WO2016205566A1 - Anticorps spécifiques anti-fzd7 et vaccins pour traiter le cancer et réguler la fonction de cellules souches - Google Patents

Anticorps spécifiques anti-fzd7 et vaccins pour traiter le cancer et réguler la fonction de cellules souches Download PDF

Info

Publication number
WO2016205566A1
WO2016205566A1 PCT/US2016/037934 US2016037934W WO2016205566A1 WO 2016205566 A1 WO2016205566 A1 WO 2016205566A1 US 2016037934 W US2016037934 W US 2016037934W WO 2016205566 A1 WO2016205566 A1 WO 2016205566A1
Authority
WO
WIPO (PCT)
Prior art keywords
limitation
fzd7
antibody
inhibitors
seq
Prior art date
Application number
PCT/US2016/037934
Other languages
English (en)
Inventor
Christelle Jost
Joseph Buechler
Gunars Valkirs
Dennis Carson
Karl WILLERT
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2016205566A1 publication Critical patent/WO2016205566A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present application generally relates to antibodies and antibody fragments that specifically bind to human frizzled receptor(s) (“FZD”), e.g,, FZD7, and block or inhibit Wnt signaling, as well as compositions containing these anti-FZD7 antibodies and anti-FZD7 antibody fragments.
  • FZD human frizzled receptor
  • anti-FZD7 antibodies or antibody fragments antagonize, inhibit, neutralize or block at least one biological effect associated with human FZD7, as discussed infra.
  • anti-FZD7 antibodies and antibody fragments preferably will not substantially interact with (bind to) other FZD family members
  • the invention relates to nucleic acids encoding said anti-FZD7 antibodies and anti ⁇ FZD7 antibody fragments. Further, the invention pertains to the use of said nucleic acids to express said anti-FZD7 antibodies and antibody fragments in desired host cells. Also, the invention pertains to anti-idiot pic antibodies produced against any of the disclosed anti-FZB7 antibodies and antibody fragments.
  • the invention further relates to therapeutic and diagnostic uses of anti-FZD7 antibodies and antr-FZD? antibody fragments, preferably anti-FZD? antibodies and anii- FZD7 antibody fragments that do not substantially interact with (bind to) other FZD family members, in the treatment, or prophylaxis of diseases, such as cancer.
  • the invention also encompasses the use of anti-FZD? antibodies and anti ⁇ FZD7 antibody fragments, preferably anti-FZD7 antibodies and anti-FZD7 antibody fragments that do not substantially interact with (bind to) other FZD family members, for tissue engineering.
  • the present invention pertains to novel peptides, compositions containing and their use in vaccines
  • WNT genes encode secreted lipid modified growth factors (Takada et al, 2006; Willert et al., 2003) that exert potent effects on stem cells (Nusse, 2008; Reya and Clevers, 2005; Reya et al., 2003; Willert et al, 2003). Binding of WNT proteins to their cognate receptors encoded by the Frizzled (FZD) gene family initiates various intracellular signaling cascades, most prominently the WNT/
  • Frizzled Frizzled
  • WNT signaling pathway is a major regulator of many developmental processes, including axis specification, tissue patterning and organogenesis, and its deregulation during embryogenesis often has catastrophic consequences for the developing organism.
  • WNT signaling is critical for homeostasis of multiple tissues, including blood, intestine, skin and liver to name a few (reviewed in (Nusse, 2008).
  • inappropriate activation of WNT signaling has been observed in multiple cancers.
  • a comprehensive analysis of human colorectal cancers found that over 90% of cancers harbor mutations in genes associated with WNT signaling (Cancer Genome Atlas, 2012). Deregulated WNT signaling has also been observed in cancers of the skin, liver, brain, breast and ovary.
  • WNT signaling pathway was first elucidated in the Drosophila developmental mutant wingless (wg) and from the murine proto-oncogene int-l , now Wntl (Nusse & Varmus : 1982, Cell 31 :99-109; Van Ooyen & Nusse, 1984, Cell 39:233-40; Cabrera et al, 1987, Cell 50:659-63; Rijsewijk et aL 1987, Cell 50:649-57). Wnt genes encode secreted lipid-modified glycoproteins of which 19 have been identified in mammals.
  • FZD Frizzled
  • LDL low-density lipoprotein
  • CRD cysteine-rich domain
  • FZD receptors have been grouped into those that activate the canonical ⁇ -catenin pathway and those that activate non-canonical pathways, all of which are described below (Miller et al., 1999, Oncogene 18:7860-72).
  • FZD receptors interact with LRP5/6, single pass transmembrane proteins with four extracellular EGF-like domains separated by six YWTD amino acid repeats (Johnson et al. s 2004, J. Bone Mineral Res, 19: 1749).
  • the canonical WNT signaling pathway activated upon receptor binding is mediated by the cytoplasmic protein Dishevelled ("DSH") interacting directly with the FZD receptor and results in the cytoplasmic stabilization and accumulation of ⁇ -catenin.
  • DSH cytoplasmic protein Dishevelled
  • ⁇ -catenin is localized to a cytoplasmic destruction complex that includes the tumor suppressor proteins adenomatous polyposis coli ("APC”) and Axin. These proteins function as critical scaffolds to allow glycogen synthase kinase (“03 ⁇ ”) ⁇ 3 ⁇ to bind and phosphorylate ⁇ -catenin, marking it for degradation via the ubiquitin/proteasome pathway. Accumulated cytoplasmic ⁇ -catenin is then transported into the nucleus where it interacts with the DNA-binding proteins of the Tcf Lef family to activate transcription.
  • WNT ' iigands also activate ⁇ - catenin-independent pathways (Veeman et al, 2003, Dev. Cell 5:367-77).
  • Non-canonical WNT signaling has been implicated in numerous processes, including gastrulation movements via a mechanism similar to the Drosophila planar cell polarity ("PCP") pathway.
  • PCP Drosophila planar cell polarity
  • Other potential mechanisms of non-canonical WNT signaling include calcium flux, JNK, and both small and heteronimerie G-proteins.
  • Antagonism is often observed between the canonical and non-canonical pathways, and some evidence indicates that non-canonical signaling can suppress cancer formation (Olson & Gibo, 1998, Exp, Cell Res. 241 : 134; Topol et al, 2003, J. Cell Biol. 162:899-908).
  • the canonical WNT signaling pathway also plays a central role in the maintenance of stem cell populations in the small intestine and colon, and the inappropriate activation of this pathway plays a prominent role in colorectal cancers (Reya & Clevers, 2005, Nature 434:843).
  • Stem cells reside in the crypts of the absorptive epithelium of the intestines and slowly divide to produce rapidly proliferating cells that give rise to all the differentiated cell populations that move up out of the crypts to occupy the intestinal villi.
  • the WNT signaling cascade plays a dominant role in controlling cell fates along the crypt- villi axis and is essential for the maintenance of the stem cell population, Disruption of WNT signaling, e.g., genetic loss of Tef7/2 by homologous recombination (Korinek et al, 1998, Nat. Genet. 19:379) or overexpression of Dickkopf-1 (Dkkl), a potent secreted Wnt antagonist (Pinto et al, 2003, Genes Dev. 17:1709-13; Kuhnert et at., 2004, Proc. Natl Acad. Sci. 101 :266-71), results in depletion of intestinal stem cell populations.
  • Dkkl Dickkopf-1
  • FZD 7 is one of ten identified human WNT receptors
  • FZD 7 is expressed in the epiblast of the developing mouse embryo (Kemp CR, et al. Expression of FrizzledS, Frizzled?, and Frizzled! 0 during early mouse development and interactions with canonical Wnt signaling. Dev Dyn. 2007;236(7):201 1-2019) and that the human homolog FZD 7 is elevated in undifferentiated human embryonic stem cells ("hESCs") (Melchior K, et al. The WNT receptor FZD7 contributes to self-renewal signaling of human embryonic stem cells. Biol Chem. 2008;389(7):897-903; and Sparger JM, et al. Gene expression patterns in human embryonic stem cells and human piuripotent germ cell tumors. Proc Natl Acad Sci USA. 2003; 100(23): 13350-13355).
  • FZ.D7 is specifically expressed in human piuripotent stem cells ("hPSCs") (Fernandez et al., 2014, PNAS, 1 1 1(4): 1409- 14), The inventors previously demonstrated that FZD7 is abundantly expressed in hPSCs and, in fact, based on RNA-seq analysis, FZD 7 is the most highly expressed FZD gene in hPSCs. Additionally, the inventors discovered that FZD7 expression declines upon differentiation, and knockdown of FZD7 expression using shRNAs disrupts the piuripotent state of hPSCs. Therefore, FZD7 is a stem cell specific WNT receptor. In addition, FZD7, along with FZD!, 5 and 8, is also expressed in mouse crypt epithelial preparations, which harbor intestinal stem ceils (Hughes et al., 201 1).
  • FZD7 expression has also been detected in several cancer cell lines and tumors, including breast cancer (Chakrabarti et ai,, 2 14; Simmons et al., 2014; Yang et al., 201 1), ovarian cancer (Asad et ah, 2014), hepatocellular carcinoma (Merle et al, 2005; Nambotin et al., 201 1 ; Nambotin et al., 2012; Song et ai., 2014; Wei et al, 201 1), Wilms 5 tumor (Pode- Shakked et al, 201 1), gastric cancers ( irikoshi et al., 2001 ) and colon cancer (Ueno et al., 2009; Vincan et al, 2010), Moreover, in view of the central role for FZD7 in stem cells (discovered by the inventors) and its limited expression in adult tissues, FZD7 may represent a unique cancer stem cell marker that
  • the present invention addresses the need for inhibitors that are specific to an individual WNT signaling pathway and provides novel antibodies and antibody fragments, e.g., Fabs, that specifically bind to human FZD7 without substantially interacting with (binding to) other FZD family members.
  • novel antibodies and antibody fragments e.g., Fabs
  • the invention generally relates to anti-human FZD7 antibodies or antibody fragments thereof.
  • the anti-human FZD7 antibody fragment comprises: (a) a variable light chain comprising a CDRl sequence consisting of SEQ ID NO:6; a CDR2 sequence consisting of SEQ ID NO:7; and a CDR3 sequence consisting of SEQ ID NO:8; and/or (b) a variable heavy chain comprising a CDRl sequence consisting of SEQ ID NO:9; a CDR2 sequence consisting of SEQ ID NO: 10; and a CDR3 sequence consisting of SEQ ID NO; i l .
  • the anti-human FZD7 antibody or antibody fragment may comprise: (a) a light chain comprising an amino acid sequence with at least. 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 4 and/or (b) a heavy chain comprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:5.
  • the anti-human FZD7 antibody or antibody fragment comprises: (a) a light chain having the amino acid sequence of SEQ ID NO;4, and/or (b) a heavy chain hav ng the amino acid sequence of SEQ ID NO:5,
  • the anti-human FZD7 antibody fragment comprises: (a) a variable light chain comprising a CDR.1 sequence consisting of SEQ ID NO: 14; a CDR2 sequence consisting of SEQ ID NO: 15; and a CDR3 sequence consisting of SEQ ID NO: 16; and/or (b) a variable heavy chain comprising a CDR1 sequence consisting of SEQ ID NO: 17; a CDR2 sequence consisting of SEQ ID NO: 18; and a CDR3 sequence consisting of SEQ ID NO: 19.
  • the anti-human FZD7 antibody or antibody fragment may comprise: (a) a light chain comprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 12 and/or (b) a heavy chain comprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 13.
  • the anti-human FZD7 antibody or antibody fragment comprises: (a) a light chain having the amino acid sequence of SEQ ID NO: 12, and/or (b) a heavy chain having the amino acid sequence of SEQ ID NO: 13.
  • the anti-human FZD7 antibody or antibody fragment thereof may be selected from the group consisting of scFvs, cameibodies, nanobodies, IgNAR, Fab fragments, Fab' fragments, MetMab like antibodies, monovalent antibody fragments, and F(ab') 2 fragments, Additionally, the anti-human FZD7 antibody or antibody fragment may comprise a human constant domain, e.g., an IgG l , IgG2, IgG3 or IgG4 isotype. Preferably, the anti-human. FZD7 antibody or antibody fragment thereof is a humanized antibody or antibody fragment.
  • the anti-human FZD7 antibody or antibody fragment may be directly or indirectly attached to a detectable label or therapeutic agent.
  • the anti-human FZD7 antibody or antibody fragment is attached to at least one effector moiety, which optionally comprises a chemical linker and a cytotoxic agent or payload.
  • the anti -human FZD7 antibody or antibody fragment thereof is attached to one or more detectable moieties, e.g., comprising a fluorescent dye, enzyme, substrate, bioluminescent material, radioactive material, chemiluminescent moiety, or mixtures thereof, in yet another embodiment, the anti-human FZD7 antibody or fragment thereof is attached to a detectable moiety.
  • the anti-human FZD7 antibody fragments when administered to a human subject, may inhibit or neutralize at least one biological effect elicited by FZD7, e.g., FZD7 signaling via Wnt3a and/or FZD7 ⁇ mediated ceil migration.
  • the anti-human FZD7 antibodies or antibody fragments does not substantially interact with (bind to) other FZD family members.
  • the anti- human FZD7 antibody or antibody fragment has higher affinity for FZD7 as compared to other FZD family members (FZDl, FZD2, FZD3, FZD4, FZD 5, FZD6, FZD8, FZD9 and FZD 10) and/or does not bind to other FZD family members (FZDl , FZD2, FZD3, FZD4, FZD 5, FZD6, FZD , FZD9 and FZD 10).
  • the invention also provides an anti-idiotypic antibody produced against the disclosed anti-human FZD7 antibody or antibody fragments, Also encompassed by the disclosure is a method of using the anti-idiotypic antibody or another antibody that specifically binds said anti-human FZD antibody to monitor the in vivo levels of said anti- FZD7 antibody or antibody fragment in a subject or to neutralize said anti-FZD7 antibody in a subject being administered said anti-FZD7 antibody or antibody fragment.
  • the invention further provides a composition suitable for therapeutic, prophylactic, or a diagnostic use comprising a therapeutically, prophyiactically or diagnostically effective amount of at least one of the disclosed anti-human FZD7 antibodies or antibody fragments or an anti-idiotypic antibody raised against at least one of the disclosed anti-human FZD7 antibodies or antibody fragments.
  • the composition may be suitable for subcutaneous administration or intravenous administration and, optionally, may further comprise a pharmaceutically acceptable diluent, carrier, soiubilizer, emulsifier, preservative, or mixture thereof
  • the composition may be lyophilized, stabilized and/or formulated for administration by injection.
  • the composition may further comprise another active agent, e.g., an immune modulator, an anti-metasiatie, a chernotherapeutic, a hormone or growth factor antagonist, an alkylating agent, a TLR agonist or a cytokine or cytokine antagonist.
  • another active agent e.g., an immune modulator, an anti-metasiatie, a chernotherapeutic, a hormone or growth factor antagonist, an alkylating agent, a TLR agonist or a cytokine or cytokine antagonist.
  • the other agent is selected from the group consisting of alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, aikyl sulfonates, nitrosoureas and triazenes); uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demefhyldopan®.
  • vincristine Vincasar®, Oncovin#
  • vindesine Eldisine®
  • vinoreibine Vineibine®
  • vinflunine Javlor®
  • platinum-based agents carboplatin (Paraplat®, Parapiatin®), cisplatin (Platinol®), oxaliplatin (Eloxatin®), nedaplatin, satraplatin, and tripladn a thracyclines: daunorubicin (Cerubidme®, Rubidomycin®).
  • doxorubicin (Adriamycin®), epirubicin (Ellence®), idarubicin (Idamycin®), mitoxantrone (Novantrone®), valrubicin (Valstar®), aclarubicin, amrubicin, liposomal doxorubicin, liposomal daunorubicin, pirarubicin, pixantrone, and zorubicin; topoisomerase inhibitors: topotecan (Hycamtin®), irinotecan (Camptosar®), etoposide (Toposar®, VePesid ), teniposide (Vumon®), iamellarin D, SN-38, caraptothecin (e.g., IT-101), beiotecan, and rubitecan; taxanes: paclitaxel (Taxol®), docetaxei (Taxotere®), larotaxel,
  • Roferon-A®, Intron®-A) or IFN-gamma (Actirnmune®)); interlenkins: IL-1, IL-2 (Proleukin®), IL-24, IL-6 (Sigosix®), IL-I 2; HSP90 inhibitors (e.g., geldanamycin or any of its derivatives).
  • the HSP90 inhibitor is selected from geidanamycin, 17-aUcylamino ⁇ 17 ⁇ desmethoxygeidanamycin (“ 17-AAG”) or 17 ⁇ (2-dimethyiaminoethyi)amino- 17- desmethoxygeldanamycin (“I7-DMAG”) anti-androgens wliich include, without limitation nilutamide (Nilandron®) and bicalutamide (Caxodex®); antiestrogens which include, without limitation tamoxifen (Nolvadex®), toremifene (Fareston®), letrozole (Femara®), iesiolactone (Tesiac D), anastrozole (Arimidex®), bicalutamide (Casodex®), exemesiane (AromasinS), flutamide (Eulexin®), fulvestrant (Fasiodex®), ral
