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WO2007084693A2 - Immunoconjugués présentant une efficacité accrue pour le traitement de maladies - Google Patents

Immunoconjugués présentant une efficacité accrue pour le traitement de maladies Download PDF

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Publication number
WO2007084693A2
WO2007084693A2 PCT/US2007/001483 US2007001483W WO2007084693A2 WO 2007084693 A2 WO2007084693 A2 WO 2007084693A2 US 2007001483 W US2007001483 W US 2007001483W WO 2007084693 A2 WO2007084693 A2 WO 2007084693A2
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WIPO (PCT)
Prior art keywords
antibody
residues
antibodies
lysine
terminal sequence
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PCT/US2007/001483
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English (en)
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WO2007084693A3 (fr
Inventor
Le Sun
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Welson Pharmaceuticals, Inc.
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Priority to EP07717984A priority Critical patent/EP1973947A2/fr
Publication of WO2007084693A2 publication Critical patent/WO2007084693A2/fr
Publication of WO2007084693A3 publication Critical patent/WO2007084693A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments

Definitions

  • the present invention relates to antibodies with modified N- or C-termini that are enriched with lysine or tyrosine residues, and methods of using these antibodies for the treatment or diagnosis of cancer and other diseases.
  • Antibodies have been used to treat cancer and immunological or angiogenic disorders.
  • the use of antibody-drug conjugates allows targeted delivery of drug moieties to tumors and other diseased tissues, where systemic administration of the unconjugated drug agents may result in unacceptable levels of toxicity to normal cells.
  • the basic unit of a native antibody is a monomer which consists of two identical heavy chains and two identical light chains linked by disulfide bonds. There are at least five different types of heavy chains - namely, ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , which provide different effector functions.
  • Heavy chains ⁇ , ⁇ and ⁇ have three constant domains (C H I, C H 2, and C H 3), and heavy chains ⁇ and ⁇ have four constant domains (C H I , C H 2, C H 3, and C H 4). Each heavy chain also has one variable domain (V H ).
  • native human antibodies can be grouped into five classes: IgG, IgA, IgM, IgD, and IgE. Several of these classes can be further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, IgA, or IgA2.
  • a typical IgG molecule is composed of two heavy chains ⁇ and two identical light chains ( ⁇ or K). Disulfide bonds connect the light chains to the heavy chains, as well as between the heavy chains.
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain, (V L ) is aligned with the variable domain of the heavy chain (V H ) to form the antigen recognition site.
  • variable domain The variability in a variable domain (V L or V H ) is not evenly distributed throughout the domain. It is typically concentrated in three segments called hypervariable regions. The more highly conserved portions of a variable domain are called the framework regions (FRs).
  • FRs framework regions
  • Each variable domain of native heavy and light chains comprises four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of the antibody.
  • Papain digestion of an antibody produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment.
  • Pepsin treatment of an antibody yields an F(ab') 2 fragment which has two antigen- binding sites joined at the hinge region between C H I and C H 2 through disulfide bonds. The reduction of the F(ab')2 fragment produces two Fab' fragments.
  • Each Fab' fragment contains at least one sulfhydryl group that can be utilized in conjugation with a toxin, a radioactive isotope, or another agent of interest.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain C H I domain.
  • Fab, Fab', F(ab')2, and other antigen-binding fragments can also be produced using recombinant host cells, as appreciated by those skilled in the art.
  • an antibody of the present invention comprises an N- or C- terminal sequence which consists of 2 to 20 amino acid residues, a substantial potion of which are lysine or tyrosine residues.
  • the substantial potion can be, for example, at least one-third, one-half, or two-third of the residues in the N- or C-terminal sequence.
  • the N- or C-terminal sequence consists of 3 to 15, 3 to 10, or 4 to 8 amino acid residues, among which at least 2, 3, 4, 5, 6, or more residues are lysine or tyrosine.
  • These 2, 3, 4, 5, 6, or more residues can be lysine residues. They can also be tyrosine residues.
  • these 2, 3, 4, 5, 6, or more residues can be a mixture of lysine and tyrosine residues.
  • the N- or C-terminal sequence consists of about 5, 6, 7, or 8 amino acid residues, among which at least 2, 3, 4, 5, or more residues are lysine or tyrosine.
  • the lysine or tyrosine residues in the N- or C- terminal sequence are covalently coupled to radioactive isotopes, chemotherapeutic agents, toxins, prodrugs, pro-drug activating enzymes, or other cytotoxic agents.
  • radioactive isotopes include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , and Lu 177 .
  • Non-limiting examples of cytotoxic agents include mitomycin C and pingyangmycin.
