WO2012172495A1 - Compositions et procédés de ciblage du tem8 par des anticorps - Google Patents

Compositions et procédés de ciblage du tem8 par des anticorps Download PDF

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WO2012172495A1
WO2012172495A1 PCT/IB2012/052990 IB2012052990W WO2012172495A1 WO 2012172495 A1 WO2012172495 A1 WO 2012172495A1 IB 2012052990 W IB2012052990 W IB 2012052990W WO 2012172495 A1 WO2012172495 A1 WO 2012172495A1
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antibody
seq
nos
antibodies
tem8
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PCT/IB2012/052990
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English (en)
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Tony Fleming
Rou-fun KWONG
Saurabh Saha
Brad St. Croix
Xiaoyan Michelle ZHANG
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Novartis Ag
National Institutes Of Health
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • This application relates to antibodies that specifically bind TEM8 and their use in the inhibition of tumor angiogenesis.
  • PHS-CRADA Public Health Service Cooperative Research and Development Agreement
  • Angiogenesis the process of developing a hemovascular network from preexisting blood vessels, is essential for the growth of solid tumors and is a component of normal wound healing and growth processes. It also has been implicated in the pathophysiology of many diseases and conditions, including atherogenesis, arthritis, psoriasis, corneal neovascularization, and diabetic retinopathy. Because angiogenesis factors play an important role in the development of malignancies (Klagsburn et ai, Cancer Res. 36: 1 10-1 14, 1976; and Brem et ai, Science 195: 880-881 , 1977), it would be advantageous to identify new anti-angiogenic agents.
  • Tumor Endothelial Marker 8 also known as Anthrax Toxin Receptor 1 (ANTXR1 ), is a single-pass cell-surface glycoprotein that is upregulated on tumor vessels of various tumor types in both mice and humans (Nanda et ai, Cancer Res., 64(3):817-820, 2004; Carson-Walter et al., Cancer Res., 61 (18):6649-6655, 2001 ), and can be expressed by the tumor cells themselves (Carson-Walter et ai Cancer Res., 61 (18):6649-6655, 2001 ; Yang et al., Biochim Biophys Acta, 1813(1 ):39-49, 201 1 ).
  • TEM8 Tumor Endothelial Marker 8
  • ANTXR1 Anthrax Toxin Receptor 1
  • the present invention relates to an isolated antibody or antigen binding fragment thereof that specifically binds to a human or mouse TEM8 with an EC50 of less than or equal to 1 nM, wherein EC50 is a measurement of the concentration of an antibody or antigen binding fragment thereof that elicits 50% of maximal binding.
  • the antibody or antigen binding fragments described herein may bind to human or mouse TEM8 with an EC50 of less than or equal to 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.6 nM, 0.4nM, 0.3nM, 0.2 nM or less than or equal to 0.1 nM.
  • An antibody or antigen binding fragment thereof can bind to human or mouse TEM8, for example, with an EC50 of less than or equal to 0.4 nM, including 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, 0.31 , 0.3, 0.29, 0.28, 0.27, 0.26, 0.25, 0.24, 0.23, 0.22, 0.21 , 0.2, 0.19, or 0.18 nM.
  • the invention also includes an isolated antibody or antigen binding fragment thereof that specifically binds to human or mouse TEM8, and cross competes with an antibody described in Table 1.
  • the antibody or antigen binding fragment thereof as described herein can be a monoclonal antibody, a human or humanized antibody, a chimeric antibody, a single chain antibody, a Fab fragment, Fv fragment, F(ab')2 fragment, or ScFv fragment, and/or an IgG isotype.
  • the antibodies of the invention can include a framework in which an amino acid has been substituted into the antibody framework from the respective human VH or VL germline sequences.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L1 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L2 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L3 in Table 1.
  • the invention also includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L5 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody K1 D2 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain variable domain sequences of antibody L1 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain variable domain sequences of antibody L2 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain variable domain sequences of antibody L3 in Table 1.
  • the invention also includes an antibody or antigen binding fragment thereof having the heavy and light chain variable domain sequences of antibody L5 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain variable domain sequences of antibody K1 D2 in Table 1.
  • the invention also relates to an isolated antibody having a heavy and light chain, each including an amino acid sequence at least 90% identical to the heavy chain of SEQ ID NOs: 9, 23, 37, 51 , or 65; and a light chain of SEQ ID NOs: 10, 24, 38, 52, and 66, wherein said antibody binds human TEM8.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a heavy chain CDR1 selected from the group consisting of SEQ ID NOs 1 , 15, 29, 43, and 57; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, and 58; and a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 17, 31 , 45, and 59, wherein said isolated antibody or antigen binding fragment thereof binds to human TEM8.
  • a heavy chain CDR1 selected from the group consisting of SEQ ID NOs 1 , 15, 29, 43, and 57
  • a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, and 58
  • a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 17, 31 , 45, and 59
  • the isolated antibody or antigen binding fragment thereof further includes a light chain CDR1 selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, and 60; a light chain CDR2 selected from the group consisting of SEQ ID NOs 5, 19, 33, 47, and 61 ; and a light chain CDR3 selected from the group consisting of SEQ ID NOs 6, 20, 34, 48, and 62.
  • a light chain CDR1 selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, and 60
  • a light chain CDR2 selected from the group consisting of SEQ ID NOs 5, 19, 33, 47, and 61
  • a light chain CDR3 selected from the group consisting of SEQ ID NOs 6, 20, 34, 48, and 62.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a light chain CDR1 selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, and 60; a light chain CDR2 selected from the group consisting of SEQ ID NOs 5, 19, 33, 47, and 61 ; and a light chain CDR3 selected from the group consisting of SEQ ID NOs 6, 20, 34, 48, and 62, wherein said isolated antibody or antigen binding fragment thereof binds to human TEM8.
  • a light chain CDR1 selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, and 60
  • a light chain CDR2 selected from the group consisting of SEQ ID NOs 5, 19, 33, 47, and 61
  • a light chain CDR3 selected from the group consisting of SEQ ID NOs 6, 20, 34, 48, and 62
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a heavy chain CDR1 , CDR2, and CDR3, wherein the sequence of heavy chain CDR1 , CDR2, and CDR3 comprise, respectively, SEQ ID NOs: 1 , 2, 3; 15, 16, 17; 29, 30, 31 ; 43, 44, 45; or 57, 58, 59, wherein said antibody or antigen binding fragment thereof binds to human TEM8.
  • the isolated antibody or antigen binding fragment thereof further includes a light chain CDR1 , CDR2, and CDR3, wherein the sequence of the light chain CDR1 , CDR2, and CDR3 comprise,
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a light chain CDR1 , CDR2, and CDR3, wherein the sequence of the light chain CDR1 , CDR2, and CDR3 comprise, respectively, SEQ ID NOs: 4, 5, 6; 18, 19, 20; 32, 33, 34; 46, 47, 48; or 60, 61 , 62, wherein said antibody or antigen binding fragment thereof binds human TEM8.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that binds TEM8 having CDRH1 , CDRH2, CDRH3 and CDRL1 , CDRL2, CDRL3, wherein CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 1 , 2, 3, and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 4, 5, 6; or CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 15, 16, 17, and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 18, 19, 20; or CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 29, 30, 31 , and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 32, 33, 34; or CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 43, 44, 45, and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 46, 47, 48; or
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a heavy chain CDR1 , CDR2, and CDR3, wherein the sequence of heavy chain CDR1 , CDR2, and CDR3 comprise, respectively, SEQ ID NOs: 71 , 72, 73; 77, 78, 79; 83, 84, 85; 89, 90, 91 ; or 95, 96, 97, wherein said antibody or antigen binding fragment thereof binds to human TEM8.
  • the isolated antibody or antigen binding fragment thereof further includes a light chain CDR1 , CDR2, and CDR3, wherein the sequence of the light chain CDR1 , CDR2, and CDR3 comprise, respectively, SEQ ID NOs: 74, 75, 76; 80, 81 , 82; 86, 87, 88; 92, 93, 94; or 98, 99, 100.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a light chain CDR1 , CDR2, and CDR3, wherein the sequence of the light chain CDR1 , CDR2, and CDR3 comprise, respectively, SEQ ID NOs: 74, 75, 76; 80, 81 , 82; 86, 87, 88; 92, 93, 94; or 98, 99, 100, wherein said antibody or antigen binding fragment thereof binds to human TEM8.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a heavy chain CDR1 selected from the group consisting of SEQ ID NOs 71 , 77, 83, 89, and 95; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 72, 78, 84, 90, and 96; and a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 73, 79, 85, 91 , and 97, wherein said isolated antibody or antigen binding fragment thereof binds to human TEM8.
  • a heavy chain CDR1 selected from the group consisting of SEQ ID NOs 71 , 77, 83, 89, and 95
  • a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 72, 78, 84, 90, and 96
  • a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 73, 79, 85, 91 , and 97
  • the isolated antibody or antigen binding fragment thereof further includes a light chain CDR1 selected from the group consisting of SEQ ID NOs: 74, 80, 86, 92, and 98; a light chain CDR2 selected from the group consisting of SEQ ID NOs 75, 81 , 87, 93, and 99; and a light chain CDR3 selected from the group consisting of SEQ ID NOs 76, 82, 88, 94, and 100.
  • a light chain CDR1 selected from the group consisting of SEQ ID NOs: 74, 80, 86, 92, and 98
  • a light chain CDR2 selected from the group consisting of SEQ ID NOs 75, 81 , 87, 93, and 99
  • a light chain CDR3 selected from the group consisting of SEQ ID NOs 76, 82, 88, 94, and 100.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a heavy chain variable domain sequence selected from the group consisting of SEQ ID NOs: 7, 21 , 35, 49, and 63.
