NZ790344A - Multispecific antibodies against CD40 and CD137 - Google Patents
Multispecific antibodies against CD40 and CD137 Download PDFInfo
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- NZ790344A NZ790344A NZ790344A NZ79034417A NZ790344A NZ 790344 A NZ790344 A NZ 790344A NZ 790344 A NZ790344 A NZ 790344A NZ 79034417 A NZ79034417 A NZ 79034417A NZ 790344 A NZ790344 A NZ 790344A
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- 102000004965 antibodies Human genes 0.000 title claims abstract 114
- 108090001123 antibodies Proteins 0.000 title claims abstract 114
- 101710040446 CD40 Proteins 0.000 title claims abstract 8
- 102100013137 CD40 Human genes 0.000 title claims abstract 8
- 102100002977 CDR1 Human genes 0.000 claims 31
- 239000000427 antigen Substances 0.000 claims 30
- 108091007172 antigens Proteins 0.000 claims 30
- 102000038129 antigens Human genes 0.000 claims 30
- 210000004027 cells Anatomy 0.000 claims 22
- 230000000875 corresponding Effects 0.000 claims 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims 15
- 108020004707 nucleic acids Proteins 0.000 claims 11
- 150000007523 nucleic acids Chemical class 0.000 claims 11
- 239000000203 mixture Substances 0.000 claims 10
- 210000001744 T-Lymphocytes Anatomy 0.000 claims 8
- 150000001413 amino acids Chemical class 0.000 claims 8
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- 230000001404 mediated Effects 0.000 claims 5
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- 230000035755 proliferation Effects 0.000 claims 5
- 210000003171 Lymphocytes, Tumor-Infiltrating Anatomy 0.000 claims 4
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- 108009000071 Non-small cell lung cancer Proteins 0.000 claims 4
- 230000035772 mutation Effects 0.000 claims 4
- 206010025650 Malignant melanoma Diseases 0.000 claims 3
- 206010028980 Neoplasm Diseases 0.000 claims 3
- 108091008153 T cell receptors Proteins 0.000 claims 3
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims 3
- 230000004663 cell proliferation Effects 0.000 claims 3
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- 210000001185 Bone Marrow Anatomy 0.000 claims 2
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- 102000000588 Interleukin-2 Human genes 0.000 claims 2
- 108010002350 Interleukin-2 Proteins 0.000 claims 2
- 210000001165 Lymph Nodes Anatomy 0.000 claims 2
- URRBLVUOXIGNQR-HXUWFJFHSA-N [(1R)-1-phenylethyl] N-(2-aminoethyl)-N-[(3-methoxy-4-phenylmethoxyphenyl)methyl]carbamate Chemical compound C1([C@@H](C)OC(=O)N(CCN)CC=2C=C(C(=CC=2)OCC=2C=CC=CC=2)OC)=CC=CC=C1 URRBLVUOXIGNQR-HXUWFJFHSA-N 0.000 claims 2
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- 241000894007 species Species 0.000 claims 2
- 206010006187 Breast cancer Diseases 0.000 claims 1
- 210000001266 CD8-Positive T-Lymphocytes Anatomy 0.000 claims 1
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Abstract
Multispecific antibodies binding to human CD40 and human CD137, methods for preparing such multispecific antibodies, and methods of using such multispecific antibodies for therapeutic or other purposes.
Description
MULTISPECIFIC DIES AGAINST CD40 AND CD137
FIELD OF THE INVENTION
The present invention relates to multispecific antibodies binding to CD40 and
CD137, and to uses of such multispecific dies, in particular to the use for
treatment of cancer.
The entire disclosure in the complete ication of our New Zealand Patent
Application No. 749274 is by this cross-reference incorporated into the present
specification.
BACKGROUND OF THE INVENTION
CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and
is known as a co-stimulatory protein found on a diversity of cell types. CD40 is
constitutively expressed by antigen-presenting cells , including tic cells
(DCs), B cells and macrophages. It can also be sed by endothelial cells, platelets,
smooth muscle cells, fibroblasts and epithelial cells. Consistent with its widespread
expression on normal cells, CD40 is also expressed on a wide range of tumor cells.
The presentation of peptide antigens in the context of MHC class II molecules to
antigen-specific CD4+ T cells, together with co-stimulatory s (from CD80 and/or
CD86), results in the activation of CD4+ T cells and the up-regulation of the DC licensing
factors CD40 ligand ) and lymphotoxin-α1β2 2). Expression of CD40L and
LTα1β2 on activated antigen-specific CD4+ T cells induces signaling through CD40 and
the LTβ receptor (LTβR), and this licenses DCs to induce CD8+ T-cell responses. CD40
signaling results in the production of interleukin-12 (IL-12) and the up-regulation of
CD70, CD86, 4-1BB ligand (4-1BBL), OX40 ligand ) and GITR ligand (GITRL),
whereas LTβR signaling leads to the production of type I interferons (IFNs). The
ing system that controls the activity of nuclear factor kappaB (NF-kB) is responsive
to virtually all TNFR superfamily members. Pathogen-associated molecular patterns
(PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these
events. Priming of CD8+ T cells by MHC class I-restricted peptides results in the upregulation
of CD27, 4-1BB, OX40 and glucocorticoid-induced TNFR-related protein
(GITR). Stimulation of these receptors on CD8+ T cells by their cognate TNF superfamily
ligands, in combination with IL-12 and type I IFNs, results in robust CD8+ T cell
activation, proliferation and effector function, as well as the ion and maintenance
of CD8+ T cell memory. CD40 antibodies can exert different actions, CD40-expressing
tumor cell kill by ion of antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated
18886264_1 (GHMatters) P44724NZ01
phagocytosis (ADCP), induction of cell signaling to induce direct apoptosis or growth
arrest, but also, independent of CD40 expression on the tumor cells, through licensing of
APCs to stimulate an anti-cancer immune se. Antibodies binding to CD40 can
trigger CD40 on APCs to prime effector cytotoxic T lymphocytes (CTLs) and induce
release of IL-2 by these cells, and indirectly activate NK cells. Antibodies stimulating
CD40 have been disclosed in the prior art, and include CP-870,893, a human IgGZ
antibody (WO 2003/040170); dacetuzumab, a humanized IgGl antibody (WO
2000/075348) and Chi Lob 7/4, a ic IgGl antibody (US 2009/0074711).
Furthermore, an nistic CD40 antibody has been disclosed, lucatumumab, a human
IgGl antibody (
CD137 (4-1BB) is also a member of the TNFR family. CD137 is a co-stimulatory
molecule on CD8+ and CD4+ T cells, regulatory T cells (Tregs), Natural Killer T cells
(NK(T) cells), B cells and phils. On T cells, CD137 is not constitutively expressed,
but induced upon T-cell or (TCR) activation (for example, on tumor infiltrating
lymphocytes (TILs) (Gros et al., J. Clin Invest 2014;124(5):2246-59)). Stimulation via
its natural ligand 4-1BBL or agonist dies leads to signaling using TRAF-2 and
TRAF-1 as adaptors. Early signaling by CD137 involves K-63 poly-ubiquitination
reactions that ultimately result in activation of the nuclear factor(NF)-kB and mitogen-
activated protein(MAP)-kinase ys. Signaling leads to increased T cell co-
ation, proliferation, cytokine production, maturation and prolonged CD8+ T-cell
survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor
l by T cells in various pre-clinical models (Murillo et al., Clin Cancer Res
2008;14(21):6895-906). Antibodies stimulating CD137 can induce survival and
proliferation of T cells, thereby enhancing the anti-tumor immune response. Antibodies
stimulating CD137 have been disclosed in the prior art, and include ab, a human
IgG4 antibody (AU2004279877) and utomilumab, a human IgGZ antibody (Fisher et al.
2012 Cancer Immunol. Immunother. 61: 1721-1733).
Westwood JA, et al., ia Research 38 (2014), 948-954 discloses
“Combination anti-CD137 and anti-CD40 antibody y in murine myc-driven
3O hematological cancers”.
U820090074711 ses “Human therapies using chimeric agonistic anti-human
CD40 antibody”.
However, despite these and other advances in the art, there is a need for
multispecific antibodies that can bind both CD40 and CD137, simultaneously g to
CD40-expressing APCs and CD137-expressing T cells, thereby bringing these cell types
in close t. This, in turn, can lead to activation of both cell types and efficient
induction of anti-tumor immunity.
SUMMARY OF THE INVENTION
The present inventors have identified multispecific antibodies that can bind both
CD40 and CD137 and elicit T cell and APC activation.
So, in one aspect, the invention s to a multispecific antibody comprising
(i) a first antigen-binding region binding to human CD40, and (ii) a second
antigen-binding region binding to human CD137.
In some embodiments, the invention relates to such a multispecific antibody
wherein the first antigen-binding region comprises heavy and light chain le region
CDR1, CDRZ and CDR3 which comprise specific amino acid sequences, optionally with
ons, or the amino acid sequences of an antibody which competes with or has the
specificity of an antibody sing such specific amino acid sequences. In specific
embodiments, the first n-binding region comprises heavy and light chain variable
sequences comprising the CDRl, CDRZ and CDR3 of anti-CD40 antibody 001, or
competes with or has the specificity of such an antibody.
In some embodiments, the invention relates to such a multispecific antibody
wherein the second antigen-binding region comprises heavy and light chain variable
sequences wherein the CDRl, CDRZ and CDR3 comprise specific amino acid sequences
or provide specific amino acid ces, optionally with mutations, or comprise the
amino acid sequences of an antibody which competes with or has the specificity of an
antibody comprising such specific amino acid sequences. In specific embodiments, the
second antigen-binding region comprises heavy and light chain variable sequences
comprising the CDR1, CDRZ and CDR3 of anti-CD137 antibody 001, 002, 003, 004, 005,
006, 007, 008, 009, 010, 011 or 012, or competes with or has the specificity of any such
antibody.
These and other aspects and embodiments, including nucleic acids encoding the
amino acid sequences of such multispecific dies; expression vectors sing
such nucleic acids; host cells comprising such nucleic acids or expression vectors;
compositions comprising such multispecific antibodies; such compositions for use in
treating cancer or other es; methods for producing such pecific antibodies;
3O and diagnostic methods and kits based on such multispecific dies, are described in
further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Sequence ents for human, African elephant and wild boar
CD137. Amino acids in African nt or wild boar CD137 that differ from those in the
human sequence are highlighted in black.
Figure 2: CD137 shuffle ucts, containing African elephant le 5) or
wild boar (shuffle 1-4, 6) CD137 domains.
Figure 3: Expression of CD137 shuffle constructs on HEK293-T17 cells. HEK293-
T17 cells were transfected with the CD137 shuffle constructs. Cell surface expression of
the constructs was measured by flow cytometry, using a polyclonal anti-CD137 antibody
that recognizes human, wild boar and African elephant CD137.
Figure 4: Binding of CD137 antibody clones to CD137 shuffle constructs
sed on HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137
shuffle constructs, and with human CD137 (hCD137 wt), African elephant of wild boar
CD137, as indicated. Binding of the different CD137 antibody clones to these constructs
expressed on HEK293-T17 cells was measured by flow cytometry. ng with
polyclonal anti-CD137 dy is shown as a control.
Figure 5: Matrix-like mixing grids used for automated bispecific antibody
ery. al antibodies can be plated as indicated. Parental antibodies can then
be combined to obtain bispecific antibodies using a simple matrix-like mixing grid.
Controlled Fab-arm exchange can then be performed to obtain bispecific antibodies.
Figure 6: Expression of CD40 and CD137 on the cell surface of stably transduced
HEK293-NFK-gfp-luc and K562 cells. NF-KB/293/GFP-Luc (A) and K562 (B) cells were
stably transduced with CD40 or CD137. Surface expression of CD40 (left panels) and
CD137 (right ) was determined by flow cytometry (white curves: l t
antibody; grey curves: antibody staining).
Figure 7: Analysis of ific antibodies simultaneously targeting CD40 and
CD137 (CD40xCD137). Bispecific dies targeting CD40 and CD137 (CD40-
FEALxCD137-FEAR) were tested in the reporter assay in duplicate (A-L: CD40-
001xCD137-001 until 01xCD137-012). Activation of CD137 was measured by
luciferase activity (relative luminescence units, RLU) of NF-KB/293/GFP-Luc transduced
with CD137 (HEK293_NFK_CD137_gfp_luc) upon incubation with the indicated bispecific
antibodies and K562 cells transduced with CD40 (K562_CD40) for trans-activation or
wildtype K562 cells (K562_wt) as a control. Activation of CD40 was measured by
3O luciferase activity (RLU) of NF-KB/293/GFP-Luc transfected with CD40
(HEK293_NFK_CD40_gfp_luc) upon incubation with the ted bispecific antibodies
and K562 transduced with CD137 (K562_CD137) for trans-activation or wildtype K562
cells (K562_wt) as a control. The two monospecific, monovalent antibodies with one
irrelevant arm (b12-FEALxCD137-FEAR, b12-FEALxCD40-FEAR) were used as control for
the bispecific CD40xCD137 antibodies.
Figure 8: Induction of CD8+ T-cell proliferation by CD40xCD137 bispecific
antibodies in a tigen-specific T cell assay. CFSE-labeled PBMCs were incubated
with CD40xCD137 bispecific antibodies or monospecific, lent control antibodies,
for four days. Proliferation of CD8+ cells was measured by flow cytometry. Data shown
are CFSE plots showing CD8+ T-cell proliferation induced by the indicated bispecific and
control antibodies at 0.02 ug/mL (A), percentages of divided cells and proliferation
indices for 01-FEALxCD137FEAR (B), CD40FEALxCD137FEAR
(C), CD40FEALxCD137FEAR (D) and 01-FEALxCD137FEAR (E),
as calculated using FlowJo software.
Figure 9: ement of CD8+ T-cell proliferation by CD40xCD137 bispecific
antibodies in an antigen-specific T-cell assay. T cells transfected with a claudinspecific
TCR and labeled with CFSE were incubated with claudin-6 IVT-RNA-electroporated
immature DCs in the presence or absence of CD40xCD137 bispecific antibodies or
l antibodies for five days. CD8+ T-cell proliferation was measured by flow
cytometry. Data shown are CFSE plots showing CD8+ T-cell proliferation induced by the
indicated bispecific antibodies and controls at 0.02 ug/mL (A), percentages divided cells
and proliferation indices for the indicated bispecific antibodies (B) and for 01-
FEALxCD137FEAR and control antibodies (C) and CD40FEALXCD137
FEAR and l antibodies (D) at the indicated concentrations, as calculated using
FlowJo software. eration index curves for the indicated bispecific antibodies at serial
dilutions ranging from 6.4 x 10'5 to 5 ug/mL are also shown (E). Curves were analyzed
by non-linear regression (sigmoidal dose-response with variable slope) using GraphPad
Prism 5 software Pad Software, San Diego, CA, USA). The EC50 values for
ion of T-cell proliferation for 01-FEALxCD137FEAR and CD40
FEALxCD137FEAR were 0.005 and 0.030 ug/mL, respectively.
Figure 10: ion of CD84r T-cell proliferation by the humanized CD40xCD137
bispecific antibody in a tigen-specific T cell assay. CFSE-labeled PBMCs were
incubated with humanized CD40xCD137 bispecific antibody, the parental bispecific
antibody or IgGl control antibody for four days. Proliferation of CD8+ T cells was
measured by flow cytometry. Data shown are percentages of divided cells and
proliferation index, as calculated by FlowJo software. (n.d. = not determined)
Figure 11: Enhancement of CD8+ T-cell proliferation by a humanized
3O CD40xCD137 bispecific antibody in an antigen-specific T-cell assay. T cells transfected
with a claudinspecific TCR and labeled with CFSE were incubated with n-6 IVT-
RNA-electroporated immature DCs in the presence or absence of a humanized
CD40xCD137 bispecific antibody -CD40H6LC1-FEALxCD137HC7LC2-
FEAR), the parental bispecific antibody or an IgGl l antibody for four days. CD8+
T-cell proliferation was measured by flow cytometry. Data shown are percentages
divided cells and proliferation indices for the indicated antibodies, as calculated using
FlowJo software. (n.d. = not determined)
Figure 12: Ex vivo expansion of TILs from a human melanoma tissue resection
by a CD40xCD137 bispecific antibody. Tumor pieces from the resected tissue were
cultured with 100 U/mL IL-2 and the indicated concentration of a CD40xCD137 bispecific
antibody -CD40FEALxCD137FEAR). After 14 days of culture, cells were
harvested and analyzed by flow cytometry. Relative viable TIL count per sample
(normalized to 1,000 measured counting beads) is shown. Each data point refers to a
single well, enting the expansion of TILs out of two tumor pieces analyzed in one
FACS tube. The line indicates the mean of five measured samples.
Figure 13: Ex vivo expansion of TILs from a human non-small cell lung cancer
(NSCLC) tissue ion by a CD40xCD137 bispecific antibody. Tumor pieces from
resected NSCLC tissue were ed with 10 U/mL IL-2 and the indicated tration
of D137 bispecific antibody (BisG1-CD40FEALxCD137FEAR). After 10
days of culture, cells were harvested and analyzed by flow cytometry. Relative viable TIL
count per sample (normalized to 1,000 measured counting beads) is shown. Each data
point refers to a single well, representing the expansion of TILs out of two tumor pieces
analyzed in one FACS tube. The line indicates the mean of five measured samples.
Table 1 - Sequences
Sequence name Type of Sequence Sequence
sequence identifier
CD40-001 VH CDR1 GYTl-‘I'EYI SEQ ID NO:1
antibody (mouse)
VH CDRZ IIPNNGGT SEQ ID NO:2
VH CDR3 TRREVYGRNYYALDY SEQ ID NO:3
VL CDR1 QGINNY SEQ ID NO:4
VL CDRZ YTS
VL CDR3 QQYSNLPYT SEQ ID NO:5
VH EVQLQQSGPDLVKPGASVKISCKTS SEQ ID NO:6
GYTl-‘I'EYIMHWVKQSHGKSLEWIG
GIIPNNGGTSYNQKFKDKATMTVDK
SSSTGYMELRSLTSEDSAVYYCTRRE
VYGRNYYALDYWGQGTLVTVSS
VL DIQMTQ'I'I'SSLSASLGDRVTITCSA SEQ ID NO:7
SQGINNYLNWYQQKPDGTVKLLIYY
TSSLHSGVPSRFSGSGSGTDYSLTIS
N LE PEDIATYYCQQYSNLPYTFGGGT
KLEIK
CD137 dy VH CDR1 GFSLSSYA SEQ ID NO:8
clone 001 (rabbit)
VH CDRZ IWNNGAT SEQ ID NO:9
VH CDR3 ARSANDAWSTYSDL SEQ ID NO:10
VL CDR1 QTITNY SEQ ID NO: 11
VL CDRZ KAS
VL CDR3 SSSGYGFV SEQ ID NO: 12
VH QSVEESGGRLVTPGTPLTLTCTVSGFS SEQ ID NO: 13
LSSYAVSWVRQAPGKGLEWIGVIWN
NGATHYATWAKGRI—‘I'ISKAS'I‘I'VDLK
VTSP‘I‘I'EDTATYFCARSANDAWSTYS
DLWGQGTLVTVSS
VL DIVMTQTPASVEAAVGGTVTIKCQASQ SEQ ID NO: 14
TITNYLSWYQQKPGQPPKLLIYKASTLT
SGVSSRFKGSGSGTEI—‘I'LTISDLESDDA
ATYYCQNYYYGSSSGYGFVFGGGTEVVV
CD137 antibody VH CDR1 GFSLTYYA SEQ ID NO:15
clone 002 (rabbit)
VH CDRZ IYDNGAT SEQ ID NO:16
VH CDR3 ARSANNAWSTYSNL SEQ ID NO:17
VL CDR1 EDISSY SEQ ID NO:18
VL CDRZ KAS
VL CDR3 QSYYSGSISGYGFV SEQ ID NO: 19
VH QSVEESGGRLVTPGTPLTLTCTVSGFS SEQ ID NO:20
LTYYAVTWVRQPPGKGLEWIGVIYDN
GATAFATWAKGRFI'MSKNSTI'VALKV
TSPTI'EDTATYFCARSANNAWSTYSN
LWGQGTLVTVSS
VL DIVMTQTPSSVSAAVGGTVTINCQAS SEQ ID NO:21
EDISSYLSWYQQKLGQPPKLLIYKAST
LESGVPSRFKGSGSGTEYTLTISDLES
DDAATYYCQSYYSGSISGYGFVFGGGT
GVVVK
CD137 antibody VH CDR1 GFI'ISSYH SEQ ID NO:22
clone 003 (rabbit)
VH CDRZ IYGGTA‘I‘I’ SEQ ID NO:23
VH CDR3 ARARYSGGSYANYVFNL SEQ ID NO:24
VL CDR1 QSISSY SEQ ID NO:25
VL CDRZ RTS
VL CDR3 QGYDWSSSNRYDNT SEQ ID NO:26
VH QSVEESGGRLVTPGTPLTLTCTAS SEQ ID NO:27
GFI'ISSYHMIWVRQAPGEGLEWI
GDIYGGTA‘I‘I’DYASWAKGRI—‘I’IS
KTS'I'I’VDLKMTSL'I‘I'EDTATYFCA
RARYSGGSYANYVFNLWGQGTLV
VL DIVMTQTPASVEAAVGGTVTIKCQ SEQ ID NO:28
ASQSISSYLAWYQQKPGQPPKLLI
ESGVPSRFKGSGSGTEI—‘I'L
TISDLESADAATYYCQGYDWSSSN
RYDNTFGGGTEVVVK
CD137 antibody VH CDR1 GFSLSRYD SEQ ID NO:29
clone 004 (rabbit)
VH CDRZ T SEQ ID NO:3O
VH CDR3 AREGDYWDFNL SEQ ID NO:31
VL CDR1 QSISNL SEQ ID NO:32
VL CDRZ GAS
VL CDR3 AGGFPGLDTVAA SEQ ID NO:33
VH QSLEESGGRLVTPGTPLTLTCTASGF SEQ ID NO:34
SLSRYDMSWVRQAPGKGLEYIGVIS
SSGGTNYANWAKGRFI'ISKTSTI'VD
LKITS PTI'E DTATYFCAREGDYWDFN
LWGPGTLVTVSS
VL AQVLTQTPSSVSAAVGGTVTINCQA SEQ ID NO:35
SQSISNLLAWYQQKPGQPPKLLIYG
ASTLASGVPSRFSGSGSGTEFI'LTIS
DLESDDAATYYCAGGFPGLDTVAAF
GGGTEAVVT
CD137 antibody VH CDRl GFI'ISDFH SEQ ID NO:36
clone 005 (rabbit)
VH CDRZ IITSASTI' SEQ ID NO:37
VH CDR3 ARSTYTDTSGYYFDF SEQ ID NO:38
VL CDRl NR SEQ ID NO:39
VL CDRZ SAS
VL CDR3 LGSYDCDSADCFA SEQ ID NO:4O
VH QSVEESGGRLVTPGTPLTLTCTASG SEQ ID NO:41
FI'IS DFHVTWVRQAPGKGLEWIGTI
ITSAS'I'I'AYATWARGRFI'IS KSSTI'V
NLKIVS PTI'EDTATYFCARSTYTDTS
GYYFDFWGQGTLVTVSS
VL AQVLTQTASPVSAAVGGTVIINCQSS SEQ ID NO:42
QSIYNGNRLSWYQQKPGQPPKLLIYS
GVSSRFKGSGSGTQFI'LAISD
VQSDDAATYYCLGSYDCDSADCFAFG
GGTEVVVE
CD137 antibody VH CDRl GFSLSSYA SEQ ID NO:43
clone 006 (rabbit)
VH CDRZ ISTSGIT SEQ ID NO:44
VH CDR3 ARLNGFDDYVRYFDF SEQ ID NO:45
VL CDR1 ESIASN SEQ ID NO:46
VL CDRZ AAS
VL CDR3 QSAFYVSSSDNA SEQ ID NO:47
VH QSVEESGGRLVTPGTPLTLTCTVSGFS SEQ ID NO:48
LSSYAMSWVRQAPGKGLEWIGIISTS
SWAKGRI—‘I'ISKTSTMVDLKIT
SP'I'I'EDTATYFCARLNGFDDYVRYFDF
WGLGTLVTVSS
VL AIELTQTPSSVSAAVGGTVTINCQASE SEQ ID NO:49
SIASNLAWYQQKPGQPPKLLIYAASYL
ASGVPSRFKGSGSGTEYTLTISGVQSA
DAATYYCQSAFYVSSSDNAFGGGTEVV
CD137 antibody VH CDR1 GFSLSSYD SEQ ID NO:50
clone 007 (rabbit)
VH CDRZ IGSDGSA SEQ ID NO:51
VH CDR3 ARDWNDYWAHDL SEQ ID NO:52
VL CDR1 QIVTSKSA SEQ ID NO:53
VL CDRZ KAS
VL CDR3 AGGYYNSGDLNP SEQ ID NO:54
VH QSLEESGGRLVTPGTPLTLTCTASGFS SEQ ID NO:55
LSSYDVSWVRQAPGKGLEYIGFIGSD
GSAHYATWVKGRI—‘I'ISKTS'I‘I'VDLKIT
SP'I'I'EDTATYFCARDWNDYWAHDLW
VL AQVLTQ'I'I'SPVSAAVGGTVTINCQAS SEQ ID NO:56
SALSWYQQKPGQPPRLLIYK
ASTLASGVPSRFSGSGSGTQFI'LTIS
DLESDDAATYYCAGGYYNSGDLNPF
WO 11421
GGGTEVVVK
CD137 dy VH CDR1 GFSLSSYD SEQ ID NO:57
clone 008 (rabbit)
VH CDRZ ISSSGNT SEQ ID NO:58
VH CDR3 AREGDYWDFNL SEQ ID NO:59
VL CDR1 QSISNL SEQ ID NO:60
VL CDRZ RAS
VL CDR3 AGGFPGLDTGAT SEQ ID NO:61
VH QSLEESGGRLVTPGTPLTLTCTASGFSL SEQ ID NO:62
SSYDMSWVRQAPGKGLEYIGYISSSG
NTYYASWAKSRI—‘I'ISKTS'I‘I’VDLKITS
EDTATYFCAREGDYWDFNLWGPG
TLVTVSS
VL AQVLTQTPSSVSAAVGGTVTINCQAS SEQ ID NO:63
QSISNLLAWYQQKPGQRPRLLIYRAS
TLASGVPSRFKGSGSGTEI—‘I'LTISDLE
SEDAATYYCAGGFPGLDTGATFGGGT
EAVVT
CD137 antibody VH CDR1 GFSLNDYW SEQ ID NO:64
clone 009 (rabbit)
VH CDRZ IDVGGSL SEQ ID NO:65
VH CDR3 ARGGLTYGFDL SEQ ID NO:66
VL CDR1 EDISSY SEQ ID NO:67
VL CDRZ GAS
VL CDR3 HYYATISGLGVA SEQ ID NO:68
VH QSLEESGGRLVTPGTPLTLTCTVSG SEQ ID NO:69
FSLNDYWMSWVRQAPGKGLEWIG
YIDVGGSLYYASWAKGRFI'ISRTST
TVDLKMTS LTI'EDTATYFCARGGLT
YGFDLWGPGTLVTVSS
VL DIVMTQTPASVSEPVGGTVTINCQA SEQ ID NO:70
YLAWYQQKPGQRPKRLIYG
ASDLASGVPSRFSASGSGTEYALTIS
DLESADAATYYCHYYATISGLGVAFG
GGTEVVVK
CD137 antibody VH CDR1 GFSLSTYA SEQ ID NO:71
clone 010 (rabbit)
VH CDRZ VYDNGYI SEQ ID NO:72
VH CDR3 ARSADGSWSTYFNL SEQ ID NO:73
VL CDR1 ESISNY SEQ ID NO:74
VL CDRZ KAS
VL CDR3 QTNYCCSSSDNGFA SEQ ID NO:75
VH QSVEESGGRLVTPGTPLTLTCTVSGFSL SEQ ID NO:76
STYAMIWVRQAPGKGLEWIGVVYDNG
YISHATWVKGRI—‘I’ISKTS'I‘I'VGLEITSP
'I'I'EDTATYFCARSADGSWSTYFNLWG
QGTLVTVSS
VL DIVMTQTPASVEAAVGGTVTIKCQAS SEQ ID NO:77
ESISNYLAWYQQKPGQPPKLLIYKAS
TLASGVSSRFKGSGSGTEI—‘I'LTISDL
ESADAATYYCQTNYCCSSSDNGFAF
GGGTEVVVK
CD137 antibody VH CDR1 GIDLSSYH SEQ ID NO:78
clone 011 (rabbit)
VH CDRZ T SEQ ID NO:79
VH CDR3 ARGYSEDSYWGL SEQ ID NO:80
VL CDR1 QNIENY SEQ ID NO:81
VL CDRZ DTS
VL CDR3 QQDYGIIFVDNI SEQ ID NO:82
VH QSLEESGGRLVTPGTPLTLTCTVSGIDL SEQ ID NO:83
SSYHMCWVRQAPGKGLEYIGYIAYGG
NTYYANWAKGRFI'IS KTS'I'I'VDLRITS
PTI'E DTATYFCARGYSEDSYWGLWGP
GTLVTVSS
VL AYDMTQTPASVEAAVGGTVTIKCQAS SEQ ID NO:84
QNIENYLAWYQQKPGQPPKLLIYDTS
KLTSGVPSRFSGSGSGTDFI'LTISGVQ
SDDAATYYCQQDYGIIFVDNIFGGGTE
CD137 antibody VH CDRl GFSLSDYY SEQ ID NO:85
clone 012 (rabbit)
VH CDRZ MSGSGST SEQ ID NO:86
VH CDR3 ARDGDYAGWGYATGAFDP SEQ ID NO:87
VL CDRl QSVVGNSL SEQ ID NO:88
VL CDRZ SAS
VL CDR3 TGRYNSDTDTFV SEQ ID NO:89
VH GGRLVTPGTPLTLTCTVSGFS L SEQ ID NO:9O
SDYYMTWVRQAPGKGLEYIGIMSGSG
STYYASWAKGRFI'ISKTSSTI'LELKITS
PTI'EDTAIYFCARDGDYAGWGYATGAF
DPWGPGTLVTVSS
VL TPSPVSAAVGGTVTISCQASQ SEQ ID NO:91
SVVGNSLLSWFQKKPGQPPKLLIYSAS
SLASGVPSRFKGSGSGTQFI'LTISDLES
DDAATYYCTGRYNSDTDTFVFGGGTEV
Human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCS SEQ ID NO:92
(TNR9_Human) FCDNNRNQICSPCPPNSFSSAG
GQRTCDICRQCKGVFRTRKECSSTSNAE
CDCTPGFHCLGAGCSMCEQDCKQGQELT
KKGCKDCCFGTFNDQKRGICRPWTNCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
FLLFFLTLRFSVVKRGRKKLLYIFKQPFM RP
EEDGCSCRFPEEEEGGCEL
Human CD137 MGNSCYNIVATLLLVLNFERTRSVPDPCS SEQ ID NO:93
shuffle 6 (amino NCSAGTFCGKNIQELCMPCPPNSFSSAG
acids 24-47 GQRTCDICRQCKGVFRTRKECSSTSNAE
replaced by wild CDCTPGFHCLGAGCSMCEQDCKQGQELT
boar CD137) KKGCKDCCFGTFNDQKRGICRPWTNCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
GGCEL
Human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCS SEQ ID NO:94
shuffle 5 (amino NCPAGTFCDNNRNQICSPCPLNSFSSTGG
acids 48-88 QMNCDMCRKCEGVFKTKRACSPTRDAEC
replaced by ECTPGFHCLGAGCSMCEQDCKQGQELTK
African elephant KGCKDCCFGTFNDQKRGICRPWTNCSLD
CD137) GKSVLVNGTKERDVVCGPSPADLSPGAS
SVTPPAPAREPGHSPQIISFFLALTSTALLF
LLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
VQITQEEDGCSCRFPEEEEGGCEL
Human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCS SEQ ID NO:95
shuffle 4 (amino NCPAGTFCDNNRNQICSPCPPNSFSSAG
acids 89-114 GQRTCDICRQCKGVFRTRKECSSTSNAE
replaced by wild CDCVPGFRCLGAGCAMCEEYCQQGQELT
boar CD137) QKGCKDCCFGTFNDQKRGICRPWTNCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
EDGCSCRFPEEEEGGCEL
Human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCS SEQ ID NO:96
shuffle 3 (amino FCDNNRNQICSPCPPNSFSSAG
acids 8 GQRTCDICRQCKGVFRTRKECSSTSNAE
replaced by wild CDCTPGFHCLGAGCSMCEQDCKQGQELT
boar CD137) KEGCKDCSFGTFNDEEHGVCRPWTDCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
VQITQEEDGCSCRFPEEEEGGCEL
Human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPA SEQ ID NO:97
shuffle 2 (amino GTFCDNNRNQICSPCPPNSFSSAGGQRTCDICR
adds 139'161_ QCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC
rep'aced by W"d SMCEQDCKQGQELTKKGCKDCCFGTFND
boar CD137)
QKRGICRPWTNCSLAGKPVLMNGTKARD
VVCGPRPADLSPGASSVTPPAPAREPGHS
PQIISFFLALTSTALLFLLFFLTLRFSVVKRG
RKKLLYIFKQPFMRPVQTIQEEDGCSCRF
Human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPA SEQ ID NO:98
shuffle 1 (amino GTFCDNNRNQICSPCPPNSFSSAGGQRTCDICR
acids 162-186
replaced by wild QCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC
boar CD137) SMCEQDCKQGQELTKKGCKDCCFGTFNDQKR
GICRPWTNCSLDGKSVLVNGTKERDVVCGPSfl'
DFSPGTPSTI'MPVPGGEPGHTSHIISFFLALTST
ALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPV
Q'ITQEEDGCSCRFPEEEEGGCEL
b12 VH CDRl GYRFSNFV SEQ ID NO:99
VH CDRZ INPYNGNK SEQ ID NO: 100
VH CDR3 ARVGPYSWDDSPQDNYYMDV SEQ ID NO: 101
VL CDR 1 HSIRSRR SEQ ID NO: 102
VL CDR 2 GVS
VL CDR 3 QWGASSYT SEQ ID NO: 103
VH QVQLVQSGAEVKKPGASVKVSCQASGYR SEQ ID NO: 104
FSNFVIHWVRQAPGQRFEWMGWINPYN
GNKEFSAKFQDRVTFI'ADTSANTAYM ELR
SLRSADTAVYYCARVGPYSWDDSPQDNY
YM DVWGKG'I'I'VIVSS
VL EIVLTQSPGTLSLSPGERATFSCRSSHSIR SEQ ID NO: 105
SRRVAWYQHKPGQAPRLVIHGVSNRASG
ISDRFSGSGSGTDFI'LTITRVEPEDFALYY
CQVYGASSYTFGQGTKLERK
a) CH3 GQPREPQVYTLPPSRDELTKNQVSLTCLV SEQ ID NO: 106
region KGFYPSDIAVEWESNGQPENNYKTI'PPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALH NHYTQKSLSLSPGK
IgGlm(f) CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLV SEQ ID NO: 107
region KGFYPSDIAVEWESNGQPENNYKTI'PPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALH SLSLSPGK
IgGlm(ax) CH3 QVYTLPPSRDELTKNQVSLTCLV SEQ ID NO: 108
region KGFYPSDIAVEWESNGQPENNYKTI'PPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEGLH NHYTQKSLSLSPGK
IgGl heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID NO: 109
constant region — PVTVSWNSGALTSGVHTFPAVLQ
WT* SSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
VFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTI'PPVLDSDGSFFLY
KSRWQQGNVFSCSVM H EALH N
HYTQKSLSLSPGK
IgGl heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID NO: 110
nt region — KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
F405L* SSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTI'PPVLDSDGSFLL
YSKLTVDKSRWQQGNVFSCSVM H EALH
NHYTQKSLSLSPGK
IgGl heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID NO:111
constant region — KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
K409R* SSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKP
STYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTI'PPVLDSDGSFFLY
SBLTVDKSRWQQGNVFSCSVM H EALH N
HYTQKSLSLSPGK
Human IgGl ASTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID NO: 112
heavy chain KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
constant sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNH
with FEAR* KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FLFPPKPKDTLMISRTPEVTCVV
VAVSHEDPEVKFNWYVDGVEVH NAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTI'PPVLDSDGSFFLY
SBLTVDKSRWQQGNVFSCSVM H EALH N
HYTQKSLSLSPGK
Human IgGl ASTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID NO: 113
heavy chain KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
constant sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNH
with FEAL* KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
EGGPSVFLFPPKPKDTLMISRTPEVTCVV
VAVSHEDPEVKFNWYVDGVEVH NAKTKP
STYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
GQPENNYKTI'PPVLDSDGSFLL
YSKLTVDKSRWQQGNVFSCSVM H EALH
NHYTQKSLSLSPGK
Human Kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLN SEQ ID NO: 114
light chain NFYPREAKVQWKVDNALQSGNSQESVTE
constant sequence QDSKDSTYSLSSTLTLSKADYEKH KVYAC
LSSPVTKSFNRGEC
Human CD40 MVRLPLQCVLWGCLLTAVH PEPPTACREK SEQ ID NO:115
QCCSLCQPGQKLVSDCTEFFETE
CLPCGESEFLDTWNRETHCHQHKYCDPN
LGLRVQQKGTSETDTICTCEEGWHCTSE
ACESCVLHRSCSPGFGVKQIATGVSDTIC
EPCPVGFFSNVSSAFEKCHPWTSCETKDL
VVQQAGTNKTDVVCGPQDRLRALVVIPII
FGILFAILLVLVFIKKVAKKPTNKAPHPKQE
PDDLPGSNTAAPVQETLHGCQPV
TQEDGKESRISVQERQ
Human IgGl ASTKGPSVFPLAPSSKSTSGGTAALGCLV SEQ ID NO:
heavy chain KDYFPEPVTVSWNSGALTSGVHTFPAVLQ 116
constant sequence SSGLYSLSSVVTVPSSSLGTQTYICNVNH
with FEA* KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
EGGPSVFLFPPKPKDTLMISRTPEVTCVV
VAVSHEDPEVKFNWYVDGVEVH NAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTI'PPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVM H EALH N
HYTQKSLSLSPGK
CD40-001 humanized antibody (HC6 and LC1):
VH-CD40 VH EVQLVQSGAEVKKPGASVKVSCKTSGYT SEQ ID NO:117
HC6 FI'EYIMHWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSS
CD40HC6 HC, IgGl EVQLVQSGAEVKKPGASVKVSCKTSGYT SEQ ID NO:118
FI'EYIM HWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNH KPSNTKVDKRVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSH EDPEVKFNWYVDG
VEVH NAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTI'PPV
WO 11421
LDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMH EALHNHYTQKSLSLSPGK
CD40HC6- HC, IgGl FEAL EVQLVQSGAEVKKPGASVKVSCKTSGYT SEQ ID NO: 119
FEAL FI'EYIM HWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPEEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVAVSH EDPEVKFNWYVDG
VEVH NAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQWTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYK'I'I'PPV
LDSDGSFLLYSKLTVDKSRWQQGNVFSC
SVMH YTQKSLSLSPGK
CD40HC6- HC, IgGl FEAR EVQLVQSGAEVKKPGASVKVSCKTSGYT SEQ ID NO:120
FEAR FI'EYIMHWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYM ELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNH KPSNTKVDKRVEPKSCDKT
PAPEEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVAVSHEDPEVKFNWYVDG
VEVH NAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYK'I'I'PPV
LDSDGSFFLYSBLTVDKSRWQQGNVFSC
SVMH YTQKSLSLSPGK
VL-CD40LC1 VL DIQMTQSPSSLSASVGDRVTITCSASQGI SEQ ID NO:121
NNYLNWYQQKPGKAVKLLIYYTSSLHSGV
PSRFSGSGSGTDYTFI'ISSLQPEDIATYYC
QQYSNLPYTFGGGTKVEIK
CD40LC1 LC, kappa DIQMTQSPSSLSASVGDRVTITCSASQGI SEQ ID NO:122
NNYLNWYQQKPGKAVKLLIYYTSSLHSGV
PSRFSGSGSGTDYTFI'ISSLQPEDIATYYC
QQYSNLPYTFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKH KVYACEVTHQGLSSP
VTKSFNRGEC
CD137-OO9 humanized dy (HC7 and LC2):
VH-CD137-OO9- VH EVQLVESGGGLVQPGRSLRLSCTASGFSL SEQ ID NO:123
HC7 NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFI'ISRDDSKSIAYLQM NSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
CD137HC7 HC, 1961 EVQLVESGGGLVQPGRSLRLSCTASGFSL SEQ ID NO:124
NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFI'ISRDDSKSIAYLQM NSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAAL
YFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSH EDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWL
CKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTI'PPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
CD137-OO9-HC7- HC, IgGl FEAR EVQLVESGGGLVQPGRSLRLSCTASGFSL SEQ ID NO:125
FEAR NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFI'ISRDDSKSIAYLQM NSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPEEGGPSVFLFPPKPKDTLMISRTPE
VTCVVVAVSH FNWYVDGVEVH N
AKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSN KALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTI'PPVLDSD
GSFFLYSBLTVDKSRWQQGNVFSCSVMH
EALH NHYTQKSLSLSPGK
CD137-OO9-HC7- HC, IgGl FEAL SGGGLVQPGRSLRLSCTASGFSL SEQ ID NO:126
FEAL NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFI'ISRDDSKSIAYLQM NSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPEEGGPSVFLFPPKPKDTLMISRTPE
AVSH EDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTI'PPVLDSD
GSFLLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
VL-CD137-OO9- VL DIVMTQSPSSLSASVGDRVTITCQASEDI SEQ ID NO:127
LC2 SSYLAWYQQKPGKAPKRLIYGASDLASG
VPSRFSASGSGTDYTFFISSLQPEDIATYY
CHYYATISGLGVAFGGGTKVEIK
CD137-OO9-LC2 LC, kappa DIVMTQSPSSLSASVGDRVTITCQASEDI SEQ ID NO:128
YQQKPGKAPKRLIYGASDLASG
VPSRFSASGSGTDYTFI'ISSLQPEDIATYY
CHYYATISGLGVAFGGGTKVEIKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKH KVYACEVTHQ
GLSSPVTKSFNRGEC
Human c0137 Amino acids LQDPCSNCPAGTFCDNNRNQICSP SEQ ID NO:129
(shuffle 6) 24-47 of
human CD137
Human c0137 Amino acids CPPNSFSSAGGQRTCDICRQCKGVFRTR SEQ ID NO:13O
(shuffle 5) 48-88 of KECSSTSNAECDC
human CD137
Human CD137 Amino acids TPGFHCLGAGCSMCEQDCKQGQELTK SEQ ID NO:131
(shuffle 4) 89-114 of
human CD137
Human c0137 Amino acids CFGTFNDQKRGICRPWTN SEQ ID NO:132
le 3) 115-138 of
human CD137
Human CD137 Amino acids CSLDGKSVLVNGTKERDVVCGPS SEQ ID NO:133
(shuffle 2) 139-161 of
human CD137
Human CD137 Amino acids PADLSPGASSVTPPAPAREPGHSPQ SEQ ID NO:134
(shuffle 1) 162-186 of
human CD137
Wild Boar CD137 MGNGYYNIVATVLLVM N FERTRSVPDPCS SEQ ID NO:135
NCSAGTFCGKNIQELCM PCPSNSFSSTSG
QKACNVCRKCEGVFRTKKECSSTSNAVC
ECVPGFRCLGAGCAMCEEYCQQGQELTQ
EGCKDCSFGTFN DEEHGVCRPWTDCSLA
GKPVLMNGTKARDVVCGPRPTDFSPGTP
S'I'I'M PVPGG EPGHTS HVII FFLALMSTAVF
VLVSYLALRFSVVQQGRKKLLYIVKQPFLK
EEDACSCRFPEEEEGECEL
African Elephant MGNGYYNMVATVLLVMNFERTGAVQDSC SEQ ID NO:136
CD137 RDCLAGTYCVKNESQICSPCPLNSFSSTG
GQMNCDMCRKCEGVFKTKRACSPTRDAE
CECVSGFHCLGAGCTMCQQDCKQGQEL
TKEGCKDCCLGTFNDQKNGICRPWTNCS
LEGKSVLANGTKKRDVVCGPPAADSFPDT
SSVTVPAPERKPDH HPQIITFFLALISAALL
FLVFFLVVRFSVAKWGRKKLLYIFKQPFIK
PVQTAQEEDGCSCRFPEEEEGDCEL
*amino acids positions 118-447 ing to EU numbering
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “CD40” as used herein, refers to CD40, also referred to as tumor
necrosis factor receptor superfamily member 5 (TNFRSFS), which is the receptor for the
ligand TNFSF5/CD4OL. CD40 is known to transduce TRAF6- and MAP3K8-mediated
signals that activate ERK in macrophages and B cells, leading to induction of
immunoglobulin secretion by the B cells. Other synonyms used for CD40 include, but are
not limited to, B-cell surface antigen CD40, Bp50, CD4OL receptor and CDw40. In one
ment, CD40 is human CD40, having UniProt ion number P25942. The
sequence of human CD40 is also shown in SEQ ID NO:115. Amino acids 1-20 of SEQ ID
NO:115 correspond to the signal peptide of human CD40; while amino acids 21-193 of
SEQ ID NO:115 correspond to the extracellular domain of human CD40; and the
remainder of the n; i.e. from amino acids 194-215 and 216-277 of SEQ ID NO:115
is transmembrane and asmic domain, respectively.