  • kinase inhibitors which include without limitation debromohymenialdisine; cyclooxygenase inhibitors which include without limitation 1H- indole-3-acetamide, l -(4-chlorobenzoyl)-5-methoxy-2-methyl-N-(2-phenyiethyl)-(9Cl), 5- alkyl substituted 2 ⁇ aiylaminophenylacetic acid and its derivatives (e.g., celecoxib (Celebrex®), rofecoxib (Vioxx®), etoricoxib (Arcoxia®), lumiracoxib (Prexige®), valdecoxib (Bextra®) or 5-alkyl-2-arylaminophenylacetic acid); cRAF kinase inhibitors which include without limitation 3-(3,5-dibromo-4 ⁇ hydroxybenzylidene)-5 ⁇ iodo-l ,3- dihydroindoi-2-one and benzamide
  • glycogen synthase kinase-3 (GSK3) inhibitors which include without limitation indirubin-3 ; -rnonooxime; hi stone deacelyiase (HDAC) inhibitors which include without limitation suberoylanilide hydroxamic acid (SAHA), [4-(2-amino-phenylcarbamoyl)-benzyl]-carbamic acid pyridine-?)- ylmeihylester and its derivatives, butyric acid, pyroxamide, trichostatin A, oxamiladn, apicidin, depsipeptide, depudecin, trapoxin, vorinostat (Zoiinza®), and compounds disclosed in WO 02/22577; I-kappa B ⁇ aipha kinase inhibitors
  • isolated nucleic acid sequences encoding the disclosed anti-human FZD7 antibodies or antibody fragments or anti-idiotypic antibodies provided, vectors containing the isolated nucleic acid sequenee(s), and host cells comprising the isolated nucleic acid sequence(s).
  • the host cell may be a mammalian, bacterial, fungal, yeast, avian or insect cell. Methods of expressing an anti-human FZD7 antibody or antibody fragment comprising cuituring the host cell under conditions that provide for expression of said antibody or antibody fragment are also provided.
  • the invention farther relates to the therapeutic and diagnostic uses of ami -human FZD7 antibodies and fragments thereof.
  • the invention provides a method of blocking, inhibiting or neutralizing FZD7 signaling (e.g., via Wnt3a) comprising administering to a subject in need an effective amount of an anti-human FZD7 antibody or antibody fragment selected from FZD7-Fab-1791 or FZD7-Fab-1291.
  • the invention further provides a method for treating or preventing a condition associated with aberrant FZD7 expression in a subject comprising admi istering to a subject in need an effective amount of an anti-human FZD7 antibody or antibody fragment selected from F2D7-Fab-1791 or FZD7-Fab-1291.
  • the condition is cancer, preferably, a solid tumor, more preferably, a cancer is selected from ovarian cancer, breast cancer, renal cancer or glioblastoma,
  • the invention also provides a method for blocking, inhibiting or neutralising FZD7-mediated cell migration in a subject comprising administering to a subject in need an effective amount of an anti-human FZD7 antibody or antibody fragment selected from FZD7- Fab-1791 or FZD7-Fab-1291.
  • the invention encompasses a method for generating a specific cell population from human pluripotent stem cells, comprising applying an effective amount of an anii-human FZD7 antibody or antibody fragment selected from FZD7-Fab-1791 or FZD7- Fab- 1291 to human pksripoient stem cells; and isolating specific cell populations of interest.
  • the anti-human FZD7 antibody fragment preferably does not substantially interact with (bind to) other FZD family members (FZD1 , FZD2, FZD3, FZD4, FZD 5, FZD6, FZD8, FZD9 and FZD 10).
  • the anti-human FZD7 antibody fragment comprises: (a) a variable light chain comprising a CDR1 sequence consisting of SEQ ID NO: 6; a CDR2 sequence consisting of SEQ ID NO:7; and a CDR3 sequence consisting of SEQ ID NO:8; and/or (b) a variable heavy chain comprising a CDRi sequence consisting of SEQ ID NO:9; a CDR2 sequence consisting of SEQ ID NO: 10; and a CDR3 sequence consisting of SEQ ID NO: I 1 .
  • the anti-human FZD7 antibody or antibody fragment comprises: (a) a light chain comprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 4 and/or (b) a heavy chain comprising an amino acid sequence with at least 80, 85, 90. 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:5.
  • the anti-human FZD7 antibody or antibody fragment comprises: (a) a light chain having the amino acid sequence of SEQ ID NO:4, and/or (b) a heavy chain having the amino acid sequence of SEQ ID NO: 5.
  • the anti-human FZD7 antibody fragment comprises: (a) a variable light chain comprising a CDRi sequence consisting of SEQ ID NO: 14; a CDR2 sequence consisting of SEQ ID NO: 15; and a CD 3 sequence consisting of SEQ ID NO: 16; and/or (b) a variable heavy chain comprising a CDR I sequence consisting of SEQ I D NO: 17; a CDR2 sequence consisting of SEQ I D NO: 18; and a CDR3 sequence consisting of SEQ ID NO: 19.
  • the anti-human FZD7 antibody or antibody fragment comprises: (a) a light chain comprising an amino acid sequence with at least 80, 85, 90, 95, 96 s 97, 98, or 99% sequence identity to SEQ ID NO: 12 and/or (b) a heavy chain comprising an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 13.
  • the anti-human FZD7 antibody or antibody fragment comprises: (a) a light chain having the amino acid sequence of SEQ ID NO: 12, and/or (b) a heavy chain having the amino acid sequence of SEQ ID NO: 13.
  • the anti-human FZD7 antibody or antibody fragment may be selected from the group consisting of scFvs, camelhodies, nanobodies, IgNAR, Fab fragments, Fab' fragments, MetMab like antibodies, monovalent antibody fragments, and F(ab')2 fragments.
  • the antibody or antibody fragment used in these methods comprises a human constant domain, e.g., an IgGl , igG2, IgG3, or lgG4 antibody.
  • the antibody or antibody fragment is a humanized antibody or antibody fragment.
  • the antibody may also be an anti-idiotypic antibody produced against an anti-human FZD7 antibody or antibody fragment.
  • the antibody or antibody fragment may be directly or indirectly attached to a detectable label (e.g., a fluorescent dye, enzyme, substrate, bioiuminescent material, radioactive material, chemiluminesceiit moiety, or mixtures thereof) or therapeutic agent, at least one effector moiety (which optionally comprises a chemical linker), or one or more functional moieties.
  • a detectable label e.g., a fluorescent dye, enzyme, substrate, bioiuminescent material, radioactive material, chemiluminesceiit moiety, or mixtures thereof
  • therapeutic agent e.g., a detectable label, enzyme, substrate, bioiuminescent material, radioactive material, chemiluminesceiit moiety, or mixtures thereof
  • at least one effector moiety which optionally comprises a chemical linker
  • the therapeutic methods further comprise administering separately or co-administering another agent, e.g., comprise another active agent, e.g., an immune modulator, an anti ⁇ metastatic, a chemotherapeutic, a hormone or growth factor antagonist, an alkylating agent, a T i .R agonist or a cytokine or cytokine antagonist.
  • another agent e.g., comprise another active agent, e.g., an immune modulator, an anti ⁇ metastatic, a chemotherapeutic, a hormone or growth factor antagonist, an alkylating agent, a T i .R agonist or a cytokine or cytokine antagonist.
  • the other agent is selected from the group consisting of alkylating agents (including, without limitation, nitrogen mustards, ethyienimine derivatives, alkyl sulfonates, nitrosoureas and triazenes); uracil mustard (Aminouracil Mustard®, Chloretbaminaeil®, Demethyldopan ⁇ , Desmeihyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen Mustard®, Uracillost®, Uraeilmostaza®, Urarnustin®, Uramustine®); bendamustine (Treakisyrn®, Ribomustin®, Treanda®); ehlormethine (Mustargen®); cyclophosphamide (Cytoxan®, eosar®, Clafen®, Endoxan®, Procytox®, Revim.muneTM); ifosfamide (Mitoxana®
  • pyrimidine analogs, purine analogs and adenosine deaminase inhibitors methotrexate (Rheumatrex®, Trexall®), 5 -fiuoro uracil (Adrucil®. Efudex®, Fiuoroplex®), floxuridine (FUDF®), carmofur.
  • cytarabine (Cytosar-U®, Tarabine PFS), 6-mercaptopurine (Puri-Nethol®)), 6-ihioguanine (Thioguanme Tabloid®), fludarabine phosphate (Fiudara®), pentostatin (Nipent®), pemetrexed (Aiimta®), raltitrexed (Tomudex®), cladribine (Leustatin®), ciofarabine (Clofarex®, Clolar®), mercaptopurine (Puri-Netho!®), capecitabine (Xeloda®), nelarabine (Arranon®), azacitidine (Vidaza®), decitabine (Dacogen®), enocitabine (Sunrabin®), sapacitabine, tegafur-uracil, tiazofurine, tioguanine, trofosfamide, and gemcitabine (G
  • the HSP90 inhibitor is selected from geldanamycin, 17-alkylamino-17- desmethoxygeldanamycin (" 17-AAG”) or 1 -(2-dimethylaminoethyl)amino- 17 ⁇ desmethoxygeldanamyctn (“17 ⁇ DMAG”): anti-androgens which include, without limitation nilutamide ( ilandron®) and bicaiutamide (Caxodex®); antiestrogens which include, without limitation tamoxifen (Nolvadex®), toremifene (Fareston®), letrozole (Femara®), testolactone (Teslac®), anastrozole (Arimidex®), bicaiutamide (Casodex®), exemestane (Aromasin®), flutamide (Eulexin®), fulvestrant (Fas!odex®), raloxifene (Evista
  • insulin tyrosine kinase inhibitors which include without limitation hydroxyl-2- naphthalenylrnethylphosphonic acid
  • c-Jun-N-terminal kinase (INK) inliibitors which include without limitation pyrazoleanthrone and epigallocatechin gallate
  • mitogen-activated protein kinase (MAP) inhibitors which include without limitation benzenesulfonan ide, N-[2-[[[[[3 ⁇ (4- chlorophenyl)-2-propenyl]methyljamino].methyl]phenyl]-N-(2-hy-droxyethyl)--4-methoxy- (9CI);
  • MDM2 inhibitors which include without limitation trans-4-iodo, 4 -boranyl-cha!cone; MEK inhibitors which include without limitation butanedinitrile, bis[amino 2- aminophenyl)thio]methylene]-(
  • MMI270B or AAJ996 mTor inhibitors which include without limitation rapamycin (Rapamune®), and analogs and derivatives thereof, AP23573 (also known as ridaforoiimus, deforolimus, or MK- 8669), CCI-779 (also known as temsirolimus) (Torisel®) and SDZ-RAD; NGFR tyrosine kinase inhibitors which include without limitation tyrphostin AG 879; p38 MAP kinase inhibitors which include without limitation Phenol, 4-[4-(4-fluorophenyI)-5-(4-pyridinyl)-lH- imidazol-2-yl]-(9Cl), and benzamide, 3-(dimethylamino) ⁇ N-[3 ⁇ [(4-hydroxylbenzoyl)amino]- 4-methylphenyl]-(9Cl); p56 tyrosine kinase inhibitors which
  • Bisindolylmaleimide IX Sphinogosine, staurosporine, and Hypericin; PKC delta kinase inhibitors which include without limitation rottlerin; polyamine synthesis inhibitors which inciude without limitation DMFO: FTP 1 B inhibitors which include wiihout limitation L ⁇ ieucinamide; protein tyrosine kinase inhibitors which include, wiihout limitation tyrphosiin Ag 216, tyrphostin Ag 1288.
  • bevacizumab and small molecules, e.g., sunitinib (Sutent®), sorafimb (Nexavar®), ZD6474 (also known as vandetanib) (ZaetimaTM), SU6668, CP-547632 and AZD2171 (also known as cediranib) (RecentinTM).
  • the anti-human FZD7 antibody or antibody fragment and the at least one other agent may be administered concurrently or, alternatively, the anti-human FZD7 antibody or antibody fragment may be administered before or after the at least one other agent.
  • Another aspect of the invention generally pertains to a human cell that may be engineered to express at least one antibody fragment as discussed herein, e.g., wherein said human cell may comprise an immune cell, e.g., a B cell, T cell, macrophage, dendritic cell, leukocyte, lymphocyte, or an N ceil,
  • an immune cell e.g., a B cell, T cell, macrophage, dendritic cell, leukocyte, lymphocyte, or an N ceil
  • Yet another aspect of the invention generally relates to a composition as discussed herein which may further include an agonistic or antagonistic antibody specific to a checkpoint inhibitor or checkpoint stimulator molecule such as PD1 , PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, K R, LAG3, PD-1 , TIM-3.
  • a checkpoint inhibitor or checkpoint stimulator molecule such as PD1 , PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, K R, LAG3, PD-1 , TIM-3.
  • Another aspect of the invention generally encompasses a transgenic mouse, a cell or tissue isolated therefrom that may express a humanized Fzd7 gene comprising SEQ ID NO: 2 with a P18SL mutation or SEQ ID NO: 44 with a P1 8L mutation.
  • Yet another embodiment of the invention generally relates to a method of using a transgenic mouse, a cell or tissue isolated therefrom that may express a humanized Fzd7 gene comprising SEQ ID NO: 2 with a PI SSL mutation or SEQ ID NO: 44 with a P188L mutation to assess the safety and/or efficacy of anli-FZD7 antibody fragments as discussed herein.
  • Another embodiment of the invention generally pertains to a method of blocking, inhibiting or neutralizing FZD7 signaling that may comprise administering to a subject in need an effective amount of an anti-human FZD7 antibody or antibody fragment selected from FZD7 ⁇ Fab-I 791 or FZD7- Fab- 1291 or a human ceil engineered to express said antibody fragment such as a T cell or NK cell, e.g., wherein said method may inhibit or block migration of, or the aggregation or single cell spheroid aggregation of cancer cells, e.g., glioblastoma, ovarian cancer, breast cancer, lung cancer, sarcoma, melanoma, lymphoma, or renal cancer cells.
  • cancer cells e.g., glioblastoma, ovarian cancer, breast cancer, lung cancer, sarcoma, melanoma, lymphoma, or renal cancer cells.
  • Yet another embodiment of the invention generally pertains to a method for treating or preventing a condition associated with aberrant FZD7 expression in a subject that may comprise administering to a subject in need an effective amount of an anti-human FZD7 antibody or antibody fragment selected from FZD7-Fab-1791 or FZD7-Fab-1291 or a human cell engineered to express said antibody fragment such as a T cell or NK cell, e.g., wherein said method may inhibit or block migration of, or the aggregation or single cell spheroid aggregation of cancer ceils, e.g., glioblastoma, ovarian cancer, breast cancer, lung cancer, sarcoma, melanoma, lymphoma, or renal cancer cells.
  • an anti-human FZD7 antibody or antibody fragment selected from FZD7-Fab-1791 or FZD7-Fab-1291 or a human cell engineered to express said antibody fragment such as a T cell or NK cell, e.g., wherein
  • Yet another aspect of the invention generally encompasses a method for blocking, inhibiting or neutralizing FZD7- mediated ceil migration in a subject that may comprise administering to a subject in need an effective amount of an anti-human FZD7 antibody or antibody fragment selected from FZD7- Fab-1791 or FZD7 ⁇ Fab-1291 or a fragment thereof or a human cell engineered to express said antibody or antibody fragment such as a T cell or NK. cell.
  • Another aspect of the invention generally relates to any of the methods disclosed herein wherein any of said methods may include the administration of an agonistic or antagonistic antibody specific to a checkpoint inhibitor or checkpoint stimulator molecule such as PD] , PD-L1 , PD-L2, CD27, CD28, CD4G, CD 137, OX40, GITR, ICOS, A2AR, B7- H3, B7-H4, BTLA, CTLA4, IDC), KIR, LAG3, PD ⁇ 1 S TI -3.
  • a checkpoint inhibitor or checkpoint stimulator molecule such as PD] , PD-L1 , PD-L2, CD27, CD28, CD4G, CD 137, OX40, GITR, ICOS, A2AR, B7- H3, B7-H4, BTLA, CTLA4, IDC), KIR, LAG3, PD ⁇ 1 S TI -3.
  • a checkpoint inhibitor or checkpoint stimulator molecule such as PD] ,
  • GSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRCiRPAFPF SEQ ID NO: 35.
  • Another aspect of the invention generally relates to a peptide that may comprise or may consist of a fragment of the following peptide or a multimer thereof: GSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPF (SEQ ID NO: 35), with the proviso that said fragment comprises the leucine contained in SEQ ID NO: 35 corresponding to the leucine at position 188 of human FZD7.
  • Said peptide specifically binds to FZD7-Fabl791 or FZD7-Fabl291 or an antibody containing the identical CDR.s as FZD7- Fab-17 1 or FZD7- Fab- 1291.
  • Said peptide may comprise at least 3 residues including the leucine contained in SEQ ID NO: 35 which corresponds to the leucine at position 188 of human FZD7.
  • said peptide may comprise at least 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 2, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues of the peptide of SEQ ID NO: 35 including the leucine contained in SEQ ID NO: 35 which corresponds to the leucine at position 1 88 of human FZD7.
