U.S. patent application number 13/107504 was filed with the patent office on 2012-01-12 for complete human monoclonal igg4lambda specific for ctla-4 and uses thereof for detection of soluble ctla-4 and isolation of regulatory cells.
Invention is credited to Li-Te CHIN, Shu-Ching Hsu.
Application Number | 20120009207 13/107504 |
Document ID | / |
Family ID | 40998524 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120009207 |
Kind Code |
A1 |
CHIN; Li-Te ; et
al. |
January 12, 2012 |
Complete human monoclonal IgG4lambda specific for CTLA-4 and uses
thereof for detection of soluble CTLA-4 and isolation of regulatory
cells
Abstract
The present invention provides a CTLA-4 non-blocking agent of a
complete human antibody nature, thus is non-immunogenic in a human.
The immunoassay method using such a non-blocking agent measures the
CTLA-4 content in a sample of a human subject. The present
invention further provides a novel method for isolating human
regulatory T cells. The resultant enriched and depleted cellular
populations are useful in treating or ameliorating of human
diseases.
Inventors: |
CHIN; Li-Te; (Hsinchu,
TW) ; Hsu; Shu-Ching; (Yanchao, TW) |
Family ID: |
40998524 |
Appl. No.: |
13/107504 |
Filed: |
May 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11943682 |
Nov 21, 2007 |
7968300 |
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13107504 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
A61K 35/26 20130101;
C07K 2317/92 20130101; G01N 33/505 20130101; C07K 16/2818 20130101;
A61K 38/17 20130101; C07K 2317/34 20130101; A61P 37/06
20180101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61P 37/06 20060101 A61P037/06 |
Claims
1. A method of isolating regulatory T cells from human samples,
said method comprising: (a) contacting a population of suspended
cells in a sample with a CTLA-4 non-blocking agent that recognizes
the extracellular domain of CTLA-4, wherein said CTLA-4
non-blocking agent is non-immunogenic in a human and comprises an
antibody or an antigen-binding fragment thereof; and (b) selecting
cells that bind to the CTLA-4 non-blocking agent, wherein the
selected cells are enriched for regulatory T cells, and (c) further
comprising transfusing the enriched population of said regulatory T
cells into a subject to suppress the autoimmune response.
2. (canceled)
3. The method of claim 1 wherein the isolated and enriched cellular
population is expanded in cell culture before transfusing into a
subject.
4. The method of claim 1 wherein the negative or exclusive cellular
populations of step (b) that do not bind to the CTLA-4 non-blocking
agent are deficient in regulatory T cells.
5. The method of claim 4 further comprising introducing said
depleted cellular population into a subject to enhance the
activation of T cells.
6. The method of claim 5 wherein the depleted cellular population
further contacts an antigen in cell culture before introducing into
a subject.
Description
[0001] The current application is a divisional application of, and
claims a priority to U.S. Ser. No. 11/943,682 filed on Nov. 21,
2007, now issued as U.S. Pat. No. 7,968,300. All references and IDS
filed with Ser. No. 11/943,682 are incorporated herewith.
FIELD OF THE INVENTION
[0002] This invention relates to the study of human CTLA-4
(cytotoxic T lymphocyte antigen-4, or CD152) that represents an
essential receptor involved in negative regulation of T cell
activation. More particularly, it relates to the identification of
an antigen specific to human CTLA-4 molecule, a complete human
monoclonal antibody which specifically binds to the antigen, and
various uses for the monoclonal antibody, such as detection and
isolation agents.
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"Complementarity determining region 1 (CDR1)- and CDR3-analogous
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IgG1 and IgE production by stimulation of the B cell CTLA-4
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2001, "Synaptic pattern formation during cellular recognition,"
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"Successful bone marrow transplantation for IPEX syndrome after
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abrogates their function in vivo," J. Immunol., 177:4376-83. [0039]
Sakaguchi, S, 2005, "Naturally arising Foxp3-expressing CD25+CD4+
regulatory T cells in immunological tolerance to self and
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"Serum soluble CTLA-4 levels are increased in diffuse cutaneous
systemic sclerosis," Rheumatology, 43:1261-6. [0041] Schwartz, J C,
et al, 2001, "Structural basis for co-stimulation by the human
CTLA-4/B7-2 complex," Nature, 410:604-8. [0042] Sharpe, A H and
Freeman, G J, 2002, "The B7-CD28 superfamily" Nat. Rev. Immunol.,
2:116-26. [0043] Stamper, C C, et al, 2001, "Crystal structure of
the B7-1/CTLA-4 complex that inhibits human immune responses,"
Nature, 410:608-11. [0044] Steiner, K, et al, 1999, "Enhanced
expression of CTLA-4 (CD152) on CD4+ T cells in HIV infection,"
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"Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory
T cells constitutively expressing cytotoxic T lymphocyte-associated
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1995, "Loss of CTLA-4 leads to massive lymphoproliferation and
fatal multiorgan tissue destruction, revealing a critical negative
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H, 2005, "Mechanisms of suppression by suppressor T cells," Nat.
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[0055] All of the publications, patents and patent applications
cited above or elsewhere in this application are herein
incorporated by reference in their entirety to the same extent as
if the disclosure of each individual publication, patent
application or patent was specifically and individually indicated
to be incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0056] On-going studies of the collaboration scenarios between T
cells and antigen-presenting cells (APCs) foresaw dramatic changes
which have revealed some therapeutic targets and medicinal
potentials in certain regards. For example, it has been shown that
the specialized and dynamic molecular machinery, present in the
tight junction between a T cell and an APC, regulates immunological
responses (Dustin, 2002; Grakoui et al., 1999; Qi et al., 2001). It
has also been inferred that the machinery, termed the immunological
synapse, correlates with a high degree of intercellular
communication controlling disparate biological process (Davis and
Dustin, 2004). A number of molecules have been confined at the
immunological synapse to ensure their interim expression and
interaction at the right time and place thus the sum and
integration of signals are relevant to evoke appropriate T cell
responses. Within this limited and m-sized area is full of
interacting molecules, of which CTLA-4 has been identified to be
responsible for inhibiting T cell responses in a T cell receptor
(TCR)-dependent manner (Chikuma and Bluestone, 2002; Egen and
Allison, 2002).
[0057] Human CTLA-4 was mapped to band q33 of chromosome 2 and was
classified into a group of immunomodulating receptors, collectively
termed as CD28 superfamily (Sharpe and Freeman, 2002). As shown in
FIG. 1, the complete cDNA sequence of human CTLA-4 (isoform A) has
the Genbank accession number L15006 and the structure has the
accession number 1AH1 in the Molecular Modeling DataBase (MMDB) of
NCBI's structure database. The region of amino acids -35 to 0 is
the signal peptide; 1-126 is the extracellular V-like domain;
123-151 is the transmembrane domain; and 152-188 is the cytoplasmic
domain. It is well established that two members of this
superfamily, CD28 and CTLA-4, have opposing functions and that
CTLA-4 represents one of the major inhibitory receptors involved in
co-stimulatory pathways regulating both humoral and cellular immune
responses (Krummel and Allison, 1996; Linsley et al., 1991; Pioli
et al., 2000). A majority of studies indicate that CD28 provides
direct enhancement signals, including up-regulation/stabilization
of cytokine gene transcription, improved cell survival, lowered
threshold for activation, and cytoskeletal effects; however,
information on the function of CTLA-4 is much less clear. Thus far,
the most compelling evidence for an inhibitory role of CTLA-4 is
derived from the deficient knockout mice (CTLA-4.sup.-/-) (Tivol et
al., 1995). These mice suffer from a fatal T-cell
lymphoproliferative disorder with splenomegaly, lymphoadenopathy
and hyper-responsive infiltration in several organs including heart
that become apparent by four weeks after birth. This fetal disorder
is presumably due to reactivities to multiple self-antigens, since
the expression of a single transgenic TCR prevents this disease.