The term “CD137” as used herein, refers to CD137 (4-lBB), also referred to as
tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the receptor
for the ligand TNFSF9/4-1BBL. CD137 (4-iBB) is believed to be involved in T-cell
activation. Other synonyms for CD137 include, but are not limited to, 4-iBB ligand
receptor, CDw137, T-cell n 4-iBB homolog and T-cell antigen ILA. In one
embodiment, CD137 (4-iBB) is human CD137 (4-iBB), having UniProt accession
number Q07011. The sequence of human CD137 is also shown in SEQ ID NO:92. Amino
acids 1-23 of SEQ ID NO:92 correspond to the signal peptide of human CD137; while
amino acids 24-186 of SEQ ID NO:92 correspond to the ellular domain of human
CD137; and the remainder of the protein, i.e. from amino acids 187-213 and 214-255 of
SEQ ID NO:92 are transmembrane and cytoplasmic domain, respectively.
The term “chimeric dy” as used herein, refers to an antibody wherein the
variable region is derived from a non-human species (e.g. derived from rodents) and the
constant region is derived from a ent species, such as human. Chimeric dies
may be generated by antibody engineering. “Antibody engineering” is a term used
generically for different kinds of modifications of dies, and processes for antibody
engineering are well-known for the skilled person. In particular, a chimeric antibody may
be ted by using standard DNA techniques as described in Sambrook et al., 1989,
Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press,
Ch. 15. Thus, the chimeric antibody may be a genetically or an enzymatically engineered
inant dy. It is within the knowledge of the skilled person to generate a
chimeric antibody, and thus, generation of the chimeric antibody may be performed by
other methods than those described herein. Chimeric monoclonal antibodies for
therapeutic applications in humans are ped to reduce anticipated antibody
immunogenicity of man antibodies, e.g. rodent antibodies. They may typically
contain non-human (e.g. murine or ) variable regions, which are specific for the
antigen of interest, and human constant antibody heavy and light chain domains. The
terms “variable region” or “variable domain” as used in the context of chimeric
antibodies, refer to a region which comprises the CDRs and framework regions of both
the heavy and light chains of an immunoglobulin, as described below.
The term ized antibody” as used herein, refers to a genetically engineered
man antibody, which contains human antibody constant domains and non-human
variable domains modified to contain a high level of sequence homology to human
variable domains. This can be achieved by grafting of the six non-human antibody CDRs,
which together form the antigen-binding site, onto a homologous human or
framework region (FR) (see WO92/22653 and EP0629240). In order to fully reconstitute
the binding affinity and specificity of the parental antibody, the substitution of
framework residues from the parental antibody (i.e. the non-human dy) into the
human framework regions (back-mutations) may be required. Structural homology
modeling may help to identify the amino acid residues in the ork regions that are
important for the binding properties of the antibody. Thus, a humanized antibody may
se non-human CDR sequences, primarily human framework regions optionally
comprising one or more amino acid back-mutations to the non-human amino acid
sequence, and fully human constant regions. Optionally, additional amino acid
modifications, which are not necessarily utations, may be applied to obtain a
humanized antibody with preferred characteristics, such as affinity and biochemical
properties and/or additional amino acid mutations may be introduced in the constant
3O region.
As used herein, a protein which is “derived from” another protein, e.g., a parent
protein, means that one or more amino acid sequences of the protein are cal or
similar to one or more amino acid ces in the other or parent protein. For
example, in an antibody, binding arm, n-binding , constant region, or the
like which is derived from another or a parent antibody, binding arm, antigen-binding
region, or constant region, one or more amino acid ces are identical or similar to
those of the other or parent antibody, binding arm, antigen-binding region, or constant
region. Examples of such one or more amino acid sequences include, but are not limited
to, those of the VH and VL CDRs and/or one or more or all of the framework regions, VH,
VL, CL, hinge, or CH regions. For e, a humanized antibody can be described
herein as “derived from” a non-human parent antibody, g that at least the VL and
VH CDR sequences are identical or similar to the VH and VL CDR sequences of said non-
human parent dy. A chimeric antibody can be bed herein as being “derived
from” a non-human parent antibody, meaning that typically the VH and VL sequences
may be identical or similar to those of the non-human parent antibody. Another example
is a binding arm or an antigen-binding region which may be described herein as being
“derived from” a particular parent antibody, meaning that said binding arm or antigen-
binding region typically comprises identical or similar VH and/or VL CDRs, or VH and/or
VL sequences to the binding arm or antigen-binding region of said parent antibody. As
described elsewhere herein, however, amino acid modifications such as mutations can
be made in the CDRs, constant regions or elsewhere in the antibody, binding arm,
antigen-binding region or the like, to introduce desired characteristics. When used in the
context of one or more sequences d from a first or parent protein, a ar”
amino acid sequence preferably has a sequence ty of at least about 50%, such as
at least about 60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, or at least about 97%, 98% or 99%.
Non-human antibodies can be generated in a number of different species, such as
mouse, rabbit, chicken, guinea pig, llama and goat.
Monoclonal antibodies can be produced by a variety of techniques, including
conventional monoclonal antibody methodology, e.g., the standard somatic cell
hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other
ques for producing monoclonal antibodies can be employed, e.g., viral or
oncogenic transformation of B-lymphocytes or phage display ques using libraries
of antibody genes, and such methods are well known to a person skilled in the art.
Hybridoma production in such non-human species is a very well established
procedure. Immunization protocols and techniques for isolation of splenocytes of
zed animals/non-human species for fusion are known in the art. Fusion partners
3O (e.g., murine myeloma cells) and fusion procedures are also known.
The term “human antibody” as used herein, refers to dies having le
and nt s derived from human germline immunoglobulin sequences. Human
antibodies may include amino acid residues not encoded by human ne
immunoglobulin sequences (e.g., mutations introduced by random or site-specific
mutagenesis in vitro or by c mutation in vivo). However, the term "human
antibody", as used herein, is not intended to include antibodies in which CDR sequences
derived from the germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
Human monoclonal antibodies can be generated using transgenic or
transchromosomal mice carrying parts of the human immune system rather than the
mouse system.
The term “immunoglobulin” refers to a class of structurally related glycoproteins
consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight
chains and one pair of heavy (H) , all four inter-connected by disulfide bonds. The
structure of globulins has been well characterized. See for instance Fundamental
Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each
heavy chain (abbreviated “HC”) typically is comprised of a heavy chain variable region
(abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein
as CH or CH). The heavy chain constant region typically is comprised of three domains,
CH1, CH2, and CH3. The heavy chain may lly r se a hinge region. Each
light chain (abbreviated “LC”) lly is comprised of a light chain variable region
(abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as
CL or CL). The light chain nt region typically is comprised of one domain, CL. The
VH and VL regions may be further subdivided into regions of hypervariability (or
ariable regions which may be hypervariable in sequence and/or form of
structurally defined loops), also termed complementarity ining regions (CDRs),
interspersed with regions that are more conserved, termed framework regions (FRs).
Each VH and VL is typically ed of three CDRs and four FRs, arranged from amino-
terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3,
FR4 (see also Chothia and Lesk J. Mol. Biol. m, 901-917 (1987)). Unless otherwise
stated or contradicted by context, CDR sequences herein are identified according to
IMGT rules using DomainGapAlign (Program version: 4.9.1; 201319) (Lefranc MP.,
c Acids Research 1999;27:209-212, and Ehrenmann F., Kaas Q. and Lefranc M.-P.
Nucleic Acids Research 2010;38, D301-307; see also internet http address
www.imgt.org/).
Unless otherwise stated or contradicted by t, reference to amino acid
positions in the constant regions in the present invention is according to the EU-
3O numbering (Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63(1):78-85; Kabat et
al., Sequences of ns of Immunological Interest, Fifth Edition. 1991 NIH Publication
No. 91-3242).
The term “antibody” (Ab) in the context of the present invention refers to a
molecule comprising at least one antibody variable domain such as an globulin
heavy chain variable region, or an immunoglobulin heavy chain variable region and a
light chain variable region, or a fragment thereof, or a derivative of either thereof, which
has the ability to specifically bind to an antigen, such as under typical physiological
conditions with a half-life of significant periods of time, such as at least about 30
minutes, at least about 45 minutes, at least about one hour, at least about two hours, at
least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours
or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other
relevant functionally-defined period (such as a time ient to induce, promote,
enhance, and/or modulate a physiological response associated with antibody binding to
the antigen and/or time sufficient for the antibody to recruit an effector activity). In
particular the antibody may be an immunoglobulin molecule, a fragment of an
immunoglobulin molecule or a derivative thereof. The variable regions of the heavy and
light chains of the immunoglobulin molecule 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, ing various cells of the immune system
(such as effector cells) and components of the complement system such as Clq, the first
component in the classical y of complement activation. As indicated above, the
term antibody herein, unless otherwise stated or clearly contradicted by context,
es fragments of an antibody that are antigen-binding nts, i.e., retain the
y to specifically bind to the antigen. It has been shown that the antigen-binding
on of an dy may be performed by fragments of a full-length antibody.
Examples of antigen-binding fragments encompassed within the term "antibody" include
(i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1
domains, or a monovalent antibody as described in W02007059782 (Genmab); (ii)
F(ab')2 fragments, nt fragments comprising two Fab fragments linked by a disulfide
bridge at the hinge ; (iii) an Fd fragment consisting essentially of the VH and CH1
domains; (iv) an Fv fragment consisting essentially of the VL and VH domains of a single
arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)),
which consists essentially of a VH domain and also called domain antibodies (Holt et al;
Trends Biotechnol. 2003 Nov;21(11):484-90); (vi) camelid or nanobodies (Revets et al;
Expert Opin Biol Ther. 2005 Jan;5(1):111-24) and (vii) an isolated complementarity
determining region (CDR). Furthermore, although the two s of the Fv fragment,
VL and VH, are coded for by te genes, they may be joined, using recombinant
3O methods, by a synthetic linker that enables them to be made as a single protein chain in
which the VL and VH regions pair to form monovalent molecules (known as single chain
antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426
(1988) and Huston et al., PNAS USA 85, 883 (1988)). Such single chain
antibodies are encompassed within the term antibody unless otherwise noted or y
indicated by context. Although such fragments are generally ed within the meaning
of antibody, they collectively and each independently are unique features of the present
invention, exhibiting different biological properties and utility. These and other useful
dy fragments in the context of the present ion, as well as bispecific formats
of such fragments, are discussed further herein. It also should be understood that the
term antibody, unless specified otherwise, also includes polyclonal antibodies,
monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric dies
and humanized antibodies, and antibody fragments retaining the ability to specifically
bind to the antigen (antigen-binding fragments) provided by any known technique, such
as enzymatic cleavage, e synthesis, and recombinant techniques. An antibody as
generated can possess any isotype and/or subclass.
Regular antibodies; e.g. antibodies raised in any species are normally
monospecific, bivalent antibodies, which means that they comprise two antigen-binding
s which bind to the same epitope.
The term “multispecific antibody” in the context of the present invention refers to
an antibody having different antigen-binding regions defined by different antibody
sequences. Thus a multispecific antibody may have two, three, four, five or more
ent n-binding regions. Examples of multispecific antibodies include antibodies
having two different antigen-binding regions; Le. a ific dy.
es of multispecific antibodies comprising three or more different antigen-
binding regions include but are not d to (i) bispecific antibodies coupled with an
additional single chain variable Fragment (scFv) at their Fc part (Weidle et al., Cancer
Genomics Proteomics. 2013 Jan-Feb;10(1):1-18), (ii) fusion proteins consisting of three
or more scFv (triabody, tetrabody; Chames et al., FEMS Microbiol Lett. 2000 Aug
1;189(1):1-8) and (iii) fusion proteins connected to scFv (Kermer et.al. Mol Cancer Ther.
2014 Jan;13(1):112-21).
The term “bispecific antibody” in the context of the t invention refers to an
antibody having two different antigen-binding regions defined by ent antibody
ces.
When used herein, unless contradicted by context, the term “Fab-arm” or “arm”
refers to one heavy chain-light chain pair and is used interchangeably with “half
molecules” herein.
The term “binding arm comprising an antigen-binding region” means an antibody
3O molecule or fragment that comprises an antigen-binding region. Thus, a g arm can
comprise, e.g., the six VH and VL CDR sequences, the VH and VL sequences, a Fab or
Fab’ fragment, or a Fab-arm.
When used herein, unless contradicted by context, the term “Fc region” refers to
an antibody region comprising at least a hinge region, a CH2 domain, and a CH3
domain.
As used herein, the term pe" refers to the specific type of immunoglobulin
encoded by the HC (for instance IgG, IgD, IgA, IgE, and IgM) or LC (kappa, K or lambda,
WO 11421
A) genes. Within each isotype, there may be several subclasses, such as IgGl, IgGZ,
IgG3, IgG4, IgA1, IgA2, etc.
The term “monovalent antibody” means in the t of the present invention
that an antibody molecule is e of binding a single molecule of the antigen, and
thus is not e of antigen cross-linking.
A “CD40 antibody” or “anti-CD40 antibody” is an antibody as described above,
which binds specifically to the antigen CD40.
A “CD137 antibody” or “anti- CD137 dy” is an antibody as bed above,
which binds specifically to the antigen CD137.
A “CD40xCD137 antibody” or “anti-CD40xCD137 antibody” is a bispecific
antibody, which comprises two different antigen-binding regions, one of which binds
specifically to the antigen CD40 and one of which binds ically to the antigen
CD137.
The term “specifically binds”, “specifically binding”, “specific binding” or other
similar wording refers to the ability of an antibody to preferentially bind to a particular
antigen compared to other antigens, or to a particular part (epitope) of an antigen
compared to other parts of the same antigen.
As used herein, the term "binding" in the t of the binding of an antibody to
a predetermined antigen or e typically is a binding with an ty corresponding
to a KB of about 10'7 M or less, such as about 10'8 M or less, such as about 10'9 M or
less, about 10'10 M or less, or about 10'11 M or even less when determined by for
instance surface plasmon nce (SPR) technology in a BIAcore 3000 instrument
using the antibody as the ligand and the antigen as the analyte (or vice versa), and
binds to the predetermined antigen with an affinity corresponding to a KD that is at least
ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower,
such as at least 10,000-fold lower, for instance at least 100,000-fold lower than the KD
for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined
antigen or a closely-related antigen. The amount with which the affinity is higher is
dependent on the KD of the dy, so that when the KD of the antibody is very low
3O (that is, the antibody is highly specific), then the amount with which the affinity for the
n is higher than the affinity for a non-specific antigen may be at least 10,000 fold.
The term “kd” (sec'l), as used herein, refers to the dissociation rate constant of a
particular antibody-antigen interaction. Said value is also referred to as the koff value.
The term "KD" (M), as used herein, refers to the dissociation equilibrium constant
of a particular antibody-antigen interaction.
Two antibodies have the “same specificity” if they bind to the same antigen and
to the same epitope. Whether an antibody to be tested recognizes the same epitope as a
certain antigen-binding antibody, i.e., the antibodies bind to the same epitope, may be
tested by different methods well known to a person skilled in the art.
The competition between the antibodies can be detected by a blocking
assay. For example, a competitive ELISA assay may be used as a cross-blocking assay.
For example, target antigen may be coated on the wells of a microtiter plate and
antigen-binding antibody and candidate competing test antibody may be added. The
amount of the n-binding antibody bound to the antigen in the well indirectly
correlates with the binding ability of the candidate competing test antibody that
competes therewith for binding to the same epitope. Specifically, the larger the affinity
of the candidate competing test antibody is for the same epitope, the smaller the
amount of the antigen-binding antibody bound to the antigen-coated well. The amount
of the antigen-binding antibody bound to the well can be ed by labeling the
antibody with detectable or measurable labeling substances.
An antibody competing for binding to an antigen with r antibody, e.g., an
antibody comprising heavy and light chain variable regions as bed herein, or an
antibody having the icity for an antigen of another antibody, e.g., an dy
comprising heavy and light chain le regions as described herein, may be an
antibody comprising variants of said heavy and/or light chain variable regions as
described herein, e.g. modifications in the CDRs and/or a n degree of identity as
bed .
An “isolated multispecific antibody” as used herein is intended to refer to a
multispecific antibody which is substantially free of other antibodies having different
nic specificities (for instance an isolated bispecific antibody that specifically binds
to CD40 and CD137 is substantially free of monospecific antibodies that specifically bind
to CD40 or CD137).
The term “epitope” means a protein determinant capable of specific g to an
dy. Epitopes y consist of surface groupings of molecules such as amino acids
or sugar side chains and usually have specific three-dimensional structural
characteristics, as well as specific charge characteristics. Conformational and non-
3O conformational epitopes are distinguished in that the binding to the former but not the
latter is lost in the presence of denaturing solvents. The epitope may comprise amino
acid residues directly involved in the binding and other amino acid residues, which are
not directly involved in the binding, such as amino acid es which are effectively
blocked or covered by the specifically antigen-binding peptide (in other words, the amino
acid residue is within the footprint of the specifically antigen-binding peptide).
The term "monoclonal antibody" as used herein refers to a preparation of
antibody molecules of single molecular composition. A monoclonal antibody composition
displays a single binding specificity and affinity for a particular epitope.
When used herein the term “heterodimeric interaction between the first and
second CH3 regions” refers to the interaction between the first CH3 region and the
second CH3 region in a first-CH3/second-CH3 heterodimeric antibody.
When used herein the term “homodimeric interactions of the first and second CH3
regions” refers to the interaction between a first CH3 region and r first CH3 region
in a CH3/first-CH3 homodimeric antibody and the interaction between a second CH3
region and another second CH3 region in a second-CH3/second-CH3 homodimeric
antibody.
When used herein the term “homodimeric antibody” refers to an antibody
comprising two first Fab-arms or half-molecules, n the amino acid sequence of
said ms or olecules is the same.
When used herein the term “heterodimeric antibody” refers to an antibody
comprising a first and a second Fab-arm or half-molecule, wherein the amino acid
sequence of said first and second Fab-arms or half-molecules are different. In particular,
the CH3 region, or the antigen-binding region, or the CH3 region and the antigen-
binding region of said first and second Fab-arms/half-molecules are ent.
The term "reducing conditions" or "reducing environment" refers to a condition or
an environment in which a substrate, such as a cysteine residue in the hinge region of
an antibody, is more likely to become reduced than oxidized.
The present invention also provides multispecific antibodies, such as bispecific
antibodies, comprising onal variants of the VL regions, VH regions, or one or more
CDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or
CDR used in the context of a bispecific antibody still allows each antigen-binding region
of the bispecific antibody to retain at least a substantial proportion (at least about 50%,
60%, 70%, 80%, 90%, 95% or more) of the affinity and/or the specificity/selectivity of
the parent ific antibody and in some cases such a bispecific antibody may be
associated with greater affinity, selectivity and/or specificity than the parent bispecific
Such functional variants typically retain significant sequence ty to the
3O parent bispecific antibody. The percent identity between two ces is a function of
the number of identical positions shared by the sequences (i.e., % homology = # of
identical positions/total # of positions x 100), taking into account the number of gaps,
and the length of each gap, which need to be uced for optimal alignment of the
two sequences. The t identity between two nucleotide or amino acid sequences
may e.g. be determined using the algorithm of E. Meyers and W. , Comput. Appl.
Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version
2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty
of 4. In addition, the percent identity between two amino acid sequences may be
ined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)
algorithm.
In the context of the present invention the following notations are, unless
otherwise indicated, used to describe a mutation; name of amino acid which is mutated,
followed by the position number which is mutated, followed by what the mutation
encompass. Thus if the mutation is a tution, the name of the amino acid which
replaces the prior amino acid is included, if the amino acid is deleted it is indicated by a
*, if the mutation is an addition the amino acid being added is included after the original
amino acid. Amino acid names may be one or three-letter codes. Thus for example;
tution of a Lysine in position 409 with an Arginine is referred to as K409R,
substitution of Lysine in position 409 with any amino acid is referred to as K409X,
deletion of Lysine in position 409 is referred to as K409* and addition of P after Lysine at
position K409 is referred to as K409KP.
Exemplary ts include those which differ from the VH and/or VL and/or CDRs
of the parent sequences mainly by conservative substitutions; for instance 12, such as
11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative
amino acid residue replacements.
In the context of the present invention, conservative substitutions may be
d by substitutions within the classes of amino acids reflected in the following:
Amino acid residue classes for conservative tutions:
Acidic Residues: Asp (D) and Glu (E)
Basic Residues: Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues: Ser (S), Thr (T), Asn (N), and Gln (Q)
Aliphatic Uncharged Residues: Gly (G), Ala (A), Val (V), Leu (L), and Ile (I)
lar Uncharged Residues: Cys (C), Met (M), and Pro (P)
Aromatic Residues: Phe (F), Tyr (Y), and Trp (W)
The first and/or second antigen-binding region of the present invention may also
3O be a variant of a first and/or second antigen-binding region, respectively, disclosed
herein.
It is well known to a person skilled in the art how to introduce modifications and
that certain amino acids of the CDR sequences may be modified; e.g. by amino acid
substitutions to e.g. increase affinity of an antibody to its target antigen, reduce
potential genicity of non-human dies to be used in humans and/or to
se the yield of dies sed by a host cell. Such modifications can be
introduced without affecting the epitope of the target antigen to which the antibody
binds.
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The term "recombinant host cell" (or simply "host cell" or “cell”), as used herein,
is intended to refer to a cell into which a nucleic acid, such as an expression vector has
been uced, e.g. a nucleic acid, such as an expression vector encoding a
multispecific antibody of the ion. Recombinant host cells include, for e,
transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO
cells, and lymphocytic cells.
The term “treatment” refers to the administration of an effective amount of a
therapeutically active multispecific antibody of the present invention with the purpose of
easing, ameliorating, arresting or eradicating (curing) ms or disease states.
The term "effective amount" or “therapeutically effective amount” refers to an
amount effective, at dosages and for periods of time necessary, to achieve a desired
therapeutic result. A therapeutically effective amount of a multispecific antibody may
vary according to factors such as the e state, age, sex, and weight of the
individual, and the ability of the multispecific antibody to elicit a desired response in the
individual. A therapeutically effective amount is also one in which any toxic or
detrimental effects of the multispecific antibody or a fragment thereof, are outweighed
by the therapeutically beneficial effects.
The term “anti-idiotypic antibody” refers to an antibody which recognizes unique
determinants generally associated with the antigen-binding site of an antibody.
In the context of the present invention the term “induce Fc-mediated or
on to a lesser extent” used in relation to an dy, including a multispecific
antibody, means that the antibody induces Fc-mediated or functions, such function
in particular being selected from the list of IgG Fc receptor (chammaR, FcyR) binding,
C1q binding, ADCC or CDC, to a lesser extent compared to a human IgGl antibody
comprising (i) the same CDR ces, in particular comprising the same first and
second antigen-binding regions, as said antibody and (ii) two heavy chains comprising
human IgGl hinge, CH2 and CH3 regions.
Fc-mediated effector on may be measured by binding to FcyRs, binding to
Clq, or induction of iated cross-linking via FcyRs.
Further aspects and embodiments of the invention
The present invention relates to a le comprising two different antigen-
binding regions, one of which has specificity for human CD40 and one of which has
specificity for human CD137.
In a particular embodiment, said molecule may be a multispecific antibody.
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Thus the present invention s to a multispecific antibody comprising (i) a
first antigen-binding region binding to human CD40; and (ii) a second antigen-binding
region binding to human CD137.
As shown by the inventors of the present invention a bispecific dy
according to the present invention may induce intracellular signaling when binding to
CD40 expressed on one cell and binding to CD137 expressed on another cell. Thus, a
multispecific antibody according to the present invention is able to trans-activate two
different cells. In humans, CD40 is expressed on a number of cells including antigenpresenting
cells (APCs), such as tic cells, whereas CD137 is expressed on T cells
and other cells. Thus, multispecific antibodies, such as bispecific antibodies, ing to
the present invention binding to CD40 and CD137 are able to bind simultaneously to
APCs and T cells expressing these receptors. Without being bound by theory,
multispecific antibodies, such as bispecific antibodies, according to the present invention
may thus (i) mediate cell-to-cell interaction between APCs and T cells by receptor
binding and (ii) activate both CD40 and CD137 at once, which is primarily induced by
linking and receptor clustering upon cell-to-cell interaction and not necessarily
dependent on agonistic activity of the parental monospecific bivalent antibodies. Thus,
these trans-activating multispecific antibodies, such as bispecific antibodies, exert co-
stimulatory activity in the context of APC:T cell ctions, and can elicit a T cell
response against tumor cells. As such, this mechanism of action can t natural T-cell
activation via antigen-presentation by activated APCs, allowing for the presentation of a
variety of tumor-specific antigens by the APCs to T cells. Without being d to theory,
the costimulatory activity may provide for one or more of (i) only specific T cells being
activated (i.e., those that are in contact with an APC) as d to any T cell and (ii)
ivation of exhausted T cells, by strong co-stimulation via activated APCs and
CD137 triggering and (iii) the priming of T cells by inducing antigen presentation by
activated APCs and at the same time triggering CD137.
Thus, a multispecific, such as a bispecific, dy of the present invention may
be used for treatment of a disease which would benefit from activation of T cells, such as
3O cancer.
In one embodiment, the multispecific antibody according to the present invention
comprises
(I) a first antigen-binding region binding to human CD40, wherein said first antigen-
binding region ses heavy and light chain variable region CDR1, CDR2, and CDR3
selected from the group consisting of:
a) heavy chain le region CDR3 having the sequence set forth in SEQ ID NO:3
or a sequence n up to four amino acids are modified in SEQ ID NO:3, and
light chain variable region CDR3 having the ce set forth in SEQ ID NO:5 or
a sequence wherein up to four amino acids are ed in SEQ ID NO:5; and
b) heavy and light chain variable region CDR3 of an antibody which (i) competes for
human CD40 binding with an antibody comprising heavy and light chain variable
region CDR3 according to a) and/or (ii) has the specificity for CD40 of an
antibody comprising heavy and light chain variable region CDR3 according to a),
(II) a second antigen-binding region binding to human CD137.
In a further embodiment, the first antigen-binding region may r comprise
heavy chain variable region CDR1 having the ce as set forth in SEQ ID NO:1 or a
sequence wherein up to 2 amino acids are modified in SEQ ID NO:1, and/or heavy chain
variable region CDRZ having the sequence as set forth in SEQ ID NO:2 or a sequence
wherein up to 2 amino acids are modified in SEQ ID NO:2; and/or light chain variable
region CDR1 having the sequence as set forth in SEQ ID NO:4 or a sequence n up
to 2 amino acids are modified in SEQ ID NO:4, and/or light chain le region CDRZ
having the ce YTS or a sequence wherein up to 2 amino acids are modified in
Thus, in one embodiment, the present invention s to a multispecific
antibody comprising
(I) a first antigen-binding region binding to human CD40, wherein said first antigen-
binding region comprises heavy and light chain variable region CDR1, CDRZ, and CDR3
selected from the group consisting of:
a) heavy chain variable region CDR1, CDRZ and CDR3 having the sequences set
forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region
CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and
, respectively;
b) heavy and light chain variable region CDR1, CDRZ and CDR3 according to a)
3O having a total of one to twelve mutations; and
c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an antibody
which (i) competes for human CD40 binding with an antibody comprising heavy
and light chain variable region CDR1, CDRZ and CDR3 according to a) or b)
and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light
chain variable region CDR1, CDRZ and CDR3 according to a) or b), and
(II) a second antigen-binding region binding to human CD137.
In a further embodiment, said first antigen-binding region comprises a first heavy
chain le (VH) sequence, and a first light chain le (VL) sequence, and said
second antigen-binding region comprises a second heavy chain variable (VH) sequence,
and a second light chain variable (VL) sequence, wherein said variable sequences each
comprises three CDR sequences, CDRl, CDRZ and CDR3, respectively, and four
framework sequences, FR1, FR2, FR3 and FR4, respectively.
In a further embodiment, said multispecific antibody comprises (I) a first binding
arm comprising said first n-binding region, and (II) a second binding arm
comprising said second antigen-binding region.
