Indian Patents
Title of Invention

FULL-LENGTH ANTIBODY FUSION PROTEIN,POLYNUCLEOTIDE AND VECTOR THEREOF
Abstract ANTIBODY TARGETING THROUGH A MODULAR RECOGNITION DOMAIN Antibodies containing one or more modular recognition domains (MRDs) used to target the antibodies to specific sites are described. The use of the antibodies containing one or more modular recognition domains to treat disease, and methods of making antibodies containing one or more modular recognition domains are also described.
Full Text The Patents Act, 1970
Act 39 of 1970
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
[See Section 10; Rule 13]
“ANTIBODY TARGETING THROUGH A MODULAR RECOGNITION
DOMAIN”
APPLICANT AND ADDRESS:
THE SCRIPPS RESEARCH INSTITUTE; Drop TPC-8, 10550 N. Torrey Pines Rd. La
Jolla, CA 92037, United States of America
NATIONALITY: US
The following specification particularly describes the invention and the manner in which it is
to be performed.
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TECHNICAL FIELD
This disclosure relates generally to antibodies containing one or more modular recognition
domains and more specifically to the use of the antibodies containing one or more modular
recognition domains to treat disease, and methods of making antibodies containing one or
more modular recognition domains.
BACKGROUND
Catalytically active monoclonal antibodies (Abs) can be used for selective prodrug activation
and chemical transformations. Monoclonal Abs with aldolase activity have emerged as
highly efficient catalysts for a number of chemical transformations, particularly aldol and
retro-aldol reactions. The retro-aldolase activity of Abs, such as 38C2 and 93F3, have
allowed researchers to design, synthesize, and evaluate prodrugs of various chemotherapeutic
agents that can be activated by retro-aldol reactions. (Construction of 38C2 was described in
WO 97/21803, herein incorporated by reference). 38C2 contains an antibody combining site
that catalyzes the aldol addition reaction between an aliphatic donor and an aldehyde
acceptor. In a syngeneic mouse model of neuroblastoma, systemic administration of an
etoposide prodrug and intra-tumor injection of 38C2 inhibited tumor growth.
One drawback in the use of catalytic Abs is that they lack a device to target the catalytic Ab
to the malignant cells. Previous studies demonstrated that in an antibody-directed enzyme
prodrug therapy (ADEPT) or antibody-directed abzyme prodrug therapy (ADAPT) approach,
enzymes or catalytic antibodies can be directed to tumor cells by chemical conjugation or
recombinant fusion to targeting antibodies. However, a more efficient alternative would be
using the catalytic antibody fused to a targeting peptide located outside the antibody
combining site, thereby leaving the active site available for the prodrug activation. For
example, the fusion of Ab 38C2 to an integrin αvβ3-binding peptide would selectively
localize the antibody to the tumor and/or the tumor vasculature and trigger prodrug activation
at that site. The potential therapy of this approach is supported by preclinical and phase Ill
clinical data suggesting that peptides can be converted into viable drugs through fusion to
antibody Fc regions.
The development of bispecific or multi-specific antibodies that target two or more cancer
targets simultaneously and or activate prodrugs offers a novel and promising solution to
attacking cancer and other diseases. Such antibodies are exemplified in Figure 1 of the
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present application. Studies of bispecific antibodies (BsAb) that simultaneously target two
tumor-associated antigens (e.g. growth factor receptors) for down-regulation of multiple cell
proliferation/survival pathways has provided support for this approach.Traditionally,
bispecific antibodies have been prepared by chemically linking two different monoclonal
antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma. Dualspecific,
tetravalent IgG-like molecules, or dual-variable-domain immunoglobins, have been
engineered from two monoclonal antibodies. Thes dual-variable-domain immunoglobins are
capable of binding both antigens in the presence of serum. However, these approaches
present challenges with respect to manufacturing, yield and purity.
A variety of recombinant methods have been developed for efficient production of small
BsAb fragments such as diabody, minibody, and Fab-scFv fusion proteins. These BsAb
fragments may possess some advantages over the full-length IgG-like molecules for certain
clinical applications, such as for tumor radio-imaging and targeting, because of better tissue
penetration and faster clearance from the circulation. On the other hand, IgG-like BsAb may
prove to be preferred over smaller BsAb fragments for other in vivo applications, specifically
for oncology indications, by providing the Fc domain that confers long serum half-life and
supports secondary immune function, such as antibody-dependent cellular cytotoxicity and
complement-mediated cytotoxicity. Unlike their fragment counterparts, engineering and
production of recombinant IgG-like BsAb has been, however, rather technically challenging
due to their large size (~150-200 kDa) and structural complexity. Success in the field, as
judged by successful application in animal models, has been very limited. Recently, with the
examination of a variety of constructs, the efficient expression of Fc domain containing BsAb
molecules in mammalian cells has made some strides.
Another approach that has been used to target antibodies is through the use of peptibodies.
Peptibodies are essentially peptide fusions with antibody Fc regions. Given the success of
studies using random peptide libraries to find high-affinity peptide ligands for a wide variety
of targets, fusion of such peptides to antibody Fc regions provides a means of making
peptides into therapeutic candidates by increasing their circulatory half-life and activity
through increased valency.
Protein interactions with other molecules are basic to biochemistry. Protein interactions
include receptor-ligand interactions, antibody-antigen interactions, cell-cell contact and
pathogen interactions with target tissues. Protein interactions can involve contact with other
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proteins, with carbohydrates, oligosaccharides, lipids, metal ions and the like materials. The
basic unit of protein interaction is the region of the protein involved in contact and
recognition, and is referred to as the binding site or target site.
Peptides derived from phage display libraries typically retain their binding characteristics
when linked to other molecules. Specific peptides of this type can be treated as modular
specificity blocks or molecular recognition domains (MRDs) that can be combined to create a
single protein with binding specificities for several defined targets.
An example of such a defined target site is integrin. Integrins are a family of transmembrane
cell adhesion receptors that are composed of α and β subunits and mediate cell attachment to
proteins within the extracellular matrix. At present, eighteen α and eight β subunits are
known; these form 24 different αβ heterodimers with different specificities for various ECM
cell-adhesive proteins. Ligands for various integrins include fibronectin, collagen, laminin,
von Willebrand factor, osteopontin, thrombospondin, and vitronectin, which are all
components of the ECM. Certain integrins can also bind to soluble ligands such as
fibrinogen or to other adhesion molecules on adjacent cells. Integrins are known to exist in
distinct activation states that exhibit different affinities for ligand. Recognition of soluble
ligands by integrins strictly depends on specific changes in receptor conformation. This
provides a molecular switch that controls the ability of cells to aggregate in an integrin
dependent manner and to arrest under the dynamic flow conditions of the vasculature. This
mechanism is well established for leukocytes and platelets that circulate within the blood
stream in a resting state while expressing non-activated integrins. Upon stimulation through
proinflammatory or prothrombotic agonists, these cell types promptly respond with a number
of molecular changes including the switch of key integrins, β2 integrins for leucocytes and
αvβ3 for platelets, from “resting” to “activated” conformations. This enables these cell types
to arrest within the vasculature, promoting cell cohesion and leading to thrombus formation.
It has been demonstrated that a metastatic subset of human breast cancer cells expresses
integrin αvβ3 in a constitutively activated form. This aberrant expression of αvβ3 plays a
role in metastasis of breast cancer as well as prostate cancer, melanoma, and neuroblastic
tumors. The activated receptor strongly promotes cancer cell migration and enables the cells
to arrest under blood flow conditions. In this way, activation of αvβ3 endows metastatic cells
with key properties likely to be critical for successful dissemination and colonization of target
organs. Tumor cells that have successfully entered a target organ may further utilize αvβ3 to
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thrive in the new environment, as αvβ3 matrix interactions can promote cell survival and
proliferation. For example, αvβ3 binding to osteopontin promotes malignancy and elevated
levels of osteopontin correlate with a poor prognosis in breast cancer.
For these reasons, and for its established role in angiogenesis, the αvβ3 integrin is one of the
most widely studied integrins. Antagonists of this molecule have significant potential for use
in targeted drug delivery. One approach that has been used to target αvβ3 integrin uses the
high binding specificity to αvβ3 of peptides containing the Arg-Gly-Asp (RGD) sequence.
This tripeptide, naturally present in extracellular matrix proteins, is the primary binding site
of the αvβ3 integrin. However, RGD based reporter probes are problematic due to fast blood
clearance, high kidney and liver uptake and fast tumor washout. Chemical modification of
cyclised RGD peptides has been shown to increase their stability and valency. These
modified peptides are then coupled to radio-isotpes and used either for tumor imaging or to
inhibit tumor growth.
lntegrin αvβ3 is one of the most well characterized integrin heterodimers and is one of several
heterodimers that have been implicated in tumor-induced angiogenesis. While sparingly
expressed in mature blood vessels, αvβ3 is significantly up-regulated during angiogenesis in
vivo. The expression of αvβ3 correlates with aggressiveness of disease in breast and cervical
cancer as well as in malignant melanoma. Recent studies suggest that αvβ3 may be useful as
a diagnostic or prognostic indicator for some tumors. Integrin αvβ3 is particularly attractive
as a therapeutic target due to its relatively limited cellular distribution. It is not generally
expressed on epithelial cells, and minimally expressed on other cell types. Furthermore,
αvβ3 antagonists, including both cyclic RGD peptides and monoclonal antibodies,
significantly inhibit cytokine-induced angiogenesis and the growth of solid tumor on the
chick chorioallantoic membrane.
Another integrin heterodimer, αvβ5, is more widely expressed on malignant tumor cells and
is likely involved in VEGF-mediated angiogenesis. It has been shown that αvβ3 and αvβ5
promote angiogenesis via distinct pathways: αvβ3 through bFGF and TNF-a, and αvβ5
through VEGF and TGF-α. It has also been shown that inhibition of Src kinase can block
VEGF-induced, but not FGF2-induced, angiogenesis. These results strongly imply that FGF2
and VEGF activate different angiogenic pathways that require αvβ3 and αvβ5, respectively.
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Integrins have also been implicated in tumor metastasis. Metastasis is the primary cause of
morbidity and mortality in cancer. Malignant progression of melanoma, glioma, ovarian and
breast cancer have all been strongly linked with the expression of the integrin αvβ3 and in
some cases with αvβ5. More recently, it has been shown that activation of integrin αvβ3 play
a significant role in metastasis in human breast cancer. A very strong correlation between
expression of αvβ3 and breast cancer metastasis has been noted where normal breast epithelia
are αvβ3 negative and approximately 50% of invasive lobular carcinomas and nearly all bone
metastases in breast cancer express αvβ3. Antagonism of αvβ3 with a cyclic peptide has been
shown to synergize with radioimmunotherapy in studies involving breast cancer xenografts.
Angiogenesis, the formation of new blood vessels from existing ones, is essential to may
physiological and pathological processes. Normally, angiogenesis is tightly regulated by proand
anti-angiogenic factors, but in the case of diseases such as cancer, ocular neovascular
disease, arthritis and psoriasis, the process can go awry. The association of angiogenesis with
disease has made the discovery of anti-angiogenic compound attractive. The most promising
phage derived anti-angiogenic peptide described to date, developed by Amgen, neutralizes
the angiogenic cytokine Ang2.
