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EP4577527A1 - Conjugués dota d'anticorps vhh - Google Patents

Conjugués dota d'anticorps vhh

Info

Publication number
EP4577527A1
EP4577527A1 EP23858213.4A EP23858213A EP4577527A1 EP 4577527 A1 EP4577527 A1 EP 4577527A1 EP 23858213 A EP23858213 A EP 23858213A EP 4577527 A1 EP4577527 A1 EP 4577527A1
Authority
EP
European Patent Office
Prior art keywords
substituted
unsubstituted
independently
pharmaceutically acceptable
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23858213.4A
Other languages
German (de)
English (en)
Inventor
Michael J. Abrams
Adam Daniel JUDGE
Alexander Laurence MANDEL
Raja Solomon Viswas
Douglas Bruce MACKAY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abdera Therapeutics Inc
Original Assignee
Abdera Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abdera Therapeutics Inc filed Critical Abdera Therapeutics Inc
Publication of EP4577527A1 publication Critical patent/EP4577527A1/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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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    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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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
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    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
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    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1054—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from lung
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    • A61K51/04—Organic compounds
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    • 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
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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
    • A61K51/1096—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 radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3023—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
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    • C07K—PEPTIDES
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    • C07K2317/52—Constant or Fc region; Isotype
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C07K2319/00—Fusion polypeptide

Definitions

  • monomeric scFv’s, heavy-chain only antibodies, or single-domain antibody fragments provides the extraordinar specificity of a full-size antibody (e.g. an IgG (-150 kDa) in a smaller format (e.g. 15 to 30 kDa) and with a much shorter serum half-life (e.g. 30 minutes to 2 hours) (Bates A, Power, C, Antibodies (Basel) 8: 28 (2019)).
  • serum half-life e.g. 30 minutes to 2 hours
  • 225 Ac is among the most cytotoxic of the a-emitting radioisotopes, and a single decay event can effectively destroy a cancer cell by causing double-strand DNA breaks and subsequent cell death.
  • the potency of a-emitting radioisotopes makes them attractive as cell killing agents, capable of overcoming the acquired resistance observed in response to other therapies.
  • numerous challenges remain with respect to systemic administration and the achievement of desired dosimetry in target versus non-target tissues as a result of decay events in different locations in vivo.
  • Key to the application of a-emitting radionuclides as targeted therapeutics is the ability to modulate the distribution of daughter nuclides in vivo so as to limit toxicity.
  • the high ionization density released by an a-emitter compromised the immunoreactivity of isotope-labeled Fab fragments via radiolysis at doses of 1,000 gray (Gy) or higher.
  • significant radiolysis of a-emitting isotope-labeled antibodies was observed at doses over 1,200 Gy (Zalutsky M et al., J Nucl Med. 42(10): 1508-15 (2001)).
  • the identification of an appropriate targeted delivery vehicle for a-emitting radioisotopes is not straightforward.
  • the present invention relates to immunoconjugates or radioimmunoconjugate, compositions comprising the same, and methods of using such immunoconjugates and compositions.
  • the immunoconjugates and compositions of the present invention have numerous uses, e.g., for delivery of a radioisotope to kill a target cell (e.g. a cancer cell expressing a target antigen bound by the radioimmunoconjugate); for detection and characterization of malignant cells within a subject (e.g. target antigen expression); and for diagnosis and treatment of a variety of diseases and conditions, such as, e.g., cancers, tumors, and other growth abnormalities involving antigen-expressing cells.
  • the present invention addresses a number of challenges inherent in the targeted delivery of alpha particle emitters in vivo through the selection and particular combination of specific delivery platform components.
  • the alpha particle emitting radioisotope-delivery platforms of the present invention provide shorter half-lives compared to traditional IgGs, but longer half-lives than smaller monomeric antibody fragment formats. Such half-lives allow for a reduction in toxicity due to the alpha emitter, while preserving the antibody fragment long enough in the body to exert therapeutic activity.
  • the alpha particle emitting radioisotopedelivery platforms of the current disclosure exhibit enhanced tumor targeting and reduced accumulation in radiosensitive tissues such as the bone-marrow and kidney.
  • the alpha particle emitting radioisotope-delivery platforms of the present invention exhibit excellent tumor binding and labeling properties for tumors with different antigen densities, which can be a limitation for some use of some immunoconjugates.
  • an immunoconjugate comprising an: a) antigen binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating agent; wherein the molecular weight of the immunoconjugate is between 60 and 110 kDa.
  • the antigen binding region comprises an scFv polypeptide or a VHH polypeptide.
  • the antigen binding region comprises an scFv polypeptide.
  • the antigen binding region comprises a VHH polypeptide.
  • the antigen binding region is humanized.
  • R 1 is a chelating moiety or a radionuclide complex thereof
  • R 2 is a moiety that is capable of reacting with an amine (-NH2) or thiol (-SH) of a tumor targeting moiety R 3 ;
  • L is a linker that is -L l -L 2 -L 3 - L 4 -L 5 -;
  • L 1 is unsubstituted or substituted Ci-Cioalkylene, unsubstituted or substituted Ci- Cioheteroalkylene, unsubstituted or substituted C2-C2oalkenylene, unsubstituted or substituted C2-C2oalkynylene, C4-C2opolyethylene glycol, -(X 3 CH2CH2)t-, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; each X 3 is independently selected from O and NR 4 ; each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;
  • the immunoconjugate is a compound of Formula (II), Formula (III), or Formula (IV), or a pharmaceutically acceptable salt thereof:
  • R 1 is a chelating moiety or a radionuclide complex thereof
  • -NH-R 3 is a tumor targeting moiety
  • L is a linker that is -L l -L 2 -L'- L 4 -L 5 -;
  • L 1 is unsubstituted or substituted Ci-Cioalkylene, unsubstituted or substituted Ci- Cioheteroalkylene, unsubstituted or substituted C2-C2oalkenylene, unsubstituted or substituted C2-C2oalkynylene, C4-C2opolyethylene glycol, -(X 3 CH2CH2)t-, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; each X 3 is independently selected from O and NR 4 ; each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;
  • -SCH2- is the thiol (-SH) of the side chain of a cysteine residue of the tumor targeting moiety R 3 .
  • the compound Formula (VII) has the structure of Formula (Vila), or a pharmaceutically acceptable salt thereof:
  • -SCH2- is the thiol (-SH) of the side chain of a cysteine residue of the tumor targeting moiety R 3 .
  • the compound Formula (VIII) has the structure of Formula (Villa), or a pharmaceutically acceptable salt thereof:
  • the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn); or the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region and alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn).
  • the antigen binding region specifically binds to HER2 or to DLL3. In certain embodiments, the antigen binding region specifically binds to HER2. In certain embodiments, the antigen binding region of the immunoconjugate comprises: a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22; and c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23 and that binds to HER2.
  • the antigen binding region of the immunoconjugate comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 20 and that binds to HER2. In certain embodiments, the antigen binding region specifically binds to DLL3. In certain embodiments, the antigen binding region of the immunoconjugate comprises: a) a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31; b) a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and c) a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33 and that binds to DLL3.
  • the antigen binding region of the immunoconjugate comprises a sequence that is at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 30 and that binds to DLL3.
  • the immunoglobulin heavy chain constant region comprises a CH2 domain of an immunoglobulin, CH3 domain of an immunoglobulin, or a CH2 and a CH3 domain of an immunoglobulin.
  • the immunoglobulin heavy chain constant region comprises a CH2 and a CH3 domain of an immunoglobulin.
  • the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region.
  • the immunoglobulin heavy chain constant region is an IgA, IgGl, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgGl isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region and alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn).
  • the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement dependent cytotoxicity (CDC), antibody-dependent cell-cytotoxicity (ADCC), antibody-dependent cellphagocytosis ADCP, or a combination thereof.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-cytotoxicity
  • ADCP antibody-dependent cellphagocytosis ADCP
  • the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected from the list consisting of: (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y, or 329R (k) 33 IS, (1) 236F or 236R, (m) 238A, 238E, 238G, 238H, 2381, 238V, 238W, or 238Y, (n) 248A, (
  • the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region comprises L234A, L235E, G237A, A330S, and P33 IS per EU numbering.
  • the amino acid alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate.
  • the alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof per EU numbering.
  • the alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 253, 254, 310, 435, 436 and combinations thereof per EU numbering.
  • the alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253 A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof per EU numbering.
  • the alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253 A, S254A, H310A, H435Q, Y436A and combinations thereof per EU numbering. In certain embodiments, the alteration to one or more amino acid residues that alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253 A, H310A, H435Q, and combinations thereof per EU numbering. In certain embodiments, the immunoconjugate has a serum half-life of less than 15 days.
  • the immunoconjugate has a serum half-life of less than 10 days. In certain embodiments, the immunoconjugate has a serum half-life of less than 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of less than 72 hours.
  • the antigen binding region is coupled to the immunoglobulin heavy chain constant region by a linker amino acid sequence or a human IgG hinge region. In certain embodiments, the antigen binding region is coupled to the immunoglobulin heavy chain constant region by a human IgG hinge region.
  • Also described herein is a method of delivering a radioisotope to a cancer cell or a tumor cell in an individual comprising administering to the individual the immunoconjugate, thereby delivering the radioisotope to the cancer cell or the tumor cell.
  • the individual is a human individual.
  • the cancer cell or the tumor cell comprises a lung cancer cell, a breast cancer cell, an ovarian cancer cell, or a neuroendocrine cancer cell.
  • the method comprises administering from 0.5 pCi to 30.0 pCi per kilogram to the individual.
  • the cancer cell or the tumor cell expresses an antigen specifically bound by the immunoconjugate.
  • the immunoconjugate for use in delivering a radioisotope to a cancer cell or a tumor cell in an individual.
  • the individual is a human individual.
  • the cancer cell or the tumor cell comprises a lung cancer cell, a breast cancer cell, an ovarian cancer, or a neuroendocrine cancer cell.
  • the cancer cell or the tumor cell expresses an antigen specifically bound by the immunoconjugate.
  • a method of imaging a tumor in an individual comprising administering to the individual the immunoconjugate.
  • the individual is a human individual.
  • the cancer or the tumor comprises lung cancer, breast cancer, ovarian cancer, or a neuroendocrine cancer.
  • the tumor expresses an antigen specifically bound by the immunoconjugate.
  • an expression vector comprises the nucleic acid.
  • a cell comprises the nucleic acid or the expression vector.
  • the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a CHO cell.
  • the invention provides a targeted imaging complex, comprising an immunoconjugate of the invention and further comprising an imaging metal.
  • the invention provides a targeted imaging complex, comprising an antibody construct of an immunoconjugate of the invention and further comprising an imaging metal.
  • the imaging metal is a radioisotope.
  • the imaging metal is selected from the group comprising: in In (Indium-111), 89 Zr (Zirconium-89), 64 Cu (Copper-64), 68 Ga (Gallium-69) and 134 Ce (Caesium-134).
  • the imaging metal is selected from the group consisting of in In, 89 Zr, 64 Cu, 68 Ga, and 134 Ce.
  • the imaging metal is in In. In one embodiment, the imaging metal is covalently bound to the immunoconjugate or antibody construct. In one embodiment, the imaging metal is associated with the chelating agent of an immunoconjugate. In one embodiment, the invention provides a method of determining the location of a cancer cell in vivo in a patient, comprising administering to the patient a targeted imaging complex of the invention. In one embodiment, the patient is a human patient.
  • the invention provides a kit for preparing a radiopharmaceutical of the invention, comprising an immunoconjugate of the invention. In one embodiment, the invention provides a kit comprising a radioimmunoconjugate of the invention. In one embodiment, the invention provides a kit for preparing a pharmaceutical composition of the invention, comprising an immunoconjugate of the invention. In one embodiment, the invention provides a kit for preparing a pharmaceutical composition of the invention, comprising a radioimmunoconjugate of the invention. In one embodiment, the invention provides a kit comprising a pharmaceutical composition of the invention.
  • the immunoconjugate or radioimmunoconjugate of the invention comprises a dimerization domain or motif.
  • the dimerization domain or motif is in the hinge region and/or the variant constant region.
  • the immunoconjugate or radioimmunoconjugate or pharmaceutical composition of the invention has a half-life in human serum of less than 96 hours. In some further embodiments, a half-life in human serum of less than 72 hours. In some further embodiments, the half-life is less than 48, 36, 24, and/or 12 hours. In some embodiments, the halflife is between 4 and 8 hours, between 6 and 12 hours, between 8 and 16 hours, between 12 and 24 hours, or between 24 and 48.
  • the invention provides an isolated nucleic acid encoding an antigen binding arm or a component thereof as provided herein. In one aspect, the invention provides an isolated nucleic acid encoding an antigen binding region of an immunoconjugate herein. In one aspect, the invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate herein. In one aspect, the invention provides an isolated nucleic acid encoding a hinge region of an immunoconjugate herein. In one aspect, the invention provides an isolated nucleic acid encoding a variant constant region of an immunoconjugate herein.
  • the invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate herein and a hinge region of an immunoconjugate herein. In one aspect, the invention provides an isolated nucleic acid encoding a VHH polypeptide of an immunoconjugate herein, a hinge region of an immunoconjugate herein, and a variant constant region of an immunoconjugate herein.
  • the invention provides methods of using an immunoconjugate, radioimmunoconjugate, targeted imaging complex or pharmaceutical composition of the present invention.
  • the invention provides a method of treating a disease, disorder, or condition, the method comprising administering to patient in need thereof a pharmaceutically effective amount of a radioimmunoconjugate or pharmaceutical composition herein.
  • a method of the invention comprises the step of administering to a subject, in need thereof, any of the radioimmunoconjugates or pharmaceutical compositions described herein.
  • the method is for inhibiting the growth and/or the killing of a cancer cell or tumor.
