WO2012044612A1 - Génération, caractérisation et utilisations d'anticorps anti-her3 - Google Patents

Génération, caractérisation et utilisations d'anticorps anti-her3 Download PDF

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WO2012044612A1
WO2012044612A1 PCT/US2011/053436 US2011053436W WO2012044612A1 WO 2012044612 A1 WO2012044612 A1 WO 2012044612A1 US 2011053436 W US2011053436 W US 2011053436W WO 2012044612 A1 WO2012044612 A1 WO 2012044612A1
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antibody
her3
seq
fragment
expression
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PCT/US2011/053436
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English (en)
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Ningyan Zhang
Leonard G. Presta
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Merck Sharp & Dohme Corp.
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Priority to JP2013531716A priority Critical patent/JP2014504850A/ja
Priority to CA2811747A priority patent/CA2811747A1/fr
Priority to EP11829799.3A priority patent/EP2621532A1/fr
Priority to AU2011307251A priority patent/AU2011307251A1/en
Publication of WO2012044612A1 publication Critical patent/WO2012044612A1/fr

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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates generally to the fields of molecular biology. More specifically, the invention concerns anti-Her3 antibodies and antigen-bindnig fragments thereof, and uses of same. Methods for the prevention and treatment of diseases and conditions associated with aberrant Her3 activity are also included. Pharmaceutical composition comprising as an active agent an inhibitor of Her3 activity, particularly a humanized anti-Her3 antibody are also included by the invention. BACKGROUND OF THE INVENTION
  • Her3 signaling is an evolutionarily conserved mechanism used to control eel! fates through local cell interactions.
  • Signaling pathways between the extracellular environment and the nucleus of a cell involve the formation of many molecular complexes in which multiple proteins are assembled to directly or indirectly induce molecular events, such as enzyme activation or de-activation, Gomperts et al, Signal Transduction (Academic Press, N.Y., 2002).
  • receptor tyrosine kinases The class of receptor tyrosine kinases is so named because when activated by dimerization, the intracellular domain of RTKs acquire tyrosine kinase activity that can, in turn, activate a variety signal transduction pathways.
  • the predominant biological activity of some receptor tyrosine kinases is the stimulation of cell growth and proliferation, while other receptor tyrosine kinases are involved in arresting growth and promoting differentiation.
  • a single tyrosine kinase can inhibit, or stimulate cell proliferation depending on the cellular environment in which it is expressed.
  • receptor tyrosine kinases are a subclass of cell-surface growth-factor receptors with an intrinsic, ligand-controlled tyrosine-kinase activity.
  • Ligand-mediated receptor tyrosine kinases are believed to function as "master switches" for a coordinated cellular communication network that regulates the normal proliferation of eukaryotic cells. This is generally accomplished by catalyzing the transfer of a phosphate group from ATP to a tyrosine residue located on a protein substrate. Schlessinger, Cell, 103: 211-225 (2000).
  • cellular growth is generally instigated via ligand-stimulated tyrosine phosphorylation of intracellular substrates.
  • these receptors are able to integrate these external stimuli with internal signal transduction pathways, and thereby instigate an intracellular web of biochemical processes with a capacity to drive dramatic cellular transitions, such as proliferation and migration.
  • a promising set of targets for therapeutic intervention in the treatment of cancer includes the members of the epidermal growth factor receptor since the reversible
  • EGFR-TKIs block a cell surface receptor responsible for triggering and/or maintaining the cell signaling pathway that induces tumor cell growth and division.
  • the epidermal growth factor family can be subdivided into four groups based on their receptor-binding specificities (Herl, Her2, Her3, and Her4). These receptors are structurally related and include three functional domains: an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (Plowman, Culouscou et al. 1993).
  • the extracellular domain can be further divided into four subdomains (I-IV), including two cysteine-rich regions (II and IV) and two flanking regions (I and ill).
  • I-IV subdomains
  • two cysteine-rich regions II and IV
  • flanking regions I and ill.
  • Her3 is inimitable because of its catalytically deficient kinase domain, its high propensity to self associate in the absence of ligand and the ability of the monomeric species of the extracellular domains of Her3 to assume a locked conformation, using an intramolecular tether.
  • ERBB receptors Under normal physiological conditions, activation of the ERBB receptors is controlled by the spatial and temporal expression of their ligands, which are members of the EGF family of growth factors. Ligand binding to ERBB receptors induces the formation of receptor homo- and heterodimers. Dimerization consequently stimulates the intrinsic tyrosine kinase activity of the receptors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which ignites a cascade of signaling pathways that include various downstream adaptor and effector. Ultimately, downstream effects on gene expression determine the biological response to receptor activation. Nature Reviews Cancer 5, 341-354 (May 2005) Nancy E. Hynes.
  • the secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation.
  • Reviews describing intracellular signal transduction include Aaronson, S. A., Science, 254:1 146-1 153, 1991 ; Schlessinger, J. Trends Biochem. ScL, 13:443-447, 1988; and Ullrich, A., and Schlessinger, J., Cell, 61 :203-212, 1990.
  • Her2 distinguishes itself in several ways.
  • Her2 is an orphan receptor. The activation of the Her2 oncogene is believed to follow the binding of a yet unidentified growth factor ligand to the Her2 receptor complex, which leads to heterodimerization, triggering a cascade of growth signals that culminates in gene activation, increasing evidence suggests that it acts mainly as a co-receptor, increasing the affinity of ligand binding to dimeric receptor complex.
  • Her2 is a preferred partner for other EGFR family members (Her 1 /EGFR, Her3, and Her4) for the formation of heterodimers, which show high ligand affinity and superior signaling activity.
  • Her2 undergoes proteolytic cleavage, releasing a soluble extracellular domain (ECD). Shedding of the ECD has been shown to represent an alternative activation mechanism of full-length Her2 both in vitro and in vivo, as it leaves a membrane- anchored fragment with kinase activity.
  • Her2/neu is a very active tyrosine kinase, but cells expressing Her2/neu alone, and not other members of the EGFR family, fail to bind heregulin.
  • the Her2 gene (c-erbB-2, neu) encodes a 185 kDa transmembrane tyrosine kinase receptor that has partial homology with other members of the epidermal growth factor receptor family [Shih, C, Padhy, L. C. commonly Murray, M., et al. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts, Nature, 290, 261-264 (1981)]. It is now known that normal human cells express a small constitutive amount of Her2 protein on the plasma membrane.
  • Her2 in EGFR family signaling correlates with its involvement in the oncogenesis of several types of cancers, such as breast, ovarian, colon, endometrium, salivary gland, lung, kidney, colon and bladder and gastric cancers, regardless of its expression level (Slamon, D., et al., 1989, Science 244:707; Hynes, N., et al, 1994, Biochem. Biophys. Acta. 1 198: 165).
  • Her2 may also render tumor cells resistant to certain chemotherapeutics (Pegram, M, et al., 1997, Oncogene 15:537). Both the erbB and erbB-2 genes have been shown to be activated as oncogenes by mechanisms involving overexpression or mutations that constitutively activate the catalytic activity of their encoded receptor proteins [Bargmann et al., Cell 45:649-657 (1986); Velu et al., Science 238: 1408-1410 (1987)]. They are frequently upregulated in solid epithelial tumors of, by way of example, the prostate, lung and breast, and are also upregulated in glioblastoma tumors.
  • EGF-related growth factors are produced either by the tumour cells themselves or are available from surrounding stromal cells, leading to constitutive EGFR activation. See Sunpaweravong et al., J, Cancer Res. Clin. Oncol. 131, 11- 119 (2005); Salomon et al., Crit. Rev. Oncol. Hematol. 19, 183-232 (1995).
  • WO 00/31048 discloses a quinazoline derivative which acts as an inhibitor of receptor tyrosine kinases such as EGFR, Her2 and Her4. An inhibition of Her3 is however not disclosed.
  • WO 00/78347 discloses methods for arresting or inhibiting cell growth, comprising preventing or reducing ErbB-2/ErbB-3 heterodimer formation.
  • the agent may be a combination of an anti-Her2 extracellular domain antibody and an anti-Her3 antibody, e.g. the Her3 antibody H3.105.5 purchased from Neomarkers. It is however not clear which type of anti-Her3 antibody is required to obtain desirable therapeutic effects.
  • U.S. Pat. No. 5,804,396 describes a method for identifying an agent for treatment of a proliferative disorder, comprising the steps of assaying a potential agent for activity in inhibition of signal transduction by a Her2 Her3 or Her2/Her4 or Her3/Her4 heterodimer.
  • the patent is innocently silent relative to specific Her3 inhibitors.
  • Her3 is a transmembrane glycoprotein encoded by the c-erbB3 gene is a member of the epidermal growth factor receptor (EGFR) subfamily of type 1 receptor protein tyrosine kinase (RTK) family, which also includes EGFR, Her2/neu, and Her4 (see, e.g. U.S. Pat. No.
  • the ErbB3 receptor is an important receptor involved in cellular growth and differentiation. Particular attention has focused on the role of ErbB3 as a coreceptor of ErbB2 in the area of cancer research.
  • Her3 distinguishes itself from other epidermal growth factor receptors in that it is a kinase defective receptor, e.g., low tyrosine kinase activity (see, e.g. Guy et al. (1994) Proc Natl Acad Sci USA 91(17), 8132-6; Carraway et al., (1994) J. Biol. Chem. 269, 14303-14306).
  • kinase defective receptor e.g., low tyrosine kinase activity
  • it makes up for this deficiency in that it functions most effectively as a ligand binding receptor for neuregulins NRG-1 and NRG-2.
  • ErbB-2 is devoid of an activating ligand, it can act only in the context of a heterodimer with a ligand-bound receptor.
  • Her2 requires Her3 in order to transform normal cells into cancer cells.1
  • Her3 signaling thus relies on the formation of signaling-competent heterodimers with other ErbB members.
  • the most mitogenic "couple" amongst the ErbB receptors is Her2 Her3. (Citri et al., 2003). Indeed, this receptor pair
  • Her2/Her3 forms the most potent signaling module of the ErbB-receptor family in terms of cell growth and transformation. [Karamouzis et al, Int'l J. of Biochemistry & Cell Biol., 39: 851-856 (2007); Citri et al, Experimental, Cell Research, 284: 54-65 (2003)].
  • both Her2 and Her3 are active only in the context of ErbB heterodimers, and ErbB-2. This dimerization enables Her2 to activate the PI3K signaling pathway. Indeed, increased expression of Her3 increases the signaling potency of Her2, whereas decreased Her3 expression results in the loss of Her2 activity. Her3 is involved in Her2-mediated tumorigenesis through dimerization with Her2.
  • Her2 and Her3 have been detected in 12-50% of invasive breast cancers, and the increased drug resistance in many Her2-overexpressing cancers depends on augmented levels of Her3 and/or EGFR [Abd El-Rehim et al. British Journal of Cancer pp. 1532-1542 (2004)].
  • Her3 has been found to be overexpressed in various organs including breast, lung, pancreas and stomach. Furthermore, its overexpression has been documented in 20-30% of invasive and in approximately one third of in situ breast carcinomas, and is associated with poor prognostic factors [Badra et al., Cancer Letters, 244: 34- 1 (2006)].
  • T Is small molecule tyrosine kinase inhibitors
  • pertuzumab Another anti-Her2 antibody, pertuzumab, has been shown in a phase II clinical trial to have activity in ovarian cancer (Gordon, M. S. conjunction et al., 2006, J. Clin. Oncol. 24:4324).
  • Her2 respond significantly to Herceptin therapy, thus limiting the number of patients suitable for therapy.
  • the development of resistance to drugs or a change in the expression or epitope sequence of Her2 on tumor cells may render even those approachable patients unreactive with the antibody and therefore abrogating its therapeutic benefits.
  • Her3 Since Her3 has no catalytic activity, it appears that Her3 promotes drug resistance by enabling autocrine or paracrine ligands ( RG1 and NRG2) to activate catalytically competent RTKs, and through its capacity to channel signaling to PI3K Akt signaling pathways. Finally, it is formally possible that Her3 affects response to ERBB inhibitors indirectly, through protection of ERBB2 kinase domain or extracellular domain in heterodimers from phosphatases or inhibitors, or by reducing formation of ERBB2 homodimers, or dimers with other receptors such as ERBB4 that may have protective value for patients.
  • Murine or chimeric Her3 antibodies have been reported, such as in U.S. Pat. No.
  • Her2/Her3 While the proposed partnership between Her2/Her3 have created opportunities for improving efficacy of ERBB-targeted pharmaceuticals, by interfering with coupling of ERBB2 to ERBB3 through dimerization inhibitors, the art is completely silent as to the identification of a Her3 selective antagonist that is therapeutically effective in treating Her3 mediated cellular proliferative disorders. Indeed, despite encouraging results, the failure of Herceptin therapies for many ERBB2-amplified breast cancers, the absence of a Her3 selective antibody that is therapeutically effective together with the eventual development of therapeutic resistance in cases where robust responses occur at first, has created a need for an alternative, more potent, Her3 inhibitor. More, in spite of the discouraging landscape attendant conventional tyrosine kinase inhibitors, in particular, Her3 antibodies, the inventors endeavored to develop effective Her3 antagonists, the details of which are disclosed herein.
  • novel Her3 selective agents e.g., antibodies that are unencumbered by the deficiencies attendant current Her2/Her3 moieties.
  • a pharmaceutical composition comprising as an active agent a specific type of inhibitor of Her3 activity, e.g., antibody and pharmaceutically acceptable carriers, diluents and/or adjuvants. Methods of using the Her3 antibodies are also disclosed.
  • Embodiments of this invention are made available by the development of antibodies and antigen-bindnig fragments thereof, in particular humanized anti-her3 antibodies that retain favorable affinity to the Her3 receptor protein, particularly human Her3 receptor protein.
  • Her3 presents as an important and advantageous therapeutic target and the
  • Her3 binding agents such as immunoconjugates, antibodies, and fragments thereof
  • Her3 binding agents provide improved therapeutic and diagnostic agents for use in targeting pathological conditions associated with expression and/or activity of the Her3 mediated signaling pathway.
  • the anti-Her3 antibodies described herein, (“Invention Antibodies”) offer an important new approach to the treatment of various disorders of cell fate, in particular hyperproliferative disorders (e.g., cancer).
  • compositions and methods based on binding Her3. Kits and articles of manufacture related to the Her3 binding are also included.
  • a broad aspect of the invention relates to at least one humanized monoclonal antibody, or binding fragment thereof described herein that binds specifically to an antigen present in various cancers mediated by or related to Her3 activation or dysregulation, wherein the antigen is Her3.
  • the monoclonal antibodies of the invention bind to the human Her3 receptor (Her3) and can thus be useful in methods to treat or diagnose pathological
  • An embodiment of this invention relates to the antibodies and antigen-binding fragments thereof described herein, including polypeptides comprising amino acid sequences of the immunoglobulin chains of the antibodies and fragments, e.g., of the VRs, FRs and CDRs polypeptides and the polynucleotides encoding them.
  • Variant antibodies exemplified by diabody, bi-specific, trivalent & tetravalent antibodies or other antibodies derived from the herein described invention antibodies are also encompassed by the invention.
  • the present invention includes isolated polypeptides (e.g., antibodies and antigen-binding fragments thereof) comprising one or more (e.g., 3) CDRs taken from the light and/or heavy chain immunoglobulin set forth herein (e.g confuse 3 light chain CDRs and 3 heavy chain CDRs) as defined by the convention set forth in Kabat, "Sequences of Proteins of Immunological Interest” (National Institutes of Health, Bethesda, Md. s 1987 and 1991) or in Chothia et al, J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol. 186:651 (1989) (Kabat or Chothia) or Al-Lazikani et al, J. Mol. Biol. 273: 927-948 (1997).
  • CDRs taken from the light and/or heavy chain immunoglobulin set forth herein (e.g confuse 3 light chain CDRs and 3 heavy chain CDRs)
  • the present invention provides antibodies and antigen-bindnig fragments thereof that bind to Her3.
  • At least one invention described herein binds to the iigand binding domain of the Her3 receptor.
  • the invention provides an antibody, in particular, a chimeric or humanized antibody that binds to the negative regulator region, resident in the extracellular domain of the Her3 receptor.
  • the invention provides an isolated antibody comprising at least one (at least 2, at least 3, at least 4, at least 5, and/or 6) hypervariable sequence(s) (ITVR(s)) comprising a sequence selected from the group consisting of HVR-L1, HVR-L2, HVR-L3, HVR-H1 , HVR-H2, and/or HVR-H3 of the antibody described herein, wherein said isolated antibody specifically binds human Her3.
  • ITVR(s) hypervariable sequence(s)
  • the invention provides an isolated antibody that binds to the same epitope on human Her3 as any one or more of the humanized antibodies disclosed herein.
  • the invention provides an isolated anti-Her3 antibody, wherein a full length IgG form of the antibody specifically binds human Her3 with a binding affinity sufficient to be therapeutically effective when administered to a patient.
  • a representative affinity is 70 pM or better.
  • binding affinity of a ligand to its receptor can be determined using any of a variety of assays, and expressed in terms of a variety of quantitative values. Accordingly, in one embodiment, the binding affinity is expressed as Kd values and reflects intrinsic binding affinity (e.g., with minimized avidity effects).
  • binding affinity is measured in vitro, whether in a cell-free or cell-associated setting.
  • Any of a number of assays known in the art, including those described herein, can be used to obtain binding affinity measurements, including, for example, Biacore, radioimmunoassay (RIA) and ELISA.
  • the invention provides an anti-Her3 antibody comprising: at least one, two, three, four, five, and/or six hypervariable region (HVR) sequences selected from the group described in Appendix II, infra.
  • HVR hypervariable region
  • the invention provides an anti-Her3 antibody comprising: at least one, two, three, four, five, and/or six hypervariable region (HVR) sequences selected from the group described in Appendix II, infra.
  • HVR-L1 comprising at least one HVR-L1 sequence from those set forth in Appendix II.
  • an antibody of the invention comprises a light chain comprising at least one, at least two or all three of HVR sequences selected from the group as set forth in Appendix II.
  • the amino acid position/boundary delineating a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art (as described below). Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria, One or more of these positions can also be found in extended hypervariable regions.
  • antibody includes “antibodies” such as one or more of the Her3 specific antibodies described herein that bind Her3. As well, it includes monoclonal, polyclonal, multivalent, bispecific, and trivalent or optimized antibodies including fragments thereof. The invention also contemplates the use of single chains such as the variable heavy and light chains of the antibodies. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. In the present case, monoclonal antibodies to Her3 receptor proteins have been generated and have been isolated and shown to have high affinity to Her3.
  • the invention also includes modifications to the invention antibodies including variants thereof which do not significantly affect their binding properties. Such variants may have enhanced or decreased activity towards its binding partner.
  • Another embodiment of the invention encompasses monoclonal antibody or an antigen-binding fragment thereof that may be Fab fragments, F(ab)2 fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fd' fragments or Fv fragments, Fv, scFv, scFv-Fc or diabodies or any functional fragment whose half-life would have been increased by a chemical modification, especially by PEGylation, or by incorporation in a liposome. It may also be an anti-idiotypic antibody. Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules.
  • a second approach is to express the therapeutic protein as a genetic fusion with a natural protein that has a long serum half-life; either 67 kDa serum albumin (SA) - Syed S., Schuyler P.D., Kulczycky M., Sheffield W.P. Blood (1997) 89:3243-3252) or the Fc portion of an antibody, which adds an additional 60-70 kDa in its natural dimeric form, depending on glycosylation (Mohler et al., J. Immunol., 151 : 1548-1561 (1993).
  • SA serum albumin
  • Glycosylated variants (Glycoforms) of the invention antibodies are also envisioned.
  • antibodies, or fragments thereof are modified to reduce or eliminate potential glycosylation sites.
  • the amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-Hnkage), serine (O-linkage), and threonine (O-!inkage) residues.
  • the sequence of the antibody is examined, for example, by using publicly available databases such as the website provided by the Center for Biological Sequence Analysis (see http://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linked glycosylation sites) and http://www.cbs.dtu.dk/services/NetOGlyc/ for predicting O-linked glycosylation sites). Additional methods for altering glycosylation sites of antibodies are described in U.S. Pat. Nos. 6,350,861 and 5,714,350, the entire content of each of which is incorporated herein in its entirety.
  • glycosylation sites of the antibody can be altered, for example, by mutagenesis (e.g., site-directed mutagenesis).
  • mutagenesis e.g., site-directed mutagenesis
  • modified antibodies having reduced glycosylation sites or carbohydrates relative to the unmodified form are referred to as
  • an "afucosylated” anti-Her3 antibody derived from one or more antibodies described herein is representative of such an antibody that falls within the scope of the invention. See Li et al., Nat. Biotechnol., 24: 210-215 ( 2006); Shields, R.L. et al. Lack of fucose on human IgGl N-linked oligosaccharide improves binding to human Fc YRIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733-26740 (2002).
  • the invention antibodies or antigen binding fragments thereof are modified to enhance glycosylation.
  • Fc engineered variants of the invention antibodies are also encompassed by the present invention.
  • Such variants include antibodies or antigen binding fragments thereof which have been engineered so as to introduce mutations or substitutions in the Fc region of the antibody molecule so as to improve or modulate the effector functions of the underlying antibody molecule relative to the unmodified antibody.
  • improved effector functions is included o refer to such activities as CDC and ADCC.
  • the invention provides Fc variants that have improved function and/or solution properties as compared to the aglycosylated form of the parent Fc polypeptide.
  • Improved functionality herein includes but is not limited to binding affinity to an Fc ligand.
  • Improved solution properties herein include but are not limited to stability and solubility.
  • the proposed Fc variants bind to an FcyR with an affinity that is within about 0.5-fold of the glycosylated form of the parent Fc polypeptide.
  • the aglycosylated Fc variants bind to an FcyR with an affinity that is comparable to the glycosylated parent Fc polypeptide.
  • the Fc variants bind to an FcyR with an affinity that is greater than the glycosylated form of the parent Fc polypeptide.
  • Another broad aspect of the invention comprises an antibody or a binding fragment thereof that comprises a light chain comprising at least one complementarity determining region CDR having an amino acid sequence as set forth in one or more appendices detailed herein (Tables 1-4) or at least one CDR whose sequence has at least 80%, preferably 85%, 90%, 95% and 98% identity, after optimum alignment, with the sequences set forth in one or more appendices described herein or a heavy chain comprising at least one CDR comprising an amino acid sequence selected from the group set forth in one or more appendices set forth herein or at least one CDR whose sequence has at least 80%, preferably 85%, 90%, 95% and 98% identity, after optimum alignment, with said at least one CDR as set forth in one or more appendices set forth herein.
  • the antibody of the invention comprises at least one heavy chain and/or a light chain comprising at least one amino acid sequence as set forth in one of Tables 1 or 2, Nucleic acid molecules comprising nucleotide sequences encoding at least one or more of the above referenced amino acid sequences are also contemplated. See Appendices I - II.
  • the light chain may likewise comprise the amino acid sequence as set forth in one or more table detailed herein, while the heavy chain may comprise the amino acid sequence as set forth in one or more table as set forth herein. See Tables 1 -6.
  • Antibodies that compete with any one or more of the antibodies described herein for binding with Her3 l re also within the scope of the invention.
  • scFv may be prepared as fusion proteins with multimerization domains.
  • the multimerization domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucine-zipper domains.
  • helix structures such as Leucine-zipper domains.
  • the interaction between the VH/VL regions of the scFv are used for the multimerization (e.g. di-, tri- and pentabodies).
  • a multivalent antibody construct comprises at least one antigen recognition site specific for Her3 receptor protein. In certain embodiments, at least one of the antigen recognition sites is located on a scFv domain, while in other embodiments; all antigen recognition sites are located on scFv domains.
  • a multivalent, multispecific antibody or fragment thereof comprising more than one antigen binding site having an affinity toward a Her3 target antigen and one or more hapten binding sites having affinity towards hapten molecules. Also preferred, the multivalent, multispecific antibody or fragment thereof further comprises a diagnostic/detection and/or therapeutic agent.
  • multivalent antibody refers to an antibody or antibody construct comprising more than one antigen recognition site.
  • a “bivalent” antibody construct has two antigen recognition sites, whereas a “tetravalent” antibody construct has four antigen recognition sites.
  • the terms “monospecific”, “bispecific”, “tri specific”, “tetraspecific”, etc. refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody construct of the invention.
  • a "monospecific” antibody constructs antigen recognition sites all bind the same epitope.
  • a “bispecific” antibody construct has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope.
  • a “multivalent monospecific” antibody construct has multiple antigen recognition sites that all bind the same epitope.
  • a “multivalent bispecific” antibody construct has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.
  • the antibody construct is monospecific.
  • the multivalent antibody is tetravalent.
  • the antibody is a monospecific tetravalent antibody, wherein the antibody comprises four Her3 antigen recognition sites.
  • the antibody construct is specific for an epitope on Her3.
  • the antibody construct is bispecific. in one embodiment, the antibody construct has two Her3-specific antigen recognition sites and two Her3 -specific recognition sites.
  • the antibody construct is a trivalent antibody construct specific for the Her3 receptor protein.
  • the invention contemplates an antibody construct having two Her3-specific antigen recognition sites and two Her3 -specific recognition sites.
  • antibody constructs may be multispecific for different epitopes on human Her3 receptor proteins.
  • at least one antigen recognition site may be located on a scFv domain, and in certain embodiments, all antigen recognition sites are located on scFv domains.
  • the invention provides an antibody fragment comprising: (i) a first polypeptide comprising a light chain variable domain (and in some embodiments further comprising a light chain constant domain), (ii) a second polypeptide comprising a heavy chain variable domain, a first Fc polypeptide sequence (and in some embodiments further comprising a non-Fc heavy chain constant domain sequence), and (iii) a third polypeptide comprising a second Fc polypeptide sequence.
  • the second polypeptide is a single polypeptide comprising a heavy chain variable domain, heavy chain constant domain (e.g., all or part of CHI) and the first Fc polypeptide.
  • the first Fc polypeptide sequence is generally linked to the heavy chain constant domain by a peptide bond [i.e., not a non-peptidyl bond].
  • the third polypeptide comprises an N-termina!ly truncated heavy chain which comprises at least a portion of a hinge sequence at its N terminus.
  • the third polypeptide comprises an N-terminally truncated heavy chain which does not comprise a functional or wild type hinge sequence at its N terminus.
  • the two Fc polypeptides of an antibody of the invention or a fragment thereof are covalently linked.
  • the two Fc polypeptides may be linked through intermolecular disulfide bonds, for instance through intermolecular disulfide bonds between cysteine residues of the hinge region.
  • the invention provides a composition comprising a population of immunoglobulins wherein at least (or at least about) 50%, 75%, 85%, 90%, 95% of the immunoglobulins are antibody fragments of the invention.
  • a composition comprising said population of immunoglobulins can be in any of a variety of forms, including but not limited to host cell lysate, cell culture medium, host cell paste, or semi-purified or purified forms thereof. Purification methods are well known in the art, some of which are described herein.
  • Another embodiment of the invention provides a Her3-specific diabody antibody.
  • diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al., 1996, PNAS 16: 5879-5883).
