NZ622050B2 - ANTIBODIES TO CD1d - Google Patents
ANTIBODIES TO CD1d Download PDFInfo
- Publication number
- NZ622050B2 NZ622050B2 NZ622050A NZ62205012A NZ622050B2 NZ 622050 B2 NZ622050 B2 NZ 622050B2 NZ 622050 A NZ622050 A NZ 622050A NZ 62205012 A NZ62205012 A NZ 62205012A NZ 622050 B2 NZ622050 B2 NZ 622050B2
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- New Zealand
- Prior art keywords
- seq
- cd1d
- antibody
- human
- antigen binding
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
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Abstract
Disclosed is an isolated antibody or antigen binding portion thereof which binds to an epitope of human CD1d wherein the epitope comprises residues 87 to 93 and 141 to 143 of SEQ ID NO: 116 (sequences as defined in the specification).
Description
ANTIBODIES TO CD1d
FILING DATA
This application is associated with and claims priority from Australian patent
application no. 2011904190 filed on 14 October 2011 and US patent application no.
61/547,307 filed on 14 October 2011, the entire contents of each of these applications are
incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to antibodies that bind CD1d and inhibit CD1d-
mediated biological functions such as activation of the CD1d-restricted T cell, natural
killer T (NKT) cells.
BACKGROUND
Bibliographic details of the publications referred to by the authors in this
specification are collected alphabetically at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as,
an acknowledgment or any form of suggestion that this prior art forms part of the common
general knowledge in any country.
CD1d is a counter-receptor essential for triggering cell populations, such as NKT
cells, to release high levels of cytokines, an activity associated with some inflammatory
diseases. Blockade of CD1d-mediated effects is therefore of potential therapeutic benefit.
[0006] CD1d protein is displayed on a number of antigen presenting cell (APC) subsets
including Langerhans cells (the major dendritic antigen-presenting cells in skin), activated
B-cells, dendritic cells in lymph nodes, and activated blood monocytes. One population of
cells stimulated via CD1d is NKT cells, a subset of T cells that express an alpha/beta (αβ)
T cell receptor (TCR) along with a variety of molecular markers typically associated with
NK cells, such as CD161 and NKG2D. NKT cells are stimulated by antigen presenting
cells (APC) via CD1d-presenting lipids or glycolipids. The majority of human CD1d-
restricted NKT cells express a semi-invariant TCR comprising Vα24Jα18 paired with
Vβ11 (Brigl, M et al., 2004 Annu. Rev. Immunol., 22:817-890). CD1d-TCR interactions
rapidly induce many Th1- or Th2-like cytokines, such as interferon (IFN)-γ and tumour
necrosis factor (TNF)-a , and interleukin (IL)-4, IL-5 and IL-13. The balance of Th1/Th2
9704459 1
cytokine responses is known to play an important role in orchestrating immune response
properties.
Five CD1 genes have thus far been identified in humans: CD1a, CD1b, CD1c,
CD1d and CD1e. CD1 proteins are expressed as large subunits (heavy chains) non-
covalently associated with β2-microglobulin (b 2M) (Van Agthoven, A., and Terhorst, C.,
1982 J. Immunol. 128:426-432; Terhorst, C., et al., 1981 Cell 23:771-780). The
extracellular domain of CD1d consists of three domains: the α1 domain (residues 20-108),
the α2 domain (residues 109-201), and the α3 domain (residues 202-295) (Pellicci, D.G.,
et. al., 2009 Immunity 31: 47-59).
[0008] A variety of lipids with different structures have been shown to bind CD1d
molecules in a unique manner that accommodates a fatty acid chain in each of the two
hydrophobic binding pockets (A′ and F) of the CD1d molecule. Lipid species capable of
binding CD1d molecules include mycolic acids, diacylglycerols, sphingolipids,
polyisoprenoids, lipopeptides, phosphomycoketides and small hydrophobic compounds
(Venkataswamy, M. M. and Porcelli, S.A., 2010 Semin Immunol 22: 68-78). The
prototypical compound used to study NKT cell activation in vitro and in vivo is KRN7000,
an α-galactosylceramide (“αGalCer”) derived from the marine sponge Agelas mauritianus.
Additional agonists include but are not restricted to isoglobotrihexosylceramide (“iGb3”),
reported to be an endogenous glycosphingolipid, as well as members of a class of
microbial-derived α-glycuronosylceramides, and a variety of human glycolipids such as
lysophophatidylcholine and lysosphingomyelin (Fox, L. M., et al., 2009 Plos Biol 7:
e1000228). Certain naturally occurring beta-linked glycosphingolipids such as the C24:1
form of β-D-glucopyranosylceramide, are also weak agonists for NKT cells (Brennan, P.
J., et al., 2011 Nat Immunol 12:1202-1211).
[0009] Excessive cytokine production by NKT cells may contribute to the pathology of
certain autoimmune or inflammatory diseases such as myasthenia gravis (Reinhardt, C. et
al., 1999 Neurology 52:1485-87), psoriasis (Bonish, B.D., et al., 2000 J. Immunol.
165:4076-85), ulcerative colitis (Saubermann, L.J., et al., 2000 Gastroenterology 119:119-
128), primary biliary cirrhosis (Kita, H., et al., 2002 Gastroenterology 123:1031-43), colitis
(Heller, F., et al. 2002 Immunity 17, 629-638), steatohepatitis (Syn, W., et al., (2010)
Hepatology, 51(6):1998-2007), autoimmune hepatitis (Santodomingo-Garzon, T. and
Swain, M.G. (2011) Autoimmunity Reviews 10:793-800), atherosclerosis (Kyriakakis, E.,
et. al., Eur J Immunol 2010 40:3268-79) and pulmonary inflammation or dysfunction
associated with sickle cell disease (Wallace et al. 2009 Blood 114:667-676). There is
increasing evidence for NKT cells to exert detrimental effects in asthma (Iwamura, C. and
Nakayama, T., 2010 Curr Opin Immunol 22:807-13).
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Asthma is a chronic inflammatory pulmonary disorder characterized by reversible
airway obstruction arising from chronic local inflammation, mucus obstruction, and
bronchospasm in response to nonspecific stimuli (Murdoch, J. R. and Lloyd, C. M. 2010
Mutat Res 690: 24-39). The high asthma prevalence, increasing incidence and enormous
associated healthcare expenditure positions asthma as a major public health problem
(Holgate, S. T. and Polosa, R. 2008 Nat Rev Immunol 8: 218-30; Bahadori, K., Doyle-
Waters M. M., et al., 2009 BMC Pulm Med 9:24). There is a significant unmet medical
need for treatments for patients suffering from severe forms of asthma, such as
corticosteroid-refractory asthma. Patients with severe asthma do not respond well to the
standard-of-care and represent approximately 5-10% of the total asthmatic population. This
comprises around 850,000 patients in the United States alone.
In mouse models of allergic asthma, NKT cells have been shown to exacerbate
disease (Akbari, O., et. al. 2003 Nat Med 9: 582-8). NKT cells may become activated by
CD1d-restricted glycolipid antigens and release cytokines such as IFN-γ, IL-4, IL-5 and
IL-13, which activate eosinophils and other cellular subsets important in asthma (Chuang,
Y. H., et al., 2011 J Immunol 186: 4687-92). By targeting NKT cells, the administration of
anti-CD1d antibodies or CD1d-dependent antagonists suppresses experimentally induced
airway inflammation (Lisbonne, M., et. al. 2003 J Immunol 171: 1637-41; Pichavant, M.,
et al. 2008 J Exp Med, 205: 385-93). NKT cells are also detrimental in non-human primate
models of asthma (Matangkasombut, P. et. al., 2008 J Allergy Clin Immunol 121: 1287-9).
Such results suggest that the low numbers of NKT cells present in the lungs may be
important for the development and perpetuation of human asthma.
Nonalcoholic fatty liver disease (NAFLD) is a condition in which excess fat
accumulates in patients without a history of alcohol abuse. NAFLD is classified into
simple steatosis and nonalcoholic steatohepatitis (NASH). In NASH, steatosis, intralobular
inflammation and hepatocellular ballooning are present, often accompanied by progressive
fibrosis. Long-standing NASH may progress to liver cirrhosis, and hepatocellular
carcinoma (HCC) may be an outcome. NAFLD is regarded as a hepatic manifestation of
metabolic syndrome. NAFLD has been increasing worldwide over recent decades in line
with the increased prevalence of obesity, type 2 diabetes, and hyperlipemia. NAFLD/
NASH is currently regarded as the most common chronic liver disease worldwide. It is
estimated that about 20% of all adults have NAFLD and 2-3% of adults have NASH.
Nonalcoholic fatty liver disease is a major cause of chronic liver disease. It encompasses a
spectrum of histopathology, including hepatic steatosis (fatty liver) and nonalcoholic
steatohepatitis (NASH).
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The liver harbors resident populations of NKT cells which may regulate innate
immune responses. For example, NKT cells with an invariant T cell receptor comprise up
to 20% of T cells in murine livers. Such cells are also enriched in human livers (up to 10%
of T cells) which harbor a more diverse repertoire of NKT cells. In both species, NKT cells
reside mainly in the hepatic sinusoids, where they provide intravascular immune
surveillance. NKT cells specifically recognize glycolipid antigens and can produce
cytokines when activated. This cell subset may contribute to the pathogenesis of NASH
(see for example Syn, W., et al., (2010) Hepatology, 51(6):1998-2007). Accordingly,
delivery of an anti-CD1d antibody that blocks the function of NKT cells in vivo may be of
therapeutic benefit.
The three main broad categories of autoimmune liver disease are autoimmune
hepatitis (AIH), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).
Each of these diseases has a relatively distinct clinical, serologic and histologic profile.
These three liver diseases also differ in the histopathological patterns of liver injury. AIH
is characterized by a progressive destruction of the hepatic parenchyma, known as
interface hepatitis. On the other hand, PBC is distinguished by specific destruction of small
intrahepatic bile ducts, whereas PSC mainly involves destruction of large bile ducts.
Despite the varied profiles of these conditions all these autoimmune hepatic diseases share
common pathways of immune-mediated liver injury involving the hepatic recruitment of T
lymphocytes which recognize and destroy hepatocytes, with the subsequent development
of liver fibrosis. NKT cells may contribute to the pathology of autoimmune liver diseases
(Santodomingo-Garzon, T. and Swain, M.G. (2011) Autoimmunity Reviews 10:793-800).
Activated NKT cells may induce hepatocyte death directly through up-regulation of cell
surface FasL expression and/or the release of tumor necrosis factor alpha (TNF-α) and
perforins/granzyme B. NKT cells may indirectly induce hepatocyte death through the
release of pro-inflammatory cytokines such as IFN-γ. NKT cells can also produce IL-4,
which induces Th2 responses and the subsequent production of autoantibodies by plasma
cells. Since activation of NKT cells can lead to hepatocyte destruction and ultimately the
development of cirrhosis, the blockade of NKT cell function by delivery of an anti-CD1d
antibody may therefore be of therapeutic benefit. In addition to cytokine release, NKT cell
effector functions which result in cell lysis, such as perforin release and granzyme release
and Fas-L mediated cell death, and other known NKT functions such as IL-2 mediated
bystander effects, may also be relevant in conditions in which NKT cells are implicated.
Blockade of the NKT cell activator CD1d, for example through administration of an anti-
CD1d antibody, may also modulate these NKT effector functions.
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SUMMARY OF THE INVENTION
There is a critical need to identify therapies that inhibit CD1d-mediated cell
activation and subsequently show benefit in the treatment of inflammatory diseases such as
severe corticosteroid refractory asthma. Fully human antibodies possess several
advantages to address the goal of developing human medicines that improve therapeutic
efficacy. They can be targeted to bind highly potent neutralizing epitopes and when
administered to humans are well-tolerated. While murine antibodies have been described
in the art that bind and interact with CD1d, the current invention describes human
antibodies that exhibit strong potency in inhibiting CD1d mediated NKT cell activation
and resultant effector function. Surprisingly, in some cases the potency of these antibodies
is orders of magnitude more potent than current state of the art antibodies. Such antibodies
with significantly enhanced potency should allow for the treatment of CD1d- mediated
diseases and should exhibit superior clinical efficacy.
Accordingly in a first aspect the present invention provides an isolated antibody or
antigen binding portion thereof which binds to human CD1d wherein the isolated antibody
or antigen binding portion thereof competes for binding to CD1d with at least one antibody
selected from the group consisting of 401.11 and 402.8.
In a second aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof binds to the same epitope of CD1d as that bound by at least
one antibody selected from the group consisting of 401.11 and 402.8.
In a third aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VH domain having a sequence selected from
the group consisting of SEQ ID NOs 1, 3, 5, 7, 8, 9, 24, 25, 26, 30, 33, 36, 40, 41, 42, 43,
44 and 45 and sequences at least 95% identical thereto.
In a fourth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VL domain having a sequence selected from
the group consisting of SEQ ID NOs 2, 4, 6, 46, 49 and 62 and sequences at least 95%
identical thereto.
In a fifth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
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antigen binding portion thereof comprises a VH domain comprising human FR1, FR2, FR3
and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the
sequence of CDR1 is DYAMH (SEQ ID NO: 124) or GYYWS (SEQ ID NO: 125).
In a sixth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VH domain comprising human FR1, FR2, FR3
and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the
sequence of CDR1 is GFTFDDY (SEQ ID NO: 135) or GGSFSGY (SEQ ID NO: 136).
In a seventh aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VL domain comprising human FR1, FR2, FR3
and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the
sequence of CDR1 is RASQHISSWLA (SEQ ID NO: 141) or ASSSGAVSSGNFPN (SEQ
ID NO: 142).
[0023] In an eighth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof binds to CD1d with an EC50 of less than 20ng/ml as
measured using a cell based potency assay. In one embodiment the isolated antibody or
antigen binding portion thereof binds to human CD1d with an EC50 of from 0.5ng/ml to
20ng/ml.
In a ninth aspect the present invention provides an isolated DNA molecule which
encodes the isolated antibody or antigen binding portion thereof of the present invention.
In a tenth aspect the present invention provides a method of treating a condition
involving NKT cell effector function in a human subject comprising administering to the
subject an isolated antibody or antigen binding portion thereof of the present invention.
In an eleventh aspect the present invention provides a method of detecting the
presence of CD1d in a sample the method comprising contacting a sample suspected to
contain CD1d with the isolated antibody or antigen binding portion thereof of the present
invention under conditions which allows the binding of the antibody or antigen binding
portion thereof to CD1d to form a complex and detecting the presence the complex in the
sample.
In a twelfth aspect the present invention provides a method of detecting the
presence of CD1d-positive cells in a cell sample the method comprising contacting a
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population of cells with an isolated antibody or antigen binding portion thereof of the
present invention to allow the binding of the antibody or antigen binding portion thereof to
CD1d-positive to form a complex and detecting the presence of the antibody or antigen
binding portion thereof-cell complex.
[0028] In a thirteenth aspect the present invention provides a method of selecting a
CD1d-binding protein which binds specifically to human CD1d and competes for binding
on CD1d with at least one antibody selected from the group consisting of 401.11, 402.8
and 401.11.158 from a plurality of CD1d-binding proteins, the method comprising:
contacting the plurality of CD1d-binding proteins to a human CD1d mutein in
which the amino acid positions 87 to 93 and 141-143 of SEQ ID NO: 116 have
been substituted with corresponding murine amino acids at these positions, under
conditions sufficient to allow binding of CD1d-binding proteins to the mutein to
form a CD1d-binding protein-human CD1d mutein complex and a depleted
plurality of CD1d-binding proteins which do not bind the human CD1d mutein,
and
collecting CD1d-binding proteins which do not bind to the human CD1d mutein
from the depleted plurality of CD1d-binding proteins,
wherein the collected CD1d-binding proteins bind specifically to human CD1d
and compete for binding on CD1d with at least one antibody selected from the
group consisting of 401.11, 402.8 and 401.11.158.
In a fourteenth aspect the present invention provides a method of selecting a
CD1d-binding protein which binds specifically to CD1d from a plurality of CD1d-binding
proteins, the method comprising:
contacting the plurality of CD1d-binding proteins to hCD1dmu (SEQ ID NO:
119) in which the amino acids located at positions 87 to 93 and 141 to 143 of
human CD1d (SEQ ID NO 116) have been replaced with the corresponding
murine sequence at these positions, under conditions sufficient to allow binding of
CD1d-binding proteins to the hCD1dmu to form a CD1d-binding protein-
hCD1dmu complex and a depleted plurality of CD1d binding proteins which do
not bind hCD1dmu, and
collecting CD1d-binding proteins which do not bind to the hCD1dmu from the
depleted plurality of CD1d-binding proteins,
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wherein the collected CD1d binding proteins bind specifically to human CD1d
(SEQ ID NO: 116) or mCD1dhu (SEQ ID NO: 118).
In one embodiment of any of the above aspects the isolated antibody or antigen
binding portion thereof also binds to cynomolgus and rhesus monkey CD1d.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Graphical Representation of results of assay demonstrating inhibition of
tetramer binding by anti-CD1d antibodies. Anti-CD1d antibodies 401.11 and 402.8 showed
improved inhibition of CD1d tetramer binding compared with antibodies 42 and 51.1, as
determined by a reduction in the mean fluorescence intensity of the signal, in an assay
using a α-Galactosylceramide (α-GalCer) lipid-loaded;) CD1d tetramer binding to J.RT3-
T3.5 cells stably transfected with an NKT cell receptor. The irrelevant specificity negative
control antibody showed no inhibition. Table 2 lists the EC50 values of all antibodies
tested.
Figure 2: Graphical Representation of results of assay demonstrating inhibition of
IL-2 release by anti-CD1d antibodies. Anti-CD1d antibodies 402.8 and 401.11 showed
improved inhibition of IL-2 release after 24 hours, as determined by ELISA, compared
with anti-CD1d antibodies 42 and 51.1 in an assay using α-GalCer-loaded CD1d-positive
U-937 cells and NKT cell receptor-stably transfected J.RT3-T3.5 cells. In all assays the
irrelevant specificity negative control antibody showed no inhibition of IL-2 release. EC50
values from representative experiments are presented in Table 3.
Figure 3: Graphical Representation of results of assay demonstrating binding of
anti-CD1d antibodies to Primary peripheral blood mononuclear cells (PBMCs) by flow
cytometry. Anti-CD1d antibody 402.8, as an example of antibodies described in this
specification, but not an irrelevant specificity negative control antibody, bound a CD1d-
positive, CD11c-positive population in primary human PBMCs, as determined by flow
cytometry.
Figure 4: Graphical Representation of results of assay demonstrating inhibition of
primary NKT cell function by anti-CD1d antibodies in Primary NKT Cell-Based Assays
using THP-1 cell line as Antigen-Presenting Cells. Antibodies 401.11 and 402.8 exhibited
up to 114-fold and up to 180-fold improved inhibition respectively of IFN-g (A), IL-4 (B),
IL-5 (C) and IL-13 (D) release after 24 hours, as determined by ELISA, compared with
anti-CD1d antibody 42. This result was from an assay using α-GalCer-expanded NKT
cells and α-GalCer-loaded THP-1 cells as CD1d-positive cells. In all assays, the irrelevant
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specificity negative control antibody did not inhibit cytokine release. EC50 values from
representative experiments are presented in Table 4.
Figure 5: Graphical Representation of results of assay demonstrating inhibition of
primary NKT cell function by anti-CD1d antibodies in Primary NKT Cell-Based Assays
using primary CD14+ monocytes as Antigen Presenting Cells. Antibodies 401.11 and
402.8 demonstrated significantly improved inhibition of IFN-g (A), IL-4 (B), IL-5 (C) and
IL-13 (D) release after 24 hours, as determined by ELISA, compared with anti-CD1d
antibodies 42 and 51.1 in an assay using α-GalCer-expanded NKT cells and α-GalCer-
loaded CD14+ monocyte-derived dendritic cells as CD1d-positive cells. In all assays, the
irrelevant specificity negative control antibody did not inhibit cytokine release. EC50
values from representative experiments are presented in Table 5.
Figure 6: Graphical representation of results of a competition ELISA
demonstrating that highly potent anti-CD1d antibodies share a similar neutralizing epitope
that is different to the epitope seen by lower-potency prior-art antibodies. As per Example
7, anti-CD1d antibody 402.8 competed with itself and with 401.11, but not with anti-CD1d
antibodies 42 and 51.1, for binding to human CD1d using a competition ELISA based
approach, as shown by absorbance readings at 450nm corresponding to the levels of
bound biotinylated 402.8 (A) and converted degree of competition (percentage) values (B).
Figure 7: Graphical Representation of results of assay demonstrating cross-
reactivity with recombinant cynomolgus macaque CD1d. As per Example 8, anti-CD1d
antibodies 401.11 and 402.8 bound human CD1d (A) and were cross-reactive with
cynomolgus macaque CD1d (B) by ELISA.
Figure 8: Graphical Representation of results of assay demonstrating cross-
reactivity with cynomolgus macaque cell-based CD1d. As per Example 9, anti-CD1d
antibody 402.8, but not an irrelevant specificity negative control antibody, bound CD1d on
PBMCs from two independent cynomolgus macaque donors as shown by flow cytometry.
Data are presented as flow cytometry histograms of gated live cells with the percentage of
CD1d-positive cells demarcated in the histogram.
Figure 9: Graphical Representation of results of assay demonstrating cell-based
inhibition of cynomolgus CD1d-mediated primary NKT expansion. As per Example 10,
anti-CD1d antibody 402.8, but not an irrelevant specificity negative control antibody,
inhibited the expansion of cynomolgus NKT cells in the presence of a GalCer-loaded
CD1d-positive PBMCs, as shown by quantification of CD3+Va 24+ cells by flow
cytometry.
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Figure 10: Sequence alignments showing sequences of variable regions of 401.11.
Boxed regions contain CDRs (as indicated) as defined by the Kabat numbering system and
the enhanced Chothia numbering system. CDRs defined by the Kabat numbering system
are shown in bold. CDRs defined by the enhanced Chothia numbering system are
underlined.
Figure 11: Sequence alignments showing sequences of variable regions of 402.8.
Boxed regions contain CDRs (as indicated) as defined by the Kabat numbering system and
the enhanced Chothia numbering system. CDRs defined by the Kabat numbering system
are shown in bold. CDRs defined by the enhanced Chothia numbering system are
underlined.
Figure 12: Alignment of Variants of 401.11. As per Example 11, an amino acid
sequence alignment of the heavy and light chains of 401.11 versus IGHV3-9.01 and
variants of 401.11 is presented.
Figure 13: Alignment of Optimized Variants of 401.11. As per Example 11, an
amino acid sequence alignment of the heavy and light chains of 401.11 and variants
thereof is presented.
Figure 14: Graphical Representation of results of assay demonstrating improved
inhibition of primary NKT cell function by enhanced variants of anti-CD1d antibody
401.11. As per Example 11, 401.11 and variants thereof were titrated from 1 μg/mL.
401.11 antibody variants demonstrated similar or improved inhibition of IFN-γ (A) and IL-
4 (B) release after 24 hours, as determined by ELISA, compared with 401.11, and
significantly improved inhibition of IFN-γ (A) and IL-4 (B) release after 24 hours, as
determined by ELISA, compared with anti-CD1d antibodies 42 and 51.1 titrated from
10μg/mL, in an assay using α-GalCer-expanded NKT cells and α-GalCer-loaded CD14+
monocyte-derived dendritic cells as CD1d-positive cells. In all assays, the irrelevant
specificity negative control antibody did not inhibit cytokine release. EC50 values from
representative experiments are presented in Table 13.
Figure 15. Alignment of optimized variants of 402.8. As per Example 11, an
amino acid sequence alignment of the heavy chain of 402.8 versus variants of 402.8 is
presented.
Figure 16. Graphical Representation of results of assay demonstrating inhibition
of primary NKT cell function by enhanced variants of anti-CD1d antibody 402.8. As per
Example 11, 402.8 and variants thereof were titrated from 10 μg/mL and demonstrated
similar inhibition of IFN-γ (A) and IL-13 (B) release after 24 hours, as determined by
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ELISA, and significantly improved inhibition of IFN-γ (A) and IL-13 (B) release after 24
hours, as determined by ELISA, compared with anti-CD1d antibody 42 titrated from
10μg/mL, in an assay using α-GalCer-expanded NKT cells and α-GalCer-loaded CD14+
monocyte-derived dendritic cells as CD1d-positive cells. In all assays, the irrelevant
specificity negative control antibody did not inhibit cytokine release. EC50 values from
representative experiments are presented in Table 18.
Figure 17. Graphical Representation of results of assay demonstrating improved
inhibition of primary NKT cell function by anti-CD1d antibodies in Primary NKT cell-
based assays using an alternative antigen to α-GalCer. As per Example 12, antibodies
401.11.158, 401.11 and 402.8 titrated from 1μg/mL, demonstrated significantly improved
inhibition of IFN-γ (A) and IL-4 (B) release after 24 hours, as determined by ELISA,
compared with anti-CD1d antibodies 42 and 51 titrated from 10μg/mL, in an assay using
α-GalCer-expanded NKT cells and C24:1 β-D-glucopyranosylceramide-loaded CD14+
monocyte-derived dendritic cells as CD1d-positive cells. In all assays, the irrelevant
specificity negative control antibody did not inhibit cytokine release. EC50 values from
representative experiments are presented in Table 20.
