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WO2023172951A1 - Methods of treating solid tumors with anti-tissue factor antibody-drug conjugates - Google Patents

Methods of treating solid tumors with anti-tissue factor antibody-drug conjugates Download PDF

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WO2023172951A1
WO2023172951A1 PCT/US2023/063927 US2023063927W WO2023172951A1 WO 2023172951 A1 WO2023172951 A1 WO 2023172951A1 US 2023063927 W US2023063927 W US 2023063927W WO 2023172951 A1 WO2023172951 A1 WO 2023172951A1
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antibody
dose
cancer
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subjects
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Raffaella Faggioni
Leo FAORO
Christian SCHEFFOLD
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Exelixis Inc
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Exelixis Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/68031—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
    • A61K47/6835—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
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • Tissue factor plays an important role in these coagulation processes.
  • TF is a cell surface receptor for the serine protease factor Vila (FVIIa).
  • FVIIa serine protease factor Vila
  • the TF/FVIla complex catalyzes conversion of the inactive protease factor X (FX) into the active protease factor Xa (FXa).
  • FXa and its co-factor FVa form the prothrombinase complex, which generates thrombin from prothrombin.
  • Thrombin converts soluble fibrinogen into insoluble strands of fibrin and catalyzes many other coagulation- related processes.
  • TF is over-expressed on multiple types of solid tumors.
  • TF/FVIla signaling can support angiogenesis, tumor progression, and metastasis.
  • an antibody-drug conjugate comprising: a. an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID NO:41), wherein the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL- CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, and b. one or more linker-toxin moieties represented by Formula I:
  • ## represents the point of attachment of the linker-toxin moiety to the TF antibody and the linker-toxin moiety is attached to the TF antibody through a covalent bond.
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, n is an integer greater than or equal to 1 , and the succinimidyl group is attached to the Ab through a covalent bond.
  • TF tissue factor
  • n is selected from the group consisting of 1 , 2, 3, 4, and 5.
  • n is selected from the group consisting of 2, 3, and 4.
  • the Ab comprises: a VH that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38. [0011] In some embodiments, the Ab comprises: a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGL EWMGWIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDT AVYYCARDAGTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKA
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3, and n is an integer greater than or equal to 1 .
  • TF tissue factor
  • n is selected from the group consisting of 1 , 2, 3, 4, and 5.
  • n is selected from the group consisting of 2, 3, and 4.
  • the Ab comprises a VH sequence that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38.
  • the Ab comprises a full heavy chain sequence that is
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISVWRQAPGQGLEWMG WIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDA GTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
  • n is selected from the group consisting of 1 , 2, 3, 4, [0019] In some embodiments, n is selected from the group consisting of 2, 3, and 4.
  • an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3; the one or more linker-toxins are attached to the Ab through a covalent bond; and
  • TF tissue factor
  • kits for treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate composition comprising the antibody-drug conjugate disclosed herein, wherein the composition comprises a multiplicity of drugantibody ratio (DAR) species, wherein the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
  • DAR drugantibody ratio
  • an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISVWRQAPGQGLEWMG WIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDA GTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
  • KSFNRGEC SEQ ID NO:40
  • linker-toxins are attached to the Ab through a covalent bond
  • an antibody-drug conjugate composition comprising the antibody-drug conjugate disclosed herein, wherein the composition comprises a multiplicity of drug- antibody ratio (DAR) species, wherein the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
  • the antibody is lgG1 kappa.
  • kits for treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate as disclosed herein and a pharmaceutically acceptable carrier in a pharmaceutical composition.
  • the method provided herein comprises administering to the subject a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg of antibody-drug conjugate disclosed herein.
  • the method provided herein comprises administering to the subject a dose of about 0.16 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.25 mg/kg, 2.5 mg/kg, 2.75 mg/kg, or about 3.0 mg/kg of antibody-drug conjugate disclosed herein. In some embodiments, the method comprises administering to the subject the antibody-drug conjugate disclosed herein intravenously (IV) every 3 weeks.
  • combination treatment methods further comprising administering to the subject an antibody that binds PD-1 , wherein the antibody that binds PD-1 comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL- CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from Nivolumab.
  • the antibody that binds PD-1 comprises a VH and a VL, wherein the VH and VL are from Nivolumab.
  • the antibody that binds PD-1 is Nivolumab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43 and a light chain comprising the amino acid sequence of SEQ ID NO:44.
  • Nivolumab is administered at a dose of about 360 mg intravenously (IV) every 3 weeks.
  • combination treatment methods further comprising administering to the subject an antibody that binds vascular endothelial growth factor (VEGF), wherein the antibody that binds VEGF comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from Bevacizumab.
  • VEGF vascular endothelial growth factor
  • the antibody that binds VEGF comprises a VH and a VL, wherein the VH and VL are from Bevacizumab.
  • the antibody that binds VEGF is Bevacizumab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:45 and a light chain comprising the amino acid sequence of SEQ ID NO:46.
  • Bevacizumab is administered at a dose of about 15 mg/kg intravenously (IV) every 3 weeks.
  • the solid tumor is a cancer.
  • the cancer is selected from the group consisting of: non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ova
  • the subject is a human subject.
  • FIGs. 1A-1D illustrates the study design as described in Examples 7 and 8.
  • FIG. 2 illustrates dose-escalation/de-escalation decisions per the i3+3 design for the Study as described in Example 7.
  • FIGs. 3A-3B provide 25A3-LT-A mean PK for the intact ADC (FIG. 3A), total antibodies (Ab, FIG. 3A), and free payload (FIG. 3B) from 0.16 to 2.0 mg/kg as described in Example 8.
  • the term “about” or “approximately” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” or “approximately” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1 %. In certain embodiments, where applicable, the term “about” or “approximately” indicates the designated value(s) ⁇ one standard deviation of that value(s).
  • tissue Factor tissue Factor
  • platelet tissue factor factor III
  • thromboplastin a cell surface receptor for the serine protease factor Vila. It is often times constitutively expressed by certain cells surrounding blood vessels and in some disease settings.
  • antibody-drug conjugate refers to a conjugate comprising an antibody conjugated to one or more cytotoxic agents, optionally through one or more linkers.
  • anti-TF antibody-drug conjugate or “anti-TF ADC” refers to a conjugate comprising an anti-TF antibody conjugated to one or more cytotoxic agents, optionally through one or more linkers.
  • TF antibody As used herein, the terms “TF antibody,” “anti-TF antibody” are synonymous.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • the cytotoxic agent can be an anti-angiogenic agent, a pro-apoptotic agent, an antimitotic agent, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioactive isotope.
  • cytotoxic agents include calicheamycin, camptothecin, carboplatin, irinotecan, SN- 38, carboplatin, camptothecan, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, doxorubicin, etoposide, idarubicin, topotecan, vinca alkaloid, maytansinoid, maytansinoid analog, pyrrolobenzodiazepine, taxoid, duocarmycin, dolastatin, auristatin, and derivatives thereof.
  • a “linker” refers to a molecule that connects one composition to another, e.g., an antibody to an agent.
  • Linkers described herein can conjugate an antibody to a cytotoxic agent.
  • Exemplary linkers include a labile linker, an acid labile linker, a photolabile linker, a charged linker, a disulfide-containing linker, a peptidasesensitive linker, a [3-glucuronide-linker, a dimethyl linker, a thio-ether linker, and a hydrophilic linker.
  • a linker can be cleavable or non-cleavable.
  • immunoglobulin refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI , CH2, and CHS. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.
  • antibody is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.
  • An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies.
  • antigen-binding domain means the portion of an antibody that is capable of specifically binding to an antigen or epitope.
  • full length antibody is an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region.
  • a “full length antibody” is an antibody that comprises two heavy chains and two light chains.
  • Fc region means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system.
  • the structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art.
  • the VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved.
  • the more conserved regions are called framework regions (FRs).
  • Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991 ) Public Health Service, National Institutes of Health, Bethesda, MD.
  • a “Complementary Determining Region (CDR)” refers to one of three hypervariable regions (H1 , H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH [3-sheet framework, or one of three hypervariable regions (L1 , L2 or L3) within the non-framework region of the antibody VL [3-sheet framework.
  • CDRs are variable region sequences interspersed within the framework region sequences. CDRs are well recognized in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains.
  • CDRs have also been defined structurally by Chothia as those residues that are not part of the conserved [3-sheet framework, and thus are able to adapt different conformations. See Chothia and Lesk, J Mol Biol, 1987, 196:901 -917. Both the Kabat and Chothia nomenclatures are well known in the art.
  • AbM, Contact and IMGT also define CDRs. CDR positions within a canonical antibody variable domain have been determined by comparison of numerous structures.
  • a number of hypervariable region delineations are in use and are included herein.
  • the Kabat CDRs are based on sequence variability and are the most commonly used. See Kabat et al. (1992) Sequences of Proteins of Immunological Interest, DIANE Publishing: 2719. Chothia refers instead to the location of the structural loops (Chothia and Lesk, supra).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • the Contact hypervariable regions are based on an analysis of the available complex crystal structures.
  • IMGT ImMunoGeneTics
  • IG immunoglobulins
  • TR T cell receptors
  • MHC major histocompatibility complex
  • the heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, E, y, and p, respectively.
  • the IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: lgG1 , lgG2, lgG3, lgG-4, lgA1 , and lgA2.
  • constant region refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the CHI , CH2 and CHS domains of the heavy chain and the CL domain of the light chain.
  • the “Ell numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.
  • the term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies.
  • a population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts.
  • a monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones.
  • the selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
  • affinity maturation is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
  • affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope).
  • affinity refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).
  • KD dissociation equilibrium constant
  • the kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).
  • the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule).
  • Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule.
  • Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.
  • kd (sec 1 ), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the koff value.
  • k a (M’ 1 xse 1 ), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the kon value.
  • affinity of an antibody is described in terms of the KD for an interaction between such antibody and its antigen. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.
  • Fc effector functions refer to those biological activities mediated by the Fc region of an antibody, which activities may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement dependent cytotoxicity (CDC), Fc receptor binding to activate antibodydependent cellular cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP).
  • CDC complement dependent cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody dependent cellular phagocytosis
  • amino acid refers to the twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).
  • treating refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • therapeutically effective amount or “effective amount” refers to an amount of an antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.
  • the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs and sheep. In certain embodiments, the subject is a human. In some embodiments the subject has a disease or condition that can be treated with an antibody provided herein. In some aspects, the disease or condition is a cancer.
  • ADCs Antibody-drug conjugates
  • mAbs monoclonal antibodies
  • payloads cytotoxic agents
  • the antibody component of the ADC is directed towards a tumor-associated antigen, allowing for tumor specificity and targeted drug delivery to the tumor site. This approach is expected to result in reduced exposure of nontarget tissues to the cytotoxic agent and enhanced antitumor potency. Due to the reduced toxicity of ADC-mediated drug delivery; the therapeutic window may be broadened in comparison to traditional chemotherapy.
  • Tissue Factor (TF; also known as coagulation factor III [F3], thromboplastin, CD142, or platelet tissue factor) is a 45-kDa, single-chain, type I transmembrane glycoprotein that is expressed under normal physiological conditions in the subendothelial tissue. TF plays a major role in regulating the extrinsic coagulation cascade. It acts as a unique cell-associated receptor for the activated form of coagulation factor VII (FVIIa) and initiates blood coagulation after a vascular injury. In addition to regulating hemostatic balance, the signaling pathways dependent on TF contribute to angiogenesis, inflammation, atherosclerosis, tumor metastasis, and immune evasion.
  • amino acid sequence of an extracellular domain of human TF is as follows: SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTE CDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTI QSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAK TNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (SEQ ID N0:41).
  • TF has been found to be aberrantly overexpressed in many solid tumors including cervical cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, lung cancer, breast, prostate and pancreatic cancer.
  • SCCHN head and neck
  • TF expression has been strongly associated with disease progression and poor prognosis in solid tumors, such as poor disease outcomes, shorter overall survival, and increased metastasis.
  • TF may facilitate cell proliferation and tumor survival through a variety of mechanisms. Activation of PAR-2 by the TF-FVIla complex has been found to induce cell proliferation, migration, and angiogenesis via expression of VEGF and interleukin-8. Additionally, direct binding of TF to [33 and [31 integrins has been found to induce angiogenesis and cell proliferation in vitro and in vivo. Given its roles in cancer biology and aberrant expression in many tumors, direct or indirect inhibition of TF activity is considered a potential therapeutic strategy for the treatment of cancer. In addition to its expression on the cell surface of cancer cells, TF undergoes efficient internalization and lysosomal targeting, making it an attractive tumor- associated antigen for ADC-mediated internalization of a cytotoxic agent into tumor cells.
  • TF-targeting ADCs engineered with human IgG mAbs may also be able to recruit immune effector cells to cancer cells by binding effector cells through their Fc receptors to the Fc region of the antibody leading to antibody-dependent cellular toxicity (ADCC) mediated by immune effector cells.
  • ADCC antibody-dependent cellular toxicity
  • release of the cytotoxic agent of ADCs after binding to the TF protein on the cancer cell surface can lead to the death of adjacent tumor cells through a bystander effect.
  • 25A3-LT-A is an ADC that has been designed to improve the therapeutic potential of ADCs targeting TF. It is composed of a human mAb (25A3) against TF that is conjugated via a cysteine residue to a novel toxin (N-acyl sulfonamide auristatin, Compound 9), using a cleavable linker.
  • the toxin (Compound 9) and its linker are designated as LT-A.
  • the toxin (Compound 9) is released by enzymatic cleavage.
  • the toxin (Compound 9) is a novel auristatin that inhibits cell division by blocking the polymerization of tubulin.
  • 25A3-LT-A did not affect the coagulation cascade as measured by Fxa conversion and thrombin generation assays, while still binding human and cynomolgus TF with high affinity.
  • HPAF-II pancreatic adenocarcinoma
  • 25A3-LT-A was more potent than the ADC generated with the same antibody conjugated to MMAE.
  • PDX patient-derived xenograft
  • cytotoxic agent is linked directly to the anti-TF antibody. In some embodiments, the cytotoxic agent is linked indirectly to the anti-TF antibody.
  • the ADCs further comprise a linker.
  • the linker links the anti-TF antibody to the cytotoxic agent.
  • the number of cytotoxic agents conjugated to an antibody in an ADC is defined as the drug-antibody ratio or DAR.
  • DAR drug-antibody ratio
  • the majority of conjugation methods yield an ADC composition that includes various DAR species, with the reported DAR being the average of the individual DAR species.
  • the ADCs described herein are defined as having a specific DAR, it is to be understood that the number provided represents the average of the individual DAR species in the ADC composition.
  • the ADCs provided herein have a drug-antibody ratio (DAR) of approximately 3.8.
  • DAR drug-antibody ratio
  • provided herein is an antibody-drug conjugate of
  • Ab is a tissue factor (TF) antibody, and n is an integer greater than or equal to 1 .
  • n is an integer from 1 to 10.
  • n is selected from the group consisting of 1 , 2, 3, 4, and 5.
  • n is an integer selected from the group consisting of 2, 3, and 4.
  • the succinimidyl group is attached to the Ab through a covalent bond.
  • the payload as used herein improves the characteristics of microtubule inhibitor payloads (e.g., by lower off-target deconjugation).
  • the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, n is an integer greater than or equal to 1.
  • the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3 as shown in Table 1 .
  • the Ab comprises a VH and a VL from the antibody designated 25A3 as shown in Table 1.
  • the Ab comprises a heavy chain (HC) and a light chain (LC) from the antibody designated 25A3 as shown in Table 2.
  • the ADCs described herein comprise an antibody that comprises a VH comprising the amino acid sequence of SEQ ID NO: 37 and a VL comprising the amino acid sequence of SEQ ID NO: 38.
  • the ADCs described herein comprise an antibody that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:39 and a light chain comprising the amino acid sequence of SEQ ID NQ:40.
  • an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH-CDR1 , a VH- CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3; the one or more linker-toxins are attached to the Ab through a covalent bond; and ## represents a point of attachment of the linker-toxin to the Ab.
  • the linker-toxin of Formula I is also referred to as “LT-A,” where ## represents a point of attachment to the tissue factor (TF) antibody, Ab.
  • the antibody-drug conjugate of Formula II where Ab comprises the VH- CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 from the antibody designated 25A3, is also referred to as “25A3-LT-A”.
  • compositions comprising an ADC comprising an antibody (Ab) and one or more linker-toxins of Formula I.
  • the composition comprises a multiplicity of drug-antibody ratio (DAR) species.
  • DAR drug-antibody ratio
  • the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
  • an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
  • Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is
  • an antibody-drug conjugate composition comprising an ADC of the present disclosure, wherein the composition comprises a multiplicity of drug-antibody ratio (DAR) species, wherein the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
  • DAR drug-antibody ratio
  • the method provided herein is for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ovarian
  • the ADC provided herein for use in treating a solid tumor selected from a group consisting of non- small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration- resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumoragnostic (TA) tissue-factor-positive (TF+) cancer.
  • NSCLC non- small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ova
  • a use of the ADC provided herein for the manufacture of a medicament for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC ova
  • the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is urothelial cancer. In some embodiments, the solid tumor is endometrial cancer (EC). In some embodiments, the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)). In some embodiments, the solid tumor is cervical cancer (e.g., with squamous cell or adenocarcinoma histology). In some embodiments, the solid tumor is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the solid tumor is pancreatic cancer.
  • NSCLC non-small cell lung cancer
  • the solid tumor is urothelial cancer.
  • the solid tumor is endometrial cancer (EC).
  • the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)).
  • EOC epithelial ovarian cancer
  • the solid tumor is cervical
  • the solid tumor is esophageal squamous-cell carcinoma (SCC).
  • the solid tumor is prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)).
  • the solid tumor is breast cancer, e.g., triplenegative breast cancer (TNBC) or hormone-receptor positive breast cancer (HR+BC).
  • the solid tumor is tumor-agnostic (TA) tissue- factor-positive (TF+) cancer.
  • the ADC provided herein (e.g., 25A3-LT-A) is administered to the subject as a single-agent therapy.
  • the dose ranges from about 1 .0 mg/kg to about 3.0 mg/kg.
  • the dose is about 0.16 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, about 2.5 mg/kg, about 2.75 mg/kg, or about 3.0 mg/kg.
  • the ADC provided herein (e.g., 25A3-LT-A) is administered to the subject intravenously (IV) every 3 weeks. 4. Combination Therapy with Nivolumab
  • PD-1 is an immune-inhibitory receptor expressed in activated T cells; it is involved in the regulation of T-cell functions, including those of effector CD8+ T cells.
  • this protein can also promote the differentiation of CD4+ T cells into T regulatory cells. It is expressed in many types of tumors including melanomas, and has demonstrated to play a role in anti-tumor immunity.
  • the antibody that binds PD-1 comprises a VH- CDR1 , VH-CDR2, and VH-CDR3 as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:43, and a VL-CDR1 , VL-CDR2, and VL-CDR3 as set forth in a light chain comprising the amino acid sequence of SEQ ID NO:44.
  • the antibody that binds PD-1 comprises a VH domain comprising the amino acid sequence as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:43, and a VL domain comprising the amino acid sequence as set forth in the heavy chain comprising the amino acid sequence of SEQ ID NO:44.
  • the antibody that binds PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43, and a light chain comprising the amino acid sequence of SEQ ID NO:44.
  • the antibody that binds PD-1 is Nivolumab (OPDIVO®), which is a fully human lgG4 antibody targeting PD-1 .
  • the heavy chain sequence of Nivolumab is
  • the combination therapy provided herein is for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ova
  • the ADC provided herein for use in combination with Nivolumab for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC ova
  • the ADC provided herein (e.g., 25A3-LT-A) for use in combination with Nivolumab for treating a solid tumor selected from a group consisting of NSCLC, SCCHN, and esophageal SCC.
  • the ADC provided herein for the manufacture of a medicament for treating a solid tumor in combination with Nivolumab
  • the solid tumor is selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)),
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ovarian
  • provided herein is a use of the ADC provided herein (e.g., 25A3-LT-A) for the manufacture of a medicament for treating a solid tumor in combination with Nivolumab, wherein the solid tumor is selected from a group consisting of NSCLC, SCCHN, and esophageal SCC.
  • the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is urothelial cancer. In some embodiments, the solid tumor is endometrial cancer (EC). In some embodiments, the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)). In some embodiments, the solid tumor is cervical cancer (e.g., with squamous cell or adenocarcinoma histology). In some embodiments, the solid tumor is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the solid tumor is pancreatic cancer.
  • NSCLC non-small cell lung cancer
  • the solid tumor is urothelial cancer.
  • the solid tumor is endometrial cancer (EC).
  • the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)).
  • EOC epithelial ovarian cancer
  • the solid tumor is cervical
  • the solid tumor is esophageal squamous-cell carcinoma (SCC).
  • the solid tumor is prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC))
  • the solid tumor is breast cancer, e.g., triplenegative breast cancer (TNBC) or hormone-receptor positive breast cancer (HR+BC).
  • the solid tumor is tumor-agnostic (TA) tissue- factor-positive (TF+) cancer.
  • the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Nivolumab at a dose of 360 mg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the ADC provided herein e.g., 25A3-LT-A
  • Nivolumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Nivolumab at a dose of 360 mg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every
  • the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose of about 0.16 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, about 2.5 mg/kg, about 2.75 mg/kg, or about 3.0 mg/kg.
  • the combination therapy further comprises administering to the subject Nivolumab at a dose of 360 mg.
  • the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every 3 weeks.
  • Nivolumab and the ADC provided herein are administered to the subject on the same day.
  • Nivolumab is administered prior to the ADC provided herein (e.g., 25A3-LT-A) on the day both are given to the subject.
  • Nivolumab and the ADC provided herein are administered at least 30 minutes apart.
  • the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the combination therapy is administered to the subject intravenously (IV) at least 21 days apart. In some embodiments, the combination therapy lasts no more than 2 years.
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • PDGF/VEGF growth factor family e.g., UniProtKB/Swiss-Prot: P15692 or NCBI gene ID: 7422.
  • VEGF is a member of the PDGF/VEGF growth factor family. It is a heparin-binding protein, which exists as a disulfide-linked homodimer. This growth factor induces proliferation and migration of vascular endothelial cells, and is essential for both physiological and pathological angiogenesis. Disruption of this gene in mice resulted in abnormal embryonic blood vessel formation.
  • the antibody that binds VEGF comprises a VH-CDR1 , VH-CDR2, and VH-CDR3 as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:45, and a VL-CDR1 , VL-CDR2, and VL-CDR3 as set forth in a light chain comprising the amino acid sequence of SEQ ID NO:46.
  • the antibody that binds VEGF comprises a VH domain comprising the amino acid sequence as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:45, and a VL domain comprising the amino acid sequence as set forth in the heavy chain comprising the amino acid sequence of SEQ ID NO:46.
  • the antibody that binds VEGF comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:45, and a light chain comprising the amino acid sequence of SEQ ID NO:46.
  • the antibody that binds VEGF is Bevacizumab (AVASTIN®), which is a humanized immunoglobulin G1 (lgG1 ) antibody targeting VEGF.
  • Bevacizumab is EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINT YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHW YFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSD
  • the combination therapy provided herein is for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ova
  • the ADC provided herein for use in combination with Bevacizumab for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC ova
  • the ADC provided herein for the manufacture of a medicament for treating a solid tumor in combination with Bevacizumab
  • the solid tumor is selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), and breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC epithelial ovarian
  • the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is urothelial cancer. In some embodiments, the solid tumor is endometrial cancer (EC). In some embodiments, the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)). In some embodiments, the solid tumor is cervical cancer (e.g., with squamous cell or adenocarcinoma histology). In some embodiments, the solid tumor is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the solid tumor is pancreatic cancer.
  • NSCLC non-small cell lung cancer
  • the solid tumor is urothelial cancer.
  • the solid tumor is endometrial cancer (EC).
  • the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)).
  • EOC epithelial ovarian cancer
  • the solid tumor is cervical
  • the solid tumor is esophageal squamous-cell carcinoma (SCC).
