NZ625046B2 - Binding molecules specific for her3 and uses thereof - Google Patents
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- NZ625046B2 NZ625046B2 NZ625046A NZ62504612A NZ625046B2 NZ 625046 B2 NZ625046 B2 NZ 625046B2 NZ 625046 A NZ625046 A NZ 625046A NZ 62504612 A NZ62504612 A NZ 62504612A NZ 625046 B2 NZ625046 B2 NZ 625046B2
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- antibody
- her3
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
Abstract
Disclosed is an antibody or an antigen-binding fragment thereof, which specifically binds to HER3, comprising an antibody VL and an antibody VH, wherein the VL comprises the amino acid sequences: X1GSX2SNIGLNYVS or SGSX2SNIGLNYVS; RNNQRPS; and AAWDDX3X4X5GEX6 or AAWDDLSPPGEA wherein (a) X1 represents amino acid residues Arginine (R) or Serine (S), (b) X2 represents amino acid residues Serine (S) or Leucine (L), (c) X3 represents amino acid residues Serine (S) or Glycine (G), (d) X4 represents amino acid residues Leucine (L) or Proline (P), (e) X5 represents amino acid residues Arginine (R), Isoleucine (I), Proline (P) or Serine (S), and (f) X6 represents amino acid residues Valine (V) or Alanine (A), and wherein the VH comprises the amino acid sequence: YYYMQ; X7IGSSGGVTNYADSVKG or YIGSSGGVTNYADSVKG; and VGLGDAFDI wherein X7 represents amino acid residues Tyrosine (Y), Isoleucine (I) or Valine (V). ents amino acid residues Arginine (R) or Serine (S), (b) X2 represents amino acid residues Serine (S) or Leucine (L), (c) X3 represents amino acid residues Serine (S) or Glycine (G), (d) X4 represents amino acid residues Leucine (L) or Proline (P), (e) X5 represents amino acid residues Arginine (R), Isoleucine (I), Proline (P) or Serine (S), and (f) X6 represents amino acid residues Valine (V) or Alanine (A), and wherein the VH comprises the amino acid sequence: YYYMQ; X7IGSSGGVTNYADSVKG or YIGSSGGVTNYADSVKG; and VGLGDAFDI wherein X7 represents amino acid residues Tyrosine (Y), Isoleucine (I) or Valine (V).
Description
BINDING MOLECULES SPECIFIC FOR HER3 AND USES THEREOF
REFERENCE TO SEQUENCE LISTING TED ELECTRONICLY
This application incorporates by reference a Sequence Listing submitted with
this application as text file entitled 100WOl_SL” created on November 12, 2012 and
having a size of 31.3 kilobytes.
FIELD OF THE INVENTION
The present invention provides compositions that specifically bind to HER3
and methods for the use of such compositions for the treatment of .
OUND ART
The human epidermal growth factor receptor 3 (HER3, also known as Erbb3)
is a receptor protein tyrosine and belongs to the epidermal growth factor receptor (EGFR)
EGFIUHER subfamily of receptor protein tyrosine kinases (RTK), consisting of EGFR
(HERl/Erbbl), HER2/Erbb2, rbb3 and HER4/Erbb4. EGFR and HER2 are among
the most stablished oncogenic RTKs driving the tumorigenesis of multiple types of
solid tumors, including major ries such as breast, colorectal, and lung cancers. The
tyrosine kinase activities of EGFR and HER2 have been shown to be essential for their
oncogenic activities.
Like the prototypical EGFR, the transmembrane receptor HER3 consists of an
extracellular -binding domain (ECD), a dimerization domain within the ECD, an
transmembrane , and intracellular protein tyrosine kinase domain (TKD) and a C-
terminal phosphorylation domain (see, e.g., Kim et al. (1998), Biochem. J. 334, 189—195;
Roepstorff et a]. (2008) Histochem. Cell Biol. 129, 563—578).
The ligand Heregulin (HRG) binds to the extracellular domain of HER3 and
activates the receptor-mediated signaling y by promoting dimerization with other
EGFR family members (e. g., other HER ors) and transphosphorylation of its
intracellular domain. HER3 has been shown to lack detectable tyrosine kinase activity, likely
due to a non—conservative replacement of certain key residues in the tyrosine kinase domain.
Therefore, a consequence of this kinase—deficiency, HER3 needs to form hetero—dimers with
WO 78191
other RTKs, especially EGFR and HER2, to undergo phosphorylation and be functionally
active.
The central role for HER3 in oncogenesis is acting as a scaffolding protein to
enable the m induction of the PI3K/AKT pathway. HER3 has been shown to n
a cluster of six C-terminal tyrosine-containing motifs that when phosphorylated, mimics the
consensus PI3K/p85 binding site. Hence by forming heterodimers with HER3, the upstream
onco-drivers, EGFR, HER2, cMET and FGFRZ, can couple most efficiently to the
PI3K/AKT pathway. Therefore, it is reasonable to expect that a loss of HER3 activity can
block cancer progression in diverse systems driven by ent RTKs. Studies have shown
that HER3 siRNA knockdown in HER2-amplified breast cancer cells led to similar anti-
proliferation effects as HER2 siRNA knockdown, further demonstrating the cancer’s critical
need for HER3.
s promoting tumor growth in unstressed conditions, HER3 has been
found to be highly involved in conferring therapeutic resistances to many targeted drugs,
including EGFR tyrosine kinase inhibitors, HER2 monoclonal antibodies such as
trastuzumab, as well as small molecule inhibitors of PI3K or AKT or MEK. This adds
another layer of attraction to HER3 as a promising cancer target for both primary tumor
debulking as well as combating cancer resistance issues that invariably come up e
initial clinical responses.
HER3 has two different ways to dimerize with its partner RTKs: dependent
(in the presence of HRG) or -independent. In terms of HER2-HER3 dimers,
it is known that in cells with low to medium HER2 expression, HER3 can only complex with
HER2 after ligand-binding; in st, in cells with amplified HER2 (HER2 IHC 3+), they
form spontaneous dimers without HRG (Junttila et a]. (2009) Cancer Cell. 15(5):429—40).
The dimers formed in the presence or absence of the ligand are structurally distinct as was
demonstrated by an earlier study showing that trastuzumab/Herceptin® (Genentech/Roche
HER2 monoclonal antibody approved for HER2 3+ breast cancers) can only disrupt the
ligand-independent dimer but not the ligand- ent dimer, whereas
pertuzumab\Omnitarg® (rhuMAb 2C4, Genentech/Roche HER2 monoclonal antibody in
phase 3 trials) can only disrupt the ligand-dependent .
Dimer formation between HER family members s the signaling
potential of HER3 and is a means not only for signal diversification but also for signal
amplification. HER3 has been shown to be phosphorylated in a variety of cellular contexts.
For example, HER3 is constitutively phosphorylated on tyrosine residues in a subset of
human breast cancer cells overexpressing HER3 (see, e.g., Kraus et al. (1993) Proc. Natl.
Acad. Sci. USA 90, 2900—2904; Kim et a]. (1998), Biochem. J. 334, 189—195; Schaefer et a].
(2004) Cancer Res. 64, 3395—3405; Schaefer et al. (2006) Neoplasia 8, 612—622).
Accordingly, therapies that ively interfere with HER3 phosphorylation are desirable.
In addition, HER3 has been found to be overexpressed and/or overactivated in
several types of cancers such as breast cancer, ovarian cancer, prostate cancer, liver cancer,
kidney and urinary bladder cancers, pancreatic cancers, brain cancers, hematopoietic
neoplasms, retinoblastomas, melanomas, ctal cancers, gastric cancers, head and neck
cancers, lung , etc. (see, e.g., Sithanandam & Anderson (2008) Cancer Gene Ther. 15,
413-448). In general, HER3 is frequently activated in EGFR, HER2, C-Met, and -
expressing cancers.
A correlation between the expression of HER2/HER3 and the progression
from a non—invasive to an invasive stage has been shown ndi et al., Oncogene 10,
1813—1821; o et al., Cancer 87, 487—498; Naidu et al., Br. J. Cancer 78, 1385—1390).
Thus, HER3 can be used as a diagnostic marker for increased tumor siveness and poor
survival. Sustained HER3 activation of P13K/AKT has been repetitively shown to account for
tumor resistance to EGFIUHER2 inhibitors.
Although the role of HER3 in the development and progression of cancer has
been ed (see, e.g., Horst et al. (2005) Int. J. Cancer 115, 519—527; Xue et a]. (2006)
Cancer Res. 66, 1418-1426), HER3 remains largely unappreciated as a target for clinical
intervention. Most current immunotherapies primarily focus on inhibiting the action of HER2
and, in particular, heterodimerization of HER2/HER3 complexes (see, e.g., Sliwkowski et al.
(1994) J. Biol. Chem. 269, 14661—14665). Thus, it is an object of the present invention to
provide improved immunotherapeutic agents that effectively inhibit HER3-mediated cell
ing that can be used for diagnosis, prognosis prediction, and treatment of a variety of
cancers .
BRIEF SUMMARY OF THE INVENTION
The disclosure provides anti—HER3 binding molecules, e.g., dies or
antigen-binding fragments thereof, e. g., monoclonal antibodies capable of suppressing HER3
activity in both -dependent and independent settings. In contrast, other anti-HER3
onal antibodies in the art (e.g., Ab #6 (International Patent Publication WC
2008/100624) and Ul-59 (International Patent Publication WO 2007077028; also referred to
herein as AMG), can only suppress ligand-dependent HER3 activity. Also disclosed are
affinity matured anti-HER3 -binding molecules with sed potency and extended half—life,
which consequently can be administered less frequently, at an increased dose interval,
and in smaller dose volumes. The disclosure also provides methods of treating diseases such
as cancer in a human subject comprising administration of an ER3 binding molecule.
In some specific aspects a 2C2-derived YTE mutant human antibody is used.
The disclosure provides an isolated binding molecule or antigen—binding
fragment thereof which specifically binds to an epitope within the extracellular domain of
HER3, wherein the binding molecule specifically binds to the same HER3 epitope as an
antibody or antigen-binding nt thereof comprising the heavy chain variable region
(VH) and light chain variable region (VL) of CL16 or 2C2. Also provided is an isolated
binding molecule or antigen-binding fragment thereof which specifically binds to HER3, and
competitively inhibits HER3 binding by an antibody or antigen-binding fragment thereof
sing the VH and VL of CL16 or 2C2.
The disclosure also provides an isolated binding molecule or antigen binding
nt thereof which ically binds to HER3 sing an antibody VL, wherein the
VL comprises the amino acid sequence:
[FW1]X1 GSX2SNIGLNYVS[FW2]RNNQRPS[FW3]AAWDDX3X4X5GEX6[FW4]
wherein [FWl], [FWZ], [FW3] and [FW4] represent VL framework s, and
wherein
(a) X1 represents amino acid es ne (R) or Serine (S),
(b) X2 ents amino acid residues Serine (S) or e (L),
(c) X3 represents amino acid residues Serine (S) or Glycine (G),
(d) X4 represents amino acid residues Leucine (L) or Proline (P),
(e) X5 represents amino acid residues Arginine (R), Isoleucine (I), Proline (P)
or Serine (S), and
(f) X6 represents amino acid residues Valine (V) or Alanine (A).
Furthermore, the disclosure provides an isolated binding molecule or antigen
binding fragment thereof which specifically binds to HER3 comprising an antibody VH,
wherein the VH comprises the amino acid sequence:
[FW5]YYYMQ[FW6]X7IGSSGGVTNYADSVKG[FW7]VGLGDAFDI[FW8]
_ 4 _
2012/066038
n [FW5], [FW6], [FW7] and [FWg] represent VH framework regions, and
wherein X7 represents amino acid residues Tyrosine (Y), Isoleucine (I) or Valine (V).
The disclosure provides an isolated binding molecule or antigen binding
fragment thereof which specifically binds to HER3 comprising an antibody VL and an
dy VH, wherein the VL comprises the amino acid sequence:
[FW1]X1GSX2SNIGLNYVS[FWflRNNQRPS[FW3]AAWDDX3X4X5GEX6[FW4]
wherein [FWl], [FWZ], [FW3] and [FW4] represent VL framework regions, and
wherein
(a) X1 represents amino acid es ne (R) or Serine (S),
(b) X2 represents amino acid residues Serine (S) or Leucine (L),
(c) X3 represents amino acid residues Serine (S) or e (G),
(d) X4 represents amino acid residues Leucine (L) or Proline (P),
(e) X5 represents amino acid residues Arginine (R), Isoleucine (I), Proline (P)
or Serine (S), and
(f) X6 represents amino acid residues Valine (V) or Alanine (A), and
wherein the VH comprises the amino acid sequence:
[FW5]YYYMQ[FW6]X7IGSSGGVTNYADSVKG[FW7]VGLGDAFDI[FW8]
wherein [FW5], [FW6] ,[FW7] and [FWg] represent VH ork regions, and
wherein X7 ents amino acid residues Tyrosine (Y), Isoleucine (I) or Valine (V).
The disclosure also provides an isolated binding molecule or antigen binding
fragment thereof which specifically binds to HER3 comprising an antibody VL, wherein the
VL comprises a VL complementarity determining region-l (VL-CDRl) amino acid sequence
identical to, or identical except for four, three, two or one amino acid tutions to: SEQ
ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. Also, the disclosure provides an isolated
binding le or antigen binding fragment thereof which specifically binds to HER3
comprising an antibody VL, wherein the VL comprises a VL complementarity determining
region-2 (VL-CDR2) amino acid sequence identical to, or identical except for four, three, two
or one amino acid substitutions to SEQ ID NO: 21.
In addition, the disclosure provides an isolated binding molecule or antigen
binding fragment thereof which specifically binds to HER3 comprising an antibody VL,
wherein the VL comprises a complementarity determining region-3 (VL-CDR3) amino acid
sequence cal to, or identical except for four, three, two, or one amino acid substitutions
to: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26,
_ 5 _
SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. Also, the disclosure
provides an ed binding molecule or n binding fragment thereof which specifically
binds to HER3 comprising an antibody VH, wherein the VH comprises a complementarity
determining region-1 (VH-CDR1) amino acid sequence identical to, or identical except for
four, three, two, or one amino acid substitutions to SEQ ID NO: 31.
Furthermore, the disclosure provides an isolated binding molecule or antigen
binding fragment f which specifically binds to HER3 comprising an antibody VH,
wherein the VH comprises a complementarity ining region-2 (VH-CDR2) amino acid
sequence identical to, or identical except for four, three, two, or one amino acid substitutions
to: SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. Also provided is an isolated binding
molecule or antigen binding fragment thereof which specifically binds to HER3 comprising an
antibody VH, wherein the VH comprises a complementarity ining region-3 R3)
amino acid sequence cal to, or identical except for four, three, two, or one amino acid
substitutions to SEQ ID NO: 35.
The disclosure provides an isolated binding le or antigen binding
fragment thereof which specifically binds to HER3 comprising an antibody VL, wherein the
VL comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acid ces identical to, or
identical except for four, three, two, or one amino acid substitutions in one or more of the VLCDRS
to: SEQ ID NOs: 18, 21 and 22, SEQ ID NOs: 18, 21, and 26, SEQ ID NOs: 18, 21, and
27 , SEQ ID NOs: 20, 21, and 22, SEQ ID NOs: 19, 21, and 22, SEQ ID NOs: 18, 21, and 25,
SEQ ID NOs: 18, 21, and 28, SEQ ID NOs: 18, 21, and 29, SEQ ID NOs: 18, 21, and 30, SEQ
ID NOs: 18, 21, and 23, SEQ ID NOs: 19, 21, and 23, SEQ ID NOs: 20, 21, and 23, SEQ ID
NOs: 18, 21, and 24, or SEQ ID NOs: 18, 21, and 25, respectively. The disclosure also provides
an isolated binding molecule or antigen binding fragment thereof which specifically binds to
HER3 sing an antibody VH, wherein the VH comprises VH-CDR1, VH-CDR2, and
VH-CDR3 amino acid sequences identical to, or identical except for four, three, two, or one
amino acid substitutions in one or more of the VH-CDRS to: SEQ ID NOs: 31, 32 and 35, SEQ
ID NOs: 31, 33, and 35, or SEQ ID NOs: 31, 34, and 35, respectively.
[0021a] In particular, the present invention relates to an dy or an antigen-binding
fragment thereof, which specifically binds to HER3, comprising an antibody variable light
chain region (VL) and an antibody le heavy chain region (VH), wherein the VL
comprises the amino acid sequence:
(Followed by page 6A)
wherein [FW5], (FW6], [FW7] and [FW8] represent VH framework regions, although
other embodiments are described herein for completeness.
In addition, the disclosure provides an isolated dy or antigen-binding
nt thereof which specifically binds to HER3 comprising a VL and a VH comprising
VL-CDR1, VL-CRD2, 3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid
sequences identical or identical except for four, three, two, or one amino acid substitutions in
-6A-
(Followed by page 7)
one or more CDRs to: SEQ ID NOs: 18, 21, 22, 31, 32, and 35, SEQ ID NOs: 18, 21, 26, 31,
32 and 35, SEQ ID NOs: 18, 21, 27, 31, 32 and 35, SEQ ID NOs: 20, 21, 22, 31, 32 and 35,
SEQ ID NOs: 19, 21, 22, 31, 32 and 35, SEQ ID NOs: 18, 21, 25, 31, 32 and 35, SEQ ID
NOs:18, 21, 28, 31, 32 and 35, SEQ ID NOs:18, 21, 29, 31, 32 and 35, SEQ ID NOs:18, 21,
, 31, 32 and 35, SEQ ID NOs:18, 21, 23, 31, 32 and 35, SEQ ID NOs:19, 21, 23, 31, 32
and 35, SEQ ID NOs: 20, 21, 23, 31, 32 and 35, SEQ ID NOs: 18, 21, 24, 31, 32 and 35, or
SEQ ID NOs: 18, 21, 25, 31, 32 and 35, respectively. Also provided is an isolated binding
molecule or antigen binding fragment thereof which specifically binds to HER3 comprising
an antibody VL and an antibody VH, wherein the VL comprises an amino acid sequence at
least about 90% to about 100% identical to a reference amino acid sequence selected from the
group ting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID
NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. The disclosure also
provides an isolated binding molecule or antigen binding fragment f which specifically
binds to HER3 comprising an antibody VL and an antibody VH, wherein the VH ses
an amino acid sequence at least about 90% to about 100% cal to a reference amino acid
sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID
NO: 13. Furthermore, the sure provides an isolated antibody or antigen binding
fragment thereof which specifically binds to HER3, wherein the antibody or antigen binding
fragment comprises a VL comprising a sequence at least about 90% to about 100% identical
to a reference amino acid sequence selected from the group ting of SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ
ID NO: 16, and SEQ ID NO: 17, and n the antibody or antigen binding fragment
comprises a VH comprising a sequence at least about 90% to about 100% cal to a
reference amino acid sequence selected from the group ting of SEQ ID NO: 2, SEQ ID
NO: 12 and SEQ ID NO: 13.
The disclosure also provides an isolated antibody or n g fragment
thereof, which comprises a VL comprising SEQ ID NO: 49 and a VH comprising SEQ ID
NO: 50. In addition, the disclosure provides an isolated antibody or antigen binding fragment
f, which comprises a VL comprising SEQ ID NO: 3 and a VH comprising SEQ ID NO:
2. Further, the disclosure provides an isolated binding molecule or antigen-binding fragment
thereof which specifically binds to an epitope within the extracellular domain of HER3,
comprising an dy VL of SEQ ID NO:3, an antibody VH of SEQ ID NO: 2, and an IgGl
constant region of SEQ ID 46. Also provided is an isolated binding molecule or antigen-
binding fragment thereof which specifically binds to an epitope within the extracellular
domain of HER3, consisting of an antibody VL of SEQ ID NO: 3, an antibody VH of SEQ
ID NO: 2, and an IgGl constant region of SEQ ID 46.
BRIEF DESCRIPTION OF THE GS/FIGURES
shows the internalization of Clone l6 anti-HER3 monoclonal
antibodies in KPL4 cells shown as depletion of surface fluorescent staining. The top panel
shows internalization at time = 0. The bottom panels show internalization after 2.5 hours.
shows a multiple sequence alignment corresponding to the VL
sequences of anti—HER3 monoclonal antibodies Clone l6 (CL16; original, parent clone),
Clone 16 (GL; germlined clone), 5H6, 8A3, 4H6, 6E.3, 2Bll, 2Dl, 3A6 and 4C4. The
on of CDRl, CDR2, and CDR3 is indicated. Amino acid residues which differ with
respect to the CL16 (GL) antibody are highlighted.
shows a multiple sequence alignment corresponding to the VH
sequences of ER3 monoclonal dies Clone l6 (CL16; parent clone), and clones
15Dl2.l (also referred to as 15Dl2.I ) and 15Dl2.2 (also referred to as 15Dl2.V). The
locations of CDRl, CDR2, and CDR3 are indicated. Amino acid residues which differ with
respect to the CL16 parent antibody are highlighted.
shows a multiple sequence alignment ponding to the VL
sequences of anti-HER3 monoclonal antibodies CL16 (original, parent clone), CL16 (GL;
germlined clone), 1A4, 2C2, 3E.l, 2F10, and 2Bll. The location of CDRl, CDR2, and
CDR3 is indicated. Amino acid residues which differ with respect to the CL16 (GL) antibody
are highlighted.
shows suppression of HER3 phosphorylation (pHER3) in —
driven MCF—7 cells, where HER3 is only activated by exogenous HRG (ligand). The 2C2
anti-HER3 monoclonal, published ER3 monoclonal dies AMG and MM, and
R347 control antibody were assayed. Maximum percentages of pHER3 inhibition and IC50's
are ted.
shows growth suppression in MDA-MB—l75 cells, an established
HRG—autocrine loop driven model n endogenous HRG drives HER3 activity and cell
growth. The 2C2 anti-HER3 monoclonal, hed anti-HER3 onal dies AMG
and MM, and R347 control antibody were assayed. Maximum percentages of growth
tion and IC50's are presented.
shows growth suppression in HMCB cells, an established HRG—
autocrine loop driven model wherein endogenous HRG drives HER3 activity and cell growth.
The 2C2 anti-HER3 monoclonal, published anti-HER3 monoclonal antibodies AMG and
MM, and R347 control antibody were assayed. ICso's are presented.
shows that 2C2 not only inhibited HMCB cell growth but also
suppressed HER3 phosphorylation (pHER3) and AKT phosphorylation (pAKT) in this ligand
dependent melanoma.
shows that 2C2 suppressed HER3 phosphorylation (pHER3) and AKT
phosphorylation (pAKT) in the ligand ent A549 NSCLC.
shows suppression of HER3 phosphorylation (pHER3) in cell models
for Lung Gastric and Breast cancer. Panel A shows suppression of pHER3 in the HCC827
cell line, a mutant riven NSCLC model with EGFIUHER3 cross-talk. Panel B shows
suppression of pHER3 in an EGFR-TKI—resistant HCC827 NSCLC model obtained through
long-term treatment with EGFR TKI. Panel C shows suppression of pHER3 in the MKN45
cell line, a cMET-amplified gastric cancer model with cMET-HER3 cross-talk. Panel D
shows suppression of pHER3 in the Kato III cell line, an FGFRZ-amplified gastric cancer
model with HER3 cross-talk. Panel E shows suppression of pHER3 in the BT-474
cell line, a HERZ-amplified breast cancer ligand-independent model (i.e., cells lack HRG
expression). The 2C2 anti-HER3 monoclonal, published anti-HER3 onal antibodies
AMG and MM, and R347 control antibody were assayed. Maximum percentages of pHER3
inhibition and IC50's are presented.
shows suppression of AKT phosphorylation (pAKT) in cell models for
gastric and breast cancer. Panel A shows suppression of pAKT in the MKN45 cell line.
Panel B shows suppression of pAKT in the Kato III cell line. Panel C shows suppression of
pAKT in the BT-474 cell line, a HERZ-amplified breast cancer ligand-independent model
(i.e., cells lack HRG expression). The 2C2 anti-HER3 monoclonal, published ER3
monoclonal antibodies AMG and MM, and R347 control antibody were assayed. Maximum
tages ofpAKT inhibition and IC50's are presented.
shows 2C2 suppresses cell signaling and proliferation in -
361 cells. Panel A shows that 2C2 suppressed HER3 phosphorylation (pHER3) in HERZ-
amplified —36l cells. Panel B shows that 2C2 suppressed cell growth in a dose
dependent manner. The t inhibition is shown for 6 and 14 day treatments (top and
bottom panels, respectively).
shows that 2C2 suppressed HER3 phosphorylation (pHER3) in
HARA-B cells expressing high levels of HRG.
shows that 2C2 and rhuMab 2C4, but not the EGFR antagonists
cetuximab or gef1tinib, inhibit HRG -dependent signaling (bottom of Panels A and B).
The top portion of Panels A and B are basal cells, SW620 (Panel A, left), SW480 (Panel A,
middle), C010205 (Panel A, right), LOVO (Panel B, left), HCTlS (Panel B, middle) and
Caco-2 (Panel B, right).
shows an HRG-HER3 ELISA binding assay measuring the direct
blocking of HRG binding to HER3 by the Clone 16, published AMG and MM anti-HER3
monoclonal antibodies, a positive control -blocking anti-HER3 monoclonal antibody,
and the R347 l dy.
shows 2C2 blocks HERZ-HER3 dimerization. Panel A shows a
HRG-inducible HERZ-HER3 dimerization assay that assesses the extent of HERZ-HER3
complex formation in T-47D cells, a ligand-dependent model g clear HRG—induced
HERZ-HER3 association, pre-treated with 2C2, CLl6, AMG and MM anti-HER3
monoclonal antibodies. All anti-HER3 antibodies blocked this ligand-induced HERZ-HER3
zation. Panel B shows a ligand-independent ER3 dimerization assay that
assesses the extent of HERZ-HER3 complex formation in BT-474 cells, pre-treated with 2C2
or CLl6 blocked this -independent HERZ-HER3 zation.
shows HER3 internalization and degradation induced by 2C2. Panel
A shows a FACS-based internalization assay that quantifies time course and extent of target
internalization in response to two different 2C2 monoclonal antibody concentrations. Panel B
shows HER3 degradation in model colorectal cancer cells Lovo, HCTlS, and SW620
pretreated with ER3 2C2 monoclonal antibody, or the R347 control antibody.
shows a FACS—based cell-cycle analysis demonstrating that in SkBR3
cells, a HERZ-amplified breast cancer cell-line similar to BT-474, both Herceptin®
(trastuzumab) and CLl6 monoclonal antibody (parental lead for the 2C2 monoclonal
antibody) caused cell—cycle arrest at the Gl—phase. Results corresponding to cells treated with
the R347 control antibody and with the rhuMAb 2C4 anti-HER2 monoclonal antibody
(pertuzumab/ Omnitarg®) are also shown.
shows inhibition of HRG induced VEGF secretion by anti-HER3
antibodies. Panel A shows changes in VEGF secretion in BT-474 breast cancer cells
ated with anti-HER3 monoclonal antibodies CL16 and Merrimack MM, anti-HER2
onal antibody Herceptin® uzumab), or the R347 control antibody. Panel B
shows changes in VEGF secretion in MCF-7 model breast cancer cells pretreated with anti—
HER3 monoclonal antibodies CL16 and ack MM, anti-HER2 monoclonal antibody
Herceptin® (trastuzumab), or the R347 l antibody.
shows that the anti-HER3 monoclonal antibody 2C2 binds to cellsurface
based cyno HER3 ectopically expressed in Ad293 cells and tes its activity.
Panel A shows a Western blot analysis of Ad293 cells transfected with a control vector (left
side) or a vector expressing cyno HER3 (right side). The cells were treated with 2C2 or a
control antibody (R347) with or without co-stimulation with HRG and probed with anti-
HER3 (middle blot), anti-pHER3 (top blot), and anti-GAPDH (bottom blot) antibodies.
Panel B represents the densitometry-based fication of pHER3 in the upper four lanes
of Panel A.
shows a dose—dependent reduction in tumor volume after
administration of the 2C2 monoclonal antibody using the human FADU head and neck
xenograft model. Panel A shows that 7 mg/kg of 2C2 administered twice per week was
maximally efficacious at 99% dTGI (tumor growth inhibition) in this model. Panel B shows
strong reduction in tumor volume after the ed administration of the 2C2 monoclonal
antibody with the anti-EGFR monoclonal antibody cetuximab using the human FADU head
and neck xenograft model. The combination treatment produced 7 out of 10 partial
sions and 2/ 10 complete sions.
shows non-linear pharmacokinetics for 2C2 after single dose and
repeat-dose administration of 5 mg/kg or 30 mg/kg to tumor-bearing mice. Data suggest that
mouse HER3 serves as a sink to bind 2C2 administered to the mice and that 30 mg/kg as a
single dose is sufficient to saturate the sink.
shows the anti-tumor benefit of a 10 mg/kg loading dose of the
monoclonal antibody 2C2 using the human FADU head and neck xenograft model.
Administration of a loading dose of 2C2 to saturate the mouse HER3 sink d 2C2 at 3
mg/kg to demonstrate strong anti-tumor activity while 3 mg/kg of 2C2 without a loading dose
has only modest activity.
shows that treatment with 2C2-YTE reduces the levels of pHER3 and
pAKT in FADU xenograft tumor extracts. In this experiment the levels of pHER3 and pAKT
were reduced by 59.5% and 51.7%, respectively. No change was seen in total HER3 levels in
this experiment.
shows a dose—dependent reduction in tumor volume after
administration of the 2C2 monoclonal antibody using the human Detroit562 head and neck
xenograft model. Panel A shows that 10 mg/kg of 2C2 administered twice per week was
maximally efficacious at 72% dTGI. Panel B shows a reduction in tumor volume after the
combined administration of the 2C2 onal antibody with the anti-EGFR monoclonal
antibody cetuximab using the human t562 head and neck aft model. The
combination treatment ed 9 out of 10 partial regressions while cetuximab alone
produced 5/10 partial regressions. The Detroit562 xenograft model contains a PIK3CA
mutation.
shows a dose dependent reduction in tumor volume after the
administration of the 2C2-YTE monoclonal antibody using the human CAL27 head and neck
xenograft model.
shows a dose—dependent reduction in tumor volume after
administration of the 2C2 monoclonal antibody using the human A549 NSCLC xenograft
model. Panel A shows that 30 mg/kg of 2C2 administered twice per week was maximally
efficacious at 91% dTGI up to the last day of the treatment phase (day 33; regrowth
afterwards). 2C2-YTE and 2C2 both at 10 mg/kg have comparable activity. Panel B shows a
ion in tumor volume after the combined administration of the 2C2 monoclonal
antibody with the anti-EGFR monoclonal antibody cetuximab using the human A549 NSCLC
xenograft model. The on of cetuximab to 2C2 increased the activity of 2C2 during the
treatment phase and d tumor regrowth during the tumor regrowth phase. The A549
xenograft model contains a KRAS mutation and a LKB-l deletion.
shows a reduction in tumor volume after administration of the 2C2-
YTE monoclonal antibody using the human HARA-B squamous cell oma xenograft
model. 30 mg/kg of 2C2-YTE administered twice per week was maximally efficacious at
64.6% dTGI. 2C2-YTE at 10 mg/kg had comparable activity while 2C2-YTE at 3 mg/kg was
not active.
shows a dose—dependent reduction in tumor volume after
administration of the 2C2 monoclonal dy using the human HT-29 colorectal xenograft
model. 30 mg/kg of 2C2 administered twice per week was maximally efficacious at 56%
dTGI up to the last day of the treatment phase (day 26; th afterwards). 2C2-YTE and
2C2 both at 30 mg/kg have comparable activity. The HT-29 aft model contains a
BRAF mutation.
shows a ion in tumor volume after administration of the 2C2
monoclonal antibody using the human HCT-ll6 colorectal xenograft model. 30 mg/kg of
2C2 administered twice per week was maximally efficacious at 43% dTGI. E and
2C2 both at 10 mg/kg have comparable activity. The HCT-ll6 xenograft model contains a
KRAS mutation.
shows a reduction in tumor volume after administration of the 2C2
monoclonal antibody using the human LOVO colorectal xenograft model. 30 mg/kg of 2C2
administered twice per week was maximally efficacious at 48% dTGI. 2C2-YTE and 2C2
both at 10 mg/kg have comparable activity. The LOVO xenograft model contains a KRAS
mutation.
shows a reduction in tumor volume after administration of the 2C2
monoclonal antibody using the human DUl45 te xenograft model. 30 mg/kg of 2C2
administered twice per week was maximally efficacious at 77% dTGI. The DUl45 xenograft
model contains a LKB-l deletion.
shows a reduction in tumor volume after administration of the 2C2
monoclonal antibody using the human BT-474 breast cancer orthotopic xenograft model.
Panel A shows 30 mg/kg of 2C2 administered twice per week was lly efficacious at
55% dTGI. Panel B shows a reduction in tumor volume after the ed administration of
the 2C2 onal antibody with the small molecule drug lapatinib using the human BT-
474 breast cancer orthotopic xenograft model. The addition of 2C2 to lapatinib increased the
ty of lapatinib during the treatment phase and modestly d tumor regrowth during
the tumor regrowth phase. 2C2-YTE and 2C2 both at 30 mg/kg have comparable activity
during the treatment phase as monoeff1cacy treatments. Panel C shows a reduction in tumor
volume after the administration of the 2C2 monoclonal dy using the human BT-474
breast cancer orthotopic xenograft model. Trastuzumab alone was very active in this model
and little enhancement was seen by the addition of 2C2 in this model. The BT-474 xenograft
model contains amplified HER2 (3+ by HercepTest).
shows that treatment with Clone l6 (2C2 precursor) reduces the
levels of pHER3 and pAKT in BT-474 xenograft tumor extracts. In this experiment the
levels were of pHER3 and pAKT were d by 50% and 46.1%, respectively. No change
was seen in total HER3 levels in this experiment.
shows a reduction in tumor volume after administration of the 2C2
monoclonal antibody using the human MCF-7 breast cancer orthotopic xenograft model.
Panel A shows 10 mg/kg of 2C2 administered twice per week was lly efficacious at
34% dTGI. 2C2-YTE and 2C2 both at 10 mg/kg have comparable activity. Panel B shows a
reduction in tumor volume after the combined administration of the 2C2 monoclonal
antibody with the small molecule drug paclitaxel using the human MCF-7 breast cancer
orthotopic xenograft model. The on of 2C2 to paclitaxel increased the activity of
paclitaxel during the treatment phase. The MCF-7 xenograft model ns low levels of
HER2 (1+ by HercepTest).
shows a reduction in tumor volume after administration of 2C2—YTE
using the human MDA-MB-36l breast cancer orthotopic xenograft model (Panels A-C). The
addition of 2C2-YTE to the onal antibody trastuzumab increased the activity of
trastuzumab during the treatment phase and delayed tumor regrowth during the tumor
regrowth phase (Panel A). The addition of 2C2-YTE to the onal antibody rhuMAb
2C4 modestly increased the activity of rhuMAb 2C4 but did not delay the regrowth of the
tumors (Panel B). Addition of 2C2-YTE to the small molecule drug lapatinib increased the
activity of lapatinib but did not delay the th of the tumors (Panel C).
shows prolonged exposure levels of the monoclonal antibody 2C2-
YTE in serum of naive human FcRn SCID transgenic mice compared to 2C2 and Clone 16—
GL after a single dose of these antibodies at 60 mg/kg.
shows HER3 protein levels increase in se to treatment with the
MEK tor (MEKi) selumetinib (indicated by a star). Treatment with the MEKi in
combination with 2C2 reduces the HER3 levels back to normal in HT-29 cells (left), LOVO
e) and Colo205 (right) cancer models. The levels of pHER3 were also examined in the
HT—29 and LOVO models and shown to respond similarly.
shows that the combination of 2C2-YTE and selumetinib increases
the anti-tumor efficacy of either agent alone in subcutaneous cancer xenograft models and
A549 (Panel A, top), HT-29 (Panel B, top), LOVO (Panel C, top). Western blot analysis
from tumor lysates (A549, HT-29 and LOVO xenograph models) of mice treated with the
combination showed that phospho-HER3 and phospho-ERK were completely ted
(Panels A—C, bottom).
