Classifications

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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • 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
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  • 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
  • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
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  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
• • A—HUMAN NECESSITIES
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  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0058—Antibodies
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57557—Immunoassay; Biospecific binding assay; Materials therefor for cancer of other specific parts of the body, e.g. brain
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70532—B7 molecules, e.g. CD80, CD86
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the antibodies or antibody fragments are antibody-drug conjugates.
• B7-H3 is a co-inhibitory ligand expressed on the surface of many tumor cells as well as in the tumor microvasculature (Suh et al., 2003; Zang et al., 2007; Wang et al., 2012). It is thought to actively inhibit the effector functions of cytotoxic T lymphocytes (CTLs) or induce the generation of regulatory T cells, all of which down-regulate immune responses (Pardoll, 2012).
• CTLs cytotoxic T lymphocytes
• B7-H3 is overexpressed on renal, pancreatic, colorectal, non-small cell lung, ovarian, bladder, melanoma, and neuroectodermal cancers (Loo et al., 2012), as well as prostate cancer cells (Zang et al., 2007; Koenig, 2014), pointing to the broad applicability of targeting B7-H3 for therapy and imaging.
• B7-H3 in prostatectomy specimens from 803 patients with localized disease, the vast majority (93%) of the prostate tumors expressed B7-H3 (Zang et al., 2007).
• peptide refers to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.
• polypeptide encompasses an antibody or a fragment thereof.
• an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
• the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv, Fd, Fd', single chain antibody (ScFv), diabody, linear antibody), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
• fragments thereof such as Fab, Fab', F(ab')2, Fv, Fd, Fd', single chain antibody (ScFv), diabody, linear antibody
• light chain refers to the smaller immunoglobulin subunit which associates with the amino terminal region of a heavy chain.
• a light chain comprises a variable region (VL) and a constant region (CL).
• Light chains are classified as either kappa or lambda (k, l) based on the amino acid sequences of their constant domains (CL). A pair of these can associate with a pair of any of the various heavy chains to form an immunoglobulin molecule.
• Antibodies may be bispecific or multispecific. “Bispecific antibodies” are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen.
• antibody variable regions with the desired binding specificities are fused to immunoglobulin constant domain sequences.
• the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, Cm, and Cm regions. It is preferred to have the first heavy-chain constant region (Cm) containing the site necessary for light chain bonding, present in at least one of the fusions.
• DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell.
• the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
• the preferred interface comprises at least a part of the CH3 domain.
• one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
• Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
• bispecific antibodies can be prepared using chemical linkage.
• Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
• the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
• the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
• bispecific antibodies have been produced using leucine zippers (Kostelny et al, J. Immunol., 148(5):1547-1553, 1992).
• the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
• the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
• a bispecific or multispecific antibody may be formed as a DOCK-AND- LOCKTM (DNLTM) complex (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400).
• DDD dimerization and docking domain
• R regulatory
• AD anchor domain
• the DDD and AD peptides may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.
• Multivalent antibodies may comprise (or consist of) three to about eight, for examle four, antigen binding sites.
• the multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions.
• the polypeptide chain(s) may comprise VDl-(Xl).sub.n-VD2-(X2) n -Fc, wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1.
• Charge modifications are particularly useful in the context of a multispecific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi-/multispecific antigen binding molecules with a VH/VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015/150447, particularly the examples therein, incorporated herein by reference in its entirety).
• a bi-specific T-cell engagers is an artificial bispecific monoclonal antibody that directs a host’s immune system, more specifically the T cells’ cytotoxic activity, to target diseased cells.
• BiTEs are fusion proteins consisting of two single chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons.
• scFvs single chain variable fragments
• One of the scFvs binds to T cells via the CD3 receptor, and the other to an infected cell via a specific molecule.
• Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate.
• the conjugate can be, for example, an antibody conjugated to another proteinatious, carbohydrate, lipid, or mixed moiety molecule(s).
• Such antibody conjugates include, but are not limited to, modifications that include linking the antibody to one or more polymers.
• an antibody may be linked to one or more water-soluble polymers. Linkage to a water-soluble polymer reduces the likelihood that the antibody will precipitate in an aqueous environment, such as a physiological environment.
• Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides.
• a reporter molecule is defined as any moiety which may be detected using an assay.
• Antibody conjugates may be used to deliver cytotoxic agents to target cells.
• Cytotoxic agents of this type may improve antibody-mediated cytotoxicity, and include such moieties as cytokines that directly or indirectly stimulate cell death, radioisotopes, chemotherapeutic drugs (including prodrugs), bacterial toxins (e.g., pseudomonas exotoxin, diphtheria toxin, etc.), plant toxins (e.g., ricin, gelonin, etc.), chemical conjugates (e.g., maytansinoid toxins, calechaemicin, etc.), radioconjugates, enzyme conjugates (e.g., RNase conjugates, granzyme antibody-directed enzyme/prodrug therapy), and the like.
• cytokines that directly or indirectly stimulate cell death
• chemotherapeutic drugs including prodrugs
• bacterial toxins e.g., pseudomonas exotoxin, diphtheria toxin, etc.
• plant toxins e.
• Antibody conjugates are also used as diagnostic agents.
• Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as “antibody-directed imaging.”
• Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509).
• the imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, MR hyperpolarized molecules, targeted ultrasound bubbles, and X-ray imaging agents.
