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WO2009002939A2 - Anticorps autophiles - Google Patents

Anticorps autophiles Download PDF

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Publication number
WO2009002939A2
WO2009002939A2 PCT/US2008/067917 US2008067917W WO2009002939A2 WO 2009002939 A2 WO2009002939 A2 WO 2009002939A2 US 2008067917 W US2008067917 W US 2008067917W WO 2009002939 A2 WO2009002939 A2 WO 2009002939A2
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WO
WIPO (PCT)
Prior art keywords
antibody
autophilic
peptide
seq
antibodies
Prior art date
Application number
PCT/US2008/067917
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English (en)
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WO2009002939A3 (fr
Inventor
Heinz Kohler
Jean Amick
Michael A. Russ
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Innexus Biotechnology International Limited
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Application filed by Innexus Biotechnology International Limited filed Critical Innexus Biotechnology International Limited
Publication of WO2009002939A2 publication Critical patent/WO2009002939A2/fr
Publication of WO2009002939A3 publication Critical patent/WO2009002939A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • the present invention relates to antibodies, methods of making the same, and methods of using the antibodies in the detection, prevention, and/or treatment of a variety of disease conditions.
  • Antibodies have emerged as a major therapeutic tool for the treatment of chronic diseases, such as cancer and autoimmune disorders. Notable success stories include Herceptin® in the treatment of breast cancer and Rituxan® in the treatment of non-Hodgkin's lymphoma.
  • Herceptin® in the treatment of breast cancer
  • Rituxan® in the treatment of non-Hodgkin's lymphoma.
  • a key advantage of antibodies in the treatment of disease lies in their ability to target disease-causing cells or molecules, while sparing healthy tissues and normal products of the body.
  • antibodies that exhibit desired specificities in laboratory studies often fail in pre-clinical and clinical evaluations because of inefficient targeting, low therapeutic efficacy, and/or unacceptable side effects.
  • Crosslinking of cellular receptors also increases the binding avidity of an antibody to its target antigen, and thus is likely to increase all cell surface-dependent therapeutic mechanisms, such as complement-mediated killing and complement-dependent opsonization and phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), as well as enhanced inhibition of cell growth or alterations in metabolic pathways within cells through increased binding to and blockade of cellular receptors when using antibodies targeted to cellular receptors.
  • ADCC antibody-dependent cellular cytotoxicity
  • a rare class of self-binding antibodies variously known as “autophilic antibodies” or “autobodies”, has been identified in Nature. They are capable of forming dimers and/or polymers through noncovalent interactions with self.
  • an autophilic antibody is TEPC- 15, which targets a normally cryptic determinant of phosphorylcholine on apoptotic cells and atherosclerotic lesions (Binder, J., et al., 2003; Kang, C-Y, et al., 1988). Dimerization or multimerization may be induced only after the modified antibody attaches to its cell surface target, i.e., "differential oligomerization". In solution, an autophilic antibody can be in equilibrium between its monomelic and dimeric forms (Kaveri S., et al., 1990).
  • Autophilic antibodies belong to a larger class of antibodies, referred to herein as "SuperAntibodiesTM.”
  • Super-antibodies exhibit one or more beneficial properties in addition to the antigen binding properties usually associated with antibodies (Kohler H., et al., 1998; Kohler H., 2000).
  • the referenced class of super- antibodies comprises antibodies having catalytic, adjuvant, membrane-penetrating, and/or autophilic properties, and includes molecules that afford superior targeting and therapeutic properties.
  • Such super-antibodies are considered chimeric and typically comprise an antibody or antibody fragment covalently linked to at least one non-antibody moiety, such as a peptide, which has catalytic, adjuvant, membrane-penetrating, and/or autophilic properties.
  • a non-antibody moiety such as a peptide, which has catalytic, adjuvant, membrane-penetrating, and/or autophilic properties.
  • the conjugation of certain peptides to antibodies has been shown to increase the potency of antibodies, e.g., in inducing apoptosis (Zhao, et al. 2001 ; Zhao, et al 2002a; Zhao, et al. 2002b).
  • the conjugation chemistry used in previous studies has utilized the nucleotide binding site (Pavlinkova, et al. 1997) or the carbohydrate moiety of antibodies as the site of specific attachment (Award, et al. 1994).
  • 6,218,160 (issued to Duan) proposes site-specific conjugation of an enzyme to an antibody by formation of a dihydrazone bridge therebetween.
  • U. S. Patent No. 5,596,081 (issued to Haley et al.) discloses a method for site-specific attachment of a purine or purine analog photoaff nity compound to an antibody molecule.
  • U.S. Patent No. 6,238,667 (issued to Kohler) proposes photochemically cross-linking an azido-peptide molecule to an antibody at a purine or tryptophan affinity site on the antibody.
  • 2005/0033033 proposes a super-antibody for inhibiting cell apoptosis, wherein the super-antibody comprises an anti-caspase antibody conjugated to a membrane transporter peptide.
  • U.S. Patent Pub. No. 2003/0103984 discloses a fusion protein comprising antibody and peptide domains in which the peptide domain can have autophilic activity.
  • U.S. Patent No. 6,482,586 (issued to Arab et al.) proposes covalent hybrid compositions for use in intracellular targeting.
  • 6,406,693 (issued to Thorpe et al.) proposes antibodies and conjugates for cancer treatment by binding to aminophospholipid on the luminal surface of tumor blood vessels.
  • U.S. Patent No. 6,780,605 (issued to Frostegard) proposes a method of diagnosing cardiovascular disease that employs antibodies specific for platelet activating factor.
  • U.S. Patent No. 6,716,410 (issued to Witztum et al.) proposes a treatment for atherosclerosis that employs a monoclonal antibody having specific binding affinity for oxidized low density lipoprotein (oxLDL), which is covalently linked to a therapeutic agent, e.g., a thrombolytic agent.
  • oxLDL oxidized low density lipoprotein
  • 2003/0143226 proposes a monoclonal antibody having specific binding affinity for an oxidized LDL receptor, which inhibits binding of oxLDL to the receptor.
  • the above approaches are proposed to enhance the antigen detection ability and/or therapeutic efficacy of antibodies, which are not sufficiently effective in locating or killing their targets in either their native or "humanized” states. Still, there continues to be a need for enhancing the detection, prevention and/or treatment of many diseases using suitably modified antibodies.
  • An object of the present invention is to address the foregoing needs with suitably prepared super- antibodies.
  • a super- antibody contemplated by the present invention comprises immunoglobulin (Ig) and non-immunoglobulin (non-Ig) domains, wherein at least one non-Ig domain is covalently attached to the Ig domain, preferably as a chemically formed hybrid molecule, i.e., an immunoconjugate.
  • the immunoglobulin domain can comprise a polyclonal antibody, monoclonal antibody, Fab fragment, or F(ab') 2 fragment, which imparts specific binding affinity for an antigenic determinant.
  • the non-Ig domain is an organic chemical moiety that imparts, or augments, autophilic, membrane-penetrating, adjuvant, and/or catalytic properties to the immunoconjugate, but which does not contain an azido, purine or pyrimidine group.
  • the non-Ig domain comprises a peptide having autophilic, membrane-penetrating, adjuvant, and/or catalytic properties.
  • Autophilic antibodies described herein behave as monomeric antibodies when not bound to an antigen. Binding of an autophilic antibody to an antigen induces dimerization and/or multimerization of autophilic antibodies, a process termed Dynamic Cross Linking
  • Another aspect of the present invention is directed to a method of making novel super-antibodies.
  • Methods of the present invention include molecular biological techniques to generate a recombinant chimeric autophilic antibody.
  • a recombinant chimeric autophilic antibody of the present invention includes at least one autophilic peptide.
  • Autophilic antibodies are provided according to embodiments of the present invention which include an immunoglobulin component and an autophilic peptide fused thereto.
  • Autophilic antibodies are provided according to embodiments of the present invention which include an immunoglobulin component having a binding affinity for a CD20 antigen an autophilic peptide fused thereto.
  • the immunoglobulin component can be an antibody heavy chain and/or an antibody light chain.
  • the immunoglobulin component is chimeric, including immunoglobulin portions derived from two or more sources or species.
  • Autophilic antibodies are provided according to embodiments of the present invention wherein immunoglobulin component and autophilic peptide are expressed as a fusion protein.
  • the autophilic peptide is preferably expressed at the C-terminus of the immunoglobulin component in particular embodiments of the present invention.
  • the autophilic peptide includes a peptide selected from SEQ ID No. 1 ,
  • the immunoglobulin component includes chimeric
  • the immunoglobulin component includes rituximab.
  • Expression vectors are provided according to embodiments of the present invention which encode a chimeric heavy chain and/or a chimeric light chain, and an autophilic peptide. At least one protein expressed from the expression vector is a fusion protein including a chimeric heavy chain and/or a chimeric light chain, fused to an autophilic peptide.
  • the chimeric heavy chain includes a variable heavy chain of an anti-CD20 antibody such as mouse monoclonal 1F5 anti-CD20 antibody and rituximab anti-CD20 antibody.
  • an anti-CD20 antibody such as mouse monoclonal 1F5 anti-CD20 antibody and rituximab anti-CD20 antibody.
  • the chimeric heavy chain includes a human gamma constant heavy chain.
  • Expression vectors are provided according to embodiments of the present invention which include a nucleic acid sequence encoding a chimeric immunoglobulin heavy chain linked to an autophilic peptide and a nucleic acid sequence encoding a chimeric light chain of an immunoglobulin.
  • the nucleic acid sequences are operably linked to a transcription promoter.
  • the nucleic acid sequence encoding the chimeric immunoglobulin heavy chain linked to an autophilic peptide is separated from the nucleic acid sequence encoding the chimeric light chain of an immunoglobulin by an internal ribosome entry site
  • the chimeric heavy chain encoded by a nucleic acid in an expression vector of the present invention includes SEQ ID No. 26, SEQ ID No. 28, or a substantially identical chimeric heavy chain.
  • the chimeric heavy chain encoded by a nucleic acid in an expression vector of the present invention includes SEQ ID No. 27, SEQ ID No. 45 or a substantially identical chimeric heavy chain-autophilic peptide fusion protein.
  • Both the chimeric light chain and the chimeric heavy chain can be expressed as fusion proteins including an autophilic peptide.
  • a method of generating a fusion protein which includes an antigen binding region and an autophilic peptide is provided according to embodiments of the present invention expressing the fusion protein from an expression construct encoding the fusion protein.
  • the fusion protein forms a heavy chain of an autophilic antibody.
  • Isolated host cells transformed with an inventive expression vector described herein are provided according to embodiments of the present invention.
  • a photoactivatable organic molecule is covalently linked to an immunoglobulin at a site on the immunoglobulin having binding affinity for the organic molecule.
  • the mutual attraction of Ig and photoactivatable organic molecule favors contact and coupling of the two entities upon exposure to activating radiation.
  • the organic molecule contains a chromophore, such as an aromatic hydrocarbon moiety, other than a purine or pyrimidine group, susceptible to photoactivation.
  • an azido group need not be present in the molecule.
  • an aromatic hydrocarbon moiety (AHM) of the invention which is photoactivatable, is a single ring or polynuclear aiyl or heterocycle. Inclusive of such moieties are substituted benzene, naphthalene, anthracene, phenanthrene, pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, thiazole, pyridine, indole, benzofuran, thionaphthene, quinoline, or isoquinoline groups.
  • an AHM is present in the photoactivatable organic molecule as part of a side chain of an amino acid residue.
  • amino acid residues are tryptophan, tyrosine, histidine, and phenylalanine, which have indole, phenol, imidazole, and phenyl side chains, respectively.
  • a tryptophan residue is most preferred.
  • a super- antibody of the invention can also be conjugated with one or more non- autophilic peptides to add functionality.
  • a super-antibody can bear a membrane- penetrating peptide sequence, which facilitates translocation of the antibody across the cell membrane where it can bind to an intracellular target.
  • the membrane-penetrating peptide comprises at least one MTS peptide or MTS -optimized peptide.
  • an autophilic super-antibody can be conjugated with a membrane- penetrating peptide sequence, thereby imparting both functionalities to the antibody.
  • a super-antibody having specific binding affinity for atherosclerotic plaques which permits detection, prevention and/or treatment of atherosclerosis.
  • an autophilic super-antibody is capable of binding an antigenic determinant of atherosclerotic plaques, e.g., ox-LDL, and can dimerize or oligomerize once specifically bound to its antigenic determinant. In this way, uptake of ox-LDL by macrophages can be effectively blocked or reduced, thereby inhibiting chronic inflammation associated with atherosclerosis.
  • an autophilic peptide of the immunoconjugate comprises a T15, T15E, T15-scr2, R24, R24-charged, or other optimized amino acid sequence.
  • the immunoglobulin and/or peptide domains of the super-antibody are humanized to improve tolerance in a patient.