ED DESCRIPTION OF THE INVENTION
The present invention es molecules and antigen—binding fragments
thereof that bind to HER3. In some aspects, such molecules are antibodies and antigen—
binding fragments thereof that specifically bind to HER3. Related polynucleotides,
compositions comprising the anti-HER3 dies or antigen-binding fragments thereof, and
methods of making the anti-HER3 antibodies and antigen-binding fragments are also
provided. s of using the novel anti-HER3 antibodies, such as methods of treating
cancer in a subject and diagnostic uses, are further provided.
In order that the present invention can be more readily understood, certain
terms are first defined. Additional definitions are set forth throughout the detailed description.
1. Definitions
Before describing the present invention in detail, it is to be understood that this
invention is not limited to specific itions or process steps, as such can vary. As used
in this specification and the appended claims, the singular forms "a", "an" and "the" include
plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well
as the terms "one or more," and "at least one" can be used interchangeably herein.
Furthermore, r" where used herein is to be taken as ic disclosure
of each of the two specified features or components with or without the other. Thus, the term
and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A
or B," "A" (alone), and "B" (alone). Likewise, the term r" as used in a phrase such as
"A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B,
or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C
(alone).
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to which this
disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei—Show, 2nd ed., 2002, CRC Press; The nary of Cell and Molecular
Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And
Molecular Biology, Revised, 2000, Oxford sity Press, provide one of skill with a
l dictionary of many of the terms used in this ion.
Units, prefixes, and symbols are denoted in their Systeme International de
Unites (SI) ed form. Numeric ranges are inclusive of the numbers defining the range.
Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy
orientation. The gs ed herein are not limitations of the various aspects which
can be had by reference to the specification as a whole. Accordingly, the terms defined
immediately below are more fully defined by reference to the specification in its entirety.
It is tood that wherever aspects are described herein with the language
"comprising," otherwise ous aspects described in terms of "consisting of‘ and/or
"consisting essentially of‘ are also provided.
Amino acids are referred to herein by either their commonly known three
letter symbols or by the one—letter symbols recommended by the IUPAC—IUB Biochemical
lature Commission. Nucleotides, likewise, are referred to by their commonly
accepted —letter codes.
The terms "HER3" and "HER3 receptor" are used hangeably , and
refer to the ErbB3 protein (also referred to as HER3, ErbB3 receptor in the literature) as
described in US. Pat No. 5,480,968 and in Plowman et a1. (1990) Proc. Natl. Acad. Sci. USA
87, 4905—4909; see also, Kani et a1. (2005) Biochemistry 44, 15842—15857, and Cho & Leahy
(2002) e 297, 1330—1333. The full—length, mature HER3 protein sequence (without
leader sequence) corresponds to the sequence shown in and SEQ ID NO: 4 of US.
Pat. No. 5,480,968 minus the 19 amino acid leader sequence that is cleaved from the mature
The terms "inhibition" and "suppression" are used interchangeably herein and
refer to any statistically significant decrease in biological activity, including full blocking of
the ty. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or 100% in biological activity. Accordingly, when the terms
"inhibition" or "suppression" are applied to describe, e. g., an effect on ligand-mediated HER3
phosphorylation, the term refers to the ability of an antibody or antigen binding fragment
thereof to statistically significantly decrease the phosphorylation of HER3 induced by an
EGF-like ligand, relative to the phosphorylation in an untreated (control) cell. The cell which
expresses HER3 can be a naturally occurring cell or cell line (e. g., a cancer cell) or can be
recombinantly produced by introducing a nucleic acid encoding HER3 into a host cell. In
one aspect, the anti-HER3 binding le, e. g., an antibody or antigen binding fragment
thereof inhibits ligand mediated phosphorylation of HER3 by at least 10%, or at least 20%, or
at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%,
or at least 905, or about 100%, as determined, for example, by Western blotting followed by
_ l6 _
probing with an anti-phosphotyrosine antibody or by ELISA, as described in the Examples
infra.
The term h suppression" of a cell expressing HER3, as used herein,
refer to the y of ER3 binding molecule, e.g., an dy or antigen-binding
fragment thereof to statistically significantly decrease proliferation of a cell expressing HER3
relative to the proliferation in the absence of the ER3 binding molecule, e.g., an
dy or antigen-binding fragment thereof In one aspect, the proliferation of a cell
expressing HER3 (e.g., a cancer cell) can be decreased by at least 10%, or at least 20%, or at
least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or
at least 90%, or about 100% when cells are contacted with an anti-HER3 binding molecule,
e. g., an antibody or antigen-binding fragment thereof of the present invention, relative to the
proliferation measured in the absence of the anti-HER3 binding molecule, e.g., an antibody or
antigen-binding fragment thereof (control conditions). Cellular proliferation can be assayed
using art recognized techniques with measure rate of cell division, the fraction of cells within
a cell population oing cell division, and/or rate of cell loss from a cell population due
to terminal differentiation or cell death (e. g., thymidine incorporation).
The terms "antibody" or "immunoglobulin," as used interchangeably herein,
include whole antibodies and any antigen binding fragment or single chains thereof
A typical antibody comprises at least two heavy (H) chains and two light (L)
chains interconnected by ide bonds. Each heavy chain is comprised of a heavy chain
variable region viated herein as VH) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2, and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein as VL) and a light chain
constant region. The light chain constant region is comprised of one domain, CL. The VH
and VL regions can be further subdivided into regions of hypervariability, termed
Complementarity Determining Regions (CDR), interspersed with regions that are more
ved, termed framework regions (FW). Each VH and VL is composed of three CDRs
and four FWs, arranged from amino-terminus to carboxy-terminus in the following order:
FWl, CDRl, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light
chains n a g domain that interacts with an antigen. The constant s of the
antibodies can mediate the binding of the immunoglobulin to host tissues or factors, ing
various cells of the immune system (e.g., effector cells) and the first component (Clq) of the
classical complement . Exemplary antibodies of the present disclosure include the
Clone 16 (CL16) anti-HER3 antibodies (original and ned), affinity optimized clones
including for example, the anti-HER3 2C2 antibody, and serum half-life-optimized anti-
HER3 antibodies ing for example the anti-HER3 2C2-YTE antibody.
The term "germlining" means that amino acids at specific positions in an
antibody are mutated back to those in the germ line. E.g., the CL16 "germlined" antibody is
generated from the original CL16 antibody by introducing three point mutations, YZS, E3V
and M201, into FWl of the VL regions.
The term "antibody" means an immunoglobulin molecule that recognizes and
specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate,
polynucleotide, lipid, or combinations of the foregoing h at least one antigen
recognition site within the variable region of the immunoglobulin molecule. As used ,
the term "antibody" asses intact polyclonal antibodies, intact monoclonal antibodies,
antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv)
mutants, multispecific antibodies such as bispecific antibodies generated from at least two
intact antibodies, ic antibodies, humanized antibodies, human antibodies, fusion
proteins comprising an antigen determination portion of an antibody, and any other modified
globulin molecule comprising an antigen recognition site so long as the antibodies
exhibit the desired biological activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (6.g. IgGl,
IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant
domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The ent
classes of immunoglobulins have different and well known subunit ures and three-
dimensional configurations. Antibodies can be naked or conjugated to other molecules such
as toxins, radioisotopes, etc.
A "blocking" antibody or an "antagonist" antibody is one which inhibits or
reduces biological activity of the antigen it binds, such as HER3. In a certain aspect blocking
dies or antagonist antibodies substantially or completely inhibit the biological activity
of the antigen. Desirably, the ical activity is reduced by 10%, 20%, 30%, 50%, 70%,
80%, 90%, 95%, or even 100%.
The term "HER3 antibody" or "an dy that binds to HER3" or "anti-
HER3" refers to an dy that is e of binding HER3 with sufficient affinity such that
the antibody is useful as a eutic agent or diagnostic reagent in targeting HER3. The
extent of binding of an anti-HER3 antibody to an unrelated, non-HER3 protein is less than
about 10% of the binding of the antibody to HER3 as measured, e. g., by a radioimmunoassay
(RIA), BIACORETM (using recombinant HER3 as the analyte and antibody as the , or
vice versa), or other binding assays known in the art. In certain s, an antibody that
binds to HER3 has a dissociation constant (KD) of :1 HM, 5100 nM, :10 nM, 51 nM, 50.1
nM, :10 pM, 51 pM, or £0.1pM.
The terms "antigen binding fragment" refers to a portion of an intact antibody
and refers to the antigenic determining le regions of an intact antibody. It is known in
the art that the antigen binding function of an antibody can be med by fragments of a
full-length antibody. es of antibody fragments include, but are not limited to Fab,
Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecif1c
antibodies formed from antibody fragments.
A "monoclonal antibody" refers to a homogeneous antibody population
involved in the highly specific recognition and binding of a single antigenic inant, or
epitope. This is in contrast to polyclonal antibodies that typically include different antibodies
directed against ent antigenic determinants. The term "monoclonal antibody"
encompasses both intact and full-length monoclonal antibodies as well as antibody fragments
(such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an
antibody portion, and any other ed immunoglobulin le comprising an n
ition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any
number of ways including, but not limited to, by hybridoma, phage selection, recombinant
sion, and transgenic animals.
The term "humanized antibody" refers to an antibody derived from a nonhuman
(e.g., murine) immunoglobulin, which has been engineered to n minimal non-
human (e. g., murine) sequences. Typically, humanized antibodies are human
immunoglobulins in which es from the complementary determining region (CDR) are
replaced by residues from the CDR of a non—human species (e.g., mouse, rat, rabbit, or
hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature,
321:522—525; Riechmann et al., 1988, Nature, 332:323—327; Verhoeyen et al., 1988, Science,
239:1534—1536). In some instances, the Fv framework region (FW) residues of a human
immunoglobulin are replaced with the corresponding residues in an antibody from a non-
human s that has the desired specificity, affinity, and lity.
The humanized antibody can be further modified by the substitution of
additional residues either in the Fv framework region and/or within the replaced non-human
residues to refine and optimize antibody specificity, affinity, and/or capability. In general,
the humanized antibody will comprise substantially all of at least one, and typically two or
three, variable domains containing all or substantially all of the CDR regions that correspond
to the non-human immunoglobulin whereas all or substantially all of the FR regions are those
of a human immunoglobulin sus ce. The humanized antibody can also comprise
at least a portion of an immunoglobulin nt region or domain (Fc), typically that of a
human immunoglobulin. Examples of methods used to generate humanized dies are
described in US. Pat. Nos. 5,225,539 or 5,639,641.
A "variable region" of an antibody refers to the le region of the antibody
light chain or the variable region of the antibody heavy chain, either alone or in combination.
The variable regions of the heavy and light chain each consist of four framework s
(FW) connected by three complementarity determining regions (CDRs) also known as
hypervariable regions. The CDRs in each chain are held together in close proximity by the
FW regions and, with the CDRs from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two techniques for determining CDRs:
(1) an approach based on cross—species sequence variability (i.e., Kabat et a]. ces of
Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda
Md.)); and (2) an approach based on crystallographic studies of n-antibody complexes
(Al—lazikani et a]. (1997) J. Molec. Biol. 273:927—948)). In addition, combinations of these
two approaches are sometimes used in the art to determine CDRs.
The Kabat ing system is generally used when referring to a residue in
the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of
the heavy chain) (e.g,, Kabat et al., ces of Immunological Interest, 5th Ed. Public
Health e, National Institutes of Health, Bethesda, Md. (1991)).
The amino acid position numbering as in Kabat, refers to the numbering
system used for heavy chain variable domains or light chain variable domains of the
compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this
numbering system, the actual linear amino acid sequence can n fewer or additional
amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable
domain. For example, a heavy chain variable domain can include a single amino acid insert
ue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues
82a, 82b, and 82c, etc. according to Kabat) after heavy chain FW residue 82.
_ 20 _
TABLE 1
The Kabat numbering of residues can be determined for a given antibody by
alignment at regions of homology of the sequence of the antibody with a "standard" Kabat
numbered sequence. Chothia refers instead to the location of the structural loops ia
and Lesk, J. Mol. Biol. 196:901—917 (1987)). The end of the Chothia CDR-H1 loop when
ed using the Kabat numbering convention varies between H32 and H34 depending on
the length of the loop (this is because the Kabat numbering scheme places the insertions at
H35A and H35B; if neither 35A nor 35B is t, the loop ends at 32; if only 35A is
present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM
hypervariable regions represent a compromise between the Kabat CDRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
IMGT oGeneTics) also provides a numbering system for the
immunoglobulin variable regions, including the CDRs. See e. g., Lefranc, M.P. et al., Dev.
Comp. Immunol. 27: 55—77(2003), which is herein incorporated by reference. The IMGT
numbering system was based on an alignment of more than 5,000 sequences, structural data,
and characterization of hypervariable loops and allows for easy comparison of the variable
and CDR regions for all s. According to the IMGT numbering schema l is at
positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102,
l is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at
positions 89 to 97.
As used throughout the specification the VH CDRs sequences described
correspond to the classical Kabat numbering locations, namely Kabat VH-CDRl is at
positions 31—35, VH—CDR2 is a positions 50—65, and VH—CDR3 is at positions 95—102. VL—
CDR2 and VL-CDR3 also correspond to classical Kabat ing locations, namely
ons 50—56 and 89—97, tively. As used herein, the terms Rl" or "light
chain CDRl" correspond to sequences located at Kabat positions 23-34 in the VL (in
st, the classical VL-CDRl location according to the Kabat numbering schema
corresponds to positions 24—34).
As used herein the Fc region includes the polypeptides comprising the
constant region of an antibody excluding the first constant region globulin domain.
Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG,
and the last three nt region immunoglobulin domains of IgE and IgM, and the flexible
hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG,
Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the
hinge between Cgammal (Cyl) and Cgamma2 . Although the boundaries of the Fc
region may vary, the human IgG heavy chain Fc region is usually defined to se
residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the
EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest,
5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ). PC may
refer to this region in isolation, or this region in the t of an antibody, dy
fragment, or PC fusion protein. Polymorphisms have been observed at a number of different
Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as
numbered by the EU index, and thus slight differences between the presented sequence and
sequences in the prior art may exist.
The term "human antibody" means an antibody produced by a human or an
antibody having an amino acid sequence corresponding to an antibody produced by a human
made using any technique known in the art. This definition of a human antibody includes
intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one
human heavy and/or light chain polypeptide such as, for e, an antibody comprising
murine light chain and human heavy chain polypeptides.
The term ric antibodies" refers to antibodies wherein the amino acid
sequence of the immunoglobulin molecule is derived from two or more species. Typically,
the variable region of both light and heavy chains corresponds to the variable region of
dies derived from one species of mammals (e.g., mouse, rat, , etc) with the
desired specif1city, affinity, and capability while the constant regions are homologous to the
sequences in antibodies derived from another (usually human) to avoid eliciting an immune
response in that species.
The terms "YTE" or "YTE " refer to a mutation in IgGl PC that results
in an increase in the g to human FcRn and improves the serum half-life of the antibody
having the mutation. A YTE mutant ses a combination of three mutations,
M252Y/S254T/T256E (EU numbering Kabat et a1. (1991) Sequences of Proteins of
Immunological Interest, US. Public Health e, National Institutes of Health,
Washington, DC), introduced into the heavy chain of an IgGl. See US. Patent No.
7,658,921, which is orated by reference herein. The YTE mutant has been shown to
increase the serum half-life of antibodies approximately four-times as compared to wild-type
ns of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514—24 (2006)). See
also US. Patent No. 7,083,784, which is hereby incorporated by reference in its entirety.
"Binding affinity" generally refers to the th of the sum total of non-
covalent interactions between a single binding site of a molecule (e. g., an antibody) and its
binding r (e.g., an n). Unless indicated otherwise, as used herein, "binding
affinity" refers to sic binding affinity which s a 1:1 interaction between members
of a binding pair (e. g., antibody and antigen). The affinity of a molecule X for its partner Y
can generally be represented by the dissociation constant (KD). Affinity can be measured by
common methods known in the art, including those described herein. finity antibodies
generally bind antigen slowly and tend to dissociate readily, s high-affinity antibodies
generally bind antigen faster and tend to remain bound longer. A variety of methods of
measuring binding affinity are known in the art, any of which can be used for purposes of the
present invention.
"Potency" is normally expressed as an IC50 value, in nM unless otherwise
stated. IC50 is the median inhibitory concentration of an antibody molecule. In onal
assays, IC50 is the concentration that reduces a biological response by 50% of its maximum.
In ligand-binding studies, IC50 is the concentration that reduces receptor binding by 50% of
maximal specific binding level. IC50 can be calculated by any number of means known in the
art. Improvement in potency can be determined by measuring, e. g., against the parent CL16
(Clone 16) monoclonal antibody.
The fold ement in potency for the antibodies or polypeptides of the
invention as compared to a Clone 16 antibody can be at least about 2-fold, at least about 4-
fold, at least about , at least about 8-fold, at least about 10-fold, at least about 20-fold,
at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at
least about 70—fold, at least about 80—fold, at least about 90—fold, at least about lOO—fold, at
least about ld, at least about 120-fold, at least about ld, at least about l40-fold,
at least about 150-fold, at least about l60-fold, at least about l70-fold, or at least about 180—
fold or more.
"Antibody—dependent cell—mediated cytotoxicity" or "ADCC" refers to a form
of xicity in which secreted 1g bound onto Fc receptors (FcRs) present on certain
cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and hages) enables these
cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently
kill the target cell with cytotoxins. Specific high-affinity IgG antibodies directed to the
surface of target cells "arm" the cytotoxic cells and are absolutely required for such killing.
Lysis of the target cell is extracellular, requires direct cell—to—cell t, and does not
involve complement. It is contemplated that, in on to antibodies, other proteins
comprising Fc regions, specifically Fc fusion proteins, having the capacity to bind
specifically to an n-bearing target cell will be able to effect cell-mediated cytotoxicity.
For simplicity, the cell-mediated cytotoxicity resulting from the activity of an Fc fusion
protein is also referred to herein as ADCC activity.
A polypeptide, dy, polynucleotide, vector, cell, or composition which is
"isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in
a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells
or compositions include those which have been purified to a degree that they are no longer in
a form in which they are found in nature. In some aspects, an antibody, polynucleotide,
vector, cell, or composition which is isolated is substantially pure.
The term "subject" refers to any animal (e. g., a mammal), including, but not
limited to humans, non-human primates, rodents, and the like, which is to be the recipient of
a ular treatment. Typically, the terms ct" and "patient" are used interchangeably
herein in reference to a human subject.
The term "pharmaceutical composition" refers to a preparation which is in
such form as to permit the biological activity of the active ient to be effective, and
which contains no additional components which are unacceptably toxic to a subject to which
the composition would be administered. Such composition can be sterile.
An "effective amount" of an antibody as disclosed herein is an amount
sufficient to carry out a specifically stated purpose. An "effective amount" can be determined
empirically and in a routine manner, in relation to the stated purpose.
The term "therapeutically effective " refers to an amount of an
dy or other drug effective to " a disease or disorder in a subject or mammal.
The word "label" when used herein refers to a able nd or
composition which is conjugated directly or indirectly to the antibody so as to te a
"labeled" antibody. The label can be detectable by itself (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a
substrate nd or composition which is able.
Terms such as "treating" or "treatment" or "to treat" or iating" or "to
alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of,
and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic
or preventative measures that prevent and/or slow the development of a targeted pathologic
condition or disorder. Thus, those in need of treatment include those already with the
disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
In certain aspects, a subject is successfully ed" for cancer according to the methods of
the present invention if the patient shows, e.g., total, partial, or transient remission of a
certain type of .
The terms r", "tumor", "cancerous", and "malignant" refer to or describe
the physiological condition in mammals that is typically characterized by unregulated cell
growth. Examples of cancers include but are not limited to, carcinoma including
adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More
particular examples of such s include squamous cell cancer, small-cell lung cancer,
non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma,
pancreatic , glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as
hepatic carcinoma and hepatoma, bladder cancer, breast cancer (including hormonally
mediated breast cancer, see, e.g., Innes et a]. (2006) Br. J. Cancer 94:1057—1065), colon
cancer, colorectal , endometrial oma, myeloma (such as multiple myeloma),
salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors,
basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular
cancer, esophageal cancer, various types of head and neck cancer and cancers of us
origins, such as, mucinous ovarian , cholangiocarcinoma (liver) and renal papillary
carcinoma.
As used herein, the term "carcinomas" refers to cancers of epithelial cells,
which are cells that cover the surface of the body, produce hormones, and make up glands.
Examples of carcinomas are cancers of the skin, lung, colon, stomach, breast, prostate and
thyroid gland.
The term "KRAS mutation," as used herein, refers to mutations found in
certain cancers in a human homolog of the v-Ki-ras2 Kirsten rat sarcoma viral oncogene.
Non—limiting examples of human KRAS gene mRNA sequences e Genbank Accession
Nos. NM004985 and NM033360. It has been reported that KRAS mutations are found in
73% of atic , 35% of colorectal tumors, 16% of ovarian tumors and 17% of lung
tumors. KRAS mutation generally occur in codons 12 or 143 of the human KRAS gene.
"Polynucleotide," or "nucleic acid," as used interchangeably herein, refer to
polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, cleotides, modif1ed nucleotides or bases, and/or their analogs, or
any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A
polynucleotide can comprise modif1ed tides, such as methylated nucleotides and their
analogs. The preceding description s to all polynucleotides referred to herein, including
RNA and DNA.
The term "vector" means a construct, which is capable of delivering, and in
some aspects, expressing, one or more gene(s) or sequence(s) of interest in a host cell.
Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors
associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in
liposomes, and certain otic cells, such as producer cells.
The terms "polypeptide," "peptide," and "protein" are used interchangeably
herein to refer to polymers of amino acids of any length. The polymer can be linear or
branched, it can comprise ed amino acids, and it can be interrupted by ino
acids. The terms also encompass an amino acid polymer that has been modified naturally or
by intervention; for example, disulfide bond formation, glycosylation, lipidation, ation,
orylation, or any other manipulation or modification, such as conjugation with a
labeling component. Also included within the tion are, for example, polypeptides
containing one or more analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other cations known in the art. It is understood that, because the
polypeptides of this invention are based upon antibodies, in certain aspects, the ptides
can occur as single chains or associated chains.
The terms "identical" or percent "identity" in the context of two or more
nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the
same or have a specified tage of nucleotides or amino acid es that are the same,
when compared and aligned (introducing gaps, if necessary) for maximum pondence,
not considering any conservative amino acid substitutions as part of the ce identity.
The percent identity can be measured using ce comparison software or algorithms or
by visual inspection. Various algorithms and software are known in the art that can be used
to obtain alignments of amino acid or tide sequences.
One such non-limiting example of a sequence alignment algorithm is the
algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci, 87:2264—2268, as modified
in Karlin et al., 1993, Proc. Natl. Acad. Sci, 90:5873—5877, and incorporated into the
NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389—3402).
In certain aspects, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic
Acids Res. 25:3389—3402. BLAST—2, WU—BLAST—2 (Altschul et al., 1996, Methods in
Enzymology, 0—480), ALIGN, ALIGN—2 (Genentech, South San Francisco, California)
or Megalign (DNASTAR) are additional publicly available re programs that can be
used to ahgn sequences In cenain aspects the tidentfiy behNeen UNO nucleofide
wwwwflskmmmwuwgflwGAPmfimmmflwGCGmmwmpmMgfiggmmga
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1,
2, 3, 4, 5, or 6). In certain alternative aspects, the GAP program in the GCG software
package, which incorporates the thm of Needleman and Wunsch (J. Mol. Biol.
(48):444—453 ) can be used to determine the percent identity between two amino acid
sequences (e. g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain aspects,
the percent identity between nucleotide or amino acid sequences is determined using the
algorithm of Myers and Miller (CABIOS, 4: 1 1-17 (1989)). For example, the percent identity
can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue
table, a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximal
ent by particular alignment software can be determined by one skilled in the art. In
certain aspects, the default parameters of the alignment software are used.
In certain s, the percentage identity "X" of a first amino acid sequence to
a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino
acid residues scored as identical matches in the alignment of the first and second sequences
(as aligned by visual inspection or a particular sequence alignment program) and Z is the total
number of residues in the second sequence. If the length of a first sequence is longer than
the second sequence, the percent identity of the first sequence to the second sequence will be
higher than the percent identity of the second ce to the first sequence.
A "conservative amino acid substitution" is one in which one amino acid
residue is ed with another amino acid residue having a similar side chain. Families of
amino acid es having similar side chains have been defined in the art, including basic
side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e. g., gine, glutamine, , threonine, tyrosine,
cysteine), ar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, nine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, phan, histidine). For
e, substitution of a phenylalanine for a tyrosine is a conservative substitution. In
certain aspects, conservative substitutions in the sequences of the polypeptides and antibodies
of the invention do not abrogate the binding of the polypeptide or antibody containing the
amino acid sequence, to the antigen(s), i.e., the HER3 to which the polypeptide or antibody
binds. Methods of identifying nucleotide and amino acid vative substitutions which do
not eliminate antigen binding are well-known in the art (see, e. g., Brummell et al., Biochem.
32: 1180—1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879—884 (1999); and Burks et
al., Proc. Natl. Acad. Sci. USA 94:.412—417 (1997)).
The term "consensus sequence," as used herein with respect to light chain
(VL) and heavy chain (VH) variable regions, refers to a composite or genericized VL or VH
sequence d based on information as to which amino acid residues within the VL or VH
chain are amenable to modification t ent to antigen binding. Thus, in a
"consensus sequence" for a VL or VH chain, certain amino acid ons are occupied by
one of multiple possible amino acid residues at that position. For example, if an arginine (R)
or a serine (S) occur at a particular position, then that particular position within the consensus
sequence can be either arginine or serine (R or S). Consensus sequences for VH and VL
chain can be defined, for example, by in vitro affinity maturation (e. g., randomizing every
amino acid position in a certain CDR using degenerate coding primers), by ng
mutagenesis (e. g., e scanning mutagenesis) of amino acid residues within the dy
CDRs, or any other methods known in the art, ed by evaluation of the binding of the
mutants to the antigen to determine whether the mutated amino acid on affects antigen
binding. In some aspects, mutations are uced in the CDR regions. In other aspects,
mutations are introduced in framework regions. In some other aspects, mutations are
introduced in CDR and framework s.
11. Anti-HER3-binding Molecules
The present invention provides HER3 binding molecules, e.g., antibodies and
antigen-binding fragments f that specifically bind HER3. The full-length amino acid
(aa) and nucleotide (nt) sequences for HER3 are known in the art (see, e.g., UniProt Acc. No.
P2186 for human HER3, or UniProt Acc. No. 088458 for mouse HER3). In some aspects,
the anti-HER3 binding molecules are human antibodies. In certain s, the HER3 binding
molecules are antibodies or antigen-binding fragments thereof In some aspects, HER3
binding molecules, e.g., antibodies or antigen-binding nts thereof comprise a Fab, a
Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, an
IgNar, an intrabody, an IgGACHZ, a minibody, a 3, a ody, a triabody, a diabody, a
single—domain antibody, DVD—Ig, Fcab, mAbZ, a (scFv)2, or a scFv—Fc. In some aspects, the
antibody is of the IgGl subtype and comprises the triple mutant YTE, as disclosed supra in
the Definitions section.
In certain aspects, anti-HER3 antibodies or antigen-binding fragments thereof
of the invention are modified compared to the parent Clone l6 (CL16) antibody. The
modifications can include mutations in the CDR regions and/or in the FW regions as
compared to CLl6. In certain aspects, an anti-HER3 antibody of the invention comprises
modifications to CDRl and/or CDR3 of the light chain of CLl6, including, but not limited to:
l) a light chain CDRl comprising the consensus sequence
X1GSX2SNIGLNYVS, wherein X1 is ed from R or S, and X2 is selected from S or L;
2) a light chain CDR3 comprising the consensus sequence
AAWDDX3X4X5GEX6, wherein X3 is selected from S or G, X4 is selected from L or P, X5 is
ed from R, I, P or S, and X6 is selected from V or A.
WO 78191
In certain aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises modifications to CDR2 of the heavy chain of CLl6, ing, but
not limited to a heavy chain CDRl comprising the consensus ce
GGVTNYADSVKG, wherein X7 is selected from Y, I or V.
In one aspect, an anti-HER3 antibody or n binding fragment f
comprises a VL region comprising the consensus amino acid sequence:
[FW1]X1GSX2SNIGLNYVS[FW2]RNNQRPS[FW3]AAWDDX3X4X5GEX6[FW4]
wherein [FWl], [FWz], [FW3] and [FW4] represent the amino acid residues of VL
framework region 1 (SEQ ID NO: 40 or 44), VL framework region 2 (SEQ ID NO:
41), VL framework region 3 (SEQ ID NO: 42) and VL framework region 4 (SEQ ID
NO: 43), and wherein X1 represents amino acid residues arginine (R) or serine (S),
X2 represents amino acid residues serine (S) or leucine (L), X3 ents amino acid
es serine (S) or glutamic acid (E), X4 represents amino acid residues leucine (L)
or proline (P), X5 represents amino acid residues arginine (R), isoleucine (I), proline
(P) or serine (S), and X6 represents amino acid residues valine (V) or arginine (R).
In one aspect, an anti-HER3 antibody or antigen binding fragment thereof
comprises a VH region comprises the consensus amino acid sequence:
[FW5]YYYMQ[FW6]X7IGSSGGVTNYADSVKG[FW7]VGLGDAFDI[FW8]
wherein [FW5], [FW6], [FW7] and [FWg] represent the amino acid residues of VH
framework region 1 (SEQ ID NO: 36), VH framework region 2 (SEQ ID NO: 37),
VH framework region 3 (SEQ ID NO: 38) and VH ork region 4 (SEQ ID NO:
39), and wherein X7 represents amino acid residues tyrosine (Y), isoleucine (I) or
valine (V).
In one aspect, an anti-HER3 antibody or antigen binding fragment thereof
comprises a VL region comprising the consensus amino acid sequence:
[FW1]X1GSX2SNIGLNYVS[FWflRNNQRPS[FW3]AAWDDX3X4X5GEX6[FW4]
wherein [FW1], [FWz], [FW3] and [FW4] represent the amino acid residues of VL
framework region 1 (SEQ ID NO: 40 or 44), VL framework region 2 (SEQ ID NO:
41), VL framework region 3 (SEQ ID NO: 42) and VL framework region 4 (SEQ ID
NO: 43), and wherein X1 ents amino acid residues ne (R) or serine (S),
X2 represents amino acid residues serine (S) or e (L), X3 represents amino acid
residues serine (S) or glutamic acid (E), X4 represents amino acid residues leucine (L)
or proline (P), X5 represents amino acid residues arginine (R), isoleucine (I), proline
(P) or serine (S), and X6 represents amino acid residues valine (V) or arginine (R);
and wherein said anti-HER3 antibody or antigen binding nt thereof further
comprises a VH region which comprises the consensus amino acid sequence:
[FW5]YYYMQ[FW6]X7IGSSGGVTNYADSVKG[FW7]VGLGDAFDI[FW8]
n [FW5], [FW6], [FW7] and [FWg] represent the amino acid residues of VH
framework region 1 (SEQ ID NO: 36), VH framework region 2 (SEQ ID NO: 37),
VH ork region 3 (SEQ ID NO: 38) and VH framework region 4 (SEQ ID NO:
39), and wherein X7 represents amino acid residues tyrosine (Y), isoleucine (I) or
valine (V).
In some aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises a VL—CDRl consisting of sequence selected from the group
ting of SEQ ID NOs: 18, 19 and 20. In some s, an anti-HER3 antibody or
antigen-binding fragment thereof of the invention comprises a VL-CDRl comprising a
sequence selected from the group consisting of SEQ ID NOs: 18, 19 and 20. In some aspects,
an anti-HER3 antibody or antigen-binding fragment thereof of the invention comprises a VL—
CDR2 ting of SEQ ID NO: 21. In some aspects, an anti-HER3 antibody or antigen-
binding fragment thereof of the invention comprises a VL-CDR2 comprising SEQ ID NO:
21. In some s, an anti-HER3 antibody or antigen-binding fragment f of the
invention comprises a VL—CDR3 consisting of a sequence selected from the group consisting
of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some aspects, an anti-HER3
antibody or n-binding fragment thereof of the invention comprises a VL-CDR3
comprising a sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26,
27, 28, 29, and 30.
In some aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises a VH-CDRl consisting of SEQ ID NO: 31. In some aspects, an
anti-HER3 antibody or antigen-binding fragment thereof of the invention comprises a VH-
CDRl comprising SEQ ID NO: 31. In some aspects, an anti-HER3 antibody or antigenbinding
fragment thereof of the invention comprises a VH—CDR2 consisting of a sequence
selected from the group consisting of SEQ ID NOs: 32, 33 and 34. In some aspects, an anti—
HER3 antibody or antigen-binding fragment thereof of the invention comprises a VH-CDR2
comprising a sequence selected from the group consisting of SEQ ID NOs: 32, 33 and 34. In
some aspects, an anti-HER3 antibody or antigen-binding fragment thereof of the invention
ses a VH—CDR3 consisting of SEQ ID NO: 35. In some aspects, an anti—HER3
antibody or antigen-binding nt thereof of the invention comprises a VH-CDR3
comprising SEQ ID NO: 35.
In some aspects, an anti-HER3 antibody or n-binding fragment thereof
of the invention comprises a VL—CDRl consisting of a sequence ed from the group
consisting of SEQ ID NOs: 18, 19 and 20, except for one, two, three or four amino acid
tutions. In some aspects, an anti-HER3 dy or antigen-binding fragment thereof of
the invention comprises a VL—CDRl comprising a ce ed from the group
consisting of SEQ ID NOs: 18, 19 and 20, except for one, two, three or four amino acid
substitutions. In some aspects, an anti-HER3 antibody or antigen-binding fragment thereof of
the invention comprises a VL-CDR2 consisting of SEQ ID NO: 21, except for one, two, three
or four amino acid substitutions. In some aspects, an ER3 antibody or antigen-binding
nt thereof of the invention ses a VL-CDR2 comprising SEQ ID NO: 21, except
for one, two, three or four amino acid substitutions. In some aspects, an anti-HER3 antibody
or antigen-binding fragment f of the invention comprises a VL-CDR3 consisting of a
sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29,
and 30, except for one, two, three or four amino acid substitutions. In some aspects, an anti—
HER3 antibody or antigen-binding fragment thereof of the invention comprises a VL-CDR3
comprising a sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26,
27, 28, 29, and 30, except for one, two, three or four amino acid tutions.
In some s, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises a VH-CDRl consisting of SEQ ID NO: 31, except for one, two,
three or four amino acid substitutions. In some aspects, an ER3 antibody or antigen-
binding fragment thereof of the invention comprises a VH-CDRl comprising SEQ ID NO:
_ 32 _
2012/066038
31, except for one, two, three or four amino acid substitutions. In some aspects, an anti-HER3
dy or antigen-binding fragment thereof of the invention comprises a 2
consisting of a sequence selected from the group consisting of SEQ ID NOs: 32, 33 and 34,
except for one, two, three or four amino acid substitutions. In some aspects, an anti—HER3
antibody or antigen-binding fragment thereof of the invention comprises a VH-CDR2
comprising a sequence selected from the group consisting of SEQ ID NOs: 32, 33 and 34,
except for one, two, three or four amino acid tutions. In some aspects, an anti—HER3
antibody or antigen-binding fragment thereof of the invention ses a VH-CDR3
consisting of SEQ ID NO: 35, except for one, two, three or four amino acid tutions. In
some aspects, an anti-HER3 antibody or antigen-binding fragment thereof of the invention
comprises a VH—CDR3 comprising SEQ ID NO: 35, except for one, two, three or four amino
acid substitutions.
In some aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises a VL—CDRl consisting of a sequence ed from the group
consisting of SEQ ID NOs: 18, 19 and 20; a VL—CDR2 consisting of SEQ ID NO: 21; and a
VL—CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 22,
23, 24, 25, 26, 27, 28, 29, and 30. In some aspects, an anti-HER3 dy or nbinding
fragment thereof of the invention comprises a VL—CDRl comprising a sequence
selected from the group consisting of SEQ ID NOs: 18, 19 and 20; a VL—CDR2 comprising
SEQ ID NO: 21; and a VL—CDR3 comprising a sequence selected from the group consisting
of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30.