The TCR-dependent activation in these knockout mice appears to
require CD28 costimulation, because CTLA-4.sup.-/-CD28.sup.-/- mice
do not suffer the lymphoproliferative disease. Likewise, treatment
of these mice perinatally with soluble CTLA-4-Ig, which competes
ligands access of cell surface CTLA-4, prevents such a disease
effectively. Nonetheless, the mechanism of CTLA-4 action is still
unclear, with no obvious central theme.
[0058] Conceptually, the interaction of CD28 on the lymphocyte with
B7 proteins on the APC provides a necessary costimulatory second
signal for a T cell to be able to fully respond to an antigen. The
original family members in the pathway consist of two B7
ligands--CD80 (B7-1) and CD86 (B7-2), which have specificities
towards the two receptors--CD28 and CTLA-4. CD28 is constitutively
expressed on the surface of T cells whereas CTLA-4 surface
expression is rapidly up-regulated to a limited extent following T
cell activation. The kinetics of expression of CD80 and CD86 also
differ. CD86 is constitutively expressed on interdigitating
dendritic cells, Langerhans cells, peripheral blood dendritic
cells, memory B cells and germinal center B cells. Furthermore,
CD86 is expressed at low levels on monocytes, but its rapid
up-regulation through IFN-.gamma. stimulation has led to the
hypothesis that CD86 functions primarily in initiating an immune
response. On the other hand, CD80, being expressed later in time,
may serve to amplify or regulate the response. Newly identified
family members of the related molecules include: the inducible
costimulatory molecule (ICOS), program death 1 (PD-1) receptor, B
and T lymphocyte attenuator (BTLA), B7-H1, B7-H2, B7-H3, B7-H4,
PD-1 ligand 1 (PD-L1) and PD-L2 (Wang and Chen, 2004). The novel
interactions among these new family members underscore additional
complexity of this costimulatory pathway in mounting an appropriate
immune response.
[0059] A shorter soluble form of CTLA-4 lacking the transmembrane
region has been achieved from RT-PCR cloning of non-activated T
cells in animals as well as humans (Magistrelli et al., 1999; Oaks
et al., 2000). Soluble CTLA-4 (sCTLA-4, CTLA-4 isoform B; FIG. 1)
seems to be a fully functional CD80 and CD86 receptor, thus likely
to affect T-cell responses in a paracrine manner. Furthermore,
immunoreactive sCTLA-4 can be detected in the serum of 14/64
healthy subjects. In addition, the presence of high concentration
of sCTLA-4 was observed in sera of patients with autoimmune thyroid
diseases such as Graves' disease (Oaks and Hallett, 2000). Finally,
recent reports show that sCTLA-4 levels are augmented in patients
with autoimmune diseases, such as type-1 diabetes (Nistico et al.,
1996), diffuse cutaneous systemic sclerosis (Sato et al., 2004),
systemic lupus erythematosus (Wong et al. (2005) Rheumatology
44:989), and allergic asthma (Wong et al. (2005) Clin. Exp.
Immunol. 141:122). It has been shown that activated T cells
suppress sCTLA-4 mRNA expression and express preferentially the
membrane-bound, full-length CTLA-4 (flCTLA-4) mRNA (Gough et al.,
2005). Thus the ratio of sCTLA-4 to flCTLA-4 may have an important
role in the regulation of immune homeostasis. The alternate
transcripts or spliced variants of sCTLA-4, which lack the
transmembrane encoding regions, were first deposited in the GenBank
Sequence Database in humans, mice, and rats (accession numbers
U90273, U90270, and U90271) in 1997 followed by a description of
the same transcript in humans, being expressed by non-stimulated
human T cells. The endogenous 174-aa soluble form, designed as
isoform B, can be retrieved under the accession number
NP.sub.--001032720.
[0060] It is also known in the field that immune reactivity is
further controlled by various types of regulatory T cells (Tregs)
(Sakaguchi, 2005). Tregs can be broadly divided into two subsets,
i.e., the natural Treg cells of CD4.sup.+CD25.sup.+ phenotype,
which constitute 5-10% of peripheral T cells, and the
stimulation-induced (or adaptive) Treg cells identified in various
models of inflammation, alloreactivity, or autoimmunity
(Prud'homme, 2004). Recent findings suggest that the suppressive
potential of CD4.sup.+CD25.sup.+ natural Tregs to other activated
effector T cells is mediated by restricting early proliferation and
the anti-effector function in inflamed tissues (von Boehmer, 2005).
The forkhead-family transcription factor gene FOXP3, encoding the
scurfin transcriptional regulator (Genbank accession number
EF534714, NCBI protein accession number ABQ15210), has been
implicated in the development and function of natural Tregs (Hori
et al., 2003). A FOXP3 mutation in scurfy mice results in the
absence of Tregs and early death from a multi-organ inflammatory
disorder similar to the CTLA-4 or TGF-.beta. deficiency. FOXP3 was
shown to function as a transcriptional repressor, targeting
composite NF-AT/AP-1 sites in cytokine gene promoters and the
region responsible for NF-AT inhibition was mapped to the amino
terminus (Lopes et al., 2006; Wu et al., 2006).
[0061] In principle, conventional techniques to isolate this rare
Treg population often involve a two-step, multiple antibody
selection procedure (Miltenyi Biotec, Bergisch Gladbach, Germany;
BD Biosciences Pharmingen, San Jose, Calif.). Briefly, CD4.sup.+ T
lymphocytes are first preserved from not binding to a cocktail of
mAbs that recognize other CD antigens expressed on erythrocytes,
platelets, monocytes and peripheral leukocytes, etc. Subsequently,
anti-human CD25 mAb positively selects the CD25.sup.+ cells from
the enriched CD4.sup.+ cells, yielding CD4.sup.+CD25.sup.+ Treg
cells. However, inevitably intermittent exposure to environmental
pathogens results in traditional effector T cell activation and
consequently expression of CD25 on human CD4.sup.+ T cells, making
identification of the Treg population a very difficult task.
Additionally, even CD4.sup.-CD8.sup.+ natural Treg cells have been
reported by Xystrakis et al. (2004) Blood 104: 3294-3301,
indicating that Treg is a heterogeneous population. Furthermore,
although FOXP3 expression is found predominantly within the Tregs,
its intracellular nuclear localization causes direct detection
impossible to live cells. Therefore, the characterization and
application of Treg cells have been hampered by a lack of specific
molecular markers on the surface of Tregs. A more complex approach
was engineered to circumvent this particular problem, in which
purified CD4.sup.+CD25.sup.+ peripheral blood mononuclear cells are
further activated with agents such as ionomycin, and the Tregs are
isolated based on binding to CTLA-4 blocking mAb (Birebent et al.,
2004). Yet an even more complicated process has evolved by using
additional surface markers like CD45RA and CD127 (WO
2007/117602).
[0062] Of interest is the association and potential synergism
between the suppressive function of Tregs and the CTLA-4
expression. Unusually for non-activated T cells, Tregs
constitutively express CTLA-4 (Takahashi et al., 2000), and CTLA-4
blockade on the Treg by specific blocking mAb can attenuate their
suppressive activity, leading to the development of autoimmune
disease in vivo (Read et al., 2006). In addition, it has been
observed not only that the reported CD4.sup.-CD8.sup.+ natural
Tregs express CTLA-4 (Xystrakis et al. 2004) but also that
CD4.sup.+CD25.sup.+ cells further purified on the basis of
recycling CTLA-4 are much more potent as regarding suppression
(Birebent, et al., 2004). More importantly, the fact that inducible
Tregs were the dominant source of sCTLA-4 was revealed in the 2007
British Society for Immunology Congress (Ward and Barker, 2007).
Together, they indicate a strong correlation between CTLA-4
expression and suppressive regulatory function, supportive of the
concept that CTLA-4 is functionally relevant to Tregs.
[0063] Because Tregs, in accompany with sCTLA-4, are involved in
preventing allograft rejection and graft versus host disease and
exert a dominant effect in controlling autoimmunity and maintaining
peripheral tolerance, specific immune therapies designed to isolate
and then expand them may improve the clinical course of various
T-cell mediated pathology. As CTLA-4 provides the most important
attenuating costimulatory signals, it will be expected by one of
skill in the art that these molecules offer new targets for
immunotherapy and diagnostics.
