WO2011116014A2 - Humanized antibodies to labyrinthin and uses thereof - Google Patents

Humanized antibodies to labyrinthin and uses thereof Download PDF

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Publication number
WO2011116014A2
WO2011116014A2 PCT/US2011/028552 US2011028552W WO2011116014A2 WO 2011116014 A2 WO2011116014 A2 WO 2011116014A2 US 2011028552 W US2011028552 W US 2011028552W WO 2011116014 A2 WO2011116014 A2 WO 2011116014A2
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Prior art keywords
antibody
seq
chain variable
antigen
labyrinthin
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PCT/US2011/028552
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French (fr)
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WO2011116014A9 (en
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James A. Radosevich
Michael Babich
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Mission Therapeutics, Inc.
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Publication of WO2011116014A2 publication Critical patent/WO2011116014A2/en
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • Cancer is a leading cause of death in men and women throughout the world. In the United States alone, over 1 million new cases are diagnosed each year, and over 0.5 million deaths are reported annually (Landis et al., 1998). Historically, tumors are grouped and treated, based in part by the tissues in which they arise, e.g.— breast cancer, colon cancer, and lung cancer, and the like. Yet, within lung cancer, for example, it is well recognized that these tumors are a very heterogeneous group of neoplasms. This is also true for tumors arising in other tissues. In part, because of this heterogeneity, there are complex and inconsistent classification schemes which are used for human tumors. Previous attempts to treat cancer have been hampered by: 1) the arbitrary classification of tumors arising within given tissues, and 2) by using microscopic methods based on how these tumors look (histological classification).
  • Labyrinthin is a cell surface protein expressed on the extracellular surface of the plasma membrane of adenocarcinomas, and is not cell cycle specific. Lab is not found in the serum of normal or tumor bearing patients, and is not shed into the culture media by Lab positive cell lines, thus Lab represents a useful marker for adenocarcinoma-type cancers.
  • Immunotherapy in humans has been limited, in part due to adverse responses to non-human monoclonal antibodies.
  • Early clinical trials using rodent antibodies revealed human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, which lead to rapid clearance of the antibody.
  • HAMA human anti-mouse antibody
  • HAA human anti-rat antibody
  • Less immunogenic antibodies have since been developed, including chimeric antibodies, humanized antibodies, PRIMATIZED ® antibodies, and human antibodies prepared using transgenic mice or phage display libraries. See Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6851 -5; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-33; Newman et al.
  • the present invention provides humanized anti-Lab antibodies and methods for their use.
  • the present invention provides antibodies, in particular, humanized antibodies, that bind to a labyrinthin protein, and that exhibit numerous desirable properties, such as high affinity binding to a labyrinthin protein.
  • the invention comprises a humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein
  • the antibody comprises: (1) a variable heavy (V H ) domain that comprises: a) a heavy chain variable region CDR1 comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof; and (2) a variable light (V L ) domain that comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human V H
  • the invention comprises a humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; and c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human V H domain.
  • the antibody comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; and c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human V H domain.
  • the invention comprises a humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human V L domain.
  • the antibody comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human
  • the invention comprises a humanized antibody, or antigen-binding portion thereof, wherein the antibody comprises: a variable heavy (V H ) domain that comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, incorporated into a human V H domain.
  • V H variable heavy
  • a humanized antibody of the invention comprises a heavy chain constant region (C H ) encoded by SEQ ID NO: 10, and/or a light chain constant region (C L ) encoded by SEQ ID NO: 9.
  • the invention comprises a pharmaceutical composition comprising any of the above antibodies and a pharmaceutically acceptable carrier.
  • the invention comprises an isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any of the above antibodies.
  • the invention comprises a method of ameliorating an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of any of the above antibodies in an amount effective to ameliorate the adenocarcinoma.
  • said administering reduces metastasis of said adenocarcinoma, such as an increase in time to metastasis (e.g. 1, 2, 3, 4, 5, or more months; or 1, 2, 3, 4, 5, or more years), or a decrease in the frequency of metastases (e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more), relative to an untreated subject.
  • said administering results in a decrease in size of the adenocarcinoma, such as a size decrease of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, relative to a pre-treatment size.
  • the invention comprises a method of ameliorating relapse of an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of any of the above antibodies in an amount effective to ameliorate relapse of the adenocarcinoma.
  • said administering reduces metastasis of said adenocarcinoma, such as an increase in time to metastasis (e.g. 1, 2, 3, 4, 5, or more months; or 1, 2, 3, 4, 5, or more years), or a decrease in the frequency of metastases (e.g.
  • said administering results in a decrease in size of the adenocarcinoma, such as a size decrease of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, relative to a pre-treatment size.
  • the invention comprises a method of ameliorating an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of any of the above antibodies, wherein the antibody binds to a cancer stem cell.
  • said administering reduces metastasis of said adenocarcinoma, such as an increase in time to metastasis (e.g. 1, 2, 3, 4, 5, or more months; or 1, 2, 3, 4, 5, or more years), or a decrease in the frequency of metastases (e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more), relative to an untreated subject.
  • said administering results in a decrease in size of the adenocarcinoma, such as a size decrease of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%), or more, relative to a pre-treatment size.
  • the cancer stem cell is a self- renewing cell.
  • the cancer stem cell is an adenocarinoma cancer stem cell.
  • the cancer stem cell expresses one or more of CD133, CD44, CD166, CD29, CD24, Lgr5, ALDH1, ESA, and b-catenin.
  • the invention comprises a kit comprising a container containing the humanized antibody of any of the above antibodies and instructions directing a user to treat an adenocarcinoma in a subject with the antibody in an effective amount.
  • Figure 1 shows the sequence of murine antibody clone X373. Shown are the VL light chain nucleic acid sequence (SEQ ID NO:l) and peptide sequence (SEQ ID NO:2), and the VH heavy chain nucleic acid sequence (SEQ ID NO:3) and peptide sequence (SEQ ID NO:4).
  • Figure 2 shows the sequence of humanized antibody clone X509. Shown are the VL light chain nucleic acid sequence (SEQ ID NO:5) and peptide sequence (SEQ ID NO:6), and the VH heavy chain nucleic acid sequence (SEQ ID NO:7) and peptide sequence (SEQ ID NO:8). Also shown are the VL delineated CDR1, CDR2, and CDR3 regions (SEQ ID NOs: 11, 12 and 13, respectively) and the VH delineated CDR1, CDR2, and CDR3 regions (SEQ ID NOs: 14, 15 and 16, respectively).
  • Figure 3 shows a CL constant light chain sequence (SEQ ID NO:9) and CHI constant heavy chain region sequence (SEQ ID NO: 10).
  • Figure 4 shows affinity measurement of the murine antibody clone X373 and humanized antibody clone X509 to a labyrinthin protein.
  • This invention provides chimeric and humanized antibodies against labyrinthin (Lab) and methods of using these antibodies.
  • the term antibody refers to an immunoglobulin protein, or antibody fragments that comprise an antigen binding site (e.g., Fab, modified Fab, Fab', F(ab') 2 or Fv fragments, or a protein having at least one immunoglobulin light chain variable region or at least one immunoglobulin heavy chain region).
  • Humanized antibodies of the invention include diabodies, tetrameric antibodies, single chain antibodies, tretravalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and domain-specific antibodies that recognize a particular epitope.
  • immune response refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region
  • V H a heavy chain constant region
  • the heavy chain constant region is comprised of three domains, C H I, C H2 and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L 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
  • Each V H and V L 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, 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 of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Lab). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the v, V H , C h and C H I domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region (see, FUNDAMENTAL IMMUOLOGY (Paul ed., 3 rd ed.
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H 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. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • an "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g. an isolated antibody that specifically binds Lab is substantially free of antibodies that specifically bind antigens other than Lab).
  • An isolated antibody that specifically binds Lab may, however, have cross-reactivity to other antigens, such as Lab molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • the terms "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.
  • “isotype” refers to the antibody class (e.g., IgM, IgG (and subclasses such as IgGl, IgG2, IgG3, IgG4), IgA, IgE, etc.) that is encoded by the heavy chain constant region genes.
  • an antibody that "specifically binds to human Lab” is intended to refer to an antibody that binds to human Lab with an affinity having a K D of preferably at least 1 x 10 ⁇ 6 M, more preferably at least 1 x 10 ⁇ 7 M, more preferably at least 5 x 10 ⁇ 8 M, more preferably at least 1 x 10 ⁇ 8 M, or more preferably at least 5 x 10 ⁇ 9 M.
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a (i.e., K ⁇ j/K a ) and is expressed as a molar concentration (M).
  • K D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore.RTM. system.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non- human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 : 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151 : 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)).
  • Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 : 2623 (1993)).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • a preferred antibody of the invention is the humanized monoclonal antibody X509.
  • the invention provides an isolated monoclonal antibody, or antigen binding portion thereof comprising:
  • the antibody specifically binds Lab, preferably human Lab.
  • the invention provides antibodies that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s of X509.
  • the amino acid sequence of the V H CDRl s of X509 is shown in SEQ ID NO: 14.
  • the amino acid sequence of the V H CDR2 of X509 is shown in SEQ ID NO: 15.
  • the amino acid sequence of the V H CDR3 of X509 is shown in SEQ ID NO: 16.
  • the amino acid sequences of the V K CDRl of X509 is shown in SEQ ID NO: 11.
  • the amino acid sequence of the V K CDR2 of X509 is shown in SEQ ID NO: 12.
  • the amino acid sequence of the V K CDR3 of X509 is shown in SEQ ID NO: 13.
  • the CDR regions are delineated using the Kabat system (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).
  • the CDR3 domain independently from the CDRl and/or CDR2 domain(s), alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, for example, Klimka et al., British J. of Cancer 83(2):252-260 (2000) (describing the production of a humanized anti-CD30 antibody using only the heavy chain variable domain CDR3 of murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.
  • the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domains from an antibody derived from a human or non- human animal, wherein the monoclonal antibody is capable of specifically binding to Lab.
  • the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody, such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to Lab.
  • inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from a non- human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental non-human antibody.
  • the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to Lab.
  • a human antibody such as, for example, a human antibody obtained from a non-human animal
  • the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a first human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to Lab and wherein the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that is lacking binding specificity for Lab to generate a second human antibody that is capable of specifically binding to Lab.
  • inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from the first human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental first human antibody.
  • the humanized antibody or affinity-matured antibody may be an antibody fragment, such as a Fab, that is optionally conjugated with one or more cytotoxic agent(s) in order to generate an
  • the humanized antibody or affinity-matured antibody may be an intact antibody, such as an intact IgGl antibody.
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • the antibody of choice is a single-chain Fv fragment (scFv). See WO 1993/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.
  • the antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibody fragments may be monospecific or bispecific.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • Exemplary bispecific antibodies may bind to two different epitopes of the Labyrinthin protein.
  • Other such antibodies may combine a Labyrinthin binding site with binding site(s) for HER-2, EGFR, ErbB, ErbB3, and/or ErbB4.
  • an anti-Labyrinthin arm may be combined with an arm that binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells that express Labyrinthin. These antibodies possess a Labyrinthin-binding arm and an arm that binds the cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate, or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).
  • antibody- variable domains with the desired binding specificities are fused to immunoglobulin constant-domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding, present in at least one of the fusions.
  • CHI first heavy-chain constant region
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy-chain- light-chain pair (providing a second binding specificity) in the other arm It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 1994/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 1991/00360, WO 1992/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed, for example, in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker that is too short to allow pairing between the two domains on the same chain.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol,. 147: 60 (1991).
  • an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-Lab antibodies of the invention.
  • the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence having SEQ ID NO: 8; (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence having SEQ ID NO: 6; and the antibody exhibits one or more of the following properties: (c) the antibody binds to human Lab with a K D of lxlO "7 M or less;(d) the antibody binds to an adenocarcinoma tumor cell line.
  • the V H and/or V L amino acid sequences may be 85%, 90%, 95%, 96%, 97%), 98%) or 99% homologous to the sequences set forth above.
  • An antibody having V H and V L regions having high (i.e., 80% or greater) homology to the V H and V L regions of the sequences set forth above, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 5 and 7, followed by testing of the encoded altered antibody for retained function (i.e., the functions set forth in (c) and (d) above) using the functional assays described herein.
  • mutagenesis e.g., site-directed or PCR-mediated mutagenesis
  • the percent homology between two amino acid sequences is equivalent to the percent identity between 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 percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (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.
  • 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.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • an antibody of the invention comprises 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 comprise specified amino acid sequences based on the preferred antibodies described herein (e.g., X509), or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-Lab antibodies of the invention.
