EP0881910A1 - Utilisation d'immunoconjugues pour accroitre l'efficacite des vaccins multietapes stimulant les reactions immunitaires en cascade - Google Patents

Utilisation d'immunoconjugues pour accroitre l'efficacite des vaccins multietapes stimulant les reactions immunitaires en cascade

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Publication number
EP0881910A1
EP0881910A1 EP96943706A EP96943706A EP0881910A1 EP 0881910 A1 EP0881910 A1 EP 0881910A1 EP 96943706 A EP96943706 A EP 96943706A EP 96943706 A EP96943706 A EP 96943706A EP 0881910 A1 EP0881910 A1 EP 0881910A1
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Prior art keywords
antibody
vaccine
cea
administering
mammal
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EP96943706A
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German (de)
English (en)
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EP0881910A4 (fr
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Hans J. Hansen
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Immunomedics Inc
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Immunomedics Inc
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Priority claimed from US08/577,106 external-priority patent/US7354587B1/en
Application filed by Immunomedics Inc filed Critical Immunomedics Inc
Priority to EP09002533A priority Critical patent/EP2057999A3/fr
Publication of EP0881910A1 publication Critical patent/EP0881910A1/fr
Publication of EP0881910A4 publication Critical patent/EP0881910A4/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • 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
    • A61K39/0005—Vertebrate antigens
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001182—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to methods for inducing humoral and cellular immune responses against malignant cells and infectious agents.
  • this invention is directed to methods for producing an integrated i munologic response against tumor cells or infectious agents using immunoconjugates compri ⁇ ing antibodies and anti-idiotype antibodies that mimic an epitope of an antigen that is associated with a tumor or infectious agent.
  • the present invention also is directed to a method for augmenting such an integrated response using immunoconjugates, antibodies, anti-idiotype antibodies and cytokines.
  • TAA tumor associated antigens
  • CEA carcinoembryonic antigen
  • Ab2 anti-idiotype antibodies
  • Goldenberg Amer. J . Med . 94 : 297 (1993).
  • Ab2 are antibodies directed against the variable regions of conventional antibodies (Abl) . Since Ab2 and antigen can bind with the same regions of the Abl-combining site, certain Ab2 (termed “Ab2 ⁇ ” or "internal-image” antibodies) can mimic the three dimensional structure of the nominal antigen. Jerne et al ., EMBO J . 1 : 243 (1982); Losman et al . , Int . J. Cancer 46 : 310 (1990); Losman et al . , Proc . Nat 'l Acad .
  • antigen mimicry properties of anti-idiotype antibodies have led to the use of Ab2 ⁇ as surrogate antigens (or idiotype vaccines) , when the nominal antigen is not readily available or when the host is tolerant to the nominal antigen.
  • immunization with Ab2fi mimicking certain TAA creates specific immunity to the TAA and protect against subsequent tumor growth. See, for example, Nepom et al . , Proc . Nat 'l Acad . Sci . USA 81 : 2864 (1984); Raychaudhuri et al., J . Immunol . 139 : 271 (1987).
  • anti- idiotype vaccines have been developed against infectious organisms, such as Streptococcus pneumoniae [McNamara et al . , Science 226 : 1325 (1984)], hepatitus B virus [Kennedy et al . , Science 223 : 930 (1984) ], Escherichia coli K13 [Stein et al ., J . Exp . Med . 160 : 1001 (1984)], Schistosomiasis man ⁇ oni [Kresina et al . , J . Clin . Invest 83 : 912 (1989)], and Moloney murine sarcoma virus [Powell et al., J. Immunol . 142 : 1318 (1989)].
  • Cancer patients receiving an anti-TAA of animal origin will usually produce antibodies to the Abl and these anti-immunoglobulin antibodies include Ab2.
  • the anti-idiotype response also may include the generation of T cells (T2) . Fager erg et al . , Cancer Immunol . Immunother. 37 : 264 (1993) .
  • Ab2 may subsequently induce a humoral and cellular anti-anti-idiotypic response, Ab3 and T3, respectively, which may recognize the same epitope as Abl. Id .
  • Another object of this invention i ⁇ to provide methods for inducing humoral and cellular immune responses in a mammal against a tumor that expresses a tumor associated antigen comprising the administration of a vaccine comprising an antibody component that binds with the HLA-DR-complex and an antigenic peptide that induces a major histocompatibility (MHC) -restricted immune response.
  • a vaccine comprising an antibody component that binds with the HLA-DR-complex and an antigenic peptide that induces a major histocompatibility (MHC) -restricted immune response.
  • MHC major histocompatibility
  • a method for inducing humoral and cellular immune responses in a mammal against a tumor that expresses a tumor associated antigen (TAA) or against a disease caused by an infectious agent comprising: (a) administering a first vaccine intradermally to the mammal, wherein the first vaccine comprises an immunoconjugate that comprises: (i) an antibody component that binds with the HLA-DR-complex, and ( ⁇ ) an antigenic peptide, wherein the antigenic peptide comprises at least one epitope of a TAA or an antigen associated with the infectious agent, and
  • the antibody component of the fir ⁇ t vaccine may be selected from the group consisting of (a) a murine monoclonal antibody; (b) a humanized antibody derived from a murine monoclonal antibody; (c) a human monoclonal antibody; and (d) an antibody fragment derived from (a) , (b) or (c) , wherein the antibody fragment is selected from the group consisting of F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, sFv and minimal recognition unit.
  • the present invention also is directed to a method further comprising administering at least one of interferon-7, interleukin-2, or interleukin-12 prior to and during the administering of the vaccine intravenously to the mammal.