  • the use of an immunoconjugate or radioimmunoconjugate described herein is provided for the manufacture of a medicament for treating a disease, disorder, or condition in a subject, such as, e.g., cancer.
  • the invention provides a process for making a radioimmunoconjugate or pharmaceutical composition of the present invention, the method comprising radiolabeling the immunoconjugate with an appropriate isotope, such as, e.g., an alpha or beta particle emitter.
  • an appropriate isotope such as, e.g., an alpha or beta particle emitter.
  • FIG. 1A and IB show binding of anti-HER2 and anti-DLL3 VHH-Fc constructs.
  • FIG. 3A and 3B show internalization of anti-HER.2 and anti-DLL3 VHH-Fc constructs in cells expressing HER2 and DLL3.
  • FIG. 4 shows self-interaction data for anti-HER2 and anti-DLL3 VHH-Fc constructs.
  • FIG. 10A, 10B and 10C show tumor: non-tum or tissue ratios for labeled anti-HER2 VHH-Fc constructs.
  • FIG. 12 shows whole body clearance of VHH-Fc (H101) and VHH-Fc variants (H105, H107, and H108) labeled with ni In.
  • FIG. 13 shows biodistribution over time for labeled anti-DLL3 VHH-Fc constructs.
  • FIG. 14 shows biodistribution for labeled anti-DLL3 VHH-Fc constructs.
  • FIG. 15A and 15B show biodistribution for 225 Ac labeled anti-HER2 (15A) and anti- DLL3 (15B) VHH-Fc constructs.
  • FIG. 16A, 16B, and 16C show the results of a toxicity study carried out with 225 Ac labeled anti-HER2 VHH-Fc constructs.
  • FIG. 17 shows the immunoreactive fraction of different anti-DDL3 VHH-Fc constructs loaded with 177 Lu.
  • FIG. 18 shows the chemical structures of certain linker chelators described herein.
  • FIG. 19A and FIG 19B show imaging experiments with ni In in naive mice to measure total body clearance of radioactivity using different conjugates (see Example B-26).
  • the present invention addresses a number of challenges inherent in the targeted delivery of radioisotopes in vivo through the selection and particular assembly of specific immunoconjugate and radioimmunoconjugate components.
  • the radioisotope-delivering platforms of the present invention provide shorter half-lives compared to traditional IgGs, but longer half-lives than smaller monomeric antibody fragment formats.
  • the subject radioisotope delivering platforms are useful for in vivo targeted delivery of radioisotopes (such as alpha- or beta-emitters) safely and effectively by, in part, reducing certain adverse effects caused by platforms having half-lives over 5 days and/or molecular weights under 60 kDa.
  • the subject radioisotope delivering platforms are useful for in vivo targeted delivery of radioisotopes (such as alpha- or beta-emitters) safely and effectively, in part, by exhibiting decreased loss of targeting capacity due to radiolysis as compared to other possible delivery platforms.
  • the subject radioisotope delivering platforms are useful for in vivo targeted delivery of radioisotopes (such as alpha- or beta-emitters) safely and effectively, in part, by exhibiting increased stability in manufacturing under the temperatures required for certain radiolabeling processes (e.g., high temperature chelation with certain chelators) as compared to other possible delivery platforms using antibody fragments.
  • radioisotopes such as alpha- or beta-emitters
  • the invention provides immunoconjugates that specifically bind to a target antigen with high affinity.
  • the present invention provides an immunoconjugate that specifically binds to a cell-surface antigen of a cancer cell.
  • the immunoconjugate comprises three, four, five, six, or more CDRs or HVRs (Kabat).
  • the immunoconjugate binds a specific antigen and/or epitope with an affinity characterized by a KD of ⁇ 1 pM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10' 8 M or less, e.g. from 10' 8 M to 10' 13 M, e.g., from 10' 9 M to 10' 13 M).
  • an immunoconjugate of the current disclosure comprises an: a) VHH antigen binding region; b) an immunoglobulin Fc region, together referred to as aVHH-Fc; and c) a chelating moiety or a radionuclide complex thereof.
  • an immunoconjugate of the current disclosure comprises an: a) VHH antigen binding region; b) an immunoglobulin Fc region; and c) a chelating moiety or a radionuclide complex thereof; wherein the molecular weight of said immunoconjugate is between 60 and 110 kDa.
  • the radioisotope delivering platforms have sizes larger than about 60 kDa, in order to avoid certain toxicities from an alpha emitting isotope cargo, such as, e.g., off-target renal toxicities. In some embodiments, the radioisotope delivering platforms have sizes less than about 110 kDa in order to improve tumor penetration. In some embodiments, the radioisotope delivering platform has size between 60 and 110 kDa due to its dimeric structure of two individual antigen binding arms each having a VHH polypeptide fused to a hinge region and a wild-type or variant constant region. In some embodiments, the variant constant region has specific amino acid substitution(s) relatively to a wildtype Fc region in order to reduce half-life and/or eliminate Fc effector function(s).
  • the tumor antigen comprises Her2, Trop2, CEA, NaPi2b, uPAR, CDCP1, MUC-1, MUC-16, CEACAM-5, MR-1, Fnl4, MAGE-3, NY-ESO-1, EGFR, PDGFR, IGF1R, CSF-1R, PSMA, PSCA, STEAP-1, FAP, TEM8, 5T4, VEGFR, NRP1, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD39, CD44, CD47, CD52, CD70, CD71, CD74, CD79b, CD132, CD133, CD138, CD166, CD205, CD276, R0R1, ROR2, Glypican 3, Trail Receptor 2 (DR5), PD-L1, Mesothein, Bombesin, EpCAM, DARPP, CSPG4, Galectin-3, Integrin avpi, Integrin avP3, Integrin avP5, Integrin avP6,
  • the tumor antigen comprises human epidermal growth factor receptor 2 (HER2), Delta-like ligand 3 (DLL3), folate receptor alpha (F0LR1), or Wnt activated inhibitory factor 1 (WAIF1).
  • the tumor antigen comprises HER2.
  • the tumor antigen comprises DLL3.
  • the tumor antigen comprises FOLR1.
  • the tumor antigen comprises WAIF1.
  • the tumor antigen comprises TROP2.
  • the tumor antigen comprises EGFR.
  • the tumor antigen comprises PSA.
  • the tumor antigen comprises MUC-1.
  • the tumor antigen comprises CEA.
  • the tumor antigen comprises NY-ESO-1.
  • the antigen binding region of the immunoconjugate comprises: a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; b) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22; and c) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23.
  • the antigen binding region of the immunoconjugate comprises: a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31; b) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32; and c) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33.
  • the immunoconjugate of the present invention comprises a synthetically engineered antibody derivate, such as, e.g. a protein or polypeptide comprising an autonomous VH domain (such as, e.g., from cam elids, murine, or human sources), single-domain antibody domain (sdAb), heavy-chain antibody domains derived from a camelid (VHH fragment or VH domain fragment), heavy-chain antibody domains derived from a camelid VHH fragments or VH domain fragments, heavy-chain antibody domain derived from a cartilaginous fish, immunoglobulin new antigen receptor (IgNAR), VNAR fragment, single-chain variable (scFv) fragment, nanobody, “camelized” or “camelised” scaffold comprising a VH domain, Fd fragment consisting of the heavy chain and CHI domains, single chain FV-CH3 minibody, Fc antigen binding domain (Fcabs), scFv-Fc fusion, multimerizing s
  • an autonomous VH domain such
  • the antigen binding regions of the immunoconjugates described herein may comprise an Fc or heavy chain constant region.
  • the antigen binding molecules can be coupled to the Fc or heavy chain constant region directly, by a suitable linker, or by an IgG hinge region.
  • the inclusion of the heavy chain constant region or Fc region confers such advantages as allowing for optimization and tuning of serum half-life, the addition of additional sites to conjugate a chelating or cytotoxic agent, and allow for purification of the immunoconjugates using standard processes and methods.
  • the addition of a heavy chain constant region also increases the size which may shift the catabolisis and elimination of the immunoconjugate to the liver from the kidney.
  • the immunoglobulin constant region comprises or consists of an Fc region.
  • the immunoglobulin heavy chain constant region comprises a CH2 domain of an immunoglobulin, CH3 domain of an immunoglobulin, or a CH2 and a CH3 domain of an immunoglobulin.
  • the immunoglobulin heavy chain constant region comprises a CH2 and a CH3 domain of an immunoglobulin.
  • the immunoglobulin heavy chain constant region may be human, preventing or reducing an endogenous immune response against the immunoconjugate.
  • the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region.
  • the immunoglobulin heavy chain constant region is an IgA, IgGl, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgGl isotype. In certain embodiments, the immunoglobulin heavy chain constant region is an IgG4 isotype.
  • the immunoglobulin heavy chain constant region can be a variant constant region that comprises one or more alterations to amino acid residue(s) that confers additional utility and advantageous properties to the immunoconjugates described herein.
  • the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or alters binding of the immunoconjugate to the neonatal Fc receptor (FcRn).
  • the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn).
  • the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region and reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn). In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces an effector function of the immunoglobulin heavy chain constant region. In certain embodiments, the immunoglobulin heavy chain constant region comprises an alteration to one or more amino acid residues that reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn).
  • the alterations to heavy chain constant regions of the immunoconjugate can reduce effector function associated with a heavy chain constant region, such as, the ability to fix complement, promote phagocytosis, or recruit other immune effector cells (e.g., NK cells) to the heavy chain constant region.
  • the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is an alteration that reduces complement dependent cytotoxicity (CDC), antibody-dependent cellcytotoxicity (ADCC), antibody-dependent cell-phagocytosis ADCP, or a combination thereof.
  • the alteration to one or more amino acid residues that reduces the effector function of the immunoglobulin heavy chain constant region is selected from the list consisting of: (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235 A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y, or 329R (k) 331 S, (1) 236F or 236R, (m) 238 A, 238E, 238G, 238H, 2381, 238V, 238W, or 238 Y, (n) 248 A
  • the alterations to heavy chain constant regions of the immunoconjugate can reduce the serum half-life of the immunoconjugate.
  • the amino acid alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) reduces the serum half-life of the immunoconjugate.
  • the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 251, 252, 253, 254, 255, 288, 309, 310, 312, 385, 386, 388, 400, 415, 433, 435, 436, 439, 447, and combinations thereof per EU numbering.
  • the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 253, 254, 310, 435, 436 and combinations thereof per EU numbering .
  • the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253 A, I253D, I253P, S254A, H310A, H310D, H310E, H310Q, H435A, H435Q, Y436A, and combinations thereof per EU numbering.
  • the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253 A, S254A, H310A, H435Q, Y436A and combinations thereof per EU numbering. In certain embodiments, the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: 1253 A, H310A, H435Q, and combinations thereof per EU numbering.
  • the alteration that alters or reduces binding of the immunoconjugate to the neonatal Fc receptor (FcRn) is to an amino acid residue selected from the list consisting of: H310A, H435Q, and combinations thereof per EU numbering.
  • a heavy chain constant regions of the immunoconjugate comprises a sequence at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, a heavy chain constant regions of the immunoconjugate comprises a sequence identical to SEQ ID NO: 1. In certain embodiments, a heavy chain constant regions of the immunoconjugate comprises a sequence at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1, wherein the heavy chain constant region comprises an 1253 A substitution per EU numbering.
  • a heavy chain constant regions of the immunoconjugate comprises a sequence at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, a heavy chain constant regions of the immunoconjugate comprises a sequence identical to SEQ ID NO: 2. In certain embodiments, a heavy chain constant regions of the immunoconjugate comprises a sequence at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2, wherein the heavy chain constant region comprises an S254A substitution per EU numbering.
  • a heavy chain constant regions of the immunoconjugate comprises a sequence at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, a heavy chain constant regions of the immunoconjugate comprises a sequence identical to SEQ ID NO: 3. In certain embodiments, a heavy chain constant regions of the immunoconjugate comprises a sequence at least 90%, 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3, wherein the heavy chain constant region comprises an H310A substitution per EU numbering.
  • the immunoconjugate has a serum half-life of about 1 day to about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 4 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days
  • the heavy chain constant region has a molecular weight of about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, or about 20 kDa to about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, or about 25 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at least about 10 kDa, about 15 kDa, or about 20 kDa. In certain embodiments, the heavy chain constant region has a molecular weight of at most about 15 kDa, about 20 kDa, or about 25 kDa.
  • the antigen binding regions and the heavy chain constant regions can be connected by a suitable hinge or linker sequence.
  • the antigen binding region is coupled to the immunoglobulin heavy chain constant region by a linker amino acid sequence or a human IgG hinge region.
  • Appropriate IgG hinge regions comprise and include IgGl or IgG4 hinge regions.
  • the hinge region is an IgGl hinge region.
  • the hinge region is an IgGl hinge regions with a with a C220S substitution per EU numbering.
  • Suitable hinge regions include those described in Wu et al., “Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange,” Protein Engineering, Design and Selection, Volume 14, Issue 12, December 2001, Pages 1025-1033; Shu et al, “Secretion of a single-gene-encoded immunoglobulin from myeloma cells.” Proceedings of the National Academy of Sciences Sep 1993, 90 (17) 7995-7999; Davis et al., “Abatacept binds to the Fc receptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity.” J Rheumatol. 2007 Nov;34(l l):2204-10.
  • Useful linkers include glycine-serine polymers, including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • linkers for linking antibody fragments or single chain variable fragments can include AAEPKSS, AAEPKSSDKTHTCPPCP, GGGG, or GGGGDKTHTCPPCP.
  • non-proteinaceous polymers including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is may find use as linkers.
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes polyoxyalkylenes
  • copolymers of polyethylene glycol and polypropylene glycol may find use as linkers, that is may find use as linkers.
  • the total size of the immunoconjugate may be such that it promotes tissue penetration, stability, and/or clearance.
