EP4577526A1 - Vhh-antikörperkonjugate - Google Patents

Vhh-antikörperkonjugate

Info

Publication number: EP4577526A1
Authority: EP; European Patent Office
Prior art keywords: substituted; unsubstituted; pharmaceutically acceptable; compound; acceptable salt
Prior art date: 2022-08-22
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

EP23858210.0A

Other languages

English (en)

French (fr)

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 United States

Original Assignee

Abdera Therapeutics Inc United States

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.)

2022-08-22

Filing date

2023-08-21

Publication date

2025-07-02

2023-08-21 Application filed by Abdera Therapeutics Inc United States filed Critical Abdera Therapeutics Inc United States

2025-07-02 Publication of EP4577526A1 publication Critical patent/EP4577526A1/de

Status Pending legal-status Critical Current

Links

Classifications

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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
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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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- A61K51/1096—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
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- 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
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- 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
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6524—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having four or more nitrogen atoms as the only ring hetero atoms
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/30—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IG], 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
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06052—Val-amino acid
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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- C07K5/0802—Tripeptides with the first amino acid being neutral
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Definitions

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 2 is a moiety that is capable of reacting with an amine (-NH2) or thiol (-SH) of a tumor targeting moiety R 3 ; and v is 1, 2, 3, or 4.
v is 1.
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:
an immunoconjugate that has the structure of 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
L is an optional linker
-NH-R 3 is a tumor targeting moiety; and v is 1, 2, 3, or 4.
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
R 2 is a moiety that is capable of reacting with a thiol (-SH) of
an immunoconjugate that has the structure of 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; and v is 1, 2, 3, or 4.
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 immunoconjugate of Formula (II) has the structure of Formula (Ila), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (III) has the structure of Formula (Illa), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (IV) has the structure of Formula (IVa), or a pharmaceutically acceptable salt thereof:
R 2 is a moiety that is capable of reacting with a thiol (-SH) of a cysteine of the tumor targeting moiety R 3 .
the immunoconjugate of Formula (V) has the structure of Formula (Va), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (VI) has the structure of Formula (Via), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (VII) has the structure of Formula (Vila), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (VIII) has the structure of Formula (Villa), 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 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.
the antigen binding region comprises an scFv polypeptide or a VHH polypeptide.
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 is an IgA, IgGl, IgG2, IgG3, or IgG4 isotype.
the antigen binding region is humanized, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region, or both.
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 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 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); or both.
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 cell -phagocytosis ADCP, or a combination thereof.
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 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 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.
the radioisotope is an alpha emitter selected from the list consisting of 225 Ac, radium-223 ( 223 Ra), radium-224 ( 224 Ra), thorium-227 ( 227 Th), lead-212 ( 212 Pb), bismuth-212 ( 212 Bi), and bismuth ( 213 Bi).
the radioisotope is 225 Ac.
the radioisotope is a beta emitter.
the radioisotope is a beta emitter selected from 177 Lu, 90 Y, copper-67 ( 67 Cu), and smathrium-153 ( 153 Sm).
a method of killing a cancer cell in an individual comprising administering to the individual the immunoconjugate, thereby killing the cancer cell.
the individual is a human individual.
the cancer cell comprises a lung cancer cell, a breast cancer cell, an ovarian cancer cell, or a neuroendocrine cancer cell.
the method comprises administering from 0.1 pCi to 30.0 pCi per kilogram to the individual.
the method comprises administering from 10 mCi to 75 mCi per meter squared of body area to the individual.
the cancer cell expresses an antigen specifically bound by the immunoconjugate.
the immunoconjugate in a method of killing a cancer cell in an individual.
the individual is a human individual.
the cancer 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 expresses an antigen specifically bound by the immunoconjugate.
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.
the immunoconjugate for use in a method of imaging a tumor in an individual.
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 subject radioisotope delivery platforms have a molecular size large enough (e.g., 60 kDa to 110 kDa) to substantially reduce off-target toxi cities, especially renal damage e.g., from an alpha emitting isotope cargo) and a small enough size for increased tissue penetration as compared to traditional IgGs, with maintained target specificity, and increased probability of first decay event in target tissue.
a molecular size large enough (e.g., 60 kDa to 110 kDa) to substantially reduce off-target toxi cities, especially renal damage e.g., from an alpha emitting isotope cargo) and a small enough size for increased tissue penetration as compared to traditional IgGs, with maintained target specificity, and increased probability of first decay event in target tissue.
Such sizes provide for preferential elimination by the liver as opposed to the kidney, sparing the kidney from radiotoxicity.
the subject radioisotope delivery platforms are useful for in vivo targeted delivery of alpha 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 delivery platforms are useful for in vivo targeted delivery of alpha emitters safely and effectively, in part, by exhibiting decreased loss of targeting capacity due to radiolysis as compared to other possible delivery platforms.
the invention provides a method of delivering an a-emitting radioisotope to a cancer cell in vivo in a patient, comprising administering a radioimmunoconjugate or pharmaceutical composition of the invention to the patient.
the patient is a human patient.
the invention provides a method of inhibiting the growth of a cancer cell, comprising contacting the cancer cell with a radioimmunoconjugate of the invention.
the cancer cell is in vivo in a patient.
the method involves administering a pharmaceutical composition of the invention to the patient.
the patient is a human patient.
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. [0056] In one embodiment, 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.
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.
kits of the invention includes a reagent or pharmaceutical device in addition to the immunoconjugate, radioimmunoconjugate or pharmaceutical composition of the invention.
the kit of the present invention is an immunoassay kit for specifically detecting an antigen in a biological sample, comprising: (a) immunoconjugate, radioimmunoconjugate or targeted imaging complex as described herein and/or a composition thereof; and (b) instructions for detecting the immunoconjugate, radioimmunoconjugate or targeted imaging complex.
the invention provides an isolated nucleic acid encoding an antigen binding arm or a component thereof as provided herein.
the invention provides an isolated nucleic acid encoding an antigen binding region of an immunoconjugate herein.
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. In one aspect, 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 a vector comprising a nucleic acid as provided herein.
the vector is an expression vector.
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.
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.
FIG. 4 shows self-interaction data for anti-HER2 and anti-DLL3 VHH-Fc constructs.
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 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) antigen binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating moiety or a radionuclide complex thereof.
an immunoconjugate of the current disclosure comprises an: a) antigen binding region; b) an immunoglobulin heavy chain constant region; and c) a chelating moiety or a radionuclide complex thereof; wherein the molecular weight of said immunoconjugate is between 60 and 110 kDa.