While the VEGFs and their receptors have been among the most extensively targeted
molecules in the angiogenesis field, preclinical efforts targeting the more recently discovered
angiopoietin-Tie2 pathway are underway. Both protein families involve ligand receptor
interactions, and both include members whose functions are largely restricted postnatally to
endothelial cells and some hematopoietic stem cell lineages. Tie-2 is a receptor tyrosine
kinase with four known ligands, angiopoietin-1 (Ang1) through angiopoietin-4 (Ang4), the
best studied being Angl and Ang2. Ang1 stimulates phosphorylation of Tie2 and the Ang2
interaction with Tie2 has been shown to both antagonize and agonize Tie2 receptor
phosphorylation. Elevated Ang2 expression at sites of normal and pathological postnatal
angiogenesis circumstantially implies a proangiogenic role for Ang2. Vessel-selective Ang2
induction associated with angiogenesis has been demonstrated in diseases including cancer.
In patients with colon carcinoma, Ang2 is expressed ubiquitously in tumor epithelium,
whereas expression of Ang1 in tumor epithelium was shown to be rare. The net gain of Ang2
activity has been suggested to be an initiating factor for tumor angiogenesis.
Other fusion proteins directed towards cellular receptors are under clinical evaluation.
Herceptin (Trastuzumab), developed by Genentech, is a recombinant humanized monoclonal
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antibody directed against the extracellular domain of the human epidermal tyrosine kinase
receptor 2 (HER2 or ErbB2). The HER2 gene is overexpressed in 25% of invasive breast
cancers, and is associated with poor prognosis and altered sensitivity to chemotherapeutic
agents. Herceptin blocks the proliferation of ErbB2-overexpressing breast cancers, and is
currently the only ErbB2 targeted antibody therapy approved by the FDA for the treatment of
ErbB2 over-expressing metastatic breast cancer (MBC). In normal adult cells, few ErbB2
molecules exist at the cell surface ~ 20,000 per cell, so few heterodimers are formed and
growth signals are relatively weak and controllable. When ErbB2 is overexpressed, ~
500,000 per cell, multiple ErbB2 heterodimers are formed and cell signaling is stronger,
resulting in enhanced responsiveness to growth factors and malignant growth. This explains
why ErbB2 overexpression is an indicator of poor prognosis in breast tumors and may be
predictive of response to treatment.
ErbB2 is a promising and validated target for breast cancer, where it is found both in primary
tumor and metastatic sites. Herceptin induces rapid removal of ErbB2 from the cell surface,
thereby reducing its availability to heterodimerize and promote growth. Mechanisms of
action of Herceptin observed in experimental in vitro and in vivo models include inhibition of
proteolysis of ErbB2's extracellular domain, disruption of downstream signaling pathways
such as phosphatidylinositiol 3-kinase (P13K) and mitogen-activated protein kinase (MAPK)
cascades, GI cell-cycle arrest, inhibition of DNA repair, suppression of angiogenesis and
induction of antibody dependent cellular cytotoxicity (ADCC). The majority of patients with
metastatic breast cancer who initially respond to Herceptin, however, demonstrate disease
progression within one year of treatment initiation.
Another target cellular receptor is type 1 insulin-like growth factor-1 receptor (IGF-1R), IGF-
1R is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and
proliferation. The IGF system is frequently deregulated in cancer cells by the establishment
of autocrine loops involving IGF-I or -II and/or IGF-1R overexpression. Moreover,
epidemiological studies have suggested a link between elevated IGF levels and the
development of major human cancers, such as breast, colon, lung and prostate cancer.
Expression of IGFs and their cognate receptors has been correlated with disease stage,
reduced survival, development of metastases and tumor de-differentiation.
Besides IGF-1R, epidermal growth factor receptor (EGFR) has also been implicated in the
tumorigenesis of numerous cancers. Effective tumor inhibition has been achieved both
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experimentally and clinically with a number of strategies that antagonize either receptor
activity. Because of the redundancy of growth signaling pathways in tumor cells, inhibition
of one receptor function (e.g. EGFR) could be effectively compensated by up-regulation of
other growth factor receptor (e.g. IGF-1R)-mediated pathways. For example, a recent study
has shown that malignant glioma cell lines expressing equivalent EGFR had significantly
different sensitivity to EGFR inhibition depending on their capability of activating IGF-1R
and its downstream signaling pathways. Other studies have also demonstrated that
overexpression and/or activation of IGF-1R in tumor cells might contribute to their resistance
to chemotherapeutic agents, radiation, or antibody therapy such as Herceptin. And
consequently, inhibition of IGF-1R signaling has resulted in increased sensitivity of tumor
cells to Herceptin.
EGFR is a receptor tyrosine kinase that is expressed on many normal tissues as well as
neoplastic lesions of most organs. Overexpression of EGFR or expression of mutant forms of
EGFR has been observed in many tumors, particularly epithelial tumors, and is associated
with poor clinical prognosis. Inhibition of signaling through this receptor induces an antitumor
effect. With the FDA approval of Cetuximab, also known as Erbitux (a mouse/human
chimeric antibody) in February of 2004, EGFR became an approved antibody drug target for
the treatment of metastatic colorectal cancer. In March of 2006, Erbitux also received FDA
approval for the treatment squamous cell carcinoma of the head and neck (SCCHN). More
recently, Vectibix, a fully human antibody directed against EGFR, was approved for
metastatic colorectal cancer. Neither drug is a stand-alone agent in colorectal cancer – they
were approved as add-ons to existing colorectal regimens. In colorectal cancer, Erbitux is
given in combination with the drug irinotecan and Vectibix is administered after disease
progression on, or following fluoropyrimidine-, oxaliplatin-, and irinotecan- containing
chemotherapy regimens. Erbitux has been approved as a single agent in recurrent or
metastatic SCCHN only where prior platinum-based chemotherapy has failed. Advanced
clinical trials which use these drugs to target non-small cell lung carcinoma are ongoing.
The sequence of Erbitux or the EGFR antibody, is well known in the art (see for example,
Goldstein, et al., Clin.Cancer Res. 1:1311, 1995; U.S. Patent No. 6,217,866), herein
incorporated by reference.
An obstacle in the utilization of a catalytic antibody for selective prodrug activation in cancer
therapy has been systemic tumor targeting. The present disclosure describes an approach
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based on the adaptation of target binding peptides, or modular recognition domains (MRDs),
which are fused to full length antibodies that effectively target tumor cells or soluble
molecules while retaining the prodrug activation capability of the catalytic antibody. Since
the MRDs are fused to the antibody so as not to significantly mitigate binding to the
antibody's traditional binding site, the antibody's specificity remains intact after MRD
addition.
As noted in Figure 2, MRDs, designated by triangles, circles, and squares, can be appended
on any of the termini of either heavy or light chains of a typical antibody. The first schematic
represents a simple peptibody with a peptide fused to the C-terminus of an Fc. This approach
provided for the preparation of bi-, tri-, tetra, and penta-specific antibodies. Display of a
single MRD at each N- and C-termini of an IgG provides for octavalent display of the MRD.
As an alternative to the construction of bi- and multifunctional antibodies through the
combination of antibody variable domains, high-affinity peptides selected from phage display
libraries or derived from natural ligands may offer a highly versatile and modular approach to
the construction of multifunctional antibodies that retain both the binding and half-life
advantages of traditional antibodies. MRDs can also extend the binding capacity of noncatalytic
antibodies, providing for an effective approach to extend the binding functionality of
antibodies, particularly for therapeutic purposes.
STATEMENT OF DISCLOSURE
Accordingly, the disclosure provides an isolated antibody comprising at least one modular
recognition domain (MRD); a peptide comprising a sequence selected from the group
consisting of: NFYQCIX1X2LX3X4X5PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein
X1 is E or D X2 is any amino acid; X3 is any amino acid; X4 is any amino acid and X5 is any
amino acid;
MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDE
LLLY (SEQ ID NO:20); NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO:48);
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO:49);
XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22);
XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25);
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XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28);
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31);
Xn AQQEECEX1X2PWTCEHMXn where n is from about 0 to 50 amino acid
residues and X, X1 and X2 are any amino acid (SEQ ID NO:57);
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO:35);
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36);
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37);
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38);
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO:39);
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO:40);
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO:41);
NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO:42);
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO:43);
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO:44);
NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO:45);
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO:46);
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO:47);
AQQEECEFAPWTCEHM (SEQ ID NO:21);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEH
MLE (SEQ ID NO:23);
AQQEECELAPWTCEHM (SEQ ID NO:24);
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEH
MLE (SEQ ID NO:26);
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AQQEECEFSPWTCEHM (SEQ ID NO:27);
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHM
LE2xConFS (SEQ ID NO:29);
AQQEECELEPWTCEHM (SEQ ID NO:30);
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHM
LE (SEQ ID NO:32);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEH
MLE (SEQ ID NO:33);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHM
LE (SEQ ID NO:34); and
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEH
MLE (SEQ ID NO:10).
An isolated polynucleotide comprising a nucleotide sequence encoding the antibody ; a
vector comprising the polynucleotide; a host cell comprising the vector or progeny of the cell
and a method for producing the antibody comprising selecting for a MRD using an MRD
binding target, wherein the MRD is derived from a phage display library or from natural
ligands.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the schematic representation of different designs of tetravalent IgG-like BsAbs.
FIG. 2A shows a typical peptibody as C-terminal fusion with Fc.
FIG.2B shows an antibody with a C-terminal MRD fusion with the light chain of the
antibody.
FIG.2C shows an antibody with an N-terminal MRD fusion with the light chain of the
antibody.
FIG. 2D shows an antibody with unique MRD peptides fused to each terminus of the
antibody.
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FIG. 3 depicts the results of an ELISA in which integrin and Ang2 were bound by an antiintegrin
antibody fused to a ang-2 targeting MRD.
FIG. 4 depicts the results of an ELISA in which integrin and Ang2 were bound by an antiintegrin
antibody fused to a ang-2 targeting MRD.
FIG. 5 depicts the results of an ELISA in which an anti-ErbB2 antibody was fused to an
MRD which targeted Ang2.
FIG. 6 depicts the results of an ELISA in which an Ang2 targeting MRD was fused to a
hepatocyte growth factor receptor binding antibody.
FIG. 7 depicts the results of an ELISA in which an integrin targeting MRD was fused to an
ErbB2 binding antibody.
FIG. 8 depicts the results of an ELISA in which an integrin targeting MRD was fused to an
hepatocyte growth factor receptor binding antibody.
FIG. 9 depicts the results of an ELISA in which an insulin-like growth factor-I receptor
targeting MRD was fused to an ErbB2 binding antibody.
FIG.10 depicts the results of an ELISA in which a VEGF-targeting MRD was fused to an
ErbB2 binding antibody.
FIG. 11 depicts the results of an ELISA in which an integrin targeting MRD was fused to a
catalytic antibody.
FIG.12 depicts the results of an ELISA in which an Ang-2-targeting MRD was fused to a
catalytic antibody.