[0064] Studies of the physiological function and practical uses of
CTLA-4 became possible with the isolation of monoclonal antibodies
(mAbs). The first reported mouse anti-human CTLA-4 mAb (clone 11D4)
suggested that blocking CTLA-4 signaling might deliver a positive
signal synergizes with that delivered by CD28 (Linsley et al.,
1992). The immune-enhancing nature of CTLA-4 antagonism has thus
opened the possibility for a readily applicable tumor immunotherapy
by temporary removal of CTLA-4-mediated inhibition using
antagonistic Abs (Egen et al., 2002). Although the mechanisms by
which CTLA-4 regulates T cell responses are not completely
understood, blocking its activity with an antagonistic or blocking
mAb offers a novel approach that holds a promise for immunotherapy.
A set of corresponding U.S. patents such as U.S. Pat. No.
5,811,097, No. 5,855,887, No. 6,051,227, No. 6,207,156 and No.
7,229,628, illustrates approach of CTLA-4 blockade to strongly
enhance antitumor responses has been highly regarded for the
treatment potentials.
[0065] The anti-CTLA4 blocking mAbs, e.g., clone BNI3 (Steiner et
al., 1999) (commercially available from BD Pharmingen) and clone
AS33 (Antibody Solutions, Mountain View, Calif.), are often in use
to detect sCTLA-4 in biological fluid (Oaks and Hallett, 2000) and
to purify Tregs from activated peripheral blood (Birebent, et al.,
2004). However, these may not be the best available strategy.
Structural analyses have shown that the human CTLA-4 protein is
composed of disulfide-linked homodimers of extracellular
immunoglobulin variable (IgV) domains, each domain consisting of
two layered (3-sheets with ten strands (A, A', B, C, C', C'', D, E,
F and G) that form three complementarity determining region
(CDR)-like regions (Schwartz et al., 2001; Stamper et al., 2001).
Together with one mutational study (Peach et al., 1994), these two
structural studies have independently pointed out that CDR1-like
(the B-C loop) and CDR3-like (the F-G loop) regions in CTLA-4
directly bind endogenous B7 ligands (CD80 and CD86), whereas CDR2's
responsibility is very trivial if there is any. Therefore, although
in the initial publications there is no definite information to
describe the CTLA-4 epitope on which the blocking mAbs bind,
antagonistic effects and the subsequent enhancement on T-cell
activation may be mediated by mAb competition that results from
specific binding with amino acid residues on or close to a room
encompassing CDR1 and CDR3. Thus uses of blocking mAbs in pair or
in combination with endogenous B7 ligands provide a possible
limitation caused by steric hindrances.
SUMMARY OF THE INVENTION
[0066] In one aspect, the present invention pertains to the
discovery herein that portions other than CDR1 and CDR3 of human
CTLA-4, such as the Met 55-cored CDR2-like sequence, do not play a
role in binding of B7 ligands. Accordingly, mAbs that recognize a
pre-determined Met 55-cored region can be used and are particularly
useful to detect sCTLA-4 and/or to purify Tregs. To explore the
effect of this Met 55-cored CDR2-like sequence, the inventors have
recently developed and had possession of a complete human
monoclonal IgG4.lamda., targeting this particular stretch (Chin et
al., 2007). As the mAb is capable, under the condition that the
binding of a natural agonist was not interrupted, to mediate high
(nanomolar) affinity binding to an extracellular constituency
encompassing the CDR2-like region of CTLA-4, whereby it can react
to activated human CD3.sup.+ cells, thus the invention can further
provide a method for detecting sCTLA-4 and/or isolation Tregs.
[0067] The method contemplated herein may lead to an increase in
sensitivity for sCTLA-4 detection and thus may be used to diagnose
those conditions in which disease activity is tightly associated
with sCTLA-4 production. Assays of interest include ELISA, RIA, FIA
and flow cytometry, etc. In one embodiment, the agent is selected
from the group consisting of: an antibody to CDR2 region of CTLA-4,
a blocking antibody to CTLA-4, or a preferred combination of an
antibody to CDR2 region of CTLA-4 and labeled human CD80. Binding
may be quantified by a variety of methods known in the art. After
an incubation period sufficient to allow the binding to reach
equilibrium, the insoluble support is washed, and the remaining
label quantified. The preferred agents in combination will enhance
the detected label in the presence of sCTLA-4 and thus increase the
detection limit.
[0068] On the other hand, the present invention may lead to an
increase in efficiency for Treg isolation and encompasses both in
vitro and in vivo methods. For in vitro uses, the cell
intrinsically possessing the CTLA-4 receptor without prior
activation, i.e., Tregs, may be purified from peripheral blood
mononuclear cells. Efficient purification strategies are known in
the art, including depletion and enrichment. Strategies of interest
include magnetic cell separation, panning, bead-based
chromatography and cytometry sorting, etc. As an example, purified
mAb can be bound to an insoluble support, e.g. microtiter plate,
magnetic beads, etc. The candidate cells are added to the support,
and the unbound components are then washed off. The target cells
are finally purified or isolated by elution. As to in vivo methods,
the Treg may be present in a mammal, especially a human subject
such as one who is suffering from declined or excessive Treg levels
and who could benefit from a respective increase or decrease in
Treg cells. Potential patients include those who have low or no
level of Treg and develop autoimmune diseases that require Treg
transfusion (Rao et al., 2007; Wildin and Freitas, 2005), or those
who undergone adoptive immunotherapy that demand Treg reduction
(Dannull et al., 2005). Thus, the invention provides a method for
repopulating Treg cells in a human comprising administering to the
human a therapeutically effective amount of a mAb.
[0069] In an embodiment, the invention provides antibodies that
specifically bind to the Met 55-cored CDR2-like region of human
CTLA-4. Preferred antibodies are monoclonal antibodies (mAbs) which
are non-immunogenic in a human and bind to an epitope in the
extracellular domain of CTLA-4. A preferred form of mAbs is human
monoclonal IgG4.lamda., or a fragment thereof. More preferably,
suitable mAbs will have an equilibrium dissociation constant (Kd)
at least about 10.sup.-6M toward human CTLA-4, more preferably at
least about 10.sup.-8M. The antibody is preferably an IgG antibody,
particularly IgG4.
[0070] According to a further aspect, the invention is concerned
with the CTLA-4 and a soluble form of this particular receptor
which is the CTLA-4 extracellular domain. The mAbs against the Met
55-cored CDR2-like region are optionally conjugated with, or fused
to, molecules which increase the serum half-lives thereof and can
be formulated as pharmaceutical compositions comprising the mAbs
and a physiologically acceptable carrier. Antibodies which bind to
the Met 55-cored CDR2-like region may optionally be fused to a
heterologous polypeptide or magnetic particles and the antibody or
fusion thereof may be used to isolate and purify CTLA-4.
[0071] In further embodiments, antibodies which bind to CTLA-4 may
optionally be fused or linked to a toxin and the antibody or fusion
thereof may be used to separate or kill Treg cells from a source of
human lymphocytes. Methods to fuse or link are known in the art,
including genetic and chemical techniques. Genetic manipulations
may include constructing an artificial nucleic acid segment
consists of amino acid residues of a toxin molecule and
antigen-binding domains derived from a mAb and producing said
construct in a proper host, as described in WO 2005/012495. The
immunotoxin may also be obtained from chemical conjugations such as
using a heterobifunctional reagent (e.g., N-succinimidyl
3-(2-pyridyldithio)propionate), carbodiimide linkage or mixed
anhydride procedure (Burstein and Knapp, 1977). The toxin moiety
can be, e.g., any of the following toxic polypeptides: ricin,
pseudomonas exotoxin, bryodin, diphtheria toxin, gelonin,
.alpha.-sarcin, aspergillin, restrictocin, angiogenin, saporin,
abrin, pokeweed antiviral protein, or a functional fragment of any
of these toxic polypeptides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1 shows the complete protein sequence of human CTLA-4
isoforms A and B with critical amino acids corresponding to CDR1, 2
and 3 are indicated. Asterisks indicate amino acid identity.
