EP1233790A2 - Procedes permettant de reduire les effets des cancers exprimant l'antigene a33 a l'aide d'immunoglobulines specifiques de l'antigene a33 - Google Patents

Procedes permettant de reduire les effets des cancers exprimant l'antigene a33 a l'aide d'immunoglobulines specifiques de l'antigene a33

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Publication number
EP1233790A2
EP1233790A2 EP00972349A EP00972349A EP1233790A2 EP 1233790 A2 EP1233790 A2 EP 1233790A2 EP 00972349 A EP00972349 A EP 00972349A EP 00972349 A EP00972349 A EP 00972349A EP 1233790 A2 EP1233790 A2 EP 1233790A2
Authority
EP
European Patent Office
Prior art keywords
seq
immunoglobulin
group
sequence selected
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00972349A
Other languages
German (de)
English (en)
Inventor
Carlos F. Barbas, Iii
Christoph Rader
Gerd Ritter
Sydney Welt
Lloyd J. Old
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
Scripps Research Institute
Memorial Sloan Kettering Cancer Center
Original Assignee
Ludwig Institute for Cancer Research Ltd
Sloan Kettering Institute for Cancer Research
Ludwig Institute for Cancer Research New York
Scripps Research Institute
Memorial Sloan Kettering Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/425,638 external-priority patent/US6342587B1/en
Priority claimed from US09/543,004 external-priority patent/US6346249B1/en
Application filed by Ludwig Institute for Cancer Research Ltd, Sloan Kettering Institute for Cancer Research, Ludwig Institute for Cancer Research New York, Scripps Research Institute, Memorial Sloan Kettering Cancer Center filed Critical Ludwig Institute for Cancer Research Ltd
Publication of EP1233790A2 publication Critical patent/EP1233790A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—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
    • C07K16/3046—Stomach, Intestines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • This invention relates to immunoglobulin products that bind with specificity to A33 antigen.
  • this invention is directed to A33 antigen specific CDRs.
  • the antibodies and antibody like proteins may be humanized.
  • antibodies as therapeutic agents is gaining acceptance as an important and valuable approach in the treatment of various conditions, such as types of cancer.
  • the specificity of antibodies makes them particularly useful in treating conditions where a "marker” or “markers” characterize abnormal cells.
  • Antibodies effectively target such cells by binding to these markers, which are molecules present in, or preferably on, the cell type of interest.
  • mice were injected with the molecule of interest. As this molecule was foreign to the mouse, an antibody response would result. The antibodies were then purified from murine blood or serum, for eventual diagnostic or therapeutic use.
  • Murine antibodies recognized as foreign by a human host, elicit the so-called “human anti- mouse antibody” or "HAMA” response. See, e.g., Schiff, et al., Cane. Res. 45:879-885 (1985).
  • HAMA human anti- mouse antibody
  • the Fc portion of murine antibodies is not as efficacious in stimulating human complement or cell mediated toxicity.
  • Chimeric antibodies contain portions of antibodies from two or more different species, such as the variable regions of a mouse antibody, and the constant regions of a human antibody.
  • the advantage of such chimeras is that they retain the specificity of murine antibodies, but also stimulate human Fc complement fixation. Such chimeras can still elicit a HAMA response, however. See, e.g., Bruggemann, et al., J. Exp. Med 170:2153-2157 (1989). Additional approaches have been sought which would alleviate these problems.
  • British Patent Applications 120694 and 125023 disclosing the general approach.
  • Chimeric antibodies contain portions of antibodies from two or more different species, such as the variable regions of a mouse antibody, and the constant regions of a human antibody.
  • the advantage of such chimeras is that they retain the specificity of murine antibodies, but also stimulate human Fc complement fixation. Such chimeras can still elicit a HAMA response, however. See, e.g., Bruggemann, et
  • Patent Application GB 2188638A and US Patent No. 5,585,089 are exemplary of technology in this area. These references disclose processes wherein recombinant antibodies are produced where the only portion of the antibody that is substituted is the complementarity determining region, or "CDR.”
  • CDR complementarity determining region
  • the CDR grafting technique has been used to generate antibodies which consist of murine CDRs, and human variable region framework and constant regions. See, e.g.,
  • humanized antibodies must include a small number of critical murine antibody residues in the human variable region. The particular residues of importance depend upon the structure of both the murine antibody and human antibody. See, e.g., WO 04381 to Harris et al.
  • U.S. Patent No. 5,958,412 describes humanized antibodies to a molecule referred to as "A33.” This molecule is known to be associated with colon cancer. See, e.g., U.S. Patent Nos. 5,643,550 and 5,160,723, incorporated by reference. Also see U.S. Patent No. 5,712,369, to Old, et al., also incorporated by reference, teaching the isolation and characterization of the A33 molecule.
  • Phage display is a methodology which has been used to express and to select recombinant antibodies. See, e.g., Vaughan, et al, Nat. Biotechnol. 16(6):535-539 (1998), incorporated by reference. This methodology is employed in the disclosure which follows.
  • the rabbit Ig gene repertoire has been well characterized. See, e.g., Knight, et al., Adv.
  • Figure 1 depicts the amino acid sequence of the V regions of rabbit anti A33 antigen antibodies.
  • three rabbit antibodies, rabbit 1, rabbit 2 and rabbit 3 are shown.
  • the V sequence of the humanized antibodies is shown.
  • the amino acid sequence of six human antibodies, labelled human A to F are listed.
  • the framework regions corresponding to about amino acids 1-22 (FR1), 35-49 (FR2), 57-88 (FR3) and 98-107 (FR4) of the VL chain and amino acids 1-30(FR1), 36-49(FR2), 66-94(FR3), 103-113 (FR4) of the VH chain.
  • the CDR regions correspond to about amino acids 24-34 (CDR1), 50-56 (CDR2), 89- 97 (CDR3) of the VL chain and about amino acids 31-35 (CDR1), 50-65 (CDR2), and 95-109 (CDR3) of the VH chain.
  • Figure 2 depicts Western blot reactivity of human Fab B with Triton X-100 extracts of human A33 antigen expressing (LIM 1215, SW1222) and nonexpressing (SW620) human colon cancer cell lines. Specific binding was detected by alkaline-phosphatase-conjugated goat anti-human F(ab') 2 polyclonal antibodies and visualized using chemiluminescence. Numbers on the left indicate molecular masses of standard proteins in kilodaltons ("kDs").
  • Figure 3 depicts flow cytometry histograms demonstrating that the selected rabbit clones
  • Human A refers to human VLA and VHA
  • Human B refers to human VLB and VHB
  • Human C refers to human VLC and VHC
  • Human D refers to human VLD and VHD
  • Human E refers to human VLE and VHE
  • Human F refers to human VLF and VHF.