In some aspects, an anti-HER3 dy or antigen-binding nt thereof
of the invention comprises a VH-CDRl consisting of SEQ ID NO: 31; a VH-CDR2
consisting of a sequence selected from the group consisting of SEQ ID NOs: 32, 33 and 34;
and a VH-CDR3 consisting of SEQ ID NO: 35. In some aspects, an anti-HER3 antibody or
antigen-binding fragment thereof of the invention comprises a VH-CDRl comprising SEQ
ID NO: 31; a VH—CDR2 comprising a sequence selected from the group consisting of SEQ
ID NOs: 32, 33 and 34; a VH—CDR3 comprising SEQ ID NO: 35.
In some aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention ses a VL—CDRl consisting of a sequence selected from the group
ting of SEQ ID NOs: 18, 19 and 20, except for one, two, three or four amino acid
substitutions; a VL-CDR2 consisting of SEQ ID NO: 21, except for one, two, three or four
amino acid substitutions; and a VL—CDR3 consisting of a ce selected from the group
2012/066038
consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30, except for one, two, three
or four amino acid tutions. In some s, an anti-HER3 antibody or antigen-binding
fragment thereof of the invention comprises a VL—CDRl sing a sequence selected
from the group consisting of SEQ ID NOs: 18, 19 and 20, except for one, two, three or four
amino acid substitutions; a VL-CDR2 comprising SEQ ID NO: 21, except for one, two, three
or four amino acid substitutions; and a VL—CDR3 comprising a sequence selected from the
group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, and 30, except for one, two,
three or four amino acid substitutions.
In some aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the ion ses a VH-CDRl consisting of SEQ ID NO: 31, except for one, two,
three or four amino acid substitutions; a VH—CDR2 consisting of a sequence selected from
the group consisting of SEQ ID NOs: 32, 33 and 34, except for one, two, three or four amino
acid substitutions; and a VH—CDR3 consisting of SEQ ID NO: 35, except for one, two, three
or four amino acid substitutions. In some s, an anti-HER3 antibody or antigen-binding
fragment thereof antibody of the invention comprises a VH-CDRl comprising SEQ ID NO:
31, except for one, two, three or four amino acid substitutions; a VH-CDR2 comprising a
sequence selected from the group consisting of SEQ ID NOs: 32, 33 and 34, except for one,
two, three or four amino acid substitutions; and VH—CDR3 comprising SEQ ID NO: 35,
except for one, two, three or four amino acid substitutions.
In certain aspects, an anti-HER3 antibody or antigen-binding fragment f
of the invention comprises modifications to CDRl, CDR2, and/or CDR3 of the heavy and/or
light chain, and further comprises modifications to FWl, FW2, FW3, and/or FW4 of the
heavy and/or light chain. In some aspects, FW1 comprises SEQ ID NO: 40 or 44, FWz
comprises SEQ ID NO: 41, FW3 comprises SEQ ID NO: 42, FW4 comprises SEQ ID NO: 43,
FW5 comprises SEQ ID NO: 36, FW6 comprises SEQ ID NO: 37, FW7 comprises SEQ ID
NO: 38, and FWg comprises SEQ ID NO: 39.
In some aspects, FW1 comprises SEQ ID NO: 40 or 44, except for one, two,
three or four amino acid substitutions; FWz comprises SEQ ID NO: 41, except for one, two,
three or four amino acid substitutions; FW3 comprises SEQ ID NO: 42, except for one, two,
three or four amino acid substitutions; FW4 ses SEQ ID NO: 43, except for one, two,
three or four amino acid substitutions; FW5 comprises SEQ ID NO: 36, except for one, two,
three or four amino acid tutions; FW6 comprises SEQ ID NO: 37, except for one, two,
three or four amino acid substitutions; FW7 comprises SEQ ID NO: 38, except for one, two,
three or four amino acid substitutions; and FWg comprises SEQ ID NO: 39, except for one,
two, three or four amino acid substitutions.
In certain aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises a VL and a VH sing VL-CDRl, VL-CRD2, VL-CDR3,
l, VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for
four, three, two, or one amino acid substitutions in one or more CDRs to: SEQ ID NOs: 18,
21, 22, 31, 32, and 35, SEQ ID NOs: 18, 21, 26, 31, 32 and 35, SEQ ID NOs: 18, 21, 27, 31,
32 and 35, SEQ ID NOs: 20, 21, 22, 31, 32 and 35, SEQ ID NOs: 19, 21, 22, 31, 32 and 35,
SEQ ID NOs: 18, 21, 25, 31, 32 and 35, SEQ ID NOs: 18, 21, 28, 31, 32 and 35, SEQ ID
, 21, 29, 31, 32 and 35, SEQ ID NOs:18, 21, 30, 31, 32 and 35, SEQ ID NOs: 18, 21,
23, 31, 32 and 35, SEQ ID NOs: 19, 21, 23, 31, 32 and 35, SEQ ID NOs: 20, 21, 23, 31, 32
and 35, SEQ ID NOs: 18, 21, 24, 31, 32 and 35, or SEQ ID NOs: 18, 21, 25, 31, 32 and 35,
respectively.
Heavy and light chain variable domains of the anti-HER3 antibody or antigen-
binding fragment thereof of the invention include the sequences listed in TABLE 2.
TABLE 2
ID Description Sequence
QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
Germlined FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL
QYELTQPPSASGTPGQRVTMSCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQR
ori_inal PSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL
CL16 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMQWVRQAPGKGLEWVSYIGS
SGGVTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGLGDAFD
IWGQGTMVTVSS
5H6 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDGLPGEVFGGGTKLTVL
8A3 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLIGEVFGGGTKLTVL
4H6 VL QSVLTQPPSASGTPGQRVTISCRGSSSNIGLN Y V SWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL
7 6E3 VL PPSASGTPGQRVTISCSGSLSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEVFGGGTKLTVL
2B 1 l VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLPGEVFGGGTKLTVL
--QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGEAFGGGTKLTVL
3A6 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPSGEVFGGGTKLTVL
4C4 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLRGEVFGGGTKLTVL
12 15D 1 2.1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMQWVRQAPGKGLEWVSIIGSS
_ 35 _
2012/066038
(15D 1 2 .I) VH GGVTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGLGDAFDI
WGQGTMVTVSS
13 15D12.2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSYYYMQWVRQAPGKGLEWVSVIGS
(15D12.V) SGGVTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGLGDAFD
VH IWGQGTMVTVSS
14 1A4 VL QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPPGEAFGGGTKLTVL
3 2C2 VL QSVLTQPPSASGTPGQRVTISCSGSLSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPPGEAFGGGTKLTVL
QSVLTQPPSASGTPGQRVTISCRGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPPGEAFGGGTKLTVL
QSVLTQPPSASGTPGQRVTISCSGSSSNIGLNYVSWYQQLPGTAPKLLISRNNQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSPSGEAFGGGTKLTVL
In certain aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises an dy VL and an antibody VH, wherein the VL ses
an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID
NO: 17.
In other s, an anti-HER3 antibody or n-binding fragment thereof
of the invention comprises an antibody VL and an antibody VH, wherein the VH comprises
an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%,
about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid
sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID
NO: 13.
In other aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprises a VL sing a sequence at least about 80%, about 85%, about
90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to
a reference amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, and SEQ ID NO: 17, and further comprises a VH comprising a sequence at least
about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about
99%, or about 100% identical to a reference amino acid sequence selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO: 13.
In some aspects, an anti-HER3 antibody or antigen-binding nt thereof
comprises a VH of TABLE 2 and a VL of TABLE 2. Antibodies are designated throughout
the specification according to their VL chains. The heavy chains of the specific antibodies
disclosed in the present ication correspond to the CL16 original heavy chain (SEQ ID
NO: 2). Thus, the "CL16 antibody" is an IgGl comprising two original CL16 light chains
(SEQ ID NO: 17) and two CL16 original heavy chains (SEQ ID NO: 2), whereas the "2C2
antibody" is an IgGl comprising two 2C2 light chains (2C2 VL (SEQ ID NO: 3) and two
CL16 original heavy chains (SEQ ID NO: 2).
In some aspects, the anti-HER3 antibody or antigen-binding fragment thereof
ses a heavy chain constant region or fragment thereof In some specific aspects, the
heavy chain constant region is an IgG constant region. The IgG constant region can comprise
a light chain constant region selected from the group ting of a kappa nt region
and a lambda constant region.
In certain aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention binds HER3 with substantially the same or better affinity as the CL16
antibody, comprising the CL16 original heavy chain (SEQ ID NO: 2) and the original CL16
light chain (SEQ ID NO: 17). In certain aspects, an anti-HER3 antibody or antigen-binding
fragment thereof of the ion binds HER3 with substantially the same or better affinity as
the 2C2 antibody, comprising the 2C2 light chain (2C2 VL (SEQ ID NO: 3) and the CL16
original heavy chain (SEQ ID NO: 2).
In one aspect of the present invention, an anti-HER3 antibody or antigen-
binding nt f specifically binds HER3 and antigenic fragments thereof with a
dissociation constant of kd (koff/kon) of less than 10’6 M, or of less than 10’7 M, or of less than
’8 M, or of less than 10’9 M, or of less than 10’10 M, or of less than 10’11 M, or of less than
’12 M, or of less than 10’13 M. In a particular aspect of the present invention, an anti-HER3
antibody or antigen-binding fragment thereof specifically binds HER3 and antigenic
nts thereof with a dissociation constant between 2><10’10 M and 6><10’10 M.
In another , an anti-HER3 antibody or antigen-binding nt thereof
of the invention binds to HER3 and/or antigenic fragments thereof with a Koff of less than
l><1073 s71, or less than 2><10’3 s71. In other aspects, an anti-HER3 antibody or antigen-
binding fragment thereof binds to HER3 and nic fragments thereof with a Koff of less
than 10’3 s71, less than 5><10’3 s71, less than 10’4 s71, less than 5><10’4 s71, less than 10’5 s71,
less than 5><10’5 s71, less than 10’6 s71, less than 5><10’6 s71, less than less than 5><10’7 s71, less
2012/066038
than 10’8 s71, less than 5><1078 s71, less than 10’9 s71, less than 5><1079 s71, or less than 10’10
s71. In a particular aspect, an anti-HER3 dy or antigen-binding fragment thereof of the
invention binds to HER3 and/or antigenic fragments f with a Koff of between 0.5><10’4
s’1 and 2.0x10’4 s71.
In another aspect, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention binds to HER3 and/or antigenic fragments thereof with an association rate
constant or k0n rate of at least 105 M7 s71, at least 5><105 M71 s71, at least 106 M71 s71, at least
><106 M71 s71, at least 107 M71 s71, at least 5><107 M71 s71, or at least 108 M71 s71, or at least
109 M71 s71. In another aspect, an anti-HER3 antibody or antigen-binding fragment thereof of
the invention binds to HER3 and/or antigenic fragments thereof with an association rate
constant or k0n rate of between l><105 M71 s71 and 6><105 M71 s71.
The VH and VL sequences disclosed in TABLE 1 can be "mixed and
matched" to create other anti-HER3 binding les of the invention. In certain aspects,
the VH ces of 15Dl2.I and 15D12.V are mixed and matched. Additionally or
alternatively, the VL sequences of 5H6, 8A3, 4H6, 6E.3, 2Bll, 2Dl, 3A6, 4C4, 1A4, 2C2,
3E.l can be mixed and matched.
In certain aspects, an ER3 antibody or antigen-binding fragment thereof
of the invention ses mutations that improve the binding to human FcRn and improve
the ife of the anti-HER3 antibody or antigen-binding fragment thereof. In some aspects,
such mutations are a methionine (M) to tyrosine (Y) mutation in position 252, a serine (S) to
threonine (T) mutation in position 254, and a threonine (T) to glutamic acid (E) mutation in
position 256, numbered according to the EU index as in Kabat (Kabat, et a]. (1991)
Sequences of ns of Immunological st, US. Public Health Service, National
Institutes of Health, Washington, DC), introduced into the constant domain of an IgGl. See
US. Patent No. 7,658,921, which is incorporated by reference herein. This type of mutant
IgG, referred to as a "YTE mutant" has been shown display approximately four-times
increased half-life as compared to ype versions of the same antibody (Dall'Acqua et al.,
J. Biol. Chem. 281:23514-24 (2006)). In some aspects, an anti-HER3 antibody or antigen-
binding fragment thereof comprising an IgG constant domain comprises one or more amino
acid substitutions of amino acid residues at positions 251—257, 0, 308—314, 9,
and 428-436, numbered according to the EU index as in Kabat, n such mutations
increase the serum half-life of the anti-HER3 antibody or antigen-binding fragment thereof.
In some aspects, a YTE mutant further comprises a substitution at position 434
of the IgG constant domain, numbered according to the EU index as in Kabat, with an amino
acid selected from the group consisting of tryptophan (W), methionine (M), tyrosine (Y), and
serine (S). In other aspects, a YTE mutant further comprises a substitution at position 434 of
the IgG constant domain, numbered according to the EU index as in Kabat, with an amino
acid selected from the group ting of phan (W), methionine (M), tyrosine (Y), and
serine (S), and substitution at on 428 of the IgG constant domain, ed ing
to the EU index as in Kabat, with an amino acid selected from the group consisting of
threonine (T), e (L), phenylalanine (F), and serine (S).
In yet other aspect, a YTE mutant further comprises a substitution at on
434 of the IgG constant domain, numbered according to the EU index as in Kabat, with
tyrosine (Y), and a substitution at position 257 of the IgG constant domain, numbered
according to the EU index as in Kabat, with leucine (L). In some s, a YTE mutant
further comprises a substitution at position 434 of the IgG constant domain, numbered
according to the EU index as in Kabat, with serine (S), and a substitution at position 428 of
the IgG constant domain, numbered according to the EU index as in Kabat, with leucine (L).
In a specific aspect, an anti-HER3 antibody or antigen-binding fragment
thereof comprises a 2C2 light chain variable region (2C2 VL; SEQ ID NO: 3), an original
CL16 heavy chain variable region (SEQ ID NO: 2), and an IgGl nt domain comprising
a methionine (M) to tyrosine (Y) mutation in position 252, a serine (S) to threonine (T)
mutation in position 254, and a threonine (T) to glutamic acid (E) mutation in position 256 of
the IgGl constant , numbered according to the EU index as in Kabat.
In certain aspects, an anti-HER3 antibody or antigen-binding fragment thereof
of the invention comprise at least one IgG constant domain amino acid tution selected
from the group consisting of:
(a) tution of the amino acid at position 252 with tyrosine (Y), phenylalanine (F),
tryptophan (W), or threonine (T),
(b) substitution of the amino acid at on 254 with threonine (T),
(c) substitution of the amino acid at position 256 with serine (S), arginine (R),
glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T),
(d) substitution of the amino acid at position 257 with leucine (L),
(e) substitution of the amino acid at position 309 with proline (P),
(f) substitution of the amino acid at position 311 with serine (S),
(g) substitution of the amino acid at position 428 with threonine (T), leucine (L),
phenylalanine (F), or serine (S),
(h) substitution of the amino acid at position 433 with ne (R), serine (S),
isoleucine (I), proline (P), or ine (Q),
(i) substitution of the amino acid at position 434 with tryptophan (W), methionine
(M), serine (S), histidine (H), phenylalanine (F), or tyrosine, and
(j) a combination of two or more of said substitutions,
wherein the positions are ed according to the EU index as in Kabat, and
wherein the modified IgG has an increased serum half-life compared to the serum
half-life of an IgG having the wild-type IgG constant domain.
In other aspects, the VH and/or VL amino acid sequences can be 85%, 90%,
95%, 96%, 97%, 98% or 99% similar to the ces set forth above, and comprise 1, 2, 3,
4, 5 or more conservative substitutions. A HER3 antibody having VH and VL regions having
high (i.e., 80% or greater) similarity to the VH regions of SEQ ID NOs: 2, 12 or 13 and/or
VL regions of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, or 17, respectively, can
be ed by mutagenesis (e. g., site—directed or PCR—mediated mutagenesis) of nucleic acid
molecules encoding SEQ ID NOs: 1—17, followed by testing of the encoded altered antibody
for retained function using the functional assays described herein.
The ty or avidity of an antibody for an antigen can be determined
experimentally using any suitable method well known in the art, e.g., flow cytometry,
enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g.,
BIACORETM analysis). Direct binding assays as well as competitive binding assay formats
can be y ed. (See, for example, Berzofsky et al., "Antibody—Antigen
Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY.
(1984); Kuby, Immunology, W. H. Freeman and Company: New York, NY. (1992); and
methods described herein. The measured affinity of a particular antibody—antigen interaction
can vary if measured under different conditions (e. g., salt concentration, pH, temperature).
Thus, measurements of affinity and other n-binding parameters (e.g., KD or Kd, Km,
Koff) are made with standardized solutions of antibody and antigen, and a standardized buffer,
as known in the art and such as the buffer described herein.
It also known in the art that affinities ed using BIACORETM analysis
can vary ing on which one of the reactants is bound to the chip. In this t, affinity
can be measured using a format in which the targeting dy (e. g., the 2C2 monoclonal
dy) is immobilized onto the chip (referred to as an "IgG down" format) or using a
format in which the target protein (e. g., HER3) is immobilized onto the chip (referred to as,
e. g., a "HER3 down" format).
111. g Molecules that Bind to the Same Epitope as anti-HER3
Antibodies and Antigen-binding Fragments Thereof of the Invention
In another aspect, the invention comprises inding molecules that bind
to the same epitope as do the various anti-HER3 antibodies described herein. The term
"epitope" as used herein refers to a protein determinant capable of binding to an antibody of
the ion. Epitopes usually consist of chemically active 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-
conformational epitopes are guished in that the binding to the former but not the latter is
lost in the presence of denaturing solvents. Such antibodies can be identified based on their
ability to cross—compete (e. g., to competitively inhibit the binding of, in a tically
significant manner) with antibodies such as the CLl6 antibody, the 2C2 antibody, or the 2C2-
YTE mutant, in rd HER3 binding . Accordingly, in one aspect, the invention
provides anti-HER3 antibodies and antigen-binding fragments thereof, e. g., human
monoclonal antibodies, that compete for binding to HER3 with another anti-HER3 antibody
or antigen-binding fragment thereof of the invention, such as the CLl6 antibody or the 2C2
antibody. The y of a test antibody to inhibit the binding of, e.g., the CLl6 antibody or
the 2C2 antibody demonstrates that the test antibody can compete with that antibody for
binding to HER3; such an antibody can, according to non-limiting theory, bind to the same or
a related (e. g., a structurally similar or spatially proximal) epitope on HER3 as the anti-HER3
antibody or antigen-binding fragment thereof with which it competes. In one aspect, the anti-
HER3 antibody or antigen-binding fragment thereof that binds to the same epitope on HER3
as, e. g., the CLl6 antibody or the 2C2 antibody, is a human monoclonal antibody.
IV. ism of Action
In some aspects, a HER3-binding molecule, e.g., an ER3 antibody or
n-binding fragment f can suppress HER3 phosphorylation. In other aspects, a
HER3-binding molecule, e.g., an ER3 antibody or antigen-binding nt thereof
can ss AKT phosphorylation. In still other aspects, a HER3 -binding molecule, e. g., an
anti-HER3 antibody or antigen-binding fragment thereof can suppress HERZ-HER3 dimer
ion. In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress cell growth. In some s, a HER3 —binding
molecule, e.g., an anti-HER3 antibody or antigen-binding nt thereof lacks ADCC
effect. In specific aspects, a HER3—binding le, e. g., an anti-HER3 antibody or antigen-
g fragment thereof can suppress HER3 phosphorylation, AKT phosphorylation, and/or
tumor colony ion Via a ligand-independent mechanism of action.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress HER3 phosphorylation in HRG-driven breast
cancer MCF—7 cells as measured by ELISA, with an IC50 lower than about 30 ng/mL, lower
than about 25 ng/mL, lower than about 20 ng/mL, lower than about 15 ng/mL, or lower than
about 10 ng/mL. In a specific aspect, a HER3-binding molecule, e. g., an ER3 antibody
or antigen-binding fragment thereof can suppress HER3 phosphorylation in HRG-driven
breast cancer MCF—7 cells as measured by ELISA, with an IC50 lower than about 20 ng/mL.
In a specific aspect, a inding molecule, e.g., an anti-HER3 dy or n-
binding fragment thereof can suppress HER3 phosphorylation in HRG-driven breast cancer
MCF—7 cells as measured by ELISA, with an IC50 lower than about 15 ng/mL. In another
specif1c aspect, a inding molecule, e. g., an anti-HER3 antibody or antigen—binding
fragment thereof can suppress HER3 phosphorylation in HRG-driven breast cancer MCF-7
cells as measured by ELISA, with an IC50 lower than about 10 ng/mL.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress cell growth in MDA-MB-175 breast cancer
cells with an IC50 lower than about 0.90 ug/mL, lower than about 0.80 ug/mL, lower than
about 0.70 ug/mL, lower than about 0.60 ug/mL, lower than about 0.50 ug/mL, lower than
about 0.40 ug/mL, lower than about 0.30 ug/mL, or lower than about 0.20 ug/mL. In a
specific aspect, a HER3-binding molecule, e. g., an anti-HER3 antibody or antigen—binding
fragment thereof can suppress cell growth in MDA-MB-175 breast cancer cells, with an IC50
lower than about 0.50 ug/mL. In a specific aspect, a HER3-binding molecule, e. g., an anti-
HER3 antibody or antigen-binding fragment f can suppress cell growth in MDA-MB-
175 breast cancer cells, with an IC50 lower than about 0.40 ug/mL. In another specific aspect,
a HER3-binding molecule, e. g., an anti-HER3 antibody or antigen-binding fragment f
can suppress cell growth in MDA-MB-175 breast cancer cells, with an IC50 lower than about
0.30 ug/mL. In another specific aspect, a HER3—binding le, e.g., an anti-HER3
antibody or antigen-binding fragment thereof can suppress cell growth in MDA-MB-175
breast cancer cells, with an IC50 lower than about 0.20 ug/mL.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress cell growth in HMCB melanoma cells with an
IC50 lower than about 0.20 ug/mL, lower than about 0.15 ug/mL, lower than about 0.10
ug/mL, lower than about 0.05 ug/mL, lower than about 0.04 ug/mL, or lower than about 0.03
ug/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress cell growth in HMCB melanoma cells, with an
IC50 lower than about 0.10 ug/mL. In a specific aspect, a HER3-binding molecule, e. g., an
ER3 antibody or antigen-binding fragment thereof can ss cell growth in HMCB
melanoma cells, with an IC50 lower than about 0.05 ug/mL. In a specific aspect, a HER3—
binding molecule, e.g., an anti-HER3 antibody or antigen-binding fragment thereof can
suppress cell growth in HMCB melanoma cells, with an IC50 lower than about 0.04 ug/mL.
In a ic aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or antigenbinding
fragment thereof can suppress cell growth in HMCB melanoma cells, with an IC50
lower than about 0.03 ug/mL.
In some s, a HER3-binding le, e.g., an ER3 dy or
antigen-binding fragment thereof can suppress HER3 phosphorylation in EGFR-driven
HCC827 lung cancer cells with an IC50 lower than about 20 ng/mL, lower than about 15
ng/mL, lower than about 10 ng/mL, lower than about 8 ng/mL, lower than about 6 ng/mL,
lower than about 4 ng/mL, or lower than about 2 ng/mL. In a specific aspect, a HER3-
binding molecule, e.g., an anti-HER3 antibody or antigen-binding fragment thereof can
suppress HER3 orylation in riven HCC827 lung cancer cells, with an IC50
lower than about 10 ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-
HER3 antibody or n-binding fragment thereof can suppress HER3 phosphorylation in
EGFR—driven HCC827 lung cancer cells, with an IC50 lower than about 8 ng/mL. In a
specific aspect, a HER3-binding molecule, e. g., an anti-HER3 dy or antigen—binding
fragment thereof can suppress HER3 phosphorylation in EGFR-driven HCC827 lung cancer
cells, with an IC50 lower than about 6 ng/mL. In a specific aspect, a HER3 -binding molecule,
e. g., an anti-HER3 antibody or antigen-binding fragment thereof can suppress HER3
phosphorylation in EGFR-driven HCC827 lung cancer cells, with an IC50 lower than about 4
ng/mL. In a ic aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress HER3 phosphorylation in EGFR-driven
HCC827 lung cancer cells, with an IC50 lower than about 2 ng/mL.
In some s, a HER3-binding molecule, e.g., an anti-HER3 dy or
antigen-binding fragment thereof can suppress HER3 phosphorylation in EGFR-driven
HCC827 lung cancer cells ant to TKI with an IC50 lower than about 30 ng/mL, lower
than about 25 ng/mL, lower than about 20 ng/mL, lower than about 15 ng/mL, lower than
about 10 ng/mL, or lower than about 5 ng/mL. In a specific aspect, a HER3-binding
molecule, e.g., an anti-HER3 antibody or antigen-binding fragment thereof can suppress
HER3 phosphorylation in EGFR-driven HCC827 lung cancer cells resistant to TKI, with an
K30bwaflwnflmMZOnglenaqmdficfipmnaHER$Mmmgnmbmfleag,mumd
HER3 antibody or n-binding fragment thereof can suppress HER3 phosphorylation in
EGFR-driven HCC827 lung cancer cells resistant to TKI, with an IC50 lower than about 15
ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress HER3 phosphorylation in EGFR-driven
HCC827 lung cancer cells resistant to TKI, with an IC50 lower than about 10 ng/mL. In a
specific aspect, a HER3-binding le, e. g., an anti-HER3 antibody or antigen—binding
fragment thereof can suppress HER3 phosphorylation in riven HCC827 lung cancer
cells resistant to TKI, with an IC50 lower than about 5 ng/mL.
In some specific aspects, a HER3—binding molecule, e. g., an anti—HER3
dy or antigen-binding fragment thereof can be used to treat TKI resistant cancers.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 dy or
antigen-binding fragment thereof can suppress HER3 phosphorylation in cMET-driven
MKN45 human gastric adenocarcinoma cells with an IC50 is lower than about 15 ng/mL,
lower than about 10 ng/mL, lower than about 9 ng/mL, lower than about 8 ng/mL, lower than
about 7 ng/mL, lower than about 6 ng/mL, lower than about 5 ng/mL, or lower than about 4
ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 dy or
antigen-binding fragment thereof can suppress HER3 phosphorylation in riven
MKN45 human gastric adenocarcinoma cells with an IC50 lower than about 10 ng/mL. In a
specific aspect, a HER3-binding molecule, e. g., an anti-HER3 antibody or antigen—binding
fragment thereof can suppress HER3 phosphorylation in cMET-driven MKN45 human
gastric adenocarcinoma cells with an IC50 lower than about 8 ng/mL. In a specific aspect, a
inding le, e.g., an anti-HER3 antibody or antigen-binding fragment thereof
can suppress HER3 phosphorylation in cMET-driven MKN45 human gastric adenocarcinoma
cells with an IC50 lower than about 6 ng/mL. In a specific aspect, a HER3-binding molecule,
e. g., an anti-HER3 antibody or antigen-binding fragment thereof can ss HER3
phosphorylation in cMET-driven MKN45 human gastric adenocarcinoma cells with an IC50
lower than about 4 ng/mL.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof of the invention can suppress pAKT in riven
MKN45 cells with an IC50 lower than about 15 ng/mL, lower than about 10 ng/mL, lower
than about 9 ng/mL, lower than about 8 ng/mL, lower than about 7 ng/mL, lower than about 6
ng/mL, lower than about 5 ng/mL, lower than about 4 ng/mL, or lower than about 3 ng/mL.
In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or antigen-
binding fragment thereof can suppress pAKT in cMET-driven MKN45 cells with an IC50
lower than about 8 ng/mL. In a specific , a HER3 -binding molecule, e.g., an ER3
antibody or n-binding fragment thereof can suppress pAKT in cMET-driven MKN45
cells with an IC50 lower than about 6 ng/mL. In a ic aspect, a HER3-binding molecule,
e.g., an anti-HER3 antibody or antigen-binding fragment thereof can ss pAKT in
cMET-driven MKN45 cells with an IC50 lower than about 4 ng/mL. In a specific aspect, a
HER3-binding molecule, e.g., an anti-HER3 antibody or antigen-binding nt thereof
can suppress pAKT in cMET-driven MKN45 cells with an IC50 lower than about 3 ng/mL.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof of the invention can ss pHER in FGFRZ-driven Kato
III human gastric signet ring oma cells with an IC50 lower than about 9 ng/mL, lower
than about 8 ng/mL, lower than about 7 ng/mL, lower than about 6 ng/mL, lower than about 5
ng/mL, lower than about 4 ng/mL, lower than about 3 ng/mL, lower than about 2 ng/mL, or
lower than about 1 ng/mL. In a specific aspect, a HER3 -binding molecule, e.g., an anti-HER3
dy or antigen-binding fragment thereof can suppress pHER in FGFRZ-driven Kato III
human gastric signet ring carcinoma cells with an IC50 lower than about 5 ng/mL. In a
specific aspect, a HER3-binding molecule, e. g., an anti-HER3 antibody or antigen—binding
fragment f can suppress pHER in FGFRZ-driven Kato III human gastric signet ring
carcinoma cells with an IC50 lower than about 4 ng/mL. In a specific aspect, a HER3 -binding
molecule, e.g., an ER3 antibody or antigen-binding fragment thereof can suppress
pHER in FGFRZ-driven Kato III human gastric signet ring carcinoma cells with an IC50
lower than about 3 ng/mL. In a specific aspect, a HER3 -binding molecule, e.g., an anti-HER3
antibody or antigen-binding fragment thereof can suppress pHER in FGFRZ-driven Kato III
human gastric signet ring carcinoma cells with an IC50 lower than about 2 ng/mL. In a
specific aspect, a HER3-binding molecule, e. g., an anti-HER3 dy or antigen—binding
fragment thereof can suppress pHER in FGFR2-driven Kato III human gastric signet ring
carcinoma cells with an IC50 lower than about 1 ng/mL.
In some aspects, a HER3-binding molecule, e.g., an ER3 antibody or
antigen-binding fragment thereof can suppress pAKT in FGFR-2 driven Kato III cells with an
IC50 lower than about 6 ng/mL, lower than about 5 ng/mL, lower than about 4 ng/mL, lower
than about 3 ng/mL, lower than about 2 ng/mL, or lower than about 1 ng/mL. In a specific
aspect, a HER3-binding molecule, e. g., an anti-HER3 antibody or antigen-binding fragment
thereof can suppress pAKT in FGFR-2 driven Kato III cells with an IC50 lower than about 4
ng/mL. In a ic aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or
n-binding nt thereof can suppress pAKT in FGFR-2 driven Kato III cells with an
IC50 lower than about 3 ng/mL. In a specific aspect, a HER3-binding le, e.g., an anti-
HER3 antibody or n-binding fragment thereof can suppress pAKT in FGFR—2 driven
Kato III cells with an IC50 lower than about 2 ng/mL. In a specific aspect, a HER3-binding
molecule, e.g., an anti-HER3 antibody or antigen-binding fragment thereof can suppress
pAKT in FGFR-2 driven Kato III cells with an IC50 lower than about 1 ng/mL.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof of the invention can suppress pHER in ligand independent
BT-474 breast cancer cells with an IC50 lower than about 10 ng/mL, lower than about 9
ng/mL, lower than about 8 ng/mL, lower than about 7 ng/mL, lower than about 6 ng/mL,
lower than about 5 ng/mL, lower than about 4 ng/mL. In a specific aspect, a HER3-binding
le, e.g., an anti-HER3 antibody or antigen-binding fragment thereof can ss
pHER in ligand independent BT—474 breast cancer cells with an IC50 lower than about 8
ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress pHER in ligand independent BT-474 breast
cancer cells with an IC50 lower than about 6 ng/mL. In a specific aspect, a inding
molecule, e.g., an ER3 antibody or antigen-binding fragment thereof can suppress
pHER in ligand independent BT-474 breast cancer cells with an IC50 lower than about 4
ng/mL.
In some aspects, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment f of the invention can suppress pAKT in ligand independent
BT-474 breast cancer cells with an IC50 lower than about 10 ng/mL, lower than about 9
ng/mL, lower than about 8 ng/mL, lower than about 7 ng/mL, lower than about 6 ng/mL,
lower than about 5 ng/mL, lower than about 4 ng/mL. In a specific , a HER3-binding
molecule, e.g., an ER3 antibody or antigen-binding fragment thereof can suppress
pAKT in ligand independent BT-474 breast cancer cells with an IC50 lower than about 8
ng/mL. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 antibody or
antigen-binding fragment thereof can suppress pAKT in ligand independent BT-474 breast
cancer cells with an IC50 lower than about 6 ng/mL. In a specific aspect, a HER3-binding
molecule, e.g., an anti-HER3 antibody or antigen-binding fragment thereof can suppress
pAKT in ligand independent BT-474 breast cancer cells with an IC50 lower than about 4
ng/mL. In some aspects, a HER3-binding le, e. g., an ER3 antibody or antigen-
binding fragment thereof can suppress pHER3, pAKT, and tumor colony formation in BT-
474 cells, a ligand independent breast cancer model.
In some aspects, a HER3-binding le, e.g., an ER3 antibody or
antigen-binding fragment thereof of the invention can suppress HRG induced VEGF
ion. In a specific aspect, a HER3-binding molecule, e.g., an anti-HER3 dy or
antigen-binding fragment f of the ion can suppress HRG induced VEGF
secretion in ligand independent BT-474 breast cancer cells and/or HRG-driven breast cancer
MCF—7 cells.
In some aspects, a HER3-binding molecule, e.g., an ER3 antibody or
antigen—binding nt thereof of the invention can cause cell cycle arrest. In a specific
aspect, a HER3-binding molecule, e. g., an anti-HER3 antibody or antigen-binding fragment
thereof of the invention can cause cell cycle arrest in breast cancer cells, including but not
limited to SKBR3 or BT474 cells.
V. Preparation of Anti-HER3 Antibodies and Antigen-Binding Fragments
Monoclonal anti-HER3 antibodies can be prepared using hybridoma methods,
such as those described by Kohler and Milstein (1975) Nature 256:495. Using the oma
method, a mouse, hamster, or other appropriate host animal, is immunized as described above
to elicit the production by cytes of antibodies that will specifically bind to an
immunizing antigen. Lymphocytes can also be immunized in vitro. ing immunization,
the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example,
polyethylene glycol, to form hybridoma cells that can then be selected away from unfused
lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed
specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting,
2012/066038
or by an in vitro g assay (e.g. radioimmunoassay (RIA); enzyme—linked
sorbent assay (ELISA)) can then be propagated either in in vitro culture using
standard s (Goding, Monoclonal Antibodies: ples and Practice, Academic Press,
1986) or in vivo as ascites tumors in an animal. The monoclonal dies can then be
purified from the culture medium or ascites fluid as described for polyclonal antibodies
above.
Alternatively anti-HER3 monoclonal antibodies can also be made using
recombinant DNA methods as described in US. Patent No. 4,816,567. The polynucleotides
ng a monoclonal dy are ed from mature B-cells or hybridoma cell, such as
by RT-PCR using oligonucleotide primers that ically amplify the genes encoding the
heavy and light chains of the antibody, and their sequence is determined using conventional
procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned
into suitable expression vectors, which when transfected into host cells such as E. coli cells,
simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not
otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host
cells. Also, recombinant anti-HER3 monoclonal antibodies or antigen-binding fragments
thereof of the desired species can be isolated from phage display libraries expressing CDRs
of the desired species as described (McCafferty et al., 1990, Nature, 348:552—554; Clarkson
et al., 1991, , 352:624—628; and Marks et al., 1991, J. Mol. Biol., 222:581—597).
The polynucleotide(s) encoding a anti-HER3 antibody or antigen-binding
nts thereof can further be ed in a number of different manners using
recombinant DNA technology to generate alternative antibodies. In some aspects, the
constant domains of the light and heavy chains of, for e, a mouse monoclonal
antibody can be substituted (1) for those regions of, for example, a human antibody to
generate a chimeric antibody or (2) for a non-immunoglobulin polypeptide to generate a
fusion antibody. In some aspects, the constant regions are truncated or removed to generate
the desired antibody fragment of a monoclonal antibody. Site-directed or high-density
mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a
monoclonal antibody.
