NZ619849B2 - Antibodies that bind to ox40 and their uses - Google Patents
Antibodies that bind to ox40 and their uses Download PDFInfo
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- NZ619849B2 NZ619849B2 NZ619849A NZ61984912A NZ619849B2 NZ 619849 B2 NZ619849 B2 NZ 619849B2 NZ 619849 A NZ619849 A NZ 619849A NZ 61984912 A NZ61984912 A NZ 61984912A NZ 619849 B2 NZ619849 B2 NZ 619849B2
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Abstract
Disclosed is an antagonist antibody or fragment thereof that binds to human OX40 comprising a heavy chain CDR1 comprising the amino acid sequence of GFSLSTSGMG, and a heavy chain CDR2 comprising the amino acid sequence of IWWDDDK, and a heavy chain CDR3 comprising the amino acid sequence of ARIDWDGFAY; and comprising a light chain CDR1 comprising the amino acid sequence of SSVSY, and a light chain CDR2 comprising the amino acid sequence of ATS and a light chain CDR3 comprising the amino acid sequence of QQWSSNPWT. Also disclosed is the use of the above-described antibody or fragment thereof in the manufacture of a medicament for treating an OX40 mediated disorder in a subject. AY; and comprising a light chain CDR1 comprising the amino acid sequence of SSVSY, and a light chain CDR2 comprising the amino acid sequence of ATS and a light chain CDR3 comprising the amino acid sequence of QQWSSNPWT. Also disclosed is the use of the above-described antibody or fragment thereof in the manufacture of a medicament for treating an OX40 mediated disorder in a subject.
Description
Antibodies that bind to OX40 and their uses
Related application
This application claims benefit of US provisional application No. 61/506,491 filed on
July 11, 2011; all of which are hereby incorporated by reference in their entirety
Field of the Invention
The present invention relates to antagonist antibodies or fragments thereof that bind to human
OX40. More specifically, the present invention relates to an antagonist antibody or fragment
thereof that binds to human OX40 comprising a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 1, and/or a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 2, and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID
NO: 3; and/or comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 4, and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5
and/or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
Background of the Invention
OX40 is a member of the TNFR-superfamily of receptors and was first identified in 1987 as a
50 kDa glycoprotein expressed on activated CD4+ T cells from the rat (Paterson DJ et al.,
(1987) Mol. Immunol. 24: 1281-90). The extracellular ligand binding domain of OX40 is
composed of 3 full cysteine-rich domains (CRDs) and a partial, fourth C-terminal CRD
(Bodmer JL et al., (2002) Trends Biochem. Sci. 27: 19-26). The ligand for OX40 is OX40L
(CD252) and 3 copies of OX40 bind to the trimeric ligand to form the OX40-OX40L complex
(Compaan DM & Hymowitz SG (2006) Structure, 14: 1321-1330). OX40 is a membrane-
bound receptor; however a soluble isoform has also been detected (Taylor L & Schwarz H
(2001) J. Immunol. Methods, 255: 67-72). Unlike CD28, OX40 is not constitutively expressed
on naïve T cells but is induced after engagement of the T-Cell Receptor (TCR). OX40 is a
secondary costimulatory molecule, expressed after 24 to 72 hours following activation; its
ligand, OX40L, is also not expressed on resting antigen presenting cells, but is following their
activation. OX40 is expressed mainly by activated CD4+ T cells and to a limited extent, by
activated CD8+ T cells (Salek-Ardakani S et al., (2006) Curr. Immunol. Rev. 2: 37-53).
Summary of the Invention
1000501383
The present disclosure relates generally to antagonist antibodies or fragments thereof that bind
to human OX40, methods for their preparation and use, including methods for treating OX40
mediated disorders. The antagonist antibodies or fragments thereof of the present invention
that bind to human OX40 are antagonistic antibodies and do not show agonistic effects and/or
activate human OX40 on binding.
In one aspect, the present disclosure provides an antagonist antibody or fragment thereof that
binds to human OX40 comprising a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 1, and/or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
NO: 2, and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3;
and/or comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4,
and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and/or a
light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
In a further aspect the present invention provides an antagonist antibody or fragment thereof
that binds to human OX40 comprising a heavy chain variable region sequence comprising the
amino acid sequence of SEQ ID NO: 7. In a further aspect the present invention provides an
antagonist antibody or fragment thereof that binds to human OX40 comprising a heavy chain
variable framework region that is the product of or derived from a human gene selected from
the group consisting of: IGHV2-70*10 (SEQ ID NO: 19), IGHV2-70*01 (SEQ ID NO: 20),
IGHV2-70*13 (SEQ ID NO: 21), IGHV2-5*09 (SEQ ID NO: 22), and IGHV2-70*11 (SEQ
ID NO: 23).
In a further aspect the present invention provides an antagonist antibody or fragment thereof
comprising a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 32
and wherein the heavy chain variable framework region comprises at least one amino acid
modification from the corresponding heavy chain variable framework region of the
corresponding murine antibody.
In a further aspect the present invention provides an antagonist antibody or fragment thereof
that binds to human OX40 comprising a light chain variable region sequence comprising the
amino acid sequence of SEQ ID NO: 8. In a further aspect the present invention provides an
antagonist antibody or fragment thereof that binds to human OX40 comprising a light chain
1000501383
variable framework region that is the product of or derived from a human gene selected from
the group consisting of: IGKV3-11*01 (SEQ ID NO: 24), IGKV1-39*01 (SEQ ID NO: 25),
IGKV1D-39*01 (SEQ ID NO: 26), IGKV3-11*02 (SEQ ID NO: 27) and IGKV3-20*01 (SEQ
ID NO: 28).
In a further aspect the present invention provides an antagonist antibody or fragment thereof
comprising a light chain variable framework region that is the product of or derived from
human gene IGKV3-11*01 (SEQ ID NO: 24) and wherein the light chain variable framework
region comprises at least one amino acid modification from the corresponding framework
region of the light chain variable region of the corresponding murine antibody.
In a further aspect the present invention provides an antagonist antibody or fragment thereof
that binds to human OX40 comprising a heavy chain sequence selected from the group
consisting of SEQ ID NOS: 32, 33, 34, 35, 36, 37 and 38. In a further aspect the present
invention provides an antagonistic antibody or fragment thereof that binds to human OX40
comprising a light chain sequence selected from the group consisting of SEQ ID NOS: 39, 40,
41, 42, 43, 44, 45, 46, 47, 48 and 49.
In a further aspect the present invention provides an antagonist antibody or fragment thereof
that binds to human OX40 comprising:
(a) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 37 or 38; and
(b) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 47.
In a further aspect the present invention provides an antagonistic antibody or fragment thereof
that binds to human OX40 comprising a heavy chain variable region comprising the amino
acid sequence selected from the group consisting of SEQ ID NOS: 58, 59, 79 and 80. In a
further aspect the present invention provides an antagonistic antibody or fragment thereof that
binds to human OX40 comprising a light chain variable region comprising the amino acid
sequence selected from the group consisting of SEQ ID NOS: 60, 86, 87 and 89.
In a further aspect the present invention provides an antagonist antibody or fragment thereof
that binds to human OX40 comprising:
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(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58 or
59; and
(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.
In a further aspect the present invention provides an antagonistic antibody or fragment thereof
that binds to human OX40, wherein the antibody comprises a human IgG4 Fc region, wherein
the antibody has no Fc-mediated cytotoxicity activity. In a further aspect the present invention
provides an antagonistic antibody or fragment thereof that binds to human OX40, wherein the
antibody comprises a human IGHG1 Fc region, wherein the antibody is competent for
cytotoxicity mechanisms such as antibody dependent cellular cytotoxicity (ADCC). In a
preferred aspect, the antagonistic antibody or fragment thereof that binds to human OX40 has
a non fucosylated IGHG1 Fc region and exhibits enhanced Fc-mediated cytotoxicity
mechanisms such as ADCC.
In another aspect, the disclosure of the present invention also describes antagonistic
humanized antibodies or fragments thereof that bind with a similar affinity to human OX40 as
the corresponding chimeric antibody e.g. retain at least 75% of the OX40 binding affinity (K )
of the corresponding chimeric antibody or have at least equivalent or higher OX40 binding
affinity (K ) when compared to the corresponding chimeric antibody. In a further aspect the
present invention provides an antagonist antibody or fragment thereof that binds to an epitope
within the second domain of human OX40 extracellular region.
The disclosure of the present invention also provides isolated nucleic acids encoding
antibodies and fragments thereof that bind to human OX40, vectors and host cells comprising
the nucleic acid or the vector. Compositions comprising the antagonist antibody or fragment
thereof and a pharmaceutically acceptable carrier and immunoconjugates comprising the
antagonist antibody or fragment thereof linked to a therapeutic agent are also provided.
The present disclosure also provides methods for treating OX40 mediated disorders. In one
aspect, in an in vitro model of alloreactive T cell activation and proliferation (mixed
lymphocyte reaction; MLR), an antagonistic antibody or fragment thereof efficiently inhibits
MLR in two different individuals (responders), with an EC value of approximately
100ng/mL. Furthermore, in a xenogenic graft versus host reaction, a model for allogenic graft
1000501383
versus host disease (GVHD) observed after bone marrow transplant in human patients, an
antagonistic antibody or fragment thereof potently suppressed the GVHD reaction.
The present disclosure also provides kits and articles of manufacture comprising the antibody
or fragments thereof, a composition or an immunoconjugate for the treatment of an OX40
mediated disorder.
As used herein, except where the context requires otherwise, the term "comprise" and
variations of the term, such as "comprising", "comprises" and "comprised", are not intended
to exclude other additives, components, integers or steps.
Reference to any prior art in the 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 New Zealand or any other jurisdiction.
Brief Description of the Figures
Figure 1: (A) Direct-binding ELISA on immobilized recombinant human OX40-his.
Binding of chimeric 2F8 and 1D4 antibodies on human OX40 was measured by direct ELISA.
Various concentrations (ranging from 10 to 0.01mg/ml) of 1D4 (black histograms) and 2F8
(white histograms) were incubated with 2mg/ml of recombinant human OX40-his tagged
protein coated overnight at 4°C in a 96-well plate. Binding of each antibody to OX40 was
detected by horseradish peroxidise (HRP)-conjugated anti human antibody. (B) Competitive
ELISA on immobilized recombinant human OX40-Fc. Inhibitory effects of chimeric 1D4
and 2F8 on OX40/OX40L interaction were evaluated by blocking ELISA. Various
concentrations (ranging from 10 to 0.01mg/ml) of 1D4 (black histograms) and 2F8 (white
histograms) were incubated with 2mg/ml of recombinant human OX40-Fc tagged protein
coated overnight at 4°C in a 96-well plate. After five minutes, a fixed concentration of
biotinylated recombinant human OX40L (0.04mg/ml) was added to each well and incubated
for 30 minutes at room temperature. Binding of OX40L to OX40 was detected using
Streptavidin-HRP.
Figure 2: One way mixed lymphocyte reaction (MLR) measured by H thymidine
incorporation. Bars show the mean H-thymidine incorporation (counts) of at least triplicates
± standard error of the mean. Isotype control (trastuzumab) and positive control (efalizumab)
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are shown. Effector stands for only effector cells. Effector + target represent a measurement
where antibodies have been omitted.
Figure 3: Flow cytometry analysis of chimeric 1D4 antibody
(A) Staining on human activated peripheral blood mononuclear cells (PBMCs) and
HPB-ALL cells. Histogram plots show the fluorescence intensity (X-axis) and relative cell
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number (% of max events – Y-Axis). The type of cells stained is indicated. Human PBMC
were activated with PHA and IL-2 for 48h prior measurements.
(B) Staining on activated cynomolgus monkey PBMCs. Binding of the chimeric 1D4
antibody to cynomolgus OX40 was evaluated by flow cytometry. Peripheral blood
mononuclear cells (PBMCs) were isolated from whole blood collected from a cynomolgus
monkey and 3x10 cells were cultured for 50 hours in the presence of 10mg/ml of PHA and
100U/ml of rhuIL-2. Activated PBMCs were incubated with 25mg/ml of either control
antibody (upper profile (i)) or biotinylated sheep anti-human OX40 antibody (middle profile
(ii)) or biotinylated chimeric 1D4 antibody (lower profile (iii)). Binding of each antibody to
cynomolgus OX40 was detected with streptavidin-APC.
Figure 4: Surface Plasmon resonance measurements of anti-OX40 antibodies. Data are
expressed as number of response (abbreviated RU; Y axis) vs. time (X axis).
- VH1/VL1 antibody vs 1D4 chimera.
- VH1, VH2, and VH3 based humanized antibodies (as indicated) vs 1D4 chimera.
- examples of poor binders: VH4/VL4, VH5/VL4, VH5/VL5, and VH5/VL6.
- examples of weak binders (VH5/VL9 and VH4/VL9) and good binders (VH6/VL9
and VH7/VL9).
- VH7 based humanized antibodies.
- VH6/VL9 has best binding properties over 1D4 chimera and humanized variant
VH7/VL9.
Figure 5: Sequence Alignment. Alignment of the heavy chain () or light chain () variable region of 1D4 with selected germline frameworks (IGHV 2-70*10 (SEQ ID NO:
19) and IGKV3-11*01 (SEQ ID NO: 24)) from IMGT and back-mutated variable region
variants (VH1 (SEQ ID NO: 29), VH2 (SEQ ID NO: 77), VH3 (SEQ ID NO: 78), VH4 (SEQ
ID NO: 79), VH5 (SEQ ID NO: 80), VH6 (SEQ ID NO: 58), VH7 (SEQ ID NO: 59), (VL1
(SEQ ID NO: 30), VL2 (SEQ ID NO: 81), VL3 (SEQ ID NO: 82), VL4 (SEQ ID NO: 83),
VL5 (SEQ ID NO: 84), VL6 (SEQ ID NO: 85), VL7 (SEQ ID NO: 86), VL8 (SEQ ID NO:
87), VL9 (SEQ ID NO: 60) VL10 (SEQ ID NO: 88), VL11 (SEQ ID NO: 89).
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Figure 6: Thermostability measurements of humanized anti-OX40 anti-antibody
VH6/VL9 FAB fragment using differential scanning calorimetry. Data are expressed as
excess molar heat capacity (abbreviated Cp [kcal/mol/ C]; Y axis) vs. temperature (X axis).
Figure 7: Epitope Characterisation. This figure shows humanized anti-OX40 anti-antibody
VH6/VL9 epitope based on ELISA assay results as described in Example 7.
Figure 8: Mixed lymphocyte reaction (MLR) measured by H thymidine incorporation.
and 8B show the results of mixed lymphocyte reaction from two unrelated donors.
The proliferation was measured by H-thymidine incorporation. The graphs show the absolute
counts values for each condition ± SEM. Responder cells were untreated PBMCs, stimulator
cells were mitomycin-treated PBMCs. All conditions with test antibodies were done with
responder cells mixed with heterologous stimulator PBMCs. The positive control was
Efalizumab (anti LFA-1 antibody).
Figure 9: Xenogeneic graft versus host reaction model. This figure shows the percent
survival within groups of eight animals for each mentioned condition. Vehicle: only PBS. The
vertical dotted line indicates the last day of treatment. No mortality and no symptoms were
observed in a group of two irradiated control animals that did not received PBMCs (not
shown).
Detailed description of the invention
The present disclosure relates to antagonist antibodies and fragments thereof that bind to
human OX40.
The term "human OX40" as used herein includes variants, isoforms, and species homologs of
human OX40. Accordingly, antibodies of this disclosure may, in certain cases, cross-react
with OX40 from species other than human. In certain embodiments, the antibodies may be
completely specific for one or more human OX40 proteins and may not exhibit species or
other types of non-human cross-reactivity. The complete amino acid sequence of an
exemplary human OX40 has Swiss-Prot accession number P43489 (TNR4_HUMAN; SEQ ID
NO: 12). OX40 is also known as CD134, TNFRSF4, ACT35 or TXGP1 L. Human OX40 is
designated GeneID: 7293 by Entrez Gene, and HGNC: 11918 by HGNC. OX40 has also been
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designated CD134 (cluster of differentiation 134). OX40 can be encoded by the gene
designated TNFRSF4/OX40.
The use of "human OX40" herein encompasses all known or as yet undiscovered alleles and
polymorphic forms of human OX40. The terms "human OX40", "OX40" or "OX40 Receptor"
are used herein equivalently and mean "human OX40" if not otherwise specifically indicated.
The term "OX40 ligand" or "OX40L" are used herein equivalently and include OX40 ligand,
specifically human OX40 ligand. OX40L is a member of the TNF superfamily and is also
known as gp34 or CD252. OX40L has also been designated CD252 (cluster of differentiation
252) and has the sequence database accession number P23510 (Swiss-Prot) or Q6FGS4
(Uniprot). OX40L is expressed on the surface of activated B cells, T cells, dendritic cells and
endothelial cells.
The term "antibody or fragment thereof that binds to human OX40" as used herein includes
antibodies or a fragment thereof that binds to human OX40 e.g. human OX40 in isolated
form, with an affinity (K ) of 500 nM or less, preferably 200nM or less, more preferably 150
nM or less, more preferably 120 nM or less, even more preferably 110 nM or less. The term
"antibody or fragment thereof that binds to human OX40" includes antibodies or antigenic
binding fragments thereof.
The terms "antagonistic antibody" or "antagonist antibody" are used herein equivalently and
include an antibody that is capable of inhibiting and/or neutralising the biological signalling
activity of OX40, for example by blocking binding or substantially reducing binding of OX40
to OX40 ligand and thus inhibiting or reducing the signalisation pathway triggered by OX40
and/or inhibiting or reducing an OX40-mediated cell response like lymphocyte proliferation,
cytokine expression, or lymphocyte survival.
The term "antibody" as referred to herein includes whole antibodies and any antigen binding
fragments or single chains thereof. An "antibody" refers to a glycoprotein comprising at least
two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an
antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable
region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain
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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 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) which are hypervariable in sequence and/or
involved in antigen recognition and/or usually form structurally defined loops, interspersed
with regions that are more conserved, termed framework regions (FR or FW). Each VH and
VL is composed of three CDRs and four FWs, arranged from amino- terminus to carboxy-
terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The amino
acid sequences of FW1, FW2, FW3, and FW4 all together constitute the “non-CDR region” or
“non-extended CDR region” of VH or VL as referred to herein.
The term “heavy chain variable framework region” as referred herein may comprise one or
more (e.g., one, two, three and/or four) heavy chain framework region sequences (e.g.,
framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)).
Preferably the heavy chain variable region framework comprises FW1, FW2 and/or FW3,
more preferably FW1, FW2 and FW3. The term “light chain variable framework region” as
referred herein may comprise one or more (e.g., one, two, three and/or four) light chain
framework region sequences (e.g., framework 1 (FW1), framework 2 (FW2), framework 3
(FW3) and/or framework 4 (FW4)). Preferably the light chain variable region framework
comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3.
The variable regions of the heavy and light chains contain a binding domain that interacts with
an antigen. The constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells of the immune system (e.g.,
effector cells) and the First component (C1q) of the classical complement system.
Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by
the constant region. Human constant light chains are classified as kappa (CK) and lambda
(Cλ) light chains. Heavy chains are classified as mu ( ), delta ( ), gamma ( ), alpha ( ), or
epsilon ( ), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Thus, "isotype" as used herein is meant any of the classes and/or subclasses of
immunoglobulins defined by the chemical and antigenic characteristics of their constant
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regions. The known human immunoglobulin isotypes are IgG1 (IGHG1), IgG2 (IGHG2),
IgG3 (IGHG3), IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD),
and IgE (IGHE). The so-called human immunoglobulin pseudo-gamma IGHGP gene
represents an additional human immunoglobulin heavy constant region gene which has been
sequenced but does not encode a protein due to an altered switch region (Bensmana M et al.,
(1988) Nucleic Acids Res. 16(7): 3108). In spite of having an altered switch region, the
human immunoglobulin pseudo-gamma IGHGP gene has open reading frames for all heavy
constant domains (CH1-CH3) and hinge. All open reading frames for its heavy constant
domains encode protein domains which align well with all human immunoglobulin constant
domains with the predicted structural features. This additional pseudo-gamma isotype is
referred herein as IgGP or IGHGP. Other pseudo immunoglobulin genes have been reported
such as the human immunoglobulin heavy constant domain epsilon P1 and P2 pseudo-genes
(IGHEP1 and IGHEP2). The IgG class is the most commonly used for therapeutic purposes.
In humans this class comprises subclasses IgG1, IgG2, IgG3 and IgG4. In mice this class
comprises subclasses IgG1, IgG2a, IgG2b, IgG2c and IgG3.
The term "chimeric antibody" as used herein includes antibodies in which the variable region
sequences are derived from one species and the constant region sequences are derived from
another species, such as an antibody in which the variable region sequences are derived from a
mouse antibody and the constant region sequences are derived from a human antibody.
The term "humanized antibody" or "humanized anti-OX40 antibody" as used herein includes
antibodies in which CDR sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences. Additional framework
region modifications may be made within the human framework sequences as well as within
the CDR sequences derived from the germline of another mammalian species.
The term "Fab" or "Fab region" as used herein includes the polypeptides that comprise the
VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or
this region in the context of a full length antibody or antibody fragment.
The term "Fc" or "Fc region", as used herein includes the polypeptide comprising the constant
region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc
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refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the
last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-
terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc
comprises immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and Cγ3) and the hinge
between C gamma l (Cγl) and C gamma 2 (Cγ2). Although the boundaries of the Fc region
may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226
or P230 to its carboxyl-terminus, wherein the numbering is according to the EU numbering
system. For human IgG1 the Fc region is herein defined to comprise residue P232 to its
carboxyl-terminus, wherein the numbering is according to the EU numbering system
(Edelman GM et al., (1969) Proc Natl Acad Sci USA, 63(1): 78-85). Fc may refer to this
region in isolation or this region in the context of an Fc polypeptide, for example an antibody.
The term "hinge" or "hinge region" or "antibody hinge region" herein includes the flexible
polypeptide comprising the amino acids between the first and second constant domains of an
antibody. The “hinge region” as referred to herein is a sequence region of 6-62 amino acids in
length, only present in IgA, IgD and IgG, which encompasses the cysteine residues that bridge
the two heavy chains. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG
CH2 domain begins at residue EU position 237. Thus for IgG the antibody hinge is herein
defined to include positions 221 (D221 in IgG1) to 231 (A231 in IgG1), wherein the
numbering is according to the EU numbering system (Edelman GM et al., supra).
