 and CD68. The term macrophage
includes both activated and un-activated macrophages. Activated
macrophages may be characterized by expression of one or more of
CD69, ENG, FCER2 and IL2RA, HLA-DR, CD86. Un-activated macrophages
have not yet received activating signals through for example TLR
receptors and therefore they express less activation markers on the
cell surface which correlates with lesser maturation. M1
macrophages may be characterized by expression of one or more of
CD16.sup.+CD32.sup.+CD64.sup.+and secrete mainly IL-23 and IL-1,
TNF, IL-6 and high levels of IL-12 and in addition effector
molecules such as iNOS and ROI. M1 macrophages have cytotoxic
features as opposed to M2 macrophages. M2 macrophages may be
characterized by expression of one or more of
SRA/B.sup.+CD163.sup.+MR.sup.+CD14.sup.+ and express TGF.beta.,
IL-10 and IL-1Ra. Tumour associated macrophages (TAMs) share many
characteristics with the M2 macrophages and are considered as M2
polarised macrophages. The M1/M2 paradigm can also be found in
monocyte subsets where CD14.sup.+CD16.sup.-CXC3R1.sup.low monocytes
are considered the "inflammatory" subset and the
CD14.sup.lowCD16.sup.+CXC3R1.sup.high are "resident" monocytes.
[0017] The three major types of lymphocyte are T cells, B cells and
natural killer (NK) cells. The term "T-lymphocyte" includes
CD4.sup.+ T cells such as T helper cells (Th1 cells and Th2 cells),
and CD8.sup.+ T cells such as cytotoxic T cells. Th1 cells may be
characterized by expression of CCR5 and/or by production of
IFN-.gamma.. Th2 cells may be characterized by expression of CCR3
and/or by production of IL-4.
[0018] The claimed methods may, in particular, target eosinophils.
Eosinophilia is an important component of certain respiratory
conditions and may be defined as the presence of more than 500
eosinophils/microlitre of blood. Thus, reducing numbers of
circulating eosinophils represents an important therapeutic
approach. Eosinophils, or eosinophil granulocytes, are white blood
cells and represent an important immune system component. Along
with mast cells, they also control mechanisms associated with
allergy and asthma. They are granulocytes that develop during
haematopoiesis in the bone marrow before migrating into blood.
[0019] The name "eosinophil" derives from the eosinophilic
"acid-loving" properties of the cell. Normally transparent, it is
this affinity that causes them to appear brick-red after staining
with eosin, a red dye, using the Romanowsky method. The staining is
concentrated in small granules within the cellular cytoplasm, which
contain many chemical mediators, such as histamines and proteins
such as eosinophil peroxidase, ribonuclease (RNase),
deoxyribonucleases, lipase, plasminogen, and major basic protein.
These mediators are released by a process called degranulation
following activation of the eosinophil, and are toxic to both
parasite and host tissues.
[0020] Eosinophils develop and mature in bone marrow. They
differentiate from myeloid precursor cells in response to the
cytokines interleukin 3 (IL-3), interleukin 5 (IL-5), and
granulocyte macrophage colony-stimulating factor (GM-CSF).
Eosinophils produce and store many secondary granule proteins prior
to their exit from the bone marrow. After maturation, eosinophils
circulate in blood and migrate to inflammatory sites in tissues in
response to chemokines such as CCL11 (eotaxin-1), CCL24
(eotaxin-2), CCLS (RANTES) and MCP1/4. Eosinophils may be activated
by Type 2 cytokines released from a specific subset of helper T
cells (Th2); IL-5, GM-CSF, and IL-3 are important for eosinophil
activation as well as maturation. CD44 and CD69 have been shown to
represent different types of cell-surface activation markers for
human eosinophils. CD69 is absent from "fresh" eosinophils but
expressed following activation (using cytokines). CD44 on the other
hand is constitutively expressed but expression is significantly
up-regulated in response to activation (Matsumoto et al., Am. J.
Respir. Cell Mol. Biol., Volume 18, Number 6, June, 1998 860-866).
Cell specific markers for eosinophils include CD9 and CDw125.
[0021] CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressed
on these aforementioned cells bind to chemokines such as CCL2
(binds CCR2), CCL3, CCL5, CCL9 (binds CCR1), CCL11 (binds CCR3),
CCL12 (binds CCR2), CCL-14 (binds CCR1), CCL16 (binds CCR1), CCL28
(binds CCR3), CCL24 (binds CCR3), CCL26 (binds CCR3) and/or CXCL8
(binds CXCR1 and CXCR2). Chemokines MIP1.gamma. (CCL9), MRP-2
(CCL10), Mlp-1.delta. (CCL15) and CCL23 appear to bind CCR1 only.
Chemokines Eotaxin (CCL11) and CCL24 (Eotaxin-2) only bind CCR3.
Chemokine MIP1.beta. (CCL4) only binds CCR5.
[0022] CXCR1 binds CXCL6, CXCL7, CXCL8.
[0023] CXCR2 binds CXCL1, 2, 3, 5, 6, 7, 8.
[0024] CCR1 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) receptor 1. The
HGNC ID for this gene is 1602. The gene is located at chromosome
position 3p21. The previous symbol and name CMKBR1, SCYAR1.
Synonyms for this gene include CD191, CKR-1, MIP1.alpha. R. The
Entrez Gene reference sequence for CCR1 is 1230 as available on 13
Jun. 2011, which is incorporated herein by reference in its
entirety.
[0025] CCR2 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) receptor 2. The
HGNC ID for this gene is 1603. The gene is located at chromosome
position 3p21. The previous symbol and name for the gene is CMKBR2.
Synonyms for this gene include CC-CKR-2, CD192, CKR2, FLJ78302,
MCP-1-R. The NCBI Reference Sequence is NM_001123041.2 as available
on 13 Jun. 2011, which is incorporated herein by reference in its
entirety.
[0026] CCR3 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) receptor 3. The
HGNC ID for this gene is 1604. The gene is located at chromosome
position 3p21.3. The previous symbol and name for the gene is
CMKBR3. Synonyms for this gene include CC-CKR-3, CD193 and CKR3.
The Genbank reference sequence for CCR3 is AF247361.1 as available
on 13 Jun. 2011, which is incorporated herein by reference in its
entirety.
[0027] CCR5 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) receptor 5. The
HGNC ID for this gene is 1605. The gene is located at chromosome
position 3p21. The previous symbol and name for the gene is CMKBRS.
Synonyms for this gene include CC-CKR-5, CD195 CKR-5, IDDM22 and
CKRS. The Entrez Gene reference sequence for CCR5 is 1234 as
available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0028] CXCR1 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) receptor 1. The
HGNC ID for this gene is 6026. The gene is located at chromosome
position 2q35. The previous symbol and name for the gene is CMKAR1,
IL8RA, "interleukin 8 receptor, alpha". Synonyms for this gene
include CD181, CDw128a, CKR-1. The Genbank reference sequence for
CXCR1 is U11870.1 as available on 13 Jun. 2011, which is
incorporated herein by reference in its entirety.
[0029] CXCR2 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) receptor 2. The
HGNC ID for this gene is 6027. The gene is located at chromosome
position 2q35. The previous symbol and name for the gene is IL8RB,
"interleukin 8 receptor, beta". Synonyms for this gene include
CD182, CMKAR2. The Genbank reference sequence for CXCR2 is U11869.1
as available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0030] CCR7 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) receptor 7. The
HGNC ID for this gene is 1608. The gene is located at chromosome
position 17q12-q21.2. The previous symbol and name for the gene is
CMKBR7, EBI1. Synonyms for this gene include BLR2, CD197 and
CDw197. The RefSeq reference sequence for CCR7 is NM_001838.3 as
available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0031] The various embodiments of the methods of the invention may
involve specific binding interactions with any one or more of these
further cell-surface markers in addition to the removal based upon
binding to CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7.
Suitable binding reagents can be prepared to specifically bind to
these cell-surface markers. The discussion of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 specific binding reagents thus
applies mutatis mutandis.
[0032] Treatment according to the various embodiments of the
invention may result in alleviation or amelioration of symptoms,
prevention of progression, regression of the condition, or complete
recovery. Measurable parameters of successful treatment include one
or more, up to all, of lungfunction with spirometry, PEF, lung
biopsy. Pulsoxymeter. paO2, paCO2. In specific embodiments, a
single treatment is sufficient to cause a depletion of around 10%,
20%, 30%, 40%, 50%, 60% or 70%, or higher up to 80%, 90%, 95% or
more, or any range of values between and including these amounts,
of one or more of a specific chemokine receptor, in particular one
or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7,
expressing cells from the peripheral blood of the patient. In
specific embodiments, at least around 50% depletion is achieved in
a single treatment. Thus, successful treatment may be defined with
reference to depletion of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells. Treatment may lead to
depletion of between approximately 100 and 500 million CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, such as
monocytes, in certain embodiments and more particularly to about
100, 150, 200, 250, 300, 350, 400, 450, or 500 million CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells.
[0033] By binding to the column through the binding
reagent-chemokine receptor interaction, chemokine receptor
expressing cells are immobilized. These immobilized cells express
further unoccupied chemokine receptors, which may be of the same or
different type to those used for capture. These additional
chemokine receptors may permit circulating chemokines which
contribute to the inflammatory condition to be captured from the
peripheral blood. Thus, a reduction in circulating (specific)
chemokine levels may provide a measure of successful treatment.
[0034] The duration of treatment may be readily determined by one
skilled in the art and will depend upon factors such as the flow
rate of the peripheral blood. Duration of treatment may be tied
into monitoring of the treatment itself, with the treatment
considered complete once a measurable parameter of treatment has
reached a defined threshold. Any suitable parameter may be employed
as discussed herein. Thus, for example, treatment may be considered
complete when a reduction in one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells, such as a 50% reduction
in one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells, has been achieved. The apheresis system may be
operated ata flow rate of around 10-80 mL/min, or more specifically
between around 20-70 mL/min, or between around 30-60 mL/min. In
specific embodiments, the treatment is performed for a period of
around 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120 etc., or
any range of values between and including these amounts, minutes.
The treatment is typically not aimed to remove all of the cells
expressing the chemokine receptor in the peripheral blood, as a
basal level of those cells is required in healthy subjects.
However, it has been found that only low blood volumes need to be
applied to the columns of the various embodiments of the invention
in order to achieve effective levels of depletion of the chemokine
receptor-expressing cells. Thus, in certain embodiments, around
10-80% or more specifically around 20, 30, 40 or 50%, or any range
of values between and including these amounts, of the patient's
blood is applied to the column in a single treatment. The volume of
blood circulated through the apheresis column or system may be in
the region of around 1000-3000 ml, such as around 1000, 1200, 1400,
1600, 1800 or 2000 ml or any range of values between and including
these amounts. The treatment may be considered complete once this
volume of blood has been circulated. The patient may be
administered anticoagulants prior to each treatment session. A
suitable solution, such as a sterile saline solution, optionally
including an anticoagulant such as Heparin, may be used for priming
the apheresis (extracorporeal) system. An additional volume of
anticoagulant may be added to the circuit at the start of each
treatment session, for example as a bolus injection.
[0035] In certain embodiments the invention relies upon a binding
reagent which is capable of specifically binding to a chemokine
receptor. This specific binding reaction permits cells expressing
the chemokine receptor to be removed from the peripheral blood of
the patient when the blood is passed over the solid support upon or
within which the binding reagent is immobilized. Specific chemokine
receptors of interest include CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7. The binding reagent can be any binding reagent capable
of specifically binding to the receptor in question. By "specific
binding" is meant that the binding reagent displays sufficient
specificity of binding and appropriate binding affinity/kinetics to
permit removal of cells expressing one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 from the peripheral blood. Whilst it
is not precluded that the binding reagent is capable of binding to
other molecules, such as other chemokine receptors, the binding
reagent will preferentially bind to cells expressing one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and in particular
to cells expressing increased levels of one or more of CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (as defined further herein).
The binding reagent capable of specifically binding to CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 may be either an agonist or an
antagonist of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7,
respectively. As the disease condition relies upon up-regulation of
expression of or signaling via CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7, in certain embodiments the binding reagent capable of
specifically binding to CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7 is an antagonist of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7, respectively. Chemokines are typically, although not
necessarily exclusively (particularly in the case of truncated or
modified forms) agonists of their cognate receptor and serve to
activate the cells expressing the relevant receptor, as would be
appreciated by one skilled in the art. Antibodies against the
relevant chemokine receptor are generally considered to be
antagonists, as would be appreciated by one skilled in the art.
Specific examples of binding reagents include proteins or
polypeptides, such as antibodies and receptor ligands, in
particular chemokines. The binding reagent may be a nucleic acid
molecule in certain embodiments. In some embodiments, the nucleic
acid is an aptamer. Nucleic acid aptamers are polynucleotides of
approximately 15-40 nucleotides long. Nucleic acid aptamers can be
made using the SELEX process (systemic evolution of ligands by
exponential enrichment) or any other process known to those of
skill in the art.
[0036] In other embodiments, the binding reagent may be a peptide,
and in certain instances, a peptide aptamer. Peptide aptamers are
artificial recognition molecules that consist of a variable peptide
sequence inserted into a constant scaffold protein (Baines I C,
Colas P. Peptide aptamers as guides for small molecule drug
discovery. Drug Discov Today. 2006;11:334-341, incorporated herein
by reference). A number of methodologies, such as phage display,
ribosome display and yeast two-hybrid screening systems are
available for screening a library of potential peptide-based
binding agents. Similarly protein scaffolds based on domains such
as fibronectins, ankyrin repeats, protein A, SH3 domains,
lipocalins and ubiquitin can be used as the binding agent. Again a
number of technologies such as phage display and ribosome display
are available for screening a library of protein--based binding
agents. Similarly, libraries of candidate chemical compounds can be
screened for specific binding to the relevant chemokine receptor
using suitable screening techniques known in the art, which may be
high throughput screens in certain embodiments. The candidate
binding agent may be immobilized on a solid support and the ability
of the agent to specifically retain cells expressing the chemokine
receptor of interest or labelled chemokine receptors determined. A
range of cell types may be applied to the solid supports to confirm
specificity of binding, or alternatively a mixed sample (such as
peripheral blood) may be applied to the solid support. Retention of
the cell type of interest (expressing the appropriate chemokine
receptor) can be confirmed to identify suitable binding agents. A
range of small-molecule antagonists of CCR-2 are discussed by Xia M
and Sui Z in Expert Opin Ther Pat. 2009 March; 19(3):295-303
--Recent developments in CCR2 antagonists, and incorporated herein
by reference.
[0037] In the context of the various embodiments of the present
invention the term "chemokine" also comprises biotinylated or
otherwise labelled chemokines. The term "chemokine" also comprises
modified and truncated versions of the chemokine and chemokine
fragments with the proviso that the modified or truncated form
retains its ability to bind to its cognate receptor (and thus
remains functional in the context of the various embodiments of the
invention). The chemokine does not necessarily need to retain
biological activity as it is specific binding affinity for CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 that is required. In
certain embodiments, the chemokine lacks biological activity, for
example in terms of activation of the (CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7) receptor. Modifications may be made to
improve protein synthesis, for example uniformity of product and
yield. As known to those skilled in the art, exemplary
modifications may comprise amino acid additions, substitutions,
deletions or other modifications to one or more amino acids in the
chemokine. Modifications may comprise substitution of the wild type
amino acid with non-natural amino acids such as norleucine (NLeu)
and derivatized amino acids such as pyroglutamic acid (pyroGlu).
Such modifications may be made to minimize side-product formation
during storage and use of the columns of the various embodiments of
the invention. Modifications may be made to improve labelling, for
example inclusion of a polyethylene glycol (PEG) spacer to
facilitate biotinylation. The biotinylation and/or conjugation with
fluorochromes or other labelling groups of the chemokine is
performed in a manner which does not substantially affect the
receptor binding capacity. Site specific biotinylation or other
labelling is preferred as non-selective labelling of chemokines may
compromise receptor binding activity. Biotinylation or other
labelling is generally preferred at or towards the C-terminus of
the protein as the inventors have found that modifications in this
area are generally well tolerated (in terms of a minimal effect on
receptor binding capability). Biotinylation may be carried out
site-specifically at any suitable amino acid. Examples of suitable
amino acids include lysine, diaminopropionic acid and ornithine.
Generally, reference may be made to Natarajan S et al, Int. J.
Pept. Protein Res., 1992, 40, 567-74; Baumeister B, Int. J. Peptide
Res. And Therapeutics, 2005, 11, 139-141; Bioconjugate techniques
2.sup.nd edition, Greg T. Hermanson, incorporated by reference
herein in its entirety.
[0038] Truncations may involve deletion of either N or C terminal
amino acids as appropriate, or both. Typically, the truncated
version will retain the residues required for the chemokine to fold
correctly, for example to retain a chemokine fold structure,
consistent with the requirement that a truncated version must
retain the ability to bind to the relevant receptor (expressed by
(on the surface of) a leukocyte). Chemokine molecules typically
include disulphide bonds between the 1.sup.st and 3.sup.rd and
2.sup.nd and 4.sup.th cysteine residues respectively, as would be
understood by one skilled in the art. Where sequences are presented
herein, it is assumed that these disulphide bonds will form in the
folded protein (unless otherwise stated). Truncated versions may
comprise anywhere between 1 and 100 less amino acids, such as 1, 2,
3, 4, 5 etc amino acids, than the wild type amino acid sequence in
certain embodiments. Of course, truncated versions may comprise
further modification as detailed herein. The modified or truncated
version may have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or more overall amino acid sequence identity with the full
length wild type chemokine (where a deletion is counted as a
difference in amino acid sequence) in certain embodiments. Over the
common sequence between the molecules (i.e the amino acids that
have not been deleted), there may be 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity in
certain embodiments. Sequence identity may be determined using
known algorithms, such as BLAST or GAP analysis (GCG Program)
(applying default settings), which are freely available. Chemokines
may lack the N-terminal signal peptide which is cleaved off during
synthesis in vivo.
[0039] Specific chemokines useful in the various embodiments of the
present invention for binding to CCR2 include CCL2 (MCP-1), MCP-2,
MCP-3, MCP-4 (CCL12) and MCP-5. Both MCP-1 and MCP-5 bind solely to
the chemokine receptor CCR2 and so these chemokines may be
preferred in some embodiments. Each chemokine is able to bind to a
chemokine receptor implicated in respiratory conditions, in
particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD). More specifically, each of MCP-1, MCP-2, MCP-3, MCP-4 and
MCP-5 are useful for removing CCR2 expressing cells from the blood
of a patient. Specific chemokines useful in the various embodiments
of the present invention for binding to CCR1, CCR3 and/or CCR5
include CCL5 (RANTES). RANTES is able to bind to chemokine
receptors implicated in respiratory conditions, in particular
sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD). More
specifically, RANTES is useful for removing CCR1, CCR3 and/or CCR5
expressing cells from the blood of a patient.
[0040] Specific chemokines useful for binding to CCR1 include CCL9,
MRP-2 (CCL10), CCL14, CCL15, CCL16 and CCL23 Specific chemokines
useful for binding to CCR3 include CCL11 (Eotaxin), CCL28, CCL24
(Eotaxin 2) and CCL26. Specific chemokines useful for binding to
CCR5 include CCL3, CCL4, CCL5, CCL8 with CCL4 binding only to CCR5.
Specific chemokines useful in the various embodiments of the
present invention for binding to CXCR1 and/or CXCR2 include CXCL8
(IL-8). IL-8 is able to bind to chemokine receptors implicated in
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). Specific chemokines useful in
the various embodiments of the present invention for binding to
CXCR1 include CXCL6, CXCL7, CXCL8. Specific chemokines useful in
the various embodiments of the present invention for binding to
CXCR2 include CXCL1, 2, 3, 5, 6, 7, 8. CCL19 is useful for the
removal of CCR7 expressing cells.
[0041] The chemokines described in greater detail herein (with
reference to the relevant figures and amino acid sequences, as set
forth in the SEQ ID NOs) may each be applied according to the
present invention.
[0042] The modified and truncated chemokines described in greater
detail herein (with reference to the relevant amino acid sequences,
as set forth in the SEQ ID NOs and accompanying experimental
examples) may each be applied according to the present invention.
Such modified forms may instruct the skilled person regarding
additional modified forms of the same and other chemokines which
may be suitable for use in the invention. Chemokines show variable
sequence homology varying from less than 20% to over 90% but all
share very similar tertiary structures consisting of a disordered
N-terminus, followed by a long loop (the N-loop) that ends in a
3.sub.10 helix, a 3-stranded .beta.-sheet and a C-terminal helix.
The overlall topology is stabilsed by disulphide bonds. This common
tertiary structure is a common feature of the chemokine protein
family (Fernandez E J annd Lolis E., Annu. Rev. Pharmacol.
Toxicol., 202, 42, 469-99; Allen S J et al, Annu. Rev. Immunol.,
2007, 25, 787-820, incorporated herein by reference).
[0043] Truncations within this N-terminal region can maintain
binding to the receptor but can lead to a change or loss of
function (for examples Zhang Y J et al, J. Biol. Chem., 1994, 269,
15918; Gong J-H and Clark-Lewis I., J. Exp. Med., 1995, 181,
631-640; Fernandez E J annd Lolis E., Annu. Rev. Pharmacol.
Toxicol., 202, 42, 469-99; Allen S J et al, Annu. Rev. Immunol.,
2007, 25, 787-820, each of which is incorporated herein by
reference). Truncations at the C-terminus of the chemokine can also
be made and maintain receptor binding activity (Treating
Inflammatory Disorders, Ola Winqvist and Graham Cotton,
WO2010/029317, incorporated herein by reference in its
entirety).