In certain aspects, the anti-HER3 dy or antigen-binding fragment thereof
is a human antibody or antigen-binding fragment thereof. Human antibodies can be directly
prepared using various techniques known in the art. Immortalized human B lymphocytes
immunized in vitro or isolated from an immunized individual that produce an antibody
WO 78191
directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J.
Immunol., 147 (1):86—95; and US. Patent 5,750,373).
Also, the anti-HER3 human dy or antigen-binding fragment thereof can
be selected from a phage library, where that phage library expresses human antibodies, as
described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309—314, Sheets et al.,
1998, Proc. Nat’l. Acad. Sci., 95:6157—6162, boom and Winter, 1991, J. Mol. Biol.,
, and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques for the generation and
use of antibody phage libraries are also described in US. Patent Nos. 5,969,108, 6,172,197,
,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068;
6,706,484; and 7,264,963; and Rothe et al., 2007, J. Mol. Bio.,
doi: 10.1016/j.jmb.2007. 12.018 (each of which is orated by reference in its entirety).
Affinity maturation strategies and chain ng strategies (Marks et al.,
1992, Bio/Technology 10:779—783, incorporated by reference in its ty) are known in the
art and can be employed to generate high affinity human antibodies or antigen-binding
fragments thereof
In some s, an anti-HER3 monoclonal antibody can be a humanized
antibody. Methods for engineering, humanizing or resurfacing non-human or human
antibodies can also be used and are well known in the art. A humanized, resurfaced or
similarly engineered dy can have one or more amino acid residues from a source that is
non-human, e. g., but not limited to, mouse, rat, rabbit, non-human primate or other mammal.
These non-human amino acid residues are ed by residues that are often referred to as
"import" residues, which are typically taken from an "import" variable, constant or other
domain of a known human sequence. Such imported sequences can be used to reduce
genicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity,
specif1city, ife, or any other suitable characteristic, as known in the art. In general, the
CDR residues are directly and most substantially involved in influencing HER3 binding.
Accordingly, part or all of the non-human or human CDR sequences are maintained while the
non—human sequences of the variable and constant regions can be replaced with human or
other amino acids.
Antibodies can also optionally be humanized, resurfaced, engineered or
human antibodies engineered with retention of high ty for the n HER3 and other
favorable biological properties. To achieve this goal, humanized (or human) or engineered
ER3 antibodies and resurfaced antibodies can be optionally prepared by a process of
analysis of the parental sequences and various conceptual humanized and engineered
products using dimensional models of the al, engineered, and zed
sequences. Three—dimensional immunoglobulin models are commonly available and are
familiar to those d in the art. Computer programs are ble which rate and
display probable three-dimensional mational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the y of the candidate immunoglobulin to bind its antigen, such
as HER3. In this way, framework (FW) residues can be selected and combined from the
consensus and import sequences so that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved.
Humanization, resurfacing or ering of anti-HER3 antibodies or antigen-
binding fragments thereof of the present invention can be performed using any known
method, such as but not limited to those described in, Jones et al., Nature 321:522 (1986);
Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims
et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter
et al., Proc. Natl. Acad. Sci. USA. 89:4285 (1992); Presta et al., J. Immunol. 151:2623
(1993), US. Pat. Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;
,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 762; 5,530,101;
,585,089; 5,225,539; 4,816,567, 7,557,189; 7,538,195; and 7,342,110; International
Application Nos. PCT/US98/l6280; PCT/US96/l8978; PCT/US9l/09630;
PCT/US91/05939; PCT/US94/01234; PCT/GB89/01334; PCT/GB91/01134;
PCT/GB92/01755; International Patent Application Publication Nos. WO90/14443;
WO90/14424; WO90/14430; and European Patent Publication No. EP ; each of which
is entirely incorporated herein by reference, including the references cited therein.
Anti-HER3 humanized antibodies and antigen-binding fragments f can
also be made in enic mice containing human immunoglobulin loci that are e
upon immunization of producing the full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. This approach is described in US. Patent Nos.
807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
In certain aspects an anti-HER3 antibody nt is provided. Various
techniques are known for the production of antibody fragments. Traditionally, these
fragments are derived via proteolytic digestion of intact antibodies (for example Morimoto et
al., 1993, l of Biochemical and Biophysical Methods 24: 107-1 17; n et al., 1985,
Science, 229:81). In certain aspects, anti-HER3 dy fragments are produced
recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted
from E. coli or other host cells, thus allowing the production of large amounts of these
fragments. Such anti-HER3 antibody fragments can also be isolated from the antibody phage
libraries sed above. The anti-HER3 antibody fragments can also be linear antibodies as
bed in US. Patent No. 5,641,870. Other techniques for the production of antibody
fragments will be apparent to the skilled tioner.
According to the present invention, techniques can be adapted for the
production of single—chain antibodies ic to HER3 (see, e.g., US. Pat. No. 4,946,778).
In addition, methods can be adapted for the construction of Fab expression libraries (see, e.g.,
Huse et al., Science 246:1275—1281 (1989)) to allow rapid and effective identification of
monoclonal Fab fragments with the desired specificity for HER3, or derivatives, fragments,
analogs or homologs f. Antibody fragments can be produced by techniques in the art
including, but not limited to: (a) a F(ab')2 fragment ed by pepsin digestion of an
antibody molecule; (b) a Fab fragment generated by reducing the disulfide bridges of an
F(ab')2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule
with papain and a reducing agent, and (d) Fv fragments.
It can further be desirable, ally in the case of antibody fragments, to
modify an anti-HER3 antibody or antigen-binding fragment thereof in order to increase its
serum half-life. This can be achieved, for example, by incorporation of a salvage or
binding epitope into the dy or antibody fragment by on of the appropriate region
in the antibody or antibody fragment or by incorporating the epitope into a peptide tag that is
then fused to the antibody or antibody fragment at either end or in the middle (e. g., by DNA
or peptide synthesis), or by YTE mutation. Other methods to se the serum half-life of
an antibody or antigen—binding fragment thereof, e. g., conjugation to a heterologous le
such as PEG are known in the art.
Heteroconjugate anti-HER3 antibodies and antigen-binding fragments thereof
are also within the scope of the t invention. Heteroconjugate antibodies are composed
of two covalently joined antibodies. Such antibodies have, for example, been proposed to
target immune cells to unwanted cells (see, e.g., US. Pat. No. 4,676,980). It is contemplated
that the heteroconjugate anti-HER3 antibodies and antigen-binding nts thereof can be
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ed in vitro using known methods in synthetic protein chemistry, including those
ing crosslinking agents. For example, immunotoxins can be constructed using a
disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents
for this purpose include iminothiolate and methylmercaptobutyrimidate.
In n aspects, the HER3-binding molecules of the invention, e. g.,
antibodies or antigen-binding fragments f can be combined with other therapeutic
agents or conjugated to other therapeutic agents or toxins to form immunoconjugates and/or
fusion proteins. Examples of such therapeutic agents and toxins include, but are not limited to
mab (Erbitux®), panitumumab (Vectibix®), lapatinib (Tykerb®/Tyverb®), and
paclitaxel ®, Abraxane®) and derivatives (e.g., docetaxel).
In some s the HER3 —binding molecules of the invention, e.g., antibodies
or antigen-binding fragments f can be conjugated to antibodies or antibody nts
targeting epidermal growth factor receptor (EGFR). In other aspects, the HER3-binding
molecules of the invention can be conjugated to tyrosine kinase tors. In some specific
aspects, the HER3-binding molecules of the invention can be conjugated to inhibitors of the
tyrosine kinase activity associated with EGFR and/or HER2/neu. In some aspects, the HER3—
binding molecules of the invention can be conjugated to antimitotic . In some specific
aspects, the HER3-binding molecules of the invention can be conjugated to agents that
stabilize the mitotic spindle microtubule assembly.
For the purposes of the present invention, it should be appreciated that
modif1ed ER3 antibodies or antigen-binding fragments thereof can comprise any type
of variable region that provides for the association of the antibody or polypeptide with HER3.
In this regard, the variable region can comprise or be derived from any type of mammal that
can be induced to mount a humoral response and generate immunoglobulins against the
desired tumor associated antigen. As such, the variable region of the modified anti-HER3
antibodies or antigen-binding fragments thereof can be, for example, of human, murine, non-
human primate (e.g., cynomolgus monkeys, macaques, etc.) or lupine origin. In some aspects
both the variable and constant regions of the modified anti-HER3 antibodies or antigen-
binding fragments thereof are human. In other s the variable regions of compatible
antibodies (usually derived from a non—human ) can be ered or specifically
tailored to improve the binding properties or reduce the immunogenicity of the molecule. In
this respect, variable regions useful in the present invention can be humanized or ise
d through the inclusion of imported amino acid sequences.
In certain s, the variable domains in both the heavy and light chains of
an anti-HER3 antibody or antigen-binding fragment thereof are altered by at least partial
replacement of one or more CDRs and, if necessary, by l framework region replacement
and sequence changing. gh the CDRs can be derived from an antibody of the same
class or even subclass as the dy from which the framework regions are derived, it is
envisaged that the CDRs will be derived from an dy of different class and in certain
aspects from an antibody from a different species. It is not necessary to e all of the
CDRs with the complete CDRs from the donor le region to transfer the antigen binding
capacity of one variable domain to another. Rather, it is only necessary to transfer those
residues that are necessary to maintain the activity of the n binding site. Given the
explanations set forth in US. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well
within the competence of those skilled in the art, either by ng out routine
experimentation or by trial and error testing to obtain a functional antibody with reduced
immunogenicity.
Alterations to the variable region notwithstanding, those skilled in the art will
appreciate that the modified anti-HER3 antibodies or antigen-binding fragments thereof of
this invention will comprise antibodies (e.g., ength antibodies or immunoreactive
fragments thereof) in which at least a fraction of one or more of the constant region s
has been deleted or otherwise altered so as to provide d biochemical characteristics
such as increased tumor localization or d serum half—life when ed with an
antibody of approximately the same immunogenicity comprising a native or unaltered
constant region. In some aspects, the constant region of the modified dies will
comprise a human constant region. Modifications to the constant region compatible with this
invention comprise additions, deletions or substitutions of one or more amino acids in one or
more domains. That is, the modified antibodies disclosed herein can comprise alterations or
modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3)
and/or to the light chain constant domain (CL). In some aspects, modified constant regions
wherein one or more domains are partially or entirely deleted are contemplated. In some
aspects, the modified antibodies will comprise domain deleted constructs or variants wherein
the entire CH2 domain has been removed (ACH2 constructs). In some aspects, the omitted
constant region domain will be replaced by a short amino acid spacer (e.g., 10 residues) that
provides some of the molecular lity typically imparted by the absent constant region.
Besides their configuration, it is known in the art that the nt region
mediates several effector functions. For example, binding of the Cl component of
complement to antibodies activates the complement . Activation of complement is
important in the opsonisation and lysis of cell ens. The activation of ment also
stimulates the atory se and can also be involved in mune
ensitivity. Further, antibodies bind to cells via the Fc , with a PC receptor site on
the antibody Fc region binding to a PC receptor (FcR) on a cell. There are a number of Fc
receptors which are specific for ent classes of antibody, including IgG (gamma
receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of
antibody to Fc receptors on cell es triggers a number of important and diverse
biological responses including engulfment and destruction of antibody-coated particles,
clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called
antibody—dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory
mediators, placental transfer and control of immunoglobulin production.
In certain aspects, a anti-HER3 antibody or an antigen-binding fragment
thereof es for altered effector functions that, in turn, affect the biological profile of the
administered antibody or antigen-binding fragment thereof. For example, the deletion or
inactivation (through point mutations or other means) of a constant region domain can reduce
Fc receptor binding of the circulating modifled antibody thereby increasing tumor
localization. In other cases it can be that constant region modiflcations, consistent with this
invention, moderate complement g and thus reduce the serum half-life and nonspecific
association of a conjugated cytotoxin. Yet other ations of the constant region can be
used to eliminate disulflde linkages or oligosaccharide moieties that allow for enhanced
localization due to increased antigen specificity or antibody flexibility. Similarly,
modifications to the constant region in accordance with this invention can easily be made
using well known biochemical or molecular ering techniques well within the purview
of the skilled artisan.
In certain aspects, a HER3-binding molecule that is an dy or antigen-
binding nt thereof does not have one or more effector functions. For instance, in some
aspects, the antibody or antigen-binding fragment thereof has no antibody-dependent cellular
cytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity (CDC) activity.
In certain aspects, the anti-HER3 antibody or antigen binding fragment thereof does not bind
to an Fc receptor and/or complement factors. In certain aspects, the antibody or antigen-
binding fragment thereof has no effector function.
It will be noted that in certain aspects, the anti-HER3 modified dies or
antigen-binding fragments f can be engineered to fuse the CH3 domain directly to the
hinge region of the respective modified antibodies or fragments thereof. In other constructs it
can be desirable to provide a peptide spacer between the hinge region and the modified CH2
and/or CH3 domains. For example, ible ucts could be expressed wherein the
CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is
joined to the hinge region with a 5—20 amino acid spacer. Such a spacer can be added, for
instance, to ensure that the regulatory elements of the constant domain remain free and
accessible or that the hinge region remains e. However, it should be noted that amino
acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune
response t the construct. Accordingly, in certain aspects, any spacer added to the
construct will be relatively non-immunogenic, or even omitted altogether, so as to maintain
the desired biochemical qualities of the modified antibodies.
Besides the deletion of whole nt region domains, it will be appreciated
that the anti-HER3 antibodies and antigen-binding fragments thereof of the present invention
can be provided by the partial deletion or substitution of a few or even a single amino acid.
For example, the on of a single amino acid in selected areas of the CH2 domain can be
enough to substantially reduce Fc binding and thereby increase tumor localization. Similarly,
it can be desirable to simply delete that part of one or more constant region domains that
control the effector function (e.g., complement ClQ binding) to be modulated. Such partial
deletions of the constant regions can improve selected characteristics of the antibody or
antigen-binding fragment f (e. g., serum half-life) while leaving other ble
functions associated with the subject constant region domain . Moreover, as alluded to
above, the constant regions of the disclosed anti—HER3 antibodies and antigen—binding
fragments thereof can be modified through the mutation or substitution of one or more amino
acids that enhances the profile of the ing construct. In this respect it is possible to
disrupt the activity provided by a conserved binding site (e. g., Fc binding) while substantially
maintaining the configuration and immunogenic profile of the modified antibody or antigen—
binding fragment thereof. n aspects can comprise the addition of one or more amino
acids to the constant region to enhance ble characteristics such as decreasing or
increasing effector function or provide for more cytotoxin or carbohydrate attachment. In
such aspects it can be desirable to insert or replicate specific sequences derived from selected
constant region domains.
The present invention further embraces variants and equivalents which are
substantially homologous to the chimeric, humanized and human anti-HER3 antibodies, or
antigen-binding fragments thereof, set forth herein. These can contain, for example,
conservative substitution mutations, i.e., the substitution of one or more amino acids by
similar amino acids. For example, conservative substitution refers to the tution of an
amino acid with another within the same general class such as, for e, one acidic amino
acid with another acidic amino acid, one basic amino acid with another basic amino acid or
one neutral amino acid by another neutral amino acid. What is intended by a conservative
amino acid substitution is well known in the art.
An anti-HER3 antibody or antigen-binding fragment thereof can be further
modified to n additional chemical moieties not ly part of the protein. Those
derivatized moieties can improve the solubility, the biological half-life or absorption of the
protein. The moieties can also reduce or eliminate any desirable side s of the proteins
and the like. An overview for those moieties can be found in Remington's ceutical
Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).
VI. Polynucleotides Encoding HER3-Binding les
In certain aspects, the invention encompasses polynucleotides comprising
nucleic acid sequences that encode a polypeptide that specifically binds HER3 or an antigen-
binding fragment thereof For example, the invention es a polynucleotide comprising a
nucleic acid sequence that encodes an anti-HER3 antibody or s an n—binding
fragment of such an antibody. The polynucleotides of the invention can be in the form of
RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and
can be double—stranded or single—stranded, and if single stranded can be the coding strand or
ding (anti-sense) strand.
In certain aspects, the cleotides are isolated. In certain aspects, the
polynucleotides are substantially pure. In certain aspects the polynucleotides se the
coding sequence for the mature polypeptide fused in the same g frame to a
polynucleotide which aids, for example, in expression and secretion of a polypeptide from a
host cell (e.g., a leader sequence which ons as a secretory sequence for controlling
transport of a polypeptide from the cell). The polypeptide having a leader sequence is a
preprotein and can have the leader sequence cleaved by the host cell to form the mature form
—56—
2012/066038
of the polypeptide. The polynucleotides can also encode for an HER3-binding proprotein
which is the mature protein plus additional 5' amino acid residues.
In certain aspects the polynucleotides comprise the coding sequence for the
mature HER3 ng polypeptide, e. g., an anti-HER3 antibody or an antigen-binding
fragment thereof fused in the same reading frame to a marker sequence that allows, for
example, for cation of the encoded polypeptide. For example, the marker sequence can
be a istidine tag supplied by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host, or the marker ce can be
a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a
mammalian host (e.g., COS—7 cells) is used.
The present invention r relates to variants of the described
polynucleotides encoding, for example, HER3 -binding fragments, analogs, and derivatives of
the HER3-binding molecules of the invention.
The polynucleotide variants can contain alterations in the coding regions, non-
coding s, or both. In some aspects the polynucleotide variants contain alterations
which produce silent substitutions, additions, or deletions, but do not alter the properties or
activities of the encoded polypeptide. In some aspects, nucleotide variants are produced by
silent substitutions due to the degeneracy of the genetic code. cleotide variants can be
produced for a variety of reasons, e.g., to optimize codon expression for a particular host
(change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
Vectors and cells comprising the polynucleotides described herein are also ed.
In some aspects a DNA sequence encoding a HER3—binding molecule, e.g., an
anti-HER3 antibody or an antigen-binding nt thereof can be constructed by chemical
synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based
on the amino acid sequence of the d polypeptide and selecting those codons that are
favored in the host cell in which the recombinant polypeptide of interest will be produced.
Standard methods can be applied to synthesize an isolated polynucleotide ce encoding
an isolated polypeptide of interest. For example, a complete amino acid sequence can be
used to construct a back—translated gene. r, a DNA oligomer containing a nucleotide
sequence coding for the particular ed polypeptide can be synthesized. For example,
l small oligonucleotides coding for portions of the desired polypeptide can be
synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3'
overhangs for complementary assembly.
Once led (by synthesis, site—directed mutagenesis or another method),
the polynucleotide sequences encoding a particular isolated polypeptide of interest will be
inserted into an expression vector and operatively linked to an expression control sequence
appropriate for expression of the protein in a desired host. Proper assembly can be confirmed
by nucleotide sequencing, restriction mapping, and expression of a ically active
polypeptide in a suitable host. As is well known in the art, in order to obtain high expression
levels of a transfected gene in a host, the gene must be operatively linked to riptional
and translational expression control sequences that are functional in the chosen expression
host.
In certain aspects, recombinant sion vectors are used to amplify and
express DNA encoding anti-HER3 antibodies or n-binding fragments thereof.
Recombinant expression vectors are replicable DNA constructs which have synthetic or
cDNA—derived DNA nts encoding a polypeptide chain of an ER3 antibody or
and antigen-binding fragment thereof, operatively linked to suitable transcriptional or
translational regulatory elements derived from mammalian, microbial, viral or insect genes.
A riptional unit generally comprises an assembly of (l) a genetic element or elements
having a regulatory role in gene expression, for e, transcriptional promoters or
enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated
into protein, and (3) appropriate transcription and translation tion and termination
sequences, as bed in detail below. Such regulatory ts can include an operator
sequence to control transcription. The ability to replicate in a host, y conferred by an
origin of replication, and a selection gene to facilitate recognition of transformants can
additionally be incorporated. DNA regions are operatively linked when they are functionally
related to each other. For example, DNA for a signal peptide (secretory leader) is operatively
linked to DNA for a polypeptide if it is expressed as a precursor which participates in the
ion of the polypeptide; a promoter is operatively linked to a coding ce if it
controls the transcription of the sequence; or a ribosome binding site is operatively linked to a
coding sequence if it is positioned so as to permit translation. Structural elements intended
for use in yeast expression systems include a leader sequence enabling extracellular secretion
of translated protein by a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it can include an inal methionine residue. This
residue can optionally be subsequently cleaved from the expressed recombinant protein to
provide a final product.
—58—
The choice of expression control sequence and expression vector will depend
upon the choice of host. A wide y of sion host/vector ations can be
employed. Useful expression vectors for otic hosts, e, for example, vectors
comprising expression control sequences from SV40, bovine oma virus, adenovirus and
cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial
plasmids, such as plasmids from E. 6012', including pCR 1, pBR322, pMB9 and their
derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA
phages.
Suitable host cells for expression of an HER3—binding molecule, e. g., an anti—
HER3 antibody or antigen-binding fragment thereof include prokaryotes, yeast, insect or
higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram
negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells
include established cell lines of ian origin as described below. ree translation
s could also be employed. Appropriate cloning and expression vectors for use with
ial, fungal, yeast, and mammalian cellular hosts are bed by Pouwels et a1.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure of
which is hereby incorporated by reference. Additional information regarding methods of
protein production, including antibody production, can be found, e.g., in US. Patent
Publication No. 2008/0187954, US. Patent Nos. 6,413,746 and 6,660,501, and International
Patent Publication No. WO 23, each of which is hereby incorporated by reference
herein in its entirety.
Various mammalian or insect cell culture systems can also be advantageously
employed to express recombinant HER3—binding molecules, e.g., anti—HER3 antibodies or
antigen-binding fragments thereof Expression of recombinant ns in mammalian cells
can be med because such proteins are generally correctly folded, appropriately
modified and completely functional. Examples of suitable mammalian host cell lines include
HEK-293 and HEK—293T, the COS—7 lines of monkey kidney cells, described by Gluzman
(Cell 23:175, 1981), and other cell lines including, for example, L cells, C127, 3T3, Chinese
hamster ovary (CHO), NSO, HeLa and BHK cell lines. ian expression vectors can
comprise nontranscribed elements such as an origin of replication, a suitable promoter and
enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed
ces, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites, and transcriptional termination
2012/066038
sequences. virus systems for production of heterologous proteins in insect cells are
reviewed by Luckow and Summers, BioTechnology 6:47 (1988).
HER3—binding molecules, e.g., anti-HER3 antibodies or antigen-binding
fragments thereof produced by a transformed host can be purified ing to any suitable
method. Such standard s include chromatography (e.g., ion exchange, affinity and
sizing column chromatography), centrifugation, differential solubility, or by any other
standard technique for protein purification. Affinity tags such as hexahistidine, maltose
binding domain, influenza coat sequence and hione-S-transferase can be attached to the
protein to allow easy purification by passage over an appropriate affinity column. Isolated
ns can also be physically characterized using such techniques as proteolysis, nuclear
magnetic resonance and x-ray crystallography.
For example, supernatants from systems which secrete recombinant protein
into culture media can be first concentrated using a commercially available protein
concentration , for example, an Amicon or Millipore Pellicon iltration unit.
Following the concentration step, the concentrate can be applied to a le cation
matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or
substrate having pendant diethylaminoethyl (DEAE) groups. The es can be
acrylamide, agarose, dextran, cellulose or other types commonly employed in protein
purification. Alternatively, a cation exchange step can be employed. le cation
gers include various insoluble matrices comprising sulfopropyl or carboxymethyl
groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl
or other aliphatic groups, can be employed to further purify an HER3-binding molecule.
Some or all of the foregoing purif1cation steps, in various combinations, can also be
employed to provide a homogeneous recombinant protein.
A recombinant HER3 -binding protein, e.g., an anti-HER3 antibody or antigen-
binding nt thereof produced in bacterial culture can be isolated, for example, by initial
extraction from cell pellets, followed by one or more tration, salting—out, aqueous ion
exchange or size exclusion chromatography steps. High performance liquid chromatography
(HPLC) can be employed for final purification steps. Microbial cells ed in expression
of a recombinant protein can be disrupted by any convenient method, including -thaw
cycling, sonication, ical disruption, or use of cell lysing agents.
2012/066038
Methods known in the art for purifying antibodies and other proteins also
include, for example, those described in US. Patent Publication Nos. 2008/0312425,
2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein
in its entirety.
In certain aspects, the HER3-binding molecule is a polypeptide that is not an
antibody. A variety of methods for identifying and producing non-antibody polypeptides that
bind with high affinity to a protein target are known in the art. See, e. g., Skerra, Curr. Opin.
Biotechnol., 18:295—304 (2007), Hosse et al., Protein Science, 15:14—27 (2006), Gill et al.,
Curr. Opin. hnol., 17:653—658 (2006), Nygren, FEBS J., 275:2668—76 (2008), and
Skerra, FEBS J., 275:2677—83 (2008), each of which is incorporated by reference herein in its
entirety. In certain aspects, phage display technology can been used to identify/produce an
HER3-binding polypeptide. In certain aspects, the polypeptide ses a protein ld
of a type selected from the group consisting of protein A, a lipocalin, a fibronectin domain,
an ankyrin consensus repeat domain, and thioredoxin.
VI. Treatment s Using Therapeutic ER3 Antibodies
Methods of the invention are directed to the use of anti-HER3 binding
molecules, e.g., antibodies, including antigen-binding fragments, variants, and derivatives
thereof, to treat patients having a disease ated with HER3 expression or HER3-
expressing cells. By "HER3-expressing cell" is meant a cell expressing HER3. Methods for
detecting HER3 expression in cells are well known in the art and include, but are not limited
to, PCR techniques, immunohistochemistry, flow cytometry, Western blot, ELISA, and the
like.
Though the following discussion refers to stic methods and treatment of
various diseases and disorders with an HER3—binding molecule of the ion, the s
described herein are also applicable to anti-HER3 antibodies, and the antigen-binding
fragments, ts, and derivatives of these anti-HER3 antibodies that retain the desired
properties of the anti-HER3 antibodies of the invention, e. g., capable of specifically binding
HER3 and neutralizing HER3 activity. In some aspects, HER3 -binding molecules are human
or zed dies that do not mediate human ADCC, or are selected from known anti-
HER3 antibodies that do not mediate ADCC, or are ER3 antibodies that are engineered
such that they do not mediate ADCC. In some aspects, the HER3-binding molecule is a
clone 16 monoclonal antibody. In other s, the HER3-binding molecule is a clone 16
YTE mutant antibody. In some aspects the HER3 -binding le is a P2Bll monoclonal
_ 61 _
antibody. In some aspects the HER3-binding molecule is a 1A4 monoclonal antibody. In
some aspects the HER3-binding molecule is a 2C2 monoclonal antibody. In some aspects the
HER3-binding molecule is a 2F10 monoclonal dy. In some aspects the HER3-binding
molecule is a 3E1 monoclonal antibody. In some aspects the HER3-binding molecule is a
P2Bll monoclonal antibody engineered to extend serum half-life. In some aspects the HER3—
binding le is a 1A4 monoclonal antibody engineered to extend serum half-life. In
some aspects the HER3 -binding molecule is a 2C2 onal antibody engineered to extend
serum half-life. In some aspects the HER3-binding molecule is a 2F10 onal antibody
engineered to extend serum half-life. In some aspects the HER3-binding molecule is a 3E1
onal antibody engineered to extend serum half-life. In other aspects the HER3-
binding molecule is a P2Bll YTE mutant antibody. In other aspects the inding
molecule is a 1A4 YTE mutant antibody. In other aspects the HER3-binding molecule is a
2C2-YTE mutant antibody. In other aspects the HER3-binding le is a 2F10 YTE
mutant antibody. In other aspects the HER3 -binding molecule is a 3E1 YTE mutant antibody.
In one aspect, treatment includes the application or administration of an anti-
HER3 binding molecule, e.g., an antibody or antigen binding nt, variant, or derivative
thereof of the current invention to a subject or patient, or application or administration of the
anti-HER3 binding molecule to an isolated tissue or cell line from a subject or patient, where
the subject or patient has a e, a symptom of a disease, or a predisposition toward a
disease. In another aspect, treatment is also intended to e the application or
administration of a pharmaceutical ition comprising the anti-HER3 binding molecule,
e. g., an antibody or antigen g fragment, variant, or derivative f of the current
invention to a t or patient, or application or administration of a pharmaceutical
composition comprising the anti-HER3 binding molecule to an isolated tissue or cell line
from a subject or patient, who has a e, a symptom of a disease, or a predisposition
toward a disease.
The anti-HER3 binding molecules, e. g., antibodies or antigen-binding
nts, variants, or derivatives thereof of the present invention are useful for the treatment
of various cancers. In one aspect, the invention relates to anti-HER binding molecules, e. g.,
antibodies or antigen-binding fragments, variants, or tives thereof for use as a
medicament, in particular for use in the ent or prophylaxis of cancer. Examples of
cancer include, but are not limited to colon cancer, lung cancer, gastric cancer, head and neck
squamous cells cancer, melanoma, pancreatic cancer, prostate , and breast cancer.
In ance with the methods of the present invention, at least one anti-
HER3 binding molecule, e.g., an antibody or antigen binding fragment, variant, or derivative
thereof as defined ere herein is used to promote a ve therapeutic response with
respect to cancer. The term "positive therapeutic response" with respect to cancer treatment
refers to an improvement in the disease in association with the activity of these anti-HER3
binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives
f, and/or an ement in the symptoms associated with the disease. Thus, for
example, an improvement in the disease can be characterized as a complete response. By
ete response" is intended an e of clinically able disease with
normalization of any previously test results. Alternatively, an improvement in the disease can
be categorized as being a partial response. A ive therapeutic response" encompasses a
reduction or inhibition of the progression and/or duration of cancer, the reduction or
amelioration of the severity of cancer, and/or the amelioration of one or more symptoms
thereof resulting from the administration of an ER3 binding molecule of the invention.
In specific aspects, such terms refer to one, two or three or more results following the
administration of anti-HER3 binding molecules of the invention: (1) a stabilization, reduction
or elimination of the cancer cell tion; (2) a stabilization or reduction in cancer growth;
(3) an impairment in the formation of cancer; (4) eradication, removal, or control of y,
regional and/or metastatic cancer; (5) a reduction in mortality; (6) an increase in disease-free,
relapse—free, progression—free, and/or overall survival, duration, or rate; (7) an increase in the
response rate, the durability of response, or number of patients who respond or are in
remission; (8) a decrease in hospitalization rate, (9) a decrease in alization s, (10)
the size of the cancer is maintained and does not increase or increases by less than 10%,
preferably less than 5%, preferably less than 4%, preferably less than 2%, and (12) an
increase in the number of patients in remission.
Clinical response can be assessed using screening techniques such as magnetic
resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan,
flow cytometry or fluorescence—activated cell sorter (FACS) analysis, histology, gross
ogy, and blood chemistry, including but not limited to changes detectable by ELISA,
RIA, tography, and the like. In addition to these positive eutic responses, the
t undergoing therapy with the anti-HER3 binding molecule, e. g., an antibody or
antigen-binding fragment, variant, or derivative thereof, can experience the beneficial effect
of an improvement in the symptoms associated with the disease.
The anti-HER3 binding molecules, e.g., antibodies or antigen-binding
fragments, ts, or derivatives thereof of the invention can be used in combination with
any known therapies for cancer, including any agent or combination of agents that are known
to be useful, or which have been used or are currently in use, for treatment of cancer, e. g.,
colon cancer, lung , gastric cancer, head and neck squamous cells cancer, and breast
. The second agent or combination of agents of the pharmaceutical combination
ation or dosing regimen preferably has complementary activities to the antibody or
polypeptide of the invention such that they do not adversely affect each other.
Anticancer agents include drugs used to treat malignancies, such as cancerous
growths. Drug therapy can be used alone, or in combination with other treatments such as
surgery or radiation therapy. Several classes of drugs can be used in cancer ent,
depending on the nature of the organ involved. For example, breast cancers are commonly
stimulated by estrogens, and can be treated with drugs which inactive the sex es.
Similarly, prostate cancer can be treated with drugs that inactivate androgens, the male sex
hormone. Anti-cancer agents for use in certain methods of the present invention include,
among others, antibodies (e. g., dies which bind IGF-lR, antibodies which bind EGFR,
antibodies which bind HER2, antibodies which bind HER3, or antibodies which bind cMET),
small molecules targeting IGFlR, small les targeting EGFR, small molecules
targeting HER2, antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule
targeting agents, kinase tors, protein synthesis inhibitors, immunotherapeutic agents,
hormonal therapies, glucocorticoids, aromatase tors, mTOR inhibitors,
chemotherapeutic agents, Protein Kinase B inhibitors, Phosphatidylinositol 3-Kinase (PI3K)
tors, Cyclin Dependent Kinase (CDK) inhibitors, RLr9, CD289, enzyme inhibitors,
anti-TRAIL, MEK inhibitors, etc.
In specific aspects the HER3-binding les of the invention, e.g.,
antibodies or antigen-binding fragments thereof, can be administered in combination with
antibodies or antibody nts targeting epidermal growth factor receptor (EGFR), e. g.,
cetuximab (Erbitux®, Imclone),panitumumab (Vectibix®, Amgen), matuzumab/EMD72000
(Merck Serono), MM-lSl oligoclonal (Merrimack), nimotuzumab (TheraCIM, InnGene
Kalbiotechy), GA201/RG7160 (Roche), Sym004 (Symphogen), 945A
(EGFlUHER3 dual specific, Genentech). In other specific aspects the HER3-binding
molecules of the invention, e.g., antibodies or antigen-binding fragments thereof, can be
administered in combination with dies or dy fragments targeting HER2, e.g.,
pertuzumab (rhuMAb 2C4/Omnitarg®, Genentech), zumab (Herceptin®,
Genentech/Roche), MM-111 (HER2/HER3 bispecific antibody, Merrimack, e.g., WC
2009/126920). In still other specific aspects the HER3-binding molecules of the invention,
e. g., antibodies or antigen-binding fragments thereof, can be administered in combination
with antibodies or antibody nts that also target HER3, e. g., MEHD—7945A/RG7597
(EGFIUHER3 dual specific, Genentech, e.g., WO 8127), MM—121 (Merrimack, e.g.,
WC 2008/100624), MM-111 (HER2/HER3 ific antibody, Merrimack, e.g., WC
2009/126920), AV—203 (Aveo, e.g., WC 2011/136911), AMG888 (Amgen, WO
2007/077028), HER3—8 (ImmunogGen, e.g., ). In further specific s
the HER3-binding molecules of the invention, e.g., antibodies or antigen-binding nts
thereof, can be administered in combination with antibodies or antibody fragments targeting
HER4. In a specific aspect, the HER3-binding molecules of the invention can be
stered in combination with an antibody that targets EGFR, or HER2 (e. g., cetuximab
or zumab). In a r specific aspect, the HER3 ng les of the invention
can be administered in combination with antibody drug conjugates that targets HER2 (e.g.,
trastuzumab emtansine, Genentech/Roche). It is contemplated that the HER3-binding
molecules of the invention enhance the alization and degradation of a co-receptor
induced by the binding of an antibody to the co-receptor and will thus, enhance the efficacy
of an antibody and/or antibody drug ate that targets EGFR, HER2 and/or HER4.
In other aspects, the HER3-binding molecules of the invention can be
administered in combination with tyrosine kinase inhibitors. In some other specific aspects,
the HER3-binding molecules of the invention can be administered in combination with
tors of the tyrosine kinase activity associated with EGFR and/or HER2/neu, e.g.,
lapatinib. In specific aspects the HER3-binding molecules of the invention, can be
administered in combination with small molecule inhibitors of the epidermal growth factor
receptor(s) (e. g., EGFR, HER2, HER4) e. g., gefitinib (lressa®, Astrazeneca); canertinib/CI-
1033 (Pfizer); lapatinib (Tykerb®, GlaxoSmithKline), erlotinib (Tarceva®, OSI Pharma),
afatinib (Tovok®/Tomtovok®, Boehringer eim), neratinib (HKI-272, Pfizer).