[0073] FIG. 2 illustrates the immunoblot screening of peptide
arrays denotes the mAb specificity against the Met 55-cored
CDR2-like region of human CTLA-4. 1 .mu.g/ml of purified mAb was
used to determine the binding epitope. Sequences of
EYASPGKATEVRVTV, KVELMYPPPYYLGIG and QYIKANSKFIGITEL indicate
CDR1-encompassing region, CDR3-encompassing region and T cell
epitope (italic) used for site-directed immunization,
respectively.
[0074] FIG. 3 demonstrates the immunological and biochemical
natures of the anti-CTLA-4 mAb. Panel A shows isotyping and
subtyping results by immobilizing anti-human Igs as indicated. The
binding profile of the mAb was subsequently revealed by
biotinylated CTLA-4-muIg and avidin-peroxidase conjugates. Results
indicate that the present mAb belongs to a type of IgG4%. Panel B
indicates the isoelectric point of the monoclonal IgG4.lamda.,
anti-CTLA-4 (lane 4 and 8), resolved by isoelectric focusing.
Monoclonal human myeloma IgG1.lamda., (lane 2 and 6) and
IgG4.lamda., (lane 3 and 7) were run in parallel for comparison.
The electrophoretic patterns were visualized by either Coomassie
brilliant blue staining (lane 1-5) or immunoblot (lane 6-8) with
anti-human IgG conjugated with peroxidase and FAST.TM. DAB (Sigma).
The calculated isoelectric points for human IgG1.lamda.,
IgG4.lamda., and anti-CTLA-4 mAb to be approximately in the range
of 7.92-8.79, 5.76-6.52 and 7.87-8.41, respectively, based on the
calibration against the linear regression of standard protein
markers. Panel C outlines the affinity determination by IAsys.
Surface plasmon resonance obtained at 25.degree. C. for increasing
concentrations of anti-CTLA-4 mAb on purified, unlabeled
CTLA-4-muIg. The straight line in the inset was obtained from the
k.sub.obs plot versus ligated Ab concentration and yielded a
k.sub.diss (the intercept) of 16.81 and a k.sub.ass value (the
gradient) of 4.20.times.10.sup.9. Therefore produced a Kd
(k.sub.diss/k.sub.ass) of 4.times.10.sup.-9 M.
[0075] FIG. 4 indicates the binding of the present anti-CTLA-4 and
CD80/CD86 agonists to human CTLA-4 are not mutually exclusive.
Results obtained from ligand competition assays that test the
ability of the complete human monoclonal anti-CTLA-4 (solid line)
and BNI3 (dashed line) anti-CTLA-4 to compete for the CD80/CD86 and
CTLA-4 interactions. Biotinylated CD80-muIg or CD86-muIg plus
indicated increasing concentrations of the mAbs (10.sup.-3-10
.mu.g/mL) were incubated in microtiter wells coated with purified
CTLA-4-muIg. Bound CD80/CD86 was detected with avidin-peroxidase
conjugate and a peroxidase substrate. The data shown are
representative of three experiments.
[0076] FIG. 5 is a representative combined data from the ELISA on
human CTLA-4 detection, using available mAbs and recombinant human
CD80 to CTLA-4. The assay was measured using commercially available
CTLA4-muIg as a standard. ELISA analysis showing the limit of
sensitivity of 0.39, 1.56 and 6.25 ng/ml for the assay using a pair
of the human monoclonal IgG4% and CD80 (.diamond-solid.), the human
IgG4% and BNI3 (.tangle-solidup.) and blocking mAbs of BNI3 and
AS-33 ( ), respectively.
[0077] FIGS. 6A and 6B present the effect of isolating Tregs based
on intrinsic CTLA-4 surface expression. In FIG. 6A, detection of
FOXP3 gene expression relative to GAPDH expression by real-time PCR
analysis in purified CD4.sup.+CD25.sup.- (.box-solid.),
CD4.sup.+CD25.sup.+ (.tangle-solidup.) and CTLA-4.sup.+ () T cells
from three normal donors is shown. Shown in FIG. 6B, pronounced
enhancements, as compared with the control group (.box-solid.), in
the kinetics of thymidine incorporation were observed when Tregs
were removed by the uses of CTLA-4.sup.+ () or
anti-CTLA-4-diphtheria toxin conjugate (.tangle-solidup.). It was
difficult to consistently recover CD4.sup.-CD25.sup.- population
and thus omitted from the analysis. The proliferation response of
CD4.sup.+CD25.sup.+ (.largecircle.) and CTLA-4.sup.+ cells
(.gradient.) is indicated. The data represent the mean of
triplicate samples.
DETAILED DESCRIPTION OF THE INVENTION
[0078] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
DEFINITIONS
[0079] A "complete human antibody" is an antibody containing
exclusively human sequences. The antibody is preferably a
monoclonal antibody.
[0080] The terms "CTLA-4" when used herein encompass the native
human sequence of CTLA-4 isoform A (FIG. 1). Optionally, the CTLA-4
is not associated with native glycosylation. "Native glycosylation"
refers to the carbohydrate moieties which are covalently attached
to CTLA-4 when it is produced in the mammalian cell from which it
is derived in nature. Accordingly, human CTLA-4 produced in a
non-human cell is an example of a CTLA-4 which is "not associated
with native glycosylation". Sometimes, CTLA-4 is unglycosylated as
a result of being produced recombinantly in a prokaryote or being
synthesized chemically.
[0081] "Soluble CTLA-4" or "sCTLA-4" is a CTLA-4 molecule which
contains neither a transmembrane domain nor a cytoplasmic tail and
represents the native human sequence of CTLA-4 isoform B (FIG. 1).
The "CTLA-4 extracellular domain" is a form of CTLA-4 which is
essentially free of the transmembrane and cytoplasmic domains of
CTLA-4. Ordinarily, the "CTLA-4 extracellular domain" will have an
amino acid sequence of least about 95% amino acid sequence identity
with the amino acid sequence of CTLA-4 isoform B indicated in FIG.
1, preferably includes CDR1-, CDR2- and CDR3-like regions.
[0082] An "antigenic function" means possession of an epitope or
antigenic site that is capable of cross-reacting with antibodies
raised against a native sequence of CTLA-4 defined by the CDR-like
regions. The principal antigenic function of a CDR2-like region is
that it does not involve in binding of CTLA-4, sCTLA-4 or CTLA-4
extracellular domain to B7 molecules.
[0083] "Immunize" a cell or an animal with an antigen refers to the
action of exposing the cell or the animal to the antigen. The cell
or animal can be immunized in any manner that leads to contact
between the cell or the animal with the antigen.
[0084] A "heteromyeloma cell line" is a cell line derived from
fusion of two different myeloma cells. The two different myeloma
cells are preferably a human myeloma cell and a murine myeloma
cell. Heteromyeloma cell lines are known in the art. For example,
U.S. Pat. No. 6,228,361 and Chin et al., 2001 describe the
preparation, characterization and use of various heteromyeloma cell
lines.
[0085] A "fusion partner" is a cell that can be used to fuse with
an antibody-producing cell for a beneficial purpose. Typically, the
fusion leads to prolonged antibody production. Thus, without fusion
to the fusion partner, the antibody-producing cell ceases to
produce antibodies in culture. Upon fusion to the fusion partner,
however, fused cells can be selected that produce antibodies in
culture for at least about 3 months, preferably at least about 6,
9, 12, 18, 24 months or more. Fusion partners include, but are not
limited to, myeloma cells and heteromyeloma cells.
[0086] An "agonist" is a molecule that can bind to cellular
receptors, e.g., CTLA-4, and thus can produce various biological
effects and initiate changes in cell function. Endogenous agonists
are generally nature-occurring ligands such as neurotransmitters
and, in the case of CTLA-4, CD80 and CD86. Exogenous agonists are
commonly found as drugs.