  • Another aspect of the invention generally relates to a peptide as discussed herein which may attached to a polypeptide or another moiety which is not an amino terminal or carboxy terminal fragment of human FZD7, and/or which may be attached to an immunogen or to a detectable label or to a moiety which improves in vivo half-life or in vivo stability, e.g., wherein the label may be GST, GFP or FLAG.
  • Another aspect of the invention generally pertains to a recombinant cell that ma - express a peptide as discussed herein, e.g., a human, immune cell such as a T or NK cell.
  • a peptide as discussed herein, e.g., a human, immune cell such as a T or NK cell.
  • Another aspect of the invention generally relates to a polyclonal or monoclonal antibody that specifically binds to the peptide or cell as discussed herein.
  • Another aspect of the invention generally relates to a method of producing antibodies specific to human FZD7 that may comprise immunizing a host or immune ceils (in vitro immunization) with at least one peptide as discussed herein.
  • a therapeutic or prophylactic cancer vaccine that may contain at least one peptide or cell as discussed herein and at.
  • Said vaccine may comprise at least one adjuvant, and further wherein said adjuvant may comprise one or more of ALUM, saponin, squaiene, a dsR A, a ssDNA, an unmethylated CFG, a TLR agonist, and a cytokine.
  • Another aspect of the invention generally pertains to a vaccine as discussed herein which may further include an agonistic or antagonistic antibody specific to a checkpoint inhibitor or checkpoint stimulator molecule such as PD1 , PD-L1, PD-L2, CD27, CD28, CD40, CD 13 GX40, GITR, iCOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-L
  • a checkpoint inhibitor or checkpoint stimulator molecule such as PD1 , PD-L1, PD-L2, CD27, CD28, CD40, CD 13 GX40, GITR, iCOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-L
  • Yet another aspect of the invention generally relates to a method of eliciting protective or therapeutic anti-cancer immunity that may comprise administering a prophylactically or therapeutically effective amount of a peptide, cell or vaccine as discussed herein, e.g., wherein said cancer may be selected from ovarian cancer, breast cancer, lung cancer, sarcoma, melanoma, lymphoma, renal cancer or glioblastoma.
  • Said method may further include an agonistic or antagonistic antibody specific to a checkpoint inhibitor or checkpoint stimulator molecule such as PD1 , PD-L1 , PD-L2, CD27, CD28, CD40, CD 137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAGS, PD-1 , or
  • Figure 1 provides (i) amino acid sequences corresponding to the light chain region (SEQ ID NO:4) and the heavy chain region (SEQ ID NO: 5) of FZD7 ⁇ Fal 17 L (ii) and amino acid sequences corresponding to the light chain region (SEQ ID NO: 12) and the heavy chain region of FZD7-Fab-1291 (SEQ ID NO: 13).
  • the light chain CDRs (SEQ ID NOs: 6-8 and SEQ ID NOs:14-I6) and heavy chain CDRs (SEQ ID NOs:9 ⁇ l 1 and SEQ ID NOs:! 7-19) are designated in bold.
  • Figure 2 provides (i) amino acid sequences corresponding to the Sight chain region (SEQ ID NO:20) and the heavy chain region (SEQ ID O:21 ) of a chimeric antibody, chFZD7-l ?91 , (ii) and amino acid sequences corresponding to the light chain region (SEQ ID NO:22) and the heavy chain region (SEQ ID NO:23) of a chimeric antibody, chFZD7-1291.
  • the light chain and heavy chain CDRs are designated in bold.
  • Figure 3 provides an alignment of the human FZD7 amino acid sequence (SEQ ID NO: I) and the mouse FZD7 amino acid sequence (SEQ ID NO: 2) as well as a consensus sequence ("majority' ' ) (SEQ ID NO: 3).
  • the amino acid residues thai differ betwee the
  • Figure 4A-D provides epitope mapping data for FZD7-Fab ⁇ 1791 and FZD7-Fab- 1291 .
  • Figure 4A FZD7-Fab-1791 reacts with human FZD7, but not with mouse FZD7 (right panel), whereas a control antibody reacts with both human and mouse FZD7 (left panel).
  • Figure 4B Schematic of the predicted FZD7 protein topology showing the region of greatest divergence between the mouse and human FZD7 amino acid sequences (green rectangle) and the C -terminal V5 tag (orange rectangle).
  • FZD7 Mutational analysis of FZD7, in which mouse FZD7 amino acid residues were substituted with the corresponding human FZD7 amino acid residues (SEQ ID NOs:24-34), identified the FZD7-Fabs' epitope (see Figure 4C and Figure 4D, in which + indicates FZD7 reactivity with the Fab, whereas - indicates a lack of reactivity between FZD7 and the Fab).
  • Figure 5 shows FZD7 expression in different ceil lines. Eighteen different cell lines were (i) untreated, (ii) treated with Wnt3 (10 nM), or (iii) treated with CHIR (1 uM) for 24 hours before total RNA was isolated from the cells and qRT-PCR was performed. All values were normalized to the sample with the lowest amount of FZD7 rnR.NA, i.e., CHIR- treated HepG2 cells. Each of the three bars for each cell line follow the order of (i). (ii), and then (iii) from left to right.
  • Figure 6A and 6B show FZD7 signaling in cancel cell lines.
  • Figure 6 A The breast cancer cell line, MDA- B-231 , was stably transduced with the Wnt reporter, TOP- Flash. Cells were treated with an increasing dose of Wnt3a (0-5 nM) in the presence of absence of FZD7-Fab-179l . The addition of FZD7-Fab ⁇ 1791 (200 nM) reduced reporter activity in the ceils by about 3-fold.
  • Figure 6E OVCAR4 cells were treated with WnOa, CHIR, FZD7-Fah ⁇ 179R or FZD7-Fab-1291 for 24 hours before cell lysate was prepared and immunoblotted for FZD7 protein. Certain treatments resulted in a decrease in FZD7 protein levels.
  • Figure 7A and 7B show the characterization of FZD7 expression in an ovarian cancer line, OVCAR4.
  • Figure 7A OVCAR4 cells were analyzed by flow cytometry for surface expression of FZD7 using FZD7 ⁇ Fab-17 1 , and sorted into cell populations with high FZD7 expression (FZD7 Hi ) and low FZD7 expression (FZD7 Lo ).
  • Figure 7B FZD7 Hi cells are enriched for FZD7 mRNA relative to FZD7 L'J cells.
  • FIG. 8A FZD7 ⁇ Fab-17 1 blocks migration of a breast cancer cell line.
  • a confluent layer MDA-MB231 a breast cancer ceil line, was "wounded '5 , i.e., a pipette tip was used to create a scratch in the confluent layer. After 24 hours, the width of the wound was measured. The amount of wound closure, i.e., cell migration, was significantly less in the presence of FZD7-Fab-1791 as compared to control (no FZD7-Fah-1791). The presence of Wnt3a had no effect on migration.
  • FIG. 8B FZD7-Fab-1791 blocks migration of an ovarian cancer cell line, OVCAR4 cells were seeded on the upper chamber of a tra swell chamber, and FZD7-Fab-1791 (500 nM) was added to both the upper and lower chambers. FZD7-Fab-1791 significantly reduced migration of ceils through the membrane as measured by fixing and staining he lower surface,
  • Figure 9 shows a significant reduction in overall survival of ovarian cancer patients (1581 subjects) with high levels of FZD7 expression.
  • Figure 11A and 11B show a correlation between FZD7 expression and cancer.
  • Figure 11 A Relative expression of FZD7 (black) and GAPDFI (gray) were analyzed in 82 PDX samples. FZD7 expression varied between individual samples. The blue dotted line indicates an expression value of 9.
  • Figure 11B Relative expression analysis of FZD7 across different cancer types showed that 50% (n ::: 8) of the breast cancer samples express high levels of FZD7.
  • Figure 12 shows the expression of FZ.D7 in glioblastoma.
  • RNA was isolated from, the indicated eel! types, labeled on the X axis, and expression of FZD7 and POU5F1 was determined by RT-qPCR.
  • HE 293 and HI cells served as negative and positive controls, respectively, for FZD7 and POU5F1 expression.
  • NHA :: normal human astrocytes.
  • Figure 13A arsd
  • Figure 13B shows a clonogenicity assay of GBM cell line.
  • the GBM line TS528 was seeded at low density in wells of a 24 well plate (600 cells per well). Each well of this 24 well plate contained 1 ,200 micro we I Is, each 300 ⁇ in diameter and 200 ⁇ in depth. Cells were allowed to grow for 10 days in the absence ( Figure 13 A, top panel, "untreated") or presence (Figure 13 A, bottom panel, "+FZD7-Ig") FZD7-speciik antibody.
  • Figure 138 shows a bar graph that provides quantitation on the number of colonies per well of a 24-well plate.
  • Figure 14A-D shows FZD7 antibody specificity.
  • GST fusions with 42 amino acids of FZD7 surrounding amino acid 188 named GST-LI 88 ( Figure 14 A, SEQ ID NO: 35) and GST-P I 88 ( Figure 14B, SEQ ID NO: 36) were expressed and immunoblotted either with the FZD7"Speeific antibody or with a GST-specific antibody.
  • LI 88 represents the wildtype sequence recognized by the FZD7-specific antibody ( Figure 1.4C).
  • PI 88 represents the mutant sequence that is not recognized by the antibody ( Figure 14C).
  • aGST served as a control ( Figure 14 ⁇ ).
  • FIG. 15 shows the specificity of the FZD7-specific antibody for mouse Fzd7- P188L.
  • Cell lysates from rn.ouse embryonic fibroblasts (MEF) generated from El 3.5 wildtype (M£F-Fzd7WT) and mutant (MEF ⁇ Fzd7P188L) embryos were immunoblotted with the FZD7-specific antibody.
  • Only ceil lysates from mouse embryos carrying the engineered mutation at position 188 (Proline to Leucine) were reactive with the antibody, HEK293 cells transfected with a human FZD 7 expression vector served as positive control.
  • the present invention provides antibodies and antibody fragments, e.g., Fabs, that specifically bind to FZD7 and block or inhibit WNT signaling through this particular FZD receptor, as well as compositions containing these anti-FZD7 antibodies and anti ⁇ FZD7 antibody fragments.
  • the invention relates to nucleic acids encoding said anti- FZD7 antibodies and anti-FZD7 antibody fragments and the use of said nucleic acids to express said anti-FZD7 antibodies and antibody fragments in desired host cells, including an i-idiotypic antibodies produced against any of the disclosed anti-FZD7 antibodies and antibody fragments.
  • the invention provides methods for using the disclosed anti-FZD7 antibodies and anti-FZD7 antibody fragments which do not substantially interact with (bind to) other FZD family members in the diagnosis, treatment or prophylaxis of diseases, such as cancer, as well as the use of the anti-FZD7 antibodies and antibody fragments for tissue engineering.
  • FZD7 refers to "frizzled class receptor 7", which is a protein encoded by the FZD7 gene.
  • FZD7 is intended to encompass the human protein (NP_0O3498) (SEQ ID NO: 3 ) as well as its orthoiogs, e.g., murine F?.d7 (also known as Fz?) (NP 032083) (SEQ ID NO:2) as well as chimpanzee, cow, rat, etc, encoded by the corresponding gene in the respective species, in a preferred embodiment, “FZD7” refers to human FZD7.
  • FZD7 includes ail precursor, mature, and variant forms thereof.
  • Amino acids 1 -32 of FZD7 SEQ ID NO: 1 and SF.Q ID NO: 2 represent a signal sequence, and it is to be understood that FZD7 additionally includes any signal sequence that can be used in place of amino acids 1-32.
  • antibody is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-eovalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, Ig , JgA, IgE. IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be ''antibodies.”
  • a preferred source for producing antibodies useful as starting material according to the invention is rabbits.
  • antibody coding sequences have been described; and others may be raised by methods well-known in the art. Examples thereof include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (such as scFvs), cameibodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small-modular immunopharmaceuticals (SMIPs), and antibody fragments such as Fabs, Fab', F(ab3 ⁇ 4 and the like. See Stre!tsov VA, et ah. Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isoiype, Protein Sci. 2005 Nov; 14(1 1 ):2901-9.
  • antibodies or antigen binding fragments may be produced by genetic engineering.
  • antibody-producing cells are sensitized to the desired antigen or immunogen.
  • the messenger R A isolated from antibody producing ceils is used as a template to make cDNA using PGR amplification.
  • a library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors.
  • a combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library.
  • Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof.
  • Variant polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function.
  • Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues, etc), Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
  • Chimeric antibodies may be made by recombinant means by combining the variable light and heavy chain regions (V L and V H ), obtained from antibody producing cells of one species with the constant light and heavy chain regions from another.
  • V L and V H variable light and heavy chain regions
  • chimeric antibodies utilize rodent or rabbi variable regions and human constant regions, in order to produce an antibody with predominantly human domains.
  • the production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated herein by reference in its entirety).
  • the human constant regions of chimeric antibodies of the invention may be selected from IgGl , IgG2, IgG3, and IgG4 constant regions.
  • Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody, and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g.. U.S. Patent No. 6,187,287, incorporated fully herein by reference.
  • immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab' )2 3 or other fragments) may be synthesized.
  • "Fragments” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques.
  • Fv immunoglobulins for use in the present invention may be produced by synthesizing a fused variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest. e.g. diabodies, which comprise two distinct. Fv specificities.
  • SMI Ps small molecule immunopharmaceuticals
  • camelbodies. nanobodies, and IgNAR. are encompassed by immunoglobulin fragments.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, eD A, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • Immunoglobulins and fragments thereof may be modified post-translationaily, e.g. to add effector moieties such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, toxins, substrates, bioiuminescent materials, radioactive materials, chemiiuminescent moieties and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions of the present invention. Examples of additional effector molecules are pro vided infra.
  • Antibodies consist of two identical light polypeptide chains of molecular weight approximately 23,000 daltons (the ''Might chain”), and two identical heavy chains of molecular weight 53,000-70,000 (the "heavy chain”).
  • the four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y” configuration.
  • the "branch" portion of the "Y” configuration is designated the F a - L , region; the stem portion of the "Y” configuration is designated the Fc region.
  • the amino acid sequence orientation runs from the N-temiinal end at the top of the "Y" configuration to the C-terminal end at the bottom of each chain.
  • the N-terminai end possesses the variable region having specificity for the antigen that elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody.
  • variable region is linked in each chain to a constant region that extends the remaining length of the chain and that within a particular class of antibody does not vary with the specificity of the antibody (i.e., the antigen eliciting it).
  • constant regions There are five known major classes of constant regions that determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE corresponding to ⁇ , ⁇ . ⁇ , ⁇ , and ⁇ (gamma, mu, alpha, delta, or epsilon) heavy chain constant regions),
  • the constant region or class determines subsequent effector function of the antibody, including activation of complement (Kabat, E.
  • variable region refers to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen.
  • Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • Each light chain has a variable domain (Vt.) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • CDR complementarity determining region
  • hypervariable region refers to one or more of the hyper-variable or complementarity determining regions (CDRs) found in the variable regions of light or heavy chains of an antibody (See Kabat, E. A. et aL, Sequences of Proteins of Immunological interest. National Institutes of Health, Bethesda, Md., (1987)), These expressions include the hypervariable regions as defined by Kabat et ah ("Sequences of Proteins of Immunological Interest," Kabat E., et a!,, US Dept.
  • SDRs selectivity determining regions