In some aspects, the HER3-binding molecules of the invention can be
administered in combination with antimitotic agents. In some specific aspects, the HER3—
binding molecules of the invention can be administered in combination with agents that
stabilize the mitotic spindle microtubule assembly, e.g, paclitaxel or docetaxel.
—65—
In some aspects, the HER3-binding molecules of the invention can be
administered in combination with MEK (mitogen-activated protein kinase (MAPK) kinase,
also known as MAPKK) inhibitors, e.g., selumetinib (AZD6244, 42866,
AstraZeneca), WX-554 (Wilex), trametinib (GlaxoSmithKline), refametinib (Ardea
Biosciences), E-6201 ), MEK-162 (Novartis). In a particular , the combination of
a MEK inhibitor and a HER3-binding molecule of the invention is more efficacious than
either agent alone. In a ic , a inding molecule of the invention is
administered in combination with selumetinib.
Where the combined therapies comprise stration of an anti-HER3
g molecule in combination with administration of another therapeutic agent, the
methods of the invention encompass nistration, using separate formulations or a single
pharmaceutical formulation, and consecutive administration in either order. In some aspects,
the anti-HER3 antibodies described herein are administered in combination with other drugs,
n the antibody or antigen-binding fragment, variant, or derivative thereof and the
therapeutic agent(s) can be administered sequentially, in either order, or simultaneously (i.e.,
concurrently or within the same time frame).
The ation therapy can provide "synergy" and prove "synergistic", i.e.,
the effect achieved when the active ingredients used together is greater than the sum of the
s that results from using the compounds separately. A synergistic effect can be attained
when the active ingredients are: (l) co—formulated and administered or delivered
simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in
parallel as separate formulations; or (3) by some other regimen. When delivered in alternation
therapy, a synergistic effect can be attained when the compounds are administered or
delivered sequentially, e. g., by different injections in separate syringes. In l, during
alternation therapy, an effective dosage of each active ingredient is administered sequentially,
i.e., serially, whereas in combination therapy, effective dosages of two or more active
ingredients are administered together.
In some aspects, the HER3-binding molecule, e. g., an anti-HER3 antibody or
antigen binding fragment thereof of the ion can be administered in a synergistic
combination with a epidermal growth factor receptor (EGFR) inhibitor. In some aspects, the
EGFR inhibitor is an antibody. In ic aspects, the EGFR inhibitor antibody is Erbitux®
(cetuximab) or panitumumab (Vectibix®). In specific aspects the HER3 —binding molecules of
the invention, e.g., antibodies or antigen-binding fragments thereof, can be administered in a
WO 78191
synergistic combination with inhibitors of the tyrosine kinase activity associated with EGFR
and/or HER2/neu, e. g., lapatinib. In some aspects, the HER3-binding molecule, e.g., an anti-
HER3 antibody or antigen binding fragment f of the invention can be administered in a
synergistic combination with a HER2 inhibitor. In some aspects, the HER2 inhibitor is an
antibody. In specific aspects, the HER2 inhibitor antibody is pertuzumab b
2C4/Omnitarg®, Genentech), trastuzumab (Herceptin®, Genentech/Roche) or trastuzumab
emtansine (Genentech/Roche). In specific aspects the HER3—binding molecules of the
invention, e.g., antibodies or antigen-binding fragments thereof, can be administered in a
synergistic combination with inhibitors of the tyrosine kinase activity associated with
HER2/neu, e.g., lapatinib. In some s, the HER3 -binding molecules of the invention can
be stered in a synergistic combination with an antimitotic agent. In some specific
aspects the totic agent stabilizes the mitotic spindle microtubule assembly. In some
specific aspects, the antimitotic agent is paclitaxel or docetaxel. In some specific
embodiments, the 2C2 antibody can be administered in a synergistic combination with a
growth factor receptor (EGFR) inhibitor. In some specific ments, the EGFR inhibitor
is an antibody. In specific embodiments, the EGFR inhibitor antibody administered
istically with the 2C2 dy is Erbitux® imab). In specific embodiments the
2C2 antibody can be administered in a synergistic combination with inhibitors of the tyrosine
kinase activity associated with EGFR and/or HER2/neu, e.g., lapatinib. In some
embodiments, the 2C2 antibody can be administered in a synergistic combination with an
antimitotic agent. In some specific embodiments, the antimitotic agent administered
synergistically with the 2C2 antibody stabilizes the c spindle microtubule assembly. In
some specific embodiments, the antimitotic agent administered istically with the 2C2
antibody is paclitaxel.
In one aspect, the cancer comprises the KRAS mutation. In ic aspects,
the KRAS on is located at codon 12 of a human KRAS gene. As trated in the
Examples section, anti-HER3 antibodies disclosed herein as capable on inhibiting the growth
of tumor cells that comprise a KRAS mutation, either when used as a single agent
(monotherapy) or in combination with another therapeutic agent.
A further aspect is the use of anti—HER3 binding molecules, e.g., antibodies or
antigen-binding fragments, variants, or derivatives thereof, for stic monitoring of
protein levels in tissue as part of a clinical testing procedure, e. g., to determine the efficacy of
a given treatment regimen. For example, ion can be facilitated by coupling the antibody
—67—
to a detectable substance. Examples of detectable substances include various enzymes,
prosthetic , fluorescent materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include horseradish peroxidase, ne
phosphatase, B-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group
xes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent al es l; examples of bioluminescent materials include
luc1ferase, luc1fer1n, and aequor1n; and examples of su1table radloact1ve mater1al 1nclude. . . . . . . . . 125
1311, 358, or 3H.
VII. Pharmaceutical Compositions and Administration Methods
Methods of preparing and administering anti-HER3 binding molecules, e. g.,
antibodies, or n-binding fragments, variants, or derivatives thereof of the invention to a
subject in need thereof are well known to or are readily ined by those skilled in the art.
The route of administration of the anti-HER3 binding molecule, e. g, antibody, or antigen-
binding fragment, variant, or derivative thereof can be, for example, oral, parenteral, by
inhalation or topical. The term eral as used herein includes, e.g., intravenous,
rterial, eritoneal, intramuscular, subcutaneous, rectal, or vaginal administration.
However, in other methods compatible with the teachings herein, anti-HER3 binding
molecules, e. g., antibodies, or n-binding fragments, variants, or derivatives thereof, of
the invention can be red ly to the site of the adverse cellular population thereby
increasing the exposure of the diseased tissue to the therapeutic agent.
As discussed herein, anti—HER3 binding molecules, e.g., antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the invention can be
administered in a pharmaceutically effective amount for the in viva treatment of HER3-
expressing cell—mediated diseases such as certain types of cancers.
The pharmaceutical compositions used in this invention can comprise
pharmaceutically acceptable carriers, including, e. g., water, ion gers, proteins, buffer
substances, and salts. Preservatives and other additives can also be present. The carrier can
be a solvent or sion . Suitable formulations for use in the therapeutic methods
disclosed herein are described in ton's Pharmaceutical Sciences (Mack Publishing
Co.) 16th ed. (1980). In some aspects, the HER3—binding molecules of the invention are
ated in a refrigerator (2-8 oC) stable composition. In a particular aspect, the
refrigerator stable composition comprises 25 mM histidine/histidine HCL, 205 mM sucrose,
0.02% polysorbate 80 at pH 6.0. In another particular aspect, the HER3-binding molecules of
the invention are formulated at 25-100 mg/ml in the refrigerator stable composition.
In any case, sterile injectable solutions can be prepared by incorporating an
active compound (e.g., an ER3 antibody, or antigen-binding nt, variant, or
tive thereof, by itself or in combination with other active agents) in the required
amount in an appropriate t followed by ed sterilization. Further, the preparations
can be packaged and sold in the form of a kit. Such articles of manufacture can have labels
or package inserts indicating that the associated compositions are useful for treating a subject
suffering from, or predisposed to a e or disorder.
eral formulations can be a single bolus dose, an on or a loading
bolus dose followed with a maintenance dose. These compositions can be administered at
specific fixed or variable intervals, e.g., once a day, or on an "as needed" basis.
The composition can be administered as a single dose, le doses or over
an established period of time in an infusion. Dosage regimens also can be adjusted to provide
the m d response (e.g., a therapeutic or prophylactic se).
Therapeutically effective doses of the compositions of the t invention,
for treatment of HER3—expressing cell—mediated diseases such as certain types of cancers
including e.g., colon cancer, lung cancer, gastric cancer, head and neck squamous cells
cancer, ma, pancreatic cancer, prostate cancer, and breast cancer, vary depending
upon many different factors, including means of administration, target site, physiological
state of the patient, whether the patient is human or an animal, other medications
administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a
human, but non-human mammals including transgenic mammals can also be treated.
Treatment s can be titrated using routine methods known to those of skill in the art to
optimize safety and cy.
The amount of at least one anti-HER3 binding molecule, e.g., antibody or
binding fragment, variant, or derivative thereof to be administered is readily determined by
one of ordinary skill in the art without undue experimentation given the disclosure of the
present invention. Factors influencing the mode of administration and the respective amount
of at least one anti-HER3 binding molecule, e. g., antibody, antigen-binding fragment, variant
or derivative thereof include, but are not limited to, the severity of the disease, the history of
the disease, and the age, height, weight, health, and physical condition of the individual
_ 69 _
WO 78191
undergoing therapy. Similarly, the amount of anti-HER3 binding molecule, e.g., antibody, or
fragment, variant, or tive thereof, to be administered will be dependent upon the mode
of administration and whether the subject will undergo a single dose or le doses of this
agent.
The present invention also provides for the use of an anti-HER3 binding
molecule, e. g., an dy or antigen-binding fragment, variant, or derivative thereof, in the
manufacture of a medicament for treating a type of cancer, including, e.g., colon cancer, lung
cancer, gastric cancer, head and neck squamous cells cancer, melanoma, pancreatic ,
prostate cancer, and breast cancer.
The invention also provides for the use of an anti-HER3 binding le,
e. g., antibody of the ion, or antigen-binding fragment, variant, or derivative f, in
the manufacture of a medicament for treating a subject for ng a type of cancer. In
certain aspects, the medicament is used in a subject that has been pretreated with at least one
other therapy. By "pretreated" or "pretreatmen " is intended the subject has received one or
more other therapies (e. g., been treated with at least one other anti-cancer therapy) prior to
ing the medicament comprising the anti-HER3 g molecule, e.g., antibody or
antigen-binding fragment, variant, or derivative thereof. It is not necessary that the subject
was a responder to pretreatment with the prior y or therapies. Thus, the subject that
receives the medicament comprising the anti-HER3 binding molecule, e. g., an antibody or
antigen—binding fragment, variant, or derivative f could have responded, or could have
failed to respond to pretreatment with the prior therapy, or to one or more of the prior
therapies where pretreatment comprised multiple therapies.
The ion also provides for the co-administration of an anti-HER3 binding
molecule, e. g., antibody of the invention, or antigen-binding fragment, variant, or derivative
thereof and at least one other therapy. The anti-HER3 antibody and the at least one other
therapy can be co-administered together in a single composition or can be co-administered
together at the same time or overlapping times in separate compositions.
The invention also provides for the use of an anti-HER3 binding molecule,
e. g., antibody of the invention, or antigen-binding fragment, variant, or derivative thereof, in
the manufacture of a ment for treating a subject for ng cancer, wherein the anti-
HER3 binding molecule is administered before a subject has been d with at least one
other therapy.
VIII. Diagnostics
The invention further provides a diagnostic method useful during diagnosis of
HER3 -expressing cell—mediated diseases such as certain types of cancer including, e.g., colon
cancer, lung cancer, gastric cancer, head and neck squamous cells cancer, melanoma,
pancreatic cancer, prostate cancer, and breast cancer, which involves measuring the
expression level of HER3 protein or transcript in tissue or other cells or body fluid from an
individual and ing the measured sion level with a standard HER3 expression
level in normal tissue or body fluid, whereby an increase in the expression level compared to
the standard is indicative of a disorder.
The anti-HER3 antibodies of the invention and antigen-binding fragments,
variants, and tives thereof, can be used to assay HER3 protein levels in a ical
sample using classical immunohistological methods known to those of skill in the art (e. g.,
see Jalkanen, et al., J. Cell. Biol. [01:976—985 (1985); Jalkanen et al., J. Cell Biol. 105:3087—
3096 (1987)). Other antibody—based methods useful for detecting HER3 protein expression
include assays, such as the enzyme linked immunosorbent assay (ELISA),
immunoprecipitation, or Western blotting. Suitable assays are described in more detail
elsewhere herein.
By "assaying the expression level of HER3 ptide" is intended
qualitatively or quantitatively measuring or estimating the level of HER3 polypeptide in a
first biological sample either directly (e.g., by determining or estimating absolute protein
level) or relatively (e.g., by comparing to the disease associated polypeptide level in a second
biological ). HER3 polypeptide expression level in the first biological sample can be
ed or estimated and compared to a standard HER3 polypeptide level, the standard
being taken from a second ical sample obtained from an individual not having the
disorder or being determined by averaging levels from a population of individuals not having
the disorder. As will be appreciated in the art, once the "standard" HER3 ptide level is
known, it can be used repeatedly as a standard for comparison.
The invention further provides a diagnostic method useful during diagnosis of
HER3—expressing cell—mediated diseases such as certain types of cancer ing, e.g., colon
cancer, lung cancer, gastric cancer, head and neck squamous cells cancer, melanoma,
pancreatic cancer, prostate , and breast cancer, which involves measuring the activity
level of HER3 n in tissue or other cells or body fluid from an individual and ing
the measured activity level with a standard HER3 activity level in normal tissue or body
fluid, whereby an increase in the activity level compared to the standard is indicative of a
disorder.
The invention further provides a diagnostic method useful during treatment of
HER3 -expressing cell—mediated diseases such as certain types of cancer including, e.g., colon
cancer, lung , gastric cancer, head and neck squamous cells cancer, melanoma,
pancreatic , prostate cancer, and breast cancer, which es measuring the activity
level of HER3 protein in tissue or other cells or body fluid from an individual during
treatment of a HER3-expressing cell-mediated disease and comparing the measured activity
level with a standard HER3 ty level in normal tissue or body fluid and/or comparing the
measured ty level with a standard HER3 ty level in tissue or body fluid obtained
from the dual prior to treatment, whereby a decrease in the activity level compared to
the standard is indicative of an inhibition of HER3 activity.
By "assaying the activity level of HER3 protein" is intended qualitatively or
quantitatively measuring or estimating the activity of HER3 protein in a first biological
sample either directly (e.g., by determining or estimating absolute activity level) or relatively
(e.g., by comparing to the ty level in a second biological sample). HER3 protein
activity level in the first biological sample can be measured or ted and compared to a
standard HER3 protein activity, the standard being taken from a second biological sample
obtained from an individual not having the disorder or being determined by averaging levels
from a population of individuals not having the disorder or from an individual prior to
treatment. As will be appreciated in the art, once the "standard" HER3 n ty level
is known, it can be used repeatedly as a standard for comparison. In certain aspects, the
activity level of HER3 in a biological sample is measured or estimated or compared by
detecting phosphorylated HER3 in a biological sample. In a specific aspect, the activity level
of HER3 in a ical sample is measured or estimated or compared by detecting
phosphorylated HER3 in a skin biopsy, wherein the skin is stimulated with HRG prior to or
after biopsy.
By "biological sample" is intended any ical sample obtained from an
individual, cell line, tissue culture, or other source of cells ially expressing HER3.
Methods for obtaining tissue biopsies and body fluids from mammals are well known in the
art.
In some aspects, the bioactivity of a HER3 inhibitor (e. g., anti-HER3 antibody
of the invention and n-binding fragments, variants and derivatives thereof)
WO 78191 2012/066038
administered to a t can be detected using an eX—vivo assay. In particular aspects the ex—
vivo assay comprises detecting the level of phosphorylated HER3 in a skin biopsy, wherein
the skin is stimulated with HRG prior to or after biopsy. In a specific aspect matched skin
biopsies are taken from a subject that has been administered the HER3 inhibitor. In a specific
aspect, HRG is ed under a first area of the skin and a control buffer is injected under a
second area of the skin of a t administered the HER3 inhibitor, wherein after a d
amount of time (e.g., 10-60 minutes) a biopsy is taken from the first and second areas of the
skin. In an alternative aspect, a first skin biopsy is treated with HRG and a second skin
biopsy is treated with a control buffer, n the first and the second biopsies are matched
skin es taken from a subject that has been administered the HER3 tor. In another
specif1c aspect, the level of phosphorylated HER3 is detected in the skin biopsies. In certain
aspects, the difference in the level of phosphorylated HER3 between the first (HRG treated)
and the second (control buffer treated) biopsy is ined. In certain aspects, the skin
biopsy is homogenized and the level of phosphorylated HER3 is detected by ELISA. In still
other s, the levels of phosphorylated HER3 in the skin biopsies from a subject that has
been administered the HER3 inhibitor is compared to the levels of phosphorylated HER3 in
skin biopsies from a control subject that has not been stered the HER3 inhibitor,
wherein a reduction in the level of phosphorylated HER3 in the skin biopsies of the subject
that has been administered the HER3 tor is a measure of the bioactivity of the HER3
inhibitor. In alternative aspects, the levels of phosphorylated HER3 in the skin biopsies from
a subject that has been administered the HER3 inhibitor is compared to the levels of
phosphorylated HER3 in skin biopsies from the same subject taken prior to the administration
of the HER3 inhibitor, wherein a reduction in the level of phosphorylated HER3 in the skin
biopsies of the subject after administration of the HER3 inhibitor is a measure of bioactivity
of the HER3 inhibitor. Other specific aspects of the methods are detailed in the Examples
section 5.15.
IX. Kits comprising HER3-binding Molecules
The present invention provides kits that comprise the HER3-binding molecule,
e. g., an anti-HER3 antibody or antigen binding fragment thereof of the invention described
herein and that can be used to perform the methods described herein. In certain aspects, a kit
comprises at least one purified anti-HER3 antibody or an n-binding fragment thereof in
one or more containers. In some aspects, the kits contain all of the components necessary
and/or sufficient to perform a detection assay, including all controls, directions for
performing assays, and any necessary software for analysis and tation of results. One
skilled in the art will readily recognize that the disclosed HER3—binding molecule, e.g., an
anti-HER3 antibody or antigen binding fragment thereof of the present invention can be
readily incorporated into one of the established kit s which are well known in the art.
X. Immunoassays
Anti-HER3 binding molecules, e.g., dies or antigen-binding fragments
thereof, variants, or derivatives thereof of the molecules of the invention can be assayed for
immunospecific binding by any method known in the art. The immunoassays that can be
used include but are not limited to competitive and non-competitive assay systems using
ques such as Western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation , precipitin
reactions, gel ion precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric , cent assays, protein
A immunoassays, to name but a few. Such assays are routine and well known in the art (see,
e. g., Ausubel et al., eds, (1994) Current ols in Molecular Biology (John Wiley & Sons,
Inc., NY) Vol. 1, which is incorporated by reference herein in its entirety).
HER3-binding molecules, e.g., anti-HER3 antibodies or antigen-binding
fragments thereof, variants, or derivatives thereof of the molecules of the invention, can be
employed histologically, as in immunofluorescence, immunoelectron microscopy or non—
immunological assays, for in situ detection of HER3 receptors or conserved variants or
peptide fragments thereof. In situ detection can be accomplished by removing a histological
specimen from a patient, and applying o a labeled HER3-binding molecule, e.g., an
anti-HER3 antibody or antigen-binding fragment thereof, variant, or derivative thereof,
ably applied by overlaying the labeled HER3-binding molecule (e.g., and antibody or
fragment) onto a biological sample. Through the use of such a procedure, it is possible to
determine not only the presence of HER3, or conserved variants or e fragments, but
also its distribution in the examined tissue. Using the present invention, those of ordinary
skill will readily perceive that any of a wide y of histological methods (such as ng
procedures) can be modified in order to achieve such in situ detection.
The binding activity of a given lot of HER3 -binding molecule, e. g., anti-HER3
dy or antigen-binding fragment thereof, variant, or derivative thereof can be
determined according to well—known methods. Those skilled in the art will be able to
ine operative and optimal assay conditions for each determination by employing
routine experimentation.
Methods and reagents suitable for determination of binding characteristics of
an isolated HER3-binding molecule, e.g., anti-HER3 antibody or n-binding fragment
thereof, variant, or an altered/mutant tive thereof, are known in the art and/or are
commercially available. Equipment and software designed for such kinetic analyses are
cially ble (e. g., BIAcore, BIAevaluation software,GE care; KinExa
Software, Sapidyne Instruments).
The practice of the present invention will employ, unless otherwise indicated,
tional techniques of cell biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and logy, which are within the skill of the art.
Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed.
(1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory
Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs
Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, s I and II; Gait, ed.
(1984) Oligonucleotide Synthesis; Mullis et al. US. Pat. No. 4,683,195; Hames and Higgins,
eds. (1984) Nucleic Acid Hybridization; Hames and s, eds. (1984) Transcription And
ation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized
Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular
Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos
eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory);
Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987)
Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir
and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John
Wiley and Sons, Baltimore, Md.).
General principles of antibody ering are set forth in Borrebaeck, ed.
(1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). l principles of protein
engineering are set forth in Rickwood et al., eds. (1995) Protein Engineering, A Practical
Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General ples of antibodies
and antibody-hapten binding are set forth in: Nisonoff (1984) Molecular logy (2nd
ed.; Sinauer Associates, land, Mass.); and Steward (1984) Antibodies, Their Structure
and Function (Chapman and Hall, New York, NY.) Additionally, standard methods in
immunology known in the art and not specifically described are generally followed as in
Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994)
Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell
and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co.,
NY).
Standard reference works setting forth general ples of immunology
include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J
Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY);
Kennett et al., eds. (1980) onal Antibodies, Hybridoma: A New Dimension in
Biological Analyses m Press, NY); Campbell (1984) "Monoclonal Antibody
Technology" in Laboratory Techniques in mistry and Molecular y, ed. Burden
et al., (Elsevere, dam); Goldsby et al., eds. (2000) Kuby Immunnology (4th ed.; H.
nd & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al.
(2005) ar and Molecular Immunology (5th ed.; ElseVier Health Sciences Division);
Kontermann and Dubel (2001) Antibody Engineering ger Verlan); Sambrook and
Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin
(2003) Genes VIII (Prentice Hall2003); Harlow and Lane (1988) Antibodies: A Laboratory
Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold
Spring Harbor Press).
All of the references cited above, as well as all references cited herein, are
incorporated herein by reference in their ties.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
Aspects of the present disclosure can be r d by reference to the
following miting examples, which describe in detail preparation of certain antibodies of
the present disclosure and methods for using antibodies of the present disclosure. It will be
apparent to those skilled in the art that many modifications, both to materials and methods,
can be practiced without departing from the scope of the present disclosure.
—76—
Example 1. Methods for Isolation/Optimization of anti-HER3 Monoclonal
Antibodies
1.1. ns and Cell Lines
Recombinant human Herl(ECD)/Fc chimera, human HER2(ECD)/Fc chimera,
human CD)/Fc chimera and human Her4(ECD)/Fc were all purchased from R&D
Systems (Minneapolis, MN) and were fused to the C—terminal 6X Histidine—tag via a linker
peptide. Recombinant mouse HER3 (ECD)/Fc chimera was generated in house. Human KPL—
4 breast cancer cells were cultured in DMEM supplemented with 5% fetal bovine serum
(FBS).
1.2. Library ion of HER3 Binders — fication of Clone 16 Antibody
(CL16)
The unlabeled and biotinylated HER3(ECD)/Fc were used as the targets for
selection of HER3 binders from Dyax's Fab 310 human Fab phage display library (Dyax,
Cambridge, MA). Two arms of panning were carried out: captured panning and in solution
panning. For the captured panning, input phage were first incubated with polyclonal human
lgG captured on immunotubes via immobilized inant Protein A/G, and then ed
with unlabeled target captured on immunotubes via immobilized recombinant Protein A/G.
In the in solution g, input phage were allowed to incubate with
onal human lgG, streptavidin-coated magnetic beads with quenched biotin for
deselection and then selected with biotinylated target with subsequent incubation with
streptavidin-coated magnetic beads to capture phage bound to the target. After removal of
unbound phage by g ively with TPBS (1X PBS/0.1% Tween—20), the bound
phage were eluted with lOOmM TEA (triethylamine). Eluted phage and the remaining phage
on beads from in solution panning were subsequently amplified, and subjected to further
rounds of selection. Three rounds of selection were carried out for each arm of selection.
The percentage of positive binding phages ranged from less than 1% using
capture panning up to 68% using three rounds of in solution panning (TABLE 3).
TABLE 3: ing of HER3 binders.
Total clones ed
Positive clones
Positive rate (%)
1.3. Screening for Human and Mouse HER3 Binders by Phage ELISA
Phage enriched from the second and the third rounds of selection were
screened by phage ELISA for human and mouse HER3 binding. 96-well half area plates were
coated with Sug/ml, 50ul per well of different ns d in 1x PBS, pH 7.4 ght
at 4°C. The coated plates were blocked with 3% (w/v) non—fat milk in TPBS for 1 hour at
room temperature, and washed two times with TPBS. The plates were then incubated for l h
with overnight phage atant. After washing ten times with TPBS, the plates were
incubated with horseradish peroxidase (HRP)—conjugated anti-M13 antibody for 1 hour, and
washed ten times. Plates were developed with tetramethylbenzidine (TMB) peroxidase
substrate solution, the reactions were stopped with 0.18M of HZSO4, and plates were read at
450 nm on an ELISA plate reader.
29 unique positive binders were identified that were cross reactive to murine
HER3 (as a HER3—Fe fusion). None of the identified binders showed cross reactivity to
HER2 or Her4 (data not shown).
1.4. atting of Fabs into Whole IgG Antibodies and Expression
The immunoglobulin variable light chain (VL) and variable heavy chain (VH)
from positive phage clones were generated by PCR and inserted into a human IgG1
expression vector containing the lambda light chain constant region and the CHl-hinge-CH2-
CH3 IgGl region. To express IgG1 antibodies, human embryonic kidney 293—F cells were
transiently transfected with the reformatted IgG s using 293fectinTM reagent
(Invitrogen, Carlsbad, CA). Conditioned media were harvested 10 days after transfection,
pooled, and sterile—filtered. IgGls were ed using protein A beads. The final eluted
IgGls were dialyzed against PBS, and IgG1 concentrations were determined by protein
quantitation assay.
Clone l6 (CLl6; SEQ ID NOs: l and 2, VL and VH amino acid sequences,
respectively) was reformatted to human IgG1
_ 78 _
1.5. Determination of Internalization of Clone 16 Antibody (CL16) by
fluorescence
Human breast cancer KPL-4 cells were labeled with Clone 16 dy
(CL16). Incubation of the cells with CL16 lead to an increase in HER3 endocytosis, which
prevented the receptor from forming active signaling complexes with HER2 at the cell
surface.
Cell surface attached CL16 antibodies were allowed to internalize by
incubating the cells under growth conditions for either zero (non-internalized) or 2.5 hours
nalized) (. All cells were then fixed with 3.7% paraformaldehyde, washed in
PBS, permeabilized with 0.5% Triton X-100 in PBS, and stained with l [Lle Alexa Fluor®
488 goat anti-human IgG (Invitrogen) prior to addition of antifade mounting media and
fluorescent microscopy examination. The CL16 antibody was found to internalize in KPL—4
cells. At time zero KPL-4 cells showed intense cell surface staining ( 0 hours, top
panel), after incubation under growth conditions for 2.5 hours the cell surface staining was
diminished and replaced by intracellular punctuate staining indicative of internalization ( 2.5 hours, bottom panels).
1.6. Construction of a Phage Vector Expressing Clone 16 Fab
DNA ng the n binding fragment (Fab) 0f the antibody Clone 16
was cloned into a modified, Ml3—based phage expression vector previously described by
Dall’Acqua et a1. (Dall’Acqua et al., 2005, Methods. 36:43—60). In this d vector, a
human lambda 0L) constant region DNA was engineered in place of the human kappa (K)
light chain. The expression of Fab fragment is under the control of the LacZ promoter and
secretion of the Fab fragment is enabled by the phage P3 signal ces fused to the N—
termini of either the VH and the VL chains of the Fab fragment. The cloning was carried out
by ization nesis as described by Kunkel (Kunkel, T. A., 1985, Proc. Natl. Acad.
Sci. USA; 82:488—492) and Wu (Wu, H., 2003, Methods M01. Bi01.207:l97—2l2).
Briefly, the variable regions of clone 16 IgG were amplified by polymerase
chain on (PCR). By hybridization followed by DNA polymerization reaction, the clone
16 variable light region was integrated in frame with the human lambda constant region, and
the variable heavy region was cloned in frame with the human heavy chain constant region 1
(CH1), respectively. The phage vector containing the Clone l6 Fab fragment was then grown
in Escherichia coli CJ236 strain to produce uridine (U) ning single stranded DNA
(ssDNA) as bed by Wu and An (Wu, H. and An, LL., 2003, Methods M01.
Biol.207:213-33). The uridine containing ssDNA was used as the template to introduce
designed mutations for improving binding affinity to HER3.
1.7. Germlining of Clone 16 (CL16)
ce analysis shows that the VH frameworks of Clone 16 (CL16) shares
100% sequence identity with VH germline gene 3-23 while VL frameworks differ at 6
positions from its closest germline gene 47*01. Site direct mutagenesis to change each and all
of the amino acids that differ from the germline gene 47*01 was med. 1cally, six
point mutations were uced into the light chain variable regions as follows: Y2S, E3V,
S18R, M21I, H3 8Q and S50Y where the first letter represents the one letter amino acid code
of the original Clone 16, the number represents the framework residue number (as per Kabat),
and the second letter ents the one letter amino acid code of the germline sequence. See
sequences in and , corresponding to the original VL CL16 and germlined
(GL) VL CL16, respectively. The resultant variants were expressed as Fab and their binding
to the recombinant HER3 protein was determined by ELISA.
The binding results showed that the H3 8Q amino acid mutation in ork
2 improved binding over the parental Clone 16 as measured by ELISA. In contrast, the S49Y
mutation in the same framework had negative impact on binding. Other point mutations
showed no impact on HER3 binding. The fully germlined mutant with all 6 non-germline
amino acids mutated showed a similar degree of reduced binding as the S50Y point mutation,
indicating that amino acid S50 participates in binding. Further testing of the clone with all the
germline point mutations except S50Y retained and/or increased binding to HER3 comparing
to the parental clone 16. This partially germlined clone, Clone 16 (GL) (also referred to here
as “GL—P6”), was used as the template for further affinity optimization.
1.8. Affinity zation of Clone 16 (CL16)
Each amino acid of all 6 complementary-determining regions (CDRs) of
germlined clone GL-P6 was individually d to other 20 amino acids using a
hybridization nesis method (Kunkel, 1985). Two sets of DNA primers, one containing
a NSS codon encoding 8 amino acids and the other containing a NWS codon encoding 12
different amino acids, were used to introduce mutations to each targeted CDR position. The
individual degenerate primers were used in hybridization mutagenesis reactions. Briefly, each
degenerate primer was orylated, then used in a 10:1 ratio with the uridinylated GL-16
_ 80 _
Fab ssDNA. The e was heated to 95°C then cooled down to 55°C over 1 hour.
Thereafter, T4 ligase and T7 DNA polymerase were added and the mix was incubated for 1.5
hours at 37°C. Synthesis products for VH and VL CDRs were pooled respectively; r,
NSS and NWS libraries were kept separate and screened independently. Typically, 1 [LL of
the pooled library DNA was electroporated into XLl-Blue for plaque formation on XLl-Blue
bacterial lawn or for production of Fab fragments (Wu and An, 2003).
1.9. Primary Screening of the Fab Library
The primary screen ted of a single point ELISA (SPE) assay which was
carried out using culture supernatant of bacteria grown in 96-well plates (deep well) and
infected with individual recombinant M13 clones as described elsewhere (Wu and An, 2003).
, this capture ELISA involved coating individual wells of a 96-well Maxisorp
plate with approximately 50 ng of a sheep anti-human Fd antibody (Biodesign
International, ME) in a ate buffer at pH 8.5 overnight at 4°C. The next day, the plate
was d with 3% BSA in PBS buffer for l h at room temperature. Fab supernatant was
then added to the plate and incubated at room temperature for 1 hr. After washing, 0.1 ug of
biotinylated HER3 protein was added to the well and the mixture was ted for 1.5 h at
room temperature. This was followed by incubation with neutravidin-horseradish dase
(HRP) conjugate (Pierce, IL) for approximately 40 min at room temperature. HRP activity
was detected with tetra-methyl- benzidine (TMB) substrate and the reaction quenched with
0.2 M H2SO4. Plates were read at 450 nm.
Clones ting an optical density (OD) signal at 450 nm greater than the
parental clone GL—P6 Fab were picked and regrown (15 mL) (Wu and An, 2003) and re—
assayed by ELISA (as described above) in duplicate to confirm positive results. Clones that
repeatedly ted a signal greater than that of the GL-P6 Fab were sequenced. The Fab
protein concentration of each clone that had a CDR change was then determined by a
quantitative Fab ELISA, where a Fab with known concentration was used as a reference. The
Fab concentration was determined by comparing the ELISA signals with the signals
generated by the reference Fab. The binding assay was repeated once more for all positive
variants under normalized Fab concentrations in order to determine the relative binding
ty of the mutant Fabs and the parental GL-P6 Fab.
The binding ELISA showed that two VH variants, designated clone l4C7 and
clone 15Dl2, which contained the Y501 or Y50V point mutations, respectively, in CDR2
displayed approximately a 5-fold improvement in HER3 binding over the parental, ned
clone GL-P6. In the VL mutagenesis campaign, l single mutations either in CDRl, e.g.,
clone 4H6 (comprising the SZ4R point mutation), clone 6E3 (comprising the SZ7L point
on) or in CDR3, e.g., clone 5H6 (comprising the S94G point mutation), clone 8A3
(comprising the S96a1 point mutation), clone 4C4 (comprising the S96aR point mutation),
clone 2Bll (comprising the S96aP point mutation) and clone 2Dl (comprising the V97A
mutation) displaying improved binding were identified.
Most notably, the substitution of amino acid S96a of VL-CDR3 with either
Isoleucine (I), Arginine (R) or Proline (P) resulted in a 3.5-fold, 8.6—fold and 32-fold binding
improvement, respectively.
1.10. Combinatorial ing of the Fab Library
The point mutations in VH and VL determined to be beneficial for binding to
HER3 were further combined to gain additional binding synergy. The combinatorial mutants
were expressed as Fab and ed using the HER3 binding ELISA. While combining either
one of the YSOI or Y50V point mutation in the VH chain of the Fab fragment with the VL
mutations appeared to have no beneficial but reduced g to HER3, combining several
VL mutations further improved binding. These combination of VL mutations include the
combinations in clone lA4 (comprising the L96P, S97P and VlOOA point mutations), clone
2C2 (comprising the SZ6L, L96P, S97P and VlOOA point mutations), clone 2F10
(comprising the S97P and VlOOA mutations) and clone 3E1 (comprising the SZ3R, L96P,
S97P and VlOOA point mutations).
1.11. Conversion of the Affinity-optimized Fab Variants to IgG Format and
Antibody Expression of
Singe mutant and compbination mutant variants displaying improved binding
were ted into IgG format for further characterization. The variable regions of each
variant were amplified by PCR using primers that encoded restriction sites to facilitate
cloning into an IgG mammalian expression vector for expression using HEK 293F cells. The
secreted, e human IgG1 proteins were purified from the conditioned media directly on
lmL HiTrap n A columns (GE Healthcare, NJ) according to the manufacturer’s
instructions. Purified human IgG1 s (typically > 95% homogeneity, as judged by
sodium l sulphate—polyacrylamine gel electrophoresis) were ed against PBS,
flash frozen, and stored at —70°C.
_ 82 _
Binding of the purified IgGs was ed using a HER3 binding ELISA.
The combination mutant IgGs showed improved g as determined by the total binding
signal, with 2C2 showing the most significant binding improvement over the parental Clone
16 and other combination mutant variants. Binding of the IgGs to murine and cynomolgus
HER3 were also tested by ELISA. The results showed improved binding of the combination
mutants to these paralogous HER3 species.