  • the solid tumor is prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC))
  • the solid tumor is breast cancer, e.g., triplenegative breast cancer (TNBC) or hormone-receptor positive breast cancer (HR+BC).
  • the solid tumor is tumor-agnostic (TA) tissue- factor-positive (TF+) cancer.
  • the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Bevacizumab at a dose of 15 mg/kg. In some embodiments, the ADC provided herein (e.g., 25A3-LT- A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the ADC provided herein e.g., 25A3-LT- A
  • Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Bevacizumab at a dose of 15 mg/kg. In some embodiments, the ADC provided herein (e.g., 25A3-LT- A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the ADC provided herein e.g., 25A3-LT- A
  • Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose of about 0.16 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, about 2.5 mg/kg, about 2.75 mg/kg, or about 3.0 mg/kg.
  • the combination therapy further comprises administering to the subject Bevacizumab at a dose of 15 mg/kg.
  • the ADC provided herein (e.g., 25A3-LT-A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
  • Bevacizumab and the ADC provided herein are administered to the subject on the same day.
  • Bevacizumab is administered prior to the ADC provided herein (e.g., 25A3-LT-A) on the day both are given to the subject.
  • Bevacizumab and the ADC provided herein are administered at least 30 minutes apart.
  • the ADC provided herein (e.g., 25A3-LT-A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
  • the combination therapy is administered to the subject intravenously (IV) at least 21 days apart. In some embodiments, the combination therapy lasts no more than 2 years.
  • the ADCs can be prepared using any suitable methods as disclosed in the art employing organic chemistry reactions, conditions, and reagents known to those skilled in the art.
  • a process for preparing an antibody-drug conjugate comprising: (A) reacting a nucleophilic or an electrophilic group on an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID NO:41 ) with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form an Ab-linker intermediate, and reacting the Ab-linker intermediate with the -NH2 group on Compound 9:
  • Ab antigen binding protein
  • TF human Tissue Factor
  • Compound 9, to provide the antibody drug conjugate or (B) reacting the -NH2 group on Compound 9 with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form a linker-toxin intermediate, and reacting the linker-toxin intermediate with a nucleophilic or an electrophilic group on an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID N0:41 ) to provide the antibody-drug conjugate.
  • Ab antigen binding protein
  • the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL- CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3; and
  • the antibody-drug conjugate comprises one or more moieties represented by Formula I:
  • the nucleophilic or electrophilic group on the Ab is a thiol or an amine.
  • the process further comprises treating the Ab with a reducing agent to reduce one or more disulfide linkages in the Ab to provide the nucleophilic thiol group.
  • the ADCs may be prepared by a method comprising (A) (i) reacting a nucleophilic or electrophilic group on the antibody with a bifunctional linker to form an antibody-linker intermediate, or (ii) reacting a nucleophilic or electrophilic group on the antibody with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form an antibody-linker intermediate, and (B) reacting the antibodylinker intermediate with the -NH2 group on Compound 9 to provide the ADC.
  • the ADCs may be prepared by a method comprising (A) (i) reacting the NH2 group on Compound 9 with a bifunctional linker to form a linker-toxin intermediate, or (ii) reacting the NH2 group on Compound 9 with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form a linker-toxin intermediate, and (B) reacting the linker-toxin intermediate with a nucleophilic or electrophilic group on the antibody to provide the antibody-drug conjugate.
  • the electrophilic or nucleophilic group on the antibody is a thiol (for example from a cysteine residue on the antibody), or an amine (for example from a lysine residue on the antibody).
  • Compound 9 and linker-toxins comprising Compound 9 may be prepared by standard synthetic organic chemistry protocols from commercially available starting materials.
  • kits comprising an TF ADC as disclosed herein or the pharmaceutical composition comprising same.
  • the kit further comprises a chemotherapy and/or an anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb).
  • an anticancer antibody e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb.
  • kits comprising the TF ADC as disclosed herein or the pharmaceutical composition comprising same, and instructions for use.
  • the kit further comprises a chemotherapy and/or an anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb).
  • an anticancer antibody e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb.
  • kits comprising the TF ADC provided herein or a pharmaceutical composition comprising same, packaged into suitable packaging material.
  • the kit further comprises a chemotherapy and/or an anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb).
  • a kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • packaging material refers to a physical structure housing the components of the kit.
  • the packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).
  • Kits provided herein can include labels or inserts.
  • Labels or inserts include “printed matter,” e.g., paper or cardboard, separate or affixed to a component, a kit or packing material (e.g., a box), or attached to, for example, an ampoule, tube, or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media, or memory type cards.
  • Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, and date.
  • Kits provided herein can additionally include other components. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Kits can also be designed for cold storage.
  • reaction was acidified with the addition of 1 M aqueous HCI (50 mL) and concentrated under reduced pressure to remove the dioxane.
  • the remaining reaction mixture was extracted with ethyl acetate (4 x 50 mL) and the organic phase was pooled, washed with brine (15 mL + 2 mL 2 M HCI), dried over MgSO4, filtered and concentrated under reduced pressure to yield a light colored oil.
  • the oil was redissolved in diethyl ether ( ⁇ 50 mL) and concentrated under reduced pressure (3x) to facilitate the removal of residual dioxane, affording the title product as a stiff oil (7.81 g 97% yield with some residual dioxane and Compound 4).
  • MS m/z obs. 606.7 (M+1 ).
  • the reaction was monitored by HPLC-MS for the consumption of starting materials. After 6 days, the reaction was complete with the total consumption of Compound 14, leaving only Compound 15 and a small amount ( ⁇ 5%) of the bis-TFP maleic amide intermediate.
  • the reaction was transferred to a separating funnel, diluted with diethyl ether (75 ml) and washed with 5% LiCI (1 x 20 mL), 1 M HCI (2 x 20 mL), sat. NaHCOs (5 x 20 mL) and brine (1 x 20 mL). The organic layer was dried over Na2SO4, filtered and evaporated to give brown crude oil with residual DMF.
  • the reaction was monitored for completion by HPLC-MS and no change to reaction progress was observed between the samples taken at 30 minutes and 1 h ( ⁇ 95% complete).
  • the reaction was allowed to stir overnight at room temperature, then 2-(2-aminoethylamino)ethanol (0.483 mL, 4.781 mmol, 4 equiv), EtOAc (10 mL) and dhhO (5 mL) were added to the stirred suspension, which underwent a color change to deep blue.
  • the suspension was stirred vigorously for 4 hr as the suspended solids gradually dissolved into the biphasic mixture.
  • This mixture was transferred to a separating funnel and diluted with EtOAc (100 mL) and brine (10 mL), and the aqueous layer was extracted using 10% IpOH/ EtOAc (4 x 50 mL). The organic layers were pooled and washed with brine (10 mL), dried over Na2SO4, and evaporated to yield a faintly blue crude solid.
  • This crude solid was dissolved in a mixture of methanol (0.5 mL) and dichloromethane (6 mL) and purified on a Biotage® SNAP Ultra 100 g silica gel column (2-20% MeOH in CH2CI2 over 10 column volumes, followed by an 8-column volume plateau at 20% MeOH).
  • ADCs Antibody-Drug Conjugates (ADCs) of anti-TF antibodies and Linker-Toxin A, as prepared in Example 1 , were prepared as described below (see also International Patent Application Publication No. WO 2021/003399, incorporated herein by reference in its entirety).
  • ADCs Antibody-Drug Conjugates
  • 5 to 10 mg/mL of 25A3 antibody (see Tables 1 and 2 for CDR and V region sequences of clone 25A3) in phosphate-buffered saline (PBS), pH 7.4 was reduced by the addition of Tris(2-carboxyethyl)phosphine (2.0-2.5 or 3.2 molar equivalents) and a final concentration of 0.8 mM diethylenetriamine-pentaacetic acid.
  • the ADC was purified through one set of PBS primed columns first, the sample was then collected and purified a second time through the other set. After the second purification, the ADC was pooled back together and sterile filtered and frozen at - 80°C.
  • Drug-antibody ratio may be measured by UV/vis spectroscopy, hydrophobic interaction chromatography (HIC), and/or reverse phase liquid chromatography separation with time-of-flight detection and mass characterization ⁇ RP-UPLC/Mass spectrometry), as described in WO 2016/041082.
  • Distribution of drug-linked forms (for example, the fraction of DAR0, DARI, DAR2, etc. species) may also be analyzed by various techniques known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF), as also described in WO 2016/041082.
  • the drug-antibody ratio (DAR) of the resulting ADCs was ⁇ 3.
  • ADCs comprising 25A3 and LT-A (25A3-LT-A), e.g. as prepared in this example, were used in the studies of Examples 3-7 below.
  • Example 3 ADC Formulation
  • HCI 1 N is used to titrate the histidine buffer to the target pH so amounts may vary from batch to batch.
  • 25A3-LT-A (synthesized as described in Example 2 and formulated as described in Example 3) was administered via IV infusion at 3, 6, 12, or 15 mg/kg/dose once every 3 weeks for a total of 4 doses. Terminal necropsy of 4 animals of each sex for each dose was on Day 65, and potential reversibility of findings was examined following a 6-week recovery period in two animals of each sex for each dose (Day 106).
  • Corneal findings were considered likely secondary to those noted in the eyelids. At recovery, there was erosion/ulcer of the cornea in one of four males (mild, with pigmented corneal epithelium) and one of four females (moderate, with dorsal erosion/ulcer and/or hyperplasia of the conjunctival epithelium) at 12 mg/kg/dose and minimal corneal epithelial atrophy was noted in the right eye of one female at 12 mg/kg/dose. The incidence and/or severity of the findings were decreased compared to findings at the end of the dosing period. This is considered consistent with an ongoing recovery process.
  • HNSTD non-severely toxic dose
  • the proposed starting dose for the Phase 1 clinical study is 0.16 mg/kg IV q3w. This starting dose is expected to have a safety margin of 6 based on the HED, 19 based on predicted human maximum serum concentration (Cmax), and 37 based on predicted human area under the curve (AUC) compared to the HNSTD in cynomolgus monkeys.
  • Cmax predicted human maximum serum concentration
  • AUC predicted human area under the curve
  • 25A3-LT-A exposures at the proposed starting dose of 0.16 mg/kg are predicted to reach ⁇ 50% target occupancy at Cmax and ⁇ 10% target occupancy 2 days after dose.
  • the estimated 25A3-LT-A efficacious exposure levels ranged from 3.6 pg/mL to 10 pg/mL.
  • Human PK simulations suggested that human doses of
  • a single-dose GLP rat study (a human non-relevant species) evaluated the acute toxicity and toxicokinetics of the toxin of 25A3-LT-A, when administered as a single dose to rats at doses of 1 , 3, and 6 mg/kg.
  • An additional control group was administered the vehicle. Animals from all groups were terminated 3 days after dose administration to assess the toxicity of the toxin of 25A3-LT-A. Additional animals from the control and the 6 mg/kg groups were euthanized following a 14-day recovery period to assess the potential for recovery.
  • Clinical pathology changes included marked decreases in reticulocytes in both sexes at 3 and 6 mg/kg and mild to moderate decreases in neutrophils and eosinophils at 6 mg/kg, which correlated with decreased hematopoietic cellularity noted microscopically. Mild to moderate decreases in lymphocytes were noted at 6 mg/kg which correlated to lymphoid depletion observed microscopically in multiple lymphoid tissues. Evidence of a mild inflammatory response was observed at 3 and 6 mg/kg as indicated by increases in fibrinogen at 3 and 6 mg/kg, increased globulin at 6 mg/kg, and minimal prolongations in prothrombin times in both sexes at 6 mg/kg.
  • Histopathologic findings were generally consistent with those anticipated from effects of the free toxin of 25A3-LT-A. These effects were characterized by reversible microscopic findings in the bone marrow, spleen, thymus, lymph nodes, and exorbital lacrimal gland.
  • the toxin of 25A3-LT-A was negative in the bacterial reverse mutation assay. Consistent with the mechanism of action for the toxin of 25A3-LT-A, which is an anti-mitotic agent that inhibits cell division by blocking the polymerization of tubulin, the toxin was positive in both the in vitro and in vivo micronucleus assays.
  • Example 7 A Dose-Escalation and Expansion Study of the Safety and Pharmacokinetics of Anti-TF ADC as Singe-Agent Therapy or in Combination Therapy in Subjects with Inoperable Locally Advanced or Metastatic Solid Tumors
  • PK pharmacokinetics
  • the MTD of 25A3-LT-A given as a single agent and in combination with nivolumab or bevacizumab was determined using an interval 3+3 (i3+3) design (Liu et al 2020).
  • the safety and preliminary efficacy of 25A3-LT-A are further evaluated in multiple tumor-specific expansion cohorts including subjects with non-small cell lung cancer [NSCLC], endometrial cancer (EC), EOC, cervical cancer, squamous cell carcinoma of the head and neck [SCCHN], pancreatic cancer, esophageal squamous-cell carcinoma (SCC), metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer (TNBC), and hormone-receptor positive breast cancer (HR+BC), and a tumor-agnostic (TA) TF-positive (TF+) cohort.
  • NSCLC non-small cell lung cancer
  • EC endometrial cancer
  • EOC cervical cancer
  • SCCHN squamous cell carcinoma of the head and neck
  • SCC esophageal squamous-cell carcinoma
  • mCRPC metastatic castration-resistant prostate cancer
  • TNBC triple-negative breast cancer
  • HR+BC hormone-receptor positive breast cancer
  • TA tumor
  • Simon’s 2 Stage design (Simon 1989) is used with the following exceptions: two dose levels are evaluated without interim futility testing in selected tumor types in order to understand the exposure-safety and exposure-response relationships more completely, and RD is evaluated without interim futility testing in the TA TF+ cohort.
  • 25A3-LT-A is evaluated as monotherapy in multiple tumor-specific cohorts and a TA TF+ cohort and as combination therapy with nivolumab in NSCLC, SCCHN, and esophageal SCC, and with bevacizumab in EOC.
  • Subjects are monitored for potential ADC-associated toxicities including but not limited to ocular, pulmonary, skin, and neurological events, as well as potential hemorrhagic events.
  • the primary objective is to determine the MTD and/or RD for further evaluation of intravenous (IV) administration of 25A3-LT- A when administered alone and in combination therapy in subjects with advanced malignancies.
  • Additional objectives of the dose-escalation stage include: to establish the preliminary safety and tolerability profile of 25A3-LT-A when administered alone and in combination therapy; to evaluate the PK of 25A3-LT-A (antibody conjugated to payload), total antibody (unconjugated and conjugated antibody), and free payload following IV administration alone and in combination therapy; to assess the immunogenicity of 25A3-LT-A; and to evaluate the anti-tumor activity of 25A3-LT-A alone and in combination therapy as measured by objective response rate (ORR), duration of response (DOR), and progression-free survival (PFS) per RECIST 1.1 as assessed by the Investigator.
  • ORR objective response rate
  • DOR duration of response
  • PFS progression-free survival
  • An exploratory objective of the dose-escalation stage is to evaluate the relationship between PK and exploratory biomarkers, preliminary efficacy, and safety outcomes.
  • the primary objective is to evaluate preliminary efficacy of 25A3-LT-A when administered alone and in combination therapy by determining the ORR per RECIST 1 .1 as assessed by the Investigator.
  • Additional objectives of the cohort expansion stage include: to evaluate the safety and tolerability of 25A3-LT-A when administered alone and in combination therapy; to further evaluate the PK of 25A3-LT-A (antibody conjugated to payload), total antibody (unconjugated and conjugated antibody), and free payload following IV administration alone and in combination therapy; to assess the immunogenicity of 25A3-LT-A; to evaluate the anti-tumor activity of 25A3-LT-A alone and in combination therapy as measured by DOR and PFS per RECIST 1.1 as assessed by the Investigator; to evaluate the anti-tumor activity of 25A3-LT-A as measured by ORR, DOR, and PFS per RECIST 1.1 as assessed by a Blinded Independent Radiology Committee (BIRC) for selected cohorts; to evaluate the BIRC
  • An exploratory objective of the cohort expansion stage is to assess the effects of 25A3-LT-A on tumor and blood biomarkers, to evaluate the exposure of nivolumab or bevacizumab in combination with 25A3-LT-A, to assess the immunogenicity of nivolumab or bevacizumab in combination with 25A3-LT-A, and to evaluate the association between TF expression and efficacy outcomes.
  • This study consists of a Dose-Escalation Stage and a Cohort-Expansion Stage for the evaluation of 25A3-LT-A as monotherapy and in combination with nivolumab or bevacizumab.
  • the safety and PK of 25A3-LT-A monotherapy is evaluated in the Dose-Escalation Stage.
  • Subjects in the Dose-Escalation Stage provide available archival tumor tissue during the screening period (if archival tissue is not available, fresh tumor biopsy material is provided if the biopsy can be safely performed per investigator discretion).
  • Subjects in the Cohort-Expansion Stage are required to provide tissue samples (archival or fresh biopsy tumor tissue) during the screening period.
  • Subjects in both stages may also provide a tumor tissue from a fresh biopsy optionally during the treatment period.
  • EC Endometrial Cancer
  • EOC epithelial ovarian cancer
  • HR+ BC HR-positive breast cancer
  • IV intravenously
  • mCRPC metastatic castration-resistant prostate cancer
  • NSCLC non-small cell lung cancer
  • RD recommended dose
  • SCC squamous cell carcinoma
  • SCCHN squamous cell carcinoma of the head and neck
  • TA tumor agnostic
  • TF+ tissue factor-positive
  • TBD to be determined
  • TNBC triple-negative breast cancer.
  • a All subjects treated at a single 25A3-LT-A dose level in the Dose-Escalation Stage are defined as a cohort (e.g., Cohort A1 includes all subjects treated at the 25A3-LT-A starting dose).
  • c Approximately 41 subjects enroll across nine targeted dose level increments. If additional intermediate doses are explored the total sample size may be expanded by up to 6 subjects per additional intermediate dose (a maximum of 2 intermediate doses may be explored). Additional subjects may also be added at any dose level being evaluated (up to 6 additional subjects for a total of 12 at that dose level) if the Cohort Review Committee concludes that additional safety data should be obtained at this dose level.
  • the TA TF+ cohort is opened at selected sites and/or countries.
  • Table 4B Enrollment Estimates for Combination-Therapy Evaluation EOC, epithelial ovarian cancer; IV, intravenously; NSCLC, non-small cell lung cancer; q3w, once every three weeks; RD, recommended dose; SCC, squamous cell carcinoma; SCCHN, squamous cell carcinoma of the head and neck; TBD, to be determined.
  • a All subjects treated at a single 25A3-LT-A dose level in the Dose-Escalation Stage are defined as a cohort (e.g., Cohort AN1 includes all subjects treated at the 25A3-LT-A plus nivolumab starting dose).
  • the starting dose level of 25A3-LT-A combination-therapy is at a dose deemed safe by the Cohort Review Committee based on the experience in the 25A3-LT-A Single-Agent Dose-Escalation Stage.
  • c Approximately 24 subjects enroll across three targeted dose level increments for each combination regimen. If additional intermediate doses are explored, the total sample size may be expanded by up to 6 subjects per additional intermediate dose (a maximum of 2 intermediate doses may be explored). Additional subjects may also be added at any dose level being evaluated (up to 6 additional subjects for a total of 12 at that dose level) if the Cohort Review Committee concludes that additional safety data should be obtained at this dose level.
  • d At the RD for 25A3-LT-A.
  • All subjects receive 25A3-LT-A as a single 30-minute IV infusion q3w. Subjects continue treatment until a discontinuation criterion is met. After the last follow-up visit subjects are followed every 12 weeks to obtain information on subsequent anticancer therapy and survival.
  • 25A3-LT-A single-agent therapy evaluation during the Single-Agent Dose-Escalation Stage, subjects with advanced solid tumors are treated with 25A3-LT-A at an estimated 9 dose levels using an i3+3 design. The MTD is established at the end of the Dose-Escalation Stage.
  • 25A3-LT-A combination therapy evaluation an additional, independent, dose-escalation evaluation is performed to establish an MTD for different combination regimens of 25A3-LT-A.
  • 25A3-LT-A combined with nivolumab or bevacizumab is evaluated in subjects with advanced solid tumors during the Combination-Therapy Dose-Escalation Stage.
  • an i3+3 design is utilized, and the starting 25A3-LT-A dose level is a dose level deemed to be safe by the Cohort Review Committee based on experience in the Single-Agent Dose- Escalation Stage.
  • Dose-Escalation Stage with 25A3-LT-A administered as a single agent and in combination with nivolumab or bevacizumab to subjects with advanced solid tumors, an i3+3 study design is used to identify the MTD of 25A3-LT-A.
  • Dose escalation consists of ascending dose levels of 25A3-LT-A administered to subjects with advanced solid tumors. Dose level decisions are made by the Cohort Review Committee after review of all available safety data. Dose escalation for combination therapy is initiated separately at a time and at a dose of 25A3-LT-A deemed safe and appropriate by the Cohort Review Committee and may occur before an MTD for single agent 25A3-LT-A is determined.
  • the starting 25A3-LT-A single agent dose level of the study is 0.16 mg/kg given every 3 weeks as an infusion (IV q3w).
  • the dose increment between two dose levels depends on emerging pharmacokinetic and safety data from the current and previous dose level cohorts.
  • the Dose- Escalation Stage evaluates 9 dose levels (0.16, 0.5, 1.0, 1.5, 2.0, 2.25, 2.5, 2.75, and 3.0 mg/kg). The dose levels above 1 mg/kg were selected based on the safety and PK profile from dose levels ⁇ 1 mg/kg.
  • the 3-week period (21 days) after the first dose comprises the DLT Evaluation Period.
  • Dose escalation of 25A3-LT-A in combination therapy is conducted in parallel with the single-agent 25A3-LT-A dose escalation and initiated at a starting dose level deemed safe by the Cohort Review Committee.
  • the dose levels of 25A3-LT-A evaluated in the Combination-Therapy Dose-Escalation Stage is informed by the same planned dose levels in the Single-Agent Dose-Escalation Stage.
  • i3+3 Dose-Escalation Study Design The i3+3 design (Liu et al 2020) is used to identify the MTD of 25A3-LT-A as a single-agent and combination therapy. Dose-escalation/de-escalation decisions are based on events occurring during the DLT-Evaluation Period and are guided using the i3+3 algorithm described below.
  • Subjects are deemed evaluable for purposes of making dose-escalation decisions if they experience a DLT or complete the DLT-Evaluation Period without experiencing a DLT; subjects who do not complete the DLT-Evaluation Period for reasons other than safety are be considered evaluable.
  • Enrollment cohorts of 3 subjects are treated and observed, and decisions about dose level escalation, de-escalation, and expansion for subsequent cohorts are based upon a comparison of observed DLT rates for all evaluable subjects treated at a given dose level against the El (although the actual algorithm is more sophisticated, accounting for the amount of available information at the time of each decision).
  • Accrual to the Single-Agent Dose- Escalation Stage ends when either 12 evaluable subjects are observed in the dose level indicated as the next step in the enrollment algorithm or 30 evaluable subjects in the standard Dose-Escalation cohorts across the last five planned dose levels (A5- A9) are treated and observed in the stage for a regimen, whichever comes first.
  • the Cohort Review Committee is allowed to stop or limit enrollment at a dose level even if the decision rules would allow to continue with enrollment of a 3-subject cohort at that dose level.
  • the decision rules serve as a guide for the Cohort Review Committee to determine the dose level for enrollment of successive cohorts.
  • Dose escalation decisions according to the i3+3 design i.e. , “E” decisions
  • E decisions
  • the Cohort Review Committee may choose to stop or limit enrollment at a dose level and de-escalate (even with fewer than 3 evaluable subjects) if emerging data indicate a safety risk at that dose level.
  • evaluation of a treatment regimen may be stopped early if it becomes clear a tolerable dose level does not emerge using available dose levels.
  • Additional subjects may be added at any dose level being evaluated if the Cohort Review Committee concludes that additional safety data should be obtained at this dose level.
  • the Cohort Review Committee may adjust the total number of enrolled subjects following the i3+3 design if there are more than nine targeted dose escalation level increments in the single-agent evaluation, more than three targeted dose levels increments in the combination therapy evaluation, or more subjects than anticipated are enrolled at an individual dose level.