  • the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising 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 CDR3 sequence comprises an amino acid sequence having SEQ ID NO: 16, or conservative modifications thereof;
  • the light chain variable region CDR3 sequence comprises an amino acid sequence having
  • the antibody exhibits one or more of the following properties:
  • (d) binds to an adenocarcinoma tumor cell line.
  • the heavy chain variable region CDR2 sequence comprises an amino acid sequence having SEQ ID NO: 15, or conservative modifications thereof; and the light chain variable region CDR2 sequence comprises an amino acid sequence having SEQ ID NO: 12, or conservative modifications thereof.
  • the heavy chain variable region CDR1 sequence comprises an amino acid sequence having SEQ ID NOs: 14, or conservative modifications thereof; and the light chain variable region CDR1 sequence comprises an amino acid sequence having SEQ ID NO: 11, or conservative modifications thereof.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) and (d) above) using the functional assays described herein.
  • the invention provides antibodies that bind to the same epitope on human Lab as any of the Lab monoclonal antibodies of the invention (i.e., antibodies that have the ability to cross-compete for binding to Lab with any of the monoclonal antibodies of the invention).
  • the reference antibody for cross-competition studies can be the monoclonal antibody X509 (having V H and V L sequences as shown in SEQ ID NOs: 8 and 6, respectively).
  • Such cross-competing antibodies can be identified based on their ability to cross-compete with X509 in standard Lab binding assays. For example, BIAcore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the antibodies of the current invention.
  • test antibody to inhibit the binding of, for example, X509, to human Lab demonstrates that the test antibody can compete with X509 for binding to human Lab and thus binds to the same epitope on human Lab as X509.
  • the antibody that binds to the same epitope on human Lab as X509 is a human monoclonal antibody.
  • human monoclonal antibodies can be prepared and isolated as described in the Examples.
  • An antibody of the invention further can be prepared using an antibody having one or more of the V H and/or V L sequences disclosed 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 (i.e., V H and/or V L ), 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.
  • another embodiment of the invention pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDRl, CD2, and CDR3 sequences having SEQ ID NOs: 14, 15, and 16, respectively, and a light chain variable region comprising CDRl, CDR2, and CDR3 sequences having SEQ ID NOs: 11, 12 and 13, respectively.
  • such antibodies contain the V H and V L CDR sequences of monoclonal antibodies X509 yet may contain different framework sequences from these antibodies.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • 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.
  • Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research 25:3389-3402), which is well known to those skilled in the art.
  • BLAST is a heuristic algorithm in that a statistically significant alignment between the antibody sequence and the database sequence is likely to contain high-scoring segment pairs (HSP) of aligned words. Segment pairs whose scores cannot be improved by extension or trimming is called a hit.
  • HSP high-scoring segment pairs
  • nucleotide sequences of VBASE origin are translated and the region between and including FR1 through FR3 framework region is retained.
  • the database sequences have an average length of 98 residues. Duplicate sequences which are exact matches over the entire length of the protein are removed.
  • the nucleotide sequences are translated in all six frames and the frame with no stop codons in the matching segment of the database sequence is considered the potential hit. This is in turn confirmed using the BLAST program tblastx, which translates the antibody sequence in all six frames and compares those translations to the VBASE nucleotide sequences dynamically translated in all six frames.
  • the identities are exact amino acid matches between the antibody sequence and the protein database over the entire length of the sequence.
  • the positives are not identical but amino acid substitutions guided by the BLOSUM62 substitution matrix. If the antibody sequence matches two of the database sequences with same identity, the hit with most positives would be decided to be the matching sequence hit.
  • variable region modification is to mutate amino acid residues within the V H and/or V K CDRl, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest.
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples.
  • Preferably conservative modifications are introduced.
  • the mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions.
  • typically no more than one, two, three, four or five residues within a CDR region are altered.
  • the invention provides isolated anti-Lab monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a V H CDRl region comprising an amino acid sequence having SEQ ID NO: 14, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 14; (b) a V H CDR2 region comprising an amino acid sequence having SEQ ID NO: 15, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 15; (c) a V H CDR3 region comprising an amino acid sequence having SEQ ID NO: 16, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 16; (d) a V K CDRl region comprising an amino acid sequence having SEQ ID NO: 11, or an amino acid sequence having one, two
  • Engineered antibodies of the invention include those in which modifications have been made to framework residues within V H and/or V K , e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, 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.
  • Another type of 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.
  • the hinge region of CHI 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. Pat. No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHI 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 binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.
  • 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. Pat. No. 6,277,375 to Ward.
  • the antibody can be altered within the CHI 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. Pat. 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 function(s) of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 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 CI component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues within amino acid positions 231 and 239 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 et 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 Fcv receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439.
  • 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.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered 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.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
  • the Ms704, Ms705, and Ms709 FUT8v-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22).
  • EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a flucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme.
  • Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked
  • the fucose residues of the antibody may be cleaved off using a fucosidase enzyme.
  • a fucosidase enzyme for example, the fucosidase alpha-L- fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516- 23).
  • an antibody can be pegylated to, for example, increase the biological (e.g., serum) half life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
  • the antibodies of the present invention may be further characterized by the various physical properties of the anti-Lab antibodies. Various assays may be used to detect and/or differentiate different classes of antibodies based on these physical properties.
  • antibodies of the present invention may contain one or more glycosylation sites in either the light or heavy chain variable region.
  • the presence of one or more glycosylation sites in the variable region may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41 :673-702; Gala F A and Morrison S L (2004) J Immunol 172:5489-94; Wallick et at (1988) J Exp Med 168:1099-109; Spiro R G (2002) Glycobiology 12:43 R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al.
  • variable region glycosylation may be tested using a Glycoblot assay, which cleaves the antibody to produce a Fab, and then tests for glycosylation using an assay that measures periodate oxidation and Schiff base formation.
  • variable region glycosylation may be tested using Dionex light chromatography (Dionex-LC), which cleaves saccharides from a Fab into monosaccharides and analyzes the individual saccharide content.
  • Dionex-LC Dionex light chromatography
  • the antibodies of the present invention do not contain asparagine isomerism sites.
  • a deamidation or isoaspartic acid effect may occur on N-G or D-G sequences, respectively.
  • the deamidation or isoaspartic acid effect results in the creation of isoaspartic acid which decreases the stability of an antibody by creating a kinked structure off a side chain carboxy terminus rather than the main chain.
  • the creation of isoaspartic acid can be measured using an iso-quant assay, which uses a reverse-phase HPLC to test for isoaspartic acid.
  • Each antibody will have a unique isoelectric point (pi), but generally antibodies will fall in the pH range of between 6 and 9.5.
  • the pi for an IgGl antibody typically falls within the pH range of 7-9.5 and the pi for an IgG4 antibody typically falls within the pH range of 6-8.
  • Antibodies may have a pi that is outside this range. Although the effects are generally unknown, there is speculation that antibodies with a pi outside the normal range may have some unfolding and instability under in vivo conditions.
  • the isoelectric point may be tested using a capillary isoelectric focusing assay, which creates a pH gradient and may utilize laser focusing for increased accuracy (Janini et al (2002) Electrophoresis 23:1605-11 ; Ma et al.
  • an anti-Lab antibody that contains a pi value that falls in the normal range. This can be achieved either by selecting antibodies with a pi in the normal range, or by mutating charged surface residues using standard techniques well known in the art.
  • Each antibody will have a melting temperature that is indicative of thermal stability
  • T M i indicates the temperature of the initial unfolding of the antibody.
  • T M 2 indicates the temperature of complete unfolding of the antibody.
  • the thermal stability of an antibody may be measure using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).
  • antibodies are selected that do not rapidly degrade. Fragmentation of an anti-Lab antibody may be measured using capillary electrophoresis (CE) and MALDI-MS, as is well understood in the art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).
  • CE capillary electrophoresis
  • MALDI-MS MALDI-MS
  • antibodies are selected that have minimal aggregation effects. Aggregation may lead to triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less and even more preferably 5% or less. Aggregation may be measured by several techniques well known in the art, including size-exclusion column (SEC) high performance liquid chromatography (HPLC), and light scattering to identify monomers, dimers, trimers or multimers.
  • SEC size-exclusion column
  • HPLC high performance liquid chromatography
  • the anti-Lab antibodies having V H and V K sequences disclosed herein can be used to create new anti-Lab antibodies by modifying the VH and/or V K sequences, or the constant region(s) attached thereto.
  • the structural features of an anti-Lab antibody of the invention e.g. X509
  • X509 are used to create structurally related anti-Lab antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human Lab.
  • one or more CDR regions of X509, or mutations thereof can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-Lab antibodies of the invention, as discussed above.
  • the starting material for the engineering method is one or more of the V H and/or V K 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 V H and/or V K 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" sequencers) derived from the original sequencers) and then the "second generation" sequence(s) is prepared and expressed as a protein.
  • the invention provides a method for preparing an anti-Lab antibody comprising: (a) providing: (i) a heavy chain variable region antibody sequence comprising a CDR1 sequence having SEQ ID NO: 14, a CDR2 sequence having SEQ ID NO: 15, and/or a CDR3 sequence having SEQ ID NO: 16; and/or (ii) a light chain variable region antibody sequence comprising a CDR1 sequence having SEQ ID NO: 11, a CDR2 sequence having SEQ ID NO: 12, and/or a CDR3 sequence having SEQ ID NO: 13; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein.
  • the antibody encoded by the altered antibody sequence(s) is one that retains one, some or all of the functional properties of the anti-Lab antibodies described herein, which functional properties include, but are not limited to: (a) the antibody binds to human Lab with a K D of 1 x 10 ⁇ 7 M or better; or (b) the antibody binds an adenocarcinoma tumor cell line.
  • 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., flow cytometry, binding assays).
  • mutations can be introduced randomly or selectively along all or part of an anti-Lab antibody coding sequence and the resulting modified anti-Lab 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 physiochemical properties of antibodies.
  • nucleic acid molecules that encode the antibodies of the invention.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.
  • a nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library.
  • Preferred nucleic acids molecules of the invention are those encoding the VH and VL sequences of the X509 monoclonal antibody. DNA sequences encoding the VH sequences of X509 is shown in SEQ ID NO: 5. DNA sequences encoding the VL sequences of X509 is shown in SEQ ID NO: 7.
  • DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these
  • VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • operatively linked is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in- frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3).
  • CHI, CH2 and CH3 DNA molecule encoding heavy chain constant regions
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., 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) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGl or IgG4 constant region.
  • the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., 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) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
  • the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser) 3 , such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser) 3
  • Monoclonal antibodies (mAbs) of the present invention 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. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
  • the preferred animal system for preparing hybridomas is the murine system.
  • Hybridoma production in the mouse is a very 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) and 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. Pat. No. 4,816,567 to Cabilly et al.).
  • the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos.
  • the antibody is able to block the activity of all endogenous Labyrinthin produced by tumors and suppressor lymphoid cells (T cells).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. a small-molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. a small-molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioactive isotope (i.e., a radioconjugate).
  • the antibody is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antibody molecule).
  • Maytansine may, for example, be converted to May-SS-Me, which may be reduced to May-SH3 and reacted with modified antibody (Chari et al., Cancer Research, 52: 127-131 (1992)) to generate a maytansinoid-antibody immunoconjugate.
  • Another immunoconjugate of interest comprises an anti-Labyrinthin antibody conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics is capable of producing double- stranded DNA breaks at sub-picomolar concentrations.
  • Structural analogues of calicheamicin include, but are not limited to, vi 1 , a-i , (X3 1 , N-acetyl-Vi 1 , PSAG and .theta.'i (Hinman et al., Cancer Research, 53 : 3336-3342 (1993) and Lode et al. Cancer Research, 58: 2925-2928 (1998)). See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001 expressly incorporated herein by reference.
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 1993/21232 published Oct. 28, 1993.
  • the present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a
  • DNase deoxyribonuclease
  • radioactive isotopes are available for the production of radioconjugated anti- Labyrinthin antibodies. Examples include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu.
  • Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-l -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as N-
  • a ricin immunotoxin can be prepared as described in Vitetta et al. Science, 238: 1098 (1987).
  • Carbon- 14-labeled l -isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 1994/1 1026.
  • the linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, dimethyl linker, or disulfide-containing linker (Chari et al. Cancer Research, 52: 127-131 (1992)) may be used.
  • a fusion protein comprising the anti-Labyrinthin antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis.
  • the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g. avidin) that is conjugated to a cytotoxic agent (e.g. a radionucleotide).
  • a "receptor” such as streptavidin
  • a ligand e.g. avidin
  • cytotoxic agent e.g. a radionucleotide
  • the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO 1981/01145) to an active anti-cancer drug.