an immunoconjugate of the current disclosure comprises an: a) VHH antigen binding region; b) an immunoglobulin heavy chain constant region; 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 heavy chain constant region; and c) a chelating moiety or a radionuclide complex thereof; wherein the molecular weight of said immunoconjugate is between 60 and 110 kDa.
an immunoconjugate of the current disclosure comprises an: a) VHH antigen binding region; b) an immunoglobulin Fc region, together referred to as a VHH-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.
an immunoconjugate of the current disclosure comprises an: a) VHH antigen binding region; b) a variant immunoglobulin Fc region; and c) a chelating moiety or a radionuclide complex thereof.
an immunoconjugate of the current disclosure comprises an: a) VHH antigen binding region; b) a variant 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 variant immunoglobulin Fc region comprises one or more amino acid alterations to reduce the serum or plasma half-life of the immunoconjugate.
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 toxi cities. 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).
At least one of the two antigen binding regions in the immunoconjugate consists of one or two heavy chain only variable (VHH) polypeptides. In a preferred embodiment at least one of the two antigen binding regions consists of one VHH polypeptide. In a preferred embodiment, each of the two antigen binding regions of the immunoconjugate consists of one VHH polypeptide, which VHH polypeptides are the same or different.
VHH heavy chain only variable
the antigen binding regions of the immunoconjugate bind to the same antigen. In one embodiment, the antigen binding regions of the immunoconjugate bind to different antigens. In one embodiment, the antigen binding regions of the immunoconjugate are the same. In one embodiment, the antigen binding regions of the immunoconjugate are different. In one embodiment, the antigen binding region of each antigen binding arm consists of one or two VHH polypeptides.
the antigen binding region of one antigen binding arm consists of two VHH polypeptides and the antigen binding region of the other antigen binding arm does not comprise a VHH polypeptide.
the two antigen binding arms bind the same antigen.
the two antigen binding arms bind different antigens.
the two VHH polypeptides are the same.
the two VHH polypeptides are different.
the immunoconjugate is bispecific.
the antigen binding region of one antigen binding arm consists of one VHH polypeptide and the antigen binding region of the other antigen binding arm consists of two VHH polypeptides.
the two antigen binding arms bind the same antigen. In one embodiment, the two antigen binding arms bind different antigens. In one embodiment, the three VHH polypeptides are the same. In one embodiment, two of the three VHH polypeptides are the same and are different from the third VHH polypeptide. In one embodiment, the three VHH polypeptides are different. In one embodiment, the immunoconjugate is bispecific.
the antigen binding region of each antigen binding arm of the immunoconjugate consists of one VHH polypeptide.
the VHH polypeptides bind to the same antigen.
the VHH polypeptides bind to different antigens.
the VHH polypeptides are the same.
the VHH polypeptides are different.
the immunoconjugate is bispecific.
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, ROR1, ROR2, Glypican 3, Trail Receptor 2 (DR5), PD-L1, Mesothein, Bombesin, EpCAM, DARPP, CSPG4, Galectin-3, Integrin avpi, Integrin avP3, Integrin avP5, Integrin avP6, Integr
the immunoconjugate has a serum half-life of about 12 hours to about 120 hours.
the immunoconjugate has a serum half-life of about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, about 12 hours to about 72 hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours, about 12 hours to about 108 hours, about 12 hours to about 120 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours, about 24 hours to about 96 hours, about 24 hours to about 108 hours, about 24 hours to about 120 hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours, about 36 hours to about 72 hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours, about 36 hours to about 108 hours, about 36 hours to about 120 hours, about 48 hours to about 60 hours, about 48 hours to about 72 hours, about 48 hours to about 84 hours, about 48 hours to about 96 hours, about 36 hours to about
the immunoconjugate has a serum halflife of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours. In certain embodiments, the immunoconjugate has a serum half-life of at least about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 108 hours. In certain embodiments, the immunoconjugate has a serum half-life of at most about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, or about 120 hours.
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 immunoconjugate has a serum half-life of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In certain embodiments, the immunoconjugate has a serum half-life of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, or about 9 days. In certain embodiments, the immunoconjugate has a serum half-life of at most about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.
the heavy chain constant region has a molecular weight of about 10 kDa to about 25 kDa. In certain embodiments, 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.
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 immunoconjugate of the present invention comprises a linker or hinge region, which is a polypeptide linking an antigen binding region to a heavy chain constant region or a variant constant region in the instant invention.
a linker or hinge region which is a polypeptide linking an antigen binding region to a heavy chain constant region or a variant constant region in the instant invention.
Naturally occurring and synthetic hinge regions linking immunoglobulin components are well known in the art and available for use in the present invention. For example, see US 8,067,548 and references therein.
the hinge regions of the immunoconjugate are the same. In one embodiment, the hinge regions of the immunoconjugate are different.
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 -dep endent cellular cytotoxicity.” J Rheumatol. 2007 Nov;34(l l):2204-10.
Appropriate hinges may also include a non-IgG based polypeptide linker.
the linker amino acid sequence may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another, and so that they retain the desired activity.
the linker is from about 1 to 50 amino acids in length or about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used.
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 sizes of the immunoconjugates and/or the heavy chain constant region variants described herein allow for an increased safety profile or therapeutic index of the immunoconjugates included herein.
a safety profile may be reflected in the reduction of accumulation of radiation in radio sensitive major tissues such as kidney and bone marrow and/or an increase in radiation accumulation in target tissues (i.e., a tumor or cancerous tissue) or more radio tolerant organs such as the liver.
the immunoconjugates of this disclosure result in a total radiation exposure per treatment as measured in Gray (Gy).
the bone marrow is exposed to 4 Gy or less per treatment.
the bone marrow is exposed to 3 Gy or less per treatment.
the bone marrow is exposed to 2 Gy or less per treatment.
the bone marrow is exposed to 1.5 Gy or less per treatment.
the bone marrow is exposed to 1.0 Gy or less per treatment.
the bone marrow is exposed to 0.5 Gy or less per treatment.
a chelating agent of the immunoconjugate is covalently linked to an antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof.
a chelating agent is covalently linked to the antigen binding region, the heavy chain constant region, the immunoglobulin Fc region, or any combination thereof directly (e.g., without the use of a spacer, stretcher or linker).
the chelating agent is covalently linked to the antigen binding arm through a linker that is covalently linked to the chelating agent and covalently linked to the antigen binding arm.
the linker is hydrophilic (e.g., a PEG chain).
the immunoconjugate comprises more than one chelating agent, which are the same or different.
an immunoconjugate having more than one chelating agent has more than one chelating agent attached to the same antigen binding arm.
an immunoconjugate having more than one chelating agent and less than eleven chelating agents has more than two chelating agents, more than three chelating agents, more than four chelating agents, more than five chelating agents, more than six chelating agents, more than seven chelating agents, more than eight chelating agents, or more than nine chelating agents.
the chelating agents are the same.
each antigen binding arm is linked directly or indirectly to more than one chelating agent.
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 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 chelator is conjugated at a predefined ratio of polypeptide (i.e., antigen binding region and/or the immunoglobulin heavy chain constant) to chelator.
the radioisotope chelating agent is coupled to the antigen binding region and/or the immunoglobulin heavy chain constant region at a ratio of 1 :1 to 8: 1.
the radioisotope chelating agent is coupled to the antigen binding region and/or the immunoglobulin heavy chain constant region at a ratio of 1 : 1 to 6: 1.