  • Figure 5 depicts analysis of purified rabbit and humanized Fab by SDS-PAGE and
  • FIG. 6 depicts representative Biacore sensorgrams obtained for the binding of rabbit Fab 1 to immobilized human A33 antigen.
  • FIG. 7 Immunohistochemical reactivity of humanized Fab B in human colon cancer tissue sections.
  • (A) Humanized Fab B showed intense staining in SW1222 xenograph.
  • Insert A DNA sequence foreign to the host, consisting of a structural gene and optionally additional DNA sequences.
  • Structural gene A nucleic acid molecule coding for a polypeptide and being in operable linkage with a suitable promoter, termination sequence and optionally other regulatory DNA sequences.
  • Promoter A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that sequence.
  • Inducible promoter A promoter where the rate of RNA polymerase binding and initiation is modulated by external stimuli. Such stimuli include light, heat, anaerobic stress, alteration in nutrient conditions, presence or absence of a metabolite, presence of a ligand, microbial attack, wounding and the like.
  • Multimeric protein A globular protein containing more than one separate polypeptide or protein chain associated with each other to form a single protein. Both heterodimeric and homodimeric proteins are multimeric proteins.
  • Polypeptide and peptide A linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • Protein A linear series of greater than about 50 amino acid residues connected one to the other as in a polypeptide.
  • Fab fragment A protein consisting of the portion of an immunoglobulin molecule containing the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen.
  • Fab fragments are typically prepared by proteolytic digestion of substantially intact immunoglobulin molecules with papain using methods that are well known in the art; however, a Fab fragment may also be prepared by expressing the desired portions of immunoglobulin heavy chain and immunoglobulin light chain in a host cell, using methods well known in the art.
  • F v fragment A protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically combining with antigen.
  • F v fragments are typically prepared by expressing the desired portions of immunoglobulin heavy chain variable region and immunoglobulin light chain variable region in a host cell using methods well known in the art.
  • V L CDR1, V L CDR2, and V, CDR3 denote immunoglobulin light chain complementarity determining region 1 , 2 and 3 respectively.
  • V H CDR 1 , V H CDR2, and V H CDR3 denote immunoglobulin heavy chain complementarity determining region 1, 2 and 3 respectively.
  • V L FR1 , V, FR2, and V L FR3 V, FR4 denote immunoglobulin light chain framework region 1, 2, 3 and 4 respectively.
  • V H FR1, V H FR2, and V H FR3 V H FR4 denote immunoglobulin heavy chain framework region 1, 2, 3 and 4 respectively.
  • Immunoglobulin superfamily molecule A molecule that has a domain size and amino acid residue sequence that is significantly similar to immunoglobulin or immunoglobulin related domains. The significance of similarity is determined statistically using a computer program such as the Align program described by Dayhoff et al., Meth Enzymol., 91 :524-545 (1983) incorporated by reference. A typical Align score of less than 3 indicates that the molecule being tested is a member of the immunoglobulin gene superfamily.
  • immunoglobulin superfamily molecules include the following members: immunoglobulin heavy chains (i.e., the heavy chain of IgM, IgD, IgG, IgA or IgE and light chains K and ⁇ ), T cell receptors ( ⁇ , ⁇ , ⁇ , X, CD3), major histocompatibility antigens (Class I H-chain, ⁇ 2 -microglobulin, Class II ( ⁇ and ⁇ )), ⁇ 2 -microglobulin associated antigens (TL H chain, Qa-2 H chain, CD 1 a H chain), T lymphocyte antigens (CD2, CD4, CD7, CD8 chain I, CD8 Chain lid, CD28 and CTLA4), haemopoietic/endothelium antigens (LFA-3, MRC OX-45), brain/lymphoid antigens (Thy-1, MRC OX-2), immunoglobulin receptors (Poly Ig R, Fc gamma 2b/gamma 1R, Fc ⁇
  • PDGF PDGF receptor
  • CSF1 colony stimulating factor-1
  • non-cell surface molecules o ⁇ B-glycoprotein, basement membrane link protein
  • A33 antigen Heaths et al., Proc Natl Acad Sci 94:469-474 (1997)) (See e.g., Williams and Barclay, in Immunglobulin Genes, p 361, Academic Press, NY (1989); and Sequences of Proteins of Immunological Interest, 4th ed., U.S. Dept. of Health and Human Serving (1987)).
  • Epitope A portion of a molecule that is specifically recognized by an immunoglobulin product. It is also referred to as the determinant or antigenic determinant.
  • Bi specific antibody A multivalent antibody containing binding sites specific for two different antigenic determinants.
  • a bispecific antibody may be chemically synthesized as antibody heteroconjugates (AHCs) by covalently attaching two whole monoclonal antibodies ("whole AHCs") (B. Karpovsky, et al. (1984) J. Exp. Med. 160(6):1686-1701) or by attaching two monoclonal antibody Fab or Fab' fragments (“monovalent AHCs”) (M. Brennan, et al., Science (1985) 229:(1708):81-83), where each antibody or antibody fragment has a different antigenic specificity.
  • AHCs antibody heteroconjugates
  • bispecific antibodies may be produced from a "hybrid hybridoma," a cell fusion of two monoclonal antibody-producing cells (C. L. Reading, in HYBRIDOMAS AND CELLULAR IMMORTALITY, B. H. Tom etal., eds., 1984, (New York: Plenum Press), p. 235; U. D. Staerz et al., Proc. Natl. Acad. Sci. (1986) 83: 1453-1457; A. Lanzavecchia et al., Eur. J. Immunol. (1987)17:105-111; D. B.
  • One embodiment of the invention is directed to a method for producing a humanized antibody as a portion of a humanized antibody which binds to a specific antigen.
  • the method comprises the steps of immunizing a rabbit with an amount of a specific antigen which is specific to provoke an immune response, isolating RNA from antibody producing cells of the rabbit, converting the RNA to cDNA, and combining a portion of the cDNA which encodes for a portion of a rabbit antibody which binds to the antigen with a cDNA molecule which encodes a non binding portion of a human antibody, to form a hybrid molecule consisting of rabbit cDNA and human cDNA. Thereafter, the hybrid molecule is inserted into a host cell, and the host cell is cultured to express a protein product of the hybrid molecule. Finally, the hybrid protein is isolated.
  • the humanized antibody or a portion of the humanized antibody may consist of rabbit
  • the humanized antibody may be a Fab fragment.
  • the antigen may be a molecule or portion of a molecule presented on a cell surface.