In one embodiment, the first binding arm comprises said first antigen-binding
region and a first heavy chain constant sequence, and the second binding arm comprises
said second-antigen-binding region and a second heavy chain constant sequence.
In a further embodiment, (i) said first binding arm comprises said first n-
binding region, wherein the first binding arm comprises a first heavy chain comprising a
first heavy chain le (VH) sequence and a first heavy chain constant (CH)
sequence, and a first light chain comprising a first light chain variable (VL) sequence,
and (ii) said second g arm comprises said second antigen-binding region, wherein
the second binding arm comprises a second heavy chain comprising a second heavy
chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a
second light chain sing a second light chain variable (VL) sequence.
In a further embodiment, said first light chain r ses a first light chain
constant (CL) sequence, and said second light chain r comprises a second light
chain constant (CL) sequence.
In one embodiment, the first g arm comprises a first Fab-arm comprising
said first antigen-binding region, and the second binding arm comprises a second Fab-
arm comprising said second antigen-binding region.
In one embodiment, said first and second antigen-binding regions of the
multispecific antibody according to the present invention are derived from a humanized
3O antibody. In one embodiment, the first and second binding arm may be derived from a
humanized antibody.
In one embodiment, the first and second binding arms of the multispecific
antibody according to the present invention are derived from a full-length antibody.
In one embodiment, the first and second binding arm of the multispecific
antibody according to the present invention are derived from a full-length IgGl,)\
(lambda) or IgGl,K (kappa) dy.
In one embodiment, the first and second binding arms are derived from a
onal antibody.
In one embodiment, the first and said second heavy chains of the multispecific
antibody according to the present invention are of an IgG isotype. The subclass of the
first and second heavy chains may, for example, be separately selected from the group
consisting of IgGl, IgGZ, IgG3, and IgG4. In one embodiment, the first and second
heavy chains are of the same IgG subclass, such as IgGl.
In one embodiment, the multispecific antibody according to the t invention
is an isolated antibody.
In a further embodiment, each of said first and second heavy chains comprises at
least a hinge region, a CH2 and a CH3 region. In a further embodiment, the CH3 regions
of said first and second heavy chains comprise asymmetrical mutations.
In one embodiment, the multispecific antibody according to the present invention
is a bispecific antibody.
In one embodiment, the multispecific antibody according to the t invention
may cross-link one cell expressing human CD40, e.g. a first cell, and another cell
expressing human CD137, e.g. a second cell.
In one embodiment, said cross-linking is determined by an assay using a first cell
line expressing human CD40 and a second cell line expressing human CD137, and
wherein either the first or the second cell line comprises a er structure resulting in
the production of a measurable reporter upon NF-KB activation.
In one embodiment said first cell may be an antigen-presenting cell and said
second cell may be a , such as a CD4+ or a CD8+ .
Different methods may be used to determine cross-linking a first cell expressing
CD40 and a second cell expressing CD137, and the present invention is not limited to
any particular method.
In one embodiment, said cross-linking may be determined by a reporter assay
e.g. as described in Example 4. In brief, said assay comprises co-culturing a reporter cell
line expressing a first target n and transduced with a reporter gene (luciferase for
instance) driven by NF-KB response elements with a second cell line expressing a second
target antigen, adding a multispecific antibody according to the present invention in
3O concentrations from 100 ng/mL to 10,000 ng/mL to the cell co-culture, and measuring
reporter gene expression, e.g. rase tion, wherein said first target antigen is
human CD40 and said second target antigen is human CD137 or vice versa.
A multispecific antibody e of inducing cross-linking of the CD40 and CD137
expressed on different cells, will in this assay result in measurable activation of the first
target antigen based on the reporter gene expression upon NF-KB y activation.
In one embodiment, the multispecific dy according to the present invention
may be able to induce reporter gene expression ed upon NF-KB activation only
upon addition of the second cell line expressing the second target antigen without the
NF-KB reporter gene.
In one embodiment, the multispecific antibody according to the t invention
may be able to induce higher reporter gene expression produced upon NF-KB activation
when the second cell line expressing the second target antigen t the NF-KB
reporter gene is added compared to addition of a second cell line not sing the
second target antigen.
In one embodiment, the multispecific antibody is a bispecific antibody, and said
bispecific antibody may, in one embodiment:
(i) induce reporter gene expression when added to a co-culture of the reporter
cell line expressing CD137 and the second cell line expressing CD40, or
(ii) induce a higher amount of the reporter gene expression when added to the
co-culture of the reporter cell line expressing CD137 and the second cell line expressing
CD40, compared to a reference bispecific antibody comprising the same second antigen-
binding region binding to human CD137, but wherein the first antigen-binding region of
the reference bispecific antibody binds to an irrelevant target antigen, e.g. wherein the
first n-binding region comprises heavy chain variable region CDR1, CDR2 and
CDR3 having the sequences set forth in SEQ ID NOs:99, 100 and 101, respectively, and
light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ
ID NOs:102, GVS and 103, respectively.
In one ment, the pecific antibody is a bispecific antibody, and said
bispecific antibody may, in one embodiment:
(i) induce reporter gene expression when added to a co-culture of the reporter
cell line expressing CD40 and the second cell line expressing CD137, or
(ii) induce a higher amount of the reporter gene expression when added to the
co-culture of the reporter cell line expressing CD40 and the second cell line expressing
CD137, compared to a reference bispecific antibody comprising the same first antigen-
binding region binding to human CD40, but wherein the second antigen-binding region
of the reference bispecific antibody comprises heavy chain variable region CDR1, CDR2
3O and CDR3 having the sequences set forth in SEQ ID , 100 and 101, respectively,
and light chain le region CDR1, CDR2 and CDR3 having the sequences set forth in
SEQ ID 2, GVS and 103, respectively.
In one embodiment, the multispecific antibody according to the present invention
s and/or enhances proliferation of T cells, e.g. n said T cells are CD4+
and/or CD8+ T cells.
Different s for determining or measuring proliferation of T cells may be
used and the present ion is not limited to any particular method.
WO 11421
In one embodiment, said induction or enhancement of proliferation of T cells is
determined by a tigen-specific T-cell proliferation assay, e.g. as described in
Example 5. Thus induction and/or enhancement of proliferation of T cells may be
ined by sub-optimal activation of T cells in a PBMC pool (peripheral blood
mononuclear lymphocyte). The sub-optimal activation may be determined by titrating
the concentration of anti-CD3 antibody added to a PBMC pool, measuring T cell
proliferation and choosing the D3 antibody concentration which results in low T cell
proliferation but allows for further ement of the T cell proliferation. This
concentration is PBMC-donor-dependent and is determined for each donor before the
assay is med.
In one embodiment, said induction or enhancement of proliferation of T cells is
determined by activating T cells in PBMCs with said timal concentration of an anti-
CD3 antibody, contacting the PBMCs with the multispecific dy and determining
proliferation of the T cells. In a further embodiment, the PBMCs may be labelled with
CFSE, contacting the PBMCs with the multispecific antibody may be performed by
tion for 4 days, and proliferation of the T cells may be measured by flow
cytometry.
Inducing a certain reaction or effect such as ing proliferation ofT cells" may
mean that there was no such reaction or effect such as proliferation of T cells before
induction, but it may also mean that there was a certain level of on or effect such
as proliferation ofT cells before induction and after induction said reaction or effect such
as proliferation ofT cells is enhanced. Thus, "inducing" also includes "enhancing".
Proliferation of T cells may also be measured by an antigen-specific T cell
proliferation assay, e.g., as described in Example 6, using a test antigen of interest.
Thus, induction and/or enhancement of T cell proliferation may be measured by co-
culturing T cells expressing a TCR specific for a peptide of the test antigen presented in
major histocompatibility complex (MHC) and DCs presenting a corresponding peptide in
MHC, which is then recognized by the TCR. For e, the T cells may be CD8+ T cells
and the MHC may be MHC Class I, or the T cells may be CD4+ T cells and the MHC may
3O be MHC Class II. T cells expressing a specific TCR may be ted by transduction
with mRNA encoding the TCR. DCs presenting the corresponding peptide may be
generated by transduction of the DCs with mRNA encoding the n. Co-culture of the
TCR-positive T cells with the antigen-presenting cells induces T-cell proliferation; the
extent of the proliferation may depend on the antigen density presented by the DCs
and/or on the strength of the costimulatory signal. In one embodiment, proliferation ofT
cells may be measured by such an antigen-specific T-cell assay using CFSE labeled T-
cells, adding antibodies to be tested and after 4 days measuring T cell proliferation by
flow cytometry.
In one embodiment, said induction or enhancement of proliferation of T cells is
determined using tumor-infiltrating lymphocytes (TILs) in an ex vivo expansion assay,
e.g., as bed in Example 11. The effect of the multispecific antibody of the invention
on the induction or enhancement of proliferation of the TILs may be assessed by
incubating a human tumor sample with interleukin-2 (IL-2) and said dy, and
retrieving and counting viable TILs after incubation for a period of about 10 to about 14
days. An induction or enhancement of proliferation of TILs can then be determined by
comparison with a le control, e.g., a human tumor sample incubated without any
multispecific antibody or with a reference (control) multispecific antibody. For example,
a sample of human tumor tissue can be ed, e.g., via a biopsy or from a surgical
specimen, washed in serum-free medium, and tumor pieces with a diameter of about 1-
2 mm placed into culture dishes or wells, e.g., 1 or 2 tumor pieces in 1 mL suitable
medium, and incubated at 37°C. A suitable medium can be, for example, a free
medium (e.g. X-VIVO 15) supplemented with 10% Human Serum Albumin, 1%
Pen/Strep, 1% Fungizone and IL-2 at a concentration ranging from 10 to 100 U/mL,
e.g., 10 U/mL or 100 U/mL. The multispecific antibody can then be added at a suitable
concentration in TIL medium. After a total e period of 10-14 days, optionally
splitting the cell culture if needed during this , TILs can be harvested and counted,
e.g., by flow cytometry, using, e.g., anti-CD3, anti-CD4, anti-CD8, anti-CD56 and 7-AAD
antibodies to permit detection of viable CD4+ and CD8+ T cells as well as NK cells.
In one embodiment, the multispecific dy is a ific antibody, which
induces and/or enhances more proliferation ofT cells compared to a reference bispecific
antibody comprising a second antigen-binding region according to any aspect or
embodiment described , but wherein the first antigen-binding region comprises
heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in
SEQ ID NOs:99, 100 and 101, respectively, and light chain variable region CDR1, CDR2
and CDR3 having the sequences set forth in SEQ ID NOs:102, GVS and 103,
respectively.
In one embodiment, the multispecific antibody is a bispecific antibody, which
3O induces and/or enhances more proliferation ofT cells compared to a reference bispecific
antibody comprising a first antigen-binding region according to any aspect or
embodiment described , but wherein the second antigen-binding region comprises
heavy chain variable region CDR1, CDR2 and CDR3 having the ces set forth in
SEQ ID NOs:99, 100 and 101, respectively, and light chain variable region CDR1, CDR2
and CDR3 having the sequences set forth in SEQ ID NOs:102, GVS and 103,
respectively.
Binding to CD40
As described above, the multispecific antibody according to the t invention
comprises a first antigen-binding region binding to human CD40.
In one embodiment, the multispecific dy according to the present invention
comprises a first antigen-binding region g to human CD40, wherein said first
antigen-binding region comprises heavy and light chain variable region CDR3 selected
from the group ting of:
a) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:3
or a sequence wherein up to four amino acids are modified in SEQ ID NO:3, and
light chain variable region CDR3 having the sequence set forth in SEQ ID NO:5 or
a sequence wherein up to four amino acids are modified in SEQ ID NO:5,
b) heavy and light chain variable region CDR3 of an antibody which (i) competes for
human CD40 binding with an antibody sing heavy and light chain le
region CDR3 according to a) and/or (ii) has the specificity for CD40 of an
antibody comprising heavy and light chain variable region CDR3 according to a).
In a r embodiment, the first antigen-binding region may further comprise a
heavy chain variable region CDR1 having the sequence as set forth in SEQ ID NO: 1 or a
sequence wherein up to 2 amino acids are modified in SEQ ID NO:1, and/or heavy chain
variable region CDRZ having the sequence as set forth in SEQ ID NO:2 or a sequence
wherein up to 2 amino acids are modified in SEQ ID NO:2; and/or light chain variable
region CDR1 having the sequence as set forth in SEQ ID NO:4 or a sequence wherein up
to 2 amino acids are modified in SEQ ID NO:4, and/or light chain variable region CDRZ
having the sequence YTS or a sequence n up to 2 amino acids are modified in
YTS.
In one embodiment, the multispecific antibody according to the present invention
comprises a first antigen-binding region binding to human CD40, wherein said first
antigen-binding region ses heavy and light chain variable region CDR1, CDRZ and
CDR3 selected from the group consisting of:
3O a) heavy chain variable region CDR1, CDRZ and CDR3 having the sequences set
forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region
CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and
, respectively,
b) heavy and light chain variable region CDR1, CDRZ and CDR3 according to a)
having a total of one to twelve mutations; and
c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an antibody
which (i) competes for human CD40 g with an antibody comprising heavy
and light chain variable region CDR1, CDRZ and CDR3 according to a) or b)
and/or (ii) has the icity for CD40 of an dy comprising heavy and light
chain variable region CDR1, CDRZ and CDR3 according to a) or b).
In one embodiment said first antigen-binding region comprises a first heavy chain
le (VH) sequence, and a first light chain variable (VL) ce, n said
variable sequences each comprises three CDR sequences, CDR1, CDRZ and CDR3,
respectively.
In one embodiment, said first antigen-binding region comprises a first heavy
chain variable (VH) sequence, and a first light chain variable (VL) sequence, and wherein
said variable sequences each comprises three CDR ces, CDR1, CDRZ and CDR3,
respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.
In one embodiment, said first antigen-binding region comprises heavy chain
variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:1,
2 and 3, respectively, and light chain variable region CDR1, CDRZ and CDR3 having the
sequences set forth in SEQ ID NOs:4, YTS and 5, respectively. Thus the first antigen-
binding region may comprise heavy and light chain variable region CDR1, CDRZ and
CDR3 having the sequences of the CD40 antibody as set forth in Table 1.
An example of an antibody comprising such a first antigen-binding region is the
chimeric antibody Chi Lob 7/4 and CD40-001 disclosed herein.
In another embodiment, said first antigen-binding region comprises heavy chain
variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:1,
2 and 3, respectively, and light chain variable region CDR1, CDRZ and CDR3 having the
ces set forth in SEQ ID NOs:4, YTS and 5, tively having a total of one to
twelve mutations, such as one to eleven mutations, one to ten mutations, one to eight
mutations, one to seven mutations, one to six mutations, one to five mutations, one to
four mutations, one to three mutations, or one to two mutations.
In one embodiment, said mutation may be an amino acid substitution, such as a
conservative amino acid substitution.
3O In one ment, said mutations may be distributed across the VH CDR1, 2
and 3 and VL CDR 1, 2 and 3 so that each of the VH and VL CDR3 comprises at the most
three mutations and each of the VH and VL CDRZ and CDRl comprises at the most two
amino acid mutations.
In a further embodiment, the first antigen-binding region comprises heavy and
light chain CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:1, 2, 3,
4, YTS and 5, respectively, having a total of one to twelve mutations and wherein the VH
and VL CDR3 each comprises up to three amino acid mutations, and the VH and VL
CDRl and CDRZ each comprises up to two amino acid mutations.
WO 11421
In a further embodiment, the first antigen-binding region comprises heavy and
light chain CDRl, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:1, 2, 3,
4, YTS and 5, respectively, having a total of one to ten mutations, such as one to eight,
and wherein the VH and VL CDR1, CDRZ, and CDR3 each comprises up to two amino
acid mutations.
In a further embodiment, the first antigen-binding region ses heavy and
light chain CDRl, CDRZ and CDR3 having the ces set forth in SEQ ID NOs:1, 2, 3,
4, YTS and 5, respectively, having a total of one to six mutations, and wherein the VH
and VL CDRl, CDRZ, and CDR3 each comprises at the most one amino acid mutation.
It is well known to a person skilled in the art how to introduce mutations and that
certain amino acids of the CDR sequences may be mutated; e.g. by amino acid
substitutions to e.g. increase affinity of the antibody to its target antigen, reduce
potential immunogenicity of non-human antibodies to be used in humans and/or to
increase the yield of dies expressed by a host cell. Such mutations can be
introduced without affecting the epitope of the target to which the dy binds.
In another embodiment, said first antigen-binding region comprises heavy and
light chain variable region CDRl, CDRZ and CDR3 of an dy which (i) competes for
human CD40 binding with an antibody comprising heavy and light chain variable region
CDRl, CDRZ and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an
antibody comprising heavy and light chain variable region CDRl, CDRZ and CDR3
ing to a) or b).
In a further embodiment, said first n-binding region comprises heavy and
light chain variable regions of an antibody which (i) competes for human CD40 binding
with an antibody comprising heavy and light chain variable region CDRl, CDRZ and
CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody
comprising heavy and light chain variable region CDRl, CDRZ and CDR3 according to a)
or b).
The term “competes” refers in this t to the competition between two
antibodies for binding to a target antigen. If two antibodies do not block each other for
3O binding to a target antigen, such antibodies are non-competing and this is an indication
that said antibodies do not bind to the same part, i.e. epitope of the target antigen. It is
well known to a person skilled in the art how to test for competition of antibodies for
binding to a target antigen. An example of such a method is a so-called cross-
competition assay, which may e.g. be performed as an ELISA or by flow-cytometry.
For example, an ELISA-based assay may be performed by g ELISA plate
wells with each of the antibodies; adding the competing antibody and His-tagged
extracellular domain of the target antigen and incubate; detecting whether the added
dy inhibited binding of the His-tagged n to the coated antibody may be
WO 11421
performed by adding biotinylated anti-His dy, ed by Streptavidin-poly-HRP,
and further developing the reaction with ABTS and measuring the absorbance at 405
nm. For e a flow-cytometry assay may be performed by incubating cells
expressing the target antigen with an excess of unlabeled antibody, incubating the cells
with a sub-optimal concentration of biotin-labelled antibody, followed by incubation with
fluorescently labeled streptavidin and analyzing by flow cytometry.
In one embodiment, said VH sequence of the first antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to
at least one of SEQ ID NO:117 and SEQ ID NO:6, such as SEQ ID NO:117.
In one ment, said VL sequence of the first antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
at least one of SEQ ID NO:121 and SEQ ID NO:7, such as SEQ ID NO:121.
In one embodiment, said VH and VL sequence of the first antigen-binding region
each comprises a sequence having at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ
ID NO:6 and SEQ ID NO:7, respectively.
In one embodiment, said VH and VL ce of the first antigen-binding region
each comprises a sequence having at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ
ID NO:117 and SEQ ID NO:121, respectively.
In one embodiment, the VH and VL sequences only deviate in the non-CDR
sequences as set forth in SEQ ID NO:6 and 7, respectively.
In one embodiment, the VH and VL sequences only deviate in the R
sequences as set forth in SEQ ID NO:117 and 121, respectively.
In one ment, the VH and VL sequences only deviate in the framework
sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences
3O of the VH and VL sequences of the first antigen-binding region has at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4
ork sequences of said VH and VL sequences.
In one embodiment, the tive FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL sequences of the first antigen-binding region has at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least
99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework
sequences of the VH sequence as set forth in SEQ ID NO:6, and VL sequence as set forth
in SEQ ID NO:7.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework ces
of the VH and VL sequences of the first antigen-binding region has at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least
99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework
sequences of the VH sequence as set forth in SEQ ID NO:6, and VL sequence as set forth
in SEQ ID NO:7, and the heavy and light chain variable region CDR1, CDRZ and CDR3 of
the first antigen-binding region has a total of one to twelve mutations compared to the
heavy and light chain variable region CDR1, CDRZ and CDR3 having the sequences as
set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively. In a further embodiment,
said mutations may be as described above.
In an even further embodiment, the respective FR1, FR2, FR3 and FR4 framework
sequences of the VH and VL sequences of the first antigen-binding region has at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, or at least 99% amino acid sequence identity to the tive FR1, FR2, FR3 and
FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:6, and VL
sequence as set forth in SEQ ID NO:7, and the first antigen-binding region comprises
heavy and light chain variable region CDR1, CDRZ and CDR3 having the sequences as
set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL sequences of the first antigen-binding region has at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least
99%, or 100% amino acid sequence ty to the respective FR1, FR2, FR3 and FR4
framework sequences of the VH sequence as set forth in SEQ ID NO:117, and VL
sequence as set forth in SEQ ID NO:121, and the heavy and light chain variable region
CDR1, CDRZ and CDR3 of the first antigen-binding region has a total of one to twelve
ons compared to the heavy and light chain variable region CDR1, CDRZ and CDR3
having the ces as set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively. In
3O a further embodiment, said mutations may be as described above.
In an even further ment, the respective FR1, FR2, FR3 and FR4 framework
ces of the VH and VL sequences of the first antigen-binding region has at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2,
FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:117,
and VL sequence as set forth in SEQ ID NO:121, and the first antigen-binding region
comprises heavy and light chain le region CDR1, CDRZ and CDR3 having the
ces as set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively.
In one embodiment, said VH sequence of the first antigen-binding region
comprises the amino acid sequence of SEQ ID .
In one embodiment, said VL sequence of the first antigen-binding region
comprises the amino acid sequence of SEQ ID NO: 121.
In a further embodiment, said VH and VL sequences of the first antigen-binding
region comprise the amino acid sequences of SEQ ID NO:117 and SEQ ID NO:121,
respectively.
In one ment, said VH sequence of the first antigen-binding region
comprises the amino acid sequence of SEQ ID NO:6.
In one embodiment, said VL sequence of the first antigen-binding region
ses the amino acid sequence of SEQ ID NO:7.
In a further embodiment, said VH and VL sequences of the first antigen-binding
region comprises the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7,
respectively.
In one embodiment, the mu|tispecific antibody according to the t invention
may comprise a first binding arm comprising said first antigen-binding region of any
aspect or ment herein.
In one embodiment, the mu|tispecific antibody according to the present invention
comprises a first binding arm comprising said first antigen-binding region and a first
heavy chain constant sequence.
In one embodiment, the mu|tispecific antibody according to the present invention
comprises a first binding arm comprising said first antigen-binding , wherein the
first binding arm comprises a first heavy chain comprising a first heavy chain variable
(VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain
comprising a first light chain variable (VL) ce.
In one embodiment, said first light chain further ses a first light chain
constant (CL) sequence.
In a further embodiment, said first heavy chain ses at least a hinge region,
3O a CH2 and a CH3 region.
In a ic embodiment, the mu|tispecific antibody according to the present
invention comprises a first Fab-arm comprising said first antigen-binding region.
In one embodiment, the first antigen-binding region may be derived from a
mouse antibody.
In one embodiment, the first n-binding region may be derived from a
chimeric antibody, such as Chi Lob 7/4.
In one embodiment, the first antigen-binding region may be derived from a
humanized antibody.
In one embodiment, the first binding arm may be derived from a full-length
antibody.
In one embodiment, the first binding arm may be derived from a ength
IgGl,)\ (lambda) or IgGl,K (kappa) antibody.
In one embodiment, the first binding arm may be derived from a onal
antibody.
In one embodiment, said first heavy chain may be of an IgG isotype, optionally
ed from the group consisting of IgGl, IgGZ, IgG3, and IgG4.
In one embodiment, the first binding arm may be derived from an antibody
comprising a HC comprising SEQ ID NO:118 and an LC comprising SEQ ID NO:122,
optionally with one or more mutations in the constant region of the HC, such as 1 to 10,
such as 1 to 5, such as 1, 2, 3, 4 or 5 mutations.
In one embodiment, the first binding arm comprises a HC comprising SEQ ID
NO:118, 119 or 120 and an LC comprising SEQ ID .
Binding to CD137
The multispecific antibody according to the present invention comprises a second
antigen-binding region binding to human CD137.
In a further embodiment, the second antigen-binding region also binds to
lgus CD137.
In one embodiment, said second antigen-binding region comprises heavy and
light chain le region CDR3 selected from the group consisting of:
a) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:1O or a sequence n up to four amino acids are modified in SEQ ID
NO:10, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:12 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:12 (CD137 clone 001),
b) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
3O NO:17 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:17, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:19 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:19 (CD137 clone 002),
c) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:24 or a sequence wherein up to four amino acids are ed in SEQ ID
NO:24, and light chain le region CDR3 having the sequence set forth in
SEQ ID NO:26 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:26 (CD137 clone 003),
d) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:31 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:31, and light chain le region CDR3 having the sequence set forth in
SEQ ID NO:33 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:33 (CD137 clone 004),
heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:38 or a ce n up to four amino acids are modified in SEQ ID
NO:38, and light chain le region CDR3 having the sequence set forth in
SEQ ID NO:4O or a sequence wherein up to four amino acids are modified in SEQ
ID NO:40 (CD137 clone 005),
f) heavy chain le region CDR3 having the sequence set forth in SEQ ID
NO:45 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:45, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:47 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:47 (CD137 clone 006),
9) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:52 or a sequence wherein up to four amino acids are ed in SEQ ID
NO:52, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:54 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:54 (CD137 clone 007),
h) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:59 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:59, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:61 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:61 (CD137 clone 008),
heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:66 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:66, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:68 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:68(CD137 clone 009),
j) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:73 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:73, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:75 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:75 (CD137 clone 010),
k) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:80 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:80, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:82 or a ce wherein up to four amino acids are modified in SEQ
ID NO:82 (CD137 clone 011),
I) heavy chain variable region CDR3 having the sequence set forth in SEQ ID
NO:87 or a sequence wherein up to four amino acids are modified in SEQ ID
NO:87, and light chain variable region CDR3 having the sequence set forth in
SEQ ID NO:89 or a sequence wherein up to four amino acids are modified in SEQ
ID NO:89 (CD137 clone 012), and
m) heavy and light chain variable region CDR3 of an antibody which (i) competes for
human CD137 g with an antibody comprising heavy and light chain
variable region CDR3 according to any of a) to l) and/or (ii) has the icity for
CD137 of an antibody comprising heavy and light chain variable CDR3 according
to any of a) to I).
In a r embodiment, said second antigen-binding region further comprises
heavy and/or light chain region CDR1 and CDRZ selected from the group consisting of:
a) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:8
or a sequence wherein up to two amino acids are modified in SEQ ID NO:8,
and/or heavy chain variable region CDRZ having the sequence set forth in SEQ
ID NO:9 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:9, and/or light chain variable region CDR1 having the sequence set forth in
SEQ ID NO:11 or a sequence wherein up to two amino acids are modified in SEQ
ID NO:11, and/or light chain variable region CDRZ having the sequence KAS or a
sequence wherein up to two amino acids are modified in KAS (CD137 clone 001),
b) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:15 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:15, and/or heavy chain variable region CDRZ having the sequence set forth
3O in SEQ ID NO:16 or a ce wherein up to two amino acids are modified in
SEQ ID NO:16, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:18 or a sequence n up to two amino acids are modified
in SEQ ID NO:18, and/or light chain variable region CDRZ having the sequence
KAS or a sequence wherein up to two amino acids are modified in KAS (CD137
clone 002),
heavy chain variable region CDR1 having the ce set forth in SEQ ID
NO:22 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:22, and/or heavy chain le region CDRZ having the sequence set forth
in SEQ ID NO:23 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:23, and/or light chain le region CDR1 having the sequence set
forth in SEQ ID NO:25 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:25, and/or light chain variable region CDRZ having the ce
RTS or a sequence wherein up to two amino acids are modified in RTS (CD137
clone 003),
d) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:29 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:29, and/or heavy chain variable region CDRZ having the sequence set forth
in SEQ ID NO:30 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:30, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:32 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:32, and/or light chain variable region CDRZ having the sequence
GAS or a ce wherein up to two amino acids are modified in GAS (CD137
clone 004),
heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:36 or a sequence wherein up to two amino acids are ed in SEQ ID
NO:36, and/or heavy chain variable region CDRZ having the sequence set forth
in SEQ ID NO:37 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:37, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:39 or a sequence n up to two amino acids are modified
in SEQ ID NO:39, and/or light chain variable region CDRZ having the sequence
SAS or a sequence wherein up to two amino acids are modified in SAS (CD137
clone 005),
f) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:43 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:43, and/or heavy chain variable region CDRZ having the sequence set forth
in SEQ ID NO:44 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:44, and/or light chain variable region CDR1 having the ce set
3O forth in SEQ ID NO:46 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:46, and/or light chain variable region CDRZ having the sequence
AAS or a sequence wherein up to two amino acids are modified in AAS (CD137
clone 006),
9) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:50 or a sequence n up to two amino acids are modified in SEQ ID
NO:50, and/or heavy chain variable region CDRZ having the sequence set forth
in SEQ ID NO:51 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:51, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:53 or a sequence wherein up to two amino acids are ed
in SEQ ID NO:53, and/or light chain variable region CDRZ having the ce
KAS or a sequence wherein up to two amino acids are modified in KAS (CD137
clone 007),
h) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:57 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:57, and/or heavy chain variable region CDRZ having the sequence set forth
in SEQ ID NO:58 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:58, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:60 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:60, and/or light chain variable region CDRZ having the sequence
RAS or a sequence wherein up to two amino acids are modified in RAS (CD137
clone 008),
heavy chain le region CDR1 having the sequence set forth in SEQ ID
NO:64 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:64, and/or heavy chain variable region CDRZ having the sequence set forth
in SEQ ID NO:65 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:65, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:67 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:67, and/or light chain variable region CDRZ having the sequence
GAS or a sequence wherein up to two amino acids are ed in GAS (CD137
clone 009),
j) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:71 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:71, and/or heavy chain le region CDRZ having the sequence set forth
in SEQ ID NO:72 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:72, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:74 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:74, and/or light chain variable region CDRZ having the sequence
3O KAS or a sequence wherein up to two amino acids are modified in KAS (CD137
clone 010),
k) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:78 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:78, and/or heavy chain le region CDRZ having the sequence set forth
in SEQ ID NO:79 or a ce wherein up to two amino acids are modified in
SEQ ID NO:79, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:81 or a sequence wherein up to two amino acids are modified
in SEQ ID NO:81, and/or light chain variable region CDRZ having the sequence
DTS or a sequence wherein up to two amino acids are modified in DTS (CD137
clone 011), and
I) heavy chain variable region CDR1 having the sequence set forth in SEQ ID
NO:85 or a sequence wherein up to two amino acids are modified in SEQ ID
NO:85, and/or heavy chain variable region CDR2 having the sequence set forth
in SEQ ID NO:86 or a sequence wherein up to two amino acids are modified in
SEQ ID NO:86, and/or light chain variable region CDR1 having the sequence set
forth in SEQ ID NO:88 or a sequence wherein up to two amino acids are ed
in SEQ ID NO:88, and/or light chain variable region CDR2 having the sequence
SAS or a sequence wherein up to two amino acids are modified in SAS (CD137
clone 012).
In one embodiment said second antigen-binding region ses heavy and light
chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:8, 9 and 10, respectively, and light chain le region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:11, KAS
and 12, respectively, (CD137 clone 001),
b) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:15, 16 and 17, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:18, KAS
and 19, respectively, (CD137 clone 002),
heavy chain le region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:22, 23, and 24, respectively, and light chain le region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:25, RTS
and 26, respectively, (CD137 clone 003),
d) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:29, 30 and 31, tively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:32, GAS
3O and 33, respectively, (CD137 clone 004),
heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS
and 40, respectively, (CD137 clone 005),
f) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:43, 44 and 45, tively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:46, AAS,
and 47, respectively, (CD137 clone 006),
g) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:50, 51 and 52, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:53, KAS
and 54, respectively, (CD137 clone 007),
h) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:57, 58 and 59, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:60, RAS
and 61, respectively, (CD137 clone 008),
i) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:64, 65 and 66, tively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS
and 68, respectively, (CD137 clone 009),
j) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:71, 72 and 73, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:74, KAS
and 75, respectively, (CD137 clone 010),
k) heavy chain le region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:78, 79 and 80, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:81, DTS
and 82, respectively, (CD137 clone 011),
l) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:85, 86 and 87, tively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:88, SAS
and 89, respectively, (CD137 clone 012),
m) heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of
a) to I) having a total of one to twelve mutations, and
n) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody
which (i) competes for human CD137 binding with an dy comprising heavy
and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to
3O m) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and
light chain variable region CDR1, CDR2 and CDR3 according to any of a) to m).
Thus, the second antigen-binding region may comprise the heavy and light chain
variable region CDR1, CDR2 and CDR3 sequences of a CD137 antibody as set forth in
Table 1; Le. CD137 clone 001, CD137 clone 002, CD137 clone 003, CD137 clone 004,
CD137 clone 005, CD137 clone 006, CD137 clone 007, CD137 clone 008, CD137 clone
009, CD137 clone 010, CD137 clone 011 or CD137 clone 012. In ular, the second
n-binding region may se the heavy and light chain variable region CDR1,
CDRZ and CDR3 sequences from the same CD137 antibody clone, optionally wherein the
framework regions are primarily human framework regions, optionally comprising one or
more amino acid back-mutations to the non-human amino acid sequence.
In a further embodiment, the second antigen-binding region ses heavy and
light chain variable regions of an antibody which (i) competes for human CD137 binding
with an antibody comprising heavy and light chain variable region CDRl, CDRZ and
CDR3 according to any of a) to m) and/or (ii) has the specificity for CD137 of an
dy comprising heavy and light chain variable region CDRl, CDRZ and CDR3
according to any of a) to m).
In one embodiment, said second antigen-binding region binds to human CD137
(SEQ ID NO:92) to a higher degree than it binds to a mutant human CD137 (SEQ ID
NO:93). The mutant human CD137 of SEQ ID NO:93 is also referred to as shuffle 6
herein.
In another embodiment, said second antigen-binding region binds to human
CD137 (SEQ ID NO:92) to a higher degree than it binds to a mutant human CD137 (SEQ
ID NO:94). The mutant human CD137 of SEQ ID NO:94 is also ed to as shuffle 5
herein.
In a further ment, said second antigen-binding region binds to human
CD137 (SEQ ID NO:92) to the same degree that it binds to a mutant human CD137
(SEQ ID NO:95). The mutant human CD137 of SEQ ID NO:95 is also referred to as
shuffle 4 herein.
In the t of the present invention “to a higher ” means that the
affinity of the second antigen-binding region is higher for human CD137 (SEQ ID NO:92)
than for a mutant human CD137 (SEQ ID NO:93 and 94, shuffle 6 and 5 respectively). If
there is no binding to the mutant CD137, the affinity for human CD137 will be infinitely
higher than for said mutant CD137. However, in case of binding to said mutant CD137
the ty may be 2-fold, such as 3-fold, or 4-fold, or 5-fold, or 6-fold higher for human
CD137 than for the respective mutant CD137.
In the context of the present invention “to the same ” means that the
3O affinity of the second antigen-binding region is similar for human CD137 (SEQ ID NO:92)
and for a mutant human CD137 (SEQ ID NO:95, shuffle 4). In particular, “similar” in this
context may mean that the affinity for human CD137 and for said mutant CD137 differs
at the most by 2.5-fold, such as 2.2-fold, or 2.0-fold, or 1.8-fold, or 1.75-fold or 1.5-
fold.
The mutant human CD137 in SEQ ID NO:93 corresponds to the amino acid
sequence of human CD137 wherein amino acids 24-47 (shuffle 6) were replaced by the
corresponding amino acids from wild boar CD137.
Thus, in one embodiment, the second antigen-binding region binds to an epitope
of human CD137 which comprises or requires one or more of the amino acids L, Q, D, P,
C, S, N, C, P, A, G, T, F, C, D, N, N, R, N, Q, I, C, S and P at positions 24-47 of SEQ ID
NO:92 sponding to SEQ ID NO:129).
The mutant human CD137 in SEQ ID NO:94 corresponds to the amino acid
sequence of human CD137 wherein amino acids 48-88 (shuffle 5) were replaced by the
corresponding amino acids from African nt CD137.
Thus, in one embodiment, the second antigen-binding region binds to an epitope
of human CD137 which comprises or requires one or more of the amino acids C, P, P, N,
S, F, S, S, A, G, G, Q, R, T, C, D, I, C, R, Q, C, K, G, V, F, R, T, R, K, E, C, S, S, T, S, N,
A, E, C, D and C at positions 48-88 of SEQ ID NO:92 (corresponding to SEQ ID NO:130).
The mutant human CD137 in SEQ ID NO:95 corresponds to the amino acid
ce of human CD137 wherein amino acids 59-114 (shuffle 4) were replaced by the
corresponding amino acids from African elephant CD137.
Thus, in one embodiment, the second antigen-binding region does not bind to an
epitope of human CD137 which comprises or requires one or more of the amino acids T,
P, G, F, H, C, L, G, A, G, C, S, M, C, E, Q, D, C, K, Q, G, Q, E, L, T and K 89-114 at
positions of SEQ ID NO:92 (corresponding to SEQ ID NO:131).