[0087] An "antagonist" is a molecule that can bind to receptors,
e.g., CTLA-4, but do not activate signal transduction mechanisms.
The biological effects of a given antagonist are derived from
preventing agonist binding and receptor activation, e.g., blocking
mAbs.
[0088] "Non-immunogenic in a human" means that upon contacting the
polypeptide of interest in a physiologically acceptable carrier and
in a therapeutically effective amount with the appropriate tissue
of a human, no state of sensitivity or resistance to the
polypeptide of interest is demonstrable upon the second
administration of the polypeptide of interest after an appropriate
latent period e.g., 8 to 14 days. It will be understood by one of
skill in the art that a polypeptide of complete human origin
typically represents "non-immunogenic in a human".
[0089] "Treating or ameliorating" a disease or medical condition
means reducing or eliminating the symptoms of the disease or
medical condition, or slowing down the progress of the
disease/medical condition. The reduction is preferably at least
about 10%, more preferably at least about 20%, 30%, 40%, 50%, 60%,
70%, 80% or 90%.
[0090] An "effective amount" is an amount of an agent that is
sufficient to result in the intended effect. For example, for an
antibody used to treat or ameliorate a disease, an effective amount
is an amount of the antibody sufficient to reduce or eliminate the
symptoms of the disease, or to slow down the progress of the
disease.
[0091] A "sample" is an aliquot or a representative portion of a
substance, material, or population. For example, a sample may be a
sample of water, sewage, oil, sand, blood, biological tissue, urine
or feces. A "biological sample" is a sample collected from or
present within a biological subject, preferably a human
subject.
[0092] "Essentially pure" protein means a composition comprising at
least about 90% by weight of the protein, based on total weight of
the composition, preferably at least about 95% by weight.
"Essentially homogeneous" protein means a composition comprising at
least about 99% by weight of protein, based on total weight of the
composition.
[0093] As used herein, the term "antibody" is used in the broadest
sense and specifically covers monoclonal antibodies, antibody
compositions with polyepitopic specificity, bispecific antibodies,
diabodies, and single-chain molecules, as well as antibody
fragments (e.g., Fab, F(ab').sub.2, and Fv), so long as they
exhibit the desired biological activity.
[0094] The term "monoclonal antibody (mAb)" as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations that may be present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigenic site (epitope). The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method.
[0095] A "functional derivative" of a mAb is a compound having a
qualitative biological property in common with a native mAb
protein. "Functional derivatives" include, but are not limited to,
fragments of native sequenced mAb, provided that they have a
biological activity in common with a corresponding native sequenced
mAb. The phrase "fragment" as used in connection with mAb
fragmented derivatives, such as immunotoxins, provided that they
have a biological activity in common with a corresponding native
sequenced mAb.
[0096] A "magnetic cell separation" or "magnetic flow sorting" is a
technique of cell selection based on achieving steady-state
isolation between magnetic and non-magnetic cell streams in a
flowing suspension. The selectivity depends upon cell tagging with
cell surface marker antibodies labeled with a magnetic colloid. The
characteristic feature of the method is its capability to
fractionate cells based on the surface antigen expression. In order
to perform this technique for the purpose of positive selection or
depletion of cells labeled with human IgG4.lamda., mAb (as
described in Example 3), 1.times.10.sup.8 single-cell suspension of
PBMC can be first labeled with the mAb at 20 .mu.g/ml reaction
buffer for 15 min at 4.degree. C. After unbound mAb is removed by
washing, 0.2 ml of mouse anti-Human IgG MicroBeads (Miltenyi
Biotec) is added and develops 15 min at 4.degree. C. Following
washing steps to remove unbound MicroBeads, the resuspended cells
are magnetically separated in a magnetic field provided by the
manufacturer (VarioMACS Separator). This isolated cell population
represents cells intrinsically express CTLA-4 on their surface, and
thus stands for Tregs.
[0097] A "thymidine incorporation assay", which evaluates the
capability of cells to expand, can be used to measure the
suppression of T cell proliferation in response to a recall antigen
by isolated Tregs. In order to perform this assay,
5.times.10.sup.5/well PMBC from vaccinated donors are activated
with 5 .mu.g/ml of tetanus toxoid (TT) as described in Example 3.
During TT-driven proliferation, the cells are plated out in 96-well
culture dishes containing a test sample with or without isolated
Tregs (such test samples are optionally diluted) and cultured for
three to seven days in a cell culture incubator at 37.degree. C. in
5% CO.sub.2 and air. Proliferation was measured by
.sup.3H-thymidine incorporation. During the final 16 hours of an
assay, 1 .mu.Ci of .sup.3H-thymidine is added to each well for the
last 16 and proliferation is measured by scintillation counting
using a Packard TopCount.RTM. Microplate Scintillation Counter
(PerkinElmer, Shelton, Conn.). Tregs are expected to induce a
statistically significant decrease (to a P value of 0.05) in
.sup.3H-thymidine uptake, relative to control. Preferred Treg cells
lead to a decrease in .sup.3H-thymidine uptake which is at least
30% of that of the control.
MODES AND METHODS FOR CARRYING OUT THE INVENTION
[0098] The present invention is based on the discovery of the human
monoclonal IgG4.lamda. mAbs against the Met 55-cored CDR2-like
region of CTLA-4. The epitope thus defined is shared by both CTLA-4
and sCTLA-4. The experiments described herein demonstrate that this
mAb is a complete human mAb which appears not to play a role in the
binding of CTLA-4 to its endogenous B7 ligands. In particular, this
antibody has been found to enhance CTLA-4-CD80 interaction in
certain conditions, thus indicating that it may be used to detect
sCTLA-4 in combination with human CD80. As the presence of nature
ligands of CTLA-4 is common in preparation of peripheral blood
mononuclear cells (PBMC), other uses for this mAb, e.g. single-step
enrichment of human Tregs, will be apparent and evident from the
following discussion. A description follows as to how such a mAb
may be prepared.
[0099] Culture materials and reagents are known in the art and may
be obtained commercially. The culture medium used is RPMI-1640
(HyClone, Logan, Utah), supplemented with 1.times. non-essential
amino acids (Life Technologies, Gaithersburg, Md.), 10% fetal
bovine serum (FBS; Life Technologies) and 50 .mu.g/ml of gentamycin
and kanamycin (Sinton Chemical & Pharmaceutical, Hsinchu,
Taiwan). Purified and biotinylated human CTLA-4-murine Ig fusion
protein (rhCTLA-4-murine Ig or CD152-muIg), CD80-muIg and CD86-muIg
(Ancell, Bayport, Minn.) are used in antigen-specific and competing
enzyme-linked immunosorbent assay (ELISA), together with
peroxidase-labeled goat antibodies against human IgG and IgM (Zymed
Laboratories, South San Francisco, Calif.) or avidin horseradish
peroxidase (eBioscience, San Diego, Calif.) as the reporting
system. The fluorochrome-conjugated mouse mAb against human IgGs
and human CD3 (UCHT1; mouse IgG1), together with rat mAb against
mouse IgG2a are commercially available from Becton Dickinson
Immunocytometry Systems (San Jose, Calif.) and Abcam (Cambridge,
UK). The anti-CD3 (OKT3; mouse IgG2a) uses for T cell activation
and the antagonistic anti-CD152 (BNI3; mouse IgG2a) can be
purchased from eBioscience and Abcam, respectively.
[0100] Complete human mAbs are produced from in vitro stimulation
and culture techniques. Generally, plasma and buffy coat samples
from healthy routine blood donors, screened negative for HIV-1/2,
HTLV-I/II, HCV, HBsAg and containing normal levels of alanine
transferase (ALT), can be obtained from local Blood Centers. PBMC
are isolated by density centrifugation on Ficoll-Paque (GE
Healthcare Bio-Sciences, Uppsala, Sweden) as described elsewhere.