  • An "epitope" or “binding site” is an area or region on an antigen to which an antigen-binding peptide (such as an antibody) specifically binds.
  • a protein epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the "footprint' 5 of the specifically antigen binding peptide).
  • the term epitope herein includes both types of amino acid binding sites in any particular region of FZD7 that specifically binds to an anti- FZD7 antibody or anti-FZD7 antibody fragment.
  • FZD7 may comprise a number of different epitopes, which may include, without limitation, (1 ) linear peptide antigenic determinants, (2) conformational antigenic determinants which consist of one or more non-contiguous amino acids located near each other in a mature FZD7 conformation; and (3) post-translational antigenic determinants which consist, either in whole or part, of molecular structures covalentiy attached to a FZD7 protein such as carbohydrate groups.
  • an antibody e.g., firs antibody
  • the epitope binding site for the first antibody comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the amino acid residues on the antigen that constitutes the epitope binding site of the second antibody.
  • a first antibody binds substantially or partially the same or overlapping epitope as a second antibody means that the first and second antibodies compete in binding to the antigen, as described above.
  • the term "binds to substantially the same epitope or determinant as" a monoclonal antibody means that an antibody ''competes " ' with the antibody.
  • the phrase "binds to the same or overlapping epitope or determinant as" an antibody of interest means that an antibody "competes" with said antibody of interest for at least one, (e.g., at least 2, at least 3, at least 4, at least 5) or all residues on FZD7 to which said antibody of interest specifically binds.
  • the identification of one or more antibodies that bind(s) to substantially or essentially the same epitope as the monoclonal antibodies described herein can be readily determined using alanine scanning. Additionally, any one of variety of immunological screening assays in which antibody competition can be assessed. A number of such assays are routinely practiced and well known in the art (see, e.g., U.S.
  • test antibodies to he examined are obtained from different source animals, or are even of a different Ig isotype
  • a simple competition assay may be employed in which the control antibody is mixed with the test antibody and then applied to a sample containing FZD7. Protocols based upon ELISAs, radioimmunoassays, Western blotting, and the use of BiAcore® analysis are suitable for use in such simple competition studies.
  • control anti ⁇ FZD7 antibody is pre-mixed with varying amounts of the test antibody (e.g., in ratios of about 1 : 1 , 1 :2, 1 : 1 0 or about 1 : 100) for a period of time prior to applying to the FZD7 antigen sample.
  • control and varying amounts of test antibody can simply be added separately and admixed during exposure to the FZD7 antigen sample.
  • bound antibodies can be distinguished from free antibodies (e.g., by using separation or washing techniques to eliminate unbound antibodies) and control antibody from the test antibody (e.g., by using species specific or isotype specific secondary antibodies or by specifically labeling the control antibody with a detectable label) it can be determined if the test antibody reduces the binding of the control antibody to the FZD7, indicating that the test antibody recognizes substantially the same epitope as the control anti ⁇ FZD7 antibody.
  • the binding of the (labeled) control antibody in the presence of a completely irrelevant antibody (that does not bind FZD7) can serve as the control high value
  • the control low value can be obtained by incubating the labeled control antibody with the same but unlabeled control antibody, where competition would occur and reduce binding of the labeled antibody.
  • a significant reduction in labeled antibody reactivity in the presence of a test antibody is indicative of a test antibody that recognizes substantially the same epitope, i.e., one that competes with the labeled control antibody.
  • any test antibody that reduces the binding of the control antibody to FZD7 by at Jeast about 5.0%, such as at least about 60%, or more preferably at least about 70% (e.g., about 65-100%), at any ratio of test antibody between about 1 : 1 or 1 : 10 and about 1 : 100 is considered to be an antibody thai binds to substantially the same or overlapping epitope or determinant as the control antibody.
  • test antibody will reduce the binding of the control antibody to FZD7 antigen preferably at least about 50%, at least about 60%, at least about 80% or at least about 90% (e.g., about 95%) of the binding of the control antibody observed in the absence of the test antibody.
  • a simple competition assay in which a test antibody is applied at saturating concentration to a surface onto which FZD7 is immobilized also may be advantageously employed.
  • the surface in the simple competition assay is preferably a BIAcoreCD chip (or other media suitable for surface plasmon resonance analysis).
  • the binding of a control antibody that binds FZD7 to the FZD7-eoated surface is measured. This binding to the FZD7- containing surface of the control antibody alone is compared with the binding of the control antibody in the presence of a test antibody.
  • test antibody that reduces the binding of control antibody by at least about 20% or more, at least about 40%, at least about 50%, at least about 70%, or more, can be considered to be an antibody that binds to substantially the same epitope or determinant as the control antibody.
  • test antibody will reduce the binding of the control antibody to FZD7 by at least about 50% (e.g., at least about 60%, at least about 70%, or more). It will be appreciated that the order of control and test antibodies can be reversed; i.e.
  • control antibody can be first bound to the surface and then the test antibody is brought into contact with the surface thereafter in a competition assay.
  • the antibody having greater affinity for FZD7 antigen is bound to the FZD7-corttaining surface first, as it will be expected that the decrease in binding seen for the second antibody (assuming the antibodies are competing) will be of greater magnitude.
  • assays are provided in e.g., Saunal and Regemxsortel, J. Immunol. Methods 183: 33-41 (1995), the disclosure of which is incorporated herein by reference,
  • an antibody binds the same or overlapping epitope(s) on FZD7 as another antibody or the epitope bound by a test antibody may in particular be determined using a western-blot based assay.
  • a library of peptides corresponding to the antigen bound by the antibody, the FZD7 protein is made, that comprise overlapping portions of the protein, typically 10-25, 10-20 or 10-15 amino acids long.
  • These different overlapping amino acid peptides encompassing the FZD7 sequence are synthesized and covalently bound to a nitrocellulose membrane. Blots are then prepared and probed according to the manufacturer's recommendations.
  • the immunoblot assay detects by iluorometric means what peptides in the library bind to the test antibody and thereby can identify what residues on the antigen, i.e., FZD7, interact with the test antibody, (See US Patent No, 7,935,340, incorporated by reference herein).
  • epitope mapping techniques are known in the art.
  • X- ray co-crystallography of the antigen and antibody; NM ; surface plasmon resonance (e.g., at 25° or 37°C); array-based oligo-peptide scanning; site-directed mutagenesis (e.g., alanine scanning); mutagenesis mapping; hydrogen-deuterium exchange: phage display; and limited proteolysis are all epitope mapping techniques that are well known in the art. See, e.g., Epitope Mapping Protocols: Second Edition, Methods in Molecular Biology (Springer Protocols), ed. Mike Schutkowski and Ulrieh Reineke and Epitope Mapping Protocols, ed. Glenn Morris (Humana Press), both of which are herein incorporated by referenced in their entirety,
  • test antibodies to be examined are obtained from different source animals, or are even of a different Ig isotype
  • a simple competition assay may be employed in which the control antibody (one of FZD7-Fab ⁇ 1791. FZD7 ⁇ Fab ⁇ i291 , chFZD7- 1791 or chFZD7-1291 , for example) is mixed with the test antibody and then applied to a sample containing FZD7, each of which is known to be bound by FZD7 ⁇ Fab-1791 , FZD7- Fab- 1291 , chFZD7- 179-1 or ehFZD7-129L Protocols based upon ELISAs, radioimmunoassays, Western blotting, and BIACOR.E analysis (as described in the Examples section herein) are suitable for use in such simple competition studies,
  • the method comprises pre-mixing the control antibody with varying amounts of the test antibody (e.g., in ratios of about 1 : 1, 1 :2, 1 : 10 or about 1 : 100) for a period of time prior to applying to the FZD7 antigen sample.
  • the control and varying amounts of test antibody can be added separately and admixed during exposure to the FZD7 antigen sample.
  • bound antibodies can be distinguished from free antibodies (e.g., by using separation or washing techniques to eliminate unbound antibodies) and control antibody from the test antibody (e.g., by using species specific or isotype specific secondary antibodies or by specifically labelling the control antibody with a detectable label), the method can be used to determine that the test antibody reduces the binding of the control antibody to the FZD7 antigen, indicating that the test antibody recognizes substantially the same epitope as the control antibody (e.g., FZD7- Fab-1791 , FZD7-Fab-1291 , chFZD7-1791 or chFZD7-1291).
  • the binding of the (labeled) control antibody in the presence of a completely irrelevant antibody (that does not bind FZD7) can serve as the control high value.
  • the control low value can be obtained by- incubating the labeled control antibody with the same but unlabeled control antibody, where competition would occur and reduce binding of the labeled antibody.
  • a significant reduction in labeled antibody reactivity in the presence of a test antibody is indicative of a test antibody that recognizes substantially the same epitope, i.e., one that competes with the labeled control antibody.
  • test antibody will reduce the binding of FZD7 ⁇ Fab-1791 , FZD7-Fab-1271, chFZD7- 1791 or chFZD7 ⁇ 1291 to FZD7 antigen at least, about 50%, at least about 60%, at least about 80% or at least about 90% (e.g., about 95%) of the binding of FZD7-Fab-1791 , FZD7-Fab- 1271 , chFZD7 ⁇ 1791 or chFZD7-1291 observed in the absence of the test antibody.
  • These methods can be adapted to identify and/or evaluate antibodies that compete with other control antibodies,
  • a simple competition assay in which a test antibody is applied at saturating concentration to a surface onto which FZD7 is immobilized also may be advantageously employed.
  • the surface in the simple competition assay is preferably of a media suitable for Octet a sd/or ProteOn.
  • the binding of a control antibody e.g., FZD7-Fab-1791 , FZD7-Fab- 127.1 , chFZD7-17 1 or chFZD7-1291 ) to the FZD7-coated surface is measured.
  • This binding to the FZD7 ⁇ containing surface of the control antibody alone is compared with the binding of the control antibody in the presence of a test antibody, A significant reduction in binding to the FZD7-containing surface by the control antibody in the presence of a test antibody indicates that the test antibody recognizes substantially the same epitope as the control antibody such that the test antibody "competes" with the control antibody.
  • Any test antibody tha reduces the binding of control antibody (such as FZD7-Fab-179i , FZD7-Fab-127L chFZD7-1791 or chFZD7-1291) to FZD7 by at least about 20% or more, at least about 40%, at least about 50%.
  • control antibody e.g., FZD7- Fab-1 91, FZD7-Fab-12 1, chFZD7-1791 or chFZD7-1291
  • test antibody will reduce the binding of the control antibody (e.g., FZD7-Fab-1791, FZD7 ⁇ .Fab-i 271 5 chFZD7-1791 or chFZD7-1291) to the FZD7 antigen by at least about 50% (e.g., at least about 60%, at least about 70%, or more), it will be appreciated that the order of control and test antibodies can be reversed; i.e.
  • control antibody can be first bound to the surface and then the test antibody is brought into contact with the surface thereafter in a competition assay.
  • the antibody having higher affinity for FZD? is bound to the FZD7- containing surface first, as it will be expected that the decrease in binding seen for the second antibody (assuming the antibodies are competing) will be of greater magnitude.
  • assays are provided in, e.g., Saunal and Regenmorte!, J. Immunol. Methods 1 83 : 33-41 (1989), the disclosure of which is incorporated herein by reference.
  • an epitope region for an anti ⁇ FZD7 antibody may be determined by epitope "footprinting" using chemical modification of the exposed amines/carboxyls in the FZD7 protein.
  • HXMS hydrogen-deuterium exchange detected by mass spectrometry
  • a hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange occurs, wherein the backbone amide groups participating in protein binding are protected from back exchange and therefore will remain deuterated.
  • Relevant regions can be identified at this point by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and/or electrospray ionization mass spectrometry. See, e.g., Ehring H, Analytical Biochemistry, 207(2)i252 ⁇ 259 (1999) and Engen, J. R. and Smith, D. ' !,., Anal. Chem.
  • NMR nuclear magnetic resonance epitope mapping
  • the antigen typically is selectively isotopically labeled with 15N so that only signals corresponding to the antigen and no signals from the antigen binding peptide are seen in the NMR-spectrum.
  • Antigen signals originating from amino acids involved in the interaction with the antigen binding peptide typically will shift position in the spectres of the complex compared to the spectres of the free antigen, and the amino acids involved in the binding can be identified that way. See, e.g., Ernst Sobering Res Found Workshop. (44): 149-67 (1989); Huang et ah Journal of Molecular Biology 281 (l):61-67 ( 1998); and Saito and Patterson, Methods 9(3):516-24 (1996).
  • Epitope mapping/characterization also can be performed using mass spectrometry methods. See, e.g., Downward, J Mass Speclrom, 35(4):493-503 (2000) and iselar and Downard, Anal Chem. 71 (9): 1792-801 ( 1999).
  • Protease digestion techniques also can be useful in the context of epitope mapping and identification. Antigenic determinant-relevant regions/sequences can be determined by protease digestion, e.g. by using trypsin in a ratio of about 1 :50 to FZD7 overnight (o/n) digestion at 37°C and pH 7-8, followed by mass spectrometry ("MS”) analysis for peptide identification.
  • MS mass spectrometry
  • the peptides protected from trypsin cleavage by the anti ⁇ FZD7 antibody can subsequently be identified by comparison of samples subjected to trypsin digestion and samples incubated with antibody and then subjected to digestion by e.g. trypsin (thereby revealing a footprint for the antibody).
  • enzymatic digestion can provide a quick method for analyzing whether a potential antigenic determinant sequence is within a region of FZD7 in the context of a FZD7 -binding polypeptide, if the polypeptide is not surface exposed, it is most likely not relevant in terms of imrnunoge icity/antigemcity. See, e.g., iVianca. A 1st Super San a. 27(1); 15-9 (1991) for a discussion of similar techniques.
  • Site-directed mutagenesis is another technique useful for elucidation of a binding epitope. For example, in “alanine-scanning", each residue within a protein segment is replaced with an alanine residue (or another residue such as valine where alanine is present in the wild-type sequence), and the consequences ' for binding affinity measured. If the mutation leads to a significant reduction in binding affinity, It is most likely involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies which do not bind the unfolded protein) can be used to verify that the alanine-replacement does not influence overall fold of the protein.
  • mutational analysis can be performed by substituting residues in the murine FZD7 receptor with corresponding residues from the human FZD7 receptor.
  • Electron microscopy can also be used for epitope "footprinfmg", For example, Wang et al., Nature 355:275-278 (1992) used coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of a Fab-fragment on the capsid surface of native cowpea mosaic virus,