  • the benefit of the approach is that it allows for additional information to be gathered at lower doses while also providing a mechanism for accounting for this information during dose escalation.
  • DLT events observed in backfill cohorts are incorporated into dose level decision.
  • a decision of De-escalate or DU i.e. , de-escalate and not use the dose level again in the study
  • the outline of the approach is characterized below.
  • the “standard Dose-Escalation cohorts” are denoted as the cohorts of subjects for the Dose-Escalation, and the “backfill cohorts” as the cohorts that are used to further characterize the safety of lower doses. When a dose has 12 or more subjects in total, that dose is deemed “full”.
  • a cohort consists of three subjects and dose d is currently used for treating subjects in the standard Dose-Escalation cohorts. After the enrollment of a cohort at dose d completes, subjects are allowed to backfill at the dose (d - 1), one level below dose d , as long as dose (d - 1) is not full and dose d has been used to treat more than three subjects (e.g., dose d has experienced a Stay decision).
  • Intermediate Dose Level Cohorts may be inserted between previously opened dose levels and evaluated at the request of the Cohort Review Committee based on emerging safety and PK data.
  • MTD Determination Once all the enrolled subjects complete their evaluation for DLTs during the DLT-Evaluation Period (or have been deemed not evaluable) and a criterion for stopping further enrollment in the standard Dose- Escalation cohorts is met, statistical analysis by isotonic regression is used to select the MTD based on the observed DLT data from all the dose levels (Liu et al., Journal of Biopharmaceutical Statistics. 30(2):294-304 (2020)).
  • the Cohort Review Committee endorses the MTD of 25A3-LT-A as a single agent and in combination with nivolumab or bevacizumab
  • RD Determination Independent of MTD determination and upon review of all available safety, PK, and preliminary clinical activity data, the Cohort Review Committee chooses one recommended dose (RD) level of 25A3-LT-A at which the corresponding expansion cohort subjects are treated. For each treatment regimen, the 25A3-LT-A RD is the MTD or a lower dose level at the Cohort Review Committee’s discretion.
  • Intra-subiect Dose-Escalation With Sponsor approval, prior to establishing the MTD the Cohort Review Committee may allow an intra-subject dose escalation of a subject to a safe 25A3-LT-A single-agent or combination therapy dose level (that is, DLT evaluation has been completed for at least 3 subjects with no reported DLTs) if the subject has received 6 doses of 25A3-LT-A study treatment at the assigned dose and has not experienced unacceptable side effects (e.g., ocular toxicity) per Investigator assessment in discussion with the Medical Monitor or a treatment-emergent toxicity > Grade 2.
  • a safe 25A3-LT-A single-agent or combination therapy dose level that is, DLT evaluation has been completed for at least 3 subjects with no reported DLTs
  • Treatment Extension Period After completing the 21 -day DLT-Evaluation Period, subjects have the opportunity to continue to receive study treatment, including the combination therapy agent, if applicable, in the Treatment Extension Period for up to 24 months, provided they do not have radiographic progressive disease (PD) or unacceptable toxicity. Subjects may continue to receive 25A3-LT-A for more than 24 months if the subject is deriving clear clinical benefit as determined by the Investigator and agreed by the Sponsor.
  • PD radiographic progressive disease
  • Dose-Limiting Toxicities Dose-limiting toxicity (DLT) is determined by the Cohort Review Committee upon review of all available data.
  • 25A3-LT-A is delayed until the toxicity resolves.
  • subjects who recover within 21 days are allowed to resume 25A3-LT-A at a maximum dose of one dose level below the dose that produced the DLT. If the reduced dose is tolerated without DLT or other unacceptable toxicity, the subject is eligible to proceed to the Treatment Extension Period at the reduced dose.
  • Dose-Escalation Stage Study Visits Subjects in the Dose-Escalation Stage visit the clinic for study assessments during study periods as follows: PreTreatment Period (Screening), DLT-Evaluation Period (Days 1-21 ), Treatment Extension Period (Day 22+), and Post-Treatment Period.
  • PreTreatment Period Screening
  • DLT-Evaluation Period Days 1-21
  • Treatment Extension Period Day 22+
  • Post-Treatment Period Post-Treatment Period.
  • DLT-Evaluation Period Days 1-21
  • DLTs are determined by the Cohort Review Committee upon review of all available data and are defined above.
  • subjects receive treatment with 25A3-LT-A as a single-agent or combination therapy and are monitored for safety (including laboratory assessments) and signs of toxicity at Study Safety Visits (SSVs).
  • Subjects may continue to receive study treatment until radiographic PD as assessed by the investigator or any other treatment discontinuation criteria are met for up to 24 months with the agreement of the Sponsor.
  • Subjects may continue to receive 25A3- LT-A for more than 24 months if the subject is deriving clear clinical benefit as determined by the Investigator and agreed by the Sponsor.
  • PostTreatment Follow-Up Visits FU
  • two post-treatment follow-up safety visits occur 30 (+14) days and 60 (+14) days after the date of the decision to discontinue study treatment.
  • Subjects administered nivolumab have a third post-treatment follow-up safety visit 100 (+14) days after the date of the decision to discontinue study treatment. If a related AE leading to study treatment discontinuation or related SAE is ongoing at the last follow-up visit, it is to be followed until considered resolved or irreversible.
  • NPACT nonprotocol anticancer therapy
  • the Investigator or designee makes contact with the subject every 12 weeks after the post-treatment follow-up visits until the subject expires, withdraws consent for such contacts, or the Sponsor decides to cease collecting these data for the study. Radiographic tumor imaging is performed per the protocol-defined schedule until disease progression per RECIST 1.1 , initiation of subsequent anticancer therapy, or death.
  • Cohort-Expansion Stage After the RD of 25A3-LT-A as a single-agent or combination therapy is determined in the Dose-Escalation Stage, the corresponding Cohort-Expansion Stage is initiated. The Cohort-Expansion Stage further explores safety, tolerability, and preliminary efficacy of 25A3-LT-A as single-agent and combination therapy in multiple tumor-specific expansion cohorts and a TA TF+ cohort.
  • Subjects with advanced NSCLC, EC, EOC, cervical cancer, SCCHN, pancreatic cancer, esophageal SCC, mCRPC, TNBC, or HR+ BC, or TA TF+ are enrolled in the Single-Agent Cohort-Expansion Stage and are treated at the RD of 25A3-LT-A monotherapy.
  • Subjects with NSCLC, SCCHN, or esophageal SCC are enrolled in the Combination Therapy Cohort-Expansion Stage and treated at the RD of 25A3-LT-A plus nivolumab combination therapy.
  • Subjects with EOC may be enrolled in the Combination Therapy Cohort-Expansion Stage and treated at the RD of 25A3-LT-A plus bevacizumab.
  • the following cohorts enroll according to a Simon’s 2-Stage design: EC, SCCHN, pancreatic cancer, esophageal SCC, mCRPC, TNBC, and HR+ BC.
  • the TA TF+ cohort evaluates tumors other than those included in the tumor-specific expansion cohorts without interim futility testing.
  • Two dose levels (RD and a dose lower than RD [RD-low]) are explored in select single-agent tumor cohorts (NSCLC, EOC, and cervical cancer) with no interim futility analysis. Subjects are enrolled into the appropriate open cohorts following the eligibility review process by the Sponsor.
  • Subjects are randomized if a subject is eligible for more than one study cohort. A cohort may be closed to enrollment at any time at the Sponsor’s discretion.
  • Cohort-Expansion Stacie Study Visits Each subject’s course of treatment in expansion cohorts consists of the following periods: Pre-Treatment Period (Screening), Treatment Period (Day 1 +), and Post-Treatment Period.
  • subjects receive treatment with 25A3-LT-A as a single-agent or combination therapy and are monitored for safety (including laboratory assessments) and signs of toxicity at study safety visits (SSVs).
  • Subjects may continue to receive study treatment until radiographic PD as assessed by the investigator or any other treatment discontinuation criteria are met for up to 24 months with the agreement of the Sponsor.
  • Subjects may continue to receive 25A3- LT-A for more than 24 months if the subject is deriving clear clinical benefit as determined by the Investigator and agreed by the Sponsor.
  • Treatment Discontinuation Criteria Subjects receive study treatment until treatment discontinuation. Subjects may discontinue study treatment and assessments or withdraw their consent to participate in the study at any time without prejudice. For subjects who discontinue study treatment, every effort is made to undertake protocol-specified follow-up procedures including end of treatment assessments, survival follow-up, and documentation of subsequent anticancer treatment(s) unless consent for non-interventional study assessments is also withdrawn.
  • Tumor Biopsies Subjects in the Dose-Escalation and Cohort-Expansion Stages provide available archival tumor tissue. If archival tumor tissue is not available, a fresh tumor biopsy may be collected prior to first dose with subject consent, and if it is safely accessible per investigator medical judgement. Subjects in the Cohort-Expansion Stage are required to provide tissue samples (archival or fresh biopsy tumor tissue) during the screening period. Biopsy locations for study purposes should obtain adequate material for histology with the lowest procedural risk to the subject.
  • Fresh tumor biopsies should follow standard clinical practice and procedural risks must be carefully assessed. These procedural risks include, but are not limited to, factors which may indicate a higher bleeding risk (i.e. , low platelet counts, anticoagulation therapy). In order to determine response per RECIST 1.1 , target lesions in these subjects must not be biopsied. During the screening period, fresh tumor biopsies should be performed at least 7 days before first dose of 25A3- LT-A treatment. For both the Dose-Escalation and Cohort-Expansion Stages, an optional on-treatment biopsy may be performed at Day 15. For on-treatment biopsies, any procedure-associated AEs must have resolved prior to the next 25A3- LT-A administration. Subjects are closely followed for adequate wound healing after the tumor biopsy.
  • Study Committees The Sponsor engages a Cohort Review Committee, Study Oversight Committee (SOC), corporate safety governance, and Blinded Independent Radiology Committee (BIRC) to review safety, PK, and efficacy data for this study.
  • SOC Study Oversight Committee
  • BIRC Blinded Independent Radiology Committee
  • End of Tria is defined as the later of two dates: the date of the last study visit or procedure for the last subject remaining or the date at which the last data point required for follow-up for the last subject is obtained.
  • Target Population Subjects in this study must have received standard life-prolonging therapies or are not qualified to receive such therapies.
  • Inclusion criteria include the presence of a cytologically or histologically and radiologically confirmed solid tumor that is inoperable, locally advanced, metastatic, or recurrent.
  • an inclusion criterion is that the subject has received standard lifeprolonging therapies unless they do not exist, or available therapies are intolerable or no longer effective.
  • an inclusion criterion is that the subject has received standard lifeprolonging therapies unless they do not exist, or available therapies are intolerable or no longer effective.
  • Cohort-Expansion Stage Cohorts B - K, BN, DB, FN, and HN
  • an inclusion criterion is that the subject has received standard lifeprolonging therapies unless they do not exist, or available therapies are intolerable or no longer effective.
  • Cohort B Non-small Cell Lung Cancer
  • Stage IV NSCLC non-squamous cell or squamous cell histology
  • Subjects without an actionable genetic alteration must have received platinum-containing chemotherapy and ICI therapy, administered concurrently or sequentially for metastatic disease.
  • Subjects who are not qualified to receive ICI therapy are eligible if they have received platinum-containing doublet chemotherapy.
  • Subjects with an actionable genetic alteration must have received at least one molecularly targeted therapy for that specific genetic alteration unless such therapy is not available or the subject is not qualified to receive such therapy.
  • Subjects must have received no more than 3 lines of prior systemic anticancer therapy for locally advanced or metastatic disease (e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination).
  • Hormonal therapy is not counted towards the maximum allowed number of prior therapies.
  • Cohort E Cervical Cancer
  • subjects with persistent, recurrent or metastatic carcinoma of the uterine cervix squamous cell or adenocarcinoma histology
  • Subjects must have received platinum-containing chemotherapy for recurrent or metastatic disease.
  • Subjects who have received no more than 2 lines of prior systemic anticancer therapy for locally advanced or metastatic disease e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination. Chemoradiation therapy is not counted towards the maximum allowed number of prior therapies.
  • Cohorts F and FN included are subjects with head and neck cancer (squamous cell histology) who have documented radiographic disease progression during or following their last systemic anticancer therapy.
  • Allowed primary tumor locations are oral cavity, oropharynx, hypopharynx, glottic larynx. Excluded are subjects with primary tumor site of the nasopharynx. Subjects must have received prior platinum-containing chemotherapy (including chemoradiation) for inoperable locally advanced, recurrent, or metastatic disease. Subjects must have also received ICI therapy and/or EGFR inhibitor therapy, if locally considered as standard of care and if eligible. Subjects who have received no more than 3 lines of prior systemic anticancer therapy for inoperable locally advanced, recurrent, or metastatic disease setting (e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination) are included.
  • prior platinum-containing chemotherapy including chemoradiation
  • ICI therapy and/or EGFR inhibitor therapy if locally considered as standard of care and if eligible.
  • Subjects who have received no more than 3 lines of prior systemic anticancer therapy for inoperable locally advanced, recurrent, or metastatic disease setting e
  • pancreatic cancer adenocarcinoma histology
  • Subjects must have received prior treatment with gemcitabine in combination with albumin-bound paclitaxel (nab-paclitaxel) or FOLFIRINOX, or have documented ineligibility for standard therapy.
  • esophageal cancer squamous cell histology
  • Subjects with esophageal adenocarcinoma and adenocarcinoma of gastroesophageal junction (GEJ) are excluded.
  • Subjects must have received prior treatment with chemotherapy such as fluorouracil/capecitabine/taxane in combination with platinum and ICI therapy, if locally considered as standard of care and if eligible, administered concurrently or sequentially for inoperable locally advanced, recurrent, or metastatic disease.
  • Subjects who have received no more than 2 lines of prior systemic anticancer therapy for inoperable locally advanced, recurrent, or metastatic disease setting e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination
  • metastatic disease setting e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination
  • PSA progression Prostate specific antigen (PSA) progression defined by a minimum of 2 rising PSA values from 3 or 4 consecutive assessments with an interval of at least 7 days between assessments.
  • the screening PSA value must be at least 2 ng/mL (2pg/L) and the oldest qualifying value must have been based on a blood sample drawn no longer than one year prior to signing of the informed consent form (ICF) with no change in systemic regimen for the treatment of prostate cancer; up to one PSA decrease is permitted as long as it is not the most recent value.
  • the study laboratory is the local laboratory at which the subject’s previous PSA blood samples were drawn, then the screening local lab PSA must be the highest, (ii) Radiographic progression in soft- tissue disease. Subjects with bone only disease are eligible if they can be evaluated by imaging either of new lesions on technetium bone scan or new I progressing osteolytic lesion on CT scan.
  • Subjects must have received at least one prior novel hormonal therapy (NHT; e.g., enzalutamide, apalutamide, darolutamide, abiraterone, or equivalent) for locally advanced or metastatic castration-sensitive prostate cancer (mCSPC) or mCRPC.
  • NHT nuclear-hydroxy-3-phosphate
  • Subjects may have received one prior taxane-based chemotherapy (e.g., docetaxel, cabazitaxel) for mCRPC or mCSPC.
  • Subjects should have received no more than 3 prior systemic therapies for mCSPC and/or mCRPC.
  • TNBC Cohort J
  • ER- estrogen receptor negative
  • PR- progesterone receptor negative
  • HER-2- human epidermal growth factor receptor 2 negative or low
  • HER-2 negativity is defined as either of the following by local laboratory assessments: in situ hybridization (ISH) non-amplified (ratio of HER-2 to CEP17 ⁇ 2.0 or single probe average HER-2 gene copy number ⁇ 4 signals/cell), or IHC 0 or IHC 1 +.
  • HER-2 low defined as an IHC score of 1 + or 2+ with a negative ISH test. If more than one test result is available and they do not all meet the inclusion criterion definition, there should be discussion with the Sponsor to establish eligibility of the subject.
  • Subjects must have received at least one line of prior systemic chemotherapy (anthracycline-, alkylator-, or taxane-based chemotherapy) but no more than three lines of prior systemic anticancer therapies for locally advanced or metastatic disease (e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy).
  • prior systemic chemotherapy anthracycline-, alkylator-, or taxane-based chemotherapy
  • prior systemic anticancer therapies for locally advanced or metastatic disease e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy.
  • HER-2 low defined as an IHC score of 1 + or 2+ with a negative ISH test. If more than one test result is available and they do not all meet the inclusion criterion definition, there should be discussion with the Sponsor to establish eligibility of the subject. Subjects must have postmenopausal status due to either surgical/natural menopause or ovarian suppression with gonadotropin-releasing hormone (GnRH) agonist (e.g., goserelin). Subjects must have received at least one prior endocrine anticancer therapy and one prior CDK4/6 inhibitor therapy either sequentially or concurrently for inoperable locally advanced or metastatic breast cancer.
  • GnRH gonadotropin-releasing hormone
  • Subjects must have received at least one line of prior systemic chemotherapy (anthracycline- alkylator- or taxane-based chemotherapy) but no more than three lines of prior systemic anticancer therapies for locally advanced or metastatic disease (e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy).
  • prior systemic chemotherapy anthracycline- alkylator- or taxane-based chemotherapy
  • therapies for locally advanced or metastatic disease e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy.
  • cytotoxic therapy e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy.
  • endocrine anticancer therapy e.g., fulvestrant (selective estrogen receptor degrader [SERD]), tamoxifen (selective estrogen receptor modulator [SERM]), exemestane (steroidal aromatase inhibitor [SAI]), and letrozole (non-steroidal aromatase inhibitor [NSAI
  • Cohort L Tumor-Agnostic TF-Positive Cancers
  • Participants in this cohort is at selected sites and countries based on site feasibility assessment. IHC should indicate that the subject’s tumor is TF positive using archival histological material available obtained within the last two years. Fresh biopsy for eligibility assessment should not be undertaken. Assessment of tumor TF- positivity should be undertaken locally using the Sponsor-designated commercially available monoclonal antibody used per manufacturer protocol or following the guidance provided in in a separate manual. Adequate amounts of the same archival material must also be available for submission to the Sponsor. Other eligibility criteria are applied as for other cohorts, but Investigators must initially consult with the Sponsor via the Medical Monitor, or their designate, to discuss suitability for enrollment.
  • Cohort L is exploratory, a signal-finding exercise to determine whether a larger study is indicated and also to evaluate what level of TF expression may be needed to enroll in a subsequent study. For this reason, TF expression, as determined by the sites locally, satisfies the criterion for TF-positivity and subsequent analysis of the same material for TF expression by the Sponsor permits both further examination of expression-response relationship and, additionally, the degree of variability between two different assessments. This informs the associated question regarding the need for a companion diagnostic assay. Beyond the criterion of TF- expression histologically, entry criteria into this cohort align with the other expansion cohorts in the study. The TA TF+ cohort is opened at selected sites and/or countries.
  • Subjects in the Cohort-Expansion Stage have measurable disease per RECIST 1.1 as determined by the investigator. Measurable disease at screening is not required for the following subjects: subjects in the Dose-Escalation Stage, subjects with prostate cancer (Cohort I) without soft tissue disease, or subjects with primary brain tumors, such as glioblastoma (RECIST assessments are not required for these subjects). Subjects in the Cohort-Expansion Stage must have available archival tumor tissue collected no more than 2 years prior to consent.
  • a fresh tumor biopsy is collected from subjects enrolled in the Dose-Escalation Stage and must be collected from subjects in the Cohort- Expansion Stage, at least 7 days (and up to 60 days) prior to first dose.
  • Specific requirements for tumor tissue samples are provided in the Laboratory Manual.
  • Subjects in the Cohort-Expansion Stage must have recovery to baseline or ⁇ Grade 1 severity (Common Terminology Criteria for Adverse Events version 5 [CTCAE v5]) from AEs, unless AEs are clinically nonsignificant (e.g., alopecia) or stable (e.g., Grade 1 peripheral neuropathy).
  • Subjects in the Cohort-Expansion Stage must be age 18 years or older on the day of consent.
  • Subjects in the Cohort-Expansion Stage must have an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0-1 .
  • Subjects in the Cohort-Expansion Stage must have an adequate organ and marrow function, based upon meeting all of the following laboratory criteria within 10 days before first dose of study treatment: absolute neutrophil count (ANC)
  • G-CSF granulocyte colony-stimulating factor
  • Female subjects of childbearing potential must not be pregnant at screening. Female subjects are considered to be of childbearing potential unless one of the following criteria is met: permanent sterilization (hysterectomy, bilateral salpingectomy, or bilateral oophorectomy) or documented postmenopausal status (defined as 12 months of amenorrhea in a woman over 45 years-of-age in the absence of other biological or physiological causes. In addition, females under 55 years-of-age must have a serum follicle stimulating hormone (FSH) level > 40 mlll/mL to confirm menopause). Documentation may include review of medical records, medical examination, or medical history interview by study site staff.
  • FSH serum follicle stimulating hormone
  • Receipt of any type of small molecule kinase inhibitor within 2 weeks before first dose of study treatment.
  • Receipt of any anticancer hormonal therapy within 2 weeks or within 5 half-lives of the agent, whichever is shorter, before first dose of study treatment.
  • LHRH luteinizing hormone-releasing hormone
  • antagonist e.g., relugolix
  • the subject has uncontrolled, significant intercurrent or recent illness including, but not limited to, the following conditions:
  • Acute ocular infection, acute or chronic ulcerative/cicatricial condition of conjunctiva or cornea including but not limited to ocular disorders from autoimmune diseases (e.g., mucous membrane pemphigoid, Sjogren’s syndrome), significant conjunctiva/cornea scarring (e.g., radiation keratopathy), severe dry eye disease (for Schirmer’s test read ⁇ 5 mm), history of corneal transplantation, monocularity.
  • autoimmune diseases e.g., mucous membrane pemphigoid, Sjogren’s syndrome
  • significant conjunctiva/cornea scarring e.g., radiation keratopathy
  • severe dry eye disease for Schirmer’s test read ⁇ 5 mm
  • Peripheral neuropathy (sensory and/or motor) CTCAE Grade > 2.
  • Bleeding disorder or a history of clinically significant bleeding event e.g., requiring invasive procedures or urgent transfusion) within 2 months before first dose.
  • Congestive heart failure New York Heart Association class 3 or 4, unstable angina pectoris, serious cardiac arrhythmias (e.g., ventricular flutter, ventricular fibrillation, torsades de pointes).
  • cardiac arrhythmias e.g., ventricular flutter, ventricular fibrillation, torsades de pointes.
  • Stroke including transient ischemic attack [TIA]
  • myocardial infarction or other ischemic within 6 months before first dose.
  • Thromboembolic events e.g., DVT or PE
  • Subjects must have received anticoagulation therapy and be asymptomatic at the time of first dose.
  • Tumors with a recognized risk of bleeding from vascular involvement e.g., lesions invading major pulmonary blood vessels or carotid arteries.
  • Pregnant or lactating females Pregnant or lactating females.
  • IPRs severe infusion-related reactions
  • 25A3-LT-A Injection Drug Product is administered IV over approximately 30 minutes every three weeks (q3w).
  • nivolumab is also administered by IV infusion q3w.
  • Dosing of 25A3-LT-A is based on actual body weight (mg/kg). For subjects with a body weight > 100 kg, the maximum total dose is calculated based on 100 kg body weight. Standard institutional dose rounding rules are applied when available. If not available, rounding should be based on the nearest milligram.
  • the starting dose level of 25A3-LT-A for subjects receiving combination therapy with nivolumab or bevacizumab in the Dose-Escalation Stage is a dose level of 25A3-LT-A that has been deemed safe by the Cohort Review Committee for single-agent 25A3-LT-A therapy.
  • the starting dose of 25A3-LT-A for subjects in the Combination Therapy Cohort- Expansion Stage is the RD of the combination therapy determined in the Dose-Escalation Stage.
  • 25A3-LT-A is administered q3w by IV infusion over approximately 30 minutes.
  • Subjects assigned to the 25A3-LT-A + nivolumab combination regimen also receive IV infusions of nivolumab (360 mg q3w) at the study site over approximately 60 minutes and thereafter 30 minutes unless an IRR was experienced with the previous infusion, then it remains as a 60-m inute infusion.