  • a pharmaceutical composition is also contemplated, which contains one or more super-antibodies and a pharmaceutically acceptable carrier. Due to its superior avidity, a super-antibody of the invention can be administered to a patient in a dosage similar to, or less than, that practicable for the corresponding non- autophilic antibody.
  • an assay of cells undergoing apoptosis can be performed by contacting the cells with a super-antibody of the invention.
  • the super-antibody specifically binds to an antigenic determinant of a cell undergoing apoptosis and can be visualized by a reporter molecule or secondary antibody.
  • antigenic determinants associated with apoptosis are membrane-bound phosphorylcholine and phosphatidylserine.
  • Figure 1 compares the internalization of MTS conjugated antibodies and non- MTS conjugated antibodies using anti-caspase 3 antibodies
  • Figure 2 depicts the effect of chemotherapeutic drug (actinomycin D) on cell death in the presence and absence of MTS-conjugated (Sab) antibody;
  • Figure 3 depicts enhanced binding of anti-CD20 antibodies conjugated with T15 peptide
  • Figure 4 depicts improved binding of anti-CD20 antibodies conjugated with Tl 5 peptide at low concentrations of antibody
  • Figure 5 depicts improved binding of anti-CD20 antibodies conjugated with Tl 5 peptide to DHL-4 cells at high concentrations of antibody
  • Figure 6 depicts enhanced induction of apoptosis of tumor cells with mouse anti- CD20 conjugated with T15 peptide
  • Figure 7 compares the binding of anti-GM2 antibody and T15 conjugated anti- GM2 antibody to ganglioside GM2;
  • Figure 8 illustrates the self-binding activity of anti-GM2 antibody and Tl 5 conjugated anti-GM2 antibody;
  • Figure 9 demonstrates binding specificity of T 15 conjugated anti-GM2 antibody to different gangliosides
  • Figure 10 depicts differences in cell surface binding of anti-GM2 antibody and
  • Figure 11 depicts the effect of anti-GM2 antibody and Tl 5 conjugated anti-GM2 antibody on Jurkat cell growth
  • Figure 12 compares the efficacy of autophilic peptide conjugation to an affinity site on an antibody (nucleotide) vs. a non-affinity site (CHO - carbohydrate) using anti-GM2;
  • Figure 13 depicts enhanced apoptosis of tumor cells using anti-GM2 antibody conjugated with T15 peptide
  • Figure 14 compares the binding of Herceptin® (upper panel) and the autophilic peptide conjugated form of Herceptin (lower panel) to small cell lung cancer cell;
  • Figure 15 depicts photo-conjugation of biotin-amino acids to monoclonal OKT3 antibody.
  • a panel of biotin-amino acids were mixed with the monoclonal antibody OKT3 at concentration from 20-50 MoI and exposed to UV for 2 minutes. The reacted mixture was dot-blotted with avidin-HRP and scanned. Color intensity is indicated at the y-axis;
  • FIG. 16 Panel A: Titration of biotin-tryptophan photo-conjugation to chimeric anti-GM2 antibody. Chimeric anti-GM2 was photo-biotinylated with Trp peptide at different molarities. ELISA wells were incubated with chimeric biotinylated anti-GM2 blocked and developed with avidin-HRP. Panel B: Photobiotinylation of humanized anti-Her2/neu
  • Figure 17 Denaturation of photo-biotinylated anti-GM2 antibody. Detection of biotin on denatured/renatured antibody in ELISA as in Fig. 16A;
  • FIG. 18 Panel A: Comparison of single versus multiple biotin anti-GM3 antibody. ELISA wells were coated with ganglioside, single and multiple biotin anti-GM3 was added and developed with avidin-HRP. Panel B: Comparison of single versus multiple biotin chimeric anti-Gm2 antibody to Gm2. Comparison of single versus multiple biotin antibody, ELISA as in Fig. 19;
  • Figure 19 compares chemically biotinylated with photo-biotinylated antibodies.
  • Figure 21 demonstrates antigen specific binding of photobiotinylated anti-glycolyl
  • Figure 22 illustrates a proposed mechanism by which an autophilic antibody of the present invention, which is immunospecific for ox -LDL, can inhibit chronic inflammation leading to atherosclerosis;
  • Figure 23 is a schematic representation of structures of the chimeric 1F5 (chlF5) and DXL 1F5 (chlF5 -DXL) antibodies;
  • Figure 24 shows a comparison of binding of chlF5 to DXL-chlF5 to JOK-I cells using FACS on fixed cells;
  • Figures 25A-25F show a comparison of induction of apoptosis by chlF5 and
  • Figures 26A-26C show a comparison of CDC using chlF5 and DXL-chlF5.
  • Figures 27A-27B show a comparison of ADCC using chlF5 and DXL-chlF5.
  • Figures 28A-28B show a comparison of inhibition of proliferation, Panel A, Raji,
  • immunoglobulins have an affinity for certain photoactivatable aromatic hydrocarbon moieties. Such affinity permits close approach and prolonged contact time between the immunoglobulin (Ig) and the aromatic hydrocarbon moiety (AHM), which in turn facilitates photolytic conjugation of the Ig to an organic molecule bearing the AHM. Without wishing to be bound to any particular theory, it is believed that the attraction between the AHM and an affinity site on the Ig is probably due to van der Waals forces and/or dipole-dipole interactions, which promote the close approach and stacking of parallel aromatic rings.
  • a photoactivatable organic compound is covalently linked to an Ig to form an immunoconjugate (super- antibody).
  • Such immunoconjugate is formed by admixing the photoactivatable organic compound and Ig, and subjecting the admixture to photoactivation conditions effective to covalently link the photoactivatable organic compound to the Ig.
  • a photoactivatable organic compound of the present invention contains at least one AHM, which has a binding affinity for the Ig.
  • the photoactivatable organic compound does not contain an azido, purine or pyrimidine group, inasmuch as such groups may interact with a different affinity site on the Ig, or may unnecessarily complicate synthesis of the photoactivatable organic compound.
  • a photoactivable organic compound in addition to an AHM, comprises a peptide having self -binding, membrane-penetrating, adjuvant, and/or enzymatic properties. Such peptide can thereby impart its properties to a subsequently formed immunoconjugate.
  • a photoactivable organic compound comprising a peptide contains from about 5 to about 30 amino acid residues.
  • a peptide contains an autophilic amino acid sequence selected from the following group: NH-ASRNKANDYTTDYSASVKGRFIVSR-COOH (SEQ ID NO: 1), NH-SKAVSRFNAKGIRYSETNVDTYAS-COOH (SEQ ID NO. 4), NH-GAAVAYISSGGSSINYA-COOH (SEQ ID NO. 5), NH-GKAVAYISSGGSSINYAE-COOH (SEQ ID NO. 6), and
  • a peptide contains a membrane-penetrating amino acid sequence selected from the following group:
  • An AHM covalently linked to a peptide in a photoactivatable organic compound is preferably located at a C- or N-terminus of the peptide so as not to interfere with the desired properties of the peptide.
  • the AHM can be present in an aromatic side chain of an amino acid, such as tryptophan, tyrosine, histidine, and phenylalanine.
  • an "immunoglobulin" can be a polyclonal antibody, monoclonal antibody, Fab fragment, or F(ab') 2 fragment.
  • mutual attraction and covalent linkage between the Ig and AHM occurs at an affinity site located in a variable domain of the immunoglobulin.
  • this can ensure close approach and noncovalent interaction between two adjacent Ig molecules on a cell surface.
  • Such coupling of Ig molecules can, in turn, facilitate crosslinking of cellular receptors and promote intracellular signaling.
  • location of the peptide adjacent a cellular receptor for the peptide can facilitate transport of an immunoconjugate into the cell. Binding affinity between the Ig and AHM can be demonstrated, as shown hereinafter, by competitive binding with an aromatic reporter molecule also having affinity for the Ig binding site.
  • a plurality of photoactivatable organic compounds can be covalently linked to the Ig.
  • any type of immunoglobulin can be employed with the present invention, such as those having specific binding affinity for a cancer-related antigen, a caspase enzyme, ox-LDL, or cellular receptor.
  • An aromatic hydrocarbon moiety (AHM) of the present invention comprises at least one aryl, polynuclear aryl, heterocycle, or polynuclear heterocycle group.
  • aryl - benzene polynuclear aryl - naphthalene, anthracene, and phenanthrene
  • heterocycle - pyrrole furan, thiophene, pyrazole, oxazole, thiazole, pyridine, and imidazole
  • a photoactivatable organic compound comprises a peptide covalently bonded to an AHM
  • the AHM can be present in an amino acid residue of the peptide, e.g., tryptophan (indole), tyrosine (substituted benzene), histidine (imidazole), and phenylalanine (benzene).
  • tryptophan indole
  • tyrosine substituted benzene
  • histidine imidazole
  • phenylalanine benzene
  • compositions that comprises a pharmacologically effective amount of an instant super-antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are saline solution, e.g., 0.15% saline solution.
  • a photoreactive biotinylated tryptophan is inserted into several antibodies to yield biotinylated antibodies.
  • This biotinylation reaction is not inhibited by the presence of ATP, which is a ligand for the conserved nucleotide binding site on antibodies (Rajagopalan, et al., 1996), and suggests that a different affinity site is involved.
  • UV energy can induce reactive radicals in heterocyclic compounds, such as tryptophan (Miles, et al. 1985).
  • UV light is used to covalently attach tryptophan-containing molecules to antibodies at a tryptophan affinity site on the antibodies.
  • AHMs such as tryptophan
  • a practical application is the use of multiple biotinylated AMHs to affinity biotinylate antibodies.
  • AHM-containing peptides having biological or chemical properties can be conveniently affinity cross-linked to antibodies to create super-antibodies.
  • Alternative methods of synthesizing antibody conjugates employ chemical or genetic engineering techniques to couple a peptide to an antibody.
  • a peptide can be attached by chemical means to an immunoglobulin (whole polyclonal or monoclonal antibody, or fragment thereof) at a carbohydrate site of the Fc portion or to an amino or sulfhydryl group of an antibody.
  • a peptide can be coupled to an antibody's variable domain structures by photo-crosslinking an azido-tryptophan or azido-purine to the antibody. In the latter approach, the peptide is believed to attach preferentially to the antibody by photoactivation of the azido group at a tryptophan or purine affinity site.
  • a chimeric antibody can be expressed, using genetic manipulation techniques, as a fusion protein of an autophilic peptide and a whole immunoglobulin, or fragment thereof. See, e.g., U.S. Patent No. 6,238,667, PCT Publ. WO 9914244, U.S. Patent RE 38,008, U.S. Patent No. 5,635,180, and U.S. Patent No. 5,106,951, the disclosures of which are incorporated herein by reference.
  • Autophilic antibodies of the present invention typically comprise antibodies conjugated with one or more peptides having an autophilic sequence. It is believed that an autophilic antibody of the invention can comprise virtually any immunoglobulin. In some embodiments, the antibodies bind to targets implicated in a disease or disorder, where binding of the target has a therapeutic effect on the disease or disorder.
  • the target antigens can include cell-surface antigens, including trans-membrane receptors.
  • the Ig component of the antibodies can comprise the monoclonal antibody 5D10 which binds human B-cell receptors, the monoclonal antibody S1C5 which binds murine B-cell receptors, anti-CD20 antibodies such as rituximab (Rituxan®) which binds CD20 on normal and malignant pre-B and mature B lymphocytes, mouse monoclonal antibody 1F5 which is specific for CD-20 on human B-cell lymphomas, tositumab (Bexxar®) which also binds CD20 on B lymphocytes, anti-GM2 which binds human ganglioside GM2 lymphocytes, trastuzumab (Herceptin®) which binds the protein HER2 that is produced by breast cells, anti-caspase antibodies which recognize the caspase proteins involved in apoptosis, humanized TEPC- 15 antibodies which are capable of binding oxidized low density lipoproteins (ox-LDL) and can prevent uptake of oxidized low density lip
  • An autophilic antibody of the present invention can comprise any autophilic peptide sequence.
  • the autophilic peptide can also comprise optimized peptide sequences, which may include sequences having enhanced functionality, such as those that act as linkers to enhance display and cross-linking activity of antibodies, or residues that enhance solubility of autophilic sequences.
  • the present invention contemplates a method of producing an autophilic conjugate of the invention in which a template peptide has been modified to enhance the crosslinking potential of the autophilic antibodies as described above.
  • such functionally enhanced peptides are determined by producing a series of synthetic peptides with substitutions at each amino acid position within the template sequence and then testing this library of peptides for autophilic binding or for binding to the original peptide sequence. Those peptides with superior binding to the original sequence are then conjugated to immunoglobulins and the resultant conjugates are tested for potency, specificity, and the unwanted ability to induce aggregation.
  • the T15 peptide sequence is altered and modified sequences are selected for enhanced function.