  • the shortening of the Linker in a scFv molecule to 5-10 amino acids leads to the formation of homodimers in which an inter-chain VH N L -superimposition takes place.
  • Diabodies may additionally be stabilized by the incorporation of disulphide bridges. Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).
  • minibody By minibody the skilled person means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably
  • IgG most preferably IgGl as the dimerization region which is connected to the scFv via a Hinge region (e.g. also from IgGl) and a Linker region.
  • Hinge region e.g. also from IgGl
  • Linker region e.g. also from IgGl
  • the disulphide bridges in the Flinge region are mostly formed in higher cells and not in prokaryotes.
  • the minibody is a Her3-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Flu et al (1996, Cancer Res. 56: 3055-61).
  • triabody By triabody the skilled person means a: trivalent homotrimeric scFv derivative ( ortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein VH-VL are fused directly without a linker sequence lead to the formation of trimers.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerization is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 0:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al uncomfortable 1995 J. Mol. Biol. 246: 28-34). Therefore, an alternative embodiment proposes a Her3 -specific multimerized molecule based on the abovementioned antibody fragments and may be, for example, a triabody, a tetravalent miniantibody or a pentabody.
  • a related aspect of the invention provides monoclonal antibodies or functional fragments thereof that specifically binds human Her3 with specified affinities.
  • these antibodies bind human Her3 with an ED 5 o in the range of about 10 pM to about 500 nM.
  • these antibodies bind human Her3 with an EDso in the range of about 500 pM to about 300 nM.
  • the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non- human, human or humanized sequence (e.g., framework and/or constant domain sequences).
  • the non-human donor is a mouse.
  • an antigen binding sequence is synthetic, e.g. obtained by mutagenesis (e.g., phage display screening, etc.).
  • a chimeric antibody of the invention has murine V regions and human C region.
  • the murine light chain V region is fused to a human kappa light chain.
  • the murine heavy chain V region is fused to a human IgGl C region.
  • Humanized antibodies of the invention include those that have amino acid substitutions in the FR and affinity maturation variants with changes in the grafted CDRs.
  • the substituted amino acids in the CDR or FR are not limited to those present in the donor or recipient antibody.
  • the antibodies of the invention further comprise changes in amino acid residues in the Fc region that lead to improved effector function including enhanced CDC and/or ADCC function and B-cell killing.
  • Other antibodies of the invention include those having specific changes that improve stability.
  • Antibodies of the invention also include fucose deficient variants having improved ADCC function in vivo. In other
  • the antibodies of the invention comprise changes in amino acid residues in the Fc region that lead to decreased effector function, e.g. decreased CDC and/or ADCC function and/or decreased B-cell killing.
  • the antibodies of the invention are characterized by decreased binding (such as absence of binding) to human complement factor Clq and/or human Fc receptor on natural killer (NK) cells.
  • the antibodies of the invention are characterized by decreased binding (such as the absence of binding) to human Fc.gamma.RI, Fc.gamma.RIIA, and/or Fc.gamma.RIIIA.
  • the invention provides anti-Her3 polypeptides comprising any of the antigen binding sequences provided herein, wherein the anti-Her3 polypeptides specifically bind to the human Her3 receptor.
  • the invention provides methods for promoting the development, proliferation, maintenance or regeneration of neurons, the methods comprising administering an effective amount of an anti-Her3 antibody or immunoconjugate (in some embodiments, an anti- Her3 antibody of the invention) to a subject in need of such treatment.
  • the invention provides methods for inhibiting angiogenesis, the methods comprising administering an effective amount of an anti-Her3 antibody or
  • the site of angiogenesis is a tumor or cancer.
  • Methods of the invention can be used to affect any suitable pathological state.
  • disorders include a cancer selected from the group consisting of small cell lung cancer, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma; and disorders characterized by colon adenomas.
  • a cancer selected from the group consisting of small cell lung cancer, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma; and disorders characterized by colon adenomas.
  • a cell that is targeted in a method of the invention is a cancer cell.
  • a cancer cell can be one selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, a papillary carcinoma cell, a colon cancer cell, a pancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell, a central nervous system cancer cell, an osteogenic sarcoma cell, a renal carcinoma cell, a hepatocellular carcinoma cell, a bladder cancer cell, a gastric carcinoma cell, a head and neck squamous carcinoma cell, a melanoma cell, a leukemia cell, and a colon adenoma cell.
  • a cell that is targeted in a method of the invention is a hyperproliferative and/or hyperplastic cell.
  • a cell that is targeted in a method of the invention is a dysplastic cell.
  • a cell that is targeted in a method of the invention is a metastatic cell.
  • the cell that is targeted is a colon adenoma cell
  • the cell that is targeted expresses on the cell membrane at least about 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; 150,000; 200,000; 250,000; 300,000; 350,000;
  • Methods of the invention can further comprise additional treatment steps.
  • a method further comprises a step wherein a targeted cell and/or tissue (for e.g., a cancer cell) is exposed to radiation treatment or a chemotherapeutic agent.
  • the invention provides methods for detection of Her3, the methods comprising detecting Her3-anti-Her3 antibody complex in the sample.
  • detection includes qualitative and/or quantitative detection (e.g., measuring levels) with or without reference to a control. See infra for more details.
  • the invention provides methods for diagnosing a disorder associated with Her3 expression and/or activity, the methods comprising detecting Her3-anti- Her3 antibody complex in a biological sample from a patient having or suspected of having the disorder.
  • the Her3 expression is increased expression or abnormal expression.
  • the disorder is a tumor, cancer, and/or a cell proliferative disorder.
  • the invention provides any of the anti-Her3 antibodies described herein, wherein the anti-Her3 antibody comprises a detectable label.
  • the invention provides a complex of any of the anti-Her3 antibodies described herein and Her3.
  • the complex is in vivo or in vitro.
  • the complex comprises a cancer cell.
  • the anti-Her3 antibody is detectably labeled.
  • Also provided by the invention is a method of treating a Her3 mediated neoplasm or malignancy characterized by aberrant Her3 signaling or expression , comprising administering to a patient suffering from the neoplasm or malignancy, a therapeutically effective amount of a Her3 bind i ng antibody .
  • the Her3 antibody is administered at dosages that are therapeutically effective. In other embodiments, the Her3 antibody is administered in conjunction with chemotherapy.
  • the anti-Her3 antibody includes chimeric and humanized antibody.
  • humanized Her3 refers to an intact antibody or antibody fragment comprising any one or more of the variable light or heavy chain sequences as set forth in Appendix II.
  • Another aspect of the invention relates to the screening of a patient suspected of having a Her3 related disease or condition to determine if such a patient would benefit from treatment with an anti-Her3 antibody.
  • detection includes both cell surface detection as well as soluble Her3 found in the serum of said patient. See infra.
  • the invention also provides an isolated cell line that produces at least one anti- Her3 antibody as described herein.
  • An embodiment of the invention thus provides, in one aspect, the use of a nucleic acid of the invention in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
  • the invention provides use of an expression vector of the invention in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
  • the invention provides use of a host cell of the invention in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
  • the invention provides compositions comprising one or more nucleic acid of the invention and a carrier.
  • the carrier is pharmaceutically acceptable.
  • the invention provides host cells comprising a nucleic acid or a vector of the invention.
  • a vector can be of any type, for example a recombinant vector such as an expression vector. Any of a variety of host cells can be used.
  • a host cell is a prokaryotic cell, for example, E, coli.
  • a host cell is a eukaryotic cell, for example a mammalian cell such as Chinese Hamster Ovary (CHO) cell.
  • the invention provides methods of making an antibody or immunoconjugate of the invention.
  • the invention provides methods of making an anti-Her3 antibody (which, as defined herein includes full length and fragments thereof) or immunoconjugate, said method comprising expressing in a suitable host cell a recombinant vector of the invention encoding said antibody (or fragment thereof), and recovering said antibody.
  • the invention further provides methods of making an anti-Her3 immunoconjugate, said method comprising expressing in a suitable host cell a recombinant vector of the invention encoding an anti-Her3 antibody (or fragment thereof) or the invention, recovering said anti-Her3 antibody, and conjugating the anti-Her3 antibody to a drug.
  • At least one antibody of the invention binds to the negative regulator region, resident in the extracellular domain of the Her3 receptor.
  • the present invention further provides an antibody-recognized surface antigen present on host cell, including but not limited to T-cell acute lymphoblastic leukemia/lymphoma, human colon cancer, melanoma, human lung cancer and human prostate cancer, the antigen being Her3 or a biologically equivalent variant or fragment thereof.
  • Antibodies to Her3 as described herein may also be used in production facilities or laboratories to isolate additional quantities of the proteins, such as by affinity
  • the antibodies of the invention may also be utilized to isolate additional amounts of Her3.
  • the invention provides isolated, purified or recombinant polypeptides having an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to an amino acid sequence as set forth in one or more appendices herein described.
  • the application provides an amino acid sequence that is at least 90%, 95%, 98%, 99%, 99.3%, 99.5% or 99.7% identical to the target amino acid sequence herein described.
  • the present invention further relates to a polynucleotide encoding an antibody of the invention.
  • the invention further provides: isolated nucleic acid encoding the inventive antibodies disclosed herein including the heavy and/or light chain or antigen-binding portions thereof.
  • an aspect of the invention provides isolated nucleic acid molecules selected from the nucleotide sequences described in any one or more of the appendices detailed herein,
  • a related aspect is drawn to (a) a nucleic aid molecule described in any one or more of the appendices detailed herein encoding one or more of the sequence of amino acids as set forth in one or more of the appendices described herein; or (b) the nucleotide sequence that hybridizes to the nucleotide sequence of (a) under moderately stringent conditions, or (c) a nucleic acid molecule comprising a nucleotide sequence that is a degenerate sequence with respect to either (a) or (b) above, or (d) splice variant cDNA sequences thereof or (e) a nucleic acid of at least 18 nucleotides capable of hybridizing under conditions of great stringency with at least one of the CDRs of nucleic acid sequence described in one or more of the appendices described herein or with
  • a vector comprising the nucleic acid molecule described above, optionally, operably linked to control sequences recognized by a host cell transformed with the vector is also provided as is a host cell transformed with the vector.
  • the cells transformed according to the invention can be used in processes for preparation of recombinant antibody disclosed herein.
  • a variety of host cells can be transformed with the nucleic acid molecules encoding the antibody or a fragment thereof.
  • the host cell can be chosen from prokaryotic or eukaryotic systems, for example bacterial cells but likewise yeast cells or animal cells, in particular mammalian cells. It is likewise possible to use insect cells or plant cells. Methods of producing a recombinant protein are well known.
  • the invention likewise concerns animals, except man, which comprise at least one cell transformed according to the invention.
  • animals except man, which comprise at least one cell transformed according to the invention.
  • non-human transgenic animals that express the heavy and/or light chain or antigen-binding portions thereof of an anti- Her3 antibody are also provided.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the monoclonal antibody, or binding fragment thereof, according to the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the pharmaceutical composition may further comprise another component, such as an anti-tumor agent or an imaging reagent.
  • Certain embodiments of the invention relate to the use of the invention antibodies as targeted delivery systems for cytotoxic agents such as chemotherapeutic drugs, peptides or radionuclides, for immunological response promoters such as cytokines, for pro-drugs or for gene therapies.
  • cytotoxic agents such as chemotherapeutic drugs, peptides or radionuclides
  • immunological response promoters such as cytokines
  • pro-drugs or for gene therapies cytokines
  • the antibodies described herein may also transport/deliver payloads such as RNAi or shRNA. These payloads may be naked or chemically modified.
  • Immunoliposomes as potential delivery vehicles are also included.
  • the antibodies of the invention or binding fragments thereof will also find use in various medical or research purposes, including staging of various pathologies associated with expression of Her3. Indeed, laboratory research may also be facilitated through use of such antibodies. Identifying patients at risk of a pathological effect of an oncogenic disorder associated with expression of Her3, particularly hyperproliferative oncogenic disorders such as, but not limited to, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), T-cell acute lymphoblastic leukemia, lymphoma, Alagille syndrome, liver disease involving aberrant vascularization; diabetes, ovarian cancer, diseases involving vascular cell fate, rheumatoid arthritis, pancreatic cancer, plasma cell neoplasms (such as multiple myeloma, plasma cell leukemia, and extramedullary plasmacytoma), and neuroblastoma is also encompassed. As would be recognized by one of ordinary skill in this
  • additional embodiments of the invention pertain to the use of the invention antibodies for detecting dysplastic or neoplastic Her3 bearing cells as well as diagnosing, assessing and treating disorders associated with expression of Her3 receptor protein or aberrant activation of the Her3 cascade.
  • Her3 is intended to include diseases and other disorders in which the presence of high levels or abnormally low levels of Her 3 receptor protein (aberrant) in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder.
  • "neoplastic cells” or “neoplasia associated with expression of Her3” or “dysplastic cells associated with expression of Her3” which are used interchangeably refer to abnormal cells or cell growth characterized by increased or decreased expression levels of Her3 relative to normal. Such transformed cells proliferate without normal homeostatic growth control resulting in a condition marked by abnormal proliferation of cells of a tissue - cancer.
  • such disorders may be evidenced by an increase in the levels of Her3 on the cell surface or in increased ICD levels in the affected cells or tissues of a subject suffering from the disorder.
  • the increase in Her3 levels may be detected, for example, using an anti- Her3 antibody as described above. More, it refers to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Alternatively, the cells may express normal levels of Her 3 but are marked by abnormal proliferation. Not all neoplastic cells are necessarily replicating cells at a given time point.
  • the cells defined as neoplastic cells consist of cells in benign neoplasms and cells in malignant (or frank) neoplasms.
  • neoplastic cells are frequently referred to as cancer, typically termed carcinoma if originating from cells of endodermal or ectodermal histological origin, or sarcoma if originating from cell types derived from mesoderm.
  • "increased expression” as it relates to Her3 refers to protein or gene expression levels that demonstrate a statistically significant increase in expression (as measured by RNA expression or protein expression) relative to a control.
  • “increased expression” is also used to encompass “increased activation of the Her3 cascade".
  • the level of expression of Her3 may not be increased relative to a control but the level of activation of the Her3 cascade may be increased relative to a control or a patient without the disease.
  • Her3 -specific antibodies of this invention may be used to detect the overexpression and, thus, to detect metastatic disease.
  • the immunodetection methods of the present invention may be of utility in the diagnosis of various disease states.
  • the invention antibodies may be exploited to detect differential expression of Her3 in metastatic cells.
  • human Her3 or ICD or any other downstream target may be detected in a number of ways such as by various assays.
  • Immunodetection techniques include but are not limited to immunohistoiogical staining, western blotting, dot blotting, precipitation, agglutination, ELISA assays, immunohistochemistry, in situ hybridization, flow cytometry or radio-immunoassay (RIA) technique or equivalent on a variety of tissues and a variety of sandwich assays. These techniques are well known in the art. See, for example, U.S. Pat. No. 5,876,949, hereby incorporated by reference,
  • Another aspect of the invention relates to the use of these antibodies in methods or assays for detecting Her3 activation or expression in patients suspected of having a Her3 related disease or disorder.
  • diseases or disorders may include, but not limited to, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
  • Alagil!e syndrome liver disease involving aberrant vascularization
  • diabetes ovarian cancer
  • diseases involving vascular cell fate CADASIL
  • neuroblastoma CADASIL
  • the antibodies described herein are particularly useful for in vitro and in vivo diagnostic and prognostic applications. Suitable conditions for which the antibody of the invention will find particular use for include the detection and diagnosis of neoplasias, such as, but not limited to cancer of the ovary, prostate, colon and skin. Inflammatory responses or disorders triggered by Her3 receptor activation or cascade area also included.
  • Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads.
  • Various types of labels and methods of conjugating the labels to the antibodies of the invention are well known to those skilled in the art, such as the ones set forth below.
  • an illustrative embodiment provides a method for detecting normal, benign, hyperplastic, and/or cancerous cells or a portion thereof in a biological sample comprising: providing a Her3 antibody or binding portion thereof which recognizes an antigen (Her3) on the surface of the cells, wherein the antibody or binding portion thereof binds to an epitope of Her3 which is also recognized by any one or more monoclonal antibodies detailed herein and wherein the antibody or binding portion thereof is bound to a label effective to permit detection of the cells or a portion thereof upon binding of the antibody or binding portion thereof to the antigen; contacting the biological sample with the antibody or binding portion thereof having a label under conditions effective to permit binding of the antibody or binding portion thereof to the antigen on any of the cells or a portion thereof in the biological sample; and detecting the presence of any of the cells or a portion thereof in the biological sample by detecting the label.
  • a Her3 antibody or binding portion thereof which recognizes an antigen (Her3) on the surface of the cells, wherein the antibody or binding portion thereof binds
  • the step of contacting the antibody is carried out in a living mammal and comprises: administering the Nocthl antibody or binding portion thereof to the mammal under conditions effective to permit binding of the antibody or binding portion thereof to the antigen on any of the cells or a portion thereof in the mammal.
  • the invention antibodies may be labeled with a detectable moiety, such as a fluorophore, a chromophore, a radionuclide, a chemiluminescent agent, a bioluminescent agent and an enzyme.
  • a detectable moiety such as a fluorophore, a chromophore, a radionuclide, a chemiluminescent agent, a bioluminescent agent and an enzyme.
  • Yet another embodiment of the invention provides a method of diagnosis, preferably in vitro, of illnesses connected with an overexpression or under expression, preferably overexpression of the Her3 receptor.
  • Samples are taken from the patient and subject to any suitable immunoassay with Her3 specific antibodies to detect the presence of Her3.
  • the biological sample is preferably tissue sample or biopsies of human origin which can be conveniently assayed for the presence of a pathological hyperproliferative oncogenic disorder associated with expression of Her3.
  • results can be compared with those of control samples, which are obtained in a manner similar to the test samples but from individuals that do not have or present with a
  • the diagnostic uses of the antibodies according to the present invention embrace primary tumors and cancers, as well as metastases.
  • the antibody, or one of its functional fragments can be present in the form of an immunoconjugate or of a labeled antibody so as to obtain a detectable and/or quantifiable signal.
  • An exemplary in vitro method of diagnosing pathological hyperproliferative oncogenic disorder comprises: (a) determining the presence or absence of Her3 bearing cells in a sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said Her3 bearing cells.
  • corresponding biological sample from a normal subject or non-cancerous tissue is generally indicative of a patient with or suspected of presenting with an Her3 mediated disorder.
  • a representative in vitro method of diagnosing the presence of cancer in a patient or a susceptibility to a pathological condition associated therewith in a subject proposes: (a) measuring the levels of Her3 receptor protein in cells or tissues of the patient; and (b) comparing the measured levels of the antigen of (a) with levels of the antigen (Her3 receptor protein) in cells or tissues from a normal human control, wherein an increase in the measured levels of the antigen in the patient versus the normal control is associated with the presence of the cancer.
  • decreased Her3 expression may be diagnostic of a pathological condition such as disorders of the skin.
  • one may measure the level of the ICD as a measure of Her3 receptor activation.
  • a representative embodiment thus provides a method of diagnosing a pathological oncogenic disorder associated with aberrant expression of Her3 or increased Her3 receptor activation (Her3 cascade) comprising the steps of: a) detecting the presence and level of Her3 in a biological sample obtained from the mammal at a plurality of time points, wherein Her3 is detected by a method selected from the group consisting of immunoblotting, western blotting,
  • a method of monitoring metastatic potential of an oncogenic disorder associated with Her3 expression in a mammal is also encompassed.
  • a method for screening for metastatic potential of solid tumors comprising: a) obtaining a sample of tumor tissue from an individual in need of screening for metastatic potential of a solid tumor; b) reacting an antibody to Her3 with tumor tissue from the patient; c) detecting the extent of binding of the Her3 antibody to the tissue and d) correlating the extent of binding of the antibody with its metastatic potential.
  • the tumor is cancer arising from large bowel (colorectal cancer), prostate, breast or skin (ovarian cancer or T-ALL).
  • step c) may be performed over a plurality of time points.
  • Her3 expression is detected by a method selected from the group consisting of immunohistochemical staining, immunoblotting, western blotting, immunoperoxidase staining, fluorescein labeling, diaminobenzadine and biotinylation.
  • the invention further provides for a method for predicting susceptibility to cancer comprising detecting the expression level of Her 3 in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of Her 3 expression correlates to the degree of susceptibility.
  • the expression of Her3 in, for example, breast tissue, prostate tissue, colon tissue, or any other tissue suspected of cells expressing Her3 is examined, with the presence of Her3 in the sample providing an indication of cancer susceptibility or the emergence or existence of a tissue specific tumor.
  • Stage determination has potential prognostic value and provides criteria for designing optimal therapy.
  • pathological staging of breast cancer for example is preferable to clinical staging because the former gives a more accurate prognosis.
  • clinical staging would be preferred if it were as accurate as pathological staging because it does not depend on an invasive procedure to obtain tissue for pathological evaluation.
  • methods for gauging tumor aggressiveness are also provided as are methods for observing the progression of a malignancy in an individual over time.
  • the invention provides an in vivo imaging reagent comprising an antibody according to the invention, or one of its functional fragments, preferably labeled, especially radiolabeled, and its use in medical imaging, in particular for the detection of Her3 mediated disorders e.g., cancer characterized by over expressing Her3 or other pathologies in which cells over express Her3.
  • Her3 mediated disorders e.g., cancer characterized by over expressing Her3 or other pathologies in which cells over express Her3.
  • the imaging reagents e.g., diagnostic reagents can be administered by intravenous injection into the body of the patient, or directly into a tissue suspected of harboring Her3 bearing cells, e.g., colon or ovary or the pancreas.
  • the dosage of reagent should be within the same ranges as for treatment methods.
  • the reagent is labeled, although in some methods, the primary reagent with affinity for Her3 is unlabelled and a secondary labeling agent is used to bind to the primary reagent.
  • the choice of label depends on the means of detection. For example, a fluorescent label is suitable for optical detection. Use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Radioactive labels can also be detected using PET or SPECT.
  • Diagnosis is performed by comparing the number, size, and/or intensity of labeled loci, to corresponding baseline values.
  • the baseline values can, as an example, represent the mean levels in a population of undiseased individuals.
  • Baseline values can also represent previous levels determined in the same patient. For example, baseline values can be determined in a patient before beginning treatment, and measured values thereafter compared with the baseline values. A decrease in values relative to baseline signals a positive response to treatment.
  • a general method embodied by the invention works by administering to a patient an imaging-effective amount of an imaging reagent such as the above described monoclonal antibodies or antigen -binding fragments which are labeled and a pharmaceutically effective carrier and then detecting the agent after it has bound to Her3 present in the sample.
  • an imaging reagent such as the above described monoclonal antibodies or antigen -binding fragments which are labeled and a pharmaceutically effective carrier
  • the method works by administering an imaging-effective amount of an imaging agent comprising a targeting moiety and an active moiety.
  • the targeting moiety may be an antibody, Fab, Fab'2, a single chain antibody or other binding agent that interacts with an epitope present in Her3.
  • the active moiety may be a radioactive agent, such as radioactive technetium, radioactive indium, or radioactive iodine.
  • the imaging agent is administered in an amount effective for diagnostic use in a mammal such as a human and the localization and accumulation of the imaging agent is then detected.
  • the localization and accumulation of the imaging agent may be detected by radionuclide imaging, radioscintigraphy, nuclear magnetic resonance imaging, computed tomography, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection.
  • the in vivo imaging methods of the present invention are also useful for providing prognoses to cancer patients.
  • the presence of Her 3 indicative of an aggressive cancer likely to metastasize or likely to respond to a certain treatment can be detected.
  • the invention provides a method for observing the progression of a malignancy in an individual over time comprising determining the level of Her3 expressed by cells in a sample of the tumor, comparing the level so determined to the level of Her3 expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of Her3 expression in the tumor sample over time provides information on the progression of the cancer.
  • the in vivo imaging methods of the present invention can further be used to detect Her3 mediated cancers e.g., one that has metastasized in other parts of the body.
  • a related embodiment relates to a pharmaceutical composition for in vivo imaging of an oncogenic disorder associated with expression of Her3 comprising the invention antibodies or binding fragment thereof which is labeled and which binds Her3 in vivo; and a
  • the antibodies disclosed herein may also be used in methods of identifying human tumors that can escape anti- Her3 treatment by observing or monitoring the growth of the tumor implanted into a rodent or rabbit after treatment with a conventional anti- Her3 antibody.
  • the antibodies of the invention can also be used to study and evaluate combination therapies with anti- Her3 antibodies of this invention and other therapeutic agents.
  • the antibodies and polypeptides of this invention can be used to study the role of Her3 in other diseases by administering the antibodies or polypeptides to an animal suffering from the disease of a similar disease and determining whether one or more symptoms of the disease are alleviated.
  • target antigen e.g., Her3, which represents a positive identification
  • background expression levels are often used to form a "cut-off 1 above which increased staining will be scored as significant or positive.
  • Significant expression may be represented by high levels of antigens in target cells or tissues or alternatively, by a high proportion of cells from within a tissue that each give a positive signal.
  • Another embodiment of the invention is directed to methods for observing a coincidence between the expression of Her3 and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample.
  • factors associated with malignancy can be utilized, such as the expression of genes or gene products associated with malignancy as well as gross cytological observations.
  • Methods for observing a coincidence between the expression of Her3 and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
  • the methods proposed herein can be useful to diagnose or confirm diagnosis of an oncogenic disorder associated with expression of Her3 or susceptibility thereof.
  • the methods can be used on a patient presenting with symptoms of an oncogenic disorder. If the patient has, for example increased expression levels of Her3 or aberrant Her3 receptor activation as evidenced by increased expression levels of any one or more downstream targets effected by or related to Her3 receptor activation or increased expression levels of ICD, then the patient is likely suffering from a cancerous disorder.
  • the methods can also be used in asymptomatic patients. Presence of higher than normal Her3 may indicate for example susceptibility to future symptomatic disease.
  • the methods are useful for monitoring progression and/or response to treatment in patients who have been previously diagnosed with an Her3 mediated cancer.
  • malignancies are characterized by either increased Her3 receptor expression, increased or aberrant Her3 receptor activation or mutations resident in the Her3 receptor protein. Malignancies characterized by aberrant or increased Her3 receptor activation may be confirmed via determination of expression levels of ICD, whose expression level may be increased in the cytoplasm following activation of the Her3 cascade. Thus, in those cases where a malignancy is characterized by increased Her3 receptor activation, one is expected to find increased expression of ICD in the cytoplasm. This increase in ICD expression can be traced to the translocation f the ICD into the cytoplasm upon Her3 receptor activation.
  • measurement of Her 3 in biopsied tissue or other biological sample can be corroborated by determining expression of downstream target expression as a means of identifying high risk patients.
  • single or multiple determinations of increased Her3 expression and/or ICD expression over time may serve as a marker for illness indicative of intervening medical intervention. A positive test can therefore supplement the clinician's judgment.
  • Her3 levels also add prognostic accuracy to established severity of illness scores. Such clinical judgments will benefit by a method of scoring diseased cells.
  • measurement of Her3 expression in a tissue sample can also be used as an indicator for additional monitoring or testing, or consideration for more aggressive treatment, especially when patients are found to have increased Her3 expression levels or increased Her3 receptor activation as reflected by increased cytoplasmic ICD levels or any other downstream target.