  • the antigen may be a molecule or portion of a molecule presented on a cell surface of a neoplastic (e.g., cancer) cell.
  • the cancer cell may be a colon cancer cell.
  • the antigen may be an A33 antigen.
  • the host cell used may be a prokaryotic cell such as an E.coli cell.
  • the humanized antibody may comprise the amino acid sequence of SEQ ID NOS: 20, 21, 22, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 67.
  • An immunoglobulin product is a polypeptide, protein or multimeric protein containing at least the immunologically active portion of an immunoglobulin heavy chain or an immunologically active portion of an immunoglobulin light chain and is thus capable of specifically combining with an antigen.
  • Exemplary immunoglobulin products are an immunoglobulin heavy chain, immunoglobulin light chain, immunoglobulin molecules, bispecific antibodies, substantially intact immunoglobulin molecules, any portion of an immunoglobulin that contains the paratope, including those portions known in the art as Fab fragments, Fab' fragment, F(ab') 2 fragment and Fv fragment.
  • the structures of immunoglobulin products are well known to those skilled in the art and described in Basic and Clinical Immunology, by Stites, et al., 4th ed., Lange Medical Publications, Los Altos, Calif.
  • Another embodiment of the invention is directed to an immunoglobulin product such as an immunoglobulin molecule that binds with specificity to an A33 antigen.
  • An immunoglobulin molecule is a multimeric protein containing the immunologically active portions of an immunoglobulin heavy chain and immunoglobulin light chain covalently coupled together and capable of specifically combining with antigen. It should be noted that the immunoglobulin molecule may be a bispecific antibody with affinity for A33 and a second non-A33 epitope.
  • Another embodiment of the invention is directed to a single-chain antigen-binding protein that binds with specificity to an A33 antigen.
  • a single chain antigen binding protein is a polypeptide composed of an immunoglobulin light-chain variable region amino acid sequence (V L ) tethered to an immunoglobulin heavy-chain variable region amino acid sequence (V H ) by a peptide that links either (1) the carboxyl terminus of the V L sequence to the amino terminus of the V H sequence or (2) the carboxyl terminus of the V H sequence to the amino terminus of the
  • a single-chain antigen-binding protein-coding gene a recombinant gene coding for a single-chain antigen-binding protein, which encodes a single-chain antigen-binding protein that bind with specificity to an A33 antigen is also contemplated by this invention.
  • the structure of single chain antigen binding proteins has been described by, e.g., Bird et al., Science, 242:423-426 (1988) and U.S. Pat. No. 4,704,692 by Ladner.
  • the immunoglobulins are a large family of molecules that include several types of molecules, such as IgD, IgG, IgA, IgM and IgE.
  • the antibody molecule typically includes two heavy (H) and two light (L) chains, each of which has a variable (V) and constant (C) region.
  • V variable
  • C constant
  • An immunoglobulin heavy chain consists of an immunoglobulin heavy chain variable region and an immunoglobulin heavy chain constant region.
  • the immunoglobulin heavy chain variable region is a polypeptide containing an antigen binding site (and antibody combining site).
  • the immunoglobulin heavy chain variable region is capable of specifically binding a particular epitope.
  • the V H will be from about 110 to about 125 amino acid residues long. The amino acid residue sequence will vary widely, depending on the particular epitope the V H is capable of binding.
  • One embodiment of the invention is directed to a method of reducing the effects of colon cancer in a subject.
  • a pharmaceutically effective amount of an anti-cancer agent is conjugated to an immunoglobulin product that binds with specificity to A33 antigens.
  • This anti-cancer agent-immunoglobulin product is conjugate is administered to a subject which has colon cancer to reduce the effects of the cancer.
  • the immunoglobulin product comprises one or more CDRs having a sequence selected from the group consisting of
  • LASEFLFNGVS (SEQ ID NO:68), LASDFLFNGVS (SEQ ID NO:69), GASNLES (SEQ ID NO:70), GASDLET (SEQ ID NO:71),
  • LGGYSGSSGLT (SEQ ID NO:72), LGGYSGSAGLT (SEQ ID NO:73), HYGIS (SEQ ID NO:74), NNGIS (SEQ ID NO:75), YIYPNYGSVDYASSVNG (SEQ ID NO:76),
  • the anti-cancer agent may be a drug selected from the group consisting of calicheamicin, BCNU, streptozoicin, vincristine and 5-fluorouracil.
  • the anti-cancer agent may be a peptide that specifically inhibits DNA activity of said colon cancer.
  • Other anti-cancer agents include a radioactive isotope such as l25 I, 131 I, "Tc, 90 Y or '"In.
  • the immunoglobulin product of this invention may also comprise an immunologically active portion of an immunoglobulin light chain which has, for example, a V L CDR1 region with a sequence of LASEFLFNGVS (SEQ ID NO:68) or LASDFLFNGVS (SEQ ID NO:69); a V L CDR2 region with sequence GASNLES (SEQ ID NO:70) or GASDLET (SEQ ID NO:71); and a V CDR3 region with a sequence consisting of LGGYSGSSGLT (SEQ ID NO:72) or LGGYSGSAGLT (SEQ ID NO:73).
  • V L CDR1 has sequence LASEFLFNGVS (SEQ ID NO:68)
  • V L CDR2 has sequence GASNLES (SEQ ID NO:70) and
  • V L CDR3 has sequence LGGYSGSSGLT (SEQ ID NO:72).
  • the immunoglobulin product may contain an immunoactive portion of an immunoglobulin light chain.
  • This light chain may contain a sequence in the V, FRl region which corresponds to one of the following sequences: ELQMTQSPSSLSASVGDRVTITC (SEQ ID NO:81),
  • EFDMTQTPPSLSASVGETVRIRC (SEQ ID NO: 82), ELVMTQTPPSLSASVGETVRIRC (SEQ ID NO: 83), or ELVLTQTPPSLSPSVGETVRIRC (SEQ ID NO:84); or a V L FR2 region which corresponds to one of the following sequences: WYQQKPGKAPKLLIY (SEQ ID NO:85),
  • WYQQKPGKAPKLLIY (SEQ ID NO:86) WYQQKPGKVPKFLIY (SEQ ID NO:87), WYQQKPGKAPKFLIY (SEQ ID NO:88), WYQQKPGKVPKLLIY (SEQ ID NO:89), WYQQKPGKPPKFLIS (SEQ ID NO:90), or
  • GVPSRFSGSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:92), GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC (SEQ ID NO:93), GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:94),
  • GVPPRFSGSGSGTDYTLTIGGVQAEDVATYYC SEQ ID NO:95
  • GVPPRFSGSGSGTDYTLTIGGVQAEDAATYYC SEQ ID NO:96
  • V, FR4 region which corresponds to one of the following sequences:
  • FGGGTKVEIK (SEQ ID NO:97) or FGAGTNVEIK.(SEQ ID NO:98).