  • bevacizumab is administered at a dose of 15 mg/kg as an IV infusion q3w.
  • the first infusion is administered over 90 minutes. If first infusion is tolerated, the second infusion is administered over 60 minutes. Subsequent infusions are administered over 30 minutes if second infusion over 60 minutes is tolerated.
  • Bevacizumab should not be given 28 days prior to elective surgery. On days when both 25A3-LT-A and combination therapy are administered, nivolumab or bevacizumab is given first followed by 25A3-LT-A. The second infusion starts after the infusion set including filter has been changed and the subject has been observed to ensure no IRR has occurred. The time in between infusions is at least 30 minutes (from the end of the nivolumab or bevacizumab infusion to the start of the 25A3-LT-A infusion) during which subjects are monitored in the clinic. The interval between study treatments must be at least 21 days. Combination therapy dosing starts at day of first dose (SSV1/Day 1 ).
  • nivolumab and 25A3-LT-A are given without premedication for potential IRRs. Premedication for infusion reaction is allowed after the initial infusion. No bolus or IV push of nivolumab or bevacizumab is allowed.
  • 25A3-LT-A Injection Drug Product is supplied as 10 mL single-use vials with a concentration of 10 mg/mL.
  • concentration of 10 mg/mL For details regarding the preparation of the infusion refer to the Study Pharmacy Manual.
  • Example 8 A phase 1 study of the anti-tissue factor antibody-drug conjugate 25A3-LT-A in patients with advanced solid tumors: Initial results from the dose-escalation stage
  • 25A3-LT-A demonstrated antitumor activity across multiple tumor types and did not interfere with coagulation in enzymatic, cellular, and animal pre-clinical models. Further provided herein are initial results from the single-agent dose-escalation stage of the phase 1 study of 25A3-LT- A in advanced solid tumors.
  • the primary objective of the dose-escalation stage is to obtain maximum tolerated dose (MTD) and/or recommended dose (RD) of 25A3-LT-A.
  • Selected secondary endpoint objectives include assessing the safety; tolerability; and pharmacokinetics (PK), such as antibody conjugated to the payload, total antibody (conjugated and unconjugated), and free payload of 25A3-LT-A.
  • 25A3-LT-A was administered IV Q3W.
  • 25A3-LT-A dose could be delayed or reduced to the next lower dose level to manage AEs.
  • Lubricating eye drops were the only prophylaxis recommended with first dose for patients with pre-existing dry eyes; prophylactic corticosteroid eyedrops were not recommended.
  • Dose-limiting toxicity (DLT) was evaluated according to criteria including treatment-emergent AEs of potential clinical significance. The DLT evaluation period was between days 1-21 after the first infusion of 25A3-LT-A. Safety was assessed on days 1 , 2, 4, 8, 15, 21 of the DLT evaluation period; days 1 and 10 for each 21 -day treatment cycle thereafter; and 30 and 60 days after study treatment discontinuation.
  • Obser tumor types include one each of epithelial appendiceal cancer, melanoma, non-small cell lung cancer, ovarian cancer, peritoneal cancer, sarcoma, thymic cancer, and uterine cancer.
  • # Patients may have been receiving more than one anticoagulant agent.
  • Anti-Tumor Activity In this dose-escalation stage evaluating low, potentially pharmacologically inactive dose levels in a heavily pretreated patient population with unknown tumor TF expression levels, no patients had objective responses, and 9 had stable disease. Efficacy evaluation was not a primary objective in the dose-escalation stage and is further evaluated in specific tumor indications in the cohort-expansion stage. 25A3-LT-A Cmax and ALICo-t increased more than or proportionately to a dose increase from 0.16 mg/kg to 2.0 mg/kg.
  • 25A3-LT-A-related ocular toxicity occurred in 42% of patients and consisted mainly of low-grade conjunctivitis and dry eye; all events were reversible with standard therapy. No corneal AEs were seen. Lubricating eye drops were the only prophylaxis recommended.
  • PK analysis confirmed 25A3-LT-A was stable with very low levels of free payload, and the exposure increased more than or proportionately to a dose increase. Dose escalation is currently ongoing.
  • ABBREVIATIONS AE, adverse event; AUC, area under the curve; Cmax, maximum serum concentration; COVID-19, Coronavirus disease 2019; DLT, doselimiting toxicity; ECOG, Eastern Cooperative Oncology Group; IV, intravenous; LLOQ, lower limit of quantification; PK, pharmacokinetics; Q3W, every three weeks;

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Abstract

Provided herein are methods of treating solid tumors with anti-tissue factor antibody-drug conjugates.

Description

METHODS OF TREATING SOLID TUMORS WITH ANTI-TISSUE FACTOR ANTIBODY-DRUG CONJUGATES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/414,869, filed October 10, 2022, U.S. Provisional Patent Application No. 63/341 ,305, filed May 12, 2022, and U.S. Provisional Patent Application No. 63/318,384, filed March 9, 2022, the disclosure of each of which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14529-127-228_SEQ_LISTING. xml”, was created on March 3, 2023 and is 50,565 bytes in size.
BACKGROUND
[0003] Blood coagulation involves a complex set of processes that result in blood clotting. Tissue factor (TF) plays an important role in these coagulation processes.
TF is a cell surface receptor for the serine protease factor Vila (FVIIa). The TF/FVIla complex catalyzes conversion of the inactive protease factor X (FX) into the active protease factor Xa (FXa). FXa and its co-factor FVa form the prothrombinase complex, which generates thrombin from prothrombin. Thrombin converts soluble fibrinogen into insoluble strands of fibrin and catalyzes many other coagulation- related processes.
[0004] TF is over-expressed on multiple types of solid tumors. In cancer, TF/FVIla signaling can support angiogenesis, tumor progression, and metastasis.
SUMMARY
[0005] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate comprising: a. an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID NO:41), wherein the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL- CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, and b. one or more linker-toxin moieties represented by Formula I:
Figure imgf000004_0001
Formula I wherein:
## represents the point of attachment of the linker-toxin moiety to the TF antibody and the linker-toxin moiety is attached to the TF antibody through a covalent bond.
[0006] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate comprising a linker-toxin moiety of the Formula
Figure imgf000004_0002
Formula I wherein ## represents the point of attachment of the linker-toxin moiety to the TF antibody and the linker-toxin moiety is attached to the TF antibody through a covalent bond.
[0007] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate of Formula II:
Figure imgf000005_0001
Formula II wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, n is an integer greater than or equal to 1 , and the succinimidyl group is attached to the Ab through a covalent bond.
[0008] In some embodiments, n is selected from the group consisting of 1 , 2, 3, 4, and 5.
[0009] In some embodiments, n is selected from the group consisting of 2, 3, and 4.
[0010] In some embodiments, the Ab comprises: a VH that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38. [0011] In some embodiments, the Ab comprises: a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGL EWMGWIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDT AVYYCARDAGTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLL IYKAYNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLP PFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:40).
[0012] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate of Formula II:
Figure imgf000006_0001
Formula II wherein: Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3, and n is an integer greater than or equal to 1 .
[0013] In some embodiments, n is selected from the group consisting of 1 , 2, 3, 4, and 5.
[0014] In some embodiments, n is selected from the group consisting of 2, 3, and 4.
[0015] In some embodiments, the Ab comprises a VH sequence that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38.
[0016] In some embodiments, the Ab comprises a full heavy chain sequence that is
QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGLEWMGWIAPY SGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDAGTYSPFGYG MDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is
DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAYNLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NQ:40).
[0017] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate of Formula II:
Figure imgf000008_0001
Formula II wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISVWRQAPGQGLEWMG WIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDA GTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAY NLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO:40), and n is an integer greater than or equal to 1 .
[0018] In some embodiments, n is selected from the group consisting of 1 , 2, 3, 4, [0019] In some embodiments, n is selected from the group consisting of 2, 3, and 4.
[0020] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
Figure imgf000009_0001
Formula I wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3; the one or more linker-toxins are attached to the Ab through a covalent bond; and
## represents a point of attachment of the linker-toxin to the Ab.
[0021] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate composition comprising the antibody-drug conjugate disclosed herein, wherein the composition comprises a multiplicity of drugantibody ratio (DAR) species, wherein the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
[0022] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
Figure imgf000010_0001
Formula I wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISVWRQAPGQGLEWMG WIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDA GTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAY NLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
KSFNRGEC (SEQ ID NO:40), and the one or more linker-toxins are attached to the Ab through a covalent bond; and
## represents a point of attachment of the linker-toxin to the Ab.
[0023] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate composition comprising the antibody-drug conjugate disclosed herein, wherein the composition comprises a multiplicity of drug- antibody ratio (DAR) species, wherein the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8. [0024] In some embodiments, the antibody is lgG1 kappa.
[0025] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate as disclosed herein and a pharmaceutically acceptable carrier in a pharmaceutical composition. In some embodiments, the method provided herein comprises administering to the subject a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg of antibody-drug conjugate disclosed herein. In some embodiments, the method provided herein comprises administering to the subject a dose of about 0.16 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.25 mg/kg, 2.5 mg/kg, 2.75 mg/kg, or about 3.0 mg/kg of antibody-drug conjugate disclosed herein. In some embodiments, the method comprises administering to the subject the antibody-drug conjugate disclosed herein intravenously (IV) every 3 weeks.
[0026] Also provided herein are combination treatment methods further comprising administering to the subject an antibody that binds PD-1 , wherein the antibody that binds PD-1 comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL- CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from Nivolumab. In some embodiments, the antibody that binds PD-1 comprises a VH and a VL, wherein the VH and VL are from Nivolumab. In some embodiments, the antibody that binds PD-1 is Nivolumab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43 and a light chain comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, Nivolumab is administered at a dose of about 360 mg intravenously (IV) every 3 weeks.
[0027] In other embodiments, provided herein are combination treatment methods further comprising administering to the subject an antibody that binds vascular endothelial growth factor (VEGF), wherein the antibody that binds VEGF comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from Bevacizumab. In some embodiments, the antibody that binds VEGF comprises a VH and a VL, wherein the VH and VL are from Bevacizumab. In some embodiments, the antibody that binds VEGF is Bevacizumab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:45 and a light chain comprising the amino acid sequence of SEQ ID NO:46. In some embodiments, Bevacizumab is administered at a dose of about 15 mg/kg intravenously (IV) every 3 weeks.
[0028] In some embodiments, the solid tumor is a cancer. In some embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
[0029] In some embodiments, the subject is a human subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] These and other features, aspects, and advantages of the present disclosure are better understood with regard to the following description, and accompanying drawings, where:
[0031] FIGs. 1A-1D illustrates the study design as described in Examples 7 and 8.
[0032] FIG. 2 illustrates dose-escalation/de-escalation decisions per the i3+3 design for the Study as described in Example 7.
[0033] FIGs. 3A-3B provide 25A3-LT-A mean PK for the intact ADC (FIG. 3A), total antibodies (Ab, FIG. 3A), and free payload (FIG. 3B) from 0.16 to 2.0 mg/kg as described in Example 8.
DETAILED DESCRIPTION
1. Definitions
[0034] Unless otherwise defined, terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art.
[0035] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.
[0036] As used herein, the term “comprising” also specifically includes embodiments “consisting of” and “consisting essentially of” the recited elements, unless specifically indicated otherwise.
[0037] The term “about” or “approximately” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” or “approximately” indicates the designated value ± 10%, ± 5%, or ± 1 %. In certain embodiments, where applicable, the term “about” or “approximately” indicates the designated value(s) ± one standard deviation of that value(s).
[0038] The terms “Tissue Factor,” “TF,” “platelet tissue factor,” “factor III,” “thromboplastin,” and “CD142” are used interchangeably to refer to TF (e.g., human TF). TF is a cell surface receptor for the serine protease factor Vila. It is often times constitutively expressed by certain cells surrounding blood vessels and in some disease settings.
[0039] The term “antibody-drug conjugate” or “ADC” refers to a conjugate comprising an antibody conjugated to one or more cytotoxic agents, optionally through one or more linkers. The term “anti-TF antibody-drug conjugate” or “anti-TF ADC” refers to a conjugate comprising an anti-TF antibody conjugated to one or more cytotoxic agents, optionally through one or more linkers.
[0040] As used herein, the terms “TF antibody,” “anti-TF antibody” are synonymous.
[0041] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. The cytotoxic agent can be an anti-angiogenic agent, a pro-apoptotic agent, an antimitotic agent, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioactive isotope. Exemplary cytotoxic agents include calicheamycin, camptothecin, carboplatin, irinotecan, SN- 38, carboplatin, camptothecan, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, doxorubicin, etoposide, idarubicin, topotecan, vinca alkaloid, maytansinoid, maytansinoid analog, pyrrolobenzodiazepine, taxoid, duocarmycin, dolastatin, auristatin, and derivatives thereof.
[0042] A “linker” refers to a molecule that connects one composition to another, e.g., an antibody to an agent. Linkers described herein can conjugate an antibody to a cytotoxic agent. Exemplary linkers include a labile linker, an acid labile linker, a photolabile linker, a charged linker, a disulfide-containing linker, a peptidasesensitive linker, a [3-glucuronide-linker, a dimethyl linker, a thio-ether linker, and a hydrophilic linker. A linker can be cleavable or non-cleavable.
[0043] The term “immunoglobulin” refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI , CH2, and CHS. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.
[0044] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies.
[0045] The term “antigen-binding domain” means the portion of an antibody that is capable of specifically binding to an antigen or epitope.
[0046] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region. For example, when used to refer to an IgG molecule, a “full length antibody” is an antibody that comprises two heavy chains and two light chains.
[0047] The term “Fc region” means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of the complement system. The structures of the Fc regions of various immunoglobulins, and the glycosylation sites contained therein, are known in the art.
[0048] The VH and VL regions may be further subdivided into regions of hypervariability (“hypervariable regions (HVRs);” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding, and influence antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991 ) Public Health Service, National Institutes of Health, Bethesda, MD.
[0049] A “Complementary Determining Region (CDR)” refers to one of three hypervariable regions (H1 , H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH [3-sheet framework, or one of three hypervariable regions (L1 , L2 or L3) within the non-framework region of the antibody VL [3-sheet framework. CDRs are variable region sequences interspersed within the framework region sequences. CDRs are well recognized in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains. See Kabat et al., J Biol Chem, 1977, 252:6609-6616 and Kabat, Adv Protein Chem, 1978, 32:1 -75. CDRs have also been defined structurally by Chothia as those residues that are not part of the conserved [3-sheet framework, and thus are able to adapt different conformations. See Chothia and Lesk, J Mol Biol, 1987, 196:901 -917. Both the Kabat and Chothia nomenclatures are well known in the art. AbM, Contact and IMGT also define CDRs. CDR positions within a canonical antibody variable domain have been determined by comparison of numerous structures. See Morea et al., Methods, 2000, 20:267-279 and Al-Lazikani et al., J Mol Biol, 1997, 273:927-48. Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme. Such terminology is well known to those skilled in the art.
[0050] A number of hypervariable region delineations are in use and are included herein. The Kabat CDRs are based on sequence variability and are the most commonly used. See Kabat et al. (1992) Sequences of Proteins of Immunological Interest, DIANE Publishing: 2719. Chothia refers instead to the location of the structural loops (Chothia and Lesk, supra). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The Contact hypervariable regions are based on an analysis of the available complex crystal structures.
[0051] More recently, a universal numbering system ImMunoGeneTics (IMGT) Information SystemTM has been developed and widely adopted. See Lefranc et al., Dev Comp Immunol, 2003, 27:55-77. IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. The IMGT CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the "location" of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. Correspondence between the Kabat, Chothia and IMGT numbering is also well known in the art (Lefranc et al., supra). An Exemplary system, shown herein, combines Kabat and Chothia CDR definitions.
Figure imgf000016_0001
[0052] The light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (A), based on the sequence of its constant domain.
[0053] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 5, E, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: lgG1 , lgG2, lgG3, lgG-4, lgA1 , and lgA2. [0054] The term "constant region" or "constant domain" refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CHI , CH2 and CHS domains of the heavy chain and the CL domain of the light chain.
[0055] The “Ell numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.
[0056] The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject. [0057] A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
[0058] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).
[0059] With regard to the binding of an antibody to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.
[0060] The term “kd” (sec1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the koff value.
[0061] The term “ka” (M’1xse 1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the kon value.
[0062] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD = kd/ka. In some embodiments, the affinity of an antibody is described in terms of the KD for an interaction between such antibody and its antigen. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.
[0063] The term “KA” (M-1 ), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction. KA = ka/kd.
[0064] “Fc effector functions” refer to those biological activities mediated by the Fc region of an antibody, which activities may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement dependent cytotoxicity (CDC), Fc receptor binding to activate antibodydependent cellular cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP).
[0065] The term “amino acid” refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).
[0066] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. [0067] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of an antibody or pharmaceutical composition provided herein that, when administered to a subject, is effective to treat a disease or disorder.
[0068] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs and sheep. In certain embodiments, the subject is a human. In some embodiments the subject has a disease or condition that can be treated with an antibody provided herein. In some aspects, the disease or condition is a cancer.
2. 25A3-LT-A Development
[0069] Antibody-drug conjugates (ADCs) are targeted cancer therapies which consist of monoclonal antibodies (mAbs) conjugated via a linker component to cytotoxic agents (payloads). The antibody component of the ADC is directed towards a tumor-associated antigen, allowing for tumor specificity and targeted drug delivery to the tumor site. This approach is expected to result in reduced exposure of nontarget tissues to the cytotoxic agent and enhanced antitumor potency. Due to the reduced toxicity of ADC-mediated drug delivery; the therapeutic window may be broadened in comparison to traditional chemotherapy.
[0070] Tissue Factor (TF; also known as coagulation factor III [F3], thromboplastin, CD142, or platelet tissue factor) is a 45-kDa, single-chain, type I transmembrane glycoprotein that is expressed under normal physiological conditions in the subendothelial tissue. TF plays a major role in regulating the extrinsic coagulation cascade. It acts as a unique cell-associated receptor for the activated form of coagulation factor VII (FVIIa) and initiates blood coagulation after a vascular injury. In addition to regulating hemostatic balance, the signaling pathways dependent on TF contribute to angiogenesis, inflammation, atherosclerosis, tumor metastasis, and immune evasion. The full-length amino acid sequence of human TF is as follows (exemplary signal sequence = italic text; exemplary extracellular domain = underline text):
METPA WPR VPRPETA VARTLLLGWVFA Q VA GASGTTNTVAAYNLTWKSTNFKTILE WE P KP VN QVYTVQ I STKS G D WKS KC FYTTDTEC D LTD E I VKDVKQTYLARVFS YPA GNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVI PSRTVNRKSTDSPVECMGQEKGEFREI FYI I GAWFVVI I LVI I LAI S LH KC R KAGVGQ SWKENSPLNVS (SEQ ID NO:42).
The amino acid sequence of an extracellular domain of human TF is as follows: SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTE CDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTI QSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAK TNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (SEQ ID N0:41).
[0071] TF has been found to be aberrantly overexpressed in many solid tumors including cervical cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, lung cancer, breast, prostate and pancreatic cancer. TF expression has been strongly associated with disease progression and poor prognosis in solid tumors, such as poor disease outcomes, shorter overall survival, and increased metastasis.
[0072] TF may facilitate cell proliferation and tumor survival through a variety of mechanisms. Activation of PAR-2 by the TF-FVIla complex has been found to induce cell proliferation, migration, and angiogenesis via expression of VEGF and interleukin-8. Additionally, direct binding of TF to [33 and [31 integrins has been found to induce angiogenesis and cell proliferation in vitro and in vivo. Given its roles in cancer biology and aberrant expression in many tumors, direct or indirect inhibition of TF activity is considered a potential therapeutic strategy for the treatment of cancer. In addition to its expression on the cell surface of cancer cells, TF undergoes efficient internalization and lysosomal targeting, making it an attractive tumor- associated antigen for ADC-mediated internalization of a cytotoxic agent into tumor cells.
[0073] Targeting of tumor cells via a TF-specific ADC may lead to enhanced uptake of the cytotoxic agent into cancer cells resulting in direct cytotoxic activity. TF-targeting ADCs engineered with human IgG mAbs may also be able to recruit immune effector cells to cancer cells by binding effector cells through their Fc receptors to the Fc region of the antibody leading to antibody-dependent cellular toxicity (ADCC) mediated by immune effector cells. In addition, release of the cytotoxic agent of ADCs after binding to the TF protein on the cancer cell surface can lead to the death of adjacent tumor cells through a bystander effect. Preliminary safety and efficacy data of a TF-targeting ADC with monomethyl auristatin E (MMAE) as the cytotoxic payload has been generated in an early stage clinical trial. Although the clinical efficacy results were promising in heavily pretreated patients with an objective response rate of approximately 15% across different tumor types, bleeding was relatively common and included severe hemorrhagic complications, indicating that this TF-targeting ADC may interfere with the coagulation cascade and therefore, may have a narrow therapeutic window. TF-directed ADCs have shown efficacy in advanced cervical cancer, but have also been associated with bleeding adverse effects, indicating the need for improved TF-directed ADCs.
[0074] 25A3-LT-A is an ADC that has been designed to improve the therapeutic potential of ADCs targeting TF. It is composed of a human mAb (25A3) against TF that is conjugated via a cysteine residue to a novel toxin (N-acyl sulfonamide auristatin, Compound 9), using a cleavable linker. The toxin (Compound 9) and its linker are designated as LT-A. Upon target-mediated internalization, the toxin (Compound 9) is released by enzymatic cleavage. The toxin (Compound 9) is a novel auristatin that inhibits cell division by blocking the polymerization of tubulin. In preclinical studies, the anti-TF antibody in 25A3-LT-A did not affect the coagulation cascade as measured by Fxa conversion and thrombin generation assays, while still binding human and cynomolgus TF with high affinity. In a human pancreatic adenocarcinoma (HPAF-II) tumor model, 25A3-LT-A was more potent than the ADC generated with the same antibody conjugated to MMAE. In patient-derived xenograft (PDX) models, 25A3-LT-A showed excellent efficacy with significant tumor growth inhibition and, in some cases, complete regression.
3. Anti-TF Antibody-Drug Conjugates for Administration
[0075] Provided herein are methods of treating subjects with a solid tumor by administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate comprising an antibody that binds specifically to TF. In some embodiments, the cytotoxic agent is linked directly to the anti-TF antibody. In some embodiments, the cytotoxic agent is linked indirectly to the anti-TF antibody.
[0076] In some embodiments, the ADCs further comprise a linker. In some embodiments, the linker links the anti-TF antibody to the cytotoxic agent.
[0077] The number of cytotoxic agents conjugated to an antibody in an ADC is defined as the drug-antibody ratio or DAR. As is known in the art, the majority of conjugation methods yield an ADC composition that includes various DAR species, with the reported DAR being the average of the individual DAR species. Thus, when the ADCs described herein are defined as having a specific DAR, it is to be understood that the number provided represents the average of the individual DAR species in the ADC composition. In some embodiments, the ADCs provided herein have a drug-antibody ratio (DAR) of approximately 3.8. [0078] In some embodiments, provided herein is an antibody-drug conjugate of
Formula II:
Figure imgf000023_0001
Formula II wherein:
Ab is a tissue factor (TF) antibody, and n is an integer greater than or equal to 1 . In some embodiments, in the ADC of Formula II, n is an integer from 1 to 10. In some embodiments of the ADC of Formula II, n is selected from the group consisting of 1 , 2, 3, 4, and 5. In some embodiments, in the ADC of Formula II, n is an integer selected from the group consisting of 2, 3, and 4. In some embodiments, in the ADC of Formula II, the succinimidyl group is attached to the Ab through a covalent bond.
[0079] In some embodiments, the payload as used herein improves the characteristics of microtubule inhibitor payloads (e.g., by lower off-target deconjugation).
[0080] In some embodiments of the ADC of Formula II, the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3, n is an integer greater than or equal to 1.
[0081] In some embodiments of the ADC of Formula II, the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3 as shown in Table 1 .