  • a prodrug e.g. a peptidyl chemotherapeutic agent, see WO 1981/01145
  • an active anti-cancer drug e.g. a peptidyl chemotherapeutic agent, see WO 1981/01145
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to convert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting nontoxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs
  • antibodies with enzymatic activity can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328:457-458 (1987)).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
  • the enzymes useful in this invention can be covalently bound to the anti-Labyrinthin antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen-binding region of an antibody of the invention linked to at least a functionally active portion of a suitable enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)).
  • the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug-delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • colloidal drug-delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the anti-Labyrinthin antibodies disclosed herein may also be formulated as immunoli osomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 1997/38731 published Oct. 23, 1997.
  • Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized
  • phosphatidylethanolamine PEG-PE
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide-interchange reaction.
  • a chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
  • the invention also provides isolated nucleic acid encoding the humanized anti-Labyrinthin antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription-termination sequence.
  • the anti-Labyrinthin antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a-factor leader (including Saccharomyces and Kluyveromyces a-factor leaders), acid-phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 1990/13646.
  • mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the anti-Labyrinthin antibody.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Expression and cloning vectors may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-Labyrinthin antibody-encoding nucleic acid, such as dihydrofolate reductase (DHFR), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • DHFR dihydrofolate reductase
  • metallothionein-I and -II preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
  • host cells particularly wild-type hosts that contain endogenous DHFR transformed or co-transformed with DNA sequences encoding anti-Labyrinthin antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
  • APH aminoglycoside 3 '-phosphotransferase
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)).
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977).
  • the presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
  • vectors derived from the 1.6- ⁇ circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts.
  • an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990).
  • Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 2: 968-975 (1991).
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-Labyrinthin antibody-encoding nucleic acid.
  • Promoters suitable for use with prokaryotic hosts include the phoA promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
  • trp tryptophan
  • Other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the anti- Labyrinthin antibody.
  • Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT -rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT (SEQ ID NO: 17) region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO: 18) sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3- phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyr
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • Anti-Labyrinthin antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hin III E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978.
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early-promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the anti-Labyrinthin antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' end, occasionally 3' end, of untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-Labyrinthin antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 1994/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g.,
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-Labyrinthin antibody-encoding vectors.
  • Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • K. lactis K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.
  • drosophilarum ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated anti-Labyrinthin antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980), including DG44 (Urlaub et al., Som.
  • mice Sertoli cells TM4, Mather, Biol. Reprod., 23:243-251 (1980)
  • monkey kidney cells CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for anti- Labyrinthin antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the anti-Labyrinthin antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCrNTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the antibody can be produced intracellularly or in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology, 10: 163-167 (1992) describes a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an AMICONTM or MILLIPORE PELLICONTM ultrafiltration unit.
  • a protease inhibitor such as phenylmethylsulphonyl fluoride (PMSF) may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.
  • PMSF phenylmethylsulphonyl fluoride
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human vl, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., EMBO J., 5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled-pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a C H 3 domain
  • BAKERBOND ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse- phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM, chromatography on an anion- or cation- exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium-sulfate precipitation are also available depending on the antibody to be recovered.
  • Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low-molecular- weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • Zn-protein complexes Zn-protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth- inhibitory agent, anti-hormonal agent, Labyrinthin-targeted drug, anti-angiogenic agent, and/or cardioprotectant.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • administration of a labyrinthin antibody of the invention is combined with the administration of an additional therapeutic agent as part of a therapeutic regimen.
  • the additional therapeutic agent can be administered before, during, or after the administration of a labyrinthin binding agent, such as an antibody.
  • Agents administered during the administration of the labyrinthin antibody can be co-administered as a single composition and delivery or delivered as part of the same procedure, administered at about the same time in separate administration events.
  • Agents administered before or after administration of the labyrinthin antibody can be administered in time frames preceding or following labyrinthin binding agent administration that include the following, without limitation: less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days; less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 weeks; less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 months; and less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 years.
  • the additional therapeutic agent is conjugated to the labyrinthin antibody.
  • radioisotopes, drugs, and toxins can be conjugated to antibodies or antibody fragments to facilitate targeting of the radioisotopes, drugs or toxins to tumor sites to enhance their therapeutic efficacy and minimize side effects.
  • these agents and methods are reviewed in Wawrzynczak and Thorpe (in Introduction to the Cellular and Molecular Biology of Cancer, L. M. Franks and N. M. Teich, eds, Chapter 18, pp. 378-410, Oxford University Press, Oxford, 1986), in Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (C.-W. Vogel, ed., 3-300, Oxford University Press, New York, 1987), in Dillman, R.O.
  • an antibody of the present invention can be combined with anti-tumor or anti-cancer therapeutics capable of decreasing or preventing a further increase in tumor growth.
  • Non- limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin; alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine,
  • trimethylolomelamine nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, CasodexTM, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, dox
  • elliptinium acetate etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;
  • mitoxantrone mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK.RTM ; razoxane; sizofiran; spirogermanium; tenuazonic acid;
  • paclitaxel TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, N.J.
  • docetaxel TAXOTERETM, Rhone-Poulenc Rorer, Antony, France
  • retinoic acid esperamicins
  • capecitabine ecitabine
  • pharmaceutically acceptable salts, acids or derivatives of any of the above TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, N.J.
  • chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti- estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C;
  • anti- estrogens including for example tamoxifen, (NolvadexTM),
  • mitoxantrone vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); topoisomerase inhibitor RFS 2000;
  • the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide,
  • anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17-demethoxygeldanamycin, Alpharadin
  • Mitomalcin Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid;
  • Nocodazole Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;
  • Teloxantrone Hydrochloride Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine;
  • Tubulozole Hydrochloride Uracil Mustard; Uredepa; Vapreotide; Verteporfm; Vinblastine Sulfate;
  • Vincristine Sulfate Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; Taxol; thiosemicarbazone derivatives; telomerase inhibitors; arsenic trioxide; planomycin; sulindac sulfide; cyclopamine; purmorphamine; gamma-secretase inhibitors; CXCR4 inhibitors; HH signaling inhibitors; Bmi-1 inhibitors; Bcl-2 inhibitors; Notch- 1 inhibitors; DNA checkpoint protein inhibitors; ABC transporter inhibitors; mitotic inhibitors; intercalating antibiotics; growth factor inhibitors; cell cycle modulators; enzymes; topoisomerase inhibitors; biological response modifiers; angiogenesis inhibitors; DNA
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug-delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and
  • nanocapsules or in macroemulsions.
  • Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained- release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L- glutamate copolymers of L-glutamic acid and ⁇ ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-(-)-3-hydroxybutyric acid poly-D-(-)-3-hydroxybutyric acid.
  • formulations to be used for in vivo administration must be sterile. This is readily
  • the anti-Labyrinthin antibodies may be used to treat various diseases or disorders.
  • Exemplary conditions or disorders include benign or malignant tumors; leukemias and lymphoid malignancies; and other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders.
  • the anti-Labyrinthin antibodies can be used to treat Labyrinthin positive cells, such as Labyrinthin-positive tumors. Examples of Labyrinthin-positive tumors include adenocarcinomas.
  • the present invention provides a method of ameliorating an adenocarcinoma in a subject.
  • Amelioration of an adenocarcinoma can be treatment, suppression, prevention, or palliation of a symptom of an adenocarcinoma.
  • amelioration can be extracorporeal (also known as phoresis) capture and/or killing of a tumor cell, such as an adenocarcinoma cell.
  • the invention can be use of an antibody of the invention for determining diagnosis and treatment of a subject (theranostics).
  • the method comprises administering to the subject in need thereof an effective amount of a labyrinthin antibody of the present invention.
  • Adenocarcinomas subject to the methods of the invention can include any that arise from tissue with gland- like properties.
  • the adenocarcinoma can be any adenocarcinoma of interest, including but not limited to lung adenocarcinoma, esophagus adenocarcinoma, gastric adenocarcinoma, renal cell adenocarcinoma, prostate adenocarcinoma, colon adenocarcinoma, pancreas adenocarcinoma, cervix adenocarcinoma, gastric adenocarcinoma, salivary adenocarcinoma or breast adenocarcinoma.
  • Adenocarcinoma can be identified using methods known in the art, including but not limited to morphological analysis, histological analysis, surface marker profiling, protein profiling, and gene expression profiling.
  • adenocarcinomas subject to the methods of the invention are specifically Labyrinthin-positive adenocarcinomas.
  • amelioration of adenocarcinoma is achieved through the use of an effective amount of a labyrinthin antibody of the present invention.
  • Labyrinthin is an integral membrane protein of approximately 40 kilodaltons, the sequence of which can be found in GenBank having accession number AR532049.
  • Labyrinthin has also been shown to respond to changes in Ca2+ concentrations.
  • Labyrinthin has further been identified as a reliable cellular marker for adenocarcinomas.
  • the invention can be a method comprising screening for the presence of labyrinthin and amelioration of adenocarcinoma is achieved through the use of an effective amount of a labyrinthin antibody of the present invention.
  • screening can be done using a labyrinthin antibody of the present invention.
  • an immunoconjugate comprising the anti-Labyrinthin antibody conjugated with a cytotoxic agent is administered to the patient.
  • the immunoconjugate and/or Labyrinthin protein to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds.
  • the cytotoxic agent targets or interferes with nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases, and DNA endonucleases.
  • the anti-Labyrinthin antibodies or immunoconjugates are administered to a human patient in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • Intravenous, intraperitoneal, or subcutaneous administration of the antibody is preferred, with subcutaneous or intraperitoneal routes being particular preferred.
  • a preferred administration schedule is about 2-3 times per week, depending on the particular mammal being treated, the type of antibody, and other factors well known to the practitioner. However, other administration schedules are operable herein.
  • Other therapeutic regimens may be combined with the administration of the anti-Labyrinthin antibody.
  • the combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • It may also be desirable to combine administration of the anti-Labyrinthin antibody or antibodies with administration of an antibody directed against another tumor-associated antigen.
  • the other antibody in this case may, for example, bind to an antigen such as HER-2, EGFR, ErbB3, ErbB4, vascular endothelial growth factor (VEGF), or a B-cell surface marker or antigen (an antigen expressed on the surface of a B cell that can be targeted with an antagonist that binds thereto), such as, for example, the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers (for descriptions, see The Leukocyte Antigen Facts Book, 2 nd Edition.
  • an antigen such as HER-2, EGFR, ErbB3, ErbB4, vascular endothelial growth factor (VEGF), or a B-cell surface marker or antigen (an antigen expressed on
  • B-cell surface markers include RP105, FcRH2, B-cell CR2, CCR6, P2 x 5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287.
  • the B-cell surface marker of particular interest is preferentially expressed on B cells compared to other non-B-cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells.
  • the preferred B- cell surface markers herein are CD20 and CD22.
  • the Labyrinthin antibody may be combined with an anti-angiogenic agent, which acts to inhibit angiogenesis.
  • An example is an antagonist to VEGF, such as an antibody, e.g., A VASTESTTM.
  • the treatment of the present invention involves the combined administration of an anti-Labyrinthin antibody (or antibodies) and one or more regulators of immune function in a mammal, such as cytokines, as well as chemotherapeutic agents or growth-inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents.
  • Preferred chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics.
  • Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such
  • the antibody may be combined with an anti-hormonal compound, e.g., an anti-estrogen compound such as tamoxifen or an aromatase inhibitor such as anastrozole; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules.
  • an anti-hormonal compound e.g., an anti-estrogen compound such as tamoxifen or an aromatase inhibitor such as anastrozole; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules.
  • an anti-hormonal compound e.g., an anti-estrogen compound such as tamoxifen or an aromatase inhibitor such as anastrozole; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as
  • Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-Labyrinthin antibody.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • the preferred dosage of the antibody will be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six doses, of the anti-Labyrinthin antibody).
  • An initial higher loading dose, followed by one or more lower doses, may be administered.
  • An exemplary dosing regimen comprises administering an initial loading dose of about 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 3-5 mg/kg or 4 mg/kg, followed by a weekly maintenance dose of about 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 2-5 mg/kg or 2 mg/kg of the anti- Labyrinthin antibody.
  • an initial loading dose of about 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 3-5 mg/kg or 4 mg/kg
  • a weekly maintenance dose of about 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 2-5 mg/kg or 2 mg/kg of the anti- Labyrinthin antibody.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the antibody is suitably administered serially or in combination with radiological treatments— irradiation or introduction of radioactive substances—such as those referred to in UICC (Ed.), Klinische Onkologie, Springer- Verlag (1982).
  • the present application contemplates administration of the antibody by gene therapy.
  • administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody”. See, for example, WO 1996/07321 published Mar. 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.
  • nucleic acid (optionally contained in a vector) into the patient's cells
  • in vivo and ex vivo the nucleic acid is injected directly into the patient, usually at the site where the antibody is required.
  • ex vivo treatment the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187).
  • techniques available for introducing nucleic acids into viable cells There are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium-phosphate precipitation method, etc.