  • the immunoglobulin product may also comprise an immunologically active portion of an immunoglobulin heavy chain which has, for example, a V H CDR1 having a sequence of HYGIS (SEQ ID NO:74) or NNGIS (SEQ ID NO:75); a V H CDR2 sequence of YIYPNYGSVDYAS SVNG (SEQ ID NO : 76), YIYPNYGSVDYASWVNG (SEQ ID NO:77), or YIYPDYGSTDYASWVNG (SEQ ID
  • V H CDR1 has a sequence of HYGIS (SEQ ID NO:74)
  • V H CDR2 has a sequence of YIYPNYGSVDYASSVNG (SEQ ID NO:76);
  • V H CDR3 has a sequence of DRGYYSGSRGTRLDL (SEQ ID NO:79).
  • the immunoglobulin product may contain an immunoactive portion of an immunoglobulin heavy chain. This heavy chain may contain a sequence in the V H FR1 region which corresponds to one of the following sequences:
  • EVQVMESGGGLVKPGGSLRLSCAASGFTFS SEQ ID NO:99
  • EVQVMESGGGLVKPGGSLRLSCAASGIDFS SEQ ID NO: 100
  • EVQVMESGGGLVKPGGSLRLSCAASGIGFS SEQ ID NO:101
  • QQQVMESGGGLVTLGGSLTLTCKASGIDFS (SEQ ID NO: 102), QEQLMESGGGLVTLGGSLKLSCKASGIDFS (SEQ ID NO: 103), or QEQVMESGGGLVTLGGSLKLSCKASGIDFS (SEQ ID NO: 104); or a V H FR2 region which corresponds to one of the following sequences: WVRQAPGKGLEWIL (SEQ ID NO: 105),
  • WVRQAPGKGLEWIA SEQ ID NO: 106
  • WVRQAPGKGLEWVS. SEQ ID NO: 107
  • V H FR3 region which corresponds to one of the following sequences:
  • RFTISRDNAKNSLYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 114)
  • RFTISRDNAKNSVYLQMNSLRAEDTAVYYCAR SEQ ID NO: 115
  • RFTISRDNAKNSVYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 116)
  • RFTISLDNAQNSLYLQMNSLRAEDTAVYYCAR SEQ ID NO: 117.
  • RFTISLDNAQNS VYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 119)
  • RFTISLDNAQNSVYLQMNSLRAEDTAVYFCAR SEQ ID NO: 120
  • RFTISSDNAQNSLYLQMNSLRAEDTAVYYCAR SEQ ID NO: 121
  • RFTISSDNAQNSLYLQMNSLRAEDTAVYFCAR SEQ ID NO: 122
  • RFTISSDNAQNSVYLQMNSLRAEDTAVYYC AR (SEQ ID NO: 123) or
  • RFTISSDNAQNSVYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 124); or a V ⁇ FR4 region which corresponds to one of the following sequences:
  • the immunoglobulin product may comprise an immunologically active portion of an immunoglobulin light chain which has (A) a V L CDR1 region with a sequence of LASEFLFNGVS (SEQ ID NO:68) or LASDFLFNGVS (SEQ ID NO:69); a V L CDR2 region with a sequence of GASNLES (SEQ ID NO:70) or GASDLET (SEQ ID NO:68) or LASDFLFNGVS (SEQ ID NO:69); a V L CDR2 region with a sequence of GASNLES (SEQ ID NO:70) or GASDLET (SEQ ID
  • V H CDR1 having a sequence of HYGIS (SEQ ID NO:74) orNNGIS (SEQ ID NO:75); a V H CDR2 sequence of YIYPNYGSVDYASSVNG (SEQ ID NO:76), YIYPNYGSVDYASWVNG (SEQ ID NO:77), or YIYPDYGSTDYASWVNG (SEQ ID NO:78); and a V H CDR3 sequence of DRGYYSGSRGTRLDL (SEQ ID NO:79) or
  • the immunoglobulin product of the invention binds to A33 antigen with an affinity which is stronger than 500 pM. More preferably, the immunoglobulin product of the invention binds to A33 antigen with an affinity which is stronger than 100 pM. Another embodiment of the invention is directed to a substantially pure immunoglobulin product that binds with specificity to A33 antigen.
  • the immunoglobulin product may comprise one or more sequences of amino acids having the sequence of
  • LASEFLFNGVS SEQ ID NO:68
  • LASDFLFNGVS SEQ ID NO:69
  • GASNLES SEQ ID NO:70
  • GASDLET SEQ ID NO:71
  • LGGYSGSSGLT SEQ ID NO:72
  • LGGYSGSAGLT SEQ ID NO:73
  • HYGIS SEQ ID NO:74
  • NNGIS SEQ ID NO:75
  • YIYPNYGSVDYASSVNG SEQ ID NO:76
  • YIYPNYGSVDYASWVNG SEQ ID NO:77
  • YIYPDYGSTDYASWVNG SEQ ID NO:78
  • DRGYYSGSRGTRLDL SEQ ID NO:79
  • DRGAYAGSRGTRLDL SEQ ID NO: 80
  • the substantially pure immunoglobulin product which binds the A33 antigen contains an immunologically active portion of an immunoglobulin light chain that in turn contains one or more light chain CDRs.
  • the immunoglobulin light chain, V L CDR1 may have sequence LASEFLFNGVS (SEQ ID NO:68) or LASDFLFNGVS (SEQ ID NO:69);
  • V L CDR2 may have sequence GASNLES (SEQ ID NO:70) or GASDLET (SEQ ID NO:71); and V L CDR3 may have a sequence LGGYSGSSGLT (SEQ ID NO:72) or LGGYSGSAGLT (SEQ ID NO: 73).
  • V L CDR1 is LASEFLFNGVS (SEQ ID NO:68)
  • V CDR2 is GASNLES (SEQ ID NO:70)
  • V L CDR3 is LGGYSGSSGLT (SEQ ID NO:72).
  • A33 antigen contains an immunologically active portion of an immunoglobulin heavy chain that in turn contains one or more CDRs of a heavy chain.
  • V H CDR1 may have sequence HYGIS (SEQ ID NO:74) or NNGIS (SEQ ID NO:75);
  • V subjectCDR2 may have sequence YIYPNYGSVDYASSVNG (SEQ ID NO:76), YIYPNYGSVDYASWVNG (SEQ ID NO: 77), or YIYPDYGSTDYASWVNG (SEQ ID NO:
  • V H CDR3 may have sequence DRGYYSGSRGTRLDL (SEQ ID NO: 79) or DRGAYAGSRGTRLDL (SEQ ID NO:80).