the radioisotope chelating agent is coupled to the antigen binding region and/or the immunoglobulin heavy chain constant region at a ratio of 2: 1 to 6: 1.
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 examples include DOTA, DTP A, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN-Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn- CHX’A”-DTPA, p-SCN-Bn-TCMC, macropa-NCS, crown, p-SCN-Ph-Et-Py4Pa, 3,2-HOPO, and TCMC.
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.
the chelating agent of an immunoconjugate or radioimmunoconjugate of the invention is selected from the group comprising bifunctional chelator, DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN- Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A”-DTPA, p-SCN-Bn-TCMC, macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56: 1 (2017)), crown (Yang H, et al. Chem. Eur. J.
the chelating agent of an immunoconjugate or radioimmunoconjugate of the invention is selected from the group consisting of bifunctional chelator, DOTA, DO3A-NHS, DOTAGA-NHS, DOTAGA-anhydride DOTAGA-TFP, p-SCN- Bn-DOTA, p-SCN-Bn-DTPA, p-SCN-Bn-CHX-A”-DTPA, p-SCN-Bn-TCMC, macropa-NCS (Thiele NA, et al. Angew. Chem. Int. Ed. 56: 1 (2017)), crown (Yang H, et al. Chem. Eur. J.
the chelator is a chelator suitable for alpha emitter chelation.
Some chelators suitable for alpha emitters are described in Yang et al, “Harnessing a-Emitting Radionuclides for Therapy: Radiolabeling Method Review.” J Nucl Med. 2022 Jan;63(l):5-13.
the, chelator suitable for alpha emitter chelation is selected from the list consisting of: DOTA 1,4,7, 10-tetraazacyclododecane-l, 4,7, 10-tetraacetic acid; DO3A l,4,7-Tris(carboxymethyl)-l,4,7,10-tetraazacyclododecane; DOTAGA a-(2- Carboxyethyi)-!
DOTAGA anhydride (2,2',2"-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-l,4,7, 10- tetraazacyclododecane- 1 ,4,7-triyl)triacetic acid; Py4Pa 6,6',6",6"'-(((pyridine-2,6-diylbis(methylene))bis(azanetriyl))- tetrakis(methylene))tetrapicolinic acid; Py4Pa-NCS is 6,6'-((((4-isothiocyanatopyridine-2,6- diyl)bis(methylene))bis((carboxymethyl)azanediyl))-bis(methylene))dipicolinic acid; Crown
the chelator is a chelator suitable for an beta- or gammaemitter chelation.
the, chelator suitable for an beta- or gamma-emitter chelation is selected from the list consisting of: DOTMA (lR,4R,7R,10R)-a, a', a", a'"- tetramethyl-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid; DOTAM (1,4,7,10- tetrakis(carbamoylmethyl)- 1 ,4,7, 10-tetraazacyclododecane); DOTP A 1 ,4,7, 10-tetraazacyclo- dodecane-l,4,7,10-tetrapropionic acid; DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)
R 1 comprises a chelating moiety selected from the list consisting of: DOTA, DO3A, DO3Apic, DOTAGA, DOTAGA anhydride, Py4Pa, Py4Pa-NCS, Crown, Macropa, Macropa-NCS, HEHA, CHXoctapa, Bispa, and Noneunpa; or a radionuclide complex thereof.
R 1 comprises a chelating moiety selected from the list consisting of: DOTMA, DOTPA, DO3Apic, DO3AM-acetic acid, DOTP, DOTMP, DOTA- 4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H40ctapa, H2Dedpa, DO2P, EDTA, DTPA-BMA, 3,2,3-LI(HOPO), 3,2-HOPO, Neunpa, Neunpa-NCS, Octapa, PyPa, Porphyrin, and Deferoxamine; or a radionuclide complex thereof.
DOTMA chelating moiety selected from the list consisting of: DOTMA, DOTPA, DO3Apic, DO3AM-acetic acid, DOTP, DOTMP, DOTA- 4AMP, CB-TE2A, NOTA, NOTP, TETPA, TETA, PEPA, H40ctapa, H2Dedpa, DO2P, ED
R 1 is a chelating moiety selected from the list consisting of:
DO3A 1.4.7.10-tetraazacyclododecane-l,4,7-triacetic acid
TTHA tritetrakis(carboxymethyl)-3, 6,9, 12-tetraazatetradecanedioic acid
R 1 comprises DOTA and is attached to the tumor targeting
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 in the immunoconjugates described herein is an Auger electron-emitting radionuclide.
the radionuclide is an a-emitting radionuclide.
the radionuclide is a P-emitting radionuclide.
the radionuclide is a y-emitting radionuclide.
the type of radionuclide used in a non-peptide targeted therapeutic compound can be tailored to the specific type of cancer, the type of targeting moiety (e.g., non-peptide ligand), etc.
Radionuclides that undergo a-decay emit a-particles (helium ions with a +2 charge) from their nuclei.
the daughter nuclide has 2 protons less and 2 neutrons less than the parent nuclide. This means that in a-decay, the proton number is reduced by 2 while the nucleon number is reduced by 4.
Radionuclides that undergo P-decay emit P-particles (electrons) from their nuclei. During P- decay, one of the neutrons changes into a proton and an electron. The proton remains in the nucleus while the electron is emitted as a P-particle.
Auger electrons are very low energy electrons that are emitted by radionuclides that decay by electron capture (EC) (e.g., m In , gallium-67 ( 67 Ga) , technetium-99m ( 99m Tc) , platinum- 195m ( 195m Pt) , iodine-125 ( 125 I), and iodine-123 ( 123 I).
EC electron capture
P-Particles are electrons emitted from the nucleus. They typically have a longer range in tissue (of the order of 1-5 mm) and are the most frequently used.
a-Particles are helium nuclei (two protons and two neutrons) that are emitted from the nucleus of a radioactive atom. Depending on their emission energy, they can travel 50-100 pm in tissue. They are positively charged and are orders of magnitude larger than electrons. The amount of energy deposited per path length travelled (designated ‘linear energy transfer’) of a-particles is approximately 400 times greater than that of electrons. This leads to substantially more damage along their path than that caused by electrons. An a-particle track leads to a preponderance of complex and largely irreparable DNA double-strand breaks. The absorbed dose required to achieve cytotoxicity relates to the number of a-particles traversing the cell nucleus.
cytotoxicity may be achieved with a range of 1 to 20 a-particle traversals of the cell nucleus.
the a-particle emitters typically used include 212 Bi , 212 Pb, 213 Bi , 225 Ac, 223 Ra, and 229 Th.
the radionuclide is a diagnostic or therapeutic radionuclide.
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, 9" 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, rhenium- 186 ( 186 Re), rhenium- 188 ( 188 Re), 64 Cu, 67 Cu, 153 Sm , 89 Sr , gold-198 ( 198 Au), erbium-169 ( 169 Er), dysprosium- 165 ( 165 Dy), " m Tc, 89 Zr, or manganese-52 ( 52 Mn).
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 ( 60 Co), palldium- 103 ( 103 Pd), cesium-137 ( 137 Cs), ytterbium-169 ( 169 Yb), iridium-192 ( 192 Ir), 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.
the length of the linker is a range with a lower limit selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and an upper limit selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, and 30. In some embodiments, the length of the linker is a range with a lower limit selected from the group consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and an upper limit selected from the group consisting of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, and 50.
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.
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
a linker comprises one or more amino acid residues. In some embodiments, the linker comprises 1 to 3, 1 to 5, 1 to 10, 5 to 10, or 5 to 20 amino acid residues. In some embodiments, one or more amino acids of the linker are unnatural amino acids.
a linker can comprise flexible and/or rigid regions.
Exemplary flexible linker regions include those comprising Gly and Ser residues (“GS” linker), glycine residues, alkylene chain, PEG chain, etc.
Exemplary rigid linker regions include those comprising alpha helix-forming sequences, proline-rich sequences, and regions rich in double and/or triple bonds.
the linker comprises a peptidyl linker.
the peptidyl linker may be between 3-20 amino acids long, such as repeats of a single amino acid residue (e.g. polyglycine) or combinations of amino acid residues to give a peptide linker which imparts favorable pharmacokinetics.
the linker comprises a peptide linkage.
the peptide linkage comprises L-amino acids and/or D-amino acids.
D-amino acids are preferred in order to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases.
Cellular uptake of oligo-D-arginine sequences is known to be as good as or better than that of oligo-L-arginines.
a linker is cleavable.
a linker is designed for cleavage in the presence of particular conditions or in a particular environment, such conditions or environments near such targeted cells, tissues, or regions.
Cleavable linkers rely on the inherent properties of a cell’s cytoplasmic compartments for selective release of the cytotoxic drug.
Such linkers mainly include chemically cleavable linkers that respond to low pH (acid-labile linkers) or reducing environment (disulfide linkers), and enzymatically cleavable linkers that are susceptible to the action of certain lysosomal enzymes (peptide linkers or P-glucuronide linkers).
a linker is cleavable under physiological conditions. In some embodiments, a linker is cleavable under intracellular conditions. In some embodiments, the linker is chemically cleavable. In some embodiments, the linker is enzymatically cleavable (e.g., protease-sensitive, peptidase-sensitive) linker. In some embodiments, the linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. For example, the pH-sensitive linker can be hydrolyzable under acidic conditions.
the linker is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
the linker is cleaved by a glycosidase, e.g., glucuronidase.
P-glucuronide linkers can be readily cleaved by the abundant lysosomal enzyme P -glucuronidase, facilitating facile and selective release of the active drug.