Alignment of the amino acid sequences of the light and heavy chain variable
s for each of the identified single mutations is shown in and ,
respectively. TABLE 4 provides the SEQ ID NOs for each clone. An ent of the light
chain variable regions for each of the combination clones is provided in .
TABLE 4
SEQ ID DESCRIPTION SEQ ID DESCRIPTION
17 Clone 16 VL aa 21 Clone 4H6 VL CDR2 aa
1 Clone 16-germlined VL aa 22 Clone 4H6 VL CDR3 aa
2 Clone l6 VH aa 7 Clone 6E.3 VL aa
18 Clone 16 VL CDRl aa 19 Clone 6E.3 VL CDRl aa
21 Clone 16 VL CDR2 aa 21 Clone 6E.3 VL CDR2 aa
22 Clone 16 VL CDR3 aa 22 Clone 6E.3 VL CDR3 aa
31 Clone 16 VH CDRl aa 9 Clone 2D1 VL aa
32 Clone 16 vH CDR2 aa 18 Clone 2D1 v4 CDRl aa
Clone 16 vH CDR3 aa 21 Clone 2D1 v4 CDR2 aa
8 Clone 2B11 VL aa 28 Clone 2D1 V4 CDR3 aa
18 Clone 2B11 VL CDRl aa 10 Clone 3A6 VL aa
21 Clone 2B11 VL CDR2 aa 18 Clone 3A6 V4 CDRl aa
Clone 2B11 VL CDR3 aa 21 Clone 3A6 V4 CDR2 aa
14 Clone 1A4 VL aa 29 Clone 3A6 V4 CDR3 aa
18 Clone 1A4 VL CDRl aa 11 Clone 4C4 VL aa
21 Clone 1A4 VL CDR2 aa 18 Clone 4C4 V4 CDRl aa
22 Clone 1A4 VL CDR3 aa 21 Clone 4C4 V4 CDR2 aa
3 Clone 2C2 VL aa 30 Clone 4C4 Vs CDR3 aa
19 Clone 2C2 VL CDRl aa 12 Clone 15Dl2.1 VH aa
21 Clone 2C2 VL CDR2 aa 31 Clone 15Dl2.1 VH CDRl aa
23 Clone 2C2 VL CDR3 aa 33 Clone 15Dl2.1 VH CDR2 aa
16 Clone 2F10 VL aa 35 Clone 15Dl2.1 VH CDR3 aa
18 Clone 2F10 VL CDRl aa 13 Clone 15Dl2.2 VH aa
21 Clone 2F10 VL CDR2 aa 31 Clone 15Dl2.2 VH CDRl aa
24 Clone 2F10 VL CDR3 aa 34 Clone 2 VH CDR2 aa
Clone 3E.1 VL aa 35 Clone 15Dl2.2 VH CDR3 aa
Clone 3E.l VL CDRl aa 36 VH FW1 33
21 Clone 3E.l VL CDR2 aa 37 VH FWZ 33
23 Clone 3E.l VL CDR3 aa 38 VH FW3 33
4 Clone 5H6 VL aa 39 VH FW4 aa
18 Clone 5H6 vL CDRl aa 40 VL FWl germlined aa
21 Clone 5H6 VL CDRZ aa 41 VL FWZ aa
26 Clone 5H6 vL CDR3 aa 42 VL FW3 aa
Clone 8A3 VL aa 43 VL FW4 aa
18 Clone 8A3 vL CDRl aa 44 VL FWl original aa
21 Clone 8A3 VL CDRZ aa 45 IgGl constant *
27 Clone 8A3 VL CDR3 aa 46 IgGl constant region* - YTE
6 Clone 4H6 VL aa 47 Clone 16 VL nt
Clone 4H6 VL CDRl aa 48 Clone 16 VH nt
* allotype differences
provided
VL aa consensus: [FWl ] XlGSXZSNIGLNYVS [FWZ] RNNQRPS [FW3] AAWDDX3X4X5GEX6 [FW4]
wherein [FWl], [FWZ], [FW3] and [FW4] represent V4 framework
regions,
wherein
(a) X1 ents amino acid residues ne (R) or Serine (S),
(b) X2 represents amino acid residues Serine (S) or Leucine (L),
(c) X3 re presents amino acid residues Serine (S) or e (G),
(d) X4 ents amino acid residues Leucine (L) or e (P),
( e) Xgrepresents amino acid residues Arginine (R), Isoleucine
( I), Proline (P) or Serine (S), and
( f) X6 represents amino acid residues Valine (V) or Alanine (A).
VH aa consensus: [FW5] YYYMQ [Fl/06] GGVTNYADSVKG [FW7 ] VGLGDAFDI [FWg]
wherein [FWS], [FW6] ,[FW7] and [FW8] represent VH framework
regions,
wherein X7 represents amino acid residues Tyrosine (Y), Isoleucine
(I) or Valine (V)
1.12. Anti-HER3 Monoclonal Antibody Binding Studies
The kinetic rate (km, koff) and equilibrium dissociation constants (KD) for the
binding of the anti-HER3 IgGs to the extracellular domain of human HER3 protein were
determined using BIAcoreTM surface plasmon resonance technology by measuring the
binding of human HER3 extracellular domain(hu HER3(ECD)) to IgG captured onto a sensor
chip surface. Individual association (km) and iation (koff) rate nts were then
calculated from the resulting binding curves using the BIAevaluation software available
through the vendor. Data were fit to a 1:1 binding model, which included a term to correct for
mass transport limited binding, should it be detected. From these rate constants, the apparent
dissociation binding constant (KD) for the interaction of IgG with the human HER3
extracellular domain protein is then calculated from the quotient of koff/kon.
From high-resolution BIAcore plots, the association and dissociation rate
constants for the binding parental IgG, Clone16, to human HER3 extracellular domain were
.29 X 105 /Ms and 73.0 X 10'4 /s, respectively, yielding an apparent KD of 14 nM. In
comparison, the association rate nts for the binding of the affinity-improved IgG
ts to human HER3 extracellular domain were similar to those measured for the parental
IgG, ranging from 3.4l><105 to 4.32><105 /Ms. These same plots were also used to determine
the ponding dissociation rate constants for the Clone l6 variants, which ranged from
'4 to 6.2l><10'4 /s. The apparent KDs for the Clone l6 variants were calculated as
described above, and ranged from 0.429 nM (2C2 clone t) to 1.44 nM. (P2Bll clone
variant). Individual errors for k0n and koff were low (S~2% of the calculated parameter), and
the overall fits to the kinetic data indicated that the use of the 1:1 interaction model was
appropriate. Also, the evaluation did not indicate the binding was mass transport-limited.
TABLE 5 summarizes the biophysical attributes of the combination
monoclonal clones provided in , including Km, Koff and KD values, as well as
sion levels and yields.
The 2C2 monoclonal dy, comprising the 2C2 VL (SEQ ID NO: 3) and
the original C16 VH (SEQ ID NO: 2) was the most affinity-improved lead with a KD of
0.4nM, representing a 32-fold improvement from the parental Clone l6 monoclonal antibody.
The KD improvement was mostly a result of decreased off—rate. The expression level and
production yields were also assessed. All of the monoclonal antibody clones were well
expressed in a 5 day transient transfection study, with the 2C2 monoclonal antibody showing
the highest level of expression in this study. All affinity optimized leads showed different
extents of affinity improvement but the 1A4 antibody d out due to lower expression
efficiency.
—85—
TABLE 5: Summary of biophysical properties of the various affinity-optimized
leads in comparison with the parental CL16 (Clone 16) antibody.
Biacore Biacore sion
Kon Yield
Clone Calculat Koff (1/5) KD (nM, KD (nM, Level on
(HMS) (mg/volum
name ed pl (XE-4) lgG Her3 Day 5
(xE+5} e ml)
down) down) (transient) '
0.74
P2311 8.21 4.32 8.21 1.53 (9x) 159 ug/ml 70/500
(2 4x)
0.838 0.493
1A4 8.2 3.41 2.86 60 ug/ml 53/1200
mm) (3-6)”
0.434 0.093
2C2 8.2 3.73 1.60 148 ug/ml 71/600
(32X) (19X)
0.818 0.326
2F10 8.2 3.54 2.90 130 ug/ml 66/600
(17x) (5x)
0.52 0.286
3E 1 8.32 3.43 1.78 125 ug/ml 59/600
(26x) 032x}
Clone 16 7.83 5.29 73.0 14 1.77 ND ND
Note: Each affinity-optimized lead ses the clone name VL chain and the
original C 16 VH
Various cell—based assays were performed to assess the functional
improvement of the various affinity optimized leads over clone 16 across ligand—independent
(human breast cancer cell line BT-474, ATCC No. HTB-ZOTM) as well as ligand-dependent
(human breast cancer cell line T-47D, ATCC No.HTB—133) models (both cell lines obtained
from ATCC), including inhibition of HER3 signaling pathway (pHER3 and pAKT),
suppression of cell growth (short—term 6—day growth assay and long—term clonogenic ,
and abrogation of HRG-induced pHER3 in T-47D cells (T-47 differentiated epithelial
substrain).
enic assays were performed as follows. BT—474 cells were plated at a
density of 1,000 well into 6-well plates. After ght attachment, cells were treated
with isotype control IgG or the indicated HER3 monoclonal antibodies following a
concentration dose curve. The medium with the proper doses of monoclonal antibodies was
refreshed once a week for three weeks. At the end of day 21, cells were processed for Cell—
titer-Glo (CTG) assay to assess the inhibition of colony formation by the various monoclonal
dies (using control IgG as base-line). 1C50 values were derived from Prizm analysis.
The BT—474 6-day growth assay was performed essentially as used for
(see Section 2.2 in Example 2, infra). The BT-474 pAKT assay was performed essentially as
used for (see Section 2.6.1 in Example 2, infra). The T—47D HRG inducible pHER3
assay was performed essentially as used for (see Section 2.1 in Example 2, infra), and
the T-47D FACS binding and internalization assay was performed using the same protocol
used for A (see Section 3.3.1 in Example 3, infra).
The IC50 values and maximal inhibition levels were compiled for comparison
es. As shown in TABLE 6, the affinity improved leads displayed a consistent 2fold
increased y across most of the assays. The parental Clone 16 and/or a representative
zed clone, e.g., Clone 2C2 antibody (also referred to simply as 2C2, or 2C2
monoclonal antibody) were further characterized in a number of in vitro and in viva assays as
described below.
In addition, mutations were uced into the Fc region of the optimized
clone 2C2 to extend half-life. Specifically, M252Y, S254T, T256E, numbered according to
the EU index as in Kabat. This half-life-optimized molecule is referred to as 2C2-YTE. It
will be understood that other mutations could be introduced instead of, or in combination
with these three, see, e.g., US. Patent No. 784; International Appl. Pub. No.
WO2009058492; Dall’Acqua et al., 2002 J. Immunol. 169:5171—80; Zalevsky et al., 2010,
Nature Biotech. 28:157-9). 2C2-YTE was show to inhibit BT-474 cell proliferation and
colony growth to the same extent as 2C2 (data not shown).
A refrigerator (2-8 oC) stable composition was obtained by ating the
antibodies (e.g., 50 mg/ml) in 25 mM ine/histine HCL, 205 mM sucrose, 0.02%
polysorbate 80 at pH 6.0.
TABLE 6: Summary of the ical properties of the affinity optimized leads in
comparison with al CL16 monoclonal antibody.
—87—
BT474 T47D HRG
clonogenic BT474 6-day BT474 pAKT inducible T47D FACS
asssay growth assay pHer3 binding
"/0 Max lnflection % Max tio % Max % Max
Clone IC50 inhibitio point inhibitio n point inhibiti IC50 inhibiti Max
name (pM) n (pM) n (pM) on (pM) on Kd (pM) GMFI
P2311 26.9 87.1 98 47.5 23.6 62 79.8 85 199 1441
1A4 30.7 81.3 133.3 54.5 28.5 62 133 84 281 1577
202 31.9 87.2 62.7 48.3 42.6 61 130.3 85 316 1583
2F10 31.2 80.4 66.7 49 46.4 62 127.2 86 306 1527
3E 1 20.8 79.2 85.3 48.1 26.2 66 59.2 86 447 1644
Clone 16-
PA 64.5 79.8 280 46 73.1 64 104.4 75 112 1055
Example 2. Characterization of Anti-HER3 Monoclonal Antibodies
2.1. HRG-induced HER3 Phosphorylation (pHER3) Assay in MCF-7 Cells
MCF-7 (ATCC No. HTB-22TM) is a human breast cancer cell line with HER3
expression but no endogenous HRG expression. MCF—7 cells were plated at a density of
,000 cells/well in a 96—well plate and were allowed to attach overnight. The cells were
then starved for 24 hours before treatment. Following serum-starvation, media was
removed and replaced with free media containing test and control antibodies, and the
cells incubated at 37°C for 1 hour. Test antibodies used in this example, and in the additional
examples provided below, include the anti—HER3 dies provided herein such as, Clone
16, 2C2, 2C2-YTE; and anti-HER3 antibodies known in the art, in particular U1-59
(International Patent Publication WO 2007077028) and Ab#6 (Patent ation WO
2008/100624) designated herein as AMG and MM, respectively. Meanwhile, heregulin
(HRGBl, R&D Systems, polis, MN) stock was prepared at 4x (80ng/ml) in serum-
free growth media. At the end of the 1 hour incubation period, HRGBl was spiked into wells
(20ng/ml final concentration) and incubated at 37°C for 20 minutes. At the end of treatment,
media was removed and cells were washed with PBS. Cells were lysed in 80ul Triton X lysis
buffer (Boston Bioproducts, Ashland, MA) with protease and phosphatase inhibitors
(Calbiochem, La Jolla, CA) and were stored at -20°C until analysis. pHER3 ELISA was then
performed following manufacturer’s ol (R&D Systems, 9) using half—volume
96—well Corning® Costar® 3690 ELISA plates (Corning Life Science, Lowell, MA) and Soul
of cell lysate per well.
HER3 activation, reflected by HER3 phosphorylation (abbreviated as pHER3),
was stimulated by cells treatment with HRGBl. Pre-treatment with anti-HER3 2C2 mAb
caused a dose—dependent suppression of the pHER3 signal in the pHER3 ELISA assay ( top). The published anti-HER3 monoclonal antibodies MM and AMG were also active in
this assay, r, 2C2 was approximately 5-fold more potent as determined by IC50
measurements ( bottom). Similar results were seen for 2C2-YTE (data not shown).
2.2. Growth suppression of MDA-MB-l75 breast cancer cells
MDA-MB-l75 (ATCC No. HTB-25TM) is an established HRG-expressing (y-
isoform) breast cancer cell-line that depends on R3 signaling pathway for growth
and survival. Cells were plated at a density of 2,000 cells/well in a 96—well walled plate
and were allowed to attach overnight. The following day, media was removed and replaced
with 100ul/well fresh complete growth medium containing test and l dies. Plates
were then incubated for a total of 6 days. To calculate relative cell number, CellTiter-GloTM
(Promega, Madison, WI) was used according to manufacturer’s protocol. After ter-
GloTM addition, plates were incubated at room temperature for 10 s and luminescence
was ed using a microplate .
The growth assay was carried out with 2C2, MM, or AMG anti-HER3
monoclonal antibodies. As shown in all three antibodies achieved anti-proliferation
effect to various extents, with 2C2 showing higher potency (IC50=0.l4ug/ml) ( top)
and higher growth suppression (72%) ( bottom).
2.3. Growth Suppression of HMCB Melanoma Cells
HMCB (ATCC No. CRL—9607TM) is an established HRG—expressing (lB-
isoform) melanoma model driven by HRG-induced HERZ-HER3 heterodimerization. HMCB
cells were plated at a density of 750 per well in 100 pl of complete medium containing 10%
heat-inactivated PBS in 96 well plates (Costar®). The next day, dy treatments were
prepared in complete . The starting concentration for all anti-HER3 monoclonal
antibodies and control IgG was 10 ug/ml, and serial dilutions were prepared in complete
. The plating medium was removed and treatments were added in 100 pl per well in
triplicates.
Plates were then incubated in 5% C02 at 37°C for 6 days. Equal volumes of
CellTiter—GloTM reagent were added to each well. Plates were rocked on a plate shaker for 10
minutes at room temperature to ensure complete cell lysis. Luminescence was measured
using a 2104 EnVision® Multilabel Reader (PerkinElmer, Waltham, MA).
As shown in 2C2 was again more potent than existing antibodies. 2C2
was 8—30 fold more potent than the published anti-HER3 onal antibodies AMG and
MM in inhibiting cell growth of the HMCB melanoma cell line.
2.4. HER3 and AKT Activity Assays in HMCB Melanoma Cells and A549
NSCLC Cells
The ability of the HER3 leads to suppress the HER3 signaling pathway in the
HRG—autocrine HMCB (ATCC No. CRL—9607TM) and A549 (ATCC No.CCL—185) models
were ed. HMCB cells were plated at 105 per well in 24—well plates and in medium
containing 10% heat—inactivated FBS and allowed to reach a confluency of 80% or more
prior to antibody treatment. The plating medium was removed and the cells were subjected to
incubation with the antibodies. ER3 monoclonal antibodies and a control IgG were
ed in te . The starting tration for all anti-HER3 antibodies was
l and serial dilutions were performed. The control IgG was only used at a
concentration of 10ug/ml. Treatments were applied following removal of plating medium.
After an incubation of 6 hours (HMCB cells) or 72 hours (A549 cells) in 5% C02 at 37°C,
cells were washed once with ice—cold PBS and then lysed by adding Laemmli Reducing
buffer (Boston BioProducts, Ashland, MA).
After a brief incubation, cell lysates were collected, equal amounts were
loaded onto Bis NuPAGE® Novex® Bis-Tris gels (Invitrogen, Carlsbad, CA) and ns
erred to PVDF membranes rogen, Carlsbad, CA). Membranes were blocked with
% nonfat dry milk and 0.1% Tween 20 (Sigma, St. Louis, MO) in TBS (pH 7.4) and
incubated overnight at 4°C with antibodies to HER3 (sc-285 antibody, Cell Signaling
Technology, Beverly, MA), pHER3 (4791 antibody, Cell Signaling Technology, Beverly,
MA), AKT (9272 antibody, Cell Signaling, Technology, y, MA), pAKT (4060
antibody, Cell Signaling Technology, Beverly, MA), neuregulin-l/HRG (NRGl/HRG)
antibody (sc—348, Santa Cruz) and GAPDH (G8795 antibody, Sigma, St. Louis, MO).
Membranes were washed in 0.1% Tween 20 in TBS and then incubated for 1
hour in horseradish peroxidase—conjugated streptaVidin secondary antibodies (GE
Healthcare). After washing, protein bands were detected in X-ray film by using SuperSignal®
West Femto Chemiluminescent Substrate and SuperSignal® West Pico Chemiluminescent
Substrate (Pierce/Thermo Scientific, Rockford, IL).
As shown in FIGS. 6 and 7, the 2C2 dy abrogated the HER3 signaling
pathway in both HMCB and A549 cells. 2C2 efficiently suppressed pHER3 and its
downstream effector molecule pAKT in a dose-dependent manner and was more potent than
either of the published anti-HER3 onal antibodies AMG or the MM in HMCB cells.
The 2C2 antibody also surpressed pHER3 and its downstream effector molecule pAKT in
A549 cells.
2.5. Assay for HER3 Phosphorylation (pHER3) in Cell Models for Lung,
Gastric, and Breast Cancer
2.5.1. pHER3 Cell Assay
Cells (HCC827 NSCLC cells, Gefltinib—resistant HCC827 NSCLC cells,
MKN45 gastric cancer cells, Kato III gastric cancer cells, or BT-474 mplif1ed breast
cancer cells) were plated at a density of 30,000 cells/well in a 96—well plate and were d
to attach overnight. The cells were then treated with test or control antibodies at the indicated
dose—curve at 37°C for 4 hours. At the end of treatment, media was d and cells were
washed with PBS. Cells were lysed in 80ul Triton X lysis buffer (Boston ducts,
Ashland, MA) with protease and phosphatase inhibitors (Calbiochem, La Jolla, CA) and were
stored at -20°C until analysis. pHER3 ELISA was then performed following manufacturer’s
protocol (R&D Systems, DYC1769, polis, MN) using half—volume 96—well ELISA
plates (Costar 3690) and 50ul of cell lysate per well.
2.5.2. Suppression of pHER3 Activity in HCC827 Cells
HCC827 cells (ATCC CRL—2868TM), a mutant EGFR-driven non-small cell
lung cancer (NSCLC) model, were treated with test or control dies as described above
in Example section 2.5.1 (see above). As shown in , the 2C2 antibody was able to
partially inhibit pHER3 signal, whereas the published anti-HER3 monoclonal antibodies
AMG and MM were less ive and 10-fold less potent than 2C2.
2.5.3. Suppression of pHER3 ty in Gefitinib-Resistant HCC827 Cells
HCC827 harbors and is driven by mutant-EGFR, which makes it highly
sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as gef1tinib. Parental HCC827 cells
were exposed to a constant toxic dose of nib and resistant clones were isolated that were
shown to harbor amplified cMET, a known mechanism for s to escape TKI therapy.
TKI-resistant HCC827 cells were treated with the anti-HER3 monoclonal antibodies as
described above in es section 2.5.1 (see above). As shown in , the anti—HER3
monoclonal antibody 2C2 suppressed HER3 activity in the mutant HCC827 made resistant to
nib. Similar to the results seen for the parental cell line, 2C2 displayed higher potency
than the AMG and MM antibodies (about 10-fold better potency) in the TKI-resistant
HCC827 cell line.
2.5.4. Suppression of pHER3 Activity in MKN45 Cells
Even though cMET is not a member of the Her-family, it has been shown to
be capable of forming dimers with HER3. The MKN45 cMET-amplified gastric cancer
model cell line was used to assess whether anti—HER3 antibodies could antagonize cMET-
driven HER3 tion. MKN45 cells were d with the anti-HER3 monoclonal
antibodies as described above in Examples n 2.5.1. As shown in , all three anti—
HER3 monoclonal antibodies (2C2, AMG and MM) were able to ss pHER3 in
MKN45 cells, but 2C2 displayed higher potency than the AMG and MM antibodies
(approximately 5—7-fold better potency).
2.5.5. Suppression of pHER3 Activity in Kato 111 Cells
Besides coupling with EGFR, HER2 and cMET, HER3 dimerizes with FGFR2
to facilitate its transforming potential. The Kato III (ATCC No. HTB-103TM) cell line, a
FGFR2-amplifed gastric cancer model, was used to assess whether anti—HER3 antibodies
could suppress driven HER3 activation. Kato III cells were treated with the anti—
HER3 monoclonal antibodies as described above in Examples section 2.5.1 (see above). In
this model, all three anti-HER3 onal antibodies (2C2, AMG, and MM) achieved
similar maximal extents of pHER3 suppression (~60%), but as measured by IC50, 2C2 was
15fold more potent than the AMG and MM antibodies, respectively ().
2.5.6. Suppression of pHER3 Activity in BT-474 Cells
HER2—HER3 dimers have been shown to be one of the most transforming
oncogenic entities in cancer. Accordingly, we investigated the anti-HER3 monoclonal
antibodies in the BT-474 cell-line (ATCC NO. HTB-ZOTM), a well-established HER2-
amplif1ed breast cancer model that does not s the ligand and is expected to be driven by
WO 78191
ligand-independent HERZ-HER3 zation. BT-474 cells were treated with the three anti-
HER3 monoclonal antibodies and also 2C2—YTE, as described above in Examples n
2.5.1. Unlike the models where all three anti-HER3 onal antibodies tested were active,
such as HCC827 cells, Gefitinib—resistant HCC827 cells, MKN45 cells, and Kato 111 cells,
2C2 (both parent 2C2 and E ) was the only one among the anti-HER3
monoclonal antibodies tested showing substantial activity suppressing pHER3 ().
These results ted that 2C2 (both parent 2C2 form and 2C2-YTE mutant) was functional
in a ligand-independent model and demonstrated the bi-functionality of 2C2 in both ligand-
dependent and ligand-independent settings.
2.6. Assay for AKT Phosphorylation (pAKT) in Cell Models for Gastric, and
Breast Cancer
2.6.1. pAKT Cell Assay
Cells (MKN45 gastric cancer cells, Kato III gastric cancer cells, or BT-474
HERZ-amplified breast cancer cells) were plated at a density of 30,000 cells/well in 96—well
plates and were allowed to attach overnight. The cells were then treated with test or control
antibodies at the indicated dose—curve at 37°C for 4 hours. At the end of treatment, media was
removed and cells were washed with PBS. Cells were lysed in 80rd of Triton X lysis buffer
(Boston BioProducts, Ashland, MA) with protease and phosphatase inhibitors (Calbiochem,
La Jolla, CA), and were stored at -20°C until analysis. AKT/pAKT were analyzed based on
the manufacturer’s protocol included in the Phospho (Ser473)/Total AKT Whole Cell Lysate
Kit (Cat. No. K15100D, cale Discovery, Gaithersburg, MD) to determine pAKT
content.
2.6.2. ssion of pAKT Activity in MKN45 Cells
To ascertain if 2C2 could suppress HER3 downstream signaling pathway in
addition to pHER3, we additionally assessed its ability to suppress AKT phosphorylation in
the amplified cMET-driven gastric cancer model MKN45. MKN45 cells were treated with
anti-HER3 monoclonal antibodies as described above in Examples section 2.6.1. In this
model system, the 2C2 monoclonal antibody ed l pAKT inhibition with higher
potency (approximately 5fold higher) than the AMG, and MM anti-HER3 monoclonal
antibodies (). This demonstrated that 2C2 not only inhibits HER3 activity but also
suppresses downstream effector molecules of HER3 such as pAKT.
2.6.3. Suppression of pAKT Activity in Kato 111 Cells
To investigate whether this activity ated into a better potency
suppressing pAKT, the effector of HER3, we analyzed pAKT inhibition by various anti-
HER3 monoclonal antibodies using in this ine a Meso—Scale Discovery assay as
described above in Examples section 2.6.1. As shown in , consistent with the pHER3
data, 2C2 suppressed pAKT in amplified FGFR2-driven gastric cancer model Kato 111 cells.
2C2 again achieved higher y (as measured by lC50) and maximal response in pAKT
inhibition than the AMG and MM antibodies.
2.6.4. Suppression of pAKT Activity in BT-474 Cells
HER2—HER3 dimers have been shown to be one of the most transforming
oncogenic entities in cancer. ingly, we investigated the activity of anti-HER3
monoclonal antibodies in the BT-474 cell-line. BT-474 cells were treated with the anti-HER3
monoclonal antibodies as described above in Examples section 2.6.1, supra, and also with the
YTE mutant form of 2C2. Unlike the models where all three anti-HER3 onal
antibodies tested (2C2, AMG and MM) were active, such as MKN45 and Katolll cells, 2C2
(both parent 2C2 form and 2C2-YTE mutant) was the only one among the anti-HER3
monoclonal antibodies tested that showed substantial activity suppressing pAKT ().
These s indicated that 2C2 (both parent 2C2 form and 2C2-YTE mutant) was onal
in a -independent model and demonstrated the bi-functionality of 2C2 (both parent 2C2
form and 2C2-YTE mutant) in both ligand-dependent and ligand-independent settings.
2.7. Suppression of HER3 Signaling and Cell Proliferation in MDA-MB-361
Cells.
To characterize the activity of 2C2-YTE in HER2-amplif1ed breast cancer
cells that are not highly responsive to trastuzumab, we focused on MDA—MB—361 (ATCC
No.HTB-27), a breast cancer model that harbors the activating mutation in PIK3CA (E545K),
which may contribute to its resistance to trastuzumab due to intrinsic activation of the PI3K
y (Junttila et al, 2009, Cancer Cell. 15:429—40). We determined the effects of 2C2 on
HER3 signaling and cell proliferation in this model.
To assess signaling the human breast cell line MDA-MB-36l was plated in
24-well plates at a density of 150,000 cells per well in RPMI (lnvitrogen) supplemented with
% heat-inactivated fetal bovine serum (FBS) (lnvitrogen). The next day, the plating
medium was removed and cells were ted to incubation with the anti-HER3 antibody
_ 94 _
2C2 or a l antibody, in complete medium at a final concentration of 30 ug/mL.. After
an incubation of 6 hours in 5% C02 at 37°C, cells were washed once with ice—cold PBS and
then lysed by adding Laemmli Reducing buffer (Boston ducts, Ashland, MA). After a
brief incubation, cell s were collected, equal amounts were loaded onto Bis NuPAGE®
Novex Bis-Tris gels (Invitrogen, Carlsbad, CA) and proteins transferred to PVDF
nes (Invitrogen, Carlsbad, CA). nes were blocked with 5% nonfat dry milk
and 0.1% Tween 20 (Sigma, St. Louis, MO) in TBS (pH 7.4) and incubated overnight at 4°C
with antibodies to HER3 (sc-285 antibody, Cell Signaling Technology, Beverly, MA) and
pHER3 (4791 antibody, Cell Signaling Technology, Beverly, MA). Membranes were washed
in 0.1% Tween 20 in TBS and then incubated for 1 hour in horseradish peroxidase-
conjugated streptavidin secondary antibodies (GE Healthcare). After washing, n bands
were ed in X—ray film by using SuperSignal® West Femto Chemiluminescent Substrate
and SuperSignal® West Pico Chemiluminescent Substrate (Pierce/Thermo Scientific,
rd, IL).
To access cell eration MDA—MB—361 cells were seeded at a density of
2,000 cells in 100 uL of medium containing 10% heat-inactivated FBS; Costar white
polystyrene tissue—culture treated 96—well plates with flat bottoms (Corning) were used. The
next day, the g medium was removed and antibodies were added in complete medium
to a final volume of 100 uL per well. Plates were then incubated in 5% CO2 at 37°C for 6 or
14 days. For the 14-day assay, fresh antibodies were applied at Day 7. Equal volumes of
CellTiter-GloR reagent (Promega) were added to each well at the end of each time point.
Plates were rocked on a plate shaker for 10 minutes at room temperature to ensure complete
cell lysis. scence was measured using an EnVision 2104 Multilabel Reader
(PerkinElmer).
2C2 reduced pHER3 levels (A) and suppressed cell growth (B) of this cell line, suggesting 2C2-YTE not only is active in trastuzumab-sensitive cancers
with HER2—amplif1cation, but also active in HER2-amplif1ed cancers that are less sensitive to
trastuzumab due to mutations on PIK3 CA.
2.8. Identification of Novel HRG-dependent Cancer Types
To identify additional novel HRG-dependent cancer times multiple lung
squamous cell carcinoma (SCC) cell lines were screened for HER3 signaling activity and
HRG expression. HARA—B (JCRB No. JCRB1080.1) and KNS—62 (JCRB No. IF050358)
2012/066038
cell lines expressed significant levels of HER3, HRG as well as pHER3 (data not shown).
Accordingly, we investigated the activity of 2C2 in the HARA-B and KNS-62 cell-lines. The
cells were treated with the anti—HER3 monoclonal antibodies ially as bed above
in Examples section 2.4 supra, 2C2 was able to reduce pHER3 levels in the HARA-B cell
line () and the KNS—62 cell line (data not shown). As shown below (Examples,
section 5.4), 2C2—YTE demonstrated dose-dependent anti-tumor efficacy in the human
squamous HARA—B NSCLC xenograft model. Thus, these criteria (i.e., expression of HER3,
HRG as well as pHER3) may be useful screening tools to identify additional cancer types
sive to anti-HER3 dies, including for e 2C2, AMG, MM as described
herein and others known in the art (see for example International Patent Publications
W02011/1369ll, W02012/019024, W02010/022814).
2.9. HER2 is a Major Driver in Certain HRG-dependent Cancer Types
In the presence of the HER3 ligand heregulin (HRG), HER3 dimerizes
with EGFR or HER2, which leads to phosphorylation of HER3 and transmission of an
oncogenic signal via phosphoinositide 3 kinase (PI3K) and protein kinase B (PKB), also
known as AKT. A collection of CRC models were characterized to determine which receptor
tyrosine kinase, EGFR or HER2, is the major driver of signaling through HER3.
Specifically, six different CRC tumor cell lines, SW620 (ATCC No.CCL—227), SW480
(ATCC No.CCL—228), C010205 (ATCC No.CCL—222), LOVO (ATCC No.CCL—229),
HCT15 (ATCC No.CCL—225), and Caco—2 (ATCC No.HTB—37), were treated with
antagonists of HER2 or EGFR alone or in combination with the HER3 antagonist 2C2.
Briefly, cells were seeded into 24—well plates at a density of 1.5 X105 cells per well. The next
day, 2 identical sets of cells were treated with the 10 ug/mL of the following antibodies: 2C2
anti-HER3 antibody, the R347 control IgG antibody, the anti-HER2 antibody 2C4 (e.g.,
Patent ation W02001/00245), the anti-EGFR antibody cetuximab or the EGFR
tyrosine kinase inhibitor gefitinib at 5 uM. After 5-6 hours of incubation at 37°C, HRG was
added at 50 ng/mL into one set of cells for 15 minutes at 37°C. All cells were then washed
with cold PBS and lysed by the addition of 60 uL of 2>< SDS m dodecyl sulfate) sample
buffer (Invitrogen). Lysates were transferred to 1.5 mL tubes and boiled for 5 minutes
followed by chilling on ice for 2 minutes. Equal volumes (20 uL) of protein samples were
resolved in NuPAGE Novex is gels (Invitrogen) before er to polyvinylidene
e (PVDF) membranes (Invitrogen). Membranes were washed in Tris-buffered saline
(KPL) containing 0.1% Tween 20 (Sigma) and incubated overnight at 4°C with antibodies to
HER3 (Santa Cruz Biotechnology), Tyr1289 (Cell Signaling Technology),
phosphorylated AKT (protein kinase B (pAKT)) (Cell Signaling Technology),
phosphorylated ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase
(pERK)) (Cell Signaling Technology), and glyceraldehyde 3—phosphate dehydrogenase
) (Sigma). Membranes were washed in Tris-buffered saline (KPL) containing 0.1%
Tween 20 (Sigma) and then incubated for 1 hour in horseradish peroxidase—conjugated
ary antibodies (GE HealthCare). After washing, protein bands were detected on X-ray
film by using SuperSignal West Pico Chemiluminescent Substrate (Pierce/Thermo
Scientific).
As seen in , both the ER3 and the anti-HER2 antibodies
reduced the levels of HER3 and pAKT in ligand stimulated cells while EGFR
, pHER3
antagonist such as cetuximab and gef1tinib treatment had no effect on these signaling
molecules. These data demonstrate that HER2 is the major driver of HRG-induced HER3
ing in all the cancer models tested.
Example 3. Mechanism of Action Studies for Anti-HER3 Monoclonal
Antibodies
3.1. Clone 16 Partially Blocked Ligand-Binding to HER3
The efficacy of anti-HER3 monoclonal antibodies to block —induced
HER3 ty can be due to their ability to directly block off ligand-binding. To igate
this scenario, we established an in vitro HRG-HER3 binding assay by coating a plate with
lin (HRG) and binding labeled recombinant HER3 protein to it.
3.1.1. HRG-HER3 Binding Assay
Microplate wells were coated with 10ng/ml heregulin (HRGBl, Cat. No. 377—
HB, R&D Systems, Minneapolis, MN) overnight at 4°C. The next day, plates were washed 4
times with PBST (PBS + 0.05% Tween 20) and blocked in PBS + lug/ml BSA at room
temperature for 1 hour. During blocking, serial dilutions of test antibodies (Clone 16, AMG,
MM and a positive control anti-HER3 ligand blocking monoclonal antibody) were ed
in a separate plate in PBSTB (PBS+0.05% Tween 20 + 0.1% BSA) and combined with
Sug/ml of inant HER3 (Cat. No. 348-RB, R&D Systems, polis, MN) at room
temperature for 30 minutes. ELISA plates were then washed 4x with PBST before addition
_ 97 _
of antibody-HER3 mixture. Plates were incubated at room temperature for 1 hour and were
subsequently washed 4 times with PBST. Anti—His HRP (Cat. No. 34460, Qiagen, Valencia,
CA) was added at room ature for 1 hour. Plates were washed 4 times with PBST
followed by detection with TMB. Plates were read at 450nm using a late .