  • a semi quantitative estimation of Her3 immunoreactivity can be performed.
  • a score can be given to each slide, considering the intensity of the stain.
  • the slides may be examined and scored independently by two investigators, and discordances may be reconciled by reexamination of the slide, and the scores then averaged.
  • the intensity of immunostaining of individual cells may be scored on a scale of 0 (no staining) to 4 (strongest intensity) and the percentage of cells with staining at each intensity estimated.
  • any scoring scheme used to compare staining intensities may be used as long as it takes into account the relative intensity of cytoplasmic staining and allows differentiation among degrees of intensity of staining, thus providing a way to grade the malignancies. Because of the novel staining aspects of the present invention which results in highly differentiated staining, the scoring or grading can be done visually, thus allowing the technique of the present invention to be widely used clinically without sophisticated equipment. It will be understood that the staining results can be analyzed by appropriate sensitive optical equipment and analyzed by computer.
  • the invention provides a method for diagnosing an oncogenic disorder associated with expression of Her3 comprising: a) measuring by radioimmunoassay, competitive-binding assay, Western blot analysis, ELISA assay, or sandwich assay the amount of Her3 protein in a biological sample, e.g., biopsied tissue obtained from a patient, using an antibody that specifically binds to Her3; and b) comparing the amount of antibody bound to said Her3 protein to a normal control tissue sample, wherein increased expression or over-expression of Her3 in the sample obtained from the patient relative to the normal control tissue sample is diagnostic of an oncogenic disorder associated with expression of Her3.
  • the Her3-specific antibody comprises sat least one antibody detailed herein.
  • the same scoring criteria e.g., score of 0 to 4 may be used to score cytoplasmic ICD staining as a means of corroborating the initial diagnosis. Consequently, cells may be stained with an antibody specific for ICD and the intensity level scored using the above criteria, where the intensity of immunostaining of individual ceils may be scored on a scale of 0 (no staining) to 4 (strongest intensity) and the percentage of cells with staining at each intensity estimated. If there is no staining, a 0 score can be given. A +1 score indicates weak staining, while a +4 score indicating strong intensity of staining.
  • a prognostic index is produced by preparing a weighted scale of expression levels of the tumor markers, Her3 related to progression observed in a representative sample of a particular tumor type, wherein the different values in the weighted scale are related to increased invasiveness or metastatic spread in the representative sample.
  • the methods of the invention are also useful for identifying a human cancer patient at risk for additional neoplastic disease, for staging malignant disease in a human cancer patient and assessing the relative risk of metastatic disease versus the risk of toxicity (such as leukocytopenia, for example) from chemotherapeutic treatment.
  • the invention thus provides methods wherein the results of the determination of the level of cell surface Her3 expression and the extent of cytoplasmic localization of ICD are used to prepare a prognostic or "risk" index for making a prognostic determination.
  • a prognostic index is prepared using the above criteria, wherein a value of 0 signifies a control, a value of +1 indicates weak staining etc, wherein a prognosis of a likelihood of progressing to metastatic disease is made when the staining intensity is scored at +4.
  • An illustrative embodiment of the invention provides a diagnostic or monitoring method comprising: a) obtaining a sample of tissue from an individual in need of diagnosis or monitoring for cancer; b) detecting levels of Her3 protein in said sample, c) scoring said sample for Her3 protein levels; and d) comparing said scoring to that obtained from a control tissue sample to determine the prognosis associated with said cancer.
  • Samples may be scored using a scale of 0 to 4, where 0 is negative (no detectable Her3 expression or level comparable to a control level), and 4 is high intensity staining in the majority of cells and wherein a score of 1 to 4 indicates a poor prognosis while a score of 0 indicates a good prognosis.
  • a related aspect of the invention pertains to a method for screening for metastatic potential of a Her3 mediated hyperproliferative disorder comprising: a) obtaining a sample of diseased or target tissue from an individual in need of screening for metastatic potential of a Notch 1 mediated tumor, b) reacting an antibody to Her3 with tumor tissue from the patient, c) detecting the extent of binding of the Her3 antibody to said tissue and d) correlating the extent of binding of said antibody with its metastatic potential.
  • any of the methods of the invention involving analysis of the levels of Her3 or ICD may be used in conjunction with additional cancer markers readily known to those of skill in the art.
  • Also provided is a method of detecting the presence and extent of cancer in a patient comprising: determining the level of the antigen (Her3) in a sample of cells or a tissue section from the patient and correlating the quantity of the antigen with the presence and extent of the cancer disease in the patient relative to a normal or control patient.
  • Her3 antagonistic moiety would predict a decrease in Her3 expression levels on tumor cells or any other cells that express this cell surface receptor, while an unfavorable outcome would predict either no change in the expression levels or an increase in expression levels of Her3.
  • the present invention exploits the ability of the Her3 antibodies of the invention to bind Her3 with high affinity to be utilized in a "biomarker strategy" for measuring Her3 activity and/or expression or tumorigenic status by specifically measuring the expression levels of Her 3 on tumor/cancer cells.
  • the present invention provides a rapid means, e.g., high affinity anti- Her3 antibodies, for assessing the nature, severity and progression of a pathological hyperproliferative oncogenic disorder associated with expression of Her3.
  • the invention provides a method for determining onset, progression, or regression, of neoplasias associated with expression of Her 3 in a subject, comprising: obtaining from a subject a first biological sample at a first time point, contacting the first sample with a effective amount of an antibody described herein under conditions allowing for binding of the antibody or a fragment thereof to Her3 suspected to be contained in the sample and determining specific binding between the antibody in the first sample and Her3 bearing cells to thereby obtain a first value, obtaining subsequently from the subject a second biological sample at a second time point, and contacting the second biological sample with the Her3 antibody and determining specific binding between the antibody and Her3 in said sample to obtain a second value, and comparing the determination of binding in the first sample to the determination of specific binding in the second sample as a determination of the onset, progression, or regression of the colon cancer, wherein an increase in expression level of Her 3 in said second or subsequent sample relative to the first sample indicative of the progression of
  • Her3 is detected by (1) adding an antibody of the invention to the sample or tissue section; (2) adding goat anti-mouse IgG antibody conjugated with peroxidase; (3) fixing with diaminobenzidene and peroxide, and (4) examining the sample or section, wherein reddish brown color indicates that the cells bear the antigen.
  • the effectiveness of a cancer treatment may be monitored by periodically measuring changes in the level of the antigen in a tissue sample taken from a patient undergoing the therapy, and correlating the change in level of the antigen with the effectiveness of the therapy, wherein a lower level of Her3 expression determined at a later time point relative to the level of Her3 determined at an earlier time point during the course of therapy indicates effectiveness of the therapy for the cancer disease.
  • the application provides methods for determining the appropriate therapeutic protocol for a subject.
  • the antibodies of the invention will be very useful for monitoring the course of amelioration of malignancy in an individual, especially in those circumstances where the subject is being treated with a Her3 antibody that does not compete with the antibodies of the invention for binding Her3.
  • presence or absence or a change in the level of Her3 expression may be indicative as to whether a subject is likely to have a relapse or a progressive neoplasia or persistent neoplasias such as cancer associated with Her3.
  • an antibody fragment of the invention is capable of specifically binding to a target molecule of interest.
  • an antibody fragment specifically binds a tumor antigen.
  • the antibody fragment specifically binds a cell surface receptor that is activated upon receptor multimerization (e.g., dimerization).
  • binding of an antibody of the invention to a target molecule inhibits binding of another molecule (such as a ligand, where the target molecule is a receptor) to said target molecule.
  • an antibody fragment of the invention when bound to a target molecule inhibits binding of a cognate binding partner to the target molecule
  • a cognate binding partner can be a ligand, or a hetero or homodimerizing molecule.
  • an antibody fragment of the invention when bound to a target molecule inhibits target molecule receptor activation.
  • binding of the antibody fragment to a cell surface receptor may inhibit dimerization of the receptor with another unit of the receptor, whereby activation of the receptor is inhibited (due at least in part to a lack of receptor dimerization).
  • an antibody fragment of the invention is capable of competing with a native Her3 receptor binding partner, e.g., delta or Serrate to the Her3 receptor.
  • an antibody of the invention or a fragment thereof is capable of competing with an endogenous Notch receptor ligand for binding to a Her3 receptor.
  • the herein described antibodies antagonize, or inhibit, Her3 mediated signaling by either blocking or inhibiting Her3 binding to its endogenous ligand or preventing or delaying Her3 cascade activation (hereinafter "Antagonist Therapeutics Antibodies") are administered for therapeutic effect. Disorders which can thus be treated can be identified by in vitro assays such as those described herein or known to one skilled in the art.
  • Such antagonist antibodies include anti-Her3 neutralizing antibodies and competitive inhibitors of EGFR protein-protein interactions as detailed infra.
  • an antibody of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neopiastic or non-malignant state into a neoplastic or a malignant state.
  • Another embodiment of the preset invention is the use of any of these antibodies for the preparation of a medicament or composition for the treatment of diseases and disorders associated with Her3 receptor activation.
  • Another embodiment of the preset invention is the use of any of these antibodies in the treatment of disorders associated with Her3 activation comprising the inhibition of said activation by, e.g., inhibiting Her3 signaling, or neutralization of the receptor by blocking ligand binding.
  • Her3 related disorders may include, but are not limited to cancers, lethal congenital contractural syndrome type 2 (LCCS2).
  • the invention provides a method for treating a disease comprising administering to a subject in need of such a treatment an effective amount for treating the disease of at least one antibody or antigenic or binding fragment thereof ("fragment') disclosed herein that binds to native human Her3 (hHer3) and abolishes or attenuates the function of the native hHer3 or Her3 Her2 heterodimer.
  • fragment' antibody or antigenic or binding fragment thereof
  • a variety of diseases may be treated with the above described methods including cancer, in particular T-cell acute
  • lymphoblasticleukemia/lymphoma human breast cancer, human colorectal cancer, melanoma, human lung cancer, human head and neck cancers and human prostate cancer, an immune or inflammatory disorder an angiogenesis disorder or any other disorder mediated by Her3 signaling (G. Sithanandam & LM Anderson (2008) Cancer Gene Therapy 15:413; Jiang et al. (2007) JBC 282:32689; Grivas et al. (2007) Eur J. Cancer 43:2602).
  • the present invention provides a method of inhibiting or killing cancer cells, comprising: providing to a patient in need thereof the monoclonal antibody, or binding fragment thereof of the present invention, under conditions and in an amount sufficient for the binding to the cancer cells, thereby causing inhibition or killing of the cancer cells by the immune cells of the patient.
  • the method is for the treatment of T-cell acute lymphoblastic leukemia/lymphoma, human colon cancer, melanoma, human lung cancer and human prostate cancer.
  • the monoclonal antibody is preferably conjugated with a cytotoxic moiety, such as a chemotherapeutic agent, a photoactivated toxin, an RNAi molecule or a radioactive agent.
  • a cytotoxic moiety such as a chemotherapeutic agent, a photoactivated toxin, an RNAi molecule or a radioactive agent.
  • the cytotoxic moiety may be a Ricin.
  • Representative disorders include an immune or inflammatory disorder like colitis or asthma, an infectious disease, an angiogenesis disorder, atherosclerosis, or a disorder of the kidney or any other disorder mediated by Her3 signaling.
  • an oligonucleotide such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an oligonucleotide, such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an oligonucleotide, such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an oligonucleotide, such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an oligonucleotide, such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an oligonucleotide, such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an oligonucleotide, such as an RNAi molecule inhibiting Her3 expression may be conjugated to, or form the therapeutic agent portion of an
  • the oligonucleotide may be administered concurrently or sequentially with a naked or conjugated anti-Her3 antibody or antibody fragment of the present invention.
  • the oligonucleotides are an antisense oligonucleotide (RNAi) that preferably is directed against Her3 expression.
  • RNA inhibition is based on antisense modulation of Her 3 in cells and tissues comprising contacting the cells and tissues with at least one Her3 antibody conjugated to a nucleic acid molecule that modulated transcription or translation of Her3 receptor protein, including but not limited to double stranded RNA, (dsRNA), small interfering RNA (siRNA), ribozymes and locked nucleic acids (LNAs), and a pharmaceutically acceptable carrier.
  • dsRNA double stranded RNA
  • siRNA small interfering RNA
  • LNAs locked nucleic acids
  • the present invention further provides a method for localizing cancer cells in a patient, comprising: (a) administering to the patient a detectably-labeled monoclonal antibody of the invention, or binding fragment thereof; (b) allowing the detectably-labeled (e.g. radiolabeled; fluorochrome labeled, or enzyme labeled, especially via ELISA) monoclonal antibody, or binding fragment thereof, to bind to the cancer cells within the patient; and (c) determining the location of the labeled monoclonal antibody or binding fragment thereof, within the patient.
  • a detectably-labeled monoclonal antibody of the invention or binding fragment thereof
  • the detectably-labeled e.g. radiolabeled; fluorochrome labeled, or enzyme labeled, especially via ELISA
  • Another embodiment of the invention relates to the use of invention antibodies, and VRs, FRs and CDRs thereof, in directed molecular evolution technologies such as phage display or bacterial or yeast cell surface display technologies in order to generate polypeptides with enhanced affinity, specificity, stability or other desired characteristics.
  • Another embodiment of the present invention is a cancer cell targeting diagnostic immunoconjugate comprising an antibody component that comprises an antibody or fragment thereof of any one of the antibodies or fragments thereof of the present invention, wherein the antibody or fragment thereof is bound to at least one diagnostic/detection agent.
  • the diagnostic/detection agent is selected from the group comprising a radionuclide, a contrast agent, and a photoactive diagnostic/detection agent.
  • the diagnostic/detection agent is a radionuclide with an energy between 20 and 4,000 keV or is a radionuclide selected from the group consisting of 1 "V "in, 177 Lu, .
  • the diagnostic/detection agent is a paramagnetic ion, such as the a metal comprising chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), or a radioopaque material, such as barium, diatrizoate, ethiodized oil, gallium citrate, meglumine, metrizamide, metrizoate, propyliodone, and thallous chloride.
  • a radioopaque material such as barium, diatrizoate, ethiodized oil, gallium citrate, meglumine, metrizamide, metrizoate, propyli
  • the diagnostic/detection agent is a fluorescent labeling compound selected from the group comprising fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, ailophycocyanin, o-phthaldehyde and fluorescamine, a chemiluminescent labeling compound selected from the group comprising luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester, or a biolummescent compound selected from the group comprising luciferin, luciferase and aequorin.
  • the diagnostic immunoconjugates of the present invention are used in intraoperative, endoscopic, or intravascular tumor diagnosis.
  • the therapeutic agent is a cytotoxic agent, such as a drug or a toxin.
  • the drug is selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, hormone antagonists, endostatin, taxols, camptothecms, SN-38, doxorubicins and their analogs, antimetabolites, alkylating agents, antimitotics, antiangiogenic, apoptotic agents, methotrexate, CPT-1 1 , and a
  • the therapeutic agent is an oligonucleotide.
  • the oligonucleotide may be an antisense oligonucleotide such as an antisense oligonucleotide against Her3 or an RNAi molecule against Her3 receptor expression.
  • the therapeutic agent is a toxin selected from the group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin and combinations thereof
  • an immunomodulator is selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), a stem cell growth factor, erythropoietin, thrombopoietin and a combinations thereof, a radionuclide and combinations thereof, or a photoactive therapeutic agent selected from the group comprising chromogens and dyes.
  • the therapeutic agent is an enzyme selected from the group comprising malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, .alpha.-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, .beta.- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the invention provides an immunoconjugate (interchangeably termed "antibody drug conjugate” or "ADC”) comprising any of the anti-Her3 antibodies disclosed herein conjugated to an agent, such as a drug.
  • ADC antibody drug conjugate
  • Another embodiment of the present invention is a cancer cell targeting therapeutic immunoconjugate comprising an antibody component that comprises an antibody or fragment thereof of any one of the antibodies, fusion proteins, or fragments thereof of the present invention, wherein the antibody, fusion protein, or fragment thereof is bound to at least one therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), radionuclide, an chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), radionuclide, an chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), radionuclide, an chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), radionuclide, an
  • immunomodulator a hormone, a hormone antagonist, an enzyme, oligonucleotide, an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxic agent, an angiogenesis inhibitor, and a combination thereof.
  • the drag is a maytansinoid, an auristatin, a dolastatin, or a calicheamicin.
  • the drug is DM1, DM3, DM4, MMAE, or MMAF.
  • an antibody (Ab) is conjugated to one or more drug moieties (D), e.g. about 1 to about 20 drug moieties per antibody, through a linker (L).
  • the linker comprises linker components selected from one or more of 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP”), valine-citrulline (“val-cit”), alanine- phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2- pyridylthio) pentanoate (“SPP”), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate (“SMCC”), and/or N-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
  • MC 6-maleimidocaproyl
  • MP maleimidoprop
  • the linker comprises MC-val-cit-PAB.
  • the immunoconjugate comprises SPP-DMl, SMCC-DM1 , BMPEO-DM1, MC-vc-PAB-MMAF, MC-vc-PAB-MMAE, MC-MMAF, or MC-MMAE.
  • the invention provides an immunoconjugate comprising an anti- Her3 antibody that kills tumor cells, in some embodiments, tumor cells are killed in vitro while in another aspect, tumor cells are killed in vivo.
  • administration of the immunoconjugate reduces tumor growth (in some embodiments, having about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or less reduction in tumor growth compared to a control tumor) or reduces time to tumor doubling.
  • the antibodies and immunoconjugates of the invention bind (such as specifically bind) Her3, and in some embodiments, may modulate one or more aspects of Her3 -associated effects, including but not limited to Her3 activation, Her3 downstream molecular signaling, Her3 ligand activation, Her3 ligand downstream molecular signaling, disruption of ligand binding to Her3, Her2 phosphorylation and/or Her3 multimerization, and/or Her3 ligand phosphorylation, and/or disruption of any biologically relevant Her3 and/or Her3 ligand biological pathway, and/or treatment and/or prevention of a tumor, cell proliferative disorder or a cancer; and/or treatment or prevention of a disorder associated with Her3 expression and/or activity (such as increased Her3 expression and/or activity).
  • the immunoconjugate of the invention specifically binds to Her3.
  • the antibody or immunoconjugate specifically binds to the Her3 extracellular domain (ECD).
  • the antibody or immunoconjugate specifically binds Her3 with a KD of 70 pM or stronger.
  • the antibody or immunoconjugate of the invention reduces, inhibits, and/or blocks Her3 activity in vivo and/or in vitro.
  • the antibody or immunoconjugate reduces, inhibits and/or blocks Her 3 autophosphorylation.
  • the antibody or immunoconjugate competes for binding with Her3-Iigand (reduces and/or blocks Her3 ligand binding to Her3).
  • the antibody or immunoconjugate competes for binding with Her3-Iigand (reduces and/or blocks Her3 ligand binding to Her3).
  • immunoconjugate is internalized upon binding to Her3 expressed on a mammalian cell.
  • the antibody or immunoconjugate inhibits, reduces or prevents Her3 her2 dimerization.
  • the invention provides use of an antibody or immunoconjugate of the invention in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
  • a disorder such as a cancer, a tumor, and/or a cell proliferative disorder.
  • the invention provides compositions comprising one or more antibodies or immunoconjugates of the invention and a carrier.
  • the carrier is pharmaceutically acceptable.
  • the invention provides use of an anti-Her3 antibody or immunoconjugate (in some embodiments, an anti-Her3 antibody or immunoconjugate of the invention) in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
  • a disorder such as a cancer, a tumor, and/or a cell proliferative disorder.
  • the invention provides methods and compositions useful for modulating disease states associated with expression and/or activity of Her3, such as increased expression and/or activity or undesired expression and/or activity or decreased expression and/or activity.
  • the invention provides methods for treating or preventing a tumor, a cancer, and/or a cell proliferative disorder associated with increased expression and/or activity of Her3, the methods comprising administering an effective amount of an anti-Her3 antibody or immunoconjugate (in some embodiments, an anti-Her3 antibody of the invention) to a subject in need of such treatment.
  • an anti-Her3 antibody or immunoconjugate in some embodiments, an anti-Her3 antibody of the invention
  • the invention provides methods for killing a cell (such as a cancer or tumor cell), the methods comprising administering an effective amount of an anti-Her3 antibody or immunoconjugate (in some embodiments, an anti-Her3 antibody of the invention) to a subject in need of such treatment.
  • a cell such as a cancer or tumor cell
  • an anti-Her3 antibody or immunoconjugate in some embodiments, an anti-Her3 antibody of the invention
  • the invention provides methods for reducing, inhibiting, blocking, or preventing growth of a tumor or cancer, the methods comprising administering an effective amount of an anti-Her3 antibody or immunoconjugate (in some embodiments, an anti-Her3 antibody of the invention) to a subject in need of such treatment.
  • an anti-Her3 antibody or immunoconjugate in some embodiments, an anti-Her3 antibody of the invention
  • the invention provides methods for treating or preventing a neuropathy or neurodegenerative disease, or repairing a damaged nerve cell, the methods comprising administering an effective amount of an anti-Her3 antibody or immunoconjugate (in some embodiments, an anti-Her3 antibody of the invention) to a subject in need of such treatment.
  • an anti-Her3 antibody or immunoconjugate in some embodiments, an anti-Her3 antibody of the invention
  • the invention concerns an article of manufacture, comprising: a container; a label on the container; and a composition contained within the container, wherein the composition comprises one or more anti-Her3 antibodies or
  • immunoconjugates of the invention wherein the composition is effective for the detection, diagnosis or prognosis of neoplasia associated with expression of Her 3 and the label on the container indicates that the composition can be used for the diagnosis or the prognosis of conditions characterized by overexpression of the Her3 protein receptor.
  • a composition comprising an antibody or immunoconjugate further comprises a carrier, which in some embodiments is pharmaceutically acceptable
  • an article of manufacture of the invention further comprises instructions for administering the composition (for e.g., the antibody) to a subject (such as instructions for any of the methods described herein).
  • the invention provides use of a kit of the invention in the preparation of a medicament for the therapeutic and/or prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a cell proliferative disorder.
  • a disorder such as a cancer, a tumor, and/or a cell proliferative disorder.
  • the article of manufacture or kit may also be used for determining whether an embedded biological sample contains human Her3 protein comprising: (a) an Her3-binding agent that specifically binds with an embedded human Her3 protein to form a binding complex; and (b) an indicator capable of signaling the formation of said binding complex, wherein the Her3 binding agent is a monoclonal antibody or a binding fragment thereof as set forth in the application. Diagnostic procedures using anti- Her3 antibody of the invention can be performed by diagnostic laboratories, experimental laboratories, practitioners, or private individuals. The clinical sample is optionally pre-treated for enrichment of the target being tested for. The user then applies a reagent contained in the kit in order to detect the changed level or alteration in the diagnostic component.
  • compositions comprising one or more of the herein described antibodies effective for use in the treating Her3 mediated hyper- proliferative disorders are also included.
  • An Her3 receptor antagonist includes antigen-binding fragments thereof that binds the Her3 receptor extracellularly and is effective in blocking cleavage of the receptor or activating the Her3 receptor mediated signaling cascade or preventing Her3 from dimerizing with Her2 and its attendant consequences.
  • the compositions can be provided in an article of manufacture or a kit.
  • Another aspect of the invention is an isolated nucleic acid encoding any one or more of the anti-Her3 antibodies of the invention, as well as a vector comprising the nucleic acid.
  • the human Her3 DNA sequence can be found using GenBank Accession Number (GenBank accession number - NM__001982). Methods of recombinant production of the invention antibodies are also within the scope of the invention.
  • Another aspect of the invention is a method of inhibiting or decreasing the proliferation of cancer cells by administering a Her3 antibody which results in blocking of the endogenous ligand to the Her3 receptor or inactivation/deactivation of Her 3 signaling.
  • Another aspect of the invention is a method of destroying cancer and tumor cells which express a Her3 receptor by administering to a patient in need thereof, a therapeutically effective amount of a composition comprising a Her3 receptor binding partner, e.g., any one or more of the Her3 specific antibodies disclosed herein effective for that purpose.
  • a further aspect of the invention is a method of alleviating cancer by administering an agonist or antagonist of Her 3 receptor.
  • the modulators of Her3 signaling can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabeled compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • the Her3 receptor binding partner useful in destroying cancer cells includes soluble ligands of the receptor, antibodies and fragments thereof that bind the Her3 receptor.
  • the binding partners can be conjugated to a cytotoxic agent.
  • the antibodies are preferably growth inhibitory antibodies.
  • the cytotoxic agent can be a toxin, antibiotic, radioactive isotope or nucleolytic enzyme.
  • a preferred cytotoxic agent is a toxin, preferably a small molecule toxin such as calicheamicin or a maytansinoid.
  • the antagonists and binding partners of Her 3 receptor can be synthetically or recombinantly produced or otherwise isolated.
  • nucleic acid or a “nucleic acid molecule” “nucleic acid molecule encoding Her3” have been used for convenience to encompass DNA encoding Her3, RNA (including pre- mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. As well it encompasses any DNA or RNA molecule, either single- or double- stranded and, if single-stranded, the molecule of its complementary sequence in either linear or circular form.
  • target nucleic acid and. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
  • nucleic acids are "isolated.”
  • This term when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • RNA the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues).
  • An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence and fragments or portions thereof, of a naturally occurring or synthetic molecule.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid
  • a protein or nucleic acid that is the predominant species present in a preparation is substantially purified.
  • an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene.
  • the term "purified” in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • "Purify” or “purification” in other embodiments means removing at least one contaminant from the composition to be purified.
  • an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • an "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes, in preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Comassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • This can be a gene and a regulatory sequence(s) which are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences(s).
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist; the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • cell means cells, cell line or cell culture
  • progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.
  • the "host cells” used in the present invention generally are prokaryotic or eukaryotic hosts. Examples of suitable host cells are described in Section B. Vectors, Host Cells and Recombinant Methods: (vii) Selection and transformation of host cells.
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extra chromosomal element or by chromosomal integration.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed.
  • transfected host cell and “transformed” refer to the introduction of
  • the cell is termed "host cell” and it may be either prokaryotic or eukaryotic.
  • Typical prokaryotic host cells include various strains of E. coli.
  • Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary or cells of human origin.
  • the introduced DNA sequence may be from the same species as the host cell or a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA.
  • modulation and modulation of expression mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the Her3 receptor or the level of the protein - Her3 or modulation of the activity attendant the native Her3 receptor - signaling cascade etc. Inhibition is often the preferred form of modulation of expression and the protein receptor is often a preferred target nucleic acid.
  • replicable expression vector and "expression vector” refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA.
  • Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell.
  • the vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (foreign) DNA may be generated.
  • vector means a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself.
  • plasmid and vector are sometimes used interchangeably, as the plasmid is the most commonly used form of vector at present.
  • Typical expression vectors for mammalian cell culture expression are based on pR 5 (EP 307,247), pSV16B (WO 91/08291), and pVL1392 (Pharmingen).
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • protein or “polypeptide” are intended to be used interchangeably. They refer to a chain of two (2) or more amino acids which are linked together with peptide or amide bonds, regardless of post-translational modification (eg., glycosylation or
  • polypeptides of this invention may comprise more than one subunit, where each subunit is encoded by a separate DNA sequence.