[0044] In other embodiments, fragments and variants of chemokines
are used in the devices and methods as disclosed herein. More
particularly, such fragments and variants retain the ability to
specifically bind to their cognate chemokine receptor. Chemokines
are known by those skilled in the art to share specific receptor
binding domains, including a similar monomeric fold, characterized,
for example, by a disordered amino-terminal domain, followed by a
conserved core region, consisting of the so called "N-loop," three
anti-parallel .beta.-strands, and a carboxyl-terminal a-helix.
While not being bound by theory, it is believed that the
chemokine-chemokine receptor interaction is a two-step mechanism,
in which the core of the chemokine interacts first with a binding
site formed by the extracellular domains of the receptor, while
another interaction is formed between the chemokine N terminus and
a second binding site on the receptor in order to trigger receptor
activation. Thus, a "fragment," such as a functional fragment of a
chemokine is intended to mean a portion of the amino acid sequence
of the protein that retains binding for its cognate receptor. The
fragment may include, for example, the monomeric fold region, or
portions thereof such as the amino-terminal domain, the conserved
core region and/or the "N-loop," the anti-parallel .beta.-strands,
and/or the carboxyl-terminal a-helix or combinations and portions
thereof.
[0045] Further, it is recognized that a polypeptide can be
considerably mutated without materially altering one or more of the
polypeptide's functions, for example, without altering specific
binding and/or the folding of the protein. The genetic code is well
known to be degenerate, and thus different codons encode the same
amino acids. Even where an amino acid substitution is introduced,
the mutation can be conservative and have no material impact on the
essential functions of a protein (see for example, Stryer,
Biochemistry 4th Ed., W. Freeman & Co., New York, N.Y., 1995).
This includes, for example, the ability of the protein to bind and
interact with other proteins, such as a truncated chemokine binding
to its cognate receptor.
[0046] In some examples, part of a polypeptide chain can be deleted
without impairing or eliminating all of its functions. For example,
the deletion of between about 1 and about 20 amino acids on the C-
and/or N-terminus, such as deletions of about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at
the C- and/or N-terminus, can result in a chemokine that retains
function, such as specific binding of its cognate receptor. Such
truncations can retain the full function of an entire protein,
and/or can allow for retained functions such as protein-protein
interactions as in the case of ligand-receptor interactions.
Chemokines having deletions of a small number of amino acids, for
example, less than about 20% (such as less than about 18%, less
than about 15%, less than about 10%, less than about 8%, less than
about 5%, less than about 2%, or less than about 1%) of the total
number of amino acids in the wild type chemokine can also be used
in the methods and devices disclosed herein. Moreover, insertions
or additions can be made in the polypeptide chain for example,
adding epitope tags, without impairing or eliminating its functions
(Ausubel et al., Current Protocols in Molecular Biology, Greene
Publ. Assoc. and Wiley-Intersciences, 1998). Other modifications
that can be made without materially impairing one or more functions
of a polypeptide include, for example, in vivo or in vitro chemical
and biochemical modifications or the incorporation of unusual amino
acids. In some examples, a functional fragment of a chemokine may
consist of about 10 or more, about 25 or more, about 50 or more,
about 75 or more, about 100 or more, about 125 or more, about 150,
about 175 or more, or about more or 200 or more amino acid residues
of a chemokine amino acid sequence.
[0047] In some examples, the chemokine or a functional fragment
thereof has an amino acid that has at least about 60% or 65%
sequence identity, about 70% or 75% sequence identity, about 80% or
85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% sequence identity over its full length as compared
to a reference sequence, such as those detailed herein, for example
using the NCBI Blast 2.0 gapped BLAST set to default parameters.
Alignment may also be performed manually by inspection. One or more
conservative amino acid modifications can also be made in the
chemokine amino acid sequence, whether an addition, deletion or
modification, that does not substantially alter the 3-dimensional
structure of the polypeptide or its ability to bind to the cognate
receptor. For example, a conservative amino acid substitution does
not affect the ability of the chemokine to specifically bind its
cognate receptor. Conservative substitution tables providing
functionally similar amino acids are well known in the art. The
following six groups each contain amino acids that are conservative
substitutions for one another: 1) Alanine (A), Serine (S),
Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3)
Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (VV).
[0048] Peptides, such as chemokines and fragments thereof, can be
modified by a variety of chemical techniques to produce derivatives
having essentially the same activity or function--such as binding
to a cognate receptor--as the unmodified peptides, and optionally
having other desirable properties. For example, carboxylic acid
groups of the protein, whether carboxyl-terminal or side chain, may
be provided in the form of a salt of a pharmaceutically-acceptable
cation or esterified to form a C1-C16 ester, or converted to an
amide of formula NR1R2 wherein R1 and R2 are each independently H
or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a
5- or 6-membered ring. Amino groups of the peptide, whether
amino-terminal or side chain, may be in the form of a
pharmaceutically-acceptable acid addition salt, such as the HCl,
HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other
organic salts, or may be modified to C1-C16 alkyl or dialkyl amino
or further converted to an amide.
[0049] Hydroxyl groups of the peptide side chains can be converted
to C1-C16 alkoxy or to a C1-C16 ester using well-recognized
techniques. Phenyl and phenolic rings of the peptide side chains
can be substituted with one or more halogen atoms, such as F, Cl,
Br or I, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and
esters thereof, or amides of such carboxylic acids. Methylene
groups of the peptide side chains can be extended to homologous
C2-C4 alkylenes. Thiols can be protected with any one of a number
of well-recognized protecting groups, such as acetamide groups.
Those skilled in the art will also recognize methods for
introducing cyclic structures into the peptides of this disclosure
to select and provide conformational constraints to the structure
that result in enhanced stability. For example, a C- or N-terminal
cysteine can be added to the peptide, so that when oxidized the
peptide will contain a disulfide bond, generating a cyclic peptide.
Other peptide cyclizing methods include the formation of thioethers
and carboxyl- and amino-terminal amides and esters.
[0050] Peptidomimetic and organomimetic embodiments are also within
the scope of the present disclosure, whereby the three-dimensional
arrangement of the chemical constituents of such peptido- and
organomimetics mimic the three-dimensional arrangement of the
peptide backbone and component amino acid side chains, resulting in
such peptido- and organomimetics of the proteins of this
disclosure. For computer modeling applications, a pharmacophore is
an idealized, three-dimensional definition of the structural
requirements for biological activity. Peptido- and organomimetics
can be designed to fit each pharmacophore with current computer
modeling software (using computer assisted drug design or CADD).
See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman
& Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm
Press: Buffalo Grove, Ill., pp. 165 174 and Principles of
Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of
techniques used in CADD. Also included within the scope of the
disclosure are mimetics prepared using such techniques.
[0051] Amino acids in a peptide, polypeptide, or protein generally
are chemically bound together via amide linkages (CONN).
Additionally, amino acids may be bound together by other chemical
bonds. For example, linkages for amino acids or amino acid analogs
can include CH2NH--, --CH2S--, --CH2-CH2 --CH.dbd.CH-- (cis and
trans), --COCH2--, --CH(OH)CH2--, and --CHH2SO-- (These and others
can be found in Spatola, in Chemistry and Biochemistry of Amino
Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker,
New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983),
Vol. 1, Issue 3, Peptide Backbone Modifications (general review);
Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson, et al., Int J
Pept Prot Res 14:177-185, 1979; Spatola et al. Life Sci
38:1243-1249, 1986; Harm J. Chem. Soc Perkin Trans. 1307-314, 1982;
Almquist et al. J. Med. Chem. 23:1392-1398, 1980; Jennings-White et
al. Tetrahedron Lett 23:2533, 1982; Holladay et al. Tetrahedron.
Lett 24:4401-4404, 1983; and Hruby Life Sci 31:189-199, 1982.
[0052] CCL2 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 2, also
known as MCP-1. The HGNC ID for this gene is 10618. The gene is
located at chromosome position 17q11.2-q21.1. The previous symbol
and name for the gene is SCYA2 "small inducible cytokine A2
(monocyte chemotatic protein 1, homologus to mouse Sig-je)".
Synonyms for this gene include GDCF-2, HC11, MCP1, MGC9434, SMC-CF,
"monocyte chemoattractant protein-1", "monocyte chemotactic and
activating factor", "monocyte chemotactic protein 1, homologous to
mouse Sig-je", "monocyte secretory protein JE", "small inducible
cytokine subfamily A (Cys-Cys), member 2". The Genbank reference
sequence for CCL2 is BC009716.1 as available on 13 Jun. 2011, which
is incorporated herein by reference in its entirety.
[0053] CCL4 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 4. The HGNC
ID for this gene is 10630. The gene is located at chromosome
position 17q12-q23. The previous symbol and name for the gene is
LAG1, SCYA4, "small inducible cytokine A4 (homologous to mouse
Mip-1b)". Synonyms for this gene include Act-2, AT744.1,
MIP-1-beta. The Genbank reference sequence for CCL4 is M23502.1 as
available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0054] CCL8 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 8, also
known as MCP-2. The HGNC ID for this gene is 10635. The gene is
located at chromosome position 17q11.2. The previous symbol and
name for the gene is SCYA8, "small inducible cytokine subfamily A
(Cys-Cys), member 8 (monocyte chemotactic protein 2)". Another
synonym for this gene is HC14. The Genbank reference sequence for
CCL8 is X99886.1 as available on 13 Jun. 2011, which is
incorporated herein by reference in its entirety.
[0055] CCL7 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 7, also
known as MCP-3. The HGNC ID for this gene is 10634. The gene is
located at chromosome position 17q11.2-q12. The previous symbol and
name for the gene is SCYA6, SCYA7, "small inducible cytokine A7
(monocyte chemotactic protein 3)". Synonyms for this gene include
FIC, MARC, MCP-3, MCP3, NC28, "monocyte chemoattractant protein 3",
"monocyte chemotactic protein 3". The Genbank reference sequence
for CCL7 is AF043338 as available on 13 Jun. 2011, which is
incorporated herein by reference in its entirety.
[0056] CCL13 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 13, also
known as MCP-4. The HGNC ID for this gene is 10611. The gene is
located at chromosome position 17q11.2. The previous symbol and
name for the gene is SCYA13, "small inducible cytokine subfamily A
(Cys-Cys), member 13". Synonyms for this gene include CKb10, MCP-4,
MGC17134, NCC-1, SCYL1. The Genbank reference sequence for CCL13 is
AJ001634 as available on 13 Jun. 2011, which is incorporated herein
by reference in its entirety.
[0057] MCP-5 is a mouse chemokine in the CC chemokine family. It is
also known as Chemokine (C-C motif) ligand 12 (CCL12) and, due to
its similarity with the human chemokine MCP-1, sometimes it is
called MCP-1-related chemokine. The gene for MCP-5 is found in a
cluster of CC chemokines on mouse chromosome 11. The NCBI reference
sequence for CCL12 is NC_000077.5. Previous symbol SCYA12 as
available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0058] CCL3 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 3, also
known as MIP-1a. The HGNC ID for this gene is 10627. The gene is
located at chromosome position 17q12. The previous symbol and name
for the gene is SCYA3, "small inducible cytokine A3 (homologous to
mouse Mip-1a)". Synonyms for this gene include G0S19-1, LD78ALPHA,
MIP-1-alpha. The Genbank reference sequence for CCL3 is M23178.1 as
available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0059] CCL5 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 5, also
known as RANTES. The HGNC ID for this gene is 10632. The gene is
located at chromosome position 17q11.2-q12. The previous symbol and
name for the gene is D17S136E, SCYA5, "small inducible cytokine A5
(RANTES)". Synonyms for this gene include "beta-chemokine RANTES",
MGC17164, RANTES, "regulated upon activation, normally T-expressed,
and presumably secreted", "SIS-delta", SISd, "small inducible
cytokine subfamily A (Cys-Cys), member 5", "T-cell specific protein
p288", "T-cell specific RANTES protein", TCP228. The Genbank
reference sequence for CCL5 is AF043341.1 as available on 13 Jun.
2011, which is incorporated herein by reference in its
entirety.
[0060] IL8 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for interleukin 8, also known as CXCL8. The
HGNC ID for this gene is 6025. The gene is located at chromosome
position 4q13-q21. Synonyms for this gene include 3-10C, AMCF-I,
b-ENAP, "chemokine (C-X-C motif) ligand 8", CXCL8, GCP-1, IL-8,
K60, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF, NAP-1, SCYB8, TSG-1. The
Genbank reference sequence for CXCL8 is Y00787.1 as available on 13
Jun. 2011, which is incorporated herein by reference in its
entirety.
[0061] CCL11 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 11, also
known as eotaxin. The HGNC ID for this gene is 10610. The gene is
located at chromosome position 17q21.1-q21.2. The previous symbol
and name for the gene is SCYA11, "small inducible cytokine
subfamily A (Cys-Cys), member 11 (eotaxin)". Synonyms for this gene
include MGC22554 and "eotaxin-1". The Genbank reference sequence
for CCL11 is AB063614.1 as available on 13 Jun. 2011, which is
incorporated herein by reference in its entirety.
[0062] CCL15 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 15, also
known as HCC-2 and Lkn-1. The HGNC ID for this gene is 10613. The
gene is located at chromosome position 17.sub.811.2. The previous
symbol and name for the gene is SCYA15, "small inducible cytokine
subfamily A (Cys-Cys), member 15". Synonyms for this gene include
"CC chemokine 3", "chemokine CC-2", HCC-2, HMRP-2B, "leukotactin
1", Lkn-1, "macrophage inflammatory protein 5", "MIP-1 delta",
MIP-1d, MIP-5, NCC-3, SCYL3. The Genbank reference sequence for
CCL15 is AF031587.1 as available on 13 Jun. 2011, which is
incorporated herein by reference in its entirety.
[0063] CCL14 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 14. The
HGNC ID for this gene is 10612. The gene is located at chromosome
position 17811.2. The previous symbol and name for the gene is
SCYA14, "small inducible cytokine subfamily A (Cys-Cys), member
14". Synonyms for this gene include CKb1, HCC-1, HCC-3, MCIF,
NCC-2, SCYL2. The Genbank reference sequence for CCL14 is Z49270.1
as available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0064] CCL16 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 16. The
HGNC ID for this gene is 10614. The gene is located at chromosome
position 17.sub.811.2. The previous symbol and name for the gene is
SCYA16, "small inducible cytokine subfamily A (Cys-Cys), member
16". Synonyms for this gene include CKb12, HCC-4, LCC-1, LEC, LMC,
Mtn-1, NCC-4, SCYL4. The Genbank reference sequence for CCL16 is
AB007454.1 as available on 13 Jun. 2011, which is incorporated
herein by reference in its entirety.
[0065] CCL18 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 18
(pulmonary and activation-regulated). The HGNC ID for this gene is
10616. The gene is located at chromosome position 17q11.2. The
previous symbol and name for the gene is SCYA18, "small inducible
cytokine subfamily A (Cys-Cys), member 18, pulmonary and
activation-regulated". Synonyms for this gene include AMAC-1, CKb7,
DC-CK1, DCCK1, MIP-4, PARC. The Genbank reference sequence for
CCL18 is Y13710.1 as available on 13 Jun. 2011, which is
incorporated herein by reference in its entirety.
[0066] CCL23 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 23. The
HGNC ID for this gene is 10622. The gene is located at chromosome
position 17q11.2. The previous symbol and name for the gene is
SCYA23, "small inducible cytokine subfamily A (Cys-Cys), member
23". Synonyms for this gene include Ckb-8, CKb8, MIP-3, MPIF-1. The
Genbank reference sequence for CCL23 is U58913.1 as available on 13
Jun. 2011, which is incorporated herein by reference in its
entirety.
[0067] CCL24 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 24, also
known as eotaxin-2 and MPIF-2. The HGNC ID for this gene is 10623.
The gene is located at chromosome position 7q11.23. The previous
symbol and name for the gene is SCYA24, "small inducible cytokine
subfamily A (Cys-Cys), member 24". Synonyms for this gene include
"CK-beta-6", Ckb-6, MPIF-2, MPIF2, "eotaxin-2", "myeloid progenitor
inhibitory factor 2". The Genbank reference sequence for CCL24 is
U85768.1 as available on 13 Jun. 2011, which is incorporated herein
by reference in its entirety.
[0068] CCL26 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 26, also
known as eotaxin-3. The HGNC ID for this gene is 10625. The gene is
located at chromosome position 7q11.22. The previous symbol and
name for the gene is SCYA26, "small inducible cytokine subfamily A
(Cys-Cys), member 26". Synonyms for this gene include "CC chemokine
IMAC", IMAC, MIP-4a, MIP-4alpha, TSC-1, "chemokine N1,"
"eotaxin-3", "macrophage inflammatory protein 4-alpha", "small
inducible cytokine A26", "thymic stroma chennokine-1". The Genbank
reference sequence for CCL26 is AF124601.1 as available on 13 Jun.
2011, which is incorporated herein by reference in its
entirety.
[0069] CXCL1 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) ligand 1
(melanoma growth stimulating activity, alpha). The HGNC ID for this
gene is 4602. The gene is located at chromosome position 4q13.3.
The previous symbol and name for the gene is "fibroblast secretory
protein", FSP, GRO1, "GRO1 oncogene (melanoma growth stimulating
activity, alpha)", MGSA. Synonyms for this gene include GROa,
MGSA-a, NAP-3, SCYB1. The Genbank reference sequence for CXCL1 is
J03561.1 as available on 13 Jun. 2011, which is incorporated herein
by reference in its entirety.
[0070] CXCL2 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) ligand 2. The
HGNC ID for this gene is 4603. The gene is located at chromosome
position 4q13.3. The previous symbol and name for the gene is GRO2,
"GRO2 oncogene". Synonyms for this gene include CINC-2a, GROb,
MGSA-b, MIP-2a, SCYB2. The Genbank reference sequence for CXCL2 is
M36820.1 as available on 13 Jun. 2011, which is incorporated herein
by reference in its entirety.
[0071] CXCL3 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) ligand 3. The
HGNC ID for this gene is 4604. The gene is located at chromosome
position 4q21. The previous symbol and name for the gene is GRO3,
"GRO3 oncogene". Synonyms for this gene include CINC-2b, GROg,
MIP-2b, SCYB3. The Genbank reference sequence for CXCL3 is M36821.1
as available on 13 Jun. 2011, which is incorporated herein by
reference in its entirety.
[0072] CXCL5 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) ligand 5. The
HGNC ID for this gene is 10642. The gene is located at chromosome
position 4q13.3. The previous symbol and name for the gene is
SCYB5, "small inducible cytokine subfamily B (Cys-X-Cys), member 5
(epithelial-derived neutrophil-activating peptide 78)". A synonym
for this gene is ENA-78. The Genbank reference sequence for CXCL5
is X78686.1 as available on 13 Jun. 2011, which is incorporated
herein by reference in its entirety.
[0073] CXCL6 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) ligand 6. The
HGNC ID for this gene is 10643. The gene is located at chromosome
position 4q13.3. The previous symbol and name for the gene is
chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein
2). Synonyms for this gene include CKA-3, GCP-2. The Genbank
reference sequence for CXCL6 is U83303.1 as available on 13 Jun.
2011, which is incorporated herein by reference in its
entirety.
[0074] PPBP is the gene symbol approved by the HUGO Gene
Nomenclature Committee for pro-platelet basic protein (chemokine
(C-X-C motif) ligand 7), also known as CXCL7. The HGNC ID for this
gene is 9240. The gene is located at chromosome position 412-q13.
The previous symbol and name for the gene is THBGB1. Synonyms for
this gene include b-TG1, Beta-TG, "beta-thromboglobulin",
"connective tissue-activating peptide III", CTAP3, CTAPIII, CXCL7,
LA-PF4, LDGF, MDGF, NAP-2, NAP-2-L1, "neutrophil-activating
peptide-2", PBP, "platelet basic protein", SCYB7, TGB, TGB1. The
Genbank reference sequence for PPBP is M54995.1 as available on 13
Jun. 2011, which is incorporated herein by reference in its
entirety.
[0075] CXCL11 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-X-C motif) ligand 11. The
HGNC ID for this gene is 10638. The gene is located at chromosome
position 4q21. The previous symbol and name for the gene is SCYB9B,
SCYB11, "small inducible cytokine subfamily B (Cys-X-Cys), member
11". Synonyms for this gene include b-R1, H174, I-TAC, IP-9. The
Genbank reference sequence for CXCL11 is U66096.1 as available on
13 Jun. 2011, which is incorporated herein by reference in its
entirety.
[0076] CCL19 is the gene symbol approved by the HUGO Gene
Nomenclature Committee for chemokine (C-C motif) ligand 19, also
known as MIP-3b. The HGNC ID for this gene is 10617. The gene is
located at chromosome position 9p13. The previous symbol and name
for the gene is SCYA19, "small inducible cytokine subfamily A
(Cys-Cys), member 19". Synonyms for this gene include "beta
chemokine exodus-3", "CC chemokine ligand 19", "CK beta-11", CKb11,
"EB11-ligand chemokine", ELC, exodus-3, "macrophage inflammatory
protein 3-beta", MIP-3b. The Genbank reference sequence for CCL19
is AB000887.1 as available on 13 Jun. 2011, which is incorporated
herein by reference in its entirety.