The term "parent antibody" or "parent immunoglobulin" as used herein includes an
unmodified antibody that is subsequently modified to generate a variant. Said parent antibody
may be a naturally occurring antibody, or a variant or engineered version of a naturally
occurring antibody. Parent antibody may refer to the antibody itself, compositions that
comprise the parent antibody, or the amino acid sequence that encodes it. By "parent anti-
OX40 antibody" as used herein is meant an antibody or immunoglobulin that binds human
OX40 and is modified to generate a variant. By “corresponding murine antibody" as used
herein is meant a murine antibody or immunoglobulin that bind to human OX40 and that can
be modified to generate a variant, specifically the murine antibody 1D4 as disclosed herein.
The term "variant antibody" or "antibody variant" as used herein includes an antibody
sequence that differs from that of a parent antibody sequence by virtue of at least one amino
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acid modification compared to the parent. The variant antibody sequence herein will
preferably possess at least about 80%, most preferably at least about 90%, more preferably at
least about 95% amino acid sequence identity with a parent antibody sequence. Antibody
variant may refer to the antibody itself, compositions comprising the antibody variant, or the
amino acid sequence that encodes it.
The term "amino acid modification" herein includes an amino acid substitution, insertion,
and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution"
herein is meant the replacement of an amino acid at a particular position in a parent
polypeptide sequence with another amino acid. For example, the substitution R94K refers to a
variant polypeptide, in this case a heavy chain variable framework region variant, in which the
arginine at position 94 is replaced with a lysine. For the preceding example, 94K indicates the
substitution of position 94 with a lysine. For the purposes herein, multiple substitutions are
typically separated by a slash. For example, R94K/L78V refers to a double variant comprising
the substitutions R94K and L78V. By "amino acid insertion" or "insertion" as used herein is
meant the addition of an amino acid at a particular position in a parent polypeptide sequence.
For example, insert -94 designates an insertion at position 94. By "amino acid deletion" or
"deletion" as used herein is meant the removal of an amino acid at a particular position in a
parent polypeptide sequence. For example, R94- designates the deletion of arginine at position
94.
As used herein, the term "conservative modifications" or "conservative sequence
modifications" is intended to refer to amino acid modifications that do not significantly affect
or alter the binding characteristics of the antibody containing the amino acid sequence. Such
conservative modifications include amino acid substitutions, insertions and deletions.
Modifications can be introduced into an antibody of the invention by standard techniques
known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions are ones in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of amino acid residues
having similar side chains have been defined in the art. These families include amino acids
with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,
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isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, one or more amino acid residues within the CDR regions or within the
framework regions of an antibody of the invention can be replaced with other amino acid
residues from the same side chain family and the altered antibody (variant antibody) can be
tested for retained function.
For all human immunoglobulin heavy chain constant domains numbering is according to the
“EU numbering system” (Edelman GM et al., (1969) Proc Natl Acad Sci USA, 63(1): 78-85).
For the human kappa immunoglobulin light chain constant domain (IGKC), numbering is
according to the “EU numbering system” (Edelman GM et al., supra).
For the human lambda immunoglobulin light chain constant domains (IGLC1, IGLC2,
IGLC3, IGLC6, and IGLC7), numbering is according to the “Kabat numbering system”
(Kabat EA et al., (1991) Sequences of proteins of immunological interest. 5th Edition - US
Department of Health and Human Services, NIH publication no. 91-3242) as described by
Dariavach P et al., (1987) Proc Natl Acad Sci USA, 84(24): 9074-8 and Frangione B et al.,
(1985) Proc Natl Acad Sci USA, 82(10): 3415-9.
The term “variable domain” refers to the domains that mediates antigen-binding and defines
specificity of a particular antibody for a particular antigen. In naturally occurring antibodies,
the antigen-binding site consists of two variable domains that define specificity: one located in
the heavy chain (VH) and the other located in the light chain (VL). In some cases, specificity
may exclusively reside in only one of the two domains as in single-domain antibodies from
heavy-chain antibodies found in camelids. The V regions are usually about 110 amino acids
long, and consist of relatively invariant stretches of amino acid sequence called framework
regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called
“hypervariable regions” that are 9-12 amino acids long. The variable domains of native heavy
and light chains comprise four FRs, largely adopting a beat-sheet configuration, connected by
three hypervariable regions, which form loops. The hypervariable regions in each chain are
held together in close proximity by FRs, and with the hypervariable regions from the other
chain, contribute to the formation of the antigen binding site of antibodies (see Kabat EA et
al., supra). The term “hypervariable region” as used herein refers to the amino acid residues
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of an antibody which are responsible for antigen binding. The hypervariable region generally
comprises amino acid residues from a “complementary determining region” or “CDR”, the
latter being of highest sequence variability and/or involved in antigen recognition. For all
variable domains numbering is according to Kabat (Kabat EA et al., supra).
A number of CDR definitions are in use and are encompassed herein. The Kabat definition is
based on sequence variability and is the most commonly used (Kabat EA et al., supra).
Chothia refers instead to the location of the structural loops (Chothia C & Lesk AM (1987) J.
Mol. Biol. 196: 901-917). The AbM definition is a compromise between the Kabat and the
Chothia definitions and is used by Oxford Molecular's AbM antibody modelling software
(Martin ACR et al., (1989) Proc. Natl Acad. Sci. USA, 86: 9268–72; Martin ACR et al.,
(1991) Methods Enzymol. 203: 121–153; Pedersen JT et al., (1992) Immunomethods, 1: 126–
136; Rees AR et al., (1996) In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford
University Press, Oxford, 141–172). The contact definition has been recently introduced
(MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745) and is based on an analysis of the
available complex structures available in the Protein Databank. The definition of the CDR by
IMGT , the international ImMunoGeneTics information system (http://www.imgt.org) is
based on the IMGT numbering for all immunoglobulin and T cell receptor V-REGIONs of all
species (IMGT , the international ImMunoGeneTics information system ; Lefranc MP et al.,
(1991) Nucleic Acids Res. 27(1): 209-12; Ruiz M et al., (2000) Nucleic Acids Res. 28(1):
219-21; Lefranc MP (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc MP (2003) Nucleic
Acids Res. 31(1): 307-10; Lefranc MP et al., (2005) Dev. Comp. Immunol. 29(3): 185-203;
Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics, 6(4): 253-64).
All Complementarity Determining Regions (CDRs) discussed in the present invention, are
defined preferably according to IMGT . The variable domain residues for each of these CDRs
are as follows (numbering according to Kabat EA, et al., supra): LCDR1: 27-32, LCDR2: 50-
52, LCDR3: 89-97, HCDR1: 26-35, HCDR2: 51-57 and HCDR3: 93-102. The “non-CDR
region” of the VL region as used herein comprise the amino acid sequences: 1-26 (FR1), 33-
49 (FR2), 53-88 (FR3), and 98- approximately 107 (FR4). The “non-CDR region” of the VH
region as used herein comprise the amino acid sequences: 1-25 (FR1), 36-50 (FR2), 58-92
(FR3), and 103- approximately 113 (FR4).
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The CDRs of the present invention may comprise "extended CDRs" which are based on the
aforementioned definitions and have variable domain residues as follows: LCDR1: 24-36,
LCDR2: 46-56, LCDR3:89-97, HCDR1: 26-36, HCDR2:47-65, HCDR3: 93-102. These
extended CDRs are numbered as well according to Kabat et al., supra. The “non-extended
CDR region” of the VL region as used herein comprise the amino acid sequences: 1-23
(FR1), 37-45 (FR2), 57-88 (FR3), and 98- approximately 107 (FR4). The “non-extended CDR
region” of the VH region as used herein comprise the amino acid sequences: 1-25 (FR1), 37-
46 (FR2), 66-92 (FR3), and 103- approximately 113 (FR4).
The term "full length antibody" as used herein includes the structure that constitutes the
natural biological form of an antibody, including variable and constant regions. For example,
in most mammals, including humans and mice, the full length antibody of the IgG class is a
tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having
one light and one heavy chain, each light chain comprising immunoglobulin domains VL and
CL, and each heavy chain comprising immunoglobulin domains VH, CH1 (Cγ1), CH2 (Cγ2),
and CH3 (Cγ3). In some mammals, for example in camels and llamas, IgG antibodies may
consist of only two heavy chains, each heavy chain comprising a variable domain attached to
the Fc region.
Antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH,
CL and CH1 domains, including Fab′ and Fab′-SH, (ii) the Fd fragment consisting of the VH
and CH1 domains, (iii) the Fv fragment consisting of the VL and VH domains of a single
antibody; (iv) the dAb fragment (Ward ES et al., (1989) Nature, 341: 544-546) which consists
of a single variable, (v) F(ab')2 fragments, a bivalent fragment comprising two linked Fab
fragments (vi) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are
linked by a peptide linker which allows the two domains to associate to form an antigen
binding site (Bird RE et al., (1988) Science 242: 423-426; Huston JS et al., (1988) Proc. Natl.
Acad. Sci. USA, 85: 5879-83), (vii) bispecific single chain Fv dimers (PCT/US92/09965),
(viii) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene
fusion (Tomlinson I & Hollinger P (2000) Methods Enzymol. 326: 461-79; WO94/13804;
Holliger P et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-48) and (ix) scFv genetically
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fused to the same or a different antibody (Coloma MJ & Morrison SL (1997) Nature
Biotechnology, 15(2): 159-163).
The term "effector function" as used herein includes a biochemical event that results from the
interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include
FcγR-mediated effector functions such as ADCC (antibody dependent cell-mediated
cytotoxicity) and ADCP (antibody dependent cell-mediated phagocytosis), and complement-
mediated effector functions such as CDC (complement dependent cytotoxicity). An effector
function of an antibody may be altered by altering, i.e. enhancing or reducing, preferably
enhancing, the affinity of the antibody for an effector molecule such as an Fc receptor or a
complement component. Binding affinity will generally be varied by modifying the effector
molecule binding site, and in this case it is appropriate to locate the site of interest and modify
at least part of the site in a suitable way. It is also envisaged that an alteration in the binding
site on the antibody for the effector molecule need not alter significantly the overall binding
affinity but may alter the geometry of the interaction rendering the effector mechanism
ineffective as in non-productive binding. It is further envisaged that an effector function may
also be altered by modifying a site not directly involved in effector molecule binding, but
otherwise involved in performance of the effector function. By altering an effector function of
an antibody it may be possible to control various aspects of the immune response, e.g.
enhancing or suppressing various reactions of the immune system, with possible beneficial
effects in diagnosis and therapy.
As used herein, the term “OX40-mediated disorder” includes conditions such as allergy,
asthma, COPD, rheumatoid arthritis, psoriasis and diseases associated with autoimmunity and
inflammation.
As used herein, the term "subject" includes any human or nonhuman animal. The term
"nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as
nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
Preferably the subject is human.
Anti-OX40 antibodies
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In a first aspect the present invention provides an antagonist antibody or fragment thereof that
binds to human OX40 comprising a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 1, and/or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID
NO: 2, and/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3;
and/or comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4,
and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and/or a
light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
In some embodiments the antagonist antibody or fragment thereof that binds to human OX40
comprises an extended heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 13, and/or an extended heavy chain CDR2 comprising the amino acid sequence of SEQ
ID NO: 14, and/or an extended heavy chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 15; and/or comprises an extended light chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 16, and/or an extended light chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 17 and/or an extended light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 18.
Preferably the antagonist antibody or fragment thereof that binds to human OX40 comprises a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain
CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 3 and/or a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid
sequence of SEQ ID NO: 5 and a light chain CDR3 comprising the amino acid sequence of
SEQ ID NO: 6. More preferably the antagonist antibody or fragment thereof that binds to
human OX40 comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID
NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a
heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a light chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising
the amino acid sequence of SEQ ID NO: 5 and a light chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 6.
It is well known in the art that the CDR3 domain, independently from the CDR1 and/or CDR2
domain(s), alone can determine the binding specificity of an antibody for a cognate antigen
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and that multiple antibodies can predictably be generated having the same binding specificity
based on a common CDR3 sequence. See, for example, Klimka A et al., (2000) Br. J. Cancer,
83(2): 252-260 (describing the production of a humanized anti-CD30 antibody using only the
heavy chain variable domain CDR3 of murine anti-CD30 antibody Ki-4); Beiboer SH et al.,
(2000) J. Mol. Biol. 296: 833-849 (describing recombinant epithelial glycoprotein-2 (EGP-2)
antibodies using only the heavy chain CDR3 sequence of the parental murine MOC-31 anti-
EGP-2 antibody); Rader C et al., (1998) Proc. Natl. Acad. Sci USA, 95: 8910-8915
(describing a panel of humanized anti-integrin αvβ3 antibodies using a heavy and light chain
variable CDR3 domain of a murine anti-integrin αvβ3 antibody LM609 wherein each member
antibody comprises a distinct sequence outside the CDR3 domain and capable of binding the
same epitope as the parental murine antibody with affinities as high or higher than the parental
murine antibody); Barbas C et al., (1994) J. Am. Chem. Soc. 116: 2161-62 (disclosing that the
CDR3 domain provides the most significant contribution to antigen binding).
Accordingly, the present invention provides antibodies and fragments thereof that bind to
human OX40 comprising one or more heavy and/or light chain CDR3 domains, in particular
comprising heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and/or
light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the
antibody is capable of binding to human OX40. Within some embodiments, such inventive
antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human
antibody (a) are capable of competing for binding with; (b) retain the functional
characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the
corresponding parental non-human e.g. murine antibody.
In a further aspect the present invention provides an antagonist antibody or fragment thereof
that binds to human OX40 comprising a heavy chain variable region sequence comprising the
amino acid sequence of SEQ ID NO: 7. In another aspect the present invention provides an
antagonist antibody or fragment thereof that binds to human OX40 comprising a light chain
variable region sequence comprising the amino acid sequence of SEQ ID NO: 8. In some
embodiments the antagonist antibody or fragment thereof that binds to human OX40
comprises a heavy chain variable region sequence comprising the amino acid sequence of
SEQ ID NO: 7 and a light chain variable region sequence comprising the amino acid sequence
of SEQ ID NO: 8.
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In another aspect the present invention provides variants of an antagonist antibody or fragment
thereof that binds to human OX40. Thus the present invention provides antibodies or
fragments thereof that have an amino acid sequence of the non-CDR regions of the heavy
and/or light chain variable region sequence which is at least 80% identical (having at least
80% amino acid sequence identity) to the amino acid sequence of the non-CDR regions of the
heavy and/or light chain variable region sequence of the parent antagonist antibody of either
the heavy or the light chain e.g. of either the heavy and light variable region sequences as in
SEQ ID NO: 7 or SEQ ID NO: 8, respectively. As well antibodies or fragments thereof that
have an amino acid sequence of the non-extended CDR regions of the heavy and/or light chain
variable region sequence which is at least 80% identical to the amino acid sequence of the
non-extended CDR regions of the heavy and/or light chain variable region sequence of the
parent antagonist antibody of either the heavy or the light chain are provided by the present
invention. Preferably the amino acid sequence identity of the non-CDR regions or of the non-
extended CDR regions of the heavy and/or light chain variable region sequence is at least
85%, more preferably at least 90%, and most preferably at least 95%, in particular 96%, more
particular 97%, even more particular 98%, most particular 99%, including for example, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, and 100%. Identity or homology with respect to an amino acid sequence is
defined herein as the percentage of amino acid residues in the candidate sequence that are
identical with the antagonist antibody or fragment thereof that binds to human OX40, after
aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent
sequence identity. Thus sequence identity can be determined by standard methods that are
commonly used to compare the similarity in position of the amino acids of two polypeptides.
Using a computer program such as BLAST or FASTA, two polypeptides are aligned for
optimal matching of their respective amino acids (either along the full length of one or both
sequences or along a pre-determined portion of one or both sequences). The programs
provide a default opening penalty and a default gap penalty, and a scoring matrix such as
PAM250 (a standard scoring matrix; see Dayhoff MO et al., (1978) in Atlas of Protein
Sequence and Structure, vol 5, supp. 3) can be used in conjunction with the computer
program. For example, the percent identity can be calculated as: the total number of identical
matches multiplied by 100 and then divided by the sum of the length of the longer sequence
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within the matched span and the number of gaps introduced into the longer sequences in order
to align the two sequences.
In some embodiments the present disclosure thus provides an antagonistic antibody or
fragment thereof that binds to human OX40, wherein the antibody or fragment thereof
comprises a heavy chain variable framework region sequence which is at least 70% identical
to the framework region sequence of SEQ ID NOS: 19, 20, 21, 22 or 23 and/or a light chain
variable framework region sequence which is at least 60% identical to the framework region
sequence of SEQ ID NOS: 24, 25, 26, 27 and 28. In some embodiments the present disclosure
provides an antagonistic antibody or fragment thereof that binds to human OX40, wherein the
antibody or fragment thereof comprises a heavy chain variable framework region sequence
which is at least 74% identical to the framework region sequence of SEQ ID NO: 19 and/or a
light chain variable framework region sequence which is at least 65% identical to the
framework region sequence of SEQ ID NO: 24.
In another aspect the present invention provides an antagonistic antibody or fragment thereof
that binds to human OX40 comprising the heavy and or light chain CDRs as described supra
and further comprising a heavy chain variable framework region that is the product of or
derived from a human gene selected from the group consisting of IGHV2-70*10 (SEQ ID NO:
19), IGHV2-70*01 (SEQ ID NO: 20), IGHV2-70*13 (SEQ ID NO: 21), IGHV2-5*09 (SEQ
ID NO: 22), and IGHV2-70*11 (SEQ ID NO: 23), preferably a heavy chain variable
framework region that is the product of or derived from human gene IGHV2-70*10 (SEQ ID
NO: 19). The heavy chain variable framework region may comprise one or more (e.g., one,
two, three and/or four) heavy chain framework region sequences (e.g., framework 1 (FW1),
framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4)) present in the product
of or derived from those human genes. Preferably the heavy chain variable region framework
comprises FW1, FW2 and/or FW3, more preferably FW1, FW2 and FW3 present in the
product of or derived from a human gene selected from the group consisting of IGHV2-70*10
(SEQ ID NO: 19), IGHV2-70*01 (SEQ ID NO: 20), IGHV2-70*13 (SEQ ID NO: 21),
IGHV2-5*09 (SEQ ID NO: 22), and IGHV2-70*11 (SEQ ID NO: 23). Heavy chain
framework region sequences as used herein include FW1 (position 1 to position 25), FW2
(position 36 to position 49), FW3 (position 66 to position 94) and FW 4 (position 103 to
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position 113), wherein the amino acid position is indicated utilizing the numbering system set
forth in Kabat.
In some embodiments the present disclosure provides an antibody or fragment thereof,
wherein the antibody or fragment thereof comprises a heavy chain variable framework region
that is the product of or derived from human gene IGHV2-70*10 (SEQ ID NO: 19) and
wherein the heavy chain variable framework region comprises at least one amino acid
modification from the corresponding heavy chain variable framework region of the
corresponding murine antibody.
In some embodiments the present disclosure provides an antibody or fragment thereof
comprising a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 32
and wherein the heavy chain variable framework region comprises at least one amino acid
modification from the corresponding heavy chain variable framework region of the
corresponding murine antibody.
Preferably the amino acid modification comprises an amino acid substitution at amino acid
position selected from the group consisting of 23, 35b, 48, 50, 60, and 62, more preferably at
amino acid positions selected from the group consisting of 23, 35b, 50, 60 and 62, most
preferred at amino acid position 35b, wherein the amino acid position of each group member
is indicated according to the Kabat numbering. Specifically the amino acid modification
comprises an amino acid substitution selected from the group consisting of 23S, 35bG, 48L,
50H, 60N, and 62A, preferably an amino acid substitution selected from the group consisting
of T23S, S35bG, I48L, R50H, S60N and S62A, whereas S35bG is the most preferred amino
acid substitution wherein the amino acid position of each group member is indicated
according to the Kabat numbering.
In another aspect the present invention provides an antagonistic antibody or fragment thereof
that binds to human OX40 comprising a light chain variable framework region that is the
product of or derived from a human gene selected from the group consisting of IGKV3-11*01
(SEQ ID NO: 24), IGKV1-39*01 (SEQ ID NO: 25), IGKV1D-39*01 (SEQ ID NO: 26),
IGKV3-11*02 (SEQ ID NO: 27) and IGKV3-20*01 (SEQ ID NO: 28), preferably a light
chain variable framework region that is the product of or derived from human gene IGKV3-
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11*01 (SEQ ID NO: 24). The light chain variable region framework region may comprise one
or more (e.g., one, two, three and/or four) light chain framework region sequences (e.g.,
framework 1 (FW1), framework 2 (FW2), framework 3 (FW3) and/or framework 4 (FW4))
present in the product of or derived from those human genes. Preferably the light chain
variable region framework comprises FW1, FW2 and/or FW3, more preferably FW1, FW2
and FW3 present in the product of or derived from a human gene selected from the group
consisting of V3-11*01 (SEQ ID NO: 24), IGKV1-39*01 (SEQ ID NO: 25), IGKV1D-39*01
(SEQ ID NO: 26), IGKV3-11*02 (SEQ ID NO: 27) and IGKV3-20*01 (SEQ ID NO: 28).
Light chain framework region sequences as used herein include FW1 (position 1 to position
23), FW2 (position 35 to position49), FW3 (position 57 to position 88) and FW 4 (position 98
to position 1 08), wherein the amino acid position is indicated utilizing the numbering system
set forth in Kabat.
In some embodiments the present disclosure provides an antibody or fragment thereof
comprising a light chain variable framework region that is the product of or derived from
human gene IGKV3-11*01 (SEQ ID NO: 24) and wherein the light chain variable framework
region comprises at least one amino acid modification from the corresponding framework
region of the light chain variable region of the corresponding murine antibody.
In some embodiments the present disclosure provides an antibody or fragment thereof
comprising a light chain sequence comprising the amino acid sequence of SEQ ID NO: 39 and
wherein the light chain variable framework region of the light chain sequence comprises at
least one amino acid modification from the corresponding light chain variable framework
region of the corresponding murine antibody.