  • the present invention is further directed to a method for inducing humoral and cellular immune responses in a mammal against a tumor that expresses a tumor associated antigen (TAA) , comprising: (a) administering a first vaccine intradermally to the mammal, wherein the first vaccine comprises an immunoconjugate that comprises: (I) an antibody component that binds with the HLA-DR-complex, and
  • the antibody component of the first vaccine is selected from the group consisting of (a) a murine monoclonal antibody; (b) a humanized antibody derived from a murine monoclonal antibody; (c) a human monoclonal antibody; and (d) an antibody fragment derived from (a) , (b) or (c) , in which the antibody fragment is selected from the group consisting of F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, sFv and minimal recognition unit.
  • a suitable antigenic peptide for example, is tetanus toxin P2 peptide.
  • the present invention also is directed to a method further comprising administering at least one of ⁇ nterferon-7, mterleuk ⁇ n-2, or inte ieukm-12 prior to and during the administering of the vaccine intravenously to the mammal.
  • the present invention is further directed to a method comprising administering a second vaccine intravenously to the mammal, wherein the second vaccine comprises an immunoconjugate that comprises:
  • the present invention also is direct to a method further comprising administering at least one cytokine selected from the group consisting of ⁇ nterleukm-2 , mterleukm-12 and interferon-7 prior to and during the administering of the second vaccine intravenously to the mammal.
  • the present invention also is directed to a method for inducing humoral and cellular immune responses in a mammal against a tumor that expresses carcinoembryonic antigen (CEA) , comprising:
  • the second vaccine comprises an anti- idiotype antibody component that mimics an epitope of the CEA, and wherein the anti- idiotype antibody component is conjugated with a soluble immunogenic carrier protein, and
  • the third vaccine comprises an immunoconjugate comprising an antigenic peptide that comprises an epitope of CEA, and an antibody component that binds with the HLA-DR complex.
  • a suitable antigenic peptide of the third vaccine comprises the A3B3 domain of CEA.
  • the antigenic peptide of the third vaccine can comprise a minimal recognition unit of an anti-idiotype antibody that mimics an epitope of CEA.
  • the antibody component of the first vaccine is selected from the group consisting of:
  • the anti-idiotype antibody component is selected from the group consisting of: (a) a polyclonal antibody that binds with the variable region of a Class III anti-CEA antibody;
  • the present invention also is directed to methods further comprising administering at lea ⁇ t one of interferon-7, interleukin-2, or interleukin-12 prior to and during the administration of the second vaccine.
  • a structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • a promoter is a DNA sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene, proximal to the transeriptional start site of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • An isolated DNA molecule is a fragment of DNA that is not integrated in the genomic DNA of an organism.
  • a cloned T cell receptor gene is a DNA fragment that has been separated from the genomic DNA of a mammalian cell.
  • Another example of an isolated DNA molecule is a chemically-synthesized DNA molecule that is not integrated in the genomic DNA of an organism.
  • An enhancer is a DNA regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • cDNA Complementary DNA
  • cDNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA refers to a double- stranded DNA molecule consisting of such a single- stranded DNA molecule and its complementary DNA strand.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • a cloning vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • An expression vector is a DNA molecule comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked to" the regulatory elements.
  • a recombinant host may be any prokaryotic or eukaryotic cell that contains either a cloning vector or expression vector. This term also include ⁇ those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • a tumor associated antigen is a protein normally not expressed, or expressed at very low levels, by a normal counterpart.
  • tumor associated antigens include ⁇ -fetoprotein and carcinoembryonic antigen (CEA) .
  • an infectious agent denotes both microbe ⁇ and parasites.
  • a "microbe” includes viruses, bacteria, rickettsia, ycoplasma, protozoa, fungi and like microorganisms.
  • a "parasite” denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to antibody-induced clearance or lytic or phagocytic destruction, such a ⁇ malarial parasites, spirochetes, and the like.
  • an anti-CEA MAb is a Class III MAb, a ⁇ described by Primu ⁇ et al . , Cancer Research 43 : 686 (1983) and by Primus et al . , U.S. patent No. 4,818,709, which are incorporated by reference.
  • an Abl is an antibody that binds with a tumor associated antigen or an antigen as ⁇ ociated with an infectious agent.
  • An anti-idiotype antibody is an antibody that binds with an Abl. Importantly, an Ab2 binds with the variable region of Abl and thus, an Ab2 mimics an epitope of a tumor associated antigen or an epitope of an infectious agent associated antigen.
  • An antibody fragment is a portion of an antibody such a ⁇ F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-CEA Mab (Abl) fragment binds with CEA, while an Ab2 fragment binds with the variable region of the Abl and mimics an epitope of CEA.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant ⁇ ingle chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("sFv proteins”) , and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of MAb have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • antibody component includes both an entire antibody and an antibody fragment.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. See , for example, Kohler and Milstein, Nature 256 : 495
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • CEA-related antigen ⁇ The major members of this family of CEA-related antigen ⁇ are (1) the normal cross-reactive antigen (NCA) , which shares a similar ti ⁇ ue di ⁇ tribution with CEA, and (2) meconium antigen (MA) , which shares almost identical physiochemical properties with CEA.
  • NCA normal cross-reactive antigen
  • MA meconium antigen
  • the first panel of monoclonal antibodie ⁇ (MAb) that defined NCA-cross-reactive, MA-cross-reactive, and CEA- ⁇ pecific epitopes on the CEA molecule were described by Primus et al . , Cancer Research 43 : 686 (1983) .
  • three classes of anti-CEA antibody were identified: 1) Class I antibodies, which react with CEA, NCA and MA;
  • Class II antibodies which react with CEA and MA, but not with NCA
  • Class III antibodies which are ⁇ pecific for CEA and do not bind with NCA or MA.
  • Methods for obtaining Class III anti-CEA MAb ⁇ are disclosed by Primus et al . , Cancer Research 43 : 686 (1983), and Primus et al . , U.S. patent No. 4,818,709.