• the radioactive isotopes contemplated for use in imaging and radiotherapy as conjugates or covalent incorporation include astatine-211, actinium-225, carbon-14, bismuth -212, chromium-51, chlorine-36, cobalt-57, cobalt-58, copper-64, copper- 67, Eu-152, florine-18, gallium-68, gallium-67, gold-198, hydrogen-3, iodine-123, iodine-125, iodine-131, indium-111, iron-52, iron-59, lead-212, lutetium-177, phosphorus-32, rhenium- 186, rhenium-188, rubidium-82, rhodium-99, selenium-75, sulphur-35, samarium-153, strontium-92, strontium-89, thallium- 201, thorium-227, technetium-94m, technitium-99m
• Radioactively labeled monoclonal antibodies and antibody fragments of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
• a chemical oxidizing agent such as sodium hypochlorite
• an enzymatic oxidizing agent such as lactoperoxidase.
• a large amount of an unlabelled antibody may be administered to the patient to administration of a therapeutic dose of labelled antibody.
• This therapeutic dose can be made to deliver a radiometric dose of 5 to 500 cGy, preferably, 25 to 150 cGy, to the whole body of the patient.
• an amount of radioactivity which would provide approximately 500 cGy to the whole body is estimated to be about 825 mCi of 1-131.
• the amounts of radioactivity to be administered depend, in part, upon the isotope chosen. For therapeutic regimens using 1-131, 5 to 1500 mCi might be employed, with preferable amounts being 5 to 800 mCi, 5 to 250 mCi being most preferable. For Y-90 therapy, 1 to 200 mCi amounts of radioactivity are considered appropriate, with preferable amounts being 1 to 150 mCi, and 1 to 100 mCi being most preferred.
• the preferred means of estimating tissue doses from the amount of administered radioactivity is to perform an imaging or other pharmacokinetic regimen with a tracer dose, so as to obtain estimates of predicted dosimetry.
• a “high-dose” protocol in the range of 200 to 600 cGy (or higher) to the whole body, typically requires the support of a bone-marrow replacement protocol, as the bone-marrow is the tissue which limits the radiation dosage due to toxicity.
• a preferable dosage is in the range of 15 to 150 cGy to the whole body, the most preferable range being 40 to 120 cGy. Using such a “low-dose” protocol, toxicity to bone marrow is much lower and we have found complete remissions are achieved without the requirement of bone marrow replacement therapies.
• Either or both the diagnostic and therapeutic administrations can be preceded by “pre-doses” of unlabeled antibody.
• pre-doses of unlabeled antibody.
• the effects of pre-dosing upon both imaging and therapy have been found to vary from patient to patient.
• the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
• a metal chelate complex employing, for example, an organic chelating agent such a diethylene- triamine-pentaacetic acid anhydride (DTP A); ethylene-diamine-tetraacetic acid; monomeric or dendrimeric l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA); 1,4,7- triazacyclononane-l,4,7-triacetic acid (NOTA); DFO; HOPO; N-chloro-p- toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody (U.S.
• DTP A diethylene- triamine-pentaacetic acid anhydride
• DOTA ethylene-diamine-tetraacetic acid
• DOTA 1,4,7- triazacyclononane-l,4,7-triacetic acid
• DFO HO
• Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light.
• 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts.
• the 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins and may be used as antibody binding agents.
• Antibody drug conjugates are a new class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with disease.
• ADCs are complex molecules composed of an antibody (a whole mAh or an antibody fragment, such as a scFv) linked, via a stable chemical linker with labile bonds, to a biological active cytotoxic/anti-viral payload or drug.
• Antibody drug conjugates are examples of bioconjugates and immunoconjugates.
• Trastuzumab emtansine is a combination of the microtubule- formation inhibitor mertansine (DM- 1), a derivative of the Maytansine, and antibody trastuzumab (Herceptin®/Genentech/Roche) attached by a stable, non-cleavable linker.
• DM-1 microtubule- formation inhibitor mertansine
• a derivative of the Maytansine a derivative of the Maytansine
• antibody trastuzumab Herceptin®/Genentech/Roche
• linker cleavable or noncleavable
• cytotoxic e.g., anti-cancer
• a non-cleavable linker keeps the drug within the cell.
• the entire antibody, linker, and cytotoxic agent enter the targeted cell where the antibody is degraded to the level of amino acids.
• the resulting complex - amino acid, linker and cytotoxic agent - now becomes the active drug.
• cleavable linkers are catalyzed by enzymes in or on the host cell, thereby releasing the cytotoxic agent. Commonly used mechanisms for linker cleavage are protease sensitivity, pH sensitivity, and glutathione sensitivity.
• cleavable linker adds an extra molecule between the cytotoxic drug and the cleavage site.
• This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics.
• researchers are also developing a new method of peptide cleavage based on Edman degradation.
• Future direction in the development of ADCs also include the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and a-emitting immunoconjugates and antibody-conjugated nanoparticles.
• TDCs site-specific conjugation
• Intrabodies or other proteins
• the two major issues impacting the implementation of intrabody therapeutics are delivery, including cell/tissue targeting, and stability.
• tissue-directed delivery use of cell-type specific promoters
• viral-based delivery use of cell-permeability/membrane translocating peptides
• exosomes delivery using exosomes.
• One means of delivery comprises the use of lipid-based nanoparticles, or exosomes, as taught in U.S. Pat. Appln. Publn.
• the approach is generally to either screen by brute force, including methods that involve phage display and may include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement/modification.