Preferably the amino acid modification comprises an amino acid substitution at amino acid
position selected from the group consisting of 1, 33, 34, 46, 47, 54, 56, and 71 and/or a
deletion at amino acid position 31, more preferably an amino acid substitution at amino acid
position selected from the group consisting of 33, 34, 46, 47, 54, 56, and 71 and/or a deletion
at amino acid position 31, most preferably an amino acid substitution at amino acid position
46 and/or 47, wherein the amino acid position of each group member is indicated according to
the Kabat numbering. Specifically the amino acid modification comprises an amino acid
substitution selected from the group consisting of 1Q, 33M, 34H, 46P, 47W, 54L, 56S, and
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71Y, and/or a deletion at T31, preferably an amino acid substitution selected from the group
consisting of a 1Q, 33M, 34H, 46P, 47W, 54L, 56S and 71Y, more preferably an amino acid
substitution selected from the group consisting of 33M, 34H, 46P, 47W and 71Y, whereas
46P, 47W are particularly preferred, wherein the amino acid position of each group member is
indicated according to the Kabat numbering.
In some embodiments the antagonistic antibody or fragment thereof that binds to human
OX40 comprises a heavy chain variable framework region that is the product of or derived
from a human gene selected from the group consisting of V2-70*10 (SEQ ID NO: 19), V2-
70*01 (SEQ ID NO: 20), V2-70*13 (SEQ ID NO: 21), V2-5*09 (SEQ ID NO: 22), and V2-
70*11 (SEQ ID NO: 23) and a light chain variable framework region that is the product of or
derived from a human gene selected from the group consisting of V3-11*01 (SEQ ID NO:
24), IGKV1-39*01 (SEQ ID NO: 25), IGKV1D-39*01 (SEQ ID NO: 26), IGKV3-11*02
(SEQ ID NO: 27) and IGKV3-20*01 (SEQ ID NO: 28), preferably a heavy chain variable
framework region that is the product of or derived from human gene V2-70*10 (SEQ ID NO:
19), and a light chain variable framework region that is the product of or derived from human
gene V3-11*01 (SEQ ID NO: 24). As well combinations of heavy chain variable framework
regions which are present in the product of or derived from different human genes mentioned
supra and/or of light chain variable region framework regions which are present in the product
of or derived from different human genes mentioned supra are encompassed by the present
invention.
Germline DNA sequences for human heavy and light chain variable region genes can be found
in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.
cam.ac.uk/vbase), as well as in Kabat EA et al., supra; Tomlinson IM et al., (1992) J. Mol.
Biol. 227: 776-798 and Cox JPL et al., (1994) Eur. J. Immunol. 24: 827-836. As another
example, the germline DNA sequences for human heavy and light chain variable region genes
can be found in the Genbank database.
In another aspect, the present disclosure also provides an antagonist antibody or fragment
thereof that binds to human OX40, wherein at least one of the heavy chain CDRs and/or at
least one of the light chain CDRs comprises at least one amino acid modification. Site-
directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the
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modification(s) and the effect on antibody binding, or other functional property of interest, can
be evaluated in in vitro or in vivo assays. Preferably conservative modifications are
introduced. The modification(s) may be amino acid substitutions, additions or deletions, but
are preferably substitutions. Typically, no more than five, preferably no more than four, more
preferably no more than three, even more preferably no more than two, most preferably no
more than one amino acid modifications are performed within a CDR region.
In certain embodiments, framework sequences can be used to engineer variable regions to
produce variant antibodies. Variant antibodies of the invention include those in which
modifications have been made to framework residues within VH and/or VK, e.g. to improve
the properties of the antibody. Typically such framework modifications are made to decrease
the immunogenicity of the antibody. For example, one approach is to "backmutate" one or
more framework residues to the corresponding murine sequence or to "backmutate" one or
more framework residues to a corresponding germline sequence.
Thus in a further aspect the present disclosure provides an antagonistic antibody or fragment
thereof that binds to human OX40, wherein at least one of the framework region sequences of
the heavy chain variable region of the humanized antibody or fragment thereof comprises at
least one amino acid modification from the corresponding framework region of the heavy
chain variable region of the corresponding murine antibody. Preferably the amino acid
modification is an amino acid substitution. Typically, no more than six, preferably no more
than five, preferably no more than four, more preferably no more than three, even more
preferably no more than two, most preferably no more than one amino acid modifications are
performed within a framework region. In some embodiments the present disclosure provides
an antagonistic antibody or fragment thereof that binds to human OX40, wherein the amino
acid modification of the framework regions of the heavy chain variable region comprise an
amino acid substitution at amino acid position selected from the group consisting of 23, 35b,
48, 50, 60 and 62, and wherein the amino acid position of each group member is indicated
according to the Kabat numbering. Preferred amino acid substitution of the framework regions
of the heavy chain variable region are at amino acid positions selected from the group
consisting of 23, 35b, 50, 60 and 62. More preferred amino acid substitutions of the
framework regions of the heavy chain variable region are selected from the group consisting
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of 23S, 35bG, 48L, 50H, 60N and 62A, whereas 35bG is the most preferred amino acid
substitution of the framework regions of the heavy chain variable region.
The present disclosure also provides an antagonistic antibody or fragment thereof that binds to
human OX40, wherein at least one of the framework region sequences of the light chain
variable region of the humanized antibody or fragment thereof comprises at least one amino
acid modification from the corresponding framework region of the light chain variable region
of the corresponding murine antibody. Preferably the amino acid modification is an amino
acid substitution and/or an amino acid deletion. Typically, no more than six, preferably no
more than five, preferably no more than four, more preferably no more than three, even more
preferably no more than two, most preferably no more than one amino acid modifications are
performed within a framework region. In some embodiments the present disclosure provides a
humanized antibody or fragment thereof, wherein the amino acid modification of the
framework regions of the light chain variable region sequence comprises an amino acid
substitution at amino acid position selected from the group consisting of 1, 33, 34, 46, 47, 54,
56 and 71 and/or a deletion at amino acid position 31. More preferred amino acid
modifications of the framework regions of the light chain variable region sequence comprise a
deletion at Y31 and/or a substitution selected from the group consisting of a 1Q, 33M, 34H,
46P, 47W, 54L, 56S and 71Y, and wherein the amino acid position of each group member is
indicated according to the Kabat numbering. Most preferred amino acid modifications of the
framework regions of the light chain variable region sequence comprise a deletion at T31
and/or a substitution selected from the group consisting of 33M, 34H, 46P, 47W and 71Y,
whereas 46P, and/or L47W are particularly preferred. In some embodiments the humanized
antibody or fragment thereof of the present invention may comprise amino acid modifications
of the framework regions of the heavy chain variable region sequence as set out above and
amino acid modifications of the framework regions of the light chain variable region sequence
as set out above.
The present disclosure also provides an antagonistic antibody or fragment thereof that binds to
human OX40 that comprises a heavy chain variable region selected from the group consisting
of SEQ ID NOS: 29, 58, 59, 77, 78, 79 and 80, preferably selected from the group consisting
of SEQ ID NOS: 58, 59, 79 and 80 and more preferably from the group consisting of SEQ ID
NOS: 58 and 59. The present disclosure also provides an antagonistic antibody or fragment
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thereof that binds to human OX40 that comprises a light chain variable region selected from
the group consisting of SEQ ID NOS: 30, 60, 81, 82, 83, 84, 85, 86, 87, 88, and 89, preferably
selected from the group consisting of SEQ ID NOS: 60, 86, 87 and 89, more preferably SEQ
ID NO: 60. In some embodiments the antagonistic antibody or fragment thereof that binds to
human OX40 comprises a heavy chain variable region selected from the group consisting of
SEQ ID NOS: 29, 58, 59, 77, 78, 79 and 80, and a light chain variable region selected from
the group consisting of SEQ ID NOS: 30, 60, 81, 82, 83, 84, 85, 86, 87, 88, and 89. Given that
each of these heavy and light chain variable region sequences can bind to human OX40, the
heavy and light chain variable region sequences can be "mixed and matched" to create anti-
OX40 binding molecules of the invention. OX40 binding of such "mixed and matched"
antibodies can be tested using the binding assays described in the examples.
In some embodiments the antagonistic antibody or fragment thereof that binds to human
OX40 comprises a heavy chain variable region selected from the group consisting of SEQ ID
NOS: 58 and 59, and a light chain variable region selected from the group consisting of SEQ
ID NOS: 60 and 89. In more preferred embodiments the antagonistic antibody or fragment
thereof that binds to human OX40 comprises a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO: 58 and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 60, a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 58 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 89, a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 59 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 60, or a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 59 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 89. Most preferred is an antagonistic antibody or fragment thereof
that binds to human OX40 comprising a heavy chain variable region selected from the group
consisting of SEQ ID NOS: 58 and 59, and a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 60.
The present disclosure also provides an antagonistic antibody or fragment thereof that binds to
human OX40 that comprises a heavy chain sequence selected from the group consisting of
SEQ ID NOS: 32, 33, 34, 35, 36, 37 and 38, preferably selected from the group consisting of
SEQ ID NOS: 35, 36, 37 and 38 and more preferably from the group consisting of SEQ ID
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NOS: 37 and 38. The present disclosure also provides an antagonistic antibody or fragment
thereof that binds to human OX40 that comprises a light chain sequence selected from the
group consisting of SEQ ID NOS: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 49, preferably
selected from the group consisting of SEQ ID NOS: 45, 46, 47 and 49, more preferably SEQ
ID NO: 47. In some embodiments the antagonistic antibody or fragment thereof that binds to
human OX40 comprises a heavy chain sequence selected from the group consisting of SEQ ID
NOS: 32, 33, 34, 35, 36, 37 and 38, and a light chain sequence selected from the group
consisting of SEQ ID NOS: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 49. Given that each of
these heavy and light chain variable region sequences can bind to human OX40, the heavy and
light chain variable region sequences can be "mixed and matched" to create anti-OX40
binding molecules of the invention. OX40 binding of such "mixed and matched" antibodies
can be tested using the binding assays described e.g. in the Examples.
In some embodiments the antagonistic antibody or fragment thereof that binds to human
OX40 comprises a heavy chain sequence selected from the group consisting of SEQ ID NOS:
37 and 38, and a light chain sequence selected from the group consisting of SEQ ID NOS: 47
and 49. In more preferred embodiments the antagonistic antibody or fragment thereof that
binds to human OX40 comprises a heavy chain sequence comprising the amino acid sequence
of SEQ ID NO: 37 and a light chain sequence comprising the amino acid sequence of SEQ ID
NO: 47, a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 37 and a
light chain sequence comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain
sequence comprising the amino acid sequence of SEQ ID NO: 38 and a light chain sequence
comprising the amino acid sequence of SEQ ID NO: 47, or a heavy chain sequence
comprising the amino acid sequence of SEQ ID NO: 38 and a light chain sequence comprising
the amino acid sequence of SEQ ID NO: 49. Most preferred is an antagonistic antibody or
fragment thereof that binds to human OX40 comprising a heavy chain sequence selected from
the group consisting of SEQ ID NOS: 37 and 38, and a light chain sequence comprising the
amino acid sequence of SEQ ID NO: 47.
In one embodiment of the present disclosure, the antagonist antibody or fragment thereof is a
murine antibody, chimeric antibody or a humanized antibody, preferably a humanized
antibody, more preferably a monoclonal murine antibody, a monoclonal chimeric antibody or
a monoclonal humanized antibody.
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The present disclosure also provides a monovalent antibody or fragment thereof that binds to
human OX40, i.e. an antibody which consists of a single antigen binding arm. The present
disclosure also provides a fragment of a antibody that binds to human OX40 selected from the
group consisting of Fab, Fab′, Fab′-SH, Fd, Fv, dAb , F(ab')2, scFv, bispecific single chain Fv
dimers, diabodies, triabodies and scFv genetically fused to the same or a different antibody.
Preferred fragments are scFv, bispecific single chain Fv dimers and diabodies. The present
disclosure also provides a full length antibody that binds to human OX40.
The present disclosure also provides an antibody or fragment thereof that binds to human
OX40 which further comprises a heavy and/or light constant region in particular a human
heavy and/or a human light constant region. Human heavy constant regions may be selected
from the group of human immunoglobulins consisting of IgG1 (IGHG1), IgG2 (IGHG2), IgG3
(IGHG3), IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), or IgE
(IGHE), whereas the human heavy constant region IgG, in particular IgG1 (IGHG1) is
preferred. Human light constant region may be selected from the group of human
immunoglobulins consisting of kappa or lambda constant regions, whereas human kappa
constant region is preferred. In a preferred embodiment the antagonistic antibody or fragment
thereof that binds to human OX40 comprises a human IgG1 (IGHG1) heavy constant domain
and a human light kappa constant domain.
In addition or alternative to modifications made within the framework regions or CDR
regions, antibodies of the invention may be engineered to include modifications within the Fc
region, typically to alter one or more functional properties of the antibody, such as serum half-
life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
Furthermore, an antibody of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified to alter its glycosylation.
Each of these embodiments is described in further detail below. Modifications within the Fc
region as outlined below are according to the EU numbering of residues in the Fc region. In
one embodiment, the hinge region of CH1 is modified such that the number of cysteine
residues in the hinge region is altered, e.g., increased or decreased. This approach is described
further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the
hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy
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chains or to increase or decrease the stability of the antibody. In another embodiment, the Fc
hinge region of an antibody is mutated to decrease the biological half life of the antibody.
More specifically, one or more amino acid mutations are introduced into the CH2-CH3
domain interface region of the Fc-hinge fragment such that the antibody has impaired
Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al. In another
embodiment, the antibody is modified to increase its biological half life. Various approaches
are possible. For example, one or more of the following mutations can be introduced: T252L,
T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase
the biological half life, the antibody can be altered within the CH1 or CL region to contain a
salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an
IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. In a further
embodiment Fc region is altered by replacing at least one amino acid residue with a different
amino acid residue to alter the effector function(s) of the antibody. For example, one or more
amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can
be replaced with a different amino acid residue such that the antibody has an altered affinity
for an effector ligand but retains the antigen- binding ability of the parent antibody. The
effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1
component of complement. This approach is described in further detail in U.S. Patent Nos.
5,624,821 and 5,648,260, both by Winter et al. In another example, one or more amino acids
selected from amino acid residues 329, 331 and 322 can be replaced with a different amino
acid residue such that the antibody has altered C1q binding and/or reduced or abolished
complement dependent cytotoxicity (CDC). This approach is described in further detail in
U.S. Patent Nos. 6,194,551 by Idusogie et al. In another example, one or more amino acid
residues within amino acid positions 231 to 238 in the N-terminal region of the CH2 domain
are altered to thereby alter the ability of the antibody to fix complement. This approach is
described further in PCT Publication WO94/29351 by Bodmer et al. In yet another example,
the Fc region is modified to increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor
by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252,
254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292,
293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330,
331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,
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434, 435, 437, 438 or 439. This approach is described further in PCT Publication
WO00/42072 by Presta.
The present disclosure also provides an antagonistic antibody or fragment thereof that binds to
human OX40 comprising human heavy and/or light constant regions, wherein the human
heavy constant region comprises an isotypic variant comprising the CH1 region, the hinge
region, the CH2 region and CH3 region from human IgG4 (IGHG4) and wherein the hinge
region comprises a substitution of serine at position 228 to proline. Preferably the humanized
antibody comprising the isotypic variant is a full length antibody. A particular preferred
humanized antibody or fragment thereof that binds to human OX40 comprising an isotypic
variant comprising the CH1 from human IgG4 (IGHG4), the hinge from human IgG4
(IGHG4), having S228P substitution and the CH2 and CH3 from human IgG4 (IGHG4)
comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 57
and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 47. It has
been found that the isotypic variant exhibits no Fc-mediated cytotoxicity mechanisms such as
ADCC compared to an antagonistic antibody or fragment thereof that binds to human OX40
which comprises a human heavy constant region from human IgG1 (IGHG1) (which is usually
a native human IgG1), i.e. as compared to an antagonistic antibody or fragment thereof that
binds to human OX40 that only differs from the isotypic variant with regard to the modified
heavy constant region.
The present disclosure also provides an antagonistic antibody or fragment thereof that binds to
human OX40 which comprises a human IgG Fc region, wherein the mature core carbohydrate
structure attached to the human IgG Fc region lacks fucose (referred herein alternatively as
“non-fucosylated”). Preferably the antibody comprises a human IgG1 (IGHG1) Fc region,
wherein the mature core carbohydrate structure attached to the human IgG1 (IGHG1) Fc
region lacks fucose. More preferred is a full-length antibody comprising a human IgG1
(IGHG1) Fc region, wherein the mature core carbohydrate structure attached to the human
IgG1 (IGHG1) Fc region lacks fucose. It is known from WO03/035835 that lack of fucose in
the mature core carbohydrate structure attached to the human IgG Fc region may enhance
ADCC. Thus in a further embodiment the antagonistic antibody or fragment thereof of the
present disclosure comprises a human IgG1 (IGHG1) Fc region, wherein the mature core
carbohydrate structure attached to the human IgG1 (IGHG1) Fc region lacks fucose, whereas
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the antibody lacking fucose exhibits enhanced ADCC compared to the parent humanized
antibody or fragment thereof not lacking fucose. Methods to generate antibodies which lack
fucose are, for example (a) use of an engineered or mutant host cell that is deficient in fucose
metabolism such that it has a reduced ability (or is unable to) fucosylate proteins expressed
therein; (b) culturing cells under conditions which prevent or reduce fucosylation; (c) post-
translational removal of fucose (e. g. with a fucosidase enzyme); (d) post-translational
addition of the desired carbohydrate, e. g. after recombinant expression of a non-glycosylated
glycoprotein; or (e) purification of the glycoprotein so as to select for product which is not
fucosylated. Preferably used are methods described in Example 14 of WO10/095031 e,g.
methods described in Longmore et al., (1982) Carbohydr. Res. 365-92 or in Imai-Nishiya et
al., (2007), BMC Biotechnol. 7: 84.
Also provided by the present invention is an antagonist antibody or fragment thereof that
binds to human OX40 and which binds to the same epitope as the antibody comprising the
heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO. 7 and/or
the light chain variable sequence comprising the amino acid sequence of SEQ ID NO. 8. Also
provided by the present invention is a specific region or epitope of human OX40, in particular
of the human OX40 receptor extracellular domain, which is bound by an antibody provided
by the present invention, in particular by an antibody comprising the heavy chain variable
sequence comprising the amino acid sequence of SEQ ID NO. 7 and/or the light chain
variable sequence comprising the amino acid sequence of SEQ ID NO. 8. This specific region
or epitope of the human OX40 polypeptide can be identified by any suitable epitope mapping
method known in the art in combination with any one of the antibodies provided by the
present invention. Examples of such methods include screening peptides of varying lengths
derived from OX40 for binding to the antibody of the present invention with the smallest
fragment that can specifically bind to the antibody containing the sequence of the epitope
recognised by the antibody. The OX40 peptides may be produced synthetically or by
proteolytic digestion of the OX40 polypeptide. Peptides that bind the antibody can be
identified by, for example, mass spectrometric analysis. In another example, NMR
spectroscopy or X-ray crystallography can be used to identify the epitope bound by an
antibody of the present invention. Once identified, the epitopic fragment which binds an
antibody of the present invention can be used, if required, as an immunogen to obtain
additional antagonist antibodies which bind the same epitope.
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Anti-OX40 antibody properties
Standard assays to evaluate the binding ability of the antibodies toward e.g. human OX40 are
known in the art, including for example, ELISAs, BIAcore , Western blots, RIAs, and flow
cytometry analysis. Suitable assays are described in detail in the Examples. The binding
kinetics (e.g., binding affinity like KD) of the antibodies also can be assessed by standard
assays known in the art, such as by Scatchard or BIAcore system analysis. The relative
binding affinity K can be assessed by standard competition assays known in the art.
In a further aspect the present invention provides antagonistic antibodies or fragment thereof
that bind to human OX40 and which block a Human Mixed Lymphocyte Reaction (MLR) in a
dose dependent manner to a higher degree than recombinant humanized antibody efalizumab.
Recombinant humanized antibody efalizumab binds to the CD11a subunit of lymphocyte
function-associated antigen 1. MLR can be carried out and measured according to Example 3.
In a further aspect the present invention provides antagonist antibodies or fragment thereof
that bind to human OX40 and which are also able to recognise cynomolgus monkey OX40.
Binding of an antagonistic anti-OX40 antibody to both human and cynomolgus peripheral
blood monocuclear cells (PBMC) can be carried out and measured according to Example 4
and shown in Fig. 3. The antibody was found to recognise OX40 expressed on the surface of
human and cynomolgus monkey activated lymphocytes indicating that this antibody has cross-
reactive properties.
In a further aspect the present invention provides antagonistic antibodies or fragment thereof
that bind to human OX40, in particular human OX40 in isolated form, with an affinity (K ) of
500 nM or less, preferably 200 nM or less, more preferably 150 nM or less, more preferably
120 nM or less, even more preferably 110 nM or less e.g. measured by Surface Plasmon
Resonance (SPR) on a BIAcore instrument (GE Healthcare Europe GmbH, Glattbrugg,
Switzerland) by capturing the antibody on a protein-A coupled CM5 research grade sensor
chip (GE Healthcare Europe GmbH, Glattbrugg, Switzerland; BR14) with a
recombinant monovalent human OX40 receptor extracellular domain (SEQ ID NO: 11) used
as analyte as detailed in Examples 5 and 6 and as illustrated in Fig. 4. “Monovalent” as used
herein in relation to affinity measurements using OX40 receptor refers to a human OX40
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receptor domain, like the extracellular domain, not artificially dimerized or multimerized as it
would be e.g. if the domain would be amino-terminally fused to an immunoglobulin Fc
portion. In a preferred aspect, the present invention provides a humanized antibody or
fragment thereof that retains at least 75% of the OX40 binding affinity (K ) of the
corresponding chimeric antibody. Preferably, the humanized antibody or fragment thereof
binds human OX40 with equivalent affinity to the corresponding chimeric antibody. By
“equivalent affinity” is meant an affinity value that is within a range of ±10% of the OX40
binding affinity of the corresponding chimeric antibody. More preferably, the present
invention provides a humanized antibody or fragment thereof that binds human OX40 with a
higher affinity than the corresponding chimeric antibody. In a preferred aspect of the present
invention, antagonistic antibodies or fragment thereof that bind to human OX40 are provided
that have a binding affinity (K ) of 110 nM or less, preferably 100 nM or less, more
preferably 90 nM or less, more preferably 80 nM or less, even more preferably 70 nM or less
e.g. measured by Surface Plasmon Resonance (SPR) on a BIAcore instrument (GE
Healthcare Europe GmbH, Glattbrugg, Switzerland) by capturing the antibody on a protein-A
coupled CM5 research grade sensor chip (GE Healthcare Europe GmbH, Glattbrugg,
Switzerland; BR14) with a recombinant monovalent human OX40 receptor
extracellular domain (SEQ ID NO: 11) used as analyte as detailed in Examples 5 and 6 and as
illustrated in Fig. 4.