  • V H CDR1 is HYGIS (SEQ ID NO:74)
  • V H CDR2 is YIYPNYGSVDYASSVNG (SEQ ID NO:76)
  • V H CDR3 is DRGYYSGSRGTRLDL (SEQ ID NO:79).
  • the immunoglobulin product comprises at least two polypeptide sequences selected from the following: rabbit VLl and rabbit VHl ; rabbit VL2 and rabbit VH2 ; rabbit VL3 and rabbit VH3 ; human VLA and human VHA, human VLB and human VHB, human VLC and human VHC, human VLD and human VHD, human VLE and human VHE, or human VLF and human VHF.
  • the substantially pure immunoglobulin product may comprise an immunologically active portion of an immunoglobulin heavy chain and an immunologically active portion of an immunoglobulin light chain.
  • V L CDR1 may have sequence LASEFLFNGVS (SEQ ID NO:68 ) or LASDFLFNGVS (SEQ ID NO:69);
  • V L CDR2 may have sequence GASNLES (SEQ ID NO:70) or GASDLET (SEQ ID NO:71); and
  • V L CDR3 may have sequence LGGYSGSSGLT
  • V H CDR1 may have sequence HYGIS (SEQ ID NO:74) or NNGIS (SEQ ID NO:75);
  • V H CDR2 may have sequence YIYPNYGSVDYASSVNG (SEQ ID NO:76), YIYPNYGSVDYASWVNG (SEQ ID NO:77), or YIYPDYGSTDYASWVNG (SEQ ID NO:78); and
  • V H CDR3 may have sequence DRGYYSGSRGTRLDL (SEQ ID NO:79) or
  • An immunoglobulin product of the invention may be an antibody, a Fv fragment, a Fab fragment, a Fab 2 fragment, or a single chain antibody or a combination or multimer thereof.
  • a multimer may be any linked combination of immunoglobulin products.
  • a multimer may contain more than 2, preferably more than 4, or even more than 6 antibodies, antibody fragments, or single chain antibodies linked together.
  • Linkage may be by covalent bonds. Methods of linking antibodies and polypeptides, and proteins are known. Further, the linkage may be ionic. For example, one antibody linked to avidin may be linked by ionic bond to another antibody linked to biotin.
  • the linked immunoglobulin products need not have the same affinity.
  • one linked immunoglobulin product may have a high affinity for A33 antigen
  • another linked immunoglobulin product may have a low affinity for A33 antigen
  • a third linked immunoglobulin product may have an affinity to a toxic or therapeutic chemical such as ricin.
  • the immunoglobulin product may be an antibody molecule such as a IgM, IgD, IgG, IgA or IgE or a fragment of these molecules.
  • the immunoglobulin product may bind A33 antigen with an affinity that is stronger than 1 pM, preferably stronger than 10 pM, more preferably stronger than 100 pM, even more preferably stronger than 300 pM such as, for example, stronger than 500 pM.
  • the immunoglobulin product may be an anti A33 antigen immunoglobulin product that is derived from a rabbit.
  • a rabbit derived anti A33 antigen immunoglobulin product may be made, for example, by injecting a rabbit with A33 antigen. Another method for producing rabbit anti A33 antigen immunoglobulin product is shown in the Example section.
  • Another embodiment of the invention is directed to a CDR peptide and proteins that contain one or more CDR peptides with a sequence of LASEFLFNGVS (SEQ ID NO:68), LASDFLFNGVS (SEQ IDNO:69), GASNLES (SEQIDNO:70), GASDLET (SEQ IDNO:71),
  • LGGYSGSSGLT (SEQ ID NO:72), LGGYSGSAGLT (SEQ ID NO:73), HYGIS (SEQ ID NO:74), NNGIS (SEQ ID NO:75), YIYPNYGSVDYASSVNG (SEQ ID NO:76), YIYPNYGSVDYASWVNG (SEQ ID NO:77), YIYPDYGSTDYASWVNG (SEQ ID NO:78), DRGYYSGSRGTRLDL (SEQ ID NO:79), or DRGAYAGSRGTRLDL (SEQ ID NO:80).
  • the immunoglobulin product of the invention may be a member of an immunoglobulin gene superfamily such as a immunoglobulin heavy chain, a T cell receptor, a major histocompatibility antigen, a ⁇ 2 -microglobulin associated antigen, a T lymphocyte antigens, a haemopoietic/endothelium antigens, a brain/lymphoid antigen, an immunoglobulin receptor, a neural molecule, a tumor antigen and the like.
  • the immunoglobulin product of the invention may contain immunologically active portion of an immunoglobulin light chain.
  • the active portion may be V L FR1 with a sequence of ELQMTQSPSSLSASVGDRVTITC (SEQ ID NO:81), EFDMTQTPPSLSASVGETVRIRC (SEQ ID NO: 82), ELVMTQTPPSLSASVGETVRIRC (SEQ ID NO:83), or ELVLTQTPPSLSPSVGETVRIRC (SEQ ID NO:84). Also, the active portion may be V L FR2 having sequence WYQQKPGKAPKLLIY (SEQ ID NO:85),
  • the active portion may be V L FR3 with a sequence GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO:92), GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC (SEQ ID NO:93),
  • the active portion may also be V, FR4 with sequence FGGGTKVEIK (SEQ ID NO:97) or FGAGTNVEIK (SEQ ID NO: 98).
  • the immunoglobulin product of the invention may contain immunologically active portion of an immunoglobulin heavy chain.
  • the active portion may be V H FR1 with a sequence of EVQVMESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO:99), EVQVMESGGGLVKPGGSLRLSCAASGIDFS (SEQ ID NO:100), EVQVMESGGGLVKPGGSLRLSCAASGIGFS (SEQ ID NO:101),
  • the active portion may also be V flankFR2 with sequence WVRQAPGKGLEWIL (SEQ ID NO: 105), WVRQAPGKGLEWIA (SEQ ID NO: 106) or WVRQAPGKGLEWVS (SEQ ID NO: 107).