  • the immunoconjugate has a molecular weight of about 60 kDa to about 120 kDa. In certain embodiments, the immunoconjugate has a molecular weight of about 60 kDa to about 65 kDa, about 60 kDa to about 70 kDa, about 60 kDa to about 75 kDa, about 60 kDa to about 80 kDa, about 60 kDa to about 90 kDa, about 60 kDa to about 100 kDa, about 60 kDa to about 110 kDa, about 60 kDa to about 120 kDa, about 65 kDa to about 70 kDa, about 65 kDa to about 75 kDa, about 65 kDa to about 80 kDa, about 65 kDa to about 90 kDa, about 65 kDa to about to
  • the immunoconjugate has a molecular weight of about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, or about 120 kDa. In certain embodiments, the immunoconjugate has a molecular weight of at least about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 90 kDa, about 100 kDa, or about 110 kDa.
  • the immunoconjugates of this disclosure result in an increased amount of radiation in the tumor compared to the blood when measured as percent injected dose per gram.
  • the ratio of tumor percent injected dose per gram to blood percent injected dose per gram is greater than 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • NK cells express FcyyRIII only, whereas monocytes express FcyyRI, FcyyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in US 5,500,362 (see e.g. Hellstrom, I . et al.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc Natl Acad Sci USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the immunoconjugate is unable to bind Clq and hence lacks CDC activity (see e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402).
  • a CDC assay may be performed (see e.g., Gazzano-Santoro etal., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Btoo 101 : 1045-1052 (2003); Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
  • Immunoconjugates with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US 7,332,581).
  • the immunoconjugate may have altered effector function by comprising the following alterations L234A, L235E, G237A, A330S, and P331S per EU numbering, which reduce Fc receptor binding. See e.g., US 8,613,926 or Andersson C, Wenander et al., ’’Rapid-onset clinical and mechanistic effects of anti-C5aR treatment in the mouse collagen-induced arthritis model.” Clin Exp Immunol. 2014 Jul; 177(1):219-33.
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US 6,194,551; WO 1999/051642; Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • FcRn neonatal Fc receptor
  • FcRn neonatal Fc receptor
  • Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues: 434 or 435, e.g., substitution of Fc region residue N434A or R435A (US 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); US 5,648,260; US 5,624,821; and WO 1994/029351 concerning other examples of Fc region variants.
  • a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example.
  • the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the chelator-linker is attached to a glutamine residue through the action of a transglutaminase enzyme.
  • a secondary reactive group is attached by transglutaminase upon which the chelator-linker is added to furnish the immunoconj ugate .
  • an immunoconjugate having more than one chelating agent has more than one chelating agent attached to the same antigen binding arm.
  • the chelating agent comprises a radioisotope chelating component and a functional group that allows for covalent attachment to the antigen binding arm.
  • the functional group is directly attached to the radioisotope chelating component.
  • the chelating agent further comprises a linker between the functional group and the radioisotope chelating component.
  • the chelating agent of an immunoconjugate is not attached to the antigen binding region in the antigen binding arm of the immunoconjugate.
  • the chelating agent of the immunoconjugate is non-covalently associated with an antigen binding arm. In a preferred embodiment, the chelator is not associated with the antigen binding region in the antigen binding arm of the immunoconjugate.
  • the chelating agent comprises DOTA or a DOTA derivative. In one embodiment, the chelating agent comprises DOTAGA. In one embodiment, the chelating agent comprises macropa or a macropa derivative. In one embodiment, the chelating agent comprises Py4Pa or a Py4Pa derivative. In one embodiment, the chelating agent comprises siderocalin or a siderocalin derivative.
  • the chelating agent is a radioisotope chelating agent.
  • the radioisotope chelating agent is selected from the list consisting of: tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), a-(2-Carboxyethyl)-l,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTAGA), or (Py4Pa).
  • the radioisotope chelating agent is DOTA.
  • the radioisotope chelating agent is DOTAGA.
  • the radioisotope chelating agent is Py4Pa. In certain embodiments, the radioisotope wherein the radioisotope chelating agent is directly coupled to the antigen binding region and/or the immunoglobulin heavy chain constant region. In certain embodiments, the radioisotope chelating agent is coupled to the antigen binding region or the immunoglobulin heavy chain constant region by a linker.
  • the linker is selected from: 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl ( PAB), and those resulting from conjugation with linker reagents: N-Succinimidyl 4-(2-pyridylthio) pentanoate forming linker moiety 4-mercaptopentanoic acid (SPP), Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate (SMCC), N-Succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-Succinimidyl (4- iodo-acetyl) aminobenzoate (SIAB), polyethylene glycol (PEG), a polyethylene glycol polymers (PEGn), and
  • the linker is selected from: polyethylene glycol (PEG), a polyethylene glycol polymers (PEG), and S-2-(4- isothiocyanatobenzyl) (SCN).
  • the linker is PEG5.
  • the linker is SCN .
  • the radioisotope chelating agent is a linker-chelator selected from the list consisting of: TFP-Ad-PEG5-D0TAGA, p-SCN-Bn-DOTA, p-SCN-Ph-Et-Py4Pa, and TFP-Ad-PEG5-Ac-Py4Pa.
  • a bifunctional chelator is used to conjugate a radioisotope to a radioisotope delivery platform of the invention to create an immunoconjugate of the invention.
  • a bifunctional chelator is used to conjugate a radioisotope to a radioisotope delivery platform of the invention to create an immunoconjugate of the invention.
  • bifunctional chelators are 1,4,7, 10-tetraazacy clododecane- 1,4, 7,10- tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTP A), and related analogs of the aforementioned.
  • DOTA diethylene triamine pentaacetic acid
  • Such chelators are suitable for coordinating metal ions like a and P-emitting radionuclides.
  • R 1 comprises DOTA and is attached to the tumor targeting moiety at the position shown:
  • the immunoconjugates described herein comprise one or more R 1 moieties. In some embodiments, the immunoconjugates described herein comprise one or more than one R 1 moieties, wherein each R 1 moiety is the same.
  • the radionuclide is an Auger electron-emitting radionuclide. In some embodiments, the radionuclide is an Auger electron-emitting radionuclide that is in In, 67 Ga, 68 Ga, " m Tc, or 195m Pt. In some embodiments, the radionuclide is an Auger electron-emitting radionuclide that is m In, 67 Ga, 68 Ga, or " m Tc.
  • the radionuclide is an a-emitting radionuclide. In some embodiments, the radionuclide is an a-emitting radionuclide that is 225 Ac, 213 Bi, 223 Ra, or 212 Pb . In some embodiments, the radionuclide is an a-emitting radionuclide that is 225 Ac. In some embodiments, the radionuclide is an P-emitting radionuclide.
  • the radionuclide is a P-emitting radionuclide that is 90 Y, 177 Lu, 186 Re (186-Rhenium), 188 Re (Rhenium- 188), 64 Cu, 67 Cu, 153 Sm (Samarium- 153), 89 Sr (Strontium-89), 198 Au (Gold-198), 169 Er (Erbium- 169), 165 Dy (Dysprosium- 165), " m Tc, 89 Zr, or 52 Mn (Manganese-52).
  • the radionuclide is a P-emitting radionuclide that is 90 Y, 177 Lu, " m Tc, or 89 Zr.
  • the radionuclide is a y-emitting radionuclide.
  • the radionuclide is a y-emitting radionuclide that is 60 Co (Cobalt-60), 103 Pd (Palldium-103), 137 Cs (Cesium-137), (169Yb) (Ytterbium- 169), 192 Ir (Iridium- 192), 212 B1, 213 Bi, or 226 Ra.
  • a linker is used that connects the tumor targeting moiety and the radionuclide chelator moiety R 1 .
  • L is a hydrophobic linker. In some other embodiments, L is a hydrophilic linker. In some embodiments, the linker is flexible. In some embodiments, the linker is rigid. In some embodiments, the linker is linear. In other embodiments, the linker is branched. In some embodiments, a branched linker allows for conjugation to more than one R 1 moieties. In some embodiments, a branched linker allows for conjugation to cellpenetrating peptides (CPPs) to enhance cell penetration. In some embodiments, the linker comprises a linear structure.
  • the linker comprises a non-linear structure. In some embodiments, the linker comprises a branched structure. In some embodiments, the linker comprises a cyclic structure. In some embodiments, the linker comprises one or more linear structures, one or more non-linear structures, one or more branched structures, one or more cyclic structures, one or more flexible moieties, one or more rigid moieties, or combinations thereof.
  • Linker length may be viewed in terms of the number of linear atoms between the radionuclide chelator moiety R 1 and the tumor targeting moiety, with cyclic moieties to be counted by taking the shortest route around the ring.
  • the linker has a linear stretch of between 1 to 100 atoms, between 1-50 atoms, between 1-40 atoms, between 1-30 atoms, in other embodiments 1-20 atoms, in still other embodiments 1-15 atoms, in still other embodiments 1-10 atoms, and in still other embodiments 1-5 atoms.
  • Linker considerations include the effect on physical or pharmacokinetic properties of the resulting radioimmunoconjugate, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, and the like.
  • An optimal linker should link the carrier mAb to the radiochelator without impairing the functionality of either, provide a stable linkage in circulation, and degrade under specific conditions. Stability in circulation has proven to constitute a particularly important factor for success using this strategy.
  • a limitation to the general use of tumor targeted 225 Ac radioimmunotherapy is the excessive radiation of normal tissues by the radioimmunoconjugate. With the exception of antibodies targeting leukemias, most (>99%) of the injected antibody dose remains circulating in the blood or is not associated with the target tumor. The prolonged circulation time of untargeted antibodies, typically days to weeks, results in radiation exposure of normal organs that catabolize or retain proteins and peptides, primarily liver and kidneys.
  • the chelated radiometal then rapidly clears the body through the kidneys, thereby reducing toxicity.
  • This strategy has been attempted using disulfide, ester, tartramide, and peptide bonds (Kukis DL, et al., Cleavable linkers to enhance selectivity of antibody-targeted therapy of cancer. Cancer Biother Radiopharm . 2001;16:457-467; Quadri SM, Vriesendorp HM. Effects of linker chemistry on the pharmacokinetics of radioimmunoconjugates. Q J Nucl Med. 1998;42:250-261).
  • An optimal linker should link the carrier mAb to the radiochelator without impairing the functionality of either, provide a stable linkage in circulation, and degrade under specific conditions. Stability in circulation has proven to constitute a particularly important factor for success using this strategy.
  • Peptide linkers which are usually more stable than esters and disulfides in serum, have been most successful in radioimmunoconjugates and in the design of antibody-drug conjugates (ADC).
  • ADC antibody-drug conjugates
  • the most extensively studied peptide linkers are sensitive to cathepsins. More precisely, ADCs incorporating the dipeptide valine-citrulline have been shown to enter the target cells and migrate to the lysosomes, where they release their drug specifically under the action of cysteine proteases.
  • cathepsin B labile radioconjugates e.g., glycylglycylglycylglycyl-L-p-isothiocyanatophenylalanine-
  • glycylglycylglycyl-L-p-isothiocyanatophenylalanine- demonstrated a decrease in the liver dose and a slightly increased tumor dose was observed
  • the immunoconjugate is a compound of Formula (II), or a pharmaceutically acceptable salt thereof: Formula (II).
  • the immunoconjugate is a compound of Formula (III), or a pharmaceutically acceptable salt thereof: Formula (III).
  • the immunoconjugate is a compound of Formula (IV), or a pharmaceutically acceptable salt thereof: Formula (IV).
  • R 2 is a moiety that is capable of reacting with an amine (-NH2) of a lysine of the tumor targeting moiety R 3 .
  • the compound Formula (II) has the structure of Formula (Ila), or a pharmaceutically acceptable salt thereof:
  • the immunoconjugate is a compound of Formula (V), Formula (VI), Formula (VII), or Formula (VIII), or a pharmaceutically acceptable salt thereof:
  • R 1 is a chelating moiety or a radionuclide complex thereof
  • -S-R 3 is a tumor targeting moiety
  • L is a linker that is -L l -L 2 -L 3 - L 4 -L 5 -;
  • L 1 is unsubstituted or substituted Ci-Cioalkylene, unsubstituted or substituted Ci- Cioheteroalkylene, unsubstituted or substituted C2-C2oalkenylene, unsubstituted or substituted C2-C2oalkynylene, C4-C2opolyethylene glycol, -(X 3 CH2CH2)t-, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene; each X 3 is independently selected from O and NR 4 ; each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;
  • the immunoconjugate is a compound of Formula (VIII), or a pharmaceutically acceptable salt thereof: Formula (VIII).
  • DO2A 1.4.7.10-tetraazacyclododecane-l,7-diacetic acid
  • DNMA 1,3,4,8,9-tetraazacyclododecane-l, 4, 7, 10-tetraacetic acid
  • R 1 is a chelating moiety selected from the group consisting of: radionuclide complex thereof.
  • R 2 is a moiety that is capable of reacting with an amine (-NH2) of the tumor targeting moiety R 3 and comprises a tetrafluorophenyl ester, pentafluorophenyl ester, dinitrophenyl ester, succinimide ester, sulfosuccinimide ester, or isothiocyanate.
  • R 2 is a moiety that is capable of reacting with an amine (-NH2) of the tumor targeting moiety R 3 and comprises:
  • X is -O-.
  • X is -S-.
  • R 2 is a moiety that is capable of reacting with a thiol (-SH) of a tumor targeting moiety R 3 and comprises a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyrdinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol group.
  • a thiol (-SH) of a tumor targeting moiety R 3 and comprises a maleimide group, a haloacetamide group, a haloacetyl group, a haloacetate group, a pyrdinylthio group, a vinylcarbonyl group, an aziridinyl group, a disulfide group, an acetylene group, a hydroxysuccinimide group or a thiol
  • the tumor targeting moiety R 3 is a polypeptide comprising an antigen binding region and an immunoglobulin heavy chain constant region, wherein the molecular weight of the polypeptide is between 60 and 110 kDa.
