ANTI-NOTCH1 ANTIBODIES
FIELD OF THE INVENTION
The present invention relates to anti-notch 1 antibodies. The present invention
further relates to the methods of using such antibodies in the treatment of cancer.
BACKGROUND
Notch receptors control normal cell growth, differentiation, and death in
multicellular organisms through a signaling pathway that is triggered by ligand-induced
proteolysis (Bray, Nat. Rev. Mol. Cell Biol. 7(9):678-689, 2006). The mature Notch
heterodimer after furin-like protease cleavage at site S 1 is held in an auto-inhibited state
by a juxtamembrane negative regulatory region (NRR) consisting of three Lin12/Notch
repeats (LNR-A, B, C) and the heterodimerization (HD) domain. The HD domain is
divided into N-terminal (HD1 ) and C-terminal (HD2) halves by cleavage at site S 1.
Through an uncertain mechanism, binding of ligands of the Delta/Serrate/Lag-2 (DSL)
family to the N-terminal, EGF-repeat region relieves this inhibition and induces two
successive additional cleavages at S2 near the C-terminal region HD-2, and S3 within
transmembrane domain in Notch that are catalyzed by ADAM-type metalloproteinase
and gamma-Secretase, respectively (Gordon, W.R., et.al, Nature Structural & Molecular
Biology, 2007, volume 14, 295-300). The latter cleavage releases the intracellular
domain of Notch (Notch ICD) , permitting it to translocate to the nucleus and activate the
transcription of target genes.
In mammalian cells, there are four known Notch receptors. Notch1-4 have broad,
overlapping patterns of expression in embryonic and adult tissues, and fulfill nonredundant
roles during hematopoietic stem cell specification, T cell development,
intestinal crypt cell specification and vascular development. Acquired abnormalities
involving specific Notchl receptors have been implicated in cancers, such as T cell
acute lymphoblastic leukemia (T-ALL), breast cancer and lung cancer. In addition,
activated Notchl is a potent inducer of leukemia in murine models and is overexpressed
in various solid tumors, including non-small cell lung cancer, breast cancer
and ovarian cancer.
Over 50% of T-ALL patients harbor mutations in the Notchl receptor some of
which result in constitutive cleavage of the receptor and production of the Notchl ICD due
in part to Notc ligand-hypersenstivity or ligand-independent activation caused by
alterations in or near the NRR auto-inhibitory domain. These mutations are categorized
into 3 major classes. Class 1 mutations are single amino acid substitutions and small inframe
deletions or insertions in HD1 . Class 2 mutations are longer insertions in the
distal region of HD2 that relocate the S2-metalloprotease cleavage site beyond the
auto-inhibitory NRR domain. Class 3 mutations, also called juxtamembrane expansion
(JMEs) mutations, occur from large insertions that displace the NRR away from the cell
membrane.
Several strategies are in development to inhibit Notch signaling for therapeutic
purposes in cancer. One approach is to block the proteolytic release of intracellular
Notch from the membrane by treatment with inhibitors of gamma-secretase (GSIs).
Although GSIs have progressed into the clinic, they cannot distinguish individual Notch
receptors and cause intestinal toxicity attributed to the inhibition of both Notchl and
Notch2. There is still a need in the art for novel anti-Notchl therapies for the treatment
of cancer while providing reduced side effects, in particular, intestinal toxicity.
SUMMARY
In one embodiment, the present invention provides for antibodies that bind to
Notchl , having a heavy chain variable region having a CDR1 region, a CDR2
region, and a CDR3 region from the heavy chain variable region comprising SEQ ID
NO: 7 1.
In another embodiment, the present invention provides for antibodies that
bind to Notchl, having a light chain variable region having a CDR1 region, a CDR2
region, and a CDR3 region from the light chain variable region comprising SEQ ID
NO: 97.
The present invention also provides for antibodies that bind to Notchl having
1) a heavy chain variable region having a CDR1 region, a CDR2 region, and a
CDR3 region from the heavy chain variable region comprising SEQ ID NO: 7 1, and
2) a light chain variable region having a CDR1 region, a CDR2 region, and a CDR3
region from the light chain variable region comprising SEQ ID NO: 97.
Also provided are antibodies that bind to Notchl having a heavy chain
variable region amino acid sequence that is at least 90% identical to SEQ ID NO: 7 1.
Further provided are antibodies that bind to Notchl having a heavy chain variable
region amino acid sequence as set forth in SEQ ID NO: 7 1.
Also provided are antibodies that bind to Notchi having a light chain variable
region amino acid sequence that is at least 90% identical to SEQ ID NO: 97. Further
provided are antibodies that bind to Notchi having a light chain variable region
amino acid sequence as set forth in SEQ ID NO: 97.
Also provided are antibodies that bind to Notchi having a heavy chain amino
acid sequence that is at least 90% identical to SEQ ID NO: 111. Further provided
are antibodies that bind to Notchi having a heavy chain amino acid sequence as set
forth in SEQ ID NO: 111.
Also provided are antibodies having a light chain amino acid sequence that is
at least 90% identical to SEQ ID NO: 113. Further provided are antibodies having a
light chain amino acid sequence as set forth in SEQ ID NO: 113.
In a further embodiment, the invention provides for antibodies that bind to
Notchi, having a heavy chain variable region amino acid sequence that is at least
90% identical to SEQ ID NO: 7 1; and a light chain variable amino acid sequence that
is at least 90% identical to SEQ ID NO: 97. Further provided are antibodies that
bind to Notchi having a heavy chain variable region amino acid sequence as forth in
SEQ ID NO: 71; and a light chain variable region amino acid sequence as set forth
in SEQ ID NO: 97.
In a further embodiment, the invention provides for antibodies that bind to
Notchi , having a heavy chain amino acid sequence that is at least 90% identical to
SEQ ID NO: 111; and a light chain amino acid sequence that is at least 90%
identical to SEQ ID NO: 113. Further provided are antibodies that bind to Notchi
having a heavy chain amino acid sequence as set forth in SEQ ID NO: 111, and a
light chain amino acid sequence as set forth in SEQ ID NO: 113.
In a further embodiment, the invention provides for antibodies, that bind to
human Notchi , wherein the antibodies bind an epitope having at least 8 amino acid
residues selected from Asn 1461, Lys 1462, Val 1463, Cys 1464, Leu 1466, Leu
1580, Tyr 1621, Gly 1622, Met 1670, Asp 1671, Val 1672, Arg 1673, Leu 1707, Ala
1708, Leu 1710, Leu 171 1, Leu 1712, Leu 1713, Leu 1716 and Leu 1718.
In another embodiment, the present invention provides for antibodies that
bind to Notchi, having a heavy chain variable region having a CDR1 region, a CDR2
region, and a CDR3 region from the heavy chain variable region comprising SEQ ID
NO: 115.
In another embodiment, the present invention provides for antibodies that
bind to Notchi, having a light chain variable region having a CDR1 region, a CDR2
region, and a CDR3 region from the light chain variable region comprising SEQ ID
NO: 129.
The present invention also provides for antibodies that bind to Notchi having
1) a heavy chain variable region having a CDR1 region, a CDR2 region, and a
CDR3 region from the heavy chain variable region comprising SEQ ID NO: 115, and
2) a light chain variable region having a CDR1 region, a CDR2 region, and a CDR3
region from the light chain variable region comprising SEQ ID NO: 129.
Also provided are antibodies that bind to Notchi having a heavy chain
variable region amino acid sequence that is at least 90% identical to SEQ ID NO:
115. Further provided are antibodies that bind to Notchi having a heavy chain
variable region amino acid sequence as set forth in SEQ ID NO: 115.
Also provided are antibodies that bind to Notchi having a light chain variable
region amino acid sequence that is at least 90% identical to SEQ ID NO: 129.
Further provided are antibodies that bind to Notchi having a light chain variable
region amino acid sequence as set forth in SEQ ID NO: 129.
Also provided are antibodies that bind to Notchi having a heavy chain amino
acid sequence that is at least 90% identical to SEQ ID NO: 149. Further provided
are antibodies that bind to Notchi having a heavy chain amino acid sequence as set
forth in SEQ ID NO: 149.
Also provided are antibodies having a light chain amino acid sequence that is
at least 90% identical to SEQ ID NO: 151 . Further provided are antibodies having a
light chain amino acid sequence as set forth in SEQ ID NO: 15 1 .
In a further embodiment, the invention provides for antibodies that bind to
Notchi, having a heavy chain variable region amino acid sequence that is at least
90% identical to SEQ ID NO: 115; and a light chain variable amino acid sequence
that is at least 90% identical to SEQ ID NO: 129. Further provided are antibodies
that bind to Notchi having a heavy chain variable region amino acid sequence as
forth in SEQ ID NO: 115; and a light chain variable region amino acid sequence as
set forth in SEQ ID NO: 129.
In a further embodiment, the invention provides for antibodies that bind to
Notchi , having a heavy chain amino acid sequence that is at least 90% identical to
SEQ ID NO: 149; and a light chain amino acid sequence that is at least 90%
identical to SEQ ID NO: 151 . Further provided are antibodies that bind to Notchi
having a heavy chain amino acid sequence as set forth in SEQ ID NO: 149, and a
light chain amino acid sequence as set forth in SEQ ID NO: 151 .
In a further embodiment, the invention provides for antibodies, that bind to
human Notchi, wherein the antibodies bind an epitope having at least 8 amino acid
residues selected from Asp 1458, Asn 1461, Val 1463, Cys 1464, Leu 1466, Leu
1580, Met 1581 , Pro 1582, Tyr 1621, Gly 1622, Arg 1623, Asp 1671, Val 1672, Arg
1673, Gly 1674, Leu 1710, Gly 171 1, Ser 1712, Leu 1713, Asn 1714, lie 1715, Pro
1716 and Lys 1718.
In another embodiment, the invention provides for antibodies that
demonstrate higher inhibition of Notchi activation of a mutant Notchi receptor
compared to inhibition of Notchi activation of a native Notchi receptor. It is further
provided that the mutant Notchi receptor has a mutation in the negative regulatory
region (NRR). In a further embodiment, the mutation in the NRR is selected from
the group consisting of a class 1, a class 2, and a class 3 mutation. In a further
embodiment, the mutation in the NRR is associated with cells having abnormal
activation of Notchi . It is further provided that the cells are T-cell acute
lymphoblastic leukemia (T-ALL) cells. It is also provided that the T-ALL cells are
selected from the group consisting of HPB-ALL, ALL-SIL, CCRF-CEM, MOLT-4 and
DND-41 cells.
Also provided are antibodies that bind to Notchi and compete for binding to
Notchi with any of the antibodies described herein.
In a further embodiment, the invention provides for antibodies that bind to
Notchi where the antibodies are of isotype IgA, IgD, IgE, IgG, or IgM. Further
provided are antibodies that bind to Notchi where the isotype is IgG, and wherein
the subclass is lgG1, lgG2, lgG3 or lgG4, or is derived therefrom. Also provided are
antibodies that bind to Notchi where the subclass is derived from lgG1 .
In a further embodiment, the invention provides nucleic acids that encode any
of the antibodies described herein, or that encode any of the heavy chains and/or
light chains of antibodies described herein. For example, in one embodiment, the
invention provides nucleic acids having the sequence as set forth in SEQ ID NO:
112. In a further embodiment, the invention provides nucleic acids having the
sequence as set forth in SEQ ID NO: 114. In another embodiment, the invention
provides nucleic acids having the sequence as set forth in SEQ ID NO: 150. In a
further embodiment, the invention provides nucleic acids having the sequence as set
forth in SEQ ID NO: 152.
In a further embodiment, the invention provides for a vector comprising any of
the nucleic acids described herein. In a further embodiment, the invention provides
for host cells comprising any of the vectors described herein. In a further
embodiment, the invention provides a process for producing any of the antibodies
described herein comprising cultivating any host cells described herein and
recovering the antibodies from the culture media. In a further embodiment, the
invention provides host cells that recombinantly produce any of the antibodies
described herein. In one embodiment, any of the host cells described herein are
isolated.
In a further embodiment, the present invention provides pharmaceutical
compositions comprising any of the antibodies described herein and
pharmaceutically acceptable carriers. In a further embodiment, the invention
provides methods of treating disorders in subjects in need thereof, comprising
administering to the subjects any of the antibodies or pharmaceutical compositions
described herein. The invention further provides methods of treating disorders that
are associated with abnormal activation of Notch 1 in subjects in need thereof,
comprising administering to the subjects any of the antibodies or pharmaceutical
compositions described herein. In a further embodiment, the invention provides
methods of treating disorders, such as T-cell acute lymphoblastic leukemia (T-ALL),
non-small cell lung cancer (NSCLC), breast cancer and colon cancer, in subjects in
need thereof, comprising administering to the subjects any of the antibodies or
pharmaceutical compositions described herein. The invention further provides for a
method of treating disorders in subjects in need thereof, comprising administering to
the subjects any of the antibodies or pharmaceutical compositions described herein
in combination with one or more therapeutic agent.