  • the active portion may also be V H FR3 with sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:108), RFTISFDNAQNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:109), RFTISLDNAQNSLYLQMNSLRAEDTAVYFCAR (SEQ ID NO:110), RFTISLDNAQNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:lll)
  • RFTISFDNAQNSVYLQMNSLRAEDTAVYYCAR (SEQ ID NO:112)
  • RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR SEQ ID NO:113
  • RFTISRDNAKNSLYLQMNSLRAEDTAVYFCAR SEQ ID NO:114
  • RFTISRDNAKNSVYLQMNSLRAEDTAVYYCAR SEQ ID NO:115
  • RFTISRDNAKNSVYLQMNSLRAEDTAVYFCAR (SEQ ID NO:116)
  • RFTISLDNAQNSLYLQMNSLRAEDTAVYYCAR SEQ ID NO: 117
  • RFTISLDNAQNSLYLQMNSLRAEDTAVYFCAR SEQ ID NO:118
  • RFTISLDNAQNSVYLQMNSLRAEDTAVYYCAR SEQ ID NO:119
  • RFTISLDNAQNSVYLQMNSLRAEDTAVYFCAR SEQ ID NO:120
  • RFTISSDNAQNSLYLQMNSLRAEDTAVYYCAR SEQ ID NO:121
  • RFTISSDNAQNSLYLQMNSLRAEDTAVYFCAR (SEQ ID NO:122) RFTISSDNAQNSVYLQMNSLRAEDTAVYYCAR (SEQ ID NO:123), or RFTISSDNAQNSVYLQMNSLRAEDTAVYFCAR (SEQ ID NO: 124).
  • the active portion may also be V H FR4 with sequence WGQGTLVTISS (SEQ ID NO: 125) or WGQGTLVTVSS (SEQ ID NO: 126).
  • the substantially pure immunoglobulin product may be a humanized immunoglobulin.
  • a nucleic acid molecule encoding an immunoglobulin product of the invention may be made using conventional techniques. For example, oligonucleotides may be synthesized and ligated together to form a functional open reading frame that encodes an immunoglobulin product of the invention.
  • the nucleic acid molecule once synthesized, may be cloned into a nucleic acid vector.
  • a nucleic acid vector such as a plasmid, cosmid, phagemid, yeast plasmid, phage vectors, TI plasmid and the like are known in the art.
  • the vector may be an expression vector. Expression vectors and expression systems are available commercially.
  • Another embodiment of the invention is directed to a cell comprising a nucleic acid of the invention.
  • a cell may be made by transfection. Methods of transfection are known and kits for transfection of prokaryotic and eukaryotic cells may be purchased from commercial sources.
  • Another embodiment of the invention is directed to a method for detecting or diagnosing a disorder comprising the steps of contacting a tissue sample from a subject to the substantially pure immunoglobulin product of the invention under condition that permits the formation of a complex between said immunoglobulin product and an A33 antigen, and determining the formation of said complex.
  • Another embodiment of the invention is directed to a method of treating a patient with a neoplastic disorder comprising administering an immunoglobulin product of invention or a nucleic acid of the invention to said patient.
  • Methods for immunotherapy for cancer are known. See for example Old, L. J. Immunotherapy for Cancer, Scientific American, September 1996, US Patent 5,851,526 and 5,712,369; all incorporated herein by reference.
  • Another embodiment is directed to a therapeutic composition comprising an immunoglobulin product of the invention.
  • the immunoglobulin products of the invention may be provided in the form of a composition comprising the immunoglobulin and a pharmaceutically acceptable carrier or diluent.
  • the therapeutic composition may be used for the treatment of disorders in a mammal such as a human.
  • the invention also provides a method for treating a mammal comprising administering a therapeutically effective amount of the immunoglobulin products of the invention to the mammal, wherein the mammal has a disorder, such as cancer,
  • the immunoglobulin product of the invention may be linked to an agent. Linkage may be by covalent bonds or by antibody-epitope bond.
  • an immunoglobulin product may be crosslinked to a second antibody wherein the second antibody may have an affinity for the agent.
  • the agent may be a cytotoxic agent.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., I25 1, 131 I, "Tc, 90 Y, l u In), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • the agent may be a chemotherapeutic agent.
  • a "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,
  • the agent may be a cytokine.
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF; platelet-growth factor; transforming growth factors (TGFs); insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon- ⁇ , - ⁇ , and - ⁇ ;
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the invention also contemplated the generation of mutants of the disclosed CDRs by mutating one or more amino acids in the sequence of one or more of the CDRs. It is known that a single amino acid substitution appropriately positioned in a CDR can be sufficient to raise the affinity.
  • researchers have used site directed mutagenesis to increase affinity of some immunoglobulin products by about 10 folds. This method of increasing or decreasing affinity of antibodies by mutating CDRs is common knowledge (see, e.g., Chapter 23, Paul, W.E.,
  • Example 1 Generation of antibodies to human A33 Antigen.
  • LIM 1215 human colon carcinoma cell line LIM 1215, which is known to express large amounts of A33 antigen. It should be noted that LIM 1215 was chosen because it expresses A33 antigen. Any other cell line which expresses A33 antigen may be substituted for LIM 1215. Subject animals received three subcutaneous injections of 10 6 LIM 1215 cells followed by three subcutaneous injections of 1 ⁇ g of extracellular domain of human A33 that had been purified from LIM 1215 cells. The A33 was administered in the form of a 1 ml emulsion of RIB I adjuvant in phosphate buffered saline.
  • Antisera from the subject animals were tested following the three injections of LIM 1215 cells, and then the three injections of antigen. Testing was carried out by combining the antisera with recombinant human A33 and alkaline phosphatase conjugated, goat anti-rabbit Fc polyclonal antibodies.
  • V ⁇ 5' sense primers V ⁇ 5' sense primers
  • V ⁇ 3' antisense primer 5'-cgagggggcagccttgggctggcctgtgacggtcagctgggtccc-3' (SEQ ID NO: 8)
  • V H 3' antisense primer 5'-cgatgggcccttggtggaggctgargagayggtgaccagggtgcc-3' (SEQ ID NO: 13) were used to amplify V H .
  • the antisense primers (SEQ ID NOS: 4-6, 8 and 13) represent hybrids of rabbit and human sequences, and were designed to permit fusion of rabbit, variable domains to human constant domains (i.e., fusion of rabbit V ⁇ or V H to human CK and C H 1).
  • the rabbi V ⁇ and human CK fragments were fused using: gaggaggagg aggaggaggc ggggcccagg cggccgagct c (SEQ ID NO: 14), and gccatggctg gttgggcagc (SEQ ID NO: 15), and rabbit V H and human C H 1 were fused using: gctgcccaac cagccatggc c (SEQ ID NO: 16) and gaggaggagg aggaggagag aagcgtagtc cggaacgtc (SEQ ID NO: 17).
  • Trypsinization was carried out for 30 minutes at 37°C. The number of washing steps increased from 5 (first round) to 10 (second round), to 15 in the third and fourth rounds.
  • Output phage pool of each round was monitored, via phage ELISA, using horseradish peroxidase labelled sheep anti-M13 phage polyclonal antibodies. Increased signal above background from round to round was observed, and output numbers increased strongly after the third and fourth rounds, indicating successful selection.