Figure 18: Graphical Representation of results of a competition ELISA
demonstrating that under revised conditions, highly potent anti-CD1d antibodies share a
similar neutralizing epitope that is different to the epitope seen by prior-art antibodies. As
per Example 13, antibody 402.8 competed with itself and with 401.11, but not with
antibodies 42 and 51.1, for binding to human CD1d, as shown by absorbance readings at
450nm (A) and converted degree of competition (percentage) values (B).
Figure 19: Graphical Representation of results of a competition ELISA
demonstrating that highly potent anti-CD1d antibodies which were variants of 401.11
shared a similar neutralizing epitope with 402.8. As per Example 13, anti-CD1d antibody
402.8 competed strongly with itself and with 401.11.160, 401.11.161 and 401.11.165 as
examples of 401.11 antibody variants for binding to human CD1d, as shown by absorbance
readings at 450nm (A) and converted degree of competition (percentage) values (B).
Figure 20: Graphical Representation of results of a competition ELISA
demonstrating that highly potent anti-CD1d antibodies derived from 402.8 share a similar
neutralizing epitope with 402.8. As per Example 13, anti-CD1d antibody 402.8 competed
strongly with itself and with 402.8.84, 402.8.86 and 402.8.87, as examples of 402.8
antibody variants for binding to human CD1d, as shown by absorbance readings at 450nm
(A) and converted degree of competition (percentage) values (B).
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Figure 21: Graphical Representation of results of a Competition ELISA
demonstrating that monoclonal anti-human CD1d antibodies do not compete with the
neutralizing epitope of 402.8. As described in Example 13, anti-CD1d antibody 402.8
competed strongly with itself but not with other monoclonal anti-human CD1d antibodies,
such as AD58E7, C3D5 and C-9, for binding to human CD1d, as shown by absorbance
readings (A) and converted degree of competition (percentage) values (B).
Figure 22: Graphical Representation of results of a c ompetition ELISA
demonstrating that monoclonal anti-mouse CD1d antibodies do not compete for the
neutralizing epitope of 402.8. As described in Example 13, anti-CD1d antibody 402.8
competed strongly with itself but not with monoclonal anti-mouse CD1d antibodies, such
as HB-321, HB-322 and HB-323, for binding to human CD1d, as shown by absorbance
readings at 450nm (A) and converted degree of competition (percentage) values (B).
Figure 23: Graphical Representation of results of a competition ELISA
demonstrating that polyclonal anti-human CD1d antibodies do not compete for the
neutralizing epitope of 402.8. As described in Example 13, anti-CD1d antibody 402.8
competed strongly with itself but not with C-19, H70 and Ab96515, as examples of
polyclonal anti-human CD1d antibodies, for binding to human CD1d, as shown by
absorbance readings at 450nm (A) and converted degree of competition (percentage)
values (B).
[0054] Figure 24: Graphical Representation of results of a competition ELISA
demonstrating that highly potent anti-CD1d antibodies share a similar neutralizing epitope
that is different to the epitopes bound by other anti-CD1d antibodies. As described in
Example 13, anti-CD1d antibody 401.11.158 competed strongly with itself and with 402.8,
but not with anti-CD1d antibodies 42 and 51.1, for binding to human CD1d, as shown by
absorbance readings at 450nm (A) and converted degree of competition (percentage)
values (B).
Figure 25: Graphical Representation of results of an ELISA demonstrating that
402.8, and 401.11.165 in the form of a FAb or a full length IgG bound to human CD1d.
Figure 26: A sequence alignment of CD1d constructs used to elucidate the
location on human CD1d to which the anti-CD1d antibodies bind.
Figure 27: Graphical Representation of results of an ELISA demonstrating that a
titration of antibodies 402.8 (A) and 401.11.158 (B) bound to human CD1d and mouse
CD1d into which human sequence had been introduced (mCD1dhu). Both antibodies did
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not bind to mouse CD1d or a human CD1d into which mouse sequence had been
introduced (hCD1dmu).
Figure 28: Graphical Representation of results of hydrogen-deuterium exchange
mapping experiments demonstrating the epitope of anti-human CD1d antibodies. (A)
Human CD1d (grey) with amino acid 89-94 and 141-142 indicated in black. Note: X-ray
structure is 3HUJ with a surface representation. (B) Human CD1d (with α-GalCer bound)
in complex with the NKT-cell receptor (α and β chains). The atoms of the epitope (amino
acids 89-94 and 141-142) of the anti-CD1d antibodies on human CD1d are coloured dark
grey. The epitope of the anti-CD1d antibodies is located in close proximity to the binding
site of the NKT-cell receptor β-chain.
Figure 29A: An alignment and consensus sequence of the V region of optimised
401.11 antibodies. Boxed regions contain CDRs (as indicated) as defined by the Kabat
numbering system and the enhanced Chothia numbering system. CDRs defined by the
Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia
numbering system are underlined.
Figure 29B: An alignment and consensus sequence of the V region of optimised
401.11 antibodies. Boxed regions contain CDRs (as indicated) as defined by the Kabat
numbering system and the enhanced Chothia numbering system. CDRs defined by the
Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia
numbering system are underlined.
Figure 30A: An alignment and consensus sequence of the V region of optimised
402.8 antibodies. Boxed regions contain CDRs (as indicated) as defined by the Kabat
numbering system and the enhanced Chothia numbering system. CDRs defined by the
Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia
numbering system are underlined.
Figure 30B: An alignment and consensus sequence of the V region of optimised
402.8 antibodies. Boxed regions contain CDRs (as indicated) as defined by the Kabat
numbering system and the enhanced Chothia numbering system. CDRs defined by the
Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia
numbering system are underlined.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to human and humanised antibodies and antigen
binding portions thereof which bind a particular epitope of CD1d. The present inventors
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have found that antibodies which bind this epitope of CD1d are particularly efficacious in
decreasing the effect of CD1d on NKT cells. Due to this effect it is believed that these
antibodies and antigen binding portions thereof will be useful in the treatment of
conditions in which NKT cell effector function, such as excessive production of cytokines
by NKT cells plays a role, such as asthma.
Accordingly in a first aspect the present invention provides an isolated antibody or
antigen binding portion thereof which binds to human CD1d wherein the isolated antibody
or antigen binding portion thereof competes for binding to CD1d with at least one antibody
selected from the group consisting of 401.11 and 402.8.
[0065] In a second aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof binds to the same epitope of CD1d as that bound by at least
one antibody selected from the group consisting of 401.11 and 402.8.
In a third aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VH domain having a sequence selected from
the group consisting of SEQ ID NOs 1, 3, 5, 7, 8, 9, 24, 25, 26, 30, 33, 36, 40, 41, 42, 43,
44 and 45 and sequences at least 95% identical thereto.
In a fourth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VL domain having a sequence selected from
the group consisting of SEQ ID NOs 2, 4, 6, 46, 49 and 62 and sequences at least 95%
identical thereto.
In a fifth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VH domain comprising human FR1, FR2, FR3
and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the
sequence of CDR1 is DYAMH (SEQ ID NO: 124) or GYYWS (SEQ ID NO: 125).
In an embodiment of this aspect of the invention the sequence of CDR3 is
DMCSSSGCPDGYFDS (SEQ ID NO: 126), DLCSSGGCPEGYFDS (SEQ ID NO: 152),
DMCSSGGCPDGYFDS (SEQ ID NO: 153), DMCSSGGCPEGYFDS (SEQ ID NO: 154),
GEIYDFWNSYMDV (SEQ ID NO: 127), GEIYDFWKSYMDV (SEQ ID NO: 128),
GEIYDFYKSYLDV (SEQ ID NO: 155), GEIYDFYKSYMDV (SEQ ID NO: 156),
GEIYDFWKSYLDV (SEQ ID NO: 129) or GEIYDFYNSYMDV (SEQ ID NO: 130). In
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a further embodiment the sequence of CDR2 is TIIWNSAIIGYADSVKG (SEQ ID NO:
131), EINHSGSTNYNPSLKS (SEQ ID NO: 132), EINPSGSTNYNPSLKS (SEQ ID NO:
133) or EINHAGSTNYNPSLKS (SEQ ID NO: 134).
In a sixth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VH domain comprising human FR1, FR2, FR3
and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the
sequence of CDR1 is GFTFDDY (SEQ ID NO: 135) or GGSFSGY (SEQ ID NO: 136).
In an embodiment of the sixth aspect of the invention the sequence of CDR3 is
DMCSSSGCPDGYFDS (SEQ ID NO: 126), DLCSSGGCPEGYFDS (SEQ ID NO: 152),
DMCSSGGCPDGYFDS (SEQ ID NO: 153), DMCSSGGCPEGYFDS (SEQ ID NO: 154),
GEIYDFWNSYMDV (SEQ ID NO: 127), GEIYDFWKSYMDV (SEQ ID NO: 128),
GEIYDFYKSYLDV (SEQ ID NO: 155), GEIYDFYKSYMDV (SEQ ID NO: 156),
GEIYDFWKSYLDV (SEQ ID NO: 129) or GEIYDFYNSYMDV (SEQ ID NO: 130). In
a further embodiment the sequence of CDR2 is IWNSAI (SEQ ID NO: 137), NHSGS
(SEQ ID NO: 138), NPSGS (SEQ ID NO: 139) or NHAGS (SEQ ID NO: 140).
In a seventh aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof comprises a VL domain comprising human FR1, FR2, FR3
and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the
sequence of CDR1 is RASQHISSWLA (SEQ ID NO: 141) or ASSSGAVSSGNFPN (SEQ
ID NO: 142).
In an embodiment of the seventh aspect of the invention the sequence of CDR3 is
QQANRFPLT (SEQ ID NO: 141) or LLYFGDTQLGV (SEQ ID NO: 142). In a further
embodiment the sequence of CDR2 is AASSLQS (SEQ ID NO: 145) or SASNKHS (SEQ
ID NO: 146).
In an eighth aspect the present invention provides an isolated antibody or antigen
binding portion thereof which binds to human CD1d wherein the isolated antibody or
antigen binding portion thereof binds to CD1d with an EC50 of less than 20ng/ml as
measured using a cell based potency assay. In an embodiment of the present invention the
isolated antibody or antigen binding portion thereof binds to human CD1d with an EC50 of
from 0.5ng/ml to 20ng/ml as measured using a cell based potency assay.
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In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
1 and SEQ ID NO: 2.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
23 and SEQ ID NO: 46.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
24 and SEQ ID NO: 47.
[0078] In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
and SEQ ID NO: 6.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
25 and SEQ ID NO: 48.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
26 and SEQ ID NO: 49
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
27 and SEQ ID NO: 50.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
28 and SEQ ID NO: 51.
[0083] In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
29 and SEQ ID NO: 52.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
30 and SEQ ID NO: 53.
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In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
31 and SEQ ID NO: 54.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
32 and SEQ ID NO: 55.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
33 and SEQ ID NO: 56.
[0088] In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
34 and SEQ ID NO: 57.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
35 and SEQ ID NO: 58.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
36 and SEQ ID NO: 59.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
37 and SEQ ID NO: 60.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
38 and SEQ ID NO: 61.
[0093] In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
40 and SEQ ID NO: 62.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
41 and SEQ ID NO: 63.
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In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
42 and SEQ ID NO: 64.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
3 and SEQ ID NO: 4.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
7 and SEQ ID NO: 4.
[0098] In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
8 and SEQ ID NO: 4.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
9 and SEQ ID NO: 4.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
43 and SEQ ID NO: 65.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of SEQ ID NO:
44 and SEQ ID NO: 66.
In an embodiment of the present invention there is provided an isolated antibody
or antigen binding portion thereof comprising a VH and VL sequence pair of and SEQ ID
NO: 45 and SEQ ID NO: 67.
[0103] In an embodiment of any one of the above aspects, the antibody or antigen
binding portion thereof binds to human CD1d (SEQ ID NO:116), but not to hCD1dmu
(SEQ ID NO:119). In an embodiment of any of the above aspects, the antibody or antigen
binding portion thereof binds to mCD1dhu (SEQ ID NO:118) but not to mCD1d (SEQ ID
NO:117).
[0104] In a ninth aspect the present invention provides an isolated DNA molecule which
encodes the isolated antibody or antigen binding portion thereof of the present invention.
In one embodiment, the isolated DNA molecule is selected from any one of SEQ ID NOS:
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, 11, 12, 13, 14, 15, 16, 17, 18, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 or a sequence at least 95% identical
thereto or a sequence which hybridises thereto under moderate to high stringency
conditions. In one embodiment, the isolated DNA molecule is selected from any one of
SEQ ID NOS: 10, 11, 12, 13, 14, 15, 16, 17, 18, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115.
In a tenth aspect the present invention provides a method of treating a condition
involving NKT cell effector function in a human subject comprising administering to the
subject an isolated antibody or antigen binding portion thereof of the present invention.
In an eleventh aspect the present invention provides a method of detecting the
presence of CD1d in a sample the method comprising contacting a sample suspected to
contain CD1d with the isolated antibody or antigen binding portion thereof of the present
invention under conditions which allows the binding of the antibody or antigen binding
portion thereof to CD1d to form a complex and detecting the presence the complex in the
sample.
In a twelfth aspect the present invention provides a method of detecting the
presence of CD1d-positive cells in a cell sample the method comprising contacting a
population of cells with an isolated antibody or antigen binding portion thereof of the
present invention to allow the binding of the antibody or antigen binding portion thereof to
CD1d-positive to form a complex and detecting the presence of the antibody or antigen
binding portion thereof cell complex.
In a thirteenth aspect the present invention provides a method of selecting a
CD1d-binding protein which binds specifically to human CD1d and competes for binding
on CD1d with at least one antibody selected from the group consisting of 401.11, 402.8
and 401.11.158 from a plurality of CD1d-binding proteins, the method comprising:
contacting the plurality of CD1d-binding proteins to a human CD1d mutein in
which the amino acid positions 87 to 93 and 141-143 of SEQ ID NO: 116 have
been substituted with corresponding murine amino acids at these positions, under
conditions sufficient to allow binding of CD1d-binding proteins to the mutein to
form a CD1d-binding protein-human CD1d mutein complex and a depleted
plurality of CD1d-binding proteins which do not bind the human CD1d mutein,
and collecting CD1d-binding proteins which do not bind to the human CD1d
mutein from the depleted plurality of CD1d-binding proteins, wherein the
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collected CD1d-binding proteins bind specifically to human CD1d and compete
for binding on CD1d with at least one antibody selected from the group consisting
of 401.11, 402.8 and 401.11.158.
In a fourteenth aspect the present invention provides a method of selecting a
CD1d-binding protein which binds specifically to CD1d from a plurality of CD1d-binding
proteins, the method comprising:
contacting the plurality of CD1d-binding proteins to hCD1dmu (SEQ ID NO:
119) in which the amino acids located at positions 87 to 93 and 141to 143 of
human CD1d (SEQ ID NO 116) have been replaced with the corresponding
murine sequence at this location, under conditions sufficient to allow binding of
CD1d-binding proteins to the hCD1dmu to form a CD1d-binding protein-
hCD1dmu complex and a depleted plurality of CD1d binding proteins which do
not bind hCD1dmu and collecting CD1d-binding proteins which do not bind to
the hCD1dmu from the depleted plurality of CD1d-binding proteins, wherein the
collected CD1d binding proteins bind specifically to human CD1d (SEQ ID NO:
116) or mCD1dhu (SEQ ID NO: 118).
The anti-CD1d antibodies of the invention may also be used to identify or select
CD1d-positive cell populations from blood. Anti-CD1d antibody may be used to detect a
population of CD1d-positive cells within the peripheral blood of a human patient,
including myeloid cells such as monocytes, or lymphoid cells such as B cells. The
antibody could be used to detect these cells in conditions where such CD1d-positive cells
contribute to disease, e.g. certain leukaemias including chronic lymphocytic leukaemia
(CLL). (Metelitsa et al., Leukemia (2003) 17, 1068–1077.; Kotsianidis et al., 2011; Am J
Clin Path 136, 400-408.)
[0111] The anti-human CD1d antibody could also be used to stain tissue sections for
immunohistochemistry using methods well known in the art.
In certain embodiments of the present invention the isolated antibody or antigen
binding portion thereof may comprises a human kappa chain constant region or a human
lambda chain constant region. In certain embodiments the isolated antibody or antigen
binding portion thereof comprises an IgG1 or IgG4 constant region. Where the antibody
comprises an IgG4 constant region this may include an S228P mutation.
The present invention also provides DNA molecules which encode the isolated
antibody or antigen binding portion thereof of the present invention. In certain
embodiments the sequence of the DNA molecule is selected from any one of the group
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consisting of SEQ ID NO. 10 to 18, SQ ID NOS 68 to 115 or a sequence at least 95%
identical thereto or a sequence which hybridises thereto under moderate to high stringency
conditions.
The present invention also provides a method of treating a condition involving
NKT cell effector function in a human subject comprising administering to the subject the
isolated antibody or antigen binding portion thereof of the present invention. Examples of
conditions involving NKT cell effector function, such as excessive cytokine production by
NKT cells, which may be treated include psoriasis, ulcerative colitis, primary biliary
cirrhosis, autoimmune hepatitis, nonalcoholic steatohepatitis, atherosclerosis, ischaemia
reperfusion injury, asthma and pulmonary inflammation or dysfunction associated with
sickle cell disease.
As is described in the following Examples the present inventors have developed
potent antibodies which bind to a particular epitope of CD1d. The determination of the
nature of this epitope is routine for persons skilled in this area armed with both the
antibody and antigen. Methods well known to those skilled in this are which can used to
determine the CD1d epitope to which the antibodies 401.11 and 402.8 bind include CD1d
alanine scanning mutagenesis, hydrogen/deuterium exchange mapping, X-ray
crystallography, nuclear magnetic resonance and photoaffinity labelling.
Alanine-scanning mutagenesis (see for example Ausubel in: Current Protocols in
Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), Chapters 8 and 15; or
Cunningham et al. 1989 Science 244 1081-5) introduces single alanine mutations at every
residue in the CD1d molecule. The resulting mutant molecules are then tested for their
ability to bind the 401.11 and/or 402.8 antibodies. A loss binding means that a particular
residue which has been changed to alanine may be involved in the epitope.
[0117] In epitope mapping using hydrogen deuterium exchange the hydrogens in CD1d
are exchanged with deuterium in solution. The 401.11 and/or 402.8 antibodies are then
bound to the CD1d which is then exchanged back in H O. In this process the deuterium
present in the epitope are protected by the binding of the antibody. A comparison of the
exchange patterns with CD1d protected by the antibody binding and unprotected reveals
the epitope as the amino acid residues of CD1d retaining deuterium.
In X-ray crystallography CD1d to which the 401.11 and/or 402.8 antibody is
bound is crystallised and the crystal examined by X-ray diffraction. This methodology
provides clear information as to the region of CD1d to which the antibody is bound.
Nuclear magnetic resonance or photoaffinity labeling may also be used as described in de
Vos et al. 1992 Science 255 306-12; and Smith et al. 1992 J Mol Biol 224 899-904.
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As will be understood by person skilled in this field the epitope recognised by the
antibody or antigen binding portion thereof of the present invention may comprise a linear
series of amino acids or may be a conformational epitope.
In one aspect the present invention is directed to antibodies which compete for
binding to human CD1d with at least one antibody selected from the group consisting of
401.11 and 402.8.
As used herein “competes” means that the antibody or antigen binding portion
thereof reduces the binding of at least one antibody selected from the group consisting of
401.11, 401.11.28, 402.8, 402.8.45, 402.8.53 and 402.8.60 to CD1d in a concentration
dependent manner. An example of the way in which this may be assessed is provided in
Example 7 set out below. In particular an antibody or antigen binding portion thereof is
said to "compete" with at least one antibody selected from the group consisting of 401.11
and 402.8 for binding to CD1d where there is a greater reduction in binding of the at least
one antibody selected from the group consisting of 401.11 and 402.8 with the test antibody
than with antibody 42 or 51.1 used at the same concentration. (The prior art antibodies 42
and 51.1 are described in Exley et al. 1997 J Exp Med 186, 109-120 and WO03/092615).
As described herein, an antibody or antigen binding portion thereof which
“competes for binding to CD1d” demonstrates at least 50% competition in normalised
results in a competition ELISA, in which 40 μg/mL of non-biotinylated test antibody is
competed with 0.2 μg/mL biotinylated anti-CD1d antibody 402.8 or 401.11 or 401.11.158
bound to 1.0 μg/mL recombinant human CD1d which is immobilized on a solid substrate
In certain embodiments the present invention provides an isolated antibody or
antigen binding portion thereof binds to CD1d with an EC50 of less than 20ng/ml as
measured using a cell based potency assay. In certain embodiments the isolated antibody
or antigen binding portion thereof binds to CD1d with an EC50 of between 0.5ng/ml to
20ng/ml. As used herein the EC50 of the antibody or antigen binding portion thereof is to
be assessed as in Example 4 as set out below.
As mentioned above the antibodies or antigen binding portions thereof
specifically bind CD1d. As used herein the term "specifically" means that the binding to
CD1d is via the VH and VL domains of the antibody or antigen binding portion thereof
and not a non- specific binding such as may occur via the Fc region.
As described in the following examples the antibodies or antigen binding portions
thereof of the present invention bind to both human and cynomolgus or rhesus CD1d. This
is in contrast to prior art antibodies 42 and 51.1.
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The amino acid sequence of human CD1d may be, for example,:
MGCLLFLLLWALLQAWGSAEVPQRLFPLRCLQISSFANSSWTRTDGLAW
LGELQTHSWSNDSDTVRSLKPWSQGTFSDQQWETLQHIFRVYRSSFTRDV
KEFAKMLRLSYPLELQVSAGCEVHPGNASNNFFHVAFQGKDILSFQGTSW
EPTQEAPLWVNLAIQVLNQDKWTRETVQWLLNGTCPQFVSGLLESGKSE
LKKQVKPKAWLSRGPSPGPGRLLLVCHVSGFYPKPVWVKWMRGEQEQQ
GTQPGDILPNADETWYLRATLDVVAGEAAGLSCRVKHSSLEGQDIVLYW
GGSYTSMGLIALAVLACLLFLLIVGFTSRFKRQTSYQGVL (SEQ ID
NO:157)
The UniProt accession number for human CD1d is P15813.
In another aspect the present invention is directed to antibodies which bind the
same epitope of CD1d as that bound by at least one antibody selected from the group
consisting of 401.11 and 402.8, (and in some embodiments 40.11.158). As described
above the epitope of CD1d bound by a particular antibody can be assessed by a number of
methodologies and this can then be compared to the epitope bound by the specified
antibody.
In an embodiment the epitope comprises residues 141 to 143 of SEQ ID NO: 116
or residues 87 to 93 and 141 to 143 of SEQ ID NO: 116.
The term "antibody", as used herein, broadly refers to any immunoglobulin (Ig)
molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L)
chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative
antibody formats are known in the art. Non-limiting embodiments of which are discussed
below.
[0130] In a full-length antibody, each heavy chain is comprised of a heavy chain variable
region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a
light chain constant region. The light chain constant region is comprised of one domain,
CL. The VH and VL regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with regions that are
more conserved, termed framework regions (FR). Each VH and VL is composed of three
CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of
9704459 1
any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2) or subclass.
The term “antigen binding portion” of an antibody, as used herein, refers to one or
more fragments of an antibody or protein that retain the ability to specifically bind to an
antigen (e.g., CD1d). It has been shown that the antigen-binding function of an antibody
can be performed by fragments of a full-length antibody. Such antibody embodiments may
also be bispecific, dual specific, or multi-specific formats; specifically binding to two or
more different antigens. Examples of binding fragments encompassed within the term
"antigen- binding portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a
bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge
region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment
consisting of the VL and VH domains of a single arm of an antibody , (v) a domain
antibody (dAb) (Ward et al., 1989 Nature 341 544-6, Winter et al., PCT publication WO
90/05144 all herein incorporated by reference), which comprises a single variable domain;
and (vi) an isolated complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form monovalent molecules
(known as single chain Fv (scFv); (see e.g., Bird et al. 1988 Science 242 423-6; Huston et
al. 1988 Proc Natl Acad Sci U S A 85 5879-83). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding portion" of an antibody.
Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies
are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single
polypeptide chain, but using a linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et
al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al., 1994, Structure
2:1121-1123). Such antibody binding portions are known in the art (Kontermann and
Dubel eds., Antibody Engineering 2001 Springer-Verlag. New York. 790 pp., ISBN 3
41354-5).