  • the production of second generation Class III anti-CEA MAbs i ⁇ disclosed by Hansen et al . , Cancer 71 : 3478 (1993) , which is incorporated by reference.
  • MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclu ⁇ ion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al . , "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992) .
  • an antibody of the present invention is a subhuman primate antibody.
  • an antibody of the present invention is a "humanized" monoclonal antibody. That is, mouse complementarity determining regions are transferred from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, followed by the replacement of some human residues in the framework regions of their murine counterparts.
  • Humanized monoclonal antibodies in accordance with this invention are suitable for use in therapeutic methods. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al . , Proc . Nat ' l Acad . Sci . USA 86 : 3833 (1989) , which i ⁇ incorporated by reference in its entirety.
  • an antibody of the present invention is a human monoclonal antibody.
  • Such antibodies are obtained from tran ⁇ genic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are de ⁇ cribed by Green et al . , Nature Genet . 7 : 13 (1994) , Lonberg et al . , Nature 368 : 856 (1994), and Taylor et al . , Int . Immun . 6 : 579 (1994), which are incorporated by reference.
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of the DNA coding for the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragment ⁇ can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • Fv fragments comprise an association of V H and V L chains. This association can be noncovalent, as described in Inbar et al . , Proc . Nat 'l Acad . Sci . USA 69 : 2659 (1972) .
  • the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde. See, for example, Sandhu, supra .
  • the Fv fragment ⁇ compri ⁇ e V H and V L chains which are connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is sub ⁇ equently introduced into a host cell, such as E . coli . The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al .
  • Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR) .
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest.
  • Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody- producing cells. See, for example, Larrick et al . , Methods : A Companion to Methods in Enzymology 2 : 106 (1991) ; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al . (eds.) , pages 166-179 (Cambridge University Pres ⁇ 1995) ; and Ward et al .
  • Polyclonal Ab2 can be prepared by immunizing animals with Abl or fragments, using standard techniques. See, for example, Green et al . , "Production of Polyclonal
  • monoclonal Ab2 can be prepared u ⁇ ing
  • humanized Ab2 or subhuman primate Ab2 can be prepared using the above-described techniques. 5. Production of Bispecific Antibodies
  • Bispecific antibodies can be used to recruit and target T cells to a tumor cell.
  • a bispecific antibody is a hybrid molecule that consists of nonidentical light and heavy chain pairs, providing two distinct antibody specificities.
  • bispecific antibodies have been produced with one binding site recognizing the CD3 signal transducing protein on T cells and a second binding site for a tumor-associated antigen. See, for example, Canevari et al . , Int . J . Cancer 42 : 18 (1988); Lanzaveccia et al . , Eur . J. Immunol . 17 : 105 (1987) ; Van Dijk et al . , Int . J . Cancer 43 : 344 (1989); and Renner et al . , Science 264 : 833 (1994) .
  • Bispecific antibodies can be made by a variety of conventional methods, e . g . , disulfide cleavage and reformation of mixtures of whole antibody or, preferably F(ab') 2 fragments, fusions of more than one hybridoma to form polyo as that produce antibodies having more than one specificity, and by genetic engineering. Bispecific antibodies have been prepared by oxidative cleavage of Fab' fragments resulting from reductive cleavage of different antibodies. See, for example, Winter et al . , Nature 349 : 293 (1991) .
  • This is advantageously carried out by mixing two different F(ab') 2 fragments produced by pepsin digestion of two different antibodies, reductive cleavage to form a mixture of Fab' fragments, followed by oxidative reformation of the disulfide linkages to produce a mixture of F(ab') 2 fragments including bispecific antibodies containing a Fab portion specific to each of the original epitopes.
  • General technique ⁇ for the preparation of ⁇ uch antibody composites may be found, for example, in Nisonhoff et al . , Arch Biochem . Biophys . 93 : 470 (1961) , Ham erling et al . , J . Exp . Med .
  • More selective linkage can be achieved by using a heterobifunctional linker such as malei ide- hydroxy ⁇ uccinimide e ⁇ ter. Reaction of the ester with an antibody or fragment will derivatize amine group ⁇ on the antibody or fragment, and the derivative can then be reacted with, e . g . , an antibody Fab fragment having free sulfhydryl groups (or, a larger fragment or intact antibody with sulfhydryl groups appended thereto by, e.g., Traut' ⁇ Reagent) . Such a linker i ⁇ less likely to crosslink groups in the same antibody and improves the selectivity of the linkage.
  • a heterobifunctional linker such as malei ide- hydroxy ⁇ uccinimide e ⁇ ter. Reaction of the ester with an antibody or fragment will derivatize amine group ⁇ on the antibody or fragment, and the derivative can then be reacted with, e . g . , an antibody Fab fragment having free s
  • a bispecific antibody comprises binding moieties for T cell ⁇ and an antigen that i ⁇ a ⁇ sociated with a tumor cell or infectious agent.
  • a CEA binding moiety can be derived from a Class III Mab and the T cell-binding moiety can be derived from anti-CD3 Mab.
  • Methods for preparing anti- CD3 antibodies are well-known to those of skill in the art. See, for example, Canevari et al . , supra , Van Dijk et al . , supra , Hansen et al .
  • anti-CD3 antibodie ⁇ can be obtained from commercial sources such as Boehringer Mannheim Corp. (Indianapolis, IN; Cat. No. 1273 485) and the American Type Culture Collection (Rockville, MD; ATCC CRL 8001 [OKT-3]) .
  • a bispecific antibody can be prepared by obtaining an F(ab') 2 fragment from an anti-CEA Class III Mab, as described above.