• insertion stabilizing sequences e.g., Fc regions, chaperone protein sequences, leucine zippers
• the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
• the first step for both of these methods is immunization of an appropriate host.
• a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
• exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
• KLH keyhole limpet hemocyanin
• BSA bovine serum albumin
• Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
• Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m- maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine.
• the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
• Exemplary and preferred adjuvants in animals include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis ), incomplete Freund’ s adjuvants and aluminum hydroxide adjuvant and in humans include alum, CpG, MFP59, and combinations of immunostimulatory molecules (“Adjuvant Systems”, such as AS01 or AS03). Additional experimental forms of inoculation to induce antigen-specific B cells are possible, including nanoparticle vaccines, or gene-encoded antigens delivered as DNA or RNA genes in a physical delivery system (such as lipid nanoparticle or on a gold biolistic bead), and delivered with needle, gene gun, or transcutaneous electroporation device.
• complete Freund’s adjuvant a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis
• incomplete Freund’ s adjuvants and aluminum hydroxide adjuvant and in humans include alum, CpG, MFP59, and
• the antigen gene also can be carried as encoded by a replication competent or defective viral vector such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively a vims-like particle.
• a replication competent or defective viral vector such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively a vims-like particle.
• Methods for generating hybrids of antibody-producing cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
• transformation of human B cells with Epstein Barr vims (EBV) as an initial step increases the size of the B cells, enhancing fusion with the relatively large-sized myeloma cells. Transformation efficiency by EBV is enhanced by using CpG and a Chk2 inhibitor dmg in the transforming medium.
• human B cells can be activated by co-culture with transfected cell lines expressing CD40 Ligand (CD 154) in medium containing additional soluble factors, such as IL-21 and human B cell Activating Factor (BAFF), a Type II member of the TNF superfamily.
• CD40 Ligand CD 1414
• BAFF human B cell Activating Factor
• Fusion methods using Sendai vims or polyethylene glycol (PEG) are also known.
• the use of electrically induced fusion methods is also appropriate. Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 6 to 1 x 10 8 , but with optimized procedures one can achieve fusion efficiencies close to 1 in 200.
• the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
• HAT medium a source of nucleotides
• azaserine the medium is supplemented with hypoxanthine.
• Ouabain is added if the B cell source is an EBV-transformed human B cell line, in order to eliminate EBV-transformed lines that have not fused to the myeloma.
• Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
• the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like.
• the selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide monoclonal antibodies.
• the cell lines may be exploited for monoclonal antibody production in two basic ways.
• a sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse).
• the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
• pristane tetramethylpentadecane
• Recombinant full-length IgG antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant.
• 293 e.g., Freestyle
• Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows.
• Antibody coding sequences can be RNA, such as native RNA or modified RNA.
• Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1 -methyl-pseudouridine (NIhiY) outperforms several other nucleoside modifications and their combinations in terms of translation capacity.
• RNA may be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle.
• Matched heavy and light chain genes from single cells also can be obtained from populations of antigen specific B cells by treating cells with cell-penetrating nanoparticles bearing RT-PCR primers and barcodes for marking transcripts with one barcode per cell.
• the antibody variable genes also can be isolated by RNA extraction of a hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector.
• combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens.
• Monoclonal antibodies produced by any means may be purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer. [00145] The antibodies of the present disclosure may be purified.
• purified is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state.
• a purified protein therefore also refers to a protein, free from the environment in which it may naturally occur.
• substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
• Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
• protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
• determining the specific activity of an active fraction or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
• Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity.
• the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
• sequences of antibodies may be modified for a variety of reasons, such as improved expression, improved cross-reactivity, or diminished off-target binding. Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides.
• hydropathic index of amino acids may be considered.
• the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
• Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 + 1), glutamate (+3.0 + 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 + 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (- 3.4), phenylalanine (-2.5), and tyrosine (-2.3).
• amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein.
• substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those that are within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.
• Amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
• the present disclosure also contemplates isotype modification.
• isotype modification By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgGi can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.
• effector functions are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
• Such effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g. , Fc binding assays, ADCC assays, CDC assays, etc.).
• a binding domain e.g., an antibody variable domain
• assays e.g. , Fc binding assays, ADCC assays, CDC assays, etc.
• a monoclonal antibody may have a novel Fc glycosylation pattern.
• Glycosylation of an Fc region is typically either N-linked or O-linked.
• N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
• O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5 -hydroxy lysine may also be used.
• the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline.
• X is any amino acid except proline.
• the glycosylation pattern may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation site(s) that are not present in the polypeptide.
• Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
• An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain.
• the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites.
• the isolated monoclonal antibody, or antigen binding fragment thereof may be present in a substantially homogenous composition represented by the GNGN or G1/G2 glycoform, which exhibits increased binding affinity for Fc gamma RI and Fc gamma Rill compared to the same antibody without the substantially homogeneous GNGN glycoform and with GO, GIF, G2F, GNF, GNGNF or GNGNFX containing glycoforms.
• Fc glycosylation plays a significant role in anti-viral and anti-cancer properties of therapeutic mAbs. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs).
• NK natural killer
• Such motifs can be eliminated by altering the synthetic gene comprising the cDNA encoding the antibodies.
• Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims.
• the epitope to which a given antibody binds may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within the antigen molecule (e.g., a linear epitope in a domain).
• the epitope may consist of a plurality of non-con!iguous amino acids (or amino acid sequences ) located within the antigen molecule (e. , a conformational epitope).