Representative results are shown in .
3.1.2. Results
The ability of l monoclonal antibodies (Clone 16, AMG, MM and a
positive control anti-HER3 ligand blocking monoclonal antibody) to interfere with the
binding of HRG to HER3 was tested. The positive control HER3 ligand-blocking monoclonal
antibody, efficiently and completely suppressed the HER3 binding to HRG. In contrast,
Clone 16 (the parental lead for 2C2, see "Affinity Optimization" Examples section 1.6 above)
was only partially effective in disrupting this binding (approximately 30% maximum
inhibition). The AMG and MM monoclonal antibodies showed similar weak, partial blocking
effect (). These findings showed that Clone 16 was unlikely to function as a direct
ligand-blocking monoclonal dy.
3.2. 2C2 Disrupts ER3 Dimerization
Due to its kinase-def1cient nature, HER3 monomer is not active and it needs to
form dimers with other RTKs to be active. The HER2:HER3 dimer has been shown to
be the most oncogenic signaling species in both ligand—dependent and independent settings
(Junttila et al, 2009, Cancer Cell. 15:429-40). The disruption of HER2-HER3 zation by
2C2 was assessed using an HRG—induced HER2—HER3 dimer formation assay in T—47D
cells, a ligand—dependent breast cancer model showing HRG—induced HER3-HER2 dimer
formation, and in ligand-independent BT-474 cells. The assay was based on HER3 —HER2 co—
precipitation.
3.2.1. Ligand-induced HER2-HER3 Dimerization Assay
T—47D cells (ATCC Cat. No. HTB—l33TM) were seeded at 1x106/well in 6 well
plates overnight. Next morning, cells were treated with 2C2, CLl6, AMG and MM
onal antibodies at a concentration of Sug/ml in full serum for 2 hours at 37°C.
ls included no antibody treatment, or ent with control R347 IgGl. Treatment
was followed by 50ng/ml HRG treatment for 10 minutes at 37°C (including a control not
treated with HRG). Cells were washed 3 times with cold PBS before adding 500ul of cell
lysis buffer, including protease and phosphatase inhibitors (Sigma, St. Louis, MO). Cells
were lysed on ice for at least 30 minutes. Lysates were harvested with a cell scraper. 50ul of
a protein A beads solution containing 25ul protein A beads conjugated with lug of anti-
HER3 mAb (Cat. No. MAB3481, R&D Systems, Minneapolis, MN) were added to 500ul of
cell lysate and erred to immunoprecipitation (1P) columns. The IP columns were rotated
overnight at 4°C. Subsequently, the 1P columns were spun down to remove the lysates, and
the beads were washed with cold cell lysis buffer. 50111 of 2X SDS sample buffer containing
50mM DTT were added to each 1P column, and columns were boiled for 4 minutes. The
bottom tip of each column was removed, and columns were spun down to collect the eluates.
20ul of eluate were separated using SDS-PAGE. Western blotting was med for both
HER2 and HER3 (with anti-HER2 antibody Cat. No. OP15L, CalBiochem, La Jolla, CA; and
anti-HER3 antibody Cat. No. SC—285, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
3.2.2. Ligand-independent ER3 Dimerization Assay
BT—474 cells (ATCC Cat. No. HTB-20TM) were plated at a density of 1 X 106
cells per well in a 6-well plate in complete RPMI 1640 cell culture media with 10% heat-
inactivated PBS. The next day, plating medium was removed and replaced with fresh
te RPMI 1640 containing saturating dose of testing antibodies. In this experiment,
CL16, the sor lower ty version of 2C2, 2C2, and R347, a control IgG, were tested
at a concentration of 5 ug/mL. Cells were incubated with antibodies for 2 hours at 37°C.
Then the medium was removed, and cells were washed once with cold PBS. The crosslinker
3, 3’—dithiobis [sulfosuccinimidylpropionate] (DTSSP) was added at a concentration of 2 mM
in 1 mL cold PBS. Cells were incubated for at least 1 hour on ice. Cells were then washed 3
times with cold PBS. Cell lysis buffer (500 uL) ning protease and phosphatase
inhibitors was added and the cells were placed on ice for at least 30 minutes to allow for lysis
before harvesting with a cell scraper. HER2 and HER3 were immunoprecipitated from cell
lysates. Cell lysates (500 uL) were combined with 50 uL protein A sepharose beads (50%
slurry; Invitrogen) njugated to 1 ug of HER3 MAb (MAB3481, R&D Systems) in a
SigmaPrep spin column (Sigma). The mixture was incubated with rotation at 4°C overnight.
The next day, beads were separated from the cell lysate by centrifugation. Beads in the
columns were washed four times with cold cell lysis buffer (Cell Signaling logies)
containing se (Sigma) and atase tors (EMD Millipore). After the wash
procedure, 50 uL 2>< SDS (sodium dodecyl sulfate) sample buffer containing 50 mM
threitol (DTT; EMD Chemicals) was added into the spin columns. The columns were
then boiled for 4 minutes. Proteins were eluted by fugation and used immediately for
immunoblotting (as done in section 2.4).
3.2.3. Results
T-47D cells treated or not treated with HRG were lysed. HER3 was
precipitated with anti-HER3 monoclonal antibody, then the proteins in the pellet were
resolved on GE and blotted for the presence of HER2 as signs of HER2—HER3
interaction. The model was ligand-inducible since the dimer only occurred after -
stimulation. A pre-treatment with 2C2 efficiently prevented dimer formation, demonstrating
its ability to impede ligand-induced ER3 dimer formation. Other anti-HER3
monoclonal dies including MM, AMG, and the parental Clone 16, were also found to
be effective (A). When the cross—linker DTSSP was used to mically stabilize
protein complexes, constitutive HER2:HER3 dimer was captured in the absence of
HRG in BT-474 cells, indicating a ligand-independent heterodimer formation. Pretreatment
of cells with 2C2 or CLl6 effectively disrupted this heterodimer formation (B).
3.3. HER3 Internalization and Degradation Induced by 2C2
Target internalization and degradation are two common mechanisms by which
monoclonal antibodies inhibit their target functions. First, we assessed the 2C2—mediated
HER3 internalization in the BT-474 breast cancer cells. Next, we ained if this rapid
2C2-induced HER3 internalization could be followed by target degradation.
3.3.1. HER3 Internalization Assay
HER3 internalization was determined using a Fluorescence Activated Cell
Sorting (FACS) assay. BT—474 cells were detached with Accutase enzyme and suspend the
cells in PBS containing 1% BSA (FACS buffer) to a cell density of lOXlO6 cells/ml. 50ul of
cells were added to each cell of a U—bottom 96 well plate. Soul of anti—HER3 onal
antibodies plus Isotope control (at 20ug/ml) were added into each well to e a lOug/ml
final concentration. The plate was incubated at 37°C for 0.5 hours, 2 hours and 4.5 hours,
respectively. Cells were washed with cold FACS buffer twice (cells were pelleted by
centrifugation at 1,500 rpm for 2 minutes). Cells were resuspended with cold FACS buffer
containing mouse anti-human HER3 monoclonal antibody (Cat. No. MAB3481, R&D
Systems, Minneapolis, MN) at l [Lle or 10 ug/ml.
— 100 —
Cells and anti-human HER3 were ted on ice for 1 hour. Cells were then
washed twice with cold FACS buffer. Cells were subsequently ended with cold FACS
buffer containing an Alexa Fluor® 488—labeled secondary antibody (Invitrogen) (1:200 v/v),
and incubated on ice for 30—45 minutes. Cells were then washed with cold FACS buffer twice
and resuspended with 100ul of cold FACS buffer. At this point, FACS was performed.
Absolute Geometric Mean of Fluorescence Intensities (GMFI) were obtained by subtracting
the GMFI from controls including only the secondary antibody. Relative HER3 surface
clearance was calculated by comparing with results obtained using an IgG control
onal antibody. Representative results are shown in A.
3.3.2. HER3 n Degradation Assay
Lovo, HCT15 and SW620 colorectal model cancer cells (ATCC Nos. CCL—
229, CCL—225 and CCL—227, respectively) were seeded at 1.5X105/W6ll in 24 well plates.
After overnight attachment, the cells were treated with 2C2 and control monoclonal antibody
for 3—4 hours. Cells were washed with cold PBS once, directly lysed with 50—60ul of 2X SDS
sample buffer and boiled at 100°C for 10 minutes. 20ul of samples were loaded into SDS—
PAGE gels, ophoretically separated, and Western blotted with antibody t HER3
(Santa Cruz Biotech) to quantitate total HER3 protein levels. Antibodies t GAPDH
(Sigma) were also used to quantitate GAPDH levels as a general n loading control.
3.3.3. Results
As shown in A, both doses of 2C2 had a very similar . A 30-
minute treatment caused a 39% loss of surface HER3 population (61% remaining), whereas a
2—hour treatment caused a 62% loss (38% remaining), suggesting a rapid target
internalization by 2C2. Additionally, when the three different colorectal cancer models were
incubated with 2C2, complete HER3 degradation was observed in SW620 cells, whereas
nearly complete degradation was observed in the other two cell-lines (B),
trating that 2C2 was capable of strong target degradation capacity.
3.4. or Functions: Antibody-Dependent Cell-Mediated Cytotoxicity
(ADCC) and Complement-Dependent Cytotoxicity (CDC)
ADCC is one recognized way through which a monoclonal antibody can
confer its anti—tumor efficacy in vivo. To assess the ADCC activity of Clone 16, we used an
in vitro PBMC—enabled ADCC assay in two mplfied breast cancer models: BT-474
—101—
and SkBR3. Herceptin/Trastuzumab was used as positive control since it has been shown to
confer ADCC effect in these type of cancers. In both models we observed significant tumor—
killing effects from Herceptin, but the remaining monoclonal antibodies tested, Clone 16,
AMG and MM, were y inactive, indicating that they lacked appreciable ADCC effect
(data not shown). 2C2-YTE was tested in CDC assays using human serum as a source of
ment. In addition, the anti-HER2 antibody trastuzumab and the anti-CD20 antibody
rituximab, were included as controls. None of the antibodies including 2C2—YTE showed
any detectable CDC activity at any concentration (data not shown). SkBR3 cells do not
s CD20. As a positive control, rituximab demonstrated substantial cell-kill ty in a
r CDC assay against Daudi cells, which s CD20 (data not shown).
3.5. ycle Arrest
3.5.1. Cell-cycle Arrest Assay in SkBR3 Breast Cancer Cells
BioSantecells (ATCC No.HTB—30) were plated at a density of 150,000
cells/well in a 6-well plate and allowed to attach overnight. The following day, media was
removed and replaced with fresh growth medium containing test and control antibodies. Cells
were then incubated at 37°C for 48 hours. At the end of the treatment, cells were trypsinized,
pooled into a 15ml conical tube, and centrifuged at 1500rpm for 5 minutes. Cell were then
washed once with PBS and fixed in ice cold 70% ethanol at —20°C overnight.
Following fixation, cells were centrifuged as described above, washed once in
PBS, and resuspended in staining solution (PBS+0.l% Triton X-100, 0.2mg/ml DNAse-free
RNAse A, and 20ug/ml ium iodide). Cells were stained for 30 minutes at room
temperature in the dark, and analyzed using an LSRII Flow Cytometer System (BD
Biosciences). Propidium iodide was detected using the Texas Red channel; data was
analyzed using the FlowJo flow cytometry analysis package (Tree Star, Inc., OR) using the
Dean/Jett/Fox Model.
3.5.2. Results
The ased cell—cycle analysis showed that in SkBR3 cells, a HERZ—
amplif1ed breast cancer cell-line r to BT-474, both Herceptin and Clone l6 tal
lead for 2C2) caused cell—cycle arrest at Gl—phase (increased Gl—population by decreasing
S/G2 populations as shown in ).
—102—
3.6. Anti-angiogenic Effects by Blocking HRG-induced VEGF Secretion
HRG has been shown to drive secretion of VEGF, a major pro—angiogenic
cytokine, in various cancer models. Therefore we assessed the inhibitory effects of 2C2 in
suppressing HRG—induced VEGF secretion in two breast cancer : MCF—7 and BT—474.
3.6.1. HRG-induced VEGF Secretion Assay
MCF—7 cells and BT—474 cells were plated at a density of 100,000 cells/well in
a 24—well plate, and were allowed to rest for 2 days. Media was then d and replaced
with 500u1 of fresh growth medium containing 2% FBS and l and test antibodies.
Following 24 hour incubation, cell culture media was collected and VEGF levels were
determined using a VEGF ELISA Kit (R&D Systems DY293B). Relative cell number in
each well was determined by adding fresh media to the cells along with Cell Titer Glo
ga, 1:1 ratio) and incubating plates for 10 minutes at room temperature.
Luminescence was read using a plate reader, and these values were used for normalization of
the data.
3.6.2. Results
HRG treatment induced ic ses in VEGF secretion in the BT-474
(A) and MCF-7 (B) both breast cancer model cell-lines ranging from 6.5-fold
to 8-fold. CL16 (Clone 16), and MM monoclonal antibodies were able to suppress most of
the increases, suggesting that these anti-HER3 monoclonal antibodies can confer additional
vascular modulation effects.
e 4. Cross Reactivity with Cynomolgus Monkey and Mouse HER3
4.1. 2C2 Binds to Cynomolgus and mouse HER3 with r Affinity as to
Human HER3
Biacore assays were performed essentially as described above to compare the
affinity of 2C2 to human, cynomolgus monkey (cyno) and mouse HER3 to enable relevant
toxicity species selection (top portion of Table 7.) Additional Biacore assays were performed
for 2C2-YTE using a higher resolution BlAcore instrument, an alternative Fc capture reagent
and a refined injection protocol to t for background binding. Briefly, Protein A capture
reagent was immobilized onto two adjacent flow cells connected in series on the same CM5
sensor chip, using a standard amine protocol as ed by the instrument’s manufacturer.
—103—
WO 78191
One of these Protein A surfaces was used as a reference e for this experiment, while the
other served as the active surface used to record IgG capture and subsequent HER3 (ECD)
binding. The final Protein A densities on the reference and active flow cell surfaces were
recorded as 1986 RUs and 1979 RUs, respectively. As configured, the method was set up
such that 2C2-YTE IgG was first ed onto only the active Protein A surface, followed
by an ion of a HER3 protein solution over both the active and nce flow cell
es. In so doing, this strategy corrects the binding curve for any non—specific g of
the HER3 analyte to the Protein A capture surface. For the IgG capture step, 2C2-YTE IgG
was prepared at 10 nM in HBS-EP+ instrument buffer (0.01 M HEPES, pH 7.4, 0.15 M
NaCl, 3 mM EDTA, and 0.05% P20), then injected over the active Protein A flow cell
surface for 30 seconds at a flow rate of 10 uL/min. Human, cyno, and murine HER3 protein
were then initially prepared at 500 nM stock ons in instrument buffer, then two-fold
serial dilution series of each were generated to provide a final concentration of 0.39 nM. The
HER3 protein was then injected over both the active and reference cell Protein A surfaces for
120 seconds, at a flow rate of 75 uL/min. Dissociation data was collected for 15 minutes,
followed by two 60—second pulses with 10 mM Gly buffer, pH 1.7, n injections to
regenerate the flow cells back to the Protein A capture surfaces. Several buffer injections
were also interspersed throughout the injection series. Select buffer injections were
subsequently used along with the reference cell data to correct the raw data sets for injection
cts and/or non-specific binding interactions, a technique commonly referred to as
“double—referencing” (Myszka, 1999). Fully corrected binding data were then globally fit to a
1:1 binding model (BIAevaluation 4.1 software) that included a term to correct for mass
transport-limited binding, should it be ed. This analysis determined the kinetic rate
(kon, koff) constants, from which the nt KD was then calculated as koff/kon (bottom
of Table 7). The variation in the K0n and Koff values between the two sets of experiments are
likely due to the differences between the two protocols as detailed above and were generally
within the ed two fold error range for measuring these kinetic parameters. As shown in
TABLE 7, the affinity of 2C2, and 2C2-YTE to cyno HER3 was virtually identical with that
to human HER3. The affinity for mouse HER3 was within 3-fold of the affinity for human
HER3.
—104—
WO 78191
TABLE 7: Biacore binding assay showing 2C2's affinity to human, cyno, and
mouse HER3.
IgG Capture 2C2 2C2 2C2
(exp34c, 34d,43b) (exp43d) (exp43l)
Receptor huHER3 (ECD)-His muHER3-His Cyno HER3-His
Format IgG (Fc) capture IgG (Fc) capture IgG (Fc) capture
K0,, (l/Ms) (x105) 4.27 (——/— 0.45) 3.26 4.66
Koff(1/S) (X104) 1.71 (--/— 0.18) 3.78 1.81
KD (nM) 0.402 (--/— 0.029) 1.16 0.389
IgG Capture 2C2-YTE 2C2-YTE E
Receptor huHER3 (ECD)-His muHER3-His Cyno HER3-His
Format IgG (Fc) capture IgG (Fc) capture IgG (Fc) capture
K0n (l/Ms) (x105) 1.61 1.11 1.52
KOff(1/S) (X104) 0.743 1.91 0.734
KD (nM) 0.461 1.721 0.483
4.2. Assay for HRG—induced Phosphorylation of Cynomolgus HER3
Ad293 cells (Stratagene No. 240085) were transiently transfected with full
length R3-expression vector following protocol provided with the Lipofectamine
2000 reagent (Invitrogen). Cells were allowed to incubate at 37°C for 48 hours before
treatment. Antibodies were added at 10ug/ml in complete growth medium for 1 hour
followed by stimulation with 20ng/ml HRGBl (R&D Systems) for 10 minutes at 37°C. At
the end of treatment, media was removed and cells were washed once with PBS. Cells were
lysed with 2x Novex Tris-glycine sample buffer (Invitrogen) and the levels of pHER3 and
total HER3 were determined by immunoblotting (Cell Signaling antibody #4791 and Santa
Cruz antibody #285, respectively). Densitometry of bands was accomplished using ImageJ
software (NIH, imagej .nih. gov/ij/).
4.3. Results
To fully establish the g and cross-modulation of cyno HER3 by 2C2, a
stable Ad293 cell-line ectopically expressing full-length cyno HER3 was established, as
demonstrated by Western Blot (A). When treated with HRG, the cyno HER3
underwent robust tion as evidenced by the induction of pHER3 signal (8).
When cells were co—treated with 2C2 but not when they were treated with the R347 control
dy, pHER3 induction was completely abrogated, demonstrating that 2C2 was not only
able to bind to cyno HER3 on urface, but also able to efficiently ablate its tion
(B). ed with the above Biacore affinity ement data showing that 2C2
—105—
displayed cal affinity to cyno HER3 as to human HER3, these results validated cyno as
a relevant toxicity species for 2C2 trials.
In Vivo studies for Anti-HER3 Monoclonal dies
4.4. Subcutaneous Human FADU Head and Neck Xenograft Model Studies
4.4.1. Method
Human FADU Head and Neck cells (ATCC No.HTB-43) were maintained at
37°C in a 5% C02 tor in RPMI 1640 medium containing 4.5g/L e, L-glutamine,
sodium pyruvate and 10% fetal bovine serum. Xenografts were established by
subcutaneously injecting 5 X 106 cells per mouse (suspended in 50% el) into the right
flanks of 4— to 6-week-old athymic nu/nu mice. Tumors were allowed to grow up to 200 mm3
before randomization for efficacy studies. 2C2, 2C2-YTE, cetuximab, control IgGl or the
combination of 2C2 with cetuximab monoclonal antibodies were administered
intraperitoneally. For dose dependency studies the 2C2 was adminstered at 3, 5, 7, and 10 mg
per am body weight (mg/kg), the control at 10 mg/kg. For the combination studies 2C2
was administered at 3 mg/kg, cetuximab at 30 mg/kg and the control antibody at 6 mg/kg.
Caliper measurements were used to calculate tumor s using the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as t delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = l — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group ed to the value
at g, and dC = change in mean tumor volume in control group compared to the
value at staging.
At the conclusion of the efficacy studies with 2C2, mice were treated with 2C2
a final time as indicated to ine pharmacokinetic values. Cardiac puncture was
performed to collect blood into Microtainer Serum Separator Tubes (SST). Tubes with blood
were vortexed gently for 10 seconds and kept at room temperature for 20 minutes to allow the
serum to clot. Samples were centrifuged at 1000 X g for 10 minutes, and the serum samples
were carefully transferred into new tubes and stored at —80°C.
An indirect Enzyme-Linked Immunosorbent Assay (ELISA) format was used
for the quantitative determination of 2C2 in mouse serum. Standards, quality controls, and
—106—
mouse serum samples were ted with goat anti—human IgG dies which were
lized on a 96-well microtiter plate. After incubation, unbound materials were
removed by a wash step and 2C2 was detected using a goat anti-human IgG with adish—
peroxidase conjugate. An acidic stopping solution was added and the degree of enzymatic
turnover of substrate was determined by measuring absorbance at 450 nm. The absorbance
measured was directly proportional to the concentration of 2C2 present in the mouse serum.
A 2C2 standard curve for the assay was used to interpolate the concentration of the serum
samples.
4.4.2. Results.
Utilizing a human FADU Head and Neck aft model grown
aneously in female nude mice, 2C2 demonstrated dose—dependent anti—tumor efficacy.
Maximal efficacy at 99% tumor growth inhibition (dTGI) was observed with 7 mg/kg
administered twice per week for the on of the study (A).
Combined administration of 3 mg/kg of 2C2 with 30 mg/kg of cetuximab
administered two times per week during the treatment phase (days 7—18) showed clear
synergistic anti-tumor efficacy in the FADU xenograft model (B). This effect was
long lasting and the tumors only started to grow back at the end of the regrowth phase at day
40. The ation treatment produced 7 out of 10 partial regressions and 2/ 10 complete
regressions.
2C2 cross—reacted with mouse HER3 and it is well established that HER3 is
expressed in many non—diseased mouse tissues. Therefore, host HER3 could serve as a sink to
absorb the 2C2 monoclonal antibody before it gets to the tumor . Using tumor-bearing
female nude mice, 2C2 at 5 mg/kg was administered either once or three times to these mice
and the exposure levels of 2C2 were followed over time. 2C2 was only detectable 1 day after
the last dose of 5 mg/kg of 2C2 and became undetectable after 3 days after the last treatment
(). On the other hand, dosing with 30 mg/kg of 2C2 using the same schedules as for 5
mg/kg led to a much more prolonged window where 2C2 could be measured in mouse serum.
These findings demonstrated non-linear pharmacokinetics for 2C2 after single dose and
repeat-dose administration of 5 mg/kg or 30 mg/kg to tumor-bearing mice. The data showed
that mouse HER3 can act as a sink to bind 2C2 administered to the mice and that 30 mg/kg as
a single dose was sufficient to saturate the sink.
—lO7—
The existence of a HER3 sink in mice for 2C2 was confirmed functionally by
administering a high loading dose of 2C2 follow by a low nance dose in mice with
FADU xenograft tumors. The anti-tumor efficacy of a 10 mg/kg loading dose and a 3 mg/kg
nance dose of 2C2 was demonstrated in the FADU tumor model. 10 mg/kg of 2C2 as a
single dose had only transient anti-tumor efficacy. 2C2 given at 3 mg/kg twice per week had
modest but continuous efficacy. The ation of the 10 mg/kg loading dose with the 3
mg/kg nance dose of 2C2 was more efficacious in blocking tumor growth compared to
either treatment schedule alone ().
The ability of 2C2 to modulate the pharmacodynamic s pHER3 and
pAKT was tested in FADU xenograft tumor extracts. 2C2 was administered twice at 30
mg/kg within 48 hours to mice g human FADU xenograft tumors and extracts were
analyzed 24 hours later. Briefly, athymic nude mice were implanted aneously with
FADU head and neck cancer cells. Animals were administered 2C2 at 30 mg/kg twice within
48 hours. Extracts were prepared 24 hours later for analysis of pHER3, pAKT and total
HER3 (, top, middle and bottom panels, respectively). R347 was used as the control
IgGl antibody. There were 6 s per treatment group. Data are presented as the mean ::
standard deviation. Here, 2C2 inhibited phosphorylation of both HER3 and AKT by 59.5%
and 51.7%, respectively, compared to tumors from control IgGl-treated mice (, top
and middle panels). No modulation of total HER3 was observed by 2C2 (, bottom
panel).
4.5. Subcutaneous Human Detroit562 Head and Neck Xenograft Model
Studies
4.5.1. Method.
Human Detroit562 Head and Neck cells (ATCC No.CCL—138) were
ined at 37°C in a 5% C02 incubator in RPMI 1640 medium containing 4.5g/L glucose,
L-glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts were established by
subcutaneously injecting 5 X 106 cells per mouse into the right flanks of 4— to 6-week-old
athymic nu/nu mice. Tumors were d to grow up to 200 mm3 before randomization for
efficacy studies. 2C2, 2C2-YTE, cetuximab, control IgGl or the combination of 2C2 with
cetuximab monoclonal antibodies were administered intraperitoneally. For dose dependency
studies the 2C2 was administered at, 1, 3,10, and 30 mg per kilogram body weight (mg/kg).
For the combination studies 2C2 was administered at 3 mg/kg, cetuximab at 30 mg/kg and
— 108 —
the control antibody at 10 mg/kg. Caliper measurements were used to calculate tumor
s using the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = l — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
At the conclusion of the efficacy studies with 2C2, mice were treated with 2C2
a final time as indicated to ine cokinetic values. Cardiac puncture was
med to collect blood into SST microtainer tubes. Tubes with blood were vortexed
gently for 10 seconds and kept at room temperature for 20 minutes to allow the serum to clot.
Samples were centrifuged at 1000 X g for 10 s, and the serum samples were carefully
transferred into new tubes and stored at —80°C.
An indirect Enzyme-Linked Immunosorbent Assay (ELISA) format was used
for the quantitative determination of 2C2 in mouse serum. Standards, quality controls, and
mouse serum samples were incubated with goat anti—human IgG antibodies which were
immobilized on a 96-well iter plate. After incubation, unbound materials were
removed by a wash step and 2C2 was detected using a goat anti-human IgG with adish—
peroxidase conjugate. An acidic stopping solution was added and the degree of enzymatic
turnover of substrate was determined by measuring absorbance at 450 nm. The absorbance
measured was directly proportional to the concentration of 2C2 present in the mouse serum.
A 2C2 standard curve for the assay was used to interpolate the concentration of the serum
samples.
4.5.2. Results
2C2 showed umor efficacy in the human Detroit562 Head and Neck
xenograft model grown subcutaneously in female nude mice. 10 mg/kg of 2C2 administered
twice per week was lly efficacious at 72% dTGI (A). The Detroit562 model
contains a PIK3CA on.
The Detroit562 tumor model was sensitive to the anti-EGFR monoclonal
antibody cetuximab which caused tumor growth inhibition at 10 mg/kg administered twice
—109—
WO 78191
per week. The combination of 3 mg/kg of 2C2 with 10 mg/kg of cetuximab added to the anti-
tumor efficacy of cetuximab and resulted in 9 out of 10 partial regressions while cetuximab
alone produced 5/10 l regressions (B).
4.6. Subcutaneous Human CAL27 Head and Neck Xenograft Model Studies
4.6.1. Method.
Human CAL27 Head and Neck cells (ATCC No.CRL—2095) were ined
at 37°C in a 5% C02 incubator in RPMI 1640 medium containing 4.5g/L glucose, L-
glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts were established by
subcutaneously injecting 5 X 106 cells per mouse into the right flanks of 4— to 6-week-old
athymic nu/nu mice. Tumors were allowed to grow up to 200 mm3 before randomization for
efficacy s. 2C2-YTE, cetuximab or l IgGl were administered intraperitoneally.
For dose dependency studies the 2C2—YTE was adminstered at 3,10, and 30 mg per kilogram
body weight (mg/kg). r measurements were used to calculate tumor volumes using the
formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as percent delta tumor
growth inhibition (TGI), which was ated as follows:
percent delta TGI = 1 — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.6.2. Results.
Dose—dependent activity of 2C2-YTE was confirmed in a third head and neck
tumor model, CAL27, using 2C2-YTE. 2C2-YTE at 3, 10 or 30 mg/kg administered twice
per week showed TGI with 26.4%, 55.2%, or 68.8%, respectively, compared to control IgGl—
treated animals ().
The CAL27 tumor model was sensitive to the anti-EGFR onal antibody
cetuximab which caused tumor growth inhibition at 30 mg/kg administered twice per week
with TGI of 75.0% ().
—110—
4.7. Subcutaneous Human KRAS Mutated A549 NSCLC Xenograft Model
Studies
4.7.1. Method
Human A549 NSCLC cells (ATCC No.CCL-185) which contain a mutation in
codon 12 of the KRAS gene (were maintained at 37°C in a 5% C02 incubator in HAM’S
F12K medium containing 4.5g/L glucose, L-glutamine, sodium pyruvate and 10% fetal
bovine serum. Xenografts were established by subcutaneously injecting 5 X 106 cells per
mouse (suspended in 50% matrigel) into the right flanks of 4— to —old athymic nu/nu
mice. Tumors were allowed to grow up to 200 mm3 before randomization for efficacy
studies. 2C2, 2C2-YTE, cetuximab, control lgGl or the combination of 2C2 with cetuximab
monoclonal antibodies were administered intraperitoneally. For dose dependency s the
2C2 was adminstered at, 3, 10 and 30 mg per kilogram body weight (mg/kg) and 2C2-YTE at
mg/kg. For the ation studies 2C2 and cetuximab were each administered at 10
mg/kg. Caliper measurements were used to calculate tumor s using the formula:
tumor volume = TE + 6(1ength >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as t delta tumor
growth inhibition (TGI), which was ated as follows:
percent delta TGI = 1 — (dT + dC) X 100,
where dT = change in mean tumor volume in ent group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.7.2. Results.
2C2 demonstrated dose—dependent anti—tumor efficacy in the human A549
NSCLC xenograft model grown subcutaneously in female nude mice. Maximal efficacy of
91% dTGl was achieved with 30 mg/kg of 2C2 administered twice per week until day 33
(A). 2C2 and 2C2-YTE given at 10 mg/kg displayed similar anti-tumor efficacy in
this A549 tumor model. Once the treatment was stopped the tumors started to grow at the
same rate as tumors in control-treated mice. The A549 xenograft model contains a KRAS
mutation and a LKB-l on.
Cetuximab at 10 mg/kg alone was not efficacious in this A549 tumor model.
However, the addition of cetuximab at 10 mg/kg to 2C2 also at 10 mg/kg resulted in additive
anti-tumor efficacy during the treatment phase compared to 2C2 alone. In addition, the
— 111 —
combination treatment group showed a slower regrowth rate of the tumors after cessation of
ent (B).
4.8. Subcutaneous Human I-LARA-B Squamous NSCLC Xenograft Model
Studies
4.8.1. Method
Human squamous HARA—B NSCLC cells which express the wild—type RAS
gene, HRG and pHER3 were maintained at 37°C in a 5% C02 incubator in RPMI 1640
medium containing 4.5g/L D. glucose, 2.383 g/L HEPES Buffer, L. Glutamine, 1.5 g/L
Sodium Bicarbinate, 110 mg/L sodium pyruvate and 10% fetal bovine serum. Xenografts
were established by subcutaneously injecting 5 X 106 cells per mouse (suspended in 50%
matrigel) into the right flanks of 4— to 6-week—old athymic nu/nu mice. Tumors were allowed
to grow up to 227 mm3 before randomization for efficacy studies. E were
administered intraperitoneally at 3, 10 and 30 mg per kilogram body weight ), the
control was at 30 mg/kg. Caliper measurements were used to calculate tumor volumes using
the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as percent delta tumor
growth inhibition (TGI), which was ated as s:
percent delta TGI = l — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at g, and dC = change in mean tumor volume in control group ed to the
value at staging.
4.8.2. Results.
2C2—YTE demonstrated dose-dependent anti—tumor efficacy in the human
squamous HARA—B NSCLC xenograft model grown subcutaneously in female nude mice.
Maximal efficacy of 64.6% dTGI was achieved with 30 mg/kg of 2C2-YTE administered
twice per week until day 29 (). 2C2-YTE given at 10 mg/kg displayed similar anti-
tumor efficacy as 30 mg/kg; however, 2C2-YTE at 3 mg/kg was not efficacious in this
HARA-B tumor model. The HARA-B xenograft model contains a wild-type RAS allele.
—ll2—
4.9. Subcutaneous Human HT-29 CRC Xenograft Model Studies
4.9.1. Method
Human HT-29 colorectal carcinoma cells (ATCC N0.HTB—3 8) were
maintained at 37°C in a 5% C02 incubator in RPMI 1640 medium containing 4.5g/L glucose,
L-glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts were established by
subcutaneously injecting 5 X 106 cells per mouse into the right flanks of 4— to 6-week-old
athymic nu/nu mice. Tumors were d to grow up to 200 mm3 before ization for
cy studies. 2C2, 2C2-YTE and control lgGl monoclonal antibodies were administered
intraperitoneally. 2C2 was administered at 2, 10 and 30 mg per am body weight
(mg/kg), while 2C2—YTE was at 30 mg/kg. Caliper measurements were used to calculate
tumor volumes using the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are sed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
t delta TGI = 1 — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.9.2. Results.
2C2 showed dose—dependent anti—tumor efficacy using the human HT-29
colorectal xenograft model subcutaneously injected into female nude mice. 30 mg/kg of 2C2
administered twice per week was maximally efficacious at 56% dTGl during the treatment
phase (). 2C2-YTE displayed the same efficacy as 2C2 both given at 30 mg/kg. Once
the treatment was stopped the tumors grew at the same rate as the control tumors. The HT-29
xenograft model contains a BRAF mutation. Cetuximab at 10 mg/kg alone had no measurable
anti-tumor activity in this model. The activity of 2C2 30 mg/kg in combination with
cetuximab at 10 mg/kg was indistinguishable from the activity of 2C2 30 mg/kg alone at the
end of treatment phase (data not shown). This indicates that this EGFR-expressing CRC
tumor model, which responds well to 2C2, was not further inhibited by the addition of 2C2-
YTE to cetuximab.
—ll3—
4.10. Subcutaneous Human HCT-116 CRC Xenograft Model Studies
4.10.1. Method
Human HCT-l 16 colorectal carcinoma cells were maintained at 37°C in a 5%
C02 incubator in RPMI 1640 medium containing 4.5g/L glucose, L-glutamine, sodium
pyruvate and 10% fetal bovine serum. Xenografts were established by subcutaneously
injecting 5 X 106 cells per mouse into the right flanks of 4- to 6-week-old athymic nu/nu
mice. Tumors were allowed to grow up to 200 mm3 before randomization for efficacy
studies. 2C2, 2C2-YTE, cetuximab and control lgGl monoclonal antibodies were
administered intraperitoneally. 2C2 was administered at 3, 10 and 30 mg per am body
weight (mg/kg) while 2C2-YTE was at 30 mg/kg. Caliper measurements were used to
calculate tumor volumes using the formula:
tumor volume = TE + th >< width >< width)
for tumors grown in mice. Antitumor effects are sed as t delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = 1 — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.10.2. Results.
2C2 at several different trations and 2C2-YTE at 10 mg/kg
administered twice per week yed modest anti-tumor efficacy in the human HCT-l l6
colorectal xenograft model injected subcutaneously into female nude mice ().
Maximal efficacy was noted at 43% dTGl for 2C2 at 10 mg/kg. The anti-EGFR monoclonal
antibody cetuximab had no efficacy at 10 mg/kg. The HCT-l l6 xenograft model contains a
KRAS mutation.