  • the self -binding potential of a peptide can be enhanced by increasing complementarity of the sequence, such as described in U.S. Patent No. 4,863,857 (issued to Blalock et al.), which is incorporated herein by reference.
  • the self- binding potential and/or toleration of a peptide can also be enhanced by humanizing a self- binding peptide sequence derived from non-human animals. Humanizing a peptide sequence involves optimizing the sequence for expression or functionality in humans.
  • an autophilic peptide comprises the T15 peptide, which originally comprised regions of CDR2 and FR3 of the murine germline-encoded S107/TEPC15 antibody.
  • the T15 peptide comprises the amino acid sequence: ASRNKANDYTTDYSASVKGRFIVSR (SEQ ID NO.: 1) (Kang C-Y, et al., 1988). Its autophilic property has been shown to be antigen-independent, thereby suggesting attachment of the peptide to monomeric antibodies can impart autophilic and increased avidity properties to the antibodies (Kaveri S., et al., 1991).
  • the T15 peptide can be photo-crosslinked to an aromatic hydrocarbon moiety or nucleotide affinity site of the immunoglobulin to produce the autophilic antibody.
  • the Tl 5 peptide can be crosslinked to a carbohydrate site of the Fc portion or to an amino or sulfhydryl group of the immunoglobulin.
  • the autophilic antibody can be conveniently expressed as a fusion protein of the T15 peptide and whole immunoglobulin, or fragment thereof.
  • an autophilic peptide can comprise the scrambled T15 sequence (T15-scr2), which comprises the amino acid sequence NH-SKAVSRFNAKGIRYSETNVDTYAS-COOH (SEQ ID NO.
  • the peptide R24 comprising the sequence NH-GAA VA YIS SGGS SINYA-COOH (SEQ ID NO. 5), the peptide R24-charged comprising the sequence NH-GKA V AYISSGGSSINYAE- COOH (SEQ ID NO. 6), and any modifications to such peptides which optimize or enhance the binding and therapeutic effectiveness of antibodies.
  • an autophilic peptide comprises the T15E peptide, NH-ASRNKANDYTTEYSASVKGRFIVSR-COOH (SEQ ID NO. 14).
  • the T15E peptide can be photo-crosslinked to an aromatic hydrocarbon moiety or nucleotide affinity site of the immunoglobulin to produce the autophilic antibody.
  • the T15E peptide can be crosslinked to a carbohydrate site of the Fc portion or to an amino or sulfhydryl group of the immunoglobulin.
  • the autophilic antibody can be conveniently expressed as a fusion protein of the T15E peptide and whole immunoglobulin, or fragment thereof.
  • the attachment of autophilic peptide to a monomeric antibody can impart autophilic and increased avidity properties to the antibody (Y. Zhao, and H. Kohler, 2002).
  • the antibody can be a humanized version of an orthologous antibody, which acquires increased or optimized binding and effectiveness when conjugated to an autophilic peptide, such as one containing the Tl 5 sequence.
  • Methods of humanizing antibodies have been previously described. See, e.g., U.S. Patent No. 5,639,641 (issued to Pedersen et al.), U.S. Patent No. 5,498,531 (issued to Jarrell), U.S. Patent Nos.
  • Autophilic antibody conjugates of the present invention can also comprise one or more other bioactive or functional peptides, which confer additional functionality on the antibody conjugates.
  • the antibody conjugate can comprise an antibody that bears a Tl 5 autophilic peptide and an MTS membrane translocation peptide (Y. Zhao et al., 2003; Y. Lin et al., 1995).
  • the MTS translocation peptide can have the amino acid sequence KGEGAAVLLPVLLAAPG (SEQ ID NO. 2).
  • the translocation peptide can be an optimized MTS peptide, comprising the amino acid sequence WKGES AA VILPVLIAS PG (SEQ ID NO. 7).
  • the T15 peptide provides autophilicity to the conjugate, and the MTS sequence facilitates entry of the antibody into cells.
  • Such a conjugate can target, for example, cancer cells for radio- immunotherapy, when its antibody region targets a primarily intracellular, tumor- associated antigen, such as carcino-embryonic antigen (CEA). See, e.g., U.S. Patent No. 6,238,667, which is incorporated herein by reference.
  • CEA carcino-embryonic antigen
  • the autophilic conjugate upon administration, targets CEA-bearing, colon carcinoma cells, is internalized by translocation of the antibody mediated by the MTS peptide, and is enabled to bind to the more prevalent intracellular form of CEA.
  • Crosslinking of CEA antibody with, for instance, a therapeutic isotope such as 131 I can be retained in a cell longer than unmodified, labeled antibody and can deliver a higher radioactive dose to the tumor.
  • therapeutic isotopes as 125 I which release beta particles of short path length and are not normally considered useful for therapy, can, when delivered intracellularly in closer proximity to the nucleus, be efficacious against certain targets, especially those of lymphoid origin and accessible in the blood and lymph tissues.
  • Autophilic antibodies conjugated with one or more other functional peptides may also be useful for targeting intracellular antigens.
  • antigens could include tumor associated antigens and viral proteins.
  • an autophilic antibody specific for viral proteins which is conjugated with a self-binding peptide and a MTS peptide can also be used to bind to intracellular viral proteins and prevent production of viruses.
  • the antibody can be internalized through the MTS peptide, and can be optimized to bind intracellular viral proteins (Zhao, Y., et al. 2003).
  • Many other functional peptides may also be conjugated to the autophilic antibodies to increase functionality.
  • the invention also relates to compositions comprising a super-antibody of the invention and a pharmaceutically acceptable carrier.
  • Conjugate autophilic antibodies can bind non-covalently with other autophilic antibodies when bound to their target antigen(s).
  • premature formation of dimers or multimers of the antibodies may lead to difficulties in manufacturing, such as during purification and concentration, as well as drawbacks in administration, which may lead to side effects.
  • compositions containing autophilic antibody-peptide conjugates of the invention are formulated to reduce this dimerizing potential and maximize monomelic properties while in solution and before administration. For example, it has been found that solution dimerization can be reduced or mitigated by using a hypertonic composition.
  • salt concentrations of 0.5M or more, low levels of SDS or other various detergents such as those of an anionic nature (see U.S. Patent No. 5,151,266, which is incorporated herein by reference), or modifications of the antibody to decrease its isoelectric point, for example through the use of succinyl anhydride (see U.S. Patent No. 5,322,678, which is incorporated herein by reference), can be used to formulate compositions.
  • Disease Detection, Prevention and Treatment can be used to formulate compositions.
  • a method of enhancing apoptosis, complement fixation, effector cell-mediated killing of targets, or preventing the development of, or enhancement of, a disease state is also contemplated, which employs a super-antibody of the invention or a composition comprising the super-antibody.
  • an autophilic conjugate of the invention, or a composition containing an autophilic conjugate of the invention is administered to a subject. Once administered, the antibodies bind to target cells and enhance apoptosis, complement fixation, effector cell-mediated killing of targets, or prevent target antigens or cells from stimulating the development of, or further enhancing, a disease state.
  • a second anti-autophilic peptide antibody can be administered.
  • an autophilic conjugate contains a non-native autophilic peptide, such as the murine Tl 5 sequence
  • an anti-T15 peptide antibody can be administered, which recognizes and binds to antibodies conjugated with the T15 sequence. This allows binding to and enhancement of apoptosis of pre-localized super-antibodies.
  • a template autophilic peptide can be modified to enhance the crosslinking potential of the autophilic antibodies as described above.
  • a method of potentiating apoptosis of targeted cells of a patient comprises administering a first autophilic antibody-peptide conjugate, or a composition containing an autophilic antibody-peptide conjugate, and a second antibody, or composition containing the second antibody, which recognizes the autophilic peptide domain of the conjugate.
  • the antibody-peptide conjugate recognizes an antigen on a target cell. Owing to its homodimerization property, the antibody- peptide conjugate can bind more avidly to the target than the corresponding antibody lacking the autophilic peptide domain. This is likely due to the ability to crosslink antigen at the surface of target cells.
  • the autophilic antibodies bind to two or more antigens, with those antigens being brought in close proximity and crosslinked, due to the autophilic property of the antibodies, an apoptosis signal within the cell can be triggered.
  • a second antibody specific for the autophilic peptide, can be administered, bind to the modified antibody, and enhance the process of crosslinking and even cause temporary clearance of the target antigen.
  • the target antigen is a receptor
  • clearance from the cell surface, endocytosis, and degradation will subsequently require synthesis of new receptor protein, meaning that the biological function of the receptor will be more effectively inhibited for a longer period than using either a simple blocking antibody or small molecule inhibitor.
  • the second antibody can bear a radiolabel or other potentially therapeutic substance, so that when administered, it can attack the targeted cells. Since the autophilic peptide is present on only a small number of immunoglobulins and may be derived from another organism, the secondary antibody should have specificity for antibodies bearing the autophilic peptide. Thus, antibody specific to the autophilic peptide will have the requisite selectivity to be used in vivo.
  • a patient who suffers from a disease or condition responsive to antibody therapy is administered at least one autophilic antibody of the invention in an amount effective to alleviate symptoms of the disease or condition.
  • a disease or condition contemplated for treatment by an antibody of the invention can be a malignancy, neoplasm, cancer, atherosclerosis, auto-immune disorder, Alzheimer's disease or other neurodegenerative condition, graft or transplantation rejection, or any other disease or condition responsive to antibody therapy.
  • Atherosclerosis is a major cause of fatal and chronic vascular diseases that include stroke, heart failure and disruption of circulation in other organs and sites.
  • atherosclerosis is a chronic inflammatory disease.
  • Oxidized phospholipids in ox-LDL are ligands for scavenger receptors on macrophages (Horkko, S., et al., 2000).
  • ox-LDL and its products including but not limited to the oxidized phospholipids and oxysterols, are initiating factors to which the artery wall and its component cells respond.
  • the classical lipid hypothesis and the new inflammation hypothesis should be jointly considered part of the pathogenetic pathway in atherosclerosis.
  • a mouse T15 antibody is "humanized" into a therapeutic antibody to treat vascular diseases in humans. Humanization of non-human antibodies may require extensive re-shaping of the antibody molecule, which can result in loss or reduction of antibody specificity and affinity.
  • an autophilic peptide to a humanized T15 antibody, its superb targeting for ox-LDL can be restored, thereby blocking uptake of ox- LDL by macrophages and inhibiting chronic inflammation associated with atherosclerosis.
  • a general method of preventing or treating atherosclerosis in a patient comprises administering to the patient a super-antibody having specific binding affinity for oxidized low density lipoprotein (ox-LDL) and autophilic properties.
  • the super- antibody is administered at a dose effective to block or reduce uptake of ox-LDL by macrophages, thereby inhibiting chronic inflammation associated with atherosclerosis.
  • the immunoconjugate specifically binds phosphorylcholine and expresses the T15 idiotype.
  • the immunoconjugate can be humanized, and preferably contains an autophilic peptide sequence, such as SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 14.
  • a super-antibody, or a composition containing a super-antibody is preferably administered in one or more dosage amounts substantially identical to, or lower than, those practicable for unmodified antibodies.
  • an autophilic antibody of the invention can be administered in one or more dose amounts substantially identical to, or less than, the doses used for rituximab or trastuzumab.
  • trastuzumab a humanized monoclonal anti-HER2/neu antibody
  • HER2 + breast cancer employs an antibody concentration of about 10 mg/ml.
  • Intravenous infusion over 90 minutes provides a total initial dose of 250 mg on day 0. Beginning at day 7, 100 mg is administered weekly for a total of 10 doses. The dosing regimen is reduced gradually from 250 mg to 100 mg to a maintenance dose of 50 mg per week. Similar or lower dosage regimens to that for trastuzumab can be employed with autophilic antibodies, with any adjustments being well within the capabilities of a skilled practitioner.
  • a super-antibody of the present invention has a specific binding affinity for oxLDL.
  • an antibody domain of the super-antibody is the monoclonal antibody 1K17, as described by U.S. Patent No. 6,716,410 (issued to Witztum et al.), the pertinent disclosure of which is incorporated herein by reference.
  • the resulting superior avidity of the autophilic antibody can enhance the binding property of the antibody absent the peptide.
  • An autophilic antibody can localize to oxLDL of atherosclerotic plaques, whereupon it can be employed to detect the situs of the plaque when used with a label, reporter molecule, or secondary antibody, and the like.
  • an autophilic antibody can be employed to coat the site of oxLDL deposition, thereby preventing further accumulation of plaque.
  • an autophilic antibody can be employed to direct an anti-plaque agent, e.g., a thrombolytic or antioxidant agent.
  • the T15 idiotype was originally described as being specific for phosphorylcholine (Lieberman, et al., 1974). Previously, it was discovered that the T15 idiotype is autophilic, i.e., they self-associate as noncovalent dimers (Kaveri, S., et al., 2000). By coupling the autophilic T15 peptide to a humanized T15/S107 antibody, the self- binding properties of the T15 antibody and its avidity can be restored.