  • flanking primers For clones from final output were grown and induced with ImM IPTG. Supernatants from the clones were tested for binding to immobilized, recombinant human A33 via ELISA, using alkaline phosphatase - conjugated goat, anti-human F(ab') 2 polyclonal antibodies. All clones gave a strong signal, above background, and were subjected to DNA fingerprinting using standard methodologies. In brief, flanking primers:
  • AAGACAGCTA TCGCGAATTG CAC (SEQ ID NO: 18) and GCCCCCTTAT TAGCCTTTGC CATC (SEQ ID NO: 19)
  • VK coding sequences VK coding sequences, and 90% identity in the V H sequence.
  • SEQ ID NO: 22 had a VK coding sequence 90% identical to SEQ ID NOS: 20 and 21, and its V H sequence was identical to that of SEQ ID NO:22.
  • the hypervariable VDJ and VJ joint regions HCDR3 and LCDR3 were highly similar, suggesting that all the selected sequences originated from a single B cell clone that had undergone diversification by somatic mutation.
  • Soluble Fabs from rabbit VHl , VLl and rabbit VH2 and VL2 were produced from E. coli, in accordance with Rader, et al., supra.
  • Fab molecules were purified from concentrated supernatants and from sonicated lysates of overnight cultures that had been induced with ImM
  • CM5 sensor chip (Biacore AB) was activated for immobilization with Nhydroxysuccinimide and N-ethyl-N'-(3— dimethylaminopropyl)carbodiimide according to standard methods.
  • Recombinant human A33 antigen was coupled at a low density to the surface by injection of 30 ⁇ l to 40 ⁇ l of a 1 ng/ ⁇ l sample in 10 mM sodium acetate (pH 3.5). Approximately 500 resonance units were immobilized. Subsequently, the sensor chip was deactivated with 1 M ethanolamine hydrochloride (pH 8.5).
  • Binding of Fab to immobilized A33 antigen was studied by injection of Fab at 5 different concentrations ranging from 75 nM to 200 nM. PBS was used as the running buffer. The sensor chip was regenerated with 20 mM HC1 and remained active for at least 50 measurements. The k on and k off values were calculated using Biacore AB evaluation software. The equilibrium dissociation constant I was calculated from k ofi k on . Data obtained from different sensor chips revealed a high consistency and were further validated according to procedure as described in Rader et al., (Rader, C, Cheresh, D. A., and Barbas, C. F., Ill (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 8910-8915, incorporated herein by reference). The binding of the Fab was very strong, i.e., with affinity in the InM range ( Figure 4).
  • Kd dissociation constant
  • the CDR sequence of SEQ ID NO: 2 was used because of high expression.
  • the six variable domains described by Kabat, et al., supra, were grafted into human framework sequences. There was a potentially immunogenic tryptophan at position 62, in rabbit "HCDR2" (Kabat et al., supra., was converted to serine.
  • Linked positions (VH 27-28 and VH 71-75, respectively) indicate a coupled diversification that limits the selection to cither all-human or all rabbit sequence.
  • Overlapping oligonucleotides were designed, synthesized, and then assembled to create synthetic V ⁇ and V H coding sequences, using PCR.
  • the procedure described, supra, for the generation of rabbit antibody library was followed, and when the final constructs were completed, they were Sfi I cloned into a vector carrying a chloramphenicol resistence gene, to avoid contamination with phage from the rabbit antibody.
  • the resulting library consisted of 1X10 7 independent transformants with a theoretical complexity of 2x10 7 .
  • L denotes primers for the V L assembly
  • H denotes primers for the V H assembly:
  • the transformants were panned as described supra, but the amount of antigen employed was decreased over the course of panning. In the first two rounds, lOOng were used, followed by two rounds at 50ng, and two rounds at 25ng. Ten washing steps were carried out for each round, using 0.5% (v/v) Tween 20 in TBS. Rounds 3 and 4, and rounds 5 and 6, were linked without phage amplification. To do this, phages from rounds 3 and 5 were eluted, using 50 ⁇ l of lOOmM HCl-glycine (pH 2.2), incubated for 10 minutes at room temperature, collected, neutralized with 3 ⁇ of 2M Tris base, and 50 l of 1% (w/v) BSA in TBS. The phages were than directly subjected to another round of panning. Phages from rounds 1 , 2, 4 and 6 were eluted by trypsinization, as described supra.
  • Seventy clones resulted from final output. All were found to be positive via ELISA. Twenty-four of the seventy clones were further analyzed via DNA sequencing.
  • Sequences for the heavy and light chain of 6 of these clones are presented as human VLA, VLB, VLC, VLD, VLE, VLF, VHA, VHB, VHC, VHD, VHE, VHF in Figure 1.
  • a consensus sequence was found for the diversified framework of V H , with positions 27 and 28 in framework 1, and positions 71 and 75 in framework 3 being found to contain original rabbit residues isoleucine, aspartic acid, leucine, and glutamine, respectively, in 16 of 24 clones.
  • Three clones contained human residues phenylalanine and threonine at positions 27 and 28, and none contained human residues at positions 71 and 75. Two of the diversified positions contained mutations.
  • Pro line a human residue, was found at position 80 in framework 3, in 18 of 24 clones, including the 5 mutated clones showing strongest reactivity via ELISA.
  • the six clones (human A to F, wherein each comprise a VH and VL as shown in
  • Figure 1 referred to supra were then produced as soluble Fab molecules via E.coli. and purified as described supra. Yields ranged from 0.5 to 2mg per 1 liter shake flash culture. When subjected to flow cytometry, all Fabs bound to cells expressing native A33 antigen. Those cells which did not express human A33 were not recognized. There were slight differences in fluorescence intensity, which correlated to differences in affinity to immobilized recombinant human A33, measured by surface plasmon resonance carried out as described supra. This suggests strongly that the antibodies, which were selected on immobilized, recombinant antigen, bind to a native epitope fully accessible on the cell surface, thereby constituting a relevant therapeutic target.
  • Example 6 Characterization of Novel A33 Antibodies-
  • the 340bp Bglll/Pstl fragment was removed from the pBlueBac4/A33 vector and the resulting plasmid was religated with the use of two overlapping oligonucleotides (gatctccctccatgaaccat catcatcatcattgactgca and gtcaatgatgatgatgatgatgatggttcatggaggga (SEQ ID NO: 127)).
  • Sf9 cells When annealed, these oligonucleotides would create Bglll and Pstl sites at the 5' and 3' end respectively and sequences encoding SPSMHHHHHH (SEQ ID NO: 128) and stop codon between both restriction sites.