[0077] Examples of suitable modified chemokines of the various
embodiments of the invention containing modifications and/or
truncations and specifically adapted for use in the invention are
described in detail herein. MCP-1 has been produced with residue
75, which may be a lysine, as the site of biotinylation on the
chemokine (numbering based upon the mature protein having the amino
acid sequence of SEQ ID NO: 2). Biotinylation permits
immobilization of MCP-1 on a solid support (via a biotin-avidin
interaction). The basic amino acid sequence of MCP-1, including a
23 amino acid leader sequence is set forth as SEQ ID NO: 1. The
amino acid sequence of the mature protein is set forth as SEQ ID
NO: 2. The inventors have determined that chemokines may display
improved binding properties where the chemokine is biotinylated via
a spacer group. The spacer may prevent the biotin group from
impacting on the binding affinity of the chemokine. Any suitable
spacer group may be employed. Further modifications may provide the
molecule with advantageous properties. The invention also relates
to derivatives of truncated MCP-1 chemokines. The amino acid
sequence of the truncated version is set forth as SED ID NO: 3.
[0078] Accordingly, in certain embodiments the invention also
provides a modified MCP-1 chemokine comprising, consisting
essentially of or consisting of the amino acid sequence set forth
as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in which one or more
of the following modifications have been made:
[0079] a) the glutamine residue 1 of SEQ ID NO: 2 has been replaced
with pyroglutamine
[0080] b) the C terminus is produced as an amide derivative (this
may be achieved by synthesis on an amide linker)
[0081] c) the (C terminal region) residue at position 98 of SEQ ID
NO: 1 or position 75 of SEQ ID NO:2 or position 67 of SEQ ID NO: 3,
which may be a lysine or ornithine residue or other residue
suitable for labelling, is biotinylated, optionally via a spacer
group, in order to permit immobilization of the chemokine on a
solid support; and/or
[0082] d) the methionine residue at position 87 of SEQ ID NO: 1 or
position 64 of SEQ ID NO: 2 or position 56 of SEQ ID NO: 3 has been
replaced with norleucine.
[0083] The (C terminal region) amino acid, which may be a lysine
residue or a functional equivalent, at position 98 of SEQ ID NO: 1
or position 75 of SEQ ID NO:2 or position 67 of SEQ ID NO: 3 may be
biotinylated via a suitable spacer group, such as a polyethylene
glycol (PEG) spacer group, in order to permit immobilization of the
chemokine on a solid support. In specific embodiments, the PEG
spacer is 3,6-dioxo aminooctanoic acid. The sequence and
biotinylation of the modified MCP-1 chemokines of the invention are
shown in FIGS. 7 to 9 respectively. The modified MCP-1 chemokines
may be agonists or antagonists of CCR2 activity. They can be tested
for activity in a suitable assay, such as cell-based assays. In
particular, agonist and antagonist properties may be determined in
functional cell-based assay on human CCR2 receptor.
[0084] MCP-5 only binds CCR2 and should be selective in its removal
of CCR2 expressing cells. The full length amino acid sequence,
including the signal peptide, is set forth as SEQ ID NO: 4. The
amino acid sequence of N-terminal processed MCP-5 chemokine is 82
amino acids long and is set forth as SEQ ID NO: 5. An amino acid
sequence alignment suggests that MCP-5 has a C-terminal extension
when compared to the amino acid sequence of MCP-1. The results of
this alignment are shown in FIG. 10. C-terminal truncated versions
of MCP-5 can thus be synthesised. This truncated version will
comprise, consist essentially of or consist of MCP-5 residues 1-76,
set forth as SEQ ID NO: 6.
[0085] Accordingly, in certain embodiments the invention also
provides a modified MCP-5 chemokine comprising the amino acid
sequence set forth as SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in
which the isoleucine residue at position 97 of SEQ ID NO: 4 or at
position 75 of SEQ ID NO: 5 or SEQ ID NO: 6 has been replaced with
lysine. In certain embodiments, the modified MCP-5 chemokine
comprises, consists essentially of or consists of the amino acid
sequence of SEQ ID NO: 7. The modified MCP-5 chemokine may be
biotinylated at the lysine (or a functional equivalent) residue at
position 97 of SEQ ID NO: 4 or at position 75 of SEQ ID NO: 5 or
SEQ ID NO: 6. Biotinylation may be via a suitable spacer group.
Specific examples of the spacer group include a PEG spacer,
optionally 3,6-dioxo aminooctanoic acid. In some embodiments, the C
terminus is produced as an amide derivative. This may be achieved
by synthesis on an amide linker. In certain embodiments, the
modified MCP-5 chemokine comprises, consists essentially of or
consists of the sequence and biotinylation shown in FIG. 11. The
modified MCP-5 chemokine may be an agonist or an antagonist of CCR2
activity. They can be tested for activity in a suitable assay, such
as cell-based assays. In particular, agonist and antagonist
properties may be determined in a functional cell-based assay on
human CCR2 receptor.
[0086] Eotaxin has been produced with Lys73 as the site of
biotinylation on the chemokine (numbering based upon the mature
protein having the amino acid sequence of SEQ ID NO: 9).
Biotinylation permits immobilization of eotaxin on a solid support
(via a biotin-avidin interaction). The basic amino acid sequence of
eotaxin, including a 23 amino acid leader sequence is set forth as
SEQ ID NO: 8. The amino acid sequence of the mature protein is set
forth as SEQ ID NO: 9. The inventors have determined that
chemokines may display improved binding properties where the
chemokine is biotinylated via a spacer group. The spacer may
prevent the biotin group from impacting on the binding affinity of
the chemokine. Any suitable spacer group may be employed. Further
modifications may provide the molecule with advantageous
properties.
[0087] Accordingly, in certain embodiments the invention also
provides a modified eoxtaxin chemokine comprising the amino acid
sequence set forth as SEQ ID NO: 8 or SEQ ID NO: 9 in which one or
more of the following modifications have been made:
[0088] a) the C terminus is produced as an amide derivative
[0089] c) the (C terminal region) lysine residue at position 96 of
SEQ ID NO: 8 or position 73 of SEQ ID NO: 9 is biotinylated,
optionally via a spacer group, in order to permit immobilization of
the chemokine on a solid support
[0090] d) the methionine residue at position 85 of SEQ ID NO: 8 or
position 62 of SEQ ID NO: 9 has been replaced with norleucine
[0091] e) the Lysine at position 96 of SEQ ID NO: 8 or position 73
of SEQ ID NO: 9 s replaced with another amino acid that can enable
biotinylation such as ornithine
[0092] The (C terminal region) lysine residue at position 96 of SEQ
ID NO: 8 or position 73 of SEQ ID NO: 9 may be biotinylated via a
suitable spacer group, such as a polyethylene glycol (PEG) spacer
group, in order to permit immobilization of the chemokine on a
solid support. In specific embodiments, the PEG spacer is 3,6-dioxo
aminooctanoic acid. The sequence and biotinylation of a modified
eotaxin chemokine of the invention is shown in FIG. 7. The modified
eoxtaxin chemokines may be agonists or antagonists of CCR3
activity. They can be tested for activity in a suitable assay, such
as cell-based assays. In particular, agonist and antagonist
properties may be determined in aequorin functional cell-based
assay on human CCR3 receptor.
[0093] An example of a chemokine of the various embodiments of the
invention containing modifications and specifically adapted for use
in the invention is described in detail herein (see Example 3
below). The modified CCL8 (MCP-2) corresponds to residues 1 to 76
of the full length mature protein (and lacks the N-terminal signal
peptide of 23 amino acids, which is cleaved off) and thus retains
the chemokine fold. The Gln at the N-terminus of the protein is
subject to pyroGlu formation under physiological conditions. Thus
Gln1 of the sequence may thus be substituted with pyroglutamine to
prevent mixed species of N-terminal Gln and pyroGlu being generated
(SEQ ID NO: 13). This improves the yield of synthesis and ensures a
homogeneous chemokine preparation through column manufacture and
use. FmocLys(ivDde)-OH is incorporated as residue 75 to facilitate
site-specific labelling at this position of the protein (SEQ ID NO:
14). The naturally occurring lysine at position 75 is modified
through biotinylation. A PEG spacer may be incorporated between the
&amino functionality and the biotin (SEQ ID NO: 15).
[0094] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of SEQ ID NO: 13:
TABLE-US-00001 XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKE
VCADPKERWVRDSMKHLDQIFQNLXP
[0095] X1=pyroGlu (but may remain as Gln in some embodiments)
[0096] X75=an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG, e.g.
K(PEG-Biotin).
[0097] Or
TABLE-US-00002 SEQ ID NO: 15
XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKE
VCADPKERWVRDSMKHLDQIFQNLXP
[0098] X1=pyroGlu (but may remain as Gln in some embodiments)
[0099] X75=K(PEG-Biotin).
[0100] A further example of a chemokine of the various embodiments
of the invention containing modifications and specifically adapted
for use in the invention is described in detail herein (see Example
4 below). The modified CCL5 (RANTES) corresponds to residues 1 to
68 of the full length mature protein (and lacks the N-terminal
signal peptide of 23 amino acids, which is cleaved off) and thus
retains the chemokine fold. The single methionine (Met67) within
the sequence is mutated to lysine, to mitigate against oxidation of
this residue during the chain assembly (SEQ ID NO: 16). This Met to
Lys substitution provides a lysine at position 67 which can be
modified through biotinylation. FmocLys(ivDde)-OH is incorporated
as residue 67 to facilitate site-specific labelling at this
position of the protein (SEQ ID NO: 17). The biotinylated version
comprises, consists essentially of or consists of the amino acid
sequence of SEQ ID NO: 18.
[0101] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of SEQ ID NO: 18:
TABLE-US-00003 SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC
ANPEKKWVREYINSLEXS
[0102] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG (e.g.
K(Biotin))
[0103] A further example of a chemokine of the various embodiments
of the invention containing modifications and specifically adapted
for use in the invention is described in detail herein (see Example
below). The modified CCL2 (MCP-1) corresponds to residues 1 to 76
of the full length mature protein (and lacks the N-terminal signal
peptide of 23 amino acids, which is cleaved off) and thus retains
the chemokine fold (SEQ ID NO: 10). The Gln at the N-terminus of
the protein (Gln1) is substituted with pyroglutamine to prevent
mixed species of N-terminal Gln and pyroGlu being generated. This
improves the yield of synthesis and ensures a homogeneous chemokine
preparation through column manufacture and use. FmocLys(ivDde)-OH
is incorporated as residue 75 to facilitate site-specific labelling
at this position of the protein (SEQ ID NO: 11). A suitable spacer,
such as a PEG spacer, may be incorporated between the &amino
functionality and the biotin. The biotinylated version comprises,
consists essentially of or consists of the amino acid sequence of
SEQ ID NO: 12.
[0104] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of:
TABLE-US-00004 SEQ ID NO: 10:
XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKE
ICADPKQKWVQDSMDHLDKQTQTPKT
[0105] X=pyroGlu or Gln
[0106] And/or
TABLE-US-00005 SEQ ID NO: 12:
XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKE
ICADPKQKWVQDSMDHLDKQTQTPXT
[0107] X1=pyroGlu or Gln
[0108] X75 is an amino acid residue that can be biotinylated, such
as lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG,
optionally K(PEG-Biotin)
[0109] A further example of a chemokine of the various embodiments
of the invention containing modifications and specifically adapted
for use in the invention is described in detail herein (see Example
9 below). The modified CCL19 (MIP-3.beta.) corresponds to residues
1 to 77 of the full length mature protein (and lacks the N-terminal
signal peptide of 21 amino acids, which is cleaved off) and thus
retains the chemokine fold. An additional lysine is inserted at the
C-terminus, at position 78. The chemokine may thus comprise,
consist essentially of or consist of the amino acid sequence of SEQ
ID NO: 19. FmocLys(ivDde)-OH is incorporated as residue 78 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 20). The .epsilon.-amino side chain functionality of
Lys(78) is modified through biotinylation. The final protein may
thus comprise, consist essentially of or consist of the amino acid
sequence of SEQ ID NO: 21.
[0110] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of SEQ ID NO: 19 or 21:
TABLE-US-00006 SEQ ID NO: 19
GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLC
APPDQPWVERIIQRLQRTSAKMKRRSSX
[0111] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated (e.g. K-biotin), optionally via a spacer molecule such
as PEG, in particular K(PEG-Biotin)
TABLE-US-00007 SEQ ID NO: 21
GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLC
APPDQPWVERIIQRLQRTSAKMKRRSSX
[0112] X is K(Biotin)
[0113] A further example of a chemokine of the various embodiments
of the invention containing modifications and specifically adapted
for use in the invention is described in detail herein (see Example
3 below). The modified CXCL8 (IL-8) corresponds to residues 1 to 77
of the full length mature protein (and lacks the N-terminal signal
peptide of 22 amino acids, which is cleaved off) and thus retains
the chemokine fold. An amino acid residue capable of biotinylation,
such as lysine or ornithine, is added as residue 78 (SEQ ID NO:
22). FmocLys(ivDde)-OH may be incorporated as residue 78 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 23). The additional amino acid, in particular lysine or
ornithine, at position 78 is modified through biotinylation. A
suitable spacer, such as a PEG spacer, may be incorporated between
the &amino functionality and the biotin (SEQ ID NO: 24).
[0114] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of SEQ ID NO: 22 or 24:
TABLE-US-00008 SEQ ID NO: 22
AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL
SDGRELCLDPKENWVQRVVEKFLKRAENSX
[0115] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG
TABLE-US-00009 SEQ ID NO: 24
AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL
SDGRELCLDPKENWVQRVVEKFLKRAENSK(PEG-Biotin)
[0116] A further example of a chemokine of the various embodiments
of the invention containing truncations and modifications and
specifically adapted for use in the invention is described in
detail herein (see Example 3 below). The modified CXCL8 (IL-8)
corresponds to residues 6 to 77 of the full length mature protein,
with the first 5 N-terminal amino acids removed, (and lacks the
N-terminal signal peptide of 22 amino acids, which is cleaved off)
and thus retains the chemokine fold. An amino acid residue capable
of biotinylation, such as lysine or ornithine, is added as residue
78 (SEQ ID NO: 25). FmocLys(ivDde)-OH may be incorporated as
residue 78 to facilitate site-specific labelling at this position
of the protein (SEQ ID NO: 26). The additional amino acid, in
particular lysine or ornithine, at position 78 is modified through
biotinylation. A suitable spacer, such as a PEG spacer, may be
incorporated between the &amino functionality and the biotin
(SEQ ID NO: 27).
[0117] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of SEQ ID NO: 25 or 27:
TABLE-US-00010 SEQ ID NO: 25
SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGREL
CLDPKENWVQRVVEKFLKRAENSX
[0118] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG
TABLE-US-00011 SEQ ID NO: 27
SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGREL
CLDPKENWVQRVVEKFLKRAENSX
[0119] X is K(PEG-Biotin)
[0120] A further example of a chemokine of the various embodiments
of the invention containing modifications and specifically adapted
for use in the invention is described in detail herein (see Example
9 below). The modified CCL11 (Eotaxin) corresponds to residues 1 to
74 of the full length mature protein (and lacks the N-terminal
signal peptide of 23 amino acids, which is cleaved off) and thus
retains the chemokine fold (SEQ ID NO: 28). The lysine at position
73 may be modified through biotinylation. FmocLys(ivDde)-OH is
incorporated as residue 73 to facilitate site-specific labelling at
this position of the protein (SEQ ID NO: 29). A suitable spacer,
such as a PEG spacer, may be incorporated between the &amino
functionality and the biotin. The biotinylated version comprises,
consists essentially of or consists of the amino acid sequence of
SEQ ID NO: 30.
[0121] Thus, in other embodiments the invention also relates to a
modified chemokine comprising, consisting essentially of or
consisting of the amino acid sequence of SEQ ID NO: 28 or SEQ ID
NO: 30:
TABLE-US-00012 SEQ ID NO: 28
GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDIC
ADPKKKWVQDSMKYLDQKSPTPXP
[0122] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG, e.g.
K(PEG-Biotin).
TABLE-US-00013 SEQ ID NO: 30
H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD
ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
[0123] X is K(PEG-Biotin)
[0124] Chemokines useful in the various embodiments of the
invention may be synthesised through any suitable means known in
the art. Preferably, the chemokines are chemically synthesised as
this facilitates modification and labelling etc. However,
recombinant DNA based approaches may also be employed in
combination with appropriate labelling and modification
technologies as required. Thus, in certain embodiments the
invention also provides a nucleic acid molecule encoding the
chemokines of the various embodiments of the invention. In certain
embodiments the invention also relates to a vector containing such
a nucleic acid molecule and a host cell containing the vector. The
vector may additionally comprise a suitable promoter operably
linked to the nucleic acid molecule, to facilitate transcription of
the corresponding mRNA molecule. The host cell may be capable of
expressing the protein by transcription and translation of the
nucleic acid molecule encoding a chemokine of the various
embodiments of the invention.
[0125] The chemokines useful in the various embodiments of the
invention can be biotinylated by methods known in the art such as
described in WO 00/50088 A2, which is incorporated herein by
reference in its entirety. As indicated above, site-specific
labelling of the chemokines of the various embodiments of the
invention is preferable, although any labelling technique which
does not significantly affect the receptor-binding capacity of the
chemokine may be employed. Various site-specifically biotinylated
chemokines and native chemokines are available commercially, for
instance from Almac, Craigavon, UK. In specific embodiments the one
or more chemokines are biotinylated via a spacer group. The spacer
may be employed to prevent the biotin group from impacting on the
activity of the chemokine, in particular binding of the chemokine
to its cognate receptor. Any suitable spacer that facilitates
retention of receptor binding properties of the chemokine may be
employed in the various embodiments of the invention. Thus, in the
specific embodiments described above, spacers other than PEG
spacers may be employed as appropriate. In specific embodiments,
the spacer is a polyethylene glycol (PEG) spacer. PEG has been
shown to be an effective spacer permitting attachment of biotin to
the chemokine (which can then be immobilized on the solid support
through interaction with streptavidin) without compromising
receptor binding capability.
[0126] In the context of the various embodiments of the present
invention the term "antibody" includes all immunoglobulins or
immunoglobulin-like molecules with specific binding affinity for
the relevant chemokine receptor (including by way of example and
without limitation, IgA, IgD, IgE, IgG and IgM, combinations
thereof, and similar molecules produced during an immune response
in any vertebrate, for example, in mammals such as humans, goats,
rabbits and mice). Specific immunoglobulins useful in the various
embodiments of the invention include IgG isotypes. The antibodies
useful in the various embodiments of the invention may be
monoclonal or polyclonal in origin, but are typically monoclonal
antibodies. Antibodies may be human antibodies, non-human
antibodies, or humanized versions of non-human antibodies, or
chimeric antibodies. Various techniques for antibody humanization
are well established and any suitable technique may be employed.
The term "antibody" also refers to a polypeptide ligand comprising
at least a light chain or heavy chain immunoglobulin variable
region which specifically recognizes and binds an epitope of an
antigen, and it extends to all antibody derivatives and fragments
that retain the ability to specifically bind to the relevant
chemokine receptor. These derivative and fragments may include Fab
fragments, F(ab').sub.2 fragments, Fv fragments, single chain
antibodies, single domain antibodies, Fc fragments etc. The term
antibody encompasses antibodies comprised of both heavy and light
chains, but also heavy chain (only) antibodies. In specific
embodiments, the antibodies may be engineered so as to be specific
for more than one chemokine receptor, for example bi-specific to
permit binding to two different chemokine receptors. Suitable
commercially available antibodies which bind to the chemokine
receptors of interest are listed in table 1 below. They may or may
not be labelled. General reference may be made to "Antibodies a
laboratory manual: By E Harlow and D Lane. pp 726. Cold Spring
Harbor Laboratory. 1988", which reference is incorporated herein in
its entirety.
TABLE-US-00014 TABLE 1 Commercially available fluorophore labelled
antibodies against specific chemokine receptors Antibody
Fluorophore Supplier CCR2 PerCP Cy5.5 Biolegend CCR1 Alexa Fluor
647 Biolegend CCR3 PE Biolegend CCR5 PE Biolegend CXCR1 APC
Biolegend CXCR2 PE Biolegend CCR7 PerCP Cy5.5 Biolegend
[0127] Anti-CCR2 antibodies are described for example in WO
2010/021697, incorporated herein by reference. Further examples of
potentially useful antibodies include MLN-1202, an anti-CCR2
monoclonal antibody currently undergoing clinical trials
(Millennium Pharmaceuticals).
[0128] The chemokine receptor expressing cells may thus be targeted
using alternative binding agents, such as antibodies or other
chemical compounds, as defined herein, rather than the natural
chemokine binding partner. This approach is a new approach to
treating inflammatory conditions.
[0129] Accordingly, in certain embodiments the invention also
provides an apheresis column loaded with a solid support comprising
a binding reagent capable of specifically binding to a chemokine
receptor immobilized directly or indirectly on the support to
permit removal of a cell expressing the chemokine receptor from the
peripheral blood of a patient, wherein the binding reagent is not a
chemokine. The binding reagent capable of specifically binding to
the chemokine receptor may be an agonist or an antagonist of the
chemokine receptor. In specific embodiments, the binding reagent
capable of specifically binding to the chemokine receptor is
selected from an antibody and a chemical compound.
[0130] In other embodiments the invention thus also provides a
method for treating an inflammatory condition comprising applying
peripheral blood from a patient/subject to an apheresis column as
defined above (an apheresis column loaded with a solid support
comprising a binding reagent capable of specifically binding to a
chemokine receptor immobilized directly or indirectly on the
support to permit removal of a cell expressing the chemokine
receptor from the peripheral blood of a patient, wherein the
binding reagent is not a chemokine) thus removing chemokine
receptor expressing cells from the peripheral blood of the
patient/subject. The method may comprise returning the blood
depleted of the chemokine receptor expressing cells to the
patient/subject.