  • Amino acids may be referred to herein either by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.
  • Naturally occurring residues may be divided into groups based on common side-chain properties:
  • H is, Lys, Arg
  • Percent (%) amino acid sequence identity with respect to the ligand or receptor polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in such a ligand or receptor sequence identified herein, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • ALIGN-2 sequence comparison computer program
  • ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in the table below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif, or can be compiled from the source code provided in the table below.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • a useful method for identification of certain residues or regions in a protein that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells Science, 244: 1081-1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with a binding target.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed variants are screened for the desired activity.
  • dihedral angle refers to a rotation about a bond. See e.g., Creighton, T. E., (1993) Protein: Structures and Molecular Properties, 2 ed., W. H. Freeman and Company, New York, N.Y.
  • phi is a dihedral angle that denotes a rotation about the N ⁇
  • substantially identical with respect to an antibody polypeptide sequence shall be construed as an antibody exhibiting at least 70%, preferably 80%, more preferably 90% and most preferably 95% sequence identity to the reference polypeptide sequence.
  • the term with respect to a nucleic acid sequence shall be construed as a sequence of nucleotides exhibiting at least about 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity to the reference nucleic acid sequence.
  • the length of the comparison sequences will generally be at least 25 amino acids.
  • nucleic acids the length will generally be at least 75 nucleotides.
  • Sequence identity may be measured using sequence analysis software (e.g, Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
  • sequence analysis software e.g, Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705
  • identity or “homology” shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a
  • a molecule is "substantially similar" to another molecule if both molecules have substantially similar structures or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical. Neither N- or C- terminal extensions nor insertions shall be construed as reducing identity or homology.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site located at www.
  • sequences are then said to be "substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man- made variants.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of one of the number of contiguous positions selected from the group consisting typically of from 20 to 600, usually about 50 to about 200, more usually about 00 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art.
  • Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • substitutions as used within this invention is meant to refer to amino acid substitutions which substitute functionally equivalent amino acids.
  • Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide.
  • one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide.
  • substitutions within a group may be considered conservative with respect to structure and function.
  • the skilled artisan will recognize that the role of a particular residue is determined by its context within the three-dimensional structure of the molecule in which it occurs. For example, Cys residues may occur in the oxidized (disulfide) form, which is less polar than the reduced (thiol) form.
  • the long aliphatic portion of the Arg side chain may constitute a critical feature of its structural or functional role, and this may be best conserved by substitution of a nonpolar, rather than another basic residue.
  • side chains containing aromatic groups Trp, Tyr, and Phe
  • substitution of one of these side chains with a member of the acidic or uncharged polar group may be conservative with respect to structure and function.
  • Residues such as Pro, Gly, and Cys (disulfide form) can have direct effects on the main chain
  • amino acid sequence variant refers to a polypeptide that has amino acid sequences that differ to some extent from a native sequence polypeptide.
  • substitutional refers to molecules with some differences in their amino acid sequences as compared to a native amino acid sequence. The substitutions may be single, where only one amino acid in the molecule as been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.
  • amino acid sequence variants of Her3 receptor will possess at least about 70% homology with the native sequence Her3 receptor, preferably, at least about 80%, more preferably at least about 85%, even more preferably at least about 90% homology, and most preferably at least 95%.
  • the amino acid sequence variants can possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
  • "Insertional" variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to either the .alpha.-carboxyl or .alpha.-amino functional group of the amino acid.
  • “Deletional” variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.
  • immunoglobulin or "antibody” (used interchangeably herein) is used in the broadest sense and is meant to encompass an immunoglobulin molecule obtained by in vitro or in vivo generation of an immunogenic response.
  • a broad scope refers to an antigen- binding protein having a basic four-pofypeptide chain structure consisting of two heavy and two light chains, said chains being stabilized, for example, by interchain disulfide bonds, which has the ability to specifically bind antigen. Both heavy and light chains are folded into domains.
  • domain refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by .beta.-pleated sheet and/or intrachain disulfide bond. Domains are further referred to herein as “constant” or “variable”, based on the relative lack of sequence variation within the domains of various class members in the case of a "constant” domain, or the significant variation within the domains of various class members in the case of a "variable” domain.
  • Constant domains on the light chain are referred to interchangeably as “light chain constant regions”, “light chain constant domains”, “CL” regions or “CL” domains)
  • Constant domains on the heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “CH” regions or “CH” domains)
  • “Variable” domains on the light chain are referred to interchangeably as “light chain variable regions”, “light chain variable domains", “VL” regions or “VL” domains).
  • “Variable” domains on the heavy chain are referred to interchangeably as “heavy chain variable regions”, “heavy chain variable domains", “VH” regions or “VH” domains).
  • bispecific antibodies so long as they exhibit the desired biological activity
  • polyclonal antibodies single chain anti-Her3 antibodies, and fragments of anti-Her3 antibodies as long as they exhibit the desired biological or immunological activity.
  • the antibodies may be genetically engineered antibodies and/or produced by recombinant DNA techniques.
  • Fully human antibodies can also be produced by phage display, gene and chromosome transfection methods, as well as by other means.
  • the L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ , and ,mu. , respectively.
  • the ⁇ and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgA l, and IgA2.
  • an “intact” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CHI , CH2 and C -
  • the constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody has one or more effector functions.
  • region refers to a part or portion of an antibody chain and includes constant or variable domains as defined herein, as well as more discrete parts or portions of said domains.
  • light chain variable domains or regions include "complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs”, as defined herein.
  • antigen as used herein, means a molecule which is reactive with a specific antibody.
  • Epitopes refers to a site on an antigen to which an antibody binds.
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x- ray crystallography and 2-dimensional nuclear magnetic resonance.
  • Antibodies of "IgG class” refers to antibodies of IgGl, IgG2, IgG3, and IgG4.
  • the numbering of the amino acid residues in the heavy and light chains is that of the EU index (Kabat, et al., "Sequences of Proteins of Immunological Interest", 5 th ed., National Institutes of Health, Bethesda, Md. (1991); the EU numbering scheme is used herein).
  • antibody mutant refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such mutant necessarily have less than 100% sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Since the method of the invention applies equally to both polypeptides, antibodies and fragments thereof, these terms are sometimes employed interchangeably.
  • mutant is interchangeable with “mutationally-altered” and "gfycosylation site altered”.
  • the terms refer to an antibody that comprises at least one immunoglobulin variable region containing at least one mutation that modifies a V region glycosylation site.
  • a mutant immunoglobulin refers to an immunoglobulin (e.g., F(ab') 2 , Fv, Fab, bifunctional antibodies, antibodies, etc.) comprising at least one immunoglobulin variable region containing at least one mutation that modifies a V region glycosylation site.
  • a mutant immunoglobulin chain has at least one mutation that modifies a V region glycosylation site, typically in the V region framework.
  • the pattern i.e., frequency and or location(s) of occurrence
  • V region glycosylation sites is altered in a mutant immunoglobulin.
  • variable domain in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 1 10-amino acid span of the variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., abat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia, C. el at. (1989), Nature 342: 877).
  • the more highly conserved portions of variable domains are called the framework (FR) of 15-30 amino acids separated by shorter "hypervariable regions" (9-12 amino acids long).
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a .beta.-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the .beta.-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. See Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
  • variable domain residue numbering as in Kabat or “amino acid position numbering as in Kabat”, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a “complementarity determining region" or
  • CDR e.g. around about residues 24-34 (Li), 50-56 (L2) and 89-97 (L3) in the V L> and around about 1-35 (HI), 50-65 (H2) and 95-102 (H3) in the V H ; Kabate et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g.
  • a “variable region” has the structure of an antibody variable region from a heavy or light chain.
  • Antibody heavy and light chain variable regions contain three complementarity determining regions ("CDRs") interspaced onto a framework ("FW").
  • CDRs complementarity determining regions
  • FW framework
  • the CDRs are primarily responsible for recognizing a particular epitope. It is well known that epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the hypervariable regions are generally referred to as complementarity determining regions ("CDR") and are interposed between more conserved flanking regions referred to as framework regions ("FW").
  • CDR complementarity determining regions
  • FW framework regions
  • Amino acids associated with framework regions and CDRs can be numbered and aligned by approaches described by Kabat et al, Sequences of Proteins of immunological Interest, U.S. Department of Health and Human Services, 1991 ; C. Chothia and A.M.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • CDRs are primarily responsible for binding to a particular epitope. Within a particular CDR, there are a few specificity determining residues (SDRs) which are of greater importance for binding to an epitope (see Kashmiri et al., Methods 36:25-34, 2005; Presta, Advanced Drug Delivery Reviews 58:640-656, 2006). SDRs can be identified, for example, through the help of target protein-antibody three-dimensional structures and mutational analysis of antibody combining sites.
  • the PD-1 binding proteins of the present invention do not always require both a variable heavy chain and light chain domain to render PD- I specificity but may only need a single CDR loop or a fragment of a functional antibody (see, e.g. , Xu and Davis, 2000, Immunity 13:37-45 and Levi et al, 1993, Proc. Natl. Acad. Sci. USA 90:4374-78 (for CDR3 specificity); Williams et a!., 1989, Proc. Natl. Acad. Sci. USA 86:5537-41 (CDR2 specificity); and, Welling et al., 1991, J. Chromatography 548:235-42 (10 amino acid miniantibody).
  • Immunoglobulins or antibodies can exist in monomeric or polymeric form.
  • the term "antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding).
  • the term “conformation” refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain or region thereof).
  • the phrase “light (or heavy) chain conformation” refers to the tertiary structure of a light (or heavy) chain variable region
  • the phrase “antibody conformation” or “antibody fragment conformation” refers to the tertiary structure of an antibody or fragment thereof.
  • the fragment exhibits qualitative biological activity in common with a full-length antibody.
  • a functional fragment or analog of an anti-IgE antibody is one which can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, Fc.epsilon.RJ.
  • Antibody fragments can be prepared by in vitro manipulation of antibodies (e.g., by limited proteolysis of an antibody), or via recombinant DNA technology (e.g., the preparation of single-chain antibodies from phage display libraries).
  • Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (U.S. Patent. No. 5,641,870, Example 2; Zapata et af., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fv, single chains, and single-chain antibodies. Other than "bispecific” or “bifunctional" immunoglobulins or antibodies, an immunoglobulin or antibody is understood to have each of its binding sites identical.
  • a functional variant of the antibody molecule according to the invention is an antibody molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar substrate specificity or cleavage of the substrate.
  • the term “functional variant” also includes “a fragment”, “an allelic variant”, “variant based on the degenerative nucleic acid code” or “chemical derivatives”. Such a “functional variant” e.g. may carry one or several point mutations, one or several nucleic acid exchanges, deletions or insertions or one or several amino acid exchanges, deletions or insertions. Said functional variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.
  • allelic variant is a variant due to the allelic variation, e.g. differences in the two alleles in humans. Said variant is still retaining its biological activity such as antibody target binding activity, at least in part or even going along with an improvement said biological activity.
  • a "variant based on the degenerative of the genetic code” is a variant due to the fact that a certain amino acid may be encoded by several different nucleotide triplets. Said variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.
  • a “fusion protein” and a “fusion polypeptide” refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property.
  • the property may be a biological property, such as activity in vitro or in vivo.
  • the property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc.
  • the two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other.
  • a "chemical derivative" according to the invention is an antibody molecule according to the invention chemically modified or containing additional chemical moieties not normally being part of the molecule. Such moieties may improve the molecule's activity such as target destruction (e.g. killing of tumor cells) or may improve its solubility, absorption, biological half life, etc.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (CH 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly
  • Fab' fragments corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fc Fc receptors
  • Fc region as used herein is meant the polypeptides comprising the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and part of the flexible hinge N-terminal to these domains.
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU numbering scheme.
  • Fc may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment.
  • Ergo, by "outside the Fc region” as used herein is meant the region of an antibody that does not comprise the Fc region of the antibody.
  • “outside the Fc region” for an IgGl antibody is herein defined to be from the N-terminus up to and including residue T225 or C229, wherein the numbering is according to the EU numbering scheme.
  • the Fab region and part of the hinge region of an antibody are outside the Fc region.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a y receptor) and includes receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyros ine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyros ine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR FcR
  • FcRn neonatal receptor
  • an “Fv” fragment is the minimum antibody fragment which comprises the variable domains of its heavy chain and light chain and thus contains a complete antigen recognition and binding site.
  • This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH -VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the H -VL dimer.
  • the six CDRs confer antigen binding specificity to the antibody.
  • One or more scFv fragments may be linked to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites.
  • single chain variable fragment or scFv refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the V H and VL domains which enables the sFv to form the desired structure for antigen binding.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerization is carried out by di- f tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology II:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).
  • minibody means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerization region which is connected to the scFv via a hinge region (e.g. also from IgGl) and a linker region. The disulphide bridges in the hinge region are mostly formed in higher cells and not in prokaryotes.
  • an antibody according to the invention is a Her3-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).
  • diabodies refers to a small antibody fragments with two antigen- binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (V L ) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • V L light chain variable domain
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11 161 ; and Hollinger et al., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993).
  • triabody By triabody the skilled person means a: trivalent homotrimeric scFv derivative ( ortt et al. 1997 Protein Engineering 10: 423-433). ScFv derivatives wherein VH-VL are fused directly without a linker sequence lead to the formation of trimers.
  • the Fab fragment also designated as F(ab) also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains have a free thiol group.
  • F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab') 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990);
  • an antibody "which binds" an antigen of interest e.g. a tumor-associated polypeptide antigen target., e.g., Her3, is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins.
  • the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).
  • the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat, No. 4,816,567;
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab*, F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity- determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity, in some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • CDR complementarity- determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity.
  • the number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc) ; typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PRIMATIZED.RTM. antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest, Methods of making humanized antibodies are known in the art.
  • Human antibodies can also be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et at, J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(l):86-95 (1991).
  • “Functional fragments” or “antigen-binding fragments: of the antibodies of the invention are those fragments that retain binding to Her3 , e.g,. that have substantially the same affinity as the intact full chain molecule from which they are derived.
  • effector or “effector moiety” or “effector component” is a molecule that is bound (or linked, or conjugated), either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, to an antibody.
  • the "effector” can be a variety of molecules including, e.g., detection moieties including radioactive compounds, fluorescent compounds, an enzyme or substrate, tags such as epitope tags, a toxin, activatable moieties, a chemotherapeutic or cytotoxic agent, a chemoattractant, a lipase; an antibiotic; or a radioisotope emitting "hard” e.g., beta radiation.
  • ADCC antibody dependent cellular cytotoxicity
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. Natural Killer (NK) cells, neutrophils, and macrophages
  • NK cells Natural Killer cells
  • neutrophils neutrophils
  • macrophages e.g., neutrophils, and macrophages
  • the antibodies “arm” the cytotoxic cells and are absolutely required for such killing.
  • the primary cells for mediating ADCC, NK cells express Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RT, Fc.gamma.RH and Fc.gamma.RIII.
  • ADCC activity of a molecule of interest is assessed in vivo, e.g., in a animal model such as that disclosed in Clynes at al. PNAS (USA) 95:652-656 (1998).
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CI q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
  • CI q first component of the complement system
  • a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human
  • a humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, is transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains.
  • the constant domains of the antibody molecule is derived from those of a human antibody, in some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PrimatizedTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • Human antibodies may be desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716, 1 1 1 ; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741 , each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes., see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;
  • European Patent No. 0 598 877 U.S. Pat. Nos. 5,413,923; 5,625, 126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 ; and 5,939,598, which are incorporated by reference herein in their entireties.
  • companies such as Abgenix, Inc. (Fremont, Calif.) and Medarex (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
  • Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • is used to guide the selection of a completely human antibody recognizing the same epitope Jespers et al., Biotechnology 12:899-903 (1988).
  • a chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule is derived from those of a human antibody.
  • the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.
  • a "species-dependent antibody,” e.g., a mammalian anti-human Her3 antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species.
  • the species-dependent antibody "bind specifically" to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1 XI 0-7 M, preferably no more than about 1 X 10-8 and most preferably no more than about 1 X.10-9 M) but has a binding affinity for homologue of the antigen from a second non-human mammalian species which is at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen.
  • the species- dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a human antibody.
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells and neutrophils
  • the effector cells may be isolated from a native source, e.g., from blood.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. At 21 1 , 1 131, 1 125, Y 90, Re 186, Re 188, Sm 153, Bi 212, P 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g.
  • methotrexate methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various anti-tumor or anticancer agents disclosed below. Other cytotoxic agents are described below.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN.TML); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trof
  • elliptinium acetate etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2 ⁇ 2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g.
  • paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTER.RTM., Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C;
  • aminopterin xeloda
  • ibandronate CPT- 11
  • topoisomerase inhibitor RFS 2000 topoisomerase inhibitor
  • DMFO dif!uoromethylornithine
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-.
  • imidazoles 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • a "giycosylation variant” "Glycoform variant” antibody herein is an antibody with one or more carbohydrate moieties attached thereto which differ from one or more carbohydrate moieties attached to a main species antibody.
  • Examples of giycosylation variants herein include antibodies with a Gl or G2 oligosaccharide structure, instead of a GO
  • oligosaccharide structure attached to an Fc region thereof, antibody with one or two
  • Glycosylation sites refer to amino acid residues which are recognized by a eukaryotic cell as locations for the attachment of sugar residues.
  • the amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage), serine (O-linkage), and threonine (Olinkage) residues.
  • the specific site of attachment is typically signaled by a sequence of amino acids, referred to herein as a "glycosylation site sequence".
  • the glycosylation site sequence for N-Hnked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X may be any of the conventional amino acids, other than proline,
  • the predominant glycosylation site sequence for O-Hnked glycosylation is: -(Thr or Ser)-X-X-Pro-, where X is any conventional amino acid.
  • the recognition sequence for glycosaminoglycans (a specific type of sulfated sugar) is -Ser-Gly-X-Gly-, where X is any conventional amino acid.
  • the terms "N-linked” and "O- linked” refer to the chemical group that serves as the attachment site between the sugar molecule and the amino acid residue.
  • N-iinked sugars are attached through an amino group; O-linked sugars are attached through a hydroxyl group.
  • glycosylation site sequences in a protein are necessarily glycosylated; some proteins are secreted in both glycosylated and nonglycosylated forms, while others are fully glycosylated at one glycosylation site sequence but contain another glycosylation site sequence that is not glycosylated. Therefore, not all glycosylation site sequences that are present in a polypeptide are necessarily glycosylation sites where sugar residues are actually attached.
  • V region glycosylation site is a position in a variable region where a carbohydrate, typically an oligosaccharide, is attached to an amino acid residue in the polypeptide chain via an N-linked or O-linked covalent bond. Since not all glycosylation site sequences are necessarily glycosylated in a particular cell, a glycosylation site is defined operationally by reference to a designated cell type in which glycosylation occurs at the site, and is readily determined by one of ordinary skill in the art. Thus, a mutant antibody has at least one mutation that adds, subtracts, or relocates a V region glycosylation site, such as, for example, an N-iinked glycosylation site sequence.
  • the mutation(s) are substitution mutations that introduce conservative amino acid substitutions, where possible, to modify a glycosylation site.
  • the parent immunoglobulin sequence contains a glycosylation site in a V region framework, particularly in a location near the antigen binding site (for example, near a CDR)
  • the glycosylation site sequence is mutated (e.g., by site-directed mutagenesis) to abolish the glycosylation site sequence, typically by producing a conservative amino acid substitution of one or more of the amino acid residues comprising the glycosylation site sequence.
  • glycosylation site in a CDR
  • that glycosylation site is preferably retained. If the parent immunoglobulin specifically binds an epitope that comprises only polypeptide, glycosylation sites occurring in a CDR are preferably eliminated by mutation (e.g., site-directed mutation).
  • glycosylation-reduced antibodies and “glycosylation-reduced immunoglobulin chains” are mutant antibodies and mutant immunoglobulin chains, respectively, in which at least one glycosylation site that is present in the parent sequence has been destroyed by mutation and is absent in the mutant sequence.
  • glycosylation-supplemented antibodies and “glycosyiation-supplemented immunoglobulin chains” are mutant antibodies and mutant immunoglobulin chains, respectively, in which at least one glycosylation site is present in the mutant sequence at a position where no glycosylation site occurs in the parent sequence.
  • glycosylation-supplemented antibodies that have a higher binding affinity for a carbohydrate-containing epitope than does the parent antibody have a glycosylation site present in a CDR where the parent antibody does not.
  • a glycosylation-supplemented antibody that specifically binds an epitope that contains polypeptide sequence but no carbohydrate have a lower affinity that the parental antibody.
  • Parent immunoglobulin sequence refer herein to a reference amino acid sequence or polynucleotide sequence, respectively.
  • a parent polynucleotide sequence may encode a naturally-occurring immunoglobulin chain of a fragment thereof wherein glycosylation site sequences, if any, present in the V region occur about at the same relative amino acid residue position(s) at which glycosylation site sequence(s) are present in naturally-occurring immunoglobulin sequence(s) from which the parent sequence(s) were derived.
  • mutations such as site-directed mutations, are introduced into a parent immunoglobulin sequence, the resultant sequence is referred to as a mutant immunoglobulin sequence (or a mutated immunoglobulin sequence).
  • a “conjugate” refers to any hybrid molecule, including fusion proteins and as well as molecules that contain both amino acid or protein portions and non-protein portions. Conjugates may be synthesized by a variety of techniques known in the art including, for example, recombinant DNA techniques, solid phase synthesis, solution phase synthesis, organic chemical synthetic techniques or a combination of these techniques. The choice of synthesis will depend upon the particular molecule to be generated. For example, a hybrid molecule not entirely "protein” in nature may be synthesized by a combination of recombinant techniques and solution phase techniques.
  • ADCs antibody drug-conjugate
  • the strategy of this approach is to deliver a toxic payload to the cancer cell via an antibody that targets a cancer-specific antigen.
  • This strategy requires that the potent drug is internalized via the antibody-antigen complex, released within the cell and specifically kills the cancer cells (Bhaskar et al, Cancer Res, 2003;
  • the potent drug is internalized via the antibody-antigen complex, released within the cell and specifically kills the cancer cells.
  • the molecular target is not expressed in essential organs that are accessible to circulating antibodies.
  • the target must be at the plasma membrane of cancer cells to allow antibody access.
  • ADCC antibody dependent cellular cytotoxicity
  • cytotoxic T lymphocytes cytotoxic T lymphocytes, natural killer cells, activated macrophages
  • effector cells then specifically kill the targeted tumor cells.
  • antibody phage library refers to the phage library used in the affinity maturation process described above and in Hawkins et at., J. Mol Biol.254: 889- 896 (1992), and in Lowman et al., Biochemistry 30(45): 10832-10838 (1991).
  • Each library comprises a hypervariable region (eg. 6-7 sites) for which all possible amino acid substitutions are generated.
  • the antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle and expressed on the exterior of the phage.
  • target molecule means any molecule, not necessarily a protein, for which it is desirable to produce an antibody or ligand.
  • the target will be a protein and most preferably the target will be an antigen -EGFR or human her3 or a Her2/Her3 dimer.
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • prodrug refers to a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • prodrug refers to a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • cancer neoplasia
  • cancer neoplasia
  • cancer neoplasia
  • cancer neoplasia
  • cancer neoplasia
  • premalignant lesions neoplasms, as well as premalignant lesions.
  • Alleviation of cancer refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of alleviation include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mamma! is "alleviated” for a Her3 receptor-expressing cancer if, after receiving a therapeutic amount of a Her3 receptor agonist according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells, reduction in the tumor size; inhibition (i.e.
  • the Her3 receptor antagonist or antibody may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.
  • Detection and measurement of these above indicators are known to those of skill in the art, including, but not limited for example, reduction in tumor burden, inhibition of tumor size, reduction in proliferation of secondary tumors, expression of genes in tumor tissue, presence of biomarkers, lymph node involvement, histologic grade, and nuclear grade.
  • terapéuticaally effective amount refers to an amount of an agonist and/or antagonist antibody effective to "alleviate" a disease or disorder in a subject or mammal.
  • a “therapeutically effective amount”, in reference to the treatment of tumor, refers to an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder.
  • a “therapeutically effective amount” of a Her3 antibody for purposes of treatment of tumor may be determined empirically and in a routine manner.
  • the term “inhibition of tumor volume” refers to any decrease or reduction in a tumor volume.
  • the term “tumor volume” refers to the total size of the tumor, which includes the tumor itself plus affected lymph nodes if applicable. Tumor volume may be determined by a variety of methods known in the art, such as, e.g. by measuring the dimensions of the tumor using calipers, computed tomography (CT) or magnetic resonance imaging (MRI) scans, and calculating the volume using equations based on, for example, the z-axis diameter, or on standard shapes such as the sphere, ellipsoid, or cube.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • biologically active indicates that a composition or compound itself has a biological effect, or that it modifies, causes, promotes, enhances, blocks, reduces, limits the production or activity of, or reacts with or binds to an endogenous molecule that has a biological effect.
  • a “biological effect” may be but is not limited to one that stimulates or causes an immunreactive response; one that impacts a biological process in an animal; one that impacts a biological process in a pathogen or parasite; one that generates or causes to be generated a detectable signal; and the like.
  • Biologically active compositions, complexes or compounds may be used in therapeutic, prophylactic and diagnostic methods and compositions.
  • Biologically active compositions, complexes or compounds act to cause or stimulate a desired effect upon an animal.
  • desired effects include, for example, preventing, treating or curing a disease or condition in an animal suffering therefrom; limiting the growth of or killing a pathogen in an animal infected thereby; augmenting or altering the phenotype or genotype of an animal; and stimulating a prophylactic immunoreactive response in an animal.
  • biologically active indicates that the composition, complex or compound has an activity that impacts an animal suffering from a disease or disorder in a positive sense and/or impacts a pathogen or parasite in a negative sense.
  • a biologically active composition, complex or compound may cause or promote a biological or biochemical activity within an animal that is detrimental to the growth and/or maintenance of a pathogen or parasite; or of cells, tissues or organs of an animal that have abnormal growth or biochemical characteristics, such as cancer cells, or cells affected by autoimmune or inflammatory disorders.
  • compositions, complex or compound may be biologically active in therapeutic, diagnostic and prophylactic applications.
  • a composition, complex or compound that is described as being “biologically active in a cell” is one that has biological activity in vitro (i.e., in a cell culture) or in vivo (i.e., in the cells of an animal).
  • a “biologically active portion” of a compound or complex is a portion thereof that is biologically active once it is liberated from the compound or complex. It should be noted, however, that such a component may also be biologically active in the context of the compound or complex.
  • invention constructs may comprise an additional moiety to facilitate internalization and/or uptake by a target cell.
  • a “patient” or “subject” or “host” refers to a mammal such as a human or non- human animal.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (R ).
  • TTP time to disease progression
  • R response rate
  • the progress of therapy can be assessed by routine methods, usually by measuring serum PSA (prostate specific antigen) levels; the higher the level of PSA in the blood, the more extensive the cancer.