The resulting PBMC are magnetically labeled with CD45RO MACS.RTM.
microbeads (Miltenyi) then separated by a VarioMACS.TM. (Miltenyi)
instrument according to the manufacturer's instructions. The
purified CD45RO.sup.+ T cells are cultured at a density of
2.times.10.sup.6 cells/ml in the culture medium supplemented with
50 .mu.M 2-mercaptoethanol and 10 .mu.g/ml pokeweed mitogen (PWM;
Sigma, St. Louis, Mo.). After 24 h, cells are removed by
400.times.g centrifugation to collect CD45RO.sup.+ T cell replacing
factor. Removal of cytotoxic cell populations is similarly
performed by using colloidal super-paramagnetic microbeads
conjugated to monoclonal anti-human CD8 and anti-CD56 antibodies
(Miltenyi). Removal of IL-10-producing cells may be achieved by
using rat anti-human IL-10 (SouthernBiotech, Birmingham, Ala.) and
goat anti-rat IgG microbeads (Miltenyi).
[0101] Site-directed in vitro immunization is preformed by using
cytotoxic cell-depleted PBMC based on a two-step principle. Primary
immunization is performed by incubating the cells for 6 days in a
medium containing 10 nM of the heterotopic peptide antigen
(QYIKANSKFIGITELAATYMMGNELTFLDDSICT; Fine Research Biochem,
Taoyuan, Taiwan), 50 .mu.M 2-mercaptoethanol, 10% heat-inactivated
human serum, 0.05 ng/ml recombinant human (rh) IL-2 (eBioscience),
and 25% (v/v) CD45RO.sup.+ T cell replacing factor. For secondary
immunization, 3.times.10.sup.7 primary-immunized cells are mixed
with the peptide in a flask that had been immobilized overnight
with 5 mg/ml of CD40L (CD154; eBioscience) together with
1.times.10.sup.7 QYIKANSKFIGITEL (Fine Research Biochem)-stimulated
CD4.sup.+ T cells and 5 ng/ml rh IL-15 (eBioscience). The cells are
cultured for 3-5 days in a medium supplemented with 5% human serum,
50 mM 2-mercaptoethanol and 10 nM heterotopic peptide antigen. The
significance of differences between treated and control cultures
can be established by a variety of statistical methods known in the
art such as Student's t test.
[0102] Subsequently, the in vitro immunized cells are infected with
Epstein-Barr virus (EBV) by virus-containing supernatant derived
from the EBV-producing marmoset cell line B95-8 (American Type
Culture Collection, ATCC CRL 1612). The infected cells are seeded
at 10.sup.5/well in 96-well plates together with mytomycin (Kyowa
Hakko Kogyo, Tokyo, Japan)-treated PBMC as feeder cells
(10.sup.4/well) for the establishment of lymphoblastoid cells and
screened for Ab production by ELISA. CTLA-4-specific ELISA can be
performed by coating 0.25 .mu.g/ml purified rhCD152-muIg, 0.5
.mu.g/ml monoclonal mouse IgG2a (mIgG2a; Ancell), 1 .mu.g/ml bovine
serum albumin (BSA; Sigma) or 1 .mu.g/ml tetanus toxoid (TT;
ADImmune, Taichung, Taiwan) onto microtitre plates overnight at
4.degree. C. Culture supernatants are diluted to the desired level
in 10 mM sodium phosphate buffer (pH 8.0), containing 0 5 M sodium
chloride and 0.1% Tween-20. Coated plates are incubated with
diluted culture supernatants, washed, incubated with
peroxidase-labeled goat antibodies against human IgG and IgM and
developed (15 min) by addition of 100 .mu.l of the chromogenic
substrate o-phenylaenediamine (OPD) (Sigma). The reaction is
stopped after 30 min by adding 1 M sulphuric acid, and the
absorbances are read at 490 nm.
[0103] Somatic cell hybridization can be generated by
electrofusion. Briefly, CTLA-4-specific EBV-infected lymphoblastoid
cells were fused with heteromyeloma cells (Chin et al., 2001) in an
isotonic medium (280 mM sorbitol, 0.5 mM magnesium acetate, 0.1 mM
calcium acetate and 1 mg/ml BSA; pH6.9-7.1). Cell fusion can be
induced by high-voltage pulses using a BTX Electro Cell Manipulator
ECM 2001 (Harvard Apparatus, Holliston, Mass.). CTLA-4-specific
hybrids were selected and cloned by limiting dilution.
[0104] Instead of fusion, the in vitro immunized cells can be used
to construct an antibody library, and the antibodies of interest
are then identified from this library. Thus, after in vitro
immunization, antibody-producing cells can be identified with the
antigen (the cells at this stage can be optionally infected with
EBV). A phage-display library is then constructed using these
antibody-producing cells, and the phages containing the antibody
fragment of interest can be identified by screening this library
with the antigen. The methods of constructing phage display
libraries are known in the art (Duenas et al., 1996).
[0105] To define the specific epitope of human CTLA-4 recognized by
the mAb, peptide arrays (Genesis Biotech, Taipei, Taiwan and Fine
Research Biochem, Taoyuan, Taiwan) containing in-situ synthesized
peptides immobilized on special membrane can be used. In brief, 1
.mu.g/mL of protein A (Proteus MIDI kit, Pro-Chem, Littleton,
Mass.)-purified mAb is incubated by shaking in room temperature for
2 h. After washing, the membrane-bound mAb can be then visualized
by diluted anti-human IgG conjugated with peroxidase (Jackson
ImmunoResearch Laboratories, West Grove, Pa.) and FAST.cndot.DAB
(Sigma). The amount of bound mAb is calculated by Image-Pro Plus
4.5 software (Media Cybernetics, Silver Spring, Md.) on the scanned
images.
[0106] Assays to determine affinity and specificity of binding are
known in the art, including competitive and non-competitive assays.
A non-competitive assay is preferred in this analysis. The affinity
of the mAb can be determined against rhCTLA-4-murine Ig fusion
protein with an IAsys.RTM. optical biosensor (Affinity Sensors,
Cambridge, UK) according to the manufacturer's instructions.
Briefly, 200 .mu.g/ml dialyzed and diluted rhCTLA-4-murine Ig is
immobilized on the activated surface of carboxymethyl dextran
cuvettes in 10 mM of sodium acetate buffer at pH 3.8. After
conditioning with 10 mM HCl, immobilization of 2 mg/mL CD152-muIg
can result in a response of 1100 arc sec. This represents the
highest immobilization response for CD152 and gives a ligate
binding capacity (R.sub.max) of 300 arc sec. Serial dilutions of
the mAb in PBS, i.e. 1.34.times.10.sup.-9 M, 6.70.times.10.sup.-9
M, 1.34.times.10.sup.-8 M, 2.68.times.10.sup.-8 M and
5.36.times.10.sup.-8 M, are added to the CD152-coated cuvettes
(final volume, 50 .mu.l). Affinity constants (Kd) are calculated
from these measurements as k.sub.diss/k.sub.ass by using the
FASTFIT.RTM. program provided by the manufacturer.
[0107] The present invention provides a novel method of measuring
human sCTLA-4 in biological samples. To this end, we develop an
immunoassay for quantification sCTLA-4, and show in Example 2 that
a soluble form of CTLA-4 can be detected at least ten times of
lower sensitivity as compared with a conventional method known in
the art. For illustration, sandwich ELISAs are used for detection
of sCTLA-4 in human serum. For this purpose, wells of a 96-well
microtiter plate were coated with anti-CTLA4 blocking mAb (clone
BNI3; BD Pharmingen) or non-blocking CTLA-4 mAb as the insoluble
support. After saturation by bovine serum albumin, 100 .mu.l of a
1:3 dilution of the test samples are applied to the wells, and the
plates are incubated for 60 min at room temperature and then wash
to remove unbound material. Next, a reporting system containing
either biotinylated anti-CTLA4 mAb (clone AS-33, Antibody
Solutions) or biotinylated CD80-muIg (Ancell) is added, and the
reactions are further incubated for 1 h. Reactions are developed
using a streptavidinperoxidase complex (Zymed) and
3,39,5,59-tetramethyl-benzidine substrate. Optical density (OD) is
read at 450 nm. A standard curve can be generated with the use of a
dilution series of a commercially available CTLA4-Ig fusion protein
(Ancell). Binding may be measured by a variety of methods known in
the art. After an incubation period sufficient to allow the binding
to reach equilibrium, the insoluble support is washed, and the
remaining label quantified. Assays of interest include ELISA, RIA,
FIA and flow cytometry, etc.