[0131] Similarly, in other embodiments the invention provides a
binding reagent capable of specifically binding to a chemokine
receptor for use in the treatment of an inflammatory condition,
wherein the binding reagent is immobilized on a solid support
contained within an apheresis column as defined above (an apheresis
column loaded with a solid support comprising a binding reagent
capable of specifically binding to a chemokine receptor immobilized
directly or indirectly on the support to permit removal of a cell
expressing the chemokine receptor from the peripheral blood of a
patient/subject, wherein the binding reagent is not a chemokine),
to which is applied peripheral blood from a patient thus removing
chemokine receptor expressing cells from the peripheral blood of
the patient.
[0132] These aspects of the various embodiments of the invention
may be integrated into the more focused therapeutic aspects of the
various embodiments of the invention (i.e. treating respiratory
conditions such as sarcoidosis and COPD and various aspects
thereof) and thus, the remainder of the disclosure, including all
specific embodiments applies mutatis mutandis.
[0133] Solid support materials for immobilizing the binding
reagents of the various embodiments of the invention are known in
the art. "Solid support" refers to, for example, materials having a
rigid or semi-rigid surface or surfaces, and may take the form of
beads, resins, gels, microspheres, or other geometric
configurations. A useful support material is one that does not
activate blood cells so as to make them coagulate or adhere to the
support as peripheral whole blood is applied to the device. In
certain embodiments, a support treated with an agent to provide it
with anti-coagulation properties, in particular a heparinized
support is employed. Alternatively, the blood of the patient may be
treated with an anti-coagulant such as heparin prior to application
to the support. Useful support materials comprise high molecular
weight carbohydrates, in particular carbohydrates having a
molecular weight of 100 kDa or more, such as agarose, in
particulate form, optionally cross-linked, and cellulose. Other
preferred support materials are polymers, such as carboxylated
polystyrene, and glass. The support of the various embodiments of
the invention may be provided in the form of particles or fibres.
The support particles may have regular form, such as spheres or
beads, or irregular form. They may be porous or non-porous. A
preferred average particle size of the support is from 50 .mu.m to
2 mm. In certain embodiments Sepharose.TM., a cross linked,
beaded-form of agarose, is used as column matrix. It is chosen for
its optimal distribution capacity and can provide a large available
area for affinity binding. Solid supports may be provided in the
form of magnetic beads, with the specific binding reagent
immobilized on the magnetic beads. Following capture of the (CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7) chemokine receptor
expressing cells from the blood, the beads can be removed from the
blood with the aid of an appropriate magnetic separator.
[0134] Methods for immobilizing binding reagents on a solid support
are known in the art. A binding reagent, such as a chemokine,
antibody, peptide, nucleic acid or chemical compound, can be
immobilized on the support in a direct or indirect manner.
Immobilization can be by means of a suitable linker in some
embodiments. A preferred method of indirect immobilization of a
binding reagent, such as a chemokine, relies upon the interaction
between biotin and avidin molecules. "Avidin" or "avidin molecule"
refers to any type of protein that specifically binds biotin to the
substantial exclusion of other (small) molecules that might be
present in a biological sample. Examples of avidin include avidins
that are naturally present in egg white, oilseed protein (e.g.,
soybean meal), and grain (e.g., corn/maize), and streptavidin,
which is a protein of bacterial origin Thus, biotinylation of the
binding reagent and use of an avidin molecule such as streptavidin
immobilized on the solid support allows reliable attachment of the
binding reagent to the solid support according to methods known in
the art. Specifically, such a method may comprise providing the
binding reagent in biotinylated form, providing a solid support
having streptavidin immobilized on its surface, contacting the
support with an aqueous solution of the biotinylated binding
reagent, and rinsing the support with an aqueous solvent. In
addition, binding pair interactions, such as antibody--antigen
interactions, may also be utilised for indirect immobilisation of
binding reagent onto a support. In such embodiments the support may
be derivatised with one member of a binding pair, such as an
antibody or fragment or derivative thereof, as defined herein,
which has known affinity for a particular peptide sequence or small
molecule hapten. Incorporating the other member of the binding
pair, such as an antigen, peptide sequence or the hapten onto or
into the binding reagent facilitates immobilisation onto a solid
support coated with the corresponding antibody or fragment or
derivative thereof. Thus, the binding reagent may be modified to
include the peptide sequence or hapten into the linear molecule or
may be added as a side chain or label. Any suitable
antibody-antigen pair may be employed. The antibody fragment or
derivative may be any fragment or derivative that retains specific
binding affinity for the appropriate antigen. Examples include Fab,
F(ab').sub.2 fragments, scFV, VH domains, single domain antibodies
(such as nanobodies), heavy chain antibodies and humanized version
of non-human antibodies etc. Other high affinity interactions can
be utilised for immobilisation of binding reagents, as long as the
binding reagent can be synthesised or derivatised with one of the
interacting partners and the solid support synthesised or
derivatised with the other interacting partner without loss of
binding activity (i.e. binding of the binding reagent to the
appropriate chemokine receptor). Immobilization may occur via
essentially the same interaction in reverse in some embodiments.
Thus, the binding reagent which may be a chemokine for example, may
be attached to an antibody as defined herein, and the solid support
derivatised with the antigen. The chemokine may be produced as a
fusion protein with the antibody.
[0135] Alternatively binding reagents, such as chemokines and
antibodies, can be immobilised directly onto a solid support using
bioconjugation techniques established in the field. For example
direct immobilisation of proteins onto cyanogen bromide activated
solid supports via amino functionalities within the primary
sequence of the protein. Alternatively, sulphydryl functionalities
within proteins can be used to directly immobilise the protein to
alkyl halide derivatised supports or supports containing free thiol
functionalities. In further embodiments, proteins containing
a-thioester functionalities can be directly immobilised on supports
containing 1,2 amino thiol moieties (eg N-terminal cysteine) using
the native chemical ligation reaction. Alternatively proteins
modified with ketones and aldehydes can be immobilised on solid
supports derivatised with hydrazinyl, hydrazide and aminoxy
functionalities using hydrazone/oxime bond forming ligation
reactions (and vice versa). Alternatively `Click` chemistry can be
used to immobilise proteins onto solid supports, whereby the
protein and the support are derivatised with the appropriate
mutually reactive chemical functionalities (azides and alkynes). In
other embodiments Staudinger ligation chemistry can be used to
immobilise appropriately derivatised proteins onto the
appropriately derivatised solid supports.
[0136] The solid support is contained within or carried by the
apheresis column. Thus, by "loaded" is meant that the column
carries or contains the solid support in a manner such that
(peripheral) blood can flow through the column in contact with the
solid support. Thus, the solid support provides a matrix within the
column through which blood flows, in continuous fashion in certain
embodiments. This permits cells expressing the specific chemokine
receptor to be removed from the blood passing through the column,
such that blood exiting the column is depleted of the specific
chemokine receptor-expressing cells. In specific embodiments, the
apheresis column is loaded with a support comprising streptavidin
immobilized on the support and one or more biotinylated binding
reagents, such as chemokines, bound to the streptavidin on the
support. The solid support may be comprised of a high-molecular
weight carbohydrate, optionally cross-linked, such as agarose.
[0137] As discussed above, the binding reagent is coupled to the
solid support. The relative amounts of binding reagent may be
controlled to ensure that coupling between the solid support and
the binding reagent will be immediate, minimising the risk of
binding reagent decoupling from the solid support. Thus, it may be
ensured that there is a relative excess of immobilization sites for
the binding reagent on the solid support. Alternatively, or
additionally, following immobilization of the binding reagent on
the solid support, the solid support may be washed to remove any
unbound binding reagent.
[0138] The apheresis column utilised in the various embodiments of
the present invention acts as a leukapheresis treatment for
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). The column acts to
specifically remove one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7-expressing monocytes or leukocytes by exploiting
the interaction between CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7 expressed on the cell surface and a specific binding reagent
immobilized on a solid support contained within or carried by the
column. The overall column typically comprises, consists of, or
consists essentially of three combined components; 1) a housing
which contains or carries 2) the solid support and 3) one or more
binding reagents (immobilized thereon) which specifically bind one
or more chemokine receptors. The housing may be manufactured from
any suitable material for clinical use. In certain embodiments the
housing is composed of a plastic material. The housing includes an
in flow site for entry of blood and an out flow site for blood
(depleted of target cells) to exit the column. The housing may be
designed to maintain a continuous blood flow through the solid
support matrix. The housing (as shown for example in FIG. 3) may
include a top portion which comprises a distribution plate (2) at
the inflow site (1) to spread the blood evenly over the entire
matrix area. The distribution plate may act as a first safety
barrier preventing larger particles flowing through the column and
into the patient. However, the distribution plate is not essential
and may be removed in some embodiments to decrease the overall
resistance in the system. The column may contain one or more safety
filter units (3 and 4) placed at the inflow (1) and/or outflow (5)
sites of the plastic housing. Such filter units may act to prevent
particles larger than blood cells passing in and/or out of the
column. The safety filter units may contain a plurality of filters,
such as two, three or four filters designed to be a robust barrier
and stop all particles larger than blood cells passing through the
column. Inclusion of safety filters (3 and 4) at both ends of the
column serves to minimize the risk of leakage of particles into the
patient, including in the event that the device is incorrectly
connected resulting in blood flow in the opposite direction to that
intended. The safety filters may comprise of any suitable pore size
to prevent particles larger than blood cells from passing through
the column, as would be readily understood by one skilled in the
art. Suitable filters are commercially available. In specific
embodiments, the pore size of the filter(s) is between
approximately 60 .mu.m and 100 .mu.m, more specifically
approximately 80 .mu.m. The solid support and binding reagent
components are discussed in further detail herein.
[0139] The volume of the housing may be varied depending upon the
blood volumes intended to pass through the column. Typically, the
volume of the housing is between approximately 40 ml and 200 ml,
more specifically 50 ml to 150 ml or 60 ml to 120 ml.
[0140] The column is generally applied in the form of an apheresis
circuit. In this context, the overall system includes the apheresis
column, tubing and an appropriate pump to pump the blood around the
circuit. The system is illustrated in FIG. 4. The patient (1) is
connected to the extracorporeal circuit via sterile needles to
veins in the right and the left arms. A saline bag (3) is also
connected and the saline solution is pumped with a suitable pump
(2). Blood is drawn from one arm of the patient through the sterile
tubing system by the blood pump (4) and passed through the column
(6) and back to the patient. The tubing system may be connected to
the column via any suitable coupling, such as standard dialysis
luer-lock couplings. The couplings on the column may be
colour-coded for correct assembly. For example, red tubing for
inflow to the red column top and blue tubing for outflow back to
the patient. An air detector (8) may be present in the circuit.
Inlet pressure (5) and/or Pven sensors (7) may additionally be
employed to monitor the pressure in the circuit.
[0141] An apheresis pump, such as the 4008 ADS pump manufactured by
Fresenius Medical Care or the Adamonitor pump, may monitor the
patient's inflow and outflow. The pump may also monitor the
pressure in the extracorporeal circulation. The pump may be able to
discriminate air by a bubble catcher and air detector. A clot
catcher filter may be positioned inside the bubble catcher. The
pump may also incorporate an optical detector to distinguish
between light, e.g. saline solution or air present in the tubing
system and dark e.g. blood present in the tubing system.
[0142] A schematic diagram of a suitable pump, showing the air
detector and optical filter is shown in FIG. 8. If the pump system
detects air bubbles and optical fluctuations or if extracorporeal
pressure values are out of the set range, then the pump may stop
immediately. Alternatively or additionally a visual/audible alarm
may be emitted.
[0143] The treatment methods of the various embodiments of the
invention may thus rely upon an extracorporeal circuit. The methods
may be considered as ex vivo or in vitro methods and be defined
solely with reference to steps performed outside of the patient. In
some embodiments, however, the method further comprises, prior to
application of the blood to the column, collecting peripheral blood
from the patient. In a further embodiment, the method further
comprises, following the application of the blood to the column,
infusing the blood depleted of (CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7) chemokine receptor expressing cells to the
patient. This is then a complete leukapheresis treatment method.
Thus, a leukaphereis method, for treating respiratory conditions,
in particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD), comprises collecting peripheral blood from the patient;
applying the peripheral blood to an apheresis column loaded with a
solid support comprising one or more binding reagents capable of
specifically binding to one or more chemokine receptors, in
particular the chemokine receptor CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7, immobilized directly or indirectly on the
support thus removing one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7 expressing cells from the peripheral blood of the
patient; and infusing the depleted blood (of chemokine receptor
expressing cells) to the patient.
[0144] The peripheral blood may be continuously collected from the
patient. Similarly, the depleted blood may be continuously infused
to the patient, through use of an appropriate circuit as described
herein. Thus, the support may be disposed in a column through which
the blood is made to flow. This may be achieved using a suitable
pump for example, as also described herein. Blood flow through the
column enables the binding reagent(s) immobilized on the solid
support to capture the cells expressing the chemokine receptor,
thus depleting them from the blood and preventing their
contribution to the inflammatory respiratory conditions, in
particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD).
[0145] The methods of the various embodiments of the invention and
binding reagents for use in the methods of the various embodiments
of the invention may require that the patient has been selected for
treatment on the basis of detecting an increase in the level of
chemokine receptor, in particular, one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells in a sample
obtained from the patient. Such companion diagnostic methods are
described in greater detail herein and are based, for example, on
the observation that CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7 expression may be elevated in patients with respiratory
conditions, in particular sarcoidosis and Chronic Obstructive
Pulmonary Disease (COPD). It is shown herein that subjects
suffering from respiratory conditions such as sarcoidosis exhibit
increased frequency of chemokine receptor expressing cells in the
peripheral blood. Subjects with sarcoidosis exhibit increased
frequency of CCR1 expressing cells such as CCR1 expressing
monocytes, compared to healthy controls. Similarly, it is shown
herein that the monocytes also express CCR2. It is also shown
herein that subjects suffering from respiratory conditions such as
sarcoidosis exhibit increased frequency of CCR7 expressing cells
such as CCR7 expressing lymphocytes, and also central memory T
cells, compared to healthy controls.
[0146] Thus, (in this context) in certain embodiments the invention
also provides a method of diagnosing, monitoring progression of, or
monitoring treatment of respiratory conditions, in particular
sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD)
comprising determining:
[0147] a) the levels of one or more of the chemokine receptor CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells
[0148] b) levels of expression of one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7; and/or
[0149] c) levels of cells with high expression of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 in a sample
obtained from a subject, wherein high levels of one or more ofCCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, high
levels of expression of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 or high levels of cells with high
expression of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 or increased levels of one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells compared to
control, increased levels of expression of one or more of CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 compared to a control or
increased levels of cells with high expression of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 compared to a
control indicate the presence or progression of respiratory
conditions, in particular sarcoidosis and Chronic Obstructive
Pulmonary Disease (COPD). Levels of chemokine receptor expression,
as opposed to cell numbers, may also be investigated as increased
levels of chemokine receptor expression per cell may also be
diagnostically relevant. In specific embodiments, the cells
relevant to diagnosis etc. of respiratory conditions such as
sarcoidosis comprise monocytes, in particular CCR1 and/or CCR2
expressing monocytes. In other embodiments cells relevant to
diagnosis etc. of respiratory conditions such as sarcoidosis
comprise lymphocytes, specifically T lymphocytes such as central
memory T cells, in particular CCR7 expressing T cells.
[0150] "Diagnosing" is defined herein to include screening for a
disease/condition or pre-indication of a disease/condition,
identifying a disease/condition or pre-indication of a
disease/condition and checking for recurrence of disease/condition
following treatment. The methods of the various embodiments of the
invention may also have prognostic value, and this is included
within the definition of the term "diagnosis". The prognostic value
of the methods of the various embodiments of the invention may be
used as a marker of potential susceptibility to respiratory
conditions, in particular sarcoidosis and Chronic Obstructive
Pulmonary Disease (COPD) by identifying levels of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression linked
to the respiratory condition. Thus patients at risk may be
identified before the disease has a chance to manifest itself in
terms of symptoms identifiable in the patient. In certain
embodiments, diagnosis may be made in conjunction with other
objective indicators of respiratory conditions, in particular
sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD): The
diagnosis of sarcoidosis may be made based upon clionical findings;
x-ray, biopsy, s-Ca, ACE activity. COPD diagnosis may also be made
by clinical findings; X-ray spirometry and blood gas analysis.
[0151] "Monitoring progression of includes performing the methods
to monitor the stage and/or the state and progression of the
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). Monitoring progression may
involve performing the diagnostic methods multiple times on the
same patient to determine whether the levels of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells
are increasing, decreasing or remaining stable over a certain time
period. This may be in the context of a treatment regime.
[0152] "Monitoring the success of a particular treatment" is
defined to include determining the levels of one or more of CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells before
and after a treatment. The treatment is generally one aimed at
treating respiratory conditions, in particular sarcoidosis and
Chronic Obstructive Pulmonary Disease (COPD) and may be a treatment
according to one of the methods of the various embodiments of the
invention as defined herein. Successful treatment may be determined
with reference to a decrease in one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells as a result of, or
following, the treatment. Thus, in such methods a level of one or
more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing
cells is determined prior to treatment. This level is recorded and
a further assessment made at a predetermined time following the
treatment. The comparison of levels of one or more of CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells permits the
success of the treatment to be monitored. In specific embodiments,
a single treatment is sufficient to cause a depletion of around
10%, 20%, 30%, 40%, 50%, 60% or 70%, or higher, up to 80%, 90%, 95%
or more, or any range of values between and including these
amounts, of one or more specific chemokine receptors, in particular
one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7,
expressing cells from the peripheral blood of the patient. In
specific embodiments, at least around 50% depletion is achieved in
a single treatment. Thus, successful treatment may be defined with
reference to depletion of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells. Treatment may lead to
depletion of between approximately 100 and 500 million of one or
more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing
cells, such as monocytes, in certain embodiments. Additional
factors may be included to determine successful treatment. For
example, a lack of increase in one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells following treatment
may indicate successful treatment in terms of preventing further
progression of the condition, optionally combined with an
improvement in other markers or staging of the respiratory
conditions, in particular sarcoidosis and Chronic Obstructive
Pulmonary Disease (COPD).
[0153] By binding to the column through the binding
reagent-chemokine receptor interaction, chemokine receptor
expressing cells are immobilized. These immobilized cells express
further unoccupied chemokine receptors, which may be of the same or
different type to those used for capture. These additional
chemokine receptors may permit circulating chemokines which
contribute to the inflammatory condition to be captured from the
peripheral blood. Thus, a reduction in circulating (specific)
chemokine levels may provide a measure of successful treatment. In
specific embodiments, the cells relevant to diagnosis etc. of
respiratory conditions such as sarcoidosis comprise monocytes, in
particular CCR1 and/or CCR2 expressing monocytes. In other
embodiments cells relevant to diagnosis etc. of respiratory
conditions such as sarcoidosis comprise lymphocytes, specifically T
lymphocytes such as central memory T cells, in particular CCR7
expressing T cells.
[0154] In specific embodiments, the respiratory conditions are
selected from sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD).
[0155] The sample in which one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cell levels, levels of
expression of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 and/or levels of cells with high expression of one or
more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined
as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) are determined may
comprise any suitable tissue sample or body fluid sample.
Generally, the test sample is obtained from a human subject.
Typically, the sample is a blood sample, in particular a peripheral
blood sample. The sample may comprise an adipose tissue biopsy in
certain embodiments. The methods may involve determining levels of
one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing monocytes, macrophages or lymphocytes in certain
embodiments. In specific embodiments, the cells relevant to
diagnosis etc. of respiratory conditions such as sarcoidosis
comprise monocytes, in particular CCR1 and/or CCR2 expressing
monocytes. In other embodiments cells relevant to diagnosis etc. of
respiratory conditions such as sarcoidosis comprise lymphocytes,
specifically T lymphocytes such as central memory T cells, in
particular CCR7 expressing T cells.
[0156] Levels of CCR2, CCR1, CCR3 or CCR5 expressing cells, levels
of expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
and/or levels of cells with high expression of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2.sup.hi,
CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi o
and/or CCR7.sup.hi) may be determined according to any suitable
method. For example, flow cytometry may be employed in order to
determine the number of cells expressing CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 in the sample, to determine levels of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression and/or
to identify levels of CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi,
CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi and/or CCR7.sup.hi cells.
Flow cytometric techniques are described herein and examples of
commercially available antibodies suitably labelled for use in flow
cytometry are set out in Table 1 for example. Alternatively, the
method may involve steps of collecting and fixing the cells in the
sample, followed by incubation with a suitable binding reagent that
binds specifically to the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 chemokine receptor expressing cells in the sample. Any
suitable binding reagent, as defined herein, may be employed. For
example, a CCR2, CCR1, CCR3, CCR5, CXCR1 or CXCR2 specific antibody
may be employed. A wash step may be adopted following an incubation
period to remove any unbound reagent. Suitable wash steps and
incubation conditions would be well known to one skilled in the
art. The binding reagent may be directly labeled in order to permit
antibody binding to be directly determined. Alternatively a
secondary binding reagent, such as an antibody, may be employed
which binds to the first binding reagent and carries a label.