  • T15 antibody is biologically equivalent to the human anti- phosphorylcholine antibodies known to bind to ox-LDL and inhibit inflammation initiated by macrophages
  • the efficacy of the T15 antibody in preventing and/or treating atherosclerosis is demonstrated.
  • a proposed mode of action of the T15 antibody is schematically indicated in Fig. 22 (modified from Steinberg, Nature Medicine, 2002, 8: 12311).
  • the present invention is also for a method of detecting a disease state, such as the presence of atherosclerotic plaques in a patient's vascular system.
  • a disease state such as the presence of atherosclerotic plaques in a patient's vascular system.
  • Such method comprises administering to a patient an immunoconjugate of the present invention, which has a specific binding affinity for oxidized low density lipoprotein (ox-LDL).
  • the immunoconjugate also has autophilic properties. Sites of immunoconjugate concentration in the patient's vascular system are then detected, thereby localizing and visualizing the atherosclerotic plaques.
  • the immunoconjugate binds phosphorylcholine and/or expresses the Tl 5 idiotype. More preferably, the immunoconjugate bears an autophilic peptide having an aforementioned amino acid sequence.
  • a method of detecting cells undergoing apoptosis is also contemplated.
  • an antigenic determinant of a cell surface is represented by membrane-bound phosphorylcholine or phosphatidylserine
  • the cell can be contacted with an autophilic immunoconjugate of the invention, which has specific binding affinity for the antigenic determinant.
  • the presence or absence of immunoconjugate bound to the cell is then detected.
  • Previously described autophilic peptides can be used.
  • Such methods as flow cytometry, fluorescent microscopy, histological staining, or in vivo imaging are particularly preferred for conducting detection.
  • the immunoconjugate may be labeled with fluorescein.
  • an in vitro assay of apoptosis can be used to screen multiple antigen- positive target cell lines, and if possible, fresh isolates of antigen-positive cells.
  • a non- modified antibody is incubated with a secondary (antiimmunoglobulin) antibody to enhance the potential for cross-linking.
  • Cells may be enumerated by pre-labeling, such as with 51 Cr or 131 I-UDR, or by FACS analysis using indicators of apoptosis. Positive results in this assay predict a positive outcome using an autophilic immunoconjugate. However, negative results in the assay do not necessarily mean that subsequent conjugation with an autophilic peptide will not improve one or more antibody effector properties.
  • Autophilic antibodies of the present invention have a higher potential for forming dimers in vitro under laboratory conditions, such as in solution with PEG. This laboratory characteristic correlates with crosslinking ability upon binding to a cell-surface target and higher therapeutic potency through such mechanisms as triggering apoptosis. This characteristic can be used to identify natural SuperAntibodies and to screen for proper conjugation of self-binding peptides to a non-autophilic antibody. Suitable animal models for testing efficacy of the aforementioned autophilic antibodies include severely compromised immunodeficient (SCID) mice or nude mice bearing human tumor xenografts.
  • SCID immunodeficient mice
  • Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined herein.
  • Antibodies, antigen binding fragments and methods for their generation are known in the art and such antibodies, antigen binding fragments and methods are described in further detail, for instance, in Antibody Engineering, Kontermann, R. and D ⁇ bel, S. (Eds.), Springer, 2001; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002, particularly chapter 11; J. D. Pound (Ed.) immunochemical Protocols, Methods in Molecular Biology, Humana Press; 2nd ed., 1998; B.K.C. Lo (Ed.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; and Kohler, G. and Milstein, C, Nature, 256:495-497 (1975).
  • a recombinant chimeric autophilic antibody which includes a fusion protein including an autophilic peptide fused to at least a portion of an immunoglobulin.
  • Figure 23 shows a schematic representation of the structures of an unmodified antibody and a "DXL" autophilic antibody including an autophilic peptide at the C-terminus of the immunoglobulin heavy chain.
  • An autophilic peptide included in a recombinant chimeric autophilic antibody is an autophilic peptide which includes the amino acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical amino acid sequence.
  • An amino acid sequence which is substantially identical to the 25-mers of SEQ ID Nos.l and 14 has at least 20 contiguous amino acids, more preferably at least 22 contiguous amino acids, having an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to 20 or more contiguous amino acids of the identified autophilic amino acid sequence.
  • An amino acid sequence which is substantially identical to the 17-mers of SEQ ID Nos.5 and 6 has at least 13 contiguous amino acids, more preferably at least 15 contiguous amino acids, having an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to 13 or more contiguous amino acids of the identified autophilic amino acid sequence.
  • a peptide which is substantially identical to an autophilic peptide retains a substantially similar or better autophilic function compared to the reference autophilic peptide with which it is substantially identical.
  • Percent identity is determined by comparison of amino acid or nucleic acid sequences, including a reference sequence and a putative homologue sequence. Algorithms used for determination of percent identity illustratively include the algorithms of S. Karlin and S. Altshul, PNAS, 90:5873-5877, 1993; T. Smith and M. Waterman, Adv. Appl. Math. 2:482-489, 1981, S. Needleman and C. Wunsch, J. MoI. Biol, 48:443-453, 1970, W. Pearson and D.
  • Multimers of autophilic peptides can be used in particular embodiments of the present invention.
  • Exemplary multimers having spacer amino acids disposed between the autophilic peptides are shown as SEQ ID No. 10, SEQ ID No. 11.
  • a nucleic acid expression construct which encodes a DNA sequence encoding an autophilic peptide inserted in-frame with a DNA sequence encoding at least a portion of an immunoglobulin for use in producing a recombinant chimeric autophilic antibody.
  • a DNA sequence encoding SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical autophilic peptide is inserted in- frame with a DNA sequence encoding an immunoglobulin heavy chain and/or immunoglobulin light chain.
  • the fusion protein expressed from the DNA sequence contains an immunoglobulin heavy chain and/or immunoglobulin light chain having SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14, or a substantially identical autophilic peptide at the C-terminus or N-terminus.
  • SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 14 or a substantially identical autophilic peptide at the C-terminus or N-terminus.
  • Recombinant chimeric autophilic antibodies provided according to embodiments of the present invention include a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain, and fused to an autophilic peptide.
  • a chimeric autophilic antibody of the invention can comprise virtually any chimeric immunoglobulin.
  • the antibodies bind to targets implicated in a disease or disorder, where binding of the target has a therapeutic effect on the disease or disorder.
  • the target antigens can include cell-surface antigens, including trans-membrane receptors.
  • a chimeric autophilic antibody of the invention includes a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain.
  • a chimeric autophilic antibody of the invention preferably includes a human constant heavy chain and/or a human constant light chain.
  • a chimeric autophilic antibody of the invention preferably includes a human gamma constant heavy chain region and/or a human kappa constant light chain region.
  • Nucleic acids encoding immunoglobulin heavy chains or immunoglobulin light chains are well-known and any of various nucleic acids encoding immunoglobulin heavy chains or immunoglobulin light chains can be used to produce a recombinant chimeric autophilic antibody of the present invention. Specific nucleic acids are described herein which encode human constant heavy chain and/or a human constant light chains, particularly human gamma constant heavy chains and human kappa constant light chains. [00112] Nucleic acids encoding human gamma constant heavy chains and/or human kappa constant light chains can be obtained from commercial sources, such as vector pAc-k- CH3, available from Progen Biotechnik GmbH. Nucleic acids encoding protein and/or peptides described herein, including human gamma constant heavy chains and/or human kappa constant light chains, can be produced using recombinant techniques such as by cloning or synthesis.
  • immunoglobulin constant heavy chains and/or immunoglobulin kappa constant light chains are described, for instance, in U.S. Patent Nos. 5,736,137; 6,194,551; 6,528,624; 6,538,124; 6,737,056; 7,122,637; 7,151,164; 7,183,387; 7,297,775; 7,332,581; 7,335,742; 7,355,008; 7,364,731 and 7,371,826.
  • a chimeric autophilic antibody of the invention includes a variable heavy chain and/or a variable light chain derived from: the monoclonal antibody 5D10 which binds human B-cell receptors, the monoclonal antibody S1C5 which binds murine B-cell receptors, anti-CD20 antibodies such as rituximab (Rituxan®) which binds CD20 on normal and malignant pre-B and mature B lymphocytes, mouse monoclonal antibody 1F5 which is specific for CD-20 on human B-cell lymphomas, tositumab (Bexxar®) which also binds CD20 on B lymphocytes, anti-GM2 which binds human ganglioside GM2 lymphocytes, trastuzumab (Herceptin®) which binds the protein HER2 that is produced by breast cells, anti-caspase antibodies which recognize the caspase proteins involved in apoptosis, humanized TEPC- 15 antibodies which are capable of
  • Particular autophilic antibodies according to embodiments of the present invention include a chimeric immunoglobulin heavy chain having a variable heavy chain of an anti- CD20 immunoglobulin.
  • a chimeric autophilic antibody of the present invention includes chimeric immunoglobulin gamma heavy chain including the variable heavy chain of monoclonal antibody 1F5 and a human gamma constant heavy chain conjugated to an autophilic peptide.
  • SEQ JD No. 28 is an amino acid sequence of a chimeric immunoglobulin heavy chain including the variable heavy chain of monoclonal antibody 1F5 and a human gamma constant heavy chain.
  • a chimeric autophilic antibody includes SEQ ID No. 28 or a substantially identical amino acid sequence.
  • a substantially identical amino acid sequence of an immunoglobulin component has an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or greater % identical to an amino acid sequence disclosed herein in particular embodiments of the present invention, wherein the substantially identical protein retains a substantially similar or better function compared to the reference protein with which it is substantially identical.
  • SEQ TD No. 26 is an amino acid sequence of a chimeric immunoglobulin heavy chain including the variable heavy chain of monoclonal antibody 1F5 and a human gamma constant heavy chain conjugated to the T15E autophilic peptide.
  • An immunoglobulin gamma heavy chain portion of an anti-CD20 antibody included in a recombinant chimeric autophilic antibody has amino acid sequence SEQ ID No. 26 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • a chimeric immunoglobulin gamma heavy chain portion of an anti-CD20 antibody included in a recombinant chimeric autophilic antibody has amino acid sequence
  • SEQ ID No. 45 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • SEQ ID NO.45 Chimeric immunoglobulin heavy chain portion of an anti-CD20 autophilic antibody including an N-terminal leader and T15E at the C-terminus
  • SEQ ID NO.47 Chimeric immunoglobulin heavy chain portion of an anti-CD20 autophilic antibody without the N-terminal leader and T15E at the C-terminus
  • a chimeric immunoglobulin kappa light chain portion of an anti-CD20 antibody included in a recombinant chimeric autophilic antibody has amino acid sequence SEQ ID No.
  • SEQ ID NO. 46 Chimeric immunoglobulin light chain kappa portion of an anti-
  • CD20 autophilic antibody including a leader.
  • ARFSGSGSGTSYSLTISRVE AEDAATYYCQQWSFNPPTFGAGTKLELKRTVAAP SVFIFPPSDEQLKSGTASWC
  • SEQ ID NO. 48 Chimeric immunoglobulin light chain kappa portion of an anti-
  • CD20 autophilic antibody without the leader CD20 autophilic antibody without the leader.
  • SEQ ID NO. 49 Variable region of the immunoglobulin light chain kappa portion of an anti-CD20 autophilic antibody.
  • An anti-CD20 antibody immunoglobulin heavy chain includes a chimeric gamma heavy chain including the variable region of monoclonal antibody 1F5 and human gamma constant heavy chain region including amino acid sequence SEQ ED No. 28 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • an anti-CD20 antibody immunoglobulin gamma heavy chain has amino acid sequence SEQ ID No. 27 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • an anti-CD20 antibody immunoglobulin heavy chain includes a gamma heavy chain variable region including amino acid sequence SEQ ID No. 33 with or without leader sequence, SEQ ID NO. 34 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • an anti-CD20 antibody immunoglobulin light chain includes a kappa light chain variable region including amino acid sequence SEQ ID No. 37 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • Met Asp Phe Gln VaI Gln lie Ile Ser Phe Leu Leu He Ser Ala Ser VaI He Met Ser Arg Gly Gln Ile VaI Leu Ser Gln Ser Pro Ala He Leu Ser Ala Ser Pro Gly Glu Lys VaI Thr Met Thr Cys Arg Ala Ser Ser Ser VaI Ser Tyr He His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp He Tyr Ala Thr Ser Asn Leu Ala Ser Gly VaI Pro VaI Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg VaI Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Pro Thr Phe Gly Gly Gly Gly Thr Lys Leu Glu He Lys
  • an anti-CD20 antibody immunoglobulin heavy chain includes a gamma heavy chain variable region including amino acid sequence SEQ ID No. 39 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • SEQ ID No. 39 amino acid sequence SEQ ID No. 39 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • an anti-CD20 antibody immunoglobulin heavy chain includes a gamma heavy chain variable region of monoclonal antibody 1F5 including amino acid sequence SEQ ID No. 41.