In one embodiment, binding to the mutant and human CD137 may be performed
as the e assay described in Example 2. Thus, binding to human CD137 (SEQ ID
NO:92) and mutant human CD137 (SEQ ID , 94 and 95) may be determined by
preparing shuffle ucts derived from human CD137 in which protein domains of the
human CD137 are ed by the corresponding domain of CD137 from ent
species, using human CD137 and the different species of CD137 as reference constructs;
transducing cells with plasmids encoding the reference construct or the shuffle
constructs, respectively, and measuring binding of the antibody to each these CD137
ucts by flow cytometry, such as FACS.
Loss of binding to certain shuffle constructs indicates that the corresponding
region is likely to be involved in the antibody epitope. Thus, protein domains of human
3O CD137 contributing to the epitope of the anti-human CD137 antibodies may thereby be
determined by the shuffle assay. The different s of CD137 used to create the
shuffle constructs should be chosen so that the monoclonal anti-human CD137
antibodies do not bind to the whole CD137 protein from these different species
(reference uct).
Determination of binding to human CD137 and mutants thereof may in particular
be performed with a monoclonal antibody comprising two second antigen-binding
regions according to the present invention.
In one embodiment, said second antigen-binding region comprises heavy and
light chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain le region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:8, 9 and 10, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:11, KAS
and 12, respectively, (CD137 clone 001),
b) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:15, 16 and 17, respectively, and light chain le region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:18, KAS
and 19, respectively, (CD137 clone 002),
c) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the ces set forth in SEQ ID , SAS
and 40, respectively, (CD137 clone 005),
d) heavy chain variable region CDR1, CDR2 and CDR3 having the ces set
forth in SEQ ID NOs:43, 44 and 45, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:46, AAS
and 47, respectively, (CD137 clone 006),
e) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS
and 68, respectively, (CD137 clone 009),
f) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:71, 72 and 73, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:74, KAS
and 75, respectively, (CD137 clone 010),
g) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:85, 86 and 87, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:88, SAS
and 89, respectively, (CD137 clone 012),
h) heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of
a) to g) having a total of one to twelve mutations, and
i) heavy and light chain variable region CDR1, CDR2 and CDR3 of an dy
which (i) competes for human CD137 binding with an antibody comprising heavy
and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to
h) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and
light chain variable region CDR1, CDR2 and CDR3 according to any of a) to h).
Hence, in one embodiment, said second antigen-binding region comprises heavy
and light chain variable region CDR1, CDRZ and CDR3 selected from the group consisting
a) heavy chain variable region CDR1, CDRZ and CDR3 having the sequences set
forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region
CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS
and 68, respectively, (CD137 clone 009),
b) heavy and light chain le region CDR1, CDRZ and CDR3 according to a)
having a total of one to twelve ons;
c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an antibody
which (i) competes for human CD137 binding with an antibody comprising heavy
and light chain variable region CDR1, CDRZ and CDR3 according to a) or b)
and/or (ii) has the icity for CD137 of an antibody comprising heavy and
light chain variable region CDR1, CDRZ and CDR3 according to a) or b).
In r embodiment, said second antigen-binding region comprises heavy and
light chain variable region CDR1, CDRZ and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDRZ and CDR3 having the sequences set
forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region
CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS
and 40, respectively, (CD137 clone 005),
b) heavy and light chain variable region CDR1, CDRZ and CDR3 according to a)
having a total of one to twelve mutations, and
c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an dy
which (i) competes for human CD137 binding with an antibody sing heavy
and light chain variable region CDR1, CDRZ and CDR3 ing to a) or b)
and/or (ii) has the specificity for CD137 of an antibody comprising heavy and
light chain variable region CDR1, CDRZ and CDR3 according to a) or b).
3O In a particular embodiment, said second antigen-binding region comprises heavy
chain variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID
NOs:64, 65 and 66, tively, and light chain variable region CDR1, CDRZ and CDR3
having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively. An example
of such an antibody includes, but is not limited to, the antibody referred to herein as
CD137 clone 009.
In another embodiment, said second antigen-binding region comprises heavy
chain variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID
NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDRZ and CDR3
having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively. An example
of such an antibody includes, but is not limited to, the antibody referred to herein as
CD137 clone 005.
In r embodiment, said second antigen-binding region comprises heavy
chain le region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID
, 65 and 66, respectively, and light chain variable region CDR1, CDRZ and CDR3
having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137
clone 009), having a total of one to twelve ons, such as one to ten mutations, or
one to eight mutations, or one to six mutations, or one to four mutations, or to two
mutations.
In another embodiment, said second antigen-binding region comprises heavy
chain le region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID
NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDRZ and CDR3
having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137
clone 005), having a total of one to twelve mutations, such as one to ten mutations, or
one to eight mutations, or one to six mutations, or one to four mutations, or one to two
mutations.
In one embodiment, said mutation may be an amino acid substitution, such as a
conservative amino acid tution.
In one embodiment, said mutations may be distributed across the VH CDR1, 2
and 3 and VL CDR 1, 2 and 3 so that each of the VH and VL CDR3 comprises at the most
three ons and each of the VH and VL CDRZ and CDRl comprises at the most two
amino acid modifications.
Hence, in a further embodiment, the second n-binding region comprises
heavy and light chain CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID
NOs:64, 65, 66, 67, GAS and 68, respectively, (CD137 clone 009), having a total of one
to twelve mutations and wherein the VH and VL CDR3 each comprises up to three amino
acid modifications, and the VH and VL CDRl and CDRZ each comprises up to two amino
acid modifications.
3O In a further embodiment, the second antigen-binding region comprises heavy and
light chain CDR1, CDRZ and CDR3 having the ces set forth in SEQ ID NOs:64, 65,
66, 67, GAS and 68, respectively, (CD137 clone 009), having a total of one to ten
mutations, such as one to eight, and wherein the VH and VL CDR1, CDRZ, and CDR3
each comprises up to two amino acid modifications.
In a further embodiment, the second antigen-binding region comprises heavy and
light chain CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:64, 65,
66, 67, GAS and 68, respectively, (CD137 clone 009), having a total of one to six
mutations, and wherein the VH and VL CDRl, CDRZ, and CDR3 each comprises at most
one amino acid modification.
In another embodiment, the second n-binding region comprises heavy and
light chain CDRl, CDRZ and CDR3 having the ces set forth in SEQ ID NOs:36, 37,
38, 39, SAS and 40, respectively, (CD137 clone 005), having a total of one to twelve
mutations and wherein the VH and VL CDR3 each comprises up to three amino acid
modifications, and the VH and VL CDR1 and CDRZ each comprises up to two amino acid
modifications.
In a further embodiment, the second antigen-binding region comprises heavy and
light chain CDRl, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:36, 37,
38, 39, SAS and 40, respectively, (CD137 clone 005), having a total of one to ten
mutations, such as one to eight, and wherein the VH and VL CDRl, CDRZ, and CDR3
each comprises up to two amino acid modifications.
In a further embodiment, the second antigen-binding region ses heavy and
light chain CDRl, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:36, 37,
38, 39, SAS and 40, respectively, (CD137 clone 005), having a total of one to six
ons, and wherein the VH and VL CDRl, CDRZ, and CDR3 each comprises at most
one amino acid modification.
In a further embodiment, there may be a total of one to twelve mutations; such
as one to ten mutations, or one to eight mutations, or one to six mutations, or one to
four mutations, or one to two mutations; and each CDR sequence comprises at the most
two amino acid substitutions.
It is well known to a person skilled in the art how to introduce mutations and that
certain amino acids of the CDR sequences may be mutated; e.g., by amino acid
substitutions to, e.g., increase affinity of the antibody to its target antigen or reducing
immunogenicity for non-human antibodies to be used for ent of humans. Such
mutations can be introduced t affecting the epitope of the target antigen to which
the antibody binds.
In one ment said second antigen-binding region comprises a second heavy
3O chain variable (VH) sequence, and a second light chain variable (VL) sequence and
wherein said variable sequences each comprises three CDR sequences, CDRl, CDRZ and
CDR3, respectively.
In one ment, said second antigen-binding region comprises a second
heavy chain variable (VH) sequence, and a second light chain variable (VL) sequence
and wherein said le sequences each comprises three CDR sequences, CDRl, CDRZ
and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4,
respectively.
In one embodiment, said VH sequence of the second antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to
an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:13 (CD137 clone 001)
c) a VH sequence as set forth in SEQ ID NO:20 (CD137 clone 002)
d) a VH sequence as set forth in SEQ ID NO:27 (CD137 clone 003)
e) a VH sequence as set forth in SEQ ID NO:34 (CD137 clone 004)
f) a VH sequence as set forth in SEQ ID NO:41 (CD137 clone 005)
g) a VH ce as set forth in SEQ ID NO:48 (CD137 clone 006)
h) a VH sequence as set forth in SEQ ID NO:55 (CD137 clone 007)
i) a VH sequence as set forth in SEQ ID NO:62 (CD137 clone 008)
j) a VH sequence as set forth in SEQ ID NO:69 (CD137 clone 009)
k) a VH ce as set forth in SEQ ID NO:76 (CD137 clone 010)
I) a VH ce as set forth in SEQ ID NO:83 (CD137 clone 011)
m) a VH sequence as set forth in SEQ ID NO:90 (CD137 clone 012)
In one embodiment, said VL sequence of the second antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
an amino acid sequence selected from the group consisting of:
a) a VL sequence as set forth in SEQ ID NO:127 (humanized CD137 clone 009)
b) a VL sequence as set forth in SEQ ID NO:14 (CD137 clone 001)
c) a VL sequence as set forth in SEQ ID NO:21 (CD137 clone 002)
d) a VL sequence as set forth in SEQ ID NO:28 (CD137 clone 003)
e) a VL ce as set forth in SEQ ID NO:35 (CD137 clone 004)
f) a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005)
g) a VL sequence as set forth in SEQ ID NO:49 (CD137 clone 006)
3O h) a VL sequence as set forth in SEQ ID NO:56 (CD137 clone 007)
i) a VL sequence as set forth in SEQ ID NO:63 (CD137 clone 008)
j) a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009)
k) a VL sequence as set forth in SEQ ID NO:77 (CD137 clone 010)
l) a VL sequence as set forth in SEQ ID NO:84 (CD137 clone 011)
m) a VL sequence as set forth in SEQ ID NO:91 (CD137 clone 012)
In one ment, said VH and VL sequences of the second antigen-binding
region each comprise an amino acid sequence having at least 70%, at least 75%, at
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least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or
100% ty to an amino acid sequence selected from the group consisting of:
a) '
a VH sequence as set forth in SEQ ID NO:123 and a VL sequence as set forth
SEQ ID NO:127 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:13 and a VL sequence as set forth in
SEQ ID NO:14 (CD137 clone 001)
C) a VH sequence as set forth in SEQ ID NO:20 and a VL sequence as set forth in
SEQ ID NO:21 (CD137 clone 002)
d) a VH sequence as set forth in SEQ ID NO:27 ancl a VL sequence as set forth in
SEQ ID NO:28 (CD137 clone 003)
a VH sequence as set forth in SEQ ID NO:34 and a VL sequence as set forth in
SEQ ID NO:35 (CD137 clone 004)
f) a VH sequence as set forth in SEQ ID NO:41 ancl a VL sequence as set forth in
SEQ ID NO:42 (CD137 clone 005)
9) a VH sequence as set forth in SEQ ID NO:48 and a VL sequence as set forth in
SEQ ID NO:49 (CD137 clone 006)
h) a VH sequence as set forth in SEQ ID NO:55 and a VL sequence as set forth in
SEQ ID NO:56 (CD137 clone 007)
a VH sequence as set forth in SEQ ID NO:62 and a VL sequence as set forth in
SEQ ID NO:63 (CD137 clone 008)
j) a VH sequence as set forth in SEQ ID NO:69 and a VL sequence as set forth in
SEQ ID NO:70 (CD137 clone 009)
k) a VH sequence as set forth in SEQ ID NO:76 and a VL ce as set forth in
SEQ ID NO:77 (CD137 clone 010)
I) a VH ce as set forth in SEQ ID NO:83 and a VL sequence as set forth in
SEQ ID NO:84 (CD137 clone 011)
m) a VH sequence as set forth in SEQ ID NO:90 and a VL sequence as set forth in
SEQ ID NO:91(CD137 clone 012).
In one embodiment, said VH sequence of the second antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:41 (CD137 clone 005)
c) a VH sequence as set forth in SEQ ID NO:69 (CD137 clone 009)
In one embodiment, said VL sequence of the second antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
an amino acid sequence selected from the group consisting of:
a) a VL sequence as set forth in SEQ ID NO:127 (humanized CD137 clone 009)
b) a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005)
c) a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009)
In one embodiment, said VH and VL sequences of the second antigen-binding
region each comprise an amino acid sequence having at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or
100% identity to an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 and a VL sequence as set forth in
SEQ ID NO:127 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:41 and a VL sequence as set forth in
SEQ ID NO:42 (CD137 clone 005)
c) a VH sequence as set forth in SEQ ID NO:69 and a VL sequence as set forth in
SEQ ID NO:70 (CD137 clone 009)
In one embodiment, said VH ce of the second antigen-binding region
ses an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID
NO:41 (CD137 clone 005).
In one embodiment, said VH sequence of the second antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97% or at least 99% ty to SEQ ID
NO:69 (CD137 clone 009).
In one embodiment, said VH sequence of the second antigen-binding region
ses an amino acid sequence having at least 70%, at least 75%, at least 80%, at
3O least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
SEQ ID NO:123 (humanized CD137 clone 009).
In one embodiment, said VL ce of the second n-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID
NO:42 (CD137 clone 005).
In one embodiment, said VL sequence of the second antigen-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID
NO:70 (CD137 clone 009).
In one embodiment said VL sequence of the second n-binding region
comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
SEQ ID NO:127 (humanized CD137 clone 009).
In one embodiment, said VH and said VL sequence of the second antigen-binding
region each comprise an amino acid sequence having at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%
ty to SEQ ID NO:41; and SEQ ID NO:42 (CD137 clone 005), respectively.
In one embodiment, said VH and VL sequence of the second antigen-binding
region each comprise an amino acid sequence having at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%
identity to SEQ ID NO:69 and SEQ ID NO:70 (CD137 clone 009), respectively.
In one ment, said VH and VL sequence of the second antigen-binding
region each comprise an amino acid sequence having at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or
100% identity to SEQ ID NO:123 and SEQ ID NO:127, respectively (humanized CD137
clone 009).
In one embodiment, said VH and VL sequences only e in the framework
sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 ork sequences
of the VH and VL sequences of the first and/or second antigen-binding region have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 99%, or 100% amino acid sequence ty to the respective FR1,
FR2, FR3 and FR4 framework sequences of said VH and VL sequences.
In one ment, the VH and VL sequences only deviate in the non-CDR
sequences as set forth in SEQ ID NO:41 and 42, respectively, (CD137 clone 005).
In one embodiment the VH and VL sequences only deviate in the non-CDR
3O sequences as set forth in SEQ ID NO:69 and 70, respectively, (CD137 clone 009).
In one embodiment the VH and VL sequences only deviate in the non-CDR
sequences as set forth in SEQ ID NO:123 and 127, respectively, (humanized CD137
clone 009).
In one embodiment, the VH and VL sequences only e in the framework
sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL sequences of the first and/or second antigen-binding region have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 99%, or 100% amino acid ce identity to the respective FR1,
FR2, FR3 and FR4 framework sequences of said VH and VL sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL sequences of the second antigen-binding region have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at
least 99% amino acid sequence identity to the tive FR1, FR2, FR3 and FR4
framework sequences of the VH sequence as set forth in SEQ ID NO:41, and VL
sequence as set forth in SEQ ID NO:42.
In one embodiment, the respective FR1, FRZ, FR3 and FR4 framework sequences
of the VH and VL sequences of the second antigen-binding region have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at
least 99% amino acid ce identity to the respective FR1, FR2, FR3 and FR4
framework sequences of the VH sequence as set forth in SEQ ID NO:41, and VL
sequence as set forth in SEQ ID NO:42, and the heavy and light chain variable region
CDRl, CDRZ and CDR3 of the second antigen-binding region have a total of one to
twelve mutations compared to the heavy and light chain variable region CDRl, CDRZ
and CDR3 having the sequences as set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40,
respectively. In a further embodiment said mutations may be as described above.
In an even r embodiment, the respective FR1, FR2, FR3 and FR4 framework
sequences of the VH and VL sequences of the second antigen-binding region have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3
and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:41, and VL
ce as set forth in SEQ ID NO:42, and the second antigen-binding region
comprises heavy and light chain variable region CDRl, CDRZ and CDR3 having the
sequences as set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40, respectively.
In one embodiment the respective FR1, FR2, FR3 and FR4 ork sequences
of the VH and VL sequences of the second antigen-binding region have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at
3O least 99% amino acid sequence identity to the tive FR1, FR2, FR3 and FR4
framework sequences of the VH sequence as set forth in SEQ ID NO:69, and VL
sequence as set forth in SEQ ID NO:70.
In one embodiment the respective FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL ces of the second antigen-binding region have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at
least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4
framework sequences of the VH sequence as set forth in SEQ ID NO:69, and VL
sequence as set forth in SEQ ID NO:70, and the heavy and light chain variable region
CDRl, CDRZ and CDR3 of the second antigen-binding region have a total of one to
twelve ons compared to the heavy and light chain variable region CDRl, CDRZ
and CDR3 having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68,
respectively. In a r embodiment said mutations may be as described above.
In an even further embodiment the respective FR1, FR2, FR3 and FR4 framework
sequences of the VH and VL sequences of the second antigen-binding region have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, or at least 99% amino acid ce identity to the respective FR1, FR2, FR3
and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:69, and VL
sequence as set forth in SEQ ID NO:70, and the second antigen-binding region
comprises heavy and light chain variable region CDRl, CDRZ and CDR3 having the
sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL sequences of the second antigen-binding region have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 99% or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and
FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:123, and VL
sequence as set forth in SEQ ID NO:127.
In one embodiment the respective FR1, FR2, FR3 and FR4 framework sequences
of the VH and VL sequences of the second antigen-binding region have at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at
least 99% or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and
FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:123, and VL
sequence as set forth in SEQ ID NO:127, and the heavy and light chain le region
CDRl, CDRZ and CDR3 of the second n-binding region have a total of one to
twelve mutations compared to the heavy and light chain variable region CDRl, CDRZ
and CDR3 having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68,
respectively. In a r embodiment said mutations may be as described above.
In an even further embodiment the respective FR1, FR2, FR3 and FR4 framework
3O sequences of the VH and VL sequences of the second antigen-binding region have at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 99% or 100% amino acid sequence ty to the respective FR1,
FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID
NO:123, and VL ce as set forth in SEQ ID NO:127, and the second antigen-
binding region comprises heavy and light chain variable region CDRl, CDRZ and CDR3
having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68,
respectively.
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In one embodiment, said VH sequence of the second antigen-binding region
comprises SEQ ID NO:123 (humanized CD137 clone 009).
In one embodiment, said VL sequence of the second antigen-binding region
comprises SEQ ID NO:127 (humanized CD137 clone 009).
In one embodiment, said VH and VL sequences of the second antigen-binding
region comprise SEQ ID NO:123 and SEQ ID NO:127, respectively.
In one embodiment, said VH sequence of the second antigen-binding region
comprises a VH sequence selected from the group consisting of:
a) SEQ ID NO:41 (CD137 clone 005)
b) SEQ ID NO:69 (CD137 clone 009)
In one embodiment, said VL sequence of the second antigen-binding region
comprises a VL sequence selected from the group ting of:
a) SEQ ID NO:42 (CD137 clone 005)
b) SEQ ID NO:70 (CD137 clone 009)
In one embodiment, said VH and VL sequences of the second antigen-binding are
selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:41 and a VL sequence as set forth in
SEQ ID No:42 (CD137 clone 005),
b) a VH sequence as set forth in SEQ ID NO:69 and a VL sequence as set forth in
SEQ ID NO:70 (CD137 clone 009).
In one embodiment, the multispecific dy according to the present invention
comprises a second binding arm comprising said second n-binding region.
In one embodiment, the multispecific antibody according to the present invention
comprises a second binding arm comprising said -antigen-binding region and a
second heavy chain constant ce.
In one embodiment, the multispecific antibody according to the present invention
comprises a second binding arm comprising said second antigen-binding region, wherein
the second binding arm comprises a second heavy chain comprising a second heavy
3O chain variable (VH) sequence and a second heavy chain constant (CH) ce, and a
second light chain comprising a second light chain variable (VL) sequence.
In one embodiment said second light chain further comprises a second light chain
constant sequence.
In a further ment, said second heavy chain comprises at least a hinge
region, a CH2 and a CH3 region.
In a ic embodiment, the multispecific antibody according to the present
invention comprises a second Fab-arm comprising said second n-binding region.
In one embodiment, the second antigen-binding region is derived from a rabbit
antibody, such as any of anti-CD137 clones 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, in
particular any of clones 5 and 9, disclosed herein.
In one embodiment, the second antigen-binding region is derived from a chimeric
antibody, such as an antibody comprising a variable region from any of the anti-CD137
clones 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, in particular any of clones 5 and 9,
disclosed .
In one embodiment, the second antigen-binding region is derived from a
humanized antibody.
In one embodiment, the second binding arm is d from a full-length
antibody.
In one embodiment the second binding arm is derived from a full-length IgGl,)\
a) or IgGl,K (kappa) antibody.
In one embodiment, the second binding arm is derived from a monoclonal
antibody.
In one embodiment, said second heavy chain is of an IgG isotype, optionally
having a subclass selected from the group consisting of IgGl, IgGZ, IgG3, and IgG4.
In one embodiment, the first binding arm may be d from an antibody
comprising a HC comprising SEQ ID NO:124 and an LC comprising SEQ ID NO:128,
optionally with one or more mutations in the constant region of the HC, such as 1 to 10,
such as 1 to 5, such as 1, 2, 3, 4 or 5 mutations.
In one embodiment, the first binding arm comprises a HC sing SEQ ID
NO:124, 125 or 126 and an LC comprising SEQ ID NO:128.
Binding to CD40 and CD137
In some embodiments, the present invention relates to a multispecific antibody
comprising:
(I) a first antigen-binding region binding to human CD40, n said first n-
binding region comprises heavy and light chain le regions selected from the group
consisting of:
3O a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and
, respectively,
b) heavy and light chain variable region CDR1, CDR2 and CDR3 ing to a)
having a total of one to twelve mutations; and
C) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody
which (i) competes for human CD40 binding with an antibody comprising heavy
and light chain variable region CDR1, CDR2 and CDR3 according to a) or b)
and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light
chain variable region CDR1, CDR2 and CDR3 according to a) or b), and
(II) a second antigen-binding region binding to human CD137, wherein said second
antigen-binding region comprises heavy and light chain variable regions selected from
the group consisting of:
X) heavy chain variable region CDR1, CDR2 and CDR3 having the ces set
forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the ces set forth in SEQ ID NOs:67, GAS
and 68, respectively, (CD137 clone 009),
y) heavy and light chain variable region CDR1, CDR2 and CDR3 according to x)
having a total of one to twelve mutations; and
heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody
which (i) competes for human CD137 binding with an antibody comprising heavy
and light chain variable region CDR1, CDR2 and CDR3 according to X) or y)
and/or (ii) has the icity for CD137 of an antibody sing heavy and
light chain variable region CDR1, CDR2 and CDR3 according to x) or y).
In another embodiment, the present invention relates to a multispecific antibody
sing:
(I) a first antigen-binding region binding to human CD40, n said first antigen-
binding region comprises heavy and light chain variable regions selected from the group
consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and
, respectively,
3O b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a)
having a total of one to twelve mutations; and
heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody
which (i) es for human CD40 binding with an antibody comprising heavy
and light chain variable region CDR1, CDR2 and CDR3 according to a) or b)
and/or (ii) has the specificity for CD40 of an dy comprising heavy and light
chain variable region CDR1, CDR2 and CDR3 according to a) or b), and
(II) a second antigen-binding region binding to human CD137, wherein said second
antigen-binding region comprises heavy and light chain le regions selected from
the group consisting of:
x) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS
and 40, respectively, (CD137 clone 005),
y) heavy and light chain variable region CDR1, CDR2 and CDR3 ing to x)
having a total of one to twelve mutations; and
2) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody
which (i) competes for human CD137 binding with an antibody comprising heavy
and light chain variable region CDR1, CDR2 and CDR3 ing to X) or y)
and/or (ii) has the specificity for CD137 of an antibody comprising heavy and
light chain variable region CDR1, CDR2 and CDR3 according to x) or y).
Hence, in one embodiment, said first antigen-binding region comprises heavy
chain variable region CDR1, CDR2 and CDR3 having the amino acid sequences set forth
in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and
CDR3 having the amino acid sequences set forth in SEQ ID NOs:4, YTS and 5,
respectively; and said second antigen-binding region comprises
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS
and 68, respectively (CD136 clone 009), or
b) heavy chain le region CDR1, CDR2 and CDR3 having the sequences set
forth in SEQ ID NOs:36, 37 and 38, tively, and light chain variable region
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS
and 40 (CD137 clone 005), respectively.
3O In another embodiment, said first antigen-binding region ses heavy chain
variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1,
2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the
sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-
binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the
sequences set forth in SEQ ID NOs:64, 65 and 66, tively, and light chain variable
region CDR1, CDR2 and CDR3 having the ces set forth in SEQ ID NOs:67, GAS
and 68, respectively, (CD137 clone 009).
In another embodiment said first antigen-binding region comprises heavy chain
variable region CDRl, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:1,
2 and 3, respectively, and light chain variable region CDRl, CDRZ and CDR3 having the
sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second n-
binding region comprises heavy chain variable region CDRl, CDRZ and CDR3 having the
sequences set forth in SEQ ID NOs:36, 37 and 38, tively, and light chain variable
region CDRl, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS
and 40, tively, (CD137 clone 005).
In a further ment, said first antigen-binding region of the multispecific
antibody according to the present invention comprises a first heavy chain le (VH)
sequence, and a first light chain variable (VL) sequence, and said second antigen-binding
region of the multispecific antibody according to the t invention comprises a
second heavy chain variable (VH) sequence, and a second light chain variable (VL)
sequence and wherein said variable sequences each comprise three CDR sequences,
CDRl, CDRZ and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and
FR4, respectively.
In a further embodiment said VH and VL sequence of the first antigen-binding
region each comprises a sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to the
amino acid sequence of the VH sequence as set forth in SEQ ID NO:6 and the VL
sequence as set forth in SEQ ID NO:7, respectively, and said VH and said VL sequence of
the second antigen-binding region each ses a sequence having at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at
least 99% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID
NO:41 and the VL sequence as set forth in SEQ ID NO:42, respectively, (CD137 clone
005).
In another further embodiment, said VH and VL sequence of the first n-
binding region each comprises a sequence having at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to
3O the amino acid sequence of the VH sequence as set forth in SEQ ID NO:6 and the VL
sequence as set forth in SEQ ID NO:7, respectively, and said VH and said VL sequence of
the second antigen-binding region each comprises a sequence having at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at
least 99% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID
NO:69 and the VL sequence as set forth in SEQ ID NO:70, respectively, (CD137 clone
009).
In another further embodiment, said VH and VL sequence of the first antigen-
binding region each comprises a sequence having at least 70%, at least 75%, at least
WO 11421
80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%
ty to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:117
and the VL sequence as set forth in SEQ ID NO:121, tively, and said VH and said
VL sequence of the second n-binding region each comprise a sequence having at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 99% or 100% identity to the amino acid sequence of the VH
sequence as set forth in SEQ ID NO:123 and the VL sequence as set forth in SEQ ID
NO:127, respectively, ized CD137 clone 009).
In a particular embodiment, the present invention relates to a ific antibody
comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy
chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first
light chain comprising a first light chain variable (VL) sequence and a first light chain
constant (CL) sequence, and wherein said heavy first chain variable sequence comprises
CDR1, CDR2 and CDR3 having the ces set forth in SEQ ID NOs:1, 2 and 3,
respectively, and said first light chain sequence comprises CDR1, CDR2 and CDR3 having
the ces set forth in SEQ ID NOs:4, YTS and 5, respectively, and
(II) a second binding arm comprising a second heavy chain comprising a second
heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence,
and a second light chain further ses a second light chain constant (CL) sequence,
and a second light chain variable (VL) sequence, wherein said second heavy chain
variable sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in
SEQ ID NOs:64, 65 and 66, respectively, and said second light chain sequence
comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67,
GAS and 68, respectively, (CD137 clone 009);
wherein the first and second heavy chain are of a human IgGl isotype and
wherein the first and second light chain is of IgGl,K, and wherein the positions
corresponding to positions L234, L235, and D265 in a human IgGl heavy chain
3O according to EU numbering of both the first and second constant heavy chain are F, E,
and A, respectively, and wherein (a) the position corresponding to F405 in a human
IgGl heavy chain according to EU numbering of the first constant heavy chain is L, and
the position corresponding to K409 in a human IgGl heavy chain according to EU
numbering of the second constant heavy chain is R; or (b) the position corresponding to
K409 in a human IgGl heavy chain according to EU numbering of the first constant
heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain
according to EU ing of the second constant heavy chain is L.
In a further ular embodiment, the t invention relates to a bispecific
antibody comprising
(I) a first binding arm comprising a first heavy chain sing a first heavy
chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first
light chain comprising a first light chain variable (VL) sequence and a first light chain
constant (CL) sequence, and wherein said heavy first chain variable sequence comprises
CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3,
respectively, and said first light chain sequence comprises CDR1, CDR2 and CDR3 having
the ces set forth in SEQ ID NOs:4, YTS and 5, respectively, and
(II) a second binding arm comprising a second heavy chain sing a second
heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence,
and a second light chain further comprises a second light chain constant (CL) sequence,
and a second light chain variable (VL) sequence, n said second heavy chain
variable sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in
SEQ ID NOs:36, 37 and 38, respectively, and said second light chain sequence
comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39,
SAS and 40, respectively, (CD137 clone 005); and
wherein the first and second heavy chain are of a human IgGl isotype and
wherein the first and second light chain is of IgGl,K, and n the positions
corresponding to positions L234, L235, and D265 in a human IgGl heavy chain
according to EU ing of both the first and second constant heavy chain are F, E,
and A, respectively, and wherein (a) the position corresponding to F405 in a human
IgGl heavy chain according to EU numbering of the first constant heavy chain is L, and
the position corresponding to K409 in a human IgGl heavy chain according to EU
numbering of the second constant heavy chain is R; or (b) the position corresponding to
K409 in a human IgGl heavy chain according to EU numbering of the first constant
heavy chain is R, and the position ponding to F405 in a human IgGl heavy chain
according to EU numbering of the second constant heavy chain is L.
3O In a specific embodiment, the present invention relates to a bispecific antibody
comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy
chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first
light chain comprising a first light chain variable (VL) sequence and a first light chain
constant (CL) sequence, and wherein said first VH and VL sequences each comprises a
sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence
of the VH sequence as set forth in SEQ ID NO:117 and the VL sequence as set forth in
SEQ ID NO:121, respectively, and
(II) a second binding arm comprising a second heavy chain sing a second
heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence,
and a second light chain further comprises a second light chain constant (CL) sequence,
and a second light chain le (VL) sequence, wherein said second VH and VL
sequences comprise a sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
the amino acid sequence of the VH ce as set forth in SEQ ID NO:123 and the VL
sequence as set forth in SEQ ID NO:127, respectively, (humanized CD137 clone 009).
In a r specific embodiment, the present invention s to a bispecific
antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy
chain variable (VH) sequence and a first heavy chain nt (CH) sequence, and a first
light chain comprising a first light chain le (VL) sequence and a first light chain
nt (CL) sequence, and wherein said first VH sequence comprises SEQ ID NO:117
and said first VL sequence comprises SEQ ID NO:121, and
(II) a second binding arm comprising a second heavy chain comprising a second
heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence,
and a second light chain further comprises a second light chain constant (CL) sequence,
and a second light chain variable (VL) sequence, wherein said second VH sequence
comprises SEQ ID NO:123 and said second VL sequence comprises SEQ ID NO:127,
ized CD137 clone 009).
In a specific embodiment, the present invention relates to a bispecific antibody
comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy
chain variable (VH) ce and a first heavy chain constant (CH) sequence, and a first
light chain comprising a first light chain variable (VL) sequence and a first light chain
constant (CL) sequence, and wherein said first VH and VL ces comprise a
3O sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence
of the VH sequence as set forth in SEQ ID NO:117 and the VL sequence as set forth in
SEQ ID NO:121, respectively, and
(II) a second g arm comprising a second heavy chain comprising a second
heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence,
and a second light chain further comprises a second light chain constant (CL) sequence,
and a second light chain variable (VL) sequence, wherein said second VH and VL
sequences comprise a sequence having at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to
the amino acid sequence of the VH sequence as set forth in SEQ ID NO:123 and the VL
sequence as set forth in SEQ ID , respectively, (humanized CD137 clone 009),
wherein the first and second heavy chain are of a human IgGl isotype and
wherein the first and second light chain is of IgGl,K, and n the positions
corresponding to positions L234, L235, and D265 in a human IgGl heavy chain
according to EU numbering of both the first and second constant heavy chain are F, E,
and A, respectively, and n (a) the position corresponding to F405 in a human
IgGl heavy chain according to EU numbering of the first constant heavy chain is L, and
the on corresponding to K409 in a human IgGl heavy chain according to EU
numbering of the second constant heavy chain is R; or (b) the position corresponding to
K409 in a human IgGl heavy chain according to EU numbering of the first constant
heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain
according to EU numbering of the second nt heavy chain is L.
In a further specific ment, the present invention relates to a bispecific
antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy
chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first
light chain comprising a first light chain variable (VL) ce and a first light chain
constant (CL) sequence, and wherein said first VH ce comprises SEQ ID NO:117
and said first VL sequence comprises SEQ ID NO:121, and
(II) a second binding arm comprising a second heavy chain comprising a second
heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence,
and a second light chain further comprises a second light chain constant (CL) sequence,
and a second light chain variable (VL) sequence, wherein said second VH sequence
comprises SEQ ID NO:123 and said second VL ce comprises SEQ ID NO:127,
(humanized CD137 clone 009),
wherein the first and second heavy chain are of a human IgGl isotype and
wherein the first and second light chain is of IgGl,K, and wherein the positions
3O corresponding to positions L234, L235, and D265 in a human IgGl heavy chain
according to EU numbering of both the first and second constant heavy chain are F, E,
and A, respectively, and wherein (a) the position corresponding to F405 in a human
IgGl heavy chain according to EU numbering of the first constant heavy chain is L, and
the position corresponding to K409 in a human IgGl heavy chain according to EU
numbering of the second nt heavy chain is R; or (b) the position corresponding to
K409 in a human IgGl heavy chain according to EU ing of the first constant
heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain
according to EU numbering of the second constant heavy chain is L.
In another aspect, the present invention relates to a bispecific antibody
sing a first binding arm binding to human CD40 and a second binding arm binding
to human CD137, wherein
(i) said first binding arm ses a heavy chain (HC) amino acid sequence
comprising or consisting of SEQ ID NO:118 and a light chain (LC) amino acid sequence
comprising or consisting of SEQ ID NO:122, and
(ii) said second binding arm comprises a HC amino acid sequence comprising or
consisting of SEQ ID NO:124 and a LC amino acid sequence comprising or consisting of
SEQ ID NO:128,
ally wherein SEQ ID NOS:118, SEQ ID NO:124 or both comprise one or
more mutations in the constant region of the HC, such as 1 to 10, such as 1 to 5, such
as 1, 2, 3, 4 or 5 mutations.
In another aspect, the present invention s to a bispecific antibody
comprising a first binding arm binding to human CD40 and a second g arm g
to human CD137, wherein
(i) said first binding arm ses a HC amino acid sequence sing or
consisting of SEQ ID NO:119 and a LC amino acid sequence comprising or ting of
SEQ ID NO:122, and
(ii) said second binding arm comprises a HC amino acid sequence comprising or
ting of SEQ ID NO:125 and a LC amino acid sequence comprising or consisting of
SEQ ID NO:128.
In another aspect, the present ion relates to a bispecific dy
comprising a first binding arm binding to human CD40 and a second binding arm binding
to human CD137, wherein
(i) said first binding arm comprises a HC amino acid sequence comprising or
consisting of SEQ ID NO:120 and a LC amino acid sequence comprising or consisting of
SEQ ID NO:122, and
(ii) said second binding arm comprises a HC amino acid sequence comprising or
consisting of SEQ ID NO:126 and a LC amino acid sequence comprising or consisting of
3O SEQ ID NO:128.
Bispecific formats
In a particular embodiment the multispecific antibody according to the present
invention is a bispecific antibody.