[0082] In some embodiments of the ADC of Formula II, the Ab comprises a VH and a VL from the antibody designated 25A3 as shown in Table 1. [0083] In some embodiments of the ADC of Formula II, the Ab comprises a heavy chain (HC) and a light chain (LC) from the antibody designated 25A3 as shown in Table 2.
able 1 : Antibody 25A3-CDR Sequences
Figure imgf000025_0001
Figure imgf000025_0002
_ Exemplary CDR sequences encompass amino acids as determined by Kabat plus Chothia
23
NAI-1535656266
Table 2: 25A3 Antibody Heavy Chain and Light Chain Sequences
(variable regions in bold; cysteines involved in drug conjugation underlined)
Figure imgf000026_0002
[0084] In an embodiment, the ADCs described herein comprise an antibody that comprises a VH comprising the amino acid sequence of SEQ ID NO: 37 and a VL comprising the amino acid sequence of SEQ ID NO: 38. In an embodiment, the ADCs described herein comprise an antibody that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:39 and a light chain comprising the amino acid sequence of SEQ ID NQ:40.
[0085] In an embodiment, described herein is an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
Figure imgf000026_0001
Formula I wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH-CDR1 , a VH- CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3 from the antibody designated 25A3; the one or more linker-toxins are attached to the Ab through a covalent bond; and ## represents a point of attachment of the linker-toxin to the Ab. As used herein, the linker-toxin of Formula I is also referred to as “LT-A,” where ## represents a point of attachment to the tissue factor (TF) antibody, Ab. As used herein, the antibody-drug conjugate of Formula II, where Ab comprises the VH- CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 from the antibody designated 25A3, is also referred to as “25A3-LT-A”.
[0086] In some embodiments, provided herein is a composition comprising an ADC comprising an antibody (Ab) and one or more linker-toxins of Formula I. In an embodiment, the composition comprises a multiplicity of drug-antibody ratio (DAR) species. In some embodiments, the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
[0087] In an embodiments, provided herein is an antibody-drug conjugate comprising an antibody (Ab) and one or more linker-toxins of Formula I:
Figure imgf000027_0001
Formula I wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is
QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGLEWMGWIAPY
SGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDAGTYSPFGYG
MDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAYNLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:40), the one or more linker-toxins are attached to the Ab through a covalent bond; and ## represents a point of attachment of the linker-toxin to the Ab.
[0088] In another embodiment, described herein is an antibody-drug conjugate composition comprising an ADC of the present disclosure, wherein the composition comprises a multiplicity of drug-antibody ratio (DAR) species, wherein the average DAR of the composition is 2-4. In some embodiments, the average DAR of the composition is approximately 3.8.
[0089] In some embodiments, the method provided herein is for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
[0090] In other embodiments, provided herein is the ADC provided herein (e.g., 25A3-LT-A) for use in treating a solid tumor selected from a group consisting of non- small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration- resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumoragnostic (TA) tissue-factor-positive (TF+) cancer. [0091] In other embodiments, provided herein is a use of the ADC provided herein (e.g., 25A3-LT-A) for the manufacture of a medicament for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer.
[0092] In some embodiments, the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is urothelial cancer. In some embodiments, the solid tumor is endometrial cancer (EC). In some embodiments, the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)). In some embodiments, the solid tumor is cervical cancer (e.g., with squamous cell or adenocarcinoma histology). In some embodiments, the solid tumor is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is esophageal squamous-cell carcinoma (SCC). In some embodiments, the solid tumor is prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)). In some embodiments, the solid tumor is breast cancer, e.g., triplenegative breast cancer (TNBC) or hormone-receptor positive breast cancer (HR+BC). In some embodiments, the solid tumor is tumor-agnostic (TA) tissue- factor-positive (TF+) cancer.
[0093] In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) is administered to the subject as a single-agent therapy. In some embodiments, the dose ranges from about 1 .0 mg/kg to about 3.0 mg/kg. In some embodiments, the dose is about 0.16 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, about 2.5 mg/kg, about 2.75 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) is administered to the subject intravenously (IV) every 3 weeks. 4. Combination Therapy with Nivolumab
[0094] In another aspect, provided herein is a combination therapy with the ADC provided herein and an antibody that binds the immune checkpoint programmed death receptor-1 (PD-1) (see, e.g., UniProtKB/Swiss-Prot: Q15116 or NCBI Gene ID: 5133). PD-1 is an immune-inhibitory receptor expressed in activated T cells; it is involved in the regulation of T-cell functions, including those of effector CD8+ T cells. In addition, this protein can also promote the differentiation of CD4+ T cells into T regulatory cells. It is expressed in many types of tumors including melanomas, and has demonstrated to play a role in anti-tumor immunity.
[0095] In some embodiments, the antibody that binds PD-1 comprises a VH- CDR1 , VH-CDR2, and VH-CDR3 as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:43, and a VL-CDR1 , VL-CDR2, and VL-CDR3 as set forth in a light chain comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the antibody that binds PD-1 comprises a VH domain comprising the amino acid sequence as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:43, and a VL domain comprising the amino acid sequence as set forth in the heavy chain comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the antibody that binds PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43, and a light chain comprising the amino acid sequence of SEQ ID NO:44. In a specific embodiment, the antibody that binds PD-1 is Nivolumab (OPDIVO®), which is a fully human lgG4 antibody targeting PD-1 . The heavy chain sequence of Nivolumab is
QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHVWRQAPGKGLEWVAVIWYD GSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:43); and the light chain sequence of Nivolumab is EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:44).
[0096] In some embodiments, the combination therapy provided herein is for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer. In some embodiments, the combination therapy provided herein is for treating a solid tumor selected from a group consisting of NSCLC, SCCHN, and esophageal SCC.
[0097] In other embodiments, provided herein is the ADC provided herein (e.g., 25A3-LT-A) for use in combination with Nivolumab for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer. In some embodiments, provided herein is the ADC provided herein (e.g., 25A3-LT-A) for use in combination with Nivolumab for treating a solid tumor selected from a group consisting of NSCLC, SCCHN, and esophageal SCC.
[0098] In other embodiments, provided herein is a use of the ADC provided herein (e.g., 25A3-LT-A) for the manufacture of a medicament for treating a solid tumor in combination with Nivolumab, wherein the solid tumor is selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer. In some embodiments, provided herein is a use of the ADC provided herein (e.g., 25A3-LT-A) for the manufacture of a medicament for treating a solid tumor in combination with Nivolumab, wherein the solid tumor is selected from a group consisting of NSCLC, SCCHN, and esophageal SCC.
[0099] In some embodiments, the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is urothelial cancer. In some embodiments, the solid tumor is endometrial cancer (EC). In some embodiments, the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)). In some embodiments, the solid tumor is cervical cancer (e.g., with squamous cell or adenocarcinoma histology). In some embodiments, the solid tumor is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is esophageal squamous-cell carcinoma (SCC). In some embodiments, the solid tumor is prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)) In some embodiments, the solid tumor is breast cancer, e.g., triplenegative breast cancer (TNBC) or hormone-receptor positive breast cancer (HR+BC). In some embodiments, the solid tumor is tumor-agnostic (TA) tissue- factor-positive (TF+) cancer.
[00100] In some embodiments, the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Nivolumab at a dose of 360 mg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every 3 weeks.
[00101] In some embodiments, the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Nivolumab at a dose of 360 mg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every
3 weeks. In some embodiments, the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose of about 0.16 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, about 2.5 mg/kg, about 2.75 mg/kg, or about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Nivolumab at a dose of 360 mg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every 3 weeks.
[00102] In some embodiments, Nivolumab and the ADC provided herein (e.g., 25A3-LT-A) are administered to the subject on the same day. In further embodiments, Nivolumab is administered prior to the ADC provided herein (e.g., 25A3-LT-A) on the day both are given to the subject. Additionally or alternatively, Nivolumab and the ADC provided herein (e.g., 25A3-LT-A) are administered at least 30 minutes apart.
[00103] In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Nivolumab are both administered to the subject intravenously (IV) every 3 weeks. In some embodiments, the combination therapy is administered to the subject intravenously (IV) at least 21 days apart. In some embodiments, the combination therapy lasts no more than 2 years.
5. Combination Therapy with Bevacizumab
[00104] In another aspect, provided herein is a combination therapy with the ADC provided herein and an antibody that binds the vascular endothelial growth factor (VEGF) (see, e.g., UniProtKB/Swiss-Prot: P15692 or NCBI gene ID: 7422). VEGF is a member of the PDGF/VEGF growth factor family. It is a heparin-binding protein, which exists as a disulfide-linked homodimer. This growth factor induces proliferation and migration of vascular endothelial cells, and is essential for both physiological and pathological angiogenesis. Disruption of this gene in mice resulted in abnormal embryonic blood vessel formation. This gene is upregulated in many known tumors and its expression is correlated with tumor stage and progression. In some embodiments, the antibody that binds VEGF comprises a VH-CDR1 , VH-CDR2, and VH-CDR3 as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:45, and a VL-CDR1 , VL-CDR2, and VL-CDR3 as set forth in a light chain comprising the amino acid sequence of SEQ ID NO:46. In some embodiments, the antibody that binds VEGF comprises a VH domain comprising the amino acid sequence as set forth in a heavy chain comprising the amino acid sequence of SEQ ID NO:45, and a VL domain comprising the amino acid sequence as set forth in the heavy chain comprising the amino acid sequence of SEQ ID NO:46. In some embodiments, the antibody that binds VEGF comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:45, and a light chain comprising the amino acid sequence of SEQ ID NO:46. In a specific embodiment, the antibody that binds VEGF is Bevacizumab (AVASTIN®), which is a humanized immunoglobulin G1 (lgG1 ) antibody targeting VEGF. The heavy chain sequence of Bevacizumab is EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINT YTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHW YFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO:45); and the light chain sequence of Bevacizumab is
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:46).
[00105] In some embodiments, the combination therapy provided herein is for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer. In some embodiments, the combination therapy provided herein is for treating EOC.
[00106] In other embodiments, provided herein is the ADC provided herein (e.g., 25A3-LT-A) for use in combination with Bevacizumab for treating a solid tumor selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)), and tumor-agnostic (TA) tissue-factor-positive (TF+) cancer. In some embodiments, provided herein is the ADC provided (e.g., 25A3-LT- A) for use in combination with Bevacizumab for treating EOC.
[00107] In other embodiments, provided herein is a use of the ADC provided herein (e.g., 25A3-LT-A) for the manufacture of a medicament for treating a solid tumor in combination with Bevacizumab, wherein the solid tumor is selected from a group consisting of non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer (e.g., epithelial ovarian cancer (EOC)), cervical cancer (e.g., with squamous cell or adenocarcinoma histology), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)), and breast cancer (e.g., triple-negative breast cancer (TNBC) and hormone-receptor positive breast cancer (HR+BC)). In some embodiments, provided herein is a use of the ADC provided herein (e.g., 25A3-LT-A) for the manufacture of a medicament for treating EOC in combination with Bevacizumab.
[00108] In some embodiments, the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is urothelial cancer. In some embodiments, the solid tumor is endometrial cancer (EC). In some embodiments, the solid tumor is ovarian cancer (e.g., epithelial ovarian cancer (EOC)). In some embodiments, the solid tumor is cervical cancer (e.g., with squamous cell or adenocarcinoma histology). In some embodiments, the solid tumor is head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is esophageal squamous-cell carcinoma (SCC). In some embodiments, the solid tumor is prostate cancer (e.g., metastatic castration-resistant prostate cancer (mCRPC)) In some embodiments, the solid tumor is breast cancer, e.g., triplenegative breast cancer (TNBC) or hormone-receptor positive breast cancer (HR+BC). In some embodiments, the solid tumor is tumor-agnostic (TA) tissue- factor-positive (TF+) cancer.
[00109] In some embodiments, the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Bevacizumab at a dose of 15 mg/kg. In some embodiments, the ADC provided herein (e.g., 25A3-LT- A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
[00110] In some embodiments, the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Bevacizumab at a dose of 15 mg/kg. In some embodiments, the ADC provided herein (e.g., 25A3-LT- A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
[00111] In some embodiments, the combination therapy provided herein comprises administering to the subject the ADC provided herein (e.g., 25A3-LT-A) at a dose of about 0.16 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, about 2.5 mg/kg, about 2.75 mg/kg, or about 3.0 mg/kg. In some embodiments, the combination therapy further comprises administering to the subject Bevacizumab at a dose of 15 mg/kg. In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks.
[00112] In some embodiments, Bevacizumab and the ADC provided herein (e.g., 25A3-LT-A) are administered to the subject on the same day. In further embodiments, Bevacizumab is administered prior to the ADC provided herein (e.g., 25A3-LT-A) on the day both are given to the subject. Additionally or alternatively, Bevacizumab and the ADC provided herein (e.g., 25A3-LT-A) are administered at least 30 minutes apart.
[00113] In some embodiments, the ADC provided herein (e.g., 25A3-LT-A) and Bevacizumab are both administered to the subject intravenously (IV) every 3 weeks. In some embodiments, the combination therapy is administered to the subject intravenously (IV) at least 21 days apart. In some embodiments, the combination therapy lasts no more than 2 years.
6. Methods for Making Antibody-Drug Conjugates
[00114] The ADCs can be prepared using any suitable methods as disclosed in the art employing organic chemistry reactions, conditions, and reagents known to those skilled in the art.
[00115] In certain embodiments, described herein is a process for preparing an antibody-drug conjugate, the process comprising: (A) reacting a nucleophilic or an electrophilic group on an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID NO:41 ) with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form an Ab-linker intermediate, and reacting the Ab-linker intermediate with the -NH2 group on Compound 9:
Figure imgf000037_0001
Compound 9, to provide the antibody drug conjugate; or (B) reacting the -NH2 group on Compound 9 with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form a linker-toxin intermediate, and reacting the linker-toxin intermediate with a nucleophilic or an electrophilic group on an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID N0:41 ) to provide the antibody-drug conjugate. In some embodiments, in (A) or (B), (a) the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL- CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3; and
(b) the antibody-drug conjugate comprises one or more moieties represented by Formula I:
Figure imgf000038_0001
Formula I wherein ## represents the point of attachment of the linker-toxin moiety to the TF antibody and the linker-toxin moiety is attached to the TF antibody through a covalent bond.
[00116] In certain embodiments, the nucleophilic or electrophilic group on the Ab is a thiol or an amine. In certain embodiments of the process for preparing the ADC, the process further comprises treating the Ab with a reducing agent to reduce one or more disulfide linkages in the Ab to provide the nucleophilic thiol group. In certain embodiments, the ADCs may be prepared by a method comprising (A) (i) reacting a nucleophilic or electrophilic group on the antibody with a bifunctional linker to form an antibody-linker intermediate, or (ii) reacting a nucleophilic or electrophilic group on the antibody with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form an antibody-linker intermediate, and (B) reacting the antibodylinker intermediate with the -NH2 group on Compound 9 to provide the ADC.
[00117] In certain embodiments in which the cytotoxic agent is Compound 9, the ADCs may be prepared by a method comprising (A) (i) reacting the NH2 group on Compound 9 with a bifunctional linker to form a linker-toxin intermediate, or (ii) reacting the NH2 group on Compound 9 with a first linker component of a bifunctional linker that comprises two or more linker components followed by sequential addition of the remaining linker component(s) to form a linker-toxin intermediate, and (B) reacting the linker-toxin intermediate with a nucleophilic or electrophilic group on the antibody to provide the antibody-drug conjugate.
[00118] In some embodiments, the electrophilic or nucleophilic group on the antibody is a thiol (for example from a cysteine residue on the antibody), or an amine (for example from a lysine residue on the antibody). Compound 9 and linker-toxins comprising Compound 9 may be prepared by standard synthetic organic chemistry protocols from commercially available starting materials.
[00119] Kits
[00120] Also provided herein is a kit comprising an TF ADC as disclosed herein or the pharmaceutical composition comprising same. In some embodiments, the kit further comprises a chemotherapy and/or an anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb).
[00121] Provided herein is a kit comprising the TF ADC as disclosed herein or the pharmaceutical composition comprising same, and instructions for use. In some embodiments, the kit further comprises a chemotherapy and/or an anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb).
[00122] Also provided herein are kits comprising the TF ADC provided herein or a pharmaceutical composition comprising same, packaged into suitable packaging material. In some embodiments, the kit further comprises a chemotherapy and/or an anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb). A kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
[00123] The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).
[00124] Kits provided herein can include labels or inserts. Labels or inserts include “printed matter,” e.g., paper or cardboard, separate or affixed to a component, a kit or packing material (e.g., a box), or attached to, for example, an ampoule, tube, or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media, or memory type cards. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, and date.
[00125] Kits provided herein can additionally include other components. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Kits can also be designed for cold storage.
EXAMPLES
[00126] The following are examples of methods and compositions of the disclosure. It is understood that various other embodiments may be practiced, given the general description provided herein.
[00127] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Example 1: Synthesis of Linker-Toxin A
[00128] The following example describes the preparation of an exemplary linkertoxin (Linker-Toxin A, also referred to as LT-A) that comprises the auristatin derivative, Compound 9:
Figure imgf000040_0001
[00129] Similar protocols may be employed to prepare linker-toxins comprising other auristatin derivatives of general Formula I as described herein (see also International Patent Application Publication No. WO 2016/041082, incorporated herein by reference in its entirety). 1.1 Ethyl (2R,3R)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanoate (Compound 1)
Figure imgf000041_0001
[00130] To a stirred solution of (2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2- yl)-3-methoxy-2-methylpropanoic acid (Boc-Dap-OH, 4.31 g, 15.0 mmol) in absolute ethanol (27.0 mL) at 0°C was added thionyl chloride (3.0 mL) in a dropwise fashion. The resulting solution was allowed to warm to room temperature and progress was monitored by HPLC-MS. After 18 h, no remaining starting material was detected and the solution was concentrated to dryness under reduced pressure. The resulting oil was suspended in toluene (10 mL) and concentrated under reduced pressure two times, then suspended in diethyl ether (5 mL) and concentrated under reduced pressure two times to afford the title compound as a white solid foam (3.78 g, quant yield%). MS m/z obs. = 216.5 (M+1 ).
1.2 (3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-dimethylbutanamido)- 3-methoxy-5-methylheptanoic acid (Compound 3)
Figure imgf000041_0002
[00131] Compound 2 was prepared as described in International Patent Application Publication No. WO 2016/041082.
[00132] To a stirred solution of Compound 2 (6.965 g, 14.14 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (5.0 mL). The reaction was monitored for completion by HPLC-MS and after 40 h no starting material remained. The reaction was concentrated under reduced pressure, co-evaporated with toluene (2 x 10 mL) and dichloromethane (2 x 10 mL) to obtain a foamy white solid (6.2 g, quant yield with residual TFA). This material was dissolved in 200 mL of hot 1 :3 EtOAc: hexanes and allowed to cool to room temperature. During cooling, a precipitate formed as well as some small crystals. 5 mL EtOAc was added and the suspension was heated once again to fully dissolve the precipitate. More crystals formed on cooling to room temperature and the flask was placed at -30°C overnight. The following morning the mother liquor was decanted and the crystals rinsed with 2 x 50 mL hexanes and dried under high vacuum. Recovered 5.67 g of the title compound as a crystalline product. MS m/z obs. = 405.7 (M+1 ).
1.3 Ethyl (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3- dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3- methoxy-2-methylpropanoate (Compound 4)
Figure imgf000042_0001
[00133] To a stirred solution of Compound 3 (6.711 g, 15.37 mmol, 1 .025 equiv) in a mixture of dichloromethane (5.0 mL) and A/,A/-dimethylformamide (5.0 mL) at room temperature was added HATLI (5.732 g, 15.07 mmol, 1.005 equiv) and N,N- diisopropylethylamine (7.84 mL, 3 equiv). After stirring for 30 minutes at room temperature, a solution of Compound 1 (3.776 g, 15.00 mmol, 1.0 equiv) in a mixture of dichloromethane (1 .0 mL) and A/,A/-dimethylformamide (1 .0 mL) was added dropwise and rinsed in residual Compound 1 with an additional 3 mL of 1 :1 dichloromethane: A/,A/-dimethylformam ide. The reaction was monitored by HPLC-MS and no remaining Compound 1 was observed after 15 minutes. The reaction was concentrated under reduced pressure, diluted with ethyl acetate (~125 mL) and the organic phase was extracted with 1 M HCI (2 x 50 mL), 1 x dF (1 x 50 mL), saturated NaHCOs (3 x 50 mL), brine (25 mL). Acidic and basic aqueous layers were both washed with 25 mL EtOAc. All organics were then pooled and dried over MgSO4, filtered and concentrated to give a red oil. The residue was dissolved in a minimal amount of dichloromethane (~10 mL), loaded on to a Biotage® SNAP Ultra 360 g silica gel column (Isolera™ Flash System; Biotage AB, Sweden) for purification (20-100% EtOAc in hexanes over 10 column volumes). Fractions containing pure product were pooled to recover 7.9 g of foamy white solid. Impure fractions were subjected to a second purification on a Biotage® SNAP Ultra 100 g silica gel column and pooled with pure product to recover the title compound as a white foam solid (8.390 g, 88.3 %). MS m/z obs. = 634.7 (M+1 ).
1.4 (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3- dimethyl butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3- methoxy-2-methylpropanoic acid (Compound 5)
Figure imgf000043_0001
[00134] To a stirred solution of Compound 4 (8.390 g, 13.24 mmol) in 1 ,4-dioxane (158 mL) was added dFhO (39.7 ml) and lithium hydroxide monohydrate (1 M in H2O, 39.7 mL, 3 equiv). The reaction was stirred at 4°C and monitored by HPLC-MS for consumption of starting material, which took 3 days until only trace Compound 4 remained. During the course of the reaction, a new product, corresponding to loss of methanol (^-elimination, <2%) formed in small percentages in addition to the desired material. The reaction was acidified with the addition of 1 M aqueous HCI (50 mL) and concentrated under reduced pressure to remove the dioxane. The remaining reaction mixture was extracted with ethyl acetate (4 x 50 mL) and the organic phase was pooled, washed with brine (15 mL + 2 mL 2 M HCI), dried over MgSO4, filtered and concentrated under reduced pressure to yield a light colored oil. The oil was redissolved in diethyl ether (~50 mL) and concentrated under reduced pressure (3x) to facilitate the removal of residual dioxane, affording the title product as a stiff oil (7.81 g 97% yield with some residual dioxane and Compound 4). MS m/z obs. = 606.7 (M+1 ).
1.5 Benzyl ((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl- 3-oxo-3-((4-(2, 2, 2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin- 1- yl)-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2- yl)carbamate (Compound 7)
Figure imgf000044_0001
[00135] Compound 6 was prepared as described in International Patent Application Publication No. WO 2016/041082.