FIG.13 depicts the results of an ELISA in which an integrin and Ang-2 targeting MRD was
fused to an ErbB2 binding antibody.
FIG.14 depicts the results of an ELISA in which an integrin targeting MRD was fused to an
ErbB2-binding antibody.
FIG.15 depicts the results of an ELISA in which an integrin, Ang-2, or insulin-like growth
factor-I receptor-targeting MRD was fused to an ErbB2 or hepatocyte growth factor receptorbinding
antibody with a short linker peptide.
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FIG.16 depicts the results of an ELISA in which an integrin, Ang-2, or insulin-like growth
factor-I receptor-targeting MRD was fused to an ErbB2 or hepatocyte growth factor receptorbinding
antibody with a long linker peptide.
DETAILED DESCRIPTION OF THE DISCLOSURE
The present disclosure is in relation to an isolated antibody comprising at least one modular
recognition domain (MRD).
In another embodiment of the disclosure, the antibody and the MRD are operably linked
through a linker peptide.
In still another embodiment of the disclosure, the linker peptide is between 2 to 20 peptides
or between 4 to 15 peptides.
In yet another embodiment of the disclosure, the linker peptide comprises a sequence
selected from the group consisting of: GGGS (SEQ ID NO:1), SSGGGGSGGGGGGSS
(SEQ ID NO:2), and SSGGGGSGGGGGGSSRSS (SEQ ID NO:19).
In yet another embodiment of the disclosure, the MRD is operably linked to the C-terminal
end of the heavy chain of the antibody, the N-terminal end of the heavy chain of the antibody,
the C-terminal end of the light chain of the antibody, or the N-terminal end of the light chain
of the antibody.
In yet another embodiment of the disclosure, two or more MRDs are operably linked to any
terminal end of the antibody or wherein two or more MRDs are operably linked to two or
more terminal ends of the antibody.
In yet another embodiment of the disclosure, the target of the MRD is selected from the
group consisting of: an integrin, an angiogenic cytokine, vascular endothelial growth factor
(VEGF), insulin-like growth factor-I receptor (IGF-IR), a tumor antigen, CD20, an epidermal
growth factor receptor (EGFR), the ErbB2 receptor, the ErbB3 receptor, tumor associated
surface antigen epithelial cell adhesion molecule (Ep-CAM), an angiogenic factor, an
angiogenic receptor, cell surface antigen, vascular homing peptide, and nerve growth factor.
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In yet another embodiment of the disclosure, the integrin-targeting MRD comprises a
sequence selected from the group consisting of:
YCRGDCT (SEQ ID NO:3),
PCRGDCL (SEQ ID NO:4),
TCRGDCY (SEQ ID NO:5), and
and LCRGDCF (SEQ ID NO:6);
the angiogenic cytokine-targeting MRD comprises a sequence selected from the group
consisting of:
MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:7),
MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID NO:8),
MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID NO:9) ,
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTC
EHMLE (SEQ ID NO:10),
AQQEECEFAPWTCEHM (SEQ ID NO:21)
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCE
HMLE (SEQ ID NO:23),
AQQEECELAPWTCEHM (SEQ ID NO:24),
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE
HMLE (SEQ ID NO:26),
AQQEECEFSPWTCEHM (SEQ ID NO:27),
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEH
MLE2xConFS (SEQ ID NO:29),
AQQEECELEPWTCEHM (SEQ ID NO:30),
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AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCE
HMLE (SEQ ID NO:32),
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE
HMLE (SEQ ID NO:33);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEH
MLE (SEQ ID NO:34),
MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQ ID NO:11), and
PXDNDXLLNY (SEQ ID NO:12), wherein X is selected from one of the 20
naturally-occurring amino acids;
the VEGF-targeting MRD comprises the sequence VEPNCDIHVMWEWECFERL (SEQ
ID NO:13);
the insulin-like growth factor-I receptor- targeting MRD is selected from the group
consisting of:
SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO:14),
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO:35),
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36),
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37),
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38),
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO:39),
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO:40),
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO:41),
NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO:42),
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO:43),
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO:44),
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NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO:45),
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO:46),
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO:47),
NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO:48), and
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO:49);
the EGFR-targeting MRD comprises a sequence selected from the group consisting of:
VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLN
DAQA PK (SEQ ID NO:16), and
VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKL
NDAQAPK (SEQ ID NO:17);
the ErbB2 receptor-targeting MRD comprises the sequence
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKL
NDAQAPK (SEQ ID NO:18); and
the vascular homing peptide-targeting MRD comprises the sequence ACDCRGDCFCG
(SEQ ID NO:15).
In yet another embodiment of the disclosure, the antibody is a chimeric or humanized
antibody.
In yet another embodiment of the disclosure, the antibody binds to a cell surface antigen
selected from the group consisting of: EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like
growth factor-I receptor, prostate specific membrane antigen, an angiogenic factor, an
angiogenic receptor, and cell surface antigen.
In yet another embodiment of the disclosure, the target of the MRD is an angiogenic factor
or an angiogenic receptor and the antibody binds to a cell surface antigen selected from the
group consisting of EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor receptor,
and prostate specific membrane antigen.
17
In yet another embodiment of the disclosure, the target of the MRD is an angiogenic
receptor and the antibody binds to an angiogenic factor or an angiogenic receptor.
In yet another embodiment of the disclosure, the target of the MRD is a cell surface antigen
and the antibody binds to a cell surface antigen, an angiogenic factor, or an angiogenic
receptor.
In yet another embodiment of the disclosure, the antibody binds to ErbB2.
In yet another embodiment of the disclosure, the antibody is Trastuzumab.
In yet another embodiment of the disclosure, the MRD target is Ang2.
In yet another embodiment of the disclosure, the MRD comprises SEQ ID NO:8.
The present disclosure is also in relation to a peptide comprising a sequence selected from the
group consisting of:
NFYQCIX1X2LX3X4X5PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X1 is E
or D X2 is any amino acid; X3 is any amino acid; X4 is any amino acid and X5 is any
amino acid;
MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMP
MDNDELLLY (SEQ ID NO:20);
NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO:48);
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO:49);
XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22);
XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25);
XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28);
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31);
Xn AQQEECEX1X2PWTCEHMXn where n is from about 0 to 50 amino acid
residues and X, X1 and X2 are any amino acid (SEQ ID NO:57);
18
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO:35);
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36);
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37);
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38);
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO:39);
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO:40);
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO:41);
NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO:42);
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO:43);
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO:44);
NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO:45);
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO:46);
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO:47);
AQQEECEFAPWTCEHM (SEQ ID NO:21);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEH
MLE (SEQ ID NO:23);
AQQEECELAPWTCEHM (SEQ ID NO:24);
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEH
MLE (SEQ ID NO:26);
AQQEECEFSPWTCEHM (SEQ ID NO:27);
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHM
LE2xConFS (SEQ ID NO:29);
AQQEECELEPWTCEHM (SEQ ID NO:30);
19
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHM
LE (SEQ ID NO:32);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEH
MLE (SEQ ID NO:33);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHM
LE (SEQ ID NO:34); and
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEH
MLE (SEQ ID NO:10).
The present disclosure is also in relation to an isolated polynucleotide comprising a
nucleotide sequence encoding the antibody.
The present disclosure is also in relation to a vector comprising the polynucleotide.
The present disclosure is also in relation to a host cell comprising the vector, or progeny of
the cell.
In yet another embodiment of the present disclosure, use of antibody or the peptide for
treating a disease.
In yet another embodiment of the present disclosure, the disease is cancer.
In yet another embodiment of the present disclosure, the antibody or peptide inhibits
angiogenesis, modulates angiogenesis, or inhibits tumor growth.
The present disclosure is also in relation to a method for producing the antibody comprising
selecting for a MRD using an MRD binding target, wherein the MRD is derived from a phage
display library or from natural ligands.
The term “antibody” used herein to refer to intact immunoglobulin molecules and includes
polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies. An
intact antibody comprises at least two heavy (H) chains and two light (L) chains interconnected
by disulfide bonds. Each heavy chain is comprised of a heavy chain variable
region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is
20
comprised of a light chain variable region (abbreviated herein as VL) and a light chain
constant region. The light chain constant region is comprised of one domain, CL. The VH and
VL regions can be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and
four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen.
A “dual-specific antibody” is used herein to refer to an immunoglobulin molecule which
contain dual-variable-domain immunoglobins, where the dual-variable-domain can be
engineered from any two monoclonal antibodies.
An “antibody combining site” is that structural portion of an antibody molecule comprised of
a heavy and light chain variable and hypervariable regions that specifically binds
(immunoreacts with) an antigen. The term “immunoreact” in its various forms means specific
binding between an antigenic determinant-containing molecule and a molecule containing an
antibody combining site such as a whole antibody molecule or a portion thereof.
The term “peptibody” refers to a peptide or polypeptide which comprises less than a
complete, intact antibody.
The term “naturally occurring” when used in connection with biological materials such as a
nucleic acid molecules, polypeptides, host cells, and the like refers to those which are found
in nature and not modified by a human being.
“Monoclonal antibody” refers to a population of antibody molecules that contain only one
species of antibody combining site capable of immunoreacting with a particular epitope. A
monoclonal antibody thus typically displays a single binding affinity for any epitope with
which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule
having a plurality of antibody combining sites, each immunospecific for a different epitope,
e.g., a bispecific monoclonal antibody.
A “modular recognition domain” (MRD) or “target binding peptide” is a molecule, such as a
protein, glycoprotein and the like, that can specifically (non-randomly) bind to a target
molecule. The amino acid sequence of a MRD site can tolerate some degree of variability
21
and still retain a degree of capacity to bind the target molecule. Furthermore, changes in the
sequence can result in changes in the binding specificity and in the binding constant between
a preselected target molecule and the binding site.
“Cell surface receptor” refers to molecules and complexes of molecules capable of receiving
a signal and the transmission of such a signal across the plasma membrane of a cell. An
example of a cell surface receptor of the present disclosure is an activated integrin receptor,
for example, an activated αvβ3 integrin receptor on a metastatic cell.
The "target binding site” or “target site” is any known, or yet to be described, amino acid
sequence having the ability to selectively bind a preselected agent. Exemplary reference
target sites are derived from the RGD-dependent integrin ligands, namely fibronectin,
fibrinogen, vitronectin, von Willebrand factor and the like, from cellular receptors such as
VEGF, ErbB2, vascular homing peptide or angiogenic cytokines, from protein hormones
receptors such as insulin-like growth factor-I receptor, epidermal growth factor receptor and
the like, and from tumor antigens.
The term “protein” is defined as a biological polymer comprising units derived from amino
acids linked via peptide bonds; a protein can be composed of two or more chains.
A “fusion polypeptide” is a polypeptide comprised of at least two polypeptides and optionally
a linking sequence to operatively link the two polypeptides into one continuous polypeptide.
The two polypeptides linked in a fusion polypeptide are typically derived from two
independent sources, and therefore a fusion polypeptide comprises two linked polypeptides
not normally found linked in nature.
The term “linker” refers to a peptide located between the antibody and the MRD. Linkers
can have from about 2 to 20 amino acids, usually 4 to 15 amino acids.