  • a commonly used vector for ex vivo delivery of the gene is a retrovirus.
  • the currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOPE) and 3 -(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Choi”), for example).
  • viral vectors such as adenovirus, Herpes simplex I virus, or adeno-associated virus
  • lipid-based systems useful lipids for lipid-mediated transfer of the gene are N-[l-(2,3-dioleyloxy
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins that bind to a cell- surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem.
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is the humanized anti-Labyrinthin antibody herein.
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the label or package insert indicates that the composition comprising the antibody can be used to treat a Labyrinthin disorder, for example, to treat cancer that expresses a Labyrinthin receptor.
  • the label or package insert may indicate that the patient to be treated is one having cancer characterized by excessive activation of a Labyrinthin receptor.
  • the label or package insert may also indicate that the composition can be used to treat cancer, wherein the cancer is not characterized by overexpression of a Labyrinthin receptor.
  • the package insert may indicate that the antibody or composition can be used to treat breast cancer (e.g. metastatic breast cancer); hormone-independent cancer; prostate cancer (e.g. androgen- independent prostate cancer); lung cancer (e.g. non-small cell lung cancer); colon, rectal or colorectal cancer; or any of the other diseases or disorders disclosed herein.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the humanized antibody herein, and (b) a second container with a composition contained therein, wherein the composition comprises a therapeutic agent other than the humanized antibody.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the first and second compositions can be used in combination to treat a Labyrinthin disorder such as cancer.
  • Such therapeutic agent may be any of the adjunct therapies described in the preceding section (e.g., a chemotherapeutic agent, an anti-angiogenic agent, an anti- hormonal compound, a cardioprotectant, and/or a regulator of immune function in a mammal, including a cytokine).
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • the antibodies e.g. the humanized anti-Labyrinthin antibodies
  • the antibodies have further non-therapeutic applications.
  • the antibodies may be used as affinity-purification agents.
  • the antibodies are immobilized on a solid phase such as a SEPHADEXTM resin or filter paper, using methods well known in the art.
  • the immobilized antibody is contacted with a sample containing the Labyrinthin protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the Labyrinthin protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the Labyrinthin protein from the antibody.
  • a suitable solvent such as glycine buffer, pH 5.0
  • Anti-Labyrinthin antibodies may also be useful in diagnostic assays for Labyrinthin protein, e.g., detecting its expression in specific cells, tissues, or serum.
  • the antibody typically will be labeled with a detectable moiety.
  • a detectable moiety Numerous labels are available that can be generally grouped into the following categories:
  • Radioisotopes such as 35 S, 14 C, 125 1, 3 H, and 13 T
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation counting.
  • Fluorescent labels such as rare-earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microper oxidase, and the like.
  • luciferases e.g., firefly luciferase and bacterial lucifer
  • enzyme-substrate combinations include, for example:
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'- tetramethyl benzidine hydrochloride
  • ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- ⁇ -D- galactosidase) or fluorogenic substrate 4-methylumbelliferyl- ⁇ -D-galactosidase.
  • a chromogenic substrate e.g., p-nitrophenyl- ⁇ -D- galactosidase
  • fluorogenic substrate 4-methylumbelliferyl- ⁇ -D-galactosidase
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin, and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin, and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten e.g., digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • the anti-Labyrinthin antibody need not be labeled, and the presence thereof can be detected using a labeled antibody that binds to the Labyrinthin antibody.
  • the antibodies of the present invention may be employed in any known assay method, such as competitive-binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
  • the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
  • the antibodies may also be used for in vivo diagnostic assays.
  • the antibody is labeled with a radionuclide (such as U 1 ln, 99 Tc, 14 C, 131 I, 125 1, 3 H, 32 P or 35 S) so that, for example, a tumor can be localized using immunoscintigraphy.
  • a radionuclide such as U 1 ln, 99 Tc, 14 C, 131 I, 125 1, 3 H, 32 P or 35 S
  • the antibodies of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or fiuorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor that provides the detectable chromophore or fiuorophore.
  • other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration.
  • the antibody herein is also useful for in vivo imaging, where the labeled antibody is administered to a host, preferably the bloodstream, and the presence and location of the labeled antibody in the host is assayed.
  • This imaging technique is suitably used in the staging and treatment of neoplasms.
  • the antibody is suitably labeled with any moiety that is detectable in a host, including non-radioactive indicators detectable by, e.g., nuclear magnetic resonance, or other means known in the art.
  • the label is a radiolabel, including iodine, e.g., I and I, selenium, bifunctional chelates, copper, e.g., Cu, technetium, e.g., 99 mTc, and rhenium, e.g., 186 Re and 188 Re.
  • the radioisotope is conjugated to the protein by any means, including metal-chelating compounds or lactoperoxidase, or iodogen techniques for iodination.
  • kits comprising the antibody compositions of the invention (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use.
  • the kit can further contain one or more additional reagents, such as an
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • Example 1 Comparison of binding affinities of murine and humanized antibodies to Lab
  • a humanized antibody to Labyrinthin (designated X509) was produced.
  • Affinity of X509 for Labyrinthin was measured using standard techniques, and these measurements were compared to measurements of Labyrinthin affinity of murine anti-Labyrinthin antibody X373.
  • affinity of mouse IgG for Labyrinthin was also measured. Results of these measurements are provided in Figure 4.
  • the Kd values indicate that humanized antibody X509 has an improved affinity for Labyrinthin, compared to the murine antibody X373.

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Abstract

This invention provides chimeric and humanized antibodies against labyrinthin (Lab) and methods of using these antibodies.

Description

HUMANIZED ANTIBODIES TO LABYRINTHIN AND USES THEREOF
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 61/314,139, filed March 15, 2010, which application is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Cancer is a leading cause of death in men and women throughout the world. In the United States alone, over 1 million new cases are diagnosed each year, and over 0.5 million deaths are reported annually (Landis et al., 1998). Historically, tumors are grouped and treated, based in part by the tissues in which they arise, e.g.— breast cancer, colon cancer, and lung cancer, and the like. Yet, within lung cancer, for example, it is well recognized that these tumors are a very heterogeneous group of neoplasms. This is also true for tumors arising in other tissues. In part, because of this heterogeneity, there are complex and inconsistent classification schemes which are used for human tumors. Previous attempts to treat cancer have been hampered by: 1) the arbitrary classification of tumors arising within given tissues, and 2) by using microscopic methods based on how these tumors look (histological classification).
[0003] Labyrinthin (Lab) is a cell surface protein expressed on the extracellular surface of the plasma membrane of adenocarcinomas, and is not cell cycle specific. Lab is not found in the serum of normal or tumor bearing patients, and is not shed into the culture media by Lab positive cell lines, thus Lab represents a useful marker for adenocarcinoma-type cancers.
[0004] Immunotherapy in humans has been limited, in part due to adverse responses to non-human monoclonal antibodies. Early clinical trials using rodent antibodies revealed human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, which lead to rapid clearance of the antibody. Less immunogenic antibodies have since been developed, including chimeric antibodies, humanized antibodies, PRIMATIZED® antibodies, and human antibodies prepared using transgenic mice or phage display libraries. See Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6851 -5; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-33; Newman et al. (1992) Biotechnology (NY) 10:1455-60; Green et al. (1994) Nat. Genet. 7: 13-21 ; Marks et al. (1991) J. Mol. Biol. 222:581 -97. Avoidance of a HAMA response permits high dose and repeated dose administration to achieve a therapeutic response. The present invention provides humanized anti-Lab antibodies and methods for their use.
SUMMARY OF THE INVENTION
[0005] The present invention provides antibodies, in particular, humanized antibodies, that bind to a labyrinthin protein, and that exhibit numerous desirable properties, such as high affinity binding to a labyrinthin protein.
[0006] In one embodiment, the invention comprises a humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: (1) a variable heavy (VH) domain that comprises: a) a heavy chain variable region CDR1 comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof; and (2) a variable light (VL) domain that comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VH domain, and the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VL domain.
[0007] In another embodiment, the invention comprises a humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; and c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VH domain.
[0008] In another embodiment, the invention comprises a humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VL domain.
[0009] In another embodiment, the invention comprises a humanized antibody, or antigen-binding portion thereof, wherein the antibody comprises: a variable heavy (VH) domain that comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, incorporated into a human VH domain.
[0010] In some embodiments, a humanized antibody of the invention comprises a heavy chain constant region (CH) encoded by SEQ ID NO: 10, and/or a light chain constant region (CL) encoded by SEQ ID NO: 9.
[0011] In another embodiment, the invention comprises a pharmaceutical composition comprising any of the above antibodies and a pharmaceutically acceptable carrier.
[0012] In another embodiment, the invention comprises an isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any of the above antibodies.
[0013] In another embodiment, the invention comprises a method of ameliorating an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of any of the above antibodies in an amount effective to ameliorate the adenocarcinoma. In some embodiments, said administering reduces metastasis of said adenocarcinoma, such as an increase in time to metastasis (e.g. 1, 2, 3, 4, 5, or more months; or 1, 2, 3, 4, 5, or more years), or a decrease in the frequency of metastases (e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more), relative to an untreated subject. In some embodiments, said administering results in a decrease in size of the adenocarcinoma, such as a size decrease of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, relative to a pre-treatment size.
[0014] In another embodiment, the invention comprises a method of ameliorating relapse of an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of any of the above antibodies in an amount effective to ameliorate relapse of the adenocarcinoma. In some embodiments, said administering reduces metastasis of said adenocarcinoma, such as an increase in time to metastasis (e.g. 1, 2, 3, 4, 5, or more months; or 1, 2, 3, 4, 5, or more years), or a decrease in the frequency of metastases (e.g. by 20%, 30%, 40%, 50%, 60%), 70%), 80%), 90%), 95%), or more), relative to an untreated subject. In some embodiments, said administering results in a decrease in size of the adenocarcinoma, such as a size decrease of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, relative to a pre-treatment size.
[0015] In another embodiment, the invention comprises a method of ameliorating an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of any of the above antibodies, wherein the antibody binds to a cancer stem cell. In some embodiments, said administering reduces metastasis of said adenocarcinoma, such as an increase in time to metastasis (e.g. 1, 2, 3, 4, 5, or more months; or 1, 2, 3, 4, 5, or more years), or a decrease in the frequency of metastases (e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more), relative to an untreated subject. In some embodiments, said administering results in a decrease in size of the adenocarcinoma, such as a size decrease of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%), or more, relative to a pre-treatment size. In some embodiments, the cancer stem cell is a self- renewing cell. In some embodiments, the cancer stem cell is an adenocarinoma cancer stem cell. In some embodiments, the cancer stem cell expresses one or more of CD133, CD44, CD166, CD29, CD24, Lgr5, ALDH1, ESA, and b-catenin.
[0016] In another embodiment, the invention comprises a kit comprising a container containing the humanized antibody of any of the above antibodies and instructions directing a user to treat an adenocarcinoma in a subject with the antibody in an effective amount.
INCORPORATION BY REFERENCE
[0017] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0019] Figure 1 shows the sequence of murine antibody clone X373. Shown are the VL light chain nucleic acid sequence (SEQ ID NO:l) and peptide sequence (SEQ ID NO:2), and the VH heavy chain nucleic acid sequence (SEQ ID NO:3) and peptide sequence (SEQ ID NO:4).
[0020] Figure 2 shows the sequence of humanized antibody clone X509. Shown are the VL light chain nucleic acid sequence (SEQ ID NO:5) and peptide sequence (SEQ ID NO:6), and the VH heavy chain nucleic acid sequence (SEQ ID NO:7) and peptide sequence (SEQ ID NO:8). Also shown are the VL delineated CDR1, CDR2, and CDR3 regions (SEQ ID NOs: 11, 12 and 13, respectively) and the VH delineated CDR1, CDR2, and CDR3 regions (SEQ ID NOs: 14, 15 and 16, respectively).
[0021] Figure 3 shows a CL constant light chain sequence (SEQ ID NO:9) and CHI constant heavy chain region sequence (SEQ ID NO: 10).
[0022] Figure 4 shows affinity measurement of the murine antibody clone X373 and humanized antibody clone X509 to a labyrinthin protein.
DETAILED DESCRIPTION OF THE INVENTION
[0023] This invention provides chimeric and humanized antibodies against labyrinthin (Lab) and methods of using these antibodies. The term antibody refers to an immunoglobulin protein, or antibody fragments that comprise an antigen binding site (e.g., Fab, modified Fab, Fab', F(ab')2 or Fv fragments, or a protein having at least one immunoglobulin light chain variable region or at least one immunoglobulin heavy chain region). Humanized antibodies of the invention include diabodies, tetrameric antibodies, single chain antibodies, tretravalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and domain-specific antibodies that recognize a particular epitope.