the linker is not cleavable.
a linker component comprises an amino acid unit.
the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes.
Exemplary amino acid units include, but are not limited to, a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); or N-methyl-valine-citrulline (Me-val-cit). These dipeptide linkers show good stability in serum, yet can be recognized and rapidly hydrolyzed by certain lysosomal proteases, such as cathepsin B, following internalization.
Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
amino acid unit may comprise amino acid residues that occur naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
Amino acid units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
the linker is cleaved by a protease, a matrix metalloproteinase, a serine protease, or a combination thereof. In some embodiments, the linker is cleaved by a reducing agent. In some embodiments, the linker is cleaved by an oxidizing agent or oxidative stress.
the linker is cleaved by an MMP.
MMPs matrix metalloproteinases
a linker includes the amino-acid sequences PLG-C(Me)-AG, PLGLAG which are cleaved by the metalloproteinase enzymes MMP -2, MMP-9, or MMP-7 (MMPs involved in cancer and inflammation).
the linker comprises one or more of di-sulfide bonds.
the linker may be a non-peptidyl linker. Typical examples of these types of linker would be those based on straight or branched chain hydrocarbons or polyethylene glycols of varying lengths. These may incorporate other groups to effect solubility, rigidity, isoelectric point, such as aromatic or non-aromatic rings, halogens, ketones, aldehydes, esters, sulfonyls, phosphate groups, and so on.
Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995, 2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94: 5815); and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55: 5867).
the immunoconjugate comprises a linker, such as, e.g., a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to an antibody (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12: 2213-5; Sun et al (2003) Bioorganic & Medicinal Chemistry 11 : 1761-8).
Dendritic linkers can increase the molar ratio of drug to antibody, i.e., loading, which is related to the potency of the ADC.
a cysteine-engineered antibody bears only one reactive cysteine thiol group, a multitude of drug moieties may be attached through a dendritic linker.
each R a is independently selected from hydrogen, and Ci-C4alkyl.
X 1 is -O-.
X 1 is absent, -S-.
X 1 is -NR a -.
X 1 is -CH 2 -, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, - CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2-X 2 -, -CH2CH2-X 2 -, -CH2CH2CH2-X 2 -, -CH2CH2CH2CH2-X 2 -, -CH2CH2CH2CH2CH2-X 2 -, -CH2CH2CH2CH2CH2-X 2 -, -CH2CH2CH2CH2CH2-X 2 -, -CH2CH2CH2CH2CH2CH2-X 2 -.
X 1 is -CH2CH2- or -CH2CH2-X 2 -;
X 4 is -NH-, -N(CH 3 )-, or -N(CH2CH 3 )-.
L 1 is absent, 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;
L is -L 1 -, -L 2 -, -L 3 -, -L 4 -, -L 5 -, -L l -L 2 -L 3 -L 4 -L 5 -, or combinations thereof;
L is -L 1 -. In some embodiments, L is -L 2 -. In some embodiments, L is -L 3 -. In some embodiments, L is -L 4 -. In some embodiments, L is -L 5 -. In some embodiments, L is -L 1 -!?-. In some embodiments, L is -L 1 -! -. In some embodiments, L is -L 1 - L 4 -. In some embodiments, L is -L 1 -! -. In some embodiments, L is -L 2 -L 3 -. In some embodiments, L is -L 2 -L 4 -.
L is -L 2 -L 5 -. In some embodiments, L is -L 3 - L 4 -. In some embodiments, L is -L 3 -L 5 -. In some embodiments, L is -L 4 -L 5 -. In some embodiments, L is -L 1 -!?-! -. In some embodiments, L is -L'-L 2 -! -. In some embodiments, L is -L 1 -!?-!?-. In some embodiments, L is -L 1 -!/-!?-. In some embodiments, L is -LkL 2 -L 3 -L 4 -.
L is -L 2 -L 3 -L 4 -L 5 -. In some embodiments, L is -LkL 2 -L 4 -L 5 -. In some embodiments, L is -LkL 2 -L 3 -L 4 -L 5 -.
L 1 is unsubstituted or substituted Ci-C2oalkylene, unsubstituted or substituted Ci-C2oheteroalkylene, C4-C2opolyethylene 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.
the immunoconjugate of Formula (II) has the structure of Formula (Ila), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (III) has the structure of Formula (Illa), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (IV) has the structure of Formula (IVa), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (V) has the structure of Formula (Va), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (VI) has the structure of Formula (Via), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (VII) has the structure of Formula (Vila), or a pharmaceutically acceptable salt thereof:
the immunoconjugate of Formula (VIII) has the structure of Formula (Villa), or a pharmaceutically acceptable salt thereof:
R 1 is a chelating moiety selected from the group consisting of:
R 1 is a chelating moiety selected from the group consisting of: or a radionuclide complex thereof.
R 1 is a chelating moiety selected from the group consisting of: radionuclide complex thereof. In some embodiments, R 1 is a chelating moiety selected from the group consisting of: radionuclide complex thereof.
X 1 is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, - CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 -X 2 -, -CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 CH 2 -X2 4 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -X 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -CH 2 -X 2 -.
L is -L 1 -, -L 2 -, -L 3 -, -L 4 -, -L 5 -, -L l -L 2 -L'-L 4 -L 5 -, or combinations thereof;
L 1 is absent, unsubstituted or substituted Ci-Cioalkylene, unsubstituted or substituted Ci- Cioheteroalkylene, unsubstituted or substituted C 2 -C 2 oalkenylene, unsubstituted or substituted C 2 - C 2 oalkynylene, C4-C 2 opolyethylene glycol, -(X 3 CH 2 CH 2 ) 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;
L 2 is absent.
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.
Exemplary compounds for use in preparing immunoconjugates described herein include the compounds depicted in is 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 13 4Ce) 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 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 invention provides a radioimmunoconjugate, comprising an immunoconjugate of the invention and an a-emitting radioisotope.
the a- emitting radioisotope of the radioimmunoconjugate is selected from the group comprising: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi.
the a-emitting radioisotope of the radioimmunoconjugate is selected from the group consisting of: 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi.
the a-emitting radioisotope of the radioimmunoconjugate is 225 Ac. In one embodiment, the a-emitting radioisotope of the radioimmunoconjugate is 223 Ra. In one embodiment, the a-emitting radioisotope of the radioimmunoconjugate is 224 Ra. In one embodiment, the a-emitting radioisotope of the radioimmunoconjugate is 227 Th. In one embodiment, the a-emitting radioisotope of the radioimmunoconjugate is 212 Pb. In one embodiment, the a-emitting radioisotope of the radioimmunoconjugate is 212 Bi. In one embodiment, the a-emitting radioisotope of the radioimmunoconjugate is 213 Bi.
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 radioisotope is an alpha emitter, such as, e.g., 225 Ac, 223 Ra, 224 Ra, 227 Th, 212 Pb, 212 Bi, and 213 Bi.
the radioisotope is a beta particle emitter, such as, e.g., 177 Lu, 90 Y, 67 Cu, 153 Sm.
the radioisotope is both an alpha particle emitter and a beta and/or gamma particle emitter.
the radioisotope is both a beta particle emitter and a gamma particle and/or photon emitter.
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, and 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.
Non-limiting examples of cytotoxic agents include aziridines, cisplatins, tetrazines, procarbazine, hexamethylmelamine, vinca alkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines, actinomycin, bleomycin, plicamycin, mitomycin, daunorubicin, epirubicin, idarubicin, dolastatins, maytansines, docetaxel, adriamycin, calicheamicin, auristatins, pyrrolobenzodiazepine, carboplatin, 5 -fluorouracil (5-FU), capecitabine, mitomycin C, paclitaxel, l,3-Bis(2-chloroethyl)-l-nitrosourea (BCNU), r
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 213 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.
immunoconjugates [00320] Further embodiments of the immunoconjugates, antigen binding regions, and heavy chain variable regions are described below:
the immunoconjugate comprises a dimerization domain or motif.
the dimerization domain or motif is in a variant constant region, linker or hinge region.
the skilled worker can engineer multimeric immunoconjugates of the present invention using approaches and methods known in the art. For example, engineered cysteine residues can form covalent bonds thereby stabilizing multimeric structures that spontaneously assemble (see e.g., Glockshuber R et al., Biochemistry 29: 1362-7 (1990)).
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.
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
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.
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
substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
a parent antibody e.g., a humanized or human antibody
the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
An illustrative substitutional variant is an affinity-matured antibody, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).
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., in
affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized.
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).
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
a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
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)).
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).
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 cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; WO 2004/056312, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see e.g., Yamane- Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., BiotechnoL Bioeng., 94(4):680- 688 (2006); W02003/085107)).
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 immunoconjugates 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- azidophenyl)dithio]propioimidate.
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.
PEG derivatized immunoconjugates of the invention may comprise linkers comprising one or more -CH2CH2O- and can be used to alter biodistribution and pharmacokinetics of the immunoconjugate.
PEGs can be prepared in a polymeric form or as discrete oligomers. Bifunctionalized versions of these polymers can link immunoconjugates with a chelating agent and/or provide additional size and/or solubility to the overall molecule.
the PEG derivatized immunoconjugates exhibit reduced immunogenicity compared to their un- derivatized parental molecules.
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 host cell comprising a nucleic acid and/or vector as provided herein.
the host cell of the present invention is isolated or purified.
the host cell of the present invention is in a cell culture medium.
the nucleic acids, expression vectors, and host cells of the invention may be used to produce a composition comprising one or more of the immunoconjugates of the invention.
the host cell is eukaryotic.
the host cell is mammalian.
the host cell is a Chinese Hamster Ovary (CHO) cell.
the host cell is prokaryotic.
the host cell is E. coli.
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.
Antigen binding domains useful as antigen binding regions herein may be identified in antibodies that are either monoclonal antibodies and/or polyclonal antibodies.
DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells (see e.g., Skerra et al., Curr. Opinion in Immunol., 5:256- 262 (1993) and Pliickthun, Immunol Revs. 130: 151-188 (1992)).
host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein
the antigen binding domains of an immunoconjugate of the present invention are isolated by screening phage libraries containing phage that display various fragments of antibody variable region (Fv, scFv, or VHH) fused to phage coat protein. Such phage libraries are screened for binding to the desired target antigen or epitope. Clones expressing Fv fragments, scFv’s, or VHH’s capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding
Ill clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.
the antibody or antibody fragments thereof are isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J Mol Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
scFv single-chain Fv
Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Naive libraries for screening can be constructed from non-immunized sources to provide high- affinity antibodies to antigens (see e.g., Griffiths et al., EMBO J, 12: 725-734 (1993)).
naive libraries constructed synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Target antigen can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries.
Techniques for screening a cDNA library are well known in the art. Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding immunoconjugate of the invention is to use PCR methodology (Sambrook et al., supra, Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).
DNA encoding an immunoconjugate of the invention may be obtained from a cDNA library prepared from tissue believed to possess the immunoconjugate of the invention mRNA and to express it at a detectable level. Accordingly, human immunoconjugate of the invention DNA can be conveniently obtained from a cDNA library prepared from human tissue.
the immunoconjugate of the invention-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
desired polynucleotide sequences encoding antibodies may be isolated and sequenced from antibody producing cells such as hybridoma cells.
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein. Any of the antibody CDRs or heavy chain variable fragments of the present invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of an antibody clone using the variable domain and/or CDRs sequences from a phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., 1991, supra.
Fc constant region
the description below relates primarily to production of the antibody constructs of the invention by culturing cells transformed or transfected with a vector-containing immunoconjugate of the invention-encoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare the antibody constructs of the invention. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J, Am. Chem. Soc., 85: 2149-54 (1963)).
solid-phase techniques e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J, Am. Chem. Soc., 85: 2149-54 (1963)).
These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody construct.
Suitable host cells for the expression of glycosylated immunoconjugate are also derived from multicellular organisms (e.g., invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which suitable for use in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (see e.g., US 5,959,177; US 6,040,498; US 6,420,548; US 7,125,978; and US 6,417,429.
Vertebrate cells may also be used as hosts.
mammalian cell lines that are adapted to grow in suspension may be useful.
useful mammalian host cell lines are monkey kidney CV 1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J Gen Viral. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
Suitable prokaryotes include but are not limited to archaebacteria and eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
Enterobacteriaceae such as E. coli.
Various A. coli strains are publicly available, such as K12 strain MM294 (ATCC 31,446); X1776 (ATCC 31,537); W3110 (ATCC 27,325) and K5 772 (ATCC 53,635).
Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coll strain W3110 is one advantageous host or parent host because it is a common host strain for recombinant DNA product fermentations.
the host cell secretes minimal amounts of proteolytic enzymes.
strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coll W3110 strain 1A2, which has the complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E.
coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 pho A El 5 (argF-lac)169 degP ompT kanr;
E. coli W3110 strain 37D6 which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr;
E. coli W3 110 strain 40B4 which is strain 37D6 with a non-kanamycin resistant degP deletion mutation;
coli W3110 strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635) and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.
Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) andE. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative rather than limiting.
E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. Full length antibodies have greater half-life in circulation. Production inE. coli is faster and more cost efficient.
TIR translation initiation region
the immunoconjugate is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.
lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacterio!., 154(2):737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus,' yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J.
Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methyl otrophs, 269 (1982).
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 (fruit fly), 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.
vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc Natl Acad Set USA 7 A2 6 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.
SV40 monkey kidney CV1 line transformed by SV40
human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)
baby hamster kidney cells BHK, ATCC C
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
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 signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990.
mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory 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 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.
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. (1999) J Bio Chem 274: 19601-5; U.S. Patent No. 6,083,715; U.S. Patent No.
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.
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 Amicon 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 mixture comprising the immunoconjugate of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and generally at low salt concentrations (e.g., from about 0-0.25M salt).
an immunoconjugate of the invention according to any of the above embodiments or described herein is conjugated to a heterologous moiety or agent, such as, e.g., as described below and including any additional exogenous material as described herein.
the invention provides immunoconjugates comprising an antibody construct of the present invention conjugated to one or more therapeutic agents or radioactive isotopes.
an immunoconjugate comprises an antibody construct as described herein conjugated to a radioactive atom to form a radioconjugate.
a variety of radioactive isotopes are available for the production of radioconjugates of the invention.