• peptide cleavage analysis high-resolution electron microscopy techniques using single particle reconstruction, cryoEM, or tomography, crystallographic studies and NMR analysis.
• methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496).
• Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry.
• the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein.
• the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface.
• amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface.
• the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem.73: 256A- 265A.
• epitope refers to a site on an antigen to which B and/or T cells respond.
• B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
• An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
• Modification-Assisted Profiling also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies.
• MAP may facilitate identification of rare hybridoma clones that produce monoclonal antibodies having the desired characteristics.
• MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes.
• the present disclosure includes antibodies that may bind to the same epitope, or a portion of the epitope.
• the antibodies may be defined by their variable sequence, which include additional “framework” regions. These are provided in Table 4 that represent full variable regions.
• the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below.
• nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about
• two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
• a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters.
• optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
• IgBLAST world-wide- web at ncbi.nlm.nih.gov/igblast/
• IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches and allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene.
• the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (/. ⁇ ? ., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (/. ⁇ ? ., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
• an antibody derivative of any of the antibodies provided herein and their antigen-binding fragments.
• a derivative antibody or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc.
• an antibody derivative will possess a similar or identical function as the parental antibody.
• an antibody derivative will exhibit an altered activity relative to the parental antibody.
• a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody.
• derivative refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (/. ⁇ ? ., DNA-encoded) or non-naturally occurring amino acid residues.
• derivative encompasses, for example, as variants having altered CHI, hinge, CH2, CH3 or CH4 regions, so as to form, for example antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics.
• derivative additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc.
• non-amino acid modifications for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc. content), acetylated, pegylated,
• the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function.
• the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art.
• Differential Scanning Calorimetry (DSC) measures the heat capacity, C p , of a molecule (the heat required to warm it, per degree) as a function of temperature.
• C p the heat capacity of a molecule (the heat required to warm it, per degree) as a function of temperature.
• DSC Differential Scanning Calorimetry
• C p heat capacity of a molecule (the heat required to warm it, per degree) as a function of temperature.
• DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAh structure, producing up to three peaks in the thermogram (from unfolding of the Fab, CH2, and CH3 domains). Typically unfolding of the Fab domain produces the strongest peak.
• DLS dynamic light scattering
• DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ.
• Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pi of the protein.
• Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument.
• IEF free solution isoelectric focusing
• cIEF capillary column
• Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pis).
• This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step.
• the technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies.
• the results identify charge heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 pg/mL.
• autoreactivity also can be surveyed using assessment of binding to tissues in tissue arrays.
• Chimeric antigen receptor (CAR) molecules are recombinant fusion proteins and are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor activation motifs (IT AMs) present in their cytoplasmic tails in order to activate genetically modified immune effector cells for killing, proliferation, and cytokine production.
• Receptor constructs utilizing an antigen-binding moiety afford the additional advantage of being “universal” in that they bind native antigen on the target cell surface in an HLA-independent fashion.
• Embodiments of the CARs described herein include nucleic acids encoding an antigen-specific CAR polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding domain.
• a CAR may recognize an epitope comprised of the shared space between one or more antigens.
• a CAR can comprise a hinge domain positioned between the transmembrane domain and the antigen binding domain.
• a CAR may further comprise a signal peptide that directs expression of the CAR to the cell surface.
• a CAR may comprise a signal peptide from GM-CSF.
• a CAR may also be co-expressed with a membrane-bound cytokine to improve persistence.
• a CAR may be co-expressed with membrane-bound IL-15.
• a chimeric antigen receptor can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques.
• a nucleic acid sequence encoding the several regions of the chimeric antigen receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning (genomic library screening, PCR, primer-assisted ligation, scFv libraries from yeast and bacteria, site-directed mutagenesis, etc.).
• the resulting coding region can be inserted into an expression vector and used to transform a suitable expression host allogeneic or autologous immune effector cells, such as a T cell or an NK cell.
• the chimeric construct may be introduced into immune effector cells as naked DNA or in a suitable vector.
• Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319.
• naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.
• a viral vector e.g. , a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector
• a retroviral vector e.g. , a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector
• Suitable vectors for use in accordance with the method of the present invention are non-replicating in the immune effector cells.
• a large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.
• A. Antigen binding domains are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.
• An antigen binding domain may comprise complementarity determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragments thereof.
• the antigen binding regions or domains may comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular mouse, human, or humanized monoclonal antibody.
• the fragment can also be any number of different antigen binding domains of an antigen- specific antibody.
• the fragment may be an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.
• VH and VL domains of a CAR are separated by a linker sequence, such as a Whitlow linker.
• the prototypical CAR encodes a scFv comprising VH and VL domains derived from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains (e.g. costimulatory domains and signaling domains).
• a CAR may comprise the LCDRl-3 sequences and the HCDRl-3 sequences of an antibody that binds to B7-H3.
• a CAR that comprises: (1) the HCDR1- 3 sequences of a first antibody that binds to the antigen; and (2) the LCDRl-3 sequences of a second antibody that binds to the antigen.
• a CAR that comprises HCDR and LCDR sequences from two different antigen binding antibodies may have the advantage of preferential binding to particular conformations of an antigen (e.g., conformations preferentially associated with cancer cells versus normal tissue).
• a CAR may be engineered using VH and VL chains derived from different mAbs to generate a panel of CAR+ immune effector cells.