The antibody described herein may be may be a humanized antibody. The term
“humanized antibody” shall be understood to refer to a protein comprising a human-like
variable region, which includes CDRs from an antibody from a non-human species (e.g.,
mouse or rat or non-human primate) grafted onto or inserted into FRs from a human
antibody (this type of antibody is also referred to a “CDR-grafted antibody”). Humanized
antibodies also include proteins in which one or more residues of the human protein are
9704459 1
modified by one or more amino acid substitutions and/or one or more FR residues of the
human protein are replaced by corresponding non-human residues. Humanized antibodies
may also comprise residues which are found in neither the human antibody or in the non-
human antibody. Any additional regions of the protein (e.g., Fc region) are generally
human. Humanization can be performed using a method known in the art, e.g.,
US5225539, US6054297, US7566771 or US5585089. The term “humanized antibody”
also encompasses a super-humanized protein, e.g., as described in US7732578.
The antibody described herein may be human. The term “human antibody” as
used herein refers to proteins having variable and, optionally, constant antibody regions
found in humans, e.g. in the human germline or somatic cells or from libraries produced
using such regions. The “human” antibodies can include amino acid residues not encoded
by human sequences, e.g. mutations introduced by random or site directed mutations in
vitro (in particular mutations which involve conservative substitutions or mutations in a
small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein).
These “human antibodies” do not necessarily need to be generated as a result of an
immune response of a human, rather, they can be generated using recombinant means (e.g.,
screening a phage display library) and/or by a transgenic animal (e.g., a mouse) comprising
nucleic acid encoding human antibody constant and/or variable regions and/or using
guided selection (e.g., as described in or US5565332). This term also encompasses affinity
matured forms of such antibodies. For the purposes of the present disclosure, a human
protein will also be considered to include a protein comprising FRs from a human antibody
or FRs comprising sequences from a consensus sequence of human FRs and in which one
or more of the CDRs are random or semi-random, e.g., as described in US6300064 and/or
US6248516.
[0134] Amino acid positions assigned to CDRs and FRs may be defined according to
Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health,
Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”).
In other embodiments, the amino acid positions assigned to CDRs and FRs are defined
according to the Enhanced Chothia Numbering Scheme
(http://www.bioinfo.org.uk/mdex.html). According to the numbering system of Kabat, VH
FRs and CDRs may be positioned as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49
(FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the
numbering system of Kabat, VL FRs and CDRs are positioned as follows: residues 1–23
(FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-
107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat
numbering system, but includes all numbering systems, including the canonical numbering
system or of Chothia and Lesk J. Mol Biol. 196:901-917, 1987; Chothia et al. Nature 342,
9704459 1
877-883, 1989; and/or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997; the numbering
system of Honnegher and Plükthun J. Mol. Biol., 309: 657-670, 2001; or the IMGT system
discussed in Giudicelli et al., Nucleic Acids Res., 25: 206-211 1997. In one example, the
CDRs are defined according to the Kabat numbering system. Optionally, heavy chain
CDR2 according to the Kabat numbering system does not comprise the five C-terminal
amino acids listed herein or any one or more of those amino acids are substituted with
another naturally-occurring amino acid. In an additional, or alternative, option, light chain
CDR1 does not comprise the four N-terminal amino acids listed herein or any one or more
of those amino acids are substituted with another naturally-occurring amino acid. In this
regard, Padlan et al., FASEB J., 9: 133-139, 1995 established that the five C-terminal
amino acids of heavy chain CDR2 and/or the four N-terminal amino acids of light chain
CDR1 are not generally involved in antigen binding.
The term "antibody construct" as used herein refers to a polypeptide comprising
one or more antigen binding portions of the invention linked to a linker polypeptide or an
immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid
residues joined by peptide bonds and are used to link one or more antigen binding portions.
Such linker polypeptides are well known in the art (see e.g. Holliger et al. 1993 Proc Natl
Acad Sci U S A 90 6444-8).
An immunoglobulin constant domain refers to a heavy or light chain constant
domain. Human IgG heavy chain and light chain constant domain amino acid sequences
are known in the art and examples are represented below.
Human heavy chain IgG1 constant domain (or derivatives thereof like NCBI
Accession No: P01857)
ASTKNPDVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:158)
Human heavy chain IgG4 constant domain (like NCBI Accession No: P01861)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK
YGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
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QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:159)
[0139] Human heavy chain IgG4 constant domain incorporating an S228P mutation
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK
YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV
FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:160)
Human heavy chain IgG4 constant domain incorporating an S228P mutation and a
YTE mutation such as described in US 7,083,784 may also be used.
[0141] Human light chain kappa constant domain (like NCBI Accession No: P01834)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
NRGEC (SEQ ID NO:161)
Human light chain lambda constant domain (like NCBI Accession No: P01842)
QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA
GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA
PTECS (SEQ ID NO: 162)
As will be appreciated the sequences developed and described in the present
invention may be modified using methods well known in the art to increase binding, by for
example, affinity maturation, or to decrease immunogenicity by removing predicted MHC
class II-binding motifs. The therapeutic utility of the sequences developed and described
herein can be further enhanced by modulating their functional characteristics, such as
antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent
cytotoxicity (CDC), serum half-life, biodistribution and binding to Fc receptors or the
combination of any of these. This modulation can be achieved by protein-engineering,
glyco-engineering or chemical methods. Depending on the therapeutic application
required, it could be advantageous to either increase or decrease any of these activities.
9704459 1
Numerous methods for affinity maturation of antibodies are known in the art.
Many of these are based on the general strategy of generating panels or libraries of variant
proteins by mutagenesis followed by selection and/or screening for improved affinity.
Mutagenesis is often performed at the DNA level, for example by error prone PCR (Thie H
2009 Methods Mol Biol. 525:309-22), by gene shuffling (Kolkman and Stemmer 2001 Nat
Biotechnol. May;19(5):423-8), by use of mutagenic chemicals or irradiation, by use of
‘mutator’ strains with error prone replication machinery (Greener 1996) or by somatic
hypermutation approaches that harness natural affinity maturation machinery (Peled,
Kuang et al. 2008). Mutagenesis can also be performed at the RNA level, for example by
use of Qβ replicase (Kopsidas, Roberts et al. 2006). Library-based methods allowing
screening for improved variant proteins can be based on various display technologies such
as phage, yeast, ribosome, bacterial or mammalian cells, and are well known in the art
(Benhar 2007). Affinity maturation can be achieved by more directed/predictive methods
for example by site-directed mutagenesis or gene synthesis guided by findings from 3D
protein modeling (see for example Queen, Schneider et al. 1989 or US patent 6,180,370 or
US patent 5,225,539).
A number of methods for modulating antibody serum half-life and biodistribution
are based on modifying the interaction between antibody and the neonatal Fc receptor
(FcRn), a receptor with a key role in protecting IgG from catabolism, and maintaining high
serum antibody concentration. Dall’Acqua et al., describe substitutions in the Fc region of
IgG1 that enhance binding affinity to FcRn, thereby increasing serum half-life
(Dall'Acqua, Woods et al., 2002) and further demonstrate enhanced bioavailability and
modulation of ADCC activity with triple substitution of M252Y/S254T/T256E (YTE
mutation) (Dall'Acqua, Kiener et al., 2006). See also U.S Pat. Nos 6,277,375; 6,821,505;
and 7,083,784. Hinton et al., have described constant domain amino acid substitutions at
positions 250 and 428 that confer increased in vivo half-life (Hinton, Johlfs et al. 2004).
(Hinton, Xiong et al. 2006). See also U.S Pat. No 7,217,797. Petkova et al have described
constant domain amino acid substitutions at positions 307, 380 and 434 that confer
increased in vivo half-life (Petkova, Akilesh et al. 2006). See also Shields et al (Shields,
Namenuk et al. 2001) and . Antibody constant regions can also be
modified so as to remove effector function. The mutation of the Asparagine (N) at position
297 to a Glutamine (Q) removes the N-linked carbohydrate that mediates binding of the Fc
to Fc receptors. Such aglycosylated antibodies do not bind to the human Fc gamma RI and
do not activate the complement pathway (Tao and Morrison 1989). Other examples of
constant domain amino acid substitutions which modulate binding to Fc receptors and
subsequent function mediated by these receptors, including FcRn binding and serum half-
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life, are described in U.S Pat. Application Nos 20090142340; 20090068175; and
20090092599.
In molecules of the present invention which comprise an Fc region, in some
embodiments it may be advantageous to engineer in the substitution L235E to reduce or
abolish Fc binding and Fc-related effector function, as described in Lund, Winter et al
(1991) J Immunology 147: 2657-2662 and Alegre et al. (1992) J Immunology 148: 3461-
3468. The antibody may be an IgG1, and IgG3 or an IgG4.
In molecules of the present invention which comprise an Fc region, in some
embodiments it may be advantageous to engineer or otherwise select for an Fc in which the
C-terminal lysine (K447) is deleted. Preferably this modification improves
manufacturability by reducing heterogeneity of expressed molecule.
The glycans linked to antibody molecules are known to influence interactions of
antibody with Fc receptors and glycan receptors and thereby influence antibody activity,
including serum half-life (Kaneko, Nimmerjahn et al. 2006; Jones, Papac et al. 2007; and
Kanda, Yamada et al. 2007). Hence, certain glycoforms that modulate desired antibody
activities can confer therapeutic advantage. Methods for generating engineered
glycoforms are known in the art and include but are not limited to those described in U.S.
Pat. Nos 6,602,684; 7,326,681; 7,388,081; and .
Extension of half-life by addition of polyethylene glycol (PEG) has been widely
used to extend the serum half-life of proteins, as reviewed, for example, by Fishburn 2008.
The invention also provides compositions comprising at least one isolated
antibody or antigen binding portion thereof of the present invention. This composition will
typically comprise at least one formulating agent selected from sterile water, sterile
buffered water, and/or at least one preservative selected from the group consisting of
phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben,
benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or
mixtures thereof in an aqueous diluent, optionally, wherein the concentration of protein is
about 0.1 mg/ml to about 200 mg/ml, further comprising at least one isotonicity agent or at
least one physiologically acceptable buffer.
[0151] The antibody compositions of the invention can optionally further comprise an
effective amount of at least one compound or protein selected from at least one of an anti-
infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug,
an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI)
tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an
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antineoplastic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a topical
drug, a nutritional drug or the like. Such drugs are well known in the art, including
formulations, indications, dosing and administration for each presented herein (see, e.g.,
Nursing 2001 Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, Pa.,
2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall,
Inc, Upper Saddle River, N.J.; Pharmcotherapy Handbook, Wells et al., ed., Appleton &
Lange, Stamford, Conn., each entirely incorporated herein by reference).
The compositions of the present invention can further comprise at least one of any
suitable auxiliary, such as, but not limited to, diluent, binder, stabiliser, buffers, salts,
lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable
auxiliaries are preferred. Non-limiting examples of, and methods of preparing such sterile
solutions are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's
Pharmaceutical Sciences, 18 th Edition, Mack Publishing Co. (Easton, Pa.) 1990.
Pharmaceutically acceptable carriers can be routinely selected that are suitable for the
mode of administration, solubility and/or stability of the antibody composition as well
known in the art or as described herein.
Pharmaceutical excipients and additives useful in the present composition include
but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g.,
sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatised sugars
such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar
polymers), which can be present singly or in combination, comprising alone or in
combination 1-99.99% by weight or volume. Exemplary protein excipients include serum
albumin, such as human serum albumin (HSA), recombinant human albumin (rHA),
gelatin, casein, and the like. Representative amino acids which can also function in a
buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid,
aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine,
aspartame, and the like. One preferred amino acid is histidine. A second preferred amino
acid is arginine.
Carbohydrate excipients suitable for use in the invention include, for example,
monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and
the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;
polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the
like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol),
myoinositol and the like. Preferred carbohydrate excipients for use in the present invention
are mannitol, trehalose, and raffinose.
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Antibody compositions can also include a buffer or a pH adjusting agent;
typically, the buffer is a salt prepared from an organic acid or base. Representative buffers
include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic
acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine
hydrochloride, or phosphate buffers. Preferred buffers for use in the present compositions
are organic acid salts, such as citrate.
Additionally, the compositions of the invention can include polymeric
excipients/additives, such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates
(e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycols,
flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents,
surfactants (e.g., polysorbates such as “TWEEN® 20” and “TWEEN® 80”), lipids (e.g.,
phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
These and additional known pharmaceutical excipients and/or additives suitable
for use in the antibody compositions according to the invention are known in the art, e.g.,
as listed in “Remington: The Science & Practice of Pharmacy”, 19 th ed., Williams &
Williams, (1995), and in the “Physician's Desk Reference”, 52 nd ed., Medical Economics,
Montvale, N.J. (1998), the disclosures of which are entirely incorporated herein by
reference. Preferred carrier or excipient materials are carbohydrates (e.g., saccharides and
alditols) and buffers (e.g., citrate) or polymeric agents.
[0158] The present invention also provides a method of treating a condition involving
NKT cell effector function comprising administering the antibody or antigen binding
portion thereof. As used herein, the term “NKT cell effector function” is intended to
encompass NKT cell functions which result from CD1d-restricted glycolipid activation of
NKT cells. Such functions include, but are not necessarily limited to, any one or more of
tumor necrosis factor alpha (TNF-α), IFN-γ, IL-4, IL-5 or IL-13 release by NKT cells, up-
regulation of NKT cell surface FasL expression, the release of a perforin, and the release of
granzyme B by NKT cells.
The route of administration may be selected from wide range of routes of
administration including parenteral, intramuscular, intravenous, bolus, intraperitoneal,
subcutaneous, respiratory, inhalation, topical, nasal, vaginal, rectal, buccal, sublingual,
intranasal, subdermal, and transdermal. It is currently believed, however, that the most
appropriate route will be parental or inhalation. Additional information regarding
inhalation of proteins can be found in Borish LC, et al 1999 Am. J. Respir. Crit. Care Med.
160(6), 1816-1823.
9704459 1
For parenteral administration, the antibody or antibody binding portion thereof
can be formulated as a solution, suspension, emulsion or lyophilised powder in association,
or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of
such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human
serum albumin. Liposomes and nonaqueous vehicles, such as fixed oils, may also be used.
The vehicle or lyophilised powder may contain additives that maintain isotonicity (e.g.,
sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The
formulation is sterilised by known or suitable techniques.
Isolated nucleic acid molecules of the present invention can include nucleic acid
molecules comprising an open reading frame (ORF), optionally with one or more introns,
e.g., but not limited to, at least one specified portion of at least one CDR, as CDR1, CDR2
and/or CDR3 of at least one heavy chain or light chain, respectively; nucleic acid
molecules comprising the coding sequence for an antibody or antibody binding portion
thereof; and nucleic acid molecules which comprise a nucleotide sequence substantially
different from those described above but which, due to the degeneracy of the genetic code,
still encode at least one antibody or antibody binding portion thereof as described herein
and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it
would be routine for one skilled in the art to generate such degenerate nucleic acid variants
that code for a specific antibody or antibody binding portion thereof of the present
invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in
the present invention.
As indicated herein, nucleic acid molecules of the present invention which
comprise a nucleic acid encoding an antibody or antibody binding portion thereof can
include, but are not limited to, those encoding the amino acid sequence of an antibody or
antibody binding portion thereof, by itself; the coding sequence for the entire antibody or
or antibody binding portion thereof; the coding sequence for an antibody or antibody
binding portion thereof as well as additional sequences, such as the coding sequence of at
least one signal leader or fusion peptide, with or without the aforementioned additional
coding sequences, such as at least one intron, together with additional, non-coding
sequences, including but not limited to, non-coding 5′ and 3′ sequences, such as the
transcribed, non-translated sequences that play a role in transcription, mRNA processing,
including splicing and polyadenylation signals (for example – ribosome binding and
stability of mRNA); an additional coding sequence that codes for additional amino acids,
such as those that provide additional functionalities. Thus, the sequence encoding an
antibody or antibody binding portion thereof can be fused to a marker sequence, such as a
sequence encoding a peptide that facilitates purification of the fused antibody or antibody
binding portion thereof.
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The present invention provides isolated nucleic acids that hybridise under
selective hybridisation conditions to a polynucleotide encoding an antibody or antibody
binding portion thereof of the present invention. Thus, the polynucleotides of this
embodiment can be used for isolating, detecting, and/or quantifying nucleic acids
comprising such polynucleotides. For example, polynucleotides of the present invention
can be used to identify, isolate, or amplify partial or full-length clones in a deposited
library. In some embodiments, the polynucleotides are genomic or cDNA sequences
isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic
acid library.
[0164] Preferably, the cDNA library comprises at least 80% full-length sequences,
preferably at least 85% or 90% full-length sequences, and more preferably at least 95%
full-length sequences. The cDNA libraries can be normalised to increase the representation
of rare sequences. Low or moderate stringency hybridisation conditions are typically, but
not exclusively, employed with sequences having a reduced sequence identity relative to
complementary sequences. Moderate and high stringency conditions can optionally be
employed for sequences of greater identity. Low stringency conditions allow selective
hybridisation of sequences having about 70% sequence identity and can be employed to
identify orthologous or paralogous sequences.
Optionally, polynucleotides of this invention will encode at least a portion of an
antibody or antigen binding portion thereof encoded by the polynucleotides described
herein. The polynucleotides of this invention embrace nucleic acid sequences that can be
employed for selective hybridisation to a polynucleotide encoding an antibody or antigen
binding portion thereof of the present invention. (See, e.g., Ausubel, supra;).
The isolated nucleic acids of the present invention can be made using (a)
recombinant methods, (b) synthetic techniques, and (c) purification techniques, or
combinations thereof, as well-known in the art.
The nucleic acids can conveniently comprise sequences in addition to a
polynucleotide of the present invention. For example, a multi-cloning site comprising one
or more endonuclease restriction sites can be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the
isolation of the translated polynucleotide of the present invention. For example, a hexa-
histidine marker sequence provides a convenient means to purify the proteins of the
present invention. The nucleic acid of the present invention, excluding the coding
sequence, is optionally a vector, adapter, or linker for cloning and/or expression of a
polynucleotide of the present invention.
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Additional sequences can be added to such cloning and/or expression sequences
to optimise their function in cloning and/or expression, to aid in isolation of the
polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of
cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See,
e.g., Ausubel, supra)
The isolated nucleic acid compositions of this invention, such as RNA, cDNA,
genomic DNA, or any combination thereof, can be obtained from biological sources using
any number of cloning methodologies known to those of skill in the art. In some
embodiments, oligonucleotide probes that selectively hybridise, under stringent conditions,
to the polynucleotides of the present invention are used to identify the desired sequence in
a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and
genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel,
supra)
A cDNA or genomic library can be screened using a probe based upon the
sequence of a polynucleotide of the present invention, such as those disclosed herein.
Probes can be used to hybridise with genomic DNA or cDNA sequences to isolate
homologous genes in the same or different organisms. Those of skill in the art will
appreciate that various degrees of stringency of hybridisation can be employed in the
assay; and either the hybridisation or the wash medium can be stringent. As the conditions
for hybridisation become more stringent, there must be a greater degree of
complementarity between the probe and the target for duplex formation to occur. The
degree of stringency can be controlled by one or more of temperature, ionic strength, pH
and the presence of a partially denaturing solvent, such as formamide. For example, the
stringency of hybridisation is conveniently varied by changing the polarity of the reactant
solution through, for example, manipulation of the concentration of formamide within the
range of 0% to 50%. The degree of complementarity (sequence identity) required for
detectable binding will vary in accordance with the stringency of the hybridisation medium
and/or wash medium. The degree of complementarity will optimally be 100%, or 90-
100%, or any range or value therein. However, it should be understood that minor
sequence variations in the probes and primers can be compensated for by reducing the
stringency of the hybridisation and/or wash medium.
Methods of amplification of RNA or DNA are well known in the art and can be
used according to the present invention without undue experimentation, based on the
teaching and guidance presented herein. Known methods of DNA or RNA amplification
include, but are not limited to, polymerase chain reaction (PCR) and related amplification
processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis,
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et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat. No. 5,142,033 to
Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat.
No. 5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No.
4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No. 4,656,134 to
Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence
as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al,
with the tradename NASBA), the entire contents of which references are incorporated
herein by reference. (See, e.g., Ausubel, supra)
For instance, PCR technology can be used to amplify the sequences of
polynucleotides of the present invention and related genes directly from genomic DNA or
cDNA libraries. PCR and other in vitro amplification methods can also be useful, for
example, to clone nucleic acid sequences that code for proteins to be expressed, to make
nucleic acids to use as probes for detecting the presence of the desired mRNA in samples,
for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to
direct persons of skill through in vitro amplification methods are found in Ausubel, supra,
as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A
Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990).
Commercially available kits for genomic PCR amplification are known in the art. See, e.g.,
Advantage®-GC Genomic PCR Kit (Clontech). The T4 gene 32 protein (Boehringer
Mannheim) can be used to improve yield of long PCR products.
The isolated nucleic acids of the present invention can also be prepared by direct
chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical
synthesis generally produces a single-stranded oligonucleotide, which can be converted
into double-stranded DNA by hybridisation with a complementary sequence, or by
polymerisation with a DNA polymerase using the single strand as a template. One of skill
in the art will recognise that while chemical synthesis of DNA can be limited to sequences
of about 100 or more bases, longer sequences can be obtained by the ligation of shorter
sequences. Synthesis of longer sequences by assembly of overlapping oligonucleotides is
routine in the art.
[0174] The present invention further provides recombinant expression cassettes
comprising a nucleic acid of the present invention. A nucleic acid sequence of the present
invention, for example, a cDNA or a genomic sequence encoding an antibody or antigen
binding portion thereof of the present invention, can be used to construct a recombinant
expression cassette that can be introduced into at least one desired host cell. A recombinant
expression cassette will typically comprise a polynucleotide of the present invention
operably linked to transcriptional initiation regulatory sequences that will direct the
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transcription of the polynucleotide in the intended host cell. Both heterologous and non-
heterologous (i.e., endogenous) promoters can be employed to direct expression of the
nucleic acids of the present invention.
In some embodiments, isolated nucleic acids that serve as promoter, enhancer, or
other elements can be introduced in the appropriate position (upstream, downstream or in
intron) of a non-heterologous form of a polynucleotide of the present invention so as to up
or down regulate expression of a polynucleotide of the present invention. For example,
endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or
substitution.
[0176] The present invention also relates to vectors that include isolated nucleic acid
molecules of the present invention, host cells that are genetically engineered with the
recombinant vectors, and the production of at least one antibody or antigen binding portion
thereof by recombinant techniques, as is well known in the art. See, e.g., Ausubel, et al.,
supra. The polynucleotides can optionally be joined to a vector containing a selectable
marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate,
such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector
is a virus, it can be packaged in vitro using an appropriate packaging cell line and then
transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter. The
expression constructs will further contain sites for transcription initiation, termination and,
in the transcribed region, a ribosome binding site for translation. The coding portion of the
mature transcripts expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG)
appropriately positioned at the end of the mRNA to be translated, with UAA and UAG
preferred for mammalian or eukaryotic cell expression.
Expression vectors will preferably but optionally include at least one selectable
marker. Such markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate
reductase (DHFR, U.S. Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636;
and 5,179,017), ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase
(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; and 5,827,739) resistance for eukaryotic cell
culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other
bacteria or prokaryotics (the above patents are entirely incorporated herein by reference).
Appropriate culture mediums and conditions for the above-described host cells are known
in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a
vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-
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dextran mediated transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection or other known methods. Such methods are described in the art,
such as Ausubel, supra, Chapters 1, 9, 13, 15, 16.
At least one antibody, or antigen binding portion thereof of the present invention
can be expressed in a modified form, such as a fusion protein, and can include not only
secretion signals, but also additional heterologous functional regions. For instance, a region
of additional amino acids, particularly charged amino acids, can be added to the N-
terminus of an antibody, or antigen binding portion thereof to improve stability and
persistence in the host cell, during purification, or during subsequent handling and storage.
Also, peptide moieties can be added to an antibody, or antigen binding portion thereof of
the present invention to facilitate purification. Such regions can be removed prior to final
preparation of an antibody or at least one fragment thereof. Such methods are described in
many standard laboratory manuals, such as Ausubel, supra, Chapters 16, 17 and 18. Those
of ordinary skill in the art are knowledgeable in the numerous expression systems available
for expression of a nucleic acid encoding a protein of the present invention.
Alternatively, nucleic acids of the present invention can be expressed in a host cell
by turning on (by manipulation) in a host cell that contains endogenous DNA encoding an
antibody, or antigen binding portion thereof of the present invention. Such methods are
well known in the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746,
and 5,733,761, entirely incorporated herein by reference.
Illustrative of cell cultures useful for the production of the antibodies, specified
portions or variants thereof, are mammalian cells. Mammalian cell systems often will be in
the form of monolayers of cells although mammalian cell suspensions or bioreactors can
also be used. A number of suitable host cell lines capable of expressing intact glycosylated
proteins have been developed in the art, and include the COS-1 (e.g., ATCC CRL 1650),
COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g.,
ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, COS-7 cells, CHOK1SV
cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells and the like, which
are readily available from, for example, American Type Culture Collection, Manassas, Va.
Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma
cells. Particularly preferred host cells are CHOK1 (ATCC: CRL-9618) or CHOK1SV (e.g.
Lonza Biologics).
Expression vectors for these cells can include one or more of the following
expression control sequences, such as, but not limited to, an origin of replication; a
promoter (e.g., late or early SV40 promoters, the CMV promoter; U.S. Pat. Nos.
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,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an
EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulin
promoter; an enhancer, and/or processing information sites, such as ribosome binding sites,
RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site),
and transcriptional terminator sequences. See, e.g., Ausubel et al., supra. Other cells useful
for production of nucleic acids or proteins of the present invention are known and/or
available, for instance, from the American Type Culture Collection Catalogue of Cell
Lines and Hybridomas (www.atcc.org) or other known or commercial sources.
When eukaryotic host cells are employed, polyadenlyation or transcription
terminator sequences are typically incorporated into the vector. An example of a terminator
sequence is the polyadenlyation sequence from the bovine growth hormone gene.
Sequences for accurate splicing of the transcript can also be included. An example of a
splicing sequence is the VP1 intron from SV40 (Sprague et al. 1983 J Virol 45 773-81).
Additionally, gene sequences to control replication in the host cell can be incorporated into
the vector, as known in the art.
As will be seen the current specification uses the term "% identical" to describe a
number of sequences. As would be understood the term "% identical" means that in a
comparison of two sequences over the specified region the two sequences have the
specified number of identical residues in the same position. The level of identity may be
determined using CLUSTALW with default parameters.
It will also be noted that the sequences are "at least 95% identical" to the
comparator sequence. In certain embodiments it is preferred that the sequence is at least
96% or at least 97% or at least 98% or at least 99% identical to the comparator sequence.
The term "moderate stringency" in relation to hybridization conditions as used
herein means hybridization and/or washing carried out in 2 x SSC buffer, 0.1% (w/v) SDS
at a temperature in the range 45°C to 65°C, or equivalent conditions. The term "high
stringency" in relation to hybridization conditions as used herein means a hybridization
and/or wash carried out in 0.1 x SSC buffer, 0.1% (w/v) SDS, or lower salt concentration,
and at a temperature of at least 65°C, or equivalent conditions. Reference herein to a
particular level of stringency encompasses equivalent conditions using wash/hybridization
solutions other than SSC known to those skilled in the art. For example, methods for
calculating the temperature at which the strands of a double stranded nucleic acid will
dissociate (also known as melting temperature, or Tm) are known in the art. A temperature
that is similar to (e.g., within 5°C or within 10°C) or equal to the Tm of a nucleic acid is
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considered to be high stringency. Medium stringency is to be considered to be within 10°C
to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.
Throughout this specification the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of a stated element,
integer or step, or group of elements, integers or steps, but not the exclusion of any other
element, integer or step, or group of elements, integers or steps.
All publications mentioned in this specification are herein incorporated by
reference. Any discussion of documents, acts, materials, devices, articles or the like which
has been included in the present specification is solely for the purpose of providing a
context for the present invention. It is not to be taken as an admission that any or all of
these matters form part of the prior art base or were common general knowledge in the
field relevant to the present invention as it existed in Australia or elsewhere before the
priority date of each claim of this application.
It must be noted that, as used in the subject specification, the singular forms "a",
"an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus,
for example, reference to "a" includes a single as well as two or more; reference to "an"
includes a single as well as two or more; reference to "the" includes a single as well as two
or more and so forth.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way of
illustration and are not intended as limiting.
EXAMPLES OF THE INVENTION
GENERAL METHODS
HEK293/pTT5 expression system
[0191] For all transfections involving the HEK293E/pTT5 expression system, HEK293E
cells were cultured in complete cell growth media (1 L of F17 medium (Invitrogen), 9 mL
of Pluronic F68 (Invitrogen), 2 mM Glutamine containing 20% (w/v) Tryptone NI
(Organotechnie) with Geneticin (50 mg/mL, Invitrogen) at 50 μL/100 mL culture). At the
day before transfection, the cells were harvested by centrifugation and resuspended in fresh
media without Geneticin. The next day DNA was mixed with a commercial transfection
reagent and the DNA transfection mix added to the culture drop-wise. The culture was
incubated overnight at 37°C, 5% CO2 and 120 rpm without Geneticin. The next day 12.5
mL of Tryptone and 250 µL of Geneticin were added per 500 mL culture. The culture was
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incubated at 37°C, 5% CO2 and 120 rpm for seven days, then the supernatants were
harvested and purified.
CD1d/b 2M proteins
Human CD1d/b 2M was produced in the mammalian HEK293E/pTT5 expression
system, using a DNA expression construct coding for the extracellular domain of CD1d
with an C-terminally located HIS tag (SEQ ID NO: 19), co-transfected with a DNA
expression construct coding for b 2M (SEQ ID NO: 20). Culture supernatant containing the
secreted CD1d/b 2M protein was harvested by centrifugation at 2000 g for 10 mins to
remove the cells. The CD1d/b 2M protein complex was purified from the supernatant via
the His8 affinity tag using a HisTrapTM HP column (GE Healthcare). The eluted protein
was buffer-exchanged into PBS using a HiLoad 16/60 Superdex 200 prep grade column
(GE Healthcare) and ~50 kDa fraction was separated by gel filtration on a HiLoad 26/60
Superdex 200 prep grade column (GE Healthcare). Human b 2M alone was produced and
purified in a similar manner. A similar method of purification was adopted for the
purification of other species CD1d (e.g murine CD1d) and synthetic constructs of CD1d
(such as hCD1dmu and mCD1dmu).
To determine the sequence of cynomolgus monkey CD1d, cDNA from monkey
spleen was obtained from Biochain. The following primers were used to amplify the CD1d
DNA based on rhesus CD1d mRNA (PubMed Accession number: NM_001033114):
F1 – GTGCCTGCTGTTTCTGCTG (SEQ ID NO: 120)
R1 – TGCCCTGATAGGAAGTTTGC (SEQ ID NO: 121)
A PCR was set up that amplified a 1 kb DNA product. This DNA was ligated into
pGEM-T Easy (Promega) and sequenced using M13 forward and reverse primers. The
sequence was aligned with that of rhesus CD1d (UniProt Accession number: Q4AD67) and
found to be identical. The gene sequence was then synthesized, a C-terminal HIS tag
added, subcloned into the pTT5 vector and expressed using the HEK-293E/pTT5 system.
The protein was purified using Ni chromatography via an introduced HIS tag.
For phage display experiments, recombinant human CD1d/b 2M was biotinlyated
using an EZ-link Sulfo-NHS-LC-biotin kit (Pierce) at a 3:1 ratio of biotin: CD1d/b 2M.
Free biotin was removed from the protein preparation by dialysis against PBS using a
Slide-A-Lyzer dialysis cassette with a 3.5 kDa molecular weight cut-off. For campaign 2,
biotinylated recombinant cynomolgus CD1d/b 2M was also prepared as described above.
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Construction of vectors expressing antibodies
VH amino acid chains were expressed with a human constant region (human IgG4
heavy chain CH1, hinge, CH2 and CH3 domains (such as NCBI accession number P01861
with the substitution at S228P). This was achieved by back-translation of amino acid
sequences into DNA sequences followed by de novo synthesis and assembly of synthetic
oligonucleotides. Following gene synthesis the whole sequence was subcloned into the
multiple cloning site of the pTT5 heavy chain vector (Durocher, Y. et al., 2002, Nucleic
Acids Res, 30, E9). VL amino acid chains were expressed with a human kappa or lambda
light chain constant region (such as NCBI accession number AAI10395 and C6KXN3) by
subcloning the sequence into the multiple cloning site of the pTT5 light chain vector.
Expression and Purification of Antibodies
Heavy and light chain DNA vectors were co-transfected into the HEK293/pTT5
expression system and cultured for seven days. The supernatants derived from these
transfections were adjusted to pH 7.4 before being loaded onto a HiTrap Protein A column
(5 mL, GE Healthcare). The column was washed with 50 mL of 1X PBS (pH 7.4). Elution
was performed using 0.1 M citric acid pH 2.5. The eluted antibody was desalted using
Zeba Desalting columns (Pierce) into 1X PBS (pH 7.4). The antibodies were analyzed
using SDS-PAGE. The concentration of the antibody was determined using the BCA assay
kit (Pierce).
Example 1 – Generation of Anti-CD1d Antibodies
Phage Display
FAbs that bind to both human and cynomolgus CD1d/b 2M were isolated from a
naive phagemid library.
Anti-CD1d/b 2M FAbs were isolated from the phage display library over the
course of two panning ‘campaigns’ (i.e. discrete phage display experiments with different
reagents or panning conditions). The general protocol followed the method outlined by
Marks et al. (Marks, J.D. & Bradbury, A., 2004, Methods Mol Biol, 248, 161-76).
Each phage display campaign involved three rounds of panning. For each round,
~1x10 phage particles were blocked by mixing 1:1 with blocking buffer (5% skim milk
in phosphate buffered saline pH 7.4) and incubating for 1 hr at room temperature. The
blocked phage library was then pre-depleted for streptavidin binders by incubation for 45
mins with 100 m L of streptavidin-coupled Dynabeads (Invitrogen), which were blocked as
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described for the library. The beads (and streptavidin binders attached to them) were
discarded after the incubation step.
Recombinant CD1d/b 2M antigen was prepared for panning by capture onto the
surface of streptavidin-coupled Dynabeads (Invitrogen). To achieve this, 10-100 pmols of
biotinylated CD1d/b 2M was incubated with 100 m L of beads for 45 mins at room
temperature. The resulting CD1d/b 2M -bead complexes were washed with PBS to remove
free CD1d/b 2M and then used in the subsequent panning reaction.
Library panning was conducted by mixing the blocked and pre-depleted library
with the CD1d/b 2M-bead complexes in a 1.5 mL microcentrifuge tube and rotating for 2
hrs at room temperature. Non-specifically bound phage was removed using a series of
washes. Each wash involved pulling the bead complexes from the solution onto the tube
wall using a magnetic rack, aspirating the supernatant and then re-suspending the beads in
fresh wash buffer. This was repeated a number of times with either PBS wash buffer (PBS
with 0.5% skim milk) or PBS-T wash buffer (PBS with 0.05% TWEEN-20 (Sigma) and
0.5% skim milk). Phage that remained bound after the washing process were eluted from
the CD1d/b 2M-bead complexes by incubation with 0.5 mL of 100 mM triethylamine
(TEA) (Merck) for 20 mins at room temperature. The eluted ‘output’ phage were
neutralized by adding 0.25 mL of 1 M Tris-HCl pH 7.4 (Sigma).
At the end of the first and second rounds of panning, the output phage were added
to a 10 mL culture of exponentially growing TG1 E. coli (yeast-tryptone (YT) growth
media) and allowed to infect the cells by incubating for 30 mins at 37°C without shaking,
then with shaking at 250 rpm for 30 mins. The phagemids encoding the phage display
output were then rescued as phage particles following a standard protocol (Marks, J.D. &
Bradbury, A., 2004, Methods Mol Biol, 248, 161-76). At the end of the third panning
round TG1 cells were infected with output phage, but the cells were plated on solid YT
growth media (supplemented with 2% glucose and 100 m g/mL carbenicillin) at a sufficient
dilution to produce discrete E. coli colonies. These colonies were used to inoculate 1 mL
liquid cultures to allow expression of FAb fragments for use in screening experiments.
ELISA-based screening of FAbs for CD1d binding
[0204] Each individual E. coli colony was used to express a FAb that was screened for
CD1d/b 2M binding activity. Colonies were inoculated into 1 mL YT starter cultures
(supplemented with 100 m g/mL carbenicillin and 2% glucose) in 96-well deepwell plates
(Costar) and incubated overnight at 30°C with shaking at 650 rpm. These starter cultures
were diluted 1:50 into a 1 mL expression culture (YT supplemented with 100 m g/mL
carbenicillin only) and grown to an optical density of 0.8-1.0 at 600 nm. FAb expression
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was induced by adding isopropyl-beta-D-thiogalactopyranoside to a final concentration of
1 mM. Cultures were incubated at 20°C for 16 hrs.
FAb samples were prepared by harvesting cells by centrifugation (2500 g, 10
mins) and performing a periplasmic extraction. The cell pellet was resuspended in 75 m L of
extraction buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA, 20% Sucrose) and shaken at
1000 rpm for 10 mins at 4°C. Extract preparation was completed by adding 225 m L of
H O, shaking at 1000 rpm for 1 hr and clearing the extract by centrifugation at 2500 g for
mins. The supernatants were recovered, filtered through Acroprep 100 kDa molecular-
weight cutoff plates (Pall Corporation) and stored at 4°C until required for further
experiments.
To screen potential human CD1d-binders yielded by phage display by ELISA,
human CD1d/b 2M (produced in HEK 293E cells and biotinylated as described above) was
captured on streptavidin-coated ELISA plates (Pierce) at 1 m g/mL. Plates were then
washed and separate FAb samples (prepared as described above) were added to individual
wells on the ELISA plates. FAbs were allowed to bind the captured CD1d/b 2M for two
hours at room temperature and then washed three times with PBS-T and three times with
PBS. Bound FAbs were detected using a HRP-conjugated antibody directed against the V5
affinity tag (Sigma) fused to the C-terminus of the FAb heavy chain. The detection
antibody was incubated for 1.5 hrs at room temperature. The plates were washed to remove
unbound antibody and the assay signal was developed by incubating with 50 m L 3,3',5,5'-
Tetramethylbenzidine (KPL) and quenched with 50 m L 1 M HCl. Assay signals were read
at A450 nm using a microplate reader (Bio-Tek). Results were expressed as the raw A450
nm value, where any signal 2-fold greater than the average assay background was defined
as ‘positive’.
[0207] In later assays, Maxisorp ELISA plates (Nunc) coated with non-biotinylated
human CD1d/b 2M, cynomolgus CD1d/b 2M or b 2M alone were prepared to test the
binding of FAb samples. Washing and detection steps were as described above.
SPR-based screening of FAbs for CD1d/b 2M binding
SPR screening was conducted using a BIAcore 4000 Biosensor (GE Healthcare)
in a single concentration analyte pass assay. Approximately 10,000 RU of antiV5 antibody
(Invitrogen cat#R960CUS) was immobilized on a CM5 Series S Sensor chip, using
standard amine coupling chemistry at pH 5.5 on spots 1, 2, 4 & 5 of each of the four flow
cells leaving spot 3 unmodified. The running buffer used was HBS-EP+ (GE Healthcare)
and all interactions measured at 25ºC and data collection rate set to 10 Hz. Crude
periplasmic preparations of V5-tagged FAbs, were diluted two-fold in running buffer
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before capturing at a flow rate of 10 m L/min for 100 sec (typically around 200 RU of FAb
was captured) on spot 1 or 5 of each flow cell. Following a short stabilization period,
human or cynomolgus CD1d/b 2M was passed over all spots of all four flow cells
simultaneously at a flow rate of 30 m L/min for 100 sec. Dissociation of the interaction was
measured for 100 sec prior to regeneration back to the anti-V5 antibody using a 30 sec
pulse of 100 mM phosphoric acid. Generated sensorgrams were referenced against an
adjacent anti-V5 antibody spot for each flow cell, and fitted using a 1:1 Langmuir equation
to determine ka, kd and KD.
Results of the Phage Display Campaigns
[0209] Over 4400 clones were screened for binding to human and cynomolgus
CD1d/b 2M by SPR assays. A total of 51 FAbs were found to have high selectivity for
human and cynomolgus CD1d.
Example 2 – Confirmation of IgG Binding to CD1d
Human-cynomolgus CD1d reactive FAbs were converted to IgG4 format,
expressed and purified as described in the General Methods. The purified antibodies were
tested for binding to human and cynomolgus CD1d by ELISA and SPR using modified
versions of the assays described in Example 1. Briefly, for ELISA assays Maxisorp ELISA
plates (Nunc) were coated with the appropriate antigen at 1 m g/mL. Plates were then
washed and purified IgG samples were added to individual wells on the ELISA plates.
IgGs were allowed to bind the captured CD1d/b 2M for one hour at room temperature and
then washed three times with PBS-T and three times with PBS. Bound IgGs were detected
using a HRP-conjugated antibody directed against human Fc (Sigma). The detection
antibody was incubated for 30 minutes at room temperature. The plates were washed to
remove unbound antibody and the assay signal was developed by incubating with 50 m L
3,3',5,5'-Tetramethylbenzidine (KPL) and quenched with 50 m L 1 M HCl. Assay signals
were read at A450 nm using a microplate reader (Bio-Tek). Results were expressed as the
raw A450 nm value, where any signal 2-fold greater than the average assay background
was defined as ‘positive’.
Purified antibodies were also subjected to full kinetic characterization using a
Biacore T100 biosensor (GE Healthcare). Approximately 10,000 RU of anti-human IgG
(Invitrogen cat# H10500) was immobilized on a CM5 Series S Sensor chip, using standard
amine coupling chemistry in flow cell (FC) 1 and FC2 (or alternatively FC3 and FC4) of
the Biacore T100 Biosensor. The running buffer used was HBS-EP+ (GE Healthcare) and
interactions measured at 25ºC. Peak purified IgGs were diluted to 10 nM in running buffer,
and captured on FC2 (or alternatively FC4) at a flow rate of 10 µL/min in order to capture
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50 - 80 RU of IgG. After an appropriate stabilization period, the target, human or
cynomolgus CD1d/b 2M was passed over FC1 and FC2 (or alternatively FC3 and FC4) at a
flow rate of 60 µL/min at concentrations ranging from 33.3 nM to 0.4 nM (using a three-
fold dilution of CD1d/b 2M). The contact time for association was 120 sec and dissociation
measured for 20 mins for the highest concentration and 240 sec for all other concentrations
in the series. The sensorgram data from FC2 was subtracted from FC1 and a buffer only
control. The curves were fitted using a 1:1 Langmuir equation to generate the ka, kd and
KD values (Table 1).
Table 1: ELISA and SPR results for Phage Display Antibodies
IgG ELISA ELISA SPR human CD1d
hCD1d cCD1d ka (1/Ms) kd (1/s) KD (M)
401.1 + + 3.30E+05 6.10E-05 1.85E-10
401.3 + - 1.55E+05 4.61E-03 2.98E-08
401.9 + + 1.69E+05 2.21E-02 1.31E-07
401.11 + + 1.80E+05 4.79E-04 2.66E-09
401.12 + + 7.84E+05 4.59E-03 5.85E-09
401.14 + + 1.37E+05 2.88E-03 2.10E-08
401.22 + + 1.92E+05 2.70E-03 1.40E-08
401.24 + + 9.02E+05 1.19E-03 1.32E-09
401.26 + + 3.75E+05 6.02E-03 1.60E-08
401.28 + - 5.10E+05 3.30E-03 6.47E-09
401.30 + + 7.51E+05 3.42E-03 4.55E-09
401.33 + + 1.16E+05 1.09E-03 9.40E-09
402.1 + + 3.15E+05 7.01E-03 2.23E-08
402.2 + - 8.31E+04 4.51E-04 5.43E-09
402.4 + - 3.05E+05 2.89E-03 9.51E-09
402.5 + - 1.80E+05 3.17E-03 1.76E-08
402.6 + + 1.58E+05 3.12E-04 1.98E-09
402.7 + + 1.71E+05 5.05E-03 2.95E-08
402.8 + + 5.30E+05 1.61E-04 3.04E-10
402.9 + + 2.56E+05 1.55E-03 6.04E-09
402.11 + + 9.46E+04 5.22E-03 5.52E-08
402.12 + + 1.18E+06 8.29E-04 7.01E-10
402.15 + + 1.99E+05 4.31E-03 2.17E-08
402.16 + + 1.96E+05 8.89E-04 4.54E-09
402.17 + + 4.61E+05 2.88E-03 6.24E-09
402.18 + + 1.25E+05 4.08E-04 3.27E-09
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Example 3 – Cell-Based CD1d Tetramer Inhibition Potency Assay
Creation of a stable NKTCR-expressing cell line
To develop cell-based assays to characterize the biological potency of anti-CD1d
antibodies, a stable cell line expressing an NKT cell receptor (NKTCR) was required. The
cell line J.RT3-T3.5 (ATCC: TIB-153) was chosen for creation of a stable NKT cell
receptor-expressing cell line. J.RT3-T3.5 is derived from the E6-1 clone of Jurkat (ATCC:
TIB 152) that lacks the b chain of the T cell antigen receptor. The cells do not express
either CD3 or the T cell receptor ba heterodimer on the surface. J.RT3-T3.5 cells were co-
electroporated with two vectors, one containing the a chain of the J3N.5 NKTCR (SEQ ID
NO: 21) and the other the b chain of the J3N.5 NKTCR (SEQ ID NO: 22) (Brigl, M., et al.,
2006 J Immunol 176: 3625-34.). This NKT cell receptor is reactive to the glycolipid
antigen a -GalCer. These vectors encoding the a and b chains of the NKTCR also express
resistance genes to geneticin and blasticidin respectively. Stable incorporation of these
vectors was achieved by propagation of these cells in culture medium containing pre-
determined concentrations of geneticin and blasticidin.
To derive a clonal line, transfected J.RT3-T3.5 cells were grown to log phase in
RPMI 1640 (Gibco) under geneticin and blasticidin selection and limiting diluted at an
average of one cell per well in 96-well flat-bottom plates (Corning). To determine stable
expression of the transfected NKTCR, viable clones were subcloned into larger volumes in
24-well plates and screened by multi-parameter flow cytometry. Clones were screened for
binding to CD1d tetramer (ProImmune), expression of Va 24Ja 18, the junctional region of
the human iNKTCR, and expression of CD3, a co-receptor for the TCR. Clones with high
expression of these markers were selected by high mean fluorescence intensity (MFI) of
each of the markers. Stability was confirmed by flow cytometry of the clones after multiple
passages into T25 flasks and revival after banking down putative clones at -180°C in a
freezing medium (90% heat-inactivated foetal bovine serum and 10% DMSO). Stable
clones were identified and used in cell-based assays to characterize functional potency of
the anti-CD1d antibodies.
CD1d Tetramer Inhibition Potency Assay
[0214] A cell-based assay to characterize potency of the anti-CD1d antibodies used the
clonal NKT cell line described above, in a flow cytometry-based CD1d tetramer inhibition
assay. This assay relied on the ability of the CD1d-tetramer loaded with α-GalCer to bind
the NKTCR stably transfected into the J.RT3-T3.5 cells. The potency of anti-CD1d
antibodies was determined by the ability of the antibody to inhibit CD1d tetramer binding
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to the NKTCR present on the stably transfected J.RT3-T3.5 line. The inhibitory antibodies
bind to a specific epitope on the CD1d molecule within the tetramer that prevents
interaction of the CD1d tetramer with the stably transfected NKTCR on the J.RT3-T3.5
cells. The readout of the assay was a reduction in the mean fluorescence intensity (MFI) of
the fluorochrome-conjugated CD1d tetramer. Approximate EC50 values were generated by
titration of the anti-CD1d antibody whilst keeping the CD1d tetramer concentration
constant. To ensure the reproducibility and reliability of the assay, optimization
experiments at different CD1d tetramer concentrations were conducted to determine the
best dynamic range. The optimal concentration of the CD1d tetramer was determined to be
at a 1:1000 dilution, corresponding to approximately 10 nM.
To perform the assay, antibodies were prepared at decreasing concentrations from
m g/mL in 0.1% bovine serum albumin (BSA) in cold 1 X PBS at pH 7.4. These
antibodies were co-incubated at room temperature in the dark at a 1:1 ratio with the anti-
CD1d tetramer at a final concentration of 10 nM for a maximum of 40 minutes. This
CD1d-tetramer/ anti-CD1d antibody mixture was used to stain NKTCR-stable transfectants
of J.RT3-T3.5 cells plated at 1 x 10 cells per well in 96-well round-bottom plates. Wash
steps were done in 0.1% BSA in 1 X PBS. Data were acquired by flow cytometry and
analyzed using flow cytometry analysis software (FlowJo).
Anti-CD1d antibodies 401.1, 401.9, 401.11, 401.12, 401.14, 401.28, 401.30,
402.1, 402.6, 402.7, 402.8, 402.16, 402.17 and 402.18 were tested in this assay. An
irrelevant specificity negative control antibody (human IgG1) was chosen as a negative
control. The anti-CD1d antibodies 42 (BD Biosciences) and 51.1 (eBioscience) were
chosen as positive controls. Of these antibodies, only 401.11, 401.28, 402.1, 402.6, 402.7,
402.8, 402.16 and 402.18 demonstrated potency in this assay similar or superior to
antibody 42 (Table 2). In comparison, the negative control antibody demonstrated
negligible inhibition of tetramer binding to the cell line. Representative data from multiple
experiments are presented in Figure 1. This result could not have been predicted by assays
that measure the direct binding of antibodies to CD1d. This demonstrates the need to select
and screen for antibodies that are capable of functionally inhibiting the CD1d- NKT
interaction.