[0024] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
[0025] The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
[0026] The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. 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, CHI, 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. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. 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.
[0027] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Lab). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the v, VH, Ch and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region (see, FUNDAMENTAL IMMUOLOGY (Paul ed., 3rd ed. 1993); (iv) a Fd fragment consisting of the VH and CHI domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic 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. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
[0028] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g. an isolated antibody that specifically binds Lab is substantially free of antibodies that specifically bind antigens other than Lab). An isolated antibody that specifically binds Lab may, however, have cross-reactivity to other antigens, such as Lab molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
[0029] The terms "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. [0030] As used herein, "isotype" refers to the antibody class (e.g., IgM, IgG (and subclasses such as IgGl, IgG2, IgG3, IgG4), IgA, IgE, etc.) that is encoded by the heavy chain constant region genes.
[0031] As used herein, an antibody that "specifically binds to human Lab" is intended to refer to an antibody that binds to human Lab with an affinity having a KD of preferably at least 1 x 10~6 M, more preferably at least 1 x 10~7 M, more preferably at least 5 x 10~8 M, more preferably at least 1 x 10~8 M, or more preferably at least 5 x 10~9 M.
[0032] The term "Κ^^" or "Ka", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdis" or as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., K<j/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore.RTM. system.
[0033] Production of Humanized Anti-Labyrinthin Antibodies
[0034] Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source that is non- human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 : 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
[0035] Another method for making humanized antibodies is described in U.S. Patent Publication 2003/0017534 published Jan. 23, 2003, wherein humanized antibodies and antibody preparations are produced from transgenic non-human animals. The non-human animals are genetically engineered to contain one or more humanized immunoglobulin loci that are capable of undergoing gene rearrangement and gene conversion in the transgenic non-human animals to produce diversified humanized
immunoglobulins .
[0036] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151 : 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 : 2623 (1993)).
[0037] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
[0038] A preferred antibody of the invention is the humanized monoclonal antibody X509. In one aspect, the invention provides an isolated monoclonal antibody, or antigen binding portion thereof comprising:
[0039] a) a heavy chain variable region comprising an amino acid sequence having SEQ ID NO: 8; and
[0040] (b) a light chain variable region comprising an amino acid sequence having SEQ ID NO: 6;
[0041] wherein the antibody specifically binds Lab, preferably human Lab.
[0042] In another aspect, the invention provides antibodies that comprise the heavy chain and light chain CDRls, CDR2s and CDR3s of X509. The amino acid sequence of the VH CDRl s of X509 is shown in SEQ ID NO: 14. The amino acid sequence of the VH CDR2 of X509 is shown in SEQ ID NO: 15. The amino acid sequence of the VH CDR3 of X509 is shown in SEQ ID NO: 16. The amino acid sequences of the VK CDRl of X509 is shown in SEQ ID NO: 11. The amino acid sequence of the VK CDR2 of X509 is shown in SEQ ID NO: 12. The amino acid sequence of the VK CDR3 of X509 is shown in SEQ ID NO: 13. The CDR regions are delineated using the Kabat system (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).
[0043] It is well known in the art that the CDR3 domain, independently from the CDRl and/or CDR2 domain(s), alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, for example, Klimka et al., British J. of Cancer 83(2):252-260 (2000) (describing the production of a humanized anti-CD30 antibody using only the heavy chain variable domain CDR3 of murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol. 296:833-849 (2000) (describing recombinant epithelial glycoprotein-2 (EGP-2) antibodies using only the heavy chain CDR3 sequence of the parental murine MOC-31 anti-EGP-2 antibody); Rader et. al., Proc. Natl. Acad. Sci. U.S.A. 95:8910- 8915 (1998) (describing a panel of humanized anti-integrin αν β 3 antibodies using a heavy and light chain variable CDR3 domain of a murine anti-integrin αν β 3 antibody LM609 wherein each member antibody comprises a distinct sequence outside the CDR3 domain and capable of binding the same epitope as the parent murine antibody with affinities as high or higher than the parent murine antibody); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994) (disclosing that the CDR3 domain provides the most significant contribution to antigen binding); Barbas et al., Proc. Natl. Acad. Sci. U.S.A. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3 sequences of three Fabs (SI-1, SI-40, and SI-32) against human placental DNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacing the existing heavy chain CDR3 and demonstrating that the CDR3 domain alone conferred binding specificity); and Ditzel et al., J. Immunol. 157:739-749 (1996) (describing grafting studies wherein transfer of only the heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chain of a monospecific IgG tetanus toxoid-binding Fab p313 antibody was sufficient to retain binding specificity of the parent Fab). Each of these references is hereby incorporated by reference in its entirety.
[0044] Accordingly, the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domains from an antibody derived from a human or non- human animal, wherein the monoclonal antibody is capable of specifically binding to Lab. Within certain aspects, the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a non-human antibody, such as a mouse or rat antibody, wherein the monoclonal antibody is capable of specifically binding to Lab. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from a non- human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental non-human antibody.
[0045] Within other aspects, the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the human antibody is capable of specifically binding to Lab. Within other aspects, the present invention provides monoclonal antibodies comprising one or more heavy and/or light chain CDR3 domain from a first human antibody, such as, for example, a human antibody obtained from a non-human animal, wherein the first human antibody is capable of specifically binding to Lab and wherein the CDR3 domain from the first human antibody replaces a CDR3 domain in a human antibody that is lacking binding specificity for Lab to generate a second human antibody that is capable of specifically binding to Lab. Within some embodiments, such inventive antibodies comprising one or more heavy and/or light chain CDR3 domain from the first human antibody (a) are capable of competing for binding with; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the corresponding parental first human antibody. Various forms of the humanized antibody or affinity-matured antibody are contemplated. For example, the humanized antibody or affinity-matured antibody may be an antibody fragment, such as a Fab, that is optionally conjugated with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody or affinity-matured antibody may be an intact antibody, such as an intact IgGl antibody.
[0046] Various techniques have been developed for the production of antibody fragments of humanized antibodies. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by, and isolated from recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 1993/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibody fragments may be monospecific or bispecific.
[0047] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Labyrinthin protein. Other such antibodies may combine a Labyrinthin binding site with binding site(s) for HER-2, EGFR, ErbB, ErbB3, and/or ErbB4. Alternatively, an anti-Labyrinthin arm may be combined with an arm that binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (Fc γ R), such as FcvRI (CD64), FcvRII (CD32) and FcvRIII (CD 16) so as to focus cellular defense mechanisms to the Labyrinthin-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells that express Labyrinthin. These antibodies possess a Labyrinthin-binding arm and an arm that binds the cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate, or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).
[0048] Methods for making bispecific antibodies are known in the art. Traditional production of full- length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-chain- light-chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 1993/08829, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991). [0049] According to a different approach, antibody- variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
[0050] In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy-chain- light-chain pair (providing a second binding specificity) in the other arm It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 1994/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
[0051] According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
[0052] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 1991/00360, WO 1992/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed, for example, in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. [0053] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
[0054] Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the Labyrinthin receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
[0055] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain.
Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
[0056] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol,. 147: 60 (1991).
[0057] Homologous Antibodies
[0058] In yet another embodiment, an antibody of the invention comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-Lab antibodies of the invention. [0059] For example, the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence having SEQ ID NO: 8; (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence having SEQ ID NO: 6; and the antibody exhibits one or more of the following properties: (c) the antibody binds to human Lab with a KD of lxlO"7 M or less;(d) the antibody binds to an adenocarcinoma tumor cell line.
[0060] In other embodiments, the VH and/or VL amino acid sequences may be 85%, 90%, 95%, 96%, 97%), 98%) or 99% homologous to the sequences set forth above. An antibody having VH and VL regions having high (i.e., 80% or greater) homology to the VH and VL regions of the sequences set forth above, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 5 and 7, followed by testing of the encoded altered antibody for retained function (i.e., the functions set forth in (c) and (d) above) using the functional assays described herein.
[0061] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # 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.
[0062] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (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. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0063] Additionally or alternatively, 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. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (See www.ncbi.nlm.nih.gov).
[0064] Antibodies with Conservative Modifications
[0065] In certain embodiments, an antibody of the invention comprises 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 comprise specified amino acid sequences based on the preferred antibodies described herein (e.g., X509), or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the anti-Lab antibodies of the invention. Accordingly, the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
[0066] (a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence having SEQ ID NO: 16, or conservative modifications thereof;
[0067] (b) the light chain variable region CDR3 sequence comprises an amino acid sequence having
SEQ ID NO: 13, or conservative modifications thereof; and
[0068] the antibody exhibits one or more of the following properties:
[0069] (c) specifically binds to human Lab; and
[0070] (d) binds to an adenocarcinoma tumor cell line.
[0071] In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence having SEQ ID NO: 15, or conservative modifications thereof; and the light chain variable region CDR2 sequence comprises an amino acid sequence having SEQ ID NO: 12, or conservative modifications thereof. In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises an amino acid sequence having SEQ ID NOs: 14, or conservative modifications thereof; and the light chain variable region CDR1 sequence comprises an amino acid sequence having SEQ ID NO: 11, or conservative modifications thereof.
[0072] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) and (d) above) using the functional assays described herein.
[0073] Antibodies that Bind to the Same Epitope as Anti-Lab Antibodies of the Invention
[0074] In another embodiment, the invention provides antibodies that bind to the same epitope on human Lab as any of the Lab monoclonal antibodies of the invention (i.e., antibodies that have the ability to cross-compete for binding to Lab with any of the monoclonal antibodies of the invention). In preferred embodiments, the reference antibody for cross-competition studies can be the monoclonal antibody X509 (having VH and VL sequences as shown in SEQ ID NOs: 8 and 6, respectively). Such cross-competing antibodies can be identified based on their ability to cross-compete with X509 in standard Lab binding assays. For example, BIAcore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the antibodies of the current invention. The ability of a test antibody to inhibit the binding of, for example, X509, to human Lab demonstrates that the test antibody can compete with X509 for binding to human Lab and thus binds to the same epitope on human Lab as X509. In a preferred embodiment, the antibody that binds to the same epitope on human Lab as X509 is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the Examples.
[0075] Engineered and Modified Antibodies
[0076] An antibody of the invention further can be prepared using an antibody having one or more of the VH and/or VL sequences disclosed 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 (i.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.
[0077] 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. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)
[0078] Accordingly, another embodiment of the invention pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDRl, CD2, and CDR3 sequences having SEQ ID NOs: 14, 15, and 16, respectively, and a light chain variable region comprising CDRl, CDR2, and CDR3 sequences having SEQ ID NOs: 11, 12 and 13, respectively. Thus, such antibodies contain the VH and VL CDR sequences of monoclonal antibodies X509 yet may contain different framework sequences from these antibodies.
[0079] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, 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) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) "A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available in the accompanying Genbank accession numbers: 1-69 (NrG0010109, NT.sub.— 024637 and
BC070333),3-33 (NG.sub.-0010109 and NT-024637) and 3-7 (NG0010109 and NT.sub.-024637). As another example, the following heavy chain germline sequences found in the HCol2 HuMAb mouse are available in the accompanying Genbank accession numbers: 1-69 (NG 0010109, NT.sub.— 024637 and BC070333), 5-51 (NG.sub.-0010109 and NT.sub.-024637), 4-34 (NG.sub.-0010109 and NT. sub. - 024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).
[0080] Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research 25:3389-3402), which is well known to those skilled in the art. BLAST is a heuristic algorithm in that a statistically significant alignment between the antibody sequence and the database sequence is likely to contain high-scoring segment pairs (HSP) of aligned words. Segment pairs whose scores cannot be improved by extension or trimming is called a hit. Briefly, the nucleotide sequences of VBASE origin (vbase.mrc-cpe.cam.ac.uk/vbasel/list2.php) are translated and the region between and including FR1 through FR3 framework region is retained. The database sequences have an average length of 98 residues. Duplicate sequences which are exact matches over the entire length of the protein are removed. A BLAST search for proteins using the program blastp with default, standard parameters except the low complexity filter, which is turned off, and the substitution matrix of BLOSUM62, filters for top 5 hits yielding sequence matches. The nucleotide sequences are translated in all six frames and the frame with no stop codons in the matching segment of the database sequence is considered the potential hit. This is in turn confirmed using the BLAST program tblastx, which translates the antibody sequence in all six frames and compares those translations to the VBASE nucleotide sequences dynamically translated in all six frames.
[0081] The identities are exact amino acid matches between the antibody sequence and the protein database over the entire length of the sequence. The positives (identities+substitution match) are not identical but amino acid substitutions guided by the BLOSUM62 substitution matrix. If the antibody sequence matches two of the database sequences with same identity, the hit with most positives would be decided to be the matching sequence hit.