  • the antibody or antigen binding fragment thereof further includes a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, and 64, wherein said isolated antibody or antigen binding fragment thereof binds to human TEM8.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that includes a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, and 64, wherein said isolated antibody or antigen binding fragment thereof binds to human TEM8.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof that binds human TEM8, having heavy and light chain variable domains comprising the sequences of SEQ ID NOs: 7 and 8; 21 and 22; 35 and 36; 49 and 50; or 63 and 64, respectively.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof, that includes a heavy chain variable domain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7, 21 , 35, 49, and 63, wherein said antibody binds to TEM8.
  • the isolated antibody or antigen binding fragment thereof also includes a light chain variable domain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 8, 22, 36, 50, and 64.
  • the invention also relates to an isolated antibody or antigen binding fragment thereof, that includes a light chain variable domain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 8, 22, 36, 50, and 64, wherein said antibody binds human TEM8.
  • the invention still further relates to an isolated antibody or antigen binding fragment thereof that includes a heavy chain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 9, 23, 37, 51 , and 65, wherein said antibody binds to human TEM8.
  • the isolated antibody or antigen binding fragment thereof also includes a light chain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 10, 24, 38, 52, and 66.
  • the invention still further relates to an isolated antibody or antigen binding fragment thereof that includes a light chain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 10, 24, 38, 52, and 66, wherein said antibody binds human TEM8.
  • the invention also includes an antibody or antigen binding fragment thereof that binds to human TEM8 having a heavy chain comprising the sequence of SEQ ID NO: 9, 23, 37, 51 , or 65.
  • the antibody also includes a light chain that can combine with such heavy chain to form an antigen binding site to human TEM8.
  • the antibody or antigen binding fragment thereof includes a light chain having a sequence comprising SEQ ID NO: 10, 24, 38, 52, or 66.
  • the invention also includes pharmaceutical compositions comprising the antibody compositions described herein as well as a pharmaceutically acceptable carrier.
  • the invention includes a pharmaceutical composition comprising an antibody or antigen binding fragment thereof of Table 1 , such as, for example antibody L1 , L2, L3, L5, or K1 D2
  • the invention also includes a pharmaceutical composition comprising a combination of two or more of the antibodies or antigen binding fragments thereof of Table 1.
  • the invention also includes an isolated nucleic acid comprising a sequence encoding a polypeptide that includes a heavy chain variable domain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 7,
  • the invention also relates to an isolated nucleic acid comprising a sequence encoding a polypeptide that includes a light chain variable domain having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOs 88,
  • the invention also includes a vector that includes one or more of the nucleic acid molecules described herein.
  • the invention also includes an isolated host cell that includes a recombinant DNA sequence encoding a heavy chain of the antibody described above, and a second recombinant DNA sequence encoding a light chain of the antibody described above, wherein said DNA sequences are operably linked to a promoter and are capable of being expressed in the host cell.
  • the antibody can be a human monoclonal antibody.
  • the host cell is a non-human mammalian cell.
  • the invention still further relates to a method of inhibiting tumor growth in a subject where the method includes the step of administering to a subject in need thereof an effective amount of a composition comprising the antibody or fragments thereof described herein. It is contemplated that the subject is a human.
  • the invention also provides a method of inhibiting angiogenesis in a tumor where the method includes the step of contacting the tumor with an effective amount of a composition comprising an antibody or antigen binding fragment as described herein.
  • the tumor is from a human subject.
  • any of the foregoing antibodies or antigen binding fragments thereof may be a monoclonal antibody or antigen binding fragment thereof.
  • angiogenesis refers to a biological process leading to the generation of new blood vessels through sprouting or growth from pre-existing blood vessels. The process involves the migration and proliferation of endothelial cells from preexisting vessels. Angiogenesis occurs during pre- and post-natal development, and in the adult. Angiogenesis occurs during the normal cycle of the female reproductive system, wound healing, and during pathological processes such as cancer, where it is essential for the growth of solid tumors (for review, see Battegay, J. Molec. Med., 73(7): 333-346, 1995; Shchors and Evan, Cancer Res., 67:1630-1633. 2007).
  • Pathological angiogenesis refers to angiogenesis associated with a tumor, for example, the generation of blood vessels in or surrounding a tumor.
  • antibody as used herein includes whole antibodies and any antigen binding fragment (/ ' . e., "antigen-binding portion") or single chains thereof.
  • a naturally occurring "antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • light chain CDR1 , CDR2 or CDR3 is synonymous with CDRL1 , CDRL2, or CDRL3, respectively.
  • heavy chain CDR1 , CDR2, or CDR3 is synonymous with
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • antigen binding portion or "antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., TEM8).
  • Antigen binding functions of an antibody can be performed by fragments of an intact antibody.
  • binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341 :544- 546), which consists of a VH domain or a VL domain; and an isolated complementarity determining region (CDR).
  • Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • an Fd fragment consisting of the V
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883).
  • Such single chain antibodies include one or more "antigen binding portions" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antigen binding portions can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1 126-1 136).
  • Antigen binding portions of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).
  • Fn3 Fibronectin type III
  • Antigen binding portions can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1 -VH-CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10):1057-1062; and U.S. Pat. No. 5,641 ,870).
  • the term “Affinity” refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody “arm” interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.
  • the term “Avidity” refers to an informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody epitope affinity; the valency of both the antigen and antibody; and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups ⁇ e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • binding specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant.
  • the combining site of the antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Binding affinity of an antibody is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody.
  • Specific binding between two entities means a binding with an equilibrium constant (K A ) of at least 1 x 10 7 M “1 , 10 8 M “1 , 10 9 M “1 , 10 10 M “1 , 10 11 M “1 , 10 12 M “1 , 10 13 M “1 .
  • K A equilibrium constant
  • the phrase “specifically (or selectively) binds” to an antibody ⁇ e.g., a TEM8-binding antibody) refers to a binding reaction that is determinative of the presence of a cognate antigen ⁇ e.g., a human or mouse TEM8) in a heterogeneous population of proteins and other biologies.
  • the phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
  • modulation or “modulate” are used interchangeably herein to refer to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating) and downregulation (i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)) of an activity or a biological process.
  • “Modulates” is intended to describe both the upregulation or downregulation of a process.
  • a process which is upregulated by a certain stimulant may be inhibited by an antagonist to that stimulant.
  • a process that is downregulated by a certain modifying agent may be inhibited by an agonist to that modifying agent.
  • the term "subject” includes any human or nonhuman animal.
  • nonhuman animal includes all nonhuman vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, rodents, rabbits, sheep, dogs, cats, horses, cows, birds, amphibians, reptiles, etc.
  • chimeric antibody is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • a mouse antibody can be modified by replacing its constant region with the constant region from a human immunoglobulin.
  • TEM8 Tumor endothelial marker 8
  • human TEM8 has the sequence as set in SEQ ID NO: 101 , and is encoded by the cDNA sequence of SEQ ID NO: 102.
  • Human TEM8 is also described at GenBank Accession No: AF279145.2.
  • Mouse TEM8 is described at GenBank Accession No: AAL1 1999.1.
  • 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 sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • 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 every possible silent variation of the nucleic acid.
  • silent variations are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation 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) can be modified to yield a functionally identical molecule.
  • each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • conservatively modified variants include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term "conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding
  • cross-block means the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to TEM in a standard competitive binding assay.
  • the ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to TEM8, and therefore whether it can be said to cross-block according to the invention, can be determined using standard competition binding assays.
  • One suitable assay involves the use of the Biacore technology ⁇ e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology.
  • Another assay for measuring cross-blocking uses an ELISA-based approach.
  • epitope means a protein determinant capable of specific binding to an antibody.
  • 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.
  • high affinity for an IgG antibody or fragment thereof refers to an antibody having a KD of 10 ⁇ 8 M or less, 10 ⁇ 9 M or less, or 10 "10 M, or 10 "11 M or less, or 10 "12 M or less, or 10 "13 M or less for a target antigen.
  • high affinity binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to an antibody having a KD of 10 "7 M or less, or 10 "8 M or less.
  • the anti-TEM-8 antibodies or antigen binding fragments thereof described herein have a KD of less than or equal to 100 nM, preferably less than or equal to 10 nM, preferably less than or equal to 1 nM, preferably less than or equal to 200 pM, more preferably less than or equal to 100 pM, and still more preferably less than or equal to 10 pM.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • the human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • the human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • the human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • the human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • the human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences.
  • monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • a “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts (i.e., the constant region as well as the framework portions of the variable region). See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 -6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988;
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same.
  • Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) 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. 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 any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:1 1 -17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol.
  • 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, for example, 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 sequence.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds TEM8 is substantially free of antibodies that specifically bind antigens other than TEM8).
  • An isolated antibody that specifically binds TEM8 may, however, have cross-reactivity to other antigens.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • isotype refers to the antibody class (e.g., IgM, IgE, IgG such as lgG1 or lgG4) that is provided by the heavy chain constant region genes. Isotype also includes modified versions of one of these classes, where modifications have been made to alter the Fc function, for example, to enhance or reduce effector functions or binding to Fc receptors.
  • Kassoc or “Ka”, as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction
  • Kdis or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M).
  • K D values for antibodies can be determined using methods well established in the art. A method for determining the K D of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore ® system.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 , 1991 ; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91 -98, 1994).
  • operably linked refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • the term, "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO) or a human cell.
  • the optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.
  • the optimized sequences herein have been engineered to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic cells or prokaryotic cells is also envisioned herein.
  • the amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant,
  • combinatorial human antibody library and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • recombinant host cell refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
  • vector is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector such as an adeno-associated viral vector (AAV, or AAV2), wherein additional DNA segments may be ligated into the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non- episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the term "inhibit" as it relates to tumor growth or angiogenesis refers to prevention of tumor growth or angiogenesis in a tumor that has been contacted with an antibody that binds TEM8 as described herein, that is statistically significant relative to tumor growth or angiogenesis in a control tumor that has not been contacted with an antibody that binds TEM8 as described herein.