“Target cell” refers to any cell in a subject (e.g., a human or animal) that can be targeted by
the antibody comprising an MRD of the disclosure. The target cell can be a cell expressing
or overexpressing the target binding site, such as activated integrin receptor.
“Patient,” “subject,” “animal” or “mammal” are used interchangeably and refer to mammals
such as human patients and non-human primates, as well as experimental animals such as
22
rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and
non-mammals, such as sheep, dogs, cows, chickens, amphibians, and reptiles.
“Treating” or “treatment” includes the administration of the antibody comprising an MRD of
the present disclosure to prevent or delay the onset of the symptoms, complications, or
biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further
development of the disease, condition, or disorder. Treatment can be prophylactic (to prevent
or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical
symptoms thereof) or therapeutic suppression or alleviation of symptoms after the
manifestation of the disease. Treatment can be with the antibody-MRD composition alone,
or it can be used in combination with an additional therapeutic agent.
As used herein, the terms “pharmaceutically acceptable,” or “physiologically tolerable” and
grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents,
are used interchangeably and represent that the materials are capable of administration to or
upon a human without the production of undesirable physiological effects such as nausea,
dizziness, gastric upset and the like.
“Modulate,” means adjustment or regulation of amplitude, frequency, degree, or activity. In
another related aspect, such modulation may be positively modulated (e.g., an increase in
frequency, degree, or activity) or negatively modulated (e.g., a decrease in frequency, degree,
or activity).
“Cancer,” “tumor,” or “malignancy” are used as synonymous terms and refer to any of a
number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the
ability of affected cells to spread locally or through the bloodstream and lymphatic system to
other parts of the body (metastasize) as well as any of a number of characteristic structural
and/or molecular features. A “cancerous,” “tumor,” or “malignant cell” is understood as a
cell having specific structural properties, lacking differentiation and being capable of
invasion and metastasis. Examples of cancers are, breast, lung, brain, bone, liver, kidney,
colon, head and neck, ovarian, hematopoietic (e.g., leukemia), and prostate cancer.
“Humanized antibody” or “chimeric antibody” includes antibodies in which CDR sequences
derived from the germline of another mammalian species, such as a mouse, have been grafted
onto human framework sequences.
23
The present disclosure describes an approach based on the adaptation of target binding
peptides or modular recognition domains (MRDs) as fusions to catalytic or non-catalytic
antibodies that provide for effective targeting of tumor cells or soluble molecules while
leaving the prodrug activation capability of the catalytic antibody intact. MRDs can also
extend the binding capacity of non-catalytic antibodies providing for an effective approach to
extend the binding functionality of antibodies, particularly for therapeutic purposes.
One aspect of the present disclosure relates to development of a full-length antibody
comprising a modular recognition domain (MRD). The interaction between a protein ligand
and its target receptor site often takes place at a relatively large interface. However, only a
few key residues at the interface contribute to most of the binding. Thus, molecules of
peptide length (generally 2 to 60 amino acids) can bind to the receptor protein of a given
large protein ligand. It is contemplated that MRDs of the present disclosure contain a peptide
sequence that bind to target sites of interests and are about 2 to 60 amino acids.
The role of integrins such as αvβ3 and αvβ5 as tumor-associated markers has been well
documented. A recent study of 25 permanent human cell lines established from advanced
ovarian cancer demonstrated that all lines were positive for αvβ5 expression and many were
positive for αvβ3 expression. Studies have also shown that αvβ3 and αvβ5 is highly
expressed on malignant human cervical tumor tissues. Integrins have also demonstrated
therapeutic effects in animal models of Kaposi's sarcoma, melanoma, and breast cancer.
A number of integrin αvβ3 and αvβ5 antagonists are in clinical development. These include
cyclic RGD peptides and synthetic small molecule RGD mimetics. Two antibody-based
integrin antagonists are currently in clinical trials for the treatment of cancer. The first is
Vitaxin, the humanized form of the murine anti-human αvβ3 antibody LM609. A doseescalating
phase I study in cancer patients demonstrated that it was safe for use in humans.
Another antibody in clinical trials is CNT095, a fully human mAb that recognizes αv
integrins. A Phase I study of CNT095 in patients with a variety of solid tumors has shown
that it is well tolerated. Cilengitide, a peptide antagonist of αvβ3 and αvβ5, has also proven
safe in phase I trials. Furthermore, there has been numerous drug targeting and imaging
studies based on the use of ligands for these receptors. These preclinical and clinical
observations demonstrate the importance of targeting αvβ3 and αvβ5 and studies involving
the use of antibodies in this strategy have consistently reported that targeting through these
integrins is safe.
24
An example of an integrin-binding MRD is an RGD tripeptide-containing binding site, and is
exemplary of the general methods described herein. Ligands having the RGD motif as a
minimum recognition domain are well known, a partial list of which includes, with the
corresponding integrin target in parenthesis, fibronectin (α3β1, α5β1, αvβ1, αIIbβ3, αvβ3,
and α3β1) fibrinogen (αMβ2 and αIIbβ1) von Willebrand factor (αIIbβ3 and αvβ3), and
vitronectin (αIIbβ3, αvβ3 and αvβ5).
Examples of RGD containing targeting MRDs useful in the present disclosure have amino
acid residue sequences shown below:
YCRGDCT (SEQ ID. NO.: 3)
PCRGDCL (SEQ ID. NO.: 4)
TCRGDCY (SEQ ID. NO.: 5)
LCRGDCF (SEQ ID. NO.: 6)
A MRD that mimics a non-RGD-dependent binding site on an integrin receptor and having
the target binding specificity of a high affinity ligand that recognizes the selected integrin is
also contemplated in the present disclosure.
Angiogenesis is essential to many physiological and pathological processes. Ang2 has been
shown to act as a proangiogenic molecule. Administration of Ang2-selective inhibitors is
sufficient to suppress both tumor angiogenesis and corneal angiogenesis. Therefore, Ang2
inhibition alone or in combination with inhibition of other angiogenic factors such as VEGF
may represent an effective antiangiogenic strategy for treating patients with solid tumors.
It is contemplated that MRDs useful in the present disclosure include those that bind to
angiogenic receptors, angiogenic factors, and/or Ang-2. Examples of angiogenic cytokine
targeting MRD sequences are listed below:
MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID. NO.: 7)
MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID. NO.: 8)
MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID. NO.: 9)
25
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.: 10) (2xCon4)
MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQ ID. NO.: 11)
PXDNDXLLNY (SEQ ID. NO.: 12) where X is one of the 20 naturally-occurring amino
acids
MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDE
LLLY (SEQ ID NO: 20)
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.: 10)
AQQEECEFAPWTCEHM ConFA (SEQ ID NO:21)
core nEFAPWTn (SEQ ID NO: 22) where n is from about 0 to 50 amino acid residues
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE
(SEQ ID NO: 23) 2xConFA
AQQEECELAPWTCEHM (SEQ ID NO: 24) ConLA
XnELAPWTXn where n is from about 0 to 50 amino acid residues and X is any amino acid
(SEQ ID NO: 25)
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE
(SEQ ID NO: 26) 2xConLA
AQQEECEFSPWTCEHM ConFS (SEQ ID NO: 27)
XnEFSPWTXn where n is from about 0 to 50 amino acid residues and X is any amino acid
(SEQ ID NO: 28)
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE
2xConFS (SEQ ID NO: 29)
AQQEECELEPWTCEHM ConLE (SEQ ID NO: 30)
26
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 31) and
wherein X is any amino acid
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE
2xConLE (SEQ ID NO: 32)
It should be understood that such peptides can be present in dimmers, trimers or other
multimers either homologous or heterologous in nature. For example, one can dimerize
identical Con-based sequences such as in 2xConFA to provide a homologous dimer, or the
Con peptides can be mixed such that ConFA is combined with ConLA to create ConFA-LA
heterodimer with the sequence:
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE
(SEQ ID NO: 33).
Another heterodimer is ConFA combined with ConFS to create ConFA-FS with the
sequence:
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE
(SEQ ID NO:34).
One of skill in the art, given the teachings herein, will appreciate that other such
combinations will create functional Ang2 binding MRDs as described herein.
In one aspect, the disclosure includes a peptide having the sequence:
NFYQCIX1X2LX3X4X5PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X1 is E or D;
X2 is any amino acid; X3 is any amino acid; X4 is any amino acid and X5 is any amino acid.
The disclosure also includes peptides having a core sequence selected from:
XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 22);
XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 25);
XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 28);
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 31); or
27
Xn AQQEECEX1X2PWTCEHMXn where n is from about 0 to 50 amino acid residues and
X, X1 and X2 are any amino acid (SEQ ID NO:57).
Phage display selections and structural studies of VEGF neutralizing peptides in complex
with VEGF have been reported. These studies have revealed that peptide v114
(VEPNCDIHVMWEWECFERL) (SEQ ID. NO.: 13) is VEGF specific, binds VEGF with 0.2
μM affinity, and neutralizes VEGF-induced proliferation of Human Umbilical Vein
Endothelial Cells (HUVEC). Since VEGF is a homodimer, the peptide occupies two identical
sites at either end of the VEGF homodimer. An antibody containing an MRD that targets
VEGF is contemplated in the present disclosure. Anti-VEGF antibodies can be found for
example in Cancer Research 57, 4593-4599, Oct. 1997; J Biol Chem 281:10 6625, 2006,
herein incorporated by reference.
Insulin-like growth factor-I receptor-specific MRDs can be used in the present disclosure.
One example of an MRD sequence that targets the insulin-like growth factor-I receptor is
SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO.: 14).
Additional IGF-1R MRDs include the following:
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO: 35)
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36)
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37)
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38)
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO: 39)
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO: 40)
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO: 41)
NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO: 42)
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO: 43)
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO: 44)
28
NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO: 45)
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO: 46)
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO: 47)
NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO: 48)
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO: 49)
A number of studies have characterized the efficacy of linking the vascular homing peptide to
other proteins like IL-I2 or drugs to direct their delivery in live animals. As such, vascular
homing MRDs are contemplated for use in the present disclosure. One example of an MRD
sequence that is a vascular homing peptide is ACDCRGDCFCG (SEQ ID NO.: 15).
Numerous other target binding sites are contemplated by the present disclosure, including
epidermal growth factor receptor (EGFR), CD20, tumor antigens, ErbB2, ErbB3, ErbB4,
insulin-like growth factor-I receptor, nerve growth factor (NGR), hepatocyte growth factor
receptor, and tumor-associated surface antigen epithelial cell adhesion molecule (Ep-CAM).
MRDs can be directed towards these target binding sites.
Examples of MRD sequences that bind to EGFR are listed below:
VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQA
PK (SEQ ID. NO.: 16).
VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQ
APK (SEQ ID. NO.: 17).
An example of an MRD sequence that binds to ErbB2 is listed below:
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQA
PK (SEQ ID. NO.: 18).