[0082] Another type of variable region modification is to mutate amino acid residues within the VH and/or VK CDRl, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Preferably conservative modifications (as discussed above) are introduced. The mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.
[0083] Accordingly, in another embodiment, the invention provides isolated anti-Lab monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising: (a) a VH CDRl region comprising an amino acid sequence having SEQ ID NO: 14, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 14; (b) a VH CDR2 region comprising an amino acid sequence having SEQ ID NO: 15, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 15; (c) a VH CDR3 region comprising an amino acid sequence having SEQ ID NO: 16, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 16; (d) a VK CDRl region comprising an amino acid sequence having SEQ ID NO: 11, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 11 ; (e) a VK CDR2 region comprising an amino acid sequence having SEQ ID NO: 12, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 12; and (f) a VK CDR3 region comprising an amino acid sequence having SEQ ID NO: 13, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions as compared to SEQ ID NO: 13.
[0084] Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VK, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, 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.
[0085] Another type of 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.
[0086] In addition or alternative to modifications made within the framework or CDR regions, 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. Furthermore, 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. Each of these embodiments is described in further detail below. The numbering of residues in the Fe region is that of the EU index of Kabat.
[0087] In one embodiment, the hinge region of CHI 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. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
[0088] In another embodiment, 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. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.
[0089] In another embodiment, 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. Pat. No. 6,277,375 to Ward. Alternatively, to increase the biological half life, the antibody can be altered within the CHI 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. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.
[0090] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 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 CI component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
[0091] In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al.
[0092] In another example, one or more amino acid residues within amino acid positions 231 and 239 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 et al.
[0093] In yet another example, 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 Fcv receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for FevRI, FcvRII, FcvRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcvRIII. Additionally, the following combination mutants were shown to improve FcvRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.
[0094] In still another embodiment, the glycosylation of an antibody is modified. For example, 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. For example, 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. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.
[0095] Additionally or alternatively, 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. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such 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. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8v-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a flucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme. Hanai et al. also describe cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L- fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516- 23).
[0096] Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
[0097] Antibody Physical Properties [0098] The antibodies of the present invention may be further characterized by the various physical properties of the anti-Lab antibodies. Various assays may be used to detect and/or differentiate different classes of antibodies based on these physical properties.
[0099] In some embodiments, antibodies of the present invention may contain one or more glycosylation sites in either the light or heavy chain variable region. The presence of one or more glycosylation sites in the variable region may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41 :673-702; Gala F A and Morrison S L (2004) J Immunol 172:5489-94; Wallick et at (1988) J Exp Med 168:1099-109; Spiro R G (2002) Glycobiology 12:43 R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence. Variable region glycosylation may be tested using a Glycoblot assay, which cleaves the antibody to produce a Fab, and then tests for glycosylation using an assay that measures periodate oxidation and Schiff base formation. Alternatively, variable region glycosylation may be tested using Dionex light chromatography (Dionex-LC), which cleaves saccharides from a Fab into monosaccharides and analyzes the individual saccharide content. In some instances, it is preferred to have an anti-Lab antibody that does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation motif using standard techniques well known in the art.
[00100] In a preferred embodiment, the antibodies of the present invention do not contain asparagine isomerism sites. A deamidation or isoaspartic acid effect may occur on N-G or D-G sequences, respectively. The deamidation or isoaspartic acid effect results in the creation of isoaspartic acid which decreases the stability of an antibody by creating a kinked structure off a side chain carboxy terminus rather than the main chain. The creation of isoaspartic acid can be measured using an iso-quant assay, which uses a reverse-phase HPLC to test for isoaspartic acid.
[00101] Each antibody will have a unique isoelectric point (pi), but generally antibodies will fall in the pH range of between 6 and 9.5. The pi for an IgGl antibody typically falls within the pH range of 7-9.5 and the pi for an IgG4 antibody typically falls within the pH range of 6-8. Antibodies may have a pi that is outside this range. Although the effects are generally unknown, there is speculation that antibodies with a pi outside the normal range may have some unfolding and instability under in vivo conditions. The isoelectric point may be tested using a capillary isoelectric focusing assay, which creates a pH gradient and may utilize laser focusing for increased accuracy (Janini et al (2002) Electrophoresis 23:1605-11 ; Ma et al. (2001) Chromatographia 53:S75-89; Hunt et al (1998) J Chromatogr A 800:355-67). In some instances, it is preferred to have an anti-Lab antibody that contains a pi value that falls in the normal range. This can be achieved either by selecting antibodies with a pi in the normal range, or by mutating charged surface residues using standard techniques well known in the art.
[00102] Each antibody will have a melting temperature that is indicative of thermal stability
(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71). A higher thermal stability indicates greater overall antibody stability in vivo. The melting point of an antibody may be measure using techniques such as differential scanning calorimetry (Chen et at (2003) Pharm Res 20: 1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). TMi indicates the temperature of the initial unfolding of the antibody. TM2 indicates the temperature of complete unfolding of the antibody. Generally, it is preferred that the TMi of an antibody of the present invention is greater than 60° C, preferably greater than 65° C, even more preferably greater than 70° C. Alternatively, the thermal stability of an antibody may be measure using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).
[00103] In a preferred embodiment, antibodies are selected that do not rapidly degrade. Fragmentation of an anti-Lab antibody may be measured using capillary electrophoresis (CE) and MALDI-MS, as is well understood in the art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).
[00104] In another preferred embodiment, antibodies are selected that have minimal aggregation effects. Aggregation may lead to triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less and even more preferably 5% or less. Aggregation may be measured by several techniques well known in the art, including size-exclusion column (SEC) high performance liquid chromatography (HPLC), and light scattering to identify monomers, dimers, trimers or multimers.
[00105] Methods of Engineering Antibodies
[00106] As discussed above, the anti-Lab antibodies having VH and VK sequences disclosed herein can be used to create new anti-Lab antibodies by modifying the VH and/or VK sequences, or the constant region(s) attached thereto. Thus, in another aspect of the invention, the structural features of an anti-Lab antibody of the invention, e.g. X509, are used to create structurally related anti-Lab antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human Lab. For example, one or more CDR regions of X509, or mutations thereof, can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-Lab antibodies of the invention, as discussed above. Other types of modifications include those described in the previous section. The starting material for the engineering method is one or more of the VH and/or VK 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 VK 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" sequencers) derived from the original sequencers) and then the "second generation" sequence(s) is prepared and expressed as a protein.
[00107] Accordingly, in another embodiment, the invention provides a method for preparing an anti-Lab antibody comprising: (a) providing: (i) a heavy chain variable region antibody sequence comprising a CDR1 sequence having SEQ ID NO: 14, a CDR2 sequence having SEQ ID NO: 15, and/or a CDR3 sequence having SEQ ID NO: 16; and/or (ii) a light chain variable region antibody sequence comprising a CDR1 sequence having SEQ ID NO: 11, a CDR2 sequence having SEQ ID NO: 12, and/or a CDR3 sequence having SEQ ID NO: 13; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and/or the light chain variable region antibody sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein.
[00108] Standard molecular biology techniques can be used to prepare and express the altered antibody sequence.
[00109] Preferably, the antibody encoded by the altered antibody sequence(s) is one that retains one, some or all of the functional properties of the anti-Lab antibodies described herein, which functional properties include, but are not limited to: (a) the antibody binds to human Lab with a KD of 1 x 10~7 M or better; or (b) the antibody binds an adenocarcinoma tumor cell line.
[00110] 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., flow cytometry, binding assays).
[00111] In certain embodiments of the methods of engineering antibodies of the invention, mutations can be introduced randomly or selectively along all or part of an anti-Lab antibody coding sequence and the resulting modified anti-Lab antibodies can be screened for binding activity and/or other functional properties as described herein. Mutational methods have been described in the art. For example, 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. Alternatively, PCT Publication WO 03/074679 by Lazar et al. describes methods of using computational screening methods to optimize physiochemical properties of antibodies.
[00112] Nucleic Acid Molecules Encoding Antibodies of the Invention
[00113] Another aspect of the invention pertains to nucleic acid molecules that encode the antibodies of the invention. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.
[00114] Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library. [00115] Preferred nucleic acids molecules of the invention are those encoding the VH and VL sequences of the X509 monoclonal antibody. DNA sequences encoding the VH sequences of X509 is shown in SEQ ID NO: 5. DNA sequences encoding the VL sequences of X509 is shown in SEQ ID NO: 7.
[00116] Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in- frame.
[00117] The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., 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) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGl or IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.
[00118] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., 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) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
[00119] To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).
[00120] Production of Monoclonal Antibodies of the Invention
[00121] Monoclonal antibodies (mAbs) of the present invention 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. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
[00122] The preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very 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) and fusion procedures are also known.
[00123] 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. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos.
5,530,101 ; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
[00124] One may assess the growth-inhibitory effects of the antibody by assessing, for example, its ability to neutralize, antagonize, or inhibit a Labyrinthin activity, e.g., substantially preventing at least one of the undesirable growth-inhibitory, immunosuppressive, stroma-forming (the stromal elements including inflammatory cells, endothelial cells, and fibroblasts), or anchorage-independent growth-promoting activities of a mature Labyrinthin, as it is defined in the literature. In some embodiments, the antibody is able to block the activity of all endogenous Labyrinthin produced by tumors and suppressor lymphoid cells (T cells).
[00125] To screen for antibodies that bind to an epitope on Labyrinthin bound by an antibody of interest, a routine cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, or additionally, epitope mapping can be performed by methods known in the art.
[00126] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. a small-molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioactive isotope (i.e., a radioconjugate).
[00127] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Conjugates of an antibody and one or more small-molecule toxins, such as a calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a trichothene, and CC1065 anti-cancer agent, are also
contemplated herein.
[00128] In one preferred embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antibody molecule).
Maytansine may, for example, be converted to May-SS-Me, which may be reduced to May-SH3 and reacted with modified antibody (Chari et al., Cancer Research, 52: 127-131 (1992)) to generate a maytansinoid-antibody immunoconjugate.
[00129] Another immunoconjugate of interest comprises an anti-Labyrinthin antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double- stranded DNA breaks at sub-picomolar concentrations. Structural analogues of calicheamicin that may be used include, but are not limited to, vi1, a-i , (X31, N-acetyl-Vi1, PSAG and .theta.'i (Hinman et al., Cancer Research, 53 : 3336-3342 (1993) and Lode et al. Cancer Research, 58: 2925-2928 (1998)). See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001 expressly incorporated herein by reference.
[00130] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 1993/21232 published Oct. 28, 1993.
[00131] The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a
deoxyribonuclease; DNase).
[00132] A variety of radioactive isotopes are available for the production of radioconjugated anti- Labyrinthin antibodies. Examples include At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu.
[00133] Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-l -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l ,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science, 238: 1098 (1987). Carbon- 14-labeled l -isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 1994/1 1026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker, or disulfide-containing linker (Chari et al. Cancer Research, 52: 127-131 (1992)) may be used.
[00134] Alternatively, a fusion protein comprising the anti-Labyrinthin antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis.
[00135] In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g. avidin) that is conjugated to a cytotoxic agent (e.g. a radionucleotide).
[00136] ADEPT Technology
[00137] The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO 1981/01145) to an active anti-cancer drug. See, for example, WO 1988/07378 and U.S. Pat. No.
4,975,278.
[00138] The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to convert it into its more active, cytotoxic form.
[00139] Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting nontoxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β -lactamase useful for converting drugs derivatized with β -lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
[00140] The enzymes useful in this invention can be covalently bound to the anti-Labyrinthin antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen-binding region of an antibody of the invention linked to at least a functionally active portion of a suitable enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)).
[00141] Other modifications of the antibody are contemplated herein. For example, the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug-delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).
[00142] The anti-Labyrinthin antibodies disclosed herein may also be formulated as immunoli osomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO 1997/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
[00143] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
[00144] Vectors, Host Cells and Recombinant Methods
[00145] The invention also provides isolated nucleic acid encoding the humanized anti-Labyrinthin antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.
[00146] For recombinant production of the antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription-termination sequence.
[00147] The anti-Labyrinthin antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native anti-Labyrinthin antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a-factor leader (including Saccharomyces and Kluyveromyces a-factor leaders), acid-phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 1990/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. [00148] In general, the DNA for such precursor region is ligated in reading frame to DNA encoding the anti-Labyrinthin antibody.
[00149] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
[00150] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
[00151] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
[00152] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-Labyrinthin antibody-encoding nucleic acid, such as dihydrofolate reductase (DHFR), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
[00153] For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
[00154] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding anti-Labyrinthin antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
[00155] A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
[00156] In addition, vectors derived from the 1.6-μιη circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 2: 968-975 (1991).
[00157] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-Labyrinthin antibody-encoding nucleic acid. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the anti- Labyrinthin antibody.