[00136] To a stirred solution of Compound 5 (7.12 g, 11 .754 mmol) in dichloromethane (20 mL) was added 2,2,2-trifluoro-N-(4-sulfamoylphenyl)acetamide (Compound 6, 4.095 g, 1.3 equiv, dissolved in 3 mL DMF), A/,A/-dimethylpyridine (1.867 g, 1.3 equiv) and A/,A/-dimethylformamide (1.5 mL) to generate a light yellow suspension. Further addition of 5 mL of DMF did not clarify the solution. A/-(3- Dimethylaminopropyl)-A/'-ethylcar odiimide hydrochloride (EDCI) (2.817 g, 1.25 equiv) was added in a single portion and the reaction was monitored by HPLC-MS. After 48 hr, reaction was no longer progressing and an additional 400 mg of EDCI was added. After 18 hr, no remaining starting material was observed and the reaction was concentrated under reduced pressure to give a yellow oil. The oil was dissolved in ethyl acetate (~150 mL) and 1 M HCI (20 mL), and the organic phase was washed with cold 2 M HCI (2 x 10 mL), saturated NaHCOs (1 x 10 mL), brine (20 mL + 5 mL 2 M HCI). Acidic and basic aqueous fractions were extracted with EtOAc (1 x 20 mL), all organic fractions were pooled, dried over MgSO4 and concentrated under reduced pressure to yield an oily crude solid (13 g). The residue was dissolved in dichloromethane (~10 mL), loaded on to a Biotage® SNAP Ultra 360 g silica gel column and purified under a 10-100% EtOAc (2% AcOH) in hexanes gradient over 12 column volumes with a 3-column volume plateau at 50% EtOAc. Fractions containing the pure product were pooled, concentrated under reduced pressure, dissolved and concentrated from toluene (2 x 10 mL) and diethyl ether (2 x 10 mL) to afford the desired product, 7.1 g of white foam solid. Impure fractions were subjected to repeat purification under shallower gradient conditions using a Biotage® SNAP Ultra 100 g silica gel column on an Isolera™ instrument. All pure fractions were pooled to recover pure product (the title compound) as a white foam solid (8.60 g, 86%). MS m/z obs. = 856.7 (M+1 ). 1.6 (S)-2-amino-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl- 3-oxo-3-((4-(2, 2, 2-trifluoroacetamido)phenyl)sulfonamido)propyl)pyrrolidin- 1- yl)-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide (Compound 7a)
Figure imgf000045_0001
[00137] Compound 7 (3.71 g, 4.33 mmol) was dissolved in 10% N,N- dimethylformamide in ethyl acetate (30 mL) in a round bottom flask containing a magnetic stirrer and fitted with a 3-way gas line adapter. The vessel was twice evacuated under reduced pressure and charged with nitrogen gas. 10% palladium on carbon (0.461 g, 0.1 equiv) was added in a single portion, the 3-way adapter was fitted to the flask, a hydrogen balloon was fitted to the adapter and the vessel twice evacuated under reduced pressure and charged with hydrogen. The reaction was allowed to stir for 2 days, over which time the hydrogen balloon was occasionally recharged. After approximately 48 h, HPLC-MS analysis indicated that no starting material remained. The reaction was diluted with methanol (20 mL) and filtered through a plug of celite. The celite was washed with methanol (2 x 50 mL). All filtrates were pooled and concentrated under reduced pressure and the resulting oil dissolved and concentrated from dichloromethane. After drying under reduced pressure, the title compound was isolated as a colorless powder (3.10 g, 99%). MS m/z obs. = 722.6 (M+1 ).
1.7 (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S, 5S)-3- methoxy-1-((S)-2-((1 R, 2R)-1-methoxy-2-methyl-3-oxo-3-((4-(2, 2, 2- trifluoroacetamido)phenyl)sulfonamido) propyl)pyrrolidin- 1-yl)-5-methyl-1- oxoheptan-4-yl)-N,3-dimethylbutanamide (Compound 8)
Figure imgf000045_0002
[00138] To a stirred solution of A/,A/-(L)-dimethylvaline (1 .696 g, 9.35 mmol) in N,N- dimethylformamide (10 mL) was added HATLI (3.216 g, 8.46 mmol) and di- isopropylethylamine (3.10 mL, 17.8 mmol). A clear yellow solution resulted after 5 minutes. Stirring was continued for an additional 10 minutes, then Compound 7a (3.213 g, 4.45 mmol) was added in a single portion. After an additional 1 h of stirring, HPLC-MS indicated that trace amounts of Compound 7a remained and the reaction was for 16 h. The reaction was then concentrated under reduced pressure, diluted with ethyl acetate (120 mL) and 40 mL 1 :1 NaHCOs (sat.): 5% LiCI and transferred to a separating funnel. The aqueous layer was removed and the organic phase was washed with LiCI (1 x 20 mL), NaHCOs (sat., 2 x 20 mL). Aqueous layers were pooled and extracted with EtOAc (3 x 50 mL). Organic layers were pooled and washed with brine (1 x 20 mL), dried over sodium sulfate, filtered and concentrated to give a DMF-laden oil which was concentrated via rotary evaporator to remove residual DMF, yielding 7 g of crude straw colored oil. The oil was dissolved in a minimal amount of 10% methanol in dichloromethane (~11 mL) and loaded onto a Biotage® SNAP Ultra 360 g silica gel column for purification (2-20% MeOH in CH2CI2 over 15 column volumes, product eluting around 10-13%). The fractions containing the desired product were pooled and concentrated under reduced pressure to afford the title compound as a colorless foam. Impure fractions were combined, evaporated and subjected to repeat purification on a Biotage® SNAP Ultra 100 g silica gel column on an Isolera™ instrument and combined with the pure product from the first column to yield a colorless foam solid (3.78 g). MS m/z obs. = 850.6 (M+1 ).
1.8 (S)-N-((3R,4S,5R)-1-((S)-2-((1R,2R)-3-((4-aminophenyl)sulfonamido)-1- methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1- oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3- dimethylbutanamide (Compound 9)
Figure imgf000046_0001
[00139] To a stirred solution of Compound 8 (0.980 g, 1.154 mmol) in 1 ,4-dioxanes (15 mL) was added water (3.5 mL) and 1 M lithium hydroxide monohydrate (3 equiv., 3.46 mL). The resulting light suspension was allowed to stir at 4°C and was monitored by HPLC-MS for consumption of the starting material. When the conversion was complete (~5 days), the reaction was neutralized with 3.46 mL of 1 M HCI and concentrated under reduced pressure to remove dioxane. The resulting aqueous phase was diluted with 60 mL EtOAc and 5 mL brine, then extracted with ethyl acetate (2 x 30 mL). The organic fractions were pooled, dried over Na2SO4, filtered and evaporated to yield the title compound as a tan solid (0.930 g). Rf = 0.5 (8% MeOH in CH2CI2). MS m/z obs. = 753.7 (M+1 ).
1.9 2,3,5,6-tetrafluorophenyl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)ethoxy)ethoxy)propanoate (Compound 15)
Figure imgf000047_0001
[00140] In a dried 50 mL conical flask, 3-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)propanoic acid (Compound 14, 1.000 g, 4.52 mmol) and maleic anhydride (0.443 g, 4.52 mmol) were dissolved in anhydrous N,N- dimethylformamide (5 mL). The reaction was stirred at room temperature for 6 hr under N2, then cooled to 0°C and syn-collidine (1 .263 mL, 2.1 eq) was added dropwise. In a separate dried 50 mL conical flask, tetrafluorophenol (3.002 g, 4 eq) was dissolved in anhydrous A/,A/-dimethylformamide (10 mL). The flask was cooled to 0°C in an ice bath and trifluoroacetic anhydride (2.548 mL, 4 eq) was added dropwise. After stirring for 15 minutes, syn-collidine (2.407 mL, 4 eq) was added dropwise. The flask was allowed to stir for another 15 minutes, and then the contents were added to the first flask dropwise, via syringe. The reaction was allowed to warm to room temperature and stirring was continued under N2. The reaction was monitored by HPLC-MS for the consumption of starting materials. After 6 days, the reaction was complete with the total consumption of Compound 14, leaving only Compound 15 and a small amount (~5%) of the bis-TFP maleic amide intermediate. The reaction was transferred to a separating funnel, diluted with diethyl ether (75 ml) and washed with 5% LiCI (1 x 20 mL), 1 M HCI (2 x 20 mL), sat. NaHCOs (5 x 20 mL) and brine (1 x 20 mL). The organic layer was dried over Na2SO4, filtered and evaporated to give brown crude oil with residual DMF. Crude oil was dissolved in 8 mL of 1 :1 DMF:H2O + 0.1% TFA, loaded onto a 60 g Biotage® SNAP Ultra C18 column (Biotage AB, Uppsala, Sweden) and purified under a linear 30-100% gradient of ACN/H2O + 0.1 % TFA over 8 column volumes. Pure fractions were pooled and diluted with brine (20 mL), then extracted 3 x 50 mL Et2O. Pooled organics were dried over MgSO4, filtered and evaporated to recover the title compound as a light-yellow oil (1 .34 g, 66% yield).
1.10
Figure imgf000048_0001
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5- methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanoyl)sulfamoyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)- 3-methyl-1-oxobutan-2-yl)carbamate (Compound 12)
Figure imgf000048_0002
[00141] Compound 11 was prepared as described in International Patent Application Publication No. WO 2016/041082.
[00142] To an empty 25 mL pear shaped flask, was added Compound 11 (1 .342 g, 3.58 mmol, 3.0 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.664 g, 3.46 mmol, 2.9 equiv) and 7-hydroxy-azabenzotriazole (HOAT) (0.472 g, 3.46 mmol, 2.9 equiv). These solids were dissolved in a mixture of N,N- dimethylformamide (0.5 mL) and dichloromethane (4.5 mL) with stirring at room temperature over 30 minutes. Separately, Compound 9 (0.900 g, 1 .20 mmol) was dissolved in a mixture of A/,A/-dimethylformamide (0.2 mL) and dichloromethane (1.8 mL) and added to the pear shaped flask, rinsing with dichloromethane (1 .0 mL). Stirring rate was increased to 1000 rpm, producing a vortex. Within 2 minutes of adding Compound 9, copper (II) chloride (0.514 g, 3.83 mmol, 3.2 equiv) was added in one portion directly into the center of the vortex through a narrow powder funnel. The initially light-yellow solution turned to a dark-brown suspension which changed over 10 minutes to a dark-green suspension. The reaction was monitored for completion by HPLC-MS and no change to reaction progress was observed between the samples taken at 30 minutes and 1 h (~95% complete). The reaction was allowed to stir overnight at room temperature, then 2-(2-aminoethylamino)ethanol (0.483 mL, 4.781 mmol, 4 equiv), EtOAc (10 mL) and dhhO (5 mL) were added to the stirred suspension, which underwent a color change to deep blue. The suspension was stirred vigorously for 4 hr as the suspended solids gradually dissolved into the biphasic mixture. This mixture was transferred to a separating funnel and diluted with EtOAc (100 mL) and brine (10 mL), and the aqueous layer was extracted using 10% IpOH/ EtOAc (4 x 50 mL). The organic layers were pooled and washed with brine (10 mL), dried over Na2SO4, and evaporated to yield a faintly blue crude solid. This crude solid was dissolved in a mixture of methanol (0.5 mL) and dichloromethane (6 mL) and purified on a Biotage® SNAP Ultra 100 g silica gel column (2-20% MeOH in CH2CI2 over 10 column volumes, followed by an 8-column volume plateau at 20% MeOH). The product eluted as a broad peak after 1-2 column volumes at ~20% MeOH in CH2CI2. Fractions containing the desired material were pooled and concentrated under reduced pressure to give the title compound as a white solid (1.105 g, 83%). MS m/z obs. = 555.9 ((M+2)/2), 1109.8 (M+1 ).
1.11 (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(N-((2R,3R)-3-((S)-1- ((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3- dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3- methoxy-2-methylpropanoyl)sulfamoyl)phenyl)-5-ureidopentanamide (Compound 13)
Figure imgf000050_0001
[00143] To a solution of Compound 12 (0.926 g, 0.834 mmol) was added a mixture of dichloromethane (10 mL) and trifluoroacetic acid (2.0 mL). The reaction was monitored by HPLC-MS for consumption of starting material (~45 minutes). The reaction was co-evaporated with acetonitrile (2 x 10 mL) and dichloromethane (2 x 10 mL) under reduced pressure to remove excess trifluoroacetic acid. The resulting residue was dissolved in a minimal amount of dichloromethane and methanol (3:1 , v/v, ~2 mL), and added to a stirred solution of diethyl ether (200 mL) and hexanes (100 mL) dropwise via pipette, producing a suspension of light white solids. The solids were filtered and dried under vacuum to afford the title compound in the form of a white powder, as the trifluoroacetate salt (1 .04 g, quantitative yield with some residual solvents). MS m/z obs. = 505.8 ((M+2)/2).
1.12 (S)-N-(4-(N-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5- methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanoyl)sulfamoyl)phenyl)-2-((S)-1-(2, 5-dioxo-2, 5-di hydro- 1H-pyrrol- 1- yl)-14-isopropyl-12-oxo-3,6,9-trioxa-13-azapentadecanamido)-5- ureidopentanamide (Linker-Toxin A) H
Figure imgf000051_0001
[00144] To a stirred solution of Compound 13 (0.722 g, 0.584 mmol) in N,N- dimethylformamide (4 mL) was added Compound 15 (0.314 g, 1.2 equiv) and diisopropylethylamine (0.305 mL, 3.0 equiv). HPLC-MS analysis at 2 h indicated no remaining starting material. The reaction was acidified with TFA (300 pL) and then diluted with dihhO + 0.1 % TFA (9 mL). The resultant solution was loaded onto a 120 g Biotage® SNAP Ultra C18 column (Biotage, Uppsala, Sweden) and purified under an ACN/H2O + 0.1 % TFA gradient: 20-60% ACN over 10 column volumes, 60-100% ACN over 5 column volumes. Product eluted near 40% ACN. Pure fractions as identified by LCMS were pooled and lyophilized. A white powder solid was recovered from the lyophilizer. The lyophilization was repeated at higher concentration (approx. 50 mg/mL in 2:1 H2O/ACN) into a vial to produce the title compound as a denser, less flocculant lyophilized solid (754.2 mg, 91 %). MS m/z obs. = 647.4 ((M+2)/2), 1292.8 (M+1 ).
Example 2: ADC Synthesis
[00145] Antibody-Drug Conjugates (ADCs) of anti-TF antibodies and Linker-Toxin A, as prepared in Example 1 , were prepared as described below (see also International Patent Application Publication No. WO 2021/003399, incorporated herein by reference in its entirety). [00146] Briefly, 5 to 10 mg/mL of 25A3 antibody (see Tables 1 and 2 for CDR and V region sequences of clone 25A3) in phosphate-buffered saline (PBS), pH 7.4 was reduced by the addition of Tris(2-carboxyethyl)phosphine (2.0-2.5 or 3.2 molar equivalents) and a final concentration of 0.8 mM diethylenetriamine-pentaacetic acid. After 2 hr at 37°C, the partially reduced antibody was cooled on ice for 10 minutes, then conjugated for 1 h with 8 molar equivalents of Linker-Toxin A on ice. The reaction was quenched with an excess of N-acetyl-L-cysteine. The quenched reaction was allowed to sit on ice for 30 minutes prior to purification. ADCs were purified through two rounds of 40kDa MWCO Zeba™ Spin Desalting Columns (10 mL Columns, Product #8772, Lot # RL240689) each, as per the manufacturer’s protocol. Prior to purification, both sets of columns was primed with sterile PBS. The ADC was purified through one set of PBS primed columns first, the sample was then collected and purified a second time through the other set. After the second purification, the ADC was pooled back together and sterile filtered and frozen at - 80°C.
[00147] Drug-antibody ratio (DAR) may be measured by UV/vis spectroscopy, hydrophobic interaction chromatography (HIC), and/or reverse phase liquid chromatography separation with time-of-flight detection and mass characterization {RP-UPLC/Mass spectrometry), as described in WO 2016/041082. Distribution of drug-linked forms (for example, the fraction of DAR0, DARI, DAR2, etc. species) may also be analyzed by various techniques known in the art, including MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF), as also described in WO 2016/041082.
[00148] For this example, the drug-antibody ratio (DAR) of the resulting ADCs was ~3. The DAR was determined by hydrophobic interaction chromatography: Average DAR = (0 x (DAR0 Area%) + 2 x (DAR2 Area%) + 4 x (DAR4 Area%) + 6 x (DAR6 Area%) + 8 x (DAR8 Area%) 1 100. Size exclusion chromatography was used to ensure the ADC preparation was at least 95% monomeric.
[00149] ADCs comprising 25A3 and LT-A (25A3-LT-A), e.g. as prepared in this example, were used in the studies of Examples 3-7 below. Example 3: ADC Formulation
[00150] A solution formulation of 25A3-LT-A (synthesized as described in Example
1 ) was prepared and used for toxicology studies and a Phase 1 clinical trial. The composition of the formulation is shown in Table 3.
Table 3: Composition of 25A3-LT-A Formulation
Figure imgf000053_0001
1HCI 1 N is used to titrate the histidine buffer to the target pH so amounts may vary from batch to batch.
Example 4: GLP Toxicology Study in Cynomolgous Monkeys
[00151] In this GLP study in male and female cynomolgus monkeys, 25A3-LT-A (synthesized as described in Example 2 and formulated as described in Example 3) was administered via IV infusion at 3, 6, 12, or 15 mg/kg/dose once every 3 weeks for a total of 4 doses. Terminal necropsy of 4 animals of each sex for each dose was on Day 65, and potential reversibility of findings was examined following a 6-week recovery period in two animals of each sex for each dose (Day 106).
[00152] There were no unscheduled deaths in this study. There were also no 25A3-LT-A-related effects on body weight, urinalysis parameters, and organ weights. Additionally, there were no qualitative effects on ECGs as determined by a board- certified veterinary cardiologist. 25A3-LT-A was tolerated at up to 12 mg/kg/dose based on adverse findings necessitating dosing interruption at 15 mg/kg/dose in one male. In this male, administration of 25A3-LT-A elicited adverse test item-related clinical observations in the eyes (discharge, eyes partly or completely closed, redness of conjunctiva, severe swelling of the eyelids and periorbital areas), which resulted in secondary observations of poor condition (hunched posture, decreased activity, and suspected dehydration) following the Day 22 administration. The observations were still present at the next dosing occasion (Day 43); therefore, the animal was not dosed on Day 43.
[00153] During the treatment period, clinical observations were noted predominantly at 12 and 15 mg/kg/dose in remaining animals with some of these occurring at 6 mg/kg/dose. These included slight to severe, soft swelling of the eyelids at > 6 mg/kg/dose and slight to severe, soft swelling of the periorbital areas at > 12 mg/kg/dose. Additional observations at > 12 mg/kg/dose included abnormal (red) eyeball color and abnormal color (yellow or clear) and/or discharge (liquid or mucoid) from the eyes. By the end of the recovery period, most of these ocular clinical observations persisted at > 12 mg/kg/dose, except for periorbital swelling. [00154] Clinical observations likely a response to the ocular effects were noted at > 12 mg/kg/dose and included hunched posture, suspected dehydration, erected fur, and decreased activity. At the end of the recovery period, dehydration, erected fur and decreased activity were no longer observed, with hunched posture persisting in males only. Thus, based on these overall observations, evidence of recovery was supported. It should be noted that in contrast to the pilot toxicology study, there were no skin findings.
[00155] Regarding food consumption effects, reduced appetite was transiently observed in all males at 15 mg/kg/dose from Days 14 to 34.
[00156] Throughout the dosing period, there were non-dose-related 25A3-LT-A- related effects on clinical pathology parameters. At > 6 mg/kg/dose, with a higher magnitude of change at > 12 mg/kg/dose, there were increases in neutrophil, lymphocyte, and monocyte counts, which were reflected in the increased total WBC count. These findings were associated with 25A3-LT-A-related increases in fibrinogen, and total protein concentrations (due to increased globulin); and decreases in albumin and albumin/globulin ratio. These changes had no clear microscopic correlates but may have been reflective of the inflammatory findings noted in the eye and lung. Sporadic individual increases in platelet counts were similarly likely associated with the inflammatory process. Increases in AST with no effects on ALT or ALP and without any microscopic correlation were noted at > 12 mg/kg/dose. There were no effects on aPTT or on PT. [00157] Following the recovery period, the hematology findings were no longer observed in males previously administered > 6 mg/kg/dose and in females previously administered 15 mg/kg/dose; however, increases in WBCs, neutrophil, and lymphocyte counts were present in females alone given < 12 mg/kg/dose. Overall, there was evidence of recovery from the effects on remaining clinical pathology parameters.
[00158] 25A3-LT-A-related histopathological findings were noted in the eyelid, eye, lung, and infusion site.
[00159] In the eyelids, there was minimal to mild erosion/ulcer of the conjunctival epithelium in males at 15 mg/kg/dose and females at > 6 mg/kg/dose accompanied by minimal to moderate hyperplasia of the conjunctival epithelium in females at > 12 mg/kg/dose and/or minimal submucosal mixed cell inflammation in 1 male at 15 mg/kg/dose and 1 female at the 12 mg/kg/dose. At the end of the recovery period, minimal erosion/ulcer, conjunctival hyperplasia, mixed cell inflammation and/or minimal to mild submucosal fibrosis were noted in the eyelids of males at > 12 mg/kg/dose and females at > 6 mg/kg/dose.
[00160] In the eyes, there was marked erosion/ulcer of the cornea in one male at 15 mg/kg/dose (with mixed cell inflammation, neovascularization and correlating with macroscopic opacity, abnormal appearance, and enlargement), minimal to mild erosion/ulcer of the conjunctiva at 15 mg/kg/dose in one male and > 12 mg/kg/dose in females. Minimal to mild hyperplasia of the conjunctival epithelium occurred in males at 12 mg/kg/dose and females at > 12 mg/kg/dose, minimal to mild epithelial atrophy of the cornea in males and females at > 3 mg/kg/dose and pigmentation of the corneal epithelium in females at > 12 mg/kg/dose. Corneal findings were considered likely secondary to those noted in the eyelids. At recovery, there was erosion/ulcer of the cornea in one of four males (mild, with pigmented corneal epithelium) and one of four females (moderate, with dorsal erosion/ulcer and/or hyperplasia of the conjunctival epithelium) at 12 mg/kg/dose and minimal corneal epithelial atrophy was noted in the right eye of one female at 12 mg/kg/dose. The incidence and/or severity of the findings were decreased compared to findings at the end of the dosing period. This is considered consistent with an ongoing recovery process.
[00161] Associated with these histopathologic findings were changes noted during ophthalmic examinations at > 6 mg/kg/dose during the dosing phase. Ophthalmic examinations were conducted at least once during each dose cycle and at the end of recovery period. 25A3-LT-A-related ophthalmology findings, first noted on Day 36, were present in eyelids, conjunctiva, and cornea at 6 mg/kg/dose, but were more pronounced at > 12 mg/kg/dose. The corneal changes (including opacities, vascularization, loss of luster, and stromal vacuoles) were most likely secondary to the adnexal changes that caused the conjunctiva, eyelids, and tear production (quantity and quality) to be abnormal. Some of the changes such as conjunctival hyperemia, adnexal discharge, corneal opacities, and vascularization, were of the type seen with inadequate tear production. Animals at 6 mg/kg/dose had fewer ocular changes than in the higher dose groups.
[00162] During the recovery period, ocular changes were still present in most eyes. 25A3-LT-A-related findings were generally similar or had improved very slightly, except for one animal, administered 12 mg/kg/dose, which had an increased seventy of corneal vascularization in its right eye. At > 12 mg/kg/dose, there were animals with irregular or abnormal appearance of the eyelids (“wavy margin” or “fold-like” or “notch-like”). These eyelid findings were likely secondary to the chronicity of the palpebral inflammation and/or irritation.
[00163] In the lungs, there was interstitial inflammation in males (minimal to mild) and females (minimal to marked) at > 6 mg/kg/dose and increased alveolar macrophages (minimal to moderate) in males at > 6 mg/kg/dose and females at > 12 mg/kg/dose. In the affected areas of inflammation, the alveolar septa were infiltrated by various degrees of mononuclear cells with lesser numbers of neutrophils and macrophages, along with alveolar type II pneumocytes hyperplasia and fibrosis. Alveolar squamous metaplasia was also noted in females at > 12 mg/kg/dose and was observed in the most severely affected animals of each respective group. [00164] At the end of the recovery period, minimal to mild interstitial inflammation and/or minimal increased alveolar macrophages in the lung were observed at > 12 mg/kg/dose. The seventy and/or incidence were decreased compared to the ones at terminal necropsy, or of similar incidence compared to controls. Additionally, there was no evidence of squamous metaplasia, which suggested nearly complete recovery from the lung findings.
[00165] At the infusion sites, there was reversible, minimal erosion/ulcer of the epidermis in males at > 6 mg/kg/dose and females at > 3 mg/kg/dose. This finding was considered related to exacerbation of the experimental procedure (IV infusion). These findings were considered non-adverse due to absence of any associated clinical observations and/or low seventy.
[00166] No gender differences in PK were observed nor was there evidence of 25A3-LT-A accumulation. Notably, exposures to the toxin of 25A3-LT-A were generally undetectable or very low in serum across all dose groups. A high incidence of ADAs against 25A3-LT-A were detected in this study; nonetheless, total antibody and intact ADC serum levels were maintained at all dose levels throughout the duration of the study. The accumulation ratios for intact ADC AUC0-21 following the last dose on Day 64 were 0.6, 0.7, 1.1 , and 1 .2 for the 3 mg/kg, 6 mg/kg, 12 mg/kg and 15 mg/kg dose groups, respectively.