  • the lysine or tyrosine residues can be enriched at the N- or C-terminus of a light chain of an antibody. These residues can also be enriched at the N- or C-terminus of a heavy chain of an antibody. Where the antibody employed is an antibody fragment (e.g., Fab, F(ab') 2 , or a genetically engineered antibody fragment such as scFv or minibody), the lysine or tyrosine residues can be enriched at the N- or C-terminus of the fragment or a subunit thereof.
  • an "antibody” encompasses not only polyclonal and monoclonal antibodies, but also multispecific antibodies (e.g.
  • bispecific antibodies chimeric antibodies, linear antibodies, reduced antibodies (e.g., rlgG), antibody fragments comprising antigen-binding sites (e.g., Fab fragments, Fab' fragments, F(ab') 2 , or Fv fragments), single- chain Fv (scFv) molecules, diabodies, triabodies, and minibodies.
  • an.antibody of the present invention specifically recognizes an antigen selected from the group consisting of a growth factor, a cytokine, a hormone, a growth factor receptor, a hormone receptor, a cytokine receptor, a neurotransmitter receptor, a tyrosine kinase receptor, a tumor-specific or tumor-associated antigen, a G-protein coupled receptor, an ion channel, and an enzyme.
  • Non-limiting examples of antibodies suitable for the present invention include anti-CD22, anti-ErbB2, anti- VEGF, anti-EGFR, anti- VEGFR, anti-Her-3, anti-Her-4, anti-CEA, anti-CTLA-4, anti-CD4, anti-CD3, anti-CD20, anti-TNF-a, anti-CD 11 a, anti-Lewis Y antigen, anti-TrailR, anti-IL2R, anti-CD30, anti-CD146, anti-CD147, anti-alpha V integrin beta, anti-CD19, anti-GD2, anti- 3Hl 1, anti -EBV, anti-HIV, anti-HBV, and anti-HCV.
  • the antibodies employed in the present invention are humanized or human antibodies.
  • the present invention also features pharmaceutical compositions which comprise an antibody of the present invention.
  • a typical pharmaceutical composition of the present invention is prepared by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, in the form of lyophilized formulations or aqueous solutions.
  • the present invention features methods of using the antibodies of the present invention to modulate cell killing activity or inhibit the growth or activity of cells or pathogens. These methods comprise contacting an antibody of the present invention with the cells/pathogens to be treated.
  • the cells/pathogens to be treated can be cancer cells or other diseased or dysfunctional cells, viruses, bacteria, yeast, fungi, or other disease-causing microbes.
  • the cells can also be healthy cells.
  • the present invention further features methods of using the antibodies of the present invention to treat cancer or other diseases. These methods comprise administering a therapeutically effective amount of an antibody of the present invention to a subject of interest (e.g., a cancer patient).
  • a subject of interest e.g., a cancer patient
  • the present invention features diagnostic kits comprising the antibodies of the present invention. These antibodies are labeled by one or more detectable moieties via the additional lysine or tyrosine residues at the N- or C-termini of the antibodies.
  • diagnostic kits comprising the antibodies of the present invention. These antibodies are labeled by one or more detectable moieties via the additional lysine or tyrosine residues at the N- or C-termini of the antibodies.
  • the present invention features therapeutic antibodies with modified C- or N- termini that are enriched with lysine or tyrosine residues. These lysine or tyrosine residues enable the coupling of more radioactive or cytotoxic agents, thereby significantly improving the therapeutic effects of the antibody conjugates. Detectable labels can also be conjugated to the lysine/tyrosine-enriched termini, thereby improving the detection sensitivity of the antibody conjugates.
  • Antibodies suitable for the present invention include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecif ⁇ c antibodies), chimeric antibodies, linear antibodies, reduced antibodies (e.g., rigG), Fab fragments, Fab' fragments, F(ab') 2 , Fv fragments, single-chain Fv (scFv) molecules, diabodies, triabodies, minibodies, and other antibody fragments that comprise antigen- binding sites.
  • the present invention features the use of any antibody isotype, e.g., IgG, IgM, IgA, IgD, or IgE.
  • an antibody of the present invention (including Fab, Fv, or other antigen-binding fragments) binds to the target antigen with an affinity of at least 10 "5 M ' ⁇ 1O -6 M '1 , lCr 7 M “1 , lCr 8 M “1 , lCr 9 MT 1 , l(r 10 ivr 1 , or stronger.
  • An antibody of the present invention can be a rat, murine, cow, dog, sheep, goat, guinea pig, rabbit, macaque, chimpanzee, chicken, or human antibody. Antibodies derived from other non-human primates, mammals, or vertebrates are also contemplated by the present invention.
  • monoclonal should not be construed as requiring production of the antibody by any particular method.