  • R 2 is a moiety that is capable of reacting with a thiol (-SH) of
  • X 1 is -NH-, -N(CH 3 )-, or -N(CH 2 CH 3 )-.
  • L 1 is unsubstituted or substituted Ci-Cealkylene, unsubstituted or substituted Ci-Cioheteroalkylene, C4-C2opolyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene;
  • L 1 is unsubstituted or substituted Ci-Cealkylene, unsubstituted or substituted Ci-Cioheteroalkylene, C4-C 2 opolyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene;
  • L 1 is unsubstituted or substituted Ci-C 2 oalkylene, unsubstituted or substituted Ci-C 2 oheteroalkylene, C4-C 2 opolyethylene glycol, unsubstituted or substituted C3- Cscycloalkylene, unsubstituted or substituted monocyclic Cs-Csheterocycloalkylene, unsubstituted or substituted phenylene, unsubstituted or substituted monocyclic heteroarylene.
  • L 1 is unsubstituted or substituted Ci-Cealkylene, unsubstituted or substituted Ci-Cioheteroalkylene, C4-C2opolyethylene glycol, unsubstituted or substituted cyclohexylene, or unsubstituted or substituted phenylene. In some embodiments, L 1 is absent.
  • L 2 is absent.
  • L 5 is absent.
  • each X 3 is independently O. In some embodiments, each X 3 is independently NR 4 .
  • each R 4 is hydrogen. In some embodiments, each R 4 is Ci- Cealkyl.
  • each R 5 is independently selected from Ci-Cioalkyl, C4- Csopolyethylene glycol, and unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene. In some embodiments, each R 5 is independently selected from Ci-Cioalkyl or C4- Csopolyethylene glycol. In some embodiments, each R 5 is independently selected from unsubstituted or substituted arylene or unsubstituted or substituted heteroarylene.
  • each R a is independently selected from hydrogen, and Ci- C4alkyl. In some embodiments, each R a is independently hydrogen. In some embodiments, each R a is independently Ci-C4alkyl. [00312] In some embodiments, each R b is independently selected from hydrogen, and Ci- C4alkyl. In some embodiments, each R b is independently hydrogen. In some embodiments, each R b is independently Ci-C4alkyl.
  • Exemplary compounds for use in preparing immunoconjugates described herein include the compounds depicted in Table A.
  • the invention provides immunoconjugates.
  • the immunoconjugates are capable of delivering a-emitters in vivo when so labeled, linked or loaded with an a-emitter.
  • the immunoconjugates are also capable of delivering other radioisotopes (P-emitters, and/or y-emitters), and/or other atoms in vivo, when so labeled, linked or loaded.
  • the immunoconjugates are capable of delivering imaging metals (e.g., m In, 89 Zr, 64 Cu, 68 Ga or 134 Ce) in vivo when so labeled, linked or loaded.
  • the immunoconjugates of the current disclosure may be loaded with a radioisotope for a therapeutic or diagnostic effect.
  • the chelator may further comprise a radioisotope.
  • the radioisotope is an alpha emitter.
  • the radioisotope is an alpha emitter selected from the list consisting of 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi.
  • the radioisotope is 225 Ac.
  • the radioisotope is an beta emitter.
  • the radioisotope is a beta emitter selected from 177 Lu, 90 Y, 67 Cu, and 153 Sm.
  • the immunoconjugate of the present invention is combined with a radioisotope to provide a radioimmunoconjugate of the invention.
  • the radioisotope is 225 Ac, 86 Y, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 213 Bi, 213 -Po, 212 Bi, 223 Ra, 224 Ra, 227 Th, 149 Tb, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 137 Cs, 212 Pb, or 103 Pd.
  • the radioimmunoconjugate is labeled, linked or loaded with, and accordingly comprises, both an a-emitter and a P-emitter.
  • the radioisotope is selected for use in radio-imaging, such as, e.g., from among 68 Ga, 64 Cu, 89 Zr, ni In, 134 Ce.
  • the immunoconjugates and radioimmunoconjugates of the invention may comprise other cargos or payloads besides a radioisotope, including various cytotoxic agents, such as, e.g., a small molecule chemotherapeutic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor.
  • cytotoxic agents such as, e.g., a small molecule chemotherapeutic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor.
  • an immunoconjugate of the invention may be used to deliver a non-radioisotope cytotoxin to a target cell.
  • a radioimmunoconjugate of the invention comprises a radioisotope selected from the group comprising 225 Ac, 86 Y, 90 Y, 177 'Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 213 Bi, 213 Po, 211 At, 212 Bi, 223 Ra, 224 Ra, 227 Th, 149 Tb, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 137 Cs, 212 Pb, and 103 Pd.
  • a radioisotope selected from the group comprising 225 Ac, 86 Y, 90 Y, 177 'Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 213 Bi, 213 Po, 211 At, 212 Bi, 223 Ra, 224 Ra, 227 Th, 149 Tb, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 137 Cs, 212 Pb, and 103 Pd.
  • a radioimmunoconjugate of the invention comprises a radioisotope selected from the group consisting of 225 Ac, 86 Y, 90 Y, 177 'Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 213 Bi, 213 Po, 211 At, 212 Bi, 223 Ra, 224 Ra, 227 Th, 149 Tb, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 137 Cs, 212 Pb, and 103 Pd.
  • the radioisotope is an alpha-particle-emitting radioisotope comprises 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, or +Bi.
  • the radioisotope is an alpha-particle-emitting radioisotope selected from the group consisting of 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi.
  • the immunoconjugate comprises a dimerization domain or motif.
  • the dimerization domain or motif is in a variant constant region, linker or hinge region.
  • cysteine residues at specific locations may be used to create disulfide stabilized structures like Cys-diabodies, scFv' multimers, VHH multimers, VNAR multimers, and IgNAR multimers such as, e.g., by adding the following amino acid residues: GGGGC and SGGGGC (Tai M et al., Biochemistry 29: 8024-30 (1990); Caron P et al., J Exp Med 176: 1191-5 (1992); Shopes B, J Immunol 148: 2918-22 (1992); Adams G et al., Cancer Res 53: 4026-34 (1993); McCartney J et al., Protein Eng 18: 301-14 (1994); Perisic O et al., Structure 2: 1217-26 (1994); George A et al., Proc Natl Acad Sci USA 92: 8358-62 (1995); Tai M et al., Cancer Res (Suppl) 55:
  • polypeptide chains may be linked together using polypeptide domains which self-associate or multimerize with each other (see e.g., US 6,329,507).
  • polypeptide domains which self-associate or multimerize with each other (see e.g., US 6,329,507).
  • carboxy -terminal multimerization domains has been used to construct multivalent proteins comprising immunoglobulin domains, such as, e.g., scFvs, autonomous VH domains, VHHS, VNARS, and IgNARs.
  • self-associating domains examples include immunoglobulin constant domains (such as knobs-into-holes, electrostatic steering, and IgG/IgA strand exchange), immunoglobulin Fab chains (e.g., (Fab-scFv)2 and (Fab’ SCFV)2), immunoglobulin Fc domains (e.g., (scDiabody-Fc)2, (scFv-Fc)2 and scFv-Fc-scFv), immunoglobulin CHX domains, immunoglobulin CHI -3 regions, immunoglobulin CH3 domains (e.g., (scDiabody-CH3)2, LD minibody, and Flex -minibody), immunoglobulin CH4 domains, CHCL domains, amphiphilic helix bundles (e.g., scFv-HLX), helix-turn-helix domains (e.g., scFv-dHlx), coiled-
  • the skilled worker can engineer multimeric immunoconjugates of the present invention using various scFv-based polypeptide interactions known in the art, such as, e.g., scFv- based dimeric, trimeric, tetrameric complexes, etc.
  • scFv-based polypeptide interactions known in the art, such as, e.g., scFv- based dimeric, trimeric, tetrameric complexes, etc.
  • the length of the linker in the scFv can affect the spontaneous assembly of non-covalent based, multimeric, multivalent structures.
  • linkers of twelve amino acids or less promote the multimerization of polypeptides or proteins comprising scFvs into higher molecular weight species via favoring intermolecular domain swapping over intra- chain domain pairing (see e.g., Dolezal O et al., Protein Eng 16: 47- 56 (2003)).
  • scFvs with no linker at all or a linker with an exemplary length of 15 amino acid residues may multimerize (Whitlow M et al., Protein Eng 6: 989-95 (1993); Desplancq D et al., Protein Eng l'.
  • amino acid sequence variants of the immunoconjugates described herein are contemplated.
  • Amino acid sequence variants of an immunoconjugate may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the immunoconjugate, or by synthesis of the desired immunoconjugate or polypeptide. Such modifications include, for example, fusion of immunoglobulin domains or polypeptide sequences; substitution of hinge, linker(s), and/or chelator components; substitution of radioisotope.
  • Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the immunoconjugate. Any combination of fusion, deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., a certain binding affinity level of antigen binding, a certain level of KD, and/or a certain level of Koff
  • Antigen binding antibody fragments and sets of CDRs are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full-length native antibody (e.g., a full-length cam elid VHH IgG2 or IgG3). Certain fragments may lack amino acid residues or domain that are not essential for a desired biological activity of the antibody or to reduce the total size of the immunoconjugate of the invention.
  • an immunoconjugate of the present invention is made to be larger by the incorporation of additional structure.
  • an immunoconjugate is linked to a heterologous moiety or readily detectable moiety.
  • the linkage comprises a proteinaceous fusion.
  • the heterologous moiety is a cytotoxic agent.
  • a carboxy-terminal lysine residue is added to provide a site-specific attachment site.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an immunoconjugate with an N-terminal methionyl residue.
  • Other insertional variants of the immunoconjugate molecule include the fusion to the N- or C-terminus of the immunoconjugate to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the immunoconjugate.
  • Nucleic acids that encode the immunoconjugate of the invention may be modified to produce chimeric or fusion immunoconjugate polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CO sequences for the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc Natl Acad Set USA 81 : 6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide).
  • CH and CO sequences for the homologous murine sequences
  • heterologous polypeptide heterologous polypeptide
  • the non-immunoglobulin polypeptide sequences can substitute for the constant domains of an immunoconjugate, or they are substituted for the variable domains of one antigen-combining site of an immunoconjugate to create a chimeric bivalent immunoconjugate comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • Variations in the antibody constructs used as antigen binding domains in the inventions described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934.
  • Variations may be a substitution, deletion or insertion of one or more codons encoding the immunoconjugate or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide.
  • the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the immunoconjugate.
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the immunoconjugate with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • Substantial modifications in function or immunological identity of an immunoconjugate of the invention are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • the variations can be made using methods known in the art, such as, e.g., oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)
  • cassette mutagenesis Wells et al., Gene, 34: 315 (1985)
  • restriction selection mutagenesis Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)
  • other known techniques can be performed on the cloned DNA to produce DNA molecules encoding an immunoconjugate variant of the invention.
  • immunoconjugate variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs of immunoglobulin variable domains as well as within the immunoglobulin constant domains.
  • Amino acid substitutions may be introduced into an immunoconjugate of interest and the products screened for a desired activity, e.g., improved/retained antigen binding, decreased/retained immunogenicity, improved/retained antibody-dependent cellular cytotoxicity (ADCC), improved/retained complement dependent cytotoxicity (CDC), improved/retained target inhibition, and/or improved/retained antibody-dependent cell-mediated phagocytosis (ADCP).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • ADCP improved/retained target inhibition
  • ADCP improved/retained antibody-dependent cell-mediated phagocytosis
  • amino acid substitutions may be introduced into an immunoconjugate of interest and the products screened for the reduction or elimination of an activity, e
  • Alterations may be made in HVRs, e.g., to improve immunoconjugate affinity.
  • Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see e.g., Chowdhury , Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • HVR “hotspots” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see e.g., Chowdhury , Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the immunoconjugate to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR “hotspots” or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen.
  • Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • the immunoconjugate of the present invention comprises an antibody construct (used as an antigen binding region herein) comprising a humanized immunoglobulin domain(s).
  • an antibody construct used as an antigen binding region herein
  • Humanized forms of non-human (e.g., camelid, murine, or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as a camelid, mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321 : 522-5 (1986); Riechmann et al., Nature, 332: 323-9 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-6 (1992)).
  • Fc immunoglobulin constant region
  • antigen binding may be restored during humanization of antibodies through the selection of repaired hypervariable regions (see, e.g., US application Ser. No. 11/061,841, filed Feb. 18, 2005).
  • the method includes incorporating non-human hypervariable regions onto an acceptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor framework sequence.
  • the introduction of one or more amino acid substitutions may be accompanied by modifications in the acceptor framework sequence.
  • cysteine residue not involved in maintaining the proper conformation of the immunoconjugate of the invention also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the immunoconjugate of the invention to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment or VHH fragment).
  • Addition or deletion of glycosylation sites to an immunoconjugate may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • Addition of glycosylation sites to the immunoconjugate of the invention 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).
  • 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 immunoconjugate of the invention (for O- linked glycosylation sites).
  • the immunoconjugate of the invention amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the immunoconjugate of the invention at preselected bases such that codons are generated that will translate into the desired amino acids.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see e.g., Wright et al. TIBTECH 15:26-32 (1997)).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an immunoconjugate of the invention may be made in order to create immunoconjugate variants with certain improved properties.
  • Another means of increasing the number of carbohydrate moieties on the immunoconjugate of the invention is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the immunoconjugate of the invention may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation.
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
  • immunoconjugate variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such immunoconjugate may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI- TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function (see e.g., US 2003/0157108; US 2004/0093621).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/01 15614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/01 10704; US 2004/01 10282; US 2004/0109865; WO 2003/0851 19; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; W02002/031 140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech.