The sequence of the MRD can be determined several ways. MRD sequences can be derived
from natural ligands or known sequences that bind to a specific target binding site can be
used. Additionally, phage display technology has emerged as a powerful method in
identifying peptides which bind to target receptors. In peptide phage display libraries,
random peptide sequences can be displayed by fusion with coat proteins of filamentous
29
phage. The methods for elucidating binding sites on polypeptides using phage display
vectors has been previously described, in particular in WO 94/18221. The methods generally
involve the use of a filamentous phage (phagemid) surface expression vector system for
cloning and expressing polypeptides that bind to the pre-selected target site of interest.
The methods of the present disclosure for preparing MRDs involve the use of phage display
vectors for their particular advantage of providing a means to screen a very large population
of expressed display proteins and thereby locate one or more specific clones that code for a
desired target binding reactivity. Once the sequence of the MRD has been elucidated, the
peptides may be prepared by any of the methods disclosed in the art.
Variants and derivatives of the MRDs are included within the scope of the present disclosure.
Included within variants are insertional, deletional, and substitutional variants as well as
variants that include MRDs presented here with additional amino acids at the N- and/or Cterminus,
including from about 0 to 50, 0 to 40, 0 to 30, 0 to 20 amino acids and the like. It is
understood that a particular MRD of the present disclosure may contain one, two, or all three
types of variants. Insertional and substitutional variants may contain natural amino acids,
unconventional amino acids, or both.
It is contemplated that catalytic and non-catalytic antibodies can be used in the present
disclosure. Antibody 38C2 is an antibody-secreting hybridoma, and has been previously
described in WO 97/21803. 38C2 contains an antibody combining site that catalyzes the
aldol addition reaction between an aliphatic donor and an aldehyde acceptor. In a syngeneic
mouse model of neuroblastoma, systemic administration of an etoposide prodrug and intratumor
injection of Ab 38C2 inhibited tumor growth.
Other antibodies of interest to this disclosure include A33 binding antibodies. Human A33
antigen is a transmembrane glycoprotein of the Ig superfamily. The function of the human
A33 antigen in normal and malignant colon tissue is not yet known, however, several
properties of the A33 antigen suggest that it is a promising target for immunotherapy of colon
cancer. These properties include (i) the highly restricted expression pattern of the A33
antigen, (ii) the expression of large amounts of the A33 antigen on colon cancer cells, (iii) the
absence of secreted or shed A33 antigen, and (iv) the fact that upon binding of antibody A33
to the A33 antigen, antibody A33 is internalized and sequestered in vesicles, and (v) the
targeting of antibody A33 to A33 antigen expressing colon cancer in preliminary clinical
30
studies. Fusion of a MRD directed toward A33 to a catalytic or non-catalytic antibody would
increase the therapeutic efficacy of A33 targeting antibodies.
The present disclosure also contemplates the preparation of mono-, bi-, tri-, tetra-, and pentaspecific
antibodies. It is contemplated that the antibodies used in the present disclosure may
be prepared by any method known in the art.
In the antibody-MRD fusion molecules prepared according to the present disclosure, the
MRD may be attached to an antibody through the peptide’s N-terminus or C-terminus. The
MRD may be attached to the antibody at the C-terminal end of the heavy chain of the
antibody, the N-terminal end of the heavy chain of the antibody, the C-terminal end of the
light chain of the antibody, or the N-terminal end of the light chain of the antibody. The
MRD may be attached to the antibody directly, or attached through an optional linker peptide,
which can be between 2 to 20 peptides long. The linker peptide can contain a short linker
peptide with the sequence GGGS (SEQ ID. NO.:1), a medium linker peptide with the
sequence SSGGGGSGGGGGGSS (SEQ ID. NO.:2), or a long linker peptide with the
sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19). The present disclosure also
provides for two or more MRDs which are linked to any terminal end of the antibody. It is
also contemplated that two or more MRDs can be directly attached or attached through a
linker peptide to two or more terminal ends of the antibody. The multiple MRDs can target
the same target binding site, or two or more different target binding sites. Additional peptide
sequences may be added to enhance the in vivo stability of the MRD.
The antibody-MRD fusion molecules can be encoded by a polynucleotice comprising a
nucleotide sequence. A vector can contain the polynucleotide sequence. The polynucleotide
sequence can also be linked with a regulatory sequence that controls expression of the
polynucleotide in a host cell. A host cell, or its progeny, can contain the polynucleotide
encoding the antibody-MRD fusion molecule.
The present disclosure contemplates therapeutic compositions useful for practicing the
therapeutic methods described herein. Therapeutic compositions of the present disclosure
contain a physiologically tolerable carrier together with at least one species of antibody
comprising an MRD as described herein, dissolved or dispersed therein as an active
ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when
administered to a human patient for therapeutic purposes.
31
The preparation of a pharmacological composition that contains active ingredients dissolved
or dispersed therein is well understood in the art. Typically such compositions are prepared
as sterile injectables either as liquid solutions or suspensions, aqueous or non-aqueous,
however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be
prepared. The preparation can also be emulsified. Thus, an antibody – MRD containing
composition can take the form of solutions, suspensions, tablets, capsules, sustained release
formulations or powders, or other compositional forms.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable
and compatible with the active ingredient and in amounts suitable for use in the therapeutic
methods described herein. Suitable excipients are, for example, water, saline, dextrose,
glycerol, ethanol or the like and combinations thereof. In addition, if desired, the
composition can contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the
active ingredient.
The therapeutic composition of the present disclosure can include pharmaceutically
acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the polypeptide) that are formed with
inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids
as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also
be derived from inorganic bases such as, for example, sodium, potassium, ammonium,
calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-
ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are
sterile aqueous solutions that contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at physiological pH value, physiological
saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain
more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose,
propylene glycol, polyethylene glycol, and other solutes.
Liquid compositions can also contain liquid phases in addition to and to the exclusion of
water.
32
Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil,
organic esters such as ethyl oleate, and water-oil emulsions.
A therapeutic composition contains an antibody comprising a MRD of the present disclosure,
typically in an amount of at least 0.1 weight percent of antibody per weight of total
therapeutic composition. A weight percent is a ratio by weight of antibody to total
composition. Thus, for example, 0.1 weight percent is 0.1 grams of antibody-MRD per 100
grams of total composition.
An antibody-containing therapeutic composition typically contains about 10 microgram (ug)
per milliliter (ml) to about 100 milligrams (mg) per ml of antibody as active ingredient per
volume of composition, and more preferably contains about 1 mg/ml to about 10 mg/ml (i.e.,
about 0.1 to 1 weight percent).
A therapeutic composition in another embodiment contains a polypeptide of the present
disclosure, typically in an amount of at least 0.1 weight percent of polypeptide per weight of
total therapeutic composition. A weight percent is a ratio by weight of polypeptide to total
composition. Thus, for example, 0.1 weight percent is 0.1 grams of polypeptide per 100
grams of total composition.
Preferably, an polypeptide-containing therapeutic composition typically contains about 10
microgram (ug) per milliliter (ml) to about 100 milligrams (mg) per ml of polypeptide as
active ingredient per volume of composition, and more preferably contains about 1 mg/ml to
about 10 mg/ml (i.e., about 0.1 to 1 weight percent).
In view of the benefit of using humanized or chimeric antibodies in vivo in human patients,
the presently described antibody-MRD molecules are particularly well suited for in vivo use
as a therapeutic reagent. The method comprises administering to the patient a therapeutically
effective amount of a physiologically tolerable composition containing an antibody
comprising a MRD of the disclosure.
The dosage ranges for the administration of the antibody comprising a MRD of the disclosure
are those large enough to produce the desired effect in which the disease symptoms mediated
by the target molecule are ameliorated. The dosage should not be so large as to cause adverse
side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure,
and the like. Generally, the dosage will vary with the age, condition, sex and extent of the
33
disease in the patient and can be determined by one of skill in the art. The dosage can be
adjusted by the individual physician in the event of any complication.
A therapeutically effective amount of an antibody comprising a MRD of this disclosure is
typically an amount of antibody such that when administered in a physiologically tolerable
composition is sufficient to achieve a plasma concentration of from about 0.1 microgram (ug)
per milliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and
usually about 5 ug/ml. Stated differently, the dosage can vary from about 0.1 mg/kg to about
300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, most preferably from about
0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several
days.
The antibody comprising a MRD of the disclosure can be administered parenterally by
injection or by gradual infusion over time. Although the target molecule can typically be
accessed in the body by systemic administration and therefore most often treated by
intravenous administration of therapeutic compositions, other tissues and delivery means are
contemplated where there is likelihood that the tissue targeted contains the target molecule.
Thus, antibodies comprising a MRD of the disclosure can be administered intravenously,
intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, and can be
delivered by peristaltic means.
The therapeutic compositions containing a human monoclonal antibody or a polypeptide of
this disclosure are conventionally administered intravenously, as by injection of a unit dose,
for example. The term “unit dose” when used in reference to a therapeutic composition of the
present disclosure refers to physically discrete units suitable as unitary dosage for the subject,
each unit containing a predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
The compositions are administered in a manner compatible with the dosage formulation, and
in a therapeutically effective amount. The quantity to be administered depends on the subject
to be treated, capacity of the subject’s system to utilize the active ingredient, and degree of
therapeutic effect desired. Precise amounts of active ingredient required to be administered
depend on the judgment of the practitioner and are peculiar to each individual. However,
suitable dosage ranges for systemic application are disclosed herein and depend on the route
of administration. Suitable regimes for administration are also variable, but are typified by an
34
initial administration followed by repeated doses at one or more hour intervals by a
subsequent injection or other administration. Alternatively, continuous intravenous infusion
sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies
are contemplated.
The present disclosure is directed towards a full length antibody comprising a modular
recognition domain (MRD). Also embodied in the present disclosure are variants and
derivitaves of such antibodies comprising a MRD.
In one aspect, the antibody and the MRD are operably linked through a linker peptide. In one
aspect, the linker peptide is between 2 to 20 peptides long, or between 4 to 10 or about 4 to
15 peptides long. In one aspect of the present disclosure, the linker peptide comprises the
sequence GGGS (SEQ ID. NO.:1), the sequence SSGGGGSGGGGGGSS (SEQ ID. NO.: 2),
or the sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19). Other linkers containing a
core sequence GGGS as shown in SEQ ID NO:1 are included herein wherein the linker
peptide is from about 4-20 amino acids.
According to another embodiment of the present disclosure, the MRD is operably linked to
the C-terminal end of the heavy chain of the antibody. In another aspect, the MRD is
operably linked to the N-terminal end of the heavy chain of the antibody. In yet another
aspect, the MRD is operably linked to the C-terminal end of the light chain of the antibody.
In another aspect, the MRD is operably linked to the N-terminal end of the light chain of the
antibody. In another aspect, two or more MRDs are operably linked to any terminal end of
the antibody. In another aspect, two or more MRDs are operably linked to two or more
terminal ends of the antibody.
In one embodiment of the present disclosure, the target of the MRD is a cellular antigen. In
one embodiment of the present disclosure, the target of the MRD is CD20.
In one embodiment of the present disclosure, the target of the MRD is an integrin. In one
aspect, the peptide sequence of the integrin targeting MRD is YCRGDCT (SEQ ID. NO.:3).