Conjugates of an immunoconjugate or antibody construct may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3 -(2 -pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate H ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (pdiazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active protein coupling agents
a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
Carbon-14- labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an illustrative chelating agent for conjugation of radionucleotide to the antibody (see e.g., WO 1994/11026).
the linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell.
an acid-labile linker peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker may be used (see e.g., Chari et al., Cancer Res. 52: 127-131 (1992); US 5,208,020).
the immunoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo- EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., obtainable from Pierce Biotechnology, Inc., Rockford, IL., U.S.).
cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SM
Bifunctional chelators such as, e.g., DOTA, DTP A, and related analogs are suitable for coordinating metal ions like a and P-emitting radionuclides.
these chelating molecules can be linked to the targeting molecule by forming a new amide bond between an amine on the antibody construct (e.g., a functional group of a lysine residue) and a carboxylate on the DOTA/DTPA.
characterization and purification of the linker addition can be part of the overall synthesis of an antibody platform or immunoconjugate for radioisotope conjugation.
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.
An immunoconjugate or radioimmunoconjugate is formulated in any suitable form for delivery to a target cell/tissue.
Pharmaceutical formulations of an immunoconjugate of the present invention are prepared by mixing such immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, diluents, and/or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
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
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,
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 10.0 p
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
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,
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/
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.
the effective amount is from 1 mCi to 100 mCi per meter squared of body surface area. In certain embodiments, the effective amount is about 1 per meter squared to about 100 per meter squared.
the effective amount is about 1 per meter squared to about 5 per meter squared, about 1 per meter squared to about 10 per meter squared, about 1 per meter squared to about 15 per meter squared, about 1 per meter squared to about 20 per meter squared, about 1 per meter squared to about 25 per meter squared, about 1 per meter squared to about 75 per meter squared, about 1 per meter squared to about 100 per meter squared, about 5 per meter squared to about 10 per meter squared, about 5 per meter squared to about 15 per meter squared, about 5 per meter squared to about 20 per meter squared, about 5 per meter squared to about 25 per meter squared, about 5 per meter squared to about 75 per meter squared, about 5 per meter squared to about 100 per meter squared, about 10 per meter squared to about 15 per meter squared, about 10 per meter squared to about 20 per meter squared, about 10 per meter squared to about 100
the effective amount is about 1 per meter squared, about 5 per meter squared, about 10 per meter squared, about 15 per meter squared, about 20 per meter squared, about 25 per meter squared, about 75 per meter squared, or about 100 per meter squared. In certain embodiments, the effective amount is at least about 1 per meter squared, about 5 per meter squared, about 10 per meter squared, about 15 per meter squared, about 20 per meter squared, about 25 per meter squared, or about 75 per meter squared.
the effective amount is at most about 5 per meter squared, about 10 per meter squared, about 15 per meter squared, about 20 per meter squared, about 25 per meter squared, about 75 per meter squared, or about 100 per meter squared.
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.
the pharmaceutical compositions may be provided as a single dose composition tailored to a specific patient, i.e., as a patient specific therapeutic composition, wherein the amount of labeled and unlabeled immunoconjugate (labeled immunoconjugate, for clarity, being the same as radioimmunoconjugate herein) in the composition may depend on at least a patient weight, height, body surface area, age, gender, and/or disease state or health status.
a total volume of the patient specific therapeutic composition may be provided in a vial that is configured to be wholly administered to the patient in one treatment session, such that little to no composition remains in the vial after administration.
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.
a biological sample is a biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, saliva, sputum, tears, perspiration, mucus, cerebrospinal fluid, urine and other constituents of the body that may contain the target antigen of interest.
the sample is a body sample from any animal. In some embodiments, 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 therapeutic antibody or antibodies that binds the same target antigen.
the sample is from a mammal. In some embodiments, the sample is from a human subject, e.g., when measuring antigen expression in a clinical sample. In some embodiments, the biological sample is from clinical patients or a patient treated with a therapy/therapeutic (e.g., an antibody therapy targeting the same target antigen). In some embodiments, the biological sample is serum or plasma. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is biopsy material. In some embodiments, the biological sample is biopsy material from a clinical patient. In some embodiments, the biological sample is serum from a clinical patient. In some embodiments, the biological sample is primary cell culture material. In some embodiments, the biological sample is primary cell culture material from a clinical patient.
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 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 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 amino acid residue
amino acid sequence amino acid sequence
amino acid sequence amino acid sequence
polypeptide sequence 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)).
the term “radioisotope” includes, but is not limited to, an alpha emitting isotope (interchangeably, a-emitting isotope), beta-emitting isotope (interchangeably, P- emitting isotope), and/or gamma-emitting isotope (interchangeably, y-emitting isotope), such as, e.g., any one of 86 Y, 90 Y, 177 Lu, 186 Re, 188 Re, 89 Sr, 153 Sm, 225 Ac, 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, and 103 Pd.
an alpha emitting isotope interchangeably, a-emitting isotope
beta-emitting isotope interchangeably, P- emitting isotope
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.
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.
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)).
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.
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.
“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
the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
Cytotoxic agents include, but are not limited to, radioactive isotopes; chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various cytotoxic agents 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.
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.
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.
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.
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.
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.
heterocycle refers to heteroaromatic rings (also known as heteroaryls) and heterocycloalkyl rings containing one to four heteroatoms in the ring(s), where each heteroatom in the ring(s) is selected from O, S and N, wherein each heterocyclic group has from 3 to 10 atoms in its ring system, and with the proviso that any ring does not contain two adjacent O or S atoms.
Non-aromatic heterocyclic groups also known as heterocycloalkyls
aromatic heterocyclic groups include rings having 5 to 10 atoms in its ring system.
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.
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.
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.
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.
Flash chromatography was performed using a Biotage IsoleraOne instrument with an appropriately sized normal phase silica gel cartridge with fraction collection at 254 nm. Final compounds were purified by an Agilent prep-HPLC (Agilent, Hanover, CT) using an acetonitrile/water (+ 0.1% TFA or 0.1% FA) gradient. NMR spectra were taken with Bruker 400 or 600 MHz NMR instruments and processed with MestReNova v.14. Where available, detailed NMR Data was compiled with the multiplet analysis function used in manual mode.
Trifluoroacetic acid (8.91 g, 78.2 mmol), followed by 1,1,1 -tri ethoxy ethane (145.79 g, 898.7 mmol) was added to phosphenous acid (25.00 g, 391.0 mmol) at 0 °C under nitrogen.
the reaction mixture was stirred at 20 °C for 2 h and concentrated.
the residue was diluted with di chloromethane (200 mL), washed with saturated sodium bicarbonate (100 mL), dried over anhydrous sodium sulfate and concentrated to dryness.