  • Immunoconjugate variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such immunoconjugate variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; US 6,602,684; US 2005/0123546. Immunoconjugate variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such immunoconjugate variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/030087; WO 1998/058964; and WO 1999/022764.
  • Covalent modifications of the immunoconjugates of the invention are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of an immunoconjugate of the invention with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the immunoconjugate.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking an immunoconjugate of the invention to a water-insoluble support matrix or surface for use in the method for purifying the immunconjugates of the invention, and vice-versa.
  • crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-[(p- azi dopheny 1 ) dithi o] propi oimi date .
  • an immunoconjugate provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the immunoconjugate include but are not limited to water soluble polymers.
  • Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3 -di oxolane, poly-1, 3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the immunoconjugate may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the immunoconjugate to be improved, whether the immunoconjugate derivative will be used in a therapy under defined conditions, etc.
  • the present invention provides a composition comprising one or more of the immunoconjugates according to any of the above embodiments or described herein.
  • the invention provides an isolated nucleic acid encoding a radioisotope delivering platform as described herein.
  • nucleic acids encoding the protein components of the immunoconjugates of the present invention, expression vectors comprising the aforementioned nucleic acid, and host cells comprising the aforementioned expression vectors.
  • the invention provides a process for making an immunoconjugate of the present invention, the method comprising culturing a host cell as provided herein under conditions suitable for the expression vector encoding the radioisotope delivery platform and recovering or purifying the radioisotope delivery platform.
  • the method further comprises radiolabeling the radioisotope delivery platform with an appropriate isotope, such as, e.g., an alpha or beta particle emitter.
  • Suitable host cells for the expression of glycosylated immunoconjugate of the invention are derived from multicellular organisms.
  • invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Host cells are transformed with the above-described expression or cloning vectors for immunoconjugate of the invention production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the nucleic acid e.g., cDNA or genomic DNA
  • DNA encoding the immunoconjugate is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody).
  • Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, suitable host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • the immunoconjugate of the invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the immunoconjugate of the invention-encoding DNA that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders.
  • the host cells used to produce the immunoconjugate of the invention of this invention may be cultured in a variety of media and culture conditions.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, di thioerythritol and dithiothreitol.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 30°C.
  • the pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism.
  • the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.
  • an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter.
  • PhoA promoters are used for controlling transcription of the polypeptides.
  • the transformed host cells are cultured in a phosphate-limiting medium for induction.
  • the phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263: 133-47).
  • inducers may be used, according to the vector construct employed, as is known in the art.
  • the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells.
  • Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
  • PAGE polyacrylamide gel electrophoresis
  • immunoconjugate production is conducted in large quantity by a fermentation process.
  • Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins.
  • Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (a preferred carbon/energy source).
  • Small-scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase.
  • a desired density e.g., an OD550 of about 180-220
  • inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12- 50 hours, although longer or shorter induction time may be used.
  • various fermentation conditions can be modified.
  • additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells.
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al.
  • host strains deficient for proteolytic enzymes can be used for the present invention.
  • host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof.
  • E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2 :63-72 (1996).
  • E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention.
  • 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Forms of immunoconjugate of the invention may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage.
  • a suitable detergent solution e.g., Triton-X 100
  • Cells employed in expression of immunoconjugate of the invention can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • immunoconjugate of the invention may be desired to purify immunoconjugate of the invention from recombinant cell proteins or polypeptides.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the immunoconjugate of the invention.
  • the immunoconjugate can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the immunoconjugate is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-7 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Ami con or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the immunoconjugate composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the immunoconjugate.
  • Protein A can be used to purify antibodies that are based on human yl, y2 or y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J.
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the immunoconjugate comprises a CH3 domain
  • the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, NJ is useful for purification.
  • the method of producing an immunoconjugate involves a click chemistry step described by Poty, S et al., Chem Commun. (Camb) 54: 2599 (2016).
  • a peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen.
  • radiolabels may be incorporated into peptide.
  • radiolabels may be linked to peptide.
  • the IODOGEN method (Fraker et al. (1978) Biochem Biophys Res Commun. 80: 49-57 can be used to incorporate iodine- 123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail.
  • Immunoconjugates of the present invention may be identified, screened for, or characterized for their phy si cal/ chemi cal properties and/or biological activities by various assays known in the art.
  • the immunoconjugates and antibody constructs of the invention may be characterized for their phy si cal/ chemi cal properties and/or biological activities by various assays known in the art.
  • Immunoconjugates of the invention can be characterized by a series of assays including, but not limited to, polypeptide sequence determination, amino acid analysis, nondenaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.
  • An immunoconjugate of the present invention may be tested for its antigen binding activity by methods known in the art, e.g., ELISA, Western blot, etc.
  • the binding affinity of an antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal Biochem. 107: 220 (1980).
  • the antigen binding ability of an immunoconjugate of the invention may be quantitated using methods known in the art, e.g., a quantitative ELISA, quantitative Western blot, surface plasmon resonance assay, and/or a Scatchard analysis.
  • the KD of an immunoconjugate is measured using a radiolabeled antigen ELISA performed with the immunoconjugate.
  • the KD is measured by using surface-plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, N.J.), e.g., using immobilized antigen CM5 chips at 25° C and 10 response units.
  • binding competition assays may be used to identify immunoconjugates that compete for binding to the same antigen, or epitope thereof.
  • a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) of an immunoconjugate of the invention (see e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).
  • the epitope and/or contact residues within an antigen bound by an immunoconjugate of the invention can be identified or mapped using methods known to the skilled worker. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology (3 rd ed., Humana Press, Totowa, NJ).
  • the invention provides a composition comprising an immunoconjugate or radioimmunoconjugate of the present invention.
  • the invention further provides pharmaceutical compositions and formulations comprising at least one immunoconjugate of the present invention and at least one pharmaceutically acceptable excipient or carrier.
  • a pharmaceutical formulation comprises (1) an immunoconjugate or radioimmunoconjugate of the invention, and (2) a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: sterile water, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3 -pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
  • compositions to be used for in vivo administration are generally sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • lyophilized antibody formulations are described in US 6,267,958.
  • Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.
  • Pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX ®, Baxter International, Inc.).
  • sHASEGP soluble neutral -active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX ®, Baxter International, Inc.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • the formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • immunoconjugates may be formulated as immunoliposomes.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Liposomes containing the immunoconjugate are prepared by methods known in the art, such as described in Epstein et al., Proc Natl Acad Sci USA 82: 3688 (1985); Hwang et al., Proc Natl Acad Sci USA IT. 4030 (1980); U.S. Pat. Nos.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A chemotherapeutic agent is optionally contained within the liposome (see Gabizon et al., J. National Cancer Inst. 81 : 1484 (1989)). Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • the invention provides a method of treating a disease, disorder, or condition in a patient in need thereof, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an immunoconjugate or radioimmunoconjugate or composition of the present invention.
  • the method is for inhibiting the growth and/or the killing of a cancer cell or tumor.
  • the invention provides for the use of an immunoconjugate described herein for the preparation and/or manufacture of a medicament for treating a disease, disorder, or condition in a subject, such as, e.g., cancer.
  • compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
  • an immunoconjugate or radioimmunoconjugate or composition of the invention can be used in a method for binding target antigen in an individual suffering from a disorder associated with increased target antigen expression and/or activity, the method comprising administering to the individual the immunoconjugate or radioimmunoconjugate or composition such that target antigen in the individual is bound.
  • the target antigen is human target antigen
  • the individual is a human individual.
  • An immunoconjugate or radioimmunoconjugate or composition of the invention can be administered to a human for therapeutic purposes.
  • an immunoconjugate or radioimmunoconjugate or composition of the invention can be administered to a non-human mammal expressing target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts (e.g., a primate, pig, rat, or mouse) for veterinary purposes or as an animal model of human disease.
  • a non-human mammal expressing target antigen with which the immunoconjugate or radioimmunoconjugate cross-reacts e.g., a primate, pig, rat, or mouse
  • animal models may be useful for evaluating the therapeutic efficacy of an immunoconjugate or radioimmunoconjugate or composition of the invention (e.g., testing of dosages and time courses of administration).
  • An immunoconjugate or radioimmunoconjugate or composition of the invention can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Immunoconjugate or radioimmunoconjugate or compositions of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the immunoconjugates of the invention are administered to a human patient, in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • intravenous or subcutaneous administration of the immunoconjugate or radioimmunoconjugate or composition of the invention is preferred.
  • the dosage and mode of administration will be chosen by the physician according to known criteria.
  • the appropriate dosage of immunoconjugate or radioimmunoconjugate or composition of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the immunoconjugate or radioimmunoconjugate or composition of the invention is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the immunoconjugate or radioimmunoconjugate or composition, and the discretion of the attending physician.
  • the immunoconjugate or radioimmunoconjugate or composition of the invention is suitably administered to the patient at one time or over a series of treatments.
  • the immunoconjugate or radioimmunoconjugate or composition is administered by intravenous infusion or by subcutaneous injections.
  • about 1 pg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of immunoconjugate or radioimmunoconjugate or composition can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the immunoconjugate or radioimmunoconjugate or composition of the invention.
  • other dosage regimens may be useful.
  • a typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.
  • the dose and administration schedule may be selected and adjusted based on the level of disease, or tolerability in the subject, which may be monitored during the course of treatment.
  • the conjugates of the present invention may administered once per day, once per week, multiple times per week, but less than once per day, multiple times per month but less than once per day, multiple times per month but less than once per week, once per month, once per five weeks, once per six weeks, once per seven weeks, once per eight weeks, once per nine weeks, once per ten weeks, or intermittently to relieve or alleviate symptoms of the disease.
  • Administration may continue at any of the disclosed intervals until remission of the tumor or symptoms of the cancer being treated.
  • Administration may continue after remission or relief of symptoms is achieved where such remission or relief is prolonged by such continued administration.
  • the effective amount of the immunoconjugate or radioimmunoconjugate or composition may be provided as a single dose.
  • the Immunoconjugates and radioimmunoconjugates of the present invention maybe used in combination with conventional and/or novel methods of treatment or therapy or separately as a monotherapy.
  • the immunoconjugates and radioimmunoconjugates of the present invention maybe used with one or more radiation sensitizer agents.
  • agents include any agent that can increase the sensitivity of cancer cells to radiation therapy.
  • immunoconjugates and radioimmunoconjugates of the present invention may be used in combination with novel and/or conventional agents that can augment the biological effects of radiotherapy.
  • Irradiation of a tumor can cause a variety of biological consequences which can be exploited by combining immunoconjugates and radioimmunoconjugates of the present invention with agents that target relevant pathways.
  • agents may reduce tumor angiogenesis, or inhibit local invasion and metastasis, or prevent repopulation, or augment the immune response, or deregulate cellular energetics, or reduce population, or alter tumor metabolism, or increase tumor damage, or reduce DNA repair.
  • agents for use in combination with immunoconjugates and radioimmunoconjugates of the present invention may comprise DDR inhibitors, e.g., PARP, ATR, Chkl, or DNA-PK; or survival signaling inhibitors, e.g., mTOR, PI3k, NF-kB; or antihypoxia agents, e.g., HIF-1 -alpha, CAP, or UPR; or metabolic inhibitors, e.g., MCT1, MCT4 inhibitors; or immunotherapeutics, e.g., anti- CTLA4, anti-PD-1; or inhibitors of growth factor signal transduction, e.g., EGFR or MAPK inhibitors; or anti-invasives, e.g., kinase inhibitors, chemokine inhibitors, or integrin inhibitors; or anti-angiogenic agents, e.g., VEGF- inhibitors.
  • DDR inhibitors e.g., PARP, ATR, Chkl, or
  • Immunoconjugates and radioimmunoconjugates of the present invention may (i) inhibit the growth or proliferation of a cell to which they bind; (ii) induce the death of a cell to which they bind; (iii) inhibit the delamination of a cell to which they bind; (iv) inhibit the metastasis of a cell to which they bind; or (v) inhibit the vascularization of a tumor comprising a cell to which they bind.
  • “inhibiting cell growth or proliferation” means decreasing a cell’s growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.
  • an immunoconjugate that inhibits the growth of a tumor cell is one that results in measurable growth inhibition of a tumor cell (e.g., a cancer cell).
  • an immunoconjugate or radioimmunoconjugate of the invention is capable of inhibiting the growth of cancer cells displaying the antigen bound by the immunoconjugate or radioimmunoconjugate.
  • Preferred growth inhibitory immunoconjugates or radioimmunoconjugates inhibit growth of antigen-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the immunoconjugate or radioimmunoconjugate being tested.
  • a majority of the immunoconjugate or radioimmunoconjugate or composition administered to a subject typically consists of non-labeled immunoconjugate, with the minority being labeled radioimmunoconjugate.
  • the ratio of labeled radioimmunoconjugate to non-labeled immunoconjugate can be adjusted using known methods.
  • the immunoconjugate/radioimmunoconjugate may be provided in a total protein amount of up to 100 mg, such as less than 60 mg, or from 5 mg to 45 mg, or a total protein amount of between 0.1 pg/kg to 1 mg/kg patient weight, such as 1 pg/kg to 1 mg/kg patient weight, or 10 pg/kg to 1 mg/kg patient weight, or 100 pg/kg to 1 mg/kg patient weight, or 0.1 pg/kg to 100 pg/kg patient weight, or 0.1 pg/kg to 50 pg/kg patient weight, or 0.1 pg/kg to 10 pg/kg patient weight, or 0.1 pg/kg to 40 pg/kg patient weight, or 1 pg/kg to 40 pg/kg patient weight, or 0.1 mg/kg to 1.0 mg/kg patient weight, such as from 0.2 mg/kg patient weight to 0.6 mg/kg patient weight.