In another aspect, the peptide sequence of the integrin targeting MRD is PCRGDCL (SEQ
ID. NO.:4). In yet another aspect, the peptide sequence of the integrin targeting MRD is
TCRGDCY (SEQ ID. NO.:5). In another aspect, the peptide sequence of the integrin
targeting MRD is LCRGDCF (SEQ ID. NO.:6).
35
In one embodiment of the present disclosure, the target of the MRD is an angiogenic
cytokine. In one aspect, the peptide sequence of the angiogenic cytokine targeting MRD is
MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID. NO.:7). In another aspect, the
peptide sequence of the angiogenic cytokine targeting MRD is
MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID. NO.:8). In yet another aspect, the
peptide sequence of the angiogenic cytokine targeting MRD is
MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID. NO.:9). In another aspect, the
peptide sequence of the angiogenic cytokine targeting MRD is
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.:10). In another aspect, the peptide sequence of the angiogenic cytokine
targeting MRD is MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQ ID. NO.: 11). In
another aspect, the peptide sequence of the angiogenic cytokine targeting MRD is
PXDNDXLLNY (SEQ ID. NO.:12), where X is one of the 20 naturally-occurring amino
acids. In another embodiment, the targeting MRD peptide has the core sequence
MGAQTNFMPMDXn (SEQ ID NO: 56), wherein X is any amino acid and n is from about 0
to 15.
In another embodiment, the targeting MRD peptide contains a core sequence selected from:
XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 22);
XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 25);
XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 28);
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO: 31); and
Xn AQQEECEX1X2PWTCEHMXn where n is from about 0 to 50 amino acid residues and
X, X1 and X2 are any amino acid (SEQ ID NO:57).
Exemplary peptides containing such core peptides include for example:
AQQEECEFAPWTCEHM (SEQ ID NO:21);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE
(SEQ ID NO: 23);
AQQEECELAPWTCEHM (SEQ ID NO: 24);
36
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE
(SEQ ID NO: 26);
AQQEECEFSPWTCEHM (SEQ ID NO: 27);
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE
2xConFS (SEQ ID NO: 29);
AQQEECELEPWTCEHM (SEQ ID NO: 30);
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE
(SEQ ID NO: 32);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE
(SEQ ID NO: 33);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE
(SEQ ID NO:34); and
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.: 10).
In one embodiment of the present disclosure, the target of the MRD is ErbB2. In one
embodiment of the present disclosure, the target of the MRD is ErbB3. In one embodiment
of the present disclosure, the target of the MRD is tumor-associated surface antigen epithelial
cell adhesion molecule (Ep-CAM).
In one embodiment of the present disclosure, the target of the MRD is VEGF. In one aspect,
the peptide sequence of the VEGF targeting MRD is VEPNCDIHVMWEWECFERL (SEQ
ID. NO.:13).
In one embodiment of the present disclosure, the target of the MRD is an insulin-like growth
factor-I receptor. In one aspect, the peptide sequence of the insulin-like growth factor-I
receptor targeting MRD is SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO:14).
Other illustrative IGF-1R targeting MRDs include, for example, a peptide with the formula
NFYQCIX1X2LX3X4X5PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X1 is E or D;
X2 is any amino acid; X3 is any amino acid; X4 is any amino acid and X5 is any amino acid.
37
Illustrative peptides that contain the formula include:
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO: 35);
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36);
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37);
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38);
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO: 39);
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO: 40);
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO: 41);
NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO: 42);
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO: 43);
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO: 44);
NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO: 45);
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO: 46);
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO: 47);
NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO: 48); and
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO: 49).
In one embodiment of the present disclosure, the target of the MRD is a tumor antigen.
In one embodiment of the present disclosure, the target of the MRD is an epidermal growth
factor receptor (EGFR). In one embodiment of the present disclosure, the target of the MRD
is an angiogenic factor. In one embodiment of the present disclosure, the target of the MRD
is an angiogenic receptor.
38
In one embodiment of the present disclosure, the MRD is a vascular homing peptide. In one
aspect, the peptide sequence of the vascular homing peptide is ACDCRGDCFCG (SEQ ID.
NO:15).
In one embodiment of the present disclosure, the target of the MRD is a nerve growth factor.
In one of the present disclosure, the antibody binds to a cell surface antigen.
In one embodiment of the present disclosure, the antibody or MRD binds to EGFR, ErbB2,
ErbB3, ErbB4, CD20, insulin-like growth factor-I receptor, or prostate specific membrane
antigen. In one aspect, the peptide sequence of the EGFR targeting MRD is
VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQA
PK (SEQ ID NO: 16). In one aspect, the peptide sequence of the EGFR targeting MRD is
VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQ
APK (SEQ ID NO: 17). In one aspect of the present disclosure, the peptide sequence of
ErbB2 targeting MRD is
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQA
PK (SEQ ID NO: 18).
In one embodiment of the present disclosure, the antibody binds to an angiogenic factor.
In one embodiment of the present disclosure, the antibody binds to an angiogenic receptor.
The present disclosure also relates to an isolated polynucleotide comprising a nucleotide
sequence of the antibody. In one aspect of the present disclosure, a vector comprises the
polynucleotide. In yet another aspect, the polynucleotide is operatively linked with a
regulatory sequence that controls expression on the polynucleotide. In one aspect, a host cell
comprises the polynucleotide or progeny.
The present disclosure also relates to a method of treating a disease a subject in need thereof
is provided, the method comprising administering an antibody comprising an MRD. In one
aspect, the disease is cancer. In another aspect, undesired angiogenesis in inhibited. In yet
another aspect, angiogenesis is modulated. In yet another aspect, tumor growth is inhibited.
In another embodiment, a method of treatment comprising administering an additional
therapeutic agent along with an antibody comprising an MRD is described.
39
The present disclosure also relates to a method of making a full length antibody comprising a
MRD is described. In one aspect, the MRD is derived from a phage display library. In
another aspect, the MRD is derived from natural ligands.
In one embodiment of the present disclosure, the antibody is chimeric or humanized.
A more complete understanding can be obtained by reference to the following specific
examples, which are provided for purposes of illustration only and are not intended to limit
the scope of the disclosure.
EXAMPLES
Example 1. Integrin Targeting Antibody-MRD Molecules
Novel antibody-MRD fusion molecules were prepared by fusion of an integrin αvβ3-targeting
peptides to catalytic antibody 38C2. Fusions at the N-termini and C-termini of the light chain
and the C-termini of the heavy chain were most effective. Using flow cytometry, the
antibody conjugates were shown to bind efficiently to integrin αvβ3-expressing human breast
cancer cells. The antibody conjugates also retained the retro-aldol activity of their parental
catalytic antibody 38C2, as measured by methodol and doxorubicin prodrug activation. This
demonstrates that cell targeting and catalytic antibody capability can be efficiently combined
for selective chemotherapy.
Example 2. Angiogenic Cytokine Targeting Antibody-MRD Molecules
Angiogenic cytokine targeting antibody – MRD fusion molecules were constructed. The
antibody used was 38C2, which was fused with a MRD containing the 2xCon4 peptide
(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.: 10)). The MRD peptide was fused to either the N- or C-terminus of the light
chain and the C-terminus of the heavy chain. Similar results were found with the other Ang-
2 MRD peptides. Additional Ang-2 MRD peptides include:
LM-2x-32
MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDE
LLLY (SEQ ID NO:20)
40
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(2xCon4) (SEQ ID. NO.: 10)
AQQEECEFAPWTCEHM ConFA (SEQ ID NO:21)
core XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22)
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE
2xConFA (SEQ ID NO:23)
AQQEECELAPWTCEHM ConLA (SEQ ID NO:24)
XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25)
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE
2xConLA (SEQ ID NO:26)
AQQEECEFSPWTCEHM ConFS (SEQ ID NO:27)
XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28)
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE
2xConFS (SEQ ID NO:29)
AQQEECELEPWTCEHM ConLE (SEQ ID NO:30)
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31)
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE
2xConLE (SEQ ID NO:32).
It should be understood that such peptides can be present in dimmers, trimers or other
multimers either homologous or heterologous in nature. For example, one can dimerize
identical Con-based sequences such as in 2xConFA to provide a homologous dimer, or the
Con peptides can be mixed such that ConFA is combined with ConLA to create ConFA-LA
heterodimer with the sequence:
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE
(SEQ ID NO:33).
41
Another illustrative heterodimer is ConFA combined with ConFS to create ConFA-FS with
the sequence:
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE
(SEQ ID NO:34).
One of skill in the art, given the teachings herein, will appreciate that other such
combinations will create functional Ang2 binding MRDs as described herein.
Example 3. Antibody-MRD Fusions with Non-catalytic Antibodies
A humanized mouse monoclonal antibody, LM609, directed towards human integrin αvβ3
has been previously described. (Rader, C. et. al., 1998. Rader C, Cheresh DA, Barbas CF
3rd. Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8910-5).
A human non-catalytic monoclonal Ab, JC7U was fused to an anti-Ang2 MRD containing
2xCon4
(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.:10)) at either the N- or C-terminus of the light chain. 2xCon4
(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE
(SEQ ID. NO.: 10)) was studied as an N-terminal fusion to the Kappa chain of the antibody
(2xCon4-JC7U) and as a C-terminal fusion (JC7U-2xCon4). Both fusions maintained
integrin and Ang2 binding. As shown in the left panel of Figure 3, both antibody constructs
(2xCon4-JC7U and JC7U-2xCon4) specifically bound to recombinant Ang2 as demonstrated
by ELISA studies. Binding to Ang2, however, is significantly higher with JC7U-2xCon4,
which has the 2xCon4 (SEQ ID. NO.:10) fusion at the C-terminus of the light chain of the
antibody. The right panel of Figure 3 depicts the binding of Ang2-JC7U and JC7U-Ang2 to
integrin αvβ3. The results show that fusion of 2xCon4 (SEQ ID. NO.: 10) to either the N- or
the C- light chain terminus does not affect mAb JC7U binding to integrin αvβ3. Figure 4
depicts another ELISA study using the same antibody-MRD fusion constructs.
Example 4. Herceptin – MRD fusion molecules
Another example of MRD fusions to a non-catalytic antibody are Herceptin – MRD fusion
constructs. The Herceptin-MRD fusions are multifunctional, both small-molecule αv integrin
42
antagonists and the chemically programmed integrin-targeting antibody show remarkable
efficacy in preventing the breast cancer metastasis by interfering with αv-mediated cell
adhesion and proliferation. MRD fusions containing Herceptin-2xCon4 (which targets
ErbB2 and ang2) and Herceptin-V114 (which targets ErbB2 and VEGF targeting) and
Herceptin-RGD-4C-2xCon4 (which targets ErbB2, ang2, and integrin targeting) are effective.
Example 5. VEGF Targeting Antibody-MRD Molecules
An antibody containing an MRD that targets VEGF was constructed. A MRD which targets
v114(SEQ ID. NO. 13) was fused at the N-terminus of the kappa chain of 38C2 and
Herceptin using the long linker sequence (SEQ ID. NO. 2). Expression and testing of the
resulting antibody-MRD fusion constructs demonstrated strong VEGF binding.