  • a substantially identical amino acid sequence has an amino acid sequence at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%, 99% or greater % identical to SEQ ID No. 41.
  • an anti-CD20 antibody immunoglobulin light chain includes a kappa light chain variable region of monoclonal antibody 1F5 including amino acid sequence SEQ ID No. 43 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • SEQ ID No. 43 amino acid sequence SEQ ID No. 43 or a substantially identical amino acid sequence in particular embodiments of the present invention.
  • compositions provided according to embodiments of the present invention include an expression vector encoding an immunoglobulin heavy chain and/or an immunoglobulin light chain; and encoding an autophilic peptide.
  • an expression construct that includes a DNA sequence encoding an autophilic peptide.
  • expression construct refers to a recombinant nucleic acid sequence including a nucleic acid sequence encoding a peptide or protein to be expressed.
  • the nucleic acid encoding a peptide or protein to be expressed is operably linked to one or more regulatory nucleic acid sequences that facilitate expression of the peptide or protein to be expressed.
  • Nucleic acid sequences are operably linked when they are in functional relationship.
  • a regulatory nucleic acid sequence is illustratively a promoter, an enhancer, a
  • An expression construct can be incorporated into a vector, such as an expression vector and/or cloning vector.
  • vector refers to a recombinant nucleic acid vehicle for transfer of a nucleic acid.
  • exemplary vectors are plasmids, cosmids, viruses and bacteriophages. Particular vectors are known in the art and one of skill in the art will recognize an appropriate vector for a specific purpose.
  • IRES internal ribosome entry site
  • IRES are well-known in the art, for example as described in Pelletier, J. et al., Nature, 334:320-325, 1988; Vagner, S. et al., EMBO Rep., 2:893-898, 2001; and Hellen, C. U. et al, Genes Dev. 15:1593-1612, 2001
  • Expression constructs according to embodiments of the present invention include, in operable linkage: a promoter, a DNA sequence encoding an autophilic peptide and a transcription termination site.
  • an expression construct including, in operable linkage: a promoter, a DNA sequence encoding an autophilic peptide and a transcription termination site is included in an expression vector.
  • an expression construct including, in operable linkage: a promoter, a DNA sequence encoding an autophilic peptide and a transcription termination site is included in a plasmid expression vector.
  • promoter is known in the art and refers to one or more DNA sequences that bind an RNA polymerase and allow for initiation of transcription.
  • a promoter nucleic acid sequences is typically positioned upstream (5') of a nucleic acid encoding a peptide or protein to be expressed.
  • One of skill in the art is familiar with various well-known promoters and is able to select a promoter suitable for use in expressing a peptide or protein in a particular environment, such as in a specified cell type. Examples of well-known promoters that can be used include mouse metallothionein-1 promoter, the long terminal repeat region of Rous Sarcoma virus (RSV promoter), the early promoter of human cytomegalovirus (CMV promoter) and the simian virus 40 (SV40) early promoter.
  • RSV promoter Rous Sarcoma virus
  • SV40 simian virus 40
  • transcription termination site refers to a DNA sequence operable to terminate transcription by an RNA polymerase.
  • a transcription termination site is generally positioned downstream (3') of a nucleic acid encoding a peptide or protein to be expressed.
  • a leader sequence can be used in conjunction with expression of one or more immunoglobulin components included in an autophilic antibody described herein. Leader sequences shown can be modified or replaced with alternative leader sequences if desired.
  • a specific DNA sequence encoding T15E autophilic peptide ASRNKANDYTTEYSASVKGRFIVSR (SEQ ID NO. 14) is:
  • a specific DNA sequence encoding T15 autophilic peptide ASRNKANDYTTD YSASVKGRFIVSR (SEQ ID NO. 1) is: [00148] 5' gca agt aga aac aaa get aat gat tat aca aca gac tac agt gca tct gtg aag ggt egg ttc ate gtc tec aga 3' (SEQ ID No. 30)
  • the degeneracy of the genetic code is such that more than one nucleic acid will encode a particular autophilic peptide and these alternative sequences are considered within the scope of the present invention.
  • one or more amino acid substitutions, additions or deletions may occur in a particular autophilic peptide amino acid sequence as long as the autophilic properties of the peptide remain.
  • an anti-CD20 antibody immunoglobulin heavy chain included in an autophilic antibody of the present invention includes a gamma heavy chain region encoded by nucleic acid sequence SEQ ID No. 31 or a homolog thereof.
  • an anti-CD20 antibody immunoglobulin light chain included in an autophilic antibody of the present invention includes a kappa light chain encoded by nucleic acid sequence SEQ ID No. 32 or a homolog thereof.
  • a homolog of a nucleic acid sequence disclosed herein encodes an amino acid sequence having at least 70%, 80%, 85%, 90% and more preferably 95%, 96%, 97%, 98%,
  • nucleic acid sequence homolog hybridizes under high stringency hybridization conditions to the reference nucleic acid sequence, or a complement thereof, in particular embodiments of the present invention.
  • hybridizing and “hybridization” refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art. High stringency hybridization conditions are those which only allow hybridization of highly complementary nucleic acids. Determination of stringent hybridization conditions is routine and is well known in the art, for instance, as described in
  • nucleic acid refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds.
  • a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" to a specified second nucleotide sequence.
  • a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or
  • nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'.
  • nucleotide sequence 3'-TCGA- is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'.
  • High stringency hybridization conditions are known in the art and one of skill in the art is able to discern high stringency conditions.
  • Exemplary high stringency conditions include 50% formamide, 5X SSC, 5OmM sodium phosphate, pH 6.8, 0.1% sodium pyrophosphate, 5X Denhardt's solution, 50 micrograms/mL salmon sperm DNA, 0.1% SDS and 10% dextran sulfate at 42°C and a high stringency wash such as a wash in 0.1X
  • an anti-CD20 antibody gamma immunoglobulin heavy chain variable region included in an autophilic antibody of the present invention includes a gamma immunoglobulin heavy chain variable region encoded by nucleic acid sequence SEQ ID No. 35 or a homolog thereof.
  • SEQ ID No. 36 encodes the exemplary leader sequence having SEQ ID NO. 34. [00160] SEQ ID No. 36
  • an anti-CD20 antibody kappa immunoglobulin light chain variable region included in an autophilic antibody of the present invention includes a kappa immunoglobulin light chain variable region encoded by nucleic acid sequence SEQ ID No. 38 or a homolog thereof. [00162] SEQ ID No. 38
  • an anti-CD20 antibody gamma immunoglobulin heavy chain variable region included in an autophilic antibody of the present invention includes a gamma immunoglobulin heavy chain variable region encoded by nucleic acid sequence SEQ ID No. 40 or a homolog thereof. [00164] SEQ ID No. 40
  • an anti-CD20 antibody gamma immunoglobulin heavy chain variable region included in an autophilic antibody of the present invention includes a monoclonal antibody 1F5 gamma immunoglobulin heavy chain variable region encoded by nucleic acid sequence SEQ ID No. 42 or a homolog thereof. [00166] SEQ ID No. 42
  • an anti-CD20 antibody kappa immunoglobulin light chain variable region included in an autophilic antibody of the present invention includes a monoclonal antibody 1F5 kappa immunoglobulin light chain variable region encoded by nucleic acid sequence SEQ ID No. 44 or a homolog thereof. [00168] SEQ ID No. 44
  • an anti-CD20 antibody kappa immunoglobulin light chain variable region included in an autophilic antibody of the present invention includes a kappa immunoglobulin light chain variable region encoded by nucleic acid sequence SEQ ID No. 50 or a homolog thereof. [00170] SEQ ID No. 50
  • compositions provided according to embodiments of the present invention include an expression construct encoding a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain, and encoding an autophilic peptide.
  • an expression construct encoding a chimeric immunoglobulin heavy chain and/or a chimeric immunoglobulin light chain includes at least a variable heavy chain and/or at least a variable light chain derived from: the monoclonal antibody 5D10 which binds human B-cell receptors, the monoclonal antibody S1C5 which binds murine B-cell receptors, anti-CD20 antibodies such as rituximab (Rituxan®) which binds CD20 on normal and malignant pre-B and mature B lymphocytes, mouse monoclonal antibody IF5 which is specific for CD-20 on human B-cell lymphomas, tositumab (Bexxar®) which also binds CD20 on B lymphocytes, anti-GM2 which binds human ganglioside GM2 lymphocytes, trastuzumab (Herceptin®) which binds the protein HER2 that is produced by breast cells, anti-caspase antibodies which recognize the caspas
  • the chimeric light and heavy chains of autophilic antibodies of the present invention can be expressed together or separately to produce autophilic antibodies.
  • expression vectors are constructed encoding chimeric light and/or heavy chains of autophilic antibodies of the present invention.
  • Chimeric light and heavy chains can be encoded by nucleic acids included separate expression vectors, such as in separate plasmids.
  • the plasmids can be used together or separately to express the encoded proteins and produce the autophilic antibodies in particular embodiments.
  • chimeric light and heavy chains of autophilic antibodies can be purified and combined to form the autophilic antibodies.
  • expressed together the expressed proteins can combine to form the autophilic antibodies.
  • compositions provided according to embodiments of the present invention include an isolated host cell transformed with an expression vector encoding an immunoglobulin heavy chain having an antigen binding domain and an autophilic peptide.
  • the isolated host cell is also transformed with an expression vector encoding an immunoglobulin light chain having an antigen binding domain and the antigen binding domain of the immunoglobulin heavy chain and the antigen binding domain of the immunoglobulin light chain together form an antigen binding site of an anti-CD20 antibody.
  • An isolated host cell for producing a recombinant autophilic antibody of the present invention is in vitro in particular embodiments of the present invention.
  • Expression systems for autophilic antibody expression illustratively include: eukaryotic cells such as mammalian cells, plant cells, insect cells, yeast, and amphibian cells; and prokaryotic expression systems such as bacteria.
  • eukaryotic cells such as mammalian cells, plant cells, insect cells, yeast, and amphibian cells
  • prokaryotic expression systems such as bacteria.
  • One of skill in the art is able to select a particular expression system for use in producing a recombinant autophilic antibody.
  • Example 1 Conjugation of T15 peptide to two Mabs specific for B-cell receptor
  • the human B-cell tumor line (Su-DHL4) and murine B-cell tumor line (38Cl 3) are grown in RPMI 1640 medium (supplemented with 10% fetal bovine serum, 2 mol/L glutamine, 10 mol/L HEPES, 50 U/mL penicillin, and 50 g/mL streptomycin, 50 mol/L 2- mercaptoethanol) at 37°C under 5% carbon dioxide.
  • Two mAbs, 5D10 and S1C5, specific for the human or murine BCR, respectively, were used in this study.
  • the antibodies are purified from the culture supernatant by protein G and protein A affinity chromatography.
  • T15H peptide ASRNKANDYTTDYSASVKGRFIVSR (SEQ ID NO. 1), a VH- derived peptide from an autophilic antibody-T15, was synthesized by Genemed Synthesis (San Francisco, CA, U.S.A.). Antibodies were dialyzed against PBS (pH 6.0) and 1/10 volume of 200 mol/L sodium periodate was added and incubated at 4°C for 30 minutes in the dark. The reaction was stopped by adding glycerol to a concentration of 30 mol/L, and the sample was dialyzed at 4°C for 30 minutes against PBS (pH 7.0).
  • T15H or scrambled T15 peptide (T15scr/T15s) SYSASRFRKNGSIRAVEATTDVNSAYAK (SEQ ID NO: 3) was added to the antibodies and incubated at 37°C for 1 hour.
  • L-Lysine was added and incubated at 37°C for 30 minutes to block the remaining aldehyde group.
  • the same oxidation reaction (except adding the peptides) was applied to antibodies used as controls. After the blocking step, the antibody conjugates were dialyzed against PBS (pH 7.2) overnight.
  • Ig Capture ELISA Ig Capture ELISA.
  • Antibody conjugate was chromatographed on a 75 mL Sephacryl 300HR column (Pharmacia, Peapack, NJ). 1:10 diluted PBS (pH 7.2) was chosen as elution buffer. Fractions (0.5 mL/each) were collected and aliquots (100 L) were assayed on antihuman IgG capture ELISA. The ELISA reading (OD 490 nm) is plotted against elution volume.
  • Viability Assay for Antibody-Treated Cells [00188] Lymphoma cells were grown in 96- well tissue culture wells in 1-mL medium. Two g of antibodies or antibody-peptide conjugates were added and incubated for various times as described herein. Ten L aliquots from the cell suspension were used to determine viability by using trypan blue exclusion. [00189] FACS Assay of the B-CeIl Lymphoma.