  • Figure 1 shows a table and a series of digital images illustrating that the antibody binding fragment of L1 , L2, L3, L5 and K1 D2 bind mouse and human TEM8.
  • A An ELISA was used to identify Fabs that were able to react with purified
  • hTEM8(ED)-Fc fusion protein The half maximum concentration (EC50) of the indicated Fabs needed to bind TEM8 are shown.
  • EC50 half maximum concentration of the indicated Fabs needed to bind TEM8 are shown.
  • B The indicated Fabs were screened for their ability to bind 293 cells expressing human TEM8 (293/Flag-hTEM8) by immunofluorescence staining.
  • Figure 2 shows the amino acid (SEQ ID NO: 101 ) and cDNA (SEQ ID NO: 102) sequences for human TEM8.
  • the present invention is based, in part, on the discovery of antibody molecules that specifically bind to both human and mouse TEM8.
  • the invention relates to both full IgG format antibodies (see, e.g., antibodies L2 and L5) as well as antigen binding fragments thereof, such as Fab fragments (e.g., see antibodies L1 , L3, and K1 D2).
  • the present invention provides antibodies that specifically bind to TEM8 (e.g., human TEM8, mouse TEM8), pharmaceutical compositions, production methods, and methods of use of such antibodies and compositions.
  • the present invention provides antibodies that specifically bind to TEM8.
  • the present invention provides antibodies that specifically bind to both human and mouse TEM8.
  • Antibodies of the invention include, but are not limited to, the human monoclonal antibodies, isolated as described, in the Examples.
  • the present invention provides antibodies that specifically bind a TEM8 protein (e.g., human and/or mouse TEM8), wherein the antibodies comprise a VH domain having an amino acid sequence of SEQ ID NO: 7, 21 , 35, 49, or 63.
  • the present invention also provides antibodies that specifically bind to a TEM8 protein, wherein the antibodies comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 1 , infra.
  • the invention provides antibodies that specifically bind to a TEM8 protein (e.g., human and/or mouse TEM8), wherein the antibodies comprise (or alternatively, consist of) one, two, three, or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 1 , infra.
  • a TEM8 protein e.g., human and/or mouse TEM8
  • the antibodies comprise (or alternatively, consist of) one, two, three, or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 1 , infra.
  • the present invention provides antibodies that specifically bind to a TEM8 protein, said antibodies comprising a VL domain having an amino acid sequence of SEQ ID NO: 8, 22, 36, 50, or 64.
  • the present invention also provides antibodies that specifically bind to a TEM8 protein (e.g., human and/or mouse TEM8), said antibodies comprising a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 1 , infra.
  • the invention provides antibodies that specifically bind to a TEM8 protein (e.g., human and/or mouse TEM8), said antibodies comprising (or alternatively, consisting of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 1 , infra.
  • a TEM8 protein e.g., human and/or mouse TEM8
  • said antibodies comprising (or alternatively, consisting of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 1 , infra.
  • antibodies of the invention include amino acids that have been mutated, yet have at least 60, 70, 80, 85, 90 or 95 percent identity in the CDR regions with the CDR regions depicted in the sequences described in Table 1. In some embodiments, it includes mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared with the CDR regions depicted in the sequence described in Table 1.
  • the present invention also provides nucleic acid sequences that encode VH, VL, the full length heavy chain, and the full length light chain of the antibodies that specifically bind to a TEM8 protein (e.g., human and/or mouse TEM8).
  • a TEM8 protein e.g., human and/or mouse TEM8
  • Such nucleic acid sequences can be optimized for expression in mammalian cells (for example, Table 1 shows the optimized nucleic acid sequences for the heavy chain and light chain of antibodies L2 and L5, as well as Fab fragments L1 , L3, and K1 D2).
  • VL 36 DIELTQPPSVSVAPGQTARISCSGDNIRSYYAHWYQQKPGQAPVLVIYGDSKR PSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSSYASHDYVFGGGTKLTVL
  • antibodies of the invention include those where the amino acids or nucleic acids encoding the amino acids have been mutated, yet have at least 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to the sequences described in Table 1.
  • it includes mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared with the variable regions depicted in the sequence described in Table 1 , while retaining substantially the same antigen binding activity.
  • VH, VL, full length light chain, and full length heavy chain sequences (amino acid sequences and the nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other TEM8-binding antibodies of the invention.
  • Such "mixed and matched" TEM8- binding antibodies can be tested using the binding assays known in the art (e.g., ELISAs, and other assays described in the Example section). When these chains are mixed and matched, a VH sequence from a particular VH/VL pairing should be replaced with a structurally similar VH sequence.
  • a full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence.
  • a VL sequence from a particular VH/VL pairing should be replaced with a structurally similar VL sequence.
  • a full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence.
  • the invention provides an isolated monoclonal antibody or antigen binding region thereof having: a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 21 , 35, 49, and 63 and a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, or 64, wherein the antibody specifically binds to TEM8 (e.g., human and/or mouse TEM8).
  • TEM8 e.g., human and/or mouse TEM8
  • the invention provides (i) an isolated monoclonal antibody having: a full length heavy chain comprising an amino acid sequence that has been optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 9, 23, 37, 51 , or 65, and a full length light chain comprising an amino acid sequence that has been optimized for expression in a mammalian cell selected from the group consisting of SEQ ID NOs: 10, 24, 38, 52, or 66; or (ii) a functional protein comprising an antigen binding portion thereof.
  • CDR complementarity determining region
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31 -35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1 ), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1 ), 52-56 (HCDR2), and 95- 102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1 ), 50-52 (LCDR2), and 91 -96 (LCDR3).
  • the CDRs consist of amino acid residues 26-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1 ), 50-56
  • LCDR2 LCDR2
  • LCDR3 89-97
  • the present invention provides TEM8-binding antibodies that comprise the heavy chain and light chain CDR1 s, CDR2s and CDR3s as described in Table 1 , or combinations thereof.
  • the amino acid sequences of the VH CDR1 s of the antibodies are shown in SEQ ID NOs: 1 , 15, 29, 43, or 57.
  • the amino acid sequences of the VH CDR2s of the antibodies and are shown in SEQ ID NOs: 2, 16, 30, 44, or 58.
  • the amino acid sequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 3, 17, 31 , 45, or 59.
  • the amino acid sequences of the VL CDR1 s of the antibodies are shown in SEQ ID NOs: 4, 18, 32, 46, or 60.
  • the amino acid sequences of the VL CDR2s of the antibodies are shown in SEQ ID NOs: 5, 19, 33, 47, or 61.
  • the amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 6, 20, 34, 48, or 62. These CDR regions are delineated using the Kabat system.
  • the amino acid sequences of the VH CDR1 s of the antibodies are shown in SEQ ID NOs: 71 , 77, 83, 89, or 95.
  • the amino acid sequences of the VH CDR2s of the antibodies are shown in SEQ ID NOs: 72, 78, 84, 90, or 96.
  • the amino acid sequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 73, 79, 85, 91 , or 97.
  • the amino acid sequences of the VL CDR1 s of the antibodies are shown in SEQ ID NOs: 74, 80, 86, 92, or 98.
  • the amino acid sequences of the VL CDR2s of the antibodies are shown in SEQ ID NOs: 75, 81 , 87, 93, or 99.
  • the amino acid sequences of the VL CDR3s of the antibodies are shown in SEQ ID NOs: 76, 82, 88, 94, or 100.
  • VH CDR1 , 2 and 3 sequences and VL CDR1 , 2 and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, although each antibody preferably contains a VH CDR1 , 2 and 3 and a VL CDR1 , 2 and 3 to create other TEM8-binding binding molecules of the invention.
  • Such "mixed and matched" TEM8-binding antibodies can be tested using the binding assays known in the art and those described in the Examples ⁇ e.g., ELISAs).
  • VH CDR sequences When VH CDR sequences are mixed and matched, the CDR1 , CDR2 and/or CDR3 sequence from a particular VH sequence should be replaced with a structurally similar CDR sequence(s).
  • VL CDR sequences when VL CDR sequences are mixed and matched, the CDR1 , CDR2 and/or CDR3 sequence from a particular VL sequence should be replaced with a structurally similar CDR sequence(s).
  • novel VH and VL sequences can be created by substituting one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences shown herein for monoclonal antibodies of the present invention.
  • the antigen binding fragments of the antibodies described herein can comprise a VH CDR1 , 2, and 3, or a VL CDR 1 , 2, and 3, wherein the fragment binds to TEM8 as a single variable domain.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L1 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L2 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L3 in Table 1.
  • the invention also includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody L5 in Table 1.