Example 6. IGF-1R Targeting Antibody-MRD Molecules
Fusion of an MRD which targets the IGF-1R (SFYSCLESLVNGPAEKSRGQWDGCRKK
(SEQ ID. NO.: 14)) to the N-terminus of the kappa chain of 38C2 and Herceptin using the
long linker sequence as a connector was studied. Expression and testing of the resulting
antibody-MRD fusion constructs demonstrated strong IGF-1R binding. Additional clones
showing high binding to IGR-1R were also identified after several rounds of mutagenesis and
screening. The preferred sequences listed below show no significant or no binding affinity to
the insulin receptor. (see Table 2).
Table 1: Template for further mutagenesis:
Rm2-2-218 GTGGAGTGCAGGGCGCCG VECRAP
SEQ ID NO: 50,
51
Rm2-2-316 GCTGAGTGCAGGGCTGGG AECRAG
SEQ ID NO: 52,
53
Rm2-2-319 CAGGAGTGCAGGACGGGG QECRTG
SEQ ID NO: 54,
55
43
Table 2:
Mutant Amino acid sequence Template
SEQ
ID
NO:
Rm4-31 NFYQCIEMLASHPAEKSRGQWQECRTGG Rm2-2-319 35
Rm4-33 NFYQCIEQLALRPAEKSRGQWQECRTGG Rm2-2-319 36
Rm4-39 NFYQCIDLLMAYPAEKSRGQWQECRTGG Rm2-2-319 37
Rm4-310 NFYQCIERLVTGPAEKSRGQWQECRTGG Rm2-2-319 38
Rm4-314 NFYQCIEYLAMKPAEKSRGQWQECRTGG Rm2-2-319 39
Rm4-316 NFYQCIEALQSRPAEKSRGQWQECRTGG Rm2-2-319 40
Rm4-319 NFYQCIEALSRSPAEKSRGQWQECRTGG Rm2-2-319 41
Rm4-44 NFYQCIEHLSGSPAEKSRGQWQECRTG Rm2-2-319 42
Rm4-45 NFYQCIESLAGGPAEKSRGQWQECRTG Rm2-2-319 43
Rm4-46 NFYQCIEALVGVPAEKSRGQWQECRTG Rm2-2-319 44
Rm4-49 NFYQCIEMLSLPPAEKSRGQWQECRTG Rm2-2-319 45
Rm4-410 NFYQCIEVFWGRPAEKSRGQWQECRTG Rm2-2-319 46
Rm4-411 NFYQCIEQLSSGPAEKSRGQWQECRTG Rm2-2-319 47
Rm4-415 NFYQCIELLSARPAEKSRGQWAECRAG Rm2-2-316 48
Rm4-417 NFYQCIEALARTPAEKSRGQWVECRAP Rm2-2-218 49
Example 7. ErbB2 Binding, Ang-2-Targeting Antibody-MRD Molecules
An antibody was constructed which contains an MRD that targets Ang-2 (L17) fused to the
light chain of an antibody which binds to ErbB2. Either the short linker sequence, the long
linker sequence, or the 4th loop in the light chain constant region was used as a linker. Figure
5 depicts the results of an ELISA using constructs containing an N-terminal fusion of Ang-2
targeting MRD with the ErbB2 antibody with the short linker peptide (GGGS (SEQ ID NO.:
1)) (L17-sL-Her), a C-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody with
the short linker peptide (Her-sL-L17), a C-terminal fusion of Ang-2 targeting MRD with the
ErbB2 antibody with the 4th loop in the light chain constant region (Her-lo-L17), or an Nterminal
fusion of Ang-2 targeting MRD with the ErbB2 antibody with the long linker
peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (L17-lL-Her). ErbB2 was bound
44
with varying degrees by all of the constructs. However, Ang-2 was bound only by Her-sLL17
and L17-lL-Her.
Example 8. Hepatocyte Growth Factor Receptor Binding, Ang-2-Targeting Antibody-
MRD Molecules
Fusion of an MRD which targets Ang-2 (L17) was made to either the N-terminus or Cterminus
of the light chain of the Met antibody, which binds to hepatocyte growth factor
receptor. Either the short linker sequence or the long linker sequence were used as a
connector. Figure 6 depicts the results of an ELISA using constructs containing N-terminal
fusion of Ang-2 targeting MRD with the Met antibody with the short linker peptide (GGGS
(SEQ ID NO.: 1)) (L17-sL-Met), N-terminal fusion of Ang-2 targeting MRD with the Met
antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (L17-
lL-Met), and C-terminal fusion of Ang-2 targeting MRD with the Met antibody with the long
linker peptide (Met-iL-L17). Expression and testing of the resulting antibody-MRD fusion
constructs demonstrated strong Ang-2 binding when the long linker peptide was used. Fusion
of the Ang-2 targeting MRD to the C-light chain terminus of the antibody resulted in slightly
higher binding to Ang-2 then fusion of the Ang-2 targeting to the N-light chain terminus of
the antibody.
Example 9. ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules
An antibody was constructed which contains an MRD that targets integrin αvβ3 (RGD4C)
fused to the light chain of an antibody Herceptin which binds to ErbB2 (Her). Either the
short linker sequence, the long linker sequence, or the 4th loop in the light chain constant
region was used as a linker. Figure 7 depicts the results of an ELISA using constructs
containing an N-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody
with the short linker peptide (GGGS (SEQ ID NO.: 1)) (RGD4C-sL-Her), a C-terminal
fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody with the short linker peptide
(Her-sL-RGD4C), a C-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2
antibody with the 4th loop in the light chain constant region (Her-lo-RGD4C), or an Nterminal
fusion of integrin αvβ3 targeting MRD with the ErbB2 antibody with the long linker
peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (RGD4C-lL-Her). ErbB2 was
bound with varying degrees by all of the constructs. However, integrin αvβ3 was bound only
by RGD4C-lL-Her.
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Example 10. Hepatocyte Growth Factor Receptor Binding, Integrin-Targeting
Antibody-MRD Molecules
An antibody was constructed which contains an MRD that targets integrin αvβ3 (RGD4C)
fused to the light chain of an antibody which binds to the hepatocyte growth factor receptor
(Met). Antibody-MRD constructs containing the long linker sequence were used. Figure 8
depicts the results of an ELISA using constructs containing an N-terminal fusion of integrin
αvβ3 targeting MRD with the hepatocyte growth factor receptor antibody (RGD4C-lL-Met),
or a C-terminal fusion of integrin αvβ3 targeting MRD with the hepatocyte growth factor
receptor antibody (Met-lL-RGD4C). The RGD4C-lL-Met demonstrated strong integrin αvβ3
binding.
Example 11. ErbB2 Binding, Insulin-like Growth Factor-I Receptor -Targeting
Antibody-MRD Molecules
Antibodies were constructed which contains an MRD that targets insulin-like growth factor-I
receptor (RP) fused to the light chain of an antibody which binds to ErbB2 (Her). Either the
short linker peptide, the long linker peptide, or the 4th loop in the light chain constant region
was used as a linker. (Carter et al., Proc Natl Acad Sci U S A. 1992 May 15;89(10):4285-9.
PMID: 1350088 [PubMed - indexed for MEDLINE]; US Patent No. 5,677,171; ATCC
Deposit 10463, all incorporated by reference herein). Figure 9 depicts the results of an
ELISA using constructs containing an N-terminal fusion of insulin-like growth factor-I
receptor targeting MRD with the ErbB2 antibody with the short (RP-sL-Her), a C-terminal
fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody and
the short linker peptide (Her-sL-RP), a C-terminal fusion of insulin-like growth factor-I
receptor targeting MRD with the ErbB2 antibody with the 4th loop in the light chain constant
region (Her-lo-RP), an N-terminal fusion of insulin-like growth factor-I receptor targeting
MRD with the ErbB2 antibody with the long linker peptide (RP-lL-Her), or a C-terminal
fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 antibody with
the long linker peptide (Her-lL-RP). ErbB2 was bound with varying degrees by all of the
constructs. Insulin-like growth factor-I receptor was bound by RP-lL-Her.
Example 12. ErbB2 Binding, VEGF-Targeting Antibody-MRD Molecules
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Fusion of an MRD which targets VEGF (V114) was made to the N-terminus of the the light
chain of a ErbB2-binding antibody (Her). A medium linker peptide (SSGGGGSGGGGGGSS
(SEQ ID NO.: 2)) was used as a connector. Figure 10 depicts the results of an ELISA using a
construct containing an N-terminal fusion of VEGF targeting MRD with the ErbB2-binding
antibody with the medium linker peptide (V114-mL-Her). Expression and testing of the
resulting antibody-MRD fusion construct demonstrated strong VEGF and ErbB2 binding.
Example 13. Integrin Targeting Antibody-MRD Molecules
Fusion of an MRD which targets integrin αvβ3 (RGD) to the N-terminus of the light chain of
38C2 using the medium linker peptide as a connector was studied. Figure 11 demonstrates
that expression and testing of the resulting antibody-MRD fusion construct had strong
integrin αvβ3 binding.
Example 14. Ang-2 Targeting Antibody-MRD Molecules
Fusion of an MRD which targets Ang-2 (L17) to the C-terminus of the light chain of 38C2
using the short linker sequence as a connector was studied. Figure 12 demonstrates that
expression and testing of the resulting antibody-MRD fusion construct had strong Ang-2
binding.
Example 15. ErbB2 Binding, Integrin and Ang-2 Targeting Antibody-MRD Molecules
An MRD which targets integrin αvβ3 (RGD4C) was connected to the N-terminus of the light
chain of an ErbB2 targeting antibody (Her) with a medium linker, and an Ang-2 (L17)
targeting MRD was connected by a short linker to the C-terminus of the same ErbB2
targeting antibody (RGD4C-mL-Her-sL-L17). Figure 13 demonstrates that the resulting
antibody-MRD fusion construct bound to integrin, Ang-2, and ErbB2.
Example 16. ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules
An antibody was constructed which contains an MRD that targets integrin αvβ3 (RGD4C)
fused to the N-terminus of the heavy chain of an antibody which binds to ErbB2 (Her) using
the medium linker as a connector (RGD4C-mL-her-heavy). Figure 14 depicts the results of
an ELISA using the construct. Both integrin and ErbB2 were bound by the construct.
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Example 17. ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin,
Ang-2 or Insulin-like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules
with the Short Linker Peptide
Antibody-MRD molecules were constructed which contain ErbB2 or hepatocyte growth
factor receptor binding antibodies, and integrin αvβ3, Ang-2 or insulin-like growth factor-I
receptor-targeting MRD regions were linked with the short linker peptide to the light chain of
the antibody. Figure 15 depicts the results of an ELISA using constructs containing an Nterminal
fusion of Ang-2 targeting MRD fused to the ErbB2 antibody (L17-sL-Her), an Nterminal
fusion of integrin-targeting MRD with the ErbB2 antibody (RGD4C-sL-Her), an Nterminal
fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2
binding antibody (RP-sL-Her), a C-terminal fusion of Ang-2 targeting MRD with the
hepatocyte growth factor receptor binding antibody (L17-sL-Met), a C-terminal fusion of
Ang-2 targeting MRD with the ErbB2 binding antibody (Her-sL-L17), a C-terminal fusion of
integrin targeting MRD with the ErbB2 binding antibody (Her-sL-RGD4C), or a C-terminal
fusion of insulin-like growth factor-I receptor targeting MRD with the ErbB2 binding
antibody (Her-sL-RP). ErbB2 was bound with varying degrees by the antibody-MRD
constructs, with the exception of the construct containing the hepatocyte growth factor
receptor-binding antibody. Antigen was bound only by the Her-sL-L17 construct.