  • ⁇ assays for detecting PSA are available, e.g, Hybritech Tandem-E® and Tandem-R® PSA assay kits, the Yang ProsCheck® polyclonal assay (Yang Labs, Bellevue, Wash.), Abbott Imx® (Abbott Labs, Abbott Park, 111.) , etc. Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
  • TRUS transrectal ultrasonography
  • TRNB transrectal needle biopsy
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Her3 encompasses all members of the Her3 receptor family and in particular, the Her3 protein comprising the aminoa cid and cDNA sequence as set forth in each of SEQ ID NO: 1 and SEQ ID NO:2 in U.S Provisional Application Serial No. 61/250,060, filed October 9 2009, which is incorporated by reference herein in its entirety.
  • the Her3 ligands include Jagged l, Jagged2, Deltal, Delta3, and Delta4.
  • "Her3" cDNA and deduced amino acid sequence is as set forth in SEQ ID NO: l .
  • a “full length” Her3 receptor protein or nucleic acid refers to a polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type Her3 polynucleotide or polypeptide sequences.
  • a full length Her3 nucleic acid will typically comprise all of the exons that encode for the full length, naturally occurring protein.
  • the “full length” may be prior to, or after, various stages of post-translation processing.
  • an “antibody that inhibits the growth of cancer cells expressing Her3 receptor or a “growth inhibitory” antibody is one which binds to and results in measurable growth inhibition of cancer cells expressing or overexpressing Her3 receptor. Growth inhibition of tumor cells in vivo can be determined in various ways. The antibody is growth inhibitory in vivo if
  • an antibody that binds to "Her3” includes an antibody that preferably binds Her3 and prevents dimerization with Her2.
  • a “Her3 receptor-expressing cancer” is a cancer comprising cells that have Her3 receptor protein present on the cell surface.
  • a "Her3 receptor-expressing cancer” produces sufficient levels of Her 3 receptor on the surface of cells thereof, such that a Her3 receptor agonist/antagonist or antibody can bind thereto and have a therapeutic effect with respect to the cancer.
  • a cancer which "overexpresses" Her3 receptor is one which has significantly higher levels of Her3 receptor at the cell surface thereof, compared to a noncancerous cell of the same tissue type.
  • Her3 receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the Her3 receptor protein present on the surface of a cell (e.g. via an immunohistochernistry assay; FACS analysis).
  • Her 3 receptor-encoding nucleic acid or mRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; see W098/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR).
  • FISH fluorescent in situ hybridization
  • PCR polymerase chain reaction
  • RT-PCR real time quantitative PCR
  • various in vivo assays are available to the skilled practitioner. For example, 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.
  • a detectable label e.g. a radioactive isotope
  • an "antagonist" of Her 3 receptor in addition to binding Her3 receptor has a direct effect on a Her3 receptor bearing cell
  • the term "antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native Her3 receptor protein.
  • the Her3 receptor agonist will bind Her3 receptor and or her3 her2 complex, and as well, initiate or mediate the signaling event associated with the Her3 receptor or the Her2/Her3 dimer.
  • the ability to induce Her3 receptor activation can be quantified using techniques known in the art such as reporter constructs such as Beta- galactosidase, chloramphenicol acetyl transferase (CAT) or luciferase.
  • the Her3 receptor antagonist will inhibit signaling transmitted from the Her3 receptor or the Her2/Her3 dimer or prevent Her3 from associating with Her2 or another member of the EGFR family.
  • Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants thereof etc.
  • Methods for identifying agonists or antagonists of a Her3 receptor polypeptide are known in the art.
  • An exemplary method proposes contacting a Her3 bearing cells or tissue with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the Her3 receptor.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
  • administering includes any method of delivery of a compound of the present invention, including but not limited to, a pharmaceutical composition or therapeutic agent, into a subject's system or to a particular region in or on a subject.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • Parenteral administration and “administered parenterally” means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • Compositions and methods of the invention include an anti-Her3 "in association with" or
  • each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at several intervals over a given period of time.
  • the separate components may be administered to a subject by the same or by a different route (e.g., wherein an anti-Her3 antibody formulation is administered parenterally and gefitinib is administered orally).
  • a "disorder" is any condition that would benefit from treatment with the polypeptide. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • Bio sample as used herein is a sample of biological tissue or cells that contains nucleic acids or polypeptides, e.g., Her3 or Her3 protein, polynucleotide or transcript. Such samples include, but are not limited to, tissue isolated from primates (e.g., humans) or from rodents (e.g., mice, and rats). Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., human.
  • Providing a biological sample means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from a human, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo,
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • label or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • useful labels include fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, colloidal gold, luminescent nanocrystals (e.g. quantum dots), haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • the radioisotope may be, for example, H, C, P, S, or 125 I. In some cases, particularly using antibod ies against the proteins of the invention, the radioisotopes are used as toxic moieties, as described below. Any method known in the art for conjugating the antibody to the label may be employed.
  • radiolabeled peptides or radiolabeled antibody compositions may be extended by the addition of substances that stabilize the radiolabeled peptide or antibody and protect it from degradation. Any substance or combination of substances that stabilize the radiolabeled antibody may be used including those substances disclosed in U.S. Pat. No. 5,961,955.
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially a Her3 expressing cancer cell, either in vitro or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of PSCA expressing cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fiuorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:
  • the taxanes are anticancer drugs both derived from the yew tree.
  • Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in ceils.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or
  • immunoglobulins include hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
  • the monoclonal antibodies herein include chimeric, hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of the antibody of interest with a constant domain (e.g. "humanized” antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with
  • heterologous proteins regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv), so long as they exhibit the desired biological activity or properties.
  • antibody fragments e.g., Fab, F(ab') 2 , and Fv.
  • the resulting "chimeric" Her3 antibodies in turn, can be humanized by techniques known to one skilled in the art.
  • the affinity of a chimeric, humanized or human anti-Her3 antibody may be evaluated using a direct binding assay or a competitive binding assay, as exemplified below.
  • Naturally occurring (wildtype) antibody molecules are Y-shaped molecules consisting of four polypeptide chains, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Both types of polypeptide chains have constant regions, which do not vary or vary minimally among antibodies of the same class (i.e., IgA, IgM, etc.), and variable regions. The variable regions are unique to a particular antibody and comprise a recognition element for an epitope.
  • the carboxy-terminai regions of both heavy and light chains are conserved in sequence and are called the constant regions (also known as C- domains).
  • the amino-terminal regions also known as V-domains
  • the antibody specifically recognizes and binds to an antigen mainly through six short complementarity-determining regions (CDRs) located in their V-domains.
  • Each light chain of an antibody is associated with one heavy chain, and the two chains are linked by a disulfide bridge formed between cysteine residues in the carboxy-terminal region of each chain, which is distal from the amino terminal region of each chain that constitutes its portion of the antigen binding domain.
  • Antibody molecules are further stabilized by disulfide bridges between the two heavy chains in an area known as the hinge region, at locations nearer the carboxy terminus of the heavy chains than the locations where the disulfide bridges between the heavy and light chains are made.
  • the hinge region also provides flexibility for the antigen-binding portions of an antibody.
  • An antibody's specificity is determined by the variable regions located in the amino terminal regions of the light and heavy chains.
  • variable regions of a light chain and associated heavy chain form an "antigen binding domain" that recognizes a specific epitope; an antibody thus has two antigen binding domains.
  • the antigen binding domains in a wild type antibody are directed to the same epitope of an immunogenic protein, and a single wild type antibody is thus capable of binding two molecules of the immunogenic protein at the same time.
  • a wild type antibody is monospecific (i.e., directed to a unique antigen) and divalent (i.e., capable of binding two molecules of antigen).
  • Polyclonal antibodies are generated in an immunogenic response to a protein having many epitopes.
  • a composition (e.g., serum) of polyclonal antibodies thus includes a variety of different antibodies directed to the same and to different epitopes within the protein.
  • Methods for producing polyclonal antibodies are known in the art (see, e.g., Cooper et al., Section III of Chapter 1 1 in; Short Protocols in Molecular Biology, 2nd Ed., Ausubel et a!., eds., John Wiley and Sons, New York, 1 92, pages 1 1-37 to 1 1-41).
  • Antipeptide antibodies are generated in a humoral response to a short (typically, 5 to 20 amino acids) immunogenic polypeptide that corresponds to a few (preferably one) isolated epitopes of the protein from which it is derived.
  • a plurality of antipeptide antibodies includes a variety of different antibodies directed to a specific portion of the protein, i.e, to an amino acid sequence that contains at least one, preferably only one, epitope.
  • a "Monoclonal antibody” is a specific antibody that recognizes a single specific epitope of an immunogenic protein. In a plurality of a monoclonal antibody, each antibody molecule is identical to the others in the plurality. In order to isolate a monoclonal antibody, a clonal cell line that expresses, displays and/or secretes a particular monoclonal antibody is first identified; this clonal cell line can be used in one method of producing the antibodies of the invention.
  • naked antibody is an antibody that lacks the Fc portion of a wildtype antibody molecule.
  • the Fc portion is not required for therapeutic function in every instance, as other mechanisms, such as apoptosis, can come into play.
  • the Fc region may be deleterious in some applications as antibodies comprising an Fc region are taken up by Fc receptor-bearing cells, thereby reducing the amount of therapeutic antibody taken up by targeted cells.
  • Vaswani and Hamilton, Humanized antibodies as potential therapeutic drugs Ann, Allergy Asthma Immunol. 81 : 105-1 19, 1998.
  • Components of the immune system may recognize and react to antibodies that are clumped together on the surface of tumor cells. It is thus envisioned that the resulting immune response will target and destroy, or at least limit the proliferation of, the tumor cells.
  • an anti-C20 antibody e.g., Rituxan
  • an anti-C22 antibody might be administered separately or together, allowed to clear so that unbound antibodies are removed from the system.
  • Naked antibodies are also of interest for therapy of diseases caused by parasites, such as malaria.
  • Vukovic et al. Immunoglobulin G3 antibodies specific for the 19-kilodalton carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein 1 transfer protection to mice deficient in Fc-RI receptors, Infect. Immun. 68:3019-22, 2000.
  • Single chain antibodies generally do not include portions of the Fc region of antibodies that are involved in effector functions and are thus naked antibodies, although methods are known for adding such regions to known scFv molecules if desired. See Helfrich et al., A rapid and versatile method for harnessing scFv antibody fragments with various biological functions, J. Immunol. Meth, 237: 131-145,2000; and de Haard et al., Creating and engineering human antibodies for immunotherapy, Adv. Drug Delivery Rev. 31 :5-31, 1998.
  • proteolytic antibody fragments produced by limited proteolysis of wild type antibodies are called proteolytic antibody fragments. These include, but are not limited to, the following.
  • F(ab') 2 fragments are released from an antibody by limited exposure of the antibody to a proteolytic enzyme, e.g., pepsin or ficin.
  • An F(ab') 2 fragment comprises two
  • arms each of which comprises a variable region that is directed to and specifically binds a common antigen.
  • the two Fab' molecules are joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules may be directed toward the same (bivalent) or different (bispecific) epitopes.
  • Fab' fragments contain a single anti-binding domain comprising a Fab and an additional portion of the heavy chain through the hinge region.
  • Fab'-SH fragments are typically produced from F(ab') 2 fragments, which are held together by disulfide bond(s) between the H chains in an F(ab') 2 fragment. Treatment with a mild reducing agent such as, by way of non-limiting example, beta-mercaptoethylamine, breaks the disulfide bond(s), and two Fab' fragments are released from one F(ab') 2 fragment. Fab'-SH fragments are monovalent and monospecific.
  • Fab fragments i.e., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond
  • a convenient method is to use papain immobilized on a resin so that the enzyme can be easily removed and the digestion terminated.
  • Fab fragments do not have the disulfide bond(s) between the H chains present in an F(ab') 2 fragment.
  • Single-chain antibodies are one type of antibody fragment.
  • the term single chain antibody is often abbreviated as “scFv” or “sFv.” These antibody fragments are produced using molecular genetics and recombinant DNA technology.
  • a single-chain antibody consists of a polypeptide chain that comprises both a VH and a VL portion.
  • a single-chain antibody is a single polypeptide that comprises an antigen-binding variable region. That is, a single-chain antibody comprises the variable, antigen- binding determinative region of a single fight and heavy chain of an antibody linked together by a chain of 10-25 amino acids.
  • the terra "single-chain antibody” includes but is not limited to a disulfide-linked
  • Fv in which two single-chain antibodies linked together by a disulfide bond; a bispecific sFv (a sFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (a dimerized sFv formed when the VH domain of a first sFv assembles with the V L domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); and a triabody (a trimerized sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains may be directed towards the same or different epitopes).
  • Fully human antibodies are human antibodies that can be produced in transgenic animals such as Xenomice.
  • XenoMouse strains are genetically engineered mice in which the murine IgH and Igk loci have been functionally replaced by their human Ig counterparts on yeast artificial YAC transgenes.
  • These human Ig transgenes can carry the majority of the human variable repertoire and can undergo class switching from IgM to IgG isotypes.
  • the immune system of the xenomouse recognizes administered human antigens as foreign and produces a strong humoral response.
  • CDR peptides are another form of an antibody fragment.
  • a CDR peptide also known as “minimal recognition unit” is a peptide corresponding to a single complementarity-determining region (CDR), and can be prepared by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106, 1991.
  • compositions including pharmaceutical compositions, comprising an anti-Her3 antibody; and polynucleotides comprising sequences encoding an anti-Her3 antibody.
  • compositions comprise one or more antibodies that bind to Her3, and/or one or more polynucleotides comprising sequences encoding one or more antibodies that bind to Her3.
  • suitable carriers such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
  • the anti-Her3 antibodies of the invention are preferably monoclonal. Also encompassed within the scope of the invention are Fab, Fab', Fab'-SH and F(ab') 2 fragments of the anti-Her3 antibodies provided herein. Single chain anti-Her3 antibodies as well as multispecific and muitivariant Her3 specific antibodies are also included. These antibody fragments can be created by traditional means, such as enzymatic digestion, or may be generated by recombinant techniques. These fragments are useful for the diagnostic and therapeutic purposes set forth below.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the anti-Her3 monoclonal antibodies of the invention are preferably made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • the binding specificity of monoclonal antibodies produced by recombinant means is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunoadsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme- linked immunoadsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980),
  • the anti-Her3 antibodies of the invention can be made by using combinatorial libraries to screen for synthetic antibody clones with the desired activity or activities.
  • synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.
  • Fv antibody variable region
  • any of the anti-Her3 antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-Her3 antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al, Sequences of Proteins of immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
  • the antigen-binding domain of an antibody is formed from two variable (V) regions of about 1 10 amino acids, one each from the light (V L ) and heavy (VH) chains, that both present three hypervariable loops or complementarity-determining regions (CDRs).
  • V variable
  • V L variable
  • VH variable
  • CDRs complementarity-determining regions
  • Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
  • scFv encoding phage clones and Fab encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones”.
  • Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J. 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Filamentous phage is used to display antibody fragments by fusion to the minor coat protein pill.
  • the antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol, 222: 581-597 (1991), or as Fab fragments, as described in Hoogenboom et al., Nucl. Acids Res, 19: 4133-4137 (1991).
  • nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans. If a library biased in favor of anti-Her3 clones is desired, the subject is immunized with Her3 to generate an antibody response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBLs) are recovered for library construction.
  • a human antibody gene fragment library biased in favor of anti-Her3 clones is obtained by generating an anti-Her3 antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that Her3 immunization gives rise to B cells producing human antibodies against Her3. The generation of human antibody-producing transgenic mice is described below.
  • Additional enrichment for anti-Her3 reactive cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing Her3-specific membrane bound antibody, e.g., by cell separation with Her3 affinity chromatography or adsorption of cells to fluorochrome-labeled Her3 followed by flow-activated cell sorting (FACS).
  • FACS flow-activated cell sorting
  • spleen cells and/or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in which Her3 is not antigenic.
  • stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments.
  • the immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, lupine, canine, feline, porcine, bovine, equine, and avian species, etc.
  • Nucleic acid encoding antibody variable gene segments are recovered from the cells of interest and amplified.
  • the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene repertoires for expression.
  • V genes can be amplified from cDNA and genomic DNA, with back primers at the 5' end of the exon encoding the mature V-domain and forward primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341 : 544-546 (1989).
  • back primers can also be based in the leader exon as described in Jones et ah, Biotechnol., 9: 88-89 (1991), and forward primers within the constant region as described in Sastry et al, Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989).
  • degeneracy can be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989).
  • the library diversity is maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and V L arrangements present in the immune cell nucleic acid sample, e.g. as described in the method of Marks et al., J. Mol. Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21 : 4491-4498 (1993).
  • rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991).
  • V genes Repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments. Most of the human Vn-gene segments have been cloned and sequenced
  • V H gene repertoires can be made with all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et a!., Proc. Natl.
  • V.kappa. and V.lamda. segments have been cloned and sequenced (reported in Williams and Winter, Eur, J. Immunol., 23: 1456- 1461 (1993)) and can be used to make synthetic light chain repertoires. Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity. Following amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways.
  • Bach repertoire can be created in different vectors, and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 1 19-126 (1993), or in vivo by combinatorial infection, e.g., the loxP system described in Waterhouse et al., Nucl. Acids Res., 21 : 2265-2266 (1993).
  • the in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency.
  • Naive V H and Vt repertoires are cloned separately, one into a phagemid and the other into a phage vector.
  • the two libraries are then combined by phage infection of phagem id- containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 10 12 clones).
  • Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions.
  • These huge libraries provide large numbers of diverse antibodies of good affinity (K ⁇ f' of about 10 "8 M).
  • the repertoires may be cloned sequentially into the same vector, e.g. as described in Barbas et al. 5 Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g. as described in Clackson et al., Nature, 352: 624-628 (1991).
  • PCR assembly can also be used to join Vn nd VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires.
  • in cell PCR assembly is used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).
  • the antibodies produced by naive libraries can be of moderate affinity (K d "! of about 10 6 to 10 7 "1 ), but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al.
  • mutation can be introduced at random in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1 : 11-15 (1989)) in the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992).
  • affinity maturation can be performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones.
  • WO 9607754 published 14 Mar.
  • Her3 nucleic acid and amino acid sequences are known in the art.
  • a representative nucleic acid and amino acid sequence of Her3 is detailed in SEQ ID NOS. 1 and 2 respectively.
  • Nucleic acid sequence encoding the Her3 can be designed using the amino acid sequence of the desired region of Her3.
  • the cDNA sequence (or fragments thereof) of GenBank Accession Nos. N ..019074- Her3 is a transmembrane protein.
  • the extracellular region contains 36 EGF-iike repeats, as well as a DSL domain that is conserved among all Her3 ligands and is necessary for receptor binding.
  • the predicted protein also contains a transmembrane region, and a cytoplasmic tail lacking any catalytic motifs.
  • Human Her3 protein is a 685 amino acid protein.
  • the accession number of human Her3 is NM generaloi9074- See Sarah J. Bray, "Her3 signaling: a simple pathway becomes complex” Nature Reviews Molecular Cell Biology, 7; 678-689 (2006), the entire content of which is incorporated by reference herein.
  • DNAs encoding Her3 can be prepared by a variety of methods known in the art. These methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al. ⁇ Agnew. Chem. Int Ed. Engl., 28: 716-734 (1989), such as the triester, phosphite, phosphoramidite and H-phosphonate methods. In one embodiment, codons preferred by the expression host cell are used in the design of the Her3 encoding DNA. Alternatively, DNA encoding the Her3 can be isolated from a genomic or cDNA library.
  • the DNA molecule is operabiy linked to an expression control sequence in an expression vector, such as a plasmid, wherein the control sequence is recognized by a host cell transformed with the vector.
  • an expression vector such as a plasmid
  • plasmid vectors contain replication and control sequences which are derived from species compatible with the host cell.
  • the vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection in transformed cells.
  • Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art and some are further described herein. Eukaryotic organisms, such as yeasts, or cells derived from multicellular organisms, such as mammals, may be used.
  • the DNA encoding the Her3 is operabiy linked to a secretory leader sequence resulting in secretion of the expression product by the host cell into the culture medium.
  • secretory leader sequences include stll, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor.
  • secretory leader sequences include stll, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor.
  • secretory leader sequences include stll, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor.
  • 36 amino acid leader sequence of protein A Abrahmsen et al., EMBO J., 4: 3901 (1985)
  • Host cells are transfected and preferably transformed with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 4 precipitation and electroporation.
  • Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Methods for transformation are well known in the art, and some are further described herein.
  • Prokaryotic host cells used to produce the Her3 can be cultured as described generally in Sambrook et al., supra.
  • the mammalian host cells used to produce the Her3 can be cultured in a variety of media, which is well known in the art and some of which is described herein.
  • the host cells referred to in this disclosure encompass cells in in vitro culture as well as cells that are within a host animal.
  • Her3 Purification of Her3 may be accomplished using art-recognized methods, some of which are described herein.
  • the purified Her3 can be attached to a suitable matrix such as agarose beads, acryfamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like, for use in the affinity chromatographic separation of phage display clones.
  • a suitable matrix such as agarose beads, acryfamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like.
  • Attachment of the Her3 protein to the matrix can be accomplished by the methods described in Methods in Enzymology, vol. 44 (1976).
  • a commonly employed technique for attaching protein ligands to polysaccharide matrices, e.g. agarose, dextran or cellulose involves activation of the carrier with cyanogen halides
  • Her3 can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavid in-coated beads, or used in any other art-known method for panning phage display libraries.
  • the phage library samples are contacted with immobilized Her3 under conditions suitable for binding of at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions.
  • the phages bound to the solid phase are washed and then eluted by acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in Marks et a!., J. Mol. Biol., 222: 581-597 (1991), or by Her3 antigen competition, e.g.
  • Phages can be enriched 20-1,000-fold in a single round of selection. Moreover, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.
  • the efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen.
  • Antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions, but favors rebinding of phage that has dissociated.
  • phages can be incubated with excess biotinylated Her3, but with the biotinylated Her3 at a concentration of lower molarity than the target molar affinity constant for Her3.
  • the high affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads.
  • streptavidin-coated paramagnetic beads Such "equilibrium capture" allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity.
  • Conditions used in washing phages bound to a solid phase can also be manipulated to discriminate on the basis of dissociation kinetics.
  • Anti-Her3 clones may be activity selected.
  • the invention provides anti-Her3 antibodies that block the binding between a Her3 receptor, preferably one of a Her3 and/or Her3 receptor and its binding partner.
  • Fv clones corresponding to such anti-Her3 antibodies can be selected by (1) isolating anti-Her3 clones from a phage library as described above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) selecting Her3 and a second protein against which blocking and non-blocking activity, respectively, is desired; (3) adsorbing the anti-Her3 phage clones to immobilized Her3; (4) using an excess of the second protein to elute any undesired clones that recognize Her3-binding determinants which overlap or are shared with the binding determinants of the second protein; and (5) eluting the clones which remain adsorbed following step (4).
  • DNA encoding, for example, phage display Fv clones of the invention is readily isolated and sequenced using conventional procedures (e.g. by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA template).
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce
  • DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g. the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains.
  • constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and igE constant regions, and that such constant regions can be obtained from any human or animal species
  • a Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for "hybrid", full length heavy chain and/or light chain is included in the definition of "chimeric” and "hybrid” antibody as used herein.
  • a Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for all human, full or partial length heavy and/or light chains.
  • An antibody functional fragment refers to a portion of an antibody which retains some or all of its target-specific binding activity.
  • Such functional fragments can include, for example, antibody functional fragments such as Fv, Fab, F(ab'), F(ab)3 ⁇ 4 F(ab') 2 , single chain Fv (scFv), diabodies, triabodies, tetrabodies and minibody.
  • Other functional fragments can include, for example, heavy (H) or light (L) chain polypeptides, variable heavy (V H ) and variable light (VL) chain region polypeptides, complementarity determining region (CDR) polypeptides, single domain antibodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to retain target-specific binding activity.
  • the present invention encompasses antibody fragments. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.
  • F(ab3 ⁇ 4 fragments can be isolated directly from recombinant host cell culture.
  • Fab and F(ab') 2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046.
  • Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
  • Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use.
  • sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed.
  • the antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
  • Target-specific monoclonal antibodies for use in a biopharmaceutical formulation of the invention can include any of such various monoclonal antibody forms, alterations and modifications. Examples of such various forms and terms as they are known in the art are set forth below.
  • Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized.
  • LH keyhole limpet hemocyanin
  • serum albumin serum albumin
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1 ⁇ 4 to ⁇ fraction (1/10) ⁇ the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
  • a suitable fusing agent such as polyethylene glycol
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
  • a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner).
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT the selective culture medium for the hybridomas typically will include hypoxanthine, aminopte in, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells.
  • Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPO 1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal, Biochem,, 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D- MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g, by i.p. injection of the cells into mice.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e. g. f using protein A or protein G-Sepharose®) or ion-exchange chromatography, hydroxyiapatite chromatography, gel electrophoresis, dialysis, etc.
  • affinity chromatography e. g. f using protein A or protein G-Sepharose®
  • ion-exchange chromatography e.g. f using protein A or protein G-Sepharose®
  • hydroxyiapatite chromatography hydroxyiapatite chromatography
  • gel electrophoresis hydroxyiapatite chromatography
  • dialysis etc.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein.
  • Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (19
  • monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol, 222:581-597 (1991 ) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • the DNA that encodes the antibody may be modified, for example, by substituting human heavy chain and light chain constant domain (C H and C sequences for the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Set USA , 81 :6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • C H and C sequences for the homologous murine sequences U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Set USA , 81 :6851 (1984)
  • the non-immunog!obulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized” antibodies are chimeric antibodies (U.S.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Inmunol., 151 :2296 (1993); Chothia et al, J. Mol. Biol., 196:901 (1987)).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993)).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Human anti-Her3 antibodies of the invention can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s) as described above.
  • human monoclonal anti-Her3 antibodies of the invention can be made by the hybridoma method.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J. Immunol., 147: 86 (1991).
  • Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:383 (1991); Marks et al, J. Mol. Biol., 222:581-597 (1991 )).
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • the homozygous deletion of the antibody heavy-chain joining region (1 ⁇ 2) gene in chimeric and germ- line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
  • phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • the phage mimics some of the properties of the B-cel!.
  • Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).
  • V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice.
  • a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self- antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol 222:581 -597 (1991), or Griffith et al, EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
  • Gene shuffling can also be used to derive human antibodies from non-human, e.g. rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody.
  • this method which is also called “epitope imprinting"
  • either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described above is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras.
  • Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens, in the present case, one of the binding specificities is for Her3 and the other is for any other antigen.
  • Exemplary bispecific antibodies may bind to two different epitopes of the Her3 protein.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Her3. These antibodies possess a Her3-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-. lpha., vinca alkaloid, rich A chain, methotrexate or radioactive isotope hapten).
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies).
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI), containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH 3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers. See Kostelny et al., J. Immunol.,
  • Bispecific antibodies include cross-linked or "heteroconjugate” antibodies. As such, heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells, e.g., U.S. Pat. No. 4,676,980, and for treatment of HIV infection, e.g., WO 91/00360; WO 92/200373; EP 03089. Heteroconjugate antibodies may be made using any convenient cross-linking methods. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • Suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
  • a diabody is a bivalent dimer formed by the non-covalent association of two scFvs, yielding two Fv binding sites.