The present invention provides bispecific CD40xCD137 antibodies which are able
of cross-linking cells expressing CD40 and cells expressing CD137; such as antigenpresenting
cells and T cells, respectively. Depending on the desired functional properties
for a particular use, particular antigen-binding s can be selected from the set of
antibodies or antigen-binding regions provided by the present invention. Many different
formats and uses of bispecific antibodies are known in the art, and were reviewed by
Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar-
Apr;4(2):182-97.
A bispecific antibody according to the present invention is not limited to any
particular bispecific format or method of producing it.
Examples of bispecific antibody molecules which may be used in the present
invention comprise (i) a single antibody that has two binding arms comprising different
antigen-binding regions; (ii) a single chain antibody that has specificity to two different
epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-
variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains
two variable domains in tandem through a short peptide linkage (Wu et al., Generation
and Characterization of a Dual Variable Domain Immunoglobulin gTM) Molecule,
In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linked
bispecific (Fab’)2 fragment; (v) a Tandab, which is a fusion of two single chain ies
resulting in a tetravalent ific antibody that has two binding sites for each of the
target antigens; (vi) a flexibody, which is a ation of scFvs with a diabody
resulting in a multivalent molecule; (vii) a so-called “dock and lock” le, based on
the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs,
can yield a trivalent bispecific g protein consisting of two identical Fab fragments
linked to a different Fab fragment; (viii) a led Scorpion molecule, comprising, e.g.,
two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.
In one embodiment, the bispecific antibody of the t invention is a diabody,
a cross-body, or a bispecific antibody obtained via a lled Fab-arm exchange (such
as described in W02011131746 (Genmab)).
Examples of different classes of ific antibodies include, but are not limited
to, (i) IgG-like les with complementary CH3 domains to force heterodimerization;
(ii) recombinant IgG-like dual targeting les, wherein the two sides of the molecule
3O each contain the Fab fragment or part of the Fab fragment of at least two different
dies; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to
extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single
chain Fv les or stabilized diabodies are fused to heavy-chain constant-domains,
ions or parts thereof; (v) Fab fusion molecules, wherein different Fab-fragments
are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof;
and (vi) ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies,
nanobodies) wherein different single chain Fv molecules or different diabodies or
different chain antibodies (e.g. domain antibodies, nanobodies) are fused to each
other or to another n or carrier molecule fused to heavy-chain constant-domains,
Fc-regions or parts thereof.
Examples of IgG-like molecules with complementary CH3 domain molecules
include, but are not limited to, the Triomab/Quadroma molecules (Trion
/Fresenius Biotech; Roche, W02011069104), the so-called Knobs-into-Holes
molecules tech, WO9850431), CrossMAbs (Roche, W02011117329) and the
electrostatically-matched molecules (Amgen, EP1870459 and W02009089004; Chugai,
U8201000155133; Oncomed, W02010129304), the LUZ-Y molecules (Genentech,
Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869.
Epub 2012 Nov 1), DIG-body and PIG-body molecules abcine, W02010134666,
W02014081202), the Strand Exchange Engineered Domain body (SEEDbody) molecules
(EMD Serono, W02007110205), the Biclonics molecules (Merus, W02013157953),
FcAAdp molecules (Regeneron, W0201015792), bispecific IgGl and IgGZ molecules
(Pfizer/Rinat, 3545), Azymetric ld molecules (Zymeworks/Merck,
W02012058768), mAb-Fv molecules (Xencor, W02011028952), bivalent bispecific
antibodies (W02009080254) and the DuoBody® molecules (Genmab A/S,
W02011131746).
Examples of recombinant IgG-like dual targeting molecules include, but are not
d to, Dual Targeting g molecules (W02009058383), Two-in-one Antibody
(Genentech; Bostrom, et al 2009. Science 323, 1610—1614.), Cross-linked Mabs
(Karmanos Cancer Center), mAb2 (F-Star, W02008003116), Zybody les
(Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), approaches with common
light chain (Crucell/Merus, US7,262,028), KABodies (Novamune, W02012023053) and
Con-body (Con/Pfizer; Doppalapudi, V.R., et al 2007. Bioorg. Med. Chem. Lett.
17,501—506.).
Examples of IgG fusion molecules include, but are not d to, Dual Variable
Domain (DVD)-Ig les (Abbott, US7,612,181), Dual domain double head
antibodies (Unilever; Sanofi Aventis, W020100226923), IgG-like Bispecific molecules
(ImClone/Eli Lilly, Lewis et al. Nat Biotechnol. 2014 (2):191-8), Ts2Ab
3O (MedImmune/AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb
molecules (Zymogenetics, W02010111625), HERCULES molecules (Biogen Idec,
USOO7951918), scFv fusion les (Novartis), scFv fusion molecules (Changzhou
Adam Biotech Inc, CN 246) and Tva les (Roche, W02012025525,
W02012025530).
Examples of Fc fusion molecules include, but are not limited to, ScFv/Fc Fusions
(Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules
(Emergent BioSolutions/Trubion, nship JW, et al. AACR 100th Annual meeting
2009 (Abstract # 5465); netics/BMS, W02010111625), Dual Affinity Retargeting
Technology (Fc-DART) molecules (MacroGenics, W02008157379, W02010080538) and
Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine — China).
Examples of Fab fusion bispecific antibodies include, but are not limited to,
F(ab)2 molecules (MedareX/AMGEN; Deo et al J Immunol. 1998 Feb (4):1677-
86.), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323,
1610—1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, W02003074569,
W02005004809), Bivalent Bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000
Dec (12):7050-7.) and Fab-Fv molecules elltech, WO 2009040562 A1).
Examples of ScFv-, diabody-based and domain antibodies include, but are not
limited to, Bispecific T Cell Engager (BiTE) molecules (Micromet, 061547),
Tandem Diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004
Apr;17(4):357-66.), Dual Affinity Retargeting Technology (DART) molecules
(MacroGenics, W02008157379, W02010080538), Single-chain Diabody molecules
(Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like Antibodies (AIT,
ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, W02010059315) and
COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010
(6):667-75.), dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010)
and dual targeting heavy chain only domain dies.
In one embodiment, each of said first and second heavy chains ses at least
a hinge region, a CH2 and a CH3 region.
In a further embodiment, the CH3 regions of the first and second heavy chains
comprise asymmetrical ons, such as trical mutations (also referred to as
modifications herein) yielding a stable heterodimeric antibody.
In one embodiment, the bispecific antibody of the invention comprises a first
heavy chain comprising a first CH3 region, and a second heavy chain comprising a
second CH3 region, wherein the sequences of the first and second CH3 regions are
different and are such that the heterodimeric interaction n said first and second
CH3 regions is stronger than each of the meric interactions of said first and
second CH3 regions. More details on these interactions and how they can be achieved
3O are ed in, e.g.,
hereby incorporated by reference.
As described further herein, a stable bispecific CD40xCD137 antibody can be
obtained at high yield using a particular method on the basis of one homodimeric
parental CD40 antibody and one homodimeric al CD137 dy containing only
a few, fairly conservative, asymmetrical mutations in the CH3 regions. Asymmetrical
mutations mean that the sequences of said first and second CH3 regions contain amino
acid substitutions at non-identical positions.
Accordingly, in one embodiment of the bispecific dy as defined in any of the
embodiments disclosed herein, the sequences of said first and second heavy chain CH3
regions contain asymmetrical ons, e.g., a mutation at the position corresponding
to position 405 in a human IgG1 heavy chain ing to EU numbering in one of the
CH3 regions, and a mutation at the position corresponding to position 409 in a human
IgGl heavy chain according to EU numbering in the other CH3 region.
In one aspect, the bispecific antibody as defined in any of the embodiments
disclosed herein comprises first and second heavy chains, wherein in said first heavy
chain at least one of the amino acids in the positions corresponding to a position
selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in
a human IgGl heavy chain according to EU numbering has been substituted, and in said
second heavy chain at least one of the amino acids in the positions corresponding to a
position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and
K409 in a human IgGl heavy chain according to EU numbering has been substituted,
and wherein said first and said second heavy chains are not substituted in the same
In one embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, the first heavy chain has an amino acid substitution at a
position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409 in
a human IgGl heavy chain according to EU numbering, and the second heavy chain has
an amino acid substitution at a position selected from the group consisting of: 366, 368,
370, 399, 405, 407 and 409 in a human IgGl heavy chain according to EU numbering,
and wherein the first and second heavy chains are not substituted in the same positions.
In one embodiment of the bispecific antibody as defined in any of the
embodiments sed , the first heavy chain has an amino acid tution at
position 366, and said second heavy chain has an amino acid substitution at a on
selected from the group ting of: 368, 370, 399, 405, 407 and 409. In one
embodiment the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val,
or Gln.
3O In one embodiment of the bispecific antibody as defined in any of the
embodiments sed herein, the first heavy chain has an amino acid substitution at
position 368, and said second heavy chain has an amino acid substitution at a position
selected from the group consisting of:366, 370, 399, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the
ments disclosed herein, the first heavy chain has an amino acid substitution at
position 370, and said second heavy chain has an amino acid substitution at a position
selected from the group consisting of:366, 368, 399, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the
ments disclosed herein, the first heavy chain has an amino acid substitution at
position 399, and said second heavy chain has an amino acid substitution at a position
selected from the group ting of:366, 368, 370, 405, 407 and 409.
In one embodiment of the bispecific antibody as d in any of the
embodiments disclosed herein, the first heavy chain has an amino acid substitution at
position 405, and said second heavy chain has an amino acid substitution at a position
selected from the group ting of:366, 368, 370, 399, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, the first heavy chain has an amino acid substitution at
position 407, and said second heavy chain has an amino acid substitution at a position
selected from the group consisting of:366, 368, 370, 399, 405, and 409.
In one embodiment of the ific antibody as defined in any of the
embodiments disclosed , the first heavy chain has an amino acid substitution at
position 409, and said second heavy chain has an amino acid substitution at a position
selected from the group consisting of:366, 368, 370, 399, 405, and 407.
Accordingly, in one ment of the bispecific antibody as defined in any of the
embodiments disclosed herein, the sequences of said first and second CH3 regions
contain asymmetrical mutations, i.e. mutations at different positions in the two CH3
regions, e.g. a mutation at position 405 in one of the CH3 s and a mutation at
position 409 in the other CH3 region.
In one embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, the first heavy chain has an amino acid other than Lys,
Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,
Tyr, or Cys, at on 409 and said second heavy chain has an amino-acid substitution
at a position ed from the group consisting of:366, 368, 370, 399, 405 and 407. In
one such embodiment, said first heavy chain has an amino acid other than Lys, Leu or
Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or
Cys, at position 409 and said second heavy chain has an amino acid other than Phe, e.g.
3O Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or
Leu, at position 405. In a further embodiment hereof, said first heavy chain has an
amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His,
Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain
has an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys,
His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405.
In another embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain comprises a Phe at position 405
and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg,
His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy
chain comprises an amino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg,
His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, at position 405 and a Lys at
position 409. In a further embodiment hereof, said first heavy chain comprises a Phe at
position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser,
Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said
second heavy chain comprises an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala,
Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405
and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain comprises a Phe at position 405
and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg,
His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy
chain comprises a Leu at position 405 and a Lys at position 409. In a further
ment hereof, said first heavy chain comprises a Phe at position 405 and an Arg at
on 409 and said second heavy chain comprises an amino acid other than Phe, Arg
or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr,
or Cys, at position 405 and a Lys at position 409. In another embodiment, said first
heavy chain comprises Phe at position 405 and an Arg at position 409 and said second
heavy chain comprises a Leu at position 405 and a Lys at position 409.
In a further embodiment of the bispecific dy as defined in any of the
embodiments disclosed , said first heavy chain comprises an amino acid other
than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln,
Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises a Lys at
position 409, a Thr at on 370 and a Leu at position 405. In a further embodiment,
said first heavy chain comprises an Arg at on 409 and said second heavy chain
comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In an even further embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain comprises a Lys at position 370, a
3O Phe at position 405 and an Arg at position 409 and said second heavy chain ses a
Lys at position 409, a Thr at position 370 and a Leu at position 405.
In r embodiment of the bispecific antibody as defined in any of the
embodiments sed herein, said first heavy chain comprises an amino acid other
than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln,
Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises a Lys at
position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at
position 370 and a Leu at position 405.
WO 11421
In another ment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain comprises an Arg at position 409
and said second heavy chain comprises a Lys at position 409 and: a) an Ile at position
350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain comprises a Thr at on 350, a
Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second
heavy chain comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at
position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as d in any of the
embodiments disclosed herein, said first heavy chain ses a Thr at position 350, a
Lys at position 370, a Phe at on 405 and an Arg at position 409 and said second
heavy chain comprises an Ile at on 350, a Thr at position 370, a Leu at position
405 and a Lys at position 409.
In one embodiment of the bispecific antibody as defined in any of the
embodiments disclosed , said first heavy chain has an amino acid other than Lys,
Leu or Met at position 409 and said second heavy chain has an amino acid other than
Phe at position 405, such as other than Phe, Arg or Gly at position 405; or said first CH3
region has an amino acid other than Lys, Leu or Met at position 409 and said second
CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at
position 407.
In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein comprises a first heavy chain having an amino acid other than Lys, Leu
or Met at position 409 and a second heavy chain having an amino acid other than Tyr,
Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at on 407.
In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein comprises a first heavy chain having a Tyr at position 407 and an amino
acid other than Lys, Leu or Met at position 409 and a second heavy chain having an
amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a
3O Lys at position 409.
In one embodiment, the bispecific antibody as defined in any of the ments
disclosed herein comprises a first heavy chain having a Tyr at position 407 and an Arg at
position 409 and a second heavy chain having an amino acid other than Tyr, Asp, Glu,
Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.
In another embodiment, said first heavy chain has an amino acid other than Lys,
Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,
Tyr, or Cys, at position 409 and said second heavy chain has an amino acid other than
Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn,
Pro, Trp, or Cys, at position 407. In another embodiment, said first heavy chain has an
amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His,
Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at on 409 and said second heavy chain
has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.
In another embodiment of the bispecific antibody as d in any of the
embodiments disclosed herein, said first heavy chain has an amino acid other than Lys,
Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,
Tyr, or Cys, at position 409 and said second heavy chain has a Gly, Leu, Met, Asn or Trp
at position 407.
In another embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an
amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His,
Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain
has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met,
Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an
amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His,
Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain
has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position
In another ment of the bispecific dy as defined in any of the
embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an
amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His,
Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain
has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the
ments disclosed herein, said first heavy chain has a Tyr at position 407 and an
Arg at position 409 and said second heavy chain has an amino acid other than Tyr, Asp,
3O Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or
Cys, at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the
embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an
Arg at position 409 and said second heavy chain has an Ala, Gly, His, Ile, Leu, Met, Asn,
Val or Trp at position 407 and a Lys at position 409.
In r embodiment of the bispecific antibody as d in any of the
embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an
Arg at position 409 and said second heavy chain has a Gly, Leu, Met, Asn or Trp at
position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the
ments sed herein, the first heavy chain has an amino acid other than Lys,
Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp,
Tyr, or Cys, at position 409, and the second heavy chain has
(i) an amino acid other than Phe, Leu and Met, e.g. Gly, Ala, Val, Ile, Ser, Thr,
Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 368, or
(ii) a Trp at position 370, or
(iii) an amino acid other than Asp, Cys, Pro, Glu or Gln, e.g. Phe, Leu, Met, Gly,
Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys, at position 399 or
(iv) an amino acid other than Lys, Arg, Ser, Thr, or Trp, e.g. Phe, Leu, Met, Ala,
Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys, at position 366.
In one embodiment, the first heavy chain has an Arg, Ala, His or Gly at position
409, and the second heavy chain has
(i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at
on 368, or
(ii) a Trp at on 370, or
(iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at
position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at
position 366.
In one embodiment, the first heavy chain has an Arg at position 409, and the
second heavy chain has
(i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) a Phe, His, Lys, Arg or Tyr at position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.
3O In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein comprises first and second heavy , wherein (i) the amino acid in
the position corresponding to F405 in a human IgG1 heavy chain according to EU
numbering is L in said first heavy chain, and the amino acid in the position
corresponding to K409 in a human IgGl heavy chain according to EU numbering is R in
said second heavy chain, or (ii) the amino acid in the position corresponding to K409 in
a human IgG1 heavy chain according to EU numbering is R in said first heavy chain, and
the amino acid in the position corresponding to F405 in a human IgG1 heavy chain
according to EU numbering is L in said second heavy chain.
In a r embodiment said first and second heavy chain are of a human IgGl
In r further embodiment said first and second heavy chain are of a human
IgGZ e.
In another further embodiment said first and second heavy chain are of a human
IgG3 isotype.
In another embodiment, the bispecific dy as defined in any of the
embodiments disclosed herein comprises first and second heavy chains of the human
IgG4 e, wherein (i) the amino acid in the position corresponding to S228 in a
human IgG4 heavy chain according to EU numbering is P in said first heavy chain, and
the amino acid in the position corresponding to S228, F405 and R409 in a human IgG4
heavy chain according to EU numbering is P, L and K, respectively, in said second heavy
chain, or (ii) the amino acid in the position corresponding to S228, F405 and R409 in a
human IgG4 heavy chain ing to EU numbering is P, L and K, respectively, in said
first heavy chain, and the amino acid in the position corresponding to S228 in a human
IgG4 heavy chain according to EU numbering is P in said second heavy chain.
If reference is made herein to amino acids at certain positions of the first heavy
chain and/or amino acids at certain positions of the second heavy chain, such reference
is to be understood to e embodiments wherein the amino acids at certain ons
of the first heavy chain are present at the corresponding positions of the second heavy
chain rather than the first heavy chain and/or the amino acids at certain ons of the
second heavy chain are present at the corresponding positions of the first heavy chain
rather than the second heavy chain.
In addition to the above-specified amino-acid substitutions, said first and second
heavy chains may contain further amino-acid substitutions, deletion or insertions relative
to wild-type heavy chain sequences.
In a further embodiment, said first and second Fab-arms (or heavy chain
constant domains) comprising the first and second heavy chains comprise, except for the
specified mutations, a CH3 sequence independently selected from the following:
3O (IgGlm(a)) (SEQ ID NO:106), (IgGlm(f)) (SEQ ID NO:107), and (IgGlm(ax)
(SEQ ID NO:108).
In one embodiment, neither said first nor said second heavy chain ses a
Cys-Pro-Ser-Cys sequence in the (core) hinge region.
In a further embodiment, both said first and said second heavy chain comprise a
Cys-Pro-Pro-Cys sequence in the (core) hinge region.
In separate and specific embodiments, one or both Fab-arms comprise a heavy-
chain constant region sequence independently selected from SEQ ID NO:109, 110, 111,
112, 113 and 116 (see Table 1).
s of preparing bispecific antibodies
Traditional methods such as the hybrid hybridoma and chemical ation
methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used in the
preparation of the bispecific antibodies of the invention. Co-expression in a host cell of
two antibodies, consisting of different heavy and light chains, leads to a mixture of
possible antibody products in addition to the desired bispecific antibody, which can then
be isolated by, e.g., affinity chromatography or similar methods.
Strategies favoring the formation of a functional bispecific product upon co-
sion of different antibody ucts can also be used, e.g., by the method
described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse
hydridomas producing different antibodies leads to a limited number of dimeric
proteins because of preferential species-restricted heavy/light chain g. Another
strategy to promote formation of heterodimers over homodimers is a “knob-into-hole”
strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a
corresponding cavity in a second heavy-chain polypeptide, such that the protuberance
can be positioned in the cavity at the interface of these two heavy chains so as to
promote heterodimer formation and hinder homodimer formation. "Protuberances" are
constructed by replacing small amino-acid side-chains from the interface of the first
polypeptide with larger side chains. Compensatory ies" of identical or similar size
to the erances are created in the interface of the second polypeptide by replacing
large amino-acid side-chains with smaller ones (US patent 5,731,168). 459
(Chugai) and WO 2009/089004 (Amgen) describe other strategies for favoring
heterodimer formation upon co-expression of ent antibody s in a host cell.
In these methods, one or more residues that make up the 3 interface in both
CH3 domains are replaced with a charged amino acid such that homodimer formation is
electrostatically rable and heterodimerization is electrostatically favorable.
W02007110205 (Merck) describe yet another strategy, wherein differences between IgA
and IgG CH3 s are ted to e heterodimerization.
A preferred method for preparing the bispecific CD40xCD137 antibodies of the
3O present invention includes the methods described in WO 2011/131746 and WO
2013/060867 (Genmab) comprising the ing steps:
a) providing a first antibody comprising an Fc region, said Fc region comprising
a first CH3 region;
b) providing a second antibody comprising a second Fc region, said Fc region
comprising a second CH3 region,
wherein the first antibody is a CD40 antibody comprising two first antigen-binding
regions as described herein and the second dy is a CD137 antibody comprising
two second antigen-binding regions as described herein, or vice versa; and wherein the
sequences of said first and second CH3 regions are different and are such that the
heterodimeric interaction between said first and second CH3 regions is stronger than
each of the homodimeric interactions of said first and second CH3 regions;
c) incubating said first antibody together with said second antibody under
reducing conditions; and
d) obtaining said bispecific CD40xCD137 antibody.
In one embodiment, said first antibody is incubated together with said second
antibody under reducing ions sufficient to allow the cysteines in the hinge region
to undergo ide-bond isomerization, wherein the heterodimeric interaction between
said first and second dies in the resulting heterodimeric antibody is such that no
Fab-arm exchange occurs at 0.5 mM GSH after 24 hours at 37° C.
Without being limited to theory, in step c), the heavy-chain disu|fide bonds in the
hinge regions of the parent antibodies (first and second antibody in step a) and b)) are
reduced and the resulting nes are then able to form inter heavy-chain disu|fide
bond with ne es of another parent antibody molecule (originally with a
different specificity). In one embodiment of this method, the reducing conditions in step
c) comprise the addition of a reducing agent, e.g. a reducing agent selected from the
group consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol '), dithioerythritol
(DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-
mercapto-ethanol, preferably a reducing agent selected from the group consisting of: 2-
toethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. In a further
embodiment, step c) comprises restoring the conditions to become non-reducing or less
reducing, for example by l of a reducing agent, e.g. by desalting. In one
particular embodiment, bispecific antibodies are generated as follows: the two al
complementary antibodies, both in the same amount, are ted with 75 mM 2-
mercaptoethylamine-HCI (2-MEA) in buffer (e.g., PBS or Tris-EDTA) at 31°C for 5 hours;
the reduction reaction is stopped by removing the reducing agent 2-MEA using spin
columns (e.g., Microcon centrifugal filters, 30k, ore) (Labrijn et al. Nature
3O Protocols, Vol 9 No 10, p2450-2463; 2014). In another particular embodiment, the
method is that of e 3.
For this method, any of the CD40 and CD137 antibodies disclosed herein may be
used. In a ular embodiment the first and second antibodies, binding to human
CD40 and CD137, respectively, may be chosen so as to obtain a bispecific antibody as
described herein.
In one ment of this method, said first and/or second antibodies are full-
length antibodies.
The Fc regions of the first and second antibodies may be of any isotype,
including, but not limited to, an IgG isotype having a ss ed from the group
consisting of IgGl, IgGZ, IgG3 and IgG4. In one embodiment of this method, the Fc
regions of both said first and said second antibodies are of the IgGl isotype. In another
ment, one of the Fc regions of said antibodies is of the IgGl isotype and the
other of the IgG4 isotype. In the latter ment, the resulting bispecific antibody
comprises an Fc region of an IgGl and an Fc region of IgG4.
In a further embodiment, one of the parental antibodies has been engineered to
not bind Protein A, thus allowing separation of the heterodimeric dy from said
parental homodimeric antibodies by passing the t over a n A column.
As described above, the ces of the first and second CH3 regions of the
meric parental antibodies are different and are such that the heterodimeric
interaction between said first and second CH3 regions is stronger than each of the
homodimeric interactions of said first and second CH3 regions. More details on these
interactions and how they can be achieved are provided in
2013/060867 (Genmab), which are hereby incorporated by reference in their entirety.
In ular, a stable bispecific CD40xCD137 antibody can be obtained at high
yield using the above method of the invention on the basis of two meric
antibodies which bind CD40 and CD137, respectively, and contain only a few, fairly
conservative, asymmetrical mutations in the CH3 regions. Asymmetrical mutations mean
that the sequences of said first and second CH3 regions contain amino acid substitutions
at non-identical positions.
The bispecific antibodies of the invention may also be obtained by co-expression
of ucts encoding the first and second polypeptides in a single cell. Thus, in a
further aspect, the ion relates to a method for producing a ific antibody, said
method comprising the following steps:
a) providing a first nucleic-acid construct encoding a first polypeptide comprising
a first Fc region and a first antigen-binding region binding to human CD40 according to
any aspect or embodiment herein, said first Fc region sing a first CH3 region,
3O b) providing a second nucleic-acid construct encoding a second polypeptide
comprising a second Fc region and a second antigen-binding region binding to human
CD137 according to any aspect or embodiment herein, said second Fc region comprising
a second CH3 region,
wherein the sequences of said first and second CH3 regions are different and are
such that the heterodimeric interaction between said first and second CH3 regions is
stronger than each of the homodimeric interactions of said first and second CH3 regions,
and wherein in said first CH3 region at least one of the amino acids in the positions
corresponding to a position selected from the group consisting of T366, L368, K370,
D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering
has been substituted, and in said second CH3 region at least one of the amino acids in
the positions corresponding to a position selected from the group consisting of T366,
L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU
numbering has been substituted, and wherein said first and said second heavy chains
are not tuted in the same positions,
optionally wherein said first and second nucleic acid constructs encode light chain
sequences of said first and second antibodies,
c) co-expressing said first and second nucleic-acid constructs in a host cell, and
d) obtaining said heterodimeric protein from the cell culture.
Thus, the present invention also relates to a inant eukaryotic or
prokaryotic host cell which produces a bispecific antibody of the present invention.
In one embodiment of the present invention, the bispecific dy is obtained
by any of the methods according to the present invention.
Suitable expression vectors, including promoters, enhancers, etc., and suitable
host cells for the production of antibodies are nown in the art. Examples of host
cells include yeast, bacterial and mammalian cells, such as CHO or HEK cells.
In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein ses first and second CH3 regions, except for the specified
mutations, comprising the sequence of SEQ ID NO:107 (IgG1m(f)).
In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein comprises a first Fc-region and a second ion, wherein neither said
first nor said second Fc-region comprises a Cys-Pro-Ser-Cys ce in the hinge
region.
In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein ses a first Fc-region and a second Fc-region, wherein both of said
first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the hinge
region.
In one embodiment, the bispecific antibody as d in any of the embodiments
3O disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first
and second Fc-regions are human antibody Fc-regions.
In one embodiment, the ific antibody as defined in any of the embodiments
disclosed herein ses a first ion and a second Fc-region, wherein said first
and second Fc region, except for the specified mutations, se a sequence
independently selected from the group consisting of SEQ ID NOS:109, 110, 111, 112,
113 and 116.
WO 11421
In one embodiment, the bispecific dy as defined in any of the embodiments
disclosed herein comprises a first Fc-region and a second Fc-region, n the first
and second antigen-binding regions are from heavy-chain dies.
In one embodiment, the bispecific antibody as defined in any of the embodiments
disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first
and second antigen-binding regions comprise a first and second light chain.
In further embodiments, the co-expression method according to the invention
comprises any of the further features described under the in vitro method above.
Inert format
The effector functions mediated by the Fc region of an antibody allow for the
destruction of foreign entities, such as the killing of pathogens and the clearance and
degradation of antigens. Antibody-dependent cell-mediated xicity (ADCC) and
antibody-dependent ediated phagocytosis (ADCP) are initiated by binding of the Fc
region to Fc receptor (FcR)-bearing cells, whereas complement-dependent cytotoxicity
(CDC) and ment-dependent cell-mediated cytotoxicity (CDCC) are initiated by
binding of the Fc region to C1q, which initiates the classical route of complement
activation.
Fc-mediated effector on, such as ADCC and ment activation, have
been suggested to contribute to the therapeutic efficacy of monoclonal antibodies used
for the treatment of cancer (Weiner et al. Cell 2012, 148: 1081-1084).
The multispecific antibody, such as a bispecific antibody, according to the present
invention binds to CD137 which is expressed on T-cells, e.g. CD4+ and/or CD8+ T-cells.
By concomitant binding of the antibody to CD40, which is expressed on e.g. antigenpresenting
cells (APCs), provides stimulation to both APCs expressing CD40 and T-cells
expressing CD137 and thereby e.g. T-cell proliferation can be increased.
In general, binding of an antibody to a target antigen expressed by a cell may
lead to interactions with effector molecules such as Fc receptors or complement ns
which may induce Fc-mediated effector functions, such as ADCC or complement
activation, which may result in killing of the cell expressing said target antigen.
3O The use of the multispecific antibody, such as a bispecific antibody, according to
the present ion is based on its ability to e co-stimulation to APCs and T cells.
It is, in a particular embodiment, preferred that the multispecific antibody does
not bind to FcRs, e.g. FcyRs, and therefore does not induce FcR-mediated cross-linking.
It is, in a further ment, preferred that the multispecific antibody does not
engage effector functions so as to avoid killing of the CD40 and/or CD137 expressing
cells.
In one aspect of the present invention, the multispecific CD40xCD137 antibody
ing to the present invention comprises (i) a first binding arm comprising a first
heavy chain and a first antigen-binding region and (ii) a second binding arm comprising
a second heavy chain and a second antigen-binding region, according to any aspect or
embodiment described .
In one embodiment the multispecific antibody according to present invention
comprises a first and a second heavy chain, n said antibody induces and/or
enhances Fc-mediated effector on to a lesser extent compared to a multispecific
antibody comprising the same first and second antigen-binding regions as said antibody,
and comprising two heavy chains comprising a human IgGl hinge, CH2 and CH3
regions.
In one ment, the multispecific dy according to present invention
ses a first and a second antigen-binding region and a first and a second heavy
chain, each of the first and second heavy chains comprising a human IgGl hinge, CH2
and CH3 regions, wherein at least one of the first and second heavy chain comprises a
modification so as to induce and/or enhance Fc-mediated effector function to a lesser
extent compared to a nce pecific antibody comprising the same first and
second antigen-binding regions as said antibody, and comprising two heavy chains
comprising a human IgGl hinge, CH2 and CH3 regions without said modification.
In one embodiment, said first and second heavy chains are modified so that the
multispecific dy induces and/or enhances Fc-mediated effector function to a lesser
extent compared to a multispecific antibody which is identical except for comprising non-
modified first and second heavy chains.
In one embodiment, said Fc-mediated effector function may be measured by
binding to Fcy-receptors, binding to Clq, or induction of Fc-mediated cross-linking of
FcRs.
In one embodiment, said Fc-mediated effector function is measured by binding to
In one embodiment, said first and second heavy and light chain constant
3O ces have been modified so that binding of Clq to said multispecific antibody is
reduced compared to a wild-type multispecific antibody by at least 70%, at least 80%,
at least 90%, at least 95%, at least 97%, or 100%, wherein Clq binding is determined
by ELISA.
Human IgGl is known for its ability to induce iated effector functions,
while other human isotypes, such as IgG4, are less able to induce Fc-mediated effector
functions.
The first and second heavy chains may each be of any isotype, including, but not
limited to, an IgGl isotype ed from the groups consisting of IgGl, IgGZ, IgG3 and
WO 11421
IgG4, and may ally comprise one or more mutations or modifications. In one
embodiment, each of the first and second heavy chains is of the IgG4 isotype or derived
therefrom, optionally with one or more ons or modifications. In one embodiment,
each of the first and second heavy chains is of the IgGl isotype or derived therefrom,
optionally with one or more mutations or cations. In another embodiment, one of
the heavy chains is of the IgGl isotype and the other of the IgG4 isotype, or is derived
from such respective isotypes, optionally with one or more mutations or modifications.
In one embodiment, one or both of the first and heavy chains are such that an
antibody comprising two first or two second heavy chains would be effector-function-
deficient. For example, the first and second heavy chains may be of an IgG4 isotype, or
a non-IgG4 type, e.g. IgG1, IgGZ or IgG3, which has been mutated such that the ability
to mediate effector functions, such as ADCC, has been reduced or even eliminated
compared to non-mutated heavy chains. Such mutations have e.g. been described in
Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.;
75(24):12161-12168 (2001). The multispecific antibody according to the present
invention may comprise modifications in the first and second heavy chains ed to
a wild type human IgGl sequence. A multispecific antibody comprising such
modifications in the Fc region of the dy may become an inert, or non-activating,
multispecific antibody. The term “inertness”, “inert” or ctivating” as used herein,
refers to an Fc region which is at least not able to bind any Fcy(gamma) receptors, bind
to C1q, or induce Fc-mediated cross-linking of FcRs. The inertness of an Fc region, or the
first and/or second heavy chain of a pecific antibody of the present ion may
be tested with a bivalent, monospecific antibody comprising said Fc region, or two first
heavy chains or two second heavy chains. It may also be tested with a multispecific
antibody comprising a first and a second heavy chain.
Several variants can be constructed to make the Fc region of an antibody inactive
for interactions with Fcy receptors and C1q for therapeutic antibody development. The
present invention is not limited to any specific mutation relevant for reducing Fc-
mediated effector ons. Examples of such variants are described herein.
3O Thus, amino acids in the Fc region that play a nt role in the interactions
with C1q and the Fcy receptors may be modified. Examples of amino acid positions that
may be modified include positions L234, L235 and P331.
Hence, in one embodiment, in at least one of said first and second heavy chains
the amino acid in at least one position corresponding to L234, L235 and P331 in a
human IgGl heavy chain according to EU numbering, may be A, A and S, tively.
(Xu et al., 2000, Cell Immunol. 200(1):16-26; Oganesyan et al., 2008, Acta Cryst.
(D64):700-4). Also, L234F and L235E amino acid substitutions can result in Fc regions
with abrogated interactions with Fcy receptors and C1q (Canfield et al., 1991, J.
Exp.Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40). Hence, in one
embodiment, in at least one of said first and second heavy chains the amino acids in the
positions corresponding to L234 and L235 in a human IgGl heavy chain according to EU
numbering, may be F and E, respectively. A D265A amino acid substitution can decrease
binding to all Fc gamma receptors and prevent ADCC (Shields et al., 2001, J. Biol.
Chem. (276):6591-604). Hence, in one embodiment, in at least one of said first and
second heavy chains the amino acid in the position corresponding to D265 in a human
IgGl heavy chain according to EU numbering, may be A. Binding to C1q can be
abrogated by mutating positions D270, K322, P329, and P331. Mutating these positions
to either D270A or K322A or P329A or P331A can make the antibody deficient in CDC
activity Idusogie EE, et al., 2000, J Immunol. 164: 4). Hence, in one
embodiment, in at least one of said first and second heavy chain, the amino acids in at
least one position corresponding to D270, K322, P329 and P331 in a human IgGl heavy
chain according to EU numbering, may be A, A, A, and A, respectively.
An ative approach to minimize the interaction of the Fc region with Fcy
ors and C1q is by removal of the glycosylation site of an antibody. Mutating
position N297 to e.g. Q, A, or E s a glycosylation site which is critical for IgG-Fcy
receptor interactions. Hence, in one ment, in at least one of said first and second
heavy , the amino acid in a position corresponding to N297, may be G, Q, A or E
in a human IgGl heavy chain according to EU numbering (Leabman et al., 2013, MAbs;
(6):896-903). Another ative approach to minimize interaction of the Fc region
with Fcy receptors may be obtained by the following ons; P238A, A327Q, P329A
or L234V/L235A/GZ36del (Shields et al., 2001, J. Biol. Chem. (276):6591-604).
Alternatively, human IgGZ and IgG4 sses are considered naturally
compromised in their interactions with C1q and Fc gamma Receptors although
interactions with Fcy receptors were reported (Parren et al., 1992, J. Clin Invest. 90:
1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725). Mutations abrogating these
al interactions can be made in both isotypes, resulting in reduction of unwanted
side-effects associated with FcR binding. For IgGZ, these include V234A and 6237A, and
3O for IgG4, L235E. Hence, in one embodiment, in at least one of said first and second
heavy chains, such as in both said first and second heavy chains, the amino acid in a
position corresponding to V234 and 6237 in a human IgGZ heavy chain according to EU
numbering, may be A and A, respectively. In one ment, the amino acid in a
on corresponding to L235 in a human IgG4 heavy chain according to EU
numbering, may be E.
Other approaches to further minimize the interaction with Fcy receptors and C1q
in IgGZ antibodies include those described in W02011066501 and Lightle, S., et al.,
2010, Protein Science (19):753-62.
The hinge region of the antibody can also be of importance with respect to
interactions with Fcy receptors and complement (Brekke et a|., 2006, J Immunol
177:1129-1138; Dall’Acqua WF, et al., 2006, J Immunol 29-1138). Accordingly,
mutations in or on of the hinge region can influence effector functions of an
antibody.