• the antigen binding domain of a CAR may contain any combination of the LCDRl-3 sequences of a first antibody and the HCDRl-3 sequences of a second antibody.
• a CAR polypeptide may include a hinge domain positioned between the antigen binding domain and the transmembrane domain.
• a hinge domain may be included in CAR polypeptides to provide adequate distance between the antigen binding domain and the cell surface or to alleviate possible steric hindrance that could adversely affect antigen binding or effector function of CAR-modified immune effector cells.
• the hinge domain may comprise a sequence that binds to an Fc receptor, such as FcyR2a or FcyRla.
• the hinge sequence may comprise an Fc domain from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD or IgE) that binds to an Fc receptor.
• a human immunoglobulin e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD or IgE
• a CAR hinge domain may comprise an Ig Fc domain that comprises at least one mutation relative to wild type Ig Fc domain that reduces Fc-receptor binding.
• the CAR hinge domain can comprise an IgG4-Fc domain that comprises at least one mutation relative to wild type IgG4-Fc domain that reduces Fc-receptor binding.
• a CAR hinge domain may comprise an IgG4-Fc domain having a mutation (such as an amino acid deletion or substitution) at a position corresponding to L235 and/or N297 relative to the wild type IgG4- Fc sequence.
• a CAR hinge domain can comprise an IgG4-Fc domain having a L235E and/or a N297Q mutation relative to the wild type IgG4-Fc sequence.
• a CAR hinge domain may comprise an IgG4-Fc domain having an amino acid substitution at position L235 for an amino acid that is hydrophilic, such as R, H, K, D, E, S, T, N or Q, or that has similar properties to an “E,” such as D.
• a CAR hinge domain may comprise an IgG4-Fc domain having an amino acid substitution at position N297 for an amino acid that has similar properties to a “Q,” such as S or T.
• the hinge domain may comprise a sequence that is about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an IgG4 hinge domain, a CD8a hinge domain, a CD28 hinge domain, or an engineered hinge domain.
• the antigen-specific extracellular domain and the intracellular signaling-domain may be linked by a transmembrane domain.
• Polypeptide sequences that can be used as part of transmembrane domain include, without limitation, the human CD4 transmembrane domain, the human CD28 transmembrane domain, the transmembrane human O ⁇ 3z domain, a cysteine mutated human € ⁇ 3z domain, or other transmembrane domains from other human transmembrane signaling proteins, such as CD16, CD8, and erythropoietin receptor.
• the transmembrane domain may comprise a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of those provided in U.S. Patent Publication No. 2014/0274909 (e.g. a CD8 and/or a CD28 transmembrane domain) or U.S. Patent No. 8,906,682 (e.g. a CD8a transmembrane domain), both incorporated herein by reference.
• Transmembrane regions may be derived from (i.e.
• the transmembrane domain can be 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a CD8a transmembrane domain or a CD28 transmembrane domain.
• the intracellular signaling domain of a CAR is responsible for activation of at least one of the normal effector functions of the immune cell engineered to express the CAR.
• effector function refers to a specialized function of a differentiated cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Effector function in a naive, memory, or memory-type T cell includes antigen-dependent proliferation.
• intracellular signaling domain refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function.
• the intracellular signaling domain may be derived from the intracellular signaling domain of a native receptor.
• Examples of such native receptors include the zeta chain of the T-cell receptor or any of its homologs (e.g. , eta, delta, gamma, or epsilon), MB1 chain, B29, Fc RIII, Fc RI, and combinations of signaling molecules, such as O ⁇ 3z and CD28, CD27, 4-1BB/CD137, ICOS/CD278, IL-2R /CD122, IL-2Ro/CD132, DAP10, DAP12, CD40, OX40/CD134, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins can be used.
• intracellular signaling domain While the entire intracellular signaling domain may be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal.
• intracellular signaling domain is thus meant to include a truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal, upon CAR binding to a target.
• the intracellular signaling domain comprises a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a € ⁇ 3z intracellular domain, a CD28 intracellular domain, a CD 137 intracellular domain, or a domain comprising a CD28 intracellular domain fused to the 4- IBB intracellular domain.
• Immune effectors cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), natural killer (NK) cells, invariant NK cells, or NKT cells. Also provided herein are methods of producing and engineering the immune effector cells as well as methods of using and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune effector cells may be used as immunotherapy, such as to target cancer cells.
• the immune effector cells may be isolated from subjects, particularly human subjects.
• the immune effector cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, a subject who is undergoing therapy for a particular disease or condition, a subject who is a healthy volunteer or healthy donor, or from a blood bank.
• Immune effector cells can be collected, enriched, and/or purified from any tissue or organ in which they reside in the subject including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, bone marrow, tissues removed and/or exposed during surgical procedures, and tissues obtained via biopsy procedures.
• the isolated immune effector cells may be used directly, or they can be stored for a period of time, such as by freezing.
• Tissues/organs from which the immune effector cells are enriched, isolated, and/or purified may be isolated from both living and non-living subjects, wherein the non-living subjects are organ donors.
• Immune effector cells isolated from cord blood may have enhanced immunomodulation capacity, such as measured by CD4- or CD8-positive T cell suppression.
• the immune effector cells may be isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulation capacity.
• the pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).
• the cells may include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4 + cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
• the cells may be allogeneic and/or autologous.
• the cells may be derived from pluripotent and/or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs).
• T cells may be separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14.
• a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells.