  • label-free assay for epitope evaluation include surface piasmon resonance (“SPR”, BIACORE) and reflectometric interference spectroscopy (“RifS”), See, e.g., Fagerstarn et aL Journal Of Molecular Recognition 3:208-14 (1990); Nice et al, J. Chromatogr. 646: 159-168 (1993); Leipert et al,, Angew. Ghent. Int. Ed. 37:3308-331 1 (1998); Kroger et al, Biosensors and Bioeleetronics 17:937-944 (2002).
  • SPR surface piasmon resonance
  • RifS reflectometric interference spectroscopy
  • framework region refers to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody (See Kabat, E. A. et al.. Sequences of Proteins of Immunological interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.
  • FZD7 is a member of the 'frizzled' gene family of 7-transmembrane domain proteins that are receptors for WNT signaling proteins. It is one of ten known human FZD receptors. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated ceils.
  • FZD7 plays an important role in stem ceil growth and, moreover, is aberrantly expressed in various human cancers, in particular solid tumors such as, but not limited to. ovarian cancer, breast cancer, renal cancer and glioblastoma,
  • the present invention provides exemplary antibodies or antibody fragments that bind to FZD7, in particular human FZD7, and do not substantially interact with (bind to) other FZD family members.
  • Other antibodies or antibody fragments that bind to FZD7, including those having different CDRs and epitopic specificity may be obtained using the disclosure of the present specification and that which is generally known in the art.
  • Such antibodies and antibody fragments specifically block or inhibit WNT-FZD7 signaling and, therefore, are useful in treating or preventing diseases or conditions associated with FZD7 overexpression and/or FZD7 signaling, including, for example, cancer.
  • the disclosed FZD7-specific antibodies and antibody fragments are useful for tissue engineering.
  • the antibody or antibody fragment according to the invention comprises one or more complementarity determining regions ("CDRs"), a variable light (“VL”) chain and/or variable heavy (“VH”) chain of the anti-FZD7 antibodies and antibody fragments described herein.
  • CDRs complementarity determining regions
  • VL variable light chain
  • VH variable heavy chain
  • an anti-FZD7 antibody or antibody fragment according to the invention will interfere with, block, reduce or modulate the interaction between FZD7 and Wnt3a
  • the anti ⁇ FZD7 antibody or antibody fragment according to the invention are chimeric, humanized or human antibodies or antigen-binding fragments thereof.
  • the antibody is a monoclonal antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment, thereof, single-chain antibody or antibody that competitively inhibits the binding of an antibody disclosed herein to its respective antigenic epitope.
  • the anti ⁇ FZD7 antibody or antibody fragment according to the invention is comprised in an antibody-drug conjugate ("ADC").
  • ADC antibody-drug conjugate
  • the antibodies or antibody fragments e.g., scFv
  • an anti-cancer drug e.g., a cell toxin or a cytotoxin, that specifically targets a certain tumor marker.
  • Antibodies of the present invention may optionally be conjugated to a growth inhibitor ⁇ ' agent or cytotoxic agent including, for example, a maytansinoid (such as DM1), ealieheamie-in, dolastatin 10, monomethyl auristatin E, an antibiotic, a radioactive isotope, a nucleolytic enzyme or the like.
  • a growth inhibitor ⁇ ' agent or cytotoxic agent including, for example, a maytansinoid (such as DM1), ealieheamie-in, dolastatin 10, monomethyl auristatin E, an antibiotic, a radioactive isotope, a nucleolytic enzyme or the like.
  • the chemical linker used in an ADC should function as a highly stable bridge (as compared to a conditionally stable linker) between the antibody and release drug (i.e., "payload"), e.g., cytotoxin, that allows efficient drug release upon delivery inside malignant ceils.
  • exemplary linkers include cleavable peptides in combination with self-immolative groups thai undergo intracellular enzymatic degradation, e.g, hydrazone, peptide, disulfide, thioester, and hydrophilic.
  • the anti ⁇ FZD7 antibody or antibody .fragment according to the invention is comprised in a bispecific antibody.
  • the anti ⁇ FZD7 antibody or antibody fragment e.g., scFv
  • a chimeric antigen receptor CAR
  • a T cell and/or an NK cell e.g., a T cell and/or an NK cell removed from a patient and engineered to express the anii-FZD7 antibody or antibody fragment.
  • the engineered CAR T cells and/or NK cells may have a modular design that includes the FZD7-targeting antibody or antibody fragments herein, and a costimulatory signaling domain, e.g., to amplify the activation of the cells, giving them a stronger signal to multiply and kill cancer cells, and/or other mechanisms to amplify or dampen (in the case of adverse reactions) T cell and/or NK cell activation, e.g.. "armored CAR" technology, dual costimulatory domain (e.g., "GoCAR-Ts").
  • Such engineered CAR T cells can be reintroduced into the patient from whom it was isolated (or other patients) to recognize and kill cancer cells.
  • the anti-FZD7 antibodies or antibody fragments according to the invention have a variety of uses.
  • the subject antibodies and fragments can be useful in therapeutic applications, e.g., cancer treatment, as well as diagnostic applications, e.g., screening patient samples for FZ.D7 expression levels.
  • the subject anti-FZD7 antibodies or antibody fragments may be used alone or in association with other active agents or drugs, i.e., as a monotherapy or in combination therapy, respectively.
  • the peptide epitope for the FZD7-speeifie antibodies can be used to make cancer vaccines using methods well known in the art.
  • the subject anti-FZD7 antibodies or antibody fragments can also be used to generate specific ceil populations from human piuripotent stem cells, including both human embryonic stem cells (“hESCs”) and induced piuripotent stem cells (“hiPSCs").
  • hESCs human embryonic stem cells
  • hiPSCs induced piuripotent stem cells
  • Exemplary anii-FZD? antibodies and antibody fragments according to the invention, and the specific CDRs thereof are identified in this section.
  • each exemplified antibody or fragment, and corresponding sequences are separately identified by a specific nomenclature, i.e., FZD7-Fab-1791, FZD7-Fab-1291, chFZD7-17 1 or chFZD7- 1291.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a light chain sequence comprising the sequence set forth below:
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a heavy chain sequence comprising the sequence set forth below:
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess the same epitopic specificity as FZD7 « Fab-I791 , i.e., which contains a light chain sequence of SEQ ID NO:4 and a heavy chain sequence of SEQ ID NO;5.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that contain one, two, or three of the polypeptide sequences of SEQ ID NO:6 (ASENVLNYVS); SEQ ID NO:7 (GASN YT); and SEQ ID NO: 8 (GQSYRYP), which correspond to CDR1 , CDR2 and CDR3, respectively, of the light chain sequence of SEQ ID NO: 4, and/or which further contain one, two, or three of the polypeptide sequences of SEQ ID NO:9 (TYAMH); SEQ ID NO: 10 (RIRSKSNNYAKNYDDSVKD); and SEQ ID NO: 1 1 (ENYGG FDY), whieh correspond to CDRL CDR2 and CDR3. respectively, of the heavy chain sequence of SEQ ID NO:5, or antibodies or fragments containing combinations of sequences which are at least 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% identical thereto,
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess the same epitopic specificity as an antibody comprising one, two, three, four, five or six CDRs of FZD7-Fab ⁇ 1 91 , i.e., SEQ ID NOs:6-l l .
  • the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the exemplified light chain and heavy chain sequences set forth above, or sequences that are at least 90% or 95% identical thereto. Methods for determining homology between amino acid sequences are well known to those of ordinary skill in the art.
  • '-Homology refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • Two nucleic acid, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95 -98%o sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified sequence.
  • identity refers to an exact nucleoti de-to-nucleoti de or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Percent identity cars be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100.
  • Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O, in Atlas of Protein Sequence and Structure M, O, Dayhoff ed., 5 Suppl.
  • nucleotide sequence identity is available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can he determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif ). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six), From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • the invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to FZD7, including the light and/or heavy chains of FZD7-Fab- 1791 as well as fragments, variants, combinations of one or more of the FRs, CD s, the variable heavy chain and variable light chain sequences, and the heavy chain and light chain sequences set forth above, including all of them or sequences which are at least 90% or 95% identical thereto.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a light chain sequence comprising the sequence set forth befow:
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a heavy chain sequence comprising the sequence set forth below:
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess the same epitopic specificity as FZD7-Fab-1291 , i.e., which contains a light chain sequence of SEQ D NO: 12 and a heavy chain sequence of SEQ ID NO: 13.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that contain one, two, or three of the polypeptide sequences of SEQ ID NO: 14 (KSSQSLLYSSNQKNYLAW); SEQ ID NO: 15 (WASTRES); and SEQ ID NO: 16 (QQYYSYP), which correspond to CDR1 , CDR2 and CDRS, respectively, of the light chain sequence of SEQ ID NO: 12, and/or which further contain one, two, or three of the polypeptide sequences of SEQ ID NO: 17 (SYAMS); SEQ ID NO: 18 (TISDGGSYTRYPDKL G); and SEQ ID NO: 19 ( V GGRRD YFD Y), which correspond to CDRL CDR2 and CDR3, respectively, of the heavy chain sequence of SEQ D NO: 13, or antibodies or fragments containing combinations of sequences which are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess the same epitopic specificity as an antibody comprising one, two, three, four, five or six CDRs of FZD7-Fab- 1291 , i.e., SEQ ID NQs: I4-19.
  • the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the exemplified heavy chain and light chain sequences set forth above, or sequences that are at least 90% or 95 > identical thereto.
  • Methods for determining homology between amino acid sequences are well known to those of ordinary skill in the art,
  • the invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to FZD7, including the heavy and/or light chains of FZD7-Fab ⁇ 12 1 as well as fragments, variants, combinations of one or more of the FRs, CDRs, the variable heavy chain and variable light chain sequences, and the heavy chain and light chain sequences set forth above, including all of them or sequences which are at least 90% or 95% identical thereto,
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a light chain sequence comprising the sequence set forth below:
  • VQWKVDNALQSGNSQESVTEQDS DSTYSLSSTLTLSKADYE-KHKVYACEVTHQGL SSPVTKSFNRGEiC SEQ ID NO: 20
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a heavy chain sequence comprising the sequence set forth below: [0 ⁇ 93] MD RVPAQLLGLU WLPGAKCEVQPVESGGGLVQP GSL XSCAASGFTF
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess the same epitopic specificity as chF2D7- 1791 , i.e., which contains a iight chain sequence of SEQ ID NO:20 and a heavy chain sequence of SEQ ID MO:21.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that contain one, two, or three of the CDRs of the light chain sequence of SEQ ID NO: 20 (i.e., CDR1, CDR2 and CDR3 correspond to SEQ ID NOs:6-8), and/or which further contain one, two, or three of the CDRs of the heavy chain sequence of SEQ ID NO: 21 (i.e., CDR1 , CDR2 and CDR3 correspond to SEQ ID NOs:9- 1 1). or antibodies or fragments containing combinations of sequences which are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the exemplified heavy chain and light chain sequences set forth above, or sequences that are at least 90% or 95% identical thereto.
  • the invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to FZD7, including the heavy and/or light chains of chFZD7-1791 as well as fragments, variants, combinations of one or more of the FRs, CDRs, the variable heavy chain and variable light chain sequences, and the heavy chain and light chain sequences set forth above, including ail of them or sequences which are at least 90% or 95% identical thereto.
  • Methods for determining homology between amino acid sequences are well known to those of ordinary skill in the art.
  • antibody fragments comprise, or alternatively consist of. Fab (fragment antigen binding) fragments having binding specificity for FZD7.
  • the Fab fragment preferably includes the variable region within the light chain sequence of SEQ ID NO: 18 and the variable region within the heavy chain sequence of SEQ ID NO: 19 or sequences that are at least 90%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • This embodiment of the invention further includes Fabs containing additions, deletions, and variants of SEQ D NO; 20 and/or SEQ ID NO: 21 that retain the binding specificity for FZD7.
  • Fab fragments may be produced by enzymatic digestion (e.g.. papain) of chFZD7-1791.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a light chain sequence comprising the sequence set forth below:
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess a heavy chain sequence comprising the sequence set forth below:
  • VTC VDVSHEDPEVKFNWYVDGVEVH AKT PI EEQYNSTYRV ' V ⁇ VLWLHQDW
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that possess the same epitopic specificity as chFZD7-1293 , i.e., which contains a light chain sequence of SEQ D NO:22 and a heavy chain sequence of SEQ ID NO:23.
  • the invention includes antibodies and antibody fragments having binding specificity to FZD7 that contain one, two, or three of the CDRs of the light chain sequence of SEQ ID NO: 22 (i.e., CDR1 , CDR2 and CDR3 correspond to SEQ ID NOs: 14-16), and/or which further contain one, two, or three of the CDRs of the heavy chain sequence of SEQ ID NO: 23 (i.e., CDR1 , CDR2 and CDR.3 correspond to SEQ ID NOs: 17- 19), or antibodies or fragments containing combinations of sequences which are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto,
  • the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the exemplified heavy chain and light chain sequences set forth above, or sequences that are at least 90% or 95% identical thereto.
  • the invention is further directed to polynucleotides encoding antibody polypeptides having binding specificity to FZD7, including the heavy and/or light chains of chFZD7-1291 as well as fragments, variants, combinations of one or more of the FRs, CDRs, the variable heavy chain and variable light chain sequences, and the heavy chain and light chain sequences set forth, above, including all of them or sequences which are at least 90% or 95% identical thereto.
  • Methods for determining homology between amino acid sequences are well known to those of ordinary skill in the art.
  • antibody fragments comprise, or alternatively consist of. Fab (fragment antigen binding) fragments having binding specificity for FZD7.
  • the Fab fragment preferably includes the variable region within the light chain sequence of SEQ ID NO: 22 and the variable region within the heavy chain sequence of SEQ ID NO: 23 or sequences that are at least 90%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • This embodiment of the invention further includes Fabs containing additions, deletions, and variants of SEQ ID NO: 22. and/or SEQ ID NO: 23 that retain the binding specificity for FZD7.
  • Fab fragments may be produced by enzymatic digestion (e.g., papain) of chFZD7-!2 1 .