  • the interchain disulfide bridges of the anti-CEA Clas ⁇ III F(ab') 2 fragment are gently reduced with cysteine, taking care to avoid light-heavy chain linkage, to form Fab'-SH fragments.
  • the SH group(s) is(are) activated with an excess of bis-maleimide linker (1, 1'- (methylenedi-4, 1- phenylene)bis-malemide) .
  • the anti-CD3 Mab is converted to Fab'-SH and then reacted with the activated anti-CEA Class III Fab'-SH fragment to obtain a bispecific antibody.
  • bispecific antibodies can be produced by fusing two hybridoma cell lines that produce anti-CD3 Mab and anti-CEA Class III Mab.
  • Techniques for producing tetradomas are described, for example, by Milstein et al . , Nature 305 : 537 (1983) and Pohl et al ., Int . J . Cancer 54 : 418 (1993) .
  • bispecific antibodies can be produced by genetic engineering.
  • plasmid ⁇ containing DNA coding for variable domains of an anti-CEA Class III Mab can be introduced into hybridomas that secrete anti- CD3 antibodie ⁇ .
  • the re ⁇ ulting "transfectoma ⁇ ” produce bispecific antibodies that bind CEA and CD3.
  • chimeric genes can be designed that encode both anti-CD3 and anti-CEA binding domains.
  • General techniques for producing bispecific antibodies by genetic engineering are described, for example, by Songsivilai et al., Biochem . Biophys . Res . Commun . 164 : 271 (1989); Traunecker et al . , EMBO J. 10 : 3655 (1991); and Weiner et al . , J . Immunol . 147 : 4035 (1991) .
  • an "immunoconjugate” is a molecule comprising an antibody component and an antigenic peptide.
  • An immunoconjugate retains the immunoreactivity of the antibody component, i.e., the antibody moiety has about the same, or slightly reduced, ability to bind the cognate antigen after conjugation as before conjugation.
  • Suitable antigenic peptides comprise either at least one epitope of a tumor associated antigen or at least one epitope of an antigen associated with an infectious agent.
  • the A3B3 epitope of CEA i ⁇ an example of a preferred tumor-associated, antigenic peptide. Jessup et al . , Int . J . Cancer 55 : 262 (1993) ; Zhou et al . , Cancer Res . 53 : 3817 (1993); and Hefta et al . , Cancer Res . 52 : 5647 (1992) .
  • Peptides containing CEA epitopes can be produced by recombinant DNA methodology. Id .
  • synthetic peptide ⁇ can be produced u ⁇ ing the general technique ⁇ described below.
  • Useful antigenic peptides also include epitopes of antigens from infectious agents, such as E . coli endotoxin core polysaccharide. See, for example, Greenman et al . , J . Am . Med . Assoc . 266 : 1097 (1991) .
  • particularly useful immunoconjugate ⁇ deliver antigenic peptide ⁇ to cell ⁇ for antigen pre ⁇ entation. See, for example, Wy ⁇ -Coray et al . , Cell . Immunol . 139 : 268 (1992) , which describes the use of an antibody-peptide construct to deliver antigenic peptides to T cells.
  • antigenic peptides include the tetanus toxoid peptide P2 with an N-terminal cysteine, CQYIKANSKFIGITEL (C + tt830-844; C-ttp2; SEQ ID NO:l), and tetanus toxoid peptide P30 with a C-terminal cysteine, FNNFTVSFWLRVPKVSASHLEC (tt947-967 + C; SEQ ID N0:2).
  • Additional antigenic peptides can be derived from single complementarity-determining regions (CDR ⁇ ) of an anti-idiotype antibody.
  • CDR ⁇ complementarity-determining regions
  • Such CDR peptides, or “minimal recognition units,” can be obtained, for example, using the polymerase chain reaction to synthe ⁇ ize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al . , Methods : A Companion to Methods in Enzymology 2 : 106 (1991) ; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodie ⁇ ," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al .
  • Minimal recognition units also can be obtained by synthesizing peptides having amino acid sequences of known antibodie ⁇ . See, for example, Kabat et al . , SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, U.S. Department of Health and Human Service ⁇ (1983) .
  • Antigenic peptides can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
  • the tetanus toxoid peptides described above were constructed with a single cysteine residue that is used to attach the peptide to an antibody component.
  • such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as W-succinyl 3-(2- pyridyldithio)proprionate (SPDP) . Yu et al . , Int . J. Cancer 56 : 244 (1994) .
  • SPDP W-succinyl 3-(2- pyridyldithio)proprionate
  • an antigenic peptide can be attached to a reduced thiol group in the hinge region of an antibody component.
  • the antigenic peptide can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
  • the carbohydrate group can be used to increase the loading of the same peptide that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different peptide.
  • the Fc region is absent if an antibody fragment is used as the antibody component of the immunoconjugate.
  • a carbohydrate moiety into the light chain variable region of an antibody or antibody fragment. See, for example, Leung et al . , J . Immunol . 154 : 5919 (1995); Hansen et al . , U.S. patent No. 5,443,953 (1995) .
  • the engineered carbohydrate moiety is used to attach the antigenic peptide. 7.
  • the present invention contemplates the therapeutic use of immunoconjugates, Abl, Ab2 generated against Abl, and fragments of either Abl or Ab2.
  • immunoconjugates, antibodies and antibody fragments can be used as vaccines to induce both humoral and cellular immune responses in the recipient mammal.
  • administration of immunoconjugates, Abl and/or bispecific antibodies can be used to amplify the integrated immune response.
  • a mammal is immunized with a vaccine comprising Abl or fragments thereof, to induce the production of Ab2 and T cells (T2 cells) .
  • T2 cells Ab2 and T cells
  • the mammal may be given Abl, or fragments thereof, by intravenous administration to expand the T2 cell mass.