In another embodiment, the invention provides for any of the antibodies
disclosed herein for use in therapy. In a further embodiment, the invention provides
the use of any of the antibodies disclosed herein for the manufacture of
medicaments for therapy. In a further embodiment, the invention provides for any of
the antibodies disclosed herein for use in treating disorders that are associated with
abnormal activation of Notch 1 in subjects in need thereof. In a further embodiment,
the invention provides for any of the antibodies disclosed herein for use in treating
disorders, such as T-cell acute lymphoblastic leukemia (T-ALL), non-small cell lung
cancer (NSCLC), breast cancer and colon cancer, in subjects in need thereof.
In a further embodiment, the invention provides for antibodies that bind to
human, mouse and cynomolgus (hereinafter "cyno") Notchi, but do not bind to
human Notch2. In another embodiment, the invention provides for antibodies that
bind to human, mouse and cyno Notchi , but do not bind to human and mouse
Notch3.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic diagram of recombinant, S1-cleaved, heterodimeric
Notchi NRR protein immunogen with Avi and His tags.
FIG. 2 shows recombinant human Notchi NRR and Notch3 NRR domain swap
chimeric constructs for epitope mapping of the anti-Notchl antibodies rat 351-mlgG1,
rat 438-mlgG1 and A2.
FIG. 3 shows a structural view of the rat 438 epitope on the human Notchi NRR.
FIG. 4 shows a structural view of the rat 351 epitope on the human Notchi NRR.
FIG. 5 shows a structural view of the A2 epitope on the human Notchi NRR.
FIG. 6 shows the superposition of the structures of Notchi NRR bound to rat 438
and A2 antibodies
FIG. 7 shows the superposition of the structures of Notchi NRR (shown as
ribbons) bound to rat 351 and A2 antibodies (shown as molecular surfaces).
FIG. 8 shows the neutralizing activity of humanized 438 VH1 .1/VL1.8, rat 438-
mlgG1 and A2 antibodies against Notchi dependent signaling in human Notchi
reporter cells.
FIG. 9 shows the neutralizing activity of humanized 438 VH1 .1/VL1.8, rat 438-
mlgG1 and A2 antibodies against Notchi dependent signaling in mouse Notchi
reporter cells.
FIG. 10 shows the neutralizing activity of rat 351 and A2 antibodies against
human Notchi signaling.
FIG. 11 shows the neutralizing activity of rat 351 and A2 antibodies against
mouse Notchi signaling.
FIG. 12 shows the neutralizing activity of humanized 351 variants, rat 351-
mlgG1 and A2 antibodies against Notchi dependent signaling in human Notchi
reporter cells.
FIG. 13 shows the neutralizing activity of humanized 351 variants, rat 351-
mlgG1 and A2 antibodies against Notchl dependent signaling in mouse Notchl
reporter cells.
FIG. 14A and 14B show structural views of the interaction interface between rat
351 and Notchl NRR in the LNR-A region.
FIG. 15 shows the neutralization activity of rat 351, mutant rat 351 and A2 in coculture
reporter gene assays.
FIG. 16 shows representative epifluorescent images of CD31-Cy3
immunostaining of HUVEC-sprouts at day 10 of treatment with rat 438, rat 351 and A2,
and control medium alone and anti-VEGF antibody.
FIG. 17 shows representative confocal images of Isolectin B4-Alexa488 staining
in a mouse retinal model of angiogenesis after treatment with rat 438-mlgG1, rat 351-
mlgG1 and A2 antibodies, and controls anti-E.tenella antibody and no treatment.
FIG. 18 shows a Western blot analysis of protein extracts generated from
CCD1076SK human fibroblasts plated on recombinant human DLL4 ligand and treated
with increasing concentrations of rat 351, rat 438 and A2, and control anti-E.tenella
antibody.
FIG. 19 shows a Western blot analysis of protein extracts generated from HBPALL
cells treated with increasing concentrations of humanized 438 VH1 .1/VL1.8, rat
438-mlgG1 , and control anti-E.tenella antibody.
FIG. 20 shows a Western blot analysis of protein extracts generated from T-ALL
cell lines treated with increasing concentrations of rat 351-mlgG1, rat 438-mlgG1 and
A2, and control anti-E.tenella antibody.
FIG. 2 1 shows immunohistochemical detection of Notchl receptors and Jaggedl
ligand in the 37622A1 NSCLC patient derived xenograft.
FIG. 22 shows a chromatogram indicating that the 37622A1 NSCLC patient
derived xenograft possessed a G13V mutation in the human K-ras gene.
FIG. 23 shows western blot analysis of protein extracts generated from 37622A1
NSCLC patient derived xenografts treated with rat 438-mlgG1, A2 and control anti-
E.tenella antibodies.
FIG. 24 shows a Western blot analysis of protein extracts generated in 87393A1
NSCLC patient derived xenografts treated with rat 351-mlgG1 and control anti-E.tenella
antibodies.
FIG. 25 shows immunohistochemical detection of involucrin expression in
87393A1 NSCLC patient derived xenografts after treatment with rat 351-mlgG1 and
control anti-E.tenella antibodies.
FIG. 26 shows a Western blot analysis of involucrin expression in 87393A1
NSCLC patient derived xenografts after treatment with rat 351-mlgG1 and control anti-
E.tenella antibodies.
FIG. 27 shows histochemical identification of secretory goblet cells using Alcian
Blue stain on the ileum section of mouse intestines from Calu-6 efficacy study treated
with rat 438-mlgG1, A2, and control anti-E.tenella antibody.
FIG. 28 shows anti-Ki67 immunohistochemistry on mouse intestinal crypts
from Calu-6 efficacy study treated with rat 438-mlgG1, A2 and control anti-E.tenella
antibody.
FIG. 29 shows anti-Ki67 immunohistochemistry on mouse intestinal crypts from
87393A1 patient derived xenograft efficacy study treated with rat 351-mlgG1 and
control anti-E.tenella antibodies.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to isolated antibodies, particularly human,
humanized, chimeric and rat monoclonal antibodies that bind to Notchl . Further, the
present disclosure provides for isolated antibodies that demonstrate higher inhibition of
Notchl activation of a mutant Notchl receptor compared to inhibition of Notchl
activation of a native Notchl receptor. The disclosure provides for isolated antibodies
and methods of making such antibodies and pharmaceutical compositions containing
the antibodies. The present disclosure further relates to immunoconjugates and
bispecific molecules containing such antibodies. The disclosure also relates to methods
of using the antibodies to inhibit Notchl activation, and treat various diseases related to
abnormal cell growth, such as cancer (e.g. T-cell acute lymphoblastic leukemia (T-ALL),
non-small cell lung cancer (NSCLC), colon cancer, breast cancer and ovarian cancer.
General Techniques
Unless otherwise indicated the methods and techniques of the present invention
are generally performed according to conventional methods well known in the art and as
described in various general and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated. See, e.g., Sambrook et
al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane
Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1990), which are incorporated herein by reference.
Definitions
"Notchi" or "Notch-1" refers to native, variants, isoforms and species homologs
of human Notchi protein. Native human Notchi protein, for example, is made up of a
leader peptide, a large epidermal growth factor (EGF)-like repeat region, three Lin12
repeats, a N terminal heterodimerization domain (HD-1), a C terminal
heterodimerization domain (HD-2), a transmembrane (TM) sequence and an
intracellular domain (Notchi ICD) . The NCBI/GenBank accession number of the full
length human Notchi is NM_017617.2
"Notchi negative regulatory region", or "Notchi NRR" as used herein, unless
otherwise indicated, refers to any native or synthetic polypeptide region of Notchi
consisting of the three Lin12 domains and the amino acid sequence or sequences
located between the three Lin12 domains, plus the HD1 and HD2 domains of Notchi .
In one embodiment, the "Notchi NRR" includes the three Lin12 domains and two
heterodimerization domains HD-1 , and HD-2, wherein the HD-1 and HD-2 domains of
Notchi are covalently bonded and not yet cleaved by the furin-like protease (before S 1
cleavage). In another embodiment, the "Notchi NRR" includes the three Lin12 domains
and the two heterodimerization domains HD-1, and HD-2, wherein the HD-1 and HD-2
domains are non-covalently bonded (after S 1 cleavage). In one aspect of this
embodiment, the S2 site within the HD-2 domain has not been cleaved by the ADAMtype
metalloproteases. In another particular aspect of this embodiment, the S2 site
within the HD-2 domain is being cleaved or has already been cleaved by the ADAMtype
metalloproteases. (Gordon, W.R., et.al, Nature Structural & Molecular Biology,
2007, volume 14, 295-300).
An "antibody" is an immunoglobulin molecule capable of specific binding to a
target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least
one antigen recognition site, located in the variable region of the immunoglobulin
molecule. As used herein, the term encompasses not only intact polyclonal or
monoclonal antibodies, but also fragments (e.g., antigen binding portions) thereof (such
as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies such as shark and
camelid antibodies), and fusion proteins comprising an antibody portion (such as
domain antibodies), and any other modified configuration of the immunoglobulin
molecule that comprises an antigen recognition site. An antibody includes an antibody
of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not
be of any particular class. Depending on the antibody amino acid sequence of the
constant domain of its heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE,
IgG, and IgM, and several of these may be further divided into subclasses, e.g., lgG 1,
lgG2, lgG3, lgG4, lgA1 and lgA2. The heavy-chain constant domains that correspond
to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and
mu, respectively. The subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
An "isolated antibody", as used herein, is intended to refer to an antibody that is
substantially free of other antibodies having different antigenic specificities (e.g., an
isolated antibody that specifically binds Notch l is substantially free of antibodies that
specifically bind antigens other than Notch l ) . An isolated antibody that specifically
binds Notch l may, however, have cross-reactivity to other antigens, such as Notch-1
molecules from other species. Moreover, an isolated antibody may be substantially free
of other cellular material and/or chemicals.
As used herein, "monoclonal antibody" refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical except for possible naturally-occurring mutations
that may be present in minor amounts. Monoclonal antibodies are highly specific, being
directed against a single antigenic site.
"Humanized" antibody refers to forms of non-human (e.g. murine) antibodies that
are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as
Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain
minimal sequence derived from non-human immunoglobulin. Preferably, humanized
antibodies are human immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are replaced by residues from
a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having
the desired specificity, affinity, and capacity. In some instances, Fv framework region
(FW) residues of the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, the humanized antibody may comprise residues that are found
neither in the recipient antibody nor in the imported CDR or framework sequences, but
are included to further refine and optimize antibody performance. In general, the
humanized antibody will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR regions correspond to
those of a non-human immunoglobulin and all or substantially all of the FW regions are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise at least a portion of an immunoglobulin constant region or
domain (Fc), typically that of a human immunoglobulin. Other forms of humanized
antibodies have one or more CDRs (L-CDR1, L-CDR2, L-CDR3, H-CDR1 , H-CDR2, or
H-CDR3) which are altered with respect to the original antibody, which are also termed
one or more CDRs "derived from" one or more CDRs from the original antibody.
"Human antibody" or "fully human antibody" is intended to include antibodies
having variable regions in which both the framework and CDR regions are derived from
human germline immunoglobulin sequences. Furthermore, if the antibody contains a
constant region, the constant region also is derived from human germline
immunoglobulin sequences. The human antibodies of the disclosure may include
amino acid residues not encoded by human germline immunoglobulin sequences (e.g.,
mutations introduced by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). This definition of a human antibody includes antibodies comprising at
least one human heavy chain polypeptide or at least one human light chain polypeptide.
Human antibodies can be produced using various techniques known in the art.
The term "chimeric antibody" is intended to refer to antibodies in which the
variable region sequences are derived from one species and the constant region
sequences are derived from another species, such as an antibody in which the variable
region sequences are derived from a mouse antibody and the constant region
sequences are derived from a human antibody.
The term "recombinant antibody", as used herein, includes all antibodies that are
prepared, expressed, created or isolated by recombinant means. Such recombinant
antibodies have variable regions in which the framework and CDR regions are derived
from germline immunoglobulin sequences. In certain embodiments, however, such
recombinant antibodies can be subjected to in vitro mutagenesis (or, when an animal
transgenic for Ig sequences is used, in vivo somatic mutagenesis) and thus the amino
acid sequences of the VH and VL regions of the recombinant antibodies are sequences
that, while derived from and related to germline VH and VL sequences, may not
naturally exist within the antibody germline repertoire in vivo.
The phrases "an antibody recognizing an antigen" and "an antibody specific for
an antigen" are used interchangeably herein with the term "an antibody which binds
specifically to an antigen."
As known in the art, the term "Fc region" is used to define a C-terminal region of
an immunoglobulin heavy chain (CH2+CH3). The "Fc region" may be a native
sequence Fc region or a variant Fc region.
A "native sequence Fc region" comprises an amino acid sequence identical to
the amino acid sequence of an Fc region found in nature. A "variant Fc region"
comprises an amino acid sequence which differs from that of a native sequence Fc
region by virtue of at least one amino acid modification, yet retains at least one function
of the native sequence Fc region.
The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to
the Fc region of an antibody. For example, the FcR can be a native sequence human
FcR. Furthermore, the FcR can be one that binds an IgG antibody (a gamma receptor)
and includes receptors of the FcyRI, FcyRII, FcyRIII, and FcyRIV subclasses, including
allelic variants and alternatively spliced forms of these receptors. FcyRII receptors
include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which
have similar amino acid sequences that differ primarily in the cytoplasmic domains
thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. As will be appreciated by those of
skill in the art, inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based
inhibition motif (ITIM) in its cytoplasmic domain. FcRs have been extensively reviewed
and are well known to those of skill in the art. Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein. The term also
includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus, and the extended half life of IgGs.
The term "binds" refers to an affinity between two molecules, for example, an
antigen and an antibody. An antibody that "specifically binds to Notchl" refers to a
preferential binding of an antibody to Notchl antigen in a sample comprising multiple
different antigens, with a difference in KD of at least 100 fold or preferably 1,000 fold.
The term "high affinity" refers to an antibody having a KD of 1 x 10 6 M or less,
more preferably having a KD of 1 x 10 8 M or less. Affinity can be measured using, for
example, surface Plasmon resonance.
"Epitope" includes any protein determinant capable of specific binding to an
immunoglobulin or T-cell receptor. Epitopic determinants 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.
The term "kon" , as used herein, is intended to refer to the on-rate, or association
rate of a particular antibody-antigen interaction, whereas the term "koff," as used herein,
is intended to refer to the off-rate, or dissociation rate of a particular antibody-antigen
interaction. The term "KD" , as used herein, is intended to refer to the equilibrium
dissociation constant, which is obtained from the ratio of k0f to kon (i.e,. k0ff/kon) and is
expressed as a molar concentration (M). KD values for antibodies can be determined
using methods well established in the art. One method for determining the KD of an
antibody is by using surface plasmon resonance, typically using a biosensor system
such as a Biacore® system.
The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used
interchangeably herein to refer to chains of amino acids of any length, preferably,
relatively short (e.g., 10-100 amino acids). The chain may be linear or branched, it may
comprise modified amino acids, and/or may be interrupted by non-amino acids. It is
understood that the polypeptides can occur as single chains or associated chains.
As known in the art, "polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to chains of nucleotides of any length, and include DNA and RNA. The
nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be incorporated into a chain by
DNA or RNA polymerase.
A "variable region" of an antibody refers to the variable region of the antibody
light chain or the variable region of the antibody heavy chain, either alone or in
combination. As known in the art, the variable regions of the heavy and light chain each
consist of four framework regions (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 FWs and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of antibodies.
A "CDR" of a variable domain are amino acid residues within the variable region
that are identified in accordance with the definitions of the Kabat, Chothia, the
accumulation of both Kabat and Chothia, AbM, contact, and/or conformational
definitions or any method of CDR determination well known in the art. Antibody CDRs
may be identified as the hypervariable regions originally defined by Kabat et al. See,
e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed.,
Public Health Service, NIH, Washington D.C. The positions of the CDRs may also be
identified as the structural loop originally described by Chothia and others. See, e.g.,
Chothia et al., 1989, Nature 342:877-883. Other approaches to CDR identification
include the "AbM definition," which is a compromise between Kabat and Chothia and is
derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or
the "contact definition" of CDRs based on observed antigen contacts, set forth in
MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In another approach, referred to
herein as the "conformational definition" of CDRs, the positions of the CDRs may be
identified as the residues that make enthalpic contributions to antigen binding. See,
e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1 156-1 166. Still other
CDR boundary definitions may not strictly follow one of the above approaches, but will
nonetheless overlap with at least a portion of the Kabat CDRs, although they may be
shortened or lengthened in light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not significantly impact antigen
binding. As used herein, a CDR may refer to CDRs defined by any approach known in
the art, including combinations of approaches. The methods used herein may utilize
CDRs defined according to any of these approaches. For any given embodiment
containing more than one CDR, the CDRs may be defined in accordance with any of
Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.
A "constant region" of an antibody refers to the constant region of the antibody
light chain or the constant region of the antibody heavy chain, either alone or in
combination.
A "host cell" includes an individual cell or cell culture that can be or has been a
recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include
progeny of a single host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the original parent cell due
to natural, accidental, or deliberate mutation. A host cell includes cells transfected in
vivo with a polynucleotide(s) of the present disclosure.
As used herein, "vector" means a construct, which is capable of delivering, and,
preferably, 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 eukaryotic cells, such as producer cells.
As used herein, "expression control sequence" means a nucleic acid sequence
that directs transcription of a nucleic acid. An expression control sequence can be a
promoter, such as a constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid sequence to be
transcribed.
As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical
acceptable excipient" includes any material which, when combined with an active
ingredient, allows the ingredient to retain biological activity and is non-reactive with the
subject's immune system. Examples include, but are not limited to, any of the standard
pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions
such as oil/water emulsion, and various types of wetting agents. Preferred diluents for
aerosol or parenteral administration are phosphate buffered saline (PBS) or normal
(0.9%) saline. Compositions comprising such carriers are formulated by well known
conventional methods.
An "individual" or a "subject" is a mammal, more preferably, a human. Mammals
also include, but are not limited to, farm animals, sport animals, pets, primates, horses,
dogs, cats, mice and rats.
An "isolated protein", "isolated polypeptide" or "isolated antibody" is a protein,
polypeptide or antibody that by virtue of its origin or source of derivation ( 1) is not
associated with naturally associated components that accompany it in its native state,
(2) is free of other proteins from the same species, (3) is expressed by a cell from a
different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically
synthesized or synthesized in a cellular system different from the cell from which it
naturally originates will be "isolated" from its naturally associated components. A protein
may also be rendered substantially free of naturally associated components by isolation,
using protein purification techniques well known in the art.
Notch 1 Receptor
Human Notchl cDNA encodes a protein of 2556 amino acid residues consisting
of a leader peptide, 36 EGF-like repeats, negative regulatory region (NRR), a
transmembrane (TM) sequence and an intracellular domain (Notchl ICD) .
Anti-Notch1 antibodies that bind to the NRR
It is within the contemplation of the current disclosure that antibodies that bind to
the Notchl domain with a high affinity may reduce Notchl signal transduction, and
therefore may demonstrate biological activity in vitro and in vivo to inhibit cancer cell
growth, in particular, T-cell acute lymphoblastic leukemia (T-ALL), non-small cell lung
cancer (NSCLC), breast cancer, colon cancer and ovarian cancer. Such antibodies
may be produced following general methods known to those of ordinary skill in the art.
In one embodiment, such antibodies can be produced through immunization of a rat
with an immunogen followed by hybridoma cloning of the antibodies thus generated and
assaying the cloned antibodies by a variety of assays. For example, solid-phase ELISA
immunoassay, immunoprecipitation, BIAcore, FACS, and Western blot analysis are
among many assays that may be used to identify an antibody that specifically reacts
with Notchl . The Notchl binding affinity of the antibodies selected according to the
ELISA assay can be measured on a surface plasma resonance Biacore® instrument.
The anti-Notchl antibodies of the current disclosure can be produced by any
other methods known in the art other than described in the above paragraph. The route
and schedule of immunization of the host animal are generally in keeping with
established and conventional techniques for antibody stimulation and production, as
further described herein. General techniques for production of human and mouse
antibodies are known in the art and/or are described herein.
Anti-Notch1 antibodies generated by hybridoma technologies.
It is within the contemplation of the current disclosure that any mammalian
subject including humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human, hybridoma cell lines.
Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally,
subcutaneously, intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
Hybridomas can be prepared from the lymphocytes and immortalized myeloma
cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C.
(1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381
(1982). Hybridomas that may be used as a source of antibodies encompass all
derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies
specific for Notch 1, or a portion thereof.
Hybridomas that produce such antibodies may be grown in vitro or in vivo using
known procedures. The monoclonal antibodies may be isolated from the culture media
or body fluids, by conventional immunoglobulin purification procedures.
Humanization of anti-Notchl antibodies generated by immunization in a host animal.
It is within the contemplation of the current disclosure that anti Notchl antibodies
of the disclosure, wherein the antibodies are generated by immunization in a host
animal can be manipulated in many ways to improve their biological activity and
pharmaceutical properties. One way of such manipulation is humanization.
Methods of humanizing antibodies are well known to those of ordinary skill in the
art. In general, there are four basic steps to humanize a monoclonal antibody. These
are: ( 1 ) determining the nucleotide and predicted amino acid sequence of the starting
antibody light and heavy variable domains (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the humanizing process (3) the
actual humanizing methodologies/techniques and (4) the transfection and expression of
the humanized antibody.
A number of "humanized" antibody molecules comprising an antigen-binding site
derived from a non-human immunoglobulin have been described in the literature,
including chimeric antibodies having rodent or modified rodent V regions and their
associated CDRs fused to human constant domains; rodent CDRs grafted into a
human supporting framework region (FR) prior to fusion with an appropriate human
antibody constant domain; and rodent CDRs supported by recombinantly engineered
rodent framework regions. Such "humanized" molecules are designed to minimize
unwanted immunological response toward rodent anti-human antibody molecules which
limits the duration and effectiveness of therapeutic applications of those moieties in
human recipients.
Human anti-Notchl antibodies
It is within the contemplation of the current disclosure that fully human anti-
Notchl antibodies may be obtained by using commercially available mice that have
been engineered to express specific human immunoglobulin proteins. Transgenic
animals that are designed to produce a more desirable (e.g., fully human antibody) or
more robust immune response may also be used for generation of humanized or human
antibodies. Examples of such technologies are Xenomouse ™from Abgenix, Inc.
(Fremont, CA) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton,
NJ).
It is also within the contemplation of the current disclosure that fully human
anti-Notchl antibodies may be obtained recombinantly following general methods of
phage display technology, as will be readily apparent to those of skill in the art.
Alternatively, the phage display technology can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V) domain gene
repertoires from unimmunized donors.
Gene shuffling can also be used to derive human antibodies from rodent
antibodies, where the human antibody has similar affinities and specificities to the
starting rodent antibody. Although the above discussion pertains to humanized and
human antibodies, the general principles discussed are applicable to customizing
antibodies for use, for example, in dogs, cats, primate, equines and bovines. One or
more aspects of humanizing an antibody described herein may be combined, e.g., CDR
grafting, framework mutation and CDR mutation.
Engineered and modified anti-Notchl antibodies made recombinantly
In general, antibodies may be made recombinantly by placing the DNA
sequences of the desired antibody into expression vectors followed by transfection and
expression in host cells, including but not limited to E. coli cells, simian COS cells,
Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein. Other host cells, such as transgenic plant cells or transgenic
milk cells may also be used.
An antibody may also be modified recombinantly. For example, the DNA of the
human heavy and light chain constant regions may be used in place of the homologous
murine sequences of the murine antibody DNA, or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence for a nonimmunoglobulin
polypeptide. In similar manner, "chimeric" or "hybrid" antibodies can be
prepared that have the binding specificity of an anti-Notch 1 monoclonal antibody herein.
Antibody variable regions can also be modified by CDR grafting. Because CDR
sequences are predominantly responsible for most antibody-antigen interactions, it is
possible to express recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors that include CDR
sequences from the specific naturally occurring antibody grafted onto framework
sequences from a different antibody with different properties.
Accordingly, another aspect of the disclosure pertains to an isolated monoclonal
antibody, comprising a heavy chain variable region comprising CDR1, CDR2, and
CDR3 sequences as described herein, and a light chain variable region comprising
CDR1, CDR2, and CDR3 sequences as described herein. Thus, such antibodies
contain the VH and VL CDR sequences of the monoclonal antibodies described herein,
yet may contain different framework sequences from these antibodies. Such framework
sequences can be obtained from public DNA databases or published references that
include germline antibody gene sequences.
Another type of variable region modification is to mutate amino acid residues
within the VH and/or VL CDR1 , CDR2 and/or CDR3 regions to thereby improve one or
more binding properties (e.g., affinity) of the antibody of interest. Site-directed
mutagenesis or PCR-mediated mutagenesis can be performed to introduce the
mutation(s) and the effect on antibody binding, or other functional property of interest,
can be evaluated using in vitro or in vivo assays as described herein. Typically,
conservative modifications (as discussed below) are introduced. The mutations may be
amino acid substitutions, additions or deletions. Moreover, typically no more than one,
two, three, four or five residues within a CDR region are modified.
Epitope Mapping
The binding epitopes of monoclonal antibodies on an antigen may be mapped by
a number of methods depending on the type of antigen-antibody interactions.
If an antibody binds to a single epitope consisting of sequential amino acid
residues in an antigen, whose binding usually is not affected by antigen conformational
changes, the binding epitope is called a linear epitope. Determining the amino acid
sequence of a linear epitope can be accomplished by utilizing techniques well known in
the art. A non-linear epitope which is constituted by several sequentially discontinuous
segments or noncontiguous residues that are brought together by the folding of the
antigen to its native structure is known as a conformational epitope.
Mapping of conformational epitopes depends on the interaction of antibody to
antigen in its native conformation. A number of techniques well known in the art are
useful in determining conformational epitopes. For example, co-crystalization of
antigen-antibody complex, X-ray diffraction and structural analysis gives direction
visualization of antigen-antibody interaction. When combined with amino acid
mutagenesis, the technologies can provide powerful evidence and a vivid picture for
antibody binding epitopes. The epitope or the set of epitopes that each of the anit-
Notchi antibodies bind to may be determined according to the above mapping methods
or others generally known in the art.
Conservative substitutions
An antibody may also be modified recombinantly by conservative substitution of
one or more of the amino acid residues of the antibody or by one or more deletions or
additions of amino acids to that of the antibody. Amino acid sequence insertions
include amino- and/or carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as intrasequence
insertions of single or multiple amino acid residues. Examples of terminal insertions
include an antibody with an N-terminal methionyl residue or the antibody fused to an
epitope tag. Other insertional variants of the antibody molecule include the fusion to the
N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the
half-life of the antibody in the blood circulation.
Substitution variants have at least one amino acid residue in the antibody
molecule removed and a different residue inserted in its place. The sites of greatest
interest for substitutional mutagenesis include the hypervariable regions, but FR
alterations are also contemplated.
Affinity matured anti-Notchl antibodies
The disclosure includes affinity matured embodiments. For example, affinity
matured antibodies can be produced by procedures known in the art (such as Marks et
al. (1992) Bio/Technology, 10:779-783; Barbas et al. (1994) Proc Nat. Acad. Sci, USA
9 1:3809-3813; Schier et al. (1995) Gene, 169:147-155; Yelton et al. (1995) J. Immunol.,
155:1994-2004; Jackson et al. (1995) J. Immunol., 154(7):3310-9; Hawkins et al. (1992)
J. Mol. Biol., 226:889-896; and PCT Publication No. WO2004/058184). Such methods
may be used for adjusting the affinity of an antibody and for characterizing a CDR.
Post translational modification of anti-Notch 1 antibodies
Antibodies can also be modified by post translational modifications, including, but
not limited to glycosylation with different sugars, acetylation, and phosphorylation by
techniques are well known in the art.
Other methods of post translational modification include using coupling
techniques known in the art, including, but not limited to, enzymatic means, oxidative
substitution and chelation. Modifications can be used, for example, for attachment of
labels for immunoassay.
Anti-Notch1 antibodies with modified constant region
In some embodiments of the disclosure, the antibody comprises a modified
constant region, such as a constant region that is immunologically inert or partially inert,
e.g., does not trigger complement mediated lysis, does not stimulate antibodydependent
cell mediated cytotoxicity (ADCC), or does not activate microglia; or have
reduced activities (compared to the unmodified antibody) in any one or more of the
following: triggering complement mediated lysis, stimulating antibody-dependent cell
mediated cytotoxicity (ADCC), or activating microglia. Different modifications of the
constant region may be used to achieve optimal level and/or combination of effector
functions. See, for example, Morgan et al., Immunology 86:319-324 (1995); Lund et al.,
J. Immunology 157:4963-9 157:4963-4969 (1996); Idusogie et al., J. Immunology
164:4178-4184 (2000); Tao et al., J. Immunology 143: 2595-2601 (1989); and Jefferis et
al., Immunological Reviews 163:59-76 (1998).
In some embodiments, the antibody comprises a human heavy chain lgG1
constant region comprising the following mutations: L234A/L235A/G237A in the lower
hinge region resulting in substantially reduced ADCC and CDC activities. See for
example US200901 55256.
Modifications within the Fc region can typically be used to alter one or more
functional properties of the antibody, such as serum half-life, complement fixation, Fc
receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an
antibody of the disclosure may be chemically modified (e.g., one or more chemical
moieties can be attached to the antibody) or be modified to alter its glycosylation
pattern, again to alter one or more functional properties of the antibody.
Another modification of the antibodies herein that is contemplated by the
disclosure is pegylation. An antibody can be pegylated to, for example, increase the
biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a
reactive ester or aldehyde derivative of PEG, under conditions in which one or more
PEG groups become attached to the antibody or antibody fragment. Typically, the
pegylation is carried out via an acylation reaction or an alkylation reaction with a
reactive PEG molecule (or an analogous reactive water-soluble polymer). As used
herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG
that have been used to derivatize other proteins, such as mono (C1 to C10) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain cases, the
antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins
are known in the art and can be applied to the antibodies of the present disclosure.
Fusion protein
The disclosure also encompasses fusion proteins comprising one or more
fragments or regions from the antibodies or polypeptides of this disclosure. In one
embodiment, a fusion polypeptide is provided that comprises at least 10 contiguous
amino acids of the variable light chain region and/or at least 10 amino acids of the
variable heavy chain region of the antibodies of the current disclosure. In other
embodiments, a fusion polypeptide is provided that comprises at least about 10, at least
about 15, at least about 20, at least about 25, or at least about 30 contiguous amino
acids of the variable light chain region and/or at least about 10, at least about 15, at
least about 20, at least about 25, or at least about 30 contiguous amino acids of the
variable heavy chain region. In another embodiment, the fusion polypeptide comprises
a light chain variable region and/or a heavy chain variable region, of the antibodies of
the current disclosure. In another embodiment, the fusion polypeptide comprises one or
more CDR(s) of the antibodies of the current disclosure. For purposes of this
disclosure, a fusion protein contains one or more antibodies and another amino acid
sequence to which it is not attached in the native molecule, for example, a heterologous
sequence or a homologous sequence from another region. Exemplary heterologous
sequences include, but are not limited to a "tag" such as a FLAG tag or a 6His tag.
A fusion polypeptide can be created by methods known in the art, for example,
synthetically or recombinantly.
Bispecific Molecules
An antibody of the disclosure, or antigen-binding portions thereof, can be
derivatized or linked to another functional molecule, e.g., another peptide or protein
(e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that
binds to at least two different binding sites or target molecules. The antibody of the
disclosure may in fact be derivatized or linked to more than one other functional
molecule to generate multispecific molecules that bind to more than two different
binding sites and/or target molecules; such multispecific molecules are also intended to
be encompassed by the term "bispecific molecule" as used herein. To create a
bispecific molecule of the disclosure, an antibody of the disclosure can be functionally
linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise)
to one or more other binding molecules, such as another antibody, antibody fragment,
peptide or binding mimetic, such that a bispecific molecule results.
Single-Chain Antibodies
An antibody of the disclosure can be a single-chain antibody (scFv) in which the
heavy and light chain variable regions (Fv region) have been connected by a flexible
linker to form a single polypeptide chain, which forms an antigen-binding region. Such
single-chain antibodies may be prepared by fusing DNA encoding a peptide linker
between DNAs encoding the two variable domain polypeptides (VL and VH). The
resulting polypeptides can fold back on themselves to form antigen-binding monomers,
or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length
of a flexible linker between the two variable domains (Kortt et al. ( 1997) Prot. Eng.
10:423; Kortt et al. (2001 ) Biomol. Eng. 18:95-1 08). By combining different VL and VHcomprising
polypeptides, one can form multimeric scFvs that bind to different epitopes
(Kriangkum et al. (2001) Biomol. Eng. 18:31-40). Single chain antibodies can be
produced using various techniques known in the art.
Immunoconjugates
An antibody of the disclosure can be an immunoconjugate or antibody-drug
conjugates (ADC). Immunoconjugates combine the binding specificity of monoclonal
antibodies with the potency of chemotherapeutic agents.
Polynucleotides encoding the anti-Notch 1 antibodies
The disclosure also provides isolated polynucleotides encoding the antibodies
and peptides of the disclosure, and vectors and host cells comprising the
polynucleotide.
In one aspect, the disclosure provides compositions, such as a pharmaceutical
composition, comprising any of the polynucleotides of the disclosure. In some
embodiments, the composition comprises an expression vector comprising a
polynucleotide encoding the antibody of the disclosure. In other embodiment, the
composition comprises an expression vector comprising a polynucleotide encoding any
of the antibodies or polypeptides of the disclosure.
In another aspect, the disclosure provides a method of making any of the
polynucleotides described herein.
Polynucleotides complementary to any such sequences are also encompassed
by the present disclosure. Polynucleotides may be single-stranded (coding or
antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA
molecules. RNA molecules include HnRNA molecules, which contain introns and
correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which
do not contain introns. Additional coding or non-coding sequences may, but need not,
be present within a polynucleotide of the present disclosure, and a polynucleotide may,
but need not, be linked to other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence
that encodes an antibody or a portion thereof) or may comprise a variant of such a
sequence. Polynucleotide variants contain one or more substitutions, additions,
deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is
not diminished, relative to a native immunoreactive molecule. The effect on the
immunoreactivity of the encoded polypeptide may generally be assessed as described
herein. Variants preferably exhibit at least about 70% identity, more preferably, at least
about 80% identity, yet more preferably, at least about 90% identity, and most
preferably, at least about 95% identity to a polynucleotide sequence that encodes a
native antibody or a portion thereof.
Two polynucleotide or polypeptide sequences are said to be "identical" if the
sequence of nucleotides or amino acids in the two sequences is the same when aligned
for maximum correspondence.
It will be appreciated by those of ordinary skill in the art that, as a result of the
degeneracy of the genetic code, there are many nucleotide sequences that encode a
polypeptide as described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides
that vary due to differences in codon usage are specifically contemplated by the present
disclosure. Further, alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present disclosure. Alleles are endogenous
genes that are altered as a result of one or more mutations, such as deletions, additions
and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified using standard
techniques (such as hybridization, amplification and/or database sequence
comparison).
The polynucleotides of this disclosure can be obtained using chemical synthesis,
recombinant methods, or PCR.
For preparing polynucleotides using recombinant methods, a polynucleotide
comprising a desired sequence can be inserted into a suitable vector, and the vector in
turn can be introduced into a suitable host cell for replication and amplification. Suitable
cloning vectors may be constructed according to standard techniques, or may be
selected from a large number of cloning vectors available in the art. While the cloning
vector selected may vary according to the host cell intended to be used, useful cloning
vectors will generally have the ability to self-replicate, may possess a single target for a
particular restriction endonuclease, and/or may carry genes for a marker that can be
used in selecting clones containing the vector.
Expression vectors generally are replicable polynucleotide constructs that
contain a polynucleotide according to the disclosure. It is implied that an expression
vector must be replicable in the host cells either as episomes or as an integral part of
the chromosomal DNA. Vector components may generally include, but are not limited
to, one or more of the following: a signal sequence; an origin of replication; one or more
marker genes; suitable transcriptional controlling elements (such as promoters,
enhancers and terminator).
The vectors containing the polynucleotides of interest can be introduced into the
host cell by any of a number of appropriate means, including electroporation,
transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAEdextran,
or other substances; microprojectile bombardment; lipofection; and infection
(e.g., where the vector is an infectious agent such as vaccinia virus). The choice of
introducing vectors or polynucleotides will often depend on features of the host cell.
The disclosure also provides host cells comprising any of the polynucleotides
described herein. Any host cells capable of over-expressing heterologous DNAs can be
used for the purpose of isolating the genes encoding the antibody, polypeptide or
protein of interest. Suitable non-mammalian host cells include prokaryotes (such as E.
coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably,
the host cells express the cDNAs at a level of about 5 fold higher, more preferably, 10
fold higher, even more preferably, 20 fold higher than that of the corresponding
endogenous antibody or protein of interest, if present, in the host cells.
Pharmaceutical Compositions
In another aspect, the present disclosure provides a composition, e.g., a
pharmaceutical composition, containing one or a combination of monoclonal antibodies,
or antigen-binding portion(s) thereof, of the present disclosure, formulated together with
a pharmaceutically acceptable carrier. Such compositions may include one or a
combination of (e.g., two or more different) antibodies, or immunoconjugates or
bispecific molecules of the disclosure. For example, a pharmaceutical composition of
the disclosure can comprise a combination of antibodies (or immunoconjugates or
bispecifics) that bind to different epitopes on the target antigen or that have
complementary activities.
Pharmaceutical compositions of the disclosure also can be administered in
combination therapy, i.e., combined with other agents. For example, the combination
therapy can include an anti-Notch 1 antibody of the present disclosure combined with at
least one other anti-inflammatory, anti-cancer or immunosuppressant agent. Examples
of therapeutic agents that can be used in combination therapy are described in greater
detail below in the section on uses of the antibodies of the disclosure.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically compatible. Typically,
the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal administration (e.g., by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody, antigen-binding portion thereof,
immunoconjuage, or bispecific molecule, may be coated in a material to protect the
compound from the action of acids and other natural conditions that may inactivate the
compound.
In certain embodiments, the antibodies of the present disclosure may be present
in a neutral form (including zwitter ionic forms) or as a positively or negatively-charged
species. In some cases, the antibodies may be complexed with a counterion to form a
pharmaceutically acceptable salt. Thus, the pharmaceutical compounds of the
disclosure may include one or more pharmaceutically acceptable salts.
A "pharmaceutically acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound (e.g. antibody) and does not impart undesired
toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). For
example, the term "pharmaceutically acceptable salt" includes a complex comprising
one or more antibodies and one or more counterions, where the counterions are derived
from pharmaceutically acceptable inorganic and organic acids and bases.
Examples of such salts include acid addition salts and base addition salts. Acid
addition salts include those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well
as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsubstituted
alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include those derived from
alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as
well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, Nmethylglucamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, procaine
and the like.
Furthermore, pharmaceutically acceptable inorganic bases include metallic ions.
Metallic ions include, but are not limited to, appropriate alkali metal salts, alkaline earth
metal salts and other physiological acceptable metal ions. Salts derived from inorganic
bases include aluminum, ammonium, calcium, cobalt, nickel, molybdenum, vanadium,
manganese, chromium, selenium, tin, copper, ferric, ferrous, lithium, magnesium,
manganic salts, manganous, potassium, rubidium, sodium, and zinc, and in their usual
valences.
Pharmaceutically acceptable acid addition salts of the antibodies of the present
disclosure can be prepared from the following acids, including, without limitation formic,
acetic, acetamidobenzoic, adipic, ascorbic, boric, propionic, benzoic, camphoric,
carbonic, cyclamic, dehydrocholic, malonic, edetic, ethylsulfuric, fendizoic,
metaphosphoric, succinic, glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric,
ascorbic, glucuronic, maleic, folic, fumaric, propionic, pyruvic, aspartic, glutamic,
benzoic, hydrochloric, hydrobromic, hydroiodic, lysine, isocitric, trifluoroacetic, pamoic,
propionic, anthranilic, mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic,
aminosalicylic, silicate, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, embonic,
sulfonic, methanesulfonic, phosphoric, phosphonic, ethanesulfonic, ethanedisulfonic,
ammonium, benzenesulfonic, pantothenic, naphthalenesulfonic, toluenesulfonic, 2-
hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuric acid monomethyl ester,
cyclohexylaminosulfonic, b-hydroxybutyric, glycine, glycylglycine, glutamic, cacodylate,
diaminohexanoic, camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal 5-
phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-L-aspartic, galactaric and
galacturonic acids.
Pharmaceutically acceptable organic bases include trimethylamine, diethylamine,
N, N'-dibenzylethylenediamine, chloroprocaine, choline, dibenzylamine, diethanolamine,
ethylenediamine, meglumine (N-methylglucamine), procaine, cyclic amines, quaternary
ammonium cations, arginine, betaine, caffeine, clemizole, 2-ethylaminoethanol, 2-
diethylaminoethanol, 2-dimethylaminoethanol, ethanediamine, butylamine,
ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, ethylglucamine,
glucamine, glucosamine, histidine, hydrabamine, imidazole, isopropylamine,
methylglucamine, morpholine, piperazine, pyridine, pyridoxine, neodymium, piperidine,
polyamine resins, procaine, purines, theobromine, triethylamine, tripropylamine,
triethanolamine, tromethamine, methylamine, taurine, cholate, 6-amino-2-methyl-2-
heptanol, 2-amino-2-methyl-1 ,3-propanediol, 2-amino-2-methyl-1-propanol, aliphatic
mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic
acids, aromatic acids, aliphatic and aromatic sulfonic acids, strontium, tricine, hydrazine,
phenylcyclohexylamine, 2-(N-morpholino)ethanesulfonic acid, bis(2-
hydroxyethyl)amino-tris(hydroxymethyl)methane, N-(2-acetamido)-2-
aminoethanesulfonic acid, 1,4-piperazinediethanesulfonic acid, 3-morpholino-2-
hydroxypropanesulfonic acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane, 4-
morpholinepropanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 2-
[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid, N,N-bis(2-
hydroxyethyl)-2-aminoethanesulfonic acid, 4-(N-morpholino)butanesulfonic acid, 3-(N,Nbis[
2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, 2-hydroxy-3-
[tris(hydroxymethyl)methylamino]-1 -propanesulfonic acid, 4-(2-hydroxyethyl)piperazine-
1-(2-hydroxypropanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid)
dihydrate, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, N,N-bis(2-
hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N-
[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, N-tris(Hydroxymethyl)methyl-
4-aminobutanesulfonic acid, N-(1 ,1-dimethyl-2-hydroxyethyl)-3-amino-2-
hydroxypropanesulfonic acid, 2-(cyclohexylamino)ethanesulfonic acid, 3-
(cyclohexylamino)-2-hydroxy-1 -propanesulfonic acid, 3-(cyclohexylamino)-1-
propanesulfonic acid, N-(2-acetamido)iminodiacetic acid, 4-(cyclohexylamino)-1-
butanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, 2-amino-2-(hydroxymethyl)-
1,3-propanediol, and trometamol.
A pharmaceutical composition of the disclosure also may include a
pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable
antioxidants include: ( 1) water soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oilsoluble
antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like;
and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid
(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Examples of suitable aqueous and nonaqueous carriers that may be employed in
the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such
as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl
oleate. Proper fluidity can be maintained, for example, by the use of coating materials,
such as lecithin, by the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of presence of
microorganisms may be ensured both by sterilization procedures, supra, and by the
inclusion of various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical form may be brought
about by the inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. The use of such media and agents for pharmaceutically active
substances is known in the art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the pharmaceutical compositions
of the disclosure is contemplated. Supplementary active compounds can also be
incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the
conditions of manufacture and storage. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by including
in the composition an agent that delays absorption, for example, monostearate salts
and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by sterilization microfiltration.
Generally, dispersions are prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the preparation of sterile
injectable solutions, methods of preparation include, but are not limited to, vacuum
drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus
any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the subject being treated, and
the particular mode of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form will generally be that
amount of the composition which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most
preferably from about 1 percent to about 30 percent of active ingredient in combination
with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be administered, several
divided doses may be administered over time or the dose may be proportionally
reduced or increased as indicated by the exigencies of the therapeutic situation. It is
especially advantageous to formulate parenteral compositions in dosage unit form for
ease of administration and uniformity of dosage. Dosage unit form as used herein
refers to physically discrete units suited as unitary dosages for the subjects to be
treated; each unit contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the disclosure are dictated by and
directly dependent on (a) the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of
compounding such an active compound for the treatment of sensitivity in individuals.
For administration of the antibody, the dosage ranges from about 0.0001 to 100
mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example
dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5
mg/kg body weight or 10 mg/kg body weight or within the range of 1 to 10 mg/kg. An
exemplary treatment regime entails administration once per week, once every two
weeks, once every three weeks, once every four weeks, once per month, once every 3
months or once every three to 6 months. Dosage regimens for an anti-Notch 1 antibody
of the disclosure include, for example, 1 mg/kg body weight or 3 mg/kg body weight via
intravenous administration, with the antibody being given using one of the following
dosing schedules: (i) every four weeks for six dosages, then every three months; (ii)
every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight
every three weeks.
In some methods, two or more monoclonal antibodies with different binding
specificities are administered simultaneously, in which case the dosage of each
antibody administered falls within the ranges indicated. Antibody is usually
administered on multiple occasions. Intervals between single dosages can be, for
example, weekly, monthly, every three months or yearly. Intervals can also be irregular
as indicated by measuring blood levels of antibody to the target antigen in the patient.
In some methods, dosage is adjusted to achieve a plasma antibody concentration of
about 1 to 1000 mg/ml and in some methods about 25 to 300 mg/ml.
Alternatively, antibody can be administered as a sustained release formulation, in
which case less frequent administration is required. Dosage and frequency vary
depending on the half-life of the antibody in the patient. In general, human antibodies
show the longest half life, followed by humanized antibodies, chimeric antibodies, and
nonhuman antibodies. The dosage and frequency of administration can vary depending
on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent intervals over a long period
of time. Some patients continue to receive treatment for the rest of their lives. In
therapeutic applications, a relatively high dosage at relatively short intervals is
sometimes required until progression of the disease is reduced or terminated, and
preferably until the patient shows partial or complete amelioration of symptoms of
disease. Thereafter, the patient can be administered a prophylactic regime.
Actual dosage levels of the active ingredients in the pharmaceutical compositions
of the present disclosure may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic response for a particular
patient, composition, and mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic factors including
the activity of the particular compositions of the present disclosure employed, or the
ester, salt or amide thereof, the route of administration, the time of administration, the
rate of excretion of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition, general health and
prior medical history of the patient being treated, and like factors well known in the
medical arts.
A "therapeutically effective dosage" of an anti-Notch antibody of the disclosure
preferably results in a decrease in severity of disease symptoms, an increase in
frequency and duration of disease symptom-free periods, or a prevention of impairment
or disability due to the disease affliction. For example, for the treatment of Notchlpositive
tumors, a "therapeutically effective dosage" preferably inhibits cell growth or
tumor growth by at least about 20%, more preferably by at least about 40%, even more
preferably by at least about 60%, and still more preferably by at least about 80% relative
to untreated subjects. The ability of a compound to inhibit tumor growth can be
evaluated in an animal model system predictive of efficacy in human tumors.
Alternatively, this property of a composition can be evaluated by examining the ability of
the compound to inhibit, such inhibition in vitro by assays known to the skilled
practitioner. A therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary
skill in the art would be able to determine such amounts based on such factors as the
subject's size, the severity of the subject's symptoms, and the particular composition or
route of administration selected.
A composition of the present disclosure can be administered via one or more
routes of administration using one or more of a variety of methods known in the art. As
will be appreciated by the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. Routes of administration for antibodies of the
disclosure include intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration, for example by
injection or infusion. The phrase "parenteral administration" as used herein means
modes of administration other than enteral and topical administration, usually by
injection, and includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,
intraspinal, epidural and intrasternal injection and infusion.
Alternatively, an antibody or antigen biding portion thereof of the disclosure can be
administered via a non-parenteral route, such as a topical, epidermal or mucosal route
of administration, for example, intranasally, orally, vaginally, rectally, sublingually or
topically.
The active compounds can be prepared with carriers that will protect the
compound against rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many
methods for the preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Uses and Methods of the Disclosure
The antibodies, particularly the human antibodies, antibody compositions and
methods of the present disclosure have numerous in vitro and in vivo diagnostic and
therapeutic utilities involving the diagnosis and treatment of Notch 1 mediated disorders.
For example, these molecules can be administered to cells in culture, in vitro or ex vivo,
or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of
disorders. As used herein, the term "subject" is intended to include human and nonhuman
animals. Non-human animals include all vertebrates, e.g., mammals and nonmammals,
such as non-human primates, sheep, dogs, cats, cows, horses, chickens,
amphibians, and reptiles. Preferred subjects include human patients having disorders
mediated by Notchl activity. The methods are particularly suitable for treating human
patients having a disorder associated with abnormal Notchl expression or activation.
When antibodies to Notchl are administered together with another agent, the two can
be administered in either order or simultaneously.
Given the specific binding of the antibodies of the disclosure for Notchl , the
antibodies of the disclosure can be used to specifically detect Notchl expression on the
surface of cells and, moreover, can be used to purify Notchl via immunoaffinity
purification.
Furthermore, the antibodies, antibody compositions and methods of the present
disclosure can be used to treat a subject with abnormal Notchl expression, e.g., a
cancer. In one particular embodiment, the cancer is T-cell acute lymphoblastic
leukemia (T-ALL). In another particular embodiment, the cancer is non-small cell lung
cancer (NSCLC), breast cancer, colon cancer or ovarian cancer.
Other types of abnormal Notch 1 expression that may be treated by the
antibodies of the disclosure include, for example, mesothelioma, hepatobilliary (hepatic
and billiary duct), a primary or secondary CNS tumor, a primary or secondary brain
tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreatic cancer, skin cancer,
cancer of the head or neck, cutaneous or intraocular melanoma, rectal cancer, cancer
of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal),
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium,
carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's
Disease, cancer of the esophagus, cancer of the small intestine, cancer of the
endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer
of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis,
prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia,
lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell
carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system
(CNS), primary CNS lymphoma, non hodgkins's lymphoma, spinal axis tumors, brain
stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiple
myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a
combination of one or more of the foregoing cancers.
Suitable routes of administering the antibody compositions (e.g., human
monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates)
of the disclosure in vivo and in vitro are well known in the art and can be selected by
those of ordinary skill. For example, the antibody compositions can be administered by
injection (e.g., intravenous or subcutaneous). Suitable dosages of the molecules used
will depend on the age and weight of the subject and the concentration and/or
formulation of the antibody composition.
Human anti-Notch 1 antibodies of the disclosure can be co-administered with one
or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent or an
immunosuppressive agent. The antibody can be linked to the agent (as an
immunocomplex) or can be administered separate from the agent. In the latter case
(separate administration), the antibody can be administered before, after or concurrently
with the agent or can be co-administered with other known therapies, e.g., an anti
cancer therapy, e.g., radiation. The antibody and the agent can be prepared for
simultaneous, sequential or separate administration. Such therapeutic agents include,
among others, anti-neoplastic agents such as docetaxel, doxorubicin (adriamycin),
cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide
hydroxyurea which, by themselves, are only effective at levels which are toxic or
subtoxic to a patient. Cisplatin can be intravenously administered as a 100 mg/dose
once every four weeks and adriamycin is intravenously administered as a 60 to 75
mg/ml dose once every 2 1 days. Co-administration of the human anti-Notchl
antibodies of the present disclosure with chemotherapeutic agents provides two ant i
cancer agents which operate via different mechanisms which yield a cytotoxic effect to
human tumor cells.
Kits
Also within the scope of the present disclosure are kits comprising the antibody
compositions of the disclosure (e.g., human antibodies, bispecific or multispecific
molecules, or immunoconjugates) and instructions for use. The kit can further contain
one or more additional reagents, such as an immunosuppressive reagent, a cytotoxic
agent or a radiotoxic agent, or one or more additional antibodies of the disclosure (e.g.,
a human antibody having a complementary activity which binds to an epitope in the
Notchl antigen distinct from the first human antibody).
Accordingly, patients treated with antibody compositions of the disclosure can be
additionally administered (prior to, simultaneously with, or following administration of a
human antibody of the disclosure) another therapeutic agent, such as a cytotoxic or
radiotoxic agent, which enhances or augments the therapeutic effect of the human
antibodies.
The present disclosure is further illustrated by the following examples which
should not be construed as further limiting. The contents of all figures and all
references, patents and published patent applications cited throughout this application
are expressly incorporated herein by reference.
EXAMPLES
Example 1
Generation of recombinant human and mouse Notchi protein immunoqens
A. Expression and purification of human and mouse Notchi NRR proteins
cDNA constructs encoding the Notchi NRR region, amino acids of SEQ ID 2 for
human Notchi and amino acids of SEQ ID 6 for mouse Notchi shown in Table 1, with a
signal peptide at the N-terminus and Avi and His6 tag at the C-terminus, were cloned
into the expression vector pSMED2. These constructs were transiently transfected into
COS or Chinese hamster ovary (CHO) cells and the secreted protein in conditioned
media were analyzed on SDS-PAGE. After processing at the S 1 cleavage site, the Nterminal
-26 kDa (LNR-A, B, C and HD1) and C-terminal -12 kDa (HD2 and AviJHis
tag) halves of the Notchi NRR domain remain associated through non-covalent
interactions to form a heterodimeric complex, as shown in FIG. 1. S 1 processing of the
Notchi NRR was determined to be about 50% or less in samples prepared from CHO
cells.
To enhance processing at the S 1 cleavage site, the Notchi NRR expression
construct was transfected into CHO-PACE cells (Harrison et, al, Semin Hematol. 1998
Apr;35(2 Suppl 2):4-10) and stable cell lines with the highest expression and complete
processing of Notchi NRR were selected. Culture of these cell lines was scaled up for
the collection of conditioned media (CM) from which Notchi NRR proteins were purified.
Concentrated CHO-PACE CM was loaded onto a 27ml Qiagen Ni-NTA
Superflow column that was equilibrated with PBS at a flow rate of 1ml/min at 4°C. After
loading, the column was washed with 10 Column Volumes (CV) of PBS, followed by
10CV of Buffer A (300mM NaCI, 50mM Na2HP0 4, pH 8.0), and followed by 10CV 4%
Buffer B (500mM imidazole, 300mM NaCI, 50mM Na2HP0 4, pH 8.0). The human
Notchi AviJHis was eluted using a linear gradient to 100% Buffer B over 10CV.
Fractions containing human Notchi AviJHis were pooled, filtered and dialyzed to PBS
calcium magnesium free (CMF). The protein was further purified with two rounds of size
exclusion chromatography on a tandem Superdex-200 and Superdex-75 columns (total
CV=600ml) equilibrated with TBS + 1mM CaCI2, 0.1 mM ZnCI2. SDS-PAGE analysis of
purified human and mouse Notchi NRR_Avi_His tag proteins show that > 90% of the
purified protein was correctly cleaved into the predicted Notchi NRR N-terminal and Cterminal
peptide sizes. Light scattering (SEC-MALs) analysis of purified human and
mouse Notchi NRR proteins showed a peak at the expected molecular weight of 40
kDa on a size exclusion column under native conditions, indicating proper formation of
an intact Notchi NRR heterodimer.
B. Expression and purification of cyno-Notchl NRR-Fc fusion protein
A cDNA construct encoding the cyno Notchi NRR region, amino acids of SEQ ID
10 for cyno Notchi shown in Table 1, with a signal peptide at the N-terminus and
human lgG1 Fc fragment at the C-terminus, was cloned into the expression vector
pSMED2. This construct was transiently co-transfected into 293 (Invitrogen) cells with a
soluble PACE overexpressing construct (Harrison et, al, Semin Hematol. 1998 Apr;35(2
Suppl 2):4-10) to ensure complete processing of the cyno-Notchl NRR region.
Conditioned medium was harvested from transfected cells and the cyno-Notchl NRRFc
fusion protein was purified via protein A affinity purification. Purified protein was then
dialyzed into TBS containing 1mM CaC - SDS-PAGE analysis showed two polypeptide
fragments at expected sizes of 12Kd (HD1) and 37Kd (HD2+Fc), with >95% purity of
the protein preparation. Analytical SEC under native conditions showed a single peak
around 50 KD, representing a heterodimer of the two fragments described above, with
minimal amount of aggregates (<1%) in the preparation.
Table 1 below provides the amino acid and nucleotide sequences of human,
mouse and cyno Notchi NRR regions.
Table 1. Amino acid and nucleotide sequences of human, mouse and cyno Notchi
Human Notchl NRR amino acid GGAGRDIPPPLIEEACELPECQEDAGNKVCSL
sequence QCNNHACGWDGGDCSLNFNDPWKNCTQSLQ
CWKYFSDGHCDSQCNSAGCLFDGFDCQRAE
GQCNPLYDQYCKDHFSDGHCDQGCNSAECE
WDGLDCAEHVPERLAAGTLVVVVLMPPEQLR
NSSFHFLRELSRVLHTNVVFKRDAHGQQMIFP
YYGREEELRKHPIKRAAEGWAAPDALLGQVKA
SLLPGGSEGGRRRRELDPMDVRGSIVYLEIDN
RQCVQASSQCFQSATDVAAFLGALASLGSLNI
PYKIEAVQSETVEPPPPAQLHFM
Human Notchl NRR nucleotide atgcctctcctcctcttgctgctcctg ctgccaagccccttacacgc
sequence (nucleotides in lower gGGTGGGGCCGGGCGCGACATCCCCCCGC
case type represent the signal CGCTGATCGAGGAGGCGTGCGAGCTGCCCG
peptide Avi_His tag coding AGTGCCAGGAGGACGCGGGCAACAAGGTCT
sequence) GCAGCCTGCAGTGCAACAACCACGCGTGCG
GCTGGGACGGCGGTGACTGCTCCCTCAACT
TCAATGACCCCTGGAAGAACTGCACGCAGTC
TCTGCAGTGCTGGAAGTACTTCAGTGACGGC
CACTGTGACAGCCAGTGCAACTCAGCCGGC
TGCCTCTTCGACGGC 111GACTGCCAGCGTG
CGGAAGGCCAGTGCAACCCCCTGTACGACC
AGTACTGCAAGGACCACTTCAGCGACGGGC
ACTGCGACCAGGGCTGCAACAGCGCGGAGT
GCGAGTGGGACGGGCTGGACTGTGCGGAG
CATGTACCCGAGAGGCTGGCGGCCGGCACG
CTGGTGGTGGTGGTGCTGATGCCGCCGGAG
CAGCTGCGCAACAGCTCCTTCCACTTCCTGC
GGGAGCTCAGCCGCGTGCTGCACACCAACG
TGGTCTTCAAGCGTGACGCACACGGCCAGC
AGATGATCTTCCCCTACTACGGCCGCGAGGA
GGAGCTGCGCAAGCACCCCATCAAGCGTGC
CGCCGAGGGCTGGGCCGCACCTGACGCCCT
GCTGGGCCAGGTGAAGGCCTCGCTGCTCCC
TGGTGGCAGCGAGGGTGGGCGGCGGCGGA
GGGAGCTGGACCCCATGGACGTCCGCGGCT
CCATCGTCTACCTGGAGATTGACAACCGGCA
GTGTGTGCAGGCCTCCTCGCAGTGCTTCCA
GAGTGCCACCGACGTGGCCGCATTCCTGGG
AGCGCTCGCCTCGCTGGGCAGCCTCAACAT
CCCCTACAAGATCGAGGCCGTGCAGAGTGA
GACCGTGGAGCCGCCCCCGCCGGCGCAGC
TGCACTTCATGggagggggaagcggaggcggactgaa
cgacatcttcgaggctcagaaaatcgaatggcacgaaggtggc
ccaccacatcatcatcatcatcac
Human Notchl NRR nucleotide GGTGGGGCCGGGCGCGACATCCCCCCGCC
sequence GCTGATCGAGGAGGCGTGCGAGCTGCCCGA
GTGCCAGGAGGACGCGGGCAACAAGGTCTG
CAGCCTGCAGTGCAACAACCACGCGTGCGG
CTGGGACGGCGGTGACTGCTCCCTCAACTT
CAATGACCCCTGGAAGAACTGCACGCAGTCT
CTGCAGTGCTGGAAGTACTTCAGTGACGGC
CACTGTGACAGCCAGTGCAACTCAGCCGGC
TGCCTCTTCGACGGC 111GACTGCCAGCGTG
CGGAAGGCCAGTGCAACCCCCTGTACGACC
AGTACTGCAAGGACCACTTCAGCGACGGGC
ACTGCGACCAGGGCTGCAACAGCGCGGAGT
GCGAGTGGGACGGGCTGGACTGTGCGGAG
CATGTACCCGAGAGGCTGGCGGCCGGCACG
CTGGTGGTGGTGGTGCTGATGCCGCCGGAG
CAGCTGCGCAACAGCTCCTTCCACTTCCTGC
GGGAGCTCAGCCGCGTGCTGCACACCAACG
TGGTCTTCAAGCGTGACGCACACGGCCAGC
AGATGATCTTCCCCTACTACGGCCGCGAGGA
GGAGCTGCGCAAGCACCCCATCAAGCGTGC
CGCCGAGGGCTGGGCCGCACCTGACGCCCT
GCTGGGCCAGGTGAAGGCCTCGCTGCTCCC
TGGTGGCAGCGAGGGTGGGCGGCGGCGGA
GGGAGCTGGACCCCATGGACGTCCGCGGCT
CCATCGTCTACCTGGAGATTGACAACCGGCA
GTGTGTGCAGGCCTCCTCGCAGTGCTTCCA
GAGTGCCACCGACGTGGCCGCATTCCTGGG
AGCGCTCGCCTCGCTGGGCAGCCTCAACAT
CCCCTACAAGATCGAGGCCGTGCAGAGTGA
GACCGTGGAGCCGCCCCCGCCGGCGCAGC
TGCACTTCATG
Mouse Notch 1 NRR amino acid
sequence (amino acids in lower DAGNKVCNLQCNNHACGWDGGDCSLNFNDP
case type represent the Gp1 b WKNCTQSLQCWKYFSDGHCDSQCNSAGCLF
signal sequence and AviJHis DGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQ
tag of purified protein) GCNSAECEWDGLDCAEHVPERLAAGTLVLVVL
LPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQ
GQQMIFPYYGHEEELRKHPI KRSTVGWATSSL
LPGTSGGRQRRELDPMDIRGSIVYLEIDNRQC
VQSSSQCFQSATDVAAFLGALASLGSLNIPYKI
EAVKSEPVEPPLPSQLHLMgggsggglndifeaqkie
wheggpphhhhhh
Mouse Notch 1 NRR amino acid GGAGRDIPPPQIEEACELPECQVDAGNKVCNL
sequence QCNNHACGWDGGDCSLNFNDPWKNCTQSLQ
CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEG
QCNPLYDQYCKDHFSDGHCDQGCNSAECEW
DGLDCAEHVPERLAAGTLVLVVLLPPDQLRNN
SFHFLRELSHVLHTNWFKRDAQGQQMIFPYY
GHEEELRKHPIKRSTVGWATSSLLPGTSGGRQ
RRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQ
SATDVAAFLGALASLGSLN IPYKIEAVKSEPVEP
PLPSQLHLM
Mouse Notchl NRR nucleotide atgcctctcctcctcttgctgctcctg ctgccaagccccttacacgc
sequence (nucleotides in lower gGGTGGCGCTGGGCGCGACATTCCCCCACC
case type represent the Gp1 b GCAGATTGAGGAGGCCTGTGAGCTGCCTGA
signal sequence and AviJHis GTGCCAGGTGGATGCAGGCAATAAGGTCTG
tag of purified protein) CAACCTGCAGTGTAATAATCACGCATGTGGC
TGGGATGGTGGCGACTGCTCCCTCAACTTCA
ATGACCCCTGGAAGAACTGCACGCAGTCTCT
ACAGTGCTGGAAGTA I I I IAGCGACGGCCAC
TGTGACAGCCAGTGCAACTCGGCCGGCTGC
CTC I I IGATGGCTTCGACTGCCAGCTCACCG
AGGGACAGTGCAACCCCCTGTATGACCAGTA
CTGCAAGGACCACTTCAGTGATGGCCACTGC
GACCAGGGCTGTAACAGTGCCGAATGTGAG
TGGGATGGCCTAGACTGTGCTGAGCATGTAC
CCGAGCGGCTGGCAGCCGGCACCCTGGTG
CTGGTGGTGCTGCTTCCACCCGACCAGCTA
CGGAACAACTCCTTCCAC I I ICTGCGGGAGC
TCAGCCACGTGCTGCACACCAACGTGGTCTT
CAAGCGTGATGCGCAAGGCCAGCAGATGAT
CTTCCCGTACTATGGCCACGAGGAAGAGCT
GCGCAAGCACCCAATCAAGCGCTCTACAGT
GGGTTGGGCCACCTCTTCACTGCTTCCTGGT
ACCAGTGGTGGGCGCCAGCGCAGGGAGCT
GGACCCCATGGACATCCGTGGCTCCATTGTC
TACCTGGAGATCGACAACCGGCAATGTGTGC
AGTCATCCTCGCAGTGCTTCCAGAGTGCCAC
CGATGTGGCTGCCTTCCTAGGTGCTCTTGCG
TCACTTGGCAGCCTCAATATTCCTTACAAGAT
TGAGGCCGTGAAGAGTGAGCCGGTGGAGCC
TCCGCTGCCCTCGCAGCTGCACCTCATGgga
gggggaagcggaggcggactgaacgacatcttcgaggctcag
aaaatcgaatggcacgaaggtggcccaccacatcatcat catca
tcac
Mouse Notchl NRR nucleotide GGTGGCGCTGGGCGCGACATTCCCCCACCG
sequence CAGATTGAGGAGGCCTGTGAGCTGCCTGAG
TGCCAGGTGGATGCAGGCAATAAGGTCTGC
AACCTGCAGTGTAATAATCACGCATGTGGCT
GGGATGGTGGCGACTGCTCCCTCAACTTCAA
TGACCCCTGGAAGAACTGCACGCAGTCTCTA
CAGTGCTGGAAGTA I I I IAGCGACGGCCACT
GTGACAGCCAGTGCAACTCGGCCGGCTGCC
TC I I IGATGGCTTCGACTGCCAGCTCACCGA
GGGACAGTGCAACCCCCTGTATGACCAGTA
CTGCAAGGACCACTTCAGTGATGGCCACTGC
GACCAGGGCTGTAACAGTGCCGAATGTGAG
TGGGATGGCCTAGACTGTGCTGAGCATGTAC
CCGAGCGGCTGGCAGCCGGCACCCTGGTG
CTGGTGGTGCTGCTTCCACCCGACCAGCTA
CGGAACAACTCCTTCCAC I I ICTGCGGGAGC
TCAGCCACGTGCTGCACACCAACGTGGTCTT
CAAGCGTGATGCGCAAGGCCAGCAGATGAT
CTTCCCGTACTATGGCCACGAGGAAGAGCT
GCGCAAGCACCCAATCAAGCGCTCTACAGT
GGGTTGGGCCACCTCTTCACTGCTTCCTGGT
ACCAGTGGTGGGCGCCAGCGCAGGGAGCT
GGACCCCATGGACATCCGTGGCTCCATTGTC
TACCTGGAGATCGACAACCGGCAATGTGTGC
AGTCATCCTCGCAGTGCTTCCAGAGTGCCAC
CGATGTGGCTGCCTTCCTAGGTGCTCTTGCG
TCACTTGGCAGCCTCAATATTCCTTACAAGAT
TGAGGCCGTGAAGAGTGAGCCGGTGGAGCC
TCCGCTGCCCTCGCAGCTGCACCTCATG
Cyno-Notch1 NRR-Fc amino mgwsciilflvatatgahsGGAGRDIPPPLIEEACELPE
acid sequence (amino acids in CQEDAGNKVCSLQCNNHACGWDGGDCSLNF
lower case type represent the NDPWKNCTQSLQCWKYFSDGHCDSQCNSAG
signal sequence and hlgG 1 Fc CLFDGFDCQRAEGQCNPLYDQYCKDHFSDGH
fragment of purified protein) CDQGCNSAECEWDGLDCAEHVPERLAAGTLV
VVVLMPPEQLRNSSFHFLRELSRVLHTNVVFK
RDAHGQQMIFPYYGREEELRKHPIKRAAEGWA
APEALLGQVKASLLPGGGGGGRRRRELDPMD
VRGSIVYLEIDNRQCVQASSQCFQSATDVAAFL
GALASLGSLNIPYKIEAVQSETVEPPPPAQLHF
Mggggsggggepkssdkthtcppcpapellggpsvflfppkpk
dtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpr
eeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektisk
akgqprepqvytlppsreemtknqvsltclvkgfypsdiavewes
ngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvm
healhnhytqkslslspgk
Cyno-Notchl NRR amino acid GGAGRDIPPPLIEEACELPECQEDAGNKVCSL
sequence QCNNHACGWDGGDCSLNFNDPWKNCTQSLQ
CWKYFSDGHCDSQCNSAGCLFDGFDCQRAE
GQCNPLYDQYCKDHFSDGHCDQGCNSAECE
WDGLDCAEHVPERLAAGTLVVVVLMPPEQLR
NSSFHFLRELSRVLHTNVVFKRDAHGQQMIFP
YYGREEELRKHPIKRAAEGWAAPEALLGQVKA
SLLPGGGGGGRRRRELDPMDVRGSIVYLEIDN
RQCVQASSQCFQSATDVAAFLGALASLGSLNI
PYKIEAVQSETVEPPPPAQLHFM
Cyno-Notchl NRR-Fc atgggatggagctgtatcatcctcttcttggtagcaacagctacag
nucleotide sequence gcgcgcactccGGTGGGGCCGGGCGCGACATCC
(nucleotides in lower case type CCCCGCCGCTGATCGAGGAGGCGTGCGAGC
represent the signal sequence TGCCCGAGTGCCAGGAGGACGCGGGCAACA
and hlgG 1 Fc fragment of AGGTCTGCAGCCTGCAGTGCAACAACCACG
purified protein) CGTGCGGCTGGGACGGCGGTGACTGCTCCC
TCAACTTCAATGACCCCTGGAAGAACTGCAC
GCAGTCTCTGCAGTGCTGGAAGTACTTCAGT
GACGGCCACTGTGACAGCCAGTGCAACTCA
GCCGGCTGCCTCTTCGACGGC I I IGACTGC
CAGCGTGCGGAAGGCCAGTGCAACCCCCTG
TACGACCAGTACTGCAAGGACCACTTCAGCG
ACGGGCACTGCGACCAGGGCTGCAACAGCG
CGGAGTGCGAGTGGGACGGGCTGGACTGTG
CGGAGCATGTACCCGAGAGGCTGGCGGCCG
GCACGCTGGTGGTGGTGGTGCTGATGCCGC
CGGAGCAGCTGCGCAACAGCTCCTTCCACTT
CCTGCGGGAGCTCAGCCGCGTGCTGCACAC
CAACGTGGTCTTCAAGCGTGACGCACACGG
CCAGCAGATGATCTTCCCCTACTACGGCCGC
GAGGAGGAGCTGCGCAAGCACCCCATCAAG
CGTGCCGCCGAGGGCTGGGCCGCACCTGAA
GCCCTGCTGGGCCAGGTGAAGGCCTCGCTG
CTCCCTGGTGGCGGTGGAGGTGGGCGGCG
GCGGAGGGAGCTGGACCCCATGGACGTCCG
CGGCTCCATCGTCTACCTGGAGATTGACAAC
CGGCAGTGTGTGCAGGCCTCCTCGCAGTGC
TTCCAGAGTGCCACCGACGTGGCCGCATTC
CTGGGAGCGCTCGCCTCGCTGGGCAGCCTC
AACATCCCCTACAAGATCGAGGCCGTGCAGA
GTGAGACCGTGGAGCCGCCCCCGCCGGCG
CAGCTGCACTTCATGggagggggcggatccggcgga
ggcggagagcccaaatcttctgacaaaactcacacatgcccac
cgtgcccagcacctgaactcctggggggaccgtcagtcttcctctt
ccccccaaaacccaaggacaccctcatgatctcccggacccct
gaggtcacatgcgtggtggtggacgtgagccacgaagaccctg
aggtcaagttcaactggtacgtggacggcgtggaggtgcataat
gccaagacaaagccgcgggaggagcagtacaacagcacgta
ccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaa
tggcaaggagtacaagtgcaaggtctccaacaaagccctccca
gcccccatcgagaaaaccatctccaaag ccaaagggcagccc
cgagaaccacaggtgtacaccctgcccccatcccgggaggag
atgaccaagaaccaggtcagcctgacctgcctggtcaaaggctt
ctatcccagcgacatcgccgtggagtgggagagcaatgggcag
ccggagaacaactacaagaccacgcctcccgtgctggactccg
acggctccttcttcctctatagcaagctcaccgtggacaagagca
ggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagg
ctctgcacaaccactacacgcagaagagcctctccctgtccccg
ggtaaa
Cyno-Notchl NRR nucleotide GGTGGGGCCGGGCGCGACATCCCCCCGCC
sequence GCTGATCGAGGAGGCGTGCGAGCTGCCCGA
GTGCCAGGAGGACGCGGGCAACAAGGTCTG
CAGCCTGCAGTGCAACAACCACGCGTGCGG
CTGGGACGGCGGTGACTGCTCCCTCAACTT
CAATGACCCCTGGAAGAACTGCACGCAGTCT
CTGCAGTGCTGGAAGTACTTCAGTGACGGC
CACTGTGACAGCCAGTGCAACTCAGCCGGC
TGCCTCTTCGACGGC 111GACTGCCAGCGTG
CGGAAGGCCAGTGCAACCCCCTGTACGACC
AGTACTGCAAGGACCACTTCAGCGACGGGC
ACTGCGACCAGGGCTGCAACAGCGCGGAGT
GCGAGTGGGACGGGCTGGACTGTGCGGAG
CATGTACCCGAGAGGCTGGCGGCCGGCACG
CTGGTGGTGGTGGTGCTGATGCCGCCGGAG
CAGCTGCGCAACAGCTCCTTCCACTTCCTGC
GGGAGCTCAGCCGCGTGCTGCACACCAACG
TGGTCTTCAAGCGTGACGCACACGGCCAGC
AGATGATCTTCCCCTACTACGGCCGCGAGGA
GGAGCTGCGCAAGCACCCCATCAAGCGTGC
CGCCGAGGGCTGGGCCGCACCTGAAGCCCT
GCTGGGCCAGGTGAAGGCCTCGCTGCTCCC
TGGTGGCGGTGGAGGTGGGCGGCGGCGGA
GGGAGCTGGACCCCATGGACGTCCGCGGCT
CCATCGTCTACCTGGAGATTGACAACCGGCA
GTGTGTGCAGGCCTCCTCGCAGTGCTTCCA
GAGTGCCACCGACGTGGCCGCATTCCTGGG
AGCGCTCGCCTCGCTGGGCAGCCTCAACAT
CCCCTACAAGATCGAGGCCGTGCAGAGTGA
GACCGTGGAGCCGCCCCCGCCGGCGCAGC
TGCACTTCATG
Example 2
Generation, Cloning and Humanization of Rat Anti-Notch 1 Inhibitory Antibodies
A. Immunization and Hybridoma Generation
The human and mouse immunogens described in Example 1 were co-injected
into Sprague-Dawley rats for the generation of hybridomas. Sprague-Dawley rats were
immunized by subcutaneous injections of a mixture containing 20 mg each of human
and mouse Notchl NRR_Avi_His recombinant proteins in Freund's complete adjuvant.
Immunizations were repeated at 2-week intervals for 12 weeks. Collected sera samples
at day 0, 35, 49, and 63 after the 1st injection were tested for circulating anti-Notch 1
antibody titer activity by enzyme-linked immunosorbent assay (ELISA), as described
below.
When optimal titers were reached, a final dose of the protein mixture was
injected intravenously (tail vein) into a rat having optimal antibody titer 4 days before it
was to be sacrificed for splenocyte collection. Total splenocytes (2 x 10E08) from the
rat were fused with mouse myeloma cell line P3X63.Ag8.653 (2.5 x 10E07) using PEG
4000. Fused cells were plated out in 96-well plates (0.2 ml/well) and subjected to HAT
selection (RPMI 1640 containing 5 x 10E-04 M Hypoxanthine, 1.6 x 10E-05 M
Thymidine, 4 x 10E-04 M Aminopterin, and 20% Heat Inactivated FCS).
Fourteen days post fusion, hybridoma supernatants were harvested and tested
for the presence of rat IgGs that exhibit binding activity to human and/or mouse Notchl
NRR recombinant protein, and full length Notchl expressed on the surface of U-2 OS
cells by ELISA, as described below. Supernatants that showed binding activity to
Notchl targets were further tested for their ability to block Notchl mediated signaling
activity in a reporter gene assay, as described below. Selected Notchl signaling
blocking clones were then subcloned for further analysis.
B. Screening and Selection of Notchl Specific Antibodies
1. Recombinant protein binding ELISA
Supernatants from hybridoma cultures were first screened for binding to
recombinant human and mouse immunogens by ELISA. Purified human or mouse
Notchl NRR_Avi_His tag proteins were coated on CoStar hi-bound 96-well ELISA
plates in 0OmI of PBS with Mg/Ca at a concentration of ^g/ml overnight. The plates
were washed with PBS-Mg/Ca and blocked for 1 hour with 1% BSA in PBS-Mg/Ca.
Blocking solution was decanted from the plate and hybridoma culture supernatants
were applied to the plate. After incubation at room temperature for 1 hour, plates were
washed again with PBS-Mg/Ca before HRP-conjugated secondary antibody diluted
( 1 :20,000) in blocking buffer was applied. When the primary antibody tested was rat
IgG, the secondary antibody was goat anti-rat IgG Fc (Bethyl Biotech); and when the
primary antibody was mouse IgG, the secondary antibody was goat anti-mouse IgG Fc
(Thermal Scientific).
After 1 hour incubation with the secondary antibody, plates were washed again,
as described above, and TMB substrate solution was added. The developing reaction
was allowed for 10 minutes before the stopping solution, 0.1 8M H2S0 4, was added.
Absorbance at O. D. 450 nM was measured and data was plotted and analyzed with
Microsoft Excel and Graphpad-Prizm software. The antibodies exhibiting binding activity
to human and/or mouse Notchl NRR were selected for further cell based ELISA, as
described below.
2. Cell based ELISA
Supernatants from clones displaying positive binding to immunogens in
recombinant Notchl NRR based ELISAs described above were then screened for cell
surface Notchl binding in a cell-based ELISA. U-2 OS cells stably overexpressing
human or mouse full length Notchl protein on cell surface were plated at 50,000
cells/well in 96 well plates (white opaque, BD/VWR) the day before ELISA assay. On
the day of the ELISA, culture media were removed from wells and serially diluted ( 1:3 in
blocking buffer) antibody solutions or hybridoma culture supernatants were applied to
the plate. Plates were incubated at room temperature for 2 hours before being washed
with PBS-Mg/Ca. HRP-conjugated secondary antibody was then applied and incubated
with cells for 1 hour as described above for recombinant protein ELISA. Plates were
washed with PBS-Mg/Ca before being developed with Pico-Chemiluminescent
developing kit (Thermal Scientific), and chemiluminescence measurements were
performed per manufacturer's instructions. Data plotting and analyses were performed
with Microsoft Excel and Graphpad-Prizm software. This data was used in screening of
hybridoma clones and the characterization of a parental rat and humanized antibodies,
as described in the Examples below.
3. Reporter gene assays
Supernatants from clones displaying positive binding to immunogens were then
screened for neutralization activity in human and mouse Notchi reporter gene coculture
assays (RGA). Results of the screening were used to select primary clones.
Human Notchi reporter cells were trypinized and harvested from culture plate in
complete McCoy's 5A media (McCoy's 5A with 10% FBS and penicillin, streptomycin,
Invitrogen) and counted. Appropriate dilutions of cells were made with the same
medium to allow for 3,000 cells/well in a total volume of dqm ΐ/well on a 96 well culture
plate (white opaque, BD/VWR), in the presence of serially diluted ( 1:3 in complete
McCoy's 5A media) antibody solutions or hybridoma culture supernatants. The mixture
of cells and antibody dilutions were incubated on the plates in a cell culture incubator
(37°C, 5% C0 2) for 1hr before 15,000/well of human DLL4-HEK293 cells were added to
each well. After addition of hDLL4-HEK293 cells, the plates were further incubated for
20hrs in the incubator and Dual-Glo Luciferase assay system (Promega) was used to
measure the firefly luciferase and internal control Renilla luciferase activity per
manufacturer's instructions. Data was plotted and analyzed using Microsoft Excel and
Graphpad-Prism software. Mouse Notchi reporter gene co-culture assay was
performed as described for human Notchi reporter gene co-culture assay, except
20,000 cells/well of mouse Notchi reporter cells were co-cultured with 40,000 cells/well
of mouse DLL4-HEK293 cells.
C. Cloning and Sequencing
Primary clones with confirmed cell surface binding or neutralizing activities were
subcloned, such as clones 438 and 351 further described below. RNAs from the
subclones were extracted and the variable region DNA sequences from the expressed
antibodies were obtained via RT-PCR cloning, as described below.

WHAT IS CLAIMED IS:
1. An antibody that binds to Notch 1, comprising:
a heavy chain variable region having a CDR1 region, a CDR2 region, and
a CDR3 region from the heavy chain variable region comprising SEQ ID NO: 7 1.
2. An antibody that binds to Notchl comprising:
a light chain variable region having a CDR1 region, a CDR2 region, and a
CDR3 region from the light chain variable region comprising SEQ ID NO: 97.
3. An antibody that binds to Notchl comprising:
a heavy chain variable region having a CDR1 region, a CDR2 region, and
a CDR3 region from the heavy chain variable region as shown in SEQ ID NO: 71,
and
a light chain variable region having a CDR1 region, a CDR2 region, and a
CDR3 region from the light chain variable region as shown in SEQ ID NO: 97.
4. The antibody according to any one of claims 1-3, comprising a heavy chain
variable region amino acid sequence that is at least 90% identical to SEQ ID NO:
7 1.
5. The antibody according to claim 4, comprising a heavy chain variable region
amino acid sequence as set forth in SEQ ID NO: 7 1.
6. The antibody according to any one of claims 1-3, comprising a light chain
variable region amino acid sequence that is at least 90% identical to SEQ ID NO:
97.
7. The antibody according to claim 6, comprising a light chain variable region amino
acid sequence as set forth in SEQ ID NO: 97.
8. The antibody according to any one of claims 1-3, comprising a heavy chain
amino acid sequence that is at least 90% identical to SEQ ID NO: 111.
9. The antibody according to claim 8, comprising a heavy chain amino acid
sequence as set forth in SEQ ID NO: 111.
10. The antibody according to any one of claims 1-3, comprising a light chain amino
acid sequence that is at least 90% identical to SEQ ID NO: 113.
11. The antibody according to claim 10, comprising a light chain amino acid
sequence as set forth in SEQ ID NO: 113.
12. An antibody that binds to Notchi, comprising:
a heavy chain variable region amino acid sequence that is at least 90%
identical to SEQ ID NO: 71; and
a light chain variable amino acid sequence that is at least 90% identical to
SEQ ID NO: 97.
13. An antibody that binds to Notchi, comprising:
a heavy chain variable region amino acid sequence as set forth in SEQ ID
NO: 71; and
a light chain variable amino acid sequence as set forth in SEQ ID NO: 97.
14. An antibody that binds to Notchi, comprising:
a heavy chain amino acid sequence that is at least 90% identical to SEQ
ID NO: 111; and
a light chain amino acid sequence that is at least 90% identical to SEQ ID
NO: 113.
15. An antibody that binds to Notchi, comprising:
a heavy chain amino acid sequence as set forth in SEQ ID NO: 111; and
a light chain amino acid sequence as set forth in SEQ ID NO: 113.
16. An antibody that binds to Notchi and competes for binding to Notchi with the
antibody of claims 1, 2 or 3.
17. An antibody or antigen binding portion thereof, that binds to Notchl , wherein the
antibody binds an epitope comprising at least 7 amino acid residues selected
from Asn 1461 , Lys 1462, Val 1463, Cys 1464, Leu 1466, Leu 1580, Tyr 1621,
Gly 1622, Met 1670, Asp 1671 , Val 1672, Arg 1673, Leu 1707, Ala 1708, Leu
1710, Leu 171 1, Leu 1712, Leu 1713, Leu 1716 and Leu 1718.
18. The antibody according to any one of claims 1-17, wherein said antibody is an
IgA, IgD, IgE, IgG, or IgM molecule, or is derived therefrom.
19. The antibody according to claim 18, wherein the antibody is an IgG, having a
subclass selected from the group consisting of lgG1, lgG2, lgG3 and lgG4, or is
derived therefrom.
20. The antibody according to claim 19, wherein the subclass is derived from lgG1 .
21. A nucleic acid that encodes the antibody according to any one of claims 1-20.
22. A nucleic acid comprising the sequence as set forth in SEQ ID NO: 112.
23. A nucleic acid comprising the sequence as set forth in SEQ ID NO: 114.
24. A vector comprising the nucleic acid according to any one of claims 21-23.
25. A host cell comprising the vector of claim 24.
26. A process for producing an antibody, comprising cultivating the host cell of claim
25 and recovering the antibody from the culture media.
27. A host cell that recombinantly produces the antibody according to any one of
claims 1-20.
28. A pharmaceutical composition comprising the antibody according to any one of
claims 1-20 and a pharmaceutically acceptable carrier.
29. A method of treating a disorder in a subject in need thereof, comprising
administering to said subject the antibody according to any one of claims 1-20, or
the pharmaceutical composition of claim 28.
30. The method of claim 29, wherein said disorder is associated with abnormal
activation of Notchi .
3 1. The method of claim 29 or 30, wherein said disorder is selected from the group
consisting of T-cell acute lymphoblastic leukemia (T-ALL), non-small cell lung
cancer (NSCLC) and breast cancer in a subject in need thereof.
32. The antibody of any one of claims 1-20 for use in therapy.
33. The use of an antibody of any one of claims 1-20 for the manufacture of a
medicament.
34. The use according to claim 32 or 33, wherein said use is for the treatment of Tcell
acute lymphoblastic leukemia (T-ALL), non-small cell lung cancer (NSCLC)
and breast cancer in a subject in need thereof.
35. An antibody that binds to Notchi, comprising:
a heavy chain variable region amino acid sequence as set forth in SEQ ID
NO: 115; and
a light chain variable amino acid sequence as set forth in SEQ ID NO:
129.
36. An antibody that binds to Notchi, comprising:
a heavy chain amino acid sequence as set forth in SEQ ID NO: 149; and
a light chain amino acid sequence as set forth in SEQ ID NO: 151 .
37. An antibody that binds to human Notchi , wherein the antibody binds an epitope
comprising at least 8 amino acid residues selected from Asp 1458, Asn 1461, Val
1463, Cys 1464, Leu 1466, Leu 1580, Met 1581, Pro 1582, Tyr 1621 , Gly 1622,
Arg 1623, Asp 1671, Val 1672, Arg 1673, Gly 1674, Leu 1710, Gly 171 1, Ser
1712, Leu 1713, Asn 1714, lie 1715, Pro 1716 and Lys 1718.
38. An antibody that demonstrates higher inhibition of Notchi activation of a mutant
Notchi receptor compared to inhibition of Notchi activation of a native Notchi
receptor.
39. A pharmaceutical composition comprising the antibody according to any one of
claims 35-38 and a pharmaceutically acceptable carrier.
40. A method of treating a disorder in a subject in need thereof, comprising
administering to said subject the antibody according to any one of claims 35-38,
or the pharmaceutical composition of claim 39.
4 1.The method of claim 40, wherein said disorder is associated with abnormal
activation of Notchi .
42. The method of claim 40 or 41, wherein said disorder is selected from the group
consisting of T-cell acute lymphoblastic leukemia (T-ALL), non-small cell lung
cancer (NSCLC) and breast cancer in a subject in need thereof.
43. The antibody of any one of claims 35-38 for use in therapy.
44. The use of an antibody of any one of claims 35-38 for the manufacture of a
medicament.
45. The use according to claim 43 or 44, wherein said use is for the treatment of Tcell
acute lymphoblastic leukemia (T-ALL), non-small cell lung cancer (NSCLC)
and breast cancer in a subject in need thereof.