4.11. Subcutaneous Human LOVO CRC aft Model s
4.11.1. Method.
Human LOVO colorectal carcinoma cells (ATCC No.CCL—229) were
maintained at 37°C in a 5% C02 incubator in HAM’S F12K medium containing 4.5g/L
glucose, L—glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts were
—114—
established by subcutaneously ing 5 X 106 cells per mouse into the right flanks of 4— to
—old c nu/nu mice. Tumors were allowed to grow up to 200 mm3 before
randomization for efficacy s. 2C2, 2C2-YTE, cetuximab and control IgGl monoclonal
antibodies were administered intraperitoneally. 2C2 was stered at 10 or 30 mg per
kilogram body weight (mg/kg), 2C2-YTE and cetuximab were administered at 10 mg/kg and
the control at 30 mg/kg. Caliper measurements were used to calculate tumor volumes using
the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = l — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.11.2. Results
2C2 at 30 mg/kg administered twice per week achieved anti—tumor efficacy of
48% dTGI in the human LOVO ctal xenograft model grown subcutaneously in female
nude mice (). 2C2, 2C2-YTE and cetuximab all at 10 mg/kg had comparable
efficacy. The LOVO xenograft model contains a KRAS on.
4.12. Subcutaneous Human DU145 Prostate Carcinoma Xenograft Model
Studies
4.12.1. .
Human DU145 prostate carcinoma cells (ATCC No.HTB-8l) were maintained
at 37°C in a 5% C02 incubator in MEM medium containing Earle’s salts, l-glutamine and
% fetal bovine serum. Xenografts were established by subcutaneously injecting 5 X 106
cells per mouse (suspended in 50% matrigel) into the right flanks of 4— to -old athymic
nu/nu mice. Tumors were allowed to grow up to 200 mm3 before randomization for efficacy
studies. 2C2, MM and AMG monoclonal antibodies were administered intraperitoneally at 30
mg per kilogram body weight. Caliper measurements were used to calculate tumor volumes
using the formula:
tumor volume = TE + 6(length >< width >< width)
—115—
for tumors grown in mice. Antitumor effects are expressed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = l — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at g.
4.12.2. Results
Using a human DU145 prostate cancer xenograft model grown subcutaneously
in male nude mice 2C2 at 30 mg/kg stered twice per week demonstrated anti-tumor
efficacy of 77% dTGI in this tumor model (). The DU145 xenograft model contains a
LKB-l deletion. The ER3 monoclonal antibodies AMG and MM used at 30 mg/kg
trated anti—tumor efficacy but they were less effective than 2C2 at the same dose of 30
mg/kg.
4.13. Orthotopic Human BT-474 Breast Cancer Xenograft Model Studies
4.13.1. .
Human BT-474 breast cancer cells were maintained at 37°C in a 5% C02
incubator in RPMI 1640 medium containing 4.5g/L glucose, L-glutamine, sodium pyruvate
and 10% fetal bovine serum. Orthotopic xenografts were established by injecting 1 X 107
cells per mouse (suspended in 50% matrigel) into the y fat pad on the right flank of 4
to 6—week—old athymic nu/nu mice. Estrogen pellets (0.36 mg) were placed under the skin of
the left flank 1—2 days before cell injection. Tumors were allowed to grow up to 200 mm3
before randomization for efficacy studies. 2C2, 2C2-YTE, and or anti-HER2 antibodies
known in the art: MM, AMG and trastuzumab (trade name Herceptin®; e.g., US. Pat. No.
,821,337) were administered intraperitoneally at 30 mg per kilogram body weight. Lapatinib
was administered by oral gavaging at 100 mg per am body weight. Caliper
measurements were used to calculate tumor volumes using the formula:
tumor volume = TE + th >< width >< width)
for tumors grown in mice. mor effects are expressed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = l — (dT + dC) X 100,
—ll6—
2012/066038
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.13.2. Results
Using a HER2-driven human breast cancer model, BT-474, injected
orthotopically into the mammary fat pad of female nude mice administration of 2C2 at 30
mg/kg ed twice per week led to a 55% dTGI in BT—474 xenografts (A). BT—474
express HER2 at very high levels of 3+ characterized by HercepTest. AMG and MM both
administered at 30 mg/kg did not show anti-tumor cy in this HER2-driven model.
Lapatinib is a small molecule drug inhibiting EGFR and HER2. Since BT-474
tumors are driven by HER2, lapatinib was tested in this model and found to cause tumor
stasis in the BT-474 tumor model. The combination treatment of 30 mg/kg of 2C2 with 100
mg/kg of lapatinib resulted in improved anti-tumor efficacy of lapatinib alone which was
most clearly visible in a delay in th of the tumors in the absence of additional
ents (B). The anti-tumor activity of E was similar to that of 2C2. The
anti—HER2 antibody trastuzumab was also was tested in this model and shown to be very
active in this HER2-driven xenograph model with a dTGI of 111.6%. There was little further
enhancement in the activity of trastuzumab at 30 mg/kg by the addition of 30 mg/kg of 2C2
which showed a dTGI of 118.5% (C).
The ability of clone 16 (the parental clone from which 2C2 was derived) to
modulate the pharmacodynamic markers pHER3 and pAKT was tested in BT-474 xenograft
tumor extracts. , female athymic nude mice were implanted orthotopically with high
HER2-expressing BT-474 breast cancer cells. Animals were administered Clone 16 at 30
mg/kg twice within 48 hours. Extracts were prepared 24 hours later for analysis of pHER3,
pAKT, and total HER3 (tHER3). The results are normalized for PBS-treated control s.
There were three animals per treatment group. As shown in , Clone 16 inhibited
orylation of both HER3 and AKT by 50.0% and 46.1%, respectively, compared to
tumors from PBS—treated mice and no modulation of total HER3 was observed by Clone 16.
—ll7—
4.14. Orthotopic Human MCF-7 Breast Cancer Xenograft Model Studies
. Method.
Human MCF—7 breast cancer cells were maintained at 37°C in a 5% C02
incubator in m medium containing glutamax, 2.4/L sodium bicarbonate, Hepes and
% fetal bovine serum. Orthotopic xenografts were established by injecting 5 X 106 cells per
mouse (suspended in 50% matrigel) into the mammary fat pad on the right flank of 4 t0 6-
ld athymic nu/nu mice. Estrogen pellets (0.36 mg) were placed under the skin of the
left flank 1—2 days before cell injection. Tumors were allowed to grow up to 200 mm3 before
randomization for efficacy studies. 2C2, 2C2-YTE and trastuzumab monoclonal dies
were administered intraperitoneally. 2C2 was administered at 10 or 30 mg per kilogram body
weight (mg/kg) 2c2—YTE and trastuzumab at 10 mg/kg. Paclitaxel was administered
intravenously at 10 mg per kilogram body weight. Caliper measurements were used to
calculate tumor volumes using the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor effects are expressed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = l — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at g.
4.14.2. Results
2C2 at either 10 mg/kg or 30 mg/kg showed modest anti—tumor efficacy of
34% dTGl in a human MCF-7 breast cancer xenograft model ed orthotopically into the
mammary fat pad of female nude mice. 2C2-YTE at 10 mg/kg had similar efficacy as 2C2 at
the same concentration (A). Trastuzumab did not demonstrate efficacy in this HER2
sing model which indicated that HER2 is not sufficient to drive tumor growth. MCF-7
tumors expressed low levels of HER2 (1+) measured by Test.
Paclitaxel showed clear anti-tumor efficacy in the MCF-7 orthotopic breast
cancer model when dosed at 10 mg/kg every second day for ten days. The addition of 10
mg/kg of 2C2 t0 the paclitaxel treatment sed the anti-tumor efficacy of paclitaxel alone
at the end of the treatment phase (B). The tumors regrew at the same rate as the
paclitaxel d tumors after the treatment was stopped.
— 118 -
4.15. opic Human MDA-MB-361 Breast Cancer Xenograft Model
Studies
4.15.1. Method.
Human MDA-MB-361 breast cancer cells were ined at 37°C in a 5%
C02 incubator in RPMI 1640 medium containing 4.5g/L glucose, L-glutamine, sodium
pyruvate and 10% fetal bovine serum. Orthotopic xenografts were established by ing 5
X 106 cells per mouse (suspended in 50% matrigel) into the mammary fat pad on the right
flank of 4 to 6-week-old athymic nu/nu mice. Estrogen pellets (0.36 mg) were placed under
the skin of the left flank 1—2 days before cell injection. Tumors were allowed to grow up to
230 mm3 before randomization for efficacy studies. 2C2—YTE, and/or anti-HER2 antibodies
known in the art, in particular trastuzumab (trade name Herceptin®; e.g., US. Pat. No.
,821,337) and RhuMAb 2C4 (e.g., Patent Publication W02001/00245) designated herein as
trastuzumab and 2C4, respectively. Trastuzumab, and 2C4 monoclonal antibodies were
stered intraperitoneally at 30 mg per kilogram body weight (2C2-YTE) or at 10 mg per
kilogram body weight (trastuzumab and 2C4). Lapatinib was administered by oral gavaging
at 100 mg per kilogram body weight. Caliper measurements were used to calculate tumor
volumes using the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor s are expressed as t delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = 1 — (dT + dC) X 100,
where dT = change in mean tumor volume in treatment group ed to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
4.15.2. Results
Using a riven human breast cancer model, MDA-MB-361 (Hercept
test +2), injected orthotopically into the mammary fat pad of female nude mice,
administration of 2C2—YTE at 30 mg/kg injected twice per week for five doses led to a 70.1%
dTGl in MDA-MB-361 xenografts (FIG. . MDA-MB-361 cells express HER2 at
medium levels of 2+ characterized by HercepTest and score positive by FISH analysis
scent in situ hybridization analysis). Trastuzumab and rhuMAb 2C4both administered
—119—
at 10 mg/kg and lapatinib at 100 mg/kg administered twice daily also showed anti-tumor
efficacy in the MDA-MB-36l tumor model.
Since MDA-MB-36l tumors are driven by HER2, 2C2-YTE was combined
with drugs that target HER2 such as trastuzumab, rhuMAb 2C4 or lapatinib. The combination
treatment of 30 mg/kg of E with 10 mg/kg of trastuzumab resulted in ve anti-
tumor efficacy compared to trastuzumab alone. An additive effect was also e in a delay
in regrowth of the tumors in the absence of additional treatments (A). The
combination of 2C2-YTE with trastuzumab was better compared to combinations of 2C2-
YTE with either rhuMAb 2C4 (B) or nib (C) in this model.
4.16. Transgenic Mice sing Human FcRn or to Study Exposure of
dies with the YTE Modification.
. Method.
Transgenic female SCID mice expressing the human FcRn receptor were
given a single dose of 60 mg/kg of Clone l6-GL, 2C2 or 2C2—YTE via the intravenous route.
Serum was collected from these mice at several time points after dosing by cardiac puncture
and the blood was collected into SST microtainer tubes. The tubes were vortexed gently for
seconds and kept at room temperature for 20 minutes to allow the serum to clot. Samples
were centrifuged at 1000 X g for 10 minutes, and the serum samples were carefully
transferred into new tubes and stored at -80°C. An indirect Enzyme-Linked Immunosorbent
Assay (ELISA) format was used for the quantitative determination of 2C2 in mouse serum.
Standards, quality controls, and mouse serum samples were incubated with goat anti-human
IgG antibodies which were immobilized on a 96-well microtiter plate. After incubation,
unbound materials were removed by a wash step and 2C2 was detected using a goat anti—
human IgG with horseradish—peroxidase conjugate. An acidic stopping solution was added
and the degree of enzymatic turnover of substrate was determined by measuring absorbance
at 450 nm. The absorbance measured was directly proportional to the concentration of 2C2 or
2C2-YTE t in the mouse serum. A 2C2 or E standard curve for the assay was
used to interpolate the concentration of the serum samples.
4.16.2. Results.
2C2—YTE, which contains the YTE mutation on the 2C2 backbone, showed
higher re levels over time compared to 2C2 or Clone l6—GL (). Fourteen days
—l20—
after the single dose of antibody to these mice the serum exposure level of 2C2—YTE was
above 100 ug/ml while both 2C2 and Clone l6—GL were below 1 ug/ml. This finding
demonstrated that YTE could extend the half-life of E ed to its parental
antibody 2C2.
4.17. MEK Inhibitor s HER3 Expression And In Combination With
Anti-HER3 Antibody Shows Additive Anti-Tumor y.
KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) and BRAF (v-raf
murine sarcoma viral oncogene Bl) mutations lead to the constitutive activation of EGFR
signaling h the oncogenic Ras/Raf/Mek/Erk pathway. Kras mutation is among the
most-frequently ing mutation events in many solid tumors, especially colorectal (CRC,
—40%) and lung s (LC, ). Braf mutation also occurs at relatively high
frequency in CRC (~15%). Due to their ability to constitutively activate the ERK pathway,
mutant Kras and Braf have been shown to confer tumor resistance to RTK therapies,
especially EGFR mAbs such as Cetuximab and Panitumumab. The effect of inhibiting
mitogen-activated protein kinase (MEK) on the HER3 pathway in CRC and LC models was
examined using the MEK inhibitor selumetinib (AstraZeneca, see for e.g., WOO3/0779l4 and
WO2007/076245) alone or in combination with 2C2 (or 2C2-YTE). A number of CRC and
LC models were examined including those ing a mut-Kras (e. g. A549, LOVO) or mut-
Braf (e.g., HT-29, Colo205) or a wild type RAS (e.g., HARA—B, KNS—62).
4.17.1. Methods.
Cell culture studies: cells were plated at 105 per well in 24—well plates and in
medium containing 10% heat—inactivated FBS and allowed to reach a confluency of 80% or
more prior to treatment. 2C2 (10 ug/mL) or control antibody, MEK inhibitor selumetinib (l
or 10 uM) or a ation of 2C2 (10 ug/mL) and selumetinib (10 uM) were prepared in
complete medium. Treatments were applied following removal of plating medium. After an
incubation of 24 hours in 5% C02 at 37°C, cells were washed once with ice—cold PBS and
then lysed by adding 60 uL of 2>< sodium dodecyl sulfate (SDS) sample buffer (Invitrogen).
The samples were heated for 5 minutes and then chilled on ice for 2 minutes. The samples
were analyzed by Western blotting essentially as described above (see es, section
2.4).
Xenograft studies: Human A549 NSCLC cells (ATCC No. CCL—185) which
contain a mutation in codon 12 of the KRAS gene (were maintained at 37°C in a 5% C02
— l2l —
incubator in HAM’S F12K medium containing 4.5g/L glucose, L-glutamine, sodium
pyruvate and 10% fetal bovine serum. Xenografts were established by subcutaneously
injecting 5 X 106 cells per mouse (suspended in 50% matrigel) into the right flanks of 4— to 6—
week-old athymic nu/nu mice. Human HT-29 colorectal carcinoma cells (ATCC No. HTB-
38) were maintained at 37°C in a 5% C02 incubator in RPMI 1640 medium containing
4.5g/L glucose, L—glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts were
established by subcutaneously injecting 5 X 106 cells per mouse into the right flanks of 4— to
6—week—old athymic nu/nu mice. Human LOVO colorectal carcinoma cells (ATCC No. CCL—
229) were maintained at 37°C in a 5% C02 incubator in HAM’S F12K medium containing
4.5g/L glucose, L—glutamine, sodium pyruvate and 10% fetal bovine serum. Xenografts were
established by subcutaneously injecting 5 X 106 cells per mouse into the right flanks of 4— to
6—week—old athymic nu/nu mice. For all three tumor models, tumors were allowed to grow up
to 200 mm3 before randomization for efficacy studies. 2C2-YTE or control IgGl were
administered eritoneally. selumetinib was administered orally. For the combination
studies 2C2-YTE and selumetinib were administered at 30 mg/kg or 75 mg/kg, respectively.
r ements were used to ate tumor volumes using the formula:
tumor volume = TE + 6(length >< width >< width)
for tumors grown in mice. Antitumor s are sed as percent delta tumor
growth inhibition (TGI), which was calculated as follows:
percent delta TGI = 1 — (dT + dC) X 100,\
where dT = change in mean tumor volume in ent group compared to the value
at staging, and dC = change in mean tumor volume in control group compared to the
value at staging.
Preparation of lysates from frozen tumors: Mice were ly euthanized by
C02 asphyxiation in accordance with our in vivo protocol and tumors were excised and
transferred to Lysing Matrix A tubes. RIPA lysis buffer (500 uL) containing protease
inhibitor cocktail and phosphatase inhibitor cocktail set I and II was added, the samples were
then homogenized using a Fast Prep e. Samples were chilled on ice for 30 minutes and
ent an additional homogenization cycle before cation by centrifugation at
14,000 rpm for 10 minutes at 4°C. Clarifled lysates were transferred to fresh 1.5 mL tubes
and protein t was measured. Lysates were then stored at -80°C until analysis. The
samples were analyzed by Western blotting essentially as described above (see Examples,
section 2.4).
—l22—
4.17.2. Results.
As shown in , both total and pHER3 protein levels increased following
treatment with the MEK inhibitor selumetinib in HT-29 colorectal cancer cells grown in
e which express mutant BRAF and in LOVO cells which express a mutant KRAS
(, left and middle blots respectively). An increase of HER3 was also observed in
Colo205 cells which express mut-BRAF and in DLD-l and HCT cells, which express mutant
KRAS (FIG 36, right blot, and data not shown), following selumetinib treatment. The
increases occurred at both the 1 uM and 10 uM doses of tinib. Activity of selumetinib
was confirmed by reduction in pERK in all cell lines at both 1 HM and 10 uM doses.
Inhibition of MEK s in an inhibition of ERK phosphorylation.. The anti-HER3
antibody, 2C2, inhibited both total and pHER3 in HT-29 and LOVO cells. 2C2 also d
HER3 in Colo205 and DLD-l cells. In addition, atment of 2C2 with selumetinib
blocked the induction of total HER3 and pHER3 by selumetinib in HT-29, LOVO and DLD-
1 cells (FIG 36, and data not shown). No detectable HER3 or pHER3 could be observed in
SW480 ctal cancer cells, which express mutant KRAS, in either untreated or
selumetinib —treated cells.
As shown in A, the combination ent of 30 mg/kg of 2C2-YTE
with 75 mg/kg of selumetinib resulted in additive anti-tumor efficacy in A549 NSCLC
xenografts compared to selumetinib alone. An additive effect was also visible in a delay in
th of the tumors in the absence of additional treatments (top panel). Western blot
analysis of tumor lysates from mice treated with the combination of 30 mg/kg of 2C2-YTE
with 75 mg/kg of selumetinib over a 4 day period showed that phospho-HER3 and o-
ERK were completely inhibited. Both markers serve as pharmacodynamic read-outs for the
action of 2C2-YTE and selumetinib. Similar findings were made with the CRC xenograft
models HT—29 ( B, upper and lower panel) and LoVo ( C, upper and lower
panel). In on, phospho-AKT was found to be reduced in HT-29 tumor lysates treated
with the combination of 30 mg/kg of 2C2-YTE with 75 mg/kg of selumetinib compared to
single treatments (B, lower panel). Treatment with selumetinib alone at 75 mg/kg
lead to an increase in phospho-HER3 in the LoVo tumor extracts which was prevented in
tumors d with the combination of 2C2-YTE and selumetinib (C, lower panel).
Similar results were seen in HARA-B (data not shown).
In cell culture the levels of HER3 protein were seen to increase in response to
MEK tor across most models examined, indicating that the HER3 pathway may play a
— 123 —
2012/066038
role in resistance to MEK inhibitors. In a number of orthotopic CRC and LC xenograft model
studies the combination of E and tinib was seen to increase the anti-tumor
efficacy of either agent alone. These data t the use of 2C2 in combination with a MEK
inhibitor like selumetinib to e anti-tumor activity and prevent resistance.
4.18. Toxicology Studies in Cynomolgus Monkey
4.18.1. Method
Twenty Male cynomolgus monkeys (Macaca fascicularis) were assigned to
four groups (5 animals per group) and a total of five doses of vehicle control or 2C2-YTE at
, 30 or 120 mg/kg were administered. Animals were dosed once weekly via 5—minute IV
infusion at a dose volume of 5 mL/kg. Three animals per group were necropsied on Day 32
(three days after the final dose stration on Day 29 of the dosing phase) and two
animals per group were necropsied on Day 43 of the recovery phase (forty—five days after the
final dose administration on Day 29 of the dosing phase). Assessment of toxicity was based
on a number of s including mortality, clinical observations, body weights, dose site
tion g, clinical and anatomic pathology evaluations.
Cynomolgus monkey plasma samples were isolated and analyzed for soluble
HER3 (sHER3) levels using an anti-HER3 sandwich format with an
electrochemiluminescence (ECL) detection system for quantitation of free sHER3. Meso
Scale Discovery (MSD) bare 96—well plates (MSD, catalog number L15XA—6/L11XA—6)
were coated with 0.5 11ng of 2C2—YTE overnight at 2 to 8°C and uently blocked with
MSD Blocker A (MSD, catalog number R93BA-l). Reference Standard and Quality controls
(QC), and cynomolgus monkey plasma undiluted test samples were added to blocked plates
for 1 hour at room temperature. Biotinylated anti-hErbB3/HER3 antibody (R&D Systems,
catalog number BAM348) followed by addition of Sulfo-TAG (MSD, catalog number
R32AD-l) resulted in light emission when electrochemically stimulated. The ECL signal was
captured and recorded on a MSD Sector Imager 2400. The amount of light generated directly
correlated with the amount of sHER3 in the cynomolgus monkey plasma samples. The raw
data (ECL counts) were exported into SOFTmax® PRO. The standard curve for recombinant
human HER3 standards was fitted using a 5-parameter fit program. Cynomolgus monkey
plasma HER3 concentrations were calculated based on the rd curve using the statistical
function of SOFTmax PRO.
—124—
In addition, skin biopsy samples were collected for bioanalysis. Briefly,
matched 10 mm s are drawn on the skin on the animal and ~100 uL of PBS or HRG at
0.1 mg/mL was injected intradermally into the center of each circle. Approximately 20
minutes later a skin sample was collected from each injection site and flash frozen.
Alternatively, matched biopsy samples are collected (without prior intradermal injection)
from each and incubated for approximately 30 min at room temperature in culture media with
or without 100 ug/mL HRG followed by two washes with ice—cold PBS. The washed sample
is then flash-frozen. The tissues were then homogenized in Lysing Matrix A tubes (MP
Biomedicals) ning RIPA lysis buffer and protease tor cocktail (Sigma-Aldrich)
and atase inhibitor cocktail set I and II (EMD-Millipore) using a Fast Prep machine
(MP icals). Samples were then ted to a freeze-thaw cycle and an additional
homogenization cycle before Clarification by fugation at 14,000 rpm for 5 minutes at
4°C. Clarif1ed lysates were erred to fresh 1.5 mL tubes and protein content was
measured. The levels of total HER3 and pHER3 are determined using a sandwich ELISA
assay.
4.18.2. Results
A non-GLP, 1-month, repeat-dose toxicity study of 2C2—YTE with a ek
recovery phase was performed in cynomolgus monkeys to evaluate the toxicity and activity
of 2C2—YTE, when administered once weekly via an IV infusion to cynomolgus monkeys for
at least 1—month (5 total doses) and to assess the reversibility, persistence, or delayed
occurrence of any effects after a 6—week recovery period. No adverse effects were noted
following once weekly IV administration (5 minute infusion) of up to 120 mg/kg/dose of
2C2—YTE, for 5 weeks (5 total doses), in male cynomolgus s.
The y of 2C2—YTE to block HRG-induced pHER3 in the skin of
cynomolgus monkeys was confirmed by in vivo and ex vivo evaluations. te
suppression of circulating soluble HER3 was observed in all animals receiving intravenous
2C2-YTE. Ex-vivo stimulation of skin biopsies with HRG resulted in an increase in the
pHER3:tHER3 ratio, demonstrating that HER3 present in the skin of cynomolgus monkeys
can be activated by HRG, the predominant ligand for HER3. te suppression of HRG—
induced pHER3 was ed in all 2C2-YTE treated groups at the end of the dosing phase
(data not shown). Thus, 2C2—YTE blocked in vivo and ex vivo HRG—induced HER3
phosphorylation in cynomolgus monkey skin biopsy samples.
—125—
INCORPORATION BY REFERENCE
All publications and patents mentioned herein are hereby incorporated by
reference in their entirety as if each individual publication or patent was specifically and
dually indicated to be incorporated by reference. In addition, US. Provisional
Application Nos.: ,092 filed November 23, 2011; 61/656,670 filed June 7, 2012; and
61/722,558 filed November 5, 2012, are orated by reference in their ty for all
purposes.
The preceding description of the specific aspects will so fully reveal the
general nature of the invention that others can, by applying knowledge within the skill of the
art, readily modify and/or adapt for various applications such specific aspects, without undue
experimentation, without departing from the general concept of the present invention.
ore, such adaptations and modifications are intended to be within the meaning and
range of equivalents of the disclosed aspects, based on the teaching and guidance presented
herein. It is to be understood that the phraseology or terminology herein is for the e of
description and not of tion, such that the terminology or phraseology of the present
specification is to be interpreted by the skilled artisan in light of the teachings and guidance.
—l26—
Claims (63)
1. An dy or an antigen-binding fragment thereof, which specifically binds to HER3, comprising an antibody variable light chain region (VL) and an antibody variable heavy chain region (VH), wherein the VL comprises the amino acid sequence: [FW1]SGSLSNIGLNYVS(SEQ ID NO:19)[FW2]RNNQRPS(SEQ ID NO:21)[FW3]AAWDDSPPGEA(SEQ ID NO:23)[FW4] wherein [FW1], [FW2], [FW3] and [FW4] represent VL framework regions, and wherein the VH comprises the amino acid sequence: [FW5]YYYMQ(SEQ ID NO:31)[FW6]YIGSSGGVTNYADSVKG(SEQ ID NO:32) [FW7]VGLGDAFDI(SEQ ID NO:35)[FW8] wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions.
2. The antibody or antigen-binding fragment of claim 1, wherein the VL comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 3, and wherein the VH ses an amino acid sequence at least 80% identical to the amino acid ce of SEQ ID NO: 2.
3. The dy or antigen-binding fragment of claim 1, wherein the VL comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and wherein the VH comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2.
4. The antibody or antigen-binding fragment of claim 1, wherein the VL comprises an amino acid sequence at least 95% identical to the amino acid ce of SEQ ID NO: 3, and wherein the VH comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2.
5. The antibody or antigen-binding nt thereof of any one of claims 1-4, wherein FW1 comprises SEQ ID NO: 40 or 44, FW2 comprises SEQ ID NO: 41, FW3 comprises SEQ ID NO: 42, FW4 ses SEQ ID NO: 43, FW5 comprises SEQ ID NO: 36, FW6 ses SEQ ID NO: 37, FW7 comprises SEQ ID NO: 38 and FW8 comprises SEQ ID NO: 39.
6. The antibody or antigen-binding fragment thereof of claim 1, which ses a VL comprising SEQ ID NO: 3 and a VH comprising SEQ ID NO: 2.
7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, which comprises a heavy chain constant region.
8. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region is a human IgG constant region.
9. The antibody or n-binding fragment thereof of claim 8, wherein the human IgG nt region is an IgG1 constant region.
10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, which comprises a light chain constant region selected from the group consisting of a human kappa constant region and a human lambda nt region.
11. The antibody or antigen-binding fragment thereof of claim 10, which comprises a human lambda constant region.
12. The antibody or antigen-binding fragment of claim 8, (i) wherein the human IgG constant region comprises amino acid substitutions relative to a wild-type human IgG constant domain at positions 252, 254, and 256, wherein the numbering is according to the EU index as set forth in Kabat, and wherein (a) the amino acid at position 252 (Methionine) is substituted with Tyrosine (Y), (b) the amino acid at position 254 (Serine) is substituted with Threonine (T), and (c) the amino acid at on 256 (Threonine) is substituted with Glutamic acid (E).
13. The antibody or n-binding fragment of any one of claim 1-6, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a multispecific antibody, or an antigen-binding fragment thereof; n the antigen-binding nt is an Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2; or wherein the antibody or antigen-binding fragment thereof is conjugated to at least one heterologous agent.
14. An antibody or an antigen-binding fragment thereof, which specifically binds to HER3, comprising an antibody VL and an antibody VH, wherein the VL comprises the amino acid sequence: [FW1]X1GSX2SNIGLNYVS[FW2]RNNQRPS[FW3]AAWDDX3X4X5GEX6[FW4] wherein [FW1], [FW2], [FW3] and [FW4] represent VL framework regions, and wherein (a) X1 represents amino acid residues Arginine (R) or Serine (S), (b) X2 represents amino acid residues Serine (S) or e (L), (c) X3 represents amino acid residues Serine (S) or Glycine (G), (d) X4 represents amino acid residues e (L) or Proline (P), (e) X5 represents amino acid residues Arginine (R), Isoleucine (I), Proline (P) or Serine (S), and (f) X6 represents amino acid residues Valine (V) or Alanine (A), and wherein the VH comprises the amino acid sequence: [FW5]YYYMQ[FW6]X7IGSSGGVTNYADSVKG[FW7]VGLGDAFDI[FW8] wherein [FW5], [FW6] ,[FW7] and [FW8] ent VH framework regions, and wherein X7 represents amino acid residues Tyrosine (Y), Isoleucine (I) or Valine (V).
15. The dy or antigen-binding fragment of claim 14, wherein the VL comprises a complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, and 20, a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, and a CDR3 comprising an amino acid sequence selected from the group ting of SEQ ID NOS: 22, 23, 24, 25, 26, 27, 28, 29, and 30, and wherein the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 comprising an amino acid ce selected from the group consisting of SEQ ID NOS: 32, 33, and 34, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 35.
16. The antibody or antigen-binding nt thereof of claim 1, comprising (i) an antibody light chain comprising an dy VL sing SEQ ID NO: 3 and a human lambda light chain constant region, and (ii) an antibody heavy chain comprising an antibody VH comprising SEQ ID NO: 2 and a human IgG1 heavy chain constant region, wherein the human IgG1 nt region comprises amino acid substitutions relative to a wild-type human IgG1 constant domain at positions 252, 254, and 256, wherein the numbering is according to the EU index as set forth in Kabat, and wherein (a) the amino acid at position 252 (Methionine) is substituted with Tyrosine (Y), (b) the amino acid at position 254 (Serine) is substituted with Threonine (T), and (c) the amino acid at position 256 (Threonine) is substituted with Glutamic acid (E).
17. The antibody or antigen-binding fragment of any one of claims 1-16, wherein the antibody is a monoclonal antibody.
18. The antibody of any one of claims 1-17, wherein the antibody is a human
19. A composition comprising the antibody of any one of claims 1-18, and a pharmaceutically acceptable carrier.
20. The composition of claim 19, n the composition is refrigerator stable, wherein the antibody or antigen-binding fragment thereof is at a concentration of 25-100 mg/mL, and wherein the composition further comprises 25 mM histidine/histidine HCl, 205 mM sucrose, and 0.02% polysorbate 80 at pH 6.0.
21. A nucleic acid comprising a ce encoding the antibody or nbinding fragment according to any one of claims 1-18.
22. A vector comprising a nucleic acid according to claim 21.
23. An in vitro or ex vivo host cell sing a nucleic acid sequence according to claim 21.
24. A method of making an antibody or antigen-binding fragment thereof, comprising (a) culturing a cell comprising the nucleic acid of claim 21; and (b) isolating the antibody or antigen-binding fragment thereof.
25. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-18 in the manufacture of a medicament for inhibiting the proliferation of a cell expressing HER3.
26. The use of claim 25, wherein said cell is a human head and neck tumor cell, a human non-small cell lung cancer cell, a human colorectal tumor cell, a human prostate tumor cell, or a human breast cancer cell.
27. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-18 in the cture of a ment for reducing tumor volume of a HER3- expressing tumor in a subject.
28. The use of claim 27, wherein said tumor is a human head and neck tumor, a human non-small cell lung cancer tumor, a human colorectal tumor, a human prostate tumor, or a human breast cancer tumor.
29. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-18 in the manufacture of a ment for ng cancer in a subject.
30. The use of claim 29, wherein the cancer is a carcinoma.
31. The use of claim 29, wherein the cancer is selected from the group consisting of colorectal cancer, lung cancer, gastric cancer, breast cancer, head and neck cancer, te cancer, pancreatic cancer, thyroid cancer, melanoma, esophageal , ovarian cancer, kidney cancer, and bladder cancer.
32. The use of claim 31, n the cancer is a squamous cell carcinoma of the head and neck.
33. The use of claim 29, wherein the cancer comprises cells comprising a KRAS mutation.
34. The use of claim 29, wherein the cancer comprises cells expressing heregulin.
35. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-18 in the manufacture of a medicament for treating cancer in a t, said ng comprising administration of the antibody or antigen binding fragment to the subject with an additional therapy.
36. The use of claim 35, wherein the additional therapy is surgery.
37. The use of claim 35, wherein the additional therapy is radiation.
38. The use of claim 35, wherein the additional therapy is a therapeutically effective amount of a second agent, wherein the second agent is an anti-cancer agent other than the antibody or antigen-binding fragment thereof.
39. The use of claim 38, wherein the second agent is an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, or a MEK inhibitor.
40. The use of claim 38, wherein the second agent is cetuximab, panitumumab, mab, nimotuzumab, MM-151, or Sym004.
41. The use of claim 38, wherein the second agent is trastuzumab, zumab emtansine, or pertuzumab.
42. The use of claim 38, wherein the second agent is a kinase inhibitor.
43. The use of claim 38, wherein the second agent is gefitinib, canertinib, lapatinib, erlotinib, ib, neratinib, selumetinib, WX-554, selumetinib, inib, refametinib, E-6201, MEK-162, or MEHD-7945 A.
44. The use of claim 38, n the cancer is a squamous cell carcinoma of the head and neck, and n the second agent is cetuximab.
45. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-18.
46. A method of diagnosing a HER3-expressing cancer in a patient, wherein the method comprises the steps of: (a) contacting a biological sample from the t with the antibody or antigenbinding fragment of any one of claims 1-18; (b) detecting binding of the antibody or n-binding fragment to HER3 to determine a HER3 protein level in the biological sample from the patient; and (c) comparing the HER3 protein level with a standard HER3 protein level.
47. A method of monitoring the HER3 protein level during treatment of a HER3- sing cancer in a patient, wherein the method ses the steps of: (a) contacting a biological sample from the patient with an antibody or nbinding fragment according to any one of claims 1 to 18; (b) detecting binding of the antibody or antigen-binding fragment to HER3 to determine a HER3 protein level in the biological sample from the patient; and (c) comparing the HER3 protein level with a standard HER3 protein level.
48. A method of monitoring HER3 protein activity level during ent of cancer in a patient being administered the antibody or antigen-binding fragment of any one of claims 1-18, wherein the method comprises the steps of: (a) contacting a biological sample from the patient with an antibody or nbinding fragment that specifically binds to orylated HER3; (b) detecting binding of the antibody or antigen-binding fragment to phosphorylated HER3 to determine a HER3 protein activity level in the biological sample from the t; (c) comparing the HER3 protein activity level with a standard HER3 n activity level.
49. An antibody according to claim 1 substantially as herein described or exemplified.
50. An antibody according to claim 14 substantially as herein described or exemplified.
51. A composition according to claim 19 substantially as herein described or exemplified.
52. A nucleic acid according to claim 21 substantially as herein described or ified.
53. A vector according to claim 22 substantially as herein bed or exemplified.
54. A host cell according to claim 23 substantially as herein described or exemplified.
55. A method according to claim 24 ntially as herein described or exemplified.
56. A use according to claim 25 substantially as herein described or exemplified.
57. A use according to claim 27 substantially as herein described or exemplified.
58. A use according to claim 29 substantially as herein described or exemplified.
59. A use according to claim 35 substantially as herein described or exemplified.
60. A kit according to claim 45 substantially as herein described or ified.
61. A method according to claim 46 substantially as herein described or exemplified.
62. A method according to claim 47 substantially as herein described or exemplified.
63. A method according to claim 48 substantially as herein described or exemplified.
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161563092P | 2011-11-23 | 2011-11-23 | |
US61/563,092 | 2011-11-23 | ||
US201261656670P | 2012-06-07 | 2012-06-07 | |
US61/656,670 | 2012-06-07 | ||
US201261722558P | 2012-11-05 | 2012-11-05 | |
US61/722,558 | 2012-11-05 | ||
PCT/US2012/066038 WO2013078191A1 (en) | 2011-11-23 | 2012-11-20 | Binding molecules specific for her3 and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ625046A NZ625046A (en) | 2016-09-30 |
NZ625046B2 true NZ625046B2 (en) | 2017-01-05 |
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