[00158] Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT -rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT (SEQ ID NO: 17) region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO: 18) sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
[00159] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
[00160] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.
[00161] Anti-Labyrinthin antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and heat-shock promoters, provided such promoters are compatible with the host cell systems.
[00162] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hin III E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature, 297:598-601 (1982) on expression of human β -interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the rous sarcoma virus long-terminal repeat can be used as the promoter.
[00163] Transcription of a DNA encoding the anti-Labyrinthin antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early-promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the anti-Labyrinthin antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
[00164] Expression vectors used in eukaryotic host cells (for example, yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' end, occasionally 3' end, of untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-Labyrinthin antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 1994/11026 and the expression vector disclosed therein.
[00165] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli XI 776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting. [00166] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-Labyrinthin antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.
drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
[00167] Suitable host cells for the expression of glycosylated anti-Labyrinthin antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
[00168] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980), including DG44 (Urlaub et al., Som. Cell and Mol. Gen., 12: 555-566 (1986)) and DP12 cell lines); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[00169] Host cells are transformed with the above-described expression or cloning vectors for anti- Labyrinthin antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
[00170] The host cells used to produce the anti-Labyrinthin antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described, for example, in Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102:255 (1980); U.S. Pat. No.
4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 1990/03430; WO 1987/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCrN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
[00171] When using recombinant techniques, the antibody can be produced intracellularly or in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology, 10: 163-167 (1992) describes a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an AMICON™ or MILLIPORE PELLICON™ ultrafiltration unit. A protease inhibitor such as phenylmethylsulphonyl fluoride (PMSF) may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.
[00172] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human vl, γ2, or γ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ 3 (Guss et al., EMBO J., 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled-pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the
BAKERBOND ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse- phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion- or cation- exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium-sulfate precipitation are also available depending on the antibody to be recovered.
[00173] Pharmaceutical Formulations
[00174] Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low-molecular- weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Preferred lyophilized anti-Labyrinthin antibody formulations are described in WO 1997/04801, expressly incorporated herein by reference.
[00175] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide antibodies that bind to HER-2, EGFR, Labyrinthin (e.g. an antibody that binds a different epitope on Labyrinthin), ErbB3, ErbB4, or vascular endothelial growth factor (VEGF) antigens in the one formulation. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth- inhibitory agent, anti-hormonal agent, Labyrinthin-targeted drug, anti-angiogenic agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
[00176] In some embodiments, administration of a labyrinthin antibody of the invention is combined with the administration of an additional therapeutic agent as part of a therapeutic regimen. The additional therapeutic agent can be administered before, during, or after the administration of a labyrinthin binding agent, such as an antibody. Agents administered during the administration of the labyrinthin antibody can be co-administered as a single composition and delivery or delivered as part of the same procedure, administered at about the same time in separate administration events. Agents administered before or after administration of the labyrinthin antibody can be administered in time frames preceding or following labyrinthin binding agent administration that include the following, without limitation: less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days; less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 weeks; less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 months; and less than, about, or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 years. In some embodiments, the additional therapeutic agent is conjugated to the labyrinthin antibody. By way of example, it is well known that radioisotopes, drugs, and toxins can be conjugated to antibodies or antibody fragments to facilitate targeting of the radioisotopes, drugs or toxins to tumor sites to enhance their therapeutic efficacy and minimize side effects. Examples of these agents and methods are reviewed in Wawrzynczak and Thorpe (in Introduction to the Cellular and Molecular Biology of Cancer, L. M. Franks and N. M. Teich, eds, Chapter 18, pp. 378-410, Oxford University Press, Oxford, 1986), in Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (C.-W. Vogel, ed., 3-300, Oxford University Press, New York, 1987), in Dillman, R.O. (CRC Critical Reviews in Oncology/Hematology 1 :357, CRC Press, Inc., 1984), in Pastan et al.(Cell 47:641, 1986), in Vitetta et al. (Science 238:1098-1104, 1987) and in Brady et al. (Int. J. Rad. Oncol. Biol. Phys. 13:1535-1544, 1987). Other examples of the use of immunoconjugates for cancer and other forms of therapy have been disclosed, inter alia, in Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459, 4,460,561 and 4,624,846, and in Rowland, U.S. Pat. No. 4,046,722, Rodwell et al., U.S. Pat. No. 4,671,958, and Shih et al., U.S. Pat. No. 4,699,784, the disclosures of all of which are incorporated herein in their entireties by reference.
[00177] In some embodiments, an antibody of the present invention can be combined with anti-tumor or anti-cancer therapeutics capable of decreasing or preventing a further increase in tumor growth. Non- limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin; alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, Casodex™, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine;
elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;
mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK.R™ ; razoxane; sizofiran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included as suitable chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti- estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C;
mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO). Where desired, the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide,
Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, cell-cycle nonspecific antineoplastic agents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesine, Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedap latin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126, and Zosuquidar; Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin;
Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine;
Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefmgol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate;
Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin;
Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa- nl ; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma- lb; Iprop latin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;
Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate;
Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;
Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid;
Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;
Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur;
Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine;
Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate;
Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;
Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfm; Vinblastine Sulfate;
Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; Taxol; thiosemicarbazone derivatives; telomerase inhibitors; arsenic trioxide; planomycin; sulindac sulfide; cyclopamine; purmorphamine; gamma-secretase inhibitors; CXCR4 inhibitors; HH signaling inhibitors; Bmi-1 inhibitors; Bcl-2 inhibitors; Notch- 1 inhibitors; DNA checkpoint protein inhibitors; ABC transporter inhibitors; mitotic inhibitors; intercalating antibiotics; growth factor inhibitors; cell cycle modulators; enzymes; topoisomerase inhibitors; biological response modifiers; angiogenesis inhibitors; DNA repair inhibitors; and small G-protein inhibitors. Combinations can be made with one or more than one of the above. In some embodiments, the therapeutic agent is an anti-cancer and/or an anti-cancer stem cell therapeutic agent.
[00178] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug-delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[00179] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained- release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
[00180] The formulations to be used for in vivo administration must be sterile. This is readily
accomplished by filtration through sterile filtration membranes.
[00181] Treatment with the Anti-Labyrinthin Antibodies
[00182] It is contemplated that, according to the present invention, the anti-Labyrinthin antibodies may be used to treat various diseases or disorders. Exemplary conditions or disorders include benign or malignant tumors; leukemias and lymphoid malignancies; and other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders. In some embodiments, the anti-Labyrinthin antibodies can be used to treat Labyrinthin positive cells, such as Labyrinthin-positive tumors. Examples of Labyrinthin-positive tumors include adenocarcinomas.
[00183] In one aspect, the present invention provides a method of ameliorating an adenocarcinoma in a subject. Amelioration of an adenocarcinoma can be treatment, suppression, prevention, or palliation of a symptom of an adenocarcinoma. In another embodiment, amelioration can be extracorporeal (also known as phoresis) capture and/or killing of a tumor cell, such as an adenocarcinoma cell. In another embodiment, the invention can be use of an antibody of the invention for determining diagnosis and treatment of a subject (theranostics). In one embodiment, the method comprises administering to the subject in need thereof an effective amount of a labyrinthin antibody of the present invention. In some embodiments the anti- labyrinthin antibody binds to a cancer stem cell. [00184] Adenocarcinomas subject to the methods of the invention can include any that arise from tissue with gland- like properties. The adenocarcinoma can be any adenocarcinoma of interest, including but not limited to lung adenocarcinoma, esophagus adenocarcinoma, gastric adenocarcinoma, renal cell adenocarcinoma, prostate adenocarcinoma, colon adenocarcinoma, pancreas adenocarcinoma, cervix adenocarcinoma, gastric adenocarcinoma, salivary adenocarcinoma or breast adenocarcinoma.
Adenocarcinoma can be identified using methods known in the art, including but not limited to morphological analysis, histological analysis, surface marker profiling, protein profiling, and gene expression profiling. In some embodiments, adenocarcinomas subject to the methods of the invention are specifically Labyrinthin-positive adenocarcinomas.
[00185] In one embodiment, amelioration of adenocarcinoma is achieved through the use of an effective amount of a labyrinthin antibody of the present invention. Labyrinthin is an integral membrane protein of approximately 40 kilodaltons, the sequence of which can be found in GenBank having accession number AR532049. Labyrinthin has also been shown to respond to changes in Ca2+ concentrations. Labyrinthin has further been identified as a reliable cellular marker for adenocarcinomas.
[00186] In some embodiments, the invention can be a method comprising screening for the presence of labyrinthin and amelioration of adenocarcinoma is achieved through the use of an effective amount of a labyrinthin antibody of the present invention. In some embodiments, screening can be done using a labyrinthin antibody of the present invention.
[00187] In certain embodiments, an immunoconjugate comprising the anti-Labyrinthin antibody conjugated with a cytotoxic agent is administered to the patient. Preferably, the immunoconjugate and/or Labyrinthin protein to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases, and DNA endonucleases.
[00188] The anti-Labyrinthin antibodies or immunoconjugates are administered to a human patient in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous, intraperitoneal, or subcutaneous administration of the antibody is preferred, with subcutaneous or intraperitoneal routes being particular preferred. A preferred administration schedule is about 2-3 times per week, depending on the particular mammal being treated, the type of antibody, and other factors well known to the practitioner. However, other administration schedules are operable herein.
[00189] Other therapeutic regimens may be combined with the administration of the anti-Labyrinthin antibody. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. [00190] It may also be desirable to combine administration of the anti-Labyrinthin antibody or antibodies with administration of an antibody directed against another tumor-associated antigen. The other antibody in this case may, for example, bind to an antigen such as HER-2, EGFR, ErbB3, ErbB4, vascular endothelial growth factor (VEGF), or a B-cell surface marker or antigen (an antigen expressed on the surface of a B cell that can be targeted with an antagonist that binds thereto), such as, for example, the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers (for descriptions, see The Leukocyte Antigen Facts Book, 2nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B-cell surface markers include RP105, FcRH2, B-cell CR2, CCR6, P2 x 5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. The B-cell surface marker of particular interest is preferentially expressed on B cells compared to other non-B-cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells. The preferred B- cell surface markers herein are CD20 and CD22. In another aspect, the Labyrinthin antibody may be combined with an anti-angiogenic agent, which acts to inhibit angiogenesis. An example is an antagonist to VEGF, such as an antibody, e.g., A VASTEST™.
[00191] In one embodiment, the treatment of the present invention involves the combined administration of an anti-Labyrinthin antibody (or antibodies) and one or more regulators of immune function in a mammal, such as cytokines, as well as chemotherapeutic agents or growth-inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Preferred chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service, Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).
[00192] The antibody may be combined with an anti-hormonal compound, e.g., an anti-estrogen compound such as tamoxifen or an aromatase inhibitor such as anastrozole; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is hormone-independent cancer, the patient may previously have been subjected to anti-hormonal therapy and, after the cancer becomes hormone independent, the anti-Labyrinthin antibody (and optionally other agents as described herein) may be administered to the patient.
[00193] Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-Labyrinthin antibody.
[00194] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.
[00195] The preferred dosage of the antibody will be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, e.g. about six doses, of the anti-Labyrinthin antibody). An initial higher loading dose, followed by one or more lower doses, may be administered. An exemplary dosing regimen comprises administering an initial loading dose of about 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 3-5 mg/kg or 4 mg/kg, followed by a weekly maintenance dose of about 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 2-5 mg/kg or 2 mg/kg of the anti- Labyrinthin antibody. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
[00196] Alternatively, the antibody is suitably administered serially or in combination with radiological treatments— irradiation or introduction of radioactive substances— such as those referred to in UICC (Ed.), Klinische Onkologie, Springer- Verlag (1982).
[00197] Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody". See, for example, WO 1996/07321 published Mar. 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.
[00198] There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells, in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium-phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retrovirus. [00199] The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOPE) and 3 -(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Choi"), for example). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins that bind to a cell- surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262:4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA, 87:3410-3414 (1990). For review of the currently known gene marking and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992). See also WO 1993/25673 and the references cited therein.
[00200] In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the humanized anti-Labyrinthin antibody herein. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer. In one embodiment, the label or package insert indicates that the composition comprising the antibody can be used to treat a Labyrinthin disorder, for example, to treat cancer that expresses a Labyrinthin receptor. In addition, the label or package insert may indicate that the patient to be treated is one having cancer characterized by excessive activation of a Labyrinthin receptor. The label or package insert may also indicate that the composition can be used to treat cancer, wherein the cancer is not characterized by overexpression of a Labyrinthin receptor. In other embodiments, the package insert may indicate that the antibody or composition can be used to treat breast cancer (e.g. metastatic breast cancer); hormone-independent cancer; prostate cancer (e.g. androgen- independent prostate cancer); lung cancer (e.g. non-small cell lung cancer); colon, rectal or colorectal cancer; or any of the other diseases or disorders disclosed herein.