  • Transfection of Sf9 cells with pBlueBac4/A33 and pBlueBac4/A33-ECD transfer vectors and isolation of recombinant viruses was performed according to the manufacturer's recommendations (Invitrogen). For large-scale expression, Sf9 cells were infected with the recombinant viruses at a multiplicity of infection (MOI) of 10. After three days of infection cells were harvested by centrifugation and used immediately for the purification of recombinant proteins.
  • MOI multiplicity of infection
  • Expressed protein was purified by immunoaffinity chromatography using mouse mAb A33 immobilized to protein A conjugated Sepharose 4B beads with dimethylpimelimidate as previously described (Moritz, R.L. et al., J. Chromatogr. A, 798: 91- 101).
  • Hemadsorption assay The protein A, rabbit anti-human F(ab') 2 mixed hemadsorption assay which detects surface bound Fab by adherence of protein A coated human RBC (blood group O) to target cells was performed as previously described (Pfreundschuh, M. et al., Proc. Natl. Acad. Sci. (Wash.), 75, 5122-5126 (1978)). Results: Fabs A, B, C, E, and F were analyzed for reactivity with A33 antigen extracted from colon cancer cell lines by Western blot assays ( Figure 2). All new Fabs reacted with a band of about 43 kD protein under non reducing conditions. No Western blot reactivity was observed using reducing conditions ( Figure 2).
  • the difference between the humanized clones were found to correlate with their differences in affinity to immobilized recombinant human A33 antigen.
  • the humanized Fab were further analyzed for reactivity with human A33 antigen extracted from colon cancer cell lines by Western blotting. As shown for humanized clone B ( Figure 2), the humanized Fab strongly reacted with a band of about 43 kD under nonreducing conditions. No reactivity was observed using reducing condition, suggesting the recognition of a conformation epitope on human A33 antigen (Catimel, B. et al., (1996) J. Biol. Chem. 271, 25664-25670). Taken together, these results demonstrate that the selected humanized antibodies bind to a native epitope on human A33 antigen that is fully accessible on the cell surface.
  • Example 7 Immunohistochemistry
  • Diaminobenzidine tetrahydrochloride (DAB, Biogenex, San Ramon, C A) was used as a chromogen. Reactivity of the humanized Fab was also evaluated in human colonic adenocarcinoma samples. In order to prevent immunoreactivity of endogenous human immunoglobulin, a special technique for the detection of humanized Fab was utilized. Prior to addition to tissue, the humanized Fab (1 ⁇ g/ml) was incubated with biotinylated goat-anti human F(ab) 2 polyclonal antibodies in a test tube. The optimal ratio of humanized Fab to secondary antibody was determined in separate titration assays. Tncubation of humanized Fab and secondary antibody was done at room temperature for 1 hour and followed by an addition of human serum in order to block the activity of unbound secondary antibody. Again, the optimal ratio of human serum to secondary antibody was determined in separate titration assays.

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Abstract

L'invention concerne des procédés permettant de réduire les effets du cancer chez un sujet par administration audit sujet d'une quantité pharmaceutiquement efficace d'un agent anticancéreux conjugué avec une immunoglobuline comprenant une ou plusieurs nouvelles régions de détermination de complémentarité et d'infrastructure.
EP00972349A 1999-10-22 2000-10-20 Procedes permettant de reduire les effets des cancers exprimant l'antigene a33 a l'aide d'immunoglobulines specifiques de l'antigene a33 Withdrawn EP1233790A2 (fr)

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US425638 1989-10-23
US09/425,638 US6342587B1 (en) 1999-10-22 1999-10-22 A33 antigen specific immunoglobulin products and uses thereof
US09/543,004 US6346249B1 (en) 1999-10-22 2000-04-04 Methods for reducing the effects of cancers that express A33 antigen using A33 antigen specific immunoglobulin products
US543004 2000-04-04
PCT/US2000/029289 WO2001030393A2 (fr) 1999-10-22 2000-10-20 Procedes permettant de reduire les effets des cancers exprimant l'antigene a33 a l'aide d'immunoglobulines specifiques de l'antigene a33

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US6652853B2 (en) * 2001-03-08 2003-11-25 Ludwig Institute For Cancer Research Method for treating cancer using A33 specific antibodies and chemotherapeutic agents
US20030040027A1 (en) * 2001-08-16 2003-02-27 Gerd Ritter Method for determining protein component in a biological sample
KR100918746B1 (ko) * 2004-09-06 2009-09-24 교와 핫꼬 기린 가부시키가이샤 항 ａ３３ 항체
AU2008246442B2 (en) * 2007-05-04 2014-07-03 Technophage, Investigacao E Desenvolvimento Em Biotecnologia, Sa Engineered rabbit antibody variable domains and uses thereof
TWI609965B (zh) * 2007-05-21 2018-01-01 艾爾德生物控股有限責任公司 新穎兔抗體人化方法以及經人化之兔抗體
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CA2876397C (fr) * 2012-06-15 2019-08-06 Pfizer Inc. Anticorps antagonistes ameliores diriges contre gdf-8 et leurs utilisations
BR112019022873A8 (pt) 2017-05-02 2023-04-11 Merck Sharp & Dohme Formulação, e, vaso ou dispositivo de injeção.
JOP20190260A1 (ar) 2017-05-02 2019-10-31 Merck Sharp & Dohme صيغ ثابتة لأجسام مضادة لمستقبل الموت المبرمج 1 (pd-1) وطرق استخدامها
MX2021005394A (es) * 2018-11-07 2021-07-06 Merck Sharp & Dohme Llc Co-formulaciones de anticuerpos anti-gen de activacion de linfocitos 3 (anti-lag3) y anticuerpos anti-muerte programada-1 (anti-pd-1).
CN110540591A (zh) * 2019-08-09 2019-12-06 无锡傲锐东源生物科技有限公司 一种抗糖蛋白A33（Glycoprotein A33）单克隆抗体及其免疫检测应用

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WO1998022149A1 (fr) * 1996-11-19 1998-05-28 Ludwig Institute For Cancer Research Procede de traitement des neoplasies a l'aide d'anticorps humanises qui se fixent a l'antigene a33
WO2001030393A2 (fr) * 1999-10-22 2001-05-03 Ludwig Institute For Cancer Research Procedes permettant de reduire les effets des cancers exprimant l'antigene a33 a l'aide d'immunoglobulines specifiques de l'antigene a33
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WO2001030393A8 (fr) 2002-06-27
WO2001030393A2 (fr) 2001-05-03
AU1102401A (en) 2001-05-08
AU1097301A (en) 2001-05-08

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