  • exemplary methods suitable for making monoclonal antibodies include, but are not limited to, the hybridoma methods (e.g., Kohler et al., NATURE, 256:495 (1975)), the recombinant DNA methods (see, e.g., U.S. Patent No.4,816,567), and the phage antibody library methods (e.g., Clackson et al., NATURE, 352:624-628 (1991), and Marks et al., J. M ⁇ L.
  • DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells used for the production of the monoclonal antibodies serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • a chimeric antibody refers to an antibody in which a portion of the heavy or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass.
  • Non-limited examples of chimeric antibodies include those described in U.S. Patent No. 4,816,567 and Morrison et al.. PROC. NATL. ACAD. SCI.
  • a chimeric antibody of the present invention is a humanized antibody.
  • Humanized antibodies are particularly desirable for therapeutic treatment of human subjects.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric full-length immunoglobulins, or chimeric antigen-binding fragments (such as Fv, Fab, or F(ab')2), which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies can be derived from human immunoglobulins in which the residues forming the complementary determining regions (CDRs) are replaced by the residues from CDRs of a non-human antibody.
  • CDRs complementary determining regions
  • Fv framework residues of the human immunoglobulin are also replaced by corresponding non-human residues.
  • Humanized antibodies may include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody can comprise at least one or two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the constant regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody of the present invention comprises at least a portion of a constant region of a human immunoglobulin (Fc).
  • the present invention also features the use of human antibodies.
  • Human antibodies can be produced using transgenic mice, which are incapable of expressing endogenous immunoglobulin heavy and light chains but can express human heavy and light chains.
  • the transgenic mice are immunized in the normal fashion with a selected antigen.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma.technology.
  • the human immunoglobulin transgenes harbored in the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Using this technique, therapeutically useful IgG, IgA, IgE, or other antibody isotypes can be prepared.
  • phage display technology can be used to produce intact human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V immunoglobulin variable
  • Phage display can be performed in a variety of formats. See, e.g., Johnson, Kevin S. and Chiswell, David J., CURRENT OPINION IN STRUCTURAL BIOLOGY, 3:564-571(1993).
  • V-gene segments can be used for phage display.
  • a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. MOL. BiOL., 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).
  • human antibodies can be generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).
  • Multispecif ⁇ c e.g., bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • 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. 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. 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 (CHl) 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.
  • 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.
  • This asymmetric structure can facilitate 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.
  • Multispecific (e.g., bispecific) antibodies also encompass cross-linked or
  • heteroconjugate antibodies For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. Multispecific antibodies can also be produced from recombinant cell culture. See, e.g., Kostelny et al., J. IMMUNOL., 148(5): 1547-1553 (1992).
  • Reduced antibodies are a reduced form of immunoglobulin composed of one complete light chain and one complete heavy chain connected by disulfide bonds. It is essentially one-half of an intact immunoglobulin molecule and contains a single antigen- binding site. Reduced antibodies can be formed by the selective reduction of disulfide bonds in the hinge region of an antibody.
  • An Fv fragment contains a complete antigen-binding site which includes a V L domain and a V H domain held together by non-covalent interactions.
  • the present invention also features Fv fragments in which the V L and V H domains are cross linked through glutaraldehyde, intermolecular disulfides, or other linkers.
  • variable domains of the heavy and light chains can be fused together to form a single chain variable fragment (scFv), which retains the original specificity of the parent immunoglobulin.
  • the V L domain is connected to the V H domain by a flexible peptide linker of 5-30 amino acids in length. More preferably, the V L domain is connected to the V H domain by a flexible peptide linker of 10-20 amino acids in length. Highly preferably, the V L domain is connected to the V H domain by a flexible peptide linker of about 15 amino acids in length. Linkers with less than 5, or more than 30, amino acid residues may also be used, provided that they enables the scFv to form the desired structure for antigen binding.
  • Diabodies are small antibody fragments with two antigen-binding sites, where each fragment comprises a variable heavy domain (V H ) connected to a variable light domain (VL) in the same polypeptide chain (V H -V L ).
  • V H variable heavy domain
  • VL variable light domain
  • Triabodies can be similarly constructed with three antigen-binding sites.
  • the basic unit of a preferred minibody comprises a V L and a V H domain. In many cases, the basis unit of a minibody also comprises one or more C H or C L domain.
  • Each minibody may include 1, 2 or more such units to form 1, 2, or more antigen-binding sites.
  • the antibodies of the present invention comprise modified C- or N-termini that are enriched with lysine or tyrosine residues, hi many embodiments, the antibodies of the present invention have an N- or C-terminal sequence consisting of 2 to 30 amino acid residues among which at least two residues are lysine or tyrosine.
  • the N- or C- terminal sequence can consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, and at least one-third, one-half, or two-third of these residues are lysine or tyrosine.