[0108] Also provided is a method of isolating Treg cells from a
sample, comprising contacting a suitable sample with the antibody,
or functional derivatives thereof, of the present invention so as
to form an antibody-antigen complex between the antibody and any
CTLA-4 present on the surface of cells in the sample, and detecting
the presence of any complex so formed, thereby isolating in the
sample the presence of extracellular CTLA-4. It is intended that
the isolation of Tregs can be performed by using only one positive
selection step, i.e., preserving cells specifically express
extracellular CTLA-4. Therefore, both populations of Treg and
non-Treg can be recovered with minimal in vitro manipulation and
maximal viability. To this end, in Example 3, 1.times.10.sup.8
single-cell suspension of PBMC can be first labeled with the mAb at
20 .mu.g/ml reaction buffer for 15 min at 4.degree. C. After
unbound mAb is removed by washing, 0.2 ml of mouse anti-Human IgG
MicroBeads (Miltenyi Biotec) is added and develops 15 min at
4.degree. C. Following washing steps to remove unbound MicroBeads,
the resuspended cells are magnetically separated in a magnetic
field. This isolated cell population thus represents Treg cells
intrinsically express CTLA-4 on their surface. Consequently,
negatively selected non-Treg population can be subsequently
stimulated, e.g., by a recall antigen, without prolonged selection
steps and thus without compromise of their survival.
[0109] Also in Example 3, a functional derivative, i.e.,
immunotoxin, is prepared from conjugation of diphtheria toxin with
purified IgG4.lamda.. Purified IgG4.lamda., mAb is mixed with six
times excess of N-succinimidyl 3-(2-pyridyldithio)propionate (GE
Healthcare Bio-Sciences) in PBS, and the mixture is allowed to
react for 30 min at room temperature and then dialyzed against PBS.
The modified IgG4.lamda., is then mixed with three times excess of
reduced diphtheria toxin (Merck Taiwan LTD., Taipei, Taiwan) and
10-fold concentrated PBS (10% of the total volume) and store for 36
h at 40.degree. C. The product is dialyzed and concentrated with
Macrosep.RTM. Centrifugal Devices (Pall Corporation, East Hills,
N.Y.) equilibrated and washed with PBS.
[0110] Suitable samples which are useful in the methods of CTLA-4
detection and Treg isolation include, but are not limited to
biological fluids from a human subject such as blood, nasal mucosal
discharge, oral mucosal discharge, vaginal mucosal discharge,
semen, purrulent exudates, anal mucosal discharge and synovial
fluid. Samples may also include lymphoid tissues such as spleen,
lymph nodes, thymus, bone marrow, tonsils and Peyer's patches. In
one embodiment, the human antibody is labeled with an immunological
retrievable marker, i.e., anti-human Ig antibody conjugated with
magnetic beads. Such a specific binding may be retrieved by a
variety of methods known in the art, including but not limited to
direct labeling of the presented mAb, cell panning, and
fluorescence-activated cell sorting.
[0111] It has been generally accepted that human
CD4.sup.+CD25.sup.+ Treg cells express FOXP3, whereas CD25.sup.- T
cells do not and the expression of FOXP3 in CD4.sup.+ T cells
correlates with their ability to function as Treg cells (Sakaguchi,
2005). In one embodiment, the expression of FOXP3 is quantified by
quantitative real-time PCR (QPCR) analysis in which RNA is first
extracted using an RNeasy Mini Kit (Qiagen, Valencia, Calif.)
according to the manufacturer's instructions, and cDNA is then
prepared with 2.5 .mu.M random hexamers (Applied Biosystems Inc.,
Foster City, Calif.). Message levels can be quantified by real-time
PCR System. Amplification is carried out in a total volume of 25
.mu.l for 40 to 50 cycles of 15 seconds at 95.degree. C., 1 minute
at 60.degree. C., and product may be detected using SYBR Green I
dye (Molecular Probes Inc., Eugene, Oreg.). Samples are run in
triplicate, and their relative expression was determined by
normalizing expression of each target to GAPDH, and then comparing
this normalized value to the normalized expression in a reference
sample to calculate a fold-change value. Primers were designed so
that amplicons spanned intron/exon boundaries to minimize
amplification of genomic DNA. Primer sequences were as follows:
TABLE-US-00001 GAPDH: 5'-CCACATCGCTCAGACACCAT-3'.quadrature. (SEQ
ID NO: 2) and 5'-GGCAACAATATCCACTTTACCAGAGT-3'; (SEQ ID NO: 3)
FOXP3: 5'-GAAACAGCACATTCCCAGAGTTC-3' (SEQ ID NO: 4) and
5'-ATGGCCCAGCGGATGAG-3'. (SEQ ID NO: 5)
[0112] The antibody or its functional derivatives, e.g.,
immunotoxins, may be administered by any suitable method known in
the art, such as via intravascular, intrathecal, intravenous,
intramuscular, parenteral, subcutaneous, intramedullar,
intraperitoneal, topical, oral, rectal, vaginal, nasal, pulmonary
and intratumoral routes.
Compositions
[0113] Another aspect of the present invention provides a
composition comprising a fully human antibody prepared according to
the present invention. Preferably, the antibody binds to its
antigen with a high affinity. The Kd is preferably about 100 nM or
less, more preferably about 40 nM or less, yet more preferably
about 10 nM or less, still more preferably about 4 nM or less, and
most preferably about 1 nM or less. In particular, the antibody is
capable of recognizing at least two related antigens, such as
microbial antigens that are only different due to antigenic
variation, or proteins encoded by alleles of the same gene.
[0114] This invention also includes pharmaceutical compositions
that contain, as the active ingredient, one or more of the
antibodies in combination with a pharmaceutically acceptable
carrier or excipients. In preparing the compositions of this
invention, the active ingredient/antibody is usually mixed with an
excipient, diluted by an excipient or enclosed within such a
carrier which can be in the form of a capsule, sachet, paper or
other container. When the pharmaceutically acceptable excipient
serves as a diluent, it can be a solid, semi-solid, or liquid
material, which acts as a vehicle, carrier or medium for the active
ingredient. Thus, the compositions can be in the form of solutions
(particularly sterile injectable solutions), tablets, pills,
powders, lozenges, sachets, cachets, elixirs, suspensions,
emulsions, syrups, aerosols (as a solid or in a liquid medium),
ointments containing, for example, up to 10% by weight of the
antibody, soft and hard gelatin capsules, suppositories, and
sterile packaged powders.
[0115] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, sterile water, syrup, and methyl cellulose. The
formulations can additionally include: lubricating agents such as
talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying and suspending agents; preserving agents such as
methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring agents. The compositions of the invention can be
formulated so as to provide quick, sustained or delayed release of
the active ingredient after administration to the patient by
employing procedures known in the art.
[0116] The liquid forms in which the novel compositions of the
present invention may be incorporated for administration by
injection include aqueous solutions (such PBS), suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as corn oil, cottonseed oil, sesame oil, coconut
oil, or peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0117] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of the present invention.