A further aspect of the present invention provides antagonistic antibodies or fragments thereof
that bind to human OX40 and which have good thermal stability. In a preferred embodiment,
an antagonistic humanized antibody or fragment thereof that binds to human OX40 has a FAB
fragment thermostability temperature greater than 70 C, preferably greater than 75 C, more
preferably greater than 80 C and even more preferably greater than 85 C. For analysis of FAB
fragment thermostability differential scanning calorimetry measurements are used, whereas a
mid-point melting temperature of the FAB fragment in context of a full-length IgG is
identified. These kind of calorimetric measurements are known to the skilled person and can
be carried out according to e.g. Garber & Demarest (2007), BBRC, 355: 751-7, as further
described in Example 6 and shown in Fig. 6.
In a further aspect the present invention describes antagonistic antibodies or fragments thereof
that bind to an epitope on the human OX40 extracellular region. As described in Example 7
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and shown in Fig. 7, one or more of the four domains of the OX40 extracellular region were
exchanged between human and rat sequences and Fc fusion proteins generated. A binding
ELISA was then performed to test the reactivity of an antagonistic humanised antibody on the
human OX40 extracellular region, rat OX40 extracellular region and four human-rat chimeric
proteins. Thus the present invention provides an antagonistic antibody or fragment thereof
which maps within the second domain of human OX40 extracellular region.
The present invention also provides antagonistic antibodies or fragments thereof which can be
used to suppress immune reactions. The effect of an antagonistic humanized anti-OX40
antibody was tested in a MLR (see Example 8) used as an in vitro model of alloreactive T cell
activation and proliferation (O’Flaherty E et al., (2000) Immunology, 100(3): 289-99; DuPont
B & Hansen JA (1976) Adv. Immunol. 23: 107-202). PBMCs from two unreleated donors
were mixed, resulting in the activation of T cells and a proliferation of T lymphocytes. In
addition, three different formats of the antagonistic humanized anti-OX40 antibody were
tested in this assay: an IgG1 (IGHG1) format, a non fucosylated IgG1 (IGHG1) format and an
IgG4 (IGHG4) format, to further determine the contribution of cytotoxic mechanisms such as
ADCC on the inhibition of MLR. The antagonistic humanized anti-OX40 antibody efficiently
inhibited MLR in two different individuals (responders) with EC50 values of approximately
100 ng/mL. However the results showed a difference depending on the format of the antibody
used. In the first individual (responder 1), T cell reactivity was efficiently inhibited by the
IgG1 (IGHG1) and IgG4 (IGHG4) antibody formats indicating that the cytotoxic mechanisms
are not critical for this individual. For the second individual (responder 2) the IgG1 (IGHG1)
format achieved more than 60% inhibition, whereas the IgG4 (IGHG4) format only poorly
blocked the MLR. In both individuals, the non fucosylated IgG1 (IGHG1) format was very
effective at inhibiting MLR. These results indicate that blocking OX40 activation may be
sufficient in some individuals to inhibit MLR but this effect can be greatly enhanced by
additional cytotoxic mechanisms. Therefore for the treatment of patients suffering from OX40
mediated disorders, where the disorder appears to be independent of the patients’ OX40
costimulatory status e.g. patients with low OX40 expression levels, administration of an
antagonistic antibody or fragment thereof that binds human OX40 and which has enhanced
cytotoxic mechanisms may be particularly effective. A preferred embodiment of the present
invention provides an antagonistic humanized antibody that binds to human OX40 for the
treatment of a patient suffering from an OX40 mediated disorder. Furthermore, the patient
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may have low expression levels of OX40. Preferably the antagonistic humanized antibody that
binds to human OX40 comprises an IgG1 (IGHG1) region. More preferably the antagonistic
humanized antibody that binds to human OX40 comprises a non-fucosylated IgG1 region.
In a further aspect of the present invention, the effect of an antagonistic antibody or fragment
thereof was demonstrated in a xenogeneic graft versus host reaction, in which SCID mice
were reconstituted with human PBMCs. This reaction provides a model for the allogenic graft
versus host disease (GVHD) observed after bone marrow transplant in human patients. In this
model, human PBMCs and T lymphocytes in particular, launch a strong response against the
mouse host cells which gives rise to severe inflammatory symptoms. As described in Example
9 and shown in Fig. 9 and Table 10, an antagonistic humanized antibody that binds to human
OX40 potently suppressed the GVHD reaction at a dose of 1 mg/kg. Surprisingly, this
antibody demonstrated a better efficacy than Enbrel , a recognised therapy for GVHD
(Xhaard A et al., (2011) Bull. Cancer, 98(8): 889-99; Simpson D (2001) Expert Opin.
Pharmacother. 2(7): 1109-17). Therefore, in a preferred embodiment, the present invention
provides an antagonistic antibody or fragment thereof that binds to human OX40 and which is
effective in the treatment of GVHD. Preferably, administration of the antibody to a subject
results in a four-fold improvement in survival median (days) compared to the administration
of vehicle. More preferably, administration of the antibody to a subject results in a two-fold
improvement in survival median (days) compared to the administration of Enbrel . The
present invention therefore provides an antagonistic antibody or fragment therefore that binds
to human OX40 that is more effective than Enbrel in treating a patient with GVHD and/or at
surpressing GVHD. In addition, it has been reported that agonistic anti-OX40 binding
antibodies worsen GVHD in allogenic mouse GVHD models (Valzasina B et al., (2005)
Blood, 105(7): 2845-51; Blazar BR et al., (2003) Blood, 101(9): 3741-8), therefore it can be
concluded from Example 9, that antagonistic antibodies and fragments thereof of the present
invention show no agonistic effects on binding human OX40, since no worsening of the
GVHD was observed in the model. Therefore, the present invention provides a humanized
antibody or fragment therefore that binds to human OX40 that does not show agonistic
activity on binding.
Nucleic acids, Vectors and Host Cells
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The present disclosure also provides isolated nucleic acids encoding the antibodies and
fragments thereof that bind to human OX40, vectors and host cells comprising the nucleic
acid or the vector. The nucleic acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered
substantially pure" when purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS
treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well
known in the art, see e.g. F. Ausubel, et al., ed. (1987) Current Protocols in Molecular
Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of the
invention can be, for example, DNA or RNA and may or may not contain intron sequences. In
a preferred embodiment, the nucleic acid is a cDNA molecule.
Nucleic acids of the invention can be obtained using standard molecular biology techniques
e.g. cDNAs encoding the light and heavy chains of the antibody or encoding VH and VL
segments can be obtained by standard PCR amplification or cDNA cloning techniques. For
antibodies obtained from an immunoglobulin gene library (e.g., using phage display
techniques), one or more nucleic acids encoding the antibody can be recovered from the
library. The methods of introducing exogenous nucleic acid into host cells are well known in
the art, and will vary with the host cell used. Techniques include but are not limited to
dextran- mediated transfection, calcium phosphate precipitation, calcium chloride treatment,
polyethylenimine mediated transfection, polybrene mediated transfection, protoplast fusion,
electroporation, viral or phage infection, encapsulation of the polynucleotide(s) in liposomes,
and direct microinjection of the DNA into nuclei. In the case of mammalian cells, transfection
may be either transient or stable.
Preferred nucleic acids molecules of the invention are those encoding the heavy chain
sequence selected from the group consisting of SEQ ID NOS: 32, 33, 34, 35, 36, 37 and 38
and/or the light chain sequence selected from the group consisting of SEQ ID NOS: 39, 40,
41, 42, 43, 44, 45, 46, 47, 48 and 49. Preferred nucleic acids molecules of the invention are
those encoding the heavy chain variable region selected from the group consisting of SEQ ID
NOS: 29, 58, 59, 77, 78, 79 and 80 and/or the light chain variable region selected from the
group consisting of SEQ ID NOS: 30, 60, 81, 82, 83, 84, 85, 86, 87, 88, and 89.
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Preferred nucleic acids molecules of the invention are those encoding the light chain variable
region of SEQ ID NO: 8 and/or the heavy chain variable region of SEQ ID NO: 7, e.g. DNA
encoding the heavy chain variable region comprising the nucleic acid sequence of SEQ ID
NO: 9 and/or DNA encoding the light chain variable region comprising the nucleic acid
sequence of SEQ ID NO: 10. More preferred nucleic acid molecules of the invention are those
encoding the heavy chain variable region of SEQ ID NOS: 58 or 59 and/or the light chain
variable region of SEQ ID NO: 60, e.g. DNA encoding the heavy chain variable region
comprising the nucleic acid sequence of SEQ ID NOS: 61 or 62 and/or DNA encoding the
light chain variable region comprising the nucleic acid sequence of SEQ ID NO: 63, which are
most preferred.
Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can
be further manipulated by standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, or to fragments genes corresponding
to the fragments described supra like Fab fragment genes or to a scFv gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA
fragment encoding another protein, such as an antibody constant region or a flexible linker.
The term "operatively linked", as used in this context, is intended to mean that the two DNA
fragments are joined such that the amino acid sequences encoded by the two DNA fragments
remain in-frame. The isolated DNA encoding the VH region can be converted to a full-length
heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule
encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat EA et al., supra) and DNA
fragments encompassing these regions can be obtained by standard PCR amplification. The
heavy chain constant region can be an IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3), IgG4
(IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), or IgE (IGHE) constant
region, but most preferably is an IgG1 (IGHG1) constant region. For a Fab fragment heavy
chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule
encoding only the heavy chain CH1 constant region. The isolated DNA encoding the VL
region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by
operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain
constant region, CL. The sequences of human light chain constant region genes are known in
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the art (see e.g., Kabat EA et al., supra.) and DNA fragments encompassing these regions can
be obtained by standard PCR amplification. In preferred embodiments, the light chain
constant region can be a kappa or lambda constant region, preferably a kappa constant region.
To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to
another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 -
Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain
protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird RE et al.,
(1988) Science, 242: 423-426; Huston JS et al., (1988) Proc. Natl. Acad. Sci. USA, 85: 5879-
83; McCafferty J et al., (1990) Nature, 348: 552-554). Various techniques have been
developed for the production of antibody fragments of antibodies. Traditionally, these
fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto K et
al., (1992) J. Biochem. & Biophysical Methods, 24: 107-117 and Brennan M et al., (1985)
Science, 229: 81-3). However, these fragments can now be produced directly by recombinant
host cells. For example, the antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E.
coli and chemically coupled to form F(ab′) fragments (Carter P et al., (1992)
Bio/Technology, 10: 163-167). According to another approach, F(ab′)2 fragments can be
isolated directly from recombinant host cell culture. Other techniques for the production of
antibody fragments will be apparent to the skilled practitioner. In other embodiments, the
antibody of choice is a single-chain Fv fragment (scFv), see e.g. ; U.S. Pat.
No. 5,571,894 and U.S. Pat. No. 5,587,458. The antibody fragment may also be a “linear
antibody”, e.g., as described in U.S. Patent No. 5,641,870, for example.
The nucleic acids that encode the antibodies of the present invention may be incorporated into
a vector, preferably an expression vector in order to express the protein. A variety of
expression vectors may be utilized for protein expression. Expression vectors may comprise
self-replicating extra- chromosomal vectors or vectors which integrate into a host genome.
Expression vectors are constructed to be compatible with the host cell type. Thus vectors,
preferably expression vectors, which find use in the present invention include but are not
limited to those which enable protein expression in mammalian cells, bacteria, insect cells,
yeast, and in in vitro systems. As is known in the art, a variety of expression vectors are
available, commercially or otherwise, that may find use in the present invention for expressing
antibodies.
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Expression vectors typically comprise a protein operably linked with control or regulatory
sequences, selectable markers, any fusion partners, and/or additional elements. By "operably
linked" herein is meant that the nucleic acid is placed into a functional relationship with
another nucleic acid sequence. The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g., polyadenylation signals)
that control the transcription or translation of the antibody chain genes. Such regulatory
sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in
Enzymology 185, Academic Press, San Diego, CA (1990)). Generally, these expression
vectors include transcriptional and translational regulatory nucleic acid operably linked to the
nucleic acid encoding the antibody, and are typically appropriate to the host cell used to
express the protein. In general, the transcriptional and translational regulatory sequences may
include promoter sequences, ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator sequences. As is also known
in the art, expression vectors typically contain a selection gene or marker to allow the
selection of transformed host cells containing the expression vector. Selection genes are well
known in the art and will vary with the host cell used. For example, typically the selectable
marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host
cell into which the vector has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection).
Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote,
yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria,
including gram-negative or gram-positive organisms, for example, Enterobacteriaceae such
as Escherichia, e.g., E. coli, Enterobacter, Klebsiella, Proteus, Salmonella, e.g., Salmonella
typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B.
subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. Suitable
E. coli cloning hosts include E. coli 294 (ATCC 31,446), E. coli B, E. coli X1776 (ATCC
31,537), and E. coli W3110 (ATCC 27,325). In addition to prokaryotes, eukaryotic microbes
such as filamentous fungi or yeast are suitable cloning or expression hosts. Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host
microorganisms. However, a number of other genera, species, and strains are commonly
available and useful, such as Schizosaccharoriyces pombe; Kluyveromyces hosts including K.
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lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC
24,178), K. WaItH (AJCC 56,500), K. drosopmarum (ATCC 36,906), K. thermotolerans, or
K. marxianusyarrowia (EP402226); Pichia pastoris (EP183070); Candida; Trichoderma
reesia (EP244234); Neurospora crassa; Schwanniomyces such as Schwanniomyces
occidentalis; and filamentous fungi including Neurospora, Penicillium, Tolypocladium, or
Aspergillus hosts such as A. nidulans or A. niger.
Suitable host cells for the expression of the antibodies of the invention are derived from
multicellular organisms. Examples of invertebrate cells include plaril and insect cells.
Numerous baculoviral strains and variants and corresponding permissive insect host cells
from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly) and Bombyx mori have been
identified. A variety of viral strains for transfection are publicly available, for example, the L-
1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses may be used, particularly for transfection of Spodoptera frugiperda cells. Plant cell
cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as
hosts.
Host cells for expressing the recombinant antibodies of the invention are preferably
mammalian host cells which include Chinese Hamster Ovary (CHO cells) (including dhfr
CHO cells, described in Urlaub G & Chasin LA (1980) Proc. Natl. Acad. Sci, USA, 77: 4216-
4220, used with a DHFR selectable marker, e.g., as described in Kaufman RJ & Sharp PA
(1982) J. Mol. Biol, 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. In
particular, for use with NSO myeloma cells, another preferred expression system is the GS
gene expression system disclosed in WO 87/04462 (to Wilson), WO 89/01036 (to
Bebbington) and EP338841 (to Bebbington). When recombinant antibody genes are
introduced into mammalian host cells, the antibodies are produced by culturing the host cells
for a period of time sufficient to allow for expression of the antibody in the host cells or, more
preferably, for secretion of the antibody into the culture medium in which the host cells are
grown. Host cells useful for producing antibodies that bind to human OX40 may be cultured
in a variety of media. Commercially available media such as Ham's F10 (Sigma-Aldrich
Chemie GmbH, Buchs, Switzerland), Minimal Essential Medium (MEM; Sigma-Aldrich
Chemie GmbH), RPMI-1640 (Sigma-Aldrich Chemie GmbH, Basel, Switzerland), and
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Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich Chemie GmbH) are suitable
for culturing the host cells. Antibodies can be recovered from the culture medium using
standard protein purification methods.
Antibodies may be operably linked to a fusion partner to enable targeting of the expressed
protein, purification, screening, display, and the like. Fusion partners may be linked to the
antibody sequence via a linker sequences. The linker sequence will generally comprise a small
number of amino acids, typically less than ten, although longer linkers may also be used.
Typically, linker sequences are selected to be flexible and resistant to degradation. As will be
appreciated by those skilled in the art, any of a wide variety of sequences may be used as
linkers. For example, a common linker sequence comprises the amino acid sequence GGGGS.
A fusion partner may be a targeting or signal sequence that directs antibody and any
associated fusion partners to a desired cellular location or to the extracellular media. As is
known in the art, certain signalling sequences may target a protein to be either secreted into
the growth media, or into the periplasmic space, located between the inner and outer
membrane of the cell. A fusion partner may also be a sequence that encodes a peptide or
protein that enables purification and/or screening. Such fusion partners include but are not
limited to polyhistidine tags (His-tags) (for example H6 and H10 or other tags for use with
Immobilized Metal Affinity Chromatography (IMAC) systems (e.g. Ni affinity columns)),
GST fusions, MBP fusions, Strep-tag, the BSP biotinylation target sequence of the bacterial
enzyme BirA, and epitope tags which are targeted by antibodies (for example c-myc tags, flag-
tags, and the like). As will be appreciated by those skilled in the art, such tags may be useful
for purification, for screening, or both.
Construction and Production of Antibodies
Antibodies generated against the OX40 polypeptide may be obtained by immunisation of an
animal i.e. by administering the polypeptides to an animal, preferably a non-human animal,
using well-known and routine protocols, see for example Handbook of Experimental
Immunology (Weir DM (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986).
Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be
immunized. However, mice, rabbits, pigs and rats in particular mice are generally most
suitable. Antibodies can be produced as well by recombinant DNA techniques known to the
skilled person. In additional antibodies can be produced by enzymatic or chemical cleavage of
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naturally occurring antibodies. Humanized antibodies of the present invention may be
constructed by transferring one or more CDRs or portions thereof from VH and/or VL regions
from a non-human animal (e.g., mouse) to one or more framework regions from human VH
and/or VL regions. Optionally, human framework residues thus present in the VH and/or VL
regions may be replaced by corresponding non-human (e.g., mouse) residues when needed or
desired for decreasing immunogenicity of the antibody and/or maintaining binding affinity.
Optionally, non-human amino acid residues present in the CDRs may be replaced with human
residues. Chimeric or humanized antibodies of the present invention can be prepared based on
the sequence of a non-human monoclonal antibody prepared as described above. DNA
encoding the heavy and light chain immunoglobulins can be obtained from the non- human
hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin
sequences using standard molecular biology techniques. For example, to create a chimeric
antibody, murine variable regions can be linked to human constant regions using methods
known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al). To create a humanized
antibody, murine CDR regions can be inserted into a human framework using methods known
in the art (see e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101;
,585,089; 5,693,762 and 6,180,370 to Queen et al).
Humanized antibodies of the present invention may be constructed wherein the human
acceptor molecule for the heavy chain variable region is selected based on homology
considerations between potential acceptor molecule variable regions and the heavy chain
variable region of the murine antibody. Germline candidate human acceptor molecules are
preferred to reduce potential immunogenicity. Germline databases are made up of antibody
sequences that read through the end of the heavy chain FW3 region and partially into the
CDR3 sequence. For selection of a FW4 region, databases of mature antibody sequences
which have been derived from the selected germline molecule can be searched or antibody
sequences which have been derived from the selected germline molecule from a human donor
can be used. Human acceptor molecules are preferably selected from the same heavy chain
class as the murine donor molecule, and of the same canonical structural class of the variable
region of the murine donor molecule. Secondary considerations for selection of the human
acceptor molecule for the heavy chain variable region elude homology in CDR length between
the murine donor molecule and the human acceptor molecule. Human acceptor antibody
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molecules are preferably selected by homology search to the V-BASE database, although
other databases such as the Kabat and the public NCBI databases may be used as well.
Humanized antibodies of the present invention may be constructed wherein the human
acceptor molecule for the light chain variable region is selected based on homology
considerations between potential acceptor molecule variable regions and with the light chain
variable region of the murine antibody. Germline candidate human acceptor molecules are
preferred to reduce potential immunogenicity. Germline databases are made up of antibody
sequences that read through the end of the heavy chain FW3 region and partially into the
CDR3 sequence. For selection of a FW4 region, databases of mature antibody sequences
which have been derived from the selected germline molecule can be searched or antibody
sequences which have been derived from the selected germline molecule from a human donor
can be used. Human acceptor molecules are preferably selected from the same light chain
class as the murine donor molecule, and of the same canonical structural class of the variable
region of the murine donor molecule. Secondary considerations for selection of the human
acceptor molecule for the light chain variable region include homology in CDR length
between the murine donor molecule and the human acceptor molecule. Human acceptor
antibody molecules are preferably selected by homology searches to the V-BASE database,
and other databases such as the Kabat and the public NCBI databases may be used as well.
Methods for humanizing a nonhuman antibody are described herein, including in Example 6,
below.
The present invention provides a method of producing an antagonistic antibody or fragment
thereof that binds to human OX40 comprising culturing a host cell comprising an isolated
nucleic acid encoding the antagonistic antibody or fragment thereof that binds to human OX40
or a vector comprising an isolated nucleic acid encoding the antagonistic antibody or fragment
thereof that binds to human OX40 so that the nucleic acid is expressed and the antibody
produced. Preferably the antibody is isolated. For host cells, nucleic acids and vectors, the
ones described above can be used. Expression of the nucleic acids can be obtained by, e.g. a
combination of recombinant DNA techniques and gene transfection methods as is well known
in the art (e.g., Morrison S (1985) Science 229: 1202) and as further outlined above. For
example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or
full-length light and heavy chains, can be obtained by standard molecular biology techniques
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(e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of
interest) and the DNAs can be inserted into vectors such as expression vectors. The expression
vector and expression control sequences are chosen to be compatible with the expression host
cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted
into separate vector or, more typically, both genes are inserted into the same expression
vector. The antibody genes are inserted into the expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt
end ligation if no restriction sites are present). The light and heavy chain variable regions of
the antibodies described herein can be used to create full-length antibody genes of any
antibody isotype by inserting them into expression vectors already encoding heavy chain
constant and light chain constant regions of the desired isotype such that the VH segment is
operatively linked to the CH1 segment(s) within the vector and the VK segment is operatively
linked to the CK segment within the vector.