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Table 2. EC50 values for Tetramer Inhibition Assay
Antibody Name EC50 (ng/mL)
401.11 283.9
402.1 387.5
402.6 601.6
402.7 791.3
402.8 164.7
402.16 351.6
402.17 Negligible Inhibition
402.18 88.2
42 1435.0
51.1 775.4
Negative Control Negligible Inhibition
Example 4 – NKT cell line IL-2 release assay
Anti-CD1d antibodies were further characterized using a cell-line based
functional potency assay. The U-937 cell line (ATCC: CRL 1593.2) is a myelomonocytic
line that is CD1d-positive. U-937 cells loaded with a GalCer are able to induce the
production of IL-2 by the stable NKTCR cell line described in Example 3. Inhibitory anti-
CD1d antibodies reduce the release of IL-2 by the NKTCR cell line in response to these
a GalCer-loaded U-937 cells. IL-2 levels were measured by standard ELISA technologies
(R&D Systems).
[0218] To perform the assay, approximately 1.5 x 10 U-937 cells were loaded with
a GalCer at a final concentration of 100 ng/mL in 96-well flat-bottom plates in RPMI 1640
(Gibco). At 60 minutes following the addition of a GalCer, anti-CD1d antibodies were
added to the cells at decreasing concentrations starting from 10 m g/mL to the cells. At sixty
minutes post antibody loading, 1.5 x 10 stable NKTCR-transfected J.RT3-T3.5 cells were
added to each well. Twenty-four hours after addition of the NKTCR-transfected J.RT3-
T3.5 cells, IL-2 levels were tested by ELISA (R&D Systems) using cell-free culture
supernatants.
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Anti-CD1d antibodies 401.1, 401.9, 401.11, 401.12, 401.14, 401.28, 402.1, 402.6,
402.7, 402.8, 402.16 and 402.18 were tested in this assay. Anti-CD1d antibodies 42 and
51.1 were chosen as positive controls. An irrelevant specificity negative control antibody
(human IgG1) was chosen as a negative control. Of these antibodies, only 401.11, 402.1,
402.6, 402.7, 402.8 and 402.16 demonstrated equivalent or stronger inhibition of IL-2
release compared with antibody 42, as determined by EC50 values (Table 3 and
representative data in Figure 2). Additionally, 401.11 and 402.8 demonstrated superior
inhibition of IL-2 release compared with the antibody 51.1 (Figure 2). In comparison, the
negative control antibody demonstrated negligible inhibition of IL-2 release. Surprisingly,
401.11 was approximately 20-fold more potent than antibody 42 and approximately 15-
fold more potent than 51.1. Similarly, 402.8 was approximately 25-fold more potent than
antibody 42 and approximately 17-fold more potent than 51.1 (Figure 2). Together, these
data reveal novel fully human anti-CD1d antibodies with significantly improved biological
potency compared with those described in the art.
Table 3: EC50 values – NKT Cell Line IL-2 Assay
Antibody Name EC50 (ng/mL)
401.1 Negligible Inhibition
401.9 286.0
401.11 5.3
401.12 576.3
401.14 Negligible Inhibition
401.28 112.4
401.30 Negligible Inhibition
402.8 4.5
42 110.7
51.1 77.3
(Negative Control) Negligible Inhibition
Example 5 – Testing the Binding of Anti-CD1d antibodies to Primary PBMCs
Anti-CD1d antibodies were characterized for the ability to bind to CD1d as
displayed on primary human somatic cells. Anti-CD1d antibodies 402.8, 401.11.158 and
an irrelevant specificity negative control antibody were adjusted to a concentration of 2
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mg/mL and conjugated to the fluorochrome Pacific Blue according to the manufacturer’s
instructions (Invitrogen).
Since it is known that CD1d is expressed on certain human cell populations
present in human blood, peripheral blood mononuclear cells (PBMCs) were used to
confirm the binding of anti-CD1d antibody 402.8 to primary CD1d+ cells. PBMCs were
isolated from buffy coats by density centrifugation over a lymphoprep gradient according
to standard protocols (Nycomed). Cells were then washed several times in 1 X PBS and
stained with anti-CD1d antibody 402.8 (10 m g/mL) or negative control human IgG1 (10
m g/mL), and co-stained with anti-human CD11c (Biolegend). Anti-CD1d antibody 402.8
bound to a distinct CD1d-positive population that was CD11c-positive (Figure 3). In
contrast, the negative control antibody demonstrated negligible binding (Figure 3). Anti-
CD1d antibody 401.11.158 (10 m g/mL) also bound this CD1d-positive population (not
shown). These data clearly indicate that anti-CD1d antibodies derived from 402.8 and
401.11, bound to a CD1d+ population in primary human cells.
Example 6 – Testing the Efficacy of Anti-CD1d antibodies in Primary NKT Cell-
Based Assays
Human NKT cells are capable of eliciting rapid effector function in response to
lipid or glycolipid antigens presented in the context of CD1d. This rapid effector function
can be demonstrated by release of cytokines such as IFN-g , IL-4, IL-5, and IL-13.
Inhibitory anti-CD1d antibodies can inhibit the function of these NKT cells by binding to
CD1d present on cells and preventing the interaction between the NKT cells and their
cognate complex of CD1d and glycolipid. Suitable antigen presenting cells may include
immortalized myeloid cell lines or primary human dendritic cells. Given the rarity of NKT
cells within the peripheral blood of human donors, successful assays require isolation and
expansion of such primary NKT cells in the first instance.
Isolation and Expansion of NKT cells
PBMCs were isolated from buffy coats over a lymphoprep (Nycomed) gradient.
NKT cells were then enriched by standard magnetic-associated cell sorting (MACS)
methods (Exley et al., 2010 Curr Protoc Immunol, Chapter 14, Unit 14:11). Briefly, NKT
cells were incubated with MACS microbeads against the Va 24-Ja 18 iNKT marker
(Miltenyi Biotec). Excess microbeads were removed by washing the cell suspension twice
in cold PBS. The cell suspension was then passed through the MACS column and the
positive fraction containing the enriched NKT cells was retained. Cells from the negative
fraction may contain CD1d-positive cells, such as monocytes and dendritic cells, and can
be used as feeders to stimulate the enriched NKT cells. The feeder cells are first treated
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with mitomycin C, an inhibitor of mitosis, for 30 min at 37° C. These cells were then
washed several times with tissue culture medium, and then loaded with a -GalCer at a final
concentration of 100 ng/mL and co-cultured at a 1:1 ratio with 1 x 10 NKT cells per well
in 96-well round bottom plates. At 16 hours post incubation at 37 C and 5% CO , IL-2 was
added to the medium at a final concentration of 10 ng/mL. The cells were left to culture for
approximately 14 days. The purity of the NKT cell population was determined by multi-
parameter flow cytometry, using fluorochome-conjugated CD1d tetramers (ProImmune),
fluorochrome-conjugated anti-Va 24Ja 18 (Miltenyi Biotec) and fluorochrome-conjugated
anti-CD3 (BD Biosciences). The purity of suitable NKT populations for use in cell-based
assays was routinely more than 70% NKT cells by flow cytometry analysis.
Assay Methodology
All primary cell-based assays were undertaken at 37°C and 5% CO unless
otherwise stated. THP-1 cells were distributed into 96-well flat bottom plates at a
concentration of 2 x 10 cells per well. After ten minutes, a -GalCer was loaded onto the
cells at a final concentration of 100 ng/mL. At 45 minutes post addition of a -GalCer, anti-
CD1d inhibitory antibodies were added at decreasing concentrations from 10 m g/mL. At 30
minutes post addition of the antibodies, NKT cells were then added at 2 x 10 cells per
well. Cell-free culture supernatants were collected at 24 hours post incubation. ELISA for
human cytokines was performed on the culture supernatants: human IFN-γ, IL-4, IL-5 and
IL-13 (all R&D Systems).
Results of the Functional Assays using Primary NKT Cells
Anti-CD1d antibodies 401.1, 401.9, 401.11, 401.12, 401.14, and 402.8 were
tested in this assay. An irrelevant specificity negative control antibody (human IgG1) was
used as a negative control. Antibodies 42 and 51.1 were used as positive controls. Similar
to the results of the IL-2 cell line assay described in Example 4, only antibodies 401.11 and
402.8 showed strong inhibition of glycolipid-antigen induced cytokine release by primary
human NKT cells in the context of cellular CD1d (Figure 4 and Table 4; see IFN-γ assay
EC50 values). By comparison, the negative control antibody demonstrated negligible
inhibition. Antibody 42 showed inhibition of cytokine release by NKT cells at high doses
(10 m g/mL) but this effect was not sustained at lower concentrations (Figure 4). Antibody
42 is considered to be a strong neutralizer of NKT cell activity in vitro and is widely
published as such (Exley, M. et al., 1997, J. Exp. Med. 186:109-120; WO 03/092615).
Compared with antibody 42, antibodies 401.11 and 402.8 demonstrated up to 114-fold and
up to 180-fold improved potency respectively.
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To establish the inhibitory potency of antibodies 401.11 and 402.8 against CD1d
present on non-immortalized human cells, a functional assay was developed using primary
human monocyte-derived dendritic cells. The dynamic range of the assay may be increased
by expanding the proportion of cells that express the CD1d antigen, thereby increasing the
level of antigen presentation to CD1d-responsive NKT cells. Monocytes were isolated
from PBMC by magnetic activated cell sorting (MACS) isolation of CD14+ cells and
culture of these cells in GM-CSF and IL-4 according to standard protocols. Dendritic cells
were cultured in 96-well flat bottom plates at 2x10 cells per well and loaded with a GalCer
at 100 ng/mL for 1 hr. Inhibitory antibodies were added to the cultures for 1 hr, prior to
addition of expanded NKT cells in a 1:1 ratio with the dendritic cells. Twenty-four hours
later, cell-free supernatants were assayed for IFN-γ, IL-4, IL-5 and IL-13 release. Anti-
CD1d antibodies 401.11 and 402.8 were tested in this assay; an irrelevant specificity
human IgG1 was used as a negative control and antibodies 42 (BD Biosciences) and 51.1
(eBioscience) used as positive controls. Only antibodies 401.11 and 402.8 demonstrated
strong inhibition of glycolipid-antigen induced cytokine release by primary human NKT
cells in this primary cell-based assay (Figure 5 and Table 5). In comparison, the negative
control antibody demonstrated negligible inhibition. Anti-CD1d antibodies 42 and 51.1
showed some inhibition of cytokine release by NKT cells at high doses (10 m g/mL) but this
effect was not sustained at lower doses. Compared with antibody 42, antibodies 401.11
and 402.8 demonstrated up to 200-fold and up to 50-fold improved potency respectively
(Figure 5 and Table 5; see IFN-γ assay EC50 values). Compared with antibody 51.1,
antibodies 401.11 and 402.8 demonstrated significantly improved potency. This result
therefore demonstrates that the anti-CD1d antibodies show potent neutralizing activity in
the context of human somatic cells that naturally express the CD1d antigen.
[0227] In summary, fully human anti-CD1d antibodies 402.8 and 401.11 were identified
and demonstrated highly potent inhibition of NKT cell activity. These antibodies exhibited
100-fold improved potency when compared with the anti-CD1d antibodies 42 and 51.1.
Table 4: EC50 values – Primary NKT Cell Line Assays using THP-1 cell line
Antibody Name IFN-γ EC50 IL-4 EC50 IL-5 EC50 IL-13 EC50
(ng/mL) (ng/mL) (ng/mL) (ng/mL)
401.11 3.8 1.9 1.7 2.5
402.8 2.4 1.7 1.2 1.5
42 429.1 76.1 64.7 153.8
Negative Control Negligible Negligible Negligible Negligible
Inhibition Inhibition Inhibition Inhibition
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Table 5: EC50 values – Primary NKT Cell Line Assays using primary CD14+ cells
Antibody Name IFN-γ EC50 IL-4 EC50 IL-5 EC50 IL-13 EC50
(ng/mL) (ng/mL) (ng/mL) (ng/mL)
401.11 6.8 5.9 6.2 12.1
402.8 28.6 4.0 6.9 39.9
51.1 168.2 DNI DNI 221.7
42 1388.0 185.7 108.4 844.0
Negative Control Negligible Negligible Negligible Negligible
Inhibition Inhibition Inhibition Inhibition
DNI – Did Not Inhibit, where the inhibitory activity of the antibody was typically less than 50% of the
maximal response by human NKT cells at 1 μg/mL.
The sequences of the VH and VL domains of antibodies 401.11 and 402.8 are as
follows:
401.11 VH SEQ ID NO 1
401.11 VL SEQ ID NO 2
402.8 VH SEQ ID NO 3
402.8 VL SEQ ID NO 4
Example 7 – 402.8 and 401.11 share a common epitope on CD1d
As shown in the above results, the phage display campaign generated anti-CD1d
antibodies 401.11 and 402.8 that showed superior biological potency compared with prior
art anti-CD1d antibodies. It was hypothesized that this significantly improved potency was
due to recognition of a highly neutralizing epitope that, once bound by the anti-CD1d
antibody, prevented the interaction between the CD1d molecule and its cognate receptor,
for example the NKT cell receptor present on NKT cells. Blockade of this interaction with
CD1d was therefore necessary and sufficient to inhibit downstream biological effects such
as activation of NKT cells and the release of pro-inflammatory cytokines. To investigate
whether the highly potent anti-CD1d antibodies generated had a different epitope
specificity compared with neutralizing anti-CD1d antibodies, a competition binding ELISA
was developed.
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Assay methodology
Anti-CD1d antibody 402.8 was biotinylated using an EZ-link Sulfo-NHS-LC-
biotin kit (Pierce) at a 3:1 ratio of biotin: 402.8. Free biotin was removed from the protein
preparation by multiple washes with PBS and concentration by centrifugation (3000 rpm)
through a centrifugal filter unit with a 30 kDa cutoff (Millipore). Maxisorp ELISA plates
(Nunc) were coated with 0.5 m g/mL human CD1d and allowed to incubate overnight at
4°C. Plates were then washed three times in PBS containing 0.1% Tween20, before the
plate was blocked in 1% BSA for 1 hr at room temperature. Biotinylated 402.8 was then
co-equilibrated for 5 minutes in a 1:1 ratio with non-biotinylated anti-CD1d antibodies
(402.8, 401.11, 42 and 51.1). These antibodies were added to the plates for 1 hour at room
temperature in decreasing concentrations from 50 m g/mL (i.e. a maximum of 500-fold
excess compared with 0.1 m g/mL biotinylated 402.8). Plates were then washed three times
in PBS containing 0.1% Tween20. Streptavidin horseradish peroxidase conjugate (BD
Biosciences) was added to the plates for 1 hour at room temperature in the dark. The plates
were washed to remove unbound streptavidin-horseradish peroxidase. The assay signal
was developed by incubating with 50 m L 3,3',5,5'-Tetramethylbenzidine (KPL) and
quenched with 50 m L 1 M HCl. Assay signals were read at A450 nm using a microplate
reader (FluoStar Galaxy). Results were expressed as the raw A450 nm value and converted
to degree of competition (percentage) values by subtracting the readings corresponding
with zero percent inhibition from raw data.
Using the above described method it was demonstrated that 401.11 and 402.8
compete with each other for binding to human CD1d, as shown by absorbance values at
450nm (Figure 6A) and degree of competition with 402.8 (Figure 6B) and therefore share
an overlapping or common epitope. In contrast, 402.8 does not share an overlapping or
common epitope with either 42 or 51.1. Taken together, these data demonstrate that the
highly potent anti-CD1d antibodies 401.11 and 402.8 bind to a similar high affinity
neutralizing epitope that is not shared by anti-CD1d antibodies 42 and 51.1.
Example 8: Cross-reactivity with cynomolgus macaque CD1d
Anti-CD1d antibodies 401.11 and 402.8 were tested for binding to cynomolgus
CD1d by ELISA using modified versions of the assays described in Example 1. Maxisorp
ELISA plates (Nunc) were coated with 1 m g/mL human or cynomolgus CD1d and allowed
to incubate overnight at 4 C. Plates were then washed three times in PBS containing 0.1%
Tween20, before the plate was blocked in 1% BSA for 1 hr at room temperature. Plates
were then washed three times in PBS containing 0.1% Tween20. Anti-CD1d antibodies
were then added at decreasing concentrations from 10 m g/mL. Plates were then washed
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three times in PBS containing 0.1% Tween20. Detection of the bound antibody was
enabled using an HRP-conjugated Fc-specific antibody (Sigma). Plates were then washed
three times in PBS containing 0.1% Tween20 to remove unbound horseradish peroxidase-
conjugated anti-Fc. The assay signal was developed by incubating with 50 m L 3,3',5,5'-
Tetramethylbenzidine (KPL) and quenched with 50 m L 1 M HCl. Assay signals were read
at A450 nm using a microplate reader (FluoStar Galaxy). The results indicate that
antibodies 401.11 and 402.8, which bind human CD1d (Figure 7A), are also cross-reactive
with cynomolgus CD1d (Figure 7B). Cross-reactivity with non-human primate CD1d is
desirable to allow for testing in non-human primate models of human diseases.
Example 9: Cell-based Cross-reactivity with Cynomolgus CD1d
To demonstrate cross-reactivity with non-human primate CD1d in a cell-based
format, human and cynomolgus macaque PBMCs were stained using cross-reactive anti-
CD1d antibody 402.8. This antibody was biotinylated in a 3:1 fold ratio of biotin to IgG as
described in Example 8. A negative control antibody (human IgG1) was biotinylated in a
similar manner. Detection of the bound biotinylated anti-CD1d antibody was achieved by
incubating the cells with phycoerythrin-conjugated streptavidin. Anti-CD1d antibody
bound CD1d-positive primary monocyte-derived DCs in both human (Figure 3) and
cynomolgus macaque species (Figure 8). These results indicate 402.8 displays human and
cynomolgus macaque cross-reactivity in a cell-based context, which is important for
testing in non-human primate models of human diseases.
Example 10: Cell-based functional inhibition of cynomolgus CD1d-mediated primary
NKT function
For cell based potency assays, cynomolgus PBMC were loaded on day 0 with
a GalCer (100 ng/mL) and with or without anti-CD1d antibodies. The cultures were
prepared in 24-well plates in a humidified incubator at 37 C, 5% CO . At day 7, IL-2 (10
U/mL) was added on day 7 and the cultures left to incubate at 37 C, 5% CO for a further
96 hours. The final readout was enumeration of NKT cells using anti-CD3 and anti-T cell
receptor Va 24 antibodies (BD Biosciences). In the absence of anti-CD1d antibody, or with
addition of isotype control antibody, NKT cells expanded in the presence of a GalCer by
approximately 10-fold. Anti-CD1d antibodies 401.11 and 402.8 potently blocked the
a GalCer-mediated expansion of CD1d-restricted cynomolgus NKT cells compared with
treatment of cultures with no antibody or human IgG negative control antibody (Figure 9).
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Example 11: Optimized variants of 401.11 and 402.8
The 401.11 and 402.8 antibodies can be further optimized through alterations to
the antibody’s sequence with the aim of yielding a positive effect on the antibody’s
biophysical properties whilst having negligible or positive impact on their potency. Firstly,
alterations that enhance the expression level of the antibody with concomitantly increased
production levels may be desirable. Secondly, removal of potentially undesirable sequence
features, such as solvent-exposed cysteine residues or N-linked glycosylation sites through
amino acid substitution may reduce potential product heterogeneity, which may further
enhance these antibodies. Thirdly, substitution of amino acid residues with the potential to
impact the stability of the antibody through oxidation or isomerization during purification
or storage may be replaced with amino acids that do not undergo such transitions (Wang et
al. 2007 Journal of Pharmaceutical Sciences 96:1-26), which may further improve these
antibodies. Lastly, rare or non-germ-line 401.11 and 402.8 amino acid residues which may
potentially contribute to immunogenicity may be substituted with other amino acids with
the aim of lowering predicted immunogenicity, which may further improve these
antibodies. The following describes the implementation of these optimization strategies on
the antibodies 401.11 and 402.8.
Enhancing antibody 401.11
The variable heavy and light chain sequences of 401.11 were compared to
corresponding human germline sequences via MegAlign (DNAstar). The most homologous
germline heavy chain variable region – IGHV3-9*01 – differed from 401.11 by seven
framework amino acids. IGKV1-12*01 shared the highest sequence homology with the
401.11 light chain, differing by two framework amino acids (Figure 12). This information
was used to generate a panel of 401.11 variants containing framework residues substituted
with the corresponding germline framework residue (Figure 12).
Antibodies 401.11 and 401.11.15 through 401.11.28 (Figure 12), were produced
by co-transfections of the heavy- and light chains into HEK-293E cells. SPR (Biacore) was
used to measure the relative expression level of each antibody and its corresponding
binding to human CD1d as measured by the equilibrium dissociation constant (KD). The
resulting data is presented in Table 6.
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Table 6
IgG Expression KD
NKT IFNg-ggg NKT IFNg-ggg
Level (pM)
EC50 (ng/mL) EC50 (ng/mL)
(Protein A
(THP-1) (moDC)
capture)
401.11 DNE N/A 3.754 0.857
401.11.14 144 791 N/D N/D
401.11.15 44 507 N/D N/D
401.11.16 150 DNB N/D N/D
401.11.17 DNE N/A N/D N/D
401.11.18 185 9970 N/D N/D
401.11.19 385 667 N/D N/D
401.11.20 109 977 N/D N/D
401.11.21 534 951 N/D N/D
401.11.22 553 754 N/D N/D
401.11.23 227 1980 N/D N/D
401.11.24 359 644 4.263 0.360
401.11.25 292 932 N/D N/D
401.11.26 358 855 3.245 1.304
401.11.27 23 DNB N/D N/D
401.11.28 176 722 1.417 0.294
Note: DNE – Did not express; DNB – Did not bind; N/D – not determined; THP-1 – THP-1 cells used as
antigen presenting cells; moDC – primary monocyte-derived dendritic cells used as antigen presenting cells.
Data are representative of 3 independent experiments.
[0238] Of the fifteen antibodies tested in this experiment, thirteen had measurable levels
of antibody in the supernatant of the transfected HEK-293E cells. Ten of these thirteen
antibodies bound to CD1d with an equilibrium dissociation constant (KD) of less than 1
nM (Table 6).
Antibodies 401.11, 401.11.24, 401.11.26 and 401.11.28 were generated and tested
for functional inhibition of CD1d mediated NKT cell cytokine release using a cell-based
potency assay (Table 6). Antibodies 401.11.24, 401.11.26 and 401.11.28 showed similar or
improved potency compared to 401.11 when either THP-1 cells or primary CD14+
dendritic cells were used as CD1d-positive antigen presenting cells (APCs).
Positions 97 through (100B) of CDR3 of 401.11 heavy chain consists of the
sequence CSSSGC. To determine the role of the cysteines present in the CDR3 each
cysteine was substituted with one of nine amino acids representing the different classes of
side chains of amino acids (Rajpal et al PNAS 2005 102: 8466-8471). Following
transfection into HEK293E cells no antibody expression was detectable for any of these
variants, resulting in no detectable binding to CD1d as measured by SPR. Substitution of
both cysteines to serine residues (401.11.164) resulted in an antibody that expressed, but
which bound to human CD1d with a lower affinity compared to 401.11 suggesting that the
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cysteines of CDR3 of the heavy chain of 401.11 are desirable for antibody expression and
high affinity binding to CD1d (Table 7).
Table 7
Substitution Light Chain
from 401.11
in variable
IgG heavy chain KD (M)
N/A 401.11
401.11 WT 9.97 E-10
Mock N/A 401.11 N/D
C97S 401.11
401.11.51 N/D
401.11.52 C97T 401.11 N/D
C97G 401.11
401.11.53 N/D
401.11.54 C97L 401.11 N/D
C97V 401.11
401.11.55 N/D
401.11.56 C97K 401.11 N/D
C97Y 401.11
401.11.57 N/D
401.11.58 C97R 401.11 N/D
C97H 401.11
401.11.59 N/D
401.11.92 C(100B)S 401.11 N/D
C(100B)T 401.11
401.11.93 N/D
401.11.94 C(100B)G 401.11 N/D
C(100B)L 401.11
401.11.95 N/D
401.11.96 C(100B)V 401.11 N/D
C(100B)K 401.11
401.11.97 N/D
401.11.98 C(100B)Y 401.11 N/D
C(100B)R 401.11
401.11.99 N/D
401.11.100 C(100B)H 401.11 N/D
C97S & N45K
401.11.164 C(100B)S 6.56E-08
N/D – not determined
[0241] The heavy chain CDR3 sequence of 401.11 was targeted for further variation to
attempt to improve the expression levels and affinity of 401.11. For this analysis, each
amino acid in CDR3 of the heavy chain of 401.11 was substituted with one of nine amino
acids representing the different classes of side chains of amino acids. The expression levels
of each resulting antibody and its binding to CD1d are given in Table 8 and Table 9.