  • the invention contemplates an isolated anti-FZD7 antibody comprising a light chain polypeptide sequence selected from: SEQ ID NO:4, SEQ ID NO: 12, SEQ ID NO:20, SEQ ID NO:22, or a variant thereof; and further comprising a heavy chain polypeptide sequence selected from: SEQ ID NO:5, SEQ ID NO: 13, SEQ ID NO:21, SEQ ID NO:23, or a variant thereof, wherein one or more of the framework residues (FR residues) and/or CDR residues in said light or heavy chain polypeptides has been substituted with another amino acid residue resulting in an anti ⁇ FZD7 antibody that retains the ability to specifically hind FZD7 without substantially interacting with (binding to) other FZD family members.
  • FR residues framework residues
  • CDR residues in said light or heavy chain polypeptides has been substituted with another amino acid residue resulting in an anti ⁇ FZD7 antibody that retains the ability to specifically hind FZD7 without substantially interacting with (binding
  • the invention provides an isolated antibody or antibody fragment that competes for binding to FZD7 with an antibody or antibody fragment, disclosed herein.
  • the invention provides an antibody or antibody fragment that selectively binds to FZD7, wherein the antibody or antibody fragment binds to FZD7 with a 3 ⁇ 4> of less than or equal to 5 1 ( F s M, 10 "5 M, 5x1 (T 6 M, liT 6 M, 5xl0 '7 , 10 '7 M, 5xl0 "8 M, 10 "s M, 5x10 '3 M, Kf 9 M, 5xHF i0 , 10 "iC M, 5xlG “H M, 10 "n M, 5x10 "!2 M, 10 "t2 M, 5x10 “i3 hi, or l if u M; preferably, with a K D of less than or equal to 5x10 '50 M, 10 "s0 M, 5xl
  • the anti-FZD? antibody or antibody fragment has no cross-reactivity or minimal ⁇ cross-reaetivity with other FZD family members.
  • the inventive antibodies and fragments thereof may be modified post- translationaliy to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • Antibodies or fragments thereof may also be chemically modified to provide additional advantages such as increased solubility, stability and circulating time (in vivo half- life) of the polypeptide, or decreased imxnunogenicity (See U.S. Pat. No. 4,179,337).
  • the chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethy!eellulose, dextran, polyvinyl alcohol and the like.
  • the antibodies and fragments thereof may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the
  • polyethylene glycol to a therapeutic protein or analog may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 1 1 ,000, 1 1,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 1 7,000, 17,500, 18,000, 18,500, 1.9,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
  • Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et aL, Appl Biochem. Biotechnol. 56:59-72 (1.996); Vorobjev et ai. Nucleosides Nucleotides 18:2745-2750 (1999); and Ca!iceti et aL, Bioconj g Chem. 1.0:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
  • amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
  • Suifhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group,
  • polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
  • polyethylene glycol can be linked to polypeptides via covending bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof).
  • albumin may be used.
  • further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta- galactosidase and luciferase.
  • Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbel iiferone, diehlorotriazinylamine, phycoerythrin, and dansy! chloride.
  • Further exemplary chemiiuminescent moieties include, but are not limited to, luminoL
  • Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin.
  • Further exemplary radioactive materials include, but are not limited to, Iodine 125 ( " " !). Carbon 14 ( 1 C), Sulfur 35 ( 55 S) ; Tritium ( 3 H) and Phosphorus 32 ( 32 P).
  • Embodiments described herein further include variants and equivalents that are substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, cameibodies, nanobodies, Ig AR, polypeptides, variable regions and CDRs set forth herein, These may contain, e.g., conservative substitution mutations, (i.e., the substitution of one or more amino acids by similar amino acids).
  • conservative substitution refers to the substitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.
  • the invention further contemplates the generation and use of antibodies that bind any of the foregoing sequences, including, but not limited to, and- idiotypic antibodies.
  • an anti-idiotypic antibody could be administered to a subject who has received an anti-FZD7 antibody to modulate, reduce, or neutralize, the effect of the anti-FZD? antibody.
  • Such antibodies could also be useful for treatment of an autoimmune disease characterized by the presence of anti-FZD7 antibodies.
  • a further exemplary use of such antibodies, e.g., anti-idiotypic antibodies, is for detection of the anti-FZD7 antibodies of the present invention, for example to monitor the levels of the anti ⁇ FZD7 antibodies present In a subject's blood or other bodily fluids.
  • the invention provides a method of using the anti-idiotypic antibody to monitor the in vivo levels of said anti-FZD7 antibody or antibody fragment in a subject or to neutralize said anti-FZD 7 antibody in a subject being administered said anti-FZD? antibody or antibody fragment.
  • the invention also includes human, humanized and chimeric forms of these antibodies.
  • Methods of producing such antibodies are well known to those of ordinary skill in the art.
  • the antibodies may optionally he produced in mammalian cells (such as CHO cells), bacterial cells, yeast cells, or other ceils or using ceil-free methods as known in the art,
  • Antibody polypeptides of the invention having FZD7 binding specificity may also be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector containing a promoter (optionally as a component of a eukaryotie or prokaryotic operon) and a DNA sequence encoding an antibody heavy chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.
  • a promoter optionally as a component of a eukaryotie or prokaryotic operon
  • DNA sequence encoding an antibody heavy chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.
  • a second expression vector is produced using the same conventional means well known to those of ordinary skill in the art, said expression vector containing a promoter (optionally as a component of a eukaryotie or prokaryotic operon) and a DNA sequence encoding an antibody light chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.
  • a promoter optionally as a component of a eukaryotie or prokaryotic operon
  • a DNA sequence encoding an antibody light chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.
  • the expression vectors are transfected into a host ceil by convention techniques well known to those of ordinary skill in the art to produce a transfected host cell, said transfected host cell cultured by conventional techniques well known to those of ordinary skill in the art to produce said antibody polypeptides.
  • the host cell may be co-transfected with the two expression vectors described above, the first expression vector containing DNA encoding a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a light chain-derived polypeptide and the second vector containing DNA encoding a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a heavy chain-derived polypeptide.
  • the two vectors contain different selectable markers, but preferably achieve substantially equal expression of the heavy arid light chain polypeptides.
  • a single vector may be used, the vector including DNA encoding both the heavy and light chain polypeptides.
  • the coding sequences for the heavy and light chains may comprise e-DNA, genomic DNA, or both.
  • the host cells used to express the antibody polypeptides may be a bacterial cell, such as E, colt, or a eukaryotic cell such as a mammalian cell of a well-defined type for this purpose, such as, e.g., a myeloma cell, a Chinese hamster ovary (CHO) cell line, a NSO cell line, or a HE 293 ceil line may be used.
  • a bacterial cell such as E, colt
  • a eukaryotic cell such as a mammalian cell of a well-defined type for this purpose, such as, e.g., a myeloma cell, a Chinese hamster ovary (CHO) cell line, a NSO cell line, or a HE 293 ceil line may be used.
  • the antibodies may optionally be produced in mammalian cells (such as CHO cells), bacterial cells, yeast cells, or other cells or using cell-free methods as known in the art.
  • the general methods by which the vectors may be constructed include conventional techniques.
  • the cell line used to produce the antibody is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. co/ -derived bacterial strain, may alternatively be used.
  • the antibody polypeptides may be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography and the like.
  • the antibody polypeptides described herein may also be used for the design and synthesis of either peptide or non-peptide mimetics that would be useful for the same therapeutic applications as the antibody polypeptides of the invention. See, for example, Saragobi et a!.. Science 253:792-795 (1991), the contents of which is herein incorporated by reference in its entirety.
  • the chimeric and humanized antibodies may include an Fc derived from IgGl , IgG2, IgG3, or IgG4 constant regions.
  • the invention is further directed to polynucleotides encoding antibodies and antibody fragments having binding specificity to FZD7.
  • the invention provides a vector comprising a nucleic acid molecule encoding an anti-FZD7 antibody or fragment thereof as disclosed herein.
  • the invention provides a host eel! comprising a nucleic acid molecule encoding an anti-FZD7 antibody or fragment thereof as disclosed herein.
  • This disclosure also provides methods of detecting a cancer cell that expresses FZD7.
  • Detecting the expression of FZD7 may he used for diagnosis and staging of cancers (e.g., in immunohistoehemistry).
  • the level or extent of expression of FZD7 may indicate the stage of cancer, may be con-elated with patient outcome, or may be predictive of the outcome of different treatment options.
  • the antibody-epitope complex may be detected by Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement-fixation assay, imnnmohistochemical assay, fluorescent immunoassay, and protein A immunoassay.
  • the sample may be sample is a tissue biopsy, lymph, urine, cerebrospinal fluid, amniotic fluid, inflammatory exudate, blood, serum, stool, or liquid collected from the colorectal tract.
  • tissue or fluid collection methods can be utilized to col lect the biological sample from the subject in order to determine the level of DMA, RNA and/or polypeptide of the marker of interest in the subject. Examples of tissue or fluid collection methods include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the marker may be determined and a diagnosis can thus be made.
  • the antibodies and antibody fragments that selectively bind an FZD7 antigen may be used in diagnostic methods for detecting the presence or absence of an FZD7 antigen, wherein the presence of the antigen is indicative of cancer including but not. limited to skin, liver, blood, brain, breast and ovary.
  • the diagnostic methods may be used with patients at risk of cancer or patients without symptoms,
  • Each marker of the present invention may be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of a cancer (e.g.. ovarian, breast, renal or glioblastoma).
  • a cancer e.g.. ovarian, breast, renal or glioblastoma.
  • the cancers that may be detected using the methods described herein include but are not limited to non-solid and solid tumors, cancer of the ovary, breast, kidney or brain, and wherein the cancer may be invasive or metastatic,
  • the FZD7 antigen may be used as a cancer biomarker, i.e.. if the marker concentration is above or below the threshold level (depending upon the marker and/or the diagnostic test being performed), the marker concentration correlates with the disease or condition.
  • the level of FZD7 in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of FZD7 in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
  • Determining the level of the same marker in normal tissues of the same origin may be effected along-side to detect an elevated expression and/or amplification and/or a decreased expression, of the marker as opposed to the normal tissues.
  • Detection of the FZD7 antigen in a biological sample may be performed by means of the anti-FZD7 antigen antibody or antigen-binding fragment thereof.
  • a biological sample e.g., tissue or serum
  • FZD7 antigen is measured (e.g., by ELISA or PGR), and compared with corresponding samples from normal subjects.
  • Measuring methods include any method of nucleic acid detection, for example in situ hybridization using antisense DNA or cRNA oligonucleotide probes, ultra-high throughput sequencing, nano string technology, microarrays, rolling circle amplification, proximity-mediated ligation, PGR, qRT-FCR Chip, ChlP-qPCR, or FZD7 antige -bin di g antibodies or fragments thereof.
  • Comparatively high levels of FZD7 Indicate the presence and/or severity of cancer, e.g., skin, liver, blood, brain, breast and ovary, and may indicate metastasis or poor cancer prognosis.
  • cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy.
  • Detection of cancer ceils using an antibody to FZD7 can be used in conjunction with one or more therapies.
  • FZD7 expression is highly restricted, with limited expression in adult tissues. Therefore, therapy may be targeted to the cancer ceils thereby promoting effective treatment and/or reducing the effect on norma! non-cancerous tissue.
  • the therapeutic course e.g..
  • Expression of FZD7 may be evaluated using an in vivo diagnostic assay, e.g., by administering an antibody that specifically binds to FZD7 and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient tor localization of the label.
  • a detectable label e.g., a radioactive isotope or a fluorescent label
  • one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.
  • various in vivo and in vitro assays for detecting the presence of a cancer-associated antigen are available to the skilled practitioner,
  • the antibodies may also be used for purification or immunoprecipitation of FZD7 from cells or other samples, for detection and quantitation of cancer-associated antigen in vitro, e.g., in an EL1SA or a Western blot, to kill and eliminate cancer-associated antigen- expressing cells from a population of mixed cells, e.g., as a step in the purification of other cells.
  • the invention provides a diagnostic kit comprising an FZD7 specific antibody or antibody fragment, such as FZD7-Fab-17 1.
  • the antibody or antibody fragment may be directly or indirectly fixed to a solid phase support, such as a bead, plate, matrix, polymer, test lube, sheet, culture dish, or test strip, in another embodiment, the solid support may be an array.
  • the invention concerns an article of manufacture comprising a container and a composition of matter contained within the container, wherein the composition of matter comprises an. anti-FZD7 antibody or antibody fragment as described herein (such as FZD7-Fab-1791, FZD7-Fab-129i, chFZD7 ⁇ 1791 , or chFZD7- 1291).
  • the article may further optionally comprise a label affixed to the container, or a package insert included with the container, that refers to the use of the composition of matter for the diagnostic detection of a tumor, preferably a solid tumor, more preferably a tumor associated with ovarian cancer, breast cancer, renal cancer or glioblastoma.
  • This disclosure further provides therapeutic methods of use for the anti-FZD7 antibodies and antibody fragments.
  • anii-FZD7 antibodies and antibody fragments described herein are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with aberrant FZD7, e.g., overexpression, expression in tissues where the gene is not ""normally” expressed, etc in a subject or individual.
  • subject or “individual” is included any member of the subphylum chordata, including, without limitation., human and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and. cats; laboratory animals including rodents such, as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
  • the invention described herein is intended for use in any of the above vertebrate species.
  • the subject or individual is human, e.g., a subject, with cancer or pre-disposed to developing cancer, or a person with a condition caused, at least in part, by WNT signaling via FZD7.
  • Anti ⁇ FZD7 antibodies described herein, or fragments thereof, as well as combinations can be administered in a therapeutically effective amount to patients in need of treatment of diseases and disorders associated with aberrant FZD7 expression, e.g., cancer, in the form of a pharmaceutical composition as described in greater detail below.
  • patient refers to a living organism suffering from or prone to a condition that can he prevented or treated by the treatment regimen the invention, and includes both humans and animals.