• Such CD4 + and CD8 + populations can be further sorted into sub populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
• CD8 + T cells may be further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
• Enrichment for central memory T (TCM) cells may be carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations.
• the cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T cells of at least about 50- fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.
• 50- fold e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater
• rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.
• T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin- 15 (IL-15), with IL-2 being preferred.
• the non-specific T-cell receptor stimulus can include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.).
• T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 IU/ml IL-2 or IL-15, with IL-2 being preferred.
• HLA-A2 human leukocyte antigen A2
• T-cell growth factor such as 300 IU/ml IL-2 or IL-15, with IL-2 being preferred.
• the in vz ' zro-induced T-cells are rapidly expanded by re- stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
• the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with i
• the autologous T-cells can be modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells.
• Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12.
• IL interleukin
• Suitable methods of modification are known in the art. See, for instance, Sambrook et ai, MOLECULAR CLONING: A LABORATORY MANUAL, 3 rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
• modified autologous T- cells express the T-cell growth factor at high levels.
• T-cell growth factor coding sequences such as that of IL-12, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high-level expression.
• the immune effector cells may be natural killer (NK) cells.
• Natural killer (NK) cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are critical effectors of the early innate immune response toward transformed and virus-infected cells. NK cells constitute about 10% of the lymphocytes in human peripheral blood. When lymphocytes are cultured in the presence of interleukin 2 (IL-2), strong cytotoxic reactivity develops. NK cells are effector cells known as large granular lymphocytes because of their larger size and the presence of characteristic azurophilic granules in their cytoplasm.
• IL-2 interleukin 2
• NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. NK cells do not express T-cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.
• Stimulation of NK cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors.
• the activation status of NK cells is regulated by a balance of intracellular signals received from an array of germ- line-encoded activating and inhibitory receptors.
• NK cells encounter an abnormal cell (e.g., tumor or virus -infected cell) and activating signals predominate, the NK cells can rapidly induce apoptosis of the target cell through directed secretion of cytolytic granules containing perforin and granzymes or engagement of death domain-containing receptors.
• Activated NK cells can also secrete type I cytokines, such as interferon-g, tumor necrosis factor-a and granulocyte- macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells as well as other cytokines.
• cytokines such as interferon-g, tumor necrosis factor-a and granulocyte- macrophage colony-stimulating factor (GM-CSF)
• GM-CSF granulocyte- macrophage colony-stimulating factor
• NK cells are central players in a regulatory crosstalk network with dendritic cells and neutrophils to promote or restrain immune responses.
• the tumor cell must bear some marker that is amenable to targeting, /. ⁇ ? ., is not present on the majority of other cells.
• Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl55.
• An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
• the immune checkpoint inhibitors may be drugs, such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (e.g., International Patent Publication W02015/016718; Pardoll, Nat Rev Cancer, 12(4): 252- 264, 2012; both incorporated herein by reference).
• Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized, or human forms of antibodies may be used.
• alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure.
• Such alternative and/or equivalent names are interchangeable in the context of the present disclosure.
• lambrolizumab is also known under the alternative and equivalent names MK- 3475 and pembrolizumab.
• a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
• the PD-1 ligand binding partners are PD-L1 and/or PD-L2.
• a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners.
• PD-L1 binding partners are PD-1 and/or B7-1.
• a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners.
• a PD-L2 binding partner is PD-1.
• the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.
• Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference.
• Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as described in U.S. Patent Application Publication Nos. 2014/0294898, 2014/022021, and 2011/0008369, all of which are incorporated herein by reference.
• Nivolumab also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO ® , is an anti- PD-1 antibody described in W02006/121168.
• Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA ® , and SCH-900475, is an anti-PD-1 antibody described in W02009/114335.
• CT-011 also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009/101611.
• AMP-224 also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
• the immune checkpoint inhibitor is an anti- CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Anti- human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in US Patent No. 8,119,129; PCT Publn. Nos.
• Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used.
• a humanized CTLA-4 antibody is described in International Patent Application No. W02001/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
• the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies.
• the antibody has an at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
• Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Patent Nos. 5844905, 5885796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins such as described in U.S. Patent No. 8329867, incorporated herein by reference.
• lymphocyte- activation gene 3 also known as CD223.
• the complete protein sequence of human LAG-3 has the Genbank accession number NP-002277.
• LAG-3 is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells.
• LAG-3 acts as an “off’ switch when bound to MHC class II on the surface of antigen-presenting cells. Inhibition of LAG-3 both activates effector T cells and inhibitor regulatory T cells.
• V-domain Ig suppressor of T cell activation (VISTA), also known as C10orf54.
• the complete protein sequence of human VISTA has the Genbank accession number NP_071436. VISTA is found on white blood cells and inhibits T cell effector function.
• the immune checkpoint inhibitor is an anti-VISTA3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Anti-human- VISTA antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
• art recognized anti- VISTA antibodies can be used.
• An exemplary anti- VISTA antibody is JNJ-61610588 (also known as onvatilimab) (see, e.g., WO 2015/097536, WO 2016/207717, WO 2017/137830, WO 2017/175058).
• VISTA can also be inhibited with the small molecule CA-170, which selectively targets both PD-L1 and VISTA (see, e.g., WO 2015/033299, WO 2015/033301).
• the immune checkpoint inhibitor is an anti-CD38 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Anti-human-CD38 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CD38 antibodies can be used.