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Table 8
Substitution
IgG VH Expression Level KD (M)
401.11 N/A 36 <0.1E-10*
Mock N/A -8 DNB
401.11.36 D95K DNE N/A
401.11.37 D95R DNE N/A
401.11.38 D95S 105 DNB
401.11.39 D95G 34 N/A
401.11.40 D95L DNE N/A
401.11.41 D95Y DNE N/A
401.11.42 D95F DNE N/A
401.11.43 D95Q DNE N/A
401.11.44 M96K 85 4.31E-10
401.11.45 M96R 63 2.49E-10
401.11.46 M96F DNE 3.03E-10
401.11.47 M96Y DNE N/A
401.11.48 M96Q DNE N/A
401.11.49 M96S 47 4.01E-10
401.11.50 M96G 29 1.87E-10
401.11.60 S98D DNE N/A
401.11.61 S98T 57.85 2.42E-18
401.11.62 S98W DNE N/A
401.11.63 S98L 23.76 1.89E-10
401.11.64 S98V 84.5 2.30E-10
401.11.65 S98K 38.36 1.67E-09
401.11.66 S98R 75.87 1.56E-10
401.11.67 S98Y DNE 4.85E-11
401.11.68 S98G 84.81 1.17E-09
401.11.69 S99D 57.92 1.44E-10
401.11.70 S99T 20.14 5.49E-10
401.11.71 S99W 63.04 <0.1E-10*
401.11.72 S99L 11.43 6.49E-10
401.11.73 S99V 61.56 5.27E-10
401.11.74 S99K 56.91 9.33E-11
401.11.75 S99R 14.06 DNB
401.11.76 S99Y DNE N/A
401.11.77 S99G 20.18 2.72E-10
401.11.78 S99D DNE N/A
401.11.79 S100T 16.81 DNB
401.11.80 S100W 29.03 2.34E-09
401.11.81 S100L 24.65 5.52E-10
401.11.82 S100V 20.35 DNB
401.11.83 S100K 45.84 1.55E-09
401.11.84 S100R 51.11 1.12E-09
401.11.85 S100Y 9.87 8.23E-08
Residue numbering according to Kabat .<0.1E-10* indicates the KD of the construct was below the limit of
detection. DNE – Did not express (below 10RU); DNB – Did not bind; N/A – Not Applicable
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Table 9
Substitution
IgG VH Expression Level KD (M)
401.11 N/A 103 1.75E-10
Mock N/A 3 DNB
401.11.86 S100G 337 1.39E-10
401.11.87 G(100A)S 24 7.13E-09
401.11.88 G(100A)A 103 1.89E-09
401.11.89 G(100A)D 40 5.30E-09
401.11.90 G(100A)K 21 <0.1E-10*
401.11.101 P(100C)S 95 <0.1E-10*
401.11.102 P(100C)T 130 4.57E-10
401.11.103 P(100C)G 115 <0.1E-10*
401.11.104 P(100C)L 44 4.87E-10
401.11.105 P(100C)F 34 1.52E-03
401.11.106 P(100C)K 64 1.93
401.11.107 P(100C)Y 38 9.96E-10
401.11.108 P(100C)R 49 <0.1E-10*
401.11.109 P(100C)W 16 DNB
401.11.110 D(100D)K 59 2.47E-10
401.11.111 D(100D)R 19 <0.1E-10*
401.11.112 D(100D)S 95 2.43E-09
401.11.113 D(100D)G 59 6.79E-10
401.11.114 D(100D)L 121 1509.04
401.11.115 D(100D)Y 86 6.67E-10
401.11.116 D(100D)F 96 3.77E-08
401.11.117 D(100D)Q 164 1.33E-09
401.11.118 G(100E)S 68 <0.1E-10*
401.11.119 G(100E)A 24 8.77E-10
401.11.120 G(100E)V DNE N/A
401.11.121 G(100E)D DNE N/A
401.11.122 G(100E)K 30 1.30E-10
401.11.123 G(100E)R 22 2.39E-10
401.11.124 Y(100F)W DNE 1.38E-09
401.11.125 Y(100F)F 33 5.35E-10
401.11.126 Y(100F)S 38 3.13E-11
401.11.127 Y(100F)Q 26 2.70E-09
401.11.128 Y(100F)E DNE N/A
401.11.129 Y(100F)R DNE N/A
401.11.130 Y(100F)K DNE N/A
401.11.131 S102D 38 2.00E-10
401.11.132 S102T 55 5.69E-10
401.11.133 S102W 53 1.67E-10
401.11.134 S102L 73 5.32E-10
401.11.135 S102V 85 4.21E-10
401.11.136 S102K 22 <0.1E-10*
401.11.137 S102R 49 9.38E-10
401.11.138 S102Y 48 1.71E-10
401.11.139 S102G 44 5.94E-10
Residue numbering according to Kabat .<0.1E-10* indicates the KD of the construct was below the limit of
detection of the machine. DNE – Did not express (below 10RU); DNB – Did not bind; N/A – Not Applicable
9704459 1
Antibody 401.11.86 expressed at over 3 times the level of 401.11 and had a higher
affinity for CD1d compared to 401.11. This antibody was purified and tested for functional
inhibition of CD1d mediated NKT cell cytokine release using a cell-based potency assay
(Table 10). The assay used primary human monocyte-derived dendritic cells or THP-1
cells as a source of CD1d-positive antigen presenting cells and a GalCer-expanded NKT
cells. The protocol was as described in Example 6.
Table 10
Antibody NKT IFN-g EC50 NKT IFN-g EC50 NKT IL-4 EC50 NKT IL-4 EC50
(ng/mL)(THP-1) (ng/mL) (moDC) (ng/mL)(THP-1) (ng/mL) (moDC)
401.11 3.754 0.857 2.885 6.862
401.11.86 1.289 0.359 0.509 1.74
Note: THP-1 – THP-1 cells used as antigen presenting cells; moDC – primary monocyte-derived dendritic
cells used as antigen presenting cells. Data are representative of 5 independent experiments.
401.11.86, which differed from 401.11 by a Serine to Glycine substitution at
position 100, was more potent compared to 401.11. Antibodies were then generated that
contained substitutions identified from the most potent antibodies described above (Figure
13).
[0244] Amino acid analysis of the variable heavy chain sequence of 401.11 identified
several amino acids that may potentially undergo oxidation or isomerization. Particular
emphasis was placed on amino acids present in the CDR sequences of the antibody as any
changes to these amino acids may, over time, impact the binding profile of the antibody. In
the variable heavy chain, M96 was identified as a potential oxidation site, and D(100D)
was identified as a potential isomerization site. Semi conservative or conservative amino
acid substitutions were used in attempts to remove these potentially problematic amino
acid residues (Figure 13). The influence of these substitutions on the binding affinity of the
resulting antibodies is shown in Table 11.
Table 11
Antibody Affinity K
(pM) SPR
401.11
401.11.151
1750
401.11.152
2830
401.11.154
1770
401.11.155
2420
401.11.156
401.11.157 173
401.11.158 189
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Antibody Affinity K
(pM) SPR
401.11.159 30
401.11.160 109
401.11.161 255
401.11.165
401.11.166
401.11.167
401.11.177 507
401.11.178
401.11.179 274
401.11.180 312
401.11.181 427
Many of the antibodies tested had improved affinity compared to the original
antibody 401.11. These antibodies were then tested in a cell-based potency assay which
measures functional inhibition of CD1d mediated NKT cell cytokine release. The protocol
was as described in Example 6. Of the 19 antibodies tested, 14 antibodies consistently
demonstrated similar or improved potency compared with the 401.11 antibody. These
antibody variants had significantly improved potency compared with anti-CD1d antibodies
42 and 51.1, both of which showed some inhibitory activity at the highest concentration of
10μg/mL but failed to show inhibition at lower antibody concentrations. The EC50 values
from representative experiments are presented in Tables 12-16 below.
Table 12
Antibody NKT IL-4 EC50 NKT IL-4 NKT IL-13 NKT IL-13
(ng/mL) (moDC) EC50 (ng/mL) EC50 (ng/mL) EC50 (ng/mL)
(moDC) (moDC) (moDC)
401.11 7.633 6.545 22.75 19.55
401.11.151 12.89 N/D 57.49 N/D
401.11.152 10.2 N/D 78.49 N/D
401.11.154 4.159 N/D 17.73 N/D
401.11.155 27.64 N/D 74.72 N/D
401.11.156 1.948 N/D 5.217 N/D
401.11.157 N/D 0.799 N/D 3.871
401.11.158 N/D 0.58 N/D 3.844
401.11.159 N/D 1.694 N/D 10.47
401.11.160 N/D 1.564 N/D 6.519
401.11.161 N/D 5.244 N/D 8.381
Negative Control DNI DNI DNI DNI
N/D – not determined; Negative Control – An IgG1 of irrelevant specificity; moDC – primary monocyte-
derived dendritic cells used as antigen presenting cells; DNI – Did Not Inhibit, where the inhibitory activity
of the antibody was typically less than 50% of the maximal response by human NKT cells at 1 μg/mL.
9704459 1
Table 13
Antibody NKT IL-4 NKT IL-5 NKT IL-13 NKT TNF
NKT IFN-g
EC50 EC50 EC50 EC50
EC50 (ng/mL)
(moDC) (ng/mL) (ng/mL) (ng/mL) (ng/mL)
(moDC) (moDC) (moDC) (moDC)
401.11 39.38 17.33 47.48 83.08 16.91
401.11.156 7.763 2.716 5.856 9.528 4.507
401.11.157 9.112 3.437 8.492 20.6 5.589
401.11.158 8.165 2.187 6.13 9.823 4.3
42 >1000 136.9 286.5 DNI DNI
51.1 >1000 DNI DNI DNI DNI
Note: moDC – primary monocyte-derived dendritic cells used as antigen presenting cells; DNI – Did Not
Inhibit, where the inhibitory activity of the antibody was typically less than 50% of the maximal response by
human NKT cells at 1 μg/mL.
Table 14
Antibody NKT IL-4 EC50 NKT IL-5 NKT IL-13 NKT TNF
NKT IFN-g EC50
(ng/mL) (THP-1) EC50 EC50 EC50
(ng/mL) (THP-1)
(ng/mL) (ng/mL) (ng/mL)
(THP-1) (THP-1) (THP-1)
401.11 55.16 2.977 6.961 27.25 9.179
401.11.156 5.811 1.993 4.446 6.909 3.846
401.11.157 5.449 1.807 4.476 6.98 3.545
401.11.158 6.404 2.662 4.502 7.776 4.141
401.11.165 6.153 1.112 3.730 5.762 3.043
401.11.166 6.983 2.745 5.379 7.253 4.022
401.11.167 6.510 2.889 4.979 7.465 4.000
Isotype Control DNI DNI DNI DNI DNI
Note: Isotype Control – An irrelevant specificity IgG4; DNI – Did Not Inhibit, where the inhibitory activity
of the antibody was typically less than 50% of the maximal response by human NKT cells at 1 μg/mL; THP-
1 – THP-1 cells used as antigen presenting cells; moDC – primary monocyte-derived dendritic cells used as
antigen presenting cells.
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Table 15
Antibody NKT IL-4 EC50 NKT IL-5 NKT IL-13
NKT IFN-g EC50
(ng/mL) (moDC) EC50 (ng/mL) EC50 (ng/mL)
(ng/mL) (moDC)
(moDC) (moDC)
401.11
12.44 1.98 2.251 2.361
401.11.158
2.797 0.4322 0.7043 0.5981
401.11.177
7.556 0.7306 1.398 1.271
401.11.178
.06 0.9685 1.381 1.764
401.11.179
.2 0.7301 2.097 2.431
401.11.180
49.55 1.312 3.353 4.092
DNI DNI DNI DNI
51.1
DNI DNI DNI DNI
Isotype Control DNI DNI DNI DNI
Note: Isotype Control – An irrelevant specificity IgG4; DNI – Did Not Inhibit, where the inhibitory activity
of the antibody was typically less than 50% of the maximal response by human NKT cells at 1 μg/mL; THP-
1 – THP-1 cells used as antigen presenting cells; moDC – primary monocyte-derived dendritic cells used as
antigen presenting cells.
Table 16
Antibody NKT IL-4 NKT IL-4 NKT IL-13
NKT IFN-g NKT IFN-g
EC50 EC50 EC50
EC50 EC50 (ng/mL)
(ng/mL) (ng/mL) (ng/mL)
(ng/mL) (THP-1)
(moDC) (THP-1) (THP-1)
(moDC)
401.11 23.5 2.335 12.83
1.36 3.286
401.11.158 3.004 2.602 1.160
2.203 4.354
401.11.181 2.634 3.255 1.102
3.195 5.04
42 DNI DNI DNI DNI
51.1 DNI DNI DNI DNI
Isotype Control DNI DNI DNI DNI
Note: Isotype Control – An irrelevant specificity IgG4; DNI – Did Not Inhibit, where the inhibitory activity
of the antibody was typically less than 50% of the maximal response by human NKT cells at 1 μg/mL; THP-
1 – THP-1 cells used as antigen presenting cells; moDC – primary monocyte-derived dendritic cells used as
antigen presenting cells.
In primary NKT cell-based potency assays using CD14+ monocyte derived
dendritic cells as antigen-presenting cells, antibodies derived from 401.11 showed
improved inhibitory activity compared with the parental antibody 401.11. This is clearly
shown by a left-shift in the inhibition curve, where antibodies derived from 401.11
required lower concentrations to achieve the same inhibition of NKT-cell mediated
cytokine release (Figure 14). For example, antibodies 401.11.156 and 401.11.158, titrated
from 1 μg/mL, showed approximately 5.1-fold improvement and 4.8-fold improvement
respectively compared with 401.11, titrated from 1 μg/mL (Figure 14 and Table 13 , see
9704459 1
IFN-γ EC50 values). In several experiments, anti-CD1d antibodies 42 and 51.1 showed
minimal inhibition such that a true EC50 value could not be calculated. In experiments
where the EC50 values could be determined, antibodies derived from 401.11, titrated from
1 μg/mL showed significantly improved potency compared with anti-CD1d antibodies 42
and 51.1. These antibodies showed inhibitory activity at the highest concentration of
10μg/mL, but failed to show inhibition at lower antibody concentrations (Figure 14 and
Table 13); see IFN-γ EC50 values).
In summary, fully human optimized anti-CD1d antibodies derived from 401.11
were identified and demonstrated highly potent inhibition of NKT cell activity in the
context of primary human cells that naturally express the CD1d antigen. These antibodies
showed significantly improved potency compared with the anti-CD1d antibodies 42 and
51.1.
Optimization of the 402.8 antibody
Amino acid analysis of the variable heavy and light chain sequence of 402.8
identified several amino acids that could potentially undergo oxidation, isomerization or
deamindation present in the heavy chain (Wang et al. 2007 Journal of Pharmaceutical
Sciences 96:1-26). These include a potential deamidation site at N(100B), a potential
isomerization site at D101 and potential oxidation sites at W(100A) and M(100E) in the
heavy chain. A potential N-linked glycosylation site was identified at N52 in the heavy
chain. To remove the potential deamidation, oxidation and isomerization sites the amino
acid substitutions were made: W(100A)Y, N(100B)K, M(100E)L, D(101)E. The potential
N-linked glycoyslation site was removed by introducing an N54A which disrupts the N-
linked glycoyslation motif NX(S/T), where X is any amino acid except proline. Antibodies
were made with combinations of these amino acid substitutions in the variable heavy chain
as shown in Figure 15. Each antibody heavy chain was co-transfected with the 402.8 light
chain (SEQ ID No: 4) into HEK-293E cells, purified by Protein A chromatography and the
affinity of each antibody measured using SPR (Table 17). These antibodies were then
tested in a cell-based potency assay using primary human monocyte-derived dendritic cells
and autologous a GalCer-expanded NKT cells (Tables 18 and 19).
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Table 17
IgG K (pM)
402.8 145
402.8.53 578
402.8.54 870
402.8.55 878
402.8.60 198
402.8.84 587
402.8.86 357
402.8.87 344
Table 18
IgG NKT IL-13
NKT IFN-g
EC50 (ng/mL) EC50 (ng/mL)
(moDC)
(moDC)
402.8 0.105 0.154
402.8.53 0.658 1.524
402.8.60 0.244 0.189
402.8.84 0.516 0.663
42 116.0 17.54
Negative Control DNI DNI
Note: Negative Control – An irrelevant specificity IgG1; DNI – Did Not Inhibit, where the inhibitory activity
of the antibody was typically less than 50% of the maximal response by human NKT cells at 1 μg/mL;
moDC – primary monocyte-derived dendritic cells used as antigen presenting cells.
Table 19
Antibody NKT IL-4 NKT IL-4 EC50
NKT IFN-g NKT IFN-g
EC50 (ng/mL) (moDC)
EC50 (ng/mL) EC50 (ng/mL)
(ng/mL)
(moDC) (moDC)
(moDC)
402.8 14.96 22.45 4.358 0.273
402.8.53 277.2 105.4 ND ND
402.8.54 137.6 109.1 ND ND
402.8.55 444.6 175 ND ND
402.8.60 24.68 26.8 ND ND
402.8.84 ND ND 30.64 0.593
402.8.86 ND ND 14.31 0.721
402.8.87 ND ND 25.91 1.008
42 DNI DNI DNI DNI
51.1 DNI DNI DNI DNI
Negative Control DNI DNI ND ND
Note: Negative Control – An irrelevant specificity IgG1; DNI – Did Not Inhibit, where the inhibitory activity
of the antibody was typically less than 50% of the maximal response by human NKT cells at 1 μg/mL;
moDC – primary monocyte-derived dendritic cells used as antigen presenting cells.
These variant antibodies derived from 402.8 demonstrated strong inhibition of
αGalCer-mediated cytokine release by primary NKT cells. Some of the variant antibodies
showed reduced potency compared with 402.8 whereas other antibodies retained similar
9704459 1
potency as determined by dose-dependent inhibition of NKT cell-driven cytokine release.
In several experiments, anti-CD1d antibodies 42 and 51.1 showed minimal inhibition such
that a true EC50 value could not be calculated (Table 19). In experiments where the EC50
values could be determined, antibodies derived from 402.8 were of significantly improved
potency compared with anti-CD1d antibody 42, which showed some inhibitory activity at
the highest concentration of 10μg/mL, but did not retain inhibitory activity at lower
concentrations of antibody (Figure 16 and Table 18). This result therefore demonstrates
that the optimised anti-CD1d antibodies based on parental antibody 402.8 showed
significant improvements in potent neutralizing activity compared with anti-CD1d
antibodies 42 and 51.1 and similar neutralizing activity compared with 402.8 in the context
of primary human cells that naturally express the CD1d antigen.
Example 12: Testing the Efficacy of Anti-CD1d antibodies in Primary NKT Cell-
Based Assays using an Alternative Antigen to α-Galactosylceramide
The cell-based potency assays described in Example 6 employed αGalCer as the
glycolipid antigen. Demonstrating that the anti-CD1d antibodies possess inhibitory activity
in the context of an alternative glycolipid antigen to αGalCer supports the concept that
such highly potent neutralizing anti-CD1d antibodies bind CD1d at a location away from
the regions where lipid and/or glycolipids may be presented. The CD1d-restricted lipid and
glycolipid antigens found in nature may differ from αGalCer in terms of chemical
structure, and consequently it may be useful to demonstrate that the anti-CD1d antibodies
described in the present invention retain inhibitory activity in the context of a glycolipid
antigen with a different chemical structure. In addition, it would be useful to characterize
the inhibitory activity of potential self antigens, i.e. those found in a mammalian context.
Only a few glycolipid antigens with activity for human NKT cells have been
characterized. These include iGb3 and lysophosphatidylcholine, however such antigens
have only weak activation of NKT cells. The C24:1 N-acyl variant of an endogenous lipid,
β-D-glucopyranosylceramide (hereafter known as C24:1 β-GluCer) was described to have
activity for human NKT cells. A cell-based potency assay was developed to characterize
the inhibitory activity of proprietary anti-CD1d antibodies in the context of this alternative
antigen to αGalCer (Brennan, P. J., et al., 2011 Nat Immunol 12:1202-1211).
Assay Methods and Results
The C24:1 N-acyl variant of β-D-glucopyranosylceramide (Avanti; D-glucosyl-ß-
1,1' N-(15Z-tetracosenoyl)-D-erythro-sphingosine N-(15Z-tetracosenoyl)ß-glucosyl-
sphingene) was solubilized in DMSO at 5mg/mL at 37°C for 2 hours before storing in
small aliquots.
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To perform the cell-based potency assays using C24:1 βGluCer, NKT cells were
expanded with αGalCer as described in Example 6. Despite being stimulated in the
presence of αGalCer, these NKT cells retained functional activity to C24:1 βGluCer,
indicating that the TCR specificity of the NKT cell lines generated was also permissive to
C24:1 β-GluCer recognition in the context of human CD1d. NKT cells were phenotyped
by flow cytometry and only used in cell-based potency assays if the purity of the NKT
cells exceeded 70%.
Monocyte-derived dendritic cells were generated as described in Example 6.
These cells were cultured in 96-well flat bottom plates at 2x10 cells per well and loaded
with C24:1 βGluCer at 10 μg/mL for 24 hours. Inhibitory anti-CD1d antibodies
401.11.158, 401.11 and 402.8 were prepared in decreasing concentration from 1 μg/mL
and 42, 51.1 and negative control antibodies were prepared in decreasing concentration
from 10 μg/mL and then added to the C24:1 βGluCer-loaded dendritic cell cultures for 1
hr. Thereafter, NKT cells were added in a 1:1 ratio with the dendritic cells. Twenty-four
hours later, cell-free supernatants were assayed for IFN-γ, IL-4, IL-5 IL-13 and TNF
release. As an example, 401.11, 401.11.158 and 402.8 were tested in this assay. An
irrelevant specificity negative control antibody was used a negative control and anti-CD1d
antibodies 42 (BD Biosciences) and 51.1 (eBioscience) used as positive controls. The 42
and 51.1 antibodies and antibodies 401.11, 401.11.158 and 402.8 demonstrated inhibition
of C24:1 βGluCer-induced cytokine release by primary human NKT cells in this primary
cell-based assay (IFN-g and IL-4 curves are shown in Figure 17). In comparison, the
negative control antibody demonstrated negligible inhibition of cytokine release by NKT
cells.
The antibodies 42 and 51.1 showed some inhibition of cytokine release by NKT
cells at high doses (10 m g/mL) but this effect was not sustained at lower doses. Compared
with antibody 42, antibodies 401.11.158, 401.11, and 402.8 demonstrated up to 216-fold,
up to 58-fold and up to 139-fold improved potency respectively (Figure 17 and Table 20).
Compared with antibody 51.1, antibodies 401.11.158, 401.11, and 402.8 demonstrated up
to 175-fold, up to 47-fold and up to 112-fold improved potency respectively (Figure 17 and
Table 20; see IFN-γ assay EC50 values).
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Table 20
IFN-γ EC50 IL-4 EC50 IL-5 EC50 IL-13 EC50 TNF-α EC50
(ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)
401.11 2.376 1.452 1.231 5.549 1.793
402.8 0.9945 0.6364 0.9292 2.938 1.507
401.11.158 0.6397 0.6822 0.8679 1.439 0.8422
42 138.5 133.1 81.89 52.33 5.692
51.1 111.8 113.1 86.43 66.77 29.72
Negative
Control DNI DNI DNI DNI DNI
Negative Control: An antibody of an IgG4 isotype directed to a target other than CD1d; DNI – Did Not
Inhibit, where the inhibitory activity of the antibody was typically less than 50% of the maximal response by
human NKT cells at 1 μg/mL.
Example 13: Antibodies derived from 402.8 and 401.11 share a common epitope on
CD1d, which is not shared by anti-CD1d antibodies
Variants of 402.8 and 401.11, and commercially sourced anti-CD1d antibodies
were tested in a competition-based ELISA based on Example 7. First, it was shown that a
biotinylated version of 402.8 competed with non-biotinylated antibody 401.11 but not with
antibodies 42 and 51.1. This work is described below.
Assay methodology
Anti-CD1d antibody 402.8 was biotinylated using an EZ-link Sulfo-NHS-LC-
biotin kit (Pierce) at a 3:1 ratio of biotin: 402.8. Free biotin was removed from the protein
preparation by multiple washes with PBS and concentration by centrifugation (3000 rpm)
through a centrifugal filter unit with a 30 kDa cutoff (Millipore). Maxisorp ELISA plates
(Nunc) were coated with 1.0 m g/mL human CD1d and incubated overnight at 4°C. Plates
were then washed three times in PBS containing 0.1% Tween20, before the plate was
blocked in 1% BSA for 1 hr at room temperature. Biotinylated 402.8 was then co-
equilibrated for 5 minutes in a 1:1 ratio with non-biotinylated anti-CD1d antibodies. These
antibodies were added to the plates for 1 hour at room temperature in two-fold decreasing
concentrations from 40 m g/mL (i.e. a maximum of 200-fold excess compared with 0.2
m g/mL biotinylated 402.8), with a blank well at the final dilution (i.e. containing only
biotinylated 402.8 antibody). Plates were then washed three times in PBS containing 0.1%
Tween20. Streptavidin horseradish peroxidase conjugate (BD Biosciences) was added to
the plates for 1 hour at room temperature in the dark. The plates were washed to remove
unbound streptavidin-horseradish peroxidase. The assay signal was developed by
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incubating with 50 m L 3,3',5,5'-Tetramethylbenzidine (KPL) and quenched with 50 m L 1 M
HCl. Assay signals were read at A450 nm using a microplate reader (FluoStar Galaxy).