  • 1 x 10 6 of lymphoma cells were placed into 24-well tissue culture wells. Four g of antibodies or antibody-peptide conjugates were added and incubated for various times as described herein. 1 x 10 6 cells were removed from the culture, re-suspended in 900 L cold PBS (pH 7.2). One hundred L of Hoechst 33342 (50 g/mL; Molecular Probe, Eugene, OR, U.S.A.) was added, the cells were incubated at 37°C for 30 minutes in the dark. The cells were centrifuged and re-suspended in 100 L PBS. Then, 4 L of MC540 solution (Molecular Probe) was added, and 20-minute incubation was performed at room temperature in the dark. The cells were pelleted, re-suspended in 1 mL cold PBS (pH 7.2), and analyzed by flow cytometry. [00193] RESULTS
  • the T15H (24-mer) peptide was crosslinked to two murine mAb (S1C5 and 5D10), using carbohydrate periodate conjugation.
  • the mAb S1C5 (IgGl) is specific for the tumor idiotype of the mouse 38C13 B-cell line and the 5D10 antibody for the human Su- DHL4 B-cell tumor. Both mAbs recognize unique idiotypes of the BCR IgM on the B-cell tumors.
  • T15H-linked monoclonal antibodies were analyzed using gel electrophoresis and sizing gel filtration.
  • the electrophoretic mobility of control and T15H peptide conjugated to S1C5 and 5D10 under reducing and non-reducing conditions show no differences, indicating the absence of chemical bonds between the antibody chains.
  • the molecular species of the peptide-conjugated antibodies (5D10-T15H) was further analyzed by size exclusion chromatography. The elution profile indicated two immunoglobulin species of different sizes. The larger first peak eluted in the position of an antibody dimer. The second smaller peak eluted in the position of non-conjugated 5D10 antibody.
  • Antibodies binding to the BCR induce crosslinking of the BCR, which, in turn, inhibits cell proliferation and produces a death signal. Furthermore, chemically dimerized antibodies directed against a B-cell tumor induce hyper-crosslinking of the BCR followed by inhibition of cell division and apoptosis of the tumor. To see if similar enhancement of the antitumor effects of dimerizing antibody were induced by noncovalent, dimerizing T 15H- linked antibodies, the two B cell tumors were cultured in the absence or presence of control and T15H-linked antibodies. Co-culture of both tumors, 38C13 and Su-DHL4, with their respective T15H-linked antibodies inhibited the cell growth significantly better compared with the control antibodies.
  • the antitumor effect of antibodies directed against the BCR of B-cell lymphomas in vitro and in vivo might be caused by the induction of apoptosis.
  • Aliquots of tumor cells (38C13 and Su-DHL-4) cultured in the presence of control or T 15H- linked antibodies were analyzed for apoptosis using a double stain FACS protocol.
  • 38Cl 3 and Su-DHL4 cells underwent a moderate amount of apoptosis without antibodies over a 6, respectively, 18-hour culture. This apoptosis was enhanced when the respective antibody was added.
  • the biologic advantage of the autophilic property is exemplified with the Sl 07/Tl 5 anti-phosphorylcholine antibody.
  • This autophilic antibody is several times more potent in protecting immune-deficient mice against infection with Pneumococci pneumoniae than non-autophilic antibodies with the same antigen specificity and affinity.
  • the autophilic antibody function can be transferred to other antibodies by chemically crosslinking a peptide derived from the Tl 5 VH germline sequence.
  • the modified antibody mimics the autophilic property of the T 15/Sl 07 antibody, producing an autophilic antibody with increased avidity and enhanced targeting. Enhancing the binding of autophilic engineered antibodies to the BCR of B-cell tumor increases the strength of the death signals leading to profound inhibition of cell proliferation in culture. Even though a doubling of apoptosis is demonstrated here, other mechanisms of growth inhibition can be involved.
  • Crosslinking the BCR of the mature murine B-cell lymphoma A20 can protect against CD95 mediated apoptosis.
  • This anti-apoptotic activity of engagement of the BCR by crosslinking antibodies is highly restricted to the time window of CD95 stimulation and is not dependent upon protein synthesis.
  • the finding that BCR hypercrosslinking per se is pro- apoptotic is not at variance with reports on the anti-apoptotic activity of the BCR engagement, because it can be due to the use of less mature B-cell lines, to different strength of delivered signals by homodimerizing antibodies, or to Fas -independent apoptosis.
  • the Fc domain was not involved in the augmentation of growth inhibition and tumor cells lacking Fc receptors were susceptible to the anti growth activity of homodimers.
  • the anti-tumor effect induced by dimerizing antibodies would not be restricted to lymphoid tumors such as non-Hodgkin's B-cell lymphoma, where anti-tumor effects require the participation of Fc- receptor-bearing effector cells.
  • the homophilic peptide is of murine origin, it might be immunogenic in humans. Thus, it could be necessary to humanize the murine peptide based on sequence and structural homology using computer modeling. The demonstration that adding a single peptide to the structure of antibodies increases the amount of antibody bound to targets and the anti-tumor activity encourages attempts to engineer recombinant antibodies expressing the autophilic activity.
  • Example 2 Internalization of Antibodies Conjugated with MTS Peptide [00214] Cell line and antibodies
  • Human Jurkat T cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotic (penicillin, streptomycin and amphotericin).
  • Monoclonal (rabbit) anti- active caspase-3 antibody (#C92-605) was purchased from BD PharMingen (San Diego, CA).
  • Mouse monoclonal antibody 3Hl (anti-CEA) was purified from cell-culture supernatant by protein G affinity chromatography.
  • MTS peptide KGEGAA VLLPVLLAAPG (SEQ ID NO. 2) is a signal peptide- based membrane translocation sequence, and was synthesized by Genemed Synthesis (San Francisco, CA).
  • Antibodies were dialyzed against PBS (pH 6.0) buffer, oxidized by adding 1/10 volume of 200 mmol/L NaIO 4 and incubating at 4°C for 30 min in the dark. Adding glycerol to a final concentration of 30 mM terminated the oxidation step. Samples were subsequently dialyzed at 4°C for 1 h against Ix PBS (pH 6.0) buffer.
  • Jurkat cells 2.5 x 10 5 ) were seeded into 96-well culture plate. After incubation with 0.5 g MTS-antibody conjugates for 6, 12, 18 and 24 hour, aliquots were removed and viability was determined by trypan blue exclusion.
  • Jurkat cells were pre-treated with antibodies or a caspase-3 inhibitor (DEVD- fmk) for 1 h, centrifuged, and incubated with fresh medium containing actinomycin D alone (1 g/ml) for 4 h. After treatment, Jurkat cells were collected, washed, and resuspended in 700 1 of HL buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100, for 15 min at room temperature. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated 24h at -20°C with 0.1 volume of 5 M NaCl and 1 volumes of isopropanol.
  • DEVD- fmk caspase-3 inhibitor
  • Jurkat cells were treated as described in the DNA fragmentation section. Equal amounts of protein of the total cell lysate were applied for caspase-3 activity assay using ApoAlert Caspase-3 Fluorescent Assay Kit according to the manufacturer's instruction.
  • Jurkat total cell lysates (10 g) were separated on a 10% SDS-PAGE gel to detect immunoreactive protein against cleaved spectrin. Ponceau staining was used to monitor the uniformity of protein transfer onto the nitrocellulose membrane. The membrane was washed with distilled water to remove excess stain and blocked in Blotto (5% milk, 10 mm Tris- HCl
  • an MTS conjugated anti-active caspase 3 antibody is internalized more rapidly than unmodified antibody.
  • actinomycin D apoptosis was triggered and the cells died (see Fig.
  • Example 3 Enhancing Binding and Apoptosis Using Peptide-Conjugated Anti-
  • the human B -cell tumor lines SU-DHL-4 and Raj were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 mol/L Hepes,
  • Mouse monoclonal antibodies 1F5 IgG2a (ATTC #HB-9645) specific for human B-cell lymphomas 5D10 and 3Hl (Zhao, Lou, et al., 2002.) were purified from cell culture supernatant by protein G or protein A affinity chromatography.
  • 8-azido-adenosine-biotin was synthesized and used to affinity cross-link biotin to antibodies.
  • the 8-azidoadenosine dialdehyde was prepared as previously described (U.S.
  • Patent No. 5,800,991 issued to Haley et al., which is incorporated herein by reference).
  • HRP Sigma-Aldrich
  • SU-DHL-4 cells were fixed using 1% paraformaldehyde, and 1 x 10 6 cells were suspended in 50 L staining buffer (Hanks, containing 0.1% NaN3 and 1.0% BSA); 1.5 g photobiotinylated 1F5-T15 conjugates, naked 1F5, and control antibodies were added and incubated for 30 minutes on ice. The cells were washed twice with staining buffer, and then avidin-FITC was added for 30 minutes on ice. After washing twice with staining buffer, the cells were resuspended in 200 L PBS for FACS analysis.
  • Ix 10 5 tumor cells were seeded in complete culture medium. At days 1, 2, and 3 of culture, aliquots were removed and viable cells were counted (trypan blue).
  • Example 4 Enhanced Binding and Apoptosis with Anti-GM2 Antibodies
  • T15 peptide ASRNKANDYTTEYSASVKGRFIVSR SEQ ID NO: 1
  • VH-derived peptide from a self -binding antibody-T15 Kaveri et al, 1991
  • T15-scr scrambled T15 peptide
  • the scrambled peptide was used as a control.
  • Antibodies were dialyzed against PBS (pH 6.0), then 1/10 volume of 200 M NaIO 4 was added and incubated at 4°C for 30min in the dark.
  • GM2 ganglioside was dissolved in methanol and 0.5 g was coated per well in 96 well polystyrene plates (Costar, Cambridge, MA) and allowed to dry overnight. The wells were blocked with 1% BSA for 2 h at room temperature and 400 g of anti-GM2 antibodies, diluted in 1% BSA, were added in the first well and then serially diluted 1:1. After incubation for 1 h, the wells were washed 5X and HRP-conjugated anti-human IgG (Sigma- Aldrich) was added at a 1 :1000 dilution and incubated for 1.5 h. After washing three times, the bound antibodies were visualized using substrate o-phenylenediamine and read at OD 492 using a spectrophotometer. [00256] Specific binding ELISA
  • Gangliosides GM2, GMl, GM3 were dissolved in DMSO in 0.5 g and coated in a 96 well polystyrene plate (Costar, Cambridge, MA) dried overnight. The wells were blocked with 1% BSA for 2 h at room temperature, 400 g of ch- -GM2 antibodies (anti- GM2-T15) were added in the first well and then serially diluted 1:1. After incubation for 1 h, the wells were washed 5 times and HRP-conjugated anti-human IgG was added and incubated for 1.5 h. After washing three times, the bound antibodies were visualized using substrate o- phenylenediamine and assayed as described previously. [00258] Antibody self-binding ELISA
  • Keuls post test Data are reported as means ⁇ SD.
  • the binding capacity of the T 15- conjugated ch- -GM2 antibody was determined using a direct binding
  • both ch- -GM2 antibody (anti-GM2) and anti-GM2-T15 antibody showed a dose- dependent increase in binding to ganglioside GM2.
  • the anti-GM2-T15 antibody demonstrated a higher binding capacity compared with the naked anti-GM2 at all the doses tested, confirming that the self-binding Tl 5 peptide had increased the antigen binding capacity of the ch- -GM2 antibody at a given antibody concentration.
  • GM2 by the T15 peptide-linked antibody was due to its self-binding feature. As seen in Fig.
  • the anti-GM2-T15 antibody demonstrated a greater dose-dependent increase in binding to the peptide-conjugated anti-GM2-T15 antibody coated on the wells, whereas it did not show significant binding to the non-peptide conjugated anti-GM2 antibody.
  • These data demonstrate that the anti-GM2-T15 antibody can bind to itself or homodimerize through the Fc- conjugated, autophilic peptide moiety.
  • Tl 5 conjugation does not change the specificity of the ch- -GM2 antibody.
  • a direct antigen-binding ELISA was used to determine the binding specificity of the anti-GM2-T15 conjugated antibody. As shown in Fig. 9, the anti-GM2-T15 antibody demonstrated a specific, dose-dependent increase in binding to ganglioside GM2, whereas no binding above background levels to gangliosides GMl or GM3 was detected. This result confirms that addition of the self -binding T15 peptide did not alter nor reduce the specificity of the ch- -GM2 antibody.
  • Enhanced surface binding of anti-GM2 antibody to target tumor cells [00276]
  • the human T-cell leukemic cell line Jurkat is known to express ganglioside GM2 (Suzuki et al, 1987).
  • the ability of the peptide-conjugated anti-GM2-T15 antibody to bind to native ganglioside GM2 expressed on the surface of Jurkat cells was compared to that of the non-conjugated anti-GM2 antibody by flow cytometry. As shown in Fig.