Characterization and Purification of Anti-OX40 antibodies
Screening for antibodies can be performed using assays to measure binding to human OX40
and/or assays to measure the ability to block the binding of OX40 to its ligand, OX40L. An
example of a binding assay is an ELISA, in particular, using a fusion protein of human OX40
and human Fc, which is immobilized on plates, and employing a conjugated secondary
antibody to detect anti-OX40 antibody bound to the fusion protein. An example of a blocking
assay is a flow cytometry based assay measuring the blocking of OX40 ligand fusion protein
binding to OX40 on human CD4 cells. A fluorescently labelled secondary antibody is used to
detect the amount of OX40 ligand fusion protein binding to the cell. This assay is looking for
a reduction in signal as the antibody in the supernatant blocks the binding of ligand fusion
protein to OX40. A further example of a blocking assay is an assay where the blocking of
costimulation of naive human T cells mediated by OX40 ligand fusion protein coated to a
plate is measured by measuring thymidine incorporation. As an assay for evaluating the
functional activity of anti-OX40 antibodies e.g. the reduction of T cell activation the human
Mixed Lymphocyte Reaction (MLR) as described in Examples 3 and 8 can be used.
Antibodies of the present invention may be isolated or purified in a variety of ways known to
those skilled in the art. Standard purification methods include chromatographic techniques,
including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and
reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as
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FPLC and HPLC. Purification methods also include electrophoretic, immunological,
precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration
techniques, in conjunction with protein concentration, are also useful. To purify OX40
antibodies, selected host cells can be grown in e.g. spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before affinity chromatography
with protein A-sepharose (Pharmacia, Piscataway, NJ). Eluted antibodies can be checked by
gel electrophoresis and high performance liquid chromatography to ensure purity. A preferred
antibody of the present invention is thus an isolated and/or purified antibody that binds to
human OX40.
Immunoconjugates
In another aspect, the present invention provides an antagonist OX40 antibody or a fragment
thereof that binds to human OX40, linked to a therapeutic agent, such as a cytotoxin, a drug
(e.g., an immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as
"immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to
as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to
(e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents also include, for example,
antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-
fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil,
melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Other
examples of therapeutic cytotoxins that can be linked to an antibody of the invention include
duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An
example of a calicheamicin antibody conjugate is commercially available (Mylotarg(R);
American Home Products). Cytotoxins can be linked to antibodies of the invention using
linker technology available in the art. Examples of linker types that have been used to
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conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers,
esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example,
susceptible to cleavage by low pH within the lysosomal compartment or susceptible to
cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as
cathepsins (e.g., cathepsins B, C, D). For further discussion of types of cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also Saito G et al., (2003) Adv.
Drug Deliv. Rev. 55: 199-215; Trail PA et al., (2003) Cancer Immunol. Immunother. 52: 328-
337; Payne G (2003) Cancer Cell, 3: 207-212; Allen TM (2002) Nat. Rev. Cancer, 2: 750-
763; Pastan I & Kreitman RJ (2002) Curr. Opin. Investig. Drugs, 3: 1089-1091; Senter PD &
Springer CJ, (2001) Adv. Drug Deliv. Rev. 53: 247-264. Antibodies of the present invention
also can be linked to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also
referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be
conjugated to antibodies for use diagnostically or therapeutically include, but are not limited
131 111 90 177
to, iodine , indium , yttrium and lutetium . Methods for preparing
radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are
commercially available, including Zevalin (EDEC Pharmaceuticals) and Bexxar (Corixa
Pharmaceuticals) and similar methods can be used to prepare radioimmunoconjugates using
the antibodies of the invention. The antibody immunoconjugates of the invention can be used
to modify a given biological response, and the drug moiety is not to be construed as limited to
classical chemical therapeutic agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins may include, for example,
an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or,
biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"),
interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Techniques for linking such therapeutic agents to antibodies are well known, see, e.g., Arnon
et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., (eds.), pp. 243- 56 (Alan R. Liss,
Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery
(2nd Ed.), Robinson et al., (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody
Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84:
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Biological And Clinical Applications, Pinchera et al., (eds.), pp. 475-506 (1985); "Analysis,
Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer
Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.,
(eds.), pp. 303-16 (Academic Press 1985), and Thorpe PE & Ross WC (1982) Immunol. Rev.
62: 119-58.
In another aspect, the present invention provides an antagonist OX40 antibody or a fragment
thereof that binds to human OX40, administered together with a therapeutic agent, such as a
cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
Pharmaceutical Compositions
In another aspect, the present invention provides a composition, e.g., a pharmaceutical
composition, comprising the antagonist antibody or fragment thereof, of the present invention,
and a pharmaceutically acceptable carrier. Such compositions may include one or a
combination of (e.g., two or more different) antibodies, and/or immunoconjugates of the
invention and/or a therapeutic agent, such as a cytotoxin, a drug (e.g., an immunosuppressant)
or a radiotoxin as described supra. For example, a pharmaceutical composition of the
invention can comprise a combination of antibodies (or immunoconjugates) that bind to
different epitopes on the target antigen or that have complementary activities. Pharmaceutical
compositions of the invention also can be administered in combination therapy, i.e., combined
with other agents. For example, the combination therapy can include an antagonist OX40
antibody of the present invention combined with at least one other anti-inflammatory or
immunosuppressant agent.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like that are physiologically compatible. Preferably, the carrier is suitable for
intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of administration, the active compound, i.e.,
antibody or immunoconjugate, may be coated in a material to protect the compound from the
action of acids and other natural conditions that may inactivate the compound.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable solutions or
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dispersion. The use of such media and agents for pharmaceutically active substances is known
in the art. Except insofar as any conventional media or agent is incompatible with the active
compound, use thereof in the pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the compositions.
In another aspect, the present invention provides a composition comprising an
immunoconjugate comprising the antagonist antibody or fragment thereof that binds to human
OX40 linked to a therapeutic agent and a pharmaceutically acceptable carrier.
Immunoconjugates and therapeutic agents which can be used are as described supra.
In another aspect, the present invention provides a composition comprising the antagonist
antibody or fragment thereof of the present invention which further comprises another
pharmaceutically active agent. Preferably the another pharmaceutically active agent is one or
more of: a) another antagonist to human OX40, b) an analgesic agent and c) an immune
suppressive agent e.g. a glucocorticoid such as prednisone.
A pharmaceutical composition of the invention may also include a pharmaceutically
acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1)
water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate,
sodium metabisulfite, sodium sulfite and the like; (2) oil- soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as
citric acid, ethylenediamine tetraacetic-acid (EDTA), sorbitol, tartaric acid, phosphoric acid,
and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in
the pharmaceutical compositions of the invention include water, ethanol, polyols (such as
glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper
fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of dispersions, and by the use of
surfactants. These compositions may also contain adjuvants such as preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms
may be ensured both by sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid,
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and the like. It may also be desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
Therapeutic and other uses
The antagonist antibodies of the present invention have numerous in vitro and in vivo
diagnostic and therapeutic utilities involving the diagnosis and treatment of OX40 mediated
disorders. For example, these molecules can be administered to cells in culture, in vitro or ex
vivo, or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of OX40-
mediated disorders. Preferred subjects are human and include patients having disorders
mediated by OX40 activity (OX40 mediated disorders). The antagonist antibodies of the
present invention can be effective in treating patients independent of their OX40
costimulatory status. More preferred subjects are human and include patients expressing a
low level of OX40.
A "patient" for the purposes of the present invention includes both humans and other animals,
preferably mammals and most preferably humans. Thus the antibodies of the present invention
have both human therapy and veterinary applications. The term "treatment" or "treating" in the
present invention is meant to include therapeutic treatment, as well as prophylactic, or
suppressive measures for a disease or disorder. Thus, for example, successful administration
of an antibody prior to onset of the disease results in treatment of the disease. As another
example, successful administration of an antibody after clinical manifestation of the disease to
combat the symptoms of the disease comprises treatment of the disease. "Treatment" and
"treating" also encompasses administration of an antibody after the appearance of the disease
in order to eradicate the disease. Successful administration of an antibody after onset and after
clinical symptoms have developed, with possible abatement of clinical symptoms and perhaps
amelioration of the disease, comprises treatment of the disease. Those "in need of treatment"
include mammals already having the disease or disorder, as well as those prone to having the
disease or disorder, including those in which the disease or disorder is to be prevented.
In a particular embodiment, the antagonist antibodies are used in vivo to treat, prevent or
diagnose a variety of OX40-mediated disorders. Thus the invention provides a method for
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treating an OX40 mediated disorder in a subject, the method comprising administering to the
subject a therapeutically effective amount of the antagonist antibody or fragment thereof.
Exemplary OX40 mediated disorders include infections (viral, bacterial, fungal and parasitic),
endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, chronic
obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's Disease,
inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coeliac
disease, gall bladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical
adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune
uveitis, immune mediated inflammatory disorders of the central and peripheral nervous
system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-
Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease,
IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus,
primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, pancreatitis,
trauma (surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular
disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis,
intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis,
hypochlorhydia and neuromyelitis optica.
Other exemplary OX40 mediated disorder include infections (viral, bacterial, fungal and
parasitic), endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma,
bronchitis, influenza, respiratory syncytial virus, pneumonia, chronic obstructive pulmonary
disease (COPD), idiopathic pulmonary fibrosis (IPF), cryptogenic fibrosing alveolitis (CFA),
idiopathic fibrosing interstitial pneumonia, emphysema, pelvic inflammatory disease,
Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis,
Peyronie's Disease, coeliac disease, gallbladder disease, Pilonidal disease, peritonitis,
psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis,
meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the
central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic
lupus erythematosus) and Guillain-Barr syndrome, Atopic dermatitis, autoimmune hepatitis,
fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura,
Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's
granulomatosis, pancreatitis, trauma (surgery), graft-versus-host disease (GVHD), transplant
rejection, cardiovascular disease including ischaemic diseases such as myocardial infarction as
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well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis,
periodontitis, hypochlorhydia and neuromyelitis optica.
Preferred OX40 mediated disorders to be treated with the antibody of the invention are
selected from the group consisting of multiple sclerosis, rheumatoid arthritis, colitis, psoriasis,
asthma, COPD, IPF, graft-versus-host-disease (GVHD), atherosclerosis and diabetes. A
particular preferred OX40 mediated disorders to be treated with the antibody of the invention
is graft-versus-host-disease (GVHD).
The present invention also provides an antibody for use in the treatment of pain, particularly
pain associated with inflammation.
In one embodiment, the antibodies of the invention can be used to detect levels of OX40, or
levels of cells which contain OX40 on their membrane surface, which levels can then be
linked to certain disease symptoms. Alternatively, the antibodies can be used to inhibit or
block OX40 function which, in turn, can be linked to the prevention or amelioration of certain
disease symptoms, thereby implicating OX40 as a mediator of the disease. This can be
achieved by contacting a sample and a control sample with the OX40 antibody under
conditions that allow for the formation of a complex between the antibody and OX40. Any
complexes formed between the antibody and OX40 are detected and compared in the sample
and the control. In light of the specific binding of the antibodies of the invention for OX40,
the antibodies of the invention can be used to specifically detect OX40 expression on the
surface of cells e.g. can be used to detect a patient having low expression level of OX40. The
antibodies of the invention can also be used to purify OX40 via immunoaffinity purification.
Thus the present invention also provides an in vitro screening method to detect a patient
having a low expression level of OX40, comprising the steps of:
(a) purifying peripheral blood mononuclear cells (PBMCs) from a patient blood sample;
(b) subjecting the PBMCs to flow cytometric analysis; and
(c) determining the number of OX40 positive cells in CD4 and/or CD8 T cells and
comparing this number to control levels.
In a preferred embodiment, a low expression level of OX40 is indicated by an increase in the
expression level of OX40 positive cells when compared to control levels of up to 10%, more
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preferably of up to 20% and even more preferably of up to 30%. The approach of determining
OX40 expression is further described in detail in Kotani A et al., (2001) Blood, 98: 3162-4
and Xiaoyan Z et al., (2005) Clin. Exp. Immunol. 143: 110-6.
In another embodiment, the antibodies of the invention can be initially tested for binding
activity associated with therapeutic or diagnostic use in vitro. For example, compositions of
the invention can be tested using flow cytometric assays.
The present disclosure further provides the use of an antagonist antibody or fragment thereof
as a medicament and the use of an antagonist antibody or fragment thereof in the preparation
of a medicament for the treatment of an OX40 mediated disorder. In a further embodiment the
present disclosure provides the antagonist antibody or fragment thereof for use as a
medicament. Also provided by the present disclosure is the antagonist antibody or fragment
thereof for use in a method for treating an OX40 mediated disorder. OX40 mediated disorders
are the ones as described supra. The antagonist antibody of the present invention may be
particularly useful for treating OX40 mediated disorders independent of the OX40
costimulatory status of a patient. In a preferred embodiment, the antagonist antibody or
fragment thereof can be used for treating an OX40 mediated disorder wherein a patient
expresses a low level of OX40.
As previously described, antagonist OX40 antibodies of the invention can be co- administered
with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an
immunosuppressive agent. The antibody can be linked to the agent (as an immunoconjugate as
described supra) or can be administered separate from the agent. In the latter case (separate
administration), the antibody can be administered before, after or concurrently with the agent
or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g.,
radiation.
For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and
more usually 0.01 to 10 mg/kg, of the host body weight. An exemplary treatment regime
entails administration once per week, once every two weeks, once every three weeks, once
every four weeks, once a month, once every 3 months or once every three to 6 months. The
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antibody is usually administered on multiple occasions. Intervals between single dosages can
be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular
as indicated by measuring blood levels of antibody to the target antigen in the patient. In some
methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000
µg/ml and in some methods about 25-300 µg/ml. Alternatively the antibody can be
administered as a sustained release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of the antibody in the patient.
The dosage and frequency of administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is
administered at relatively infrequent intervals over a long period of time. Some patients
continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively
high dosage at relatively short intervals is sometimes required until progression of the disease
is reduced or terminated.
Actual dosage levels of the active ingredients, i.e. the antibody in the pharmaceutical
compositions of the present invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention employed, the route of
administration, the time of administration, the rate of excretion of the particular antibody
being employed, the duration of the treatment, other drugs, compounds and/or materials used
in combination with the particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being treated, and like factors well
known in the medical arts.
A "therapeutically effective amount" of an OX40 antibody of the invention preferably results
in a decrease in severity of disease symptoms, an increase in frequency and duration of disease
symptom-free periods, and/or a prevention of impairment or disability due to the disease
affliction. The ability of a compound for the treatment of an OX40 mediated disorder can be
evaluated in an animal model system predictive of efficacy in human. Alternatively, this
property of a composition can be evaluated by examining the ability of the compound to
inhibit cell growth, such inhibition can be measured in vitro by assays known to the skilled
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practitioner. One of ordinary skill in the art would be able to determine such amounts based
on such factors as the subject's size, the severity of the subject's symptoms, and the particular
composition or route of administration selected.
The antibody or the composition of the present invention can be administered via one or more
routes of administration using one or more of a variety of methods known in the art. As will
be appreciated by the skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of administration include intravenous,
intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of
administration, for example by injection or infusion. More preferred routes of administration
are intravenous or subcutaneous. The phrase "parenteral administration" as used herein means
modes of administration other than enteral and topical administration, usually by injection,
and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and
intrasternal injection and infusion. Alternatively, an antibody of the invention can be
administered via a non- parenteral route, such as a topical, epidermal or mucosal route of
administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
Article of manufacture and kit
In another embodiment of the disclosure, an article of manufacture comprising the antagonist
antibody or fragment thereof, the composition or the immunoconjugate of the invention for
the treatment of a OX40 mediated disorder is provided. The article of manufacture may
comprise a container and a label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials or syringes. The containers may be
formed from a variety of materials such as glass or plastic. The container holds a composition
that may be effective for treating the condition and may have a sterile access port (e.g., the
container may be an intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the composition may be the
antagonist antibody described herein. The label or package insert may indicate that the
composition may be used for treating the condition of choice, such as cancer. In one
embodiment, the label or package insert may indicate that the composition comprising the
antagonist antibody may be used to treat an OX40-mediated disorder.
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Moreover, the article of manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises the antagonist antibody herein, and (b)
a second container with a composition contained therein, wherein the composition comprises
a therapeutic agent other than the antagonist antibody. The article of manufacture in this
embodiment of the disclosure may further comprise a package insert indicating that the first
and second compositions can be used in combination to treat a OX40 mediated disease or
disorder. Such therapeutic agent may be any of the adjunct therapies described in the
preceding section (e.g., a thrombolytic agent, an anti-platelet agent, a chemotherapeutic agent,
an anti-angiogenic agent, an anti-hormonal compound, a cardioprotectant, and/or a regulator
of immune function in a mammal, including a cytokine). Alternatively, or additionally, the
article of manufacture may further comprise a second (or third) container comprising a
pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI),
phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include
other materials desirable from a commercial and user standpoint, including other buffers,
diluents, filters, needles, and syringes.
Also within the scope of the present invention are kits comprising the antibody, the
compositions or the immunoconjugates of the invention and instructions for use. The kit can
further contain one more additional reagents, such as an immunosuppressive reagent, a
cytotoxic agent or a radiotoxic agent, or one or more additional antagonist antibodies of the
invention (e.g., an antagonist antibody having a complementary activity which binds to an
epitope in the OX40 antigen distinct from the first antagonist antibody).
Without further description, it is believed that one of ordinary skill in the art may, using the
preceding description and the following illustrative examples, make and utilize the agents of
the present disclosure and practice the claimed methods. The following working examples are
provided to facilitate the practice of the present disclosure, and are not to be construed as
limiting in any way the remainder of the disclosure.
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Examples
Example 1:
Generation and screening of mouse anti-human OX40 antibodies
To produce the recombinant human OX40-Fc protein, a cDNA for the human TNFRSF4 was
purchased from imaGenes (clone number: RZPDB737H0329D; Berlin, Germany). This
cDNA was used as a template to PCR-amplify the DNA coding region of the human
TNFRSF4 extracellular domain (SEQ ID NO: 11). In a separate PCR reaction, the Fc region
of a human IgG1 (EU positions 223-451) was amplified by PCR adding a 5’ GSGGG linker
and a 3’ SA-6xHis linker and restriction sites for cloning. The two resulting products were
then fused using overlap extension PCR with flanking primers, adding restriction sites for
subsequent cloning into a modified mammalian expression vector based on the pcDNA3.1(-)
plasmid from Invitrogen (Invitrogen AG, Basel, Switzerland, Cat. No. V795-20), containing
the human CMV promoter with the Ig donor acceptor fragment (first intron) described in US
Patent 5924939, the OriP sequence (Koons MD et al., (2001) J Virol. 75(22): 10582-92), the
SV40 enhancer, and the SV40 polyA fused to the gastrin terminator as described by Kim D, et
al., (2003) Biotechnol. Prog. 19(5): 1620-2. This recombinant plasmid allowed for expression
of the human TNFRSF4 extracellular domain – Fc fusion protein in mammalian cells with
secretion into the cell culture medium driven by the native signal peptide of the human
TNFRSF4 protein. For recombinant protein production, the aforementioned recombinant
vector was transfected into suspension-adapted HEK 293 cells (ATCC number CRL 1573)
using jetPEI transfection reagent (Polyplus-transfection S.A., Strasbourg, France;
distributor: Brunschwig, Basel, Switzerland). The cell culture supernatant was collected after
five days and further purified using a Protein A affinity purification column (HiTrap Protein A
sepharose column; GE Healthcare Europe GmbH, Glattbrugg, Switzerland) operated on an
ÄKTA FPLC system (GE Healthcare Europe GmbH, Glattbrugg, Switzerland).
To produce the recombinant human OX40-his protein, the extracellular region of human
TNFRSF4 (SEQ ID NO: 11) was amplified by PCR adding a 3’ GSG-6xHis linker and
restriction sites for cloning. The PCR product was subsequently cloned in the modified
pcDNA3.1(-) plasmid described above. This recombinant plasmid allowed for the expression
of the human OX40-his protein in mammalian cells with secretion into the cell culture media
driven by the native signal peptide of the human TNFRSF4. For protein production, the
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recombinant vector was transfected into suspension-adapted HEK 293 cells (ATCC number
CRL 1573) using jetPEI transfection reagent (Polyplus-transfection S.A., Strasbourg,
France; distributor: Brunschwig, Basel, Switzerland). The cell culture supernatant was
collected five days after transfection and purified using a Ni -NTA affinity purification
column (HiTrap Ni -NTA sepharose column; GE Healthcare Europe GmbH, Glattbrugg,
Switzerland) operated on an ÄKTA FPLC system (GE Healthcare Europe GmbH, Glattbrugg,
Switzerland). Recombinant human OX40-Fc and OX40-his proteins were found to be 95%
pure as judged by SDS-PAGE, and further buffered exchanged into phosphate buffer saline
(PBS) prior use.
Recombinant human OX40-Fc protein dissolved in PBS was mixed with an equal volume of
Stimune adjuvant (Prionics, Switzerland, ref: 7925000) and an emulsion was prepared. The
emulsion was transferred to 0.5 mL insulin syringes (BD Pharmingen, Allschwil, Switzerland)
and BALB/c animals (Harlan, Netherlands) were immunized sub-cutaneously in the back
footpads, the base of the tail and the neck with 50 µg of the emulsified protein. The
immunization was repeated two weeks later with the same amount of antigen and the same
route of injection.
The presence of circulating anti-human OX40 antibodies in the immunized mouse sera was
evaluated by direct ELISA using plates coated with the recombinant human OX40-his protein.
A serial dilution (from 1:10 to 1:10 ) of the different mouse sera was added to the plates and
the bound antibodies were detected using a goat anti-mouse H+L whole molecule-HRP
(Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). A final sub-cutaneous boost with 50 µg
of antigen without adjuvant was performed in animals displaying the best anti-human OX40
IgG serum titer three days before sacrifice.
Animals were euthanized and the inguinal, axillary, brachial, popliteal and sciatic lymph
nodes were collected to prepare a single cell suspension by disturbing the lymph node
architecture with two 25G needles in a DNAse (Roche Diagnostics (Schweiz) AG, Rotkreuz,
Switzerland) and collagenase (Roche Diagnostics (Schweiz) AG, Rotkreuz, Switzerland)
solution. Single cell suspensions were fused to a myeloma cell line X63AG8.653 (mouse
BALB/c myeloma cell line; ATCC accession number: CRL 1580; Kearney JF et al., (1979) J.