Again, suitable incubation conditions and wash steps would be
apparent to one skilled in the art. The primary and secondary
binding reagents may form two halves of a binding pair. The binding
interaction should not prevent the primary binding reagent binding
to the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 receptor
expressing cells, unless a competition assay is being employed. The
two halves of a binding pair may comprise an antigen-antibody,
antibody-antibody, receptor-ligand, biotin-streptavidin pair etc.
in certain embodiments. Other techniques used to quantify chemokine
(CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7) receptor
expressing cell levels, to quantify levels of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expression and/or to quantify levels
of CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hi and/or CCR7.sup.hi cells include
PCR-based techniques such as QT-PCR and protein based methods such
as western blot. Quantitation may be achieved with reference to
fixed cell lines carrying known numbers of various receptor
expressing cells and/or known levels of receptor expression per
cell. Such fixed cell lines are available commercially (for example
ChemiScreen.TM. cell lines from Millipore). Methods analogous to
the treatment methods of the various embodiments of the invention
may also be employed, with binding of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells to the solid support
being determined following peripheral blood being passed through
the column.
[0157] The levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7 expressing cells, levels of expression of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 and/or levels of cells with high
expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
(defined as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hu or CCR7.sup.hi) may be determined
relative to a suitable control. When diagnosing a respiratory
condition, a threshold level of cells, level of expression of CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/or level of cells
with high expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7 (defined as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi,
CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) may be set
at or over which a positive diagnosis is made. This threshold may
be determined based upon measuring levels of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, levels of
expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
and/or levels of cells with high expression of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2.sup.hi,
CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi
or CCR7.sup.hi) in samples obtained from diseased patients and
comparing these levels with levels of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells, levels of expression of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/or levels of
cells with high expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 (defined as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi,
CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) in samples
obtained from healthy subjects.
[0158] In certain embodiments, a respiratory disorder is diagnosed
on the basis of levels of chemokine receptor expressing cells. A
positive diagnosis may be made in subjects based upon the presence
of greater than about 10%, greater than about 20%, greater than
about 30%, greater than about 40%, greater than about 50%, greater
than about 55%, greater than about 60%, greater than about 65%,
greater than about 70%, greater than about 75%, greater than about
80%, greater than about 85%, greater than about 90%, greater than
about 95%, or more chemokine receptor expressing cells in the
sample, as a percentage of total cells in the sample. In other
embodiments, a respiratory disorder is diagnosed on the basis of
the presence of a about a 1.2 fold or greater increase, such as
about a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or
greater fold increase in chemokine receptor expressing cells,
relative to healthy controls.
[0159] In specific embodiments, sarcoidosis is diagnosed on the
basis of levels of CCR1 or CCR7 expressing cells. A positive
diagnosis may be made in subjects based upon the presence of
greater than about 10%, greater than about 15% or greater than
about 20% CCR7 expressing central memory T cells in the sample, as
a percentage of total cells in the sample. A positive diagnosis may
be made in subjects based upon the presence of greater than about
40%, greater than about 45%, greater than about 50%, greater than
about 55%, greater than about 60%, greater than about 65%, greater
than about 70%, greater than about 75%, greater than about 80% or
greater than about 85% or more CCR7 expressing T cells in the
sample, as a percentage of total cells in the sample. A positive
diagnosis may be made in subjects based upon the presence of
greater than about 60%, greater than about 65%, greater than about
70%, greater than about 75%, greater than about 80%, greater than
about 85% or greater than about 90% CCR1 expressing monocytes in
the sample, as a percentage of total cells in the sample.
Sarcoidosis may also be diagnosed on the basis of the presence of a
about a 1.2 fold or greater increase, such as about a 1.5, 2, 2.5,
3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in
the specific chemokine receptor expressing cells, relative to
healthy controls.
[0160] In certain embodiments, progression of a respiratory
disorder, which may be in the context of a treatment regime, is
monitored on the basis of levels of chemokine receptor expressing
cells at different time points. Progression of a respiratory
disorder may be indicated in subjects based upon an increase of
greater than about 10%, such as an increase of greater than about
15%, greater than about 20%, greater than about 25%, greater than
about 30%, greater than about 35%, greater than about 40%, greater
than about 45%, greater than about 50%, greater than about 55%,
greater than about 60%, greater than about 65%, greater than about
70%, greater than about 75% or more chemokine receptor expressing
cells in the sample, as a percentage of total cells in the sample,
compared to a sample taken from the same subject at an earlier time
point. In other embodiments, progression of a respiratory disorder
is confirmed on the basis of the presence of a about a 1.2 fold or
greater increase, such as about a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,
10, 20, 50 or 100 or greater fold increase in chemokine receptor
expressing cells, relative to a sample taken from the same subject
at an earlier time point.
[0161] In specific embodiments, sarcoidosis is monitored on the
basis of levels of CCR1 or CCR7 expressing cells such as CCR7
expressing central memory T, CCR7 expressing T cells or CCR1
expressing monocytes. Progression of the disease, which may be in
the context of a treatment regime, may be indicated in subjects
based upon the presence of an increase of greater than about 10%,
such as greater than about 15%, greater than about 20%, greater
than about 25%, greater than about 30%, greater than about 35%,
greater than about 40%, greater than about 45%, greater than about
50%, greater than about 55%, greater than about 60%, greater than
about 65%, greater than about 70%, greater than about 75% or more
chemokine receptor expressing cells in the sample, as a percentage
of total cells in the sample, compared to a sample taken from the
same subject at an earlier time point.
[0162] Regression or successful treatment may be monitored based
upon similar decreases over various time points. For example,
regression or successful treatment may be indicated in subjects
based upon a decrease of about 10%, such as a decrease of about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75% or more chemokine receptor expressing cells in the sample, as a
percentage of total cells in the sample, compared to a sample taken
from the same subject at an earlier time point. In other
embodiments, regression of respiratory disorders is confirmed on
the basis of the presence of a about a 1.2 fold or greater
decrease, such as about a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20,
50 or 100 or greater fold decrease in chemokine receptor expressing
cells, relative to a sample taken from the same subject at an
earlier time point.
[0163] In specific embodiments, sarcoidosis is monitored on the
basis of levels of CCR1 or CCR7 expressing cells such as CCR7
expressing central memory T, CCR7 expressing T cells or CCR1
expressing monocytes. Regression or successful treatment of the
disease may be made in subjects based upon a decrase of about 50%,
such as such as a decrease of about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or
more CCR1 or CCR7 expressing cells in the sample, as a percentage
of total cells in the sample or by a decrease of about 10%, such as
a decrease of about 15%, about 20%, about 25%, about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about
65%, about 70%, about 75% or more chemokine receptor expressing
cells in the sample, as a percentage of total cells in the sample,
compared to a sample taken from the same subject at an earlier time
point.
[0164] Suitable software is freely available (such as the R project
for statistical computing) to perform the necessary statistical
analysis of the data obtained to calculate a useful threshold. The
threshold may be set to maximize sensitivity and/or specificity of
the test. Performance of the test in these respects may be measured
by plotting a receiver operating characteristics (ROC) curve
(sensitivity versus specificity). The area under the curve provides
an indication of the overall performance of the test. Thus, once
thresholds have been set for diagnosing the condition, a separate
control experiment does not necessarily have to be run each time a
sample is tested. Rather reference can simply be made to the
pre-existing thresholds to determine the diagnosis. However, in
certain embodiments, the sample is tested together with a control
sample taken from a healthy subject to provide a comparator based
upon essentially identical experimental conditions. The test sample
is generally tested in parallel with the control sample. The test
sample level of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells, levels of expression of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 and/or levels of cells with high
expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
(defined as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) can then be compared
with that of the control sample to make the diagnosis. A control
sample from a disease patient may also be tested in certain
embodiments. Reference to controls permits relative levels ("high",
"low" etc.) of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells in the test sample to be readily identified and
the significance thereof interpreted. Reference to controls also
permits relative levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 expression ("high", "low" etc.) within the cell
population to be determined and the significance thereof
interpreted. Such determination may, for example, indicate the
average levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expression per cell in the test sample.
[0165] Thus, in specific embodiments, high or higher levels of one
or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells or high or higher levels of one or more of CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression, for example
average CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression
per cell, or high or higher levels of one or more of CCR2.sup.hi,
CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi
and/or CCR7.sup.hi cells correlate with active disease or more
active disease. Similarly, lower or low levels of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells,
or low or lower levels of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expression, for example average CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression per cell, or
low or lower levels of one or more of CCR2.sup.hi, CCR1.sup.hi,
CCR3.sup.hi, CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi and/or
CCR7.sup.hi cells may correlate with a lack of active inflammation
or disease. This may be defined as "less active disease". It can
readily be envisaged that control samples may be assessed and
levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells, levels of expression of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 and/or levels of cells with high
expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
(defined as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) determined across the
range of severities of respiratory conditions. This may assist in
correlating the levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 expressing cells, levels of expression of CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/or levels of cells with
high expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
(defined as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) in the test sample with
the relative severity of the condition.
[0166] When monitoring progression of, or monitoring treatment of
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD), the control samples may be
taken from the subject at an earlier time point. They may, however,
be based upon known reference values as discussed above. Thus,
relative levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells, relative levels of CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7 expression including relative levels of average
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression per
cell or relative levels of CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi,
CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi and/or CCR7.sup.hi cells
may be with reference to samples taken from the same subject at a
different point in time. In certain embodiments, decreased levels
of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells, decreased relative levels of one or more of CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression including
decreased relative levels of average CCR2, CCR1, CCR3, CCR5, CXCR1,
CXCR2 and/or CCR7 expression per cell, or decreased relative levels
of one or more of CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi,
CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi and/or CCR7.sup.hi cells
correlate with successful treatment. The treatment may be any
suitable treatment, but in specific embodiments is a treatment
according to the various embodiments of the invention.
[0167] When monitoring progression of respiratory conditions, in
particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD), increased levels of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells increased relative levels
of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expression including increased relative levels of average CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression per cell or
increased relative levels of one or more of CCR2.sup.hi,
CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi
and/or CCR7.sup.hi cells may indicate the progression of condition
or disease. Thus, if levels of one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, levels of
expression of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 and/or levels of cells with high expression of one or
more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined
as CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi, CCR5.sup.hi,
CXCR1.sup.hi, CXCR2.sup.hi or CCR7.sup.hi) are increased in a
sample taken later than a sample from the same patient this may
indicate progression of the condition.
[0168] Since the levels of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells, levels of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression or
levels of one or more of CCR2.sup.hi, CCR1.sup.hi, CCR3.sup.hi,
CCR5.sup.hi, CXCR1.sup.hi, CXCR2.sup.hi and/or CCR7.sup.hi cells
are diagnostically relevant, determining such levels in a sample
obtained from a subject may influence treatment selection for that
subject. Accordingly, in certain embodiments the invention provides
a method of selecting a suitable treatment for respiratory
conditions, in particular sarcoidosis and Chronic Obstructive
Pulmonary Disease (COPD) comprising determining:
[0169] a) the levels of one or more of the chemokine receptor CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells
[0170] b) levels of expression of one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7; and/or
[0171] c) levels of cells with high expression of one or more of
CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
[0172] in a sample obtained from a subject, wherein high levels of
one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7
expressing cells, high levels of expression of one or more of CCR2,
CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 or high levels of cells
with high expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or
CCR7 or increased levels of one or more of CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells compared to control,
increased levels of expression of one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 compared to a control or increased
levels of cells with high expression of one or more of CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 compared to a control, result
in selection of a treatment as defined herein for treatment of the
respiratory conditions, in particular sarcoidosis and Chronic
Obstructive Pulmonary Disease (COPD). In certain embodiments, the
chemokine receptor expressing cells are high chemokine receptor
expressing cells, in particular, high CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7 expressing cells. In specific embodiments,
the cells comprise monocytes, in particular CCR1 and/or CCR2
expressing monocytes. In other embodiments the cells comprise
lymphocytes, specifically T lymphocytes such as central memory T
cells, in particular CCR7 expressing T cells.
[0173] In specific embodiments, a respiratory disorder is treated
on the basis of measuring levels of chemokine receptor expressing
cells. Thus, a treatment according to the various embodiments of
the invention may be applied based upon the presence of greater
than about 10%, greater than about 20%, greater than about 30%,
greater than about 40%, greater than about 50%, greater than about
55%, greater than about 60%, greater than about 65%, greater than
about 70%, greater than about 75%, greater than about 80%, greater
than about 85%, greater than about 90%, greater than about 95%, or
more chemokine receptor expressing cells in the sample, as a
percentage of total cells in the sample. In other embodiments, a
respiratory disorder is treated according to the various
embodiments of the invention on the basis of the presence of a
about a 1.5 fold or greater increase, such as about a 2, 2.5, 3,
3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in
chemokine receptor expressing cells, relative to healthy
controls.
[0174] In specific embodiments, sarcoidosis is treated on the basis
of levels of CCR1 or CCR7 expressing cells. Thus, a treatment
according to the various embodiments of the invention may be
applied based upon the presence of greater than about 10%, greater
than about 15% or greater than about 20% CCR7 expressing central
memory T cells in the sample, as a percentage of total cells in the
sample. Thus, a treatment according to the various embodiments of
the invention may be applied based upon the presence of greater
than about 40%, greater than about 45%, greater than about 50%,
greater than about 55%, greater than about 60%, greater than about
65%, greater than about 70%, greater than about 75%, greater than
about 80% or greater than about 85% or more CCR7 expressing T cells
in the sample, as a percentage of total cells in the sample. Thus,
a treatment according to the various embodiments of the invention
may be applied based upon the presence of greater than about 60%,
greater than about 65%, greater than about 70%, greater than about
75%, greater than about 80%, greater than about 85% or greater than
about 90% CCR1 expressing monocytes in the sample, as a percentage
of total cells in the sample. Sarcoidosis may also be treated on
the basis of the presence of a about a 1.2 fold or greater
increase, such as about a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20,
50 or 100 or greater fold increase in the specific chemokine
receptor expressing cells, relative to healthy controls.
[0175] For the avoidance of doubt, all embodiments described in
respect of the methods of the various embodiments of the invention
apply to these aspects mutatis mutandis and are not repeated for
reasons of conciseness. Specifically, respiratory conditions, in
particular sarcoidosis and Chronic Obstructive Pulmonary Disease
(COPD) may be indicated in conjunction with one or more of the
following indicators: The diagnosis of sarcoidosis may be made
based upon clionical findings; x-ray, biopsy, s-Ca, ACE activity.
COPD diagnosis may also be made by clinical findings; X-ray
spirometry and blood gas analysis. In specific embodiments, the
sample is a peripheral blood sample.
[0176] The methods and medical uses of the various embodiments of
the invention thus can be tailored to the need of individual
patients or groups of patients on the basis of the various
diagnostic methods of the various embodiments of the invention. By
removing from the circulation one or more of CCR2, CCR1, CCR3,
CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, such as monocytes,
macrophages and lymphocytes, in particular monocytes, upregulated
in various inflammatory conditions associated with respiratory
conditions, in particular sarcoidosis and Chronic Obstructive
Pulmonary Disease (COPD), an important factor in the inflammatory
process of such conditions can be controlled.
[0177] The various embodiments of the invention will now be
described in more detail by reference to the following non-limiting
embodiments and examples:
DESCRIPTION OF THE FIGURES
[0178] FIG. 1a, 1b & 1c--the binding of biotinylized
MIP-1.alpha. by CD4+, CD8+T-cells and CD14+ monocytes respectively,
obtained from peripheral blood of a healthy donor;
[0179] FIGS. 2a, 2b & 2c--the binding of biotinylized MCP-1 by
CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from
peripheral blood of a healthy donor;
[0180] FIGS. 3a, 3b & 3c--the binding of IL-8 by by CD4+, CD8+
T-cells and CD16+ monocytes respectively, obtained from peripheral
blood of a healthy donor
[0181] FIG. 4a--binding of MCP-1 to monocytes (dashed line) in
peripheral blood taken from IBD patients. The graph represents a
summary of four tests.
[0182] FIG. 4b--binding of CCR2-antibody to monocytes (line) in
peripheral blood taken from IBD patients. The graph represents a
summary of four tests.
[0183] FIG. 5a--binding of eotaxin to neutrophils/eosinophils
(dashed line) in peripheral blood. The graph represents a summary
of four tests.
[0184] FIG. 5b--binding of CCR3-antibody to neutrophils/eosinophils
(line) in peripheral blood. The graph represents a summary of four
tests.
[0185] FIG. 6--The plastic house and top showing the distribution
plate (2) and safety filter units (3 and 4).
[0186] FIG. 7--The overall leukapheresis system.
[0187] FIG. 8--The pump with air detector and optical detector
(4).
[0188] FIG. 9a--Results of in vitro depletion tests performed on
the bMCP-1 coupled matrix showing ability to eliminate
CCR2-expressing cells from blood from three healthy donors.
[0189] FIG. 9b--Results of in vitro depletion tests performed on
the biotinylated RANTES coupled matrix showing ability to eliminate
chemokine receptor-expressing cells from peripheral blood taken
from a healthy donor.
[0190] FIG. 9c--Results of in vitro depletion tests performed on
the biotinylated eotaxin coupled matrix showing ability to
eliminate CCR3-expressing cells from blood from a healthy
donor.
[0191] FIG. 10--Sequence and biotinylation, via a spacer group, of
mature protein MCP-1 derivative containing Gln to pyroGlu
modification.
[0192] FIG. 11--Sequence and biotinylation, via a spacer group, of
mature protein MCP-1 derivative containing Gln to pyroGlu
modification and Met to Norleu substitution.
[0193] FIG. 12--Sequence and biotinylation, via a spacer group, of
truncated MCP-1 derivative containing Met to Norleu
substitution.
[0194] FIG. 13--Alignment of MCP-1 and MCP-5 amino acid
sequences.
[0195] FIG. 14--Sequence and biotinylation, via a spacer group, of
(C-terminal) truncated MCP-5 derivative containing Ile to Lys
modification.
[0196] FIG. 15--Sequence and biotinylation, of RANTES
derivative.
[0197] FIG. 16--Sequence and biotinylation, via a spacer group, of
mature protein eotaxin derivative containing C-terminal amide.
[0198] FIG. 17--Example of gating criteria for CCR2 expressing
monocytes
[0199] FIG. 18--Frequency of CCR1 expressing monocytes in 20
healthy controls and 2 patients with sarcoidosis. The expression of
chemokine receptors and specific cell markers were analysed with
flow cytometry.
[0200] FIG. 19--Expression of CCR1 compared to binding of bRANTES
to blood monocytes from a patient with sarcoidosis. The expression
of chemokine receptors, binding of chemokine, and specific cell
markers were analysed with flow cytometry.
[0201] FIG. 20--Depletion of CCR1 expressing monocytes with
Sepharose Streptavidin-matrix conjugated with bRANTES. Blood cells
from a healthy control were incubated with biotinylated
chemokine-Sepharose Streptavidin-matrix. Unbound cells were
retrieved by washing the matrix. The cells (After Depletion) were
then analysed with flow cytometry and compared with cells that had
not been incubated with biotinylated-chemokine-matrix (Before
Depletion).
[0202] FIG. 21--Expression of CCR2 on monocytes from two patients
with sarcoidosis. The expression of chemokine receptors, binding of
chemokine and specific cell markers were analysed with flow
cytometry.
[0203] FIG. 22--Binding of the chemokine bMCP-1 to monocytes. Bars
represent frequency of MCP-1 binding monocytes and CCR2 expressing
monocytes in blood from a patient with sarcoidosis. Blood was
incubated with biotinylated chemokine and analysed with flow
cytometry.
[0204] FIG. 23--Depletion of CCR2 expressing monocytes with
Sepharose Streptavidin-matrix conjugated with bMCP-1. Blood cells
from a healthy control were incubated with biotinylated
chemokine-Sepharose Streptavidin-matrix. Unbound cells were
retrieved by washing the matrix. The cells (After Depletion) were
then analysed with flow cytometry and compared with cells that had
not been incubated with bchemokine-matrix (Before Depletion).
[0205] FIG. 24a--Frequency of CCR7 expressing T cells. Bars
represent frequency of T cells that express CCR7 in 2 patients and
20 healthy controls. The expression of chemokine receptors and
specific cell markers were analysed with flow cytometry. The T
cells were characterized as CD3 positive.
[0206] FIG. 24b--Frequency of central memory T cells in one patient
with sarcoidosis. The central memory T cells were characterized as
CD3 positive, CD4 positive, CD45RA negative, CCR7 positive
cells.
[0207] FIG. 25--Depletion of CCR7 expressing T cells with Sepharose
Streptavidin-matrix conjugated with bMIP3b. Blood cells from a
patient with Sarciodosis were incubated with biotinylated
chemokine-Sepharose Streptavidin-matrix. Unbound cells were
retrieved by washing the matrix. The cells (After Depletion) were
then analysed with flow cytometry and compared with cells that had
not been incubated with bchemokine-matrix (Before Depletion).
DESCRIPTION OF PREFERRED EMBODIMENTS
[0208] Plasma levels of MCP-1 and MIP-1.alpha. were monitored in 26
patients with active pulmonary sarcoidosis over a two year period.