  • the N- or C-terminal sequence consists of 2-20, 2-15, 2-10, 2-8, 2-6, 3-20, 3-15, 3-10, 3-8, 3-6, 4-20, 4-15, 4-10, 4- 8, 4-6, 5-20, 5-15, 5- 10, 5-8, 6-20, 6-15, 6-10, or 6-8 amino acid residues, among which at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues are lysine or tyrosine, hi another example, the N- or C-terminal sequence consists of 3, 4, 5, 6, 7, or 8 amino acid residues, among which at least 2, 3, 4, 5, 6, 7, or 8 residues are lysine or tyrosine.
  • the majority of the residues (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues) in the N- or C-terminal sequence are lysine.
  • the majority of the residues (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues) in the N- or C-terminal sequence are tyrosine.
  • the majority of the residues in the N- or C-terminal sequence consists of a mixture of lysine and tyrosine residues (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues).
  • terminal sequences can also be added to the antigen- binding fragments of intact antibodies.
  • the lysine/tyrosine-enriched sequence is introduced to a C-terminus of an antibody of the present invention.
  • the lysine/tyrosine- enriched sequence can also be added to an N-terminus of the antibody, provided that the addition of the sequence does not significantly change the antigen-binding property of the antibody.
  • Methods suitable for adding sequences to the N- or C-termini of antibodies or their fragments are well known in the art. Suitable methods include standard recombinant DNA methods, or the use of transgenic hosts (e.g., rat, mice, cow, dog, sheep, goat, guinea pig, rabbit, macaque, chimpanzee, or other non-human primates) in which one or more genes encoding the light or heavy chain(s) are modified.
  • the lysine/tyrosine-enriched terminal sequences can also be chemically linked to the ends of antibodies with desired antigen- binding specificities.
  • a peptide linker can be fused between the lysine/tyrosine-enriched sequence and the antibody terminus to increase the accessibility of the terminal lysine or tyrosine residues.
  • a peptide linker has about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues.
  • a lysine/tyrosine-enriched sequence of the present invention is added within the sequence of an antibody.
  • a lysine/tyrosine- enriched sequence can be introduced into the junction region between a variable domain and a constant domain, or between two constant domains.
  • a lysine/tyrosine-enriched sequence can also be introduced within a variable or constant domain, provided that the added sequence does not significantly affect the antigen-binding property or the stability of the antibody.
  • the lysine/tyrosine-enriched terminal sequences can be conjugated with radioactive isotopes, cytotoxic agents, or prodrugs.
  • Suitable radioactive isotopes include, but are not limited to, At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , and Lu 177 .
  • Suitable cytotoxic agents include, but are not limited to, chemotherapeutic agents, toxins, or other substances that can inhibit or prevent the function of cells or causes destruction of cells.
  • Non-limiting examples of chemotherapeutic agents include 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, 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 car
  • paclitaxel (TAXOL ® , Bristol-Myers Squibb Oncology, Princeton, NJ.) and docetaxel (TAXOTERE ® , Sanofi- aventis); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-I l; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; hormones (such as steroids); anthracycline; vinca alkaloids;
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide.
  • Toxins suitable for the present invention include small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • Nonrlimiting examples of suitable toxins include mitomycin C, pingyangmycin, calicheamicin, maytansine (U.S. Patent No. 5,208,020), trichothene, and CC 1065.
  • the lysine/tyrosine-enriched terminal sequences can also be used to conjugate prodrugs.
  • a prodrug is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., U.S. Patent No. 4,975,287, which is incorporated herein by reference in its entirety.
  • Non-limiting examples of prodrugs include phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate- eontaining prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
  • the lysine/tyrosine-enriched terminal sequences can be used to conjugate prodrug-activating enzymes which convert prodrugs (e.g. apeptidyl chemotherapeutic agent) to active anti-cancer drugs.
  • prodrugs e.g. apeptidyl chemotherapeutic agent
  • Enzymes that are suitable for this purpose 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 non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-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, ⁇ -lac
  • the lysine/tyrosine-enriched terminal sequences can be conjugated with one type of therapeutic agents (e.g., radioactive isotopes, chemotherapeutic agents, toxins, or prodrugs).
  • the lysine/tyrosine-enriched terminal sequences can also be conjugated with two or more different types of therapeutic agents (e.g., radioactive isotopes + chemotherapeutic agents, radioactive isotopes + toxin, radioactive isotopes + prodrugs, chemotherapeutic agents/toxins + prodrugs, or radioactive isotopes + chemotherapeutic agents/toxins + prodrugs).
  • two or more different agents of the same type can be coupled to a lysine/tyrosine-enriched terminal sequence.
  • Conjugation of lysine/tyrosine residues with desired agents can be made using a variety of bifunctional or multifunctional cross-linking agents.