  • An additional advantage of this second administration is that the antibodies or fragments bind with cognate antigen on cancer cell ⁇ or infectiou ⁇ organisms and thus, serve as targets for T2 cells.
  • Methods for detecting the production of T cells that react with specific antibodies are well-known to those of ordinary skill in the art. See, for example, Fagerberg et al . , Cancer Immunol . Immunother . 37 : 264 (1993) , which is incorporated by reference.
  • a mammal is subsequently immunized with a vaccine comprising Ab2, or fragments thereof, to induce the formation of Ab3 and T cells that recognize Ab2 (T3 cells) .
  • T3 cells T cells that recognize Ab2
  • An advantage of this subsequent Ab2 vaccination is that cells expressing a tumor associated antigen or infectious agent antigen are destroyed by T3 cells directed to the antigen, and by T2 cells directed to Ab3 , which also is bound by the antigen.
  • Example 4 illustrate ⁇ a method of treatment compri ⁇ ing the administration of an Abl vaccine, Abl (or fragments) , and an Ab2 vaccine.
  • a MAb conjugated to a cytokine or lymphokine is administered by intravenous injection subsequent to immunization with Abl.
  • This step amplifies the cytotoxic lymphocyte clones that are induced by the intradermal immunization with Abl and accrete in the targeted cells.
  • the MAb portion of the conjugate can be directed to the same antigen as Abl used in the immunization, or to a different antigen. If the MAb is directed to the same epitope or antigenic determinant on the antigen as the Abl used in the immunization, there should be no cross- reactivity between the idiotype of the Abl used in the vaccine and the idiotype of the MAb in the conjugate.
  • MN-14 and NP-4 are both Class III, anti-CEA MAb that react with the same epitope on CEA, but the two have different idiotypes.
  • the cytokine or lymphokine to which the MAb is conjugated is one that drives induction of immune cytotoxic lymphocytes.
  • exemplary MAb-cytokine/lymphokine conjugates include IL-1, IL-2, 11-12, IL-15, CSF and
  • GM-CSF GM-CSF, with IL-2 and IL-15 being particularly preferred.
  • the T2 response may be further amplified by the intravenous administration of Abl antibodies or fragments after Ab2 vaccination. It is possible that the efficacy of an Ab2 vaccine may be decreased by the presence of circulating Abl antibody components, which have been administered intravenously. Therefore, it is advantageous to clear circulating Abl components prior to the administration of an Ab2 vaccine.
  • One method that can be used to achieve Abl clearance is to use Abl antibodies that have been conjugated with biotin. In this way, circulating biotinylated Abl can be cleared prior to Ab2 vaccination by the intravenous administration of avidin. Preferably, clearance with avidin is performed one to two days after the intravenous administration of Abl (or fragments thereof) . This antibody clearance technique is described by Goldenberg, international application publication No. WO 94/04702 (1994) .
  • an antibody or antibody fragment is conjugated with a peptide capable of inducing a strong major histocompatibility complex (MHC)- restricted immune response.
  • MHC major histocompatibility complex
  • An example of a suitable antigenic peptide is the tetanus toxin P2 peptide, described above.
  • Such a peptide can be conjugated, for example, to the IMMU-LL1 (EPB-1) antibody, which binds with the HLA-DR-complex on the plasma membrane of macrophages, monocytes, and B-lymphocytes. Palak- Byczkow ⁇ ka et al . , Cancer Res. 49 : 4568 (1989) .
  • An IMMU- LL1 vaccine fir ⁇ t is injected intradermally to establish primary sensitization and then, the vaccine is administered intravenously to boost the immune response.
  • an immunoconjugate such as an IMMU-LL1-P2 vaccine
  • the mammal can be treated with an immunoconjugate that directs the immune response to tumor cells.
  • an immunoconjugate comprising humanized LL2 and P2 can be used to target CD22-bearing tumor cells.
  • LL2 is described by Goldenberg et al . , J . Clin . Oncol . 9 : 548 (1991) , and by Murthy et al . , Eur . J . Nucl . Med . 19 : 394 (1992).
  • the sensitizing peptide e . g . , P2
  • the sensitizing peptide is cleaved from the antibody component after internalization, bound to class II MHC heterodimers, and transported to the cell surface.
  • Cytotoxic T cells generated with the LL1-P2 vaccine will then recognize the HLA-II-peptide complex on the cellular membrane and destroy the tumor cell.
  • This general approach can be used to treat other tumors that expres ⁇ the HLA-DR complex, or to treat autoimmune disease ⁇ that are caused by cells expressing the HLA-DR complex.
  • Immunoconjugates also can be used to induce or to boost the immune response to a tumor cell or to an infectious agent using a peptide that contains a suitable epitope.
  • a peptide containing the A3B3 domain of CEA can be conjugated to IMMU-LLl antibody (or fragment) and injected subcutaneously to establish primary sensitization against CEA, or injected intravenously to boost the immune response to CEA.
  • immunoconjugates comprising CDR ⁇ of anti- idiotype antibodie ⁇ can be used to induce or to boost the immune response.
  • a peptide containing the amino acid sequence of a CDR is conjugated with an antibody or antibody fragment.
  • the minimal recognition unit of IMMU-14 Ab2 antibody can be conjugated with IMMU-LLl antibody or antibody fragment.
  • the immune response is further amplified by the administration of cytokines.
  • cytokines include the interferons (INF ⁇ ) , interleukin ⁇ (IL ⁇ ) and tumor necrosis factors.
  • INF-7 induces macrophages, as well a ⁇ cell-surface class II histocompatibility antigen ⁇ on lymphoid cell ⁇ and monocyte ⁇ . See, for example, Kleger an et al . , "Lymphokines and Monokines," in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al . (eds.) , pages 53-70 (Chapman & Hall 1993) , and Roitt et al .