  • anti-FZD7 antibodies described herein, or fragments thereof are useful (either alone or in combination with another agent) for ameliorating or reducing the symptoms of, or treating, or preventing cancer.
  • Cancer refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor.
  • the anti-FZD7 antibodies and antigen-binding fragments thereof can be used for ameliorating or reducing the symptoms of, or treating, or preventing solid tumors and non- solid tumors.
  • the anti ⁇ FZD7 antibodies and antigen-binding fragments thereof can. be used for ameliorating or reducing the symptoms of, or treating, or preventing ovarian cancer, breast cancer, renal cancer and/or glioblastoma.
  • the anti-FZD7 antibodies described herein, or antigen-binding fragments thereof, as well as combinations of said antibodies or antibody fragments are administered to a subject at a concentration of between 0.1 mg/ml and about any one of 0.5, 1 , 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml +/ ⁇ 10% error.
  • the anti-FZD7 antibodies and fragments thereof described herein are administered to a subject at a dose of between about 0,01 and 100,0 or 200,0 mg/kg of body weight of the recipient subject.
  • a dose of between about 0,01 and 100,0 or 200,0 mg/kg of body weight of the recipient subject is administered to a subject at a dose of between about 0,01 and 100,0 or 200,0 mg/kg of body weight of the recipient subject.
  • about 1 ⁇ ' ⁇ to 50 mg/kg (e.g., 0.1 -20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • about 1 to 15 mg/kg (e.g., 0, 1 mg/kg- 10 mg/kg) of antibody is an initial candidate dosage for administration to the patient.
  • a typical daily dosage might range from about 1 ,ug/kg to 100 mg/kg or more, depending on several factors, e.g., the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. However, other dosage regimens may be useful.
  • the subject anti-FZD7 antibodies and fragments thereof can be administered to a subject at an absolute dose (mg). Accordingly, in one embodiment of the invention, the anti-FZD7 antibodies and fragments thereof described herein are administered to a subject at a dose of between about 1 microgram and about 100 milligrams regardless of the route of administration.
  • the antibody containing medicament or pharmaceutical composition is peripherally administered to a subject via a route selected from one or more of; orally, sublingually, buccally, topically, rectally, via inhalation, irarssdermaily, subcutaneously, intravenously, intra-arterially or intramuscularly, via intracardiac administration, intraosseously, intradermally, intraperitoneaiiy, transmucosally, vaginally, imraviireally, epicuianeously, intra-articularly, peri-articularly or locally,
  • the concentration of the antibody or Fab administered to a given patient may be greater or lower than the exemplar/ administration concentrations set forth above.
  • the anti-FZD7 antibodies described herein, or FZD7 binding fragments thereof, as well as combinations of said antibodies or antibody fragments are administered to a subject in a pharmaceutical formulation.
  • the subject is a human.
  • a "pharmaceutical composition” or “medicament” refers to a chemical or biological composition suitable for administration to a subject, preferably a mammal, more preferably a human. Such compositions may be specifically formulated for administration via one or more of a number of routes, including but not limited to buccal, epi cutaneous, epidural, inhalation, intraarterial, intracardiac intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, rectally via an enema or suppository, subcutaneous, subdermai, sublingual, transdermal, and transmucosai. in addition, administration car's occur by means of injection, powder, liquid, gel, drops, or other means of administration.
  • the anti-FZD7 antibodies described herein, or FZD7 binding fragments thereof, as well as combinations of said antibodies or antibody fragments may be optionally administered in combination with one or more active anticancer agents.
  • exemplary anti-cancer/chemotherapeutic agents include, but are not limited to, the following: alkylating agents (including, without limitation, nitrogen mustards, ethylcnimme derivatives, aikyl sulfonates, nitrosoureas and triazenes); uracil mustard (Aminouracil Mustard®, Ch!orethammaei).®, Dernethyldopan®, Desrncthykiopan®, Haemanthamine®, Nordopan®, Uracil nitrogen Mustard®, Uraciilost®, Uraciimostaza®, Uraroustin®, Uramustine®); bendamustine (Treakisym®, Ri
  • the HSP90 inhibitor is selected from geldanamycin, 17-alky!amino- 17-desmethoxygeldanamycin ("17- A AG") or 17-(2- dimethylaminoetliyl)amino- 17-desmethoxygeldanamyein (“ 17-DMAG”); anti -androgens which include, without limitation nilutamide (Nilandron®) and bicalutamide (Caxodex®); antiestrogens which include, without limitation tamoxifen (Nolvadex®), toremifene (Fareston®), letrozoie (Femara®), testolactone (Teslac®), anastrozole (Arimidex®), bicalutamide (Casodex®), exemesiane (Aromasin®), ilutamide (Eulexin®), fulvestxant (Faslodex®), raloxifene
  • cysteine protease inhibitors which include without limitation 4-morpholinecarboxamide, N-[( ⁇ S)-3-f3uoro-2-oxo- 1 -(2- phenylethyl)propyljamino] ⁇ 2-oxo- 1 -(phenylmeth-yl)ethyl]-(9Ci); DNA intercalators which include without limitation plicamycin (Miihratin®) and daptomycin (Cubicm®); DNA strand breakers which include without limitation bleomycin (B!enoxane®); E3 ligase inhibitors which include without limitation N-((3 ,3 > 3 -trifiuoro-2- tri fl uoromethyi)propion !suifani ⁇ amide; EOF Pathway inhibitors which include, without limitation tyrphostin 46
  • AP23573 also known as ridaforolimus, deforoiimus, or MK- 8669
  • CCI-779 also known as temsirolimus
  • SDZ-RAD SDZ-RAD
  • NGFR tyrosine kinase inhibitors which include without limitation tyrphostin AG 879
  • p38 MAP kinase inhibitors which include without limitation Phenol, 4-[4-(4-fluorophenyl)-5 ⁇ (4-pyridinyl) ⁇ lH- irnidazoi-2 ⁇ yi] ⁇ (9Cl), and benzamide, 3 -(dimethylami no)- - [3 - [(4-hydroxylbenzoyI)amino] - 4 ⁇ methylphenyl]-(9Ci);
  • p56 tyrosine kinase inhibitors which include without limitation damnacanthal and tyrphostin 46: PDG F pathway inhibitors
  • SRC family tyrosine kinase inhibitors which include without limitation PPl and PP2; Syk tyrosine kinase inhibitors which include without limitation piceatarmol; Janus (JAK-2 and/or JA -3) tyrosine kinase inhibitors which include without limitation iyrphostin AG 490 and 2-naphth l vinyl ketone; retinoids which include without limitation isotretinoin (Accutane®, Amnesteeni®, Cistane®.
  • Ciaravis®, Sotret®) and tretinoin (Aberel®, Aknoten®, A vita®, Renova®, Retin- A®, Retin-A MICRO®, Vesanoid®); RNA polymerase II elongation inhibitors which include without limitation 5,6-dichloro-l-beta-D-rihGti!ranosylbenzimidazole; serine/Threonine kinase inhibitors which include without limitation 2-ammopurine; sterol biosynthesis inhibitors which include without limitation squalene epoxidase and CYP2D6; VEGF pathway inhibitors, which include without limitation anti-VEGF antibodies, e.g., bevacizumab, and small molecules, e.g., sunitinib (Sutent®), sorafinib (Nexavar®), ZD6474 (also known as vandetanib) (ZaetimaTM), SU
  • chemotherapeutic agents are also described in the scientific and patent, literature, see, e.g., Bulinski (1997) J. Cell Sci. 11:3055-3064; Panda (1997) Proc. Natl. Acad. Sci. USA 94: 10560-10564; Muhlradi (1997) Cancer Res. 57:3344-3346; Nicolaou (1 997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985; Panda (1996) J. Biol Chem. 271 :29807-29812.
  • exemplary anti-cancer agents include alitretinon, altretamine, aminopterin, aminolevulinic acid, amsacrine (Amsidine®), asparaginase (erisantaspase, Erwinase®), atrasentan, bexarotene (Targretin®), carboquone, demecolcine, efaproxiral, elsamitrucin, etoglucid, hydroxycarbamide, leucovorin, lonidamine, lucanthone, masoprocof, methyl aminolevulinate, mitoguazone, mi to lane (Lysodren®), oblimersen, omacetaxine (Genasense®), pegaspargase (Oncaspar®), portimer sodium (Photofrin®), prednimustine, sitimagene ceradenovec (Cerepro®), talaporfm, temoporfm
  • a "pharmaceutical excipient” or a “pharmaceutical ly acceptable excipient” is a carrier, usually a Liquid, in which an active therapeutic agent is formulated.
  • the active therapeutic agent is a humanized antibody described herein, or one or more fragments thereof.
  • the excipient generally does not provide any pharmacological activity to the formulation, though it may provide chemical and/or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19 Ed,, Grennaro, A., Ed., 1995, which is incorporated by reference.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible
  • the carrier is suitable for parenteral administration.
  • the carrier can be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and steriie powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the invention contemplates that the pharmaceutical composition is present in lyophilized form.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, poiyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • the invention further contemplates the inclusion of a stabilizer in the pharmaceutical composition.
  • the proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohois such as rnamiitol, sorbitol, or sodium chloride in the composition.
  • Absorption of the injectable compositions can be prolonged by including an agent which delays absorption, for example, monostearate salts and gelatin.
  • the alkaline polypeptide can be formulated in a time-release formulation, for example in a composition which includes a slow release polymer.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polyiactie, polygiyco!ic copolymers (PLG). Many methods for the preparation of such formulations are known to those skilled in the art.
  • the compounds can be administered by a variety of dosage forms. Any biologically acceptable dosage form known to persons of ordinary skill in the art, and combinations thereof, are contemplated. Examples of such dosage forms include, without limitation, reconstitutabie powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, injectables (including subcutaneous, intramuscular, intravenous, and intradermal), infusions, and combinations thereof,
  • Human FZD7 and mouse Fzd7 are 96.5% identical in their amino acid sequences (see Figure 3).
  • the Cysteine-rich domain (CRD, amino acids 46-181), the transmembrane (TM) domain region (amino acids 254-549), and the intracellular domain (amino acids 550- 574) are identical, with the exception of residue P (Pro) at position 521 between TM6 and TM7.
  • the only notable regions of divergence between human and mouse Fzd7 are in the amino terminus, which encompasses the signal sequence and is likely cleaved co ⁇ translationally, and the membrane proximal region between the CRD and the first TM domain (amino acids 172-205). This 34 amino acid region harbors 9 amino acid differences between human and mouse Fzd7.
  • FZD7-Fabl291 and FZD7-Fabl791 react with human FZD7, but not with mouse Fzd7 protein (see Figure 4A). Together with the sequence alignment, this data suggests that the binding domain for these two Fabs resides in the membrane proximal region between amino acids 172-205 (domain marked by a green rectangle in Figure 4B).
  • a series of point mutations to substitute amino acids in mouse Fzd7 demonstrates that a single amino acid substitution (Proline at 188 to Leucine, P188L) is sufficient to recreate the FZD7-Fab binding domain in mouse Fzd7 (see Figure 4C and D), Therefore, the epitope of the FZD7 antibody encompasses the region that contains residue LI 88.
  • FZD7 Fabs block Wnt3a signaling and r sul in FZD7 protein duw regulation in cancer eell fines.
  • FZD7 mRNA was detected by reverse transcription quantitative PGR (RT-qPCR) in various cell lines (see Figure 5). Activation of WNT signaling in these cells with either Wnt3a or the GS 3 inhibitor CHIR98014 (CHIR) had minor effects on FZD7 mRNA levels, [000189
  • FZD7 expression is variable within cancer cell lines.
  • the FZD7-Fabs and antibodies detect FZD7 protein by flow cytometry in human piuripotent stem cells (Fernandez et al. 2014) and in the ovarian carcinoma cell line OVCAR- 4 (see Figure 7 A).
  • QPCR of R A isolated from these two cell populations confirmed that FZD7 Hi ceils are enriched for FZD7 mRNA relative to FZD7 Lo cells (see Figure 7B).
  • the CRISPK/Cas9 system will be used to introduce a targeted mutation in the Fzd7 gene, which will result in the replacement of Proline at position 188 of SEQ ID NO: 2 to Leucine.
  • the nuclease Cas9 is guided via RNA to a specific location in the genome and introduces a double stranded break. Parameters for design of optimal guide R As are available on line (http://crispr.mit.edu/). Simultaneously, cells will be transduced with a single stranded repair oligo spanning the double-stranded break site.
  • This oligo (50-80 bases in length) is designed to include the desired change, in our case the introduction of a single base change to encode a Leucine rather than a Proline at position 188 of Fzd7. in addition, a silent change will be introduced to create a unique restriction site that will facilitate screening for the presence of the mutation,
  • This method is highly efficient and has been successfully used to generate "knock-in mice” by directly injecting the CRISPR/Cas9 system along with repair oligo into mouse blastocyst (see, e.g., Wang et al, 2013; Yang et. al.. 2013). Blastocysts will then be transferred into pseudo pregnant mice and live pups will be screened for the presence of the introduced mutation. Targeting efficiencies are often sufficiently high to target both alleles; however, in this case, a single targeted allele would be sufficient.
  • mice carrying the mutated Fzd7 gene will be dosed weekly with antibody at lOmg/kg. Unless overt toxic effects are observed in the mutant mice (e.g., reduced body size, hair loss, death, etc.), antibody injections will be performed for up to 90 days, at which point animals will be sacrificed and carefully assessed to identify potential adverse effects. Given the observed bone-related adverse effects with the current clinical trial with Vantietumab (OncoMed, see section "Introduction and Review of Relevant Literature”), close attention will be paid to bone mass and density.
  • mice will also be valuable for studying FZD7 expression domains during development and adult life, which may yield important insights into FZD7 function during development and adult hie. Few highly specific antibodies to FZD proteins have been developed, and all of these-antibodies are eross-reacdve. The subject and-FZD? antibodies and antibody fragments, which are only reactive with human FZD7, represent a unique tool to interrogate FZD 7 expression and function in the transgenic mouse,
  • Blastocyst stage mouse embryos were injected with mRNA encoding Cas9, a guide NA targeting Fzd7 near the codon encoding Pi 88, and a double-stranded DNA repair oligo (sequence provided below), and subsequently transferred into pseudopregnant recipients.
  • the genotypes of F0 pups at the site of the engineered mutation were determined by sequencing of PGR products obtained from genomic DNA isolated from ear punch biopsies. Animals homozygous for the desired mutation were viable and were crossed to maintain a colony of mice carrying the homozygous mutation that produces a Fzd7 protein with a substitution from proline to leucine at position 188,
  • VJ .5 embryos from breeding mice homozygous for the Fzd7 P188L mutation were dissociated and plated in standard cell culture conditions to establish a mouse embryonic fibroblast (MEF) culture, Cell lysates of passage 2 MEFs were imraunobiotted with the FZD7-specific antibody. Only cell lysates from mouse embryos carrying the engineered mutation at position 188 (Proline to Leucine) were reactive with the antibody ( Figure 15). HEK293 cells transfected with a human FZD7 expression vector served as positive control.
  • MEF mouse embryonic fibroblast
  • Figure 1 were expressed and punned using Glutathione Sepharose beads. Purified GST fission proteins were immunoblotted with the FZD7-specific antibody.
  • LI 88 represents the wildtype sequence recognized by the FZD7-specifie antibody
  • Human cell lines were seeded at a density of 600 cells per well of a 24-well plate.
  • FZD7 drives in vitro aggressiveness in Stem- A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway.
  • Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448-452, PM ID: 12717451