  • the ch- - GM2 antibody demonstrated a GM2 specific binding signal three times greater than background levels, whereas the binding demonstrated by the T15-conjugated anti-GM2 antibody was 2-fold higher than that of the non-peptide conjugated antibody. This result suggests that the enhanced binding demonstrated by the peptide-conjugated Ab is due to self- aggregation of this antibody. [00277] Inhibition of tumor growth
  • Antibodies binding to the B cell receptor have been shown to induce crosslinking of the BCR, which, in turn, inhibits cell proliferation (Ward et al, 1988) and produces a death signal (Hasbold et al, 1990; Wallen-Ohman et al, 1993).
  • chemically dimerized antibodies directed against a B-cell tumor induce hyper-crosslinking of the BCR followed by inhibition of cell division and induction of apoptosis of the tumor cells (Ghetie et al, 1994; Ghetie et al, 1997).
  • Example 5 Generation of Autophilic Peptide Sequences T15-scr, T15-scr2, R24. and R24-Charged
  • the peptide derived from R24 is difficult to solubilize except in DMSO or alcohol. Using such solubilizers can not only denature the antibody but also makes it difficult to conjugate to hydrophilic regions of the antibody. To overcome this solubility problem the addition and changes of sequence to charged amino acids, as shown in Table 3, were undertaken.
  • the resultant modified peptide (R24-Charged) was soluble in aqueous buffer, was able to be conjugated to the tryptophan or nucleotide binding sites and preserved self- binding as well as induced apoptosis when conjugated to anti-GM2 antibody.
  • T15scr The same amino acids present in the T15 sequence were randomly re-arranged and used to construct a further synthetic peptide; this scrambled sequence (T15scr or T15s), had no self-binding and when conjugated to anti-GM2 antibody did not induce apoptosis (see Example 4, Table 2).
  • T15scr2 a second, randomly selected sequence, derived from the amino acids of the T15 sequence, was used to generate a synthetic peptide (T15scr2).
  • this peptide demonstrated self-binding and when conjugated to anti-GM2 antibody, induced apoptosis in levels higher than the original Tl 5 sequence.
  • the parental antibody demonstrated a high level of apoptosis and killing of cancer cells.
  • the antibody was also effective in inhibiting growth of cancers in nude mouse models (not shown).
  • the antibody was "humanized” via cloning the heavy and light chain CDR's into the context of a human IgGl. Despite retention of affinity and specificity (not shown), the humanized antibody demonstrated much reduced ability to trigger apoptosis.
  • the humanized antibody, conjugated to a self-binding peptide (Sab) demonstrated high levels of apoptosis, similar to that of the parental antibody.
  • a further example is of a murine antibody, R24, which targets the GD3 ganglioside on human melanoma cells.
  • R24 a murine antibody
  • this antibody When naturally expressed, this antibody has self- binding and therapeutic activity in patients, but as a humanized antibody it loses avidity, self- binding and therapeutic activity (Chapman et al., 1994). Restoration of therapeutic activity of the humanized R24 antibody can also be achieved by conjugation of a self -binding peptide to the antibody.
  • the humanized versions of antibody TEPC- 15 and T 15/Sl 07 can also benefit from conjugation with a self -binding peptide to restore or enhance self-binding and therapeutic activity.
  • Example 8 Enhanced binding and tumor recognition by Herceptin® SuperAntibody.
  • Herceptin® (monoclonal antibody to HER2/neu), has been approved by the FDA for treatment of breast cancer. The antigen is expressed in approximately 30% of breast cancers but in only about half of those patients is the level of expression sufficient to trigger therapeutic effects. In fact, patients are normally pre-screened in a diagnostic test to determine their suitability for treatment. HER2/neu is also expressed on other cancers, such as non-small cell lung cancer but typically in only low levels, making this type of cancer unsuitable for treatment. An autophilic peptide was conjugated to Herceptin and tested for ability to bind non-small cell lung cancer. As shown in Fig. 14 (top panel), Herceptin reacts very weakly to this cancer; only 0.5% of cells are positive compared to an irrelevant antibody.
  • Anti-human IgG (whole molecule)-peroxidase-conjugated secondary antibody, avidin-conjugated peroxidase, anti-human IgG (whole molecule) antibody, monoganglioside GM2 were purchased from Sigma-Aldrich.
  • Anti-GM2 antibody, Herceptin and anti-GM3 were obtained from Corixa (Seattle, WA), Genentech (San Francisco, CA) and CMI (Havana, Cuba), respectively.
  • Trp-biotin peptides Two kinds of Trp-biotin peptides were designed: KAAGW (SEQ ID NO: 8) containing a biotin molecule on the alpha amino group [single biotin-peptide], and KAAKGEAKAAGW (SEQ ID NO: 9) containing biotin molecules on the alpha and epsilon amino groups of lysine [Multiple biotin-peptide]. These peptides were synthesized by . Genemed Synthesis, Inc. (San Francisco, CA).
  • GMl, 2 and 3 were obtained from Sigma-Aldrich, glycolytic GM3 was obtained from Alexis USA (San Diego, CA). [00298] Photobiotinylation using the tryptophan site.
  • AU antibodies were incubated with the tryptophan-containing peptides for 1 hr at room temperature.
  • the antibodies were photo-biotinylated at 200, 100, 50, 25, 10 and 1 M concentrations of biotin-peptide.
  • Photo-crosslinking was done using UV crosslinker FP- UVXL-1000 (Fisher Scientific) on the optimum setting at 100 j/cm .
  • the samples were dialyzed against PBS (pH 7.4) buffer.
  • the antibody concentration was determined using Comassie Plus Protein Assay (Pierce). Chemical biotinylation was performed with NHS- biotin (Pierce Chemical, Rockford, IL). Chimeric anti-GM3 glycolytic (CEM AB, Havana, Cuba) was biotinylated with 15 molar excess of NHS-biotin according to the manufacturer's protocol.
  • Photobiotinylated antibody was coated by adding 2 g to the first well and serially diluted and incubated overnight at 4°C. The wells are washed 3X and blocked with 3% BSA dissolved in PBS, pH 7.4 for 2 hours. The plate was washed 3X and 100 L of a 1/1000 dilution of avidin peroxidase conjugate was added per well. After incubating for 1 hour at room temperature, the wells were washed 3X with washing solution. 100 L of OPD solution
  • Goat anti-human IgG whole molecule was coated at a 1/100 dilution per well, overnight at 4°C. The plate was washed 3X and blocked 2 hours at room temperature with
  • the plate was washed 3X and 2 g of the photobiotinylated antibody was added to the first well, serially diluted and incubated for 2 hours at room temperature or 4°C, overnight.
  • the plate was washed 3X and 100 L of a 1/1000 dilution of avidin peroxidase conjugate was added per well. After incubating for 1 hour at room temperature, the wells were washed 3X with washing solution. 100 L of OPD solution
  • GMl, GM2, GM3 and glycolytic GM3 monoganglioside were dissolved in methanol and coated overnight by drying on polystyrene microtiter plates at 0.5 g per well.
  • the wells were blocked with 1% BSA for 2 hours.
  • GM2 tryptophan T15 conjugate was added to 1 % BSA to a concentration of 2 g/ 1 and 200 L was added to the first row of wells and serially diluted. After incubation at room temperature for 1 hr, the wells were washed 5X with washing solution. The plate was washed 3X and 100 L of a 1/1000 dilution of avidin peroxidase conjugate was added per well. After incubating for 1 hr at room temperature, the wells were washed 3X with washing solution. 100 L of OPD solution (OPD buffer, o- phenylenediamine and 1 L of 30% hydrogen peroxide/ml) were added to each well. The color development was stopped by adding 30 L of 4N H 2 SO 4 and the optical density was determined by scanning each well at 492 nm (Fisher Scientific Multiskan RC plate reader).
  • the antibodies were incubated with 100 M biotin peptide at pHs 5, 6, 7, 8, 9, 10 for 1 hour at room temperature and UV-crosslinked.
  • the samples were dialyzed against PBS pH 7.4 and analyzed by capture ELISA.
  • biotinylated amino acids were mixed with a monoclonal antibody, OKT3, and exposed to UV. The mixture was then dot-blotted and developed with avidin-HRP. The dots were scanned and the relative color intensity was recorded. As shown in Fig. 15, OKT3 photolyzed with biotinylated tryptophan yielded the strongest reaction with avidin followed by biotin-tyrosine. OKT3 photolyzed with other biotin amino acid gave only background reaction with avidin.
  • UV did not react with avidin in the ELISA.
  • Trp-biotin peptides that contain terminal Trp was examined.
  • Two kinds of Trp-biotin peptides were synthesized: 1) KAAGW containing a biotin molecule on the alpha amino group [single biotin-peptide] and 2) KA AKGE AKA AGW containing biotin molecules on the alpha and epsilon amino groups of lysine [multiple biotin-peptide].
  • Fig. 18 A the single biotin-peptide humanized anti-GM3 was compared to insolubilized ganglioside with the multiple biotin-peptide anti-GM3.
  • the multiple biotin antibody produced stronger ELSIA signals with avidin-HRP. Similar differences (Fig. 18B) between a single and the multiple biotinylated antibody were seen with the chimeric anti-
  • Conjugating peptides with biological or chemical properties is an attractive method to enhance the potency of antibodies or endow antibodies with diagnostic and therapeutic utility [Zhao, et al (2001); Zhao, et al (2002)a; Zhao, et al (2002)b].
  • the targeting of antibodies has been increased by conjugating autophilic peptides to produce dimerizing antibodies with enhanced targeting and induction of apoptosis.
  • membrane transporting sequence (MTS) was conjugated to antibodies and demonstrated that such MTS -antibodies penetrate the cellular membranes of living cells without harming the cells [Zhao, et al (2001)].
  • MTS antibodies against caspase-3 enzyme can inhibit induction of apoptosis in tumor cells. Attaching a peptide from the C3d complement fragment enhances the immune response to antibody vaccines creating a molecular adjuvant vaccine [Lou (1998)].
  • affinity crosslinking technique for peptides based on the discovery that antibodies can be photo-crosslinked to aromatic hydrocarbon moieties (AHMs), including heterocyclic amino acids, such as tryptophan.
  • AHMs aromatic hydrocarbon moieties
  • peptides that contain terminal tryptophan are affinity photo-cros slinking reagents for antibodies.
  • affinity conjugation methods have been demonstrated using biotinylated peptides. Exposing UV energy to a mixture of antibody and Trp-biotin peptides produces a biotin antibody that can be used in ELISA and other biotin-based detection methods.
  • Such affinity-biotinylated antibodies have a defined number of biotins attached that are less than conventional biotinylation chemistries, but sufficient to produce useful signals in ELISA.
  • Trp-affinity photo-crosslinking method is used to attach peptides with biological and chemical properties similar to those previously published [Lou et al. (1998); Zhao, et al (2001); Zhao, et al (2002)a; Zhao, et al (2002)b].
  • Example 11 Inhibition of chronic inflammation in atherosclerosis.
  • Chronic inflammation leading to atherosclerosis can be inhibited by the capacity of super-antibodies to bind avidly to ox-LDL, thereby blocking or reducing uptake of ox- LDL by macrophages.
  • Humanized autophilic antibodies having specificity for ox-LDL are administered to a patient according to the regimen described hereinabove.
  • the self-binding property of the autophilic antibodies increases their affinity for ox-LDL over that of unconjugated antibodies, and reduces recognition of the LDL particles by macrophages. Macrophage binding to ox-LDL should be effectively inhibited greater than 50% in the presence of the immunoconjugate.
  • Example 12 Cell Lines
  • SV-DHL-4 (DHL-4) cells were a kind gift of Dr. Ron Levy, JOK-I cells were a gift of Affimed Inc.
  • DHL-4 and JOK-I cells are grown in RPMH640 with Glutamax (Gibco), supplemented with 10% FBS-Premium-HI (Aleken Biologicals), and 1% Penicillin/Streptomycin (Gibco).
  • 1F5 hybridoma, Raji, and Ramos, cells are obtained from the American Type Culture Collection (ATCC), numbers HB-9645, CCL-86, CRL-1596, and TIB- 152, respectively.
  • Raji and Ramos cells are maintained in RPMI-1640 Medium with HEPES (ATCC), supplemented with 10% FBS-Premium-HI (Aleken Biologicals), and 1% Penicillin/Streptomycin (Gibco).
  • 1F5 cells are maintained in RPMI-1640 Medium with HEPES (ATCC), supplemented with 10% FBS-low-IgG (Gibco), 1% Penicillin/Streptomycin (Gibco), and 0.5% Glutamax (Gibco).