  • Non-limiting examples of suitable cross-linking agents include SPDP (N-succinimidyl-3-(2-pyridyldithiol)propionate), EDC (l-Emyl-S-P-dimethylaminopropylfcarbodiimide Hydrochloride), AEDP (3-[(2- Aminoethyl)dithio]propionic acid «HCl), ASBA (4-[p-Azidosalicylamido]butylamine), DCC, BMPS (JV-[ ⁇ -Maleimidopropyloxy]succinimide ester), EMCS ([N-e- Maleimidocaproyloxy]succinimide ester), LC-SMCC (Succinimidyl-4-[N- Maleimidomethyl]cyclohexane-l-carboxy-[6-amidocaproate]), LC-SPDP (Succinimidyl 6-(3- [2-pyridyl
  • the hetero-bifunctional cross-linkers employed contain two reactive groups: one generally reacting with primary amine group (e.g., N-hydroxy succinimide) and the other generally reacting with a thiol group (e.g., pyridyl disulfide, maleimides, or halogens.).
  • primary amine group e.g., N-hydroxy succinimide
  • a thiol group e.g., pyridyl disulfide, maleimides, or halogens.
  • the cross-linker can react with the lysine residue(s) of the antibody and through the thiol reactive group, the cross- linker, already tied up to the antibody, reacts with the cysteine residue (or other free sulfhydryl group) on a desired agent (e.g. a cytotoxic or anti-cellular agent).
  • Antibodies and the agents to be coupled generally have, or are derivatized to have, functional groups available for cross-linking purposes. This.requirement is not considered to be limiting in that a wide variety of groups can be used in this manner. For example, primary or secondary amine groups, hydrazide or hydrazine groups, carboxyl f alcohol, phosphate, or alkylating groups maybe used for binding or cross-linking.
  • Lysinyl (or amino-terminal) residues are reactive with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing ammo-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.
  • Modification of tyrosyl residues can be made by reaction with aromatic diazonium compounds or tetranitromethane. For instance, N- acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3- nitro derivatives, respectively.
  • Tyrosyl residues can also be iodinated using I 125 or I 131 .
  • the lysine/tyrosine residues employed in the present invention can be replaced by cysteine, histidine, arginine, aspartate, or glutamate residues.
  • Cross-linking agents suitable for the conjugation of these residues to the desired therapeutic or diagnostic agents include, but are not limited to, APG (p-Azidophenyl glyoxal monohydrate), ABH (p- Azidobenzoyl hydrazide), TFCS (N-[e-Trifluoroacetylcaproyloxy]succinimide ester), MPBH 4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride, PDPH (3-(2- Pyridyldithio)propionyl hydrazide), and EMCH ([N-e-Maleimidocaproic acid]hydrazide).
  • APG p-Azidophenyl glyoxal monohydrate
  • ABH p- Azidobenzoyl hydrazide
  • TFCS N-[e-Trifluoroacetylcaproyloxy]succinimide ester
  • the antibodies of the present invention can be purified to remove contaminants such as unconjugated cytotoxic agents or antibodies. In many cases, it is important to remove unconjugated cytotoxic agents because of the possibility of increased toxicity. Moreover, unconjugated antibodies may be removed to avoid the possibility of competition for the antigen between conjugated and unconjugated species. Numerous purification techniques can be used to provide conjugates to a sufficient degree of purity to render them clinically useful.
  • the present invention also features antibodies that are conjugated to a
  • receptor such as streptavidin
  • a "ligand” e.g., avidin
  • cytotoxic agent e.g., a radio nuclide
  • the antibodies of the present invention can be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., PROC. NATL. ACAD. SCL USA, 82:3688 (1985); Hwang et al., PROC. NATL. ACAD. SCI.
  • an immunoliposome of the present invention is 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.
  • Antibody fragments e.g., Fab'
  • an antibody of the present invention specifically recognizes an antigen selected from the group consisting of tumor-associated antigens, e.g., CEA (carcinoembryonic antigen), CAl 9-9 (cancer antigen 19-9), CAl 25 (cancer antigen 125), PSA (prostate specific antigen), beta-HCG (Human chorionic gonadotropin), AFP (Alpha-fetoprotein), AFP-L3 (a lectin-reactive AFP), or thyroglobulin); cell-surface receptors (e.g., growth factor receptors, hormone receptors, G-protein coupled receptors, neurotransmitter receptors, tyrosine kinase receptors, guanylyl cyclase receptors, or ionotropic receptors); ion channels (e.g.