• An exemplary anti-CD38 antibody is daratumumab (see, e.g., U.S. Pat. No. 7,829,673).
• T cell immunoreceptor with Ig and ITIM domains T cell immunoreceptor with Ig and ITIM domains (TIGIT).
• TIGIT T cell immunoreceptor with Ig and ITIM domains
• the complete protein sequence of human TIGIT has Genbank accession number NP_776160.
• the immune checkpoint inhibitor is an anti-TIGIT antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Anti-human- TIGIT antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIGIT antibodies can be used.
• An exemplary anti-TIGIT antibody is MK-7684 (see, e.g., WO 2017/030823, WO 2016/028656).
• the immune checkpoint inhibitor is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Anti-human- 0X40 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-OX40 antibodies can be used.
• GITR glucocorticoid-induced tumour necrosis factor receptor-related protein
• AITR glucocorticoid-induced tumour necrosis factor receptor-related protein
• the complete protein sequence of human GITR has Genbank accession number NP_004186.
• the immune checkpoint inhibitor is an anti-GITR antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
• Anti-human-GITR antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-GITR antibodies can be used.
• An exemplary anti-GITR antibody is TRX518 (see, e.g., WO 2006/105021).
• the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully.
• the signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).
• the present application provides for a combination therapy for the treatment of cancer wherein the combination therapy comprises adoptive T cell therapy and a checkpoint inhibitor.
• the adoptive T cell therapy comprises autologous and/or allogenic T-cells.
• the autologous and/or allogenic T-cells are targeted against tumor antigens.
• Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
• Tumor resection refers to physical removal of at least part of a tumor.
• treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
• a cavity may be formed in the body.
• Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
• agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
• additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
• cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti- hyperproliferative efficacy of the treatments.
• Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
• Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
• the present disclosure concerns immunodetection methods for detecting expression of B7-H3.
• assay formats are contemplated for detecting protein products, including immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, dot blotting, FACS analyses, mass cytometry (CyTOF), imaging mass cytometry (IMC), and Western blot to mention a few.
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• immunoradiometric assay immunoradiometric assay
• fluoroimmunoassay fluoroimmunoassay
• chemiluminescent assay chemiluminescent assay
• bioluminescent assay bioluminescent assay
• dot blotting FACS analyses
• mass cytometry CyTOF
• IMC imaging mass
• the immunobinding methods include obtaining a sample, and contacting the sample with an antibody specific for the protein to be detected, as the case may be, under conditions effective to allow the formation of immunocomplexes.
• the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, metallic, fluorescent, biological and enzymatic tags.
• the detection methods may involve imaging ex vivo, such as CyTOF or IMC.
• the detection methods may involve imaging in vivo, such as PET or SPECT imaging.
• a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.
• the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
• the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
• the second binding ligand may be linked to a detectable label.
• the second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody.
• the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
• the secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
• the present disclosure provides methods of imaging using the compounds and compositions of the present disclosure.
• the imaging is positron-emission tomography.
• Positron emission tomography (PET) imaging is based on detecting two time-coincident high-energy photons from the emission of a positron- emitting radioisotope.
• PET imaging is unique in its very high sensitivity and accurate estimation of the in vivo concentration of the radiotracer. PET imaging has been widely adopted as an important clinical modality for oncological, cardiovascular, and neurological applications. PET imaging has also become an important tool in preclinical studies, particularly for investigating murine models of disease and other small-animal models.
• a sample may be obtained and processed using well-known and routine clinical methods (e.g., procedures for drawing and processing whole blood).
• an exemplary sample may be peripheral blood drawn from a subject with cancer.
• the biological sample comprises a plurality of cells.
• the biological sample comprises fresh or frozen tissue.
• the biological sample comprises formalin fixed, paraffin embedded tissue.
• the biological sample is a tissue biopsy, fine needle aspirate, blood, serum, plasma, cerebral spinal fluid, urine, stool, saliva, circulating tumor cells, exosomes, or aspirates and bodily secretions, such as sweat.
• the biological sample contains cell-free DNA.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
• kits are envisioned containing therapeutic agents and/or other therapeutic and delivery agents.
• kits are provided for preparing and/or administering a therapy of the embodiments.
• the kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments.
• the kit may include, for example, at least one B7-H3 antibody or B7- H3-specific CAR construct, as well as reagents to prepare, formulate, and/or administer the components of the embodiments or perform one or more steps of the inventive methods.
• the kit may also comprise a suitable container, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
• a suitable container which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
• the container may be made from sterilizable materials such as plastic or glass.
• the kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill in the art.
• the instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.
• a MIL33B hybridoma pellet was frozen and sent to a sequencing core facility (Vanderbilt Sequencing Center, Arlington, TN) for sequencing of the hypervariable regions of both the heavy and light chains.
• the isotype of MIL33B has been determined to be IgG2a.
• the complementarity-determining regions (CDR) and variable regions of the MIL33B antibody are provided in Tables 1-3.
• Example 2 - MIL33B binds human 4Ig-B7-H3 with pM affinity
• the in vitro binding affinity of MIL33B for human 4Ig-B7-H3 and mouse 2Ig-B7-H3 was assessed by ELISA.
• the extracellular domains of human 4Ig-B7-H3 and murine 2Ig-B7-H3 were purchased and plated down onto 96- well plates. The plates were then incubated with varying concentrations of purified MIL33B. Plates were then washed and assessed for antibody binding through absorbance measurements. This experiment was completed for three independent productions and purifications of MIL33B.