During this period, the authors show that levels of these cytokines
were closely related to the clinical course of the disease. The
authors conclude that plasma MCP-1 and MIP-1.alpha. levels are
useful indicators of clinical severity of sarcoidosis, and that
levels "may reflect subclinical evidence of extrathoracic
sarcoidosis and may play a role in initiating monocyte migration
into the tissue".Hashimoto S et al, Clin Exp Immunol, 1998
[0209] Serum MCP-1 levels were measured in 47 sarcoidosis patients
and 10 healthy controls. Chemokine levels were significantly higher
in the patient group, and more specifically, correlated positively
with patients in early disease stages. Furthermore, MCP-1 was shown
to be specifically expressed by macrophages associated with sarcoid
lymph nodes. lyonaga K et al, Sarcoidosis Vasc Diffuse Lung Dis,
1998
[0210] The inflammatory cytokines TNF-a, IL-8, MCP-1, MMP9 and
GRO-a were measured in 100 COPD patients and 50 matched healthy
smokers. These values were subsequently correlated to the BODE
index of COPD disease severity. The largest difference in these
biomarkers what observed in serum levels of MCP-1 which were
significantly increased in the COPD group. The authors conclude
that serum MCP-1 levels may be a clinical candidate for
distinguishing between healthy smokers and patients with stable
COPD. Liu S F et al, Respirology, 2009
[0211] TNF-alpha, IL-8, MMP-9, MCP-1, TIMP-1 and TIMP-2 were
measured in 20 COPD patients, 10 asymptomatic smokers and 10
non-smoker healthy controls. The authors found highly reproducible
and statistically significant elevations of plasma IL-8 among the
COPD patients compared to the other groups. No other correlations
were observed. Shaker S B et al, Clin Respir J, 2008.
[0212] It is shown herein that subjects suffering from respiratory
conditions such as sarcoidosis exhibit increased frequency of
chemokine receptor expressing cells in the peripheral blood.
Subjects with sarcoidosis exhibit increased frequency of CCR1
expressing cells such as CCR1 expressing monocytes, compared to
healthy controls. It is also shown herein that the CCR1 expressing
cells can be removed using a suitable binding reagent, in
particular RANTES (in biotinylated form) immobilized on a suitable
matrix. Similarly, it is shown herein that the monocytes also
express CCR2. The CCR2 expressing monocytes can be depleted in
sarcoidosis patients using a suitable binding reagent, in
particular MCP-1, in biotinylated form, immobilized on a suitable
matrix. It is also shown herein that subjects suffering from
respiratory conditions such as sarcoidosis exhibit increased
frequency of CCR7 expressing cells such as CCR7 expressing
lymphocytes, and also central memory T cells,compared to healthy
controls. It is also shown herein that the CCR7 expressing cells
can be removed using a suitable binding reagent, in particular
MIP3b (in biotinylated form) immobilized on a suitable matrix.
[0213] On this basis the inventors have selected a range of
chemokine receptors to use as targets for treatment according to
the methods of the invention.
EXAMPLES 1 to 9
[0214] Materials and Methods
[0215] Isolation of peripheral blood leukocytes. Heparinized
peripheral blood from healthy blood donors or inflammatory bowel
disease (IBD) patients was fixed with 4% paraformaldehyde for 4
minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride
solution and washed twice in FACS buffer to obtain a suspension of
blood leukocytes.
[0216] Chemokines. The leukocytes were incubated for 30 min in the
dark at 4.degree. C. with biotinylated and Alexa647 Fluor.RTM.
labelled chemokine (CCLS, CCL2, CXCL8) (in concentrations 10
ng/.mu.L and 50 ng/.mu.L). The cells were then washed with
FACS-buffer and analyzed by flow cytometry. All chemokines used in
the Examples were provided by Almac Sciences Scotland Ltd,
Edinburgh, Scotland.
[0217] Flow cytometry assay. The flow cytometry assay was performed
on a two laser FACS Calibur cytometer (BD Immunocytometry systems,
San Jose, Calif., USA). Ten thousand cells were counted and
analysed in each sample. For data analyses, Cell Quest Pro software
from Becton Dickinson was used.
Example 1
[0218] Binding of monocytes to MIP-1.alpha.. In the experiment with
biotinylated MIP-1.alpha. it was found that about 90% of the
monocytes obtained from peripheral blood of healthy donors had
bound to the cytokine after 30 min of incubation (FIG. 1c), whereas
CD4+ and CD8+ lymphocytes had not bound (FIG. 1a and 1b).
Example 2
[0219] Binding of monocytes to MCP-1. In the experiment with
biotinylated MCP-1 it was found that about 90% of the monocytes
obtained from peripheral blood of healthy donors had bound to the
cytokine after 30 min of incubation (FIG. 2a), whereas CD4+ and
CD8+ lymphocytes had not bound (FIGS. 2b and 2c).
Example 3
[0220] Affinity of blood cells to biotinylated IL-8. In FIG. 3 the
binding to biotinylated IL-8 (CXCL8) of CD4+ lymphocytes (FIG. 3a),
CD8+ lymphocytes (FIG. 3b) and CD16+ neutrophils (FIG. 3c) obtained
from healthy donors is shown. After 30 min of incubation all CD16+
neutrophils bound to IL-8. In contrast no binding was observed with
CD4+ lymphocytes and CD8+ lymphocytes.
Example 4
[0221] Monocytes were investigated for their expression of CCR2
(FIG. 4b) and their ability to bind MCP-1 (FIG. 4a). CCR2
expression was noted an all monocytes with the majority of
monocytes expressing high levels, using an anti-CCR2 antibody (FIG.
4b). The MCP-1 binding to monocytes shown in FIG. 2a corresponds to
the CCR2.sup.hi expressing population shown in FIG. 4b. Thus, MCP-1
binds favourably to CCR2.sup.hi expressing cells.
Example 5
[0222] Neutrophils/eosinophils were investigated for their
expression of CCR3, (FIG. 1b) and their ability to bind eotaxin
(FIG. 5a). CCR3, expression was noted in all
neutrophils/eosinophils with the majority of
neutrophils/eosinophils expressing high levels, using an anti-CCR3,
antibody (FIG. 5b). The eotaxin binding to neutrophils/eosinophils
shown in FIG. 5a corresponds to the CCR3.sup.hi expressing
population shown in FIG. 5b. Thus, eotaxin binds favourably to
CCR3.sup.hi expressing cells.
Example 6
[0223] Preparation of a chemokine column for blood cell apheresis.
To streptavidin cross-linked agarose (ProZyme, San Leandro, Calif.,
U.S.A.) beads in the range from 75 .mu.m to 300 .mu. suspended (200
ml, .about.50%, v/v) in an aqueous solution of 25 mM sodium
phosphate (pH 7.0) and 150 mM NaCl was added a solution of 75 .mu.g
biotinylated MIP-1.alpha. (Almac Sciences) in the same buffer at
22.degree. C. and slowly stirred by hand for 3 min. After standing
for another 20 min, the support was filtered off, washed thrice
with neutral aqueous sodium phosphate/sodium chloride and filled
into a glass column (i.d. 25 mm, length 12 cm).
Example 7
[0224] Separation of monocytes from peripheral blood of a healthy
donor with the chemokine column of Example 5. Heparinized
peripheral blood from a healthy male donor was analyzed by flow
cytometry for CD4+ lymphocytes, CD8+ lymphocytes and CD14
monocytes. 100 ml of the blood was filtered through the column at a
rate of about 8 ml per min and washed with FACS buffer. The
filtered blood was analyzed for the same cells. It was found that
about 95% of the monocytes had been retained by the column whereas
more than 90% each of CD4+ and CD8+ lymphocytes had been
recovered.
Example 8
[0225] Preparation of streptavidin conjugated magnetic beads
complexed with biotinylated MIP-la. An aqueous suspension of
streptavidin conjugated magnetic beads (MagCellect Streptavidin
Ferrofluid, 1 ml; R&D Systems, Minneapolis, Minn., U.S.A.) was
mixed with 30 .mu.g of MIP-1.alpha. (Almac Sciences) in 50 ml of 25
mM sodium phosphate (pH 7.0) and 150 mM NaCl and slowly stirred for
1 hour. The particles were washed thrice with 20 ml portions the
same solvent and stored in suspension at 4.degree. C.
Example 9
[0226] Separation of CD14+ monocytes from peripheral blood of a
healthy donor with the streptavidin magnetic beads of Example 7.
100 ml of heparinized blood from the healthy donor of Example 7 was
mixed with the streptavidin conjugated magnetic beads complexed
with biotinylated MIP-1.alpha. and slowly stirred for 40 min. The
particles were separated from the blood by a magnetic separator,
and the blood analyzed for CD14+ monocytes and CD4+ and CD8+
lymphocytes. While essentially no CD14+ monocytes could be
detected, CD4+ and CD8+ lymphocytes were present in roughly the
original amounts.
Example 10`
Tailored leukapheresis
[0227] Column Design and Properties
[0228] Introduction
[0229] Apheresis is an established treatment used for depletion of
blood components, such as antibodies, low-density lipoproteins
(LDL) and blood cells. Leukapheresis is the apheresis treatment
used for removal of white blood cells, leukocytes. The patient is
connected to an extracorporeal blood circulating system; the blood
is drawn from a vein in one arm, passed through a column device and
returned into the other arm of the patient. Side effects of
leukapheresis treatments are varying from mild events like
headache, dizziness, hypotension, palpitation and flush seen in 0.1
to 5% of treated patients.
[0230] The Column
[0231] The column is intended to be used as a leukapheresis
treatment for respiratory conditions, in particular sarcoidosis and
Chronic Obstructive Pulmonary Disease (COPD). It will specifically
remove CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7-expressing
leukocytes, in particular monocytes, through the use of a binding
reagent containing resin, exploiting the CCR2, CCR1, CCR3, CCR5,
CXCR1, CXCR2 and/or CCR7-chemokine interaction. The column consists
of three combined components, the plastic house, the streptavidin
(SA) Sepharose.TM. BigBeads matrix and one or more biotinylated
chemokine bound to the matrix. The treatment is conducted using the
same techniques as a standard apheresis procedure.
[0232] The plastic house (FIG. 6)
[0233] The plastic house, designed to keep a continuous blood flow
through the matrix, consists of a transparent body and red-coloured
top. The top has a distribution plate (2) at the inflow site (1) to
spread the blood evenly over the entire matrix area. The plate is
the first safety barrier preventing larger particles flowing
through the column and into the patient. Safety filter units (3 and
4) are placed at the inflow (1) and outflow (5) sites of the
plastic housing. The safety filter unit contains three filters
designed to be a robust barrier and stop all particles larger than
blood cells passing through the column. The plastic housing design
is shown in FIG. 4. The design with safety filters (3 and 4) at
both ends of the column device will minimize the risk of leakage of
particles into the patient, including in the event that the device
is placed up side down with the blood flow in the opposite
direction to that anticipated.
[0234] Streptavidin Sepharose.TM. BigBeads
[0235] The second component in the device is the affinity matrix
called streptavidin Sepharose.TM. BigBeads (Sepharose.TM. GE
Healthcare, Sweden). Sepharose.TM. is a cross linked, beaded-form
of agarose, which is a polysaccharide extracted from seaweed.
Sepharose.TM. and agarose are commonly used as column matrices in
biomedical affinity techniques. It is chosen for its optimal
distribution capacity and can provide a large available area for
affinity binding.
[0236] Binding Reagent
[0237] Coupled to the matrix is the third component of the device,
one or more binding reagents that bind specifically to CCR2, CCR1,
CCR3, CCR5, CXCR1, CXCR2 and/or CCR7. One or more chemokines may be
employed. These peptides may be synthetic, engineered versions of
the human chemokine, which are truncated and biotinylated, but
retain binding activity to the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2
and/or CCR7 receptor. By biotinylating the engineered chemokine, it
is able to bind to the streptavidin molecules in the Sepharose.TM.
matrix. The biotin-streptavidin binding is known be one of the
strongest biological interactions with a Kd in the order of
4.times.10.sup.-14 M. The calculated ratio of streptavidin:biotin
binding sites in the column is 10:1. Therefore, the coupling
between the matrix and chemokine will be immediate, minimising the
risk of chemokine decoupling from the matrix.
[0238] The Apheresis System
[0239] To conduct the leukapheresis the following components are
needed; the column, tubing system, and a 4008 ADS pump (Fresenius
Medical Care).
[0240] The Circuit
[0241] The system is illustrated in FIG. 7. The patient (1) is
connected to the extracorporeal circuit via sterile Venflon needles
to veins in the right and the left arms. A saline bag (3) is also
connected and the saline solution is pumped with an ACD pump (2).
Blood is drawn from one arm of the patient through the sterile
tubing system by the blood pump (4) and passed through the column
(6) and back to the patient. The tubing system is connected to the
column via standard dialysis luer-lock couplings. The couplings on
the column are colour-coded for correct assembly; red tubing for
inflow to the red column top and blue tubing for outflow back to
the patient. An air detector (8) is present. Inlet pressure(5) and
Pven sensors (7) are employed to monitor the pressure in the
circuit.
[0242] The 4008 ADS pump
[0243] An apheresis pump, from Fresenius Medical Care, monitors the
patient's inflow and outflow, the pressure in the extracorporeal
circulation and can discriminate air by a bubble catcher and air
detector. A clot catcher filter is placed inside the bubble
catcher. The pump also has an optical detector to distinguish
between light, e.g. saline solution or air present in the tubing
system and dark e.g. blood present in the tubing system.
[0244] A schematic diagram of the pump, showing the air detector
and optical filter is shown in FIG. 8. If the pump system detects
air bubbles and optical fluctuations or if extracorporeal pressure
values are out of the set range, then the pump stops immediately
and a visual/audible alarm are emitted.
[0245] Legend for FIG. 8: [0246] 1. Monitor [0247] 2. Holder for
waste bag [0248] 3. Modules (left to right--Blood pump, ACD pump,
Air detector) [0249] 4. Reserve places for further modules [0250]
5. Absorber holder [0251] 6. Drip detector [0252] 7. IV pole
[0253] Preparation of the Patient
[0254] The patient will be administered anticoagulants prior to
each treatment session. A sterile saline solution with 5000 IE
Heparin will be used for priming the extracorporeal system,
thereafter a bolus injection with 4000 IE Heparin will be added
into the circuit at the start of each treatment session.
[0255] Leukapheresis Time and Flow Rate
[0256] The apheresis system should be operated at a flow rate of
30-60 mL/min. A treatment is finalised after 1800 mL of blood has
been circulated.
[0257] Storage Conditions
[0258] The column devices should be stored between 1 and 25.degree.
C. avoiding freezing and more elevated temperatures. Stability data
>3 months indicate no difference in functionality over time or
by temperature (room temperature and refrigerated). The columns
will be kept in refrigerated conditions until use. Mechanical
damage as those resulting from violent vibrations and trauma should
be avoided. Column stored outside of these recommendations should
not be used.
[0259] Transport Conditions
[0260] The column devices will be transported under refrigerated
condition, avoiding freezing and more elevated temperatures.
Mechanical damage such as those resulting from violent vibrations
and trauma should be avoided.
[0261] In-Vitro Depletion of Target Cell Populations
[0262] To investigate the ability to eliminate CCR2-expressing
cells, in vitro tests have been performed on the bMCP-1 coupled
matrix. Blood was collected from blood donors and passed through
the column device containing bMCP-1 coupled matrix. Blood samples
were taken before and after column passage and analyzed by flow
cytometry (FACS) for the depletion of CCR2-expressing cells.
[0263] The results demonstrate significant depletion of the target
population CCR2-expressing monocytes post matrix perfusion.
Depletion tests were performed on blood from three healthy donors.
The results are shown in FIG. 9a.
[0264] The in-vitro results demonstrate a specific reduction of up
to 80% of the CCR2-expressing cells by the column. Notably,
individuals with fewer CCR2 expressing cells initially achieved
lower depletion. The remaining levels of monocytes were around
20-30% in each case, irrespective of the starting point.
Non-CCR2-expressing cells remained unaffected (data not shown).
[0265] To investigate the ability to eliminate CCR1, 3 and
5-expressing cells, in vitro tests have been performed on the
biotinylated RANTES coupled matrix. Blood was collected from blood
donors and passed through the column device containing biotinylated
RANTES coupled matrix. Blood samples were taken before and after
column passage and analyzed by flow cytometry (FACS) for the
depletion of CCR1, 3 or 5-expressing cells.
[0266] The RANTES molecule was synthesized by Almac. The amino acid
sequence of the biotinylated RANTES molecule is set forth as SEQ ID
NO: 16:
TABLE-US-00015 H2N-
SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC
ANPEKKWVREYINSLEKS-CO2H
[0267] This molecule has the naturally occurring methionine at
position 67 replaced with lysine to facilitate biotinylation at
position 67.
[0268] The side-chain of Lys 67 was directly biotinylated to given
the protein primary structure shown in FIG. 15. The protein was
folded and disulphide bonds formed between the first and third
cysteine in the sequence and between the 2nd and 4th cysteines.
[0269] The results demonstrate significant depletion of the target
population chemokine receptor-expressing cells post matrix
perfusion. Depletion tests were performed on blood from a healthy
donor. The results are shown in FIG. 9b.
[0270] The in-vitro results demonstrate a specific reduction of
around 20% of the chemokine receptor-expressing cells by the
column. Non-CCR1, 3 and 5-expressing cells remained unaffected
(data not shown).
[0271] In-Vitro Depletion of Target Cell Populations
[0272] To investigate the ability to eliminate CCR3-expressing
cells, in vitro tests have been performed on the eotaxin coupled
matrix. Blood was collected from blood donors and passed through
the column device (including a magnetic separator) containing
eotaxin coupled matrix (MACS beads). Blood samples were taken
before and after column passage and analyzed by flow cytometry
(FACS) for the depletion of CCR3-expressing cells. The results
demonstrate significant depletion of the target population
CCR3-expressing neutrophils/eosinophils post matrix perfusion.
Depletion tests were performed on blood from a healthy donor. The
results are shown in FIG. 9a.
[0273] In conclusion, the in-vitro results demonstrate a specific
reduction of around 25% of the CCR3-expressing cells by the column.
Non-CCR3-expressing cells remained unaffected (data not shown).
Example 11
[0274] MCP1 Derivatives
[0275] MCP-1 has been produced with residue 75 as the site of
biotinylation on the chemokine (numbering based upon the mature
protein having the amino acid sequence of SEQ ID NO: 2).
Biotinylation permits immobilization of MCP-1 on a solid support
(via a biotin-avidin interaction). The basic amino acid sequence of
MCP-1, including a 23 amino acid leader sequence is set forth as
SEQ ID NO: 1,
TABLE-US-00016 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN
RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL
DKQTQTPKT
[0276] The amino acid sequence of the mature protein is set forth
as SEQ ID NO: 2,
TABLE-US-00017 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV
AKEICADPKQ KWVQDSMDHL DKQTQTPKT
[0277] The inventors have determined that chemokines may display
improved binding properties where the chemokine is biotinylated via
a spacer group. The spacer may prevent the biotin group from
impacting on the binding affinity of the chemokine.
[0278] Thus, MCP-1 derivatised at the &amino side chain
functionality of Lys75 with PEG-Biotin (TFA salt) will be
synthesised. The PEG spacer will be 3,6, -dioxoaminooctanoic acid.
The Gln at the N-terminus of the proteins is subject to pyroGlu
formation under physiological conditions. Thus the first glutamine
(Gln1) of the sequence will be substituted with pyroglutamine. The
molecule will be synthesised as a C-terminal amide (via synthesis
on an amide linker). The molecule is shown schematically in FIG.
10.
[0279] A biotinMCP-1 Met to Nleu analogue will also be synthesised.
The single methionine within the sequence will be altered to
Norleucine, to mitigate against oxidation of this residue during
the chain assembly and improve stability of the final product. This
molecule is shown schematically in FIG. 11.
[0280] Once synthesised, the activity of the various biotinMCP-1
derivatives will be determined in cell-based assays. In particular,
agonist and antagonist properties will be determined in aequorin
functional cell-based assay on human CCR2 receptor.
Example 12
[0281] Synthesis of a CCR2 Antagonist Biotin MCP-1 which Binds to
the Receptor without Activation
[0282] Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp.
Med., 1995, 181, 63) has been shown for an MCP-1 derivative
truncated at the N-terminus. In particular, deletion of residues
1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was
unable to cause chemotaxis of CCR2 positive cells. (inhibition of
chemotaxis IC50 20 nM)
[0283] The amino acid sequence of the truncated version is set
forth as SED ID NO:3:
TABLE-US-00018 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV
AKEICADPKQ KWVQDSMDHL DKQTQTPKT
[0284] A derivative of this truncated version will be synthesised
comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with
Met64 to Nleu substitution and derivatised at the .epsilon.-amino
side chain functionality of Lys75 with PEG-Biotin (TFA salt). This
molecule is shown schematically in FIG. 12. The PEG spacer will be
3, 6, -dioxoaminooctanoic acid.
[0285] Once synthesised, the activity of the various biotinMCP-1
derivatives will be determined in cell-based assays. In particular,
agonist and antagonist properties will be determined in aequorin
functional cell-based assay on human CCR2 receptor.
Example 13
Demonstrate Removal of CCR2 Expressing Cells using an Alternative
Chemokine Ligand To MCP-1
[0286] CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and
HCC-4 in addition to MCP-1. MCP-5 only binds CCR2 and should be
selective in its removal of CCR2 expressing cells. MCP5 is a mouse
chemokine shown to chemotact human CCR2 cells with EC50 <3
nM.
[0287] The full length amino acid sequence, including the signal
peptide, is set forth as SEQ ID NO: 4
TABLE-US-00019 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ
KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP
SCLG
[0288] The amino acid sequence of N-terminal processed MCP-5
chemokine is 82 amino acids long and is set forth as SEQ ID NO:
5
TABLE-US-00020 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD
KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG
[0289] An amino acid sequence alignment suggests that MCP-5 has a
C-terminal extension when compared to the amino acid sequence of
MCP-1. The results of this alignment are shown in FIG. 13. On this
basis a C-terminal truncated version of MCP-5 will be synthesised.