Immunol. 123(4): 1548-1550) at a ratio of 7:1 (fusion partner-to-harvested lymph node cells)
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with polyethylene glycol 1500 (Roche Diagnostics (Schweiz) AG, Rotkreuz, Switzerland).
The fused cells were plated into 96 well flat bottom plates containing mouse macrophages in
DMEM-10 medium (Invitrogen AG, Basel, Switzerland) supplemented with 10% fetal bovine
serum (FBS, PAA Laboratories, Pasching, Austria), 2mM L-glutamine, 100U/ml (Biochrom
AG, Germany) penicillin, 100 µg/ml streptomycin (Biochrom AG, Germany), 10mM HEPES
(Invitrogen AG, Basel, Switzerland), 50 µM β-mercaptoethanol (Sigma-Aldrich Chemie
GmbH, Buchs, Switzerland), HAT (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and
1% Growth factor (Hybridokine, Interchim/Uptima, Montluçon, France).
Approximately 800 wells from the fusions were screened by ELISA for the presence of mouse
IgG that recognized human OX40 and blocked the binding of human OX40L on its receptor.
Positive wells were expanded and subjected to two rounds of subcloning. Cells were collected
and the heavy and light chains were cloned and sequenced.
Example 2:
Cloning and sequencing of the VH and VL chains of the anti-OX40 antibodies from
hybridoma cells
For each positively selected hybridoma, total RNA was prepared, reverse-transcribed into
cDNA and VH and VL genes were respectively amplified by PCR. These PCR products were
ligated into a rescue-vector (pDrive vector; QIAGEN AG, Hombrechtikon, Switzerland; Cat.
No. 231124), allowing for the DNA sequencing of individual PCR products and the
determination of mono- or poly-clonality of the selected hybridomas. This vector allowed for
blue/white selection on LB-agar plates containing IPTG and X-gal (colonies with no insert
were blue because of the degradation of X-gal by the LacZ α -peptide). Recombinant plasmids
from positive (white) bacterial clones were prepared and sequenced using standard DNA
sequencing primers specific for the vector backbone (M13rev, M13fwd, T7 or SP6). DNA
sequences were finally subcloned into an expression vector for recombinant expression of the
antibody of interest in mammalian cells.
RNA isolation
Total RNA was isolated from 2-10x10 cells using the RNeasy Mini Kit from QIAGEN
(QIAGEN AG, Hombrechtikon, Switzerland; Cat. No. 74106) according to the manufacturer’s
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protocol; samples were quantified using a NanoDrop ND-1000 spectrophotometer (WITEC
AG, Littau, Switzerland).
One step RT-PCR
The total RNA preparations described above were further reverse-transcribed into cDNA, and
the VH and VL fragments were amplified by PCR using two different mixtures of degenerated
primers, each one allowing the recovery of all the different subfamilies of mouse
immunoglobulin heavy chain variable fragments and variable heavy chain junction regions or
the recovery of all mouse immunoglobulin light chain kappa variable fragments and variable
light chain kappa junction regions. The primers used for reverse transcription and
amplification were synthesized by Microsynth (Balgach, Switzerland), and were HPLC
purified (Tables 1-4). Both reverse-transcription and PCR amplification were performed
simultaneously using the QIAGEN one step RT-PCR kit (QIAGEN AG, Hombrechtikon,
Switzerland; Cat. No. 210212). Since the technique used specific primers, each mRNA
sample was then treated in duplicate allowing for the individual reverse-transcription and
amplification of either the VH or the VL fragments. 2µg of total RNA dissolved into RNase-
free water to a final volume of 30µl were mixed with: 10µl of a 5x stock solution of QIAGEN
OneStep RT-PCR Buffer, 2µl of a dNTPs mix at a concentration of 10mM, 3µl of primer mix
at a concentration of 10µM and 2µl of QIAGEN OneStep RT-PCR Enzyme Mix. The final
mixture was then placed in a PCR tube, and cycled in a PCR-themocycler (BioRad iCycler
version 4.006, Bio-Rad Laboratories AG, Reinach, Switzerland) using the following settings:
min at 50 °C
min at 95 °C
40 cycles: 30 sec at 94 °C
sec at 55 °C
1 min at 72 °C
10 min at 72 °C
Hold at 4 °C
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pDrive cloning
PCR products were run onto 2% agarose gels. Following DNA electrophoresis, the fragments
of interest (~450bp) were excised from the agarose gels, and further extracted using the
Macherey-Nagel NucloSpin Extract II kit 250 (Macherey-Nagel,Oensingen, Switzerland; Cat.
No. 740609.250). For DNA sequencing, the extracted PCR products were cloned into the
rescue-vector described above (pDrive vector, QIAGEN AG, Hombrechtikon, Switzerland;
Cat. No. 231124) and transformed into the E. coli TOP10 strain (Invitrogen AG, Basel,
Switzerland; Cat. No. C404006)
Miniprep extraction
Positive colonies were cultured overnight at 37 C (shaking 250 RPM) in 1.5ml of Luria
Bertani (LB) medium supplemented with 100µg/ml ampicillin seeded in Macherey-Nagel
Square-well Block plates (Macherey-Nagel, Oensingen, Switzerland; Cat. No. 740488.24).
The next day DNA miniprep extractions were performed using the NucleoSpin Multi-8
Plasmid kit (Macherey-Nagel, Oensingen, Switzerland; Cat. No. 740620.5).
Sequencing and Sequence analysis
Samples were sent for DNA sequencing to the DNA sequencing service company Fasteris
(Plan-les-Ouates, Switzerland). The standard primers: M13rev, M13fwd, T7, SP6 were used
(Table 5). To analyse the DNA sequences, the Clone Manager 9 Professional Edition
(Scientific & Educational Software, NC, USA) and the BioEdit Sequence Alignment Editor
(Hall, TA (1999) Nucl. Acids. Symp. Ser. 41: 95-98) were used.
Cloning of expression vector for recombinant chimeric antibody expression
For recombinant expression in mammalian cells, the isolated murine VH and VL fragments
were formatted as chimeric immunoglobulins using assembly-based PCR methods. These
chimeric antibodies consist of a heavy chain where the murine heavy chain variable domain is
fused to the human IgG1 heavy chain constant domains (γ1, hinge, γ2, and γ3 regions) and a
light chain where the murine light chain variable domain is fused to a human kappa constant
domain (Cκ). PCR- assembled murine variable and human constant parts were subsequently
cloned into a modified mammalian expression vector based on the modified pcDNA3.1(-)
vector from Invitrogen mentioned in Example 1 with the difference that a human
immunoglobulin light chain kappa leader peptide was employed to drive protein secretion. For
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protein production of the immunoglobulin candidates, equal quantities of heavy and light
chain vector DNA were co-transfected into suspension-adapted HEK-293 (ATCC number:
CRL-1573). The cell culture supernatant was collected after five days and purified using a
Protein A affinity purification column (HiTrap Protein A sepharose column) operated on an
ÄKTA FPLC system (both from GE Healthcare Europe GmbH, Glattbrugg, Switzerland).
Table 1: primer Mix VH - back
GTGATC GCC ATG GCG TCG ACC GAK GTR MAG CTT CAG GAG TC
GTGATC GCC ATG GCG TCG ACC GAG GTB CAG CTB CAG CAG TC
GTGATC GCC ATG GCG TCG ACC CAG GTG CAG CTG AAG SAR TC
GTGATC GCC ATG GCG TCG ACC GAG GTC CAR CTG CAA CAR TC
GTGATC GCC ATG GCG TCG ACC CAG GTY CAG CTB CAG CAR TC
GTGATC GCC ATG GCG TCG ACC CAG GTY CAR CTG CAG CAR TC
GTGATC GCC ATG GCG TCG ACC CAG GTC CAC GTG AAG CAR TC
GTGATC GCC ATG GCG TCG ACC GAG GTG AAS STG GTG GAR TC
GTGATC GCC ATG GCG TCG ACC GAV GTG AWG STG GTG GAG TC
GTGATC GCC ATG GCG TCG ACC GAG GTG CAG STG GTG GAR TC
GTGATC GCC ATG GCG TCG ACC GAK GTG CAM CTG GTG GAR TC
GTGATC GCC ATG GCG TCG ACC GAG GTG AAG CTG ATG GAR TC
GTGATC GCC ATG GCG TCG ACC GAG GTG CAR CTT GTT GAR TC
GTGATC GCC ATG GCG TCG ACC GAR GTR AAG CTT CTC GAR TC
GTGATC GCC ATG GCG TCG ACC GAA GTG AAR STT GAG GAR TC
GTGATC GCC ATG GCG TCG ACC CAG GTT ACT CTR AAA SAR TC
GTGATC GCC ATG GCG TCG ACC CAG GTC CAA CTV CAG CAR CC
GTGATC GCC ATG GCG TCG ACC GAT GTG AAC TTG GAA SAR TC
GTGATC GCC ATG GCG TCG ACC GAG GTG AAG GTC ATC GAR TC
Table 2: primer Mix VH - forward
CCTCCACCACTCGAGCC CGA GGA AAC GGT GAC CGT GGT
CCTCCACCACTCGAGCC CGA GGA GAC TGT GAG AGT GGT
CCTCCACCACTCGAGCC CGC AGA GAC AGT GAC CAG AGT
CCTCCACCACTCGAGCC CGA GGA GAC GGT GAC TGA GGT
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Table 3: primer Mix VL - back
GGCGGTGGC GCT AGC GAY ATC CAG CTG ACT CAG CC
GGCGGTGGC GCT AGC CAA ATT GTT CTC ACC CAG TC
GGCGGTGGCGCT AGC GAY ATT GTG MTM ACT CAG TC
GGCGGTGGC GCT AGC GAY ATT GTG YTR ACA CAG TC
GGCGGTGGC GCT AGC GAY ATT GTR ATG ACM CAG TC
GGCGGTGGC GCT AGC GAY ATT MAG ATR AMC CAG TC
GGCGGTGGC GCT AGC GAY ATT CAG ATG AYD CAG TC
GGCGGTGGCGCT AGC GAY ATY CAG ATG ACA CAG AC
GGCGGTGGC GCT AGC GAY ATT GTT CTC AWC CAG TC
GGCGGTGGCGCT AGC GAY ATT GWG CTS ACC CAA TC
GGCGGTGGC GCT AGC GAY ATT STR ATG ACC CAR TC
GGCGGTGGC GCT AGC GAY RTT KTG ATG ACC CAR AC
GGCGGTGGCGCT AGC GAY ATT GTG ATG ACB CAG KC
GGCGGTGGC GCT AGC GAY ATT GTG ATA ACY CAG GA
GGCGGTGGC GCT AGC GAY ATT GTG ATG ACC CAG WT
GGCGGTGGC GCT AGC GAY ATT GTG ATG ACA CAA CC
GGCGGTGGCGCT AGC GAY ATT TTG CTG ACT CAG TC
GGCGGTGGC GCT AGC GAA ACA ACT GTG ACC CAG TC
GGCGGTGGCGCT AGC GAA AAT GTK CTS ACC CAG TC
GGCGGTGGCGCT AGC CAG GCT GTT GTG ACT CAG GAA TC
Table 4: primer Mix VL - forward
ATGCTGAC GC GGC CGC ACG TTT KAT TTC CAG CTT GG
ATGCTGAC GC GGC CGC ACG TTT TAT TTC CAA CTT TG
ATGCTGAC GC GGC CGC ACG TTT CAG CTC CAG CTT GG
ATGCTGAC GC GGC CGC ACC TAG GAC AGT CAG TTT GG
Table 5: sequencing primers
M13-Fwd GTAAAACGACGGCCAGT
M13-Rev AACAGCTATGACCATG
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T7 TAATACGACTCACTATAGG
SP6 GATTTAGGTGACACTATAG
Example 3:
Biological characterization of anti-human OX40 antibodies
OX40-specific Antibody Detection ELISA:
Antibody titers, specificity and production by hybridomas and recombinant antibody
candidates were determined by a direct ELISA. Briefly, 96 well-microtiter plates (Costar
USA, distributor VWR AG, Nyon, Switzerland) were coated with 100 µl of recombinant
human OX40-his at 2 µg/ml in PBS (see Example 1 for the generation of the OX40-his
protein). Plates were incubated overnight at 4°C and were then blocked with PBS 2% BSA
(Bovine Serum Albumine, PAA Laboratories, Pasching, Austria) at room temperature (RT)
for one hour. The blocking solution was removed and the hybridoma supernatants or purified
antibodies were added. The plates were incubated at RT for 30 minutes, then washed nine
times with PBS 0.01% Tween-20 (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and a
Horseradish Peroxidase (HRP) labelled-Goat anti-mouse H+L-detection antibody (Sigma-
Aldrich Chemie GmbH, Buchs, Switzerland) was added at a dilution of 1:1000. To detect
recombinant chimeric antibodies (see Example 2) that possess a human Fc, a HRP-labeled
rabbit anti human IgG antibody (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) at a
dilution of 1:1000 was used as the detection antibody. Plates were incubated for 30 minutes at
room temperature (RT), washed nine times with PBS 0.01% Tween-20 and the TMB substrate
(Bio-rad Laboratories AG, Reinach, Switzerland) was added to the plates and the reaction
stopped after six minutes by adding H SO . Absorbance was then read at 450 nm by a
microplate reader (Biotek, USA; distributor: WITTEC AG, Littau, Switzerland). Figure 1A
shows that the chimeric 1D4 antibody and the chimeric 2F8 antibody recognize the OX40-his
coated protein.
OX40L blocking ELISA:
The recombinant human OX40 ligand protein (OX40L) was generated as followed: the cDNA
for human TNFSF4 (clone name: IOH46203) was purchased from imaGenes (Berlin,
Germany) and the extracellular portion (amino acids 51-183) of human TNFSF4 ligand
(numbering according to the Uniprot Q6FGS4 sequence) was amplified with flanking
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restriction sites. The resulting PCR product encompassing an ASA linker and a 8-His tag
sequence at its 5’ end was subsequently cloned into a modified version of the pREP4 vector
from Invitrogen (Invitrogen AG, Basel, Switzerland) carrying a CMV promoter, a Bovine
Growth Hormone poly-adenylation, and the murine VJ2C leader peptide to drive the secretion
of the recombinant protein. For recombinant protein production, the reconbinant vector was
transfected into suspension-adapted HEK 293 cells (ATCC number CRL 1573) using
jetPEI transfection reagent (Polyplus-transfection S.A., Strasbourg, France; distributor:
Brunschwig, Basel, Switzerland). Cell culture supernatant was collected after five days and
purified using a Protein A affinity purification column (HiTrap Protein A sepharose column;
GE Healthcare Europe GmbH, Glattbrugg, Switzerland) operated on an ÄKTA FPLC system
(GE Healthcare Europe GmbH, Glattbrugg, Switzerland).
In order to determine if the generated anti-OX40 antibodies can block the binding of OX40L
to the OX40 receptor, a blocking ELISA was developed. Ninety-six well-microtiter plates
(Costar, USA; distributor VWR AG, Nyon, Switzerland) were coated with 100 µl of
recombinant human OX40-Fc (see Example 1) at 2 µg/ml in PBS. Plates were incubated
overnight at 4°C and were then blocked with PBS 2% BSA at RT for one hour. The blocking
solution was removed and the hybridoma supernatants or purified antibodies were added to
the plate. Five minutes later, 50 µl of biotinylated-recombinant human OX40L at 0.04 mg/ml
was added to each well. Plates were incubated at RT for 60 minutes, then washed nine times
with PBS 0.01% Tween-20 and HRP-streptavidin (Sigma-Aldrich Chemie GmbH, Buchs,
Switzerland) was added at a dilution of 1:2000. Plates were incubated for 30 minutes at RT,
washed 9 times with PBS 0.01% Tween-20 and the TMB substrate (Bio-rad Laboratories AG,
Reinach, Switzerland) was added to the plates and the reaction stopped after 6 minutes by
adding H SO . Absorbance was then read at 450 nm by a microplate reader (Biotek, USA;
distributor: WITTEC AG, Littau, Switzerland). Figure 1B shows that the chimeric 1D4
antibody is able to block the interaction between OX40 and OX40L in a dose dependent
manner, whereas the chimeric 2F8 antibody is not able to block the interaction between OX40
and OX40L.
Human Mixed Lymphocyte Reaction (MLR)
Blood from two different donors was collected in three 10 mL S-Monovette with citrate as an
anti-coagulant (Sarstedt, Nümbrecht, Germany). Cells from donor No. 1 were used as effector
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cells whereas cells from donor No. 2 were used as target cells. PBMCs (peripheral blood
mononuclear cells) from the 2 donors were purified using 50 mL Blood-Sep-Filter Tubes
(distributor: Brunschwig, Basel, Switzerland) following the manufacturer’s instructions. Cells
were washed 2 times with Roswell Park Memorial Institute (RPMI, PAA Laboratories,
Pasching, Austria) medium without FBS. The target cells were incubated with 50 µg/ml of
mitomycin C (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) for 30 minutes at 37°C.
Cells were then washed 3 times with RPMI without FBS and resuspended at 1x10 cell/mL in
RPMI, 10% FBS (PAA Laboratories, Pasching, Austria), 2mM L-glutamine (Lonza, Leuven,
Belgium), 100U/ml penicillin, 100 µg/ml streptomycin (Biochrom AG, Berlin, Germany). In
96 well U bottom micro-plates (TPP, Trasadingen, Switzerland), 50’000 target cells and
80’000 effector cells were distributed in a final volume of 100 µl to each well. One hundred
µl of antibody dilutions was added to the wells. Plates were incubated for 7 days at 37°C in
% CO incubator. Seven days after the start of the MLR, cells were pulsed with 0.5 µCi of
H thymidine (Perkin Elmer). 18 hours after pulsing, cells were harvested and incorporated
radioactivity was quantified on a Wallac beta counter. Figure 2 shows that the chimeric 1D4
antibody is able to block the MLR in a dose dependent manner to a higher degree than the
positive control.
Example 4:
Binding of anti-human OX40 antibodies on human and other animal species activated
peripheral blood mononuclear cells (PBMC) by flow cytometry
Human cells
Filters containing human leukocytes were collected from the Blood Collection Center from La
Chaux-de-Fonds, Switzerland (Centre de Transfusion Sanguine et Laboratoire de Sérologie,
rue Sophie-Mairet 29, CH-2300). Cells were removed from the filters by backflushing with 60
mL of PBS containing 10U/mL of liquemin (Drossapharm AG, Lucern, Switzerland). PBMCs
were then purified with 50mL Blood-Sep-Filter Tubes (distributor: Brunschwig, Basel,
Switzerland) following manufacturer’s instructions. Cells were washed 3 times with Roswell
Park Memorial Institute (RPMI, PAA Laboratories, Pasching, Austria) medium with FBS
(PAA Laboratories, Pasching, Austria). Cells were resuspended at 3x10 cells/ml in RPMI,
% FBS (PAA Laboratories, Pasching, Austria ), 2mM Ultraglutamine (Lonza, Leuven,
Belgium), 100U/ml penicillin, 100 µg/ml streptomycin (Biochrom AG, Berlin, Germany), 10
µg/ml of Phytohemagglutinin (PHA; Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) +
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100 U/mL of rHu IL-2 (Proleukin, Novartis, Basel, Switzerland) in a 24 well plate (TPP,
Trasadingen, Switzerland). Forty-eight hours later, cells were collected and analyzed by flow
cytometry as described below.
HPB-ALL cells (T acute lymphoid leukemia cell line, from Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were cultured in RPMI,
% FBS. 2x10 cells were distributed in a 96 well V bottom plate (TPP, Trasadingen,
Switzerland), and centrifuged for three minutes at 1300 rpm; supernatants were discarded,
cells were collected and analyzed by flow cytometry as described below.
PBMCs and HPB-ALL cells prepared as described above were resuspended in 50 µl of FACS
buffer (PBS, 2% FBS, 10% Versene (Invitrogen, USA) with 5µg/mL of chimeric 1D4
antibody or 5µg/mL or an appropriate isotype control or 20 µl of a PE-labelled commercial
anti-human OX40 antibody (clone L106, BD Biosciences, Allschwil, Switzerland). Cells were
incubated for 30 minutes on ice, washed two times and resuspended in 50 µl of FACS buffer.
An anti-human IgG-Phycoerithrin-PE (BD Biosciences, Allschwil, Switzerland) diluted 1/200
was used to detect the chimeric 1D4 antibody and the isotype control antibody. Cells were
incubated for 15 minutes on ice, washed once, resuspended in 400 µl of FACS buffer and
analyzed on the FACS instrument (Cyan, Beckman Coulter International S.A., Nyon,
Switzerland).
Cynomolgus monkey primary cells
Whole blood from Cynomolgus monkeys (obtained from Professor Eric Rouiller, Laboratory
of Neurophysiology, University of Fribourg, Fribourg, Switzerland), was collected in citrate
tubes (BD Biosciences, Allschwil, Switzerland). Two mL of PBS was mixed with 3 mL of
blood and the mixture was layered on the top of 10 ml of a 85:15 Ficoll: PBS mixture (GE
Healthcare Europe GmbH, Glattbrugg, Switzerland). Samples were centrifuged for 20 minutes
at room temperature without break. The PBMC layer was collected and washed three times
with PBS. Cells were resuspended at 3x10 cells/mL in Dulbecco’s Modified Eagle Medium
(DMEM, PAA Laboratories, Pasching, Austria), 10% FBS (PAA Laboratories, Pasching,
Austria), Non-essential amino acids (PAA Laboratories, Pasching, Austria) 1mM Sodium
Pyruvate (PAA Laboratories, Pasching, Austria), 2mM Ultraglutamine (Lonza, Belgium),
100U/ml penicillin (Biochrom AG, Germany), 100 µg/ml streptomycin (Biochrom AG,
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Germany). One mL of the cell suspension was distributed in a 24 well plate (TPP,
Trasadingen, Switzerland) and 10ug/ml of PHA (PHA/M, Sigma-Aldrich Chemie GmbH,
Buchs, Switzerland) 100 U/mL of rHu IL-2 (Proleukin, Novartis, Basel, Switzerland) were
added. Cells were incubated for 50 hours at 37 °C in 5% CO incubator. Activated PBMC
were collected and resuspended in PBS/2.5% FBS (FACS buffer). Fifty thousand cells in 50
µl of FACS buffer were distributed in a 96 well V bottom plate and biotinylated anti-human
OX40-chimeric 1D4 antibody or biotinylated isotype control antibody or biotinylated
commercial anti-human OX40 raised in sheep (BD Biosciences, Allschwil, Switzerland) were
added to the wells at 25 µg/ml. Samples were incubated for 20 minutes on ice and then cells
were washed two times with cold FACS buffer and then incubated with Streptavidin-PE (BD
Biosciences, Allschwil, Switzerland) at a 1:20 dilution for 15 minutes on ice. Cells were
washed once with FACS buffer and then resuspended in 300 µl of FACS buffer. Propidium
Iodide at a volume of 2 µl (PI; Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was added
in each sample to exclude dead cells. Cells were analyzed by flow cytometry (Cyan, Beckman
Coulter International S.A., Nyon, Switzerland).