  • the invention includes an antibody or antigen binding fragment thereof having the heavy and light chain sequences of antibody K1 D2 in Table 1.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO:1 ; a heavy chain variable region CDR2 of SEQ ID NO: 2; a heavy chain variable region CDR3 of SEQ ID NO: 3; a light chain variable region CDR1 of SEQ ID NO: 4; a light chain variable region CDR2 of SEQ ID NO: 5; and a light chain variable region CDR3 of SEQ ID NO: 6.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 15; a heavy chain variable region CDR2 of SEQ ID NO: 16; a heavy chain variable region CDR3 of SEQ ID NO: 17; a light chain variable region CDR1 of SEQ ID NO: 18; a light chain variable region CDR2 of SEQ ID NO: 19; and a light chain variable region CDR3 of SEQ ID NO: 20.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 29; a heavy chain variable region CDR2 of SEQ ID NO: 30; a heavy chain variable region CDR3 of SEQ ID NO: 31 ; a light chain variable region CDR1 of SEQ ID NO: 32 a light chain variable region CDR2 of SEQ ID NO: 33; and a light chain variable region CDR3 of SEQ ID NO: 34.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 43; a heavy chain variable region CDR2 of SEQ ID NO: 44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a light chain variable region CDR1 of SEQ ID NO: 46; a light chain variable region CDR2 of SEQ ID NO: 47; and a light chain variable region CDR3 of SEQ ID NO: 48.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 57; a heavy chain variable region CDR2 of SEQ ID NO: 58; a heavy chain variable region CDR3 of SEQ ID NO: 59; a light chain variable region CDR1 of SEQ ID NO: 60; a light chain variable region CDR2 of SEQ ID NO: 61 ; and a light chain variable region CDR3 of SEQ ID NO: 62.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO:71 ; a heavy chain variable region CDR2 of SEQ ID NO: 72; a heavy chain variable region CDR3 of SEQ ID NO: 73; a light chain variable region CDR1 of SEQ ID NO: 74; a light chain variable region CDR2 of SEQ ID NO: 75; and a light chain variable region CDR3 of SEQ ID NO: 76.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 77; a heavy chain variable region CDR2 of SEQ ID NO: 78; a heavy chain variable region CDR3 of SEQ ID NO: 79; a light chain variable region CDR1 of SEQ ID NO: 80; a light chain variable region CDR2 of SEQ ID NO: 81 ; and a light chain variable region CDR3 of SEQ ID NO: 82.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 83; a heavy chain variable region CDR2 of SEQ ID NO: 84; a heavy chain variable region CDR3 of SEQ ID NO: 85; a light chain variable region CDR1 of SEQ ID NO: 86 a light chain variable region CDR2 of SEQ ID NO: 87; and a light chain variable region CDR3 of SEQ ID NO: 88.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 89; a heavy chain variable region CDR2 of SEQ ID NO: 90; a heavy chain variable region CDR3 of SEQ ID NO: 91 ; a light chain variable region CDR1 of SEQ ID NO: 92; a light chain variable region CDR2 of SEQ ID NO: 93; and a light chain variable region CDR3 of SEQ ID NO: 94.
  • an antibody that specifically binds to TEM8 comprising a heavy chain variable region CDR1 of SEQ ID NO: 95; a heavy chain variable region CDR2 of SEQ ID NO: 96; a heavy chain variable region CDR3 of SEQ ID NO: 97; a light chain variable region CDR1 of SEQ ID NO: 98; a light chain variable region CDR2 of SEQ ID NO: 99; and a light chain variable region CDR3 of SEQ ID NO: 100.
  • an antibody that specifically binds to TEM8 is an antibody that is described in Table 1.
  • the antibody that binds TEM8 is antibody L1.
  • the antibody that binds TEM8 is antibody L2.
  • the antibody that binds TEM8 is antibody L3.
  • the antibody that binds TEM8 is antibody L5.
  • the antibody that binds TEM8 is antibody K1 D2.
  • a human antibody comprises heavy or light chain variable regions or full length heavy or light chains that are "the product of” or “derived from” a particular germline sequence if the variable regions or full length chains of the antibody are obtained from a system that uses human germline immunoglobulin genes.
  • Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest.
  • a human antibody that is "the product of or "derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody.
  • a human antibody that is "the product of or "derived from” a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally occurring somatic mutations or intentional introduction of site-directed mutations.
  • a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
  • a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid
  • human antibody will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene in the VH or VL framework regions. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
  • human germline immunoglobulin genes include, but are not limited to the variable domain germline fragments described below, as well as DP47 and DPK9.
  • the present invention provides an antibody or an antigen-binding fragment thereof comprising amino acid sequences that are homologous to the sequences described in Table 1 , and said antibody binds to a TEM8 protein ⁇ e.g., human and/or mouse TEM8), and retains the desired functional properties of those antibodies described in Table 1 .
  • a TEM8 protein ⁇ e.g., human and/or mouse TEM8
  • the invention provides an isolated monoclonal antibody (or a functional antigen binding fragment thereof) comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at lest 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 21 , 35, 49, or 63 the light chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, or 64; the antibody specifically binds to TEM8 (e.g., human and/or mouse TEM8).
  • TEM8 e.g., human and/or mouse TEM8
  • the VH and/or VL amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth in Table 1 . In other embodiments, the VH and/or VL amino acid sequences may be identical except an amino acid substitution in no more than 1 ,2,3,4 or 5 amino acid position.
  • telomere sequence identity e.g., telomere sequence identity to the VH and VL regions of those described in Table 1 can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 7, 21 , 35, 49, 63, 8, 22, 36, 50, or 64, respectively, followed by testing of the encoded altered antibody for retained function using the functional assays described herein.
  • mutagenesis e.g., site-directed or PCR-mediated mutagenesis
  • the full length heavy chain and/or full length light chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth in Table 1 .
  • An antibody having a full length heavy chain and full length light chain having high (i.e., 80% or greater) identity to the full length heavy chains of any of SEQ ID NOs : 9, 23, 37, 51 , or 65, and full length light chains of any of SEQ ID NOs 10, 24, 38, 52, or 66, respectively, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides respectively, followed by testing of the encoded altered antibody for retained function using the functional assays described herein.
  • mutagenesis e.g., site-directed or PCR-mediated mutagenesis
  • the full length heavy chain and/or full length light chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth above.
  • variable regions of heavy chain and/or light chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forth above
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity equals number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences.
  • search can be performed using the BLAST program (version 2.0) of Altschul, et al., 1990 J.Mol. Biol. 215:403-10.
  • an antibody of the invention has a heavy chain variable region comprising CDR1 , CDR2, and CDR3 sequences and a light chain variable region comprising CDR1 , CDR2, and CDR3 sequences, wherein one or more of these CDR sequences have specified amino acid sequences based on the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the TEM8-binding antibodies of the invention.
  • the invention provides an isolated monoclonal antibody, or a functional antigen binding fragment thereof, consisting of a heavy chain variable region comprising CDR1 , CDR2, and CDR3 sequences and a light chain variable region comprising CDR1 , CDR2, and CDR3 sequences, wherein: the heavy chain variable region CDR1 amino acid sequences are selected from the group consisting of SEQ ID NOs: 1 , 15, 29, 43, and 57, and conservative modifications thereof; the heavy chain variable region CDR2 amino acid sequences are selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, and 58, and conservative modifications thereof; the heavy chain variable region CDR3 amino acid sequences are selected from the group consisting of SEQ ID NOs: 3, 17, 31 , 45, 59, and conservative modifications thereof; the light chain variable regions CDR1 amino acid sequences are selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, 60, and conservative modifications thereof; the light chain variable regions CDR2 amino acid sequences are selected from the group consisting
  • an antibody of the invention optimized for expression in a mammalian cell has a full length heavy chain sequence and a full length light chain sequence, wherein one or more of these sequences have specified amino acid sequences based on the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the TEM8- binding antibodies of the invention.
  • the invention provides an isolated monoclonal antibody optimized for expression in a mammalian cell consisting of a full length heavy chain and a full length light chain wherein: the full length heavy chain has amino acid sequences selected from the group of SEQ ID NOs: : 9, 23, 37, 51 , and 65, and conservative modifications thereof; and the full length light chain has amino acid sequences selected from the group of SEQ ID NOs: 10, 24, 38, 52, and 66, and conservative modifications thereof; the antibody specifically binds to TEM8 (e.g., human and/or mouse TEM8).
  • TEM8 e.g., human and/or mouse TEM8
  • the present invention provides antibodies that bind to the same epitope as do the TEM8-binding antibodies described in Table 1. Additional antibodies can therefore be identified based on their ability to cross-compete ⁇ e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in TEM8 binding assays (such as those described in the Examples).
  • the ability of a test antibody to inhibit the binding of antibodies of the present invention to a TEM8 protein demonstrates that the test antibody can compete with that antibody for binding to TEM8; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the TEM8 protein as the antibody with which it competes.
  • the antibody that binds to the same epitope on TEM8 as the antibodies of the present invention is a human monoclonal antibody.
  • human monoclonal antibodies can be prepared and isolated as described herein.
  • an antibody "competes" for binding when the competing antibody inhibits TEM8 binding of an antibody of the invention by more than 50%, in the presence of competing antibody concentrations higher than 10 6 x K D of the competing antibody.
  • An antibody of the invention further can be prepared using an antibody having one or more of the VH and/or VL sequences shown herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody.
  • An antibody can be engineered by modifying one or more residues within one or both variable regions (/ ' . e., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.
  • CDR grafting One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323- 327; Jones, P. et al., 1986 Nature 321 :522-525; Queen, C. et al., 1989 Proc. Natl.
  • another embodiment of the invention pertains to an isolated monoclonal antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 15, 29, 43, and 57; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, and 58; CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 17, 31 , 45, and 59, respectively; and a light chain variable region having CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, and 60; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 19, 33, 47, and 61 ; and CDR3 sequences consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 20, 34, 48, and 62, respectively.
  • another embodiment of the invention pertains to an isolated monoclonal antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 , 77, 83, 89, and 95; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 78, 84, 90, and 96; CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 79, 85, 91 , and 97, respectively; and a light chain variable region having CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 80, 86, 92, and 98; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 81 , 87, 93, and 99; and CDR3 sequences consisting of an amino acid sequence selected from the group consisting
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc- cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242; Tomlinson, I. M., et al., 1992 J. Mol. Biol.
  • framework sequences for use in the antibodies of the invention are those that are structurally similar to the framework sequences used by selected antibodies of the invention, e.g., consensus sequences and/or framework sequences used by monoclonal antibodies of the invention.
  • the VH CDR1 , 2 and 3 sequences, and the VL CDR1 , 2 and 3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences.