This truncated version will comprise MCP-5 residues 1-76, set forth
as SEQ ID NO: 6:
TABLE-US-00021 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD
KEICADPKEK WVKNSINHLD KTSQTFIL
[0290] In the truncated version, Ile75 to be substituted with Lys,
set forth as SEQ ID NO: 7:
TABLE-US-00022 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD
KEICADPKEK WVKNSINHLD KTSQTFKL
[0291] Following substitution, the substituted version will be
biotinylated at position 75, a lysine or other suitable residue
such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,
-dioxoaminooctanoic acid). The protein will be synthesised on an
amide linker to yield a C-terminal amide derivative. This molecule
is shown schematically in FIG. 14.
EXAMPLE 14
[0292] Eotaxin Derivatives
[0293] Eotaxin has been produced with residue 73 (thought to be a
lysine) as the site of biotinylation on the chemokine (numbering
based upon the mature protein having the amino acid sequence of SEQ
ID NO: 9). Biotinylation permits immobilization of eotaxin on a
solid support (via a biotin-avidin interaction). The basic amino
acid sequence of eoxtaxin, including a 23 amino acid leader
sequence (signal peptide) is set forth as SEQ ID NO: 8,
TABLE-US-00023 MKVSAALLWL LLIAAAFSPQ GLAGPASVPT TCCFNLANRK
IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP
[0294] The amino acid sequence of the mature protein is set forth
as SEQ ID NO: 9,
TABLE-US-00024 GPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK
DICADPKKKW VQDSMKYLDQ KSPTPKP
[0295] The inventors have determined that chemokines may display
improved binding properties where the chemokine is biotinylated via
a spacer group. The spacer may prevent the biotin group from
impacting on the binding affinity of the chemokine.
[0296] Thus, eotaxin derivatised at the .epsilon.-amino side chain
functionality of Lys73 with PEG-Biotin (TFA salt) will be
synthesised. The PEG spacer will be 3,6, -dioxoaminooctanoic acid.
The molecule will be synthesised as a C-terminal amide (via
synthesis on an amide linker) to avoid diketopiperazine formation
during the synthesis.. The molecule is shown schematically in FIG.
16.
[0297] A biotin eotaxin Met to Nleu analogue will also be
synthesised. The single methionine within the sequence will be
altered to Norleucine, to mitigate against oxidation of this
residue during the chain assembly and improve stability of the
final product.
[0298] Once synthesised, the activity of the various eoxtaxin
derivatives will be determined in cell-based assays. In particular,
agonist and antagonist properties will be determined in functional
cell-based assay on human CCR3 receptor.
[0299] Once synthesised, the activity of the various biotinMCP-5
derivatives will be determined in cell-based assays. In particular,
agonist and antagonist properties will be determined in functional
cell-based assay on human CCR2 receptor.
Example 15 to 21
[0300] Chemokine Synthesis--General Protocols
[0301] Assembly:
[0302] Chemical synthesis of chemokines was performed using
standard Fmoc solid phase peptides synthesis (SPPS) techniques on
an ABI 433 peptide synthesiser. DIC (0.5 M in DMF) and OxymaPure
(0.5 M in DMF) were used for activation, acetic anhydride (0.5 M in
DMF) for capping, and 20% piperidine in DMF for Fmoc deprotection.
Rink Amide resin was utilised for the generation of C-terminal
amide chemokines and Wang resin for C-terminal acid chemokines.
After assembly, the resin was washed with DMF and DCM and then
dried in vacuo.
[0303] Removal of Dde Protection:
[0304] The Dde protecting group was removed by treatment of resin
with a solution of 2.5% hydrazine in DMF (200 ml) over a 2 hour
period. The resin was then washed with DMF.
[0305] Labelling Steps:
[0306] 1. Couple Fmoc-8-amino-3,6-dioctanoic acid (PEG) Resin was
swollen in DMF and then a solution of Fmoc-8-amino-3,6-dioctanoic
acid (0.38 g, 1 mmol), DIC solution (2 ml, 0.5 M in DMF) and
OxymaPure solution (2 ml, 0.5 M in DMF) was added. The mixture was
sonicated for 3 hours and then washed with DMF.
[0307] 2. Capping
[0308] The resin was capped with acetic anhydride solution (0.5 M
in DMF, 10 ml) for 5 minutes and then washed with DMF.
[0309] 3. Fmoc Deprotection
[0310] Fmoc deprotection was carried out by treatment with 20%
piperidine in DMF solution (2.times.50 ml) for 15 minutes each. The
resin was washed with DMF.
[0311] 4. Couple Biotin-OSu
[0312] A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348
.mu.l) in DMF (10 ml) was added to the resin and the mixture was
sonicated for 3 hours. The resin was washed thoroughly with DMF and
DCM then dried in vacuo.
[0313] Cleavage:
[0314] Dry resin was treated with TFA (10 ml) containing a
scavenger cocktail consisting of TIS (500 thioanisole (500 .mu.l),
water (500 .mu.l), DMS (500 .mu.l), EDT (250 .mu.l), NH.sub.4I (500
.mu.g) and phenol (500 .mu.g) and the mixture was stirred at room
temperature for 5 hours. The solution was filtered into cold ether
and the resin rinsed with TFA. The precipitated peptide was
centrifuged, washed with ether, centrifuged and lyophilised.
[0315] Purification Protocol:
[0316] The crude peptide was purified by reverse phase HPLC
(RP-HPLC) using a Jupiter C18, 250.times.21 mm column, 9 ml/min,
eluting with an optimised gradient [Buffer A: water containing 0.1%
TFA, Buffer B: acetonitrile containing 0.1% TFA].
[0317] Folding Protocol:
[0318] Pure peptide (10mg) was dissolved into 6M GnHCl (16 ml) and
then rapidly diluted to 2M GnHCl concentration by the addition of
50 mM TRIS pH 8.5 (84 ml) containing 0.3 mM GSSG and 3 mM GSH. The
mixture was stirred at room temperature for 24 hours and then
analysed by RP-HPLC (Jupiter C18, 250.times.4.6 mm column, 10-60% B
over 30 minutes. Purification by RP-HPLC using an optimised
gradient afforded the desired product.
Example 15
BiotinMCP-1 (CCL2)
[0319] Target Molecule: MCP-1 derivatised at the .epsilon.-amino
side chain functionality of Lys(75) with PEG-Biotin (TFA salt)
[0320] Modifications: Human MCP-1 corresponding to residues 1-76,
is initially expressed as 99 amino acids comprising the chemokine
fold, and a 23 amino acid signal peptide which is cleaved off. The
Gln at the N-terminus of the protein is subject to pyroGlu
formation under physiological conditions. Thus Gln1 of the sequence
was substituted with pyroglutamine to prevent mixed species of
N-terminal Gln and pyroGlu being generated. This improves the yield
of synthesis and ensures a homogeneous chemokine preparation
through column manufacture and use. The naturally occurring lysine
at position 75 was modified through biotinylation on the resin. A
PEG spacer was incorporated between the .epsilon.-amino
functionality and the biotin.
[0321] The linear amino acid sequence (SEQ ID NO: 10) is shown,
prior to attachment of the PEG spacer and biotin molecules at amino
acid 75 (K):
TABLE-US-00025 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV
AKEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
[0322] X=pyroGlu or Gln
[0323] The engineered MCP-1 sequence was assembled on a solid
support (Rink Amide resin), using Fmoc protocols for solid-phase
peptide synthesis as described in the general protocols
section:
TABLE-US-00026 SEQ ID NO: 11
H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA
KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
[0324] X1=pyroGlu or Gln
[0325] X75=K(ivDde)
[0326] FmocLys(ivDde)-OH was incorporated as residue 75 to
facilitate site-specific labelling at this position of the protein.
Subsequent removal of the ivDde protecting group, followed by
coupling of the PEG spacer and Biotin, was carried out as described
in the general protocol section. Cleavage, purification and folding
protocols were carried out as described to furnish the desired
active chemokine.
TABLE-US-00027 SEQ ID NO: 12
H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA
KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
[0327] X1=pyroGlu or Gln
[0328] X75 is an amino acid residue that can be biotinylated, such
as lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG,
optionally K(PEG-Biotin)
[0329] Electrospray ionisation with tandem mass spectrometry
(ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8
Da; expected 9034.4 Da.
[0330] Functional Assay Data:
[0331] biotinMCP-1 was tested for agonist activity in an Aequorin
assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was
reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.
Example 16
biotinRANTES (CCL5)
[0332] Target Molecule: RANTES derivatised at the .epsilon.-amino
side chain functionality of Lys(67) with Biotin (TFA salt)
[0333] Modifications: Human RANTES corresponding to residues 1-68,
is initially expressed as 91 amino acids comprising the chemokine
fold, and a 23 amino acid signal peptide which is cleaved off. The
single methionine (Met67) within the sequence was mutated to
lysine, to mitigate against oxidation of this residue during the
chain assembly, which was observed during the synthesis of the
natural sequence derivative. This Met to Lys substitution provided
a lysine at position 67 which was modified through biotinylation on
the resin.
[0334] The linear amino acid sequence (SEQ ID NO: 16) is shown,
prior to attachment of the biotin molecule at amino acid 67
(K):
TABLE-US-00028 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ
VCANPEKKWVREYINSLEKS-OH
[0335] The engineered RANTES sequence was assembled on a solid
support (Wang resin), using Fmoc protocols for solid-phase peptide
synthesis as described in the general protocols section:
TABLE-US-00029 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ
VCANPEKKWVREYINSLEXS-RESIN
[0336] X is K(ivDde)
[0337] FmocLys(ivDde)-OH was incorporated as residue 67 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 17). Subsequent removal of the ivDde protecting group,
followed by coupling of the Biotin, was carried out as described in
the general protocol section. Cleavage, purification and folding
protocols were carried out as described to furnish the desired
active chemokine (SEQ ID NO: 18).
TABLE-US-00030 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ
VCANPEKKWVREYINSLEXS-OH
[0338] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG (e.g.
K(Biotin))
[0339] Electrospray ionisation with tandem mass spectrometry
(ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9
Da; expected 8070.2 Da.
[0340] Functional Assay Data:
[0341] BiotinRANTES was tested for agonist activity in an Aequorin
assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was
reported.
Example 17
BiotinMCP-2 (CCL8)
[0342] Target Molecule: MCP-2 derivatised at the s-amino side chain
functionality of Lys(75) with PEG-Biotin (TFA salt)
[0343] Modifications: Human MCP-2 corresponding to residues 1-76,
is initially expressed as 99 amino acids comprising the chemokine
fold, and a 23 amino acid signal peptide which is cleaved off. The
Gln at the N-terminus of the protein is subject to pyroGlu
formation under physiological conditions. Thus Gln1 of the sequence
was substituted with pyroglutamine to prevent mixed species of
N-terminal Gln and pyroGlu being generated. This improves the yield
of synthesis and ensures a homogeneous chemokine preparation
through column manufacture and use. The naturally occurring lysine
at position 75 was modified through biotinylation on the resin. A
PEG spacer was incorporated between the &amino functionality
and the biotin.
[0344] The linear amino acid sequence (SEQ ID NO: 13) is shown,
prior to attachment of the PEG spacer and biotin molecules at amino
acid 75 (K):
TABLE-US-00031 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG
KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
[0345] X=pyroGlu or Gln
[0346] The engineered MCP-2 sequence was assembled on a solid
support (Rink Amide resin), using Fmoc protocols for solid-phase
peptide synthesis as described in the general protocols
section:
TABLE-US-00032 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG
KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
[0347] X1=pyroGlu or Gln
[0348] X75=K(ivDde)
[0349] FmocLys(ivDde)-OH was incorporated as residue 75 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 14). Subsequent removal of the ivDde protecting group,
followed by coupling of the PEG spacer and Biotin, was carried out
as described in the general protocol section. Cleavage,
purification and folding protocols were carried out as described to
furnish the desired active chemokine (SEQ ID NO: 15):
TABLE-US-00033 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG
KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
[0350] X1=pyroGlu or Gln
[0351] X75=an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG, e.g.
K(PEG-Biotin).
[0352] Electrospray ionisation with tandem mass spectrometry
(ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6
Da; expected 9263.8 Da.
[0353] Functional Assay Data:
[0354] biotinMCP-2 was tested for activity in an Aequorin assay
against hCCR2b, (Euroscreen) and was shown to be a partial agonist
with an EC50 value of 50.9 nM. c.f. EC50 for recombinant native
MCP-2 is 23.5 nM (partial agonist).
Example 18
BiotinMIP-3.beta. (CCL19)
[0355] Target Molecule: MIP-3.beta. derivatised at the
.epsilon.-amino side chain functionality of Lys(78) with Biotin
(TFA salt)
[0356] Modifications: Human MIP-3.beta. corresponding to residues
1-77, is initially expressed as 98 amino acids comprising the
chemokine fold, and a 21 amino acid signal peptide which is cleaved
off. An additional lysine was inserted at the C-terminus, at
position 78, and modified through biotinylation on the resin.
[0357] The linear amino acid sequence (SEQ ID NO: 19) is shown,
prior to attachment of the biotin molecule at amino acid 78
(K):
TABLE-US-00034 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ
LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
[0358] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated (e.g. K-biotin), optionally via a spacer molecule such
as PEG, in particular K(PEG-Biotin)
[0359] The engineered MIP-3.beta. sequence was assembled on a solid
support (Rink Amide resin), using Fmoc protocols for solid-phase
peptide synthesis as described in the general protocols
section:
TABLE-US-00035 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ
LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN
[0360] X is FmocLys(ivDde)
[0361] FmocLys(ivDde)-OH was incorporated as residue 78 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 20). Subsequent removal of the ivDde protecting group,
followed by coupling of the Biotin, was carried out as described in
the general protocol section. Cleavage, purification and folding
protocols were carried out as described to furnish the desired
active chemokine (SEQ ID NO: 21).
TABLE-US-00036 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ
LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
[0362] X is K(Biotin)
[0363] Electrospray ionisation with tandem mass spectrometry
(ESI-TOF-MS) data of purified folded biotinMlP-3.beta.: obtained 32
9148.8 Da; expected 9149.7 Da.
[0364] Functional Assay Data:
[0365] biotinMip-3.beta. was tested for agonist activity in an
Aequorin assay against hCCR7 , (Euroscreen) and an EC50 value of
11.0 nM was reported. c.f. EC50 for recombinant native MIP-3.beta.
is 1.6 nM.
Example 19
biotinlL-8 (CXCL8)
[0366] Target Molecule: IL-8 derivatised at the s-amino side chain
functionality of Lys(78) with PEG-Biotin (TFA salt)
[0367] Modifications: Human IL-8 corresponding to residues 1-77, is
initially expressed as 99 amino acids comprising the chemokine
fold, and a 22 amino acid signal peptide which is cleaved off. An
additional lysine was inserted at the C-terminus at position 78,
and modified through biotinylation on the resin. A PEG spacer was
incorporated between the s-amino functionality and the biotin.
[0368] The linear amino acid sequence (SEQ ID NO: 22) is shown,
prior to attachment of the PEG spacer and biotin molecules:
TABLE-US-00037 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL
SDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
[0369] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG, e.g.
K(PEG-Biotin)
[0370] The engineered IL-8 sequence was assembled on a solid
support (Rink Amide resin), using Fmoc protocols for solid-phase
peptide synthesis as described in the general protocols
section:
TABLE-US-00038 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL
SDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN
[0371] X is K(ivDde)
[0372] FmocLys(ivDde)-OH was incorporated as residue 78 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 23). Subsequent removal of the ivDde protecting group,
followed by coupling of the PEG spacer and Biotin, was carried out
as described in the general protocol section. Cleavage,
purification and folding protocols were carried out as described to
furnish the desired active chemokine (SEQ ID NO: 24):
TABLE-US-00039 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL
SDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
[0373] X is K(PEG-Biotin)
[0374] Electrospray ionisation with tandem mass spectrometry
(ESI-TOF-MS) data of purified folded biotinlL-8: obtained=9416.9
Da; expected 9417.0 Da.
[0375] Functional Assay Data:
[0376] BiotinlL-8 was tested for agonist activity in an Aequorin
assay against hCXCR1, (Euroscreen) and an EC50 value of 18.9 nM was
reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.
Example 20
[0377] BiotinlL-8 (6-78)
[0378] Target Molecule: IL-8 (6-78) derivatised at the s-amino side
chain functionality of Lys(78) with PEG-Biotin (TFA salt)
[0379] Modifications: Truncated form of IL-8 corresponding to
residues 6-77, the first five N-terminal residues have been removed
and an additional lysine was inserted at the C-terminus at position
78, and modified through biotinylation on the resin. A PEG spacer
was incorporated between the s-amino functionality and the
biotin.
[0380] The linear amino acid sequence (SEQ ID NO: 25) is shown,
prior to attachment of the PEG spacer and biotin molecules:
TABLE-US-00040 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE
LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
[0381] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG
[0382] The engineered IL-8 sequence was assembled on a solid
support (Rink Amide resin), using Fmoc protocols for solid-phase
peptide synthesis as described in the general protocols
section:
TABLE-US-00041 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE
LCLDPKENWVQRVVEKFLKRAENSX-RESIN
[0383] X is K(ivDde)
[0384] FmocLys(ivDde)-OH was incorporated as residue 78 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 26). Subsequent removal of the ivDde protecting group,
followed by coupling of the PEG spacer and Biotin, was carried out
as described in the general protocol section. Cleavage,
purification and folding protocols were carried out as described to
furnish the desired active chemokine (SEQ ID NO: 27):
TABLE-US-00042 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE
LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
[0385] X is K(PEG-Biotin)
[0386] Electrospray ionisation with tandem mass spectrometry
(ESI-TOF-MS) data of purified folded biotinlL-8 (6-78):
obtained=8880.50 Da; expected 8880.4 Da.
[0387] Functional Assay Data:
[0388] BiotinlL-8 (6-78) was tested for agonist activity in an
Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of
6.1 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2
nM.
Example 21
[0389] BiotinEotaxin (CCL11)
[0390] Target Molecule: Eotaxin derivatised at the s-amino side
chain functionality of Lys(73) with PEG-Biotin (TFA salt)
[0391] Modifications: Human eotaxin corresponding to residues 1-74,
is initially expressed as 97 amino acids comprising the chemokine
fold, and a 23 amino acid signal peptide which is cleaved off. The
naturally occurring lysine at position 73 was modified through
biotinylation on the resin. A PEG spacer was incorporated between
the &amino functionality and the biotin.
[0392] The linear amino acid sequence (SEQ ID NO: 28) is shown,
prior to attachment of the PEG spacer and biotin molecules at amino
acid 73 (K):
TABLE-US-00043 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD
ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
[0393] X is an amino acid residue that can be biotinylated, such as
lysine, ornithine or diaminopropionic acid and optionally is
biotinylated, optionally via a spacer molecule such as PEG, e.g.
K(PEG-Biotin).
[0394] The engineered eotaxin sequence was assembled on a solid
support (Rink Amide resin), using Fmoc protocols for solid-phase
peptide synthesis as described in the general protocols
section:
TABLE-US-00044 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD
ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
[0395] X is K(ivDde)
[0396] FmocLys(ivDde)-OH was incorporated as residue 73 to
facilitate site-specific labelling at this position of the protein
(SEQ ID NO: 29). Subsequent removal of the ivDde protecting group,
followed by coupling of the PEG spacer and Biotin, was carried out
as described in the general protocol section. Cleavage,
purification and folding protocols were carried out as described to
furnish the desired active chemokine (SEQ ID NO: 30):
TABLE-US-00045 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD
ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
[0397] X is K(PEG-Biotin)
[0398] Electrospray ionisation with tandem mass spectrometry
(ESi-TOF-MS) data of purified folded biotinEotaxin: obtained=8731.3
Da; expected 8731.3 Da.
[0399] Functional Assay Data:
[0400] biotinEotaxin was tested for activity in an Aequorin assay
against hCCR3, (Euroscreen) and was shown to be an antagonist with
an EC50 value of 211.8 nM. c.f. EC50 for recombinant native eotaxin
is 10.7 nM (agonist).
Example 22
[0401] Diagnosis and Treatment of Sarcoidosis
[0402] Materials and Methods
[0403] 1. Flow cytometric analysis of peripheral blood
[0404] Peripheral blood from patients and healthy controls was
collected in heparin tubes. The red blood cells were lysed using
Fix Buffer (Phosphate Buffer Saline (PBS) citrate with 4%
paraformaldehyde) for four minutes at 37.degree. C. and Lysing
buffer (PBS with 10 mM Tris and 160 mM NH4Cl, pH 7.5) for 15 min at
37.degree. C. The cells were washed in PBS with 2% Bovine Growth
Serum, incubated with 10% human serum for 15min at room temperature
(RT) and stained with antibodies (Table 2) at 4.degree. C. for 30
min. The cells were analysed by flow cytometry on a FACS Canto flow
cytometer with the FACSDiva software (BD Biosciences).
TABLE-US-00046 TABLE 2 List of antibodies for flow cytometric
analysis. Antibody Fluorophore Supplier CCR1 Alexa flour 647
Biolegend CCR2 PerCPCy5.5 Biolegend CCR7 PerCpCy5.5 Biolegend CD4
V500 BD CD3 Horizon V450 BD Streptavdin APC BD CD14 FITC Beckman
Coulter CD45RA PECy7 BD
[0405] 2. Chemokine Binding Test
[0406] Peripheral blood from patients and healthy controls was
collected in heparin tubes. The red blood cells were lysed using
Fix Buffer (Phosphate Buffer Saline (PBS) citrate with 4%
paraformaldehyde) for four minutes at 37.degree. C. and Lysing
buffer (PBS with 10 mM Tris and 160 mM NH4Cl, pH 7.5) for 15 min at
37.degree. C. The cells were washed in PBS with 2% Bovine Growth
Serum, incubated with 10% human serum 15 min at room temperature
(RT) and stained with cell specific antibodies together with
biotinylated chemokine (1 .mu.M) or the corresponding chemokine
receptor antibody at 4.degree. C. for 30 min (Table 1). The
biotinylated chemokine was detected via the interaction between
biotin and a fluorophore conjugated Streptavidin. The samples were
analysed by flow cytometry on a FACS Canto flow cytometer with the
FACSDiva software (BD Biosciences).