  • tumor-associated antigens e.g., CEA (carcinoembryonic antigen), CAl 9-9 (cancer antigen 19-9), CAl 25 (cancer antigen 125
  • Non-limited examples of preferred antigens that can be recognized by the antibodies of the present invention include antigens from the following tumors: B-cell lymphoma (e.g., CD19, CD20, CD22, CD25, or HLA DR); Hodgkin's lymphoma (e.g., CD30); acute myeloid leukaemia (e.g., CD33 or CD45); lymphoid disorders (B-cell and T-cell) (e.g., CD52); epithelial tumors (e.g., carcinoembryonic antigen); epithelial tumors (breast, lung, and colon) (e.g., tumor-associated glycoprotein TAG72, epithelial-cell adhesion molecule, or polymorphic epithelial mucin MUCl); colorectal cancer (e.g., A33); clear-cell renal carcinoma (e.g., G250); neuroectodermal tumors (e.g., gangliosides GD2,
  • An antibody of the present invention can also be selected to be specific to a pathogen, such as virus, bacterium, yeast, fungus, parasite, or other pathogenic or infectious microbes or organisms.
  • the present invention features antibodies against the antigens of the following non-viral organisms: Streptococcus (e.g., Streptococcus agalactiae, Streptococcus pyogenes, Group C streptococci (beta hemolytic, occasionally alpha or gamma, e.g., Streptococcus anginosus or Streptococcus equismilis), Group D streptococci (alpha or gamma hemolytic, occasionally beta, e.g., Streptococcus bovis), Group E streptococci, Group F streptococci (beta hemolytic, e.g., Streptococcus anginosus), Group G streptococci (beta hemolytic,
  • Salmonella e.g., Salmonella typhi or Salmonella typhimurium
  • Shigella e.g., Shigella dysenteriae or Shigella boydii
  • Treponema e.g., Treponema pallidum
  • Vibrio e.g., Vibrio cholerae
  • Yersinia e.g., Yersinia enterocolitica or Yersinia pestis
  • protozoan species selected from Eimeria, Anaplasma, Giardia, Babesia, Trichomonas, Entamoeba, Balantidium, Plasmodium, Leishmania, Toxoplasma, Trypanosoma, Entamoeba, Trichomonas, Toxoplasmosa, or Pneumocystis
  • fungal species selected from Blastomyces, Microsporum, Aspergillis, Candida, Coccidiodes, Cryptococcus, Histoplasm
  • the present invention also features antibodies against antigens of the following viruses: Paramyxoviridae (e.g., pneumovirus, morbillivirus, metapneum ⁇ virus, respirovirus or rubulavirus); Adenoviridae (e.g., adenovirus); Arenaviridae (e.g., arenavirus such as lymphocytic choriomeningitis virus); Arteriviridae (e.g., porcine respiratory and reproductive syndrome virus or equine arteritis virus); Bunyaviridae (e.g., phlebovirus or hantavirus); Caliciviridae (e.g., Norwalk virus); Coronaviridae (e.g., coronavirus or torovirus); Filoviridae (e.g., Ebola-like viruses); Flaviviridae (e.g., hepacivirus or flavivirus); Herpesviridae (e.g., simplex
  • Preferred viral antigens include, but are not limited to, antigens from human immunodeficiency virus (HIV), human respiratory syncytial virus, influenza, herpes simplex virus 1. and 2, measles virus, hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus (HCV), smallpox virus, polio virus, west Nile virus, coronavirus associated with severe acute respiratory syndrome (SARS), rotavirus, papilloma virus, papillomaviruses, Epstein-Barr virus (EBV), human T- cell lymphotropic virus type I, and Kaposi's sarcoma-associated herpesvirus.
  • HCV human immunodeficiency virus
  • HCV hepatitis B virus
  • HCV hepatitis C virus
  • smallpox virus smallpox virus
  • polio virus west Nile virus
  • rotavirus pap
  • an antibody of the present invention specifically recognizes a surface or envelope antigen on the targeted pathogen, such as hepatitis B virus surface antigen (HBVsAg), HIV gpl20 or gp41, and SARS associated coronavirus envelope protein.
  • a surface or envelope antigen on the targeted pathogen such as hepatitis B virus surface antigen (HBVsAg), HIV gpl20 or gp41, and SARS associated coronavirus envelope protein.
  • Antibodies against other viral surface/envelope antigens as described in the U.S. provisional application Serial No. 60/760,383, filed January 20, 2006, and entitled "hnmunoconjugates for Treatment of Infectious Diseases," the entire content of which is incorporated herein by reference, are also contemplated by the present invention.