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EP23858210.0A 2022-08-22 2023-08-21 Vhh-antikörperkonjugate Pending EP4577526A1 (de)

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PCT/US2023/072585 WO2024044549A1 (en)	2022-08-22	2023-08-21	Vhh antibody conjugates

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EP (1)	EP4577526A1 (de)
JP (1)	JP2025529865A (de)
KR (1)	KR20250099461A (de)
CN (2)	CN120051461A (de)
AU (1)	AU2023330109A1 (de)
CA (1)	CA3265394A1 (de)
IL (1)	IL319119A (de)
MX (1)	MX2025002013A (de)
TW (1)	TW202421115A (de)
WO (1)	WO2024044549A1 (de)

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WO2025145255A1 (en) *	2024-01-05	2025-07-10	Aiga Therapeutics Inc.	Lat-1 targeting amino acid derivatives and uses thereof
TW202600607A (zh) *	2024-02-21	2026-01-01	美商雅博得樂醫療公司	Ｄｄｌ３結合免疫接合物及其用途
WO2025255396A1 (en) *	2024-06-05	2025-12-11	Ariceum Therapeutics Gmbh	Radiopharmaceutical composition and methods
WO2026024644A1 (en)	2024-07-23	2026-01-29	Abdera Therapeutics Inc.	5t4 binding polypeptides and uses thereof