Abstract

La présente invention concerne des anticorps et des fragments correspondants présentant une spécificité de liaison pour FZD7, qui, de préférence, n'interagiront pas sensiblement avec (ne se lieront pas sensiblement à) d'autres membres de la famille des FZD, et des procédés de fabrication desdits anticorps anti-FZD7 et des fragments de liaison correspondants. Un autre mode de réalisation de la présente invention concerne les anticorps et leurs fragments de liaison, comprenant les séquences des polypeptides VH, VL et/ou CDR décrits dans la description. L'invention concerne également des conjugués d'anticorps anti-FZD7 et de fragments de liaison correspondants, par exemple, des conjugués anticorps-médicament, ainsi que des lymphocytes T à récepteurs antigéniques chimères et des cellules NK basées sur les anticorps anti-FZD7 et leurs fragments de liaison. L'invention concerne en outre l'utilisation des anticorps anti-FZD7 et des fragments de liaison correspondants pour le génie tissulaire. Un autre mode de réalisation de la présente invention concerne une souris transgénique exprimant l'épitope des anticorps anti-FZD7 et des fragments de liaison correspondants. Des modes de réalisation de l'invention concernent également l'utilisation des anticorps anti-FZD7 et des fragments de liaison correspondants (ainsi que de l'épitope FZD7 identifié) pour le diagnostic, l'évaluation, la prévention et/ou le traitement de maladies et de troubles associés à une expression aberrante de FZD7, tels que le cancer.
PCT/US2016/037934 2015-06-16 2016-06-16 Anticorps spécifiques anti-fzd7 et vaccins pour traiter le cancer et réguler la fonction de cellules souches WO2016205566A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562180370P 2015-06-16 2015-06-16
US62/180,370 2015-06-16

Publications (1)

Publication Number Publication Date
WO2016205566A1 true WO2016205566A1 (fr) 2016-12-22

Family

ID=57546777

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/037934 WO2016205566A1 (fr) 2015-06-16 2016-06-16 Anticorps spécifiques anti-fzd7 et vaccins pour traiter le cancer et réguler la fonction de cellules souches

Country Status (1)

Country Link
WO (1) WO2016205566A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
CN110167967A (zh) * 2017-01-18 2019-08-23 豪夫迈·罗氏有限公司 针对抗pd-l1抗体的独特型抗体及其用途
WO2019193375A1 (fr) * 2018-04-04 2019-10-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation d'inhibiteurs de fzd7 pour traiter la néovascularisation rétinienne
CN110505884A (zh) * 2017-04-05 2019-11-26 勃林格殷格翰国际有限公司 抗癌组合疗法
CN110835372A (zh) * 2019-11-06 2020-02-25 上海健康医学院 一种靶向Frizzled7单克隆抗体及其制备方法与应用
CN111065410A (zh) * 2017-09-06 2020-04-24 弗雷德哈钦森癌症研究中心 用于改善过继细胞疗法的方法
US10675358B2 (en) 2016-07-07 2020-06-09 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
CN112159474A (zh) * 2019-11-06 2021-01-01 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2
US11746150B2 (en) 2017-12-19 2023-09-05 Surrozen Operating, Inc. Anti-LRP5/6 antibodies and methods of use
US11773171B2 (en) 2017-12-19 2023-10-03 Surrozen Operating, Inc. WNT surrogate molecules and uses thereof

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030044409A1 (en) * 2001-05-01 2003-03-06 Carson Dennis A. Immunologic compositions and methods for studying and treating cancers expressing frizzled antigens
US20030165500A1 (en) * 2001-05-01 2003-09-04 Regents Of The University Of California Wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas
WO2006036173A2 (fr) * 2004-09-21 2006-04-06 Rhode Island Hospital, A Lifespan-Partner Proteines frizzled et detection et traitement du cancer
WO2007143098A2 (fr) * 2006-06-02 2007-12-13 Aveo Pharmaceuticals, Inc. Protéines de liaison au facteur de croissance des cellules hépatiques (hgf)
JP2011087466A (ja) * 2009-10-20 2011-05-06 Nippon Menaade Keshohin Kk 幹細胞の検出及び分離培養方法
US20110237514A1 (en) * 2008-09-30 2011-09-29 Kyowa Hakko Kirin Co., Ltd. PHARMACEUTICAL COMPOSITION FOR TREATING BONE DISEASES WHICH COMPRISES PROTEIN COMPRISING Frizzled1, Frizzled2 OR Frizzled7 EXTRACELLULAR CYSTEINE-RICH DOMAIN
US20120177664A1 (en) * 2009-08-06 2012-07-12 Immunas Pharma, Inc. Antibodies That Specifically Bind to A Beta Oligomers and Use Thereof
US20140134159A1 (en) * 2012-10-23 2014-05-15 Oncomed Pharmaceuticals, Inc. Methods of treating neuroendocrine tumors using frizzled-binding agents
WO2014130965A1 (fr) * 2013-02-22 2014-08-28 Amgen Inc. Protéines liant la gastrokine-1 (gkn1) et procédés de traitement
US20140377781A1 (en) * 2006-12-14 2014-12-25 Chugai Seiyaku Kabushiki Kaisha Anti-Claudin 3 Monoclonal Antibody and Treatment and Diagnosis of Cancer Using the Same
WO2015023851A1 (fr) * 2013-08-14 2015-02-19 The Governing Council Of The University Of Toronto Anticorps contre les protéines frizzled et leurs méthodes d'utilisation

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030044409A1 (en) * 2001-05-01 2003-03-06 Carson Dennis A. Immunologic compositions and methods for studying and treating cancers expressing frizzled antigens
US20030165500A1 (en) * 2001-05-01 2003-09-04 Regents Of The University Of California Wnt and frizzled receptors as targets for immunotherapy in head and neck squamous cell carcinomas
WO2006036173A2 (fr) * 2004-09-21 2006-04-06 Rhode Island Hospital, A Lifespan-Partner Proteines frizzled et detection et traitement du cancer
WO2007143098A2 (fr) * 2006-06-02 2007-12-13 Aveo Pharmaceuticals, Inc. Protéines de liaison au facteur de croissance des cellules hépatiques (hgf)
US20140377781A1 (en) * 2006-12-14 2014-12-25 Chugai Seiyaku Kabushiki Kaisha Anti-Claudin 3 Monoclonal Antibody and Treatment and Diagnosis of Cancer Using the Same
US20110237514A1 (en) * 2008-09-30 2011-09-29 Kyowa Hakko Kirin Co., Ltd. PHARMACEUTICAL COMPOSITION FOR TREATING BONE DISEASES WHICH COMPRISES PROTEIN COMPRISING Frizzled1, Frizzled2 OR Frizzled7 EXTRACELLULAR CYSTEINE-RICH DOMAIN
US20120177664A1 (en) * 2009-08-06 2012-07-12 Immunas Pharma, Inc. Antibodies That Specifically Bind to A Beta Oligomers and Use Thereof
JP2011087466A (ja) * 2009-10-20 2011-05-06 Nippon Menaade Keshohin Kk 幹細胞の検出及び分離培養方法
US20140134159A1 (en) * 2012-10-23 2014-05-15 Oncomed Pharmaceuticals, Inc. Methods of treating neuroendocrine tumors using frizzled-binding agents
WO2014130965A1 (fr) * 2013-02-22 2014-08-28 Amgen Inc. Protéines liant la gastrokine-1 (gkn1) et procédés de traitement
WO2015023851A1 (fr) * 2013-08-14 2015-02-19 The Governing Council Of The University Of Toronto Anticorps contre les protéines frizzled et leurs méthodes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KALINKE, U ET AL.: "Virus Neutralization by Germ-line vs. Hypermutated Antibodies.", PNAS., vol. 97, no. 18, 29 August 2000 (2000-08-29), pages 10126 - 10131, XP002563641 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10287353B2 (en) 2016-05-11 2019-05-14 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US10385131B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10385130B2 (en) 2016-05-11 2019-08-20 Huya Bioscience International, Llc Combination therapies of HDAC inhibitors and PD-1 inhibitors
US11535670B2 (en) 2016-05-11 2022-12-27 Huyabio International, Llc Combination therapies of HDAC inhibitors and PD-L1 inhibitors
US10675358B2 (en) 2016-07-07 2020-06-09 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
US11547761B1 (en) 2016-07-07 2023-01-10 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
US11110178B2 (en) 2016-07-07 2021-09-07 The Board Of Trustees Of The Leland Standford Junior University Antibody adjuvant conjugates
US11279762B2 (en) 2017-01-18 2022-03-22 Genentech, Inc. Idiotypic antibodies against anti-PD-L1 antibodies and uses thereof
CN110167967A (zh) * 2017-01-18 2019-08-23 豪夫迈·罗氏有限公司 针对抗pd-l1抗体的独特型抗体及其用途
CN110505884A (zh) * 2017-04-05 2019-11-26 勃林格殷格翰国际有限公司 抗癌组合疗法
CN110505884B (zh) * 2017-04-05 2022-08-12 勃林格殷格翰国际有限公司 抗癌组合疗法
CN111065410A (zh) * 2017-09-06 2020-04-24 弗雷德哈钦森癌症研究中心 用于改善过继细胞疗法的方法
US11773171B2 (en) 2017-12-19 2023-10-03 Surrozen Operating, Inc. WNT surrogate molecules and uses thereof
US11746150B2 (en) 2017-12-19 2023-09-05 Surrozen Operating, Inc. Anti-LRP5/6 antibodies and methods of use
WO2019193375A1 (fr) * 2018-04-04 2019-10-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation d'inhibiteurs de fzd7 pour traiter la néovascularisation rétinienne
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2
CN112175083A (zh) * 2019-11-06 2021-01-05 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
CN112159474B (zh) * 2019-11-06 2021-11-19 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
CN112175083B (zh) * 2019-11-06 2021-11-19 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
CN112210011A (zh) * 2019-11-06 2021-01-12 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
CN112175084A (zh) * 2019-11-06 2021-01-05 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
CN112159474A (zh) * 2019-11-06 2021-01-01 上海健康医学院 一种靶向Frizzled7人源化抗体及其制备方法与应用
CN110835372A (zh) * 2019-11-06 2020-02-25 上海健康医学院 一种靶向Frizzled7单克隆抗体及其制备方法与应用

Similar Documents

Publication Publication Date Title
US10766962B2 (en) FZD7 specific antibodies and vaccines to treat cancer and control stem cell function
WO2016205566A1 (fr) Anticorps spécifiques anti-fzd7 et vaccins pour traiter le cancer et réguler la fonction de cellules souches
JP6968698B2 (ja) 抗ｍｕｃ１６抗体及びその使用
CN108473575B (zh) 抗-bcma多肽和蛋白质
TWI564306B (zh) 雙特異性抗體
CA2798778C (fr) Anticorps anti-trop-2 humain ayant une activite anticancereuse in vivo
RU2623161C2 (ru) Полипептиды и полинуклеотиды и их применение для лечения иммунологических нарушений и рака
KR102577551B1 (ko) TGFβ 항체, 방법 및 용도
US20180002437A1 (en) Humanized anti-trop-2 monoclonal antibodies and uses thereof
JP2018104409A (ja) 新規モジュレーターと使用の方法
KR102166083B1 (ko) 브라디키닌 b1 수용체 리간드에 대한 항체
CN106573979A (zh) 抗axl抗体
CN105143263A (zh) Bcma抗原结合蛋白
CN107531786A (zh) 抗axl拮抗抗体
CN109311997A (zh) 抗axl拮抗抗体
EA011866B1 (ru) Агенты, специфически связывающие ангиопоэтин-2
WO2009116670A1 (fr) Anticorps anti-humain spécifiques de dlk1 présentant une activité anti-tumorale in vivo
JP2017526676A (ja) がんの処置のための併用療法
CN103534268A (zh) 抗体
KR102652664B1 (ko) 조절 t 세포 표면 항원의 에피토프 및 이에 특이적으로 결합하는 항체
TR201906781T4 (tr) Anti-transglutaminaz 2 antikorları.
JP2023500248A (ja) 制御性ｔ細胞表面抗原のエピトープおよびこれに特異的に結合する抗体
WO2024012539A1 (fr) Anticorps anti-nectine-4 et son utilisation
CN114641310A (zh) 治疗癌症和其他疾病的靶向α3β1整合素
TW202110891A (zh) 抗psgl-1組合物及其用於調節骨髓細胞發炎表型之方法及用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16812472

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16812472

Country of ref document: EP

Kind code of ref document: A1