  • Frameworks that can be utilized as scaffolds on which to build the antibodies and antigen binding fragments described herein include, but are not limited to VH1A, VH1 B, VH3, Vk1 , VI2, and Vk2.
  • an embodiment of the invention relates to isolated TEM8-binding monoclonal antibodies, or an antigen binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 21 , 35, 49, and 63 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions in the framework region of such sequences, and further comprising a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, and 64 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions in the framework region of such sequences.
  • variable region modification is to mutate amino acid residues within the VH and/or VL CDR1 , CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation.”
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Conservative modifications (as discussed above) can be introduced.
  • the mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.
  • the invention provides isolated TEM8- binding monoclonal antibodies, or an antigen binding fragment thereof, consisting of a heavy chain variable region having: a VH CDR1 region consisting of an amino acid sequence selected from the group having SEQ ID NOs: 1 , 15, 29, 43, and 57 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 1 , 15, 29, 43, or 57; a VH CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, and 58 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NOs: 2, 16, 30, 44, or 58; a VH CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 17, 31 , 45, and 59, or an amino acid sequence having one, two, three, four or five amino acid
  • antibody/ immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to TEM8.
  • Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof, and include
  • immunoglobulins of other animal species preferably having humanized aspects.
  • Single heavy-chain antibodies such as those identified in camelids are of particular interest in this regard.
  • Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.
  • the invention pertains to generating non-immunoglobulin based antibodies using non- immunoglobulin scaffolds onto which CDRs of the invention can be grafted.
  • non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target TEM8 protein.
  • Non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising,
  • the fibronectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).
  • the fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). These fibronectin-based scaffolds are not an
  • immunoglobulin although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies.
  • These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo.
  • These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.
  • the ankyrin technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets.
  • the ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel a-helices and a ⁇ -turn. Binding of the variable regions is mostly optimized by using ribosome display.
  • Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A-domain” monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. Patent Application Publication Nos. 20040175756; 20050053973;
  • Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A.
  • Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (See e.g., US 5,831 ,012).
  • Affibody molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.
  • Anticalins are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains.
  • the binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity.
  • One protein of lipocalin family the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops.
  • BBP bilin-binding protein
  • One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.
  • Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules.
  • New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins.
  • two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is "ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in WO200104144 and examples of "ubiquitin-like" proteins are described in WO2004106368.
  • PEM Protein epitope mimetics
  • the present invention provides fully human antibodies that specifically bind to a TEM8 protein. Compared to the chimeric or humanized antibodies, the human TEM8- binding antibodies of the invention have further reduced antigenicity when administered to human subjects.
  • the human TEM8-binding antibodies can be generated using methods that are known in the art. For example, the humaneering technology used to converting non- human antibodies into engineered human antibodies. U.S. Patent Publication No.
  • 20050008625 describes an in vivo method for replacing a nonhuman antibody variable region with a human variable region in an antibody while maintaining the same or providing better binding characteristics relative to that of the nonhuman antibody.
  • the method relies on epitope guided replacement of variable regions of a non-human reference antibody with a fully human antibody.
  • the resulting human antibody is generally unrelated structurally to the reference nonhuman antibody, but binds to the same epitope on the same antigen as the reference antibody.
  • the serial epitope-guided complementarity replacement approach is enabled by setting up a competition in cells between a "competitor” and a library of diverse hybrids of the reference antibody ("test antibodies") for binding to limiting amounts of antigen in the presence of a reporter system which responds to the binding of test antibody to antigen.
  • the competitor can be the reference antibody or derivative thereof such as a single- chain Fv fragment.
  • the competitor can also be a natural or artificial ligand of the antigen which binds to the same epitope as the reference antibody.
  • test antibodies have one antigen-binding V-region in common from the nonhuman reference antibody, and the other V-region selected at random from a diverse source such as a repertoire library of human antibodies.
  • the common V-region from the reference antibody serves as a guide, positioning the test antibodies on the same epitope on the antigen, and in the same orientation, so that selection is biased toward the highest antigen-binding fidelity to the reference antibody.
  • reporter system can be used to detect desired interactions between test antibodies and antigen.
  • complementing reporter fragments may be linked to antigen and test antibody, respectively, so that reporter activation by fragment complementation only occurs when the test antibody binds to the antigen.
  • reporter activation becomes dependent on the ability of the test antibody to compete with the competitor, which is proportional to the affinity of the test antibody for the antigen.
  • Other reporter systems include the reactivator of an auto- inhibited reporter reactivation system (RAIR) as disclosed in U.S. Patent Application Ser. No. 10/208,730 (Publication No. 20030198971 ), or competitive activation system disclosed in U.S. Patent Application Ser. No. 10/076,845 (Publication No.
  • test antibodies are initially selected on the basis of their activity relative to that of the reference antibody when expressed as the test antibody.
  • the result of the first round of selection is a set of "hybrid" antibodies, each of which is comprised of the same non-human V-region from the reference antibody and a human V-region from the library, and each of which binds to the same epitope on the antigen as the reference antibody.
  • One of more of the hybrid antibodies selected in the first round will have an affinity for the antigen comparable to or higher than that of the reference antibody.
  • the human V-regions selected in the first step are used as guide for the selection of human replacements for the remaining non-human reference antibody V-region with a diverse library of cognate human V- regions.
  • the hybrid antibodies selected in the first round may also be used as competitors for the second round of selection.
  • the result of the second round of selection is a set of fully human antibodies which differ structurally from the reference antibody, but which compete with the reference antibody for binding to the same antigen.
  • Some of the selected human antibodies bind to the same epitope on the same antigen as the reference antibody.
  • one or more binds to the same epitope with an affinity which is comparable to or higher than that of the reference antibody.
  • mouse or chimeric TEM8-binding antibodies described above as the reference antibody, this method can be readily employed to generate human antibodies that bind to human TEM8 with the same binding specificity and the same or better binding affinity.
  • human TEM8-binding antibodies can also be commercially obtained from companies which customarily produce human antibodies, e.g., KaloBios, Inc. (Mountain View, CA).
  • Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT/EP93/02214 (WO 94/04678 published 3 March 1994).
  • a region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody”.
  • VHH camelid antibody
  • the camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers.
  • One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents.
  • a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.
  • camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with
  • a feature of the present invention is a camelid antibody or nanobody having high affinity for TEM8.
  • the camelid antibody or nanobody is naturally produced in the camelid animal, i.e., is produced by the camelid following immunization with TEM8 or a peptide fragment thereof, using techniques described herein for other antibodies.
  • the TEM8-binding camelid nanobody is engineered, i.e., produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody proteins using panning procedures with TEM8 as a target as described in the examples herein.
  • Engineered nanobodies can further be customized by genetic engineering to have a half life in a recipient subject of from 45 minutes to two weeks.
  • the camelid antibody or nanobody is obtained by grafting the CDRs sequences of the heavy or light chain of the human antibodies of the invention into nanobody or single domain antibody framework sequences, as described for example in PCT/EP93/02214.
  • Bispecific Molecules and Multivalent Antibodies are described for example in PCT/EP93/02214.
  • the present invention features bispecific or multispecific molecules comprising a TEM8-binding antibody, or a fragment thereof, of the invention.
  • An antibody of the invention, or antigen-binding regions thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein ⁇ e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • the antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multi- specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein.
  • an antibody of the invention can be functionally linked ⁇ e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.
  • the present invention includes bispecific molecules comprising at least one first binding specificity for TEM8 and a second binding specificity for a second target epitope.
  • the second target epitope is another epitope of TEM8 different from the first target epitope.
  • the molecule can further include a third binding specificity, in addition to the first and second target epitope.
  • the bispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., a Fab, Fab', F(ab')2, Fv, or a single chain Fv.
  • the antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778.
  • Diabodies are bivalent, bispecific molecules in which VH and VL domains are expressed on a single polypeptide chain, connected by a linker that is too short to allow for pairing between the two domains on the same chain.
  • VH and VL domains pair with complementary domains of another chain, thereby creating two antigen binding sites (see e.g., Holliger et a/., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et a/., 1994 Structure 2:1 121 -1 123).
  • Diabodies can be produced by expressing two polypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VL
  • VLA-VHB and VLB-VHA VL-VH configuration
  • Most of them can be expressed in soluble form in bacteria.
  • Single chain diabodies (scDb) are produced by connecting the two diabody-forming polypeptide chains with linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4): 128-30; Wu et ai, 1996 Immunotechnology, 2(1 ):21 -36).
  • scDb can be expressed in bacteria in soluble, active monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34): 128-30; Wu et ai., 1996
  • a diabody can be fused to Fc to generate a "di-diabody" (see Lu et ai., 2004 J. Biol. Chem., 279(4):2856-65).
  • antibodies which can be employed in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.
  • the bispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation.
  • cross-linking agents examples include protein A, carbodiimide, N-succinimidyl-S- acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o- phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et ai., 1984 J. Exp. Med.
  • Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).
  • the binding specificities are antibodies, they can be conjugated by sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains.
  • the hinge region is modified to contain an odd number of sulfhydryl residues, for example one, prior to conjugation.
  • both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell.
  • This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein.
  • a bispecific molecule of the invention can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants.
  • Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881 ,175; U.S.
  • Patent Number 5,132,405 U.S. Patent Number 5,091 ,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.
  • Binding of the bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay.
  • ELISA enzyme-linked immunosorbent assay
  • REA radioimmunoassay
  • FACS analysis FACS analysis
  • bioassay e.g., growth inhibition
  • Western Blot assay Western Blot assay.
  • Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.
  • the present invention provides multivalent compounds comprising at least two identical or different antigen-binding portions of the antibodies of the invention binding to TEM8.
  • the antigen-binding portions can be linked together via protein fusion or covalent or non covalent linkage.