[00201] Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the humanized antibody herein, and (b) a second container with a composition contained therein, wherein the composition comprises a therapeutic agent other than the humanized antibody. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the first and second compositions can be used in combination to treat a Labyrinthin disorder such as cancer. Such therapeutic agent may be any of the adjunct therapies described in the preceding section (e.g., a chemotherapeutic agent, an anti-angiogenic agent, an anti- hormonal compound, a cardioprotectant, and/or a regulator of immune function in a mammal, including a cytokine). Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
[00202] Non-therapeutic Uses for the Anti-Labyrinthin Antibody
[00203] The antibodies (e.g. the humanized anti-Labyrinthin antibodies) of the invention have further non-therapeutic applications.
[00204] For example, the antibodies may be used as affinity-purification agents. In this process, the antibodies are immobilized on a solid phase such as a SEPHADEX™ resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the Labyrinthin protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the Labyrinthin protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the Labyrinthin protein from the antibody.
[00205] Anti-Labyrinthin antibodies may also be useful in diagnostic assays for Labyrinthin protein, e.g., detecting its expression in specific cells, tissues, or serum.
[00206] For diagnostic applications, the antibody typically will be labeled with a detectable moiety. Numerous labels are available that can be generally grouped into the following categories:
[00207] a) Radioisotopes, such as 35S, 14C, 1251, 3H, and 13 T The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), for example, and radioactivity can be measured using scintillation counting.
[00208] b) Fluorescent labels such as rare-earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
[00209] c) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microper oxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., "Methods for the Preparation of Enzyme- Antibody Conjugates for use in Enzyme Immunoassay," in Methods in Enzym. (Ed., J. Langone & H. Van Vunakis), Academic Press, New York, 73:147-166 (1981).
[00210] Examples of enzyme-substrate combinations include, for example:
[00211] i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'- tetramethyl benzidine hydrochloride (TMB));
[00212] ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate; and
[00213] iii) β -D-galactosidase (β -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- β -D- galactosidase) or fluorogenic substrate 4-methylumbelliferyl- β -D-galactosidase.
[00214] Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.
[00215] Sometimes, the label is indirectly conjugated with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin, and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin, and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label with the antibody can be achieved.
[00216] In another embodiment of the invention, the anti-Labyrinthin antibody need not be labeled, and the presence thereof can be detected using a labeled antibody that binds to the Labyrinthin antibody.
[00217] The antibodies of the present invention may be employed in any known assay method, such as competitive-binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
[00218] For immunohistochemistry, the tumor sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example.
[00219] The antibodies may also be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionuclide (such as U 1ln, 99Tc, 14C, 131I, 1251, 3H, 32P or 35S) so that, for example, a tumor can be localized using immunoscintigraphy. [00220] As a matter of convenience, the antibodies of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or fiuorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration.
[00221] The antibody herein is also useful for in vivo imaging, where the labeled antibody is administered to a host, preferably the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is suitably used in the staging and treatment of neoplasms. The antibody is suitably labeled with any moiety that is detectable in a host, including non-radioactive indicators detectable by, e.g., nuclear magnetic resonance, or other means known in the art. Preferably, however, the label is a radiolabel, including iodine, e.g., I and I, selenium, bifunctional chelates, copper, e.g., Cu, technetium, e.g., 99 mTc, and rhenium, e.g., 186Re and 188Re. The radioisotope is conjugated to the protein by any means, including metal-chelating compounds or lactoperoxidase, or iodogen techniques for iodination.
[00222] Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit can further contain one or more additional reagents, such as an
immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope in the Lab antigen distinct from the first human antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
EXAMPLES
Example 1 : Comparison of binding affinities of murine and humanized antibodies to Lab
[00223] A humanized antibody to Labyrinthin (designated X509) was produced. The amino acid sequences for the VL light chain and VH heavy chain, and the nucleic acid sequences encoding them, are presented in Figure 2. Affinity of X509 for Labyrinthin was measured using standard techniques, and these measurements were compared to measurements of Labyrinthin affinity of murine anti-Labyrinthin antibody X373. As a control, affinity of mouse IgG for Labyrinthin was also measured. Results of these measurements are provided in Figure 4. The Kd values indicate that humanized antibody X509 has an improved affinity for Labyrinthin, compared to the murine antibody X373. [00224] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises:
(1) a variable heavy (VH) domain that comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof; and
(2) a variable light (VL) domain that comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VH domain, and the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VL domain.
2. The humanized antibody, or antigen-binding portion thereof of claim 1, wherein the antibody comprises: (1) a variable heavy (VH) domain that comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 14; b) a heavy chain variable region CDR2 comprising SEQ ID NO:15; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16; and (2) a variable light (VL) domain that comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1 ; b) a light chain variable region CDR2 comprising SEQ ID NO:12; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VH domain, and the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VL domain.
3. A humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: a) a heavy chain variable region CDRl comprising SEQ ID NO: 14, or conservative modifications thereof; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 15, or conservative modifications thereof; and c) a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, wherein the heavy chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VH domain.
4. A humanized antibody, or antigen-binding portion thereof, that binds a labyrinthin protein, wherein the antibody comprises: a) a light chain variable region CDRl comprising SEQ ID NO:l 1, or conservative modifications thereof; b) a light chain variable region CDR2 comprising SEQ ID NO:12, or conservative modifications thereof; and c) a light chain variable region CDR3 comprising SEQ ID NO: 13, or conservative modifications thereof, wherein the light chain variable CDRl, CDR2, and CDR3 regions are incorporated into a human VL domain.
5. A humanized antibody, or antigen-binding portion thereof, wherein the antibody comprises: a variable heavy (VH) domain that comprises a heavy chain variable region CDR3 comprising SEQ ID NO: 16, or conservative modifications thereof, incorporated into a human VH domain.
6. The humanized antibody of claims 1, 2, 3, 4 or 5, wherein the antibody is an IgGl, IgG2, IgG3 or IgG4 isotype.
7. The humanized antibody of claims 1, 2, 3, 4 or 5, wherein the antibody comprises a heavy chain constant region (CH) encoded by SEQ ID NO: 10.
8. The humanized antibody of claims 1, 2, 3, 4 or 5, wherein the antibody comprises a light chain constant region (CL) encoded by SEQ ID NO: 9.
9. The humanized antibody of claim 8, wherein the antibody further comprises a heavy chain constant region (CH) encoded by SEQ ID NO: 10.
10. The humanized antibody or antigen-binding portion thereof of claims 1, 2, 3, 4 or 5 that is a single chain antibody.
11. The humanized antibody or antigen-binding portion thereof of claims 1, 2, 3, 4 or 5 that is a Fab fragment.
12. The humanized antibody or antigen-binding portion thereof of claims 1, 2, 3, 4 or 5 that is conjugated with a cytotoxic agent or a radioactive isotype.
13. The humanized antibody or antigen-binding portion thereof of claims 1, 2, 3, 4 or 5, wherein the antibody binds to labyrinthin with an affinity of at least 10"7 M or better.
14. The humanized antibody or antigen-binding portion thereof of claim 13, wherein the antibody binds to labyrinthin with an affinity of at least 5x10"8 M or better.
15. The humanized antibody or antigen-binding portion thereof of claims 1, 2, 3, 4 or 5, wherein the antibody comprises a variable heavy (VH) domain having SEQ ID NO: 8.
16. The humanized antibody or antigen-binding portion thereof of claims 1, 2, 3, 4 or 5, wherein the antibody comprises a variable light (VL) domain having SEQ ID NO: 6.
17. The humanized antibody of claim 16, wherein the antibody further comprises a variable heavy (VH) domain having SEQ ID NO: 8.
18. An isolated monoclonal antibody, or an antigen-binding portion thereof, wherein the antibody cross-competes for binding to labyrinthin with the humanized antibody of claims 1, 2, 3, 4 or 5.
19. A pharmaceutical composition comprising a humanized antibody, or antigen-binding portion thereof of any one of claims 1, 2, 3, 4 or 5, and a pharmaceutically acceptable carrier.
20. The composition of claim 19, further comprising an adjuvant.
21. The composition of claim 19, wherein the antibody is an IgGl, IgG2, IgG3 or IgG4 isotype.
22. The composition of claim 19, wherein the antibody comprises a heavy chain constant region (CH) comprising SEQ ID NO: 10.
23. The composition of claim 19, wherein the antibody comprises a light chain constant region (CL) comprising SEQ ID NO: 9.
24. The composition of claim 23, wherein the antibody further comprises a heavy chain constant region (CH) comprising SEQ ID NO: 10.
25. The composition of claim 19, wherein the antibody is conjugated with a cytotoxic agent or a radioactive isotype.
26. The composition of claim 19, wherein the antibody binds to labyrinthin with an affinity of at least 10"7 M or better.
27. The composition of claim 26, wherein the antibody binds to labyrinthin with an affinity of at least 5x10"8 M or better.
28. The composition of claim 19, wherein the antibody comprises a variable heavy (VH) domain having SEQ ID NO: 8.
29. The composition of claim 19, wherein the antibody comprises a variable light (VL) domain having SEQ ID NO: 6.
30. The composition of claim 29, wherein the antibody further comprises a variable heavy (VH) domain having SEQ ID NO: 8.
31. An isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof, of claim 1, 2, 3, 4 or 5.
32. The nucleic acid molecule of claim 31, wherein the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 5.
33. The nucleic acid molecule of claim 31, wherein the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 7.
34. An expression vector comprising the nucleic acid molecule of claim 32 or 33.
35. A host cell comprising the expression vector of claim 34.
36. A method for preparing an anti-labyrinthin antibody which comprises expressing the antibody in the host cell of claim 35 and isolating the antibody from the host cell.
37. A method of ameliorating an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of claim 1, 2, 3, 4, or 5 in an amount effective to ameliorate the adenocarcinoma.
38. A method of ameliorating relapse of an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of claim 1, 2, 3, 4, or 5 in an amount effective to ameliorate relapse of the
adenocarcinoma.
39. The method of claims 37 or 38, wherein said administration reduces metastasis of said adenocarcinoma.
40. The method of claims 37 or 38, wherein the administration of the labyrinthin binding agent results in a decrease in size of the adenocarcinoma.
41. A method of ameliorating an adenocarcinoma in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding portion thereof of claim 1, 2, 3, 4, or 5, wherein the antibody binds to a cancer stem cell.
42. The method of claim 41, wherein said administration reduces metastasis of said adenocarcinoma.
43. The method of claim 41, wherein the administration of the labyrinthin binding agent results in a decrease in size of the adenocarcinoma.
44. The method of claim 41, wherein the cancer stem cell is a self-renewing cell.
45. The method of claim 41, wherein the cancer stem cell is an adenocarcinoma cancer stem cell.
46. The method of claim 41, wherein the cancer stem cell expresses CD 133, CD44, CD 166, CD29, CD24, Lgr5, ALDH1, ESA, and/or b-catenin.
47. The method of claim 37, 38, or 41, further comprising administering a therapeutic agent that is not the antibody or antigen-binding portion thereof of claim 1, 2, 3, 4, or 5.
48. The method of claim 47, wherein the therapeutic agent is a cytotoxic agent.
49. The method of claim 47, wherein the therapeutic agent is an anti-cancer stem cell therapeutic agent.
50. The method of claim 49, wherein the therapeutic agent is an anti-cancer therapeutic agent.
51. A kit comprising a container containing the humanized antibody of claim 1, 2, 3, 4 or 5, and instructions directing a user to treat an adenocarcinoma in a subject with the antibody in an effective amount.
52. The kit of claim 42 additionally comprising a container containing a therapeutic agent other than the antibody, wherein the instructions direct the user to treat the adenocarcinoma with the antibody in combination with the agent in effective amounts.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020146413A1 (en) * 2019-01-08 2020-07-16 Labyrx Immunologic Therapeutics (Usa) Limited Constructs targeting labyrinthin or a portion thereof and uses thereof
CN113412275A (en) * 2018-12-13 2021-09-17 莱比锡免疫治疗(美国)有限公司 Labyrinth protein-based peptides for cancer immunotherapy and uses thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113412275A (en) * 2018-12-13 2021-09-17 莱比锡免疫治疗(美国)有限公司 Labyrinth protein-based peptides for cancer immunotherapy and uses thereof
WO2020146413A1 (en) * 2019-01-08 2020-07-16 Labyrx Immunologic Therapeutics (Usa) Limited Constructs targeting labyrinthin or a portion thereof and uses thereof
CN113993892A (en) * 2019-01-08 2022-01-28 莱比锡免疫治疗(美国)有限公司 Constructs targeting labyrinth proteins or parts thereof and uses of said constructs

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