[0407] 3. Cell depletion by matrix conjugated with biotinylated
chemokine
[0408] Cells were prepared from peripheral blood (section 1). 1 mL
Sepharose BigBeads matrix conjugated with 0.4 mg/mL Streptavidin
(GE Healthcare) was washed in 50 mL PBS and added to a 5 mL
polystyrene tube (BD Falcon.TM.). Biotinylated chemokine was added
to the tube and incubated for 20 min at RT to enable immobilization
of the chemokine on the matrix via the biotin-streptavidin
interaction. Next, the cells were added to the chemokine-matrix and
incubated for 20 min at RT. The cells that did not bind to the
matrix were removed by washing the matrix with PBS in a sterile 40
um nylon filter (BD Falcon.TM. Cell Strainer). The flow through
cells were stained with antibodies (Table 2), analysed by flow
cytometry and compared with cells from peripheral blood that had
not been incubated with the chemokine-matrix.
[0409] Results and Discussion
[0410] White blood cells from 2 patients with sarcoidosis were
analysed for the expression of chemokine receptors with flow
cytometry. The patients exhibited increased frequency of monocytes
that expressed the receptor CCR1 based upon flow cytometry data and
binding of an anti-CCR1 antibody (FIG. 18).
[0411] The ligand for CCR1 is the chemokine RANTES that also binds
to CCR5 expressed on T cells. RANTES is expressed in the lungs
where it mediates migration of inflammatory cells. The monocytes
bind biotinylated RANTES to the same extent as the chemokine
receptor expression (FIG. 19).
[0412] The CCR1 expressing monocytes could be efficiently depleted
with bRANTES-conjugated Sepharose Streptavidin Matrix (FIG.
20).
[0413] In addition to CCR1, the monocytes express the chemokine
receptor CCR2 (FIG. 21), based upon flow cytometry data and binding
of an anti-CCR2 antibody.
[0414] The ligand for CCR2 is MCP-1 that mediate migration of
monocytes in inflammation. In accordance with the CCR2 expression,
biotinylated MCP-1 (bMCP-1) could bind to blood monocytes from a
sarcoidosis patient (FIG. 22).
[0415] The CCR2 expressing monocytes could be depleted with
bMCP1-conjugated Sepharose Streptavidin Matrix (FIG. 23).
[0416] The sarcoidosis patients exhibit an increased frequency of
circulating T cells that express the chemokine receptor CCR7 (FIG.
24a), based upon flow cytometry data and binding by an anti-CCR7
antibody. Furthermore, the frequency of central memory T cells,
which are characterized as CCR7 positive, is increased in
sarcoidosis. (FIG. 24b). Central memory T cells contribute to
inflammation by mounting a fast and strong immune response the
second time the inflammation is triggered, and may be responsible
for relapsing sarcoidosis.
[0417] The ligand for CCR7 is MIP3b. The CCR7 expressing T cells
could be efficiently depleted with bMIP3b-conjugated Sepharose
Streptavidin Matrix (FIG. 25)
[0418] We conclude that the frequency of CCR1 expressing monocytes
and T cells that express CCR7 is enhanced in Sarcoidosis. The CCR2
receptor is expressed on monocytes from sarcoidosis patients to the
same extent as in the healthy controls, but the CCR2 expressing
cells could differ in their pro-inflammatory profile in the
patients compared to healthy controls. Both monocytes and T cells
bind the chemokines that corresponded with the expression of the
chemokine receptor, and could be efficiently depleted with the
corresponding biotinylated chemokine-Sepharose
Streptavidin-matrix.
[0419] The present various embodiments of the invention is not to
be limited in scope by the specific embodiments described herein.
Indeed, various modifications of the various embodiments of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims. Moreover, all embodiments
described herein are considered to be broadly applicable and
combinable with any and all other consistent embodiments, as
appropriate.
[0420] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
30199PRTArtificial sequenceSynthetic 1Met Lys Val Ser Ala Ala Leu
Leu Cys Leu Leu Leu Ile Ala Ala Thr1 5 10 15Phe Ile Pro Gln Gly Leu
Ala Gln Pro Asp Ala Ile Asn Ala Pro Val 20 25 30Thr Cys Cys Tyr Asn
Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu 35 40 45Ala Ser Tyr Arg
Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60Ile Phe Lys
Thr Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln65 70 75 80Lys
Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90
95Pro Lys Thr276PRTArtificial sequenceSynthetic 2Gln Pro Asp Ala
Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr1 5 10 15Asn Arg Lys
Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr 20 25 30Ser Ser
Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala 35 40 45Lys
Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met 50 55
60Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr65 70
75368PRTArtificial sequenceSynthetic 3Val Thr Cys Cys Tyr Asn Phe
Thr Asn Arg Lys Ile Ser Val Gln Arg1 5 10 15Leu Ala Ser Tyr Arg Arg
Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala 20 25 30Val Ile Phe Lys Thr
Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys 35 40 45Gln Lys Trp Val
Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln 50 55 60Thr Pro Lys
Thr654104PRTArtificial sequenceSynthetic 4Met Lys Ile Ser Thr Leu
Leu Cys Leu Leu Leu Ile Ala Thr Thr Ile1 5 10 15Ser Pro Gln Val Leu
Ala Gly Pro Asp Ala Val Ser Thr Pro Val Thr 20 25 30Cys Cys Tyr Asn
Val Val Lys Gln Lys Ile His Val Arg Lys Leu Lys 35 40 45Ser Tyr Arg
Arg Ile Thr Ser Ser Gln Cys Pro Arg Glu Ala Val Ile 50 55 60Phe Arg
Thr Ile Leu Asp Lys Glu Ile Cys Ala Asp Pro Lys Glu Lys65 70 75
80Trp Val Lys Asn Ser Ile Asn His Leu Asp Lys Thr Ser Gln Thr Phe
85 90 95Ile Leu Glu Pro Ser Cys Leu Gly 100582PRTArtificial
sequenceSynthetic 5Gly Pro Asp Ala Val Ser Thr Pro Val Thr Cys Cys
Tyr Asn Val Val1 5 10 15Lys Gln Lys Ile His Val Arg Lys Leu Lys Ser
Tyr Arg Arg Ile Thr 20 25 30Ser Ser Gln Cys Pro Arg Glu Ala Val Ile
Phe Arg Thr Ile Leu Asp 35 40 45Lys Glu Ile Cys Ala Asp Pro Lys Glu
Lys Trp Val Lys Asn Ser Ile 50 55 60Asn His Leu Asp Lys Thr Ser Gln
Thr Phe Ile Leu Glu Pro Ser Cys65 70 75 80Leu Gly676PRTArtificial
sequenceSynthetic 6Gly Pro Asp Ala Val Ser Thr Pro Val Thr Cys Cys
Tyr Asn Val Val1 5 10 15Lys Gln Lys Ile His Val Arg Lys Leu Lys Ser
Tyr Arg Arg Ile Thr 20 25 30Ser Ser Gln Cys Pro Arg Glu Ala Val Ile
Phe Arg Thr Ile Leu Asp 35 40 45Lys Glu Ile Cys Ala Asp Pro Lys Glu
Lys Trp Val Lys Asn Ser Ile 50 55 60Asn His Leu Asp Lys Thr Ser Gln
Thr Phe Ile Leu65 70 75776PRTArtificial sequenceSynthetic 7Gly Pro
Asp Ala Val Ser Thr Pro Val Thr Cys Cys Tyr Asn Val Val1 5 10 15Lys
Gln Lys Ile His Val Arg Lys Leu Lys Ser Tyr Arg Arg Ile Thr 20 25
30Ser Ser Gln Cys Pro Arg Glu Ala Val Ile Phe Arg Thr Ile Leu Asp
35 40 45Lys Glu Ile Cys Ala Asp Pro Lys Glu Lys Trp Val Lys Asn Ser
Ile 50 55 60Asn His Leu Asp Lys Thr Ser Gln Thr Phe Lys Leu65 70
75897PRTArtificial sequenceSynthetic 8Met Lys Val Ser Ala Ala Leu
Leu Trp Leu Leu Leu Ile Ala Ala Ala1 5 10 15Phe Ser Pro Gln Gly Leu
Ala Gly Pro Ala Ser Val Pro Thr Thr Cys 20 25 30Cys Phe Asn Leu Ala
Asn Arg Lys Ile Pro Leu Gln Arg Leu Glu Ser 35 40 45Tyr Arg Arg Ile
Thr Ser Gly Lys Cys Pro Gln Lys Ala Val Ile Phe 50 55 60Lys Thr Lys
Leu Ala Lys Asp Ile Cys Ala Asp Pro Lys Lys Lys Trp65 70 75 80Val
Gln Asp Ser Met Lys Tyr Leu Asp Gln Lys Ser Pro Thr Pro Lys 85 90
95Pro974PRTArtificial sequenceSynthetic 9Gly Pro Ala Ser Val Pro
Thr Thr Cys Cys Phe Asn Leu Ala Asn Arg1 5 10 15Lys Ile Pro Leu Gln
Arg Leu Glu Ser Tyr Arg Arg Ile Thr Ser Gly 20 25 30Lys Cys Pro Gln
Lys Ala Val Ile Phe Lys Thr Lys Leu Ala Lys Asp 35 40 45Ile Cys Ala
Asp Pro Lys Lys Lys Trp Val Gln Asp Ser Met Lys Tyr 50 55 60Leu Asp
Gln Lys Ser Pro Thr Pro Lys Pro65 701076PRTArtificial
sequenceSyntheticmisc_feature(1)..(1)Xaa is pyroGlu or Gln 10Xaa
Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr1 5 10
15Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr
20 25 30Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val
Ala 35 40 45Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp
Ser Met 50 55 60Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr65
70 751176PRTArtificial sequenceSyntheticmisc_feature(1)..(1)Xaa is
pyroGlu or Glnmisc_feature(75)..(75)Xaa is K(ivDde) 11Xaa Pro Asp
Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr1 5 10 15Asn Arg
Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr 20 25 30Ser
Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala 35 40
45Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met
50 55 60Asp His Leu Asp Lys Gln Thr Gln Thr Pro Xaa Thr65 70
751276PRTArtificial sequenceSyntheticmisc_feature(1)..(1)Xaa is
pyroGlu or Glnmisc_feature(75)..(75)Xaa is an amino acid residue
that can be biotinylated, such as lysine, ornithine or
diaminopropionic acid and optionally is biotinylated, optionally
via a spacer molecule such as PEG, e.g. K(PEG-Biotin) 12Xaa Pro Asp
Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr1 5 10 15Asn Arg
Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr 20 25 30Ser
Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala 35 40
45Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met
50 55 60Asp His Leu Asp Lys Gln Thr Gln Thr Pro Xaa Thr65 70
751376PRTArtificial sequenceSyntheticmisc_feature(1)..(1)Xaa is
pyroGlu or Gln 13Xaa Pro Asp Ser Val Ser Ile Pro Ile Thr Cys Cys
Phe Asn Val Ile1 5 10 15Asn Arg Lys Ile Pro Ile Gln Arg Leu Glu Ser
Tyr Thr Arg Ile Thr 20 25 30Asn Ile Gln Cys Pro Lys Glu Ala Val Ile
Phe Lys Thr Lys Arg Gly 35 40 45Lys Glu Val Cys Ala Asp Pro Lys Glu
Arg Trp Val Arg Asp Ser Met 50 55 60Lys His Leu Asp Gln Ile Phe Gln
Asn Leu Lys Pro65 70 751476PRTArtificial
sequenceSyntheticmisc_feature(1)..(1)Xaa is pyroGlu or
Glnmisc_feature(75)..(75)Xaa is K(ivDde) 14Xaa Pro Asp Ser Val Ser
Ile Pro Ile Thr Cys Cys Phe Asn Val Ile1 5 10 15Asn Arg Lys Ile Pro
Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr 20 25 30Asn Ile Gln Cys
Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly 35 40 45Lys Glu Val
Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met 50 55 60Lys His
Leu Asp Gln Ile Phe Gln Asn Leu Xaa Pro65 70 751576PRTArtificial
sequenceSyntheticmisc_feature(1)..(1)Xaa is pyroGlu or
Glnmisc_feature(75)..(75)Xaa is an amino acid residue that can be
biotinylated, such as lysine, ornithine or diaminopropionic acid
and optionally is biotinylated, optionally via a spacer molecule
such as PEG, e.g. K(PEG-Biotin) 15Xaa Pro Asp Ser Val Ser Ile Pro
Ile Thr Cys Cys Phe Asn Val Ile1 5 10 15Asn Arg Lys Ile Pro Ile Gln
Arg Leu Glu Ser Tyr Thr Arg Ile Thr 20 25 30Asn Ile Gln Cys Pro Lys
Glu Ala Val Ile Phe Lys Thr Lys Arg Gly 35 40 45Lys Glu Val Cys Ala
Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met 50 55 60Lys His Leu Asp
Gln Ile Phe Gln Asn Leu Xaa Pro65 70 751668PRTArtificial
sequenceSynthetic 16Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe
Ala Tyr Ile Ala1 5 10 15Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr
Phe Tyr Thr Ser Gly 20 25 30Lys Cys Ser Asn Pro Ala Val Val Phe Val
Thr Arg Lys Asn Arg Gln 35 40 45Val Cys Ala Asn Pro Glu Lys Lys Trp
Val Arg Glu Tyr Ile Asn Ser 50 55 60Leu Glu Lys
Ser651768PRTArtificial sequenceSyntheticmisc_feature(67)..(67)Xaa
is K(ivDde) 17Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala
Tyr Ile Ala1 5 10 15Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe
Tyr Thr Ser Gly 20 25 30Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr
Arg Lys Asn Arg Gln 35 40 45Val Cys Ala Asn Pro Glu Lys Lys Trp Val
Arg Glu Tyr Ile Asn Ser 50 55 60Leu Glu Xaa Ser651868PRTArtificial
sequenceSyntheticmisc_feature(67)..(67)Xaa is an amino acid residue
that can be biotinylated, such as lysine, ornithine or
diaminopropionic acid and optionally is biotinylated, optionally
via a spacer molecule such as PEG (e.g. K(Biotin)) 18Ser Pro Tyr
Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala1 5 10 15Arg Pro
Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly 20 25 30Lys
Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln 35 40
45Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser
50 55 60Leu Glu Xaa Ser651978PRTArtificial
sequenceSyntheticmisc_feature(78)..(78)Xaa is an amino acid residue
that can be biotinylated, such as lysine, ornithine or
diaminopropionic acid and optionally is biotinylated (e.g.
K-biotin), optionally via a spacer molecule such as PEG, in
particular K(PEG-Biotin) 19Gly Thr Asn Asp Ala Glu Asp Cys Cys Leu
Ser Val Thr Gln Lys Pro1 5 10 15Ile Pro Gly Tyr Ile Val Arg Asn Phe
His Tyr Leu Leu Ile Lys Asp 20 25 30Gly Cys Arg Val Pro Ala Val Val
Phe Thr Thr Leu Arg Gly Arg Gln 35 40 45Leu Cys Ala Pro Pro Asp Gln
Pro Trp Val Glu Arg Ile Ile Gln Arg 50 55 60Leu Gln Arg Thr Ser Ala
Lys Met Lys Arg Arg Ser Ser Xaa65 70 752078PRTArtificial
sequenceSyntheticmisc_feature(78)..(78)Xaa is K(ivDde) 20Gly Thr
Asn Asp Ala Glu Asp Cys Cys Leu Ser Val Thr Gln Lys Pro1 5 10 15Ile
Pro Gly Tyr Ile Val Arg Asn Phe His Tyr Leu Leu Ile Lys Asp 20 25
30Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr Leu Arg Gly Arg Gln
35 40 45Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu Arg Ile Ile Gln
Arg 50 55 60Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg Ser Ser
Xaa65 70 752178PRTArtificial
sequenceSyntheticmisc_feature(78)..(78)Xaa is K(Biotin) 21Gly Thr
Asn Asp Ala Glu Asp Cys Cys Leu Ser Val Thr Gln Lys Pro1 5 10 15Ile
Pro Gly Tyr Ile Val Arg Asn Phe His Tyr Leu Leu Ile Lys Asp 20 25
30Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr Leu Arg Gly Arg Gln
35 40 45Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu Arg Ile Ile Gln
Arg 50 55 60Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg Ser Ser
Xaa65 70 752278PRTArtificial
sequenceSyntheticmisc_feature(78)..(78)Xaa is an amino acid residue
that can be biotinylated, such as lysine, ornithine or
diaminopropionic acid and optionally is biotinylated, optionally
via a spacer molecule such as PEG, e.g. K(PEG-Biotin) 22Ala Val Leu
Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys1 5 10 15Thr Tyr
Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val 20 25 30Ile
Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu 35 40
45Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln
50 55 60Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser Xaa65
70 752378PRTArtificial sequenceSyntheticmisc_feature(78)..(78)Xaa
is K(ivDde) 23Ala Val Leu Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln
Cys Ile Lys1 5 10 15Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys
Glu Leu Arg Val 20 25 30Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu
Ile Ile Val Lys Leu 35 40 45Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro
Lys Glu Asn Trp Val Gln 50 55 60Arg Val Val Glu Lys Phe Leu Lys Arg
Ala Glu Asn Ser Xaa65 70 752478PRTArtificial
sequenceSyntheticmisc_feature(78)..(78)Xaa is K(PEG-Biotin) 24Ala
Val Leu Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys1 5 10
15Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val
20 25 30Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val Lys
Leu 35 40 45Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp
Val Gln 50 55 60Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser
Xaa65 70 752573PRTArtificial
sequenceSyntheticmisc_feature(73)..(73)Xaa is an amino acid residue
that can be biotinylated, such as lysine, ornithine or
diaminopropionic acid and optionally is biotinylated, optionally
via a spacer molecule such as PEG, e.g. K(PEG-Biotin) 25Ser Ala Lys
Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro1 5 10 15Phe His
Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro 20 25 30His
Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu 35 40
45Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys
50 55 60Phe Leu Lys Arg Ala Glu Asn Ser Xaa65 702673PRTArtificial
sequenceSyntheticmisc_feature(73)..(73)Xaa is K(ivDde) 26Ser Ala
Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro1 5 10 15Phe
His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro 20 25
30His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu
35 40 45Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln Arg Val Val Glu
Lys 50 55 60Phe Leu Lys Arg Ala Glu Asn Ser Xaa65
702773PRTArtificial sequenceSyntheticmisc_feature(73)..(73)Xaa is
K(PEG-Biotin) 27Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys Thr Tyr
Ser Lys Pro1 5 10 15Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val Ile
Glu Ser Gly Pro 20 25 30His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu
Ser Asp Gly Arg Glu 35 40 45Leu Cys Leu Asp Pro Lys Glu Asn Trp Val
Gln Arg Val Val Glu Lys 50 55
60Phe Leu Lys Arg Ala Glu Asn Ser Xaa65 702874PRTArtificial
sequenceSyntheticmisc_feature(73)..(73)Xaa is an amino acid residue
that can be biotinylated, such as lysine, ornithine or
diaminopropionic acid and optionally is biotinylated, optionally
via a spacer molecule such as PEG, e.g. K(PEG-Biotin) 28Gly Pro Ala
Ser Val Pro Thr Thr Cys Cys Phe Asn Leu Ala Asn Arg1 5 10 15Lys Ile
Pro Leu Gln Arg Leu Glu Ser Tyr Arg Arg Ile Thr Ser Gly 20 25 30Lys
Cys Pro Gln Lys Ala Val Ile Phe Lys Thr Lys Leu Ala Lys Asp 35 40
45Ile Cys Ala Asp Pro Lys Lys Lys Trp Val Gln Asp Ser Met Lys Tyr
50 55 60Leu Asp Gln Lys Ser Pro Thr Pro Xaa Pro65
702974PRTArtificial sequenceSyntheticmisc_feature(73)..(73)Xaa is
K(ivDde) 29Gly Pro Ala Ser Val Pro Thr Thr Cys Cys Phe Asn Leu Ala
Asn Arg1 5 10 15Lys Ile Pro Leu Gln Arg Leu Glu Ser Tyr Arg Arg Ile
Thr Ser Gly 20 25 30Lys Cys Pro Gln Lys Ala Val Ile Phe Lys Thr Lys
Leu Ala Lys Asp 35 40 45Ile Cys Ala Asp Pro Lys Lys Lys Trp Val Gln
Asp Ser Met Lys Tyr 50 55 60Leu Asp Gln Lys Ser Pro Thr Pro Xaa
Pro65 703074PRTArtificial
sequenceSyntheticmisc_feature(73)..(73)Xaa is K(PEG-Biotin) 30Gly
Pro Ala Ser Val Pro Thr Thr Cys Cys Phe Asn Leu Ala Asn Arg1 5 10
15Lys Ile Pro Leu Gln Arg Leu Glu Ser Tyr Arg Arg Ile Thr Ser Gly
20 25 30Lys Cys Pro Gln Lys Ala Val Ile Phe Lys Thr Lys Leu Ala Lys
Asp 35 40 45Ile Cys Ala Asp Pro Lys Lys Lys Trp Val Gln Asp Ser Met
Lys Tyr 50 55 60Leu Asp Gln Lys Ser Pro Thr Pro Xaa Pro65 70
* * * * *