  • methods of linkage have been described for the bispecfic molecules.
  • Tetravalent compounds can be obtained for example by cross-linking antibodies of the antibodies of the invention with an antibody that binds to the constant regions of the antibodies of the invention, for example the Fc or hinge region.
  • Trimerizing domain are described for example in Borean patent EP 1 012 280B1. Pentamerizing modules are described for example in PCT/EP97/05897.
  • the present invention provides for antibodies that specifically bind to TEM8 protein which have an extended half-life in vivo.
  • kidney filtration kidney filtration, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenic responses (e.g., protein neutralization by antibodies and uptake by macrophages and dentritic cells).
  • proteolytic enzymes proteolytic enzymes
  • immunogenic responses e.g., protein neutralization by antibodies and uptake by macrophages and dentritic cells.
  • a variety of strategies can be used to extend the half life of the antibodies of the present invention.
  • polyethyleneglycol PEG
  • PEG polyethyleneglycol
  • PSA polysialic acid
  • HES hydroxyethyl starch
  • albumin-binding ligands and carbohydrate shields
  • genetic fusion to proteins binding to serum proteins such as albumin, IgG, FcRn, and transferring
  • other binding moieties that bind to serum proteins, such as nanoboies, Fabs, DARPins, avimers, affibodies, and anticalins
  • genetic fusion to rPEG, albumin, domain of albumin, albumin-binding proteins, and Fc or by incorporation into nancarriers, slow release formulations, or medical devices.
  • inert polymer molecules such as high molecular weight PEG can be attached to the antibodies or a fragment thereof with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the antibodies or via epsilon-amino groups present on lysine residues.
  • PEG polyethylene glycol
  • 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.
  • the pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • a reactive PEG molecule or an analogous reactive water-soluble polymer.
  • 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-maleimide.
  • the antibody to be pegylated is an antibody to be pegylated.
  • aglycosylated antibody Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by size-exclusion or by ion-exchange chromatography. PEG-derivatized antibodies can be tested for binding activity as well as for in vivo efficacy using methods well-known to those of skill in the art, for example, by immunoassays described herein. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
  • modified pegylation technologies include reconstituting chemically orthogonal directed engineering technology (ReCODE PEG), which incorporates chemically specified side chains into biosynthetic proteins via a reconstituted system that includes tRNA synthetase and tRNA.
  • ReCODE PEG chemically orthogonal directed engineering technology
  • This technology enables incorporation of more than 30 new amino acids into biosynthetic proteins in E.coli, yeast, and
  • the tRNA incorporates a nonnative amino acid any place an amber codon is positioned, converting the amber from a stop codon to one that signals incorporation of the chemically specified amino acid.
  • Recombinant pegylation technology can also be used for serum halflife extension. This technology involves genetically fusing a 300-600 amino acid
  • unstructured protein tail to an existing pharmaceutical protein. Because the apparent molecular weight of such an unstructured protein chain is about 15-fold larger than its actual molecular weight, the serum halflife of the protein is greatly increased. In contrast to traditional PEGylation, which requires chemical conjugation and repurification, the manufacturing process is greatly simplified and the product is homogeneous.
  • PSA polymer polysialic acid
  • PSA is a polymer of sialic acid (a sugar).
  • sialic acid a sugar
  • polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system.
  • the PSA polymer is naturally found in the human body. It was adopted by certain bacteria which evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, by virtue of molecular mimicry, to foil the body's defense system. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, as it is chemically identical to PSA in the human body.
  • HES hydroxyethyl starch
  • Hesylation of an antibody enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity.
  • parameters such as the molecular weight of HES, a wide range of HES antibody conjugates can be customized.
  • Antibodies having an increased half-life in vivo can also be generated introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or FcRn binding fragment thereof (preferably a Fc or hinge Fc domain fragment). See, e.g., International Publication No. WO 98/23289; International Publication No. WO 97/34631 ; and U.S. Patent No. 6,277,375. Further, antibodies can be conjugated to albumin (e.g., human serum albumin; HSA) in order to make the antibody or antibody fragment more stable in vivo or have a longer half life in vivo.
  • albumin e.g., human serum albumin; HSA
  • the specificities of the antibody can be designed such that one binding domain of the antibody binds to TEM8 while a second binding domain of the antibody binds to serum albumin, preferably HSA.
  • the strategies for increasing half life is especially useful in nanobodies, fibronectin-based binders, and other antibodies or proteins for which increased in vivo half life is desired.
  • the present invention provides antibodies or fragments thereof that specifically bind to a TEM8 protein recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the invention provides fusion proteins comprising an antigen-binding fragment of an antibody described herein ⁇ e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide.
  • Methods for fusing or conjugating proteins, polypeptides, or peptides to an antibody or an antibody fragment are known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851 , and 5,1 12,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570;
  • Additional fusion proteins may be generated through the techniques of gene- shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling").
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,81 1 ,238, 5,830,721 , 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof may be altered by being subjected to random mutagenesis by error- prone PCR, random nucleotide insertion or other methods prior to recombination.
  • a polynucleotide encoding an antibody or fragment thereof that specifically binds to a TEM8 protein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • antibodies or fragments thereof can be fused to marker
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1 ), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et ai, 1984, Cell 37:767), and the "flag" tag.
  • HA hemagglutinin
  • antibodies of the present invention or fragments thereof conjugated to a diagnostic or detectable agent can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidinlbiotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as, but not limited to, iodine (1311, 1251, 1231, and 1211,), carbon
  • the present invention further encompasses uses of antibodies or fragments thereof conjugated to a therapeutic moiety.
  • An antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety or drug moiety that modifies a given biological response.
  • Therapeutic moieties or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, an anti- angiogenic agent; or, a biological response modifier such as, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, an anti- angiogenic agent; or, a biological response modifier such as, for example, a
  • an antibody can be conjugated to therapeutic moieties such as a radioactive metal ion, such as alph-emiters such as 213Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 131 In, 131 LU, 131Y, 131 Ho, 131 Sm, to polypeptides.
  • the macrocyclic chelator is 1 ,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • linker molecules are commonly known in the art and described in Denardo et ai, 1998, Clin Cancer Res. 4(10):2483-90; Peterson et ai, 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et ai, 1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the invention provides substantially purified nucleic acid molecules which encode polypeptides comprising segments or domains of the TEM8-binding antibody chains described above.
  • Some of the nucleic acids of the invention comprise the nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 7, 21 , 35, 49, or 63, , and/or the nucleotide sequence encoding the light chain variable region shown in SEQ ID NO: 8, 22, 36, 50, or 64.
  • the nucleic acid molecules are those identified in Table 1.
  • Some other nucleic acid molecules of the invention comprise nucleotide sequences that are substantially identical (e.g., at least 65, 80%, 95%, or 99%) to the nucleotide sequences of those identified in Table 1. When expressed from appropriate expression vectors, polypeptides encoded by these polynucleotides are capable of exhibiting TEM8 antigen binding capacity.
  • polynucleotides which encode at least one CDR region and usually all three CDR regions from the heavy or light chain of the TEM8-binding antibody set forth above. Some other polynucleotides encode all or substantially all of the variable region sequence of the heavy chain and/or the light chain of the TEM8-binding antibody set forth above. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each of the immunoglobulin amino acid sequences.
  • nucleic acid molecules of the invention can encode both a variable region and a constant region of the antibody.
  • nucleic acid sequences of the invention comprise nucleotides encoding a mature heavy chain sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the mature heavy chain sequence set forth in SEQ ID NO: 9, 23, 37, 51 , or 65.
  • nucleic acid sequences comprising nucleotide encoding a mature light chain sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the mature light chain sequence set forth in SEQ ID NO: 10, 24, 38, 52, or 66.
  • the polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below) encoding a TEM8-binding antibody or its binding fragment.
  • Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981 ; and the solid support method of U.S.
  • Patent No. 4,458,066 Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991 ; and Eckert et al., PCR Methods and Applications 1 :17, 1991.
  • Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997).
  • nonviral vectors useful for expression of the TEMS- binding polynucleotides and polypeptides in mammalian ⁇ e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins.
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.
  • expression vector depends on the intended host cells in which the vector is to be expressed.
  • the expression vectors contain a promoter and other regulatory sequences ⁇ e.g., enhancers) that are operably linked to the
  • an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions.
  • Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells.
  • other regulatory elements may also be required or desired for efficient expression of a TEM8-binding antibody chain or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences.
  • the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987).
  • enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987).
  • the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.
  • the expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted TEM8-binding antibody sequences. More often, the inserted TEM8-binding antibody sequences are linked to a signal sequences before inclusion in the vector.
  • Vectors to be used to receive sequences encoding TEM8-binding antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.
  • the host cells for harboring and expressing the TEM8-binding antibody chains can be either prokaryotic or eukaryotic.
  • E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes, such as yeast can also be employed to express TEM8-binding polypeptides of the invention. Insect cells in combination with baculovirus vectors can also be used.
  • mammalian host cells are used to express and produce the TEM8-binding polypeptides of the present invention.
  • they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., the 1 D6.C9 myeloma hybridoma clone as described in the Examples) or a mammalian cell line harboring an exogenous expression vector (e.g., the SP2/0 myeloma cells exemplified below). These include any normal mortal or normal or abnormal immortal animal or human cell.
  • a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed including the CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas.
  • the use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987.
  • Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., Immunol. Rev.
  • expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses.
  • Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable.
  • Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pollll promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
  • Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook, et al., supra).
  • Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired.
  • cell lines which stably express TEM8- binding antibody chains or binding fragments can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media.
  • Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, 1975 Nature 256: 495. Many techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic
  • hybridomas An animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art.
  • Fusion partners ⁇ e.g., murine myeloma cells
  • fusion procedures are also known.
  • Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine ⁇ e.g.,. human) immunoglobulin sequences using standard molecular biology techniques.
  • the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et a/.).
  • the murine CDR regions can be inserted into a human framework using methods known in the art. See e.g., U.S. Patent No. 5225539 to Winter, and U.S. Patent Nos. 5530101 ; 5585089; 5693762 and
  • the antibodies of the invention are human monoclonal antibodies.
  • Such human monoclonal antibodies directed against TEM8 can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.
  • transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice.”
  • the HuMAb mouse ® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode un-rearranged human heavy ( ⁇ and ⁇ ) and ⁇ light chain
  • mice exhibit reduced expression of mouse IgM or ⁇ , and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology 1 13:49-101 ; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol.13: 65-93, and Harding, F. and Lonberg, N., 1995 Ann. N. Y. Acad. Sci.
  • human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and
  • transchomosomes such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
  • KM mice Such mice, referred to herein as "KM mice", are described in detail in PCT Publication WO 02/43478 to Ishida ei al.
  • immunoglobulin genes are available in the art and can be used to raise TEM8-binding antibodies of the invention.
  • an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used.
  • Such mice are described in, e.g., U.S. Patent Nos. 5,939,598; 6,075,181 ; 6,1 14,598; 6, 150,584 and 6,162,963 to Kucherlapati ei al.
  • mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome referred to as "TC mice” can be used; such mice are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97:722-727.
  • cows carrying human heavy and light chain are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97:722-727.
  • cows carrying human heavy and light chain are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97:722-727.
  • Human monoclonal antibodies of the invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art or described in the examples below. See for example: U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571 ,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793; 6,521 ,404; 6,544,731 ; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.
  • Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization.
  • SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization.
  • Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
  • Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody.
  • one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis. Such
  • framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell - epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.
  • antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • chemically modified e.g., one or more chemical moieties can be attached to the antibody
  • modify its glycosylation again to alter one or more functional properties of the antibody.
  • the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the antibody is modified to increase its biological half- life.
  • Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward.
  • the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1 q binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer ei al.
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • 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.
  • Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et ai.
  • 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 glycosylation 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.
  • EP 1 ,176,195 by Hang et ai. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation.
  • glycoprotein-modifying glycosyl transferases e.g., beta(1 ,4)-N acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • the TEM8-binding antibodies having VH and VL sequences or full length heavy and light chain sequences shown herein can be used to create new TEM8-binding antibodies by modifying full length heavy chain and/or light chain sequences, VH and/or VL sequences, or the constant region(s) attached thereto.
  • the structural features of a TEM8-binding antibody of the invention are used to create structurally related TEM8-binding antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human TEM8 and also inhibiting one or more functional properties of TEM8 (e.g., inhibit red blood cell lysis in a hemolytic assay).
  • one or more CDR regions of the antibodies of the present invention can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, TEMS- binding antibodies of the invention, as discussed above.
  • Other types of modifications include those described in the previous section.
  • VH and/or VL sequences provided herein or one or more CDR regions thereof.
  • To create the engineered antibody it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a "second generation" sequence(s) derived from the original sequence(s) and then the "second generation" sequence(s) is prepared and expressed as a protein.
  • the invention provides a method for preparing a TEM8-binding antibody consisting of: a heavy chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 1 , 15, 29, 43, and 57, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 16, 30, 44, and 58, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 17, 31 , 45, and 59; and a light chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 4, 18, 32, 46, and 60, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 5, 19, 33, 47, and 61 , and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 6, 20, 34, 48, and 62; altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence
  • the invention provides a method for preparing a TEM8-binding antibody optimized for expression in a mammalian cell consisting of: a full length heavy chain antibody sequence having a sequence selected from the group of SEQ ID NOs: 9, 23, 37, 51 , and 65; and a full length light chain antibody sequence having a sequence selected from the group of SEQ ID NOs: 10, 24, 38, 52, and 66; altering at least one amino acid residue within the full length heavy chain antibody sequence and/or the full length light chain antibody sequence to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein.
  • the alteration of the heavy or light chain is in the framework region of the heavy or light chain.
  • the altered antibody sequence can also be prepared by screening antibody libraries having fixed CDR3 sequences or minimal essential binding determinants as described in US20050255552 and diversity on CDR1 and CDR2 sequences.
  • the screening can be performed according to any screening technology appropriate for screening antibodies from antibody libraries, such as phage display technology.
  • Standard molecular biology techniques can be used to prepare and express the altered antibody sequence.
  • the antibody encoded by the altered antibody sequence(s) is one that retains one, some or all of the functional properties of the TEM8-binding antibodies described herein, which functional properties include, but are not limited to, specifically binding to human and/or mouse TEM8; and the antibody inhibit red blood cell lysis in a hemolytic assay.
  • the functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein, such as those set forth in the Examples (e.g., ELISAs).
  • mutations can be introduced randomly or selectively along all or part of an TEM8-binding antibody coding sequence and the resulting modified TEM8-binding antibodies can be screened for binding activity and/or other functional properties as described herein.
  • Mutational methods have been described in the art.
  • PCT Publication WO 02/092780 by Short describes methods for creating and screening antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof.
  • PCT Publication WO 03/074679 by Lazar et al. describes methods of using computational screening methods to optimize
  • Antibodies that binds TEM8 as described herein can be used at a therapeutically useful concentration for the treatment of a tumor; that is, to inhibit tumor growth, or inhibit angiogenesis (e.g., pathological or tumor angiogenesis) such as that which can be found in numerous types of cancer such as breast, colorectal, lung, or other solid tumor cancers. Suitable subjects for such therapeutic intervention include those diagnosed with that are suspecting of having solid tumor cancer.
  • angiogenesis e.g., pathological or tumor angiogenesis
  • Suitable subjects for such therapeutic intervention include those diagnosed with that are suspecting of having solid tumor cancer.
  • a therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement in the underlying pathology as noted by the clinician or other qualified observer using methods well known in the art.
  • a therapeutically effective amount of a conjugate or antibody is the amount necessary to inhibit tumor growth, pathological angiogenesis, or the amount that is effective at reducing a sign or a symptom of the tumor.
  • the therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, therapeutic amounts are amounts which eliminate or reduce the patient's tumor burden, or which prevent or reduce the proliferation of metastatic cells, or which prevent or reduce pathological angiogenesis.
  • any therapeutically relevant mode of administration can be used for the disclosed antibodies, including local, topical, oral, intravascular such as intravenous,
  • intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, subcutaneous, or systemic administration is of use with the antibodies and conjugates disclosed herein.
  • administration via inhalation or via suppository is of use with the antibodies and conjugates disclosed herein.
  • the particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic).
  • the invention encompasses diagnostic assays for determining TEM8 protein and/or nucleic acid expression as well as TEM8 protein function, in the context of a biological sample (e.g., blood, serum, cells, tissue) or from individuals afflicted with a disease or disorder, or is at risk of developing a disorder associated with pathological angiogenesis.
  • a biological sample e.g., blood, serum, cells, tissue
  • a biological sample e.g., blood, serum, cells, tissue
  • Diagnostic assays such as competitive assays rely on the ability of a labelled analogue (the "tracer") to compete with the test sample analyte for a limited number of binding sites on a common binding partner.
  • the binding partner generally is
  • test sample analyte is inversely proportional to the amount of bound tracer as measured by the amount of marker substance.
  • Dose-response curves with known amounts of analyte are prepared and compared with the test results in order to quantitatively determine the amount of analyte present in the test sample. These assays are called ELISA systems when enzymes are used as the detectable markers.
  • TEM8 protein preferably the TEM8 epitopes of the invention, being a measure of antibodies in the serum sample, most particularly, neutralising antibodies in the serum sample.
  • a significant advantage of the assay is that measurement is made of neutralising antibodies directly (i.e., those which interfere with binding of TEM8 protein, specifically, epitopes). Such an assay, particularly in the form of an ELISA test has considerable applications in the clinical environment and in routine blood screening.
  • the detection of TEM8 proteins in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with disorders associated with solid tumors and/or pathological angiogenesis.
  • these methods generally comprise administering or introducing to a patient a diagnostically effective amount of a TEM8 binding molecule that is operatively attached to a marker or label that is detectable by non-invasive methods.
  • the antibody-marker conjugate is allowed sufficient time to localize and bind to TEM8 proteins in the subject; such as on a tumor cell of a subject.
  • the patient is then exposed to a detection device to identify the detectable marker, thus forming an image of the location of the TEM8 binding molecules in the patient.
  • the presence of TEM8 binding antbody or an antigen-binding fragment thereof is detected by determining whether an antibody-marker binds to tumor in the patient. Detection of an increased level of TEM8 in comparison to a normal individual without tumor or pathological angiogenesis is indicative of a predisposition for and/or on-set of disorders associated with solid tumor burden and/or pathological
  • angiogenesis is also preferred for use in imaging methods and combined angiogenic diagnostic and treatment methods.
  • the invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with pathological angiogenesis. For example, mutations in a TEM8 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with TEM8 protein, nucleic acid expression or activity.
  • Another aspect of the invention provides methods for determining TEM8 nucleic acid expression or TEM8 protein activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as
  • Pharmacogenomics allows for the selection of agents ⁇ e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual ⁇ e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.
  • Yet another aspect of the invention pertains to monitoring the influence of agents ⁇ e.g., drugs) on the expression or activity of TEM8 protein in clinical trials.
  • agents e.g., drugs
  • compositions comprising the TEM8- binding antibodies (intact or binding fragments) formulated together with a
  • compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing, for example, pathological angiogeneis or tumor growth.
  • Pharmaceutically acceptable carriers enhance or stabilize the composition, or can be used to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • a pharmaceutical composition of the present invention can be administered by a variety of methods known in the art.