  • antibodies of the present invention include antibodies against the following antigens: ErbB receptors (e.g., EGFR/ErbBl, HER2/ErbB2/pl85 neu , HER3/ErbB3, or HER4/ErbB4/tyro2); BMPRlB (bone morphogenetic protein receptor-type IB); E16 (LATl or SLC7A5); STEAPl (six transmembrane epithelial antigen of prostate); 0772P (CA125 or MUCl 6); MPF (MSLN, SMR, or megakaryocyte potentiating factor); Napi3b (NAPI-3B, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, or type II sodium-dependent phosphate transporter 3b); Sema 5b (FLJl 0372, KIAAl 445, Mm.42015, or SEMA5B); PSCA hlg (270005 OC 12Rik or C
  • tumor necrosis factor receptor superfamily, member 13C CD22; CD 79a; CXCR5; beta subunit of MHC class II molecule (Ia antigen); P2X5 (purinergic receptor P2X, ligand-gated ion channel, 5); CD72; CDl 80; FcRHl (Fc receptor-like protein 1); IRTA2 (FCRL5 or Fc receptor-like 5); or TENB2 (TMEFF2, or transmembrane protein with EGF-like and two follistatin-like domains 2).
  • antibodies of the present invention include anti-VEGF, anti-VEGFR, anti-CEA, anti-CTLA-4, anti-CD4, anti-CD3, anti-CD20, anti-TNF-a, anti-CD 1 Ia, anti-Lewis Y antigen, anti-TrailR, anti-IL2R, anti- CD30, anti-CD 146, anti-CD 147, anti-alpha V integrin beta, anti-CD 19, and anti-GD2, anti- EBV, anti-HIV, anti-HBV, and anti-HCV.
  • the present invention further features pharmaceutical compositions comprising the antibodies of the present invention.
  • a typical pharmaceutical composition of the present invention can be prepared by mixing an antibody of the present invention having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, 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) polypeptid
  • 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
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • Supplementary active ingredients also can be incorporated into the pharmaceutical compositions of the present invention.
  • a pharmaceutical composition of the invention includes a therapeutically effective amount of an antibody comprising a modified N- or C-terminal sequence enriched with lysine or tyrosine residues.
  • a "therapeutically effective amount” refers to an amount of an antibody effective to treat a disease or disorder in a mammal, hi the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (e.g., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; inhibit to some extent tumor growth; or relieve to some extent one or more of the symptoms associated with the cancer.
  • an antibody of the present invention may prevent growth or kill existing cancer cells, it maybe cytostatic or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) or determining the response rate (RR).
  • Cancers that can be treated using the antibodies of the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, and lymphoid malignancies.
  • Specific examples of cancers include squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer
  • the antibodies of the present invention can be combined with other traditional therapies to treat cancers.
  • Agents or factors suitable for use in a combined therapy include many chemical compounds or treatment methods that induce DNA damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
  • Unconjugated chemotherapeutic agents can also be used in combination with an antibody of the present invention.
  • the active ingredient(s) in a pharmaceutical composition of the present invention can 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.
  • 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.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), 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 ⁇ oly-D-(-)-3- hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)
  • polylactides U.S. Patent No. 3,773,919
  • copolymers of L-glutamic acid and ⁇ -ethyl-L-glutamate non-de
  • a pharmaceutical composition of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts formed with the free amino groups of the protein or formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • compositions or solutions Upon formulation, compositions or solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution can be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure-
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES (15th Edition), pages 1035- 1038 and 1570- 1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • Administration of a pharmaceutical composition of the present invention can be by way of any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intratumoral, circumferentially, catheterization, or intravenous injection.
  • a pharmaceutical composition can also be administered to a subject of interest
  • Solutions of the active compounds as free base or pharmacologically-acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form is sterile and fluid to the extent that easy syringability exists. It is preferably also stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, or vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, or by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium phosphate, sodium sorbic acid, thimerosal or the like.
  • isotonic agents for example, sugars or. sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating an antibody of the present invention in the required amount in an appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle, which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • an antibody of the present invention can be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • an antibody of the present invention may be incorporated into an anti-septic wash containing sodium borate, glycerin and potassium bicarbonate.
  • An antibody of the present invention may also be dispersed in dentifrices, including: gels, pastes, powders, or slurries.
  • An antibody of the present invention may be added in a therapeutically or prophylactically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, or humectants.
  • the cells to be treated can be cancer cells or other disease or dysfunctional cells.
  • the cells to be treated can also be healthy cells.
  • the cells or tissues to be treated can also be infected with pathogens, such as viruses, bacteria, parasites, yeast, fungi, or other disease-causing microbes, germs, or worms.
  • the antibodies employed can specifically recognize an antigen of the target cell or pathogen.
  • the antigen is a surface antigen, such as an epitope in a cell surface protein, lipid, or saccharide, or in a viral envelope or surface protein.
  • the antibodies of the present invention can also be used to kill viruses, bacteria, or other pathogens in vitro, through contacting or binding to these pathogens.
  • the antibodies of the present invention can be used in vitro to kill or modulate the growth or activity of cells infected with these pathogens.
  • the present invention also features diagnostic kits comprising the antibodies of the present invention.
  • the lysine/tyrosine-enriched end(s) of these antibodies can be used to conjugate detectable labels.