[00167] Given the overall persistence of ocular findings at the end of the treatment- free period at > 6 mg/kg/dose, the highest non-severely toxic dose (HNSTD) was determined to be 3 mg/kg/dose. This dose resulted in minimal histopathological findings in the eye during the dosing phase, and no 25A3-LT-A-related histopathological or ophthalmological findings by the end of the recovery period.
[00168] In this GLP study in cynomolgus monkeys, findings of potential clinical significance were noted in the eye and eyelid during in-life observations and during serial ophthalmic evaluations (e.g., swelling, discharge, dry eye, conjunctival hyperemia, corneal opacities, and/or corneal vascularization) that also correlated with histopathological findings in the eye and in the eyelid (e.g., ulceration/erosion, hyperplasia, and/or mixed cell inflammation in conjunctival epithelium, and with secondary corneal pigmentation and/or atrophy). Additional histopathological findings were also present in the lung (e.g., interstitial inflammation).
[00169] In a pilot toxicology study in the cynomolgus monkey, in contrast to the GLP toxicology study (this Example 3), findings in the skin were noted (dry or red skin).
[00170] The proposed starting dose for the Phase 1 clinical study is 0.16 mg/kg IV q3w. This starting dose is expected to have a safety margin of 6 based on the HED, 19 based on predicted human maximum serum concentration (Cmax), and 37 based on predicted human area under the curve (AUC) compared to the HNSTD in cynomolgus monkeys. Based on PK modeling, 25A3-LT-A exposures at the proposed starting dose of 0.16 mg/kg are predicted to reach ~50% target occupancy at Cmax and < 10% target occupancy 2 days after dose. Based on mouse tumor modeling, the estimated 25A3-LT-A efficacious exposure levels ranged from 3.6 pg/mL to 10 pg/mL. Human PK simulations suggested that human doses of
2.4 mg/kg q3w are expected to achieve the average concentrations (~7 pg/mL) for tumor growth inhibition.
Example 5: Single-Dose Toxicity in Rats
[00171] A single-dose GLP rat study (a human non-relevant species) evaluated the acute toxicity and toxicokinetics of the toxin of 25A3-LT-A, when administered as a single dose to rats at doses of 1 , 3, and 6 mg/kg. An additional control group was administered the vehicle. Animals from all groups were terminated 3 days after dose administration to assess the toxicity of the toxin of 25A3-LT-A. Additional animals from the control and the 6 mg/kg groups were euthanized following a 14-day recovery period to assess the potential for recovery.
[00172] There were no mortalities and no toxin-related effects on urinalysis parameters, ophthalmic examinations, or upon gross examination at necropsy. [00173] Clinical observations were limited to two of five female rats that received the toxin of 25A3-LT-A at a dose of 6 mg/kg and included red discolored hair on the face, scabbed forefeet, red discolored skin on forefeet and hind feet, red discolored tail, and/or black/red material around the right eye. These findings were reversible by the end of the 14-day recovery period.
[00174] Clinical pathology changes included marked decreases in reticulocytes in both sexes at 3 and 6 mg/kg and mild to moderate decreases in neutrophils and eosinophils at 6 mg/kg, which correlated with decreased hematopoietic cellularity noted microscopically. Mild to moderate decreases in lymphocytes were noted at 6 mg/kg which correlated to lymphoid depletion observed microscopically in multiple lymphoid tissues. Evidence of a mild inflammatory response was observed at 3 and 6 mg/kg as indicated by increases in fibrinogen at 3 and 6 mg/kg, increased globulin at 6 mg/kg, and minimal prolongations in prothrombin times in both sexes at 6 mg/kg.
[00175] Histopathologic findings were generally consistent with those anticipated from effects of the free toxin of 25A3-LT-A. These effects were characterized by reversible microscopic findings in the bone marrow, spleen, thymus, lymph nodes, and exorbital lacrimal gland.
[00176] In the bone marrow, decreased hematopoietic cellularity affecting all hematopoietic cell lines was observed in both sexes at 6 mg/kg and correlated with decreased reticulocytes, neutrophils, lymphocytes, and eosinophils in both sexes. Decreased extramedullary hematopoiesis was observed in the spleen in males only at 6 mg/kg. In the thymus, generalized lymphoid depletion characterized by a relative reduction in the number of lymphocytes in all lymphoid compartments was observed in both sexes at > 3 mg/kg and in the mandibular lymph node of both sexes at 6 mg/kg and mesenteric lymph node of males at 6 mg/kg and in females at > 3 mg/kg. In the exorbital lacrimal glands of males at > 3 mg/kg and in females at > 1 mg/kg, findings included mild to moderate atrophy and minimal to mild single-cell necrosis. All of these findings were considered reversible, as there were no microscopic findings in any tissues at the recovery necropsy.
Example 6: Genotoxicity
[00177] The toxin of 25A3-LT-A was negative in the bacterial reverse mutation assay. Consistent with the mechanism of action for the toxin of 25A3-LT-A, which is an anti-mitotic agent that inhibits cell division by blocking the polymerization of tubulin, the toxin was positive in both the in vitro and in vivo micronucleus assays.
Example 7: A Dose-Escalation and Expansion Study of the Safety and Pharmacokinetics of Anti-TF ADC as Singe-Agent Therapy or in Combination Therapy in Subjects with Inoperable Locally Advanced or Metastatic Solid Tumors
[00178] This First-in-Human (FIH) Phase 1 clinical study evaluates the safety, tolerability, pharmacokinetics (PK), and preliminary antitumor activity of 25A3-LT-A with or without nivolumab or bevacizumab in subjects with advanced solid tumors for whom life-prolonging therapies do not exist or available therapies are intolerable or no longer effective.
[00179] The initial dose and schedule of 25A3-LT-A for this FIH Phase 1 study have been calculated based on nonclinical PK and toxicity data in animals. In accordance with International Conference on Harmonization (ICH) S9 Guidance, to estimate the starting dose a safety factor of 6 was applied to the human equivalent dose (HED) of the HNSTD of 3 mg/kg/dose from the 10-week GLP repeat-dose toxicology study in cynomolgus monkeys. The starting dose in this FIH Phase 1 clinical study is 0.16 mg/kg given intravenously (IV) every 3 weeks (q3w). The Dose- Escalation Stage evaluates dose levels up to 3 mg/kg. At dose levels < 1 mg/kg, the maximum tolerated dose (MTD) was not reached, and the safety and PK profile supported evaluation of 25A3-LT-A at dose levels > 1 .0 mg/kg, as determined by the Cohort Review Committee.
[00180] In the initial Dose-Escalation Stage, the MTD of 25A3-LT-A given as a single agent and in combination with nivolumab or bevacizumab was determined using an interval 3+3 (i3+3) design (Liu et al 2020). After 25A3-LT-A had been administered to 14 subjects across 4 dose-level cohorts in the Dose-Escalation Stage: Cohort A1 : n = 3, 0.16 mg/kg IV q3w; Cohort A2: n = 3, 0.5 mg/kg IV q3w; Cohort A3: n = 6, 1.0 mg/kg IV q3w; and Cohort A4: n = 2, 1.5 mg/kg IV q3w, no dose-limiting toxicities (DLTs) were observed. Cumulative safety data observed showed that the majority (71%) of reported adverse events (AEs) were of low grade seventy (Grade 1-2). The only AEs reported in more than 1 subject were Grade 1 nausea, which was reported for 3 subjects (1 subject in Cohort A2 and 2 subjects in Cohort A3), and Grade 1 abdominal pain, which was reported for 2 subject (1 subject each in Cohorts A1 and A3). No DLTs were observed. Three subjects experienced a Grade 3 AE. Grade 3 hypertension was reported for 1 subject in Cohort A1 with a history of hypertension and considered unrelated to study treatment. Grade 3 COVID-19 pneumonia and Grade 3 diarrhea were reported for 1 subject each in Cohort A3 (1.0 mg/kg) after the dose-limiting toxicity (DLT)-Evaluation Period and was considered unrelated to study treatment. These AEs resulted in hospitalization and, as such, were considered serious AEs (SAEs). Four subjects (29%) experienced AEs that were considered related to study treatment: Grade 1 chills (Cohort A2), Grade 1 nausea (Cohort A3), Grade 1 fatigue (Cohort A4), and Grade 1 dry eye, Grade 1 conjunctivitis, and Grade 2 pseudomembrane (reported for the same subject in Cohort A3). In addition, two subjects experienced AEs that led to discontinuation of study treatment. One subject in Cohort A3 discontinued study treatment due to Grade 3 diarrhea (not related to study treatment), and another subject in Cohort A3 discontinued study treatment due to Grade 1 conjunctivitis and Grade 2 pseudomembrane (both related to study treatment). One subject (in Cohort A3) had a fatal outcome, assessed by the Investigator to be due to disease progression, which occurred> 60 days after last dose of study treatment. In addition, 6 subjects were actively receiving treatment with 25A3-LT-A.
[00181] This preclinical data showed no bleeding abnormalities and there have been no bleeding events in the Dose-Escalation Stage. In aggregate, this suggests that 25A3-LT-A does not exacerbate the bleeding risk of bevacizumab and is a safe combination partner for bevacizumab. Further, it suggests that bevacizumab may combine well with another microtubule inhibitor, such as 25A3-LT-A as disclosed herein.
[00182] After a recommended dose (RD) for the Cohort-Expansion Stages is determined, the safety and preliminary efficacy of 25A3-LT-A are further evaluated in multiple tumor-specific expansion cohorts including subjects with non-small cell lung cancer [NSCLC], endometrial cancer (EC), EOC, cervical cancer, squamous cell carcinoma of the head and neck [SCCHN], pancreatic cancer, esophageal squamous-cell carcinoma (SCC), metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer (TNBC), and hormone-receptor positive breast cancer (HR+BC), and a tumor-agnostic (TA) TF-positive (TF+) cohort.
Simon’s 2 Stage design (Simon 1989) is used with the following exceptions: two dose levels are evaluated without interim futility testing in selected tumor types in order to understand the exposure-safety and exposure-response relationships more completely, and RD is evaluated without interim futility testing in the TA TF+ cohort. 25A3-LT-A is evaluated as monotherapy in multiple tumor-specific cohorts and a TA TF+ cohort and as combination therapy with nivolumab in NSCLC, SCCHN, and esophageal SCC, and with bevacizumab in EOC. Subjects are monitored for potential ADC-associated toxicities including but not limited to ocular, pulmonary, skin, and neurological events, as well as potential hemorrhagic events.
Dose-Escalation Stage
[00183] In the dose-escalation stage, the primary objective is to determine the MTD and/or RD for further evaluation of intravenous (IV) administration of 25A3-LT- A when administered alone and in combination therapy in subjects with advanced malignancies.
[00184] Additional objectives of the dose-escalation stage include: to establish the preliminary safety and tolerability profile of 25A3-LT-A when administered alone and in combination therapy; to evaluate the PK of 25A3-LT-A (antibody conjugated to payload), total antibody (unconjugated and conjugated antibody), and free payload following IV administration alone and in combination therapy; to assess the immunogenicity of 25A3-LT-A; and to evaluate the anti-tumor activity of 25A3-LT-A alone and in combination therapy as measured by objective response rate (ORR), duration of response (DOR), and progression-free survival (PFS) per RECIST 1.1 as assessed by the Investigator.
[00185] An exploratory objective of the dose-escalation stage is to evaluate the relationship between PK and exploratory biomarkers, preliminary efficacy, and safety outcomes.
Cohort Expansion Stage
[00186] In the cohort expansion stage, the primary objective is to evaluate preliminary efficacy of 25A3-LT-A when administered alone and in combination therapy by determining the ORR per RECIST 1 .1 as assessed by the Investigator. [00187] Additional objectives of the cohort expansion stage include: to evaluate the safety and tolerability of 25A3-LT-A when administered alone and in combination therapy; to further evaluate the PK of 25A3-LT-A (antibody conjugated to payload), total antibody (unconjugated and conjugated antibody), and free payload following IV administration alone and in combination therapy; to assess the immunogenicity of 25A3-LT-A; to evaluate the anti-tumor activity of 25A3-LT-A alone and in combination therapy as measured by DOR and PFS per RECIST 1.1 as assessed by the Investigator; to evaluate the anti-tumor activity of 25A3-LT-A as measured by ORR, DOR, and PFS per RECIST 1.1 as assessed by a Blinded Independent Radiology Committee (BIRC) for selected cohorts; to evaluate overall survival; to evaluate changes in tumor markers from baseline for selected tumor indications.
[00188] An exploratory objective of the cohort expansion stage is to assess the effects of 25A3-LT-A on tumor and blood biomarkers, to evaluate the exposure of nivolumab or bevacizumab in combination with 25A3-LT-A, to assess the immunogenicity of nivolumab or bevacizumab in combination with 25A3-LT-A, and to evaluate the association between TF expression and efficacy outcomes.
Study Design-Overview
[00189] This is a Phase 1 , open-label, multicenter, dose-escalation and expansion study evaluating the safety, tolerability, PK, pharmacodynamics, and clinical antitumor activity of 25A3-LT-A administered IV q3w as a monotherapy and in combination with nivolumab or bevacizumab to subjects with advanced solid tumors. [00190] This study consists of a Dose-Escalation Stage and a Cohort-Expansion Stage for the evaluation of 25A3-LT-A as monotherapy and in combination with nivolumab or bevacizumab. First, the safety and PK of 25A3-LT-A monotherapy is evaluated in the Dose-Escalation Stage. In this stage, subjects with advanced solid tumors are treated with 25A3-LT-A in dose-escalation cohorts using the i3+3 design (Liu et al 2020). Dose-escalation steps of 25A3-LT-A are tailored by monitoring safety and PK data. The safety and PK of 25A3-LT-A combination therapy is evaluated in the Combination Therapy Cohort-Expansion Stage for nivolumab or bevacizumab. A Cohort Review Committee determines the MTD and the RD of 25A3-LT-A from both Dose Escalation Stages (monotherapy and combination therapy) for use in the corresponding subsequent Cohort-Expansion Stages. After the RD of 25A3-LT-A is identified in the Dose-Escalation Stage, safety and efficacy of 25A3-LT-A, both as monotherapy and in combination with nivolumab or bevacizumab, are further evaluated in the Cohort-Expansion Stage. Enrollment into the Cohort-Expansion Stages is initiated after the Dose-Escalation Stages are completed. Opening of each expansion cohort is determined by the Sponsor. The TA TF+ cohort is opened at selected sites and/or countries. An overview of the study design is provided in the schematic shown in FIGs. 1A-1D and summaries of planned enrollment for the study are shown in Table 4A and Table 4B below.
[00191] Subjects in the Dose-Escalation Stage provide available archival tumor tissue during the screening period (if archival tissue is not available, fresh tumor biopsy material is provided if the biopsy can be safely performed per investigator discretion). Subjects in the Cohort-Expansion Stage are required to provide tissue samples (archival or fresh biopsy tumor tissue) during the screening period. Subjects in both stages may also provide a tumor tissue from a fresh biopsy optionally during the treatment period.
Table 4A. Enrollment Estimates for 25A3-LT-A Single-Agent Evaluation
Figure imgf000064_0001
EC, Endometrial Cancer; EOC, epithelial ovarian cancer; HR+ BC, HR-positive breast cancer; IV, intravenously; mCRPC, metastatic castration-resistant prostate cancer; NSCLC, non-small cell lung cancer; q3w, once every three weeks; RD, recommended dose; SCC, squamous cell carcinoma; SCCHN, squamous cell carcinoma of the head and neck; TA, tumor agnostic; TF+, tissue factor-positive; TBD, to be determined; TNBC, triple-negative breast cancer. a All subjects treated at a single 25A3-LT-A dose level in the Dose-Escalation Stage are defined as a cohort (e.g., Cohort A1 includes all subjects treated at the 25A3-LT-A starting dose). b Starting dose level of 25A3-LT-A. c Approximately 41 subjects enroll across nine targeted dose level increments. If additional intermediate doses are explored the total sample size may be expanded by up to 6 subjects per additional intermediate dose (a maximum of 2 intermediate doses may be explored). Additional subjects may also be added at any dose level being evaluated (up to 6 additional subjects for a total of 12 at that dose level) if the Cohort Review Committee concludes that additional safety data should be obtained at this dose level. d At the RD for 25A3-LT-A. e The TA TF+ cohort is opened at selected sites and/or countries.
Table 4B. Enrollment Estimates for Combination-Therapy Evaluation
Figure imgf000065_0001
EOC, epithelial ovarian cancer; IV, intravenously; NSCLC, non-small cell lung cancer; q3w, once every three weeks; RD, recommended dose; SCC, squamous cell carcinoma; SCCHN, squamous cell carcinoma of the head and neck; TBD, to be determined. a All subjects treated at a single 25A3-LT-A dose level in the Dose-Escalation Stage are defined as a cohort (e.g., Cohort AN1 includes all subjects treated at the 25A3-LT-A plus nivolumab starting dose). b The starting dose level of 25A3-LT-A combination-therapy is at a dose deemed safe by the Cohort Review Committee based on the experience in the 25A3-LT-A Single-Agent Dose-Escalation Stage. c Approximately 24 subjects enroll across three targeted dose level increments for each combination regimen. If additional intermediate doses are explored, the total sample size may be expanded by up to 6 subjects per additional intermediate dose (a maximum of 2 intermediate doses may be explored). Additional subjects may also be added at any dose level being evaluated (up to 6 additional subjects for a total of 12 at that dose level) if the Cohort Review Committee concludes that additional safety data should be obtained at this dose level. d At the RD for 25A3-LT-A.
[00192] All subjects receive 25A3-LT-A as a single 30-minute IV infusion q3w. Subjects continue treatment until a discontinuation criterion is met. After the last follow-up visit subjects are followed every 12 weeks to obtain information on subsequent anticancer therapy and survival. For 25A3-LT-A single-agent therapy evaluation, during the Single-Agent Dose-Escalation Stage, subjects with advanced solid tumors are treated with 25A3-LT-A at an estimated 9 dose levels using an i3+3 design. The MTD is established at the end of the Dose-Escalation Stage.
[00193] After an RD has been identified, safety and efficacy of 25A3-LT-A as single-agent therapy is evaluated in multiple tumor-specific expansion cohorts and an TA TF+ cohort in the Single-Agent Cohort-Expansion Stage (FIG. 1 B). A Simon’s 2-Stage design is used for the following cohorts: EC, SCCHN, pancreatic cancer, esophageal SCC, mCRPC, TNBC, and HR+ BC. The TA TF+ cohort evaluates tumors other than those included in the tumor-specific expansion cohorts without interim futility testing. In this cohort, subjects with a detectable level of TF expression by immunohistochemistry (IHC) is treated with the RD. [00194] Two dose levels (the RD and a dose lower than the RD [RD-low]) are explored in select single-agent tumor cohorts (NSCLC, EOC, and cervical cancer). The RD-low dose is approved by the Cohort Review Committee. Enrollment to RD or RD-low cohorts is randomized and a futility analysis is omitted in these cohorts. Subjects are randomized if a subject is eligible for more than one study cohort. A cohort may be opened or closed to enrollment at any time at the Sponsor’s discretion.
[00195] For 25A3-LT-A combination therapy evaluation, an additional, independent, dose-escalation evaluation is performed to establish an MTD for different combination regimens of 25A3-LT-A. 25A3-LT-A combined with nivolumab or bevacizumab is evaluated in subjects with advanced solid tumors during the Combination-Therapy Dose-Escalation Stage. For this evaluation, an i3+3 design is utilized, and the starting 25A3-LT-A dose level is a dose level deemed to be safe by the Cohort Review Committee based on experience in the Single-Agent Dose- Escalation Stage. After the RDs for 25A3-LT-A are identified for the combination regimens, safety and efficacy is further evaluated in three tumor-specific expansion cohorts for 25A3-LT-A plus nivolumab (NSCLC, SCCHN, and esophageal SCC) and in one tumor-specific expansion cohort for 25A3-LT-A plus bevacizumab (EOC) using a Simon’s optimal 2-stage design in the Combination Therapy Cohort- Expansion Stage.
[00196] For the Dose-Escalation Stage with 25A3-LT-A administered as a single agent and in combination with nivolumab or bevacizumab to subjects with advanced solid tumors, an i3+3 study design is used to identify the MTD of 25A3-LT-A. Dose escalation consists of ascending dose levels of 25A3-LT-A administered to subjects with advanced solid tumors. Dose level decisions are made by the Cohort Review Committee after review of all available safety data. Dose escalation for combination therapy is initiated separately at a time and at a dose of 25A3-LT-A deemed safe and appropriate by the Cohort Review Committee and may occur before an MTD for single agent 25A3-LT-A is determined. Safety and pharmacokinetic data are closely monitored at each dose level. The starting 25A3-LT-A single agent dose level of the study is 0.16 mg/kg given every 3 weeks as an infusion (IV q3w). The dose increment between two dose levels depends on emerging pharmacokinetic and safety data from the current and previous dose level cohorts. Based on the predicted human safety margins for Cmax and AUC relative to the cynomolgus monkey HNSTD together with the robust safety evaluations implemented in this study, the Dose- Escalation Stage evaluates 9 dose levels (0.16, 0.5, 1.0, 1.5, 2.0, 2.25, 2.5, 2.75, and 3.0 mg/kg). The dose levels above 1 mg/kg were selected based on the safety and PK profile from dose levels < 1 mg/kg. These dose levels were defined in a manner more conservative than a modified Fibonacci series in order to minimize the risk of exposing subjects to doses that may be too toxic. The 3-week period (21 days) after the first dose comprises the DLT Evaluation Period. Dose escalation of 25A3-LT-A in combination therapy is conducted in parallel with the single-agent 25A3-LT-A dose escalation and initiated at a starting dose level deemed safe by the Cohort Review Committee. The dose levels of 25A3-LT-A evaluated in the Combination-Therapy Dose-Escalation Stage is informed by the same planned dose levels in the Single-Agent Dose-Escalation Stage. Intermediate dose level cohorts may be inserted between previously opened dose levels and evaluated at the request of the Cohort Review Committee based on emerging safety and PK data. [00197] i3+3 Dose-Escalation Study Design. The i3+3 design (Liu et al 2020) is used to identify the MTD of 25A3-LT-A as a single-agent and combination therapy. Dose-escalation/de-escalation decisions are based on events occurring during the DLT-Evaluation Period and are guided using the i3+3 algorithm described below. Subjects are deemed evaluable for purposes of making dose-escalation decisions if they experience a DLT or complete the DLT-Evaluation Period without experiencing a DLT; subjects who do not complete the DLT-Evaluation Period for reasons other than safety are be considered evaluable.
[00198] The target toxicity probability of the MTD of 25A3-LT-A as a single-agent and combination therapy in this study is set at a target toxicity rate of 25% (PT = 0.25) and an equivalence interval (El) = [0.20, 0.30], The El provides more flexibility for defining and locating the MTD. Doses with a toxicity probability between 0.20 and 0.30 may be deemed acceptable to be the MTD. The values selected for PT and El are moderately more conservative than toxicity rates deemed acceptable for MTDs in older standard dose-escalation study designs (e.g., the 3+3 study design). [00199] The i3+3 Design Algorithm. Enrollment cohorts of 3 subjects are treated and observed, and decisions about dose level escalation, de-escalation, and expansion for subsequent cohorts are based upon a comparison of observed DLT rates for all evaluable subjects treated at a given dose level against the El (although the actual algorithm is more sophisticated, accounting for the amount of available information at the time of each decision). Accrual to the Single-Agent Dose- Escalation Stage ends when either 12 evaluable subjects are observed in the dose level indicated as the next step in the enrollment algorithm or 30 evaluable subjects in the standard Dose-Escalation cohorts across the last five planned dose levels (A5- A9) are treated and observed in the stage for a regimen, whichever comes first. Similarly, accrual to each regimen in the Combination Dose-Escalation Stage ends when either 12 evaluable subjects are observed in the dose level indicated as the next step in the enrollment algorithm or approximately 24 evaluable subjects in the standard Dose-Escalation cohorts (excluding the backfill cohorts) across three targeted dose level increments are treated and observed in the stage for each combination regimen, whichever comes first.