  • the route and/or mode of administration vary depending upon the desired results. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target.
  • the pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration ⁇ e.g., by injection or infusion).
  • the active compound i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the composition should be sterile and fluid.
  • Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the TEM8-binding antibody is employed in the pharmaceutical compositions of the invention. The TEM8-binding antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • a physician or veterinarian can start doses of the antibodies of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • effective doses of the compositions of the present invention, for the treatment of an allergic inflammatory disorder described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 15 mg/kg, of the host body weight.
  • An exemplary treatment regime entails systemic administration once per every two weeks or once a month or once every 3 to 6 months.
  • An exemplary treatment regime entails systemic
  • Antibody is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of TEM8-binding antibody in the patient. In some methods of systemic administration, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 ⁇ g/ml and in some methods 25-500 ⁇ g/ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent
  • Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show longer half life than that of chimeric antibodies and nonhuman antibodies.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • a fully human phage display library was used to generate the anti-human TEM8 antibodies described herein.
  • This library being totally synthetic, is not subject to tolerance mechanisms found in normal immune responses and allowed the generation of antibodies against regions of the TEM8 extracellular domain (ED) that are 100% conserved between mouse and human.
  • ED extracellular domain
  • In vitro selection of the phage display library involved two rounds of sequential panning on biotinylated, purified recombinant TEM8(ED)-Fc fusion proteins and one round of panning on HEK293 cells transfected with human TEM8.
  • Two of the TEM8-binding clones (L2 and L5) were reformatted to generate mouse/human chimeric IgGs by fusing the CH2/CH3 domains of mouse lgG2a to the VH/CH1 of the Fab heavy chain and the VL of the lambda light chain gene.
  • the murine constant regions were added for the preclinical mouse studies to reduce potential immune responses, increase overall stability and maximize the in vivo half-lives of the antibodies.
  • Vectors containing the chimeric heavy (geneticin-resistance gene) and light (zeocin-resistance gene) chains were stably co-transfected into HEK293T cells.
  • Clones were tested for expression of the chimeric IgG utilizing a mouse IgG-Fc ELISA (Bethyl, 000-338-9579).
  • Anti-TEM8 antibodies were collected from culture supernatants grown in serum free medium and purified by Protein A and size exclusion chromatography.
  • Antibody preparations used for in vivo studies possessed ⁇ 5% aggregates and endotoxin levels were below 10 EU/mg.
  • the DC101 hybridoma was obtained from ATCC and DC101 was purified from conditioned medium by Protein A chromatography.
  • Fab fragments for L1 , L2, L3, L5 and K1 D2 were purified to >95% homogeneity by tandem immobilized metal chelating chromatography and size exclusion
  • Fab chromatography For ELISA, Nunc Immulon plates were coated overnight with 1 ug/ml neutravidin. The next day, plates were washed with PBST, blocked with 1 % BSA and loaded with 25 ng biotinylated human TEM8-Fc. Plates were then incubated with various concentrations of Fab preparations for 1 hour, washed times with PBST and Fab binding was detected by the addition of goat anti-human kappa light chain (Sigma, A7164) and lambda light chain (Sigma A5175) antibodies conjugated to HRP, followed by washes, addition of substrate and measurement of absorbance at A450. EC50 is a measure of the concentration of a Fab fragment that elicits 50% of maximal binding in this assay.
  • HEK-293 cells and HEK-293 transfected with human TEM8 were incubated with 10 ⁇ g/ml Fab fragment in 5% fetal bovine serum/DMEM containing 0.04% sodium azide to prevent antibody internalization for 1 hour at room temperature. Cells were then washed several times with buffer and incubated with anti-human Fab secondary antibody coupled to FITC (Jackson Immunoresearch 109-096-097) for 1 hour at room temperature. After washing with buffer, cells were examined using a fluorescence microscope and photographed. Results are shown in Figure 1 B. In brief, the results demonstrate that each of the anti-TEM8 antibodies were able to specifically bind TEM8 in vitro.
  • Example 4 Treatment of tumor in a subject
  • This example describes a particular method that can be used to treat a primary or metastatic tumor in humans by administration of one or more antibodies that specifically bind TEM8.
  • Human patients are treated intravenously with at least 1 ⁇ g (such as 0.001 -1000 mg) of one or more of the antibodies described herein (e.g., the antibodies of Table 1 ) that specifically bind TEM8, for example, for a period of at least 1 day, 1 week, 1 month, at least 2 months, at least 3 months, at least 6 months, at least one year, at least 2 years, or at least five years or more or less time.
  • Administration of the antibody can be used in conjunction with normal anti-tumor therapy (for example rather than replacing the therapy).
  • the anti-TEM8 can be added to the standard of care for the particular tumor type.
  • the method includes screening subjects to determine if they have a tumor, such as a primary or metastatic tumor, followed by treatment and follow-up.
  • a tumor such as a primary or metastatic tumor
  • the subject is first screened to determine if they have a tumor.
  • methods that can be used to screen for tumors include a combination of ultrasound, tissue biopsy, or detection of tumor-associated vasculature.
  • pre-screening is not required prior to administration of the anti-TEM8 antibodies disclosed herein.
  • Pre-treatment of subjects The subject is treated prior to administration of an antibody that specifically binds TEM8.
  • an antibody that specifically binds TEM8 is not always required, and can be determined by a skilled clinician.
  • the tumor can be surgically excised (in total or in part) prior to administration of one or more antibodies.
  • the subject can be treated with the standard of care for the particular tumor present.
  • Administration can be achieved by any sufficient method known in the art, but is typically intravenous administration.
  • the antibody or conjugate is typically administered to any sufficient method known in the art, but is typically intravenous administration.
  • the antibody or conjugate is typically administered to any sufficient method known in the art, but is typically intravenous administration.
  • the antibody or conjugate is typically administered to any sufficient method known in the art, but is typically intravenous administration.
  • the antibody or conjugate is typically administered to the antibody or conjugate.
  • composition including the antibody and a
  • a therapeutically effective amount of the antibody is administered to the subject.
  • the amount of antibody administered is sufficient to treat a subject having a tumor.
  • a therapeutically effective amount can being readily determined by one skilled in the art, for example using routine trials establishing dose response curves.
  • particular exemplary dosages are provided above.
  • the antibody can be administered in a single dose delivery, via continuous delivery over an extended time period, in a repeated administration protocol (for example, by a daily, weekly, or monthly repeated administration protocol).
  • subjects having a tumor can be monitored for tumor treatment, such as regression or reduction in tumor burden (for example, reduction in metastatic lesions).
  • tumor treatment such as regression or reduction in tumor burden (for example, reduction in metastatic lesions).
  • subjects are analyzed one or more times, starting seven days following treatment
  • diagnostic imaging can be used (such as x-rays, CT scans, MRIs, ultrasound, fiber optic
  • treatment with an anti-TEM8 antibody of the invention will result in at least a 10% decrease in tumor size, degree or extent of angiogenesis, or tumor metastasis relative to a subject having a similar tumor that does not receive an anti-TEM8 antibody therapeutic.
  • the present invention relates to, but is not limited to, the following embodiments:
  • An isolated antibody having a heavy and light chain, each comprising an amino acid sequence at least 90% identical to heavy chain of SEQ ID NOs: 9, 23, 37, 51 or 65 and light chain of SEQ ID NOs 10, 24, 38, 52, and 66 respectively, wherein said antibody binds to human TEM8.
  • CDRL1 , CDRL2, and CDRL3 comprising SEQ ID NOs: 4, 5, 6; or 18, 19, 20; or 32, 33, 34; or 46, 47, 48; or 60, 61 , 62, respectively.
  • An isolated antibody or antigen binding fragment thereof that binds to human TEM8 comprising CDRL1 , CDRL2, and CDRL3 comprising SEQ ID NOs: 4, 5, 6; or 18, 19, 20; or 32, 33, 34; or 46, 47, 48; or 60, 61 , 62, respectively
  • CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 15, 16, 17, and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 18, 19, 20; or
  • CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 29, 30, 31 , and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 32, 33, 34; or
  • CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 43, 44, 45, and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 46, 47, 48; or
  • CDRH1 , CDRH2, and CDRH3 comprises SEQ ID NOs 57, 58, 59, and CDRL1 , CDRL2, CDRL3 comprises SEQ ID NOs: 60, 61 , 62.
  • VI domain comprising SEQ ID NOs: 8, 22, 36, 50, or 64.
  • An isolated antibody or antigen binding fragment thereof that binds human TEM8, having a VI domain comprising SEQ ID NOs: 8, 22, 36, 50, or 64.
  • An isolated antibody or antigen binding fragment thereof that binds human TEM8, having Vh and VI domains comprising SEQ ID NOs: 7 and 8; 21 , and 22; 35 and 36; 49 and 50; or 63 and 64; respectively.
  • a vector comprising the nucleic acid sequence of the preceding embodiment.
  • An isolated host cell comprising a recombinant DNA sequence encoding a heavy chain of the antibody or antigen binding fragment thereof of any preceding claim, and a second recombinant DNA sequence encoding a light chain of the antibody or antigen binding fragment thereof of any preceding claim , wherein said DNA sequences are operably linked to a promoter and are capable of being expressed in the host cell.
  • a method of inhibiting tumor growth in a patient in need thereof comprising contacting a tumor cell from said patient with an antibody of any of the preceding claims.
  • a method of inhibiting angiogenesis in a solid tumor comprising contacting said tumor with an antibody or antigen binding fragment thereof of any preceding claim.

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Abstract

La présente invention concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci qui se lient à la fois au TEM8 de l'humain et de la souris, ainsi que leurs compositions et procédés d'utilisation.
PCT/IB2012/052990 2011-06-14 2012-06-13 Compositions et procédés de ciblage du tem8 par des anticorps WO2012172495A1 (fr)

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