Results were expressed as the raw A450 nm value and converted to degree of competition
(percentage) values by subtracting the readings corresponding with zero percent inhibition
from raw data.
To establish that this method generated similar results as described in Example 7,
biotinylated antibody 402.8 was competed with 401.11, and anti-CD1d antibodies 42 and
51.1 for binding to human CD1d. Under these assay conditions 402.8 and 401.11 competed
for binding to human CD1d, as shown by absorbance values at 450nm (Figure 18A) and
degree of competition with 402.8 (Figure 18B), and consequently it was apparent that these
antibodies shared an overlapping or common epitope on hCD1d. In contrast, 402.8 did not
share an overlapping or common epitope with either 42 or 51.1. Taken together, this assay
demonstrated that the highly potent anti-CD1d antibodies 402.8 and 401.11 bind to a
similar high affinity neutralizing epitope on CD1d that is not shared by antibodies 42 and
51.1.
Using this modified assay, biotinylated antibody 402.8 was competed with the
following antibodies (described in Example 11) for binding to recombinant human CD1d:
401.11.24, 401.11.26, 401.11.28, 401.11.86, 401.11.151, 401.11.152, 401.11.154,
401.11.155, 401.11.156, 401.11.157, 401.11.158, 401.11.159, 401.11.160, 401.11.161,
401.11.165, 401.11.166, 401.11.167, 401.11.179, 401.11.180, 401.11.181, 402.8.45,
402.8.53, 402.8.60, 402.8.84, 402.8.86 and 402.8.87. All of these antibodies competed
with biotinylated 402.8 for binding to human CD1d, as shown by absorbance values at
450nm and converted percentage competition with 402.8. In particular, antibodies
401.11.160, 401.11.161 and 401.11.165 strongly competed with biotinylated 402.8 as
evidenced by figures demonstrating absorbance values at 450nm (Figure 19A)) and degree
of competition (Figure 19B), as did antibodies 402.8.84, 402.8.86 and 402.8.87, as
evidenced by figures demonstrating absorbance values at 450nm (Figure 20A) and degree
of competition (Figure 20B).
Testing Competition with Additional Anti-CD1d Antibodies
[0260] To investigate whether antibodies derived from 402.8 or 401.11 competed with
other anti-CD1d antibodies, a total of 23 commercially-sourced anti-CD1d antibodies were
tested. These antibodies included anti-human CD1d monoclonal antibodies, anti-mouse
CD1d monoclonal antibodies, and polyclonal anti-human CD1d antibodies. The details of
these antibodies are described in Table 21. Rat anti-mouse antibody hybridomas HB-321,
HB-322, HB-323, HB-326 and HB-327 were sourced from American Type Culture
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Collection and passaged according to the supplier’s instructions. Antibodies derived from
these cell lines were purified by Protein G affinity chromatography and verified for
binding to mouse CD1d (not shown).
None of the 23 anti-CD1d antibodies described in Table 21 competed with 402.8
for binding to human CD1d, as shown by absorbance values at 450nm and percentage
competition with 402.8. Examples of these results are presented in Figures 21-23. The
anti-human CD1d antibodies AD58E7, C3D5 and C-9 did not compete with 402.8 for
binding to human CD1d, as shown by absorbance readings at 450nm (Figure 21A) and
converted degree of competition values (Figure 21B). As another example of these results,
anti-mouse CD1d antibodies HB-321, HB-322 and HB-323 did not compete with 402.8 for
binding to human CD1d, as demonstrated by absorbance readings at 450nm (Figure 22A)
and converted degree of competition values (Figure 22B). Similarly, as an example the
polyclonal anti-human CD1d antibodies C-19, H70 and Ab96515 did not compete with
402.8 for binding to human CD1d, as shown by absorbance readings at 450nm (Figure
23A) and converted degree of competition values (Figure 23B). It is notable that none of
the polyclonal anti-CD1d antibodies tested competed with 402.8 for binding to human
CD1d. These data demonstrate that the highly potent anti-CD1d antibody 402.8, as an
example of the highly potent novel anti-CD1d antibodies tested, bound to a similar high
affinity neutralizing epitope that is not shared by the extensive list of anti-CD1d antibodies
tested.
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Table 21.
Antibody Clone or Isotype Specificity Source
Number Catalog
Number
1 NOR3.2 Mouse monoclonal Human CD1d Pierce
2 AD5-8E7 Mouse monoclonal Human CD1d Miltenyi Biotec
3 C3D5 Mouse monoclonal Human CD1d Santa Cruz Biotechnology
4 C-9 Mouse monoclonal Human CD1d Santa Cruz Biotechnology
G10 Mouse monoclonal Human CD1d Santa Cruz Biotechnology
6 3H649 Mouse monoclonal Human CD1d Santa Cruz Biotechnology
7 LS-C122839 Mouse monoclonal Human CD1d Lifespan Biosciences, Inc.
8 LS-C4448 Mouse monoclonal Human CD1d Lifespan Biosciences, Inc.
9 LS-C4449 Mouse monoclonal Human CD1d Lifespan Biosciences, Inc.
LS-C122840 Rat monoclonal Mouse CD1d LifeSpan Biosciences, Inc.
11 1B1 Rat monoclonal Mouse CD1d BD Biosciences
12 HB-321 Rat monoclonal Mouse CD1d American Type Culture Collection
(clone 19F8)
13 HB-322 Rat monoclonal Mouse CD1d American Type Culture Collection
(clone 15F7)
14 HB323 Rat monoclonal Mouse CD1d American Type Culture Collection
(clone
20H2)
HB-326 Rat monoclonal Mouse CD1d American Type Culture Collection
(clone
15C6)
16 HB-327 Rat monoclonal Mouse CD1d American Type Culture Collection
(clone 4C4)
17 3C11 Rat monoclonal Mouse CD1d BD Biosciences
18 K253 Mouse monoclonal Mouse CD1d Biolegend
19 C-19 Goat Polyclonal Human CD1a, Santa Cruz Biotechnology
CD1b, CD1d
H70 Rabbit Polyclonal Human CD1d Santa Cruz Biotechnology
21 Ab96515 Rabbit Polyclonal Human CD1d AbCam
22 GTX104898 Rabbit Polyclonal Human CD1d GeneTex
23 1401052 Mouse Polyclonal Human CD1d Sigma Aldrich
As an adjunct to the above examples, extended experiments were conducted using
a biotinylated version of an antibody derived from 401.11. This experiment was done to
demonstrate that an antibody which competes with 402.8 for binding to human CD1d
could also be used as a biotinylated reagent in an amended assay, and show the same
result. In the following description, the assay was amended such that 401.11.158, an
example of the 401.11-derived antibodies, was biotinylated and competed with 402.8 and
other anti-CD1d antibodies 42 and 51.1.
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Assay methodology
Anti-CD1d antibody 401.11.158 was biotinylated using an EZ-link Sulfo-NHS-
LC-biotin kit (Pierce) at a 3:1 ratio of biotin: 401.11.158. Free biotin was removed from
the protein preparation by multiple washes with PBS and concentration by centrifugation
(3000 rpm) through a centrifugal filter unit with a 30 kDa cutoff (Millipore). Anti-CD1d
antibody Maxisorp ELISA plates (Nunc) were coated with 1.0 m g/mL human CD1d and
allowed to incubate overnight at 4°C. Plates were then washed three times in PBS
containing 0.1% Tween20, before the plate was blocked in 1% BSA for 1 hr at room
temperature. Biotinylated 401.11.158 was then co-equilibrated for 5 minutes in a 1:1 ratio
with non-biotinylated anti-CD1d antibodies (401.11.158, 402.8, 401.11, 42 and 51.1).
These antibodies were added to the plates for 1 hour at room temperature in decreasing
concentrations from 40 m g/mL (i.e. a maximum of 200-fold excess compared with 0.2
m g/mL biotinylated 401.11.158). Plates were then washed three times in PBS containing
0.1% Tween20. Streptavidin horseradish peroxidase conjugate (BD Biosciences) was
added to the plates for 1 hour at room temperature in the dark. The plates were washed to
remove unbound streptavidin-horseradish peroxidase. The assay signal was developed by
incubating with 50 m L 3,3',5,5'-Tetramethylbenzidine (KPL) and quenched with 50 m L 1 M
HCl. Assay signals were read at A450 nm using a microplate reader (FluoStar Galaxy).
Results were expressed as the raw A450 nm value and converted to degree of competition
(percentage) values by subtracting the readings corresponding with zero percent inhibition
from raw data.
Using the above described method, 401.11.158 and 402.8 competed for binding to
human CD1d, as shown by absorbance values at 450nm (Figure 24A) and degree of
competition with 401.11.158 (Figure 24B) and therefore share an overlapping or common
epitope. In contrast, 401.11.158 does not share an overlapping or common epitope with
either 42 or 51.1. Taken together, these data demonstrate that the highly potent anti-CD1d
antibodies 401.11.158 and 402.8 may bind to a similar high affinity neutralizing epitope
that is not shared by prior art antibodies 42 and 51.1 with lesser potency.
Example 14: Epitope Mapping Experiments
Methods
Preparation of FAbs
FAbs of anti-CD1d antibodies, 402.8 and 401.11.165 were prepared by Papain
digest using the FAb Preparation Kit (Pierce) according to the manufacturer’s instructions.
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The intact FAb was removed from Fc (Fragment crystallisable) containing protein by
running the sample over a Protein A column equilibrated with Phosphate buffered saline (1
X PBS) pH 7.0 and collecting the flow-through. The FAbs were then analysed by size
exclusion chromatography (SEC) using a TSK gel G3000SWx1 column (TOSOH) at 0.5
ml/min with 1X PBS as a running buffer. The results indicated that the FAbs were >95%
pure.
FAb – CD1d binding ELISA
To confirm binding of the FAbs to human CD1d, an ELISA was performed in
which human CD1d was coated at 1 μg/mL in PBS onto a Maxisorp plate (NUNC)
overnight at 4°C. The wells were then washed with 3 separate washes with 1X PBS with
0.05% Tween-20 (Sigma). The wells were blocked with 1% BSA in PBS for 1 hour at
room temperature. The wells were then washed with 1X PBS as described above. A
titration of FAb or full-length antibody was then performed starting from a concentration
of 10 μg/mL and dilutions performed at 1:4 across the plate. A PBS only control was
included. The plate was incubated for 1 hour at room temperature and then the well were
washed as described previous. 100 μL per well of secondary antibody (Goat Anti-human
Kappa F+B HRPO Conjugate, Invitrogen) was added at a dilution of 1:2000 in 1X PBS
and incubated for 1 hour at room temperature, and then the wells were washed as described
previously. 50 μL of TMB (Sigma) was added and the plate was incubated until colour
development. The reaction was stopped by adding 50 μL of 1M HCl to each well. The
absorbance was read at 450 nm (referenced at 620 nm).
H/D exchange experiments
Human CD1d was diluted in 1 X PBS (pH 7) to a concentration of 12.8 μM. It
was then mixed with a FAb fragment of 402.8 or 401.11.165 at a concentration of 14.1
μM. 14 μL of this solution was mixed with 26 µL of 50 mM Phosphate pH 7 in D2O
(where D is deuterium). Separate solutions were prepared and incubated for 30, 100, 300
or 1000s at 23°C. At the end of the incubation period 20 μL of 2 M urea, 1M TCEP pH 3.0
was added to each solution. The sample was then passed over an immobilized pepsin
column at 200 μL/min in 0.05% trifluoracetic acid (TFA) in H2O. Peptic fragments were
loaded onto a reversed-phase trap column and desalted with 0.05% TFA in H2O for 3
mins. Peptides were separated by a C18 column with a linear gradient of 13% to 35% of
buffer comprising 95% acetonitrile, 5% H2O, 0.0025% TFA) over 23 mins. The peptides
were then analysed by mass spectrometry in profile mode. Fully deuterated control
samples were prepared by mixing 32.2 μL of 0.615 mg/mL CD1d with 59.8 μL of 100 mM
TCEP in D2O and incubate at 60°C for 3 hours.
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CD1d Mutein ELISA
All solutions were added in 50 μL/well. CD1d (wild-type) and related constructs
(such as muteins or CD1d with amino acid substitutions) were coated on 96-well Maxisorp
ELISA Plates (NUNC) at 1 μg/mL in PBS overnight at 4°C. The wells were then washed
with wash buffer (PBS + 0.05 % Tween-20) 3 times. The plates were blocked for 1 hr at
room temperature in blocking buffer (PBS + 1% BSA) before being washed 3 times.
Antibody in antibody diluent (PBS + 1% BSA + 0.05% Tween-20) was added to the wells
in a half log titration starting from 10 μg/ml, and no antibody (0 μg/mL) was included as a
negative control. The plate was then incubated at room temperature for 1 hour. The plate
was then washed as described above. Secondary antibodies (HRP-goat anti human IgG
(H+L), Invitrogen) was added at 1:2000 dilution in antibody diluent and incubated for 1
hour at room temperature. After washing the plate, 50 μL of TMB (Sigma) was added to
each well. 50 μL of 1M HCl was added after colour development to stop the reaction. The
absorbance of each well of the plate was read at 450 nm (referenced at 620nm).
Results:
Production of FAbs of 402.8 and 401.11.165
Using papain digest, FAbs of 402.8 and 401.11.165 were isolated. As measured
by size exclusion chromatography (SEC) the constructs had a purity of >95%. When tested
in an ELISA the FAbs retained binding to human CD1d (Figure 25).
H/D Exchange experiments
Using the method for H/D exchange described above, antibodies 402.8 and
401.11.165, as FAbs, were complexed to human CD1d, then incubated in D2O. This
exchanges hydrogen for deuterium on the CD1d molecule except in the locations in which
the antibody has bound to the CD1d and to which the D2O has no access. Separately
human CD1d was incubated with D2O in the absence of antibody. CD1d from both
samples underwent trypic digest and was then analysed via mass spectrometry. The
difference in deuteration levels in each segment of CD1d on exchange experiments with
and without FAbs 402.8 and 401.11.165 are shown in the following Tables 22 and 23
respectively. The sequence of the extracellular domain of CD1d used in these experiments
is listed below:
EVPQRLFPLRCLQISSFANSSWTRTDGLAWLGELQTHSWSNDSDTVRSLKPWSQG
TFSDQQWETLQHIFRVYRSSFTRDVKEFAKMLRLSYPLELQVSAGCEVHPGNASN
NFFHVAFQGKDILSFQGTSWEPTQEAPLWVNLAIQVLNQDKWTRETVQWLLNGT
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CPQFVSGLLESGKSELKKQVKPKAWLSRGPSPGPGRLLLVCHVSGFYPKPVWVK
WMRGEQEQQGTQPGDILPNADETWYLRATLDVVAGEAAGLSCRVKHSSLEGQDI
VLYWGGSYTSGSLVPRGSGSKRVHHHHHHHH (SEQ ID No: 19)
Table 22: Difference in deuteration levels in each segment of CD1d on exchange
experiments with and without FAb 402.8
Peptide Incubation Time (s) Peptide Incubation Time (s)
Start End 30 100 300 1,000 Avg Start End 30 100 300 1,000 Avg
3 11 -3% -1% -2% 0% -2% 204 216 1% 0% 2% 0% 1%
3 16 -3% -3% 0% -3% -2% 206 216 2% 1% 4% 0% 2%
12 16 -2% -6% 4% -8% -3% 219 224 1% 1% 1% 2% 1%
16 18 5% -12% -18% -9% -9% 219 239 1% 2% -2% 1% 0%
16 28 -3% -9% -15% -10% -9% 219 242 0% 1% -1% 1% 0%
19 28 -6% -8% -13% -10% -9% 225 242 0% 1% -2% 0% 0%
31 34 -2% 0% -1% 2% 0% 244 248 1% 0% -2% 0% 0%
31 36 1% -4% -1% 1% -1% 245 248 1% 1% -1% 0% 0%
36 7% -12% 0% -1% -2% 251 259 3% -1% 0% 4% 1%
48 61 -2% 1% -1% 2% 0% 251 271 0% 3% -2% -1% 0%
48 62 -1% 2% 2% 2% 1% 261 271 -3% -4% -2% 2% -2%
64 76 3% -2% 2% -1% 1% 274 303 -3% 0% -2% 0% -1%
65 76 4% -2% 0% -1% 0% 277 303 -5% 0% -2% 0% -2%
79 86 -1% -6% -14% -22% -11% 326 332 -2% 1% -2% 0% -1%
89 94 -50% -46% -44% -44% -46% 326 345 0% 0% -2% 0% -1%
89 95 -58% -61% -58% -52% -57% 335 345 -1% 0% -2% 1% -1%
114 119 0% -9% -19% -16% -11% 348 359 0% -1% 0% -1% -1%
114 123 -3% -6% -12% -10% -8% 351 359 1% -2% 0% 1% 0%
115 123 -5% -11% -15% -12% -11% 360 362 1% 4% 1% 1% 1%
120 123 -7% -2% -1% -2% -3% 360 377 0% 1% -4% -3% -1%
126 139 -7% -3% -3% -3% -4% 365 377 0% 0% -2% 2% 0%
126 142 -13% -9% -10% -9% -10% 380 385 -3% -4% -4% -1% -3%
141 142 -43% -45% -36% -25% -37% 382 385 -5% -3% -3% -2% -3%
145 155 -7% -8% -9% -10% -9% 388 389 0% 0% 0% -1% 0%
146 155 -7% -5% -16% -13% -10% 388 390 0% 0% -1% -1% 0%
158 172 -3% -5% -6% -7% -5% 392 401 3% 1% -2% 1% 1%
175 188 2% 1% -2% -1% 0% 392 402 3% 1% -3% 0% 0%
175 190 2% 2% -3% 1% 0% 404 422 -1% 0% -2% 0% -1%
191 201 -1% 1% -3% 1% -1% 406 422 0% 0% -3% -2% -1%
193 201 2% -1% -4% 3% 0%
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The mean ± standard deviation (S.D.) % deuterium difference was calculated
across 50% of the peptides with the lowest % deuterium difference. A value lower then the
mean - 3 S.D. was considered significant. The mean (50%) - 3 S.D. % deuterium
difference across the data set was 0%. The CD1d sequences that had the greatest protected
regions upon complexing with 402.8 were:
89-95 of SEQ ID NO:116 - LSYPLEL
141-142 of SEQ ID NO:116 - NL
Table 23: Difference in deuteration levels in each segment of CD1d on exchange
experiments with and without FAb 401.11.165
Peptide Incubation Time (s) Peptide Incubation Time (s)
Start End 30 100 300 1,000 Avg Start End 30 100 300 1,000 Avg
3 11 1% -1% -1% -1% 0% 193 201 4% 2% -4% -2% 0%
3 16 2% -3% 2% -2% 0% 204 216 1% 2% 1% 0% 1%
12 16 3% -6% 5% -3% 0% 206 216 3% 2% 2% -1% 2%
16 18 3% -1% 4% 3% 2% 219 224 2% 2% 2% 2% 2%
16 28 1% 0% 0% 0% 0% 219 239 -1% 2% 0% -1% 0%
19 28 1% 0% -1% -1% 0% 219 242 2% 1% 0% -1% 0%
31 34 1% 0% 0% 1% 1% 225 242 2% 0% -1% -2% 0%
31 36 1% 0% 1% -1% 0% 244 248 1% 0% -1% 0% 0%
36 1% -1% 1% -6% -1% 245 248 2% 1% -1% -1% 0%
48 61 - - - - - 251 259 6% 0% 2% -1% 2%
48 62 4% 0% 0% -4% 0% 251 271 0% 4% 0% -4% 0%
64 76 6% 2% 5% 2% 4% 261 271 1% -2% 3% 4% 1%
65 76 4% 2% 3% -1% 2% 274 303 1% 0% 0% -1% 0%
79 86 2% 2% -3% -5% -1% 277 303 0% 0% -1% -1% 0%
89 94 -14% -10% -12% -19% -14% 326 332 2% 1% -1% -2% 0%
89 95 - - - - - 326 345 2% 0% -1% -1% 0%
114 119 - - - - - 335 345 2% -1% -1% -1% 0%
114 123 6% 9% 6% 6% 7% 348 359 3% -1% 1% -3% 0%
115 123 11% 10% 9% 10% 10% 351 359 2% 0% 1% -2% 0%
120 123 - - - - - 360 362 2% 4% 2% -1% 2%
126 139 -13% -14% -10% -10% -12% 360 377 2% 1% 0% -1% 1%
126 142 -23% -19% -19% -22% -20% 365 377 3% 0% -1% 0% 1%
141 142 -69% -70% -61% -67% -67% 380 385 -2% -5% -4% -4% -4%
145 155 0% 4% -2% -6% -1% 382 385 -3% -3% -3% -3% -3%
146 155 - - - - - 388 389 0% 0% 0% -1% 0%
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Peptide Incubation Time (s) Peptide Incubation Time (s)
Start End 30 100 300 1,000 Avg Start End 30 100 300 1,000 Avg
158 172 4% 6% 5% 1% 4% 388 390 1% 0% 0% -1% 0%
175 188 1% 3% 1% 0% 1% 392 401 5% 1% -1% -1% 1%
175 190 2% 1% -3% -2% 0% 392 402 4% 1% -3% -1% 0%
191 201 1% 1% -1% 0% 0% 404 422 2% 0% 0% -1% 0%
406 422 3% 1% -1% -1% 0%
Similarly for 401.11.165, the mean ± standard deviation (S.D.) % deuterium
difference was calculated across 50% of the peptides with the lowest % deuterium
difference. A value lower then the mean - 3 S.D. was considered significant. The mean
(50%) - 3 S.D. % deuterium difference across the data set was -5%. The CD1d sequences
that had the greatest protected regions upon complexing with 401.11.165 were:
89-94 of SEQ ID NO 116 - LSYPLE
126-142 of SEQ ID NO 116 - QGTSWEPTQEAPLWVNL
Though having different primary amino acid sequences, both 401.11.165 and
402.8, when bound to human CD1d, protect similar regions of the molecule. These regions,
collectively known as the epitope, include the region of CD1d around the sequence
LSYPLE (89-94 of SEQ ID NO 116). The region QGTSWEPTQEAPLWVNL (126-142 of
SEQ ID NO 116) is also protected in the above H/D exchange experiments and several
amino acids, NL (141-142 of SEQ ID NO 116), within this larger region are highly
protected.
[0274] In order to confirm if the sequence LSYPLE (89-94 of SEQ ID NO 116) and NL
(141-142 of SEQ ID NO 116) on human CD1d were important for the binding of the anti-
CD1d antibodies described within, a series of CD1d constructs were produced. These
constructs are listed in Figure 26 and are described below:
• • • • hCD1dmu (SEQ ID NO:119) – Human CD1d in which amino acids located
between positions 87 to 93 (LRLSYPL)and 141 to 143 (NLA) have been
replaced with murine CD1d sequences (MSPKEDYPI and DLP
respectively) with positions numbered according to human CD1d (SEQ ID
NO:116).
• • • • mCD1dhu (SEQ ID NO:118) – Murine CD1d in which the amino acids
located between positions 85 to 93 (MSPKEDYPI) and 141 to 143 (DLP)
have been replaced with human CD1d sequence (LRLSYPL and NLA
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respectively) with positions numbered according to murine CD1d (SEQ ID
NO:117).
All CD1d constructs were expressed in HEK-293E cells and were detected by
polyclonal antibodies against human or mouse CD1d in an ELISA format. Human CD1d,
mouse CD1d, mCD1dhu and hCD1dmu were coated onto an ELISA plate and the binding
of antibodies 402.8 and 401.11.158 to these CD1d constructs was determined. Both
antibodies bound to human CD1d but did not bind to mouse CD1d (Figure 27). They both
bound to mouse CD1d into which the human sequence had been introduced (mCD1dhu)
indicating these human sequence amino acids are crucial for the binding of the antibodies
to human CD1d (Figure 27). Likewise, when these amino acids on human CD1d were
replaced by mouse sequence at the corresponding locations (hCD1dmu), the antibodies no
longer bound this CD1d construct (Figure 27). Taken together these results indicate that
the sequence of human CD1d between 89-95 and 141-143 are within the epitope to which
these anti-CD1d antibodies bind.