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WO2009138207A2 (de) *	2008-04-28	2009-11-19	Kuehn Andreas	Verfahren und anordnung zur steuerung von messsystemen, sowie ein entsprechendes computerprogramm und ein entsprechendes computerlesbares speichermedium
US20230181770A1 (en) *	2018-03-02	2023-06-15	Neurosn, Inc.	COMPOSITIONS AND METHODS OF TARGETING AND IMAGING AGED MICROGLIA WITH Aß PEPTIDE AMINO ACID RESIDUES FOR V-DOMAIN BINDING OF RAGE
EP3856261A1 (de) *	2018-09-28	2021-08-04	Universität Heidelberg	Markierte inhibitoren des prostataspezifischen membranantigens (psma), deren verwendung als kontrastmittel und pharmazeutika zur behandlung von psma-exprimierenden krebsen
MX2021008230A (es) *	2019-01-07	2021-08-11	Astellas Pharma Inc	Conjugado que comprende ligando y fragmento fab de anticuerpo ceacam5.
EP3958874A4 (de) *	2019-04-23	2023-09-06	The Trustees of The University of Pennsylvania	Dota-bindender chimärer antigenrezeptor für die zelluläre therapie
BR112022013678A2 (pt) *	2020-01-10	2022-11-16	Fusion Pharmaceuticals Inc	Quelatos macrocíclicos e seus usos
EP4294845A4 (de) *	2021-02-22	2026-01-07	Abdera Therapeutics Inc	Immunkonjugate für gezielte radioisotoptherapie
US20240299596A1 (en) *	2021-06-17	2024-09-12	Mayo Foundation For Medical Education And Research	Methods and materials for combining biologics with multiple chelators

2023
- 2023-08-21 EP EP23858210.0A patent/EP4577526A1/de active Pending
- 2023-08-21 IL IL319119A patent/IL319119A/en unknown
- 2023-08-21 CN CN202380073140.9A patent/CN120051461A/zh active Pending
- 2023-08-21 AU AU2023330109A patent/AU2023330109A1/en active Pending
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- 2023-08-21 WO PCT/US2023/072585 patent/WO2024044549A1/en not_active Ceased
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IL319119A (en)	2025-04-01
JP2025529865A (ja)	2025-09-09
MX2025002013A (es)	2025-06-02
TW202421115A (zh)	2024-06-01
CN120051461A (zh)	2025-05-27
CN121102519A (zh)	2025-12-12
KR20250099461A (ko)	2025-07-01
CA3265394A1 (en)	2024-02-29
WO2024044549A1 (en)	2024-02-29

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