Figure 3A and 3B shows that the chimeric 1D4 antibody is able to recognize OX40 expressed
on the surface of human and cynomolgus monkey activated lymphocytes, respectively, thus
provides for cross-reactivity properties highly desired for drug development.
Example 5:
Kinetic binding affinity constants of the chimeric 1D4 antibody for human OX40
receptor extracellular domain by surface plasmon resonance (SPR)
Kinetic binding affinity constants (KD) were measured on protein-A captured antibody using
recombinant histidine tagged human OX40 receptor extracellular domain as described in
Example 1 as analyte. Measurements were conducted on a BIAcore 2000 (GE Healthcare -
BIAcore, Uppsala, Sweden) at room temperature, and analyzed with the BiaEvaluation
software (BIAcore; v4.1).
A CM5 research grade sensor chip (GE Healthcare Europe GmbH, Glattbrugg, Switzerland;
BR14) was activated by injecting 35 µl of a 1:1 N-hydroxysulfosuccinimide (NHS)/ 1-
Ethyl[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC) solution (v/v; 5 µl/min
flow-rate; on flow paths 1 and 2). Protein-A (ref. P7837; Sigma-Aldrich Chemie GmbH,
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Buchs, Switzerland) was diluted to a final concentration of 50 µg/ml in acetate buffer pH 4.5
(GE, BR50; one pH unit below pI) and subsequently immobilized on the previously
activated CM5 sensor chip by injecting 35 µl on both flow path 1 and 2 (5 µl/min); this
corresponded to approximately 1500 response units (RUs). The protein-A-CM5 sensor chip
was then deactivated by injecting 35 µl of ethanolamine solution (5µl/min). Finally, two
injections of 10 µl of glycine solution (GE, ref. BR54; 10 mM; pH 1.5) were performed
to release non-crosslinked protein-A molecules.
Before affinity measurements, a mass transfer limitation test was performed by injecting a
fixed concentration of analyte onto a fixed quantity of protein-A captured antibody at different
flow-rates (5, 15, 30, 50, 75 µl/min for 2 min). Analysis of the on-rate slopes at different
flow-rates indicated mass transfer.
For affinity measurements, the chimeric 1D4 antibody stored in 1x PBS buffer was diluted to
a final concentration of 15 nM in HBS-EP buffer (GE, ref. BR88; 0.01 M Hepes, 0.15
M NaCl, EDTA 3 mM, 0.005% Surfactant P20, pH 7.4). 10 µl of this diluted stock were
subsequently injected on the flow-path 2 of the protein-A CM5 chip (30 µl/min) to reach 200-
250 RUs. Following this capture step, the recombinant histidine tagged human OX40
receptor extracellular domain was injected at different concentrations (50 nM to 0.4 µM) on
the flow-path 1 and 2 (flow-path 1 being used as reference) at a 30 µl/min flow rate. After
each binding event, surface was regenerated with glycine buffer pH 1.5 injected for 1 min (10
µl/min).
Measurements (sensorgram: fc2-fc1) were best fitted with a 2:1 bivalent analyte model with
mass transfer. To account for the experimental variations in protein-A captured antibody at
the beginning of each measurement, the Rmax value was set to local in all fits. Dissociation
times were of at least 300-600 seconds. Measurements were performed in duplicate and
included zero-concentration samples for referencing. The Chi2 value represents the sum of
squared differences between the experimental data and reference data at each point; while the
plots of residuals indicate the difference between the experimental and reference data for each
point in the fit. Both Chi2 and residual values were used to evaluate the quality of a fit
between the experimental data and individual binding models.
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Measurements were performed in duplicates with the captured chimeric 1D4 anti-human
OX40 antibody immobilized onto the protein-A sensor chip and the recombinant histidine
tagged human OX40 receptor extracellular domain as analyte. KD value was between 91 and
116 nM with Chi2 values < 1.25.
Example 6:
Humanization of mouse monoclonal antibody 1D4
Humanizing the anti-human OX40 mouse antibody 1D4 including selection of human
acceptor frameworks, back mutations, and mutations that substantially retain and/or improve
the binding properties of human CDR-grafted acceptor frameworks is described herein.
Design of the reshaped variable regions
Homology matching was used to choose human acceptor frameworks to graft 1D4 CDRs.
Databases e.g. a database of germline variable genes from the immunoglobulin loci of human
and mouse (the IMGT database (the international ImMunoGeneTics information system ;
Lefranc MP et al., (1999) Nucleic Acids Res. 27(1): 209-12; Ruiz M et al., (2000) Nucleic
Acids Res. 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res. 29(1): 207-9; Lefranc MP
(2003) Nucleic Acids Res. 31(1): 307-10; Lefranc MP et al., (2005) Dev. Comp. Immunol.
29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics, 6(4):
253-64) or the VBASE2 (Retter I et al., (2005) Nucleic Acids Res. 33, Database issue D671-
D674) or the Kabat database (Johnson G et al., (2000) Nucleic Acids Res. 28: 214-218)) or
publications (e.g., Kabat EA et al., supra) may be used to identify the human subfamilies to
which the murine heavy and light chain V regions belong and determine the best-fit human
germlime framework to use as the acceptor molecule. Selection of heavy and light chain
variable sequences (VH and VL) within these subfamilies to be used as acceptor may be based
upon sequence homology and/or a match of structure of the CDR1 and CDR2 regions to help
preserve the appropriate relative presentation of the six CDRs after grafting.
For example, use of the IMGT database indicates good homology between the 1D4 heavy
chain variable domain framework and the members of the human heavy chain variable domain
subfamily 2. Highest homologies and identities of both CDRs and framework sequences were
observed for germline sequences: IGHV 2-70*10 (SEQ ID NO: 19), IGHV2-70*01 (SEQ ID
NO: 20), IGHV2-70*13 (SEQ ID NO: 21), IGHV2-5*09 (SEQ ID NO: 22), and IGHV2-
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70*11 (SEQ ID NO: 23), all of which having sequence identity above 73% for the whole
sequence up to CDR3. IGHV 2-70*10, IGHV2-70*01, and IGHV2-70*13 have a sequence
identity of 74%; while IGHV2-5*09, and IGHV2-70*11 have a sequence identity of 73.5%
and 73%, respectively.
Using the same approach, 1D4 light chain variable domain sequence showed good homology
to the members of the human light chain variable domain kappa subfamily 3. Highest
homologies and identities of both CDRs and framework sequences were observed for
germline sequences: IGKV3-11*01 (SEQ ID NO: 24) (65.3% identity), IGKV1-39*01 (SEQ
ID NO: 25) (64.9% identity), IGKV1D-39*01 (SEQ ID NO: 26) (64.9% identity), IGKV3-
11*02 (SEQ ID NO: 27) (64.2% identity), and IGKV3-20*01 (SEQ ID NO: 28) (62.5%
identity).
As starting point to the humanization process, human IGHV 2-70*10 (SEQ ID NO: 19), and
IGKV3-11*01 (SEQ ID NO: 24) variable domains were selected as acceptors to the 1D4
CDRs. IGHV 2-70*10 was selected over other human heavy chain variable domains for its
superior homology with 1D4 in its framework one region.
A first humanized antibody of human gamma one isotype was prepared (see below). The
antibody encompassed a human-mouse hybrid heavy chain variable domain and a human-
mouse hybrid light chain variable domain. The hybrid heavy chain variable domain was based
on the human heavy chain variable domain IGHV 2-70*10 wherein germline CDR1 and 2
where respectively replaced for 1D4 heavy chain CDR1 and 2. Best matching JH segment
sequence to the human acceptor framework was identified from the IMGT searches mentioned
above. The resulting human-mouse hybrid heavy chain variable sequence having human
IGHV 2-70*10 framework regions, 1D4 mouse CDRs, and best matching JH to human
acceptor is refereed herein as heavy chain variable domain VH1 with SEQ ID NO: 29.
Similarly, the human-mouse hybrid light chain variable domain used for this first humanized
antibody candidate had human IGKV3-11*01 framework regions, 1D4 mouse CDRs, and best
matching JK to human acceptor, and is refereed herein as light chain variable domain VL1
with SEQ ID NO: 30. The first humanized antibody encompassing VH1 and VL1 is
abbreviated herein VH1/VL1 antibody.
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Production of the first humanized antibody prototype
Coding DNA sequences (cDNAs) for VH1 and VL1 were synthesized in a scFv format by
GENEART AG (Regensburg, Germany) thereby allowing for a single DNA sequence to
encompass both variable domains (SEQ ID NO: 31). Individual variable domain cDNAs were
retrieved from this scFv construct by PCR, and further assembled upstream of their respective
constant domain cDNA sequence(s) using PCR assembly techniques. Finally, the complete
heavy and light chain cDNAs were ligated in independent vectors that are based on a modified
pcDNA3.1 vector (Invitrogen, CA, USA) carrying the CMV promoter and a Bovine Growth
Hormone poly-adenylation signal. The light chain specific vector allowed expression of
human kappa isotype light chains by ligation of the light chain variable domain cDNA of
interest in front of the kappa light chain constant domain cDNA using BamHI and BsiWI
restriction enzyme sites; while the heavy chain specific vector was engineered to allow
ligation of the heavy chain variable domain cDNA of interest in front of the cDNA sequence
encoding the human IGHG1 CH1, IGHG1 hinge region, IGHG1 CH2, and IGHG1 CH3
constant domains using BamHI and SalI restriction enzyme sites. In both heavy and light
chain expression vectors, secretion was driven by the mouse VJ2C leader peptide containing
the BamHI site. The BsiWI restriction enzyme site is located in the kappa constant domain;
whereas the SalI restriction enzyme site is found in the IGHG1 CH1 domain.
The VH1/VL1 antibody was transiently produced by co-transfecting equal quantities of heavy
and light chains vectors into suspension-adapted HEK293-EBNA1 cells (ATCC catalogue
number: CRL-10852) using polyethylenimine (PEI, Sigma, Buchs, Switzerland). Typically,
100 ml of cells in suspension at a density of 0.8-1.2 million cells per ml is transfected with a
DNA-PEI mixture containing 50 µg of expression vector encoding the heavy chain and 50 µg
of expression vector encoding the light chain. When recombinant expression vectors encoding
antibody genes are introduced into the host cells, antibodies are produced by further culturing
the cells for a period of 4 to 5 days to allow for secretion into the culture medium (EX-CELL
293, HEK293-serum-free medium; Sigma, Buchs, Switzerland), supplemented with 0.1%
pluronic acid, 4 mM glutamine, and 0.25 µg/ml geneticin).
The VH1/VL1 antibody was purified from cell-free supernatant using recombinant protein-A
streamline media (GE Healthcare Europe GmbH, Glattbrugg, Switzerland), and buffered
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exchanged into phosphate buffer saline prior to assays. Binding to human OX40 was
measured by SPR as described in Example 5.
Back mutations of grafted human frameworks
Since straight grafting of CDRs from 1D4 mouse antibody led to a candidate having no
binding to human OX40 (Table 6 and , mutagenesis wherein human residues are
substituted for mouse residues was initiated. This process is called back-mutation and is the
most unpredictable procedure in the humanization of monoclonal antibodies. It necessitates
the identification and the selection of critical framework residues from the mouse antibody
that need to be retained in order to preserve affinity while at the same time minimizing
potential immunogenicity in the humanized antibody. Table 7, Table 8, and show
residues (Kabat numbering) that differ between mouse and human antibody frameworks.
Residues which may affect the conformations of CDRs or inter-variable domain packing are
of particular interest since these may have the highest impact on antibody affinity.
To identify residues that may impact the most CDR conformation and/or inter-variable
domain packing, a 3D model for the VH1-VL1 pair of variable domains was calculated using
the structure homology-modelling server SWISS-MODEL (Arnold K et al., (2006)
Bioinformatics, 22(2): 195-201; http://swissmodel.expasy.org) set in automated mode. Model
analysis allowed the selection of a subset of positions based on their putative influence on
CDR regions and/or heavy chain-light chain variable domain packing. This subset of positions
consisted of variable heavy chain positions: 23, 35b, 48, 50, 60, and 62 as well as variable
light chain positions: 1, 33, 34, 46, 47, 54, 56, and 71 (Kabat numbering). In addition to these
back mutations, light chain position Y31 found in the VH1/VL1 antibody was deleted in some
candidates.
Further humanized candidates based on various combinations of heavy and light chain
substitutions were prepared in the context of the VH1/VL1 antibody sequence using standard
mutagenesis and methods described above. Humanized antibody candidates were assayed for
their binding affinity by SPR as described in Example 5.
Production yields and binding properties of some of the humanized antibodies based on these
single or combination of substitutions are shown in Table 6. Out of the 28 antibodies shown,
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nine candidates did not show any binding to human OX40, and another group of nine had
weak to poor binding. VL9 based humanized antibodies showed the most consistently to
weakly bind human OX40 by SPR. Only two antibodies, VH6/VL9 and VH7/VL9 showed
good binding to human OX40. Both humanized antibodies had back mutations at variable
heavy chain positions: 23, 35b, 50, 60, and 62 and variable light chain positions: 33, 34, 46,
47 and 71 (Kabat numbering). In addition to these back mutations, both VH6/VL9 and
VH7/VL9 benefited from the removal of light chain position 31. Surprisingly VH7/VL9 had
improved affinity for human OX40 over 1D4 chimeric antibody and the VH6/VL9 variant.
The binding affinities of these humanized antibodies are summarized in Table 9.
Thermostability of selected humanized anti-OX40 antibodies by differential scanning
calorimetry
The thermal stabilities of the humanized antibodies were measured using differential scanning
calorimetry (DSC). Monoclonal antibodies melting profiles are characteristic of their isotypes
(Garber E & Demarest SJ (2007) Biochem. Biophys. Res. Commun. 355: 751-7), however the
mid-point melting temperature of the FAB fragment can be easily identified even in the
context of a full-length IgG. Such mid-point melting of FAB portion was used to monitor
monoclonal stability of the humanized candidates.
Calorimetric measurements were carried out on a VP-DSC differential scanning
microcalorimeter (MicroCal, Northampton, UK). The cell volume was 0.128 ml, the heating
rate was 200°C/h, and the excess pressure was kept at 65 p.s.i. All antibodies were used at a
concentration of 1 mg/ml in PBS (pH 7.4). The molar heat capacity of antibody was estimated
by comparison with duplicate samples containing identical buffer from which the antibody
had been omitted. The partial molar heat capacities and melting curves were analyzed using
standard procedures. Thermograms were baseline corrected and concentration normalized
before being further analyzed using a Non-Two State model in the software Origin v7.0.
Humanized variant VH6/VL9 FAB fragment displayed a single transition at 76.3°C with a
shape and amplitude consistent with a cooperative unfolding which is generally observed for a
compactly folded FAB fragments indicating that the engineering process was successful at
retaining FAB stability. Overall the humanized variant showed a good thermal stability.
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Table 6: humanized anti human OX40 antibodies
Humanized SEQ ID Mutations Transient Binding
antibody NOs VH/VL expression to
variant (mg/l) human
(IGHG1) OX40
VH1/VL1 32, 39 N.A./N.A. 40 No
VH1/VL2 32, 40 N.A./L33M 21 No
VH1/VL3 32, 41 N.A./F71Y 17 No
VH2/VL1 33, 39 T23S/N.A. 13 No
VH2/VL2 33, 40 T23S/L33M 17 No
VH2/VL3 33, 41 T23S/F71Y 14 No
VH3/VL1 34, 39 R50H/N.A. 23 No
VH3/VL2 34, 40 R50H /L33M 22 No
VH3/VL3 34, 41 R50H /F71Y 18 No
VH4/VL4 35, 42 T23S-R50H/L33M-F71Y 15 Poor
VH4/VL9 35, 47 T23S-R50H/ 3 Weak
Y31deletion-L33M-A34H-L46P-L47W-F71Y
VH5/VL4 36, 42 T23S-R50H-S60N-S62A/L33M-F71Y 15 Poor
VH5/VL5 36, 43 T23S-R50H-S60N-S62A/ 2 Poor
L33M-L46P-L47W-F71Y
VH5/VL6 36, 44 T23S-R50H-S60N-S62A/ 2 Poor
E1Q-L33M-L46P-L47W-F71Y
VH5/VL9 36, 47 T23S-R50H-S60N-S62A/ 6 Weak
Y31deletion-L33M-A34H-L46P-L47W-F71Y
VH6/VL5 37, 43 T23S-S35bG-R50H-S60N-S62A/ 0 N.D.
L33M-L46P-L47W-F71Y
VH6/VL6 37, 44 T23S-S35bG-R50H-S60N-S62A/ 0.5 N.D.
E1Q-L33M-L46P-L47W-F71Y
VH6/VL7 37, 45 T23S-S35bG-R50H-S60N-S62A/ 14 N.D.
Y31deletion-L33M-F71Y
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Table 6 (cont.): humanized anti human OX40 antibodies
Humanized SEQ ID Mutations Transient Binding
antibody NOs VH/VL expression to
variant (mg/l) human
(IGHG1) OX40
VH6/VL8 37, 46 T23S-S35bG-R50H-S60N-S62A/ 7 N.D.
Y31deletion-L33M-A34H-F71Y
VH6/VL9 37, 47 T23S-S35bG-R50H-S60N-S62A/ 3.5 Good
Y31deletion-L33M-A34H-L46P-L47W-F71Y
VH6/VL10 37, 48 T23S-S35bG-R50H-S60N-S62A/ 0.5 N.D.
Y31deletion-L33M –R54L-T56S-F71Y
VH6/VL11 37, 49 T23S-S35bG-R50H-S60N-S62A/ 5.5 N.D.
Y31deletion-L33M-A34H-R54L-T56S-F71Y
VH7/VL5 38, 43 T23S-S35bG-I48L-R50H-S60N-S62A/ 1 N.D.
L33M-L46P-L47W-F71Y
VH7/VL6 38, 44 T23S-S35bG-I48L-R50H-S60N-S62A/ 1 N.D.
E1Q-L33M-L46P-L47W-F71Y
VH7/VL7 38, 45 T23S-S35bG-I48L-R50H-S60N-S62A/ 1.5 Weak
Y31deletion-L33M-F71Y
VH7/VL8 38, 46 T23S-S35bG-I48L-R50H-S60N-S62A/ 10 Weak
Y31deletion-L33M-A34H-F71Y
VH7/VL9 38, 47 T23S-S35bG-I48L-R50H-S60N-S62A/ 3 Good
Y31deletion-L33M-A34H-L46P-L47W-F71Y
VH7/VL11 38, 49 T23S-S35bG-I48L-R50H-S60N-S62A/ 11.5 Weak/
Good
Y31deletion-L33M-A34H-R54L-T56S-F71Y
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Table 7: comparison of 1D4 and human acceptor heavy chain variable IGHV 2-70*10
frameworks
Kabat 1D4 CDR grafted
position IGHV
2-70*10
G A
11 I L
12 L V
13 Q K
S T
19 S T
23 S T
35b G S
41 S P
44 G A
48 L I
50 H R
60 N S
62 A S
65 S T
66 G R
79 F V
81 K T
82 I M
82a A T
82b S N
82c Y M
84 T P
85 T V
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Table 8: comparison of 1D4 and human acceptor light chain variable IGKV 3-11*01
frameworks
Kabat 1D4 CDR grafted
position IGKV
3-11*01
1 Q E
I T
13 A L
18 K R
19 V A
21 M L
22 T S
33 M L
34 H A
42 S Q
43 S A
45 K R
46 P L
47 W L
54 L R
56 S T
58 V I
70 S D
71 Y F
72 S T
76 N S
77 R S
78 V L
80 A P
83 A F
85 T V
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Table 9: binding characteristics of selected humanized and chimeric anti OX40
antibodies.
Humanized variants SEQ ID k (1/Ms) k (1/s) K (nM)
on off D
4 -3
1D4 chimera 50, 51 3.4x10 3.08x10 91
4 -3
VH6/VL9 37, 47 3.54x10 3.56x10 101
4 -3
VH7/VL9 38, 47 4.45x10 3.12x10 70
Example 7:
Epitope characterization of humanized anti-OX40 antibodies.
To characterize the epitope of the humanized anti-OX40 antibodies, the VH6/VL9 antibody
was mapped to a define domain of the human OX40 extracellular region using various
human-rat OX40 chimeric proteins.
Preparation of human-rat OX40 chimeric proteins and ELISA
Rat and human-rat OX40 proteins were formatted as Fc fusions proteins according to the
method described in Example 1. For the ELISA, OX40 proteins were coated at 2μg/mL, in
PBS, overnight at 4°C on high binding 96-well plates (Coastar). The plates were blocked with
PBS 2% Bovine Serum Albumin (BSA) before incubation with the VH6/VL9 antibody or
isotype control antibody. The plates were then washed and incubated with goat-anti human Ig
F(ab’)2 fragment specific-HRP (Jackson ImmunoResearch Europe Ltd, Newmarket, UK).
After washing, the plates were incubated with TMB substrate (Bio-Rad Laboratories AG,
Reinach, Switzerland) to reveal antibody binding. The reaction was stopped by adding 2M
H SO and the optical density was read at 450 nM (OD 450 nM) on a Synergy HT2
spectrophotometer (Biotek, USA; distributor: WITTEC AG, Littau, Switzerland).
Results
Regardless of its origin, the OX40 extracellular region has been divided into four structural
modules referred to as domain 1, 2, 3 and 4 (Compaan DM & Hymowitz SG (2006) Structure,
14(8): 1321-30). Chimeric OX40 proteins corresponding to the extracellular region of human
OX40 (amino acids 29-214 of human TNFRSF4, numbering according to the Uniprot P43489
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sequence)were constructed by exchanging one or more of the four domains between human
and rat sequences. For example, the chimeric RHRR OX40 protein corresponds to the rat
OX40 extracellular region wherein the second domain has been replaced by the corresponding
human domain sequence.