• the EC50 for each curve for each lot was calculated and plotted (log plot) (FIG. 1).
• MIL33B has picomolar affinity for human 4Ig-B7-H3 and nanomolar affinity for mouse 2Ig-B7-H3.
• Example 3 - MIL33B selectively binds to human 4Ig-B7-H3
• the extracellular domains of the indicated B7 family proteins were purchased from R&D Systems. All proteins were validated by the vendor to be both pure and functional. Kd values were determined utilizing capture biolayer interferometry (Octet, Molecular Devices) in “affinity” mode, wherein the MIL33B antibody was captured on the tip of the probe and placed into solutions containing different concentrations of target extracellular domain. Kd, Kd error, and R 2 values are reported in Table 6. An exemplary fitting view of the binding of MIL33B to human 4Ig-B7-H3 is shown in FIG. 2. These data show that MIL33B binds to 4Ig-B7-H3 at least 1000-fold better than to other B7 family members.
• MIL33B labeled with Alexa594 fluorophore was used in live cell immuno-fluorescence microscopy to demonstrate selective, blockable binding to both human and murine B7-H3 expressed on tumor cells.
• Murine breast tumor, 4T1 cells ATCC
• 4Ig-B7-H3 or empty vector using lenti viral constructs ATCC
• Cells were selected that demonstrated levels of 4Ig-B7-H3 expression that matched those found endogenously in human solid tumors known to express B7-H3, such as cervical cancer (HeLa) or colorectal cancer (HCT 116) (FIG. 3).
• MIL33B or isotype control IgG2a were labeled with Alexa594 fluorophore utilizing commercially available kits (Life Technologies) at 1 mg/mL and purified via size exclusion column. The resulting products were designated MIL33B-A594 or IgG2a- A594. Labeling yields were quantified via UV/Vis spectrometry.
• Cells expressing human 4Ig-B7-H3 were incubated either alone, with labeled MIL33B (1 pg/mL) or murine isotype control IgG2a (1 pg/mF) for 1 hr at 37 °C.
• Pan02 cells a pancreatic tumor of murine origin known to express murine 2Ig-B7-H3, were similarly imaged with MIL33B-A594 using 100 pg/mL of MIL33B- A594, reflecting the ⁇ 100-fold shift in Kd from human 4Ig-B7-H3 to murine 2Ig-B7-H3 (FIG. 4C).
• Example 5 Flagellin-conjugated MIL33B induces the NF-KB pathway via TFR5
• MIF33B conjugated to the TFR5 agonist, flagellin, maintained nM binding affinity and was capable of inducting inflammatory pathways, such as NF-KB via TFR5, in live cell assays.
• HCT116 cells expressing endogenous human 4Ig-B7-H3 and TFR5, the native cognate receptor for flagellin, were stably transduced with a icB-RE -IkBa- Flue reporter.
• This cell line enables the study of both the activation of IKK through the initial degradation of the IkBa-luciferase (IkBa-Fluc) fusion protein as read out by loss of light production and the subsequent activation of NF-KB transcriptional targets through the re-synthesis of IkBa-Fluc as read out by a secondary increase in light production from the KB response element (RE 5 ) (FIGS. 6A-B).
• MIF33B-flgn conjugate was able to elicit activation of NF-KB via TFR5 at 18.2 nM with a response capacity similar to 1.8 nM of purified flagellin (FIGS. 6C-D). Therefore, MIF33B conjugates can be used for tumor specific-delivery of immune-stimulants to the tumor microenvironment.
• Example 6 - MIL33B mAb can be conjugated with DFO and radiolabeled with 89 Zr to detect tumor expression of human 4Ig-B7-H3 selectively in syngeneic animal tumor models by PET imaging
• MIL33B was radiolabeled with Zr-89 for PET imaging of tumors expressing B7-H3, showing robust signal-to-noise ratios in two different immuno-competent syngeneic murine tumor models.
• MIL33B (blue) or IgG2a isotype control (red) antibodies were conjugated with DFO using standard conjugation methods.
• MIL33B-DFO or IgG2a-DFO were radio-labeled with 89 Zr by incubating in acetate buffer (pH 7) for 1 hr, and purified through size exclusion chromatography. Fabeling was confirmed via radio-TFC.
• Immune competent mice bearing subcutaneous 4T1 tumors expressing human 4Ig-B7-H3 or negative vector were utilized. 4T1 tumors also have low but detectable levels of murine 2Ig (see Western blots in FIG. 3).
• Mice were injected i.v. with ⁇ 30 pCi of radiolabeled MIF33B antibody or IgG2a control antibody and imaged at 24 and 72 hr post injection on a dedicated small animal PET/SPECT/CT scanner (Albira, Bruker Corp).
• B16F10 cells were transduced with human 4Ig-B7-H3 or empty vector.
• MIF33B was superior, both qualitatively and quantitatively, to isotype control for binding to tumors expressing human 4Ig-B7-H3, and gave the correct rank order of binding for the negative vector as predicted from their low background murine 2Ig-B7-H3 expression levels (FIG. 7).
• mice were then followed until day 150 post-inoculation. Mice were euthanized when the largest tumor axis was >1.5 cm, there was >3 mm of tumor ulceration, or if the mice were moribund as determined by independent veterinary staff. Overall survival curves and individual tumor growth curves are provided in FIG. 8.
• B7x a widely expressed B7 family member that inhibits T cell activation

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