  • IL-2 is a T cell growth factor and a stimulator of natural killer cells and tumor-reactive T cells. Id .
  • INF-7 and IL-2 are preferred cytokines for the augmentation of the immune response.
  • IL-12 is another preferred cytokine for enhancing the immune response to the immunoconjugates of the present invention.
  • This cytokine is produced by phagocytic cells in response to bacteria, bacterial products and intracellular parasites. See, for example, Trinchieri, Annu . Rev . Immunol . 13 : 251 (1995) .
  • IL-12 induces cytokine production, primarily INF-7, by natural killer cells and by T cells, and IL-12 acts a ⁇ a growth factor for activated natural killer cells and T cell ⁇ , enhances the cytotoxic activity of natural killer cells, and stimulates cytotoxic T cell generation. Jd.
  • IL-12 has been used to treat Schistosoma mansoni, Mycobacterium avium, Histoplas a capsulatum , as well as sarcoma, lung metastases.
  • Wynn et al . Nature 376 : 594 (1995) ; Castro et al . , J . Immunol . 155 : 2013 (1995) ; Zhou et al . , J . Immunol . 155 : 785 (1995) ; Zitvogel et al . , J . Immunol . 155 : 1393 (1995) .
  • the antibodies and fragments of the present invention can be used as vaccines by conjugating the antibodie ⁇ or fragments to a soluble immunogenic carrier protein.
  • Suitable carrier proteins include keyhole lympet hemocyanin, which is the preferred carrier protein.
  • the antibodies and fragments can be conjugated to the carrier protein using standard methods. See, for example, Hancock et al , "Synthesis of Peptide ⁇ for Use as Immunogens," in METHODS IN MOLECULAR BIOLOGY: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 23-32 (Humana Press 1992) . Immunoconjugates comprising one of the above-described antigenic peptides do not require the addition of an immunogenic carrier protein.
  • a preferred vaccination composition compri ⁇ es an antibody conjugate or fragment conjugate, and an adjuvant.
  • suitable adjuvants include aluminum hydroxide and lipid.
  • Methods of formulating vaccine compositions are well-known to those of ordinary skill in the art. See, for example, Rola, "Immunizing Agents and Diagnostic Skin Antigens," in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro (ed.) , pages 1389-1404 (Mack Publishing Company 1990) . Additional pharmaceutical methods may be employed to control the duration of action of a vaccine in a therapeutic application. Control release preparations can be prepared through the use of polymers to complex or adsorb the immunoconjugates, antibodies or fragments.
  • biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al . , Bio /Technology 10: 1446 (1992) . The rate of release of an immunoconjugate, antibody or antibody fragment from such a matrix depends upon the molecular weight of the immunoconjugate, antibody or antibody fragment, the amount of immunoconjugate, antibody or antibody fragment within the matrix, and the size of dispersed particles. Saltzman et al . , Biophys . J . 55: 163 (1989); Sherwood et al . , supra .
  • the therapeutic preparations of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby immunoconjugates, antibodies or antibody fragments are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient mammal.
  • Sterile phosphate- buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well- known to those in the art. See, for example, Ansel et al.
  • the immunoconjugates, antibodies or fragments may be administered to a mammal intravenously or subcutaneously. Moreover, the administration may be by continuous infusion or by single or multiple boluse ⁇ . Preferably, an antibody vaccine is administered subcutaneously, while an antibody preparation that is not a vaccine i ⁇ administered intravenously. In general, the dosage of administered immunoconjugates, antibodies or fragments for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history.
  • immunoconjugates, antibodies or fragments which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient) , although a lower or higher dosage also may be administered as circumstances dictate.
  • immunoconjugates, antibodie ⁇ or fragments are administered to a mammal in a therapeutically effective amount.
  • An antibody preparation is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient mammal.
  • an antibody preparation of the present invention is physiologically significant if it ⁇ presence invokes a humoral and/or cellular immune response in the recipient mammal.
  • a cytokine such as INF-7, IL-2 , or IL-12 may be administered before and during the administration of an Abl vaccine or an Ab2 vaccine.
  • cytokines may be administered together before and during the administration of an antibody vaccine.
  • Cytokines are administered to the mammal intravenously, intramuscularly or subcutaneously.
  • recombinant IL-2 may be administered intravenously as a bolus at 6 x io 1 iu/kg or a ⁇ a continuou ⁇ infu ⁇ ion at a dose of 18 x 10 6 IU/m 2 /d.
  • recombinant IL-2 may be administered subcutaneously at a dose of 12 x 10 6 IU. Vogelzang et al . , J. Clin . Oncol . 11 : 1809 (1993) .
  • INF-7 may be administered subcutaneously at a dose of 1.5 x IO 6 U. Lienard et al . , J. Clin . Oncol . 10 : 52 (1992) .
  • Nadeau et al . , J. Pharmacol . Exp . Ther. 274 : 78 (1995) have shown that a single intravenous dose of recombinant IL-12 (42.5 ⁇ g/kilogram) elevated IFN-7 levels in rhesus monkeys.
  • Suitable IL-2 formulations include PROLEUKIN (Chiron Corp./Cetus Oncology Corp.; Emeryville, CA) and TECELEUKIN (Hoffman-La Roche, Inc.; Nutley, NJ) .
  • ACTIMMUNE Genentech, Inc.; South San Francisco, CA is a suitable INF-7 preparation.
  • bispecific antibodies may be administered after the initial Abl treatment .
  • the function of the bispecific antibodies is to bridge lymphocytes with CEA-bearing tumor cells and to trigger the lymphocyte-mediated cytolysis.
  • Bispecific antibodies can be administered according to above-described general guidelines. However, bispecific antibodies, unlike antibody vaccines, are not conjugated with immunogens.