A binding ELISA was performed to test the reactivity of the VH6/VL9 antibody on human
OX40 extracellular region (abbreviated HHHH with SEQ ID NO: 11), rat OX40 extracellular
region (abbreviated RRRR with SEQ ID NO: 52), and on four human-rat chimeric proteins:
RHRR (SEQ ID NO: 53), HRRR (SEQ ID NO: 54), HHRR (SEQ ID NO: 55), and RRHH
(SEQ ID NO: 56). The result of this ELISA is shown in As a prerequisite to this
epitope mapping experiment, the VH6/VL9 antibody was shown to bind the human OX40
protein but not the rat OX40 protein, indicating that there was no cross-reactivity to rat OX40.
It was found that the VH6/VL9 antibody bound RHRR and HHRR but not HRRR or RRHH,
indicating that VH6/VL9 epitope maps within the second domain of human OX40
extracellular region.
Example 8:
VH6/VL9 antibody blocks human mix lymphocyte reaction by killing and blocking
mechanisms
The potency of VH6/VL9 antibody to suppress in vitro immune reactions was tested in a one-
way allogeneic mixed lymphocyte reaction (MLR). The MLR is an in vitro model of
alloreactive T cell activation and proliferation (O’Flaherty E et al., (2000) Immunology,
100(3): 289-99; DuPont B & Hansen JA (1976) Adv. Immunol. 23: 107-202). When
peripheral blood mononuclear cells (PBMCs) from two unrelated donors are mixed, T cells
get activated through recognition of allogeneic major histocompatibility (MHC) molecules.
This activation results in proliferation of T lymphocytes. The MLR reaction has been widely
used to demonstrate the effect of T-cell targeting immunosuppressive drugs (Bromelow KV et
al., (2001) J. Immunol. Methods, 247(1-2): 1-8). Immunosuppressive drugs, such as
cyclosporine work mainly through inhibiting T cell activation. Besides testing the blocking
effect by the VH6/VL9 antibody, the contribution of cytotoxic mechanisms such as antibody
dependent cellular cytotoxicity (ADCC) on the inhibition of MLR was also investigated.
Three different antibody formats of the VH6/VL9 antibody were tested in this assay: an
IGHG1 format (referred herein as VH6/VL9), a non fucosylated IGHG1 (IgG1) format
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(referred herein as non fucosylated VH6/VL9), and an IGHG4 (IgG4) format (referred herein
as VH6/VL9 IGHG4 S228P). IGHG1 (IgG1) antibodies are known to be competent for
cytotoxicity mechanism such as ADCC. Non fucosylated IGHG1 antibodies are known to
exhibit enhanced ADCC activity due to a higher affinity for the FcγRIIIa expressed on
cytotoxic cells such as natural killer cells (NK cells) (Mizushima T et al., (2011) Genes Cells,
16(11): 1071-80). In contrast, IGHG4 (IgG4) antibodies are known to have no such Fc-
mediated cytotoxicity mechanisms such as ADCC.
Formatting of the VH6/VL9 antibody
IGHG4 immunoglobulin formatting having substitution S228P was achieved by replacing the
cDNA sequence encoding the IGHG1 CH1, IGHG1 hinge region, IGHG1 CH2, and IGHG1
CH3 constant domains for a cDNA sequence encoding the IGHG4 CH1, IGHG4 hinge region
having S228P substitution, IGHG4 CH2 and IGHG4 CH3 constant domains in the heavy
chain specific vector described in Example 6. Substitution S228P was introduced in a human
IGHG4 heavy chain cDNA template by standard PCR mutagenesis techniques. The resulting
heavy chain has SEQ ID NO: 57. Production of the non-fucosylated VH6/VL9 IGHG1
antibody followed the protocol described in Example 14 of the WO2010/095031 publication.
Mixed lymphocyte reaction
Blood from two different human donors was collected in three 10 mL S-Monovette with
citrate as an anti-coagulant (Sarstedt, Nümbrecht, Germany). Peripheral blood mononuclear
cells (PBMCs) from the two human donors were purified using 50ML Blood-Sep-Filter Tubes
(distributor: Brunschwig, Basel, Switzerland) following the Manufacturer’s instructions. Cells
were washed two times with Roswell Park Memorial Institute (RPMI, PAA Laboratories,
Pasching, Austria) medium without FBS. Stimulator cells from the two donors were prepared
by incubation with 50 µg/ml of mitomycin C (Sigma-Aldrich Chemie GmbH, Buchs,
Switzerland) for 30 minutes at 37°C. Cells were then washed three times with RPMI without
FBS and resuspended at 1x10 cell/mL in RPMI, 10% FBS (PAA Laboratories, Pasching,
Austria), 2mM L-glutamine (Lonza, Leuven, Belgium), 100U/ml penicillin and 100 µg/ml
streptomycin (Biochrom AG, Berlin, Germany). Responder cells were resuspended in RPMI,
%FBS, L-glutamine, 100U/ml penicillin and 100 µg/ml streptomycin in 96 well U bottom
micro-plates (TPP, Trasadingen, Switzerland). 50’000 stimulator cells and 80’000 responder
cells were distributed in a final volume of 100 µl to each well. 100 µl of antibody dilutions (or
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medium only) was added to the wells. Plates were incubated for 7 days at 37°C in 5% CO
incubator. The cells were pulsed with 0.5 µCi of H thymidine (Perkin Elmer, Basel
Switzerland) during the last 18 hours. Cells were harvested on a filtermat filter (Perkin
Elmer) and incorporated radioactivity was quantified on a Wallac beta counter (Perkin Elmer).
Results
The results shown on demonstrate that the VH6/VL9 antibody is able to efficiently
inhibit MLR for two different individuals (responders) with EC values around 100 ng/mL.
The results also show a different response depending on the antibody format used and a
difference in the contribution of blocking and cytotoxic mechanisms is observed in the MLR
from different individuals.
The reactivity of T cells from responder 1 () was efficiently inhibited by the IGHG1
and IGHG4 formats indicating that cytotoxic mechanisms are not critical for responder 1. In
contrast, the IGHG4 format was only poorly able to block the MLR from responder 2 () at high concentration and lost very rapidly its effect at lower concentrations, whereas the
IGHG1 format could achieve more than 60% of inhibition, implying that, for responder 2,
killing mechanisms are accounting for most of the inhibitory effect.
This difference in mode of action likely arises from the fact that activation, proliferation and
survival of T cells in a MLR reaction is variably reliant on OX40 costimulatory signals
between individuals, depending on the extent of allergenic reactivity and possibly other
costimulatory signals. For individuals poorly dependant on OX40-derived costimulatory
signals, elimination of activated T cells by ADCC mechanism is the main mechanism of
action of the VH6/VL9.
Surprisingly, the non fucosylated IGHG1 format displayed a very potent ability to inhibit
MLR for both responders. This observation highlights the fact that even if a blocking
mechanism may be sufficient to achieve inhibition of MLR, addition or enhancement of
killing mechanisms improves the inhibitory effect of anti-OX40 antibodies. Such
enhancement is particularly useful when in treating OX40 mediated disorders regardless of the
OX40 costimulatory status of patients, e.g when patients have low OX40 expression levels.
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Example 9:
VH6/VL9 antibody blocks xenogeneic graft-versus-host-disease
Xenogeneic graft versus host (GVH) reaction is a model for the allogeneic graft versus host
disease (GVHD) observed after bone marrow transplant in human patients. The GVH reaction
is an acute immune response mediated by grafted immune cells which attack the host
environment as a consequence of an allogeneic or xenogeneic MHC recognition (Murphy WJ
et al., (1996) Semin. Immunol. 8(4): 233-41). T lymphocytes are the main effector cells of
GVH reactions. The immunosuppressive potency of the VH6/VL9 antibody was tested in a
xenogeneic GVHD model based on the reconstitution of SCID mice with human PBMCs. In
this model, human PBMCs and primarily the T lymphocytes launch a strong response against
the mouse host cells. The reaction leads notably to severe skin and intestinal inflammation
accompanied by weight loss. The most relevant readout of this model is the survival of the
animals.
Method
Animals (SCID mice) were sub-lethally irradiated before being reconstituted with 30 million
human PBMCs intraperitoneally. The mice were also depleted for mouse NK cells by twice
weekly injections with the TMbeta1 antibody. The treatment with VH6/VL9 antibody,
Enbrel or vehicle was given i.v. weekly for five consecutive doses and started two days
before the PBMC injection. Animals were treated either with vehicle (PBS) or the VH6/VL9
antibody at 10 mg/kg or 1 mg/kg, or Enbrel (a fusion protein of the human soluble TNF
receptor 2 fused to the Fc component of human IgG1, Amgen-Pfizer) at 8 mg/kg. The animals
were checked and scored three times weekly for GVHD symptoms including weight loss,
diarrhoea, fur aspect and general behaviour. Animals were ethically sacrificed if symptoms
were considered too severe.
Results
shows that the VH6/VL9 antibody very potently suppressed the GVHD reaction even
at the lower, 1 mg/kg dose. Surprisingly, the VH6/VL9 antibody demonstrated improved
efficacy over Enbrel , which is a recognized therapy for GVHD in human (Xhaard A et al.,
(2011) Bull. Cancer, 98(8): 889-99; Simpson D (2001) Expert Opin. Pharmacother. 2(7):
1109-17). The median survival time of animals treated with the VH6/VL9 antibody at 1 or 10
mg/kg was four-fold longer compared to the vehicle treated group (Table 10) and two-fold
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longer compared to Enbrel . In addition this result highlights that the VH6/VL9 antibody
possesses no agonistic activity, since an agonistic anti-OX40 antibody has been reported to
worsen GVHD in allogeneic mouse GVHD models (Valzasina B et al., (2005) Blood, 105(7):
2845-51; Blazar BR et al., (2003) Blood, 101(9): 3741-8), an event that was not observed in
the present study.
Table 10: Median survival time (in days) of the indicated treatment groups
1D4 1D4
Treatment Vehicle Enbrel
(10mg/kg)
(1mg/kg)
Survival median 11.5 20.5 42 47.5
(days)
Vehicle: only PBS. 1D4: GBR 830-1D4 antibody; Enbrel was the clinical product.
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Claims (50)
1. An antagonist antibody or fragment thereof that binds to human OX40 comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, and a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
2. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof is a murine antibody, chimeric antibody or a humanized antibody.
3. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof is a humanized antibody.
4. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a heavy chain variable region sequence comprising: i. the amino acid sequence of SEQ ID NO: 7; or ii. the amino acid sequence selected from the group consisting of SEQ ID NOS: 58, 59, 79 and 80.
5. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a non-CDR region of a heavy chain variable region sequence which is at least 80 % identical to: i. the non-CDR region of the heavy chain variable region sequence of SEQ ID NO: 7; or ii. the non-CDR region of the heavy chain variable region sequence selected from the group consisting of SEQ ID NOS: 58, 59, 79 and 80.
6. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a heavy chain sequence comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 35, 36, 37 and 38. 1001283242
7. The antibody or fragment thereof of claim 6, wherein the heavy chain sequence comprises a non-CDR region which is at least 80 % identical to the non-CDR region of the heavy chain variable region sequence of the heavy chain sequence selected from the group consisting of SEQ ID NOS: 35, 36, 37 or 38.
8. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a heavy chain variable framework region that is the product of or derived from: i. a human gene selected from the group consisting of IGHV2-70*10 (SEQ ID NO: 19), IGHV2-70*01 (SEQ ID NO: 20), IGHV2-70*13 (SEQ ID NO: 21), IGHV2-5*09 (SEQ ID NO: 22), and IGHV2-70*11 (SEQ ID NO: 23); or ii. human gene IGHV2-70*10 (SEQ ID NO: 19) and wherein the heavy chain variable framework region comprises at least one amino acid modification from the corresponding heavy chain variable framework region of the corresponding murine antibody.
9. The antibody or fragment thereof of claim 8, wherein the antibody or fragment thereof comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 32 and wherein the heavy chain variable framework region comprises at least one amino acid modification from the corresponding heavy chain variable framework region of the corresponding murine antibody.
10. The humanized antibody or fragment thereof of claim 8 or 9, wherein the amino acid modification comprises: i. an amino acid substitution at amino acid position selected from the group consisting of 23, 35b, 48, 50, 60, and 62, wherein the amino acid position of each group member is indicated according to the Kabat numbering; or ii. an amino acid substitution selected from the group consisting of 23S, 35bG, 48L, 50H, 60N, and 62A, wherein the amino acid position of each group member is indicated according to the Kabat numbering
11. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a light chain variable region sequence comprising: i. the amino acid sequence of SEQ ID NO: 8; or 1001283242 ii. the amino acid sequence selected from the group consisting of SEQ ID NOS: 60, 86, 87 and 89.
12. The antibody or fragment thereof of claims 1, wherein the antibody or fragment thereof comprises a non-CDR region of a light chain variable region sequence which is at least 80 % identical to: i. the non-CDR region of the light chain variable region sequence of SEQ ID NO: 8; or ii. the non-CDR region of the heavy chain variable region sequence selected from the group consisting of SEQ ID NOS: 60, 86, 87 and 89.
13. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a light chain sequence comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 45, 46, 47 and 49.
14. The antibody or fragment thereof of claim 13, wherein the light chain sequence comprises a non-CDR region which is at least 80% identical to the non-CDR region of the light chain variable region sequence of the light chain sequence selected from the group consisting of SEQ ID NO: 45, 46, 47 or 49.
15. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises a light chain variable framework region that is the product of or derived from: i. a human gene selected from the group consisting of IGKV3-11*01 (SEQ ID NO: 24), IGKV1-39*01 (SEQ ID NO: 25), IGKV1D-39*01 (SEQ ID NO: 26), IGKV3-11*02 (SEQ ID NO: 27) and IGKV3-20*01 (SEQ ID NO: 28); or ii. human gene IGKV3-11*01 (SEQ ID NO: 24) and wherein the light chain variable framework region comprises at least one amino acid modification from the corresponding framework region of the light chain variable region of the corresponding murine antibody.
16. The antibody or fragment thereof of claim 15, wherein the antibody or fragment thereof comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 39 and wherein the light chain variable framework region comprises at least one amino acid modification from the corresponding light chain variable framework region of the 1001283242 corresponding murine antibody.
17. The humanized antibody or fragment thereof of claim 15 or 16, wherein the amino acid modification comprises: i. an amino acid substitution at amino acid position selected from the group consisting of 1, 33, 34, 46, 47, 54, 56, and 71, wherein the amino acid position of each group member is indicated according to the Kabat numbering; ii. an amino acid substitution selected from the group consisting of 1Q, 33M, 34H, 46P, 47W, 54L, 56S, and 71Y, wherein the amino acid position of each group member is indicated according to the Kabat numbering; or iii. an amino acid deletion at amino acid position 31, wherein the amino acid position is indicated according to the Kabat numbering.
18. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises: (a) a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38; and (b) a light chain sequence comprising the amino acid sequence of SEQ ID NO: 47.
19. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof comprises: (a) a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 58 or SEQ ID NO: 59; and (b) a light chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 60.
20. The antibody or fragment thereof of any one of claims 1 to 19, wherein at least one of the heavy chain CDRs and/or at least one of the light chain CDRs comprises at least one amino acid modification.
21. The antibody or fragment thereof of any one of claims 1 to 20, further comprising heavy and/or light constant regions and wherein the human heavy constant region is selected from the group of human immunoglobulins consisting of IGHG1, non fucosylated IGHG1 and IGHG4. 1001283242
22. The antibody or fragment thereof of any one of claims 1 to 21, wherein the antibody is a full length antibody.
23. The antibody or fragment thereof of any one of claims 1 to 21, wherein the antibody is an antibody fragment selected from the group consisting of Fab, Fab′, Fab′-SH, Fd, Fv, dAb, F(ab')2, scFv, bispecific single chain Fv dimers, diabodies, triabodies and scFv genetically fused to the same or a different antibody.
24. The antibody or fragment thereof of any one of claims 1 to 21, wherein the antibody comprises a variant Fc region which comprises at least one amino acid modification relative to the Fc region of the parent antibody, whereas the antibody comprising the variant Fc region exhibits altered effector function compared to the parent antibody.
25. The antibody or fragment thereof of any one of claims 1 to 24, wherein the antibody or fragment thereof: i. binds to human OX40 with a K of 110 nM or less; ii. retains at least 75% of the OX40 K of the corresponding chimeric antibody; or iii. has equivalent or higher OX40 K when compared to the corresponding chimeric antibody.
26. The antibody or fragment thereof of any one of claims 1 to 24, wherein the antibody has a FAB fragment thermostability temperature greater than 75°C.
27. An isolated nucleic acid encoding the antibody or fragment thereof of any one of claims 1 to 26.
28. The isolated nucleic acid of claim 27 comprising DNA encoding the heavy chain variable region comprising the nucleotide acid sequence of SEQ ID NO: 61 or 62; and/or DNA encoding the light chain variable region comprising the nucleotide acid sequence of SEQ ID NO: 63.
29. A vector comprising the isolated nucleic acid of claim 27 or 28. 1001283242
30. A non-human host cell comprising the isolated nucleic acid of claim 27 or 28 or the vector of claim 31.
31. A method of producing an antibody or fragment thereof that binds to human OX40 comprising culturing the host cell of claim 30 so that the nucleic acid is expressed and the antibody produced.
32. An antibody or fragment thereof that binds to human OX40 encoded by the isolated nucleic acid of claim 27 or 28.
33. A composition comprising the antibody or fragment thereof of any one of claims 1 to 26 and a pharmaceutically acceptable carrier.
34. An immunoconjugate comprising the antibody or fragment thereof of any one of claims 1 to 26 linked to a therapeutic agent.
35. The antibody or fragment thereof of any one of claims 1 to 26 for use as a medicament.
36. The antibody or fragment thereof of any one of claims 1 to 26 for use in a method for treating an OX40 mediated disorder in a subject.
37. The antibody or fragment thereof of claim 36 for use in a method of treating an OX40 mediated disorder in a subject, wherein the OX40 mediated disorder is selected from the group consisting of infections, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, bronchitis, influenza, respiratory syncytial virus, pneumonia, COPD, idiopathic pulmonary fibrosis (IPF), cryptogenic fibrosing alveolitis (CFA), idiopathic fibrosing interstitial pneumonia, emphysema, pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coehac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain- Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, 1001283242 primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, trauma (such as surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and neuromyelitis optica.
38. The antibody or fragment thereof of claim 37 for use in a method of treating an OX40 mediated disorder in a subject, wherein the OX40 mediated disorder is selected from the group consisting of viral infections, bacterial infections, fungal infections and parasitic infections.
39. The antibody or fragment thereof of claim 35 or claim 36 for use in treating an OX40 mediated disorder in a subject, wherein the subject has a low expression level of OX40, indicated by an increase in the expression level of OX40 positive cells when compared to control levels of up to 10%.
40. The antibody or fragment thereof of claim 35 or claim 36 for use in treating an OX40 mediated disorder in a subject, wherein the antibody has enhanced cytotoxicity as compared to the antibody having human heavy chain constant region IGHG1 determined in a one-way allogenic mixed lymphocyte reaction (MLR) as described in Example 8.
41. The antibody or fragment thereof of any one of claims 1 to 26 for use in a method for treating an OX40 mediated disorder, wherein the OX40 mediated disorder is GVHD and wherein the antibody or fragment thereof is more effective than a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 at suppressing GVHD.
42. A kit comprising the antibody or fragment thereof of any one of claims 1 to 26, the composition of claim 33 or the immunoconjugate of claim 34 for the treatment of an OX40 mediated disorder.
43. An in vitro screening method to detect a patient having a low expression level of OX40, using antibody or fragment thereof of any one of claims 1 to 26 comprising the steps of: (a) purifying peripheral blood mononuclear cells (PBMCs) from a patient blood sample; (b) subjecting the PBMCs to flow cytometric analysis; and 1001283242 (c) determining the number of OX40 positive cells in CD4 and/or CD8 T cells and comparing this number to control levels, wherein a low expression level of OX40 is indicated by an increase in the expression level of OX40 positive cells when compared to control levels of up to 10 %.
44. Use of the antibody or fragment thereof of any one of claims 1 to 26 in the manufacture of a medicament for treating an OX40 mediated disorder in a subject.
45. Use of claim 44, wherein the OX40 mediated disorder is selected from the group consisting of infections, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, asthma, bronchitis, influenza, respiratory syncytial virus, pneumonia, COPD, idiopathic pulmonary fibrosis (IPF), cryptogenic fibrosing alveolitis (CFA), idiopathic fibrosing interstitial pneumonia, emphysema, pelvic inflammatory disease, Alzheimer's Disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's Disease, coehac disease, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain- Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, trauma (such as surgery), graft-versus-host disease (GVHD), transplant rejection, cardiovascular disease including ischaemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and neuromyelitis optica.
46. Use of claim 45, wherein the OX40 mediated disorder is selected from the group consisting of viral infections, bacterial infections, fungal infections and parasitic infections.
47. Use of any one of claims 44 to 46, wherein the subject has a low expression level of OX40, indicated by an increase in the expression level of OX40 positive cells when compared to control levels of up to 10%. 1001283242
48. Use of any one of claims 44 to 47, wherein the antibody has enhanced cytotoxicity as compared to the antibody having human heavy chain constant region IGHG1 determined in a one-way allogenic mixed lymphocyte reaction (MLR) as described in Example 8.
49. Use of the antibody or fragment thereof of any one of claims 1 to 26 in the manufacture of a medicament for treating an OX40 mediated disorder, wherein the OX40 mediated disorder is GVHD and wherein the antibody or fragment thereof is more effective than a fusion protein of the human soluble TNF receptor 2 fused to the Fc component of human IgG1 at suppressing GVHD.
50. An antibody or fragment thereof according to claim 1, substantially as hereinbefore described. 1001283242
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201161506491P | 2011-07-11 | 2011-07-11 | |
US61/506,491 | 2011-07-11 | ||
PCT/IB2012/053502 WO2013008171A1 (en) | 2011-07-11 | 2012-07-09 | Antibodies that bind to ox40 and their uses |
Publications (2)
Publication Number | Publication Date |
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NZ619849A NZ619849A (en) | 2016-01-29 |
NZ619849B2 true NZ619849B2 (en) | 2016-05-03 |
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