[00200] Operationally, these decisions are codified into tables of rules as shown in FIG. 2 and Table 5. In this fashion the i3+3 design is more flexible than the conventional 3+3 design with regard to the number of subjects enrolled and observed at a given dose level and allows easier customization of the target toxicity levels (via specification of the El).
Figure imgf000070_0001
Figure imgf000071_0001
a Upon safety and PK data review, the Cohort Review Committee is allowed to stop or limit enrollment at a dose level even if the decision rules would allow to continue with enrollment of a 3-subject cohort at that dose level.
[00201] The decision rules serve as a guide for the Cohort Review Committee to determine the dose level for enrollment of successive cohorts. Dose escalation decisions according to the i3+3 design (i.e. , “E” decisions) must be based on the data from at least 3 evaluable subjects at a dose level, but the Cohort Review Committee may choose to stop or limit enrollment at a dose level and de-escalate (even with fewer than 3 evaluable subjects) if emerging data indicate a safety risk at that dose level. Furthermore, evaluation of a treatment regimen may be stopped early if it becomes clear a tolerable dose level does not emerge using available dose levels.
[00202] Additional subjects (up to a total of 12) may be added at any dose level being evaluated if the Cohort Review Committee concludes that additional safety data should be obtained at this dose level.
[00203] The Cohort Review Committee may adjust the total number of enrolled subjects following the i3+3 design if there are more than nine targeted dose escalation level increments in the single-agent evaluation, more than three targeted dose levels increments in the combination therapy evaluation, or more subjects than anticipated are enrolled at an individual dose level.
[00204] Backfill Cohorts. The Cohort Review Committee may decide to gather more data at lower doses using a backfill approach before the final decision about the MTD at the end of the Dose-Escalation Stage. This approach allows for simultaneous backfilling at lower dose cohorts and Dose-Escalation at higher dose cohorts. A hybrid version of the i3+3 algorithm along with the Probability-of-Decision Toxicity Probability Interval (PoD-TPI) design (Zhou et al, Statistics in Biosciences. 2020 Jul; 12(2): 124-45. 2020) was used to allow for backfill cohorts. The benefit of the approach is that it allows for additional information to be gathered at lower doses while also providing a mechanism for accounting for this information during dose escalation. DLT events observed in backfill cohorts are incorporated into dose level decision. In a backfill cohort, a decision of De-escalate or DU (i.e. , de-escalate and not use the dose level again in the study) can potentially override the outcome of a higher dose level if a sufficient number of DLT events occur in that lower dose level cohort. The outline of the approach is characterized below.
[00205] The “standard Dose-Escalation cohorts” are denoted as the cohorts of subjects for the Dose-Escalation, and the “backfill cohorts” as the cohorts that are used to further characterize the safety of lower doses. When a dose has 12 or more subjects in total, that dose is deemed “full”.
[00206] Assume a cohort consists of three subjects and dose d is currently used for treating subjects in the standard Dose-Escalation cohorts. After the enrollment of a cohort at dose d completes, subjects are allowed to backfill at the dose (d - 1), one level below dose d , as long as dose (d - 1) is not full and dose d has been used to treat more than three subjects (e.g., dose d has experienced a Stay decision).
[00207] After the outcomes of subjects treated at dose level d are fully evaluated, then, (a) if there are no pending outcomes, apply the i3+3 decision to calculate the decisions of all doses lower than dose d; or (b) if there are pending outcomes from the backfill cohorts at any doses lower than the current dose d, use the PoD-TPI (Zhou et al 2020) design to calculate the probabilities of decisions (PoD) y',) at these doses. [00208] Any decision based on the backfill algorithm is assessed by the Cohort Review Committee based on emerging safety and PK data.
[00209] Intermediate Dose Level Cohorts. Intermediate dose level cohorts may be inserted between previously opened dose levels and evaluated at the request of the Cohort Review Committee based on emerging safety and PK data.
[00210] MTD Determination. Once all the enrolled subjects complete their evaluation for DLTs during the DLT-Evaluation Period (or have been deemed not evaluable) and a criterion for stopping further enrollment in the standard Dose- Escalation cohorts is met, statistical analysis by isotonic regression is used to select the MTD based on the observed DLT data from all the dose levels (Liu et al., Journal of Biopharmaceutical Statistics. 30(2):294-304 (2020)).
[00211] Specifically, the highest tested dose for which the toxicity rate is closest to the target toxicity rate of 25% (PT = 0.25) and does not exceed the upper bound of the El (i.e. , 0.30) is selected as the MTD. The Cohort Review Committee endorses the MTD of 25A3-LT-A as a single agent and in combination with nivolumab or bevacizumab
[00212] RD Determination. Independent of MTD determination and upon review of all available safety, PK, and preliminary clinical activity data, the Cohort Review Committee chooses one recommended dose (RD) level of 25A3-LT-A at which the corresponding expansion cohort subjects are treated. For each treatment regimen, the 25A3-LT-A RD is the MTD or a lower dose level at the Cohort Review Committee’s discretion.
[00213] Magnitude of Dose-Escalation. Dose escalation to the next higher dose level proceeds only if at least three subjects enrolled under i3+3 design are evaluated at the current dose level and a reason for discontinuing enrollment is not reached as discussed above. Dose level decisions are made by the Cohort Review Committee after review of all available safety data. Safety and pharmacokinetic data are closely monitored at each dose level. The dose increment between two dose levels depends on emerging pharmacokinetic and safety data from the current and previous dose level cohorts.
[00214] Regimen Re-evaluation. If the Cohort Review Committee is unable to select an acceptable RD for further evaluation of a regimen in the Cohort-Expansion Stage (e.g., early termination for safety, insufficient toxicity data have been reviewed), alternative treatment schedules may be evaluated in a new Dose- Escalation Stage. Under these circumstances, data from the first dose-escalation evaluation are used to inform the dose level and/or treatment schedule choices, but enrollment and escalation/de-escalation decisions follow the i3+3 design independent of the prior evaluation.
[00215] Intra-subiect Dose-Escalation. With Sponsor approval, prior to establishing the MTD the Cohort Review Committee may allow an intra-subject dose escalation of a subject to a safe 25A3-LT-A single-agent or combination therapy dose level (that is, DLT evaluation has been completed for at least 3 subjects with no reported DLTs) if the subject has received 6 doses of 25A3-LT-A study treatment at the assigned dose and has not experienced unacceptable side effects (e.g., ocular toxicity) per Investigator assessment in discussion with the Medical Monitor or a treatment-emergent toxicity > Grade 2.
[00216] Subjects who are allowed to dose escalate do not contribute to the DLT Evaluation at that higher dose.
[00217] Treatment Extension Period. After completing the 21 -day DLT-Evaluation Period, subjects have the opportunity to continue to receive study treatment, including the combination therapy agent, if applicable, in the Treatment Extension Period for up to 24 months, provided they do not have radiographic progressive disease (PD) or unacceptable toxicity. Subjects may continue to receive 25A3-LT-A for more than 24 months if the subject is deriving clear clinical benefit as determined by the Investigator and agreed by the Sponsor.
[00218] Dose-Limiting Toxicities. Dose-limiting toxicity (DLT) is determined by the Cohort Review Committee upon review of all available data.
[00219] For subjects who experience a DLT, 25A3-LT-A is delayed until the toxicity resolves. With the agreement of the Sponsor, subjects who recover within 21 days are allowed to resume 25A3-LT-A at a maximum dose of one dose level below the dose that produced the DLT. If the reduced dose is tolerated without DLT or other unacceptable toxicity, the subject is eligible to proceed to the Treatment Extension Period at the reduced dose.
[00220] Dose-Escalation Stage Study Visits. Subjects in the Dose-Escalation Stage visit the clinic for study assessments during study periods as follows: PreTreatment Period (Screening), DLT-Evaluation Period (Days 1-21 ), Treatment Extension Period (Day 22+), and Post-Treatment Period. [00221] During the Pre-Treatment Period (Screening), subjects are consented and undergo screening and baseline evaluations to be qualified for the study. During the DLT-Evaluation Period (Days 1-21 ), DLTs are determined by the Cohort Review Committee upon review of all available data and are defined above. During the Treatment Extension Period (Day 22+), subjects receive treatment with 25A3-LT-A as a single-agent or combination therapy and are monitored for safety (including laboratory assessments) and signs of toxicity at Study Safety Visits (SSVs). Subjects may continue to receive study treatment until radiographic PD as assessed by the investigator or any other treatment discontinuation criteria are met for up to 24 months with the agreement of the Sponsor. Subjects may continue to receive 25A3- LT-A for more than 24 months if the subject is deriving clear clinical benefit as determined by the Investigator and agreed by the Sponsor. With respect to the PostTreatment Follow-Up Visits (FU), two post-treatment follow-up safety visits occur 30 (+14) days and 60 (+14) days after the date of the decision to discontinue study treatment. Subjects administered nivolumab have a third post-treatment follow-up safety visit 100 (+14) days after the date of the decision to discontinue study treatment. If a related AE leading to study treatment discontinuation or related SAE is ongoing at the last follow-up visit, it is to be followed until considered resolved or irreversible. For Extended Follow-Up (Every 12 weeks [± 14 days] after last FU), after the last post-treatment follow-up visit, each subject continues to be followed for survival and receipt of nonprotocol anticancer therapy (NPACT). The Investigator (or designee) makes contact with the subject every 12 weeks after the post-treatment follow-up visits until the subject expires, withdraws consent for such contacts, or the Sponsor decides to cease collecting these data for the study. Radiographic tumor imaging is performed per the protocol-defined schedule until disease progression per RECIST 1.1 , initiation of subsequent anticancer therapy, or death.
[00222] Cohort-Expansion Stage. After the RD of 25A3-LT-A as a single-agent or combination therapy is determined in the Dose-Escalation Stage, the corresponding Cohort-Expansion Stage is initiated. The Cohort-Expansion Stage further explores safety, tolerability, and preliminary efficacy of 25A3-LT-A as single-agent and combination therapy in multiple tumor-specific expansion cohorts and a TA TF+ cohort. Subjects with advanced NSCLC, EC, EOC, cervical cancer, SCCHN, pancreatic cancer, esophageal SCC, mCRPC, TNBC, or HR+ BC, or TA TF+ are enrolled in the Single-Agent Cohort-Expansion Stage and are treated at the RD of 25A3-LT-A monotherapy. Subjects with NSCLC, SCCHN, or esophageal SCC are enrolled in the Combination Therapy Cohort-Expansion Stage and treated at the RD of 25A3-LT-A plus nivolumab combination therapy. Subjects with EOC may be enrolled in the Combination Therapy Cohort-Expansion Stage and treated at the RD of 25A3-LT-A plus bevacizumab. The following cohorts enroll according to a Simon’s 2-Stage design: EC, SCCHN, pancreatic cancer, esophageal SCC, mCRPC, TNBC, and HR+ BC. In this design, a minimum number of subjects must experience radiographic responses in the first stage for the second stage to be opened for enrollment. The TA TF+ cohort evaluates tumors other than those included in the tumor-specific expansion cohorts without interim futility testing. Two dose levels (RD and a dose lower than RD [RD-low]) are explored in select single-agent tumor cohorts (NSCLC, EOC, and cervical cancer) with no interim futility analysis. Subjects are enrolled into the appropriate open cohorts following the eligibility review process by the Sponsor.
[00223] In addition, two dose levels (RD and a dose lower than RD [RD-low]) are explored in select single-agent tumor cohorts (NSCLC, EOC, and cervical cancer). The additional RD-low dose is approved by the Cohort Review Committee. The enrollment to RD or RD-low cohorts is randomized and a futility analysis is omitted in these cohorts.
[00224] Subjects are randomized if a subject is eligible for more than one study cohort. A cohort may be closed to enrollment at any time at the Sponsor’s discretion. [00225] Cohort-Expansion Stacie Study Visits. Each subject’s course of treatment in expansion cohorts consists of the following periods: Pre-Treatment Period (Screening), Treatment Period (Day 1 +), and Post-Treatment Period.
[00226] During the Pre-Treatment Period (Screening), subjects are consented and undergo screening and baseline evaluations to be qualified for the study.
[00227] During the Treatment Period (Day 1 +), subjects receive treatment with 25A3-LT-A as a single-agent or combination therapy and are monitored for safety (including laboratory assessments) and signs of toxicity at study safety visits (SSVs). Subjects may continue to receive study treatment until radiographic PD as assessed by the investigator or any other treatment discontinuation criteria are met for up to 24 months with the agreement of the Sponsor. Subjects may continue to receive 25A3- LT-A for more than 24 months if the subject is deriving clear clinical benefit as determined by the Investigator and agreed by the Sponsor. [00228] During the Post-Treatment Follow-Up Visit (FU), two post-treatment followup safety visits occur 30 (+14) days and 60 (+14) days after the date of the decision to discontinue study treatment. Subjects administered nivolumab have a third posttreatment follow-up safety visit 100 (+14) days after the date of the decision to discontinue study treatment. If a related AE leading to study treatment discontinuation or related SAE is ongoing at the last follow-up visit, it is followed until considered resolved or irreversible.
[00229] For Extended Follow-Up (Every 12 weeks [± 14 days] after last FU), after the last post-treatment follow-up visit, each subject continues to be followed for survival and receipt of NPACT. The investigator (or designee) makes contact with the subject every 12 weeks after the post-treatment follow-up visits until the subject expires, withdraws consent for such contacts, or the Sponsor decides to cease collecting these data for the study. Radiographic tumor imaging is performed per the protocol-defined schedule until disease progression per RECIST 1.1 , initiation of subsequent anticancer therapy, or death.
[00230] Treatment Discontinuation Criteria. Subjects receive study treatment until treatment discontinuation. Subjects may discontinue study treatment and assessments or withdraw their consent to participate in the study at any time without prejudice. For subjects who discontinue study treatment, every effort is made to undertake protocol-specified follow-up procedures including end of treatment assessments, survival follow-up, and documentation of subsequent anticancer treatment(s) unless consent for non-interventional study assessments is also withdrawn.
[00231] Tumor Biopsies. Subjects in the Dose-Escalation and Cohort-Expansion Stages provide available archival tumor tissue. If archival tumor tissue is not available, a fresh tumor biopsy may be collected prior to first dose with subject consent, and if it is safely accessible per investigator medical judgement. Subjects in the Cohort-Expansion Stage are required to provide tissue samples (archival or fresh biopsy tumor tissue) during the screening period. Biopsy locations for study purposes should obtain adequate material for histology with the lowest procedural risk to the subject.
[00232] Fresh tumor biopsies should follow standard clinical practice and procedural risks must be carefully assessed. These procedural risks include, but are not limited to, factors which may indicate a higher bleeding risk (i.e. , low platelet counts, anticoagulation therapy). In order to determine response per RECIST 1.1 , target lesions in these subjects must not be biopsied. During the screening period, fresh tumor biopsies should be performed at least 7 days before first dose of 25A3- LT-A treatment. For both the Dose-Escalation and Cohort-Expansion Stages, an optional on-treatment biopsy may be performed at Day 15. For on-treatment biopsies, any procedure-associated AEs must have resolved prior to the next 25A3- LT-A administration. Subjects are closely followed for adequate wound healing after the tumor biopsy.
[00233] Study Committees. The Sponsor engages a Cohort Review Committee, Study Oversight Committee (SOC), corporate safety governance, and Blinded Independent Radiology Committee (BIRC) to review safety, PK, and efficacy data for this study.
[00234] End of Tria . End of trial is defined as the later of two dates: the date of the last study visit or procedure for the last subject remaining or the date at which the last data point required for follow-up for the last subject is obtained.
[00235] Number of Subjects. The estimated number of subjects enrolled in this Phase 1 study evaluating 25A3-LT-A as a single agent therapy and in combination therapy in subjects with advanced solid tumors is shown in Table 4A and Table 4B. [00236] Subjects are accrued for the study at approximately 10 sites in the US for the Dose-Escalation Stage and at approximately 80 sites globally for the Cohort- Expansion Stage.
[00237] Target Population. Subjects in this study must have received standard life-prolonging therapies or are not qualified to receive such therapies.
[00238] To be eligible for the study, the subjects meet all of the inclusion and none of the exclusion criteria. The Sponsor does not grant exceptions to these eligibility criteria.
[00239] Note: For determining numbers of prior lines of therapy, neoadjuvant, adjuvant, intraperitoneal, and maintenance therapy are not counted towards the maximum allowed number of prior therapies, and re-exposure to the same agent is not considered an additional line of therapy.
Inclusion Criteria
[00240] Inclusion criteria include the presence of a cytologically or histologically and radiologically confirmed solid tumor that is inoperable, locally advanced, metastatic, or recurrent. For the Dose-Escalation Stage Cohorts A, AB, and AN (Solid Tumors), an inclusion criterion is that the subject has received standard lifeprolonging therapies unless they do not exist, or available therapies are intolerable or no longer effective. For the Cohort-Expansion Stage (Cohorts B - K, BN, DB, FN, and HN), an inclusion criterion is that the subject has received standard lifeprolonging therapies unless they do not exist, or available therapies are intolerable or no longer effective.
[00241] With respect to Cohort B (Non-small Cell Lung Cancer), included are subjects with Stage IV NSCLC (non-squamous cell or squamous cell histology) who have documented radiographic disease progression during or following their last systemic anticancer therapy. Subjects without an actionable genetic alteration must have received platinum-containing chemotherapy and ICI therapy, administered concurrently or sequentially for metastatic disease. Subjects who are not qualified to receive ICI therapy are eligible if they have received platinum-containing doublet chemotherapy. Subjects with an actionable genetic alteration (e.g., EGFR sensitizing mutation, ALK rearrangement, ROS1 rearrangement, BRAF V600E mutation, MET Exon 14 skipping mutation, RET rearrangement) must have received at least one molecularly targeted therapy for that specific genetic alteration unless such therapy is not available or the subject is not qualified to receive such therapy. Subjects must have received no more than 3 lines of prior systemic anticancer therapy for locally advanced or metastatic disease (e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination).
[00242] With respect to Cohort C (Endometrial Cancer), included are subjects with histologically confirmed, advanced, recurrent or metastatic endometrial cancer who have documented radiographic disease progression during or following their last systemic anticancer therapy. Subjects must have received platinum-containing chemotherapy in the adjuvant, locally advanced, or metastatic disease setting. Subjects must have received no more than 3 lines of prior systemic anticancer therapy for locally advanced or metastatic disease (e.g., chemotherapy, immunotherapy including ICI, molecular targeted therapy given either as single agents or in combination).
[00243] With respect to Cohorts D and DB (Epithelial Ovarian Cancer), included are subjects with high-grade serious EOC, including primary peritoneal cancer (PPC) and fallopian tube cancer (FTC) who have platinum-resistant disease following treatment with platinum-containing chemotherapy. Platinum-resistant disease is defined as disease progression <6 months after receiving the last dose of platinumbased chemotherapy. Subjects who discontinue platinum-containing chemotherapy due to an AE cannot be defined as platinum-resistant and are therefore not eligible for the study. Note: Ovarian borderline epithelial tumors (low malignant potential), mucinous and endometrioid tumors are not eligible. Subjects must have previously received bevacizumab, if locally considered as standard of care and if eligible. Platinum-refractory disease (progression on first-line treatment or within 4 weeks of completion) are excluded. Subjects must have received no more than 3 lines of prior systemic anticancer therapy for locally advanced or metastatic disease
(e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination). Note: Hormonal therapy is not counted towards the maximum allowed number of prior therapies.
[00244] With respect to Cohort E (Cervical Cancer), included are subjects with persistent, recurrent or metastatic carcinoma of the uterine cervix (squamous cell or adenocarcinoma histology) who have documented radiographic disease progression during or following their last systemic anticancer therapy. Subjects must have received platinum-containing chemotherapy for recurrent or metastatic disease. Subjects who have received no more than 2 lines of prior systemic anticancer therapy for locally advanced or metastatic disease (e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination). Chemoradiation therapy is not counted towards the maximum allowed number of prior therapies.
[00245] With respect to Cohorts F and FN (SCCHN), included are subjects with head and neck cancer (squamous cell histology) who have documented radiographic disease progression during or following their last systemic anticancer therapy.
Allowed primary tumor locations are oral cavity, oropharynx, hypopharynx, glottic larynx. Excluded are subjects with primary tumor site of the nasopharynx. Subjects must have received prior platinum-containing chemotherapy (including chemoradiation) for inoperable locally advanced, recurrent, or metastatic disease. Subjects must have also received ICI therapy and/or EGFR inhibitor therapy, if locally considered as standard of care and if eligible. Subjects who have received no more than 3 lines of prior systemic anticancer therapy for inoperable locally advanced, recurrent, or metastatic disease setting (e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination) are included.
[00246] With respect to Cohort G (Pancreatic Cancer), included are subjects with pancreatic cancer (adenocarcinoma histology) who have documented radiographic disease progression during or following their last systemic anticancer therapy. Subjects must have received prior treatment with gemcitabine in combination with albumin-bound paclitaxel (nab-paclitaxel) or FOLFIRINOX, or have documented ineligibility for standard therapy. Subjects who have received at least 1 and no more than 2 prior lines of systemic anticancer therapy for inoperable locally advanced, recurrent, or metastatic disease, are included .
[00247] With respect to Cohorts H and HN (Esophageal SCC), included are subjects with esophageal cancer (squamous cell histology) who have documented radiographic disease progression during or following their last systemic anticancer therapy. Subjects with esophageal adenocarcinoma and adenocarcinoma of gastroesophageal junction (GEJ) are excluded. Subjects must have received prior treatment with chemotherapy such as fluorouracil/capecitabine/taxane in combination with platinum and ICI therapy, if locally considered as standard of care and if eligible, administered concurrently or sequentially for inoperable locally advanced, recurrent, or metastatic disease. Subjects who have received no more than 2 lines of prior systemic anticancer therapy for inoperable locally advanced, recurrent, or metastatic disease setting (e.g., chemotherapy, immunotherapy, molecular targeted therapy given either as single agents or in combination) are included.
[00248] With respect to Cohort I (mCRPC), included are subjects with metastatic, castration resistant adenocarcinoma of the prostate. Neuroendocrine differentiation and other histological features are permitted if adenocarcinoma is the primary histology. Subjects must have progression of prostate cancer documented by any of the following: (i) PSA progression: Prostate specific antigen (PSA) progression defined by a minimum of 2 rising PSA values from 3 or 4 consecutive assessments with an interval of at least 7 days between assessments. If qualifying solely by PSA progression, the screening PSA value must be at least 2 ng/mL (2pg/L) and the oldest qualifying value must have been based on a blood sample drawn no longer than one year prior to signing of the informed consent form (ICF) with no change in systemic regimen for the treatment of prostate cancer; up to one PSA decrease is permitted as long as it is not the most recent value. If the study laboratory is the local laboratory at which the subject’s previous PSA blood samples were drawn, then the screening local lab PSA must be the highest, (ii) Radiographic progression in soft- tissue disease. Subjects with bone only disease are eligible if they can be evaluated by imaging either of new lesions on technetium bone scan or new I progressing osteolytic lesion on CT scan. Subjects must have received at least one prior novel hormonal therapy (NHT; e.g., enzalutamide, apalutamide, darolutamide, abiraterone, or equivalent) for locally advanced or metastatic castration-sensitive prostate cancer (mCSPC) or mCRPC. Subjects may have received one prior taxane-based chemotherapy (e.g., docetaxel, cabazitaxel) for mCRPC or mCSPC. Subjects should have received no more than 3 prior systemic therapies for mCSPC and/or mCRPC. [00249] With respect to Cohort J (TNBC), included are subjects with triple-negative (estrogen receptor negative [ER-]/progesterone receptor negative [PR-] and human epidermal growth factor receptor 2 negative or low [HER-2-]) breast cancer who have documented radiographic disease progression during or following their last systemic anticancer therapy for inoperable locally advanced or metastatic disease. Regarding triple-negative breast cancer, estrogