Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/07—Tetrapeptides
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
  • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge

Definitions

• the present disclosure provides an antibody drug conjugate (ADC) having an IgG antibody that binds to a CD38 target conjugated at a Cys site in the hinge region of an IgG antibody.
• ADC antibody drug conjugate
• the present disclosure further provides a method for treating a multiple myeloma comprising providing an effective amount of a CD38 ADC.
• CDS 8 is a 45 kD type II transmembrane glycoprotein with a long C-ierminal extracellular domain and a short N-terminal cytoplasmic domain.
• the CD38 protein is a bifunctional ectoenzyme that can catalyze the conversion of NAD + into cyclic ADP-ribose (cADPR) and also hydrolyze cADPR into ADP-ribose.
• cADPR cyclic ADP-ribose
• CD38 appears on CD34 + committed stem cells and lineage -committed progenitors of lymphoid, erythroid and myeloid cells.
• CD38 expression persists mostly in the lymphoid lineage with varying expression levels at different stages of T and B cell development.
• CD38 is upregula ed in many hematopoeidc malignancies and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).
• NHL non-Hodgkin's lymphoma
• BL Burkitt's lymphoma
• MM multiple myeloma
• B-CLL B and T acute lymphocytic leukemia
• TCL T cell lymphoma
• AML acute myeloid leukemia
• HCL hairy cell
• CD38 has been reported to be involved in Ca 2+ mobilization (Morra et a!., 1998, FASEB J., 12: 581-592; Zilber et al., 2000, Proc. Natl. Acad. Sci USA, 97: 2840-2845) and in the signal transduction through tyrosine phosphorylation of numerous signaling molecules, including phospholipase C- ⁇ , ZAP-70, syk, and c-cbl, in lymphoid and myeloid cells or cell lines (Funaro et al., 1993, Eur. J.
• CD38 was proposed to be an important signaling molecule in the maturation and activation of lymphoid and myeloid cells during their normal development.
• CDS 8 Evidence for the function of CDS 8 comes from CD387 knockout mice, which have a defect in their innate immunity and a reduced T-cell depeiideiit humoral response due to a defect in dendritic cell migration (Partida-Sanchez et al., 2004, Immunity ' , 20: 279-291; Partida-Sanchez et al., 2001, Nat. Med., 7: 1209-1216).
• mice are identical to that in humans since the CD38 expression pattern during hematopoiesis differs greatly between human and mouse: a) unlike immature progenitor stem cells in humans, similar progenitor stem cells in mice express a high level of CD38 (Randall et al., 1996, Blood, 87:4057-4067; Dagher et al., 1998, Biol. Blood Marrow Transplant, 4:69-74), b) while during the human B cell development, high levels of CD38 expression are found in germinal center B cells and plasma cells (Uckun. 1990, Blood, 76: 1908-1923; Kumagai et al., 1995, J. Exp. Med., 181: 1101-1110), in the mouse, the CD38 expression levels in the corresponding cells are low (Oliver et al., 1997, J.
• a chimeric O T10 antibody with mouse Fab and human IgGl Fc mediates antibody-dependent cell-mediated cytotoxicity (ADCC) very efficiently against lymphoma cells in the presence of peripheral blood mononuclear effector cells from either MM patients or normal individuals (Stevenson et al., 1991, Blood, 77: 1071 -1079).
• a CD -grafted humanized version of the anti- CD38 antibody AT13/5 has been shown to have potent ADCC activity against CD38-positive cell lines.
• Some of the antibodies of the prior art have been shown to be able to trigger apoptosis in CD38 1" B cells. However, t ey can only do so in the presence of stroma cells or stroma-derived cytokines.
• An agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis of human germinal center (GC) B cells (Zupo et al. 1994, Eur. J. Immunol., 24: 1218-1222), and to induce proliferation of KG- 1 and HL-60 AML cells (Konopleva et al. 1998, /.
• T16 induced apoptosis of immature lymphoid cells and leukemic lymphoblast cells from an ALL patient (Kumagai et al. 1995, J. Exp. Med., 181: 1 101-11 10), and of leukemic myeloblast cells from AML patients (Todisco et al. 2000, Blood, 95:535-542), but T16 induced apoptosis only in the presence of stroma cells or stroma-derived cytokines (IL-7, IL-3, stem cell factor).
• IL-7 stroma-derived cytokines
• ADCs antibody drug conjugates
• the present disclosure provides and antibody drug conjugate (ADC) having an IgG antibody that binds to a CD38 target conjugated at Cys sites in the hinge region of an IgG antibody.
• ADC antibody drug conjugate
• the present disclosure further provides a method for treating multiple myeloma comprising providing an effective amount of a CD38 ADC.
• an anti-CD38 ADC composition comprising:
• a drug or toxin moiety that is a is a tubulin inhibitor or a doxorubicin analog
• a heavy chain hinge region of an IgG antibody may be mutated such that the heavy chain hinge region contains only one Cys residue.
• the drug or toxin moiety is selected from the group consisting of Dl, D2, D3, D4, D5, and combinations thereof, wherein the structures of Dl, D2, D3, D4 and D5 are:
• the conjugation linker moiety comprises a linker moiety and a conjugation moiety. In some embodiments, the conjugation linker moiety comprises one or more of the structures:
• the conjugation moiety is w h ere i n the wavy line indicates the point of attachment to the conjugation linker moiety.
• conjugation linker moiety is selected from the group consisting of:
• the conjugation moiety is ⁇ -" ⁇ - N -" ⁇ - / ⁇ wherein the wavy line indicates the point of attachment.
• an antibody drug conjugate (ADC) composition comprising an IgG antibody that binds to CD38, a linker moiety conjugated to one Cys residue in a hinge region of an IgG antibody mutated to have only one Cys residue, and a toxin moiety conjugated to the linker moiety.
• ADC antibody drug conjugate
• an anti-CD38 ADC composition comprising:
• a drug or toxin moiety that is a is a tubulin inhibitor or a doxorubicin analog
• the conjugation linker comprises a linker and a conjugation moiety which covalently binds to a single Cys residue in a hinge region of an IgG antibody, and wherein a heavy chain hinge region of an IgG antibody may be mutated such that the heavy chain hinge region contains only one Cys residue.
• the drug or toxin moiety is selected from the group consisting of Dl, D2, D3, D4, D5, and combinations thereof, wherein the structures of Dl, D2, D3, D4 and D5 are:
• the linker is selected from the group consisting of:
• the present disclosure further provides a method for treating multiple myeloma, comprising administering an effective amount of an anti-CD38 ADC composition comprising:
• a drug or toxin moiety that is a is a tubulin inhibitor or a doxorubicin analog
• a heavy chain hinge region of an IgG antibody may be mutated such that the heavy chain hinge region contains only one Cys residue.
• the drug or toxin moiety is selected from the group consisting of Dl, D2, D3, D4, D5, and combinations thereof, wherein the structures are:
• the conjugation linker moiety comprises a linker moiety and a conjugation moiety. In some embodiments, the conjugation linker moiety comprises one or more of the structures:
• the conjugation moiety is w h ere i n the wavy line indicates the point of attachment to the conjugation linker moiety.
• the linker moiety of the conjugation linker moiety is selected from the group consisting of:
• the conjugation moiety is ⁇ -" ⁇ - N -" ⁇ - / ⁇ wherein the wavy line indicates the point of attachment.
• a method for treating multiple myeloma comprising providing a therapeutically effective amount of an anti-CD38 ADC composition comprising:
• a drug or toxin moiety that is a is a tubulin inhibitor or a doxorubicin analog
• conjugation linker comprises a linker and a conjugation moiety which covalently binds to a single Cys residue in a hinge region of an IgG antibody, and wherein a heavy chain hinge region of an IgG antibody may be mutated such that the heavy chain hinge region contains only one Cys residue.
• the drug or toxin moiety is selected from the group consisting of Dl, D2, D3, D4, D5, and combinations thereof, wherein the structures are:
• the linker is selected from the group consisting of:
• the antibody moiety is a variant of the CD38A2 wild type antibody disclosed and claimed in US Serial Number 15/094,384, filed 08 April 2016, the disclosure of which is incorporated by reference herein.
• the CD38A2 wild type variable region sequence is disclosed herein as heavy chain SREQ ID NO. 1 and light chain SEQ ID NO. 2. More specifically, the variant sequence alters the second and third amino acids from the N terminus of the light chain variable region.
• the antibody moiety comprises CD38A2-SV (SV variant) having heavy chain SEQ ID NO. 1 and light chain SEQ ID NO. 3.
• Figure 1A shows an in vivo study of anti-CD38 ADCs on Burkitt lymphoma model. In the study, 10 million of Daudi cells were injected s.c. to Nu Nu mice. ADC #45 was iv injected to tumor bearing mice after the average tumor volume reached 200 mm .
• Figure IB shows body weight measure of mice treated with ADC #45 at three different indicated doses.
• Figure 2A shows an in vivo study of anti-CD38 ADCs on Burkitt lymphoma model.
• 10 million of Ramos cells were s.c injected to Nu Nu mice.
• ADC #45 was iv injected to tumor bearing mice at the dosages indicated after the average tumor volume reached 200 mm .
• Figure 2B shows body weight measure of mice treated with anti-CD38-ADC.
• Figure 3 shows an in vivo study of anti-CD38 ADCs on Burkitt lymphoma model.
• 10 million of Daudi-luc cells were iv injected to NOD-SCID mice.
• Anti-CD38 antibody (A2) and two anti-CD38 ADCs made with the same A2 antibody were iv injected to tumor bearing mice 14 days after injection of tumors
• Figure 4 shows an in vivo study of anti-CD38 ADCs on Burkitt lymphoma model.
• 10 million of Daudi-luc cells were iv injected to NOD-SCID mice.
• ADC #45 and ADC#41 were iv injected to tumor bearing mice 14 days after injection of tumors.
• Figure 5 shows an in vivo study of anti-CD38 ADCs on Burkitt lymphoma model.
• 10 million of Daudi-luc cells were iv injected to NOD-SCID mice.
• Anti-CD38 antibody and anti-CD38-ADC were iv injected to tumor bearing mice 14 days after injection of tumors. Mice images were taken once a week.
• Figure 6 shows an in vivo study of anti-CD38 ADCs on Burkitt lymphoma model.
• 10 million of Daudi-luc cells were iv injected to NOD-SCID mice.
• ADC #45 and ADC #41 were iv injected to tumor bearing mice 14 days after injection of tumors. The imagines of mice were taken once a week.
• Figure 7 shows CD38 expressing cancer cell lines, Ramos, Raji and RPMI8226, along with a CD38 negative cell line, PC-3 were plated in 96 well plate and treated with serial diluted
• ADC#45, ADC#41, and ADC#46 starting at 100 nM.
• the cells were treated for 3-5 days, depending on the nature of the conjugated payload. At the end of the treatment, the cells were stained with CelltitreGloTM luminescent kit from Promega and the signals were captured by a luminescent plate reader.
• the activity of the ADCs on tumor cell growth inhibition were expressed as the concentration required for 50% cell growth inhibition, the so called EC50 (in nM).
• Figure 8. shows the HIC-HPLC overlay of starting anti-CD38 antibody and purified ADC46 conjugate at 280 nm detection.
• Figure 9. shows the HIC-HPLC overlay of starting anti-CD38 antibody and purified ADC41 conjugate at 280 nm detection.
• Figure 10A shows an in vivo study of anti-CD38 ADC46 on Burkitt lymphoma model.
• 10 million of Daudi cells were injected s.c. to Nu Nu mice.
• ADC 46 was iv injected to tumor bearing mice after the average tumor volume reached 200 mm .
• Figure 10B shows bodyweight measure of mice treated with ADC 46 at three different indicated doses.
• the present disclosure provides antibody drug conjugates containing a novel human anti- CD38 antibody (A2) (described in United States Patent application 2016/0297888 serial number 15/094,384 filed 08 April 2016, the disclosure of which is incorporated by reference herein) with toxin moieties described herein including a tubulin inhibitor or a DNA damaging agent, such as doxorubicin analogs.
• A2 novel human anti- CD38 antibody
• toxin moieties described herein including a tubulin inhibitor or a DNA damaging agent, such as doxorubicin analogs.
• the ADC conjugates retained binding affinity and showed potent cell killing in a variety of CD38 positive cell lines and in vivo.
• the present disclosure provides an antibody drug conjugate (ADC) composition
• ADC antibody drug conjugate
• an IgG antibody that binds to CD38
• a conjugation linker moiety that binds to single Cys residue in the hinge region of an IgG antibody, wherein the hinge region may be mutated such that the heavy chain hinge region contains only one Cys residue and not two
• a toxin moiety selected from the group consisting of derivatives of anthracyclines and Dolastatins.
• the toxin moiety is a tubulin inhibitor or a doxorubicin analog.
• the antibody is an IgG antibody called human C38A2 (heavy/light SEQ ID NOs 3/4 in US patent application 2016/0297888 or SEQ ID NOs.
• conjugated toxin with linker structure is selected from the group consisting of:
• the present disclosure provides a method for treating multiple myeloma, comprising administering an effective amount of an antibody drug conjugate (ADC) composition comprising an IgG antibody that binds to CD38, a conjugation linker moiety that binds to Cys residues in the hinge region of an IgG antibody and to a toxin moiety.
• ADC antibody drug conjugate
• binds to Cys residues it is meant that the conjugation linker moiety may be covalently bound to the sulfur atoms of Cys residues in the hinge region of the IgG antibody.
• the toxin moiety is a tubulin inhibitor or a
• the antibody is an IgG antibody called human C38A2 (heavy/light SEQ ID NOs 3/4 in US patent application 2016/0297888 or SEQ ID NOs. 1/2 for heavy/light chain variable regions herein) family or is a C38D8 (heavy/light SEQ ID NOs 21/22 in US patent application 2016/0297888 or SEQ ID NOs. 3/4 for heavy/light chain variable regions herein).
• toxin moieties as disclosed herein, conjugated to a linker and a conjugation moiety as disclosed herein represent intermediate toxin linker conjugates, which, when covalently bound (conjugated to) the IgG antibody as disclosed herein, are ADCs as disclosed herein.
• the conjugated toxin with linker structure is selected from the group consisting of (with each compound number indicated):
• Dl and D2 are anthracycline derivatives.
• D3, D4, and D5 are tubulin inhibitors.
• the wavy line indicates the point of attachment to the linker.
• the drug linker conjugate comprises a linker L2 and a conjugation moiety, wherein the linker L2 is covalently bound to the conjugation moiety; the conjugation moiety is capable of reacting with free cysteine thiol groups in the hinge region of an IgG antibody.
• the conjugation moiety has the structure ⁇ - ⁇ N ' ("conjugation method LI").
• Affinity purified anti-CD38 antibody was buffer exchanged into 50 mM sodium phosphate buffer, pH 7.0-7.2 with 4 mM EDTA at a concentration of 5-10 mg/mL
• TCEP tris(2- carboxyethyl)phosphine)
• L014-077 was first dissolved in a 3:2 acetonitrile/water mixture at a concentration of 5 mM. An aliquot of this freshly prepared toxin-linker solution was then added to a portion of the reduced, purified anti-CD38 antibody intermediate in 4.5-5 fold molar excess. After thorough mixing and incubation at ambient temperature for >1 h, the crude conjugation reaction was analyzed by HIC-HPLC chromatography to confirm reaction completion (disappearance of starting antibody peak) at 280 nm wavelength detection.
• ADC46 Purification of ADC46 was then carried out by gel-filtration chromatography using an AKTA system equipped with a Superdex 200 pg column (GE Healthcare) equilibrated with PBS.
• the drug-to-antibody ratio (DAR) was calculated based on UV-VIS and HIC-HPLC.
• Figure 8 shows a representative HIC-HPLC comparison of starting anti-CD38 antibody and purified ADC46. Confirmation of low percent ( ⁇ 5%) high molecular weight (HMW) aggregates for the resulting ADC46 was determined using analytical SEC-HPLC.
• Reduction and analysis of anti-CD38 antibody for ADC41 was conducted in a manner identical to the procedure used to generate ADC46.
• Compound 22 was first dissolved in a 2:3 acetonitrile/water mixture at a concentration of 5 mM.
• Propylene glycol (PG) was then added to an aliquot of the reduced, purified anti-CD38 antibody to give a final concentration of 10-30% (v/v) PG before addition of the freshly prepared compound 22 solution in 4.5-5-fold molar excess.
• Subsequent analysis and purification of ADC41 was carried out in a manner identical to the procedure for ADC46.
• Figure 9 shows a representative HIC-HPLC comparison of starting anti-CD38 antibody and purified ADC41.
• mice were housed 5 mice per cage in a room with a controlled environment. Animals were provided rodent chow and water ad libitum. Acclimation of the mice to laboratory conditions was at least 72 hours prior to the start of cell administration and dosing. During the acclimation period, the animals' health status was determined. Only animals that are observed to be healthy prior to study initiation were used.
• This example provides an in vivo experiment comparing treatment of mice with control (PBS), anti-CD38 IgGl antibody (STI-0602 and STI-0607) and an ADC variant of both antibodies.
• the procedure first does a tumor cell inoculation & establishment of tumors:
• U87 cells were cultured with 10% FBS U87 medium (EMEM) and harvested with 0.05% trypsin. Cells were washed 2 times with serum-free EMEM, counted, and resuspended at 5 xlO 6 cells in 0.2 mL or, 25 xlO 6 cells/mL in a 1: 1 mix of serum-free EMEM and matrigel and injected subcutaneously into the upper right flank of each mouse.
• EMEM FBS U87 medium
• Tumor growth was monitored by tumor volume measurement using a digital caliper starting Day 6-9 after inoculation, 2 times per week thereafter and prior to study termination.
• mice were randomized and mice with very large or small tumors culled. Mice were divided into 8 groups of 10 mice each, randomized by tumor volume.
• mice were treated with either vehicle or Test Article according to Figure 4. Mice received a total of 5 doses.
• This example is an in vivo experiment comparing two disclosed CD38 ADCs in vivo with mice
• 10 million of Daudi-fluc cells were injected iv into NOD-SCID mice. 4 days after tumor established in mice, anti-CD38 antibody and ADCs were injected to mice by IV. The inhibition of tumor growth by antibody or ADCs was monitored by the luminesce intensity change of the tumor ( Figures 3, 4, 5 and 6). ADC#45 and ADC# 41 were tested. Both ADC' s use the same A2 antibody.
• the Daudi and Ramos cell line was obtained from ATCC. The cells were cultured in RPMI 1640 IX medium with 10% FBS and at 37 °C in a 5% carbon dioxide humidified environment.
• Daudi-luc cells were cultured in RPMI 1640 IX medium with 10% FBS and 0.2ug/ml puromycin at 37 °C in a 5% carbon dioxide humidified environment. Cells cultured for a period of 2 weeks and were passaged 3 times before harvest. Prior to injection, Daudi-luc cells were resuspended in HBSS. 10 million cells per 0.2 ml were injected intravenously in to the tail vein of each mouse.
• mice Female NOD SCID mice aged 6 weeks (Charles River) were used for Daudi subcutaneous xenografts and Daudi-luc intravenous xenografts.
• Tumor growth was monitored by measurement of tumor width and length using a digital caliper starting day 5-7 after inoculation, and followed twice per week until tumor volume reached
• TGI [(Last Volume Measurement of PBS Group - Volume of Treatment group on the same day as the PBS control)/ (Last Volume
• There was no body weight loss in the testing animals with a single dose of intravenously administrated ADC#45 at 10 mg/kg or lower dose Figure 2B).
• ADC#45 and ADC#41 at 10 mg/kg single dose, significantly inhibited tumor growth, while at 3 mg/kg, or 1 mg/kg, both did not show significant tumor inhibition in Daudi and Ramos subcutaneously injected xenograft tumor model in mice.
• ADC#45 at 3 mg/kg single dose completely inhibited tumor growth with a 100% survival up to 48 days in Daudi-luc intravenously injected tumor model in female NOD SCID mice.
• ADC#41 at 3 mg/kg single dose significantly inhibited tumor growth, and prolonged survival in Daudi-luc intravenously injected tumor model in female NOD SCID mice.
• Dose response was observed for ADC#45 and ADC#41 in this study.
• ADC#45 showed better tumor growth inhibition effect than ADC#41 with the same (10 mg/kg, or 3 mg/kg) dose regime. No treatment-related body weight loss was observed during the study for all treatment groups.

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