In one embodiment, the multispecific antibody comprises a first and a second
heavy chain, wherein in at least one of said first and second immunoglobulin heavy
chains one or more amino acids in the positions corresponding to positions L234, L235,
D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not
L, L, D, N, and P, respectively.
In one embodiment, in both the first and second heavy chains one or more amino
acids in the on ponding to positions L234, L235, D265, N297, and P331 in a
human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P,
respectively.
In another embodiment, in at least one of the first and second heavy chains one
or more amino acids in the positions corresponding to positions L234, L235 and D265 in
a human IgG1 heavy chain according to EU ing, are not L, L and D, respectively,
and the amino acids in the positions corresponding to N297 and P331 in a human IgG1
heavy chain according to EU ing, are N and P, tively.
In one embodiment, one or both of the heavy chains comprise a mutation
removing the acceptor site for Asn-linked glycosylation or is otherwise manipulated to
change the glycosylation properties. For example, in an IgG1 Fc-region, an N297Q
mutation can be used to remove an Asn-linked glycosylation site. Accordingly, in a
specific embodiment, one or both heavy chains comprise an IgGl wildtype sequence
with an N297Q mutation.
The term “amino acid corresponding to position” as used herein refers to an
amino acid on number in a human IgG1 heavy chain. Corresponding amino acid
positions in other immunoglobulins may be found by alignment with human IgG1. Unless
otherwise stated or contradicted by context, the amino acids of the constant region
3O sequences are herein numbered according to the EU-index of numbering (described in
Kabat, EA. et a|., 1991, Sequences of proteins of immunological st. 5th Edition -
US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,
680, 689). Thus, an amino acid or segment in one sequence that “corresponds to” an
amino acid or segment in another sequence is one that aligns with the other amino acid
or segment using a rd sequence alignment program such as ALIGN, ClustalW or
similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or
at least 95% identity to a human IgG1 heavy chain. It is considered well-known in the
art how to align a sequence or segment in a sequence and thereby determine the
corresponding position in a sequence to an amino acid position according to the present
invention.
In the context of the present invention, the amino acid position may be defined
as described above.
The term “the amino acid is not” or r wording when ing to amino acids
in a heavy chain is to be understood to mean that the amino acid is any other amino
acid than the specific amino acid mentioned. For example, the amino acid in the on
corresponding to L234 in a human IgG1 heavy chain is not L, means that the amino acid
may be any of the other naturally or non-naturally occurring amino acids than L.
In one embodiment, in said first and second heavy chains the amino acid in the
position corresponding to on D265 in a human IgG1 heavy chain according to EU
numbering, is not D.
In one embodiment, in said first and second heavy chains the amino acid in the
position corresponding to D265 in a human IgG1 heavy chain according to EU
numbering, is not D, and the amino acids in the positions corresponding to positions
N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P,
respectively.
In one embodiment, in said first and second heavy chains the amino acids in the
positions corresponding to position D265 in a human IgG1 heavy chain according to EU
numbering is hydrophobic or polar amino acids.
The term phobic” as used herein in relation to an amino acid residue,
refers to an amino acid residue selected from the group consisting of: A, C, F, G, H, I, L,
M, R, T, V, W, and Y.
Thus, in one embodiment, in said first and second heavy chains the amino acid in
the on ponding to position D265 in a human IgG1 heavy chain according to
EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L,
M, R, T, V, W and Y.
The term “polar” as used herein in relation to amino acid residues, refers to any
amino acid residue selected from the group consisting of: C, D, E, H, K, N, Q, R, S, and
3O T. Thus, in one embodiment, in said first and second heavy chains the amino acid in the
position corresponding to position D265 in a human IgG1 heavy chain ing to EU
numbering is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.
In another embodiment, in said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain ing to
EU numbering is an aliphatic uncharged, aromatic or acidic amino acid.
The term “aliphatic uncharged” as used herein in relation to amino acid residues,
refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V.
Thus, in one embodiment, in said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is selected from the group consisting of: A, G, I, L, and V.
The term tic” as used herein in relation to amino acid residues, refers to
any amino acid residue selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is ed from the group consisting of: F, T, and W.
The term “acidic” as used herein in relation to amino acid residues, refers to any
amino acid residue chosen from the group consisting of: D and E.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acid in the position corresponding to position D265 in a human IgG1 heavy
chain according to EU numbering is ed from the group ting of: D and E.
Thus, in one embodiment, in both said first and second heavy chains the amino
acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is ed from the group consisting of: D and E.
In a particular embodiment, in at least one of said first and second heavy chains
the amino acid in the on corresponding to position D265 in a human IgG1 heavy
chain according to EU numbering is selected from the group consisting of: A, E, F, G, I,
L, T, V, and W.
In a particular embodiment, in both said first and second heavy chains the amino
acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V,
and W.
In one embodiment, in at least one of said first and second heavy chains the
amino acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering, is not D.
In one embodiment, in both said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain according to
3O EU numbering, is not D.
In one embodiment, in at least one of said first and second heavy chains the
amino acid in the position corresponding to D265 in a human IgG1 heavy chain
according to EU numbering, is not D, and the amino acids in the positions ponding
to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering,
are N and P, respectively.
In one ment, in both said first and second heavy chains the amino acid in
the position corresponding to D265 in a human IgG1 heavy chain according to EU
numbering, is not D, and the amino acids in the positions corresponding to positions
N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P,
respectively.
In one embodiment, in at least one of said first and second heavy chains the
amino acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is hydrophobic or polar amino acid.
In one embodiment, in both said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is hydrophobic or polar amino acid.
Thus, in one ment, in at least one of said first and second heavy chains
the amino acid in the position corresponding to on D265 in a human IgG1 heavy
chain according to EU numbering is selected from the group of amino acids consisting of:
A, C, F, G, H, I, L, M, R, T, V, W and Y.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acid in the position corresponding to position D265 in a human heavy chain
according to EU numbering is selected from the group consisting of: C, E, H, K, N, Q, R,
S, and T. In one embodiment, in both said first and second heavy chains the amino acid
in the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is selected from the group of amino acids ting of: A, C, F, G, H, I, L,
M, R, T, V, W and Y.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to position D265 in a human heavy chain according to EU
ing is selected from the group ting of: C, E, H, K, N, Q, R, S, and T.
In r embodiment, in at least one of said first and second heavy chains the
amino acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is aliphatic uncharged, aromatic or acidic amino acids.
Thus, in one embodiment, in least one of said first and second heavy chains the
amino acid in the on corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is ed from the group consisting of: A, G, I, L, and V.
Thus, in one embodiment, in least one of said first and second heavy chains the
3O amino acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is selected from the group consisting of: F, T, and W.
Thus, in one ment, in least one of said first and second heavy chains the
amino acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering are selected from the group consisting of: D and E.
In a particular embodiment, in least one of said first and second heavy chains the
amino acid in the position corresponding to position D265 in a human IgG1 heavy chain
according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V,
and W.
In another embodiment, in both said first and second heavy chains the amino
acid in the position corresponding to on D265 in a human IgGl heavy chain
according to EU numbering is aliphatic uncharged, aromatic or acidic amino acids.
Thus, in one embodiment, in both said first and second heavy chains the amino
acid in the on corresponding to on D265 in a human IgGl heavy chain
according to EU numbering is selected from the group consisting of: A, G, I, L, and V.
Thus, in one embodiment, in both said first and second heavy chains the amino
acid in the position corresponding to position D265 in a human IgGl heavy chain
according to EU numbering is selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in both said first and second heavy chains the amino
acid in the position ponding to position D265 in a human IgGl heavy chain
according to EU numbering are selected from the group consisting of: D and E.
In a particular embodiment, in both said first and second heavy chains the amino
acid in the position corresponding to position D265 in a human IgGl heavy chain
according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V,
and W.
In further embodiment, in at least one of said first and second heavy chains the
amino acid in the on corresponding to position N297 in a human IgGl heavy chain
according to EU numbering, is not N.
In one embodiment, in at least one of the first and second heavy chains the
amino acid in the position corresponding to N297 in a human IgGl heavy chain
according to EU numbering, is not N, and the amino acid in the position corresponding to
position P331 in a human IgGl heavy chain according to EU numbering, is P.
In one embodiment, in said first and second heavy chains the amino acid in the
on corresponding to positions N297 in a human IgGl heavy chain according to EU
numbering, is not N.
In one embodiment, in both the first and second heavy chains the amino acid in
the on corresponding to N297 in a human IgGl heavy chain according to EU
numbering, is not N, and the amino acid in the on corresponding to position P331
3O in a human IgGl heavy chain according to EU numbering, is P.
In further embodiment, in at least one of said first and second heavy chains the
amino acids in the positions corresponding to positions L234 and L235 in a human IgGl
heavy chain according to EU numbering, are not L and L, respectively.
In one embodiment, in at least one of the first and second heavy chains the
amino acids in the positions corresponding to L234 and L235 in a human IgGl heavy
chain according to EU numbering, are not L and L, respectively, and the amino acids in
the positions ponding to positions N297 and P331 in a human IgGl heavy chain
according to EU numbering, are N and P, respectively.
In one embodiment, in at least one of said first and second heavy chains the
amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU ing are selected from the group consisting of: A, C, D, E, F, G, H,
I, K, M, N, P, Q, R, s, T, Y, v.
In one embodiment, in at least one of said first and second heavy chains the
amino acids in the positions ponding to positions L234 and L235 in a human IgG1
heavy according to EU ing chain are hydrophobic or polar amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234 and L235 in a human
IgG1 heavy chain according to EU numbering are each selected from the group
consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234 and L235 in a human
IgG1 heavy chain ing to EU numbering are each selected from the group of amino
acids consisting of: C, D, E, H, K, N, Q, R, S, and T.
In a particular embodiment, in at least one of said first and second heavy chains
the amino acids in the positions ponding to positions L234 and L235 in a human
IgG1 heavy chain according to EU numbering are each selected from the group
consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU numbering, are not L and L, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in
the positions ponding to L234 and L235 in a human IgG1 heavy chain according to
EU numbering, are not L and L, respectively, and the amino acids in the ons
corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU
numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to
3O EU numbering are hydrophobic or polar amino acids.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions ponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU numbering are each selected from the group consisting of: A, C, F, G, H,
I, M, R, T, v, w, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU numbering are each selected from the group of amino acids consisting
of: C, D, E, H, K, N, Q, R, S, and T.
In a particular embodiment, in both said first and second heavy chains the amino
acids in the ons corresponding to positions L234 and L235 in a human IgG1 heavy
chain according to EU numbering are each selected from the group consisting of: A, C,
D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.
In another embodiment, in at least one of said first and second heavy chains the
amino acids in the ons corresponding to positions L234 and L235 in a human IgG1
heavy according to EU numbering chain are aliphatic uncharged, aromatic or acidic
amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to ons L234 and L235 in a human
IgG1 heavy chain according to EU numbering are each selected from the group
consisting of: A, G, I, and V.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to ons L234 and L235 in a human
IgG1 heavy chain according to EU numbering are each selected from the group
consisting of: F, T, and W.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234 and L235 in a human
IgG1 heavy chain according to EU ing are each selected from the group
consisting of; D and E.
In a particular embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to L234 and L235 are each selected from
the group consisting of: A, D, E, F, G, I, T, V, and W.
In one embodiment, in at least one of said first and second heavy chains the
amino acids in the positions corresponding to positions L234 and L235 in a human IgG1
heavy chain according to EU numbering, are F and E; or A and A, respectively.
In one embodiment, in at least one of the first and second heavy chains the
amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy
chain according to EU numbering, are F and E; or A and A, respectively, and the amino
3O acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy
chain ing to EU numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in
the ons corresponding to positions L234 and L235 in a human IgG1 heavy chain
ing to EU numbering, are F and E; or A and A, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in
the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to
EU numbering, are F and E; or A and A, respectively, and the amino acids in the
positions corresponding to positions N297 and P331 in a human IgG1 heavy chain
according to EU numbering, are N and P, respectively.
In a particular embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234 and L235 in a human
IgG1 heavy chain according to EU numbering, are F and E, respectively.
In one ment, in both said first and second heavy chains the amino acids in
the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU numbering, are F and E, respectively.
In one ment, in at least one of said first and second heavy chains at least
the amino acids in the positions corresponding to ons L234 and L235 in a human
IgG1 heavy chain according to EU numbering, are A and A, respectively.
In one embodiment, in both said first and second heavy chains at least the amino
acids in the ons corresponding to positions L234 and L235 in a human IgG1 heavy
chain according to EU numbering, are A and A, respectively.
In one embodiment, in at least one of said first and second heavy chains the
amino acids in the positions corresponding to positions L234, L235, and D265 in a
human IgG1 heavy chain according to EU numbering, are not L, L, and D, respectively.
In one embodiment, in at least one of the first and second heavy chains the
amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1
heavy chain according to EU numbering, are not L, L and D, respectively, and the amino
acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy
chain according to EU numbering, are N and P, respectively.
In one ment, in at least one of said first and second heavy chains the
amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU numbering are selected from the group consisting of: A, C, D, E, F, G, H,
I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acid corresponding to position D265
is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y,
V, and W.
In one ment, in at least one of said first and second heavy chains the
3O amino acids in the positions corresponding to positions L234, L235 and D265 in a human
IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acid in the position ponding to position D265 in a human IgG1 heavy
chain according to EU ing is selected from the group of amino acids consisting of:
A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in the positions
corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU
numbering are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V,
W, and Y.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions ponding to positions L234 and L235 in a human
IgG1 heavy chain according to EU numbering are each selected from the group of amino
acids consisting of: C, D, E, H, K, N, Q, R, S, and T, the amino acid in the on
corresponding to on D265 in a human heavy chain according to EU numbering is
selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.
In a ular embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234 and L235 in a human
IgG1 heavy chain according to EU ing are each selected from the group
consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid
in the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is selected from the group ting of: A, C, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain
according to EU numbering are hydrophobic or polar amino acids.
In one ment, in both said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L,
M, R, T, V, W and Y, and the amino acids in the positions corresponding to positions
L234 and L235 in a human IgG1 heavy chain according to EU numbering are each
selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain
according to EU numbering are each selected from the group of amino acids consisting
of: C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to
position D265 in a human heavy chain according to EU numbering is selected from the
group consisting of: C, E, H, K, N, Q, R, S, and T.
In a ular embodiment, in both said first and second heavy chains the amino
3O acids in the positions ponding to positions L234 and L235 in a human IgG1 heavy
chain according to EU numbering are each selected from the group consisting of: A, C,
D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position
corresponding to position D265 in a human IgG1 heavy chain according to EU numbering
is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W,
and Y.
In another embodiment, in at least one of said first and second heavy chains the
amino acids in the positions corresponding to positions L234, L235 and D265 in a human
IgG1 heavy chain according to EU numbering are aliphatic uncharged, aromatic or acidic
amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acid in the position corresponding to position D265 in a human IgG1 heavy
chain according to EU numbering is selected from the group consisting of: A, G, I, L, and
V, and the amino acids in the positions corresponding to positions L234 and L235 in a
human IgG1 heavy chain according to EU numbering are each selected from the group
consisting of: A, G, I, and V.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234, L235 and D265 in a
human IgG1 heavy chain according to EU numbering are each ed from the group
consisting of: F, T, and W.
Thus, in one embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to ons L234, L235, and D265 in a
human IgG1 heavy chain according to EU numbering are each selected from the group
consisting of: D and E.
In a particular embodiment, in at least one of said first and second heavy chains
the amino acid in the position corresponding to on D265 in a human IgG1 heavy
chain according to EU numbering is selected from the group consisting of: A, E, F, G, I,
L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are
each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy
chain according to EU ing, are not L, L, and D, tively.
In one embodiment, in both the first and second heavy chains the amino acids in
the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain
according to EU numbering, are not L, L, and D, respectively, and the amino acids in the
positions corresponding to ons N297 and P331 in a human IgG1 heavy chain
according to EU numbering, are N and P, respectively.
3O In one embodiment, in both said first and second heavy chains the amino acids in
the positions ponding to L234, L235, and D265 in a human IgG1 heavy chain
according to EU numbering are aliphatic uncharged, aromatic or acidic amino acids.
In one embodiment, in both said first and second heavy chains the amino acid in
the position corresponding to position D265 in a human IgG1 heavy chain according to
EU numbering is selected from the group consisting of; A, G, I, L, and V, and the amino
acids in the positions ponding to positions L234 and L235 in a human IgG1 heavy
chain according to EU numbering are each selected from the group consisting of; A, G, I,
and V.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions ponding to positions L234, L235, and D265 in a human IgGl heavy
chain according to EU numbering are each selected from the group consisting of; D and
In a particular embodiment, in both said first and second heavy chains the amino
acid in the position corresponding to position D265 in a human IgGl heavy chain
ing to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V,
and W, and the amino acids in the positions corresponding to L234 and L235 are each
selected from the group consisting of: A, D, E, F, G, I, T, V, and W.
In one embodiment, in at least one of said first and second heavy chains the
amino acids in the positions corresponding to positions L234, L235, and D265 in a
human IgGl heavy chain according to EU ing, are F, E, and A; or A, A, and A,
respectively.
In one embodiment, in at least one of the first and second heavy chains the
amino acids in the positions corresponding to L234, L235, and D265 in a human IgGl
heavy chain according to EU numbering, are F, E, and A; or A, A, and A, respectively,
and the amino acids in the positions corresponding to positions N297 and P331 in a
human IgGl heavy chain according to EU ing, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy
chain ing to EU numbering, are F, E, and A; or A, A, and A, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in
the positions corresponding to L234, L235, and D265 in a human IgGl heavy chain
according to EU numbering, are F, E, and A; or A, A, and A, respectively, and the amino
acids in the positions corresponding to positions N297 and P331 in a human IgGl heavy
chain according to EU numbering, are N and P, tively.
In a particular embodiment, in at least one of said first and second heavy chains
the amino acids in the positions corresponding to positions L234, L235, and D265 in a
human IgGl heavy chain according to EU numbering, are F, E, and A, respectively.
3O In a ularly red embodiment, in both said first and second heavy
chains the amino acids in the positions corresponding to positions L234, L235, and D265
in a human IgGl heavy chain according to EU numbering, are F, E, and A, respectively.
In one embodiment, in at least one of said first and second heavy chains the
amino acids in the positions corresponding to positions L234, L235, and D265 in a
human IgGl heavy chain according to EU numbering, are A, A, and A, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in
the positions corresponding to ons L234, L235, and D265 in a human IgGl heavy
chain according to EU numbering, are A, A, and A, respectively.
In another embodiment, in at least one of said first and second heavy chains the
amino acids in the positions ponding to positions L234, L235, D265, N297, and
P331 in a human IgGl heavy chain according to EU numbering, are F, E, A, Q, and S,
respectively.
In one embodiment, in both said first and second heavy chains the amino acids in
the ons corresponding to positions L234, L235, D265, N297, and P331 in a human
IgGl heavy chain according to EU numbering, are F, E, A, Q, and S, respectively.
In a particular embodiment said first antigen-binding region comprises heavy
chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID
NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3
having the ces set forth in SEQ ID NOs:4, YTS and 5, respectively; and said
second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and
CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and
light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ
ID NOs:67, GAS and 68, respectively, (CD137 clone 009), and in at least one of the first
and second heavy chains, such as both said first and second heavy chain, the amino
acids in positions corresponding to positions L234, L235, and D265 in a human IgGl
heavy chain, are F, E, and A, respectively.
In another embodiment said first antigen-binding region comprises heavy chain
variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1,
2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the
sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-
binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the
sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable
region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS
and 40, tively, (CD137 clone 005), and in at least one of the first and second
heavy chains, such as both said first and second heavy chain, the amino acids in
positions corresponding to positions L234, L235, and D265 in a human IgGl heavy
chain, are F, E, and A, respectively.
3O A tivating Fc region prevents the antibody from interacting with Fc-
receptors present on blood cells, such as tes, or with Clq to activate the classical
complement pathway. Reduction of the Fc activity was tested in antibody variants that
contain different combinations of amino acid substitutions in the Fc region. Three amino
acid tutions were introduced in the parental antibodies of the present invention,
which include the ons L234F, L235E, and D265A. Substitutions in these three
amino acid positions were introduced in the K409R and/or F405L IgGl backbone. The
resulting non-activating antibody variant is termed with the suffix “FEAR” or “FEAL”,
respectively. Said al antibodies were used in to generate bispecific antibodies of
the present invention as bed in the examples.
In one aspect, the multispecific antibodies according to the invention may be
modified in the light chain and/or heavy chain to increase the expression level and/or
tion yield. In one ment, the antibodies according to the invention may be
modified in the light chain. Such modifications are known in the art and may be
performed according to the methods described in e.g. Zheng, L., Goddard, J.-P.,
Baumann, U., & Reymond, J.-L. (2004). Expression improvement and mechanistic study
of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis.
Journal of Molecular Biology, 341(3), 807—14.
In a further embodiment of the invention, one or both of the antibodies forming
part of the multispecific antibody of the invention have been engineered to reduce or
increase the binding to the neonatal Fc or (FcRn) in order to manipulate the serum
half-life of the multispecific antibody. ques for increasing or reducing the serum
half-life are well-known in the art. See for example Dall’Acqua et al. 2006, J. Biol.
Chem., 281:23514-24; Hinton et al. 2006,]. Immunol., 176:346-56; and Zalevsky et al.
2010 Nat. Biotechnol., 28:157-9.
In one aspect, the multispecific antibody as defined in any of the embodiments
disclosed herein comprises a first constant heavy chain (HC) and a first constant light
chain (LC), wherein the positions corresponding to positions L234, L235, and D265 in
the human IgGl heavy chain of SEQ ID NO:109 of both the first heavy chain and the
second heavy chain are F, E, and A, respectively.
In one embodiment, the pecific antibody as d in any of the
embodiments sed herein comprises a first and second constant heavy chain (HC)
and a first and second nt light chain (LC), wherein the positions corresponding to
positions L234 and L235 in the human IgGl heavy chain of SEQ ID NO:109 of both the
first heavy chain and the second heavy chain are F and E, respectively.
In one embodiment, the multispecific antibody comprises a first and a second
heavy chain, wherein the positions corresponding to positions L234 and L235 in a human
3O IgGl heavy chain according to EU ing of both the first heavy chain and the
second heavy chain are F and E, tively, and wherein (i) the position corresponding
to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain
is L, and the position corresponding to K409 in a human IgGl heavy chain according to
EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409
in a human IgGl heavy chain according to EU numbering of the first heavy chain is R,
and the position ponding to F405 in a human IgGl heavy chain according to EU
numbering of the second heavy chain is L.
In one embodiment, the multispecific antibody comprises a first and a second
heavy chain, wherein the positions corresponding to positions L234, L235, and D265 in a
human IgGl heavy chain according to EU ing of both the first and the second
heavy chain are F, E, and A, tively, and n the position corresponding to
F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain is
L, and the position corresponding to K409 in a human IgGl heavy chain according to EU
numbering of the second heavy chain is R. Thus in a further ment, said first
heavy chain ses the constant heavy chain sequence as set forth in SEQ ID
NO:113; and the second heavy chain comprises the constant heavy chain ce as
set forth in SEQ ID NO:112.
In one embodiment, the multispecific antibody comprises a first and a second
heavy chain, wherein the positions corresponding to positions L234, L235, and D265 in a
human IgGl heavy chain ing to EU numbering of both the first and second heavy
chain are F, E, and A, respectively, and wherein the position corresponding to K409 in a
human IgGl heavy chain according to EU numbering of the first heavy chain is R, and
the position corresponding to F405 in a human IgGl heavy chain according to EU
numbering of the second heavy chain is L. Thus in a further ment, said first
heavy chain comprises the constant heavy chain sequence as set forth in SEQ ID
NO:112; and the second heavy chain comprises the constant heavy chain sequence as
set forth in SEQ ID NO:113.
Nucleic acids
The present invention also relates to a nucleic acid encoding one or more amino
acid sequences according to any aspect or embodiment disclosed herein.
The present invention also relates to a nucleic acid encoding a multispecific
antibody as defined in any aspect or embodiment disclosed herein.
The present invention also relates to an expression vector comprising a nucleic
acid of the present invention.
The t invention also relates to a host cell comprising a c acid or an
expression vector according to the present invention.
3O In one embodiment said host cell is a recombinant eukaryotic, recombinant
prokaryotic, or recombinant microbial host cell.
In a further embodiment, the expression vector further comprises a nucleotide
sequence encoding the constant region of a light chain, a heavy chain or both light and
heavy chains of an dy, e.g. a human antibody.
An sion vector in the context of the present invention may be any suitable
vector, including chromosomal, non-chromosomal, and tic nucleic acid vectors (a
c acid sequence comprising a suitable set of sion control elements).
Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA,
virus, yeast plasmids, vectors obtained from combinations of plasmids and phage
DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid is
comprised in a naked DNA or RNA , including, for example, a linear expression
element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355 59
), a compacted nucleic acid vector (as described in for instance US 6,077, 835
and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a
"midge" minimally-sized nucleic acid vector (as described in for instance Schakowski et
al., Mol Ther 3, 793 800 (2001)), or as a precipitated nucleic acid vector construct, such
as a CaP04-precipitated construct (as described in for instance W0200046147,
Benvenisty and , PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725
(1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic
acid vectors and the usage thereof are well known in the art (see for instance US
5,589,466 and US 5,973,972).
In one embodiment, the vector is suitable for expression of the CD40 antibody
and/or the CD137 antibody in a ial cell. Examples of such vectors include
sion vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster,
J Biol Chem 264, 5503 5509 , pET vectors (Novagen, Madison WI) and the like).
An expression vector may also or alternatively be a vector suitable for expression
in a yeast system. Any vector suitable for expression in a yeast system may be
employed. Suitable vectors include, for example, vectors comprising tutive or
inducible ers such as alpha factor, alcohol oxidase and PGH (reviewed in: F.
Ausubel et al., ed. Current Protocols in Molecular Biology, Greene hing and Wiley
InterScience New York , and Grant et al., Methods in Enzymol 153, 516 544
(1987)).
An expression vector may also or atively be a vector suitable for expression
in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable
marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10:169-
3O 175.
A nucleic acid and/or vector may also comprises a nucleic acid sequence encoding
a secretion/localization sequence, which can target a polypeptide, such as a nascent
polypeptide chain, to the periplasmic space or into cell culture media. Such sequences
are known in the art, and include secretion leader or signal peptides.
The expression vector may comprise or be associated with any suitable promoter,
enhancer, and other expression-facilitating elements. Examples of such elements e
strong expression promoters (e. g., human CMV IE promoter/enhancer as well as RSV,
SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences,
an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as
selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids
may also comprise an inducible promoter as d to a constitutive promoter such as
CMV IE.
In one ment, the CD40 and/or CD137 antibody-encoding expression
vector may be oned in and/or delivered to the host cell or host animal via a viral
vector.
In an even further aspect, the invention relates to a host cell sing the first
and second nucleic-acid constructs specified herein above.
Thus the present invention also relates to a recombinant eukaryotic or
prokaryotic host cell which produces a multispecific antibody of the present invention,
such as a transfectoma.
The first, CD40-specific, antibody may be expressed in a recombinant eukaryotic
or prokaryotic host cell, such as a transfectoma, which produces an antibody as defined
herein. The second, CD137-specific, antibody may likewise be sed in a
recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces
an antibody. Such antibodies may be used to prepare a multispecific antibody according
to the t ion. A multispecific antibody according to the present invention may
also be expressed in a inant eukaryotic or prokaryotic host cell, such as a
transfectoma.
Examples of host cells include yeast, bacterial, plant and mammalian cells, such
as CHO, CHO-S, HEK, , HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytic
cells. For example, in one embodiment, the host cell may comprise a first and second
nucleic acid construct stably integrated into the cellular . In another
embodiment, the present invention provides a cell comprising a non-integrated nucleic
acid, such as a plasmid, cosmid, phagemid, or linear expression element, which
comprises a first and second nucleic acid construct as specified above.
In an even further aspect, the invention s to a transgenic non-human
animal or plant comprising nucleic acids encoding one or two sets of a human heavy
3O chain and a human light chain, wherein the animal or plant produces a multispecific
antibody of the invention.
The first, CD40-specific, antibody and/or second, CD137-specific, antibody may
also be produced by a hybridoma, a transgenic non-human animal or plant comprising
nucleic acids encoding one or two sets of a human heavy chain and a human light chain,
wherein the animal or plant es an antibody for use in a multispecific antibody or a
multispecific dy of the invention.
In one aspect, the invention relates to a nucleic acid encoding one or more amino
acid sequences set out in Table 1.
In one aspect, the invention relates to an expression vector sing
(i) a nucleic acid ce encoding a heavy chain ce of a first binding
arm according to any one of the embodiments disclosed herein;
(ii) a nucleic acid sequence encoding a light chain sequence of a first binding
arm according to any one of the embodiments disclosed herein;
(iii) a nucleic acid sequence ng a heavy chain sequence of a second
binding arm according to any one of the embodiments disclosed herein;
(iv) a nucleic acid sequence encoding a light chain sequence of a second binding
arm according to any one of the of the embodiments disclosed ;
(v) the c acid set forth in (i) and the nucleic acid set forth in (ii);
(vi) the nucleic acid set forth in (iii) and the nucleic acid set forth in (iv).
(vii) the nucleic acid set forth in (i), (ii), (iii) and (iv).
In a particular embodiment, the nucleic acid may encode a heavy chain variable
region comprising the VH CDR1, CDRZ and CDR3 of the CD40 antibody listed in Table 1
and encoding a human IgGl heavy chain having a sequence selected from the group
consisting of SEQ ID NO:110, 111, 112, 113 and 116.
In another ment, the nucleic acid may encode a heavy chain variable
region comprising the VH CDR1, CDRZ and CDR3 of one the CD137 antibodies listed in
Table 1, Le. any one of clones 001-012, and encoding a human IgGl heavy chain
having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113
and 116.
In separate and specific embodiments, a nucleic acid, nucleic acid construct, a
combination of a first and a second c acid construct, an expression , or a
combination of a first and a second expression vector according to the present invention
may encode
(a) a HC comprising (i) a VH comprising the VH CDR1, CDRZ and CDR3 of the
CD40 antibody in Table 1, and primarily human framework regions, optionally
3O comprising one or more amino acid back-mutations to the non-human amino acid
sequence, and (ii) a human IgGl heavy chain having a sequence selected from the
group consisting of SEQ ID NO:110, 111, 112,113 and 116;
(b) a HC comprising (i) a VH comprising the VH CDR1, CDRZ and CDR3 of one
the CD137 antibodies listed in Table 1, Le. any one of clones 2, and primarily
human framework regions, optionally comprising one or more amino acid back-
mutations to the non-human amino acid sequence, and (ii) a human IgGl heavy chain
having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113
and 116;
(c) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of the
CD40 antibody in Table 1, and primarily human framework s, optionally
comprising one or more amino acid back-mutations to the non-human amino acid
ce, and (ii) a light chain nt region having the sequence of SEQ ID NO:114;
(d) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of one
the CD137 antibodies listed in Table 1, Le. any of clones 001-012, and primarily human
framework regions, optionally comprising one or more amino acid back-mutations to the
non-human amino acid sequence, and (ii) a light chain nt region having the
sequence of SEQ ID NO:114;
(e) both (a) and (b);
(f) both (a) and (c);
(g) both (b) and (d);
(h) both (c) and (d); or
(i) both (a), (b), (c) and (d).
In other separate and specific embodiments, a nucleic acid, nucleic acid
construct, a combination of a first and a second nucleic acid construct, an sion
vector, or a combination of a first and a second expression vector according to the
present invention may encode
(a) a HC comprising (i) a VH sing the VH CDR1, CDR2 and CDR3 of SEQ ID
NOS:1, 2 and 3, and ily human framework regions, optionally comprising one or
more amino acid back-mutations to the non-human amino acid sequence, and (ii) a
human IgGl heavy chain having a sequence selected from the group consisting of SEQ
ID NO:110, 111, 112, 113 and 116;
(b) a HC comprising (i) a VH comprising the VH CDR1, CDR2 and CDR3 of SEQ ID
NO: 64, 65 and 66, and primarily human framework regions, optionally comprising one
or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a
human IgGl heavy chain having a sequence selected from the group consisting of SEQ
ID NO:110, 111, 112, 113 and 116;
3O (c) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of SEQ ID
NO:4, YTS and SEQ ID NO:5, and primarily human framework regions, optionally
comprising one or more amino acid back-mutations to the non-human amino acid
sequence, and (ii) a light chain constant region having the sequence of SEQ ID ;
(d) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of SEQ ID
NO: 67, GAS and SEQ ID NO:68, and primarily human framework regions, optionally
comprising one or more amino acid back-mutations to the non-human amino acid
ce, and (ii) a light chain constant region having the sequence of SEQ ID NO:114;
(e) both (a) and (b);
(f) both (a) and (c);
(g) both (b) and (d);
(h) both (c) and (d); or
(i) both (a), (b), (c) and (d).
In other separate and ic embodiments, a nucleic acid, nucleic acid
construct, a ation of a first and a second nucleic acid construct, an expression
vector, or a combination of a first and a second sion vector according to the
t invention may encode
(a) a HC comprising a VH comprising SEQ ID NO:117 and a human IgGl heavy
chain having a sequence selected from the group consisting of SEQ ID NO:110, 111,
112, 113 and 116;
(b) a HC comprising a VH comprising SEQ ID NO:123 and a human IgGl heavy
chain having a sequence selected from the group consisting of SEQ ID NO:110, 111,
112, 113 and 116;
(c) an LC sing a VL comprising SEQ ID NO:121 and a light chain constant
region having the sequence of SEQ ID NO:114;
(d) an LC comprising a VL comprising SEQ ID NO:127 and a light chain constant
region having the sequence of SEQ ID NO:114;
(e) both (a) and (b);
(f) both (a) and (c);
(g) both (b) and (d);
(h) both (c) and (d); or
(i) both (a), (b), (c) and (d).
In other separate and specific embodiments, a nucleic acid, a nucleic acid
construct, a combination of a first and a second nucleic acid construct, an expression
vector, or a combination of a first and a second expression vector according to the
present invention may encode
3O (a) a HC comprising SEQ ID NO:118 (CD40HC6, IgGl);
(b) a HC comprising SEQ ID NO:119 (CD40HC6-FEAL);
(c) a HC comprising SEQ ID NO:120 (CD40HC6-FEAR);
(d) a HC comprising SEQ ID NO:124 (CD137HC7);
(e) a HC comprising SEQ ID NO:125 HC7-FEAR);
(f) a HC comprising SEQ ID NO:126 (CD137HC7-FEAL);
(g) an LC comprising SEQ ID NO:122 (CD40LC1);
(h) an LC comprising SEQ ID NO:128 (CD137LC2);
(i) both (a) and (g);
WO 11421
1 1 1
0') both (b) and (9);
(k) both (c) and (g);
(I) both (d) and (h);
(m) both (e) and (h);
(n) both (f) and (h);
(0) both (b) and (e);
(p) both (c) and (f);
(Cl) both (9) and (h);
(r) both (b), (e), (9) and (h);
(5) both (c), (f), (g) and (h).
In one aspect, the ion relates to a method for producing a bispecific
antibody according to any one of the embodiments as disclosed herein, comprising the
steps of
a) culturing a host cell as disclosed herein sing an expression vector as
disclosed herein expressing the first antibody as disclosed herein and purifying said
antibody from the culture media;
b) culturing a host cell as sed herein comprising an sion vector as
disclosed herein expressing the second antibody as disclosed herein and purifying said
dy from the culture media;
c) incubating said first antibody together with said second antibody under
reducing ions sufficient to allow the cysteines in the hinge region to undergo
disulfide-bond isomerization, and
d) obtaining said bispecific antibody.
In one aspect, the invention relates to a host cell comprising an expression vector
as defined above. In one embodiment, the host cell is a recombinant eukaryotic,
inant prokaryotic, or recombinant microbial host cell.
Compositions
The present ion also relates to a composition comprising a multispecific
3O antibody according to the present invention, a nucleic acid ing to the present
invention, an expression vector to the present invention or a host cell according to the
present invention.
In a further embodiment the composition according to the present invention is a
pharmaceutical composition.
In an even further embodiment, the pharmaceutical composition according to the
present invention further comprises a pharmaceutically acceptable carrier.
In a further aspect, the invention relates to a pharmaceutical composition
comprising:
- a multispecific CD40xCD137 antibody as defined in any of the embodiments
disclosed herein, and
- a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention may contain one
multispecific antibody of the present invention or a combination of different multispecific
antibodies of the t ion.