  • a diabody refers to an engineered antibody construct prepared by isolating the binding domains (both heavy and light chain) of a binding antibody, and supplying a linking moiety which joins or operably links the heavy and light chains on the same polypeptide chain thereby preserving the binding function (see, Holliger et al. (1993) Proc. Natl. Acad. Sci.
  • Diabodies are built like scFv molecules, but usually have a short (less than 10, preferably 1-5 amino acids) peptide linker connecting both V-domains, whereby both domains can not interact intramolecular, and are forced to interact intermolecular (Holliger et al., 1993) (U.S. Pat. No. 5,837,242).
  • a diabody thus may consist of a V H -V L chain that interacts with a similar VH-VL chain to form a dimer of the formula VH-VL:V H -VL.
  • the diabody chain dimers bind the antigen specified by VH and V L bivalent.
  • Antibodies with more than two valences are contemplated.
  • trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
  • Tetravalent bispecific antibodies can be created by chemical cross-linking of two monoclonal antibodies (Bs(IgG)2) (Karpovsky et al., 1984) (U.S. Pat. No. 4,676,980). Problems related to their rapid clearance in vivo via the kidney due to their small size may be circumvented by, for example, increasing their molecular weight size thereby increasing their serum permanence and product efficacy.
  • Bs(IgG)2 monoclonal antibodies
  • problems related to their rapid clearance in vivo via the kidney due to their small size may be circumvented by, for example, increasing their molecular weight size thereby increasing their serum permanence and product efficacy.
  • Tumor localization of anti-CEA single-chain Fvs improved targeting by non-covalent dimers.
  • Peptibodies which consist of an immunoglobulin constant region domain (Fc) linked to two binding peptides through either the carboxyl- or amino termini of the Fc domain, also are included herein as an antibody functional fragment.
  • Fc immunoglobulin constant region domain
  • Such antibody binding fragments can be found described in, for example, Harlow and Lane, supra; Molec, Biology and
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • the preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fe region.
  • the preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites.
  • the multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VDl-(Xl)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1.
  • the polypeptide chain(s) may comprise: VH-CH1 -flexible linker-V H -CHl-Fc region chain; or VH-CH1-V H -CH1- Fc region chain.
  • the multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides.
  • the multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made,
  • a useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or poly alanine) to affect the interaction of the amino acids with antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of the antibody molecule include the fusion of the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5 -hydroxy lysine may also be used.
  • Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-Hnked glycosylation sites). See section marked "Effector Function Engineering", infra,
  • the carbohydrate attached thereto may be altered.
  • antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in US Pat Appl No US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Antibodies with a bisecting N-acetyl glucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003/01 1878, Jean-Mairet et ai. and U.S. Pat. No. 6,602,684, Umana et al.
  • Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody are reported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US 2005/0123546 (Umana et al.) on antigen-binding molecules with modified glycosylation.
  • At least one glycosylation variant herein comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function.
  • the Fc region further comprises one or more amino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and/or 334 of the Fc region (Eu numbering of residues).
  • Examples of cell lines producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al , Presta, L; and WO 2004/056312 Al , Adams et al., especially at Example 1 1), and knockout cell lines, such as alpha- 1 ,6-fucosy!transferase gene, FUT8, knockout CHO cells (Yamane- Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). For further details, see “Effector Function Engineering” infra.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • Exemplary Preferred Residue Substitutions Ala (A) Val; Leu; He Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg He (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; He; Val; He Met; Ala; Phe Lys ( ) Arg; Gin; Asn Arg Met (M) Leu; Phe; He Leu Phe (F) Trp; Leu; Val; He; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobic ity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. human antibody).
  • a parent antibody e.g. human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.
  • the antibodies thus generated are displayed from filamentous phage particles as fusions to the gene ill product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody. Effector Function Engineering
  • therapeutic antibodies can exert potent biological functions through two major non-exclusive mechanisms: (i) they can block interactions between receptors and their ligands due to the extraordinar epitope specificity of their variable domains
  • neutralizing/antagonist antibodies or trigger potent biological responses such as apoptosis or cell proliferation once they are bound to surface molecules (“agonist antibodies”); (ii) induce effector functions against pathogens and tumor cells following their interactions with the complement component Clq and/or with receptors for Fc region (FcyR). See Cragg et al. f Curr Opin Immunol 1 1 :541-547 (1999); Glennie et al., Immunol Today 21 :403-410 (2000).
  • immunoglobulins e.g., IgG, which is the most common immunoglobulin
  • IgG immunoglobulins
  • FcyRs cell surface Fc receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl , IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions including that of a hinge cysteine.
  • a human Fc region sequence e.g., a human IgGl , IgG2, IgG3 or IgG4 Fc region
  • an amino acid modification e.g. a substitution
  • ADCC involves the recognition of the antibody by immune cells that engage the antibody-marked cells and either through their direct action, or through the recruitment of other cell types, leads to the tagged-cell's death.
  • CDC is a process where a cascade of different complement proteins become activated, usually when several IgGs are in close proximity to each other, either with one direct outcome being cell lysis, or one indirect outcome being attracting other immune cells to this location for effector cell function.
  • a promising means for enhancing the anti-tumor potency of antibodies is via enhancement of their ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC,
  • cytotoxic effector functions such as ADCC, ADCP, and CDC
  • the importance of ADCC as a cytotoxic mechanism of anti-tumor mAbs has been demonstrated in animal studies.
  • Ravetch et al, Annu. Rev. Immunol. 1 :421-432 (1998) showed that the tumoricidal effect of a humanized anti-Her2/neu mAb (epithelial growth factor receptor 2; Trastuzumab) was significantly reduced in FcyR knockout nude mice as compared to wild-type nude mice.
  • FcyRIIIa polymorphism and also a polymorphism in FcyRIIa are associated with the response rate to therapeutic mAbs.
  • W.K. Weng and R. Levy Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J. Clin. Oncol. 21 :3940-3947 (2003).
  • FcyRs can mediate antigen uptake and processing by antigen presenting cells, enhanced FcyR affinity may also improve the capacity of antibody therapeutics to elicit an adaptive immune response.
  • Fc variants have been successfully engineered with selectively enhanced binding to FcyRs, and furthermore these Fc variants provide enhanced potency and efficacy in cell-based effector function assays. See for example U.S. Ser. No. 10/672,280, U.S. Ser. No. 10/822,231, entitled “Optimized Fc Variants and Methods for their Generation”, U.S. Ser. No. 60/627,774, entitled “Optimized Fc Variants", and U.S. Ser. No. 60/642,477, entitled “Improved Fc Variants", and references cited therein.
  • ADCC antibody-dependent cellular cytotoxicity
  • Complement initiates three mechanisms that can be used against mAb-coated tumor cells.
  • the first is direct complement killing of tumor cells by the membrane attack complex (MAC), a process usually called 'complement-dependent cytotoxicity' (CDC).
  • MAC membrane attack complex
  • CDC 'complement-dependent cytotoxicity'
  • the second mechanism is complement receptor-dependent enhancement of ADCC.
  • CR3 binds to iC3b, thus enhancing FcyR-mediated effector cell binding.
  • a third mechanism used for killing microorganisms CR3-dependent cellular cytotoxicity (CR3-DCC) is usually not activated with tumors.
  • the residue Pro331 has been implicated in CI q binding by analysis of the ability of human IgG subclasses to carry out complement mediated cell lysis. Mutation of Ser331 to
  • IgG ability of IgG to bind Clq and activate the complement cascade also depends on the presence, absence, or modification of the carbohydrate moiety positioned between the two CH2 domains (which is normally anchored at Asn297).
  • U.S. Pat. No. 6, 1 5,745 discloses a method of producing an antibody with a decreased biological half-life by introducing a utation into the DNA segment encoding the antibody.
  • the mutation includes an amino acid substitution at position 253, 310, 31 1, 433, or 434 of the Fc-hinge domain.
  • U.S. Pat. No. 6,277,375 Bl discloses a composition comprising a mutant IgG molecule having an increased serum half-life relative to the wild-type IgG, wherein the mutant IgG molecule comprises the amino acid substitutions: threonine to leucine at position 252, threonine to serine at position 254, or threonine to phenylalanine at position 256.
  • a mutant IgG with an amino acid substitution at position 433, 435, or 436 is also disclosed.
  • U.S. Pat. No. 6,528,624 discloses a variant of an antibody comprising a human IgG Fc region, which variant comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 331, 333, and 334 of the human IgG Fc region.
  • an antibody used in methods of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, e.g. in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in W099/51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No.
  • any particular antibody e.g., any one or more of the ami- antibodies disclosed herein, to mediate lysis of the target cell by complement activation and/or ADCC can be assayed.
  • Functional assays for identifying potent Fc variants of any one or more of the anti- Her3 antibodies of the invention are well known to one skilled in the art. See, for example, U.S Patent Application Publications 2005/0037000 and 2005/0064514, and International Patent Application Publication WO 04/063351 (each of which is hereby incorporated by reference in its entirety); that describe yeast display technology for characterizing an antibody with a variant Fc region.
  • R-Fc binding assays are disclosed in U.S Patent Application Publications 2005/0037000 and 2005/0064514, and International Patent Application Publication WO
  • effector cell functions include but are not limited to, antibody-dependent cell mediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis, Clq binding, and complement dependent cell mediated cytotoxicity.
  • Any cell-based or cell free assay known to those skilled in the art for determining effector cell function activity can be used (For effector cell assays, see Perussia et al. t 2000, Methods Mol. Biol. 121 : 179-92; Baggiolini et al., 1998 Experientia, 44(10): 841-8; Lehmann et al., 2000 J. Immunol.
  • the cells of interest are grown and labeled in vitro; the target antibody is added to the cell culture in combination with either serum complement or immune cells which may be activated by the antigen-antibody complexes. Cytolysis of the target cells is detected by the release of label from the lysed cells.
  • antibodies can be screened using the patient's own serum as a source of complement and/or immune cells. The antibody that is capable of activating complement or mediating ADCC in the in vitro test can then be used therapeutically in that particular patient.
  • the effector cells used in the ADCC assays of the invention are peripheral blood mononuclear cells (PBMC) that are preferably purified from normal human blood, using standard methods known to one skilled in the art, e.g. using Ficoll-Paque density gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • An exemplary assay for determining ADCC activity of such anti-Her3 antibodies with variant Fc regions is based on a 51Cr release assay comprising of: labeling target cells with [51Cr]Na2Cr04 (this cell-membrane permeable molecule is commonly used for labeling since it binds cytoplasmic proteins and although spontaneously released from the cells with slow kinetics, it is released massively following target cell necrosis); opsonizing the target cells with the anti-antibodies with variant Fc region(s) of the invention; combining the opsonized radiolabeled target cells with effector cells in a microtitre plate at an appropriate ratio of target cells to effector cells; incubating the mixture of cells for 16-18 hours at 37°C; collecting supernatants; and analyzing radioactivity.
  • the cytotoxicity of the anti-antibodies with variant Fc regions can then be determined using known formulae, for example using the following formula: % cpm-target leak cpm)/(detergent lysis cpm-target leak cpm)xl00%.
  • % lysis (ADCC-AICC)/(maximum release-spontaneous release).
  • a graph can be generated by varying either the target:effector cell ratio or antibody concentration. Perussia et al, 2000, Methods Mol. Biol. 121 : 179-92.
  • the affinities and binding properties of anti-antibodies with variant Fc regions for an FcyR may initially be determined using in vitro assays (biochemical or immunological based assays) known in the art for determining Fc-FcyR interactions, i.e., specific binding of an Fc region to an FcyR including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays.
  • the binding properties of the anti-antibodies with variant Fc regions in accordance with the invention may also be characterized by in vitro functional assays for determining one or more FcyR mediator effector cell functions.
  • the anti-Her3 Fc variants of the invention have similar binding properties in in vivo models as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • DNA encoding an amino acid sequence variant of any one or more of the herein disclosed starting anti- Her3 antibodies may be prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the antibody.
  • a nucleic acid encoding an Fc region of a parent antibody is available and this nucleic acid sequence is altered to generate a variant nucleic acid sequence encoding the Fc region variant
  • Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and unkel et al, Proc. Natl. Acad. Sci. USA 82:488 (1985)). Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA.
  • a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template.
  • the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.
  • PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Valiette et al., Nuc. Acids Res. 17:723-733 (1989).
  • primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
  • the starting material is the plasmid (or other vector) comprising the starting polypeptide DNA to be mutated.
  • the codon(s) in the starting DNA to be mutated are identified. There must be a unique restriction
  • the desired amino acid sequence encoding an anti- Fc variant can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically.
  • the modification entails one or more amino acid substitutions.
  • the substitution may, for example, be a "conservative substitution”.
  • the molecules of the invention with altered affinities for activating and/or inhibitory receptors having variant Fc regions have one or more amino acid modifications.
  • the Fc regions of any one or more of the herein disclosed anti-antibodies may be optimized for a variety of properties. Properties that may be optimized include but are not limited to enhanced or reduced affinity for an FcyR, In one embodiment, the Fc variants are optimized to possess enhanced affinity for a human activating FcyR, preferably FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and FcyROIb, most preferably FcyRIIIa. In an alternative embodiment, the Fc region is optimized to possess reduced affinity for the human inhibitory receptor FcyRIIb. These embodiments are anticipated to provide anti-antibodies with enhanced therapeutic properties in humans, for example enhanced effector function and greater anti-cancer potency.
  • the Fc variants of the present invention are optimized to have reduced or ablated affinity for a human FcyR, including but not limited to FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb.
  • FcyRI FcyRIIa
  • FcyRIIb FcyRIIc
  • FcyRIIIa FcyRIIIb
  • FcyRIIIb FcyRIIIb.
  • the Fc variants of the present invention may also be optimized for enhanced functionality and/or solution properties in aglycosylated form.
  • the aglycosylated Fc variants of the present invention bind an Fc Hgand with greater affinity than the aglycosylated form of the parent Fc polypeptide.
  • Exemplary Fc ligands include but are not limited to FcyRs, Clq, FcRn, and proteins A and G, and may be from any source, preferably human.
  • the Fc variants of the invention are optimized to be more stable and/or more soluble than the aglycosylated form of the parent Fc polypeptide.
  • Certain aspects of this invention thus involve antibodies which are (a) directed against a particular antigen and (b) belong to a subclass or isotype that is capable of mediating the lysis of cells to which the antibody molecule binds. More specifically, these antibodies should belong to a subclass or isotype that, upon complexing with cell surface proteins, activates serum complement and/or mediates antibody dependent cellular cytotoxicity (ADCC) by activating effector cells such as natural killer cells or macrophages. Towards this end, it may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example.
  • ADCC antibody dependent cellular cytotoxicity
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC).
  • ADCC antibody- dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1 93).
  • any one or more of the anti- antibodies of the invention can be engineered with dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).
  • the antibodies with variant Fc region(s) of the invention are characterized for antibody dependent cellular cytotoxicity (ADCC) see, e.g., Ding et al, Immunity, 1998, 8:403-1 1 ; which is incorporated herein by reference in its entirety.
  • ADCC antibody dependent cellular cytotoxicity
  • one or more amino acids in the Fc region can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,1 4,551 by Idusogie et al.
  • a broad aspect of the invention thus relates to immunoglobulins (e.g., anti- antibodies disclosed herein), comprising a variant Fc region, having one or more amino acid modifications (e.g., substitutions, but also including insertions or deletions) in one or more regions, which modifications alter, e.g., increase or decrease, the affinity of the variant Fc region for an FcyR.
  • one or more amino acid modification e.g., substitutions, but also including insertions or deletions
  • said one or more amino acid modification increases the affinity of the variant Fc region for FcyRIIIA and/or FcyRIIA.
  • the herein described anti-antibodies with a variant Fc region further specifically bind FcyRIIB (via the Fc region) with a lower affinity than a comparable antibody molecule (i.e., having the same amino acid sequence as the antibody with a variant Fc region except for the one or more amino acid modifications in the Fc region) comprising the wild-type Fc region binds FcyRIIB.
  • the invention encompasses molecules comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region does not bind any FcyR or binds with a reduced affinity, relative to a comparable molecule comprising the wild-type Fc region, as determined by standard assays (e.g., in vitro assays) known to one skilled in the art.
  • the invention encompasses molecules comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcyR, wherein the FcyR is Fc RIIIA.
  • the invention encompasses molecules comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcyR wherein the FcyR is FcyRIIA.
  • the invention encompasses an anti-antibody molecule comprising a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcyR wherein the FcyR is FcyRIIB.
  • At least one or more anti- antibodies which comprises an antigen binding region and a variant Fe region, wherein the variant Fc region: (A) differs from a wild-type Fc region by comprising at least one amino acid modification according to the EU index as in Kabat, relative to the wild-type Fc region
  • Fc variants of the present invention may be combined with other Fc modifications, including but not limited to modifications that alter effector function or interaction with one or more Fc ligands. Such combination may provide additive, synergistic, or novel properties in antibodies or Fc fusions.
  • the Fc variants of the present invention may be combined with other known Fc variants (Duncan et al., 1988, Nature 332:563- 564; Lund et al., 1991, J Immunol 147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation 57: 1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci USA 92: 11 80-1 1984; Jefferis et al., 1995, Immunol Left 44: 11 1-1 17; Lund et al., 1995, Faseb J9.T 15-119; Jefferis et al., 1996, Immunol Left 54: 101-104; Lund et al., 1996, J Immunol 1 7:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et
  • the Fc variants of the present invention are incorporated into an antibody or Fc fusion that comprises one or more engineered glycoforms (infra).
  • Fc variants of the present invention are contemplated with the goal of generating novel antibodies or Fc fusions with optimized properties.
  • the invention additionally, encompasses anti-antibodies including fragments thereof which are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by trypsin, papain, metabolic synthesis in the presence of tunicamycin etc.
  • Antibodies are glycoproteins containing carbohydrate structures at conserved positions in the heavy chain constant regions, with each isotype possessing a distinct array of N- linked carbohydrate structures, which variably affect protein assembly, secretion or functional activity.
  • the structure of the attached N-linked carbohydrate varies considerably and can include high-mannose, multiply-branched as well as biantennary complex oligosaccharides. (Wright, A, and Morrison, S. L, Trends Biotech. 15:26-32 (1997)).
  • the major carbohydrate units are attached to amino acid residues of the constant region of the antibody.
  • Carbohydrate is also known to attach to the antigen binding sites of some antibodies and may affect the antibody- binding characteristics by limiting access of the antigen to the antibody binding site.
  • variable domain of the antibody binds to a specific tumor receptor on the surface of the target cell. This is followed by recruitment of effector cells such as natural killer (NK) cells that bind to the constant region (Fc) of the antibody and destroy cells to which the antibody has bound.
  • NK natural killer
  • Fc constant region
  • ADCC antibody-dependent cell cytotoxicity
  • Hyperglycosylated proteins have been shown to exhibit increased serum half-life, are less sensitive to proteolysis and more heat-stable compared with the non-glycosylated forms. (Leatherbarrow et al., Mol. Immunol. 22:407 (1985)).
  • IgGl type antibodies which represent the most commonly used antibodies in cancer immunotherapy, are glycoproteins that have a conserved N-l inked glycosylation site at Asn297 in each CH2 domain.
  • ADCC antibody dependent cellular cytotoxicity
  • glycosylation of IgM at asparagine 402 in the CH 3 domain is necessary for proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, J. Immunol. 142:695 (1989)).
  • removal of glycosylation sites as positions 162 and 419 in the CHI and CH3 domain of an IgA antibody has been shown to lead to intracellular degradation and at least 90% inhibition of secretion (Taylor and Wall, Mol. Cell. Biol. 8:41 7(1988)).
  • V region Glycosylation of immunoglobulins in the variable (V) region has also been observed. Sox and Hood, Proc. Natl. Acad. Sci. USA 66:975 (1970), reported that about 20% of human antibodies are glycosylated in the V region. Glycosylation of the V domain is believed to arise from fortuitous occurrences of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence and has not been recognized in the art as playing an important role in immunoglobulin function.
  • glycosylation at CDR2 of the heavy chain, in the antigen binding site, of a murine antibody specific for ,alpha.-(l-6)dextran increases its affinity for dextran (Wallick et at, J. Exp. Med. 168: 1099 (1988) and Wright et al, EMBO J. 10:2717 (1991)). See Patent No. 6,933,368.
  • Some classes and subclasses also have O-linked sugars, often in the hinge region, eg. IgD and IgA from some species.
  • the present invention in related embodiments, provides "Engineered Glycoforms" of any one or more of the anti-antibodies disclosed herein including fragments thereof, wherein the glycosylation profiles of the antibody are altered to enhance their use in the treatment of specific types of cancers or other disease states.
  • engineered glycoform as used herein is meant a carbohydrate composition that is covalentiy attached to an Fc polypeptide, wherein the carbohydrate composition differs chemically from that of a parent Fc polypeptide.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
  • Engineered glycoforms may be generated by a variety of methods known in the art (Umana et al., 1999, Nat Biotechnol 17: 176-180; Davies et al, 2001, Biotechnol Bioeng 74:288-294;
  • Engineered glycoform typically refers to the different carbohydrate or oligosaccharide; thus an Fc polypeptide, for example an antibody or Fc fusion, may comprise an engineered glycoform.
  • engineered glycoform may refer to the Fc polypeptide that comprises the different carbohydrate or oligosaccharide.
  • Covalent modification of the target antibody included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in the native target antibody, and/or adding one or more glycosylation sites that are not present in the native target antibody.
  • the glycosylation of an antibody is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery.
  • Cells with altered g!ycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation.
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).
  • PCT Publication WO 99/54342 by Umana et al.
  • glycoprotein-modifying glycosyl transferases e.g., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIil)
  • GnTIil glycoprotein-modifying glycosyl transferases
  • Antibodies disclosed herein can be glycosylated in both the C-regions and in the V-regions. Obviously C-region glycosylation is dependent on the particular sequence which by definition defines the class and subclass of the antibody. As noted elsewhere, many classes of antibody have conserved N-linked glycosylation sites in the constant domains. For example all IgG antibodies have a conserved N-linked glycosylation site in the CH2 domain at residue Asn297.
  • O-linked glycosylation is initiated by the attachment of N-acetyi- galactosamine to a serine or threonine residue in the peptide backbone of the therapeutic protein.
  • the proximal carbohydrate is the target for glycosyltransferases to fonn a mature O-glycan structure. It is difficult to predict where O-linked glycosylation will occur in the protein as there is no clear consensus amino acid sequence for O-linked glycosylation (3, 4). However, O-linked glycosylation is affected by secondary structural elements such as an extended ⁇ -turn. In contrast, consensus amino acid sequences are known for N-glycosylation.
  • N-glycosylation occurs at a specific sequence motif, Asn-X-Thr/Ser (sequon or consensus sequence; where X is any amino acid except proline), and this consensus sequence must be accessible to the precursor transferring the enzyme.
  • Asn-X-Thr/Ser sequence motif
  • Asn-XTM Thr/Ser sequences in ⁇ -turns can influence the protein conformation by N-linked glycosylation. As glycosylation precedes final protein folding, the structure of the resultant therapeutic protein may be altered, resulting in differences in activity or stability compared with the non- glycosylated form.
  • recombinant antibodies of the invention can be modified to recreate or create additional glycosylation sites if desired, which is simply achieved by engineering the appropriate amino acid sequences (such as Asn-X-Ser, Asn-X-Thr, Ser, or Thr) into the primary sequence of the antibody.
  • Glycosylation of polypeptides is typically either N-iinked or O-linked, N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5- hydroxylysine may also be used.
  • a mutant anti-antibody that exhibits a higher affinity for its antigen e.g., receptor or endogenous binding partner, than a parent antibody that comprises a parent immunoglobulin chain, wherein the mutant
  • immunoglobulin chain comprises an amino acid substitution that eliminates a variable region glycosylation site of the parent immunoglobulin chain, said elimination having the effect of increasing the affinity of the mutant antibody relative to the parent antibody.
  • Alternative embodiments contemplate variants that are "aglycosylated.”
  • Glycosylation sites refer to amino acid residues which are recognized by a eukaryotic cell as locations for the attachment of sugar residues.
  • the amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage), serine (O-linkage), and threonine (O-finkage) residues.
  • the sequence of the antibody is examined, for example, by using publicly available databases such as the website provided by the Center for Biological Sequence Analysis (see http://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N- linked glycosylation sites) and http://www.cbs, dtu.dk/services NetOGlyc/ for predicting O- linked glycosylation sites). Additional methods for altering glycosylation sites of antibodies are described in U.S. Pat Nos. 6,350,861 and 5,714,350.
  • Glycosylation can be achieved by methods known in the art, e.g., by producing the antibody in a mammalian host cell such as Chinese Hamster Ovary (CHO) cell or in yeast.
  • Addition of glycosylation sites to the target antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threoni e residues to the native target antibody sequence (for O-linked glycosylation sites).
  • the target antibody amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the target antibody at preselected bases such that codons are generated that will translate into the desired amino acids.
  • the DNA mutation(s) may be made using methods described above under the heading of "Amino Acid Sequence Variants of Target antibody”.
  • Yeast provides substantia! advantages over bacteria for the production of immunoglobulin H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides) (Hitzman, et al. dislike 1 1th International Conference on Yeast, Genetics and Molecular Biology, Montpelier, France, Sep. 13-17, 1982).
  • sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of
  • the main species antibody or variant thereof may further comprise glycosylation variations, non-limiting examples of which include antibody comprising a Gl or G2 oligosaccharide structure attached to the Fc region thereof, antibody comprising a carbohydrate moiety attached to a light chain thereof (e.g. one or two carbohydrate moieties, such as glucose or galactose, attached to one or two light chains of the antibody, for instance attached to one or more lysine residues), antibody comprising one or two non-glycosylated heavy chains, or antibody comprising a sialylated oligosaccharide attached to one or two heavy chains thereof etc.
  • glycosylation variations non-limiting examples of which include antibody comprising a Gl or G2 oligosaccharide structure attached to the Fc region thereof, antibody comprising a carbohydrate moiety attached to a light chain thereof (e.g. one or two carbohydrate moieties, such as glucose or galactose, attached to one or two light chains of the antibody, for
  • antibodies or fragments thereof are altered wherein the constant region of the antibody is modified to reduce at least one constant region- mediated biological effector function relative to an unmodified antibody.
  • modified antibodies are often referred to as "aglycosylated" antibodies.
  • the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J. Exp. Med. 173:1483-1491 ; and Lund, J.
  • Reduction in FcR binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity, glycosylation sites of the antibody can be altered, for example, by mutagenesis (e.g., site-directed mutagenesis).
  • mutagenesis e.g., site-directed mutagenesis
  • such antibodies do not exhibit substantial immune effector functions that are dependent on glycosylation of the Fc region.
  • an aglycosylated antibody of the invention does not exhibit substantial immune effector functions except for binding to FcRn.
  • an antibody of the invention or a fragment thereof does not possess substantial or completely lacks effector functions other than FcRn binding.
  • said effector function is complement lysis.
  • said effector function is antibody dependent cell cytotoxicity (ADCC).