  • the above-described methods can be used to provide prophylaxis against infectious agents.
  • the present invention contemplates the use of methods described herein to provide protection to a mammal before exposure to an infectious agent.
  • MN-14 a Class III, anti-CEA MAb
  • MN-14 a Class III, anti-CEA MAb
  • a 20 gram BALB/c female mouse was immunized subcutaneou ⁇ ly with 7.5 ⁇ g of partially-purified CEA in complete Freund adjuvant.
  • the mouse was boosted subcutaneously with 7.5 ⁇ g of CEA in incomplete Freund adjuvant and then, the mouse was boosted intravenously with 7.5 ⁇ g of CEA in saline on days 6 and 9.
  • the mouse On day 278, the mouse was given 65 ⁇ g of CEA intravenously in saline and 90 ⁇ g of CEA in saline on day 404.
  • the mouse On day 407, the mouse was sacrificed, a cell suspension of the spleen was prepared, the spleen cells were fused with murine myeloma cells, SP2/0-Ag 14 (ATCC CRL 1581) using polyethylene glycol, and the cells were cultured in medium containing 8- azaguanine.
  • Hybridoma supematants were screened for CEA-reactive antibody using an 125 I-CEA radioimmunoas ⁇ ay (Roche; Nutley, NJ) . Positive clones were recloned.
  • MN-14 One clone, designated MN-14 , had properties similar to the Cla ⁇ III anti-CEA- ⁇ pecific MAb, NP-4, being unreactive with normal cros ⁇ -reactive antigen and meconium antigen. However, MN-14, compared with NP-4 , demonstrated significantly superior tumor targeting in a human colon tumor xenograft model and consistently stronger staining of frozen section ⁇ of colon cancer.
  • a modified antibody was prepared in which the complementarity determining regions (CDR) of MN-14 were engrafted to the framework regions of human IgG ! antibody.
  • CDR-grafted (“humanized") MN-14 antibody was designated "hMN-14.”
  • General techniques for producing humanized antibodies are described, for example, by Jones et al . , Nature 321 : 522 (1986), Riechmann et al . , Nature 332 : 323 (1988) , Verhoeyen et al . , Science 239 : 1534 (1988), Carter et al ., Proc . Nat 'l Acad . Sci . USA 89 : 4285 (1992), Sandhu, Crit . Rev . Biotech . 12 : 437 (1992), and Singer et al . , J . Immun . 150 : 2844 (1993) .
  • hMN-14 was conjugated with keyhole lympet hemocyanin. Typically, patients are immunized with subcutaneous injections of the conjugate
  • Rat Ab2 to MN-14 was prepared as described by Losman et al . , Int . J . Cancer 56 : 580 (1994) , which is incorporated by reference. Briefly, female 3-week-old Copenhagen rats were injected intraperitoneally with 200 ⁇ g of MN-14 F(ab') 2 fragments emulsified in Freund's complete adjuvant. Animals were boosted at days 200, 230, and 235 with the same amount of antigen in Freund's incomplete adjuvant. Four days after the last injection, animals were sacrificed, spleen cell su ⁇ pen ⁇ ions were prepared, and the cells were fused with murine non- secreting plasmocytoma SP2/0 using standard techniques. Hybridoma cells were cultured in the presence of rat peritoneal feeder cells (10,000 cells/200 ⁇ l culture well) .
  • WI2 is an IgG u Ab2 which is specific for MN-14 and does not react with other isotype-matched anti-CEA MABs. Immunization of mice or rabbits with WI2 (but not with control rat IgG) induced the production of Abl' anti-CEA antibodies. Thus, WI2 can be used as an idiotype vaccine for patients with CEA-producing tumors.
  • WI2 vaccine is prepared from WI2 as de ⁇ cribed for the preparation of hMN-14 vaccine.
  • a patient with Dukes C colon carcinoma underwent a primary tumor resection for cure and then, was placed on fluorouracil and Levamisole adjuvant therapy.
  • the pre- operative CEA titer was 15.5 ng/ml.
  • Three months after primary surgery, the CEA titer was in the normal range, that is, below 2.5 ng/ml.
  • the patient wa ⁇ found to have a CEA titer of 25 ng/ml and a CAT scan showed a 5 cm tumor in the left lobe of liver and a 2 cm tumor in the right lobe.
  • the CEA titer was 25 ng/ml and the patient was immunized subcutaneou ⁇ ly with 2 mg of hAbl vaccine (day 0) . Immunization was repeated at day 7.
  • the patient wa ⁇ found to have lymphocytes reactive with the Abl (T2 cells) .
  • the patient was given 100 mg of the hAbl intravenously.
  • the CEA titer was 5 ng/ml and a CAT scan showed that the left lobe tumor had decreased to 2 cm in size, while the right lobe tumor had completely regres ⁇ ed.
  • the left lobe tumor had increa ⁇ ed in size, and a large tumor mass was found in the abdomen, as confirmed by needle biopsy.
  • the CEA titer had increased to 50 ng/ml.
  • the CEA titer was found to be less than 2.5 ng/ml, and the left lobe tumor had completely resolved.
  • the mass in the abdomen was reduced in size and a needle biopsy failed to reveal the presence of a tumor, demonstrating only fibrou ⁇ ti ⁇ ue infiltrated with lymphocytes.
  • IMMU-LLl (EPB-1) is a murine monoclonal antibody that binds with the HLA-DR complex on the plasma membrane of macrophages, monocytes, and B-lymphocytes and then, rapidly internalizes.
  • the preparation of IMMU-LLl is described by Pawak-Byczkowska et al . , Cancer Res . 49 : 4568 (1989) .