The pharmaceutical compositions may be formulated in accordance with
conventional techniques such as those disclosed in Remington: The Science and Practice
of Pharmacy, 19th Edition, o, Ed., Mack Publishing Co., Easton, PA, 1995. A
pharmaceutical composition of the present invention may e.g. include diluents, fillers,
salts, buffers, detergents (e.g., a ic detergent, such as Tween-20 or 80),
stabilizers (e. g., sugars or n-free amino acids), vatives, tissue fixatives,
solubilizers, and/or other materials le for inclusion in a pharmaceutical
ition.
ceutically acceptable carriers include any and all suitable solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents,
antioxidants and absorption delaying agents, and the like that are physiologically
compatible with a multispecific dy of the present invention. Examples of suitable
aqueous and nonaqueous carriers which may be employed in the pharmaceutical
compositions of the present invention include water, saline, ate buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene , and
the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal
solutions, tragacanth gum and injectable organic , such as ethyl oleate, and/or
s buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. Proper fluidity may be ined, for example, by the use of
coating materials, such as lecithin, by the maintenance of the required particle size in
3O the case of dispersions, and by the use of surfactants.
ceutical compositions of the t invention may also se
pharmaceutically acceptable antioxidants for instance (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, butylated hydroxytoluene, lecithin, propyl gallate, alphatocopherol
, and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
Pharmaceutical compositions of the t invention may also comprise
isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or
sodium chloride in the compositions.
The ceutical compositions of the present invention may also contain one
or more adjuvants appropriate for the chosen route of administration such as
preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or
buffers, which may enhance the shelf life or effectiveness of the pharmaceutical
composition. The ceutical composition of the present invention may be prepared
with carriers that will protect the multispecific antibody t rapid release, such as a
controlled release formulation, including implants, ermal patches, and
microencapsulated delivery systems. Such carriers may include gelatin, glyceryl
monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as
ethylene vinyl e, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid alone or with a wax, or other materials well known in the art. Methods for
the preparation of such formulations are generally known to those skilled in the art.
Sterile injectable solutions may be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one or a combination of
ingredients e.g. as enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion medium and the required
other ingredients e.g. from those enumerated above. In the case of sterile s for
the preparation of sterile injectable solutions, examples of methods of ation are
vacuum drying and freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ient from a previously sterile-filtered
solution thereof.
The actual dosage levels of the active ingredients in the pharmaceutical
compositions may be varied so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a ular patient,
composition, and mode of administration, t being toxic to the patient. The
3O selected dosage level will depend upon a variety of pharmacokinetic factors including the
activity of the ular itions of the present invention employed, the route of
administration, the time of administration, the rate of excretion of the particular
compound being employed, the duration of the treatment, other drugs, compounds
and/or materials used in ation with the particular compositions employed, the
age, sex, , condition, l health and prior medical y of the patient being
treated, and like factors well known in the medical arts.
The pharmaceutical composition may be administered by any suitable route and
mode. In one embodiment, a pharmaceutical composition of the present invention is
administered erally. "Administered parenterally" as used herein means modes of
administration other than enteral and topical administration, y by injection, and
include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,
intracranial, intrathoracic, epidural and intrasternal injection and infusion.
In one embodiment the pharmaceutical composition is stered by
intravenous or subcutaneous injection or on.
The t invention also relates to the multispecific antibody according to the
present invention, the nucleic acid according to the present invention, the expression
vector according to the present invention, the host cell according to the present
invention, the composition according to the t invention, or the pharmaceutical
composition according to the present invention for use as a medicament.
The present invention also relates to the multispecific antibody according to the
present invention, the nucleic acid according to the present invention, the expression
vector according to the present invention, the host cell according to the present
invention, the composition according to the present invention, or the pharmaceutical
composition according to the present invention for use in the treatment of a disease,
such as cancer or an infectious e.
According to the invention, the term “disease” refers to any pathological state, in
particular cancer, infectious diseases, inflammatory diseases, lic diseases,
autoimmune disorders, rative diseases, apoptosis-associated diseases and
transplant ions.
As used herein, the term “cancer” includes a disease characterized by aberrantly
regulated cellular growth, proliferation, differentiation, adhesion, and/or migration. By
r cel III is meant an abnormal cell that grows by a rapid, uncontrolled cellular
eration and continues to grow after the stimuli that initiated the new growth cease.
The term r” according to the invention comprises leukemias, seminomas,
3O melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer,
trial cancer, kidney , adrenal cancer, thyroid cancer, blood cancer, skin
cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer,
h cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph
node cancer, esophagus , colorectal cancer, pancreas cancer, ear, nose and throat
(ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and
lung cancer and the metastases thereof. es thereof are lung carcinomas, mamma
1 1 5
carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical
carcinomas, or metastases of the cancer types or tumors described above.
The term “cancer” according to the invention also comprises cancer metastases.
By "metastasis" is meant the spread of cancer cells from its original site to another part
of the body. The ion of metastasis is a very complex process and s on
detachment of ant cells from the y tumor, invasion of the extracellular
matrix, penetration of the endothelial basement membranes to enter the body cavity
and vessels, and then, after being transported by the blood, ration of target organs.
Finally, the growth of a new tumor, Le. a secondary tumor or atic tumor, at the
target site depends on angiogenesis. Tumor metastasis often occurs even after the
removal of the primary tumor e tumor cells or components may remain and
develop atic potential. In one embodiment, the term "metastasis" according to
the invention relates to "distant metastasis" which relates to a metastasis which is
remote from the primary tumor and the regional lymph node system.
The term “infectious disease” refers to any disease which can be transmitted from
individual to individual or from organism to organism, and is caused by a microbial agent
(e.g. common cold).
Examples of infectious diseases include viral infectious diseases, such as AIDS
(HIV), hepatitis A, B or C, herpes, herpes zoster (chicken-pox), German measles (rubella
virus), yellow fever, dengue etc. flaviviruses, influenza viruses, hemorrhagic infectious
diseases (Marburg or Ebola viruses), and severe acute respiratory syndrome (SARS),
bacterial infectious es, such as Legionnaire's e (Legionella), sexually
transmitted diseases (e.g. chlamydia or gonorrhea), gastric ulcer (Helicobacter), cholera
(Vibrio), tuberculosis, diphtheria, infections by E. coli, Staphylococci, Salmonella or
ococci (tetanus); infections by protozoan pathogens such as malaria, sleeping
ss, leishmaniasis; toxoplasmosis, i.e. infections by Plasmodium, Trypanosoma,
Leishmania and Toxoplasma; or fungal infections, which are caused, e.g., by
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces
dermatitidis or Candida albicans.
3O The term “inflammatory disease” refers to any disease, which is characterized by
or associated with high levels of inflammation in tissues, in particular tive tissues,
or ration of these tissues. A chronic inflammatory disease is a medical condition
which is characterized by persistent inflammation. es of (chronic) inflammatory
diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease
(COPD), irritable bowel e, atherosclerosis, arthritis, ankylosing spondylitis, Crohn’s
disease, colitis, c active hepatitis, dermatitis and psoriasis.
The term “metabolic e” refers to any disease or disorder that disrupts
normal metabolism. Examples include cystinosis, diabetes, dyslipidemia,
hyperthyroidism, hypothyroidism, hyperlipidemia, hypolipidemia, galactosemia,
Gaucher’s disease, obesity and phenylketonuria.
The term “autoimmune disorder” refers to any disease/disorder in which the body
produces an immunogenic (i.e. immune system) response to some tuent of its own
tissue. In other words, the immune system loses its y to recognize some tissue or
system within the body as self and targets and attacks it as if it were foreign.
Autoimmune diseases can be classified into those in which predominantly one organ is
affected (e.g. hemolytic anemia and anti-immune thyroiditis), and those in which the
autoimmune disease process is diffused through many tissues (e.g. systemic lupus
erythematosus). For example, multiple sclerosis is thought to be caused by T cells
attacking the sheaths that surround the nerve fibers of the brain and spinal cord. This
results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are
known in the art and include, for instance, Hashimoto's thyroiditis, Grave's disease,
lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis,
systemic lupus matosus, celiac disease, Crohn's disease, colitis, diabetes,
scleroderma, psoriasis, and the like.
The term “degenerative e” refers to any disease in which the function or
structure of the affected tissues or organs will increasingly deteriorate over time.
Examples e Alzheimer’s disease, Parkinson’s disease, amyotrophic l sclerosis
(ALS), Huntington’s disease, macular degeneration, multiple sclerosis, muscular
phy, Niemann Pick disease, orosis and rheumatoid tis.
The term “apoptosis-associated diseases” refers to any disease in which
alterations of apoptosis are involved. Examples include , ogical disorders,
such as Alzheimer’s e, Parkinson’s disease, Huntington’s disease, amyotrophic
lateral sis (ALS) and stroke, heart es, such as ischemia reperfusion and
chronic heart failure, infectious diseases and autoimmune diseases.
The term “transplant rejection” refers to the rejection of a transplanted tissue or
organ by the recipient’s immune system, which may, ultimately, destroy the
transplanted tissue or organ.
3O In one embodiment, the use of the multispecific antibody, nucleic acid,
expression vector, host cell or composition for use according to the present invention
may be for treating cancer.
In one embodiment, the use of the multispecific antibody, nucleic acid,
sion vector, host cell or composition for use according to the t invention
may be for treating an infectious disease.
The t invention also s to a method of treatment of a disease, such as
cancer or an infectious disease, comprising administering the multispecific antibody
according to the present invention, the nucleic acid ing to the present invention,
the expression vector according to the present invention, the host cell according to claim
the present invention, the composition according to the present invention, or the
ceutical composition according to the t invention to a subject in need
thereof.
The present invention also relates to use of a multispecific antibody according to
the present invention, the nucleic acid according to the t invention, the expression
vector according to the present invention, the host cell according to the present
invention, the composition according to the present invention, or the pharmaceutical
composition ing to the present invention for the manufacture of a medicament.
In one embodiment the method or use according to the present invention is for
use in combination with one or more further therapeutic agent, such as a
chemotherapeutic agent.
In one aspect, the invention relates to the multispecific antibody, such as a
bispecific antibody, according to any one of the embodiments disclosed herein, the
composition as disclosed herein, or the pharmaceutical composition as disclosed herein
for use as a medicament.
In r aspect, the present invention s to the use of a multispecific
antibody according to the present invention in the manufacture of a medicament for the
treatment of a disease, such as cancer or an infectious disease.
In one aspect, the invention s to the pecific antibody according to any
one of the embodiments disclosed herein, the composition as disclosed , or the
pharmaceutical composition as disclosed herein for use in the treatment of a disease,
such as cancer or an infectious e.
In one aspect, the invention relates to a method of treatment of a disease
comprising administering the multispecific antibody according to any one of the
embodiments disclosed herein, the composition as disclosed herein, or the
ceutical composition as disclosed herein to a subject in need thereof.
The multispecific antibodies of the invention may be used for a number of
3O purposes. In particular, the multispecific antibodies of the invention may be used for the
treatment of various forms of cancer, including metastatic cancer and refractory cancer.
In one ment the use according to the present invention is in combination
with one or more further therapeutic agent, such as a chemotherapeutic agent.
In particular, the multispecific antibodies according to the ion may be
useful in therapeutic gs in which increasing proliferation of T cells is relevant. An
example of such a therapeutic g includes but is not limited to cancer or tumors,
such as hematological and solid tumors, e.g., melanoma, lung , breast cancer,
non-small cell lung cancer (NSCLC), colon cancer, renal cancer, cervical cancer and
prostate cancer, such as melanoma or NSCLC. Examples thereof are lung carcinomas,
mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas,
cervical carcinomas, or metastases of such cancer types or tumors.
The present ion also relates to a method for treating cancer, sing
a) selecting a t suffering from a cancer, and
b) administering to the subject the multispecific dy of the present
invention or a pharmaceutical composition of the present invention.
Also, the invention relates to the use of a multispecific antibody that binds to
human CD40 and human CD137 for the preparation of a medicament for the treatment
of cancer, such as one of the specific cancer indications mentioned herein.
The invention further relates to a multispecific antibody for use in the treatment
of cancer, such as one of the cancer indications mentioned above.
In one ment the method or use ing to the present invention is for
use in combination with one or more further therapeutic agent, such as a
chemotherapeutic agent.
For the above mentioned uses any multispecific dy, such as a bispecific
antibody, of the present invention may be used.
In one aspect, the invention relates to a diagnostic composition comprising a
multispecific antibody according to any one of the embodiments as disclosed herein.
In one embodiment, the diagnostic composition is a companion stic which
is used to screen and select those patients who will benefit from ent with the
multispecific antibody.
In a further ment of the methods of treatment of the present invention,
the efficacy of the treatment is being monitored during the therapy, e.g. at predefined
points in time, by determining tumor burden.
Dosage regimens in the above methods of treatment and uses are adjusted to
provide the optimum desired se (e.g., a therapeutic response). For example, a
3O single bolus may be stered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated by the exigencies of
the therapeutic situation. Parenteral compositions may be formulated in dosage unit
form for ease of administration and uniformity of dosage.
The efficient dosages and the dosage ns for the multispecific antibodies
depend on the disease or condition to be treated and may be determined by the s
skilled in the art. An exemplary, non-limiting range for a therapeutically effective
amount of a multispecific antibody of the present invention is about 0.001-30 mg/kg.
A physician or veterinarian having ordinary skill in the art may y ine
and prescribe the effective amount of the pharmaceutical composition required. For
example, the physician or veterinarian could start doses of the multispecific antibody
employed in the pharmaceutical composition at levels lower than that required in order
to achieve the d therapeutic effect and gradually increase the dosage until the
desired effect is achieved. In general, a le daily dose of a multispecific antibody of
the present invention will be that amount of the compound which is the lowest dose
effective to produce a therapeutic effect. Administration may e.g. be parenteral, such as
intravenous, intramuscular or aneous. In one embodiment, the multispecific
antibodies may be administered by infusion in a weekly dosage of calculated by mg/mz.
Such dosages can, for example, be based on the mg/kg s provided above
according to the following: dose ) x 70: 1.8. Such administration may be
repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed
by continuous infusion over a period of from 2 to 24 hours, such as from 2 to 12 hours.
In one embodiment, the multispecific antibodies may be administered by slow
uous infusion over a long period, such as more than 24 hours, in order to reduce
toxic side effects.
In one embodiment the multispecific antibodies may be administered in a weekly
dosage of calculated as a fixed dose for up to 8 times, such as from 4 to 6 times when
given once a week. Such regimen may be repeated one or more times as necessary, for
example, after 6 months or 12 months. Such fixed dosages can, for example, be based
on the mg/kg dosages provided above, with a body weight estimate of 70 kg. The
dosage may be ined or adjusted by measuring the amount of multispecific
antibody of the present invention in the blood upon administration by for instance taking
out a biological sample and using anti-idiotypic antibodies which target the CD137
antigen antigen-binding region of the multispecific dies of the present invention.
In one embodiment, the multispecific antibodies may be administered as
maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
A multispecific antibody may also be stered lactically in order to
3O reduce the risk of developing cancer, delay the onset of the occurrence of an event in
cancer ssion, and/or reduce the risk of recurrence when a cancer is in remission.
The multispecific antibodies of the invention may also be administered in
combination therapy, i.e., combined with other therapeutic agents relevant for the
disease or ion to be treated. Accordingly, in one embodiment, the multispecific
antibody-containing medicament is for combination with one or more further therapeutic
agents, such as a cytotoxic, chemotherapeutic or anti-angiogenic agent.
Such combined administration may be simultaneous, separate or sequential. For
simultaneous administration the agents may be administered as one composition or as
separate compositions, as appropriate. The present invention thus also provides
methods for ng a disorder, which methods comprise administration of a
multispecific antibody of the present invention combined with one or more additional
therapeutic agents as described below.
In one embodiment, the t invention provides a method for treating a
disorder, which method comprises administration of a therapeutically effective amount of
a multispecific antibody of the present invention, and optionally at least one additional
therapeutic agent, to a subject in need thereof.
In one embodiment, the present invention provides a method for treating or
preventing cancer, which method ses administration of a eutically effective
amount of a multispecific antibody of the present invention and at least one additional
therapeutic agent to a subject in need thereof.
Pharmaceutical compositions of the invention can also be stered in
ation therapy, i.e., combined with other , or combined with other
treatment regimen. For example the multispecific antibodies may be combined with
radiotherapy and/or surgery and/or autologous or neic peripheral stem cell or bone
marrow transplantation.
Biomarkers
Thus, in one aspect, the present invention also relates to use of the pecific
antibody as a biomarker.
In another aspect, the invention relates to a kit for ing cross-linking
between CD40- and CD137-expressing cells, in a sample obtained from a patient, such
as a blood sample, lymph node sample or bone marrow sample, comprising
i) a multispecific antibody according to any one of the embodiments as
disclosed herein; and
ii) instructions for use of said kit.
In a further aspect, the invention relates to a method for ing whether
linking between CD40- and CD137-expressing cells occurs in a sample obtained
from a patient, such as a blood sample, lymph node sample or bone marrow sample,
3O upon administration of a bispecific antibody according to any one of the embodiments as
disclosed herein, comprising the steps of:
(i) contacting the sample with a multispecific antibody ing to any one of
the embodiments as disclosed herein under conditions that allow for formation of a
complex between said multispecific antibody and the CD40- and CD137-expressing cells;
and
(ii) analyzing whether a complex has been formed.
Detection of the complex can be done by methods known in the art, such as
performed in Example 4, 5, 6, 10, 11 or 12.
Anti-idiotypic antibodies
In another aspect, the invention relates to an anti-idiotypic antibody which binds
to the first and/or second antigen-binding region as defined in any one of the
embodiments disclosed herein.
In a ular embodiment, the anti-idiotypic antibody binds to the first and/or
second antigen-binding region of a multispecific antibody, n
- the first antigen-binding region comprises a VH sequence as set forth in SEQ
ID NO:117 and a VL sequence as set forth in SEQ ID NO:121, and
- the second antigen-binding region comprises a VH sequence comprising SEQ
ID NO:123 and a VL sequence comprising SEQ ID NO:127.
In one embodiment, the anti-idiotypic antibody binds to the first antigen-binding
region defined in any one of the ments disclosed herein. In a ic
embodiment, the first antigen-binding region comprises a VH sequence comprising SEQ
ID NO:117 and a VL sequence comprising SEQ ID NO:121.
In another ment, the anti-idiotypic antibody binds to the second antigen-
g region defined in any one of the embodiments disclosed herein. In a ic
embodiment, the second antigen-binding region comprises a VH sequence comprising
SEQ ID NO:123 and a VL sequence comprising SEQ ID NO:127.
The present invention is further illustrated by the following examples, which
should not be construed as limiting the scope of the invention.
EXAMPLES
Example 1: Generation of antibodies
The CD40 and each of the CD137 antibodies (i.e. clones 1-12) mentioned in
Table 1 above were produced with the VH and VL sequences described in Table 1 and
with a human K light chain, and with a human IgGl heavy chain. The CD40 antibody was
produced with two ent human IgGl heavy ; 1) a human IgGl heavy chain
containing the following amino acid mutations: L234F, L235E, D265A and F405L (FEAL)
wherein the amino acid position number is according to EU numbering (corresponding to
SEQ ID NO:113); and 2) a human IgGl heavy chain containing the following amino acid
mutations: L234F, L235E, D265A and K409R (FEAR) wherein the amino acid position
number is according to EU numbering sponding to SEQ ID NO:112).
The CD137 dies were all produced with a human IgGl heavy chain
ning the following amino acid mutations: L234F, L235E, D265A and K409R (FEAR)
wherein the amino acid position number is according to EU numbering (correspond to
SEQ ID NO:112).
rly, a b12 antibody was produced comprising the VH and VL sequences
mentioned in Table 1, and with a human IgGl light chain and a human IgG heavy chain
containing the following amino acid mutations: L234F, L235E, D265A and F405L (FEAL)
wherein the amino acid position number is ing to EU numbering (correspond to
SEQ ID NO:113).
Antibodies were produced under serum-free conditions by co-transfecting
relevant heavy and light chain sion vectors in Expi293FTM cells (ThermoFisher
catalogue number A14527), using ExpiFectamineTM 293 (ThermoFisher catalogue
number A14525), according to the manufacturer’s instructions. Antibodies were purified
by protein A affinity chromatography and buffer exchanged into 12.6 mM NaH2PO4, 140
mM NaCl, pH 7.4 buffer (B.Braun or Thermo Fisher). After buffer exchange, samples
were sterile filtered over 0.2 pm nd filters. Purified proteins were analyzed by CE-
SDS and HP-SEC. Concentration was measured by absorbance at 280 nm. Purified
antibodies were stored at 2-8°C.
Example 2: DNA shuffling between wild boar or elephant and human C0137 to
determine domains important for binding of anti C0137 antibodies
To ine domains important for binding of anti CD137 antibodies to human
CD137, DNA shuffling was med between human and wild boar CD137 (sus scrofa;
XP_005665023) or between human and n nt CD137 (loxodonta africana;
XP_OO3413533). Shuffle constructs were prepared from DNA encoding human CD137,
by replacing human domains with wild boar (shuffle construct 1-4, 6) or elephant
le construct 5) domains. If a domain in human CD137 is important for g of
an anti CD137 antibody, binding will be lost upon replacement of that domain with the
3O wild boar or African elephant domain. Requirement is that the dy does not bind to
the whole CD137 elephant or wild boar sequence Homology between human and wild
boar and between human and African elephant CD137 is 70.2 and 74.5%, respectively.
Figure 1 shows sequence alignments of human, wild boar and African elephant CD137.
Figure 2 shows the constructs for human CD137 containing wild boar CD137 or African
elephant domains, as indicated.
3 x 106 HEK293T-17 cells were seeded in T75 culture flasks (Greiner Bio-One,
cat. no. ) in 20 mL RPMI 1640 AX medium containing 10% FCS
(Biochrom, cat. no. S 0115). After O/N incubation, cells were transiently transduced with
expression vectors encoding the e constructs or the wild boar, African elephant or
human CD137 downstream of a constitutively active human elongation factor-1 alpha
(EF-1 alpha) promotor using TransIT®-LT1 Transfection Reagent, Mirus Bio (VWR
International, cat. no. 29), according to the manufacturer’s instructions. The next
day, cells were ted using 1.5 mL Accutase (Sigma Aldrich, cat. no. A6964)
(incubation at 37°C for 5 min.) and flow cytometry was performed, essentially as
described in Example 4, to measure surface expression of the shuffle constructs and the
human, African elephant and wild boar CD137 and to measure binding of the antibody
clones to the different shuffle constructs. To measure cell surface expression of the
constructs, transduced cells were incubated with 1 ug/mL goat polyclonal anti-human
CD137 (R&D Systems, cat. no. AF838) in FACS buffer (D-PBS supplemented with 5 mM
EDTA [Sigma Aldrich, cat. no. 03690] and 5% (v/v) fetal bovine serum [FBS, Biochrom,
cat. no. S 0115]) (4°C, 20 min.), followed by incubation with APC-labeled anti-goat IgG
(H+L) (R&D Systems, cat. no. F0108) (4°C, 20 min.). g of the different CD137
antibody clones to cells expressing the shuffle constructs was measured by incubation of
the transduced cells with 1 ug/mL of the antibody clones, followed by APC-labeled
AffiniPure 2 Fragment (1:50 final on; Jackson, cat. no. 109127).
All CD137 shuffle constructs, as well as human, African elephant and wild boar
CD137, were expressed on the cell surface with similar expression levels (Figure 3).
Table 2 and Figure 4 show that all , except for clone 1, showed loss of
binding to at least African elephant or wild boar CD137. Clones 2, 3, 4, 5, 6, 7, 8, 9, 10
and 11 showed loss of binding to at least one of the shuffle constructs. Clone 1 showed
reduced binding to African elephant and shuffle uct 5, as compared to binding to
human CD137. Clone 12 did not show loss of binding to any of the shuffle constructs,
but showed reduced binding to shuffle construct 5. None of the clones showed loss of
binding or reduced binding to e constructs 1 and 2.
Table 2: Summary of binding of the CD137 antibodies to the shuffle constructs
g similar to g decreased No binding
human CD137 compared to human
binding CD137 binding
Wild boar CD137 Clone 1, 2, 6, 10 None Clone 3, 4, 5,
7, 8, 9, 11, 12
African elephant None Clone 1,3 Clone 2, 4, 5,
CD137 6, 7-12
Shuffle 1 (aa 162- Clone 1-12 None None
Shuffle 2(139-161) Clone 1-12 None None
Shuffle 3 (115-138) Clone 1, 2, 5, 6, 9, Clone 3, 7, 8 Clone 4, 11
, 12
Shuffle 4 (89-114) Clone 1, 2, 5, 6, 9, None Clone 3, 4, 7,
, 12 8, 11
Shuffle 5 (48-88) Clone 3, 4, 5, 7, 8, Clone 1, 12 Clone 2, 6, 9,
11 10
e 6 (aa 24-47) Clone 1, 2, 3, 4, 6, None Clone 5
Example 3: Generation of bispecific antibodies by 2-MEA-induced Fab-arm exchange
A method to produce stable IgGl-based bispecific antibodies is described in
W02011131746 (Genmab). The bispecific antibody product generated by this method
described below will no longer participate in m exchange. The basis for this
method was the use of complimentary CH3 domains, which promote the formation of
heterodimers under specific assay conditions. To enable the production of bispecific
antibodies by this method, IgGl molecules carrying certain mutations in the CH3 domain
were generated: in one parental IgGl antibody T350I, K37OT and F405L mutations (or
minimally F405L), in the other parental IgGl antibody a K409R mutation.
The concentrations of parental IgGl antibodies that minimally contained either an
F405L or a K409R point on were ed using their absorption at 280 nm.
Specific extinction coefficients based upon the amino acid sequence were used to infer
the n concentration.
The Cube system is Genmab’s flexible robotic work cell. The system was designed
and built in collaboration with Peak Analysis and Automation (PAA), rough UK.
Bispecific antibodies were generated by combining the following antibodies from
Example 1:
- CD40-FEAL antibody combined with each of the CD137-FEAR antibodies,
- CD40-FEAR antibody combined with the b12-FEAL dy, and
- Each of the CD137-FEAR antibodies combined with the b12-FEAL antibody
The bispecific antibody discovery process is performed in an automated fashion
on the Cube system, as shown in Figure 5 and described below.
To te ific antibodies, the following ated) steps are performed:
- Depending on the volume required, deep well source plates (96 well clear
V-bottom 2 mL polypropylene deep well plate, Corning, cat. no. 3960; 48 well Riplate®
SW 5mL, Ritter, cat. no. 43001-1062 ; 24 well Riplate SW 10 mL, Ritter, cat. no. 43001-
1066) are filled with parental antibodies - and containing antibodies in
different plates) at a concentration of 1.0 mg/mL (in 1x PBS, B.Braun) (Figure 5, left
plates).
- From these source plates, the pre-grid plates (96 well V-bottom, Corning)
are prepared by the Cube, according to Figure 5, middle . For each combination
with a parental antibody in the exchange grid, 67.5 uL al antibody is added to the
appropriate pre-grid plate.
- After the pre-grid, the exchange is performed. Here, two parental
antibodies (67.5 pL, 1.0 mg/mL each), each from a different pre-grid plate are added to
an exchange plate (96 well round-bottom polypropylene plate, Greiner, cat. no.
650293), each antibody at a final concentration of 0.5 mg/mL (equimolar concentration)
(Figure 5, right ).
- The exchange reaction is d by adding 15 pL 75 mM 2-
mercaptoethylamine-HCI (2-MEA) (in 1x PBS, B.Braun) to the exchange plate. The total
volume in the exchange plate is now 150 pL (final concentration 2-MEA 7.5 mM)
- The exchange plates are incubated at 31°C for 5 hours in the Cytomat 6000
automated incubator (Thermo Scientific).
- The reducing agent 2-MEA is removed by using ing columns (PhyTip
ing columns, 600 uL resin, PhyNexus, cat. no. PDR 9106), for which flow is
based on gravity.
0 The s are conditioned by placing an adapter with 96 columns on a
waste position, adding two times 450 pL 1x PBS (B.Braun) and allowing the ons to
flow through the columns into the waste.
0 After conditioning, 100 pL sample from the exchange plate is added,
thereby pushing the remaining PBS out of the columns.
0 After allowing the solutions to flow through the columns into the waste, the
adapter with columns is placed on a desalting (or destination) plate (96 well round-
bottom, Greiner).
o The ing sample from the exchange plate is added to the columns.
0 After allowing the samples to flow h the columns into the desalting
3O plate, 225 pL 1x PBS (B.Braun) is added to the columns and the sample is eluted into
the desalting plate.
0 The 2-MEA remains inside the columns. Where appropriate, the s
can be regenerated by washing with 1x PBS (B.Braun).
- The desalting plates are stored in the Cytomat 6001 ted incubator
(Thermo Scientific) at 8°C. These plates now contain the bispecific antibodies.
The final bispecific antibody samples were filtered over 0.2 pm dead-end filters
and the absorbance at 280 nm (A280) of bispecific products was measured to determine
the final concentration. s were stored at 2-8 0C for at least 24 hours before
further use.
The bispecific antibody exchange efficiency was quantified using High Pressure
Liquid Chromatography (HPLC) — hydrophobic interaction chromatography (HIC) using a
Butyl-NPR, 2.5 pm, 4.6 x 35 mm HIC-HPLC column (Tosoh Bioscience) with a flow rate
of 1 mL/min. Parental antibodies and analysis samples were normalized in concentration
and diluted two-fold with HIC eluent A (15.4 mM KZHPO4, 9.6 mM KH2PO4, 1.5 M
SO4; pH 7.0). 50 pL of sample was injected and n was performed with a
12-min gradient of HIC eluent A to HIC eluent B (15.4 mM KZHPO4, 9.6 mM KHZPO4; pH
7.0) with detection at 280 nm. Alternatively, HPLC - analytical cation exchange
chromatography (CIEX) was used to quantify the bispecific antibody ge efficiency.
Parental antibodies and analysis samples at 1 mg/mL in mobile Phase A (10 mM NaPO4,
pH 7.0) were injected onto the HPLC. The differently charged IgG molecules were
separated by using a ProPac WCX-10, 4 mm x 250 mm, analytical column with a flow
rate of 1 . 50 uL of sample was injected and elution was performed with a
gradient of Mobile Phase A (10 mM NaPO4, pH 7.0; prepared from a 0.1 M stock of
sodium phosphate buffer, that was obtained by adding 10.3 g NazHPO4-2HZO and 5.07 g
NaHZPO4 per liter Milli-Q) to Mobile Phase B (10 mM NaPO4, pH 7.0, 0.25 M NaCl) with
detection at 280 nm. Empower 3 software (Waters) was used to assign peaks as
parental antibodies or bispecific reaction products, and to integrate peak areas to
quantify extent of the bispecific antibody exchange reaction. Bispecific antibody reaction
products were further analyzed using analytical size exclusion chromatography, using a
TSK HP-SEC column (G3000Sle; Tosoh Biosciences, via Omnilabo, Breda, The
Netherlands) and Capillary Electrophoresis — Sodium Dodecyl Sulfate (CE-SDS) using a
LabChip GXII er Life Sciences, MA) on a HT Protein Express LabChip (Caliper Life
Sciences, MA) under reducing and non-reducing conditions according to manufacturer’s
instructions.
Example 4: Reporter assay measuring trans-activation by bispecific antibodies binding to
CD40 and CD137
3O CD40 is predominantly sed on antigen-presenting cells (APCs), such as
dendritic cells, whereas CD137 is predominantly expressed on activated T cells. Thus,
ific dies binding to CD40 and CD137 can bind aneously to APCs and T
cells expressing these receptors. Thereby, these bispecific antibodies can e cell-
cell contact between APCs and T cells by receptor binding and activate both ors.
This receptor activation can be induced by linking and receptor clustering upon
cell-cell interaction and is not necessarily dependent on tic activity of the parental
monospecific nt dies. Thus, these trans-activating bispecific antibodies can
exert co-stimulatory activity in the context of interactions between APCs and T cells.
A reporter assay system was established to measure activation of each receptor
by the bispecific antibodies. NF-KB/293/GFP-LucTM Transcriptional Reporter Cell Line
(System Biosciences; cat. no. TR860A-1) is a reporter cell line designed for monitoring
the NF-KB signal transduction pathway in vitro. Activation of the NF-KB y can be
monitored by the ion of green scent protein (GFP) fluorescence as well as
luciferase activity for quantitative transcription activation reporter assays. NF-
KB/293/GFP-Luc cells were stably transduced with expression vectors encoding full
length human CD40 or CD137 downstream of a constitutively active human tion
factor-1 alpha (EF-1 alpha) promotor, using TransIT®-LT1 Transfection t, Mirus
Bio (VWR International, cat. no. 731-0029), ing to the manufacturer’s
instructions. Stable clones were selected using 10 mg/mL blasticidin (Invivogen, cat. no.
ant-bl-1). In addition, K562 cells were stably transduced, as described supra, with
human CD40 and CD137 to generate cell lines that can provide the corresponding target
antigen for the other arm of the bispecific antibody. Cell surface expression of the
receptors was measured by flow cytometry. 0.3 x 106 cells were spun down (460 x g, 5
min.) and washed in FACS buffer (D-PBS supplemented with 5 mM EDTA [Sigma Aldrich,
cat. no. 03690] and 5% (v/v) fetal bovine serum [FBS, Biochrom, cat. no. S 0115]) (460
x g, 5 min.). 50 uL of 1:50 diluted allophycocyanin (APC)-labeled anti-human CD40 (BD
Biosciences, clone 5C3, cat. no. ) or phycoerythrin (PE)-labeled anti-human
CD137 (BD Biosciences, clone 4B4-1, cat. no. 555956) was added to the cell pellet and
ted at 4°C in the dark for 20 minutes. After washing three times with FACS buffer,
cells were ended in 100 uL FACS buffer and binding of the antibodies was
detected by flow cytometry on a FACSCanto II (BD Biosciences). Cell surface expression
of CD40 and CD137 on transduced NF-KB/293/GFP-Luc cells (Figure 6A) and K562 cells
(Figure 6B) was nicely shown.
The reporter assay measuring trans-activation was set up as follows: NF-
KB/293/GFP-Luc cells expressing one of the two indicated TNF receptors were seeded at
3O 10,000 cells/well in 30 uL RPMI 1640 medium with GlutaMAX supplement (Life
Technologies, cat. no. 61870) in white opaque 384-well cell culture plates (PerkinElmer,
cat. no. 6007680). Bispecific antibodies binding with one arm to CD40 and with the
other arm to CD137 and the corresponding monospecific, monovalent (containing one
irrelevant control arm [b12]) control antibodies were added in 10 uL/well to the reporter
cells in serial dilutions (in medium), g from 0.078 ug/mL to 10 ug/mL final
tration, ing a buffer control. 17,000 K562 cells expressing the second TNF
receptor or wildtype K562 (K562_wt) cells were added in 10 uL medium to each well and
incubated at 37°C and 5% C02 for 18 hours. Thus, the bispecific antibodies are able to
bind to the first TNF receptor on the 293/GFP-Luc cell line and, at the same time,
to the second TNF receptor on the K562 cell line. Only receptor activation of the first TNF
receptor on 293/GFP-Luc cells is measured by luciferase activity induced upon NF-
KB signaling. Thus, bispecific antibodies ing CD40 and CD137 were analyzed by
two reporter assays: the first assay measuring CD137 activation induced by
simultaneous binding of CD137 on the reporter cell line and CD40 on the K562 cells
(HEK293_NFK_CD137_gfp_luc + K562_CD40) and the second assay ing CD40
activation induced by simultaneous binding of CD40 on the reporter cell line and CD137
on the K562 cells (HEK293_NFK_CD40_gfp_luc + K562_CD137). Luciferase activity was
measured as relative luminescence units on an Envision plate reader (PerkinElmer) after
addition of 50 uL/well Steady-Glo® reagent (Promega; cat. no. E2520) reconstituted in
Glo Lysis Buffer ga; cat. no. E266A) and incubation at room temperature for 30
Only the bispecific CD40xCD137 antibodies (Figure 7 A-L, lower panels, first and
third graph) d luciferase activity (at concentrations of about 100 ng/mL and
higher) in NF-KB/293/GFP-Luc cells transduced either with CD137 or with CD40, under
trans-activation ions (incubation with K562-CD40 or K562-CD137, respectively).
None of the monospecific, monovalent (containing one irrelevant control arm [b12])
control antibodies induced luciferase activity in the transduced NF-KB/293/GFP-Luc cells
(upper panels). Furthermore, in the absence of activation conditions (using
wildtype K562 cells) no luciferase activity was induced by the bispecific CD40xCD137
antibodies (lower panels, second and fourth panel).
Example 5: Non-antigen-specific T-cell proliferation assay to measure trans-activation by
bispecific antibodies binding to CD40 and CD137
To measure tigen-specific proliferation, T cells in a peripheral blood
mononuclear cell (PBMC) population were ted with a sub-optimal concentration of
anti-CD3 antibody (clone UCHT1), combined with CD40xCD137 bispecific or l
antibodies. Within this PBMC population, n-presenting cells expressing CD40 can
be bound by the CD40-specific arm of the bispecific antibody, whereas the T cells in the
3O population can be bound by the CD137-specific arm. activation of the T cells
induced by cross-linking to the antigen-presenting cells via the bispecific dy is
measured as T-cell proliferation.