  • CHO-S cells are purchased from Invitrogen, and are grown in CD CHO medium, supplemented with 1% HT supplement (Gibco), 2% Glutamax (Gibco), and 100 U/ml pen/strep (Gibco). After introduction of vector DNA, CHO-S cells are grown as above with the addition of 1.2 mg/ml G418 (Invivogen) for selection. All cells are maintained at 37°C and 5% CO 2 . [00335] Example 13. Construction of chimeric antibody genes
  • Total RNA is isolated from about 7xlO 6 1F5 hybridoma cells using an RNeasy kit (Qiagen) according to the manufacturer's instructions. First strand cDNA synthesis, cDNA amplification by Long-Distance PCR (LD-PCR), and Proteinase K digestion are carried out using the materials and protocol of the Creator SMART cDNA library kit (Clontech). The 1F5 heavy chain variable regions are amplified from the cDNA pool by PCR using primers modVHlF5fwd (SEQ ID No. 15) and modVHlF5rev (SEQ ID No. 16).
  • the 1F5 light chain variable regions are amplified from the cDNA pool by PCR using primers modVLlF5fwd (SEQ ID No. 17) and modVLlF5rev (SEQ ID No. 18).
  • the heavy chain and light chain PCR products are cloned into the Xhol-Nhel and Sacl-Hindlll sites, respectively, of vector pAc-k- CH3 (Progen Biotechnik GmbH), to form pAc-k-lF5H and pAc-k-lF5K. Clones are verified by sequencing in both directions. All restriction enzymes are purchased from Takara or New England Biolabs. Taq polymerase (Promega) is used for all PCR. All enzymatic reactions are carried out using manufacturers' protocols.
  • Oligos LongT15fwd (SEQ ID No. 19), LongT15rev (SEQ ID No. 20), and PrimerB (SEQ ID No. 21) are used in a nested PCR similar to Horton, R.M., 1995, MoI Biotechnol 3: 93-99, to construct a DNA sequence that encodes the T15E peptide.
  • the resulting PCR product is cloned into the Sall-Notl sites in MCS B of pIRES (Clontech) to form pDXL.
  • the complete heavy and light chains of pAc-k-lF5H and pAc-k-lF5K are PCR amplified using primers modVHXfwd (SEQ ID No. 22) and modVHXrev (SEQ ID No. 23), or VKXfwd (SEQ ID No. 24) and VKXrev (SEQ ID No. 25), respectively.
  • the light chain is cloned into the Nhel-Xhol sites of MCS A of vector pDXL, and the heavy chain is cloned into the Sall-Notl sites of the resulting vector to form pchlF5-DXL. Clones are verified by sequencing in both directions.
  • pchlF5-DXL and pIRES are digested with Notl and CIaI.
  • Resulting DNA fragments of ⁇ 6 Kb from pchlF5-DXL, and -2.2 Kb from pIRES are each gel purified from a 1% agarose gel using a Qiaquick kit (Qiagen), and ligated together to form pchlF5. Clones are verified by sequencing in both directions. Oligo DNA sequences are provided in Table 5. All oligos are purchased from Operon.
  • 3xlO 5 per well of Raji, Ramos, DHL-4, JOK-I, or Jurkat cells are seeded in a 24 well plate and incubated overnight at 37°C and 5% CO 2 . Cells are then harvested and washed twice with PBS. Cells are resuspended in ImL PBS and are incubated with either chlF5 or chlF5-DXL at increasing concentrations (1 ⁇ g, 5 ⁇ g, 10 ⁇ g/mL, 20 ⁇ g/mL) and incubated at 4°C for 30 minutes.
  • Figure 24 shows the ability of the recombinant chlF5 and chIF5-DXL antibodies to bind to cells from the human B-cell JOK-I line using fluorescence activated cell sorting (FACS).
  • the dotted line shows the mean fluorescence intensity (MFI) of staining with the chlF5-DXL antibody, while the solid line represents the staining using the chlF5, non-DXL antibody. Binding of the chlF5-DXL antibody is approximately four-fold higher than binding of chlF5.
  • Results are consistence with dependence of induction of apoptosis by DXL antibodies on receptor cross-linking.
  • Figure 25 shows a comparison of induction of apoptosis by treatment with chlF5 or chlF5-DXL on Raji (panels A-C) and Ramos (panels D-F) cells. Results of analysis of untreated cells is shown in panels A and D, cells treated with chlF5 in panels B and E, and cells treated with chlF5-DXL in panels C and F.
  • the x-axis of the graph shows the intensity of annexin-V binding
  • the y-axis refers to the intensity of propidium iodide staining.
  • Addition of 20 ⁇ g of chlF5 induces apoptosis in approximately 30% of the cells ( Figure 25B versus Figure 25A).
  • the DXL chimeric antibody induces significantly more apoptosis than the non-DXL chimeric antibody (compare Figure 25C to Figure 25B).
  • the DXL antibody is a more potent inducer of apoptosis in Ramos cells at a concentration of 10 ⁇ g (compare Figure 25F to 25D and 25E).
  • Example 18 Complement Dependent Cytotoxicity (CDC) Assay
  • the CDC activity of the chlF5 and chlF5-DXL is compared in this example. 2xlO 5 cells are seeded into a 24 well plate and incubated overnight at 37°C and 5% CO 2 . Cells are then treated with increasing concentrations of Abs for 2 hours at 37°C in the presence of 5% rabbit HLA-ABC complement enriched sera (Sigma). Cells are harvested and washed once with PBS, resuspended in 200 ⁇ L of PBS containing 50 nM calcein-AM (Biochemica) and 4 ⁇ g/mL propidium iodide (Sigma).
  • CDC is induced after binding of complement components to the Fc region of an antibody, and is potent in the IgGl isotype, which is the isotype of the DXL construct.
  • An enhancing effect is observed in all cell lines.
  • Figure 26 shows graphs relating number of apoptotic cells to antibody concentration. Error bars show the standard deviation of the mean of two or more experiments. Student's t-test (two-tail) is used to test for statistical significance, *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 26A for example, at 5 ⁇ g/mL there is virtually no CDC activity in Raji cells with the non-DXL chimeric antibody. However, 35% of cells are killed with the DXL chimeric antibody.
  • PBMC Peripheral blood mononuclear cells
  • Example 20 Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) Assay
  • Target cells (Raji, Ramos, DHL-4, or JOK-I) are harvested from T75 flasks and resuspended in ImL of media containing 400 nM calcein-AM (Biochemica) and 8 ⁇ L of TFL2 dye (Oncolmmunin), used according to manufacturer's instructions.
  • Target cells are labeled for 45 minutes at 37°C, washed twice in media, and resuspended to a density of 6xlO 5 cells/mL.
  • Effector cells are then harvested from T75 flasks and resuspended to a density of 1.2xlO 7 cells/mL.
  • Target cells (T) and effector cells (E) are mixed at an E:T ratio of 20:1.
  • 250 ⁇ L of the cell mixture is aliquoted into individual 5mL round bottom tubes and incubated with increasing concentrations of Abs for 2 hours at 37°C.
  • CDC can be used as a criterion to divide different anti-CD20 antibodies into two types, as described in Cragg, M.S. et al., Blood, 103:2738-2743, 2004.
  • Type I anti-CD20 activates complement efficiently, while type II mediates ADCC not CDC.
  • the 1F5 anti-CD20 activates complement efficiently, while type II mediates ADCC not CDC.
  • CD20 belongs together with Rituxan to the type I class. Even though the parental 1F5 anti-
  • CD20 belongs to the type I class, the DXL version shows a significant increase of ADCC activity, therefore gaining type II properties. This creates a new class of therapeutic antibodies, designated here as type III.
  • Figure 27 shows graphs relating number of apoptotic cells to antibody concentration. Error bars show the standard deviation of the mean of two or more experiments. Student's t-test (two-tail) is used to test for statistical significance, *,
  • the DXL antibody induces significantly more ADCC than chlF5 in Raji and Ramos cells at 1 ⁇ g/ml and 3 ⁇ g/ml, but the increase in potency is not significant at 7.5 ⁇ g/ml.
  • the anti-proliferative effects of the chlF5 and chlF5-DXL antibodies is determined in Raji and Ramos cell lines to approximate the in vivo killing potential of these anti-CD20 antibodies on tumor cells.
  • the assay measures the level of fluorescence dye binding to nucleic acid. 5x10 3 cells per well of Raji or Ramos cells are seeded into a 96 well plate and treated with decreasing concentrations of Abs. Cells are incubated for 6 days at 37
  • DNA encoding the rituximab heavy chain is synthesized by PCR using overlapping primers to produce SEQ ID No. 31.
  • DNA encoding the rituximab light chain is synthesized by PCR using overlapping primers to produce SEQ ED No. 32.
  • the light chain is cloned into the Xhol-EcoRI sites of Multiple Cloning Site (MCS) A of vector pDXL, and the heavy chain is cloned into the Xbal-Sall sites of MCS B the same vector to form the bicistronic plasmid pRituximab-DXL having DNA sequences encoding the chimeric heavy chain and light chain separated by the IRES.
  • MCS Multiple Cloning Site
  • pRituximab-DXL is introduced into E. coli (XL-10 cells, from Stratagene) using the provided heat shock protocols. Plasmids are purified from 3 ml of overnight bacterial culture using a Qiagen mini-prep kit.
  • Vector pRituximab-DXL is electroporated into CHO-S cells using a 4 mm gap cuvette in an Eppendorf Multiporator set to 580 V and 40 ⁇ s. Two days of recovery are allowed before the start of selection.
  • Recombinant autophilic antibodies which include the rituximab heavy chain fused to the T15E autophilic peptide are purified and tested as described herein.
  • compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.
  • Binder J., et al. "Pneumococcal vaccinaton decreases atherosclerotic lesions formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL," Nature Medicine, 2003, 195: 771.

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Abstract

L'invention concerne des anticorps autophiles comprenant un composant d'immunoglobuline et un peptide autophile fusionné avec celui-ci. Les anticorps autophiles particuliers décrits ici comprennent une chaîne lourde d'immunoglobuline gamma chimérique et un peptide autophile exprimé en tant que protéine de fusion. Le peptide autophile est exprimé de préférence au niveau de la terminaison C du composant d'immunoglobuline. Des vecteurs d'expression selon des modes de réalisation de la présente invention, destinés à être utilisés dans la génération d'anticorps autophiles et qui comprennent une première séquence d'acides nucléiques codant un peptide autophile lié de manière fonctionnelle à un promoteur de transcription, sont fournis. Dans des modes de réalisation particuliers, une seconde séquence d'acides nucléiques codant une chaîne lourde chimérique d'une immunoglobuline est liée de manière fonctionnelle au promoteur de transcription et reliée à la première séquence d'acides nucléiques de sorte que l'expression des première et seconde séquences d'acides nucléiques produit une protéine de fusion de la chaîne lourde chimérique et du peptide autophile.
PCT/US2008/067917 2007-06-23 2008-06-23 Anticorps autophiles WO2009002939A2 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
US8080975B2 (en) 2008-05-09 2011-12-20 Ipowerup, Inc. Portable and universal hybrid-charging apparatus for portable electronic devices
CN103333717A (zh) * 2013-06-13 2013-10-02 中石化宁波工程有限公司 一种气化炉工艺烧嘴结构
US10973826B2 (en) 2015-10-29 2021-04-13 Novartis Ag Antibody conjugates comprising toll-like receptor agonist
WO2024152112A1 (fr) * 2023-01-18 2024-07-25 Morgan Alton C Anticorps du récepteur couplé à la protéine g (gpcr) couplés à des peptides de dimérisation homologues (hd)
WO2024152113A1 (fr) * 2023-01-18 2024-07-25 Morgan Alton C Anticorps et fragments de liaison à l'antigène couplés à des peptides de dimérisation homologue (hd) pour la prévention et le traitement d'une maladie infectieuse

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ZHAO, Y. ET AL.: 'Enhanced anti-B-cell tumor effects with anti-CD20 superantibody.' JOURNAL OF IMMUNOTHERAPY. vol. 25, no. 1, January 2002, pages 57 - 62 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8080975B2 (en) 2008-05-09 2011-12-20 Ipowerup, Inc. Portable and universal hybrid-charging apparatus for portable electronic devices
CN103333717A (zh) * 2013-06-13 2013-10-02 中石化宁波工程有限公司 一种气化炉工艺烧嘴结构
US10973826B2 (en) 2015-10-29 2021-04-13 Novartis Ag Antibody conjugates comprising toll-like receptor agonist
WO2024152112A1 (fr) * 2023-01-18 2024-07-25 Morgan Alton C Anticorps du récepteur couplé à la protéine g (gpcr) couplés à des peptides de dimérisation homologues (hd)
WO2024152113A1 (fr) * 2023-01-18 2024-07-25 Morgan Alton C Anticorps et fragments de liaison à l'antigène couplés à des peptides de dimérisation homologue (hd) pour la prévention et le traitement d'une maladie infectieuse

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