[0276] Collectively these regions form a possible binding site, or epitope, to the anti-
CD1d antibodies bind to human CD1d. When this regions are analysed on a X-ray crystal
structure of human CD1d (such as 3HUJ: PDB database) it can be seen that although these
individual regions are distant in terms of the primary amino acid sequence they are located
within close proximity to each other in the protein’s tertiary structure (Figure 28A). Both
LSYPLE (89-94) and NL (141-142) are located on or about the alpha helices that are
present within CD1d and which facilitate the presentation of lipids to the NKT cell
receptor. This epitope is located within close proximity to the binding site of the NKT cell
receptor β-chain on human CD1d (Figure 28B). An antibody that binds to this location on
CD1d would be capable of competing and inhibiting CD1d-NKT cell receptor β-chain
interaction. Such inhibition would prevent formation of the CD1d and the NKT cell
receptor complex and thus prevent downstream activation of the NKT cell.
Example 15: Ascaris suum model of asthma in cynomolgus monkeys
The efficacy of anti-CD1d antibodies is examined in a cynomolgus macaque
model of asthma. In this protocol, animals sensitized to the nematode parasite Ascaris
suum are challenged with aerosolized A. suum extract to induce acute bronchoconstriction
followed by pulmonary eosinophilic inflammation and airway hyperactivity in a manner
that closely mimics the aetiology of acute asthma exacerbations in humans. Such protocols
have been described (Hart, T. et al., (2001) J Allergy Clin Immunol 108:250-257). This
model is characterised by many features of chronic human asthma, including mucous cell
hyperplasia, subepithelial fibrosis, basement cell membrane thickening, and persistent
9704459 1
baseline hyperreactivity to methacholine. Antibody treatment is given prior to challenge
with A. suum extract and effect of treatment on airway resistance and compliance, PC50,
PCO2 and O2 levels, serum IgE and broncho-alveolar lavage (BAL) measurements, such
as IL-4, IL-5 & IL-13 concentrations and relative frequencies of leukocyte subsets such as
neutrophils, eosinosphils, mast cells basophils and lymphocytes, can be assessed.
Example 16: Alternative Animal Models of Pulmonary Inflammation
Rhesus Macaque Model of Airway Hyper-reactivity
The efficacy and safety of anti-CD1d antibodies may be tested in models of
airway hyper-reactivity. For example, airway hyper-reactivity can be induced in rhesus
macaques (Macaca mulatta) by sequential challenges with house dust mite antigen (from
Dermatophagoides farninae). (Seshasayee, D., et al., 2007 J Clin Invest 117, 3868-78).
Asthma exacerbations, characterised by cough, rapid shallow breathing and increased
airway resistance, are induced by challenge with house dust mite extract. Symptoms can be
reversed by pharmacological intervention, such as albuterol aerosol treatment. Pulmonary
function, e.g. airways resistance and dynamic compliance in response to methacholine
challenge, can be measured. Antibody treatment is given prior to re-challenge with house
dust mite antigen and pulmonary function as determined by methocholine challenge is
assessed.
Evaluation of anti-CD1d antibodies in a Cynomolgus Macaque model of Airway Hyper-
reactivity induced by α-GalCer
The efficacy and safety of anti-CD1d antibodies may be tested using a disclosed
cynomolgus macaque model of airway hyper-reactivity (Matangkasombut, P., et al., 2008 J
Allergy Clin Immunol, 121, 1287-9). In this model, cynomolgus macaques that have been
sensitized to Ascaris suum are dosed with the specific NKT cell agonist α-
galactosylceramide, (α-GalCer), via pulmonary nebuliser. The α-GalCer treatment induces
airway hyper-reactivity, as determined by methacholine challenge as described above.
Antibody treatment is given prior to re-challenge with α-GalCer and pulmonary
function assessed by methacholine challenge. In addition, bronchoalveolar lavage (BAL)
fluid samples is analysed for the presence of mediators associated with airway
inflammation, e.g. the concentrations of IL-4, IL-5 and IL-13 in the BAL may be
evaluated. Moreover, the cellular infiltrate in the BAL is examined by standard clinical
pathology techniques, such as cellular analysis using an automated haematology analyser
(for example, Advia systems) to determine the relative frequencies of major leukocyte
subsets, such as neutrophils, lymphocytes, eosinophils, mast cells and basophils.
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Example 17: Alternative Animal Models of Inflammation Driven by NKT Cells
Ulcerative Colitis
The efficacy and safety of anti-CD1d antibodies are tested in various disclosed
models of human inflammatory bowel disease (Wirtz et al., Nat Protoc 2: 541-546, 2007).
For example, intrarectal administration of oxazolone induces localized ulceration and
inflammation that histologically resembles human ulcerative colitis and is also
characterised by diarrhoea, occult blood, weight loss and occasionally rectal prolapse. The
pathology of oxazolone-induced colitis may be mediated by NKT cells, particularly via
NKT-cell driven secretion of IL-13, and thus disease may be ameliorated by treatment with
an anti-CD1d antibody (Heller, F., et al. 2002 Immunity 17, 629-638). In this model,
antibody treatment is commenced at the start of colitis induction and effects on weight,
stool consistency, occult blood and colon architecture are assessed.
Additionally, anti-CD1d antibodies are tested in a model of murine colitis induced
by adoptive transfer of activated (CD4+CD45RBhigh) T cells (Ostanin, D.V., et al (2009)
Am J Physiol Gastrointest Liver Physiol 296:G135-G146). In this model,
CD4+CD45RBhigh T cells are transferred into immunodeficient mice, resulting in weight
loss and diarrhoea, generalised colonic infiltration and inflammation, loss of goblet cells
and hyperplasia of colonic epithelium. Antibodies are tested for their ability to reduce
weight loss and diarrhoea and to ameliorate colon inflammation and histological changes.
[0283] Additionally, anti-CD1d antibodies are tested in a murine model of colitis induced
by administration of dextran sulphate sodium (DSS) in drinking water (Wirtz et al., Nat
Protoc 2: 541-546, 2007). DSS administration induces robust generalized inflammation of
the intestinal tract characterized by erosive lesions and inflammatory infiltrate. Symptoms
usually include diarrhoea, occult blood, weight loss and occasionally rectal prolapse. DSS-
induced colitis models may be either acute, involving administration of DSS for 7 days, or
chronic, involving administration of DSS for longer periods of time.
Antibodies are tested either prophylactically or therapeutically. In a prophylactic
model, antibody treatment is commenced at the start of administration of DSS. In a
therapeutic model, antibody treatment is commenced several days after commencement of
induction. The effect of the treatment on weight and stool consistency, as well as
microscopic effects on epithelial integrity and degree of inflammatory infiltrate is
determined.
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Non Alcoholic Steatohepatitis
Anti-CD1d antibodies are additionally tested in rodent models of Non-Alcoholic
Steatohepatitis (NASH), described for example in Takahashi et al (2012) World Journal of
Gastroenterology 18(19): 2300-2308. For example, in C57BL/6 mice, neonatal delivery of
streptozotocin (STZ), a hepatotoxic compound, followed by a high-fat diet leads to the
development of cardinal features of NASH. As another example, the provision of a high fat
diet in C57BL/6 or ob/ob mice can lead to induction and maintenance of NASH. In yet
another example, NASH may be generated in rats by a high fat diet plus recurrent and
intermittent hypoxemic stress. In one or several of these models, the efficacy of the anti-
CD1d antibodies in treated rodents is determined by the effects on total liver weight, serum
aminotransferase levels, serum triglyceride levels, blood glucose levels, improvements in
liver histopathology and alterations to gene expression patterns.
Autoimmune Liver Diseases
Anti-CD1d antibodies are additionally tested in animal models of autoimmune
liver disease. For example, in mice, hepatitis induced by intravenous injection with
concanavalin A (conA) has been described (Takeda, K. et al. (2000) PNAS 97(10):5498-
5503). ConA is injected into mice through the tail vein. Serum samples are obtained 20 h
after Con A injection. Serum aminotransferase [alanine aminotransferase (ALT) and
aspartate aminotransferase (AST)] levels are measured using standard photometric
techniques. Additionally, liver pathology is assessed by macroscopic and microscopic
examination of liver morphology. Antibodies are assessed for effects on serum ALT and
AST, and liver histopathology.
Example 18: Methods for generating binding proteins
Selection from an Antibody Library Using the 402.8 or 401.11 Antibody
[0287] A phage display protocol is used where a first panning round is conducted using
an antigen density (i.e. biotinlyated CD1d) of about 100 pmol and a TEA-based elution
step as described previously. The second and third rounds use a reduced antigen density
(e.g., about 50 pmol). Phage are eluted by adding the 402.8 or 401.11 (or related
antibodies) IgG at a 10-fold molar excess and incubating the reactions at room temperature
for 2, 5, 10 or 20 mins. The IgG is expected to specifically displace and elute phage
expressing FAbs that bound to the 402.8/401.11 epitope. Non-specific binders and phage
bound to other regions on the CD1d surface are less likely to elute under these conditions.
9704459 1
The washing regimen comprises six washes with M-PBS for round 1 and 2. For
round 3 the washes are three washes with PBST and then three washes with PBS.
Eluted phage are used to infect TG1 E. coli for phagemid rescue or generation of
colonies for screening as described for other phage display experiments.
Selection/Production of Antibodies using synthetic CD1d constructs
Using synthetic CD1d constructs such as hCD1dmu or mCD1dhu as panning
reagents for phage display, antibodies that recognize an epitope similar to that of 402.8 and
401.11 may be obtained. A phage display library may be depleted of antibodies that
recognize CD1d with a construct like hCD1dmu (SEQ ID NO: 119) (human CD1d in
which the key amino acids comprising the epitope are replaced by their corresponding
murine amino acid). The library may then be panned against human CD1d. The resultant
isolated antibodies will likely bind to amino acids between 89 to 94 and 141 and 142 of
human CD1d (SEQ ID NO 116).
Immunization approach
[0291] Peptide or protein mimics of the 402.8/401.11 antibody epitope on CD1d are used
as antigen in place of CD1d in library display technologies or immunization/hybridoma
approaches. For example, a chimeric CD1d molecule is constructed to contain the
402.8/401.11 epitope of human CD1d in a framework which is otherwise mouse CD1d (for
example mCD1dhu (SEQ ID NO: 118). When such a construct is used as an immunogen
in mice, the immune response is expected to be focused towards the non-murine sequence.
9704459 1
Antibody Sequence ID concordance
Antibody VH amino acid VH nucleotide VL amino acid VL nucleotide
(SEQ ID No) (SEQ ID No) (SEQ ID No) (SEQ ID No)
401.11 1 10 2 11
401.11.24 23 68 46 91
401.11.26 24 69 47 92
401.11.28 5 14 6 15
401.11.86 25 70 48 93
401.11.151 26 71 49 94
401.11.152 27 72 50 95
401.11.154 28 73 51 96
401.11.155 29 74 52 97
401.11.156 30 75 53 98
401.11.157 31 76 54 99
401.11.158 32 77 55 100
401.11.159 33 78 56 101
401.11.160 34 79 57 102
401.11.161 35 80 58 103
401.11.165 36 81 59 104
401.11.166 37 82 60 105
401.11.167 38 83 61 106
401.11.179 40 85 62 107
401.11.180 41 86 63 108
401.11.181 42 87 64 109
402.8 3 12 4 13
402.8.45 7 16 4 110
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Antibody VH amino acid VH nucleotide VL amino acid VL nucleotide
(SEQ ID No) (SEQ ID No) (SEQ ID No) (SEQ ID No)
402.8.53 8 17 4 111
402.8.60 9 18 4 112
402.8.84 43 88 65 113
402.8.86 44 89 66 114
402.8.87 45 90 67 115
Other Sequence Descriptions
SEQ ID NO Description
19 human CD1d synthetic construct
human betamicroglobulin
21 TCR alpha chain clone J3N.5
22 TCR beta chain clone J3N.5
117 murine CD1d extracellular domain construct
118 mCD1dhu CD1d synthetic construct
119 hCD1dmu CD1d synthetic construct
120 Forward primer
121 Reverse primer
122 89-94 of CD1d
123 126-142 of CD1d
124 VH CDR1 (401.11)
125 VH CDR1 (402.8)
126 VH CDR3 (401.11)
127 VH CDR3 (402.8)
128 VH CDR3 (402.8)
129 VH CDR3 (402.8)
130 VH CDR3 (402.8)
131 VH CDR2 (401.11)
132 VH CDR2 (402.8)
133 VH CDR2 (402.8)
134 VH CDR2 (402.8)
135 VH CDR1 (401.11)
136 VH CDR1 (402.8)
137 VH CDR2 (401.11)
138 VH CDR2 (402.8)
139 VH CDR2 (402.8)
140 VH CDR2 (402.8)
141 VL CDR1 (401.11)
142 VL CDR1 (402.8)
143 VL CDR3 (401.11)
144 VL CDR3 (402.8)
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SEQ ID NO Description
145 VL CDR2 (401.11)
146 VL CDR2 (402.8)
147 89-95 of CD1d
148 401.11 VH consensus sequence
149 401.11VL consensus sequence
150 402.8 VH consensus sequence
151 402.8 VL consensus sequence
152 VH CDR3 (401.11)
153 VH CDR3 (401.11)
154 VH CDR3 (401.11)
155 VH CDR3 (402.8)
156 VH CDR3 (402.8)
157 Human CD1d
158 Human heavy chain IgG1 constant domain
159 Human heavy chain IgG4 constant domain
160 Human heavy chain IgG4 constant domain (S228P)
161 Human light chain kappa constant domain
162 Human light chain lambda constant domain
9704459 1
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9704459 1
Claims (29)
1. An isolated antibody or antigen binding portion thereof which binds to an epitope of human CD1d wherein the epitope comprises residues 87 to 93 and 141 to 143 of SEQ ID NO: 116.
2. An isolated antibody or antigen binding portion thereof which binds to human CD1d wherein the isolated antibody or antigen binding portion thereof comprises a V domain comprising human FR1, FR2, FR3 and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the sequence of CDR1 is DYAMH (SEQ ID NO: 124) or GYYWS (SEQ ID NO: 125), the sequence of CDR2 is TIIWNSAIIGYADSVKG (SEQ ID NO: 131), EINHSGSTNYNPSLKS (SEQ ID NO: 132), EINPSGSTNYNPSLKS (SEQ ID NO: 133) or EINHAGSTNYNPSLKS (SEQ ID NO: 134), and the sequence of CDR3 is DMCSSSGCPDGYFDS (SEQ ID NO: 126), DLCSSGGCPEGYFDS (SEQ ID NO: 152), DMCSSGGCPDGYFDS (SEQ ID NO: 153), DMCSSGGCPEGYFDS (SEQ ID NO: 154), GEIYDFWNSYMDV (SEQ ID NO: 127), GEIYDFWKSYMDV (SEQ ID NO: 128), GEIYDFYKSYLDV (SEQ ID NO: 155), GEIYDFYKSYMDV (SEQ ID NO: 156), GEIYDFWKSYLDV (SEQ ID NO: 129) or GEIYDFYNSYMDV (SEQ ID NO: 130) and a V domain comprising human FR1, FR2, FR3 and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the sequence of CDR1 is RASQHISSWLA (SEQ ID NO: 141) or ASSSGAVSSGNFPN (SEQ ID NO: 142), the sequence of CDR2 is AASSLQS (SEQ ID NO: 145) or SASNKHS (SEQ ID NO: 146), and the sequence of CDR3 is QQANRFPLT (SEQ ID NO: 143) or LLYFGDTQLGV (SEQ ID NO: 144)..
3. An isolated antibody or antigen binding portion thereof which binds to human CD1d wherein the isolated antibody or antigen binding portion thereof comprises a V domain comprising human FR1, FR2, FR3 and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the sequence of CDR1 is GFTFDDY (SEQ ID NO: 135) or GGSFSGY (SEQ ID NO: 136), the sequence of CDR2 is IWNSAI (SEQ ID NO: 137), NHSGS (SEQ ID NO: 138), NPSGS (SEQ ID NO: 139) or NHAGS (SEQ ID NO: 140), and the sequence of CDR3 is DMCSSSGCPDGYFDS (SEQ ID NO: 126), DLCSSGGCPEGYFDS (SEQ ID NO: 152), DMCSSGGCPDGYFDS (SEQ ID NO: 153), DMCSSGGCPEGYFDS (SEQ ID NO: 154), 9704459 1 GEIYDFWNSYMDV (SEQ ID NO: 127), GEIYDFWKSYMDV (SEQ ID NO: 128), GEIYDFYKSYLDV (SEQ ID NO: 155), GEIYDFYKSYMDV (SEQ ID NO: 156), GEIYDFWKSYLDV (SEQ ID NO: 129) or GEIYDFYNSYMDV (SEQ ID NO: 130) and a V domain comprising human FR1, FR2, FR3 and FR4 framework sequences and CDR1, CDR2 and CDR3 sequences and wherein the sequence of CDR1 is RASQHISSWLA (SEQ ID NO: 141) or ASSSGAVSSGNFPN (SEQ ID NO: 142), the sequence of CDR2 is AASSLQS (SEQ ID NO: 145) or SASNKHS (SEQ ID NO: 146), and the sequence of CDR3 is QQANRFPLT (SEQ ID NO: 143) or LLYFGDTQLGV (SEQ ID NO: 144).
4. An isolated antibody or antigen binding portion thereof which binds to human CD1d as claimed in any one of claims 1 to 3 wherein the isolated antibody or antigen binding portion thereof comprises a V domain having a sequence selected from the group consisting of SEQ ID NOs 1, 3, 5, 7, 8, 9, 24, 25, 26, 30, 33, 36, 40, 41, 42, 43, 44 and 45 and sequences at least 95% identical thereto.
5. An isolated antibody or antigen binding portion thereof as claimed in claim 4 in which the sequence of the V domain is SEQ ID NO: 148 or SEQ ID NO: 150.
6. An isolated antibody or antigen binding portion thereof which binds to human CD1d as claimed in any one of claims 1 to 5 wherein the isolated antibody or antigen binding portion thereof comprises a V domain having a sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 46, 49 and 62 and sequences at least 95% identical thereto.
7. An isolated antibody or antigen binding portion thereof as claimed in claim 6 in which the sequence of the V domain is SEQ ID NO: 149 or SEQ ID NO:
8. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 7 in which the isolated antibody or antigen binding portion thereof comprises a V and V sequence pair selected from the group consisting SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 23 and SEQ ID NO: 46, SEQ ID NO: 24 and SEQ ID NO: 47, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 25 and SEQ ID NO: 48, SEQ ID NO: 26 and SEQ ID NO: 49, SEQ ID NO: 27 and SEQ ID NO: 50, SEQ ID NO: 28 and SEQ ID 9704459 1 NO: 51, SEQ ID NO: 29 and SEQ ID NO: 52, SEQ ID NO: 30 and SEQ ID NO: 53, SEQ ID NO: 31 and SEQ ID NO: 54, SEQ ID NO: 32 and SEQ ID NO: 55, SEQ ID NO: 33 and SEQ ID NO: 56, SEQ ID NO: 34 and SEQ ID NO: 57, SEQ ID NO: 35 and SEQ ID NO: 58, SEQ ID NO: 36 and SEQ ID NO: 59, SEQ ID NO: 37 and SEQ ID NO: 60, SEQ ID NO: 38 and SEQ ID NO: 61, SEQ ID NO: 40 and SEQ ID NO: 62, SEQ ID NO: 41 and SEQ ID NO: 63, SEQ ID NO: 42 and SEQ ID NO: 64, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO: 4, SEQ ID NO: 43 and SEQ ID NO: 65, SEQ ID NO: 44 and SEQ ID NO: 66, and SEQ ID NO: 45 and SEQ ID NO:
9. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 8 wherein the isolated antibody or antigen binding portion thereof binds to CD1d with an EC50 of from 0.5ng/ml to 20ng/ml as measured using a cell based potency assay.
10. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 9 wherein the isolated antibody or antigen binding portion thereof comprises a human kappa chain constant region.
11. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 10 wherein the isolated antibody or antigen binding portion thereof comprises a human lambda chain constant region.
12. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 11 wherein the isolated antibody or antigen binding portion thereof comprises an IgG1 or IgG4 constant region.
13. An isolated antibody or antigen binding portion thereof as claimed in claim 12 wherein the isolated antibody or antigen binding portion thereof comprises an IgG4 constant region which includes an S228P mutation.
14. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 13 wherein the isolated antibody or antigen binding portion thereof is an antibody.
15. An isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 14 wherein the antibody or antigen binding portion thereof is modified to modulate a functional characteristic selected from the group 9704459 1 consisting of antibody-dependent cellular cytotoxicity, complement- dependent cytotoxicity, serum half-life, biodistribution and binding to Fc receptors.
16. A composition comprising an isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15 and a pharmaceutically acceptable carrier.
17. An isolated DNA molecule which encodes the isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15.
18. An isolated DNA molecule as claimed in claim 17 wherein the sequence of the DNA molecule is selected from the group consisting of SEQ ID NOs. 10 to 18 and 68 to 115, or a sequence at least 95% identical thereto or a sequence which hybridises thereto under moderate to high stringency conditions.
19. An isolated transformed cell which produces an antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15.
20. An isolated transformed cell as claimed in claim 19 in which the cell is transformed with a DNA molecule as claimed in claim 17 or 18.
21. The use of an isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15 in the preparation of a medicament for the treatment of a condition involving NKT cell effector function.
22. The use as claimed in claim 21 in which the condition is selected from the group consisting of psoriasis, ulcerative colitis, primary biliary cirrhosis, atherosclerosis, non-alcoholic steatohepatitis, autoimmune hepatitis, ischaemia-reperfusion injury, pulmonary inflammation or dysfunction associated with sickle cell disease, and asthma.
23. An ex-vivo method of detecting the presence of human or cynomolgus CD1d in a sample the method comprising contacting a sample suspected to contain CD1d with the isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15 under conditions which allows the binding of the antibody or antigen binding portion thereof to CD1d to form a complex and detecting the presence the complex in the sample. 9704459 1
24. The ex- vivo method according to claim 23, wherein the CD1d in a sample is cell membrane bound CD1d.
25. The isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15 for use in the treatment of a condition involving NKT cell effector function.
26. A method of detecting the presence of human CD1d-positive cells in a cell sample the method comprising contacting a population of cells with an isolated antibody or antigen binding portion thereof as claimed in any one of claims 1 to 15 to allow the binding of the antibody or antigen binding portion thereof to CD1d-positive cells to form a complex and detecting the presence of the antibody or antigen binding portion thereof -cell complex.
27. A method as claimed in claim 26 wherein the cell sample is a peripheral blood sample or a cell line sample.
28. A method of selecting a CD1d-binding protein which binds specifically to human CD1d and competes for binding on CD1d with at least one antibody selected from the group consisting of 401.11 (SEQ ID NO: 1 and SEQ ID NO: 2), 402.8 (SEQ ID NO: 3 and SEQ ID NO: 4) and 401.11.158 (SEQ ID NO: 32 and SEQ ID NO: 55) from a plurality of CD1d-binding proteins, the method comprising: contacting the plurality of CD1d-binding proteins to a human CD1d mutein in which the amino acids located at positions 87 to 93 and 141to 143 of SEQ ID NO: 116 have been substituted with corresponding murine amino acids at these positions, under conditions sufficient to allow binding of CD1d-binding proteins to the mutein to form a CD1d-binding protein-human CD1d mutein complex and a depleted plurality of CD1d-binding proteins which do not bind the human CD1d mutein, and collecting CD1d-binding proteins which do not bind to the human CD1d mutein from the depleted plurality of CD1d-binding proteins, wherein the collected CD1d-binding proteins bind specifically to human CD1d and compete for binding on CD1d with at least one antibody selected from the group consisting of 401.11, 402.8 and 401.11.158. 9704459 1
29. A method of selecting a CD1d-binding protein which binds specifically to human CD1d from a plurality of CD1d-binding proteins, the method comprising: contacting the plurality of CD1d-binding proteins to hCD1dmu (SEQ ID NO: 119) in which the amino acids located at positions 87 to 93 and 141 to 143 of human CD1d (SEQ ID NO 116) have been replaced with the corresponding murine sequence at this location, under conditions sufficient to allow binding of CD1d-binding proteins to the hCD1dmu to form a CD1d-binding protein-hCD1dmu complex and a depleted plurality of CD1d binding proteins which do not bind hCD1dmu and collecting CD1d-binding proteins which do not bind to the hCD1dmu from the depleted plurality of CD1d-binding proteins, wherein the collected CD1d binding proteins bind specifically to human CD1d (SEQ ID NO: 116) or mCD1dhu (SEQ ID NO: 118). 9704459 1
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161547307P | 2011-10-14 | 2011-10-14 | |
AU2011904190 | 2011-10-14 | ||
US61/547,307 | 2011-10-14 | ||
AU2011904190A AU2011904190A0 (en) | 2011-10-14 | Antibodies to CD1d | |
PCT/AU2012/001247 WO2013053021A1 (en) | 2011-10-14 | 2012-10-15 | ANTIBODIES TO CD1d |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ622050A NZ622050A (en) | 2016-07-29 |
NZ622050B2 true NZ622050B2 (en) | 2016-11-01 |
Family
ID=
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