  • Detectable labels suitable for this purpose include a variety of compositions that are detectable by spectroscopic, photochemical, biochemical, immnunochemical, electrical, optical or chemical means.
  • Non-limiting examples include • magnetic beads, fluorescent dyes (e.g., fluorescein isofhiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., H 3 , 1 125 , S 35 , C 14 , or P 32 ), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isofhiocyanate, Texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels e.g., H 3 , 1 125 , S 35 , C 14 , or P 32
  • enzymes e.g., horse radish peroxidase, alkaline phosphat
  • Radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • the detection typically involves contacting an antibody of the present invention with a biological sample or a tissue, followed by the detection of binding of the antibody to the target antigen to determine the level of the antigen in the biological sample or tissue.
  • Suitable biological samples can be prepared from biological tissues or fluids.
  • Such samples include, but are not limited to, tissue from biopsy, sputum, amniotic fluid, blood, and blood cells (e.g., white cells).
  • Biological samples can also include sections of tissues, such as frozen sections taken for histological purposes.
  • a biological sample is typically obtained from a multicellular eukaryote, preferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and most preferably a primate such as macaques, chimpanzees, or humans.
  • a diagnostic kit of the present invention further comprises other reagents (including buffers or control reagents) for conducting immunoassays.

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Abstract

L'invention concerne des anticorps thérapeutiques ou diagnostiques comportant des séquences N-terminales ou C-terminales modifiées qui sont enrichies en résidus lysine ou tyrosine. Ces résidus lysine ou tyrosine peuvent être utilisés pour coupler des radio-isotopes, des agents cytotoxiques ou des marqueurs décelables. Les rapports stoechiométriques accrus de ces agents dans les conjugués d'anticorps permettent d'améliorer l'efficacité thérapeutique ou d'augmenter la sensibilité de détection. Des exemples non limitatifs d'anticorps adaptés pour la présente invention comprennent les anticorps anti-CD22, anti-ErbB2, anti-VEGF, anti-EGFR, anti-VEGFR, anti-Her-3, anti-Her-4, anti-CEA, anti-CTLA-4, anti-CD4, anti-CD3, anti-CD20, anti-TNF-a, anti-CD11a, anti-antigène Lewis Y, anti-TrailR, anti-IL2R, anti-CD30, anti-CD146, anti-CD147, anti-intégrine alpha V bêta, anti-CD19, anti-GD2, anti-3H11, anti-EBV, anti-HIV, anti-HBV, anti-HCV, ainsi que d'autres anticorps spécifiques de maladies.
PCT/US2007/001483 2006-01-20 2007-01-19 Immunoconjugués présentant une efficacité accrue pour le traitement de maladies WO2007084693A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2211904A2 (fr) * 2007-10-19 2010-08-04 Seattle Genetics, Inc. Agents de liaison au cd19 et utilisations de ceux-ci
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WO2022153898A1 (fr) * 2021-01-13 2022-07-21 デンカ株式会社 Procédé de mesure d'antigène cible, particules insolubles et kit de mesure d'antigène cible utilisé dans ce procédé

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EP2211904A2 (fr) * 2007-10-19 2010-08-04 Seattle Genetics, Inc. Agents de liaison au cd19 et utilisations de ceux-ci
CN101903403A (zh) * 2007-10-19 2010-12-01 西雅图基因公司 Cd19结合剂及其应用
JP2011500725A (ja) * 2007-10-19 2011-01-06 シアトル ジェネティクス，インコーポレーテッド Ｃｄ１９結合性物質およびその使用
EP2211904A4 (fr) * 2007-10-19 2011-10-19 Seattle Genetics Inc Agents de liaison au cd19 et utilisations de ceux-ci
US8242252B2 (en) 2007-10-19 2012-08-14 Seattle Genetics, Inc. CD19 binding agents and uses thereof
US9073993B2 (en) 2007-10-19 2015-07-07 Seattle Genetics, Inc. CD19 binding agents and uses thereof
JP2015129131A (ja) * 2007-10-19 2015-07-16 シアトル ジェネティクス，インコーポレーテッド Ｃｄ１９結合性物質およびその使用
US11331394B2 (en) 2007-10-19 2022-05-17 Seagen Inc. CD19 binding agents and uses thereof
RU2537175C2 (ru) * 2013-03-26 2014-12-27 Российская Федерация, От Имени Которой Выступает Министерство Образования И Науки Российской Федерации Способ получения радиоиммунного препарата для диагностики и терапии онкологических заболеваний
WO2022153898A1 (fr) * 2021-01-13 2022-07-21 デンカ株式会社 Procédé de mesure d'antigène cible, particules insolubles et kit de mesure d'antigène cible utilisé dans ce procédé

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