  • the immunoconjugate/radioimmunoconjugate may be administered from about 0.5 mg/kg to about 30 mg/kg. In certain embodiments, the immunoconjugate/radioimmunoconjugate may be administered from about 0.5 mg/kg to about 1 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 3 mg/kg, about 0.5 mg/kg to about 4 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 20 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 2
  • the immunoconjugate/radioimmunoconjugate may be administered at about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, or about 30 mg/kg. In certain embodiments, the immunoconjugate/radioimmunoconjugate may be administered at least about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, or about 20 mg/kg.
  • the immunoconjugate/radioimmunoconjugate may be administered at most about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, or about 30 mg/kg.
  • the method comprises administering the effective amount of a radioimmunoconjugate comprising 225 Ac that is from 0.01 to 0.1 mCi, or 0.1 mCi to 1.0 mCi, or from 1.0 mCi to 2.0 mCi, or from 2.0 mCi to 4.0 mCi.
  • the method comprises administering the effective amount of a radioimmunoconjugate comprising 225-Ac that is from 0.1 pCi/kg to 2.0 pCi/kg subject weight, or from 0.1 pCi/kg to 1.0 pCi/kg subject weight, or from 1.0 pCi/kg to 3.0 pCi/kg subject weight, or from 3.0 pCi/kg to 10.0 pCi/kg subject weight, or from 10.0 pCi/kg to 20.0 pCi/kg subject weight, or from 10.0 pCi/kg to 30.0 pCi/kg subject weight.
  • a radioimmunoconjugate comprising 225-Ac that is from 0.1 pCi/kg to 2.0 pCi/kg subject weight, or from 0.1 pCi/kg to 1.0 pCi/kg subject weight, or from 1.0 pCi/kg to 3.0 pCi/kg subject weight, or from 3.0 pCi/kg to
  • the effective amount of 225 Ac is about 0.1 microcurie to about 20 microcurie. In certain embodiments, the effective amount of 225 Ac is about 0.1 microcurie to about 0.2 microcurie, about 0.1 microcurie to about 0.5 microcurie, about 0.1 microcurie to about 1 microcurie, about 0.1 microcurie to about 2 microcurie, about 0.1 microcurie to about 3 microcurie, about 0.1 microcurie to about 4 microcurie, about 0.1 microcurie to about 5 microcurie, about 0.1 microcurie to about 10 microcurie, about 0.1 microcurie to about 20 microcurie, about 0.2 microcurie to about 0.5 microcurie, about 0.2 microcurie to about 1 microcurie, about 0.2 microcurie to about 2 microcurie, about 0.2 microcurie to about 3 microcurie, about 0.2 microcurie to about 4 microcurie, about 0.2 microcurie to about 5 microcurie, about 0.2 microcurie to about 10 microcurie, about 0.1 microcurie to about 20
  • the effective amount of 225 Ac is about 0.1 microcurie, about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, about 10 microcurie, or about 20 microcurie. In certain embodiments, the effective amount of 225 Ac is at least about 0.1 microcurie, about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, or about 10 microcurie.
  • the effective amount of 225 Ac is at most about 0.2 microcurie, about 0.5 microcurie, about 1 microcurie, about 2 microcurie, about 3 microcurie, about 4 microcurie, about 5 microcurie, about 10 microcurie, or about 20 microcurie.
  • the radioisotope of the radioimmunoconjugate is m In
  • the effective amount is below, for example, 15.0 mCi (i.e., where the amount of in In administered to the subject delivers a total body radiation dose of below 15.0 mCi).
  • the effective amount is below 15.0 mCi, below 14.0 mCi, below 13.0 mCi, below 12.0 mCi, below 11.0 mCi, below 10.0 mCi., below 9.0 mCi, below 8.0 mCi, below 7.0 mCi, below 6.0 mCi, below 5.0 mCi, below 4.0 mCi, below 3.5 mCi, below 3.0 mCi, below 2.5 mCi, below 2.0 mCi, below 1.5 mCi, below 1.0 mCi, below 0.5 mCi, below 0.4 mCi, below 0.3 mCi, below 0.2 mCi, or below 0.1 mCi.
  • the effective amount is from 0.1 mCi to 1.0 mCi, from 0.1 mCi to 2.0 mCi, from 1.0 mCi to 2.0 mCi, from 1.0 mCi to 3.0 mCi, from 1.0 mCi to 4.0 mCi, from 1.0 mCi to 5.0 mCi, from 1.0 mCi to 10.0 mCi, from 1.0 mCi to 15.0 mCi, from 1.0 mCi to 20.0 mCi, from 2.0 mCi to 3.0 mCi, from 3.0 mCi to 4.0 mCi, from 4.0 mCi to 5.0 mCi, from 5.0 mCi to 10.0 mCi, from 5.0 mCi to 15.0 mCi, from 5.0 mCi to 20.0 mC
  • the effective amount is 15.0 mCi, 14.0 mCi, 13.0 mCi, 12.0 mCi, 11.0 mCi, 10.0 mCi, 9.0 mCi, 8.0 mCi, 7.0 mCi, 6.0 mCi, 5.0 mCi, 4.0 mCi, 3.5 mCi, 3.0 mCi, 2.5 mCi, 2.0 mCi, 1.5 mCi, 1.0 mCi, 0.5 mCi, 0.4 mCi, 0.3 mCi, 0.2 mCi, or 0.1 mCi.
  • the effective amount is below, for example, 30.0 pCi/kg (i.e., where the amount of 225 Ac administered to the subject delivers a radiation dose of below 30.0 pCi per kilogram of subject’s body weight).
  • the effective amount is below 30 pCi/kg, 25 pCi/kg, 20 pCi/kg, 17.5 pCi/kg, 15.0 pCi/kg, 12.5 pCi/kg, 10.0 pCi/kg, 9 pCi/kg, 8 pCi/kg, 7 pCi/kg, 6 pCi/kg, 5 pCi/kg, 4.5 pCi/kg, 4.0 pCi/kg, 3.5 pCi/kg, 3.0 pCi/kg, 2.5 pCi/kg, 2.0 pCi/kg, 1.5 pCi/kg, 1.0 pCi/kg, 0.9 pCi/kg, 0.8 pCi/kg,
  • the effective amount is from 0.05 pCi/kg to 0 .1 pCi/kg, from 0 .1 pCi/kg to 0.2 pCi/kg, from 0.2 pCi/kg to 0.3 pCi/kg, from 0.3 pCi/kg to 0.4 pCi/kg, from 0.4 pCi/kg to 0.5 pCi/kg, from 0.5 pCi/kg to 0.6 pCi/kg, from 0.6 pCi/kg to 0.7 pCi/kg, from 0.7 pCi/kg to 0.8 pCi/kg, from 0.8 pCi/kg to 0.9 pCi/kg, from 0.9 pCi/kg to 1.0 pCi/kg, from 1.0 pCi/kg to 1.5 pCi/kg, from 1.5 pCi/kg, from 1.5 pCi/kg, from 1.5 pCi/
  • the effective amount is 0.05 pCi/kg, 0.1 pCi/kg, 0.2 pCi/kg, 0.3 pCi/kg, 0.4 pCi/kg, 0.5 pCi/kg, 0.6 pCi/kg, 0.7 pCi/kg, 0.8 pCi/kg, 0.9 pCi/kg, 1.0 pCi/kg, 1.5 pCi/kg, 2.0 pCi/kg, 2.5 pCi/kg, 3.0 pCi/kg, 3.5 pCi/kg, 4.0 pCi/kg or 4.5 pCi/kg, 5.0 pCi/kg, 6.0 pCi/kg, 7.0 pCi/kg, 8.0 pCi/kg, 9.0 pCi/kg, 10.0 pCi/kg, 12.5 pCi/kg
  • the effective amount is from 0.1 pCi to 100 mCi per meter squared of body surface area.
  • a preparation of radioimmunoconjugate of the invention may comprise a radiolabeled fraction (radioimmunoconjugate) and an unlabeled fraction (immunoconjugate), wherein the ratio of lab eled: unlab eled may be from about 1 : 1000 to 1 : 1.
  • cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy.
  • Therapy using radioimmunoconjugate of the invention may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness.
  • therapy using radiolabeled immunoconjugate of the invention are useful to alleviate target antigen-expressing cancers upon initial diagnosis of the disease or during relapse.
  • determining whether a cancer is amenable to treatment by methods disclosed herein involves detecting the presence of the target antigen in a subject or in a sample from a subject.
  • various detection assays are available.
  • target antigen overexpression is analyzed by immunohistochemistry (IHC). Parrafin embedded tissue sections from a tumor biopsy are subjected to the IHC assay and accorded a target antigen staining intensity criteria.
  • FISH assays such as the INFORM® (sold by Ventana, AZ, U.S.A.) or PATHVISION® (Vysis, IL, U.S.A.) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of target antigen overexpression in the tumor.
  • an immunoconjugate or radioimmunoconjugate of the invention is used to treat or prevent a cell proliferative disorder.
  • the cell proliferative disorder comprises a solid tumor cancer.
  • a solid tumor cancer is a cancer comprising an abnormal mass of tissue, e.g., carcinomas and sarcomas.
  • the cell proliferative disorder comprises a liquid tumor cancer or hematological cancer, Used interchangeably, such cancers present in the body fluid, e.g., leukemias and lymphomas.
  • the cell proliferative disorder is associated with increased expression and/or activity of a target antigen.
  • the invention provides methods for treating a cell proliferative disorder comprising administering to an individual an effective amount of an immunoconjugate or radioimmunoconjugate of the invention.
  • Non-limiting examples of cargos include cytotoxic agents, detection-promoting agents, and small molecule chemotherapeutic agents.
  • cargos include cytotoxic agents, detection-promoting agents, and small molecule chemotherapeutic agents.
  • compositions are also useful for detection and quantitation of a target epitope in vitro, e.g., in an ELISA or a Western blot, as well as purification or immunoprecipitation of a target antigen from cells or a tissue sample.
  • the immunoconjugate or radioimmunoconjugate of the invention is used in a method to detect the presence of or level of an antigen, such as, e.g., in vitro in a biological sample or in vivo using an imagine technique.
  • Immunoconjugate and radioimmunoconjugate detection can be achieved via different techniques known to the skilled worker and as described herein, e.g., IHC and PET imaging.
  • an immunoconjugate or radiolabeled immunoconjugate of the invention may comprise a radioactive atom for scintigraphic studies, for example 99m-Tc or 111-In.
  • Labelled immunoconjugates of the invention are useful as imaging biomarkers and probes by the various methods and techniques of biomedical and molecular imaging such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al Bioconjugate Chem. 15: 41-9 (2004); (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound.
  • MRI magnetic resonance imaging
  • MicroCT computerized tomography
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • Immunoscintigraphy is an imaging procedure in which antibodies labeled with radioactive substances are administered to an animal or human patient and a picture is taken of sites in the body where the antibody localizes (US 6528624). Imaging biomarkers may be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.
  • Another aspect of the present invention is a method of determining the presence of a target antigen in a sample suspected of containing the target antigen, wherein the method comprises exposing the sample to an immunoconjugate that binds to the target antigen and determining binding of the immunoconjugate to the target antigen in the sample, wherein the presence of such binding is indicative of the presence of the target antigen in the sample.
  • the sample may contain cells (which may be cancer cells) suspected of expressing the target antigen.
  • the immunoconjugate employed in the method may optionally be detectably labeled, attached to a solid support, or the like.
  • the immunoconjugates of the present invention are useful for detecting the presence of a target antigen, e.g., in vivo or in a biological sample.
  • a target antigen e.g., in vivo or in a biological sample.
  • the immunoconjugates of the invention can be used in a variety of different assays, including but not limited to ELISA, beadbased immunoassays, and mass spectrometry.
  • the sample is from a human subject.
  • the biological sample is serum from a clinical patient.
  • the biological sample is biopsy material.
  • the biological sample is biopsy material from a clinical patient.
  • the biological sample is serum from a clinical patient.
  • the biological sample is primary cell culture material.
  • the biological sample is primary cell culture material from a clinical patient.
  • the biological sample is from clinical patients or patients treated with a therapeutic antibody or antibodies that binds the same target antigen.
  • compositions comprising ‘labeled’ immunoconjugates are provided.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase, luciferin, 2,3- dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, J3-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microper
  • the amount of bound immunoconjugate is determined by removing excess unbound labeled immunoconjugate through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and correlating the measured amount with the amount of the immunoconjugate of interest in the biological sample.
  • the amount of color developed and measured will be a direct measurement of the amount of the immunoconjugate of interest present.
  • HRP is the label
  • the color may be detected using the substrate TMD, using a 450 nm read wavelength and a 620 or 630 nm reference wavelength.
  • the method involves a bead-based immunoassay, an ELISA assay, or a mass spectrometric technique.
  • the mass analyzers of such mass spectrometers include, but are not limited to, quadrupole (Q), time of flight (TOF), ion trap, magnetic sector or Fourier transform ion cyclotron resonance (FT-ICR) or combinations thereof.
  • the ion source of the mass spectrometer should yield mainly sample molecular ions, or pseudo- molecular ions, and certain characterizable fragment ions.
  • ion sources include atmospheric pressure ionization sources, e.g., electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and Matrix Assisted Laser Desorption Ionization (MALDI).
  • ESI and MALDI are the two most commonly employed methods to ionize proteins for mass spectrometric analysis of small molecules, such as, e.g., by liquid chromatography mass spectrometry (LC/MS) (Lee, M., LC/MS Applications in Drug Development (2002) J. Wiley & Sons, New York).
  • LC/MS liquid chromatography mass spectrometry
  • SELDI is a surface-based ionization technique that allows for high-throughput mass spectrometry.
  • SELDI is used to analyze complex mixtures of proteins and other biomolecules.
  • SELDI employs a chemically reactive surface such as a “protein chip” to interact with analytes, e.g., proteins, in solution.
  • analytes e.g., proteins
  • Such surfaces selectively interact with analytes and immobilize them thereon.