  • ADCC antibody dependent cell cytotoxicity
  • the antibody fragment binds FcRn.
  • Aglycosylated antibodies can be produced by a variety of methods known in the art.
  • a convenient method comprises expressing the antibody in a prokaryotic host cell such as E. coli.
  • the amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage), serine (O-lbikage), and threonine (O-linkage) residues.
  • N-linkage N-linkage
  • serine O-lbikage
  • O-linkage threonine residues.
  • the sequence of the antibody is examined, for example, by using publicly available databases such as the website provided by the Center for Biological Sequence Analysis (see
  • Removal of carbohydrate moieties present on the native target antibody may also be accomplished chemically or enzymatically.
  • Chemical deglycosylation requires exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N- acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact.
  • Chemical deglycosylation is described by Hakimuddin et al. (Arch. Biochem. Biophys., 259:52 [1987]) and by Edge et al. (Anal. Biochem., 1 18: 131 [1981]).
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-g!ycosidases as described by Thotakura et al. (Meth. Enzymol. 138:350 [1987]). Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin et al. (J. Biol. Chem., 257:3105 [1982]). Tunicamycin blocks the formation of protein-N- glycoside linkages.
  • the antibodies of the invention or an antigen-binding fragment thereof is modified to reduce or eliminate potential glycosylation sites.
  • the constant region of the antibody, or fragment thereof of the invention is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody.
  • an antibody fragment rather than an intact antibody, to increase tumor penetration, for example.
  • a systematic method for preparing such an antibody variant having an increased in vivo half-life comprises several steps. The first involves identifying the sequence and conformation of a salvage receptor binding epitope of an Fc region of an IgG molecule. Once this epitope is identified, the sequence of the antibody of interest is modified to include the sequence and conformation of the identified binding epitope. After the sequence is mutated, the antibody variant is tested to see if it has a longer in vivo half-life than that of the original antibody. If the antibody variant does not have a longer in vivo half-life upon testing, its sequence is further altered to include the sequence and conformation of the identified binding epitope. The altered antibody is tested for longer in vivo half-life, and this process is continued until a molecule is obtained that exhibits a longer in vivo half-life.
  • the salvage receptor binding epitope being thus incorporated into the antibody of interest is any suitable such epitope as defined above, and its nature will depend, e.g., on the type of antibody being modified.
  • the transfer is made such that the antibody of interest still possesses the biological activities described herein.
  • the epitope generally constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CHI, CH3, or VH region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the Q, region or V L region, or both, of the antibody fragment.
  • the antibodies of the present invention can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethyl ene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l ,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyo
  • PEG polyethylene glycol
  • Polyethylene glycol propionaidehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example,
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Another type of covalent modification of the target antibody e.g., any one or more of the anti-Her3 antibodies of the invention comprises linking the target antibody to various nonproteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301, 144; 4,670,417; 4,791,192 or 4, 179,337.
  • the antibodies and antibody fragments of the invention may be chemically modified to provide a desired effect such as increased solubility, stability and circulating time of the polypeptide, or decreased
  • the antibody or fragments thereof polypeptides 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,
  • PEGylation of antibodies and antibody fragments of the invention may be carried out by any of the PEGylation reactions known in the art, See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992). (each of which is incorporated by reference herein in its entirety).
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • the term "polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-ma!eimide.
  • the antibody to be PEGylated is an aglycosylated antibody.
  • a single immunoglobulin variable domain derived from an invention antibody containing composition is stabilized in vivo by linkage or association with a (non-polypeptide) polymeric stabilizing moiety.
  • a (non-polypeptide) polymeric stabilizing moiety examples of this type of stabilization are described, for example, in WO99/64460 (Chapman et al.) and EP1, 160,255 (King et al.), each of which is incorporated herein by reference.
  • these references describe the use of synthetic or naturally-occurring polymer molecules, such as polyalkylene, polyalkenylenes, polyoxyalkylenes or polysaccharides, to increase the in vivo half-life of immunoglobulin polypeptides.
  • a typical example of a stabilizing moiety is polyethylene glycol, or PEG, a polyalkylene.
  • PEG polyethylene glycol
  • PEGylation The process of linking PEG to an immunoglobulin polypeptide is described in these references and is referred to herein as "PEGylation.” As described therein, an
  • immunoglobulin polypeptide can be PEGylated randomly, as by attachment of PEG to lysine or other amino acids on the surface of the protein, or site-specifically, e.g., through PEG attachment to an artificially introduced surface cysteine residue.
  • it may be preferred to use a non-random method of polymer attachment because random attachment, by attaching in or near the antigen-binding site or sites on the molecule often alters the affinity or specificity of the molecule for its target antigen.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No.
  • Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with ⁇ , ⁇ -carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG-succinate derivatives.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (C1S0 2 CH 2 CF 3 ).
  • MPEG monmethoxy polyethylene glycol
  • C1S0 2 CH 2 CF 3 tresylchloride
  • polyethylene glycol is directly attached to amine groups of the protein.
  • the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethane sulphonyl group.
  • a general method for preparing PEGylated antibodies and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or antibody fragment with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby the antibody or antibody fragment becomes attached to one or more PEG groups, and (b) obtaining the reaction products.
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the 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.
  • pegylation of the proteins of the invention may be accomplished by any number of means.
  • polyethylene glycol may be attached to the protein either directly or by an intervening linker.
  • Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al. Intern. J. of Hematol. 68: 1-18 (1998); U.S. Pat No. 4,002,531 ; U.S. Pat. No.
  • polyethylene glycol can be linked to a protein via covalent 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) of the protein or to more than one type of amino acid residue (e.g, lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of PEGylation reaction to be performed, and the method of obtaining the selected N-terminally PEGylated protein.
  • the method of obtaining the N-terminally PEGylated preparation i.e., separating this moiety from other mono-PEGylated moieties if necerney
  • Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation which exploits differentia! reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N- terminus with a carbonyl group containing polymer is achieved.
  • the polymer may be of any molecular weight, and may be branched or unbranched. Branched polyethylene glycols are described, for example, in U.S. Pat. No.
  • 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.
  • the addition of PEG or another polymer does not interfere with the antigen-binding affinity or specificity of the antibody variable domain polypeptide.
  • does not interfere with the antigen-binding affinity or specificity is meant that the PEG-linked antibody single variable domain has an !C 5 o or ND 50 which is no more than 10% greater than the ICso or NDso, respectively, of a non-PEG-linked antibody variable domain having the same antibody single variable domain.
  • the phrase "does not interfere with the antigen-binding affinity or specificity” means that the PEG-linked form of an antibody single variable domain retains at least 90% of the antigen binding activity of the non-PEGylated form of the polypeptide.
  • PEGylated antibodies and antibody fragments may generally be used to treat conditions that may be alleviated or modulated by administration of the antibodies and antibody fragments described herein. Generally the PEGylated antibodies and antibody fragments have increased half-life, as compared to the non-PEGylated antibodies and antibody fragments. The PEGylated antibodies and antibody fragments may be employed alone, together, or in combination with other pharmaceutical compositions.
  • the target antibody may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example,
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules.
  • Liposomal formuiations are often used in therapeutics and pharmaceuticals.
  • any one of the antibodies or fragments thereof disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et ai. f J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See Gabizon et al., J.
  • Stealth liposomes have also been proposed for use in delivering cytotoxic agents to tumors in cancer patients.
  • a range of drugs have been incorporated into stealth liposomes, including cisplatin (Rosenthal et al, 2002), TNF.alpha. (Kim et al, 2002), doxorubicin (Symon et al., 1999) and adriamycin (Singh et al., 1999), each reference being specifically incorporated herein by reference.
  • recent reports have indicated unexpected low efficacy of stealth liposomal doxorubicin and vinorelbine in the treatment of metastatic breast cancer (Rimassa et al., 2003). See also U.S Patent Application No.
  • the invention provides improved stealthed liposome formulations, in which the stealthed liposomes are functionally associated or "coated” with an antibody that binds to an aminophospholipid or anionic phospholipid, preferably to PS or PE.
  • the 9D2, 3G4 (ATCC 4545) and like, competing antibodies of the invention are preferred for such uses, although any antibody, or antigen binding region thereof, which binds to an aminophospholipid or anionic phospholipid may be used.
  • Any stealthed liposome may form the basis of the new liposomal formulations, and preferably a PEGylated liposome will be employed.
  • the stealthed liposomes are "coated", i.e., operatively or functionally associated with the antibody that binds to an aminophospholipid or anionic phospholipid.
  • the operative or functional association is made such that the antibody retains the ability to specifically bind to the target aminophospholipid or anionic phospholipid, preferably PS or PE, thereby delivering or targeting the stealthed liposome and any contents thereof to PS- and/or PE-positive cells, such as tumor cells and tumor vascular endothelial cells.
  • the antibody-coated stealthed liposomes of the invention may be used alone.
  • such liposomes will also contain one or more second therapeutic agents, such as anti-cancer or chemotherapeutic agents (the first therapeutic agent being the antibody itself).
  • the second therapeutic agents are generally described as being within the "core" of the liposome. Any one or more of the second, anti-cancer or chemotherapeutic agents known in the art and/or described herein for conjugation to antibodies, or for combination therapies, may be used in the antibody-coated stealthed liposomes of the invention, for example, any combination therapies.
  • chemotherapeutic or radiotherapeutic agent cytokine, anti-angiogenic agent or apoptosis- inducing agent.
  • preferred chemotherapeutic agents are anti-tubulin drugs, docetaxel and paclitaxel.
  • the antibody-coated stealthed liposomes of the invention may also be loaded with one or more anti-viral drugs for use in treating viral infections and diseases.
  • anti-viral drugs any one or more of the second, anti-viral drugs known in the art and/or described herein for conjugation to antibodies, or for combination therapies, may be used in the antibody-coated stealthed liposomes of the invention.
  • antibodies or antigen-binding fragments thereof are conjugated to albumen using art recognized techniques.
  • a salvage receptor binding epitope refers to an epitope of the Fe region of an IgG molecule (e.g., IgGi, IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the anti-Her3 antibody of the invention including binding fragments thereof demonstrates both species and molecule selectivity.
  • the anti-Her3 antibody of the invention binds to human Her3.
  • a therapeutically effective amount of a naked fully human anti-Her3 antibody, or fragments thereof can be formulated in a pharmaceutically acceptable excipient.
  • the efficacy of the naked fully human Her3 antibodies and their fragments can also be enhanced by
  • Her3 antibodies conjugated with one or more therapeutic agents, including drugs, toxins, immunomodulators, hormones, oligonucleotides, hormone antagonists, enzymes, enzyme inhibitors, therapeutic radionuclides, an angiogenesis inhibitor, etc., administered concurrently or sequentially or according to a prescribed dosing regimen, with the Her3 antibodies or fragments thereof.
  • therapeutic agents including drugs, toxins, immunomodulators, hormones, oligonucleotides, hormone antagonists, enzymes, enzyme inhibitors, therapeutic radionuclides, an angiogenesis inhibitor, etc.
  • the anti-Her3 antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et a!., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction.
  • a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst.81 ( 19)1484 (1989)
  • the antibody of the present invention may also be used in ADEPT by conjugating the antibody to a prod ug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01 145) to an active anti-cancer drug.
  • a prodrug e.g., a peptidyl chemotherapeutic agent, see WO81/01 145.
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to convert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sul fate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alany!carboxylpeptidascs, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as beta-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; beta
  • antibodies with enzymatic activity can be used to convert to prodrugs of the invention into free active drugs (Massey, Nature 328: 457-458 (1987)).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
  • the enzymes of this invention can be covalently bound to the antibody mutant by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art
  • Antibodies with enzymatic activity can also be employed to convert prodrugs into active drugs.
  • Abzymes based upon the antibodies of the invention preferably the 9D2 and 3G4 and like antibodies, thus form another aspect of the present invention.
  • the technical capacity to make abzymes also exists within those of ordinary skill in the art, as exemplified by Massey et al. (1987), specifically incorporated herein by reference for purposes of supplementing the abzyme teaching.
  • Catalytic antibodies capable of catalyzing the breakdown of a prodrug at the carbamate position such as a nitrogen mustard aryl carbamate, are further contemplated, as described in EP 745,673, specifically incorporated herein by reference. Screening for Antibodies with Desired Properties
  • antibodies of the present invention can be characterized for their physical/chemical properties and biological functions by various assays known in the art.
  • antibodies are characterized for any one or more of binding to Her3 receptor protein, and/or reduction or blocking of Her 3 receptor activation; and/or reduction or blocking of Her 3 receptor downstream molecular signaling; and/or disruption or blocking of Her3 receptor binding to its native ligand, e.g, serrate or delta etc ; and/or promotion of endothelial cell proliferation; and/or inhibition of endothelial cell differentiation; and/or inhibition of arterial differentiation; and/or inhibition of tumor vascular perfusion; and/or treatment and/or prevention of a tumor, cell proliferative disorder or a cancer; and/or treatment or prevention of a disorder associated with Her3 expression and/or activity; and/or treatment or prevention of a disorder associated with Her3 receptor expression and/or activity.
  • antibodies may be selected based upon certain biological characteristics such as for example assessing the growth inhibitory effects of an anti-Her3 antibody of the invention. This property may be assessed by methods known in the art, e.g., using cells which express Her3 receptor either endogenously or following transfeetion with the Her3 receptor gene. For example, tumor cell lines and Her3 receptor-transfected cells may be treated with an anti-Her3 receptor monoclonal antibody of the invention at various biological characteristics
  • concentrations for a few days e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay.
  • Another method of measuring proliferation would be by comparing 3 H-thymidine uptake by the cells treated in the presence or absence of an anti-Her3 receptor antibody of the invention. After antibody treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter.
  • Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line.
  • the Her3 receptor agonist will inhibit cell proliferation of a Her3 receptor-expressing tumor cell in vitro or in vivo by about 25- 100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, at an antibody concentration of about 0.5 to 30 ⁇ g mL Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 g/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody.
  • the antibody is growth inhibitory in vivo if administration of the anti-Her3 receptor antibody at about 1 ⁇ g/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody.
  • the purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.
  • assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.
  • the assaying method for detecting Her3 using the antibodies of the invention or binding fragments thereof are not particularly limited. Any assaying method can be used, so long as the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (e.g., the level of Her3) in a fluid to be tested can be detected by chemical or physical means and the amount of the antigen can be calculated from a standard curve prepared from standard solutions containing known amounts of the antigen. Representative
  • immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Pat. Nos. 4,367,1 10 (double monoclonal antibody sandwich assay); Wide et al., irkham and Hunter, eds. Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970); U.S. Pat. No. 4,452,901 (western blot); Brown et al, J. Biol. Chem. 255: 4980-4983 (1980) (immunoprecipitation of labeled ligand); and Brooks et al., Clin. Exp. Immunol.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected.
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the immobilized antibody of the present invention is reacted with a test fluid (primary reaction), then with a labeled form of antibody of the present invention (secondary reaction), and the activity of the labeling agent on the immobilizing carrier is measured, whereby the Her3 level in the test fluid can be quantified.
  • the primary and secondary reactions may be performed simultaneously or with some time intervals.
  • the methods of labeling and immobilization can be performed by modifications of those methods described above.
  • the antibody used for immobilized or labeled antibody is not necessarily from one species, but a mixture of two or more species of antibodies may be used to increase the measurement sensitivity, etc.
  • the antibodies used in the primary reaction recognize the partial peptides at the C-terminal region of Her3
  • the antibodies used in the secondary reaction are preferably those recognizing partial peptides other than the C-terminal region (i.e., the N- terminal region).
  • antibodies recognizing partial peptides other than the N-terminal region are preferably employed.
  • a simultaneous assay involves a single incubation step wherein the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay,
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period, is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward” assays.
  • a combination of antibodies of the present invention specific for separate epitopes can be used to construct a sensitive three-site immunoradiometric assay.
  • the sandwich assay includes:
  • a method for quantifying expression levels of Her3 in a test fluid comprising reacting the antibody specifically reacting with a partial peptide at the N-terminal region of the Her3 immobilized on a carrier, a labeled form of the antibody specifically reacting with a partial peptide at the C-terminal region and the test fluid, and measuring the activity of the label; or
  • a method for quantifying Her3 expression in a test fluid comprising reacting the antibody specifically reacting with a partial peptide at the C-terminal region of the Her3 immobilized onto a carrier, the antibody specifically reacting with a partial peptide at the N- terminal region of a labeled form of the Her3 and the test fluid, and measuring the activity of the label; etc.
  • one skilled in the art may combine and/or competitively react antibodies of the invention or fragments thereof, a test fluid and a labeled form of Her3, measure a ratio of the labeled Her3 bound to the antibodies or fragments thereof b to thereby quantify the Her3 in the test fluid.
  • an antigen in a test fluid and a solid phase antigen are competitively reacted with a given amount of a labeled form of the antibody of the present invention followed by separating the solid phase from the liquid phase; or an antigen in a test fluid and an excess amount of labeled form of the antibody of the present invention are reacted, then a solid phase antigen is added to bind an unreacted labeled form of the antibody of the present invention to the solid phase and the solid phase is then separated from the liquid phase. Thereafter, the labeled amount of any of the phases is measured to determine the antigen level in the test fluid.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the Her3 from the sample by formation of a binary solid phase antibody-Her3 complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted Her3, if any, and then contacted with the solution containing a known quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the Her3 bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
  • This type of forward sandwich assay can be a simple "yes/no” assay to determine whether Her3 is present or can be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of Her3.
  • Such "two-site” or “sandwich” assays are described by Wide (Radioimmune Assay Method, Kirkham, ed., Livingstone, Edinburgh, 1970, pp. 199 206).
  • the amount of insoluble sediment which is produced as a result of the antigen-antibody reaction in a gel or in a solution, is measured. Even when the amount of an antigen in a test fluid is small and only a small amount of the sediment is obtained, a laser nephrometry utilizing laser scattering can be suitably used.
  • labeling agents which may be used in the above referenced assay methods (1) to (4) using labeling agents, include radioisotopes (e.g., !25 L 131 I, 3 H, 1 C, 32 P, 33 P, 3S S, etc., fluorescent substances, e.g., cyanine fluorescent dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7), fluorescamine, fluorescein isothiocyanate, etc., enzymes (e.g., .beta.-galactosidase, .beta.- glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (e.g., luminol, a luminol derivative, luciferin, lucigenin, etc.), biotin, lanthanides, etc.
  • a biotin-avidin system may be used as well for binding an antibody to a
  • the carrier include insoluble polysaccharides such as agarose, dextran, cellulose, etc.; synthetic resins such as polystyrene, polyacrylamide, silicone, etc.; or glass; and the like.
  • the antibodies of the present invention are tested for their antigen binding activity.
  • the antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.
  • Illustrative antigen binding assay are provided herein.
  • the binding affinity of anti Her3 antibodies is determined.
  • Antibodies of the invention preferably have a binding affinity(Kp) to Her3 of at least about l 10 "7 M, more preferably at least about 1x10 "8 M, more preferably at least about l lO "9 M, and most preferably at least about IxIO "10 M.
  • Preferred antibody-producing cells of the invention produce substantially only antibodies having a binding affinity to Her3 of at least about l lO "7 M, more preferably at least about lxl 0 "8 M, more preferably at least about lxlO "9 M, and most preferably at least about lxlO '10 M
  • Preferred compositions of the invention comprise substantially only antibodies having a binding affinity to Her3 of at least about lxl 0 "7 M, more preferably at least about lxl 0 "8 M, more preferably at least about lxl 0 "9 M, and most preferably at least about lxl0 "l0 M.
  • the antibodies of the invention bind to Her3 with substantially the same K D as an antibody that comprises one of the amino acid sequences selected from the group as set forth in one of Appendix I or II.
  • the antibody binds to Her3 with substantially the same KD as an antibody that comprises one or more CDRs from an antibody that comprises one of the amino acid sequences set forth herein.
  • Anti-Her3 antibodies according to the invention or identified using the methods disclosed herein have a low dissociation rate.
  • the anti-Her3 antibody has a o f r of 1 XlO ⁇ or lower, preferably a K 0 ff that is 5 X10 "5 or lower.
  • the antibodies of the invention or those identified or produced using the methods of the invention bind to Her3 with substantially the same K 0 as an antibody that comprises one or more CDRs disclosed herein.
  • Illustrative assays for affinity analysis are described herein.
  • Affinity Analysis for Epitope(s) Affinity can be either absolute or relative.
  • absolute affinity it is meant that the assay for affinity gives defined numerical determinations of the affinity of one compound for another. Comparison of the affinity of the complex being tested to that of a reference compound whose binding affinity is known allows for the determination of relative binding affinity of the test ligand.
  • affinity of one molecule for another can be measured by any method known in the art.
  • methods include competition assays, surface piasmon resonance, half-maximal binding assays, competition assays, Scatchard analysis, direct force techniques (Wong et al., Direct force measurements of the streptavidin-biotin interaction, Biomol. Eng. 16:45-55, 1999), and mass spectrometry (Downard, Contributions of mass spectrometry to structural immunology, J. Mass Spectrom. 35:493-503, 2000).
  • the binding affinity and dissociation rate of an antibody to Her3 may be determined by any method known in the art.
  • the binding affinity can be measured by competitive ELISAs, RIAs or surface piasmon resonance, such as BIAcore.
  • the dissociation rate can also be measured by surface piasmon resonance.
  • the binding affinity and dissociation rate is measured by surface piasmon resonance. More, the binding affinity and dissociation rate is measured using a BIAcore. See below for a brief description, it being understood the invention is not limited to the specific assays detailed herein.
  • low affinity refers to binding wherein the dissociation constant ( D ) between two molecules is about 10 "5 M to 10 "7 M.
  • Mode affinity refers to binding wherein the dissociation constant (KD) between two molecules is at least about 10 "7 M to 10 "8 M.
  • High affinity refers to a binding wherein the association constant between the two molecules is at least about 10 "8 M to about 10 " 1 M, and preferably about 10 "9 M to about 10 "!4 M, more preferably about 10 "10 M to about 10 "14 M, and most preferably greater than about 10 ",4 M.
  • the dissociation constant, K D is an equilibrium constant for the dissociation of one species into two, e.g., the dissociation of a complex of two or more molecules into its components, for example, dissociation of a substrate from an enzyme.
  • Exemplary K values for compositions of the present invention are from about 10 "7 M (100 nM) to about 10 "12 M (0.001 nM).
  • the stability constant is an equilibrium constant that expresses the propensity of a species to form from its component parts. The larger the stability constant, the more stable is the species.
  • the stability constant (formation constant) is the reciprocal of the instability constant (dissociation constant).
  • the affinity of an invention antibody for a target epitope, or the affinity of a bi- specific antibody for a carrier epitope, is driven by non-covalent interactions.
  • Non-covalent interactions can, but rarely do, have the strength of a covalent linkage (i.e., a chemical bond).
  • a covalent linkage i.e., a chemical bond.
  • the affinity of the invention antibody for a target epitope, although driven by non-cova!ent interactions, is so high as to approach the strength of a covalent bond. This provides for invention antibodies that are very stable relative to other Her3 receptor antibodies of the invention.
  • the affinity of an invention antibody for its cognate target epitope is a KD of about 100 nM to about 0.01 nM; more preferably, greater than about 100 nM, or greater than about 10 nM; most preferably, greater than about 1 nM, or greater than about 0.1 nM.
  • Typical D for target epitopes are from about 0.1 nM to 100 nM, preferably from about 0.1 nM to 10 nM, more preferably from about 0.5 nM to 5 nM, or about 1 nM.
  • the affinity of an antibody for its cognate carrier epitope may be greater than the affinity of an antibody for a free carrier epitope or for a monovalent antibody comprising the carrier epitope.
  • a multivalent targetable construct having x carrier epitopes has a greater affinity for its target epitope than would x number of constructs.
  • the compositions of the invention also provides for synergistic, rather than merely additive, binding effects.
  • Binding parameters such as D niay be measured using surface plasmon resonance on a chip, for example, with a BIAcoreTM chip coated with immobilized binding components.
  • Surface plasmon resonance is used to characterize the microscopic association and dissociation constants of reaction between an antibody or antibody fragment and its ligand.
  • Such methods are generally described in the following references which are incorporated herein by reference. (Vely et al., BIAcore analysis to test phosphopeptide-SH2 domain interactions, Meth. Mol. Biol. 121 :313-21, 2000; Liparoto et al., Biosensor analysis of the interleukin-2 receptor complex, J. Mol. Recog.
  • BIAcoreTM uses the optical properties of surface plasmon resonance (SPR) to detect alterations in protein concentration bound within to a dextran matrix lying on the surface of a gold/glass sensor chip interface, a dextran biosensor matrix.
  • SPR surface plasmon resonance
  • proteins are covalently bound to the dextran matrix at a known concentration and a ligand for the protein (e.g., antibody) is injected through the dextran matrix.
  • a ligand for the protein e.g., antibody
  • Near infrared light, directed onto the opposite side of the sensor chip surface is reflected and also induces an evanescent wave in the gold film, which in turn, causes an intensity dip in the reflected light at a particular angle known as the resonance angle.
  • the refractive index of the sensor chip surface is altered (e.g., by ligand binding to the bound protein) a shift occurs in the resonance angle.
  • This angle shift can be measured and is expressed as resonance units (RUs) such that 1000 RUs is equivalent to a change in surface protein concentration of 1 ng/mm 2. These changes are displayed with respect to time along the y-axis of a sensorgram, which depicts the association and dissociation of any biological reaction.
  • Relative Affinity Affinity may also be defined in relative terms, e.g., by IC 5 0.
  • the IC 5 0 of a compound is the concentration of that compound at which 50% of a reference ligand is displaced from a target epitope in vitro or targeted tissue in vivo.
  • IC50 is determined by competitive ELISA.
  • the invention provides anti-Her3 monoclonal antibodies that compete with a conventional anti ⁇ Her3 antibody for binding to Her3 receptor protein.
  • Such competitor antibodies include antibodies that recognize a Her3 epitope that is the same as or overlaps with the Her3 epitope recognized by any one of a conventional antibody.
  • Such competitor antibodies can be obtained by assay well known to one skilled in the art.
  • they can b obtained by screening anti-Her3 hybridoma supernatants for binding to immobilized Her3 in competition with labeled 26.6, 26.14, 26.20, 26.34, and/or 26.82 antibodies.
  • they can be used in a binding assay A hybridoma supernatant containing competitor antibody will reduce the amount of bound, labeled antibody detected in the subject competition binding mixture as compared to the amount of bound, labeled antibody detected in a control binding mixture containing irrelevant (or no) antibody.
  • Any of the competition binding assays described herein are suitable for use in the foregoing procedure.
  • Anti-Her3 antibodies of the invention possessing the unique properties described herein can be obtained by screening anti-Her3 hybridoma clones for the desired properties by any convenient method. For example, if an anti-Her3 monoclonal antibody that blocks or does not block the binding of Her3 receptors to its binding partner e.g., a Her3 ligand is desired, the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, wherein plate wells are coated with the binding partner, and a solution of antibody in an excess of the Her3 receptor of interest is layered onto the coated plates, and bound antibody is detected enzymatically, e.g.