PBMCs were obtained from buffy coats of healthy donors (Transfusionszentrale,
University Hospital, Mainz, Germany) using a Ficoll gradient (VWR, cat. no. 1702).
PBMCs were labeled using 1.6 uM carboxyfluorescein succinimidyl ester (CFSE) (Thermo
Fisher, cat. no. C34564) in PBS, according to the manufacturer's instructions. 75,000
CFSE-labeled PBMCs were seeded per well in a 96-well round-bottom plate (Sigma
Aldrich, CLS3799-50EA) and incubated with a sub-optimal concentration of anti-CD3
antibody (R&D Systems, clone UCHT1, cat. no. MABlOO; 0,01-0,1 ug/mL final
concentration) that was pre-determined for each donor, and bispecific or control
antibodies, in 150 uL IMDM GlutaMAX supplemented with 5% human AB serum, at 37°C,
% C02, for four days. Proliferation of CD4+ and CD8+ T cells was analyzed by flow
cytometry, essentially as described supra. 30 uL containing PE-labeled CD4 antibody (BD
ences, cat. no. 555347; 1:80 final dilution), PE-Cy7-labeled CD80 dy (clone
RPA-T8, eBioscience, cat. no. 2541; 1:80 final dilution) APC-labeled CD56
antibody (eBiosciences, cat. no. 17—0567; 1:80 final dilution) and 7-AAD (Beckman
Coulter, cat. no. AO7704; 1:80 final dilution) in FACS buffer was used to stain the cells
and exclude natural killer (NK) cells (CD56) and dead cells (7-AAD) from the analysis.
Samples were measured on a FACSCanto II (BD Biosciences). Detailed es of T-cell
proliferation based on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5
software. Mean percentages of T cells that went into division (% divided cells) and the
average number of divisions of cells that went into on (proliferation index) were
calculated.
Figure 8A shows that only the CD40xCD137 bispecific antibodies efficiently
enhanced proliferation of CD8+ T cells. The control monospecific, monovalent antibodies
(b12xCD40; b12xCD137) and the combination of ecific, monovalent CD40 with
monospecific, monovalent CD137 antibodies D40 + b12xCD137) did not induce
more proliferation than observed in the control (only the weakly activated PBMCs, ctrl,
w/o). The flow cytometry histograms, for the different antibodies at ent
concentrations, were quantified to indicate percentage of divided cells e 8B) and
eration index (Figure 8C), as described supra. These figures show that only the
bispecific antibodies were capable of inducing proliferation of CD8+ cells, with an
optimum between 0.04 and 0.2 ug/mL.
Example 6: Antigen-specific CDBJr T cell proliferation assay to measure trans-activation
by ific antibodies binding to CD40 and C0137
3O To e induction of proliferation by the bispecific antibodies in an antigen-
ic assay, dendritic cells (DCs) were ected with claudin 6 in vitro-transcribed
RNA (IVT-RNA,) to express the claudin 6 antigen. T cells were ected with the
nspecific, HLA-AZ-restricted T cell receptor (TCR). This TCR can recognize the
claudinderived epitope presented in HLA-AZ on the DC. The CD40xCD137 bispecific
antibody can cross-link CD40 on the dendritic cell and CD137 on the T cell, leading to
activation of the DC and a co-stimulatory signal to the T cell, ing in T-cell
proliferation.
HLA-AZJr PBMCs were obtained from healthy donors (Transfusionszentrale,
University Hospital, Mainz, Germany). tes were isolated from PBMCs by
magnetic-activated cell sorting (MACS) technology using D14 MicroBeads
(Miltenyi; cat. no. 130201), according to the manufacturer’s instructions. The
peripheral blood lymphocytes (PBLs, egative fraction) were frozen for future T-
cell isolation. For differentiation into immature DCs (iDCs), 1x106 monocytes/mL were
cultured in RPMI GlutaMAX (Life technologies GmbH, cat. no. 61870-044) containing 5%
human AB serum (Sigma-Aldrich Chemie GmbH, cat. no. H4522-100ML), sodium
pyruvate (Life technologies GmbH, cat. no. 11360-039), non-essential amino acids (Life
technologies GmbH, cat. no. 11140-035), 100 IU/mL penicillin-streptomycin (Life
technologies GmbH, cat. no.15140-122) 1000 IU/mL granulocyte-macrophage colony-
stimulating factor (GM-CSF; Miltenyi, cat. no. 130868) and 1000 IU/mL interleukin
4 (IL-4; Miltenyi, cat. no. 130924), for five days. Once during these five days, half
of the medium was replaced with fresh medium. iDCs were harvested by collecting non-
adherent cells; adherent cells were detached by incubation with PBS ning 2mM
EDTA for 10 min at 37°. After washing, iDCs were frozen in RPMI GlutaMAX containing
% v/v DMSO (AppliChem GmbH, cat. no A3672,0050) and 50% v/v human AB serum
for future antigen-specific T cell assays.
One day before T-cell assays were started, frozen PBLs and iDCs, from the same
donor, were thawed. PBLs were used for isolation of CD8+ T cells by MACS technology
using anti-CD8 MicroBeads (Miltenyi, cat. no. 130201), according to the
manufacturer’s instructions. About 10-15 x 106 CD8+ T cells were oporated with 10
ug IVT-RNA encoding the alpha-chain plus 10 ug of IVT-RNA encoding the beta-chain of
a claudinspecific murine TCR (HLA-A2-restricted; bed in WO 2015150327 A1) in
250 uL X-Viv015 (Biozym Scientific GmbH, cat. no.881026) in a 4-mm electroporation
cuvette (VWR International GmbH, cat. no. 732-0023) using the BTX ECM® 830
Electroporation System device (BTX; 500 V, 1 x 3 ms pulse). Immediately after
3O electroporation, cells were transferred into fresh IMDM medium (Life logies
GmbH, cat. no. 12440-061) supplemented with 5% human AB serum and rested at
37°C, 5% C02 for at least 1 hour. T cells were d using 1,6 uM carboxyfluorescein
succinimidyl ester (CFSE; ogen, cat. no. C34564) in PBS, according to the
manufacturer's instructions, and incubated in IMDM medium supplemented with 5%
human AB serum, O/N.
Up to 5 X 106 thawed iDCs were electroporated with 0,4—5 ug IVT-RNA encoding
full length n-6 (Uniprot P56747), in 250 uL X-Viv015 medium, using the
electroporation system as described above (300 V, 1x12 ms pulse) and incubated in
IMDM medium supplemented with 5% human AB serum, O/N.
The next day, cells were harvested. Cell surface expression of claudin-6 on DCs
and TCR on T cells were checked by flow cytometry. Therefore, DCs were stained with an
Alexa647-conjugated CLDN6-specific antibody (not cially available; in-house
production) and T cells were stained with an anti-mouse TCR [3 Chain antibody (Becton
Dickinson GmbH, cat. no. ). 5,000 electroporated DCs were incubated with
50,000 electroporated, CFSE-labeled T cells in the presence of ific or control
antibodies in IMDM GlutaMAX (Life Technologies, cat. no. 12440-061) supplemented
with 5% human AB serum in a l round-bottom plate. T-cell proliferation was
measured after 5 days by flow cytometry. Detailed analyses of T-cell proliferation based
on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5 software. Mean
percentages of T cells that went into division (% divided cells) and the average number
of divisions of cells that went into division (proliferation index) were calculated.
Figure 9A shows that only the CD40xCD137 bispecific antibodies efficiently
ed proliferation of CD8+ T cells. The control ecific, monovalent antibodies
(b12xCD40; b12xCD137) and the combination of monospecific, monovalent CD40 with
monospecific, lent CD137 antibodies (b12xCD40 + b12xCD137) did not induce
more proliferation than observed in the control (only the weakly activated PBMCs, ctrl,
w/o). The same is also reflected in the percentage of divided cells (Figure 9B) and is
very clear from the proliferation index e 9C). Figure 9D shows that the induction
of antigen-specific proliferation by the CD40xCD137 bispecific antibodies was
concentration dependent, with an optimum around 0.1 ug/mL in this assay.
Example 7: Humanization of murine and rabbit antibodies
Humanized dy sequences from the antibodies mouse anti-CD40-001 and
rabbit anti-CD137-009 were generated at Antitope (Cambridge, UK). Humanized
antibody sequences were generated using germline humanization (CDR-grafting)
technology. Humanized V region genes were designed based upon human germline
sequences with closest homology to the VH and VK amino acid sequences of the murine
3O and rabbit antibodies. A series of four to six VH and four or five VK (VL) germline
zed V-region genes were designed for each of the non-human parental
antibodies. Structural models of the non-human parental antibody V regions were
produced using Swiss PDB and analyzed in order to identify amino acids in the V region
frameworks that may be important for the binding properties of the dy. These
amino acids were noted for incorporation into one or more variant CDR-grafted
antibodies. The closest matching germ|ine ces used as the basis for the
humanized designs are shown in Table 3.
Table 3: Closest matching human germ|ine V segment and J segment sequences.
Antibody Heavy chain Light chain (K)
Human V region Human J region Human V region Human J region
germ|ine germ|ine germ|ine germ|ine
segment segment segment segment
Mouse anti- hIGHV1-46*01 hIGHJ4 hIGKV1-33*01 IGKJ4
CD40-001
Rabbit anti- hIGHV3-49*04 hIGHJ4 hIGKV1-33*01 IGKJ4
CD137-009
t sequences with the lowest incidence of potential T cell epitopes were then
selected using Antitope’s proprietary in silico technologies, iTopeTM and TCEDTM (T Cell
Epitope Database) (Perry, L.C.A, Jones, TD. and Baker, M.P. New Approaches to
Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development
(2008). Drugs in R&D 9 (6): 385-396; 20; , C.J., Jones, TD. and Baker, M.P.
tion of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24 (1):1-8).
y, the nucleotide sequences of the designed variants were codon-optimized for
expression in human cells.
The variable region sequences of the humanized CD40 and CD137 antibodies are
shown in the Sequence Listing and in Table 1 above.
Example 8: Expression ucts for dies, transient expression and purification
For antibody sion the VH and VL sequences were cloned in expression
vectors (pcDNA3.3) containing, in case of the VH, the relevant constant heavy chain
(HC), in certain cases ning a F405L or K409R mutation, and/or L234F, L235E and
D265A, and, in case of the VL, light chain (LC) regions. Antibodies were expressed as
IgGl,K. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were
transiently transfected in 3F cells (Life technologies, USA) using 293fectin (Life
technologies) essentially as described by Vink et al. (Vink et al., Methods, 65 (1), 5-10
2014). Next, antibodies were purified by immobilized protein G chromatography.
Example 9: Non-specific T-cell proliferation assay to test the onality of a
zed bispecific antibody binding to CD40 and CD137
To measure the functionality of a humanized bispecific antibody binding to CD40
and CD137, a non-antigen-specific T-cell proliferation assay was performed as bed
supra. In short, PBMCs of one donor were CFSE-Iabeled and incubated with a sub-
optimal concentration of anti-CD3 antibody (clone UCHT1; 0.01 pg/mL as determined for
this donor) and 0.008, 0.04, 0.2 or 1 pg/mL humanized CD40xCD137 bispecific
antibody, the parental bispecific antibody or IgGl control antibody.
Proliferation of CD8+ T cells was analyzed by flow cytometry, essentially as
described supra. Detailed analyses of T-cell proliferation based on CFSE-peaks indicating
cell divisions were made by FlowJo 7.6.5 software. Mean percentages of T cells that went
into division (% divided cells) and the average number of divisions of cells that went into
on (proliferation index) were calculated.
Figure 10 shows that the humanized CD40xCD137 bispecific antibody (BisGl-
CD40HC6LC1-FEALxCD137HC7LC2-FEAR) efficiently enhanced eration of
CD8+ T cells. The humanized ific antibody enhanced both the percentage of
divided cells and the average number of divisions of CD8+ cells. Efficacy of the
humanized bispecific antibody was comparable to that of the parental bispecific dy
(CD40-001xCD137-009).
Example 10: Antigen-specific CD8Jr T-cell eration assay to test the functionality of
the zed ific antibody binding to CD40 and CD137
To measure the functionality of the humanized bispecific antibody binding to
CD40 and CD137, an antigen-specific CD8+ T-cell proliferation assay was performed as
described supra. In short, CFSE-labeled, CLDN6-TCR transfected CD8+ T-cells were
incubated with CLDN6 ectroporated DCs in the presence of humanized
CD40xCD137 bispecific antibody, the parental antibody or IgGl l antibody. T-cell
proliferation was measured by flow cytometry after 4 days. Detailed analyses of T-cell
eration based on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5
software. Mean percentages of T cells that went into division (% divided cells) and the
3O average number of divisions of cells that went into division (proliferation index) were
calculated.
Figure 11 shows that the humanized CD40xCD137 bispecific dy (BisGl-
CD40H6LC1-FEALXCD137HC7LC2-FEAR) efficiently enhanced proliferation of
CD8+ T cells. Efficacy of the humanized bispecific antibody was comparable to that of the
parental bispecific antibody (CD40-001xCD137-009). Both the humanized and the
WO 11421
parental bispecific antibody enhanced the percentage of divided cells as well as the
eration index of the CD8+ cells in this assay.
e 11: Ex vivo TIL expansion assay to evaluate the effects of CD40xCD137
ific antibodies on tumor infiltrating lymphocytes
To evaluate the effects of CD40xCD137 bispecific antibody (BisGl-CD40
FEAL/CD137FEAR) on tumor infiltrating lymphocytes (TIL), ex vivo culture of
human tumor tissue was performed as follows. Freshly human tumor tissue resections
were washed three times by transferring the isolated tumor chunks from one wash
medium-containing well of a six-well plate (Fisher Scientific cat. no. 10110151) to the
next using a a or gical pipette. Wash medium was composed of X-VIVO 15
m, cat. no. 881024) supplemented with 1% Pen/Strep (Thermo Fisher, cat. no.
15140-122) and 1% Fungizone (Thermo Fisher, cat. no. 15290-026). Next, the tumor
was dissected with a surgical knife (Braun/Roth, cat. no. 5518091 BA223) and cut into
tumor pieces with a diameter of about 1-2 mm. Two pieces each were put into one well
of a l plate (VWR international, cat. no. 701605) containing 1 mL TIL medium (X-
VIVO 15, 10% Human Serum Albumin (HSA, CSL Behring, cat. no. 46518), 1%
Pen/Strep, 1% Fungizone and IL-2 (Proleukin®S, Novartis Pharma, cat. no. 02238131)
at the indicated concentration. Bispecific antibody binding to CD40 and CD137 was
added at the indicated final concentrations. Culture plates were incubated at 37°C and
5% C02 for 72 hours and 1 mL fresh TIL medium containing the indicated IL-2
concentration and the indicated concentration of bispecific antibody was added to each
well. Wells were monitored for the occurrence of TIL clusters using a Leica DMi1
cope equipped with a 5.0 megapixel camera, every other day. Wells were split on
an individual basis, when more than 25 TIL microclusters were detected. To split TIL
cultures, cells were re-suspended and erred to a well of a 6-well plate and
supplemented with another 2 mL of TIL medium.
After a total culture period of 10-14 days, TILs were harvested and analyzed by
flow cytometry. To allow for quantitative comparison of the different treatment ,
cell pellets were re-suspended after the last washing step with FACS-buffer
3O supplemented with BDTM CompBeads (BD ences, cat. no. 519001291). Flow
cytometric analysis was med on a BD FACSCantoTM II flow cytometer (Becton
Dickinson) and acquired data was analyzed using FlowJo 7.6.5 software. The relative
viable TIL count (7-AAD-negative cells) per 1,000 beads was calculated for each well.
Figure 12 shows the analysis of a TIL expansion from a human melanoma
tissue. Here, 100 U/mL IL-2 was used as supplement for the TIL medium. Moreover, the
following concentrations of the bispecific antibody binding to CD40 and CD137 (BisGl-
CD40FEAL/CD137FEAR) were added: 0.016, 0.08, 0.4, 2.0 and 10.0 ug/mL;
wells without antibody addition served as negative control. After 14 days of culture, TILs
were harvested and analyzed by flow try. Five samples for each antibody
concentration, derived from different wells of the 24-well plate, were measured. In all
samples cultured with the ific antibody binding to CD40 and CD137 the viable
count of TIL was substantially increased in comparison to the control samples without
antibody. Overall, about a 100-fold increase of the mean relative viable TIL count was
observed (Figure 12).
Figure 13 shows the is of a TIL expansion from a non-small cell lung
cancer (NSCLC) tissue. Here, 10 U/mL IL-2 was used as supplement for the TIL medium.
In addition, the following final concentrations of the bispecific antibody binding to CD40
and CD137 -CD40FEAL/CD137FEAR) were administered: 0.01, 0.1,
and 1.0 ug/mL; wells without antibody addition served as ve l. After 10 days
of culture, TILs were harvested and analyzed by flow cytometry. Five samples for each
antibody concentration, derived from ent wells of the l plate, were
measured. In all samples cultured with the bispecific antibody binding to CD40 and
CD137, the viable count of TIL was substantially increased in comparison to the control
samples without antibody. Overall, an up to 10-fold increase of the mean relative viable
TIL count was observed at 0.1 or 1 ug/mL (Figure 13).
Claims (82)
1. A multispecific antibody comprising (I) a first n-binding region binding to human CD40, n said first antigenbinding region ses heavy and light chain variable region CDR1, CDR2, and 5 CDR3 selected from the group consisting of: a) heavy chain variable region CDR1, CDRZ and CDR3 having the amino acid sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDRZ and CDR3 having the amino acid sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; 10 b) heavy and light chain variable region CDR1, CDRZ and CDR3 according to a) having a total of one to twelve mutations; and c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDRZ and CDR3 according to a) or b) 15 and/or (ii) has the specificity for CD40 of an dy comprising heavy and light chain variable region CDR1, CDRZ and CDR3 according to a) or b), (II) a second antigen-binding region binding to human CD137. 20 2. The multispecific antibody according to claim 1, wherein said first antigen-binding region comprises a first heavy chain variable (VH) sequence and a first light chain variable (VL) sequence, said second antigen-binding region comprises a second VH sequence and a second VL sequence, and said first and second VH and VL sequences each comprises three CDR sequences, CDR1, CDRZ and CDR3, respectively, and four 25 ork sequences, FR1, FR
2. , FR3 and FR4, respectively.
3. The multispecific dy according to any one of the preceding claims, wherein said first antigen-binding region comprises heavy chain variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and 30 light chain variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively.
4. The multispecific antibody according to any one of claims 2-3, wherein said VH ce of the first antigen-binding region comprises an amino acid sequence 35 having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NOS:117 and 6. .
5. The multispecific antibody according to any one of claims 2-4, wherein said VL sequence of the first antigen-binding region ses an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to at least one of SEQ ID NOS:121 and 7. .
6. The multispecific antibody ing to any one of claims 2-5, wherein said VH sequence of the first n-binding region comprises an amino acid sequence 10 having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to SEQ ID NO:117; and wherein said VL sequence of the first n-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to SEQ ID 15 NOS:121. .
7. The multispecific antibody according to any one of the preceding claims, wherein the second antigen-binding region binds to cynomolgus CD137. 20 .
8. The multispecific antibody according to any one of the preceding claims, wherein said second antigen-binding region binds to human CD137 (SEQ ID NO:92) to a higher degree than it binds to a mutant human CD137 (SEQ ID NO:93). .
9. The multispecific antibody according to any one of claims 1-7, wherein said second 25 antigen-binding region binds to human CD137 (SEQ ID NO:92) to a higher degree than it binds to a mutant human CD137 (SEQ ID NO:94).
10. The pecific antibody according to any one of preceding claims, wherein said second antigen-binding region binds to human CD137 (SEQ ID NO:92) to the same 3O degree that it binds to a mutant human CD137 (SEQ ID NO:95).
11. The multispecific antibody according to any one of claims 8-10, wherein binding to said human CD137 (SEQ ID NO:92) and said mutant human CD137 (SEQ ID NO:93, 94 and 95, respectively) is determined by preparing shuffle ucts derived from 35 human CD137 in which protein domains of the human CD137 are replaced by the corresponding domain of CD137 from ent species, using human CD137 and the different species of CD137 as reference constructs; transducing cells with plasmids encoding the reference construct or the shuffle constructs, respectively, and measuring binding of the antibody to each these CD137 constructs by flow cytometry.
12.The multispecific antibody according to any one of claims 1-8 and 10-11, wherein said second antigen-binding region comprises heavy and light chain variable region CDR1, CDRZ and CDR3 selected from the group consisting of: a) heavy chain le region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, tively, and light chain variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS 10 and 68, respectively, and b) heavy and light chain variable region CDR1, CDRZ and CDR3 according to a) having a total of one to twelve mutations. c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy 15 and light chain variable region CDR1, CDRZ and CDR3 according to a) or b) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDRZ and CDR3 according to a), or b).
13. The multispecific antibody according to any one of claims 1-8 and 10-12, wherein 20 said VH sequence of the second n-binding region ses an amino acid ce having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% ty to at least one of SEQ ID NOS:123 and 69. 25
14. The multispecific antibody according to any one of claims 1-8 and 10-13, wherein said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to at least one of SEQ ID NO:127 and 70.
15. The multispecific antibody according to any one of claims 1-8 and 10-14, wherein said VH and said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% ty to SEQ ID 35 NO:123 and SEQ ID NO:127, respectively.
16.The multispecific antibody according to any one of claims 1-7 and 9-11, n said second antigen-binding region comprises heavy and light chain variable region CDR1, CDRZ and CDR3 selected from the group consisting of: a) heavy chain variable region CDR1, CDRZ and CDR3 having the sequences set 5 forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, and b) heavy and light chain variable region CDR1, CDRZ and CDR3 according to a) having a total of one to twelve mutations; 10 c) heavy and light chain variable region CDR1, CDRZ and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody sing heavy and light chain le region CDR1, CDRZ and CDR3 according to a), or b) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDRZ and CDR3 according to a), or b).
17.The multispecific antibody according to any one of claims 1-7, 9-11, and 16 wherein said VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:41.
18.The multispecific antibody according to any one of claims 1-7, 9-11, and 16-17, wherein said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% ty to SEQ ID NO:42.
19.The multispecific antibody according to any one of claims 1-7, 9-11, and 16-18, wherein said VH and VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:41; 30 and SEQ ID NO:42, respectively.
20.The multispecific antibody according to any one of claims 4-19, wherein said first n-binding region comprises heavy chain variable region CDR1, CDRZ and CDR3 having the amino acid sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, 35 and light chain le region CDR1, CDRZ and CDR3 having the amino acid sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-binding region comprises a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, or b) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively. 10
21. The multispecific antibody according to any one of claims 4-20, wherein the tive FR1, FR2, FR3 and FR4 framework sequences of the VH and VL ces of the first and/or second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework 15 sequences of said VH and VL sequences.
22. The multispecific antibody according to any one of the preceding claims, wherein said antibody comprises (I) a first binding arm comprising said first antigen-binding region, and (II) a second binding arm comprising said second antigen-binding region.
23. The multispecific antibody according to claim 22, wherein said first g arm ses a first heavy chain constant sequence, and said second binding arm comprises a second heavy chain constant ce. 25
24. The multispecific antibody according to any one of claims 22-23, wherein (I) said first binding arm comprises a first heavy chain comprising a first heavy chain le (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence, and (II) said second binding arm comprises a second heavy chain comprising a second heavy chain variable (VH) 3O sequence and a second heavy chain constant (CH) sequence, and a second light chain comprising a second light chain variable (VL) sequence.
25. The pecific antibody according to claim 24, wherein said first light chain further comprises a first light chain constant (CL) sequence, and said second light chain 35 further comprises a second light chain constant (CL) sequence.
26. The multispecific antibody ing to any one of the preceding claims, wherein the first n-binding region is d from a mouse antibody.
27. The multispecific antibody according to any one of the preceding claims, wherein the first antigen-binding region is derived from a humanized antibody.
28. The multispecific antibody according to any one of claims 22-27, wherein the first binding arm is d from a full-length antibody.
29. The multispecific dy according to any one of claims 22-28, wherein the first binding arm is d from a full-length IgGl,)x (lambda) or IgGl, K (kappa) 10 dy.
30. The multispecific antibody according to any one of the preceding claims, wherein the second antigen-binding region is derived from a rabbit antibody. 15
31. The multispecific antibody according to any one of the preceding claims, wherein the second antigen-binding region is derived from a humanized antibody.
32. The multispecific antibody according to any one of claims 22-31, wherein the second binding arm is derived from a full-length antibody.
33. The multispecific antibody ing to any one of claims 22-32, wherein the second binding arm is derived from a ength x (lambda) or IgGl, K (kappa) 25
34. The multispecific dy according to any one of the preceding claims, wherein each of the first and second antigen-binding region is derived from a humanized antibody.
35. The multispecific antibody according to any one of claims 22-34, wherein each of the 3O first and second binding arm is derived from a full-length antibody.
36. The multispecific antibody according to any one of claims 22-35, wherein each of the first and second binding arm is derived from a full-length IgGl,)x (lambda) or IgGl, K (kappa) dy.
37. The multispecific antibody according to any one of claims 22-36, wherein said first and said second heavy chains are of an IgG isotype, having a subclass selected from the group consisting of IgGl, IgGZ, IgG3, and IgG4. WO 11421
38.The multispecific antibody according to any of claims 24-37, wherein each of said first and second heavy chains comprise at least a hinge region, a CH2 and a CH3 region.
39.The multispecific antibody according to claim 38, wherein the CH3 regions of the first and second heavy chains comprise asymmetrical mutations.
40.The pecific antibody ing to any one of claims 24-39, wherein in said first 10 heavy chain at least one of the amino acids in the ons corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, 15 L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and n said first and said second heavy chains are not substituted in the same positions.
41.The multispecific dy according to claim 40, wherein (i) the amino acid in the 20 position ponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgGl heavy chain according to EU numbering is R in said second heavy chain, or (ii) the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said first 25 heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said second heavy chain.
42.The multispecific dy according to any one of the preceding claims, wherein said antibody comprises a first and a second heavy chain, and wherein said antibody 30 induces Fc-mediated effector function to a lesser extent compared to a pecific antibody comprising the same first and second antigen-binding regions and two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.
43.The multispecific antibody according to claim 42, wherein said first and second heavy 35 chains are modified so that the multispecific antibody induces Fc-mediated effector function to a lesser extent ed to a multispecific antibody which is identical except for comprising non-modified first and second heavy chains.
44.The multispecific antibody according to any one of claims 42-43, wherein said Fc- mediated effector function is measured by binding to Fcy receptors, binding to Clq, or induction of Fc-mediated cross-linking of FcRs. 5
45.The multispecific antibody according to claim 44, wherein said Fc-mediated effector function is measured by binding to Clq.
46.The multispecific antibody according to claim 45, and wherein said first and second heavy and light chain constant sequences have been modified so that binding of Clq 10 to said multispecific antibody is reduced compared to a wild-type bispecific antibody by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein Clq binding is determined by ELISA.
47.The multispecific dy according to any one of the ing claims, wherein said 15 antibody ses a first and a second heavy chain, wherein in at least one of said first and second heavy chains one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgGl heavy chain according to EU numbering, are not L, L, D, N, and P, respectively. 20
48.The multispecific antibody according to claim 47, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU ing are F and E, respectively, in said first and second heavy chains.
49.The pecific antibody according to claim 48, wherein the positions corresponding 25 to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chains.
50.The multispecific antibody according to claim 49, wherein the positions corresponding to ons L234, L235, and D265 in a human IgGl heavy chain according to EU 30 numbering of both the first heavy chain and the second heavy chain are F, E, and A, respectively, and wherein (i) the on corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain is R, or (ii) the on ponding to K409 in a human 35 IgGl heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain ing to EU numbering of the second heavy chain is L.
51. The multispecific antibody according to claim 48, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering of both the first heavy chain and the second heavy chain are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain is L, and the on corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU 10 numbering of the second heavy chain is L.
52. The multispecific antibody according to any of the preceding claims, wherein said dy is able to cross-link a first cell expressing human CD40 and a second cell expressing human CD137.
53. The multispecific antibody according to claim 52, wherein said linking is determined by an assay using a first cell line expressing human CD40 and a second cell line expressing human CD137, and n either the first or the second cell line comprises a er structure resulting in the production of a able reporter 20 upon NF-KB activation.
54. The multispecific antibody according to any one of the preceding claims, wherein said antibody induces and/or enhances proliferation of T cells. 25
55. The multispecific antibody according to claim 54, wherein said T cells are CD4+ T cells and/or CD8+ T cells.
56.The multispecific antibody according to any one of claims 54-55, wherein said induction or enhancement of proliferation of T cells is ined by sub-optimal 3O activating ofT cells in a PBMC pool.
57.The pecific antibody according to claim 56, wherein sub-optimal activation is determined by titrating the tration of anti-CD3 dy added to a PBMC pool, measuring T cell proliferation and choosing the anti-CD3 antibody concentration 35 which results in low T cell proliferation but allows for further enhancement of the T cell proliferation.
58. The pecific antibody according to any one of claims 54-55, wherein proliferation of T cells is measured by co-culturing T-cells expressing a specific T-cell receptor (TCR) with dendritic cells (DCs) presenting the corresponding antigen on the major histocompatibility complex, which is recognized by the TCR.
59. The multispecific antibody according to any one of claims 54-55, wherein said T cells are tumor-infiltrating lymphocytes (TILs), and said induction or enhancement of proliferation of the TILs is determined by incubating a human tumor sample with interleukin-2 (IL-2) and said antibody, and retrieving and counting TILs after 10 incubation for a period of about 10 to about 14 days.
60. The multispecific antibody of claim 59, wherein said human tumor is a melanoma or a non-small-cell lung cancer (NSCLC) tumor. 15
61. The pecific dy according to any one of claims 54-60, wherein said antibody induces or enhances more proliferation of T cells compared to a bispecific antibody sing a second antigen-binding region according to any one of claims 1-2, but wherein the first antigen-binding region comprises heavy chain variable region CDRl, CDRZ and CDR3 having the sequences set forth in SEQ ID NOs:99, 100 20 and 101, respectively, and light chain variable region CDRl, CDRZ and CDR3 having the ces set forth in SEQ ID NOs:102, GVS and 103, respectively.
62. The antibody according to any one of the preceding claims, wherein said antibody is a bispecific dy.
63. A c acid encoding one or more amino acid sequences of claims 1-62.
64. A nucleic acid encoding a multispecific antibody as defined in any one of claims 1-62. 3O
65. An expression vector comprising a nucleic acid according to any one of claims 63-64.
66. A host cell comprising a nucleic acid according to any one of claims 63 or 64, or an sion vector of claim 65. 35
67. The host cell according to claim 66, wherein said host cell is a inant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.
68. A composition comprising a multispecific antibody ing to any one of claims 1- 62, a nucleic acid according to any one of claims 63 and 64, an expression vector ing to claim 65 or a host cell according to any one of claims 66 and 67.
69. The composition according to claim 68, which is a pharmaceutical composition.
70. The pharmaceutical ition ing to claim 69, which further comprises a pharmaceutically acceptable carrier. 10
71. The multispecific antibody according to any one of claims 1-62, the nucleic acid according to claim 63 or 64, the expression vector according to claim 65, the host cell according to claim 66 or 67, the composition according to claim 68, or the pharmaceutical composition according to claim 69 or 70 for use as a medicament. 15
72. The multispecific antibody according to any one of claims 1-62, the nucleic acid according to claim 63 or 64, the expression vector according to claim 65, the host cell according to claim 66 or 67, the composition according to claim 68, or the pharmaceutical composition according to claim 69 or 70 for use in the treatment of a disease.
73. The multispecific dy, nucleic acid, expression vector, host cell or composition for use according to claim 72 wherein the e is an infectious disease.
74. The multispecific antibody, nucleic acid, expression vector, host cell or composition 25 for use ing to claim 72, wherein the disease is cancer.
75. The multispecific antibody, nucleic acid, expression vector, host cell or composition for use according to claim 74, wherein the cancer is ed from the group consisting of melanoma, lung cancer, breast cancer, colon cancer, renal cancer, 3O cervical cancer and prostate cancer, such as non-small cell lung cancer (NSCLC) or melanoma.
76. A method of treatment of a disease sing administering the multispecific antibody according to any one of claims 1-62, the nucleic acid ing to claim 63 35 or 64, the expression vector according to claim 65, the host cell according to claim 66 or 67, the composition according to claim 68, or the pharmaceutical composition according to claim 69 or 70 to a subject in need thereof.
77. Use of a pecific antibody according to any one of claims 1-62, the nucleic acid according to claim 63 or 64, the expression vector according to claim 65, the host cell according to claim 66 or 67, the composition according to claim 68, or the pharmaceutical composition according to claim 69 or 70 for the manufacture of a ment.
78. The method or use according to any one of claims 71-77, n the method or use is for use in combination with one or more further therapeutic agent, such as a herapeutic agent.
79. A method for producing a ific antibody according to claim 62, comprising the steps of a) providing a first antibody comprising an Fc region, said Fc region comprising a first CH3 region; 15 b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region, wherein the first antibody is a CD40 antibody comprising two first antigen- binding regions according to any of claims 1-62, and the second antibody is a CD137 antibody comprising two second antigen-binding regions according to any 20 of claims 1-62, or vice versa; and wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing 25 conditions; and d) obtaining said bispecific CD40xCD137 antibody.
80. A method for detecting whether linking between CD40- and CD137-expressing cells occurs in a sample obtained from a patient, such as a blood sample, lymph node 3O sample or bone marrow sample, upon stration of a multispecific antibody according to any one of claims 1-62, comprising the steps of: (i) ting the sample with a multispecific antibody according to any one of claims 1-62, under conditions that allow for ion of a complex between said multispecific antibody and the CD40- and expressing cells; and 35 (ii) analyzing whether a complex has been formed.
81.A kit for detecting linking between CD40- and CD137-expressing cells, in a sample obtained from a patient such as a blood sample, lymph node sample or bone marrow sample, sing i)a multispecific antibody according to any one of claims 1-62; and ii) instructions for use of said kit.
82.An anti-idiotypic antibody which binds to the second or first and second antigen- binding region as defined in any of claims 1-62. ngre 1 Human (TNR9_HUMAN) GNSCYNIVATT. . .VT.NFER"RST.QDPCSNCPAGr‘FCDNNRNQIC Elephant (XP_003413533) MQDFIMGNIYYNMVATE . .VMNF'TRTGNQDICRECLAG"IICVfiNQIC Wild Boar (XP_005665023) MQDFIMGNIYYNIVATE . .VMNF'ERr‘RSfiPDPCSNCEAG'TC-NQLIC 51 100 Human (TNR9_HUMAN) S PC PPNSFSSAGGQRTCD I CRQCKGVFR'1RKECSS”SNAECDCTPGFHCE Elephant 3413533) S PCPLNSFSSIGGQMNCDCRECIIGVF'‘EACSE'TEAECICEGFHCE Wild Boar (XP_005665023) MPCPINISFSS-“GQCCRECGVFR'‘IIKECSS'ENAVCCEPGFECE 1 0 1 150 Human (TNR9_HUMAN) GAGCSMCEQDCKQGQE KDCCFG'‘FNDQKRGICRPwr‘NCSEDGKS Elephant (XP_003413533) GAGCIMCQQDCKQGQE .“KIGCKDCCIG'‘FNDQKIGIICRPW‘NCSEGKS Wild Boar (XP_005665023) GAGCflMCEIYCQQGQE .“IGICKDCFG'‘FND-GfiCRPwr‘ECSEAGKE 1 5 1 Human (TNR9_HUMAN) (146) V.VNG"KERDVVCGPSPADLSPGASSV"PPAPAR QIISFF-A-T Elephant (XP_003413533) (151) V.I“NG"KIRDVVCGPEAADSFP-SSVTVPAP:EREPEHHPQI IIFF .A .I Wild Boar (XP_005665023) (151) VEINHKARDWCGPRPDSPGISIITMPMPGEPGHTS IIFF-A-M 201 250 Human (TNR9_HUMAN) (196) S”A . .F .T.FF .T .RFSVVKRGRKK . .YIFKQPFMRPVQTTQ.flDGCSCRF Elephant (XP_003413533) (201) SEA . .F EFF .fiRFSVQKWGRKK . .YIFKQPFIPVQTQQ.flDGCSCRF Wild Boar (XP_005665023) (201) STAfiv .fiSI fl GRKK . .YIVKQPFIPQQTMQ.«DflCSCRF Human (TNR9_HUMAN) (246) P4.4 4 *IGGC‘ Elephant (XP_003413533) (251) pflaaflgaca Wild Boar (XP_005665023) (251) P4.4 4 4.64IC4
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