  • the analytes of the invention can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing multiple reactive moieties at different sites on a substrate surface, throughput may be increased.
  • the invention provides a method for detecting in a biological sample an antigen, the method comprising: (a) contacting the biological sample with an immunoconjugate described herein to allow forming an immunocomplex; (b) detecting or measuring the level of the immunoconjugate bound to the sample.
  • the immunoconjugate is immobilized to a solid support.
  • the immobilized immunoconjugate is conjugated to biotin and bound to a streptavidin coated microtiter plate.
  • Another aspect of the present invention is an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of diseases and disorders characterized by target antigen-expressing cells (e.g., a cancer cell).
  • the article of manufacture of the invention comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating, preventing and/or diagnosing the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an immunoconjugate of the invention.
  • the label or package insert indicates that the composition is used for treating cancer.
  • the label or package insert will further comprise instructions for administering the immunoconjugate composition to the cancer patient.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer’s solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate- buffered saline such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer’s solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • the article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • the invention provides a kit comprising any of the immunoconjugates described herein and an additional reagent or pharmaceutical device.
  • the kit comprises a composition as provided herein (e.g., a pharmaceutical or diagnostic composition).
  • Another aspect of the present invention is a kit useful for various purposes, e.g., target antigen-expressing cell killing; for target antigen-expressing cell detection; quantification, purification, or immunoprecipitation of target antigen from cells.
  • the kit of the invention is an immunoassay kit for specifically detecting an antigen in a biological sample, comprising: (a) an immunoconjugate as described herein and/or a composition thereof; and (b) instructions for detecting said immunoconjugate.
  • a target antigen detection assays of the present invention can be provided in the form of a kit.
  • such a kit comprises an immunoconjugate of the present invention, or a composition comprising the aforementioned, such as one described herein.
  • the kit may further comprise a solid support for the capture reagents, which may be provided as a separate element or to which the capture reagents are already immobilized.
  • the kit may contain an immunoconjugate of the invention coupled to beads (e.g., sepharose beads).
  • beads e.g., sepharose beads.
  • the invention provides kits that contain an antibody for the detection and/or quantitation of target antigen in vitro, e.g., in an ELISA or a Western blot.
  • the capture reagents e.g., the immunoconjugate of the invention
  • a solid material e.g., to beads, a microtiter plate, or a comb.
  • the detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies, such as, e.g., antibodies raised in a different species.
  • the label is an enzyme
  • the kit will ordinarily include substrates and cofactors required by the enzyme; where the label is a fluorophore, a dye precursor that provides the detectable chromophore; and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or P-galactosidase with MUG.
  • the kit of the invention comprises a container and a label or package insert on or associated with the container.
  • the container holds a composition comprising at least one immunoconjugate of the invention.
  • Additional containers may be included that contain, e.g., diluents and buffers, control immunconjugates or antibodies.
  • the label or package insert may provide a description of the composition as well as instructions for the intended in vitro or detection use.
  • the kit also typically contains additives such as stabilizers, washing and incubation buffers, and the like for performing the assay method(s).
  • the components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay(s).
  • the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
  • immunoconjugates comprising the aforementioned structures and functions, in particular platforms having VHH polypeptides, a molecular weight between 60 and 110 kDa, a serum halflife of less than 96 hours, which in some embodiments exhibit enhanced stability during the temperatures required for certain radiolabeling processes relative to other antibody fragment platforms, and which in some embodiments exhibit decreased loss of targeting capacity due to radiolysis as compared to other possible delivery platforms.
  • amino acid residue or “amino acid” includes reference to an amino acid that is incorporated into a protein, polypeptide, and/or peptide.
  • polypeptide includes any polymer of amino acids or amino acid residues.
  • polypeptide sequence refers to a series of amino acids or amino acid residues which physically comprise a polypeptide.
  • a “protein” is a macromolecule comprising one or more polypeptides or polypeptide “chains.”
  • a “peptide” is a small polypeptide of a size of 2 to 20 amino acid residues.
  • amino acid sequence refers to a series of amino acids or amino acid residues which physically comprise a peptide or polypeptide depending on the length. Unless otherwise indicated, polypeptide and protein sequences disclosed herein are written from left to right representing their order from an amino terminus to a carboxy terminus.
  • amino acid amino acid residue
  • amino acid sequence amino acid sequence
  • proteins include naturally occurring amino acids (including L and D isosteriomers) and, unless otherwise limited, also include known analogs of natural amino acids that can function in a similar manner as the common natural amino acids, such as selenocysteine, pyrrolysine, N- formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine, pyroglutamic acid, and selenomethionine (see, e.g., Ho J et al., ACS Synth Biol 5: 163-71 (2016); Wang Y, Tsao M, Chembiochem 17: 2234-9 (2016)).
  • radioconjugate is used interchangeably with the term “radioimmunoconjugate” herein.
  • the radioisotope is associated with a chelating agent of the radioimmunoconjugate. In one embodiment, the radioisotope is directly linked to the immunoconjugate.
  • the term “immunoconjugate” refers to a molecular complex comprising an at least one antigen binding region derived from an antibody (e.g., variable regions or complementarity determining regions) further coupled to at least one non-antibody derived molecule, such as a chelator or cytotoxic agent.
  • Non-antibody derived molecules may for example be conjugated to one or more lysine or cysteine resides of the antigen binding region or to a constant region coupled (by peptide linkage or otherwise) to the antigen binding region.
  • the immunoconjugate further comprises a chelating agent (interchangeably, “chelator”).
  • an immunoconjugate comprises an antibody construct of the invention linked directly or indirectly to a cytotoxic agent or radioisotope.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full- length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antibody can comprise a human IgGl constant region.
  • the antibody can comprise a human IgG4 constant region.
  • FR-H1, FR-H2, FR-H3, and FR-H4 there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
  • FR-H1, FR-H2, FR-H3, and FR-H4 four FRs in each full-length heavy chain variable region
  • FR-L1, FR-L2, FR-L3, and FR-L4 four FRs in each full-length light chain variable region.
  • the precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed.
  • the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91(2007)).
  • FRs conserved framework regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (See e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).
  • the antigen binding regions of the immunoconjugates described herein may be humanized. “Humanized” in reference to an immunoconjugate refers to an antigen binding region in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • a humanized immunoconjugate optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • human immunoconjugates are human immunoconjugates.
  • a “human immunoconjugates” is an immunoconjugates possessing an antigen binding region with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non- human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries.
  • the term excludes humanized forms of non- human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.
  • antigen binding arm refers to a single polypeptide chain, comprising an “antigen binding region”, a hinge region, and a variant constant region.
  • Other elements e.g., a chelating agent; an imaging metal
  • Immunoconjugates of the invention comprise two antigen binding arms that are covalently linked together.
  • the antigen binding arms are linked through the hinge region.
  • the antigen binding arms are linked through an immunoglobulin heavy chain constant region.
  • the antigen binding arms are linked through the variant constant region.
  • the antigen binding arms are linked via a disulfide linkage (e.g., via a cysteine residue in a hinge region).
  • antigen binding region refers to the region of an immunoconjugate responsible for specific binding to an antigen, such region one or more antigen binding domains comprising complementarity determining regions, variable regions and framework regions, which may be derived from, modeled on, or may mimic, antibodies or fragments thereof, as are known by the person of ordinary skill in the art.
  • the “antigen binding region’ of an antigen binding arm contains one or two antigen binding domains.
  • the “antigen binding region” of an antigen binding arm consists of a single antigen binding domain, which antigen binding domain is preferably a VHH polypeptide.
  • the antigen binding regions of both antigen binding arms of an immunoconjugate independently consist of a single antigen binding domain, which antigen binding domain is preferably a VHH polypeptide, which VHH polypeptides are the same or different.
  • VHH polypeptide encompasses natural and synthetic compositions and refers to a polypeptide constituting a VHH fragment as it is known in the art, z.e., a polypeptide that constitutes a single domain heavy chain only variable domain fragment, or a polypeptide that structurally and functionally resembles a VHH fragment, as such structure is further described below and has the ability to specifically bind antigen is described below, and as both are well known in the art.
  • the VHH polypeptides comprise a heavy chain variable region comprising three heavy chain CDR’s; in one embodiment the VHH polypeptide is derived from a camelid; in another embodiment the VHH polypeptide is derived from a library; VHH polypeptides bind to antigens with specificity and high affinity.
  • the VHH polypeptide is a single heavy chain variable domain comprising the arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • VHH polypeptides may be obtained, for example, as the antigen binding fragments of heavy chain only antibodies generated in vivo (e.g., in camelids).
  • VHH polypeptides may also be obtained from synthetic libraries, e.g., phage display libraries.
  • synthetic libraries e.g., phage display libraries.
  • phage display libraries For example, see McMahon et al., Nature Structural & Molecular Biology
  • VHH humanization see, for example, Vincke C, Loris R, Saerens D, Martinez- Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009 Jan 30;284(5):3273-84. doi: 10.1074/jbc.M806889200. Epub 2008 Nov 14. PMID: 19010777.
  • a “linker” herein is also referred to as “linker sequence” “spacer” “tethering sequence” or grammatical equivalents thereof.
  • a “linker” as referred herein connects two distinct molecules that by themselves possess target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides or comprise separate domains of the same polypeptide. For example, two distinct binding moieties or a heavy-chain/light-chain pair or an antigen binding region and an immunoglobulin heavy chain constant region. A number of strategies may be used to covalently link molecules together.
  • Linkers described herein may be utilized to join a light chain variable region and a heavy chain variable region in an scFv molecule; or may be used to tether an scFv or other antigen binding fragment on the N- or C- terminus of an antibody heavy chain. These include but are not limited to polypeptide linkages between N- and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents.
  • the linker is a peptide bond, generated by recombinant techniques or peptide synthesis.
  • An antibody that “binds” an antigen or epitope of interest is one that binds the antigen or epitope with sufficient affinity that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. [00512] “Specific binding” refers to an antibody or immunoconjugate that is capable of binding antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting that antigen.
  • the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to its antigen as measured, e.g., by a radioimmunoassay.
  • An “antigen specific” antibody or immunoconjugate, as used herein, is one that specifically binds to the antigen with sufficient specificity and affinity to be useful in targeting a therapeutic, targeting diagnostic, or method of detecting the antigen in a biological sample from a subject.
  • an immunoconjugate or antibody construct or target imaging complex or radioimmunoconjugate that binds to its target antigen has a dissociation constant (KD) of ⁇ 1 pM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10' 8 M or less, e.g., from 10' 8 M to 10' 13 M, e.g., from 10' 9 M to 10' 13 M).
  • KD dissociation constant
  • an immunoconjugate or antibody construct or target imaging complex or radioimmunoconjugate of the present invention binds to multiple antigens, such as, e.g., an epitope conserved among homologs from different species, such as wherein the amino acid identity of the epitope is nonidentical in different species.
  • variable constant region refers to a polypeptide comprising of a portion of an immunoglobulin heavy chain constant region that has been modified from native immunoglobulin amino acid sequence, preferably at from one to several amino acid positions.
  • EU numbering system also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) Modifications to Fc regions for various purposes are well known in the art. For example, see Kevin O. Saunders, Frontiers in Immunology, June 2019
  • Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using available computer software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative embodiments for measuring binding affinity are described herein.
  • antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of antigen. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, or derivatives thereof.
  • a “blocking” antibody or an “antagonist” antibody is an antibody that inhibits or reduces biological activity of the antigen it binds or a protein complex comprising the antigen. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen or protein complex comprising the antigen.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” as used herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), skin cancer, melanoma, lung cancer including small-cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high grade serous ovarian carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uter
  • cancer include, without limitation, retinoblastoma, thecomas, arrhenoblastomas, hepatoma, hematologic malignancies including non-Hodgkins lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial or uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma, salivary gland carcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi’s sarcoma, melanoma, skin carcinomas, Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, anaplastic astrocytoma, basal cell carcinoma (
  • metastatic cancer means the state of cancer where the cancer cells of a tissue of origin are transmitted from the original site to one or more sites elsewhere in the body, by the blood vessels or lymphatics, to form one or more secondary tumors in one or more organs besides the tissue of origin.
  • a prominent example is a metastatic breast cancer.
  • association refers to the state of two or more components of a molecule being joined, attached, connected, or otherwise coupled to form a single molecule (or single molecular complex) or the act of making two molecules associated with each other to form a single molecule (or single molecular complex) by creating an association, linkage, attachment, and/or any other connection between the two molecules.
  • linked may refer to two or more components associated by one or more atomic interactions such that a single molecule is formed and wherein the individual atomic interactions may be covalent or non-covalent.
  • Non-limiting examples of covalent associations between two components include peptide bonds and cysteine-cysteine disulfide bonds.
  • Non-limiting examples of non-covalent associations between two molecular components include ionic bonds.
  • the term “fused” refers to two or more proteinaceous components associated by at least one covalent bond which is a peptide bond, regardless of whether the peptide bond involves the participation of a carbon atom of a carboxyl acid group or involves another carbon atom, such as, e.g., the a-carbon, P-carbon, y-carbon, c- carbon, etc.
  • a “bispecific” antibody refers to an antibody that has binding specificities for at least two different epitopes, regardless of whether the plurality of epitopes are in the same molecule and/or partially overlapping.
  • the bispecific immunoconjugate of the present invention binds to two different epitopes of a single antigen described herein.
  • the terms “expressed,” “expressing,” or “expresses,” and grammatical variants thereof, refer to translation of a polynucleotide or nucleic acid into a protein.
  • the expressed protein may remain intracellular, become a component of the cell surface membrane or be secreted into an extracellular space.
  • the phrase “derived from” when referring to a polypeptide or polypeptide region means that the polypeptide or polypeptide region comprises highly similar amino acid sequences originally found in a “parental” protein and which may now comprise certain amino acid residue additions, deletions, truncations, rearrangements, or other alterations relative to the original polypeptide or polypeptide region as long as a certain function(s) (e.g., antigen binding affinity) and a structure(s) of the “parental” molecule are substantially conserved.