  • a binding competition assay such as a competitive binding ELISA
  • HRP-conjugated anti-Ig antibody or biotinylated anti-Ig antibody e.g. by developing plates with streptavidin-HRP and/or hydrogen peroxide and detecting the HRP color reaction by spectrophotometry at 490 nm with an ELISA plate reader.
  • the present invention contemplates an altered antibody that possesses some but not all effector functions, which make it a desired candidate for many applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • the Fc activities of the produced immunoglobulin are measured to ensure that only the desired properties are maintained.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express FcyRIII only, whereas monocytes express Fc yRI, FcyRH and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and inet, Annu. Rev. Immunol 9:457-92 (1991 ).
  • An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. A 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity.
  • a CDC assay e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
  • Fc n binding and in vivo clearance/half life determinations can also be performed using methods known in the art, e.g. those described in the Examples section.
  • test antibody binds to the same epitope as an anti-
  • Her3 antibody by binding the anti-Her3 antibody to Her3 receptor protein under saturating conditions, and then measuring the ability of the test antibody to bind to Her3. If the test antibody, e.g., anti-Her3 antibodies derived from the invention antibodies or in accordance with the methods of the invention is able to bind to the Her3 receptor protein at the same time as the reference anti-Her3 antibody, then the test antibody binds to a different epitope as the anti-Her3 antibody. However, if the test antibody is not able to bind to Her3 receptor protein at the same time, then the test antibody binds to the same epitope as the human anti-Her3 antibody. This experiment may be performed using ELISA, RIA or surface plasm on resonance.
  • the experiment is performed using surface plasmon resonance, supra.
  • BIAcore is used, see supra.
  • the diagnostic method may also be used to determine whether a tumor is potentially cancerous, if it expresses high levels of Her3, or benign, if it expresses low levels of Her3.
  • biological samples obtained from patients suspected of exhibiting an oncogenic disorder mediated by Her3 may be assayed for the presence of Her 3 expressing cells.
  • the anti-Her3 antibodies of the invention may be used to determine the levels of Her3 receptor protein in a tissue or in cells derived from the tissue.
  • the tissue is a diseased tissue.
  • the tissue is a tumor or a biopsy thereof.
  • a tissue or a biopsy thereof is excised from a patient.
  • the tissue or biopsy is then used in an immunoassay to determine, e.g., Her3 levels, cell surface levels of Her3, levels of tyrosine phosphorylation of Her3, or localization of Her 3 by the methods discussed herein.
  • the method can be used to determine tumors that express Her3.
  • the present invention provides methods for diagnosing cancers by assaying for changes in the level of Her3 in cells, tissues or body fluids compared with the levels in cells, tissues, or body fluids, preferably of the same type in a control sample.
  • a change, especially an increase, in levels of Her3 in the patient versus the control is associated with the presence of cancer.
  • a positive result indicating that the patient being tested has cancer is one in which levels of Her3 in or on cells, tissues or body fluid are at least two times higher, and preferably three to five times higher, or greater, than the levels of the antigens in or on the same cells, tissues, or body fluid of the control.
  • Normal controls include a human without cancer and/or non-cancerous samples from the patient.
  • the in vitro diagnostic methods may include any method known to one skilled in the art including immunohistological or immunohistochemical detection of tumor cells (e.g., on human tissue, or on cells dissociated from excised tumor specimens), or serological detection of tumor associated antigens (e.g., in blood samples or other biological fluids).
  • immunohistological or immunohistochemical detection of tumor cells e.g., on human tissue, or on cells dissociated from excised tumor specimens
  • serological detection of tumor associated antigens e.g., in blood samples or other biological fluids.
  • Immunohistochemical techniques involve staining a biological specimen, such as a tissue specimen, with one or more of the antibodies of the invention and then detecting the presence on the specimen of antibody-antigen complexes comprising antibodies bound to the cognate antigen. The formation of such antibody-antigen complexes with the specimen indicates the presence of cancer in the tissue.
  • Detection of the antibody on the specimen can be accomplished using techniques known in the art such as immunoenzymatic techniques, e.g., immunoperoxidase staining technique, or the avidin-biotin technique, or immunofluorescence techniques (see, e.g., Ciocca et at, 1986, "Immunohistochemical Techniques Using Monoclonal Antibodies", Meth. Enzymol., 121 :562 79 and Introduction to Immunology, Ed. Kimball, (2 nd Ed), Macmillan Publishing Company, 1986, pp. 1 13 1 17). Those skilled in the art can determine operative and optimal assay conditions by routine experimentation.
  • immunoenzymatic techniques e.g., immunoperoxidase staining technique, or the avidin-biotin technique
  • immunofluorescence techniques see, e.g., Ciocca et at, 1986, "Immunohistochemical Techniques Using Monoclonal Antibodies", Meth. Enzymol.,
  • the present invention assists in the diagnosis of cancers and tumors by the identification and measurement of the Her3 receptor protein levels in biological samples. If Her3 receptor protein is normally present, and the development of the oncogenic disorder is caused by an abnormal quantity of the cell surface receptor (Her3), e.g., expression relative to normal, the assay should compare Her3 levels in the biological sample to the range expected in normal, non-oncogenic tissue of the same cell type. Thus, a statistically significant increase in the amount of Her3 bearing cells or Her3 expression level in the subject relative to the control subject or subject's baseline, can be a factor that may lead to a diagnosis of an oncogenic disorder that is progressing or at risk for such a disorder.
  • Her3 receptor protein is normally present, and the development of the oncogenic disorder is caused by an abnormal quantity of the cell surface receptor (Her3), e.g., expression relative to normal
  • the assay should compare Her3 levels in the biological sample to the range expected in normal, non-oncogenic tissue of the same cell type.
  • Her3 indicative of cancers likely to metastasize
  • the presence of high levels of Her3 indicative of cancers likely to metastasize can also be detected.
  • the ability to detect the antigen provides early diagnosis, thereby affording the opportunity for early treatment. Early detection is especially important for cancers difficult to diagnose in their early stages.
  • the level of antigen detected and measured in a body fluid sample such as for example diseased tissue provides a means for monitoring the course of therapy for the cancer or tumor, including, but not limited to, surgery, chemotherapy, radiation therapy, the therapeutic methods of the present invention, and combinations thereof.
  • the level of such antigen can be used to indicate successful removal of the primary tumor, cancer, and/or metastases, for example, as well as to indicate and/or monitor the effectiveness of other therapies over time. For example, a decrease in the level of the cancer or tumor-specific antigen over time indicates a reduced tumor burden in the patient. By contrast, no change, or an increase, in the level of antigen over time indicates ineffectiveness of therapy, or the continued growth of the tumor or cancer.
  • a typical in vitro immunoassay for detecting Her3 comprises incubating a biological sample in the presence of a detectably labeled anti-Her3 antibody or antigen binding fragment of the present invention capable of selectively binding to Her3, and detecting the labeled fragment or antibody which is bound in a sample.
  • the antibody is bound to a label effective to permit detection of the cells or portions (e.g., Her3 or fragments thereof liberated from hyperplastic, dysplastic and/or cancerous cells) thereof upon binding of the antibody to the cells or portions thereof.
  • the presence of any cells or portions thereof in the biological sample is detected by detection of the label.
  • the biological sample may be brought into contact with, and immobilized onto, a solid phase support or carrier, such as nitrocellulose, or other solid support or matrix, which is capable of immobilizing cells, cell particles, membranes, or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support or matrix, which is capable of immobilizing cells, cell particles, membranes, or soluble proteins.
  • the support may then be washed with suitable buffers, followed by treatment with the detectably-labeled anti- Her3 antibody.
  • the solid phase support may then be washed with buffer a second time to remove unbound antibody.
  • the araount of bound label on the solid support may then be detected by conventional means. Accordingly, in another embodiment of the present invention, compositions are provided comprising the monoclonal antibodies, or binding fragments thereof, bound to a solid phase support, such as described herein.
  • In vitro assays in accordance with the present invention also include the use of isolated membranes from cells expressing a recombinant Her3, soluble fragments comprising the Hgand binding segments of Her3, or fragments attached to solid phase substrates. These assays allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or ligand mutations and modifications, e.g., ligand analogues.
  • the monoclonal antibodies and binding fragments thereof of the present invention may be used in in vitro assays designed to screen compounds for binding affinity to Her3. See Fodor et al. Science 251 : 767-773 (1991), incorporated herein by reference.
  • the invention contemplates a competitive drug screening assay, where the monoclonal antibodies or fragments thereof of the invention compete with a test compound for binding to Her3. in this manner the monoclonal antibodies and fragments thereof are used to detect the presence of any polypeptide which shares one or more binding sites of the Her3 and can be used to occupy binding sites on the receptor which might otherwise be occupied by the antibody.
  • the anti-Her3 antibodies of the invention may be used to determine or quantify the amount of Her3 on the cell surface after treatment of the cells with various compounds.
  • This method can be used to test compounds that may be used to activate or inhibit Her3.
  • one sample of cells is treated with a test compound for a period of time while another sample is left untreated. If the total level of Her3 is to be measured, the cells are lysed and the total Her3 level is measured using one of the immunoassays described herein.
  • a preferred immunoassay for measuring total Her3 receptor protein levels is an ELISA or Western blot. If only the cell surface level of Her3 is to be measured, the cells are not lysed, and the cell surface levels of Her3 are measured using any one or more of the assays known to the skilled artisan, e.g., one of the immunoassays described herein.
  • a preferred immunoassay for determining cell surface levels of Her3 includes the steps of labeling the cell surface proteins with a detectable label, such as biotin or 125 I, immunoprecipitating the Her3 with an anti-Her3 antibody and then detecting the labeled Her3.
  • Another preferred immunoassay for determining the localization of Her3, e.g., cell surface levels, is by using immunohistochemistry.
  • a method to determine whether a conventional anti- Her3 antibody decreases Her3 expression on a target tumor tissue or cell is provided herein.
  • inventions "conventional Her3 antagonist” "conventional treatment with a Her3 moiety” is used interchangeably to mean Her3 specific monoclonal antibodies currently available that specifically target Her3 expression and do not bind to the same epitope as the antibodies of the invention.
  • a further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer mediated by Her3.
  • the method comprises the steps of measuring the level of expression of Her3 in a cell or tissue of interest, incubating the cell or tissue with an anti-Her3 antibody or antigen-binding portion thereof, then re-measuring the level of Her 3 expression with an anti-Her3 antibody or antigen binding fragment of the invention in the cell or tissue.
  • ICD expression levels may be measured in the above example.
  • a diagnosis that levels of Her3 are low could be used for predicting that the patient is responding to treatment with the conventional anti-Her3 antibody regiment.
  • the anti-Her3 antibodies of the invention may be used in the above diagnostic assays either simultaneously with administration of the conventional Her3 antibody or after treatment with the conventional anti-Her3 antibody.
  • the conventional Her3 antibody does not compete with the anti-Her3 antibody of the invention for binding Her3 protein.
  • the above assays can be performed iteratively over a period of time to assess the therapeutic efficacy of a conventional anti-Her3 antibody based therapeutic protocol.
  • the anti-Her3 antibody of the invention can be used as a "negative biomarker" allowing it to be used to assess the treatment and therapeutic protocol of a conventional anti- Her3 antibody based therapy.
  • the invention also includes nucleic acids encoding the heavy chain and/or light chain of the anti-Her3 antibodies of the invention.
  • Nucleic acids of the invention also include fragments of the nucleic acids of the invention.
  • a “fragment” refers to a nucleic acid sequence that is preferably of sufficient length to encode a functionally active fragment of the invention antibodies, e.g., light or heavy chain.
  • a “fragment” can also mean the whole coding sequence of a gene and may include 5' and 3' untranslated regions.
  • Constructs of any one or more polynucleotides having sequences as set forth herein can be generated synthetically.
  • single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides is described by, e.g., Stemmer et al., Gene (Amsterdam) (1995) 164(l):49-53.
  • assembly PCR the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides
  • DNA shuffling Stepmmer, Nature (1994) 370:389-391).
  • polynucleotide of the invention is expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems.
  • Vectors, host cells and methods for obtaining expression in same are well known in the art. Suitable vectors and host cells are described in U.S. Pat. No. 5,654, 173.
  • Polynucleotide molecules comprising a polynucleotide sequence provided herein are generally propagated by placing the molecule in a vector. Viral and non-viral vectors are used, including plasmids.
  • plasmid will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially. Methods for preparation of vectors comprising a desired sequence are well known in the art.
  • polynucleotides set forth in any one or more of SEQ ID NOs set forth in one or more appendices disclosed herein or their corresponding full-length polynucleotides are linked to regulatory sequences as appropriate to obtain the desired expression properties. These can include promoters (attached either at the 5' end of the sense strand or at the 3' end of the antisense strand), enhancers, terminators, operators, repressors, and inducers.
  • the promoters can be regulated or constitutive. In some situations it may be desirable to use conditionally active promoters, such as tissue-specific or developmental stage-specific promoters.
  • conditionally active promoters such as tissue-specific or developmental stage-specific promoters.
  • the resulting replicated nucleic acid, RNA, expressed protein or polypeptide is within the scope of the invention as a product of the host cell or organism.
  • the product is recovered by any appropriate means known in the art.
  • a target gene e.g., corresponding to any one or more of the nucleic acid molecules set forth herein can be regulated in the cell to which the gene is native.
  • an endogenous gene of a cell can be regulated by an exogenous regulatory sequence as disclosed in U.S. Pat. No. 5,641,670.
  • the encoded antibody heavy chain preferably comprises an amino acid sequence selected from the group consisting of SEQ ID Nos as set forth in one of Appendix I-III.
  • the encoded antibody light chain preferably comprises an amino acid sequence a set forth in one of the appendices set forth herein.
  • the invention provides nucleic acids encoding both a heavy chain and a light chain of an antibody of the invention.
  • a nucleic acid of the invention may comprise a nucleic acid sequence (Appendix I) encoding an amino acid sequence as set forth in one of Appendix li or III and a nucleic acid sequence (Appendix I) encoding an amino acid sequence as set forth in one of Appendix II or III.
  • Nucleic acids of the invention include nucleic acids having at least 80%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% homology to nucleic acids of the invention.
  • the terms "percent similarity”, “percent identity” and “percent homology” when referring to a particular sequence, are used as set forth in the University of Wisconsin GCG software program.
  • Nucleic acids of the invention also include complementary nucleic acids. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 20% mismatch in the sequences.
  • the invention also provides a nucleic acid molecule encoding the variable region of the light chain (VL) as described herein as well as an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences encoding a V L as described herein, particularly to a V L that comprises an amino acid sequence of one of the sequences as set forth in Appendix II or III.
  • the invention also provides a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence of one of the sequences as set forth in Appendix I.
  • the nucleic acid molecule encoding a V L is one that hybridizes under highly stringent conditions to a nucleic acid sequence encoding a V L as described above.
  • the invention also provides a nucleic acid molecule encoding the variable region of the heavy chain (VH) as described herein as well as an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences encoding a VH as described herein, particularly to a VH that comprises an amino acid sequence of one of the sequences set forth in Appendix II or III.
  • the invention also provides a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence of any one or more of the sequences set forth in Appendix I.
  • the nucleic acid molecule encoding a VH is one that hybridizes under highly stringent conditions to a nucleic acid sequence encoding a VH as described above.
  • hybridization means the pairing of complementary strands of oligomeric compounds.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • nucleobases complementary nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances.
  • selectively hybridize means to detectably and specifically bind.
  • Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • “High stringency” or “highly stringent” conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein.
  • high stringency or “highly stringent” conditions is a method of incubating a polynucleotide with another polynucleotide, wherein one polynucleotide may be affixed to a solid surface such as a membrane, in a hybridization buffer of 6x.
  • SSPE or SSC 50% formamide, 5x.
  • Denhardfs reagent 0.5% SDS, 100 g ml denatured, fragmented salmon sperm DNA at a hybridization temperature of 42°C. for 12-16 hours, followed by twice washing at 55°C. using a wash buffer of lxSSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.
  • the nucleic acid molecule encoding either or both of the entire heavy and light chains of an anti-Her3 antibodies or the variable regions thereof may be obtained from any source that produces an anti-Her3 antibody.
  • Methods of isolating mRNA encoding an antibody are well-known in the art (See, e.g., Sambrook et ai.)
  • the mRNA may be used to produce cDNA for use in the polymerase chain reaction (PCR) or cDNA cloning of antibody genes.
  • a nucleic acid molecule encoding the entire heavy chain of an anti-Her3 antibody disclosed herein may be constructed by fusing a nucleic acid molecule encoding the variable domain of a heavy chain or an antigen-binding domain thereof with a constant domain of a heavy chain.
  • a nucleic acid molecule encoding the light chain of the anti-Her3 antibody of the invention may be constructed by fusing a nucleic acid molecule encoding the variable domain of a light chain or an antigen-binding domain thereof with a constant domain of a light chain.
  • the nucleic acid molecules encoding the VH and V L chain may be converted to full-length antibody genes by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the heavy chain constant region (CH) segment(s) within the vector and the VL segment is operatively linked to the light chain constant region (CL) segment within the vector.
  • the nucleic acid molecules encoding the V H or VL chains are converted into full-length antibody genes by linking, e.g., ligating, the nucleic acid molecule encoding a VH chain to a nucleic acid molecule encoding a CH chain using standard molecular biological techniques.
  • nucleic acid molecules encoding VL and CL chains may be expressed from a cell into which they have been introduced and the anti-Her3 antibody isolated.
  • nucleic acid molecules may be used to recombinantly express large quantities of anti-Her3 antibodies using techniques known to one skilled in the art of recombinant biologist.
  • the herein described nucleic acid molecules can also be used to recombinantly produce any one of the anti-Her3 antibody variants, mutants, fragments thereof or derivatives, including single chain antibodies, bispecific, scFv etc immunoadhesins, diabodies, mutated antibodies and antibody derivatives, as described further below.
  • the nucleic acid molecules of the invention may be used as probes or PCR primers for specific antibody sequences.
  • a nucleic acid molecule probe may be used in diagnostic methods or a nucleic acid molecule PCR primer may be used to amplify regions of DNA that could be used, inter alia, to isolate nucleic acid sequences for use in producing variable domains of anti-Her3 antibodies.
  • the nucleic acid molecules are oligonucleotides.
  • the oligonucleotides are from highly variable regions of the heavy and light chains of the antibody of interest
  • the oligonucleotides encode all or a part of one or more of the CD s,
  • a “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus, into which another genetic sequence or element (either DNA or RNA) may be inserted so as to bring about the replication of the attached sequence or element.
  • a “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) or virus, which is capable of replication largely under its own control.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • the expression vector contains a constitutively active promoter segment (such as but not limited to CMV, SV40, Elongation Factor or LTR sequences) or an inducible promoter sequence such as the steroid inducible pIND vector (Invitrogen), where the expression of the nucleic acid can be regulated.
  • a constitutively active promoter segment such as but not limited to CMV, SV40, Elongation Factor or LTR sequences
  • an inducible promoter sequence such as the steroid inducible pIND vector (Invitrogen)
  • the expression vector can be introduced into a cell by transfection,
  • the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • polyoma and strong mammalian promoters such as native
  • the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,21 , 4,634,665 and 5, 179,017, all by Axel et al.).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • neo gene for G418 selection.
  • DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used.
  • preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. a. Generating Antibodies Using Prokaryotic Host Cells
  • Polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention.
  • Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector.
  • Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.
  • the vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes encoding ampiciliin (Amp), kanamycin (Kn) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells.
  • pBR322 its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
  • promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.
  • a convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any V H or V L sequence can be easily inserted and expressed, as described above.
  • splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions.
  • the recombinant expression vector can also encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as GEM 1 M -1 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • the expression vector of the invention may comprise two or more promoter- cistron pairs, encoding each of the polypeptide components.
  • a promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression.
  • Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.
  • promoters recognized by a variety of potential host cells are well known.
  • the selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention.
  • Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes.
  • heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter,
  • Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the .beta.-galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter.
  • trp tryptophan
  • other promoters that are functional in bacteria such as other known bacterial or phage promoters
  • Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.
  • each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane.
  • the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector.
  • the signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin 0 (STII) leaders, LamB, PhoE, PelB, OmpA and MBP.
  • STII heat-stable enterotoxin 0
  • the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.
  • the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron.
  • immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm.
  • Certain host strains e.g., the E. coli trxB-strains
  • Prokaryotic host cells suitable for expressing antibodies of the invention include
  • Archaebacteria and Eubacteria such as Gram-negative or Gram-positive organisms.
  • useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. sublilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, R izobia, Vitreoscilla, or Paracoccus.
  • gram- negative cells are used.
  • E. coli cells are used as hosts for the invention. Examples of E.
  • coli strains include strain W31 10 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCCTM Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W31 10 .DELTA. fhu.DELTA. (.DELTA.tonA) ptr3 lac Iq lacL8 .DELTA.ompT.DELTA. (nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635).
  • Other strains and derivatives thereof such as E. coli 294 (ATCC 31,446), E. coli B, E.
  • E. coli .lambda. 1776 ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable, These examples are illustrative rather than limiting.
  • Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1 90). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E.
  • coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
  • Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Transformation means introducing DNA into the prokaryotic host so that the DNA is rep!icable, either as an extrachromosomal element or by chromosomal integrant.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers.
  • Another method for transformation employs polyethylene glycol/DMSO.
  • Yet another technique used is electroporation.
  • Prokaryotic cells used to produce any one or more of the anti ⁇ Her3 antibodies of the invention are grown in media known in the art and suitable for culture of the selected host cells.
  • suitable media include Luria broth (LB) plus necessary nutrient supplements.
  • the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 30°C.
  • the pFI of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism.
  • the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.
  • an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.
  • PhoA promoters are used for controlling transcription of the polypeptides.
  • the transformed host cells are cultured in a phosphate-limiting medium for induction.
  • the phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263: 133-147).
  • a variety of other inducers may be used, according to the vector construct employed, as is known in the art.
  • the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells.
  • Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
  • PAGE polyacrylamide gel electrophoresis
  • Another aspect of the invention contemplates antibody production in large quantity by a fermentation process.
  • Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins.
  • Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source).
  • Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase.
  • a desired density e.g., an OD550 of about 180-220
  • inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used.
  • various fermentation conditions can be modified.
  • additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a)
  • peptidylprolyl cis,trans-isomerase with chaperone activity can be used to co-transform the host prokaryotic cells.
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274: 19601-19605; Georgiou et a!., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-171 13; Arie et l. (2001) Mol. Microbiol. 39: 199- 210.
  • host strains deficient for proteolytic enzymes can be used for the present invention.
  • host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof.
  • E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; Georgiou et al, U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al, Microbial Drug Resistance, 2:63-72 (1996).
  • E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and
  • phenylmethylsulfonylfluoride over about 30 min. Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicofin® or Millipore Pellicon® ultrafiltration unit, A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

Abstract

La présente invention concerne des anticorps humanisés spécifiques de Her3 et des parties de liaison à un antigène de ceux-ci. L'invention concerne également des molécules d'acide nucléique codant pour les anticorps anti-Her3, ainsi que des procédés d'utilisation de ceux-ci. L'invention concerne également des compositions pharmaceutiques comportant ces anticorps, et des procédés d'utilisation des anticorps et des compositions de ceux-ci pour le traitement et le diagnostic de troubles oncogènes hyperprolifératifs pathologiques, associés à une expression aberrante de Her3 ou de Her2, comprenant une activation aberrante de chacun de ces récepteurs.
PCT/US2011/053436 2010-09-30 2011-09-27 Génération, caractérisation et utilisations d'anticorps anti-her3 WO2012044612A1 (fr)

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WO2014108484A2 (fr) 2013-01-11 2014-07-17 F. Hoffmann-La Roche Ag Thérapie de combinaison avec des anticorps anti-her3
US8859737B2 (en) 2009-12-22 2014-10-14 Roche Glycart Ag Anti-HER3 antibodies and uses thereof
CN105209493A (zh) * 2013-03-14 2015-12-30 德克萨斯州大学系统董事会 用于诊断和治疗用途的her3特异性单克隆抗体
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CN113150165A (zh) * 2014-12-22 2021-07-23 西雅图免疫公司 双特异性四价抗体及其制造和使用方法
CN113144169A (zh) * 2021-02-10 2021-07-23 渤海大学 一种黄嘌呤氧化酶抑制肽及其应用
CN115372513A (zh) * 2022-08-19 2022-11-22 珠海市丽珠单抗生物技术有限公司 糖蛋白中二硫键的差异烷基化鉴定方法
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US9611331B2 (en) 2009-12-22 2017-04-04 Roche Glycart Ag Anti-HER3 antibodies and uses thereof
US8859737B2 (en) 2009-12-22 2014-10-14 Roche Glycart Ag Anti-HER3 antibodies and uses thereof
US10196456B2 (en) 2009-12-22 2019-02-05 Roche Glycart Ag Anti-HER3 antibodies and uses thereof
US9180185B2 (en) 2013-01-11 2015-11-10 Hoffman-La Roche Inc. Combination therapy of anti-HER3 antibodies
WO2014108484A2 (fr) 2013-01-11 2014-07-17 F. Hoffmann-La Roche Ag Thérapie de combinaison avec des anticorps anti-her3
CN105209493B (zh) * 2013-03-14 2019-05-03 德克萨斯州大学系统董事会 用于诊断和治疗用途的her3特异性单克隆抗体
US9725520B2 (en) 2013-03-14 2017-08-08 The Board Of Regents Of The University Of Texas System HER3 specific monoclonal antibodies for diagnostic and therapeutic use
EP2970494A4 (fr) * 2013-03-14 2016-09-14 Univ Texas Anticorps monoclonaux spécifiques de her3 pour l'utilisation dans le diagnostic et thérapeutique
CN105209493A (zh) * 2013-03-14 2015-12-30 德克萨斯州大学系统董事会 用于诊断和治疗用途的her3特异性单克隆抗体
US10358501B2 (en) 2013-03-14 2019-07-23 The Board Of Regents Of The University Of Texas System HER3 specific monoclonal antibodies for diagnostic and therapeutic use
US11174320B2 (en) 2013-03-14 2021-11-16 The Board Of Regents Of The University Of Texas System HER3 specific monoclonal antibodies for diagnostic and therapeutic use
US10973922B2 (en) 2013-05-02 2021-04-13 Glykos Finland Oy Glycoprotein-toxic payload conjugates
CN113150165A (zh) * 2014-12-22 2021-07-23 西雅图免疫公司 双特异性四价抗体及其制造和使用方法
US11618789B2 (en) 2016-03-04 2023-04-04 Morphosys Ag Clinical assessment of M-protein response in multiple myeloma
CN113144169A (zh) * 2021-02-10 2021-07-23 渤海大学 一种黄嘌呤氧化酶抑制肽及其应用
CN113144169B (zh) * 2021-02-10 2023-05-30 渤海大学 一种黄嘌呤氧化酶抑制肽及其应用
CN115372513A (zh) * 2022-08-19 2022-11-22 珠海市丽珠单抗生物技术有限公司 糖蛋白中二硫键的差异烷基化鉴定方法
CN115372513B (zh) * 2022-08-19 2023-09-15 珠海市丽珠单抗生物技术有限公司 糖蛋白中二硫键的差异烷基化鉴定方法

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