EXAMPLES
[0118] In the examples below, the following abbreviations have the
following meanings. Abbreviations not defined have their generally
accepted meanings. [0119] .degree. C.=degree Celsius [0120] h=hour
[0121] min=minute [0122] .mu.M=micromolar [0123] mM=millimolar
[0124] M=molar [0125] ml=milliliter [0126] .mu.l=microliter [0127]
mg=milligram [0128] .mu.g=microgram [0129] ALT=alanine transferase
[0130] CDR=complementarity determining region [0131]
CTLA-4=cytotoxic T lymphocyte antigen-4 [0132] ELISA=enzyme-linked
immunosorbent assay [0133] FBS=fetal bovine serum [0134]
HBsAg=hepatitis B surface antigen [0135] HCV=hepatitis C virus
[0136] HIV=human immunodeficiency virus [0137] HTLV=human T-cell
leukemia virus [0138] IEF=isoelectric focusing [0139]
Ig=immunoglobulin [0140] Kd=equilibrium dissociation (affinity)
constant; [0141] mAb=monoclonal antibody [0142] PBMC=peripheral
blood mononuclear cells [0143] PBS=phosphate buffered saline (20 mM
phosphate buffer, pH 7.2 containing 145 mM NaCl) [0144]
rh=recombinant human [0145] Tregs=regulatory T cells [0146]
TT=tetanus toxoid
Example 1
Characterization of Anti-Human CTLA-4 mAb
a) Epitope Mapping
[0147] To characterize the nature of complete human mAb binding,
epitope mapping was performed by the Western blotting method with
arrays containing the overlapping pendecapeptides, encompassing
CDR1-like (the B-C loop), CDR3-like (the F-G loop) and the Met
55-cored sequence localized between the C' and D strands of the
CD152 extracellular portion. FIG. 2 depicts that only the peptide
corresponding to the C-terminus of the Met 55-cored sequence
(.sup.54YMMGNELTFLDDSIC.sup.68; SEQ ID NO:1) was best recognized by
the mAb, and thus representing the epitope, while neither the
promiscuous T-cell epitope used to boost in vitro stimulation, nor
CDR1-like or CDR3-like region contributes to the binding. From this
result, it can be concluded that the Ala 51, Ala 52, Thr 53 and Thr
69 are not essential for mAb recognition.
b) Immunological and Biochemical Natures of the mAb
[0148] The essentially pure mAb was isotyped and subtyped by solid
phase ELISA, utilizing its reactivity with human CTLA-4 and
appropriate immobilized typing Abs. The binding depicts the
simultaneous presence of .gamma.4 and .lamda. chains in the mAb
while other Ig chains are absent (FIG. 3A), thus the mAb has a
.gamma.4.lamda. human Ig phenotype. Additionally, to compare the
clonal nature with existing human monoclonal IgG1.lamda., and
IgG4.lamda., derived from purified myeloma proteins, the
isoelectric focusing (IEF) patterns were subsequently visualized.
The resolvable bands, as shown in FIG. 3B, indicate that the
presented mAb has a slightly lower yet basic pI similar to the
IgG1.lamda., but in contrast to the acidic IgG4.lamda., myeloma
protein or to the anionic (pI 4.5-5.0) species of proteins commonly
described for polyclonal IgG4. Western blot confirmed the purity of
the Ab samples as illustrated by the anti-.lamda., staining
configurations. As shown in FIG. 3B, the corresponding pI of the
myeloid IgG1.lamda., IgG4.lamda. and the presented mAb obtained
with linear regression of pH gradient were 7.92-8.79, 5.76-6.52 and
7.87-8.41, respectively. The equilibrium dissociation constant (Kd)
for the purified intact mAb was determined by an IAsys analysis.
The rate constant was evaluated directly from the sensogram using
five cycles of soluble mAb binding to the immobilized
rhCTLA-4-muIg. FIG. 3D reveals that, with the analysis of extent
and association in single phase, the Kd was deduced to be
4.times.10.sup.-9M.
c) Little or no Competition to B7-CD152 Binding of the mAb
[0149] Although the CDR2-containing epitope does not seem to be
involved in the binding of endogenous cognate ligands (CD80 and
CD86), the epitope might present an allosteric site for
non-competitive inhibition. To investigate this possibility,
rhCTLA-4-muIg was immobilized onto wells and either biotinylated
CD80-muIg or CD86-muIg was used as a binding ligand in the presence
of the mAb or BNI3, i.e., a CTLA-4 blocking mouse IgG2a mAb
(Steiner, et al., 1999). FIG. 4 shows, in contrast with the
expected dose-dependent inhibition of specific receptor binding by
the antagonistic BNI3, the mAb could not compete binding
significantly with either CD80-muIg or CD86-muIg. Surprisingly,
high doses of the mAb display synergism with the natural ligand
CD80 but not CD86 with a consequence up to 50% enrichment of CD80
binding to rhCTLA-4-muIg.
Example 2
Using CDR2-Specific Agent Increase the Detection Limits of Human
CTLA-4
[0150] In practice, immunoassays based on the use of pairs of
blocking Abs, which reactive with epitopes within the B7-binding
region of the molecule; confront a primary limitation of steric
hindrance. Blocking Abs may compete with each other and with
endogenous CD80 and CD86 for the restricted CDR1- and CDR3-like
regions, making them difficult to distinguish native sCTLA-4 in an
assay system. To determine whether the above CTLA-4 binding
synergism with the natural ligand CD80 displayed by the mAb may
contribute a lower sensitivity; the mAb was coupled with CD80 in
detecting sCTLA-4. In brief, the ELISA system utilizing the
IgG4.lamda.-coated plate for capture and biotinylated CD80-muIg
recombinant protein (Ancell) for detection, followed by
streptavidin-peroxidase for color reaction. FIG. 5 shows a
representative experiment. This combination predominantly resolves
a range of CTLA-4 from 0.39 to 50 ng/ml, while the blocking mAb
pair identifies 6.25 to 50 ng/ml under similar experimental
conditions. Likewise, a higher resolution of 1.56 to 50 ng/ml was
also observed when the present mAb was in use together with a
blocking mAb. This is not only consistent with the predicted
respective three-dimensional binding sites based on the previous
studies (Linsley, et al., 1992; Schwartz, et al., 2001; Stamper, et
al., 2001) but also demonstrative that CTLA-4 mAb with CDR2-like
region specificity, in combination with agents of CDR1- and
CDR3-like specificities, increases the detection limits of
CTLA-4.
Example 3
Isolation of the Intrinsic CTLA-4.sup.+ Populations Resulted in
Increased FOXP3 Expression and Suppression
[0151] One of the major unresolved problems in the field of using
Tregs as a promising alternative to the standard immunosuppression
regime is how to develop a reliable method for their isolation.
Most prior arts reported to date exploit a negative selection
non-CD.sup.+ cells and a positive selection CD25.sup.+ cells, which
is time-consuming and experience-dependent to manipulate human
PBMC. However, having acknowledged the importance of CTLA-4 in
Treg's function (Read, et al., 2006; Ward and Barker, 2007), the
present invention turned the attention to utilize intrinsic CTLA-4
surface expression for Treg isolation. In particular, we asked if
the non-blocking anti-CTLA-4 mAb provides a single positive
selection for Teg cells. To address this question, we employed a
thymidine incorporation assay to detect the suppression of built-in
Tregs and the enhancement of Treg removal.
[0152] By analyzing the subsets from the in vitro tetanus toxoid
stimulation for proliferation, removal of CTLA-4.sup.+ population
is of great benefit to a recall response (FIG. 6B), further arguing
that this is a Treg population. In some donors, the majority of
CD4.sup.- CD25.sup.- cells could be recovered from the bound
magnetic beads, but not from other donors. It is not known what
causes the variability among donors. The identity of the population
that upregulated CTLA-4 surface expression was also determined. The
cells which upregulated CTLA-4 expression were enriched for Treg
cells, and this supported by the fact that these cells retained
antigen unresponsiveness similar to the purified
CD4.sup.+CD25.sup.+ population. Because CD4.sup.+CD25.sup.+ cells
predominantly represent Tregs and CTLA-4 surface expression is
associated with an even higher FOXP3 expression, it was thus
confirmed that CTLA-4.sup.+ cells symbolize regulatory T cells.
Sequence CWU 1
1
5115PRTHomo sapiens 1Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp
Asp Ser Ile Cys1 5 10 15220DNAArtificial SequenceHuman 2ccacatcgct
cagacaccat 20326DNAArtificial SequenceHuman 3ggcaacaata tccactttac
cagagt 26423DNAArtificial SequenceHuman 4gaaacagcac attcccagag ttc
23517DNAArtificial SequenceHuman 5atggcccagc ggatgag 17
* * * * *