  • a certain function(s) e.g., antigen binding affinity
  • a parental molecule e.g., an antibody sequence
  • a polypeptide or polypeptide region e.g., a VHH polypeptide, CDR, HVR, VH, and/or VL
  • cells which express an extracellular target biomolecule or antigen on at least one cellular surface are “target positive cells” or “target+ cells” and are cells physically coupled to the specified, extracellular target biomolecule. Additional target biomolecule description is provided below. “Target biomolecule”, “target antigen molecule”, “target antigen”, “antigen of interest”, and grammatical variants and equivalents are used interchangeably herein as will be recognized by the person of ordinary skill in the art viewing the context of usage, and include the molecular determinants of antibody binding. Such antigens can be bound by the immunoconjugates described herein though the antigen binding region or antigen binding arm of the immunoconjugate.
  • selective cytotoxicity with regard to the cytotoxic activity of a molecule refers to the relative level of cytotoxicity between a biomolecule target positive cell population (e.g., a targeted cell-type) and a non-targeted bystander cell population (e.g., a biomolecule target negative cell-type), which can be expressed as a ratio of the half-maximal cytotoxic concentration (CD50) for a targeted cell-type over the CD50 for an untargeted cell-type to provide a metric of cytotoxic selectivity or indication of the preferentiality of killing of a targeted cell versus an untargeted cell.
  • a biomolecule target positive cell population e.g., a targeted cell-type
  • a non-targeted bystander cell population e.g., a biomolecule target negative cell-type
  • pharmaceutical formulation or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • an “isolated” antibody or immunoconjugate or radio immunoconjugate is one which has been separated from a component of its natural environment or artificial production.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC. Routine methods for assessment of antibody purity in a composition are known to the skilled worker, see e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
  • unwanted components (contaminants) to be purified away from are such components that would interfere with desired uses for the antibody, such as, e.g., a therapeutic use, and may include, inter alia, bacterial factors, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present at extrachromosomal location or at a chromosomal location that is different from its natural chromosomal location.
  • the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term “administer”, with respect to an immunoconjugate or composition thereof means to deliver the immunoconjugate, or composition thereof, to a subject’s body via any known method suitable for delivery of immunoconjugate or composition thereof.
  • Specific modes of administration include, without limitation, intravenous, transdermal, subcutaneous, intraperitoneal and intrathecal administration.
  • an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • radioimmunoconjugates of the invention are used to delay development of a disease or to slow the progression of a disease.
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement or prevention of a particular disorder.
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of a composition of the invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition of the invention are outweighed by the therapeutically beneficial effects.
  • the terms “predictive” and “prognostic” as used herein are interchangeable.
  • the methods for prediction or prognostication are to allow the person practicing a predictive/prognostic method of the invention to select patients that are deemed (usually in advance of treatment, but not necessarily) more likely to respond to treatment with an immunoconjugate of the present invention or a composition of the aforementioned (e.g., a pharmaceutical composition).
  • the term “detecting” is used in the broadest sense to include both qualitative and quantitative measurements of a target antigen molecule.
  • the detecting method as described herein is used to identify the mere presence of the antigen of interest in a biological sample.
  • the method is used to test whether the antigen of interest in a sample is present at a detectable level.
  • the method can be used to quantify the amount of the antigen of interest in a sample and further to compare the antigen levels from different samples.
  • the method can be used in vivo to determine the location of a target cell, for example, using a targeted imaging complex of the invention.
  • biological sample refers to any biological substance that might contain an antigen of interest.
  • a sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, itreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, and other constituents of the body that might contain the antigen of interest.
  • the sample is a biological sample from any animal.
  • the sample is from a mammal.
  • the sample is from a human subject.
  • the biological sample is serum from a clinical patient.
  • the biological sample is biopsy material.
  • the biological sample is biopsy material from a clinical patient.
  • the biological sample is serum from a clinical patient.
  • the biological sample is primary cell culture material.
  • the biological sample is primary cell culture material from a clinical patient.
  • the biological sample is from clinical patients or patients treated with a composition of the invention e.g., a radioimmunoconjugate, or treated with a different therapeutic agent, such as an antibody -drug conjugate targeting the antigen of interest or P-irradiation or a small molecule therapeutics.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • Ci-C x includes C1-C2, C1-C3 . . . Ci-C x .
  • a group designated as "Ci-Ce" indicates that there are one to six carbon atoms in the moiety, i.e. groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms.
  • C1-C4 alkyl indicates that there are one to four carbon atoms in the alkyl group, i.e., the alkyl group is selected from among methyl, ethyl, propyl, Ao-propyl, //-butyl, iso- butyl, ec-butyl, and /-butyl.
  • an “alkyl” group refers to an aliphatic hydrocarbon group.
  • the alkyl group is branched or straight chain.
  • the “alkyl” group has 1 to 10 carbon atoms, i.e. a Ci- Cioalkyl.
  • a numerical range such as “1 to 10” refers to each integer in the given range; e.g., “1 to 10 carbon atoms” means that the alkyl group consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated.
  • an alkyl is a Ci-Cealkyl.
  • the alkyl is methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, or t-butyl.
  • Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, or hexyl.
  • alkoxy refers to a (alkyl)O- group, where alkyl is as defined herein.
  • alkylamine refers to the -N(alkyl) x H y group, where x is 0 and y is 2, or where x is 1 and y is 1, or where x is 2 and y is 0.
  • An “hydroxyalkyl” refers to an alkyl in which one hydrogen atom is replaced by a hydroxyl.
  • a hydroxyalkyl is a Ci-C4hydroxyalkyl.
  • Typical hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, - CH2CH2CH2CH2OH, and the like.
  • aminoalkyl refers to an alkyl in which one hydrogen atom is replaced by an amino.
  • aminoalkyl is a Ci-C4aminoalkyl.
  • Typical aminoalkyl groups include, but are not limited to, -CH2NH2, -CH 2 CH 2 NH 2 , -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, and the like.
  • alkenyl refers to a type of alkyl group in which at least one carbon-carbon double bond is present.
  • R is H or an alkyl.
  • an alkenyl is selected from ethenyl (z.e., vinyl), propenyl (z.e., allyl), butenyl, pentenyl, pentadienyl, and the like.
  • alkynyl refers to a type of alkyl group in which at least one carbon-carbon triple bond is present.
  • R is H or an alkyl.
  • an alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • heteroalkyl refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g. -NH-, -N(alkyl)- , sulfur, or combinations thereof.
  • a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl.
  • aromatic refers to a planar ring having a delocalized 71-electron system containing 4n+271 electrons, where n is an integer.
  • aromatic includes both carbocyclic aryl (“aryl”, e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine).
  • aryl e.g., phenyl
  • heterocyclic aryl or “heteroaryl” or “heteroaromatic” groups
  • pyridine e.g., pyridine
  • the term includes monocyclic or fused-ring polycyclic (z.e., rings which share adjacent pairs of carbon atoms) groups.
  • Carbocyclic refers to a ring or ring system where the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from “heterocyclic” rings or “heterocycles” in which the ring backbone contains at least one atom which is different from carbon. In some embodiments, at least one of the two rings of a bicyclic carbocycle is aromatic. In some embodiments, both rings of a bicyclic carbocycle are aromatic. Carbocycles include aryls and cycloalkyls.
  • aryl refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom.
  • aryl is phenyl or a naphthyl.
  • an aryl is a phenyl.
  • an aryl is a phenyl, naphthyl, indanyl, indenyl, or tetrahyodronaphthyl.
  • an aryl is a Ce-Cioaryl.
  • an aryl group is a monoradical or a diradical (i.e., an arylene group).
  • cycloalkyl refers to a monocyclic or polycyclic aliphatic, non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • cycloalkyls are spirocyclic or bridged compounds.
  • cycloalkyls are optionally fused with an aromatic ring, and the point of attachment is at a carbon that is not an aromatic ring carbon atom.
  • Cycloalkyl groups include groups having from 3 to 10 ring atoms.
  • cycloalkyl groups are selected from among cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, spiro[2.2]pentyl, norbornyl and bicycle[l.l .l]pentyl.
  • a cycloalkyl is a C3- Cecycloalkyl.
  • halo or, alternatively, “halogen” or “halide” means fluoro, chloro, bromo or iodo. In some embodiments, halo is fluoro, chloro, or bromo.
  • fluoroalkyl refers to an alkyl in which one or more hydrogen atoms are replaced by a fluorine atom.
  • a fluoroalkyl is a Ci-Cefluoroalkyl.
  • the heterocyclic groups include benzo-fused ring systems.
  • non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-
  • aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinox
  • the foregoing groups are either C-attached (or C-linked) or TV-attached where such is possible.
  • a group derived from pyrrole includes both pyrrol- 1-yl (TV-attached) or pyrrol-3-yl (C-attached).
  • a group derived from imidazole includes imidazol-l-yl or imidazol-3-yl (both TV-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached).
  • the heterocyclic groups include benzo-fused ring systems.
  • at least one of the two rings of a bicyclic heterocycle is aromatic.
  • both rings of a bicyclic heterocycle are aromatic.
  • heteroaryl or, alternatively, “heteroaromatic” refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur.
  • heteroaryl groups include monocyclic heteroaryls and bicyclcic heteroaryls.
  • Monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl.
  • Monocyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine.
  • a heteroaryl contains 0-4 N atoms in the ring.
  • a heteroaryl contains 1-4 N atoms in the ring.
  • a heteroaryl contains 0-4 N atoms, 0-1 0 atoms, and 0-1 S atoms in the ring.
  • a heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring.
  • heteroaryl is a Ci- Cgheteroaryl.
  • monocyclic heteroaryl is a Ci-Csheteroaryl.
  • monocyclic heteroaryl is a 5-membered or 6-membered heteroaryl.
  • bicyclic heteroaryl is a Ce-Cgheteroaryl.
  • a “heterocycloalkyl” group refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen and sulfur. In some embodiments, a heterocycloalkyl is fused with an aryl or heteroaryl.
  • the heterocycloalkyl is oxazolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidin-2-onyl, pyrrolidine-2, 5- dithionyl, pyrrolidine-2, 5-dionyl, pyrrolidinonyl, imidazolidinyl, imidazolidin-2-onyl, or thiazolidin-2-onyl.
  • heterocycloalkyl also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the oligosaccharides.
  • a heterocycloalkyl is a C2-Cioheterocycloalkyl.
  • a heterocycloalkyl is a C4-Cioheterocycloalkyl.
  • a heterocycloalkyl contains 0-2 N atoms in the ring.
  • a heterocycloalkyl contains 0-2 N atoms, 0-2 O atoms and 0-1 S atoms in the ring.
  • bond refers to a chemical bond between two atoms, or two moi eties when the atoms joined by the bond are considered to be part of larger substructure.
  • bond when a group described herein is a bond, the referenced group is absent thereby allowing a bond to be formed between the remaining identified groups.
  • moiety refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
  • optional substituents are independently selected from halogen, -CN, -NH2, -OH, -NH(CH 3 ), -N(CH 3 )2, -CH 3 , -CH2CH 3 , -CF 3 , -OCH 3 , and -OCF 3 .
  • substituted groups are substituted with one or two of the preceding groups.
  • Example 7 Compound 1-7 1,19, 24, 31-tetraoxo-l-(2, 3,5, 6-tetrafluorophenoxy)-4, 7, 10,13, 16-pentaoxa-20, 25,30- triazatetratriacontan-34-yl)-l,4, 7,10-tetraazacyclododecane-l,4, 7-triyl) triacetic acid
  • Example 11 Compound 1-11 Synthesis of 2,2 ',2 ' '-(10-(22-carboxy-l-(3,4-dibromo-2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) ⁇ 19-oxo-3, 6,9,12,15-pentaoxa-l 8-azadocosan-22-yl)-l,4, 7,10-tetraazacyclododecane-l,4, 7- triyl)triacetic acid
  • Example 12 Compound 1-12 l-yl)ethoxy)ethoxy)ethyl)amino)-4-oxobutyl)-l,4, 7,10-tetraazacyclododecane-l,4, 7- triyl)triacetic acid
  • the title compound was prepared by standard solid phase peptide synthesis methods using trityl chloride resin (3.01 g, 2.29 mmol loading) and commercially available Fmoc protected amino acids.
  • Fmoc-Tyr-OH was loaded by treating the swelled resin with thionyl chloride, washing with DCM and then adding 3 eq of the amino acid and 6 eq of N,N- diisopropylethylamine. Subsequent amino acids were loaded by deprotection with 20% 4- methylpiperidine in DMF, then activating the next amino acid (3 eq) with HBTU (3 eq) and DIPEA (6 eq).
  • N,N-diisopropylethylamine (0.073 mL, 54.2 mg, 0.419 mmol) was added, causing the solution to turn deep green.
  • a solution of EDTA (acid form) was added, then potassium carbonate was added.
  • the mixture was extracted with ethyl acetate (125 mL) and the organic layers formed a sort of gel.
  • the gel was wasted with saturated ammonium chloride, tetrasodium EDTA solution, then the gelled organic layer was treated with MeOH and sonicated.

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Abstract

L'invention concerne des immunoconjugués comprenant : a) région de liaison à l'antigène ; b) une région constante de chaîne lourde d'immunoglobuline ; et c) un agent chélatant de radio-isotope ; le poids moléculaire dudit immunoconjugué étant compris entre 60 et 110 kDa. Les immunoconjugués peuvent être utilisés pour administrer des émetteurs alpha et bêta en vue du traitement de tumeurs ou du cancer.
EP23858213.4A 2022-08-22 2023-08-21 Conjugués dota d'anticorps vhh Pending EP4577527A1 (fr)

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