  • F(ab') 2 fragments are prepared from intact IMMU-LLl by conventional proteolysi ⁇ techniques, and conjugated with the P2 peptide [SEQ ID NO: 1] of tetanus toxin at the hinge region, a ⁇ de ⁇ cribed above.
  • the P2 peptide i ⁇ conjugated via an engineered carbohydrate moiety on the light chain of the antibody fragment ⁇ using the techniques of Leung et al . , J . Immunol 154 : 5919 (1995) .
  • the IMMU-LL1-P2 vaccine is administered ⁇ ubcutaneously to establish primary sen ⁇ itization due to the strong MHC-restricted immune response induced by the
  • the IMMU-LL1-P2 vaccine also can be administered intravenously to boost the immune response.
  • LL2 is a murine monoclonal antibody that binds with
  • CD22 on B-cell lymphomas See, for example, Goldenberg et al . , J . Clin . Oncol . 9 : 548 (1991) ; Murthy et al . ,
  • Humanized LL2 is prepared as described by Leung et al . , Hybridoma 13:469 (1994), and antibody fragments of humanized LL2 are prepared using standard techniques.
  • An LL2-P2 conjugate is prepared as described above and administered intravenously to the sensitized subject to direct the immune response against tumor cells bearing the CD22 antigen.
  • the A3B3 epitope of CEA is produced recombinantly or by peptide synthesis using the known amino acid sequence. Jessup et al . , int . J . Cancer 55 : 262 (1993); Zhou et al . , Cancer Res . 53 : 3817 (1993); and Hefta et al . , Cancer Res . 52 : 5647 (1992) .
  • A3B3 peptides are conjugated to IMMU-LLl antibody or fragment using standard techniques described above.
  • the IMMU-LL1-A3B3 vaccine is administered subcutaneously to induce the immune response against CEA- bearing tumor cells.
  • the vaccine also can be administered intravenously to boost the immune response against such tumor cells.
  • Peptides having the amino acid sequence of minimal recognition units of the Ab2 antibody described in Example 2 are prepared using the techniques described in section 6 above.
  • the peptides are conjugated with IMMU- LL1 antibodies or fragments to produce immunoconjugates that are suitable for inducing (via subcutaneous administration) or boosting (via intravenous administration) the immune response.
  • a patient with an adenocarcinoma of the lung undergoes resection of the primary tumor, and CEA is demonstrated to be present on the cancer cells by immunohistology.
  • the blood CEA increases from 5 ng/ml to 20 ng/ml and a bone scan demonstrates recurrent carcinoma at numberous sites. Standard chemotherapy is given, however, the CEA titer continues to rise, and a repeat bone scan demonstrates tumor progression.
  • the patient then is immunized with hMN14 and hW12 intradermally, as in Example 4.
  • IL-2 is administered during the immunization to divert the immune response to the Tl helper pathway.
  • a conjugate of IL-15 and hNP-4, a Class III anti-CEA- specific MAb (hNP-4-IL-15) is administered by intravenous infusion.
  • hNP-4-IL-15 a Class III anti-CEA- specific MAb
  • a hNP-4-IL-2 conjugate is administered by intravenous infusion.

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Abstract

On induit des réponses immunitaires humorales et cellulaires contre les cellules tumorales et les agents infectieux chez un mammifère à l'aide d'un vaccin contenant des immunoconjugués qui renferment des anticorps et des anticorps anti-idiotypes mimant un épitope d'un antigène associé à une tumeur ou à un agent infectieux. Ces immunoconjugués renferment également un peptide contenant un épitope d'un antigène associé à une tumeur ou d'un antigène d'un agent infectieux, un peptide contenant une unité de reconnaissance minimale d'un anticorps anti-idiotype, ou un peptide induisant une forte réponse immunitaire restreinte au complexe majeur d'histocompatibilité. On peut également utiliser des anticorps et des cytokines pour amplifier la cascade immunitaire.
EP96943706A 1995-12-22 1996-12-20 Utilisation d'immunoconjugues pour accroitre l'efficacite des vaccins multietapes stimulant les reactions immunitaires en cascade Ceased EP0881910A4 (fr)

Priority Applications (1)

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EP09002533A EP2057999A3 (fr) 1995-12-22 1996-12-20 Utilisation d'immunoconjugues pour accroitre l'efficacite des vaccins multietapes stimulant les reactions immunitaires en cascade

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US577106 1995-12-22
US08/577,106 US7354587B1 (en) 1994-07-06 1995-12-22 Use of immunoconjugates to enhance the efficacy of multi-stage cascade boosting vaccines
PCT/US1996/019755 WO1997023237A1 (fr) 1995-12-22 1996-12-20 Utilisation d'immunoconjugues pour accroitre l'efficacite des vaccins multietapes stimulant les reactions immunitaires en cascade

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EP0881910A4 EP0881910A4 (fr) 2006-05-03

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EP09002533A Ceased EP2057999A3 (fr) 1995-12-22 1996-12-20 Utilisation d'immunoconjugues pour accroitre l'efficacite des vaccins multietapes stimulant les reactions immunitaires en cascade

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EP (2) EP0881910A4 (fr)
JP (2) JP2000503003A (fr)
AU (1) AU721927B2 (fr)
CA (1) CA2240834C (fr)
WO (1) WO1997023237A1 (fr)

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WO1997023237A1 (fr) 1997-07-03
CA2240834C (fr) 2013-12-03
JP2008115196A (ja) 2008-05-22
JP2000503003A (ja) 2000-03-14
CA2240834A1 (fr) 1997-07-03
EP2057999A2 (fr) 2009-05-13
EP2057999A3 (fr) 2009-07-29
EP0881910A4 (fr) 2006-05-03
AU1287197A (en) 1997-07-17
AU721927B2 (en) 2000-07-20

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