Example 18. ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin,
Ang-2 or Insulin-like Growth Factor-I Receptor-Targeting Antibody-MRD Molecules
with the Long Linker Peptide
Antibody-MRD molecules were constructed which contain ErbB2 or hepatocyte growth
factor receptor binding antibodies, and integrin αvβ3, Ang-2 or insulin-like growth factor-I
receptor-targeting MRD regions linked with the long linker peptide to the light chain of the
antibody. Figure 16 depicts the results of an ELISA using constructs containing an Nterminal
fusion of Ang-2 targeting MRD fused to the ErbB2 antibody (L17-lL-Her), an Nterminal
fusion of integrin-targeting MRD with the ErbB2 antibody (RGD4C-lL-Her), an Nterminal
fusion of insulin-like growth factor-I receptor- targeting MRD with the ErbB2
binding antibody (RP-lL-Her), a C-terminal fusion of Ang-2 targeting MRD with the
hepatocyte growth factor receptor binding antibody (L17-lL-Met), a C-terminal fusion of
integrin targeting MRD with the hepatocyte growth factor receptor binding antibody
(RGD4C-lL-Met), a C-terminal fusion of Ang-2 targeting MRD with the insulin-like growth
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factor-I receptor binding antibody (Her-lL-RP), a C-terminal fusion of Ang-2 targeting MRD
with the the hepatocyte growth factor receptor binding antibody (Met-lL-L17), or a Cterminal
fusion of integrin targeting MRD with the the hepatocyte growth factor receptor
binding antibody (Met-lL-RGD4C). As shown in Figure 16, antibody-MRD fusions are
effective to bind antigen and ErbB2. Lu et al. J Biol Chem. 2005 May 20;280(20):19665-72.
Epub 2005 Mar 9;.; Lu et al. J Biol Chem. 2004 Jan 23;279(4):2856-65. Epub 2003 Oct 23,
Although the disclosure has been described with reference to the above examples, it will be
understood that modifications and variations are encompassed within the spirit and scope of
the disclosure. Accordingly, the disclosure is limited only by the following claims.
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WE CLAIM
1. An isolated antibody comprising at least one modular recognition domain (MRD).
2. The antibody of claim 1, wherein the antibody and the MRD are operably linked through a
linker peptide.
3. The antibody of claim 2, wherein the linker peptide is between 2 to 20 peptides or between
4 to 15 peptides.
4. The antibody of claim 2 or 3, wherein the linker peptide comprises a sequence selected
from the group consisting of: GGGS (SEQ ID NO:1), SSGGGGSGGGGGGSS (SEQ ID
NO:2), and SSGGGGSGGGGGGSSRSS (SEQ ID NO:19).
5. The antibody of any one of claims 1-4 , wherein the MRD is operably linked to the Cterminal
end of the heavy chain of the antibody, the N-terminal end of the heavy chain of the
antibody, the C-terminal end of the light chain of the antibody, or the N-terminal end of the
light chain of the antibody.
6. The antibody of any one of claims 1-5, wherein two or more MRDs are operably linked to
any terminal end of the antibody or wherein two or more MRDs are operably linked to two or
more terminal ends of the antibody.
7. The antibody of any one of claims 1-6, wherein the target of the MRD is selected from the
group consisting of: an integrin, an angiogenic cytokine, vascular endothelial growth factor
(VEGF), insulin-like growth factor-I receptor (IGF-IR), a tumor antigen, CD20, an epidermal
growth factor receptor (EGFR), the ErbB2 receptor, the ErbB3 receptor, tumor associated
surface antigen epithelial cell adhesion molecule (Ep-CAM), an angiogenic factor, an
angiogenic receptor, cell surface antigen, vascular homing peptide, and nerve growth factor.
8. The antibody of claim 7, wherein
the integrin-targeting MRD comprises a sequence selected from the group consisting of:
YCRGDCT (SEQ ID NO:3
PCRGDCL (SEQ ID NO:4),
TCRGDCY (SEQ ID NO:5), and
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LCRGDCF (SEQ ID NO:6);
the angiogenic cytokine-targeting MRD comprises a sequence selected from the group
consisting of:
MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:7),
MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID NO:8),
MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID NO:9) ,
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTC
EHMLE (SEQ ID NO:10),
AQQEECEFAPWTCEHM (SEQ ID NO:21)
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCE
HMLE (SEQ ID NO:23),
AQQEECELAPWTCEHM (SEQ ID NO:24),
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE
HMLE (SEQ ID NO:26),
AQQEECEFSPWTCEHM (SEQ ID NO:27),
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEH
MLE2xConFS (SEQ ID NO:29),
AQQEECELEPWTCEHM (SEQ ID NO:30),
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCE
HMLE (SEQ ID NO:32),
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE
HMLE (SEQ ID NO:33);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEH
MLE (SEQ ID NO:34),
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MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQ ID NO:11), and
PXDNDXLLNY (SEQ ID NO:12), wherein X is selected from one of the 20
naturally-occurring amino acids;
the VEGF-targeting MRD comprises the sequence VEPNCDIHVMWEWECFERL (SEQ
ID NO:13);
the insulin-like growth factor-I receptor- targeting MRD is selected from the group
consisting of:
SFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO:14),
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO:35),
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36),
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37),
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38),
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO:39),
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO:40),
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO:41),
NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO:42),
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO:43),
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO:44),
NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO:45),
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO:46),
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO:47),
NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO:48), and
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO:49);
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the EGFR-targeting MRD comprises a sequence selected from the group consisting of:
VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLN
DAQA PK (SEQ ID NO:16), and
VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKL
NDAQAPK (SEQ ID NO:17);
the ErbB2 receptor-targeting MRD comprises the sequence
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKL
NDAQAPK (SEQ ID NO:18); and
the vascular homing peptide-targeting MRD comprises the sequence ACDCRGDCFCG
(SEQ ID NO:15).
9. The antibody of any one of claims 1-8, wherein the antibody is a chimeric or humanized
antibody.
10. The antibody of any one of claims 1-9 wherein the antibody binds to a cell surface
antigen selected from the group consisting of: EGFR, ErbB2, ErbB3, ErbB4, CD20, insulinlike
growth factor-I receptor, prostate specific membrane antigen, an angiogenic factor, an
angiogenic receptor, and cell surface antigen.
11. The antibody of any one of claims 1-10, wherein the target of the MRD is an angiogenic
factor or an angiogenic receptor and the antibody binds to a cell surface antigen selected from
the group consisting of EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor
receptor, and prostate specific membrane antigen.
12. The antibody of any one of claims 1-11, wherein the target of the MRD is an angiogenic
receptor and the antibody binds to an angiogenic factor or an angiogenic receptor.
13. The antibody of any one of claims 1-12, wherein the target of the MRD is a cell surface
antigen and the antibody binds to a cell surface antigen, an angiogenic factor, or an
angiogenic receptor.
14. The antibody of any one of claims 1-13, wherein the antibody binds to ErbB2.
15. The antibody of claim 14, wherein the antibody is Trastuzumab.
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16. The antibody of claim 14 or 15, wherein the MRD target is Ang2.
17. The antibody of claim 16, wherein the MRD comprises SEQ ID NO:8.
18. A peptide comprising a sequence selected from the group consisting of:
NFYQCIX1X2LX3X4X5PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X1 is E
or D X2 is any amino acid; X3 is any amino acid; X4 is any amino acid and X5 is any
amino acid;
MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMP
MDNDELLLY (SEQ ID NO:20);
NFYQCIELLSARPAEKSRGQWAECRAG (SEQ ID NO:48);
NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO:49);
XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22);
XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25);
XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28);
XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31);
Xn AQQEECEX1X2PWTCEHMXn where n is from about 0 to 50 amino acid
residues and X, X1 and X2 are any amino acid (SEQ ID NO:57);
NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO:35);
NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO:36);
NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO:37);
NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO:38);
NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO:39);
NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO:40);
NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO:41);
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NFYQCIEHLSGSPAEKSRGQWQECRTG (SEQ ID NO:42);
NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO:43);
NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO:44);
NFYQCIEMLSLPPAEKSRGQWQECRTG (SEQ ID NO:45);
NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO:46);
NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO:47);
AQQEECEFAPWTCEHM (SEQ ID NO:21);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEH
MLE (SEQ ID NO:23);
AQQEECELAPWTCEHM (SEQ ID NO:24);
AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEH
MLE (SEQ ID NO:26);
AQQEECEFSPWTCEHM (SEQ ID NO:27);
AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHM
LE2xConFS (SEQ ID NO:29);
AQQEECELEPWTCEHM (SEQ ID NO:30);
AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHM
LE (SEQ ID NO:32);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEH
MLE (SEQ ID NO:33);
AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHM
LE (SEQ ID NO:34); and
AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEH
MLE (SEQ ID NO:10).
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19. An isolated polynucleotide comprising a nucleotide sequence encoding the antibody of
any of claims 1-17 or the peptide of claim 18.
20. A vector comprising the polynucleotide of claim 19.
21. A host cell comprising the vector of claim 20, or progeny of the cell.
22. The antibody of any of claims 1-17 or the peptide of claim 18 for use in treating a
disease.
23. The antibody or peptide of claim 22, wherein the disease is cancer.
24. The antibody or peptide of claim 22 or 23, wherein the antibody or peptide inhibits
angiogenesis, modulates angiogenesis, or inhibits tumor growth.
25. A method for producing the antibody of any one of claims 1-17 comprising selecting for
a MRD using an MRD binding target, wherein the MRD is derived from a phage display
library or from natural ligands.

Dated this 08th day of July, 2010
Signature:
Name: Dr. K. Rama
To: K&S Partners
The Controller of Patent Agent for the Applicant
The Patent Office, at Chennai

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Patent Number 278286
Indian Patent Application Number 4257/CHENP/2010
PG Journal Number 53/2016
Publication Date 23-Dec-2016
Grant Date 20-Dec-2016
Date of Filing 08-Jul-2010
Name of Patentee THE SCRIPPS RESEARCH INSTITUTE
Applicant Address Drop TPC-8 10550 N. Torrey Pines Rd. La Jolla CA 92037 United States of America
Inventors:
# Inventor's Name Inventor's Address
1 BARBAS III Carlos F. 7081 Walter Street San Diego CA 92122 United States of America
PCT International Classification Number C12P 21/08
PCT International Application Number PCT/US2008/088337
PCT International Filing date 2008-12-24
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 61/022,767 2008-01-22 U.S.A.
2 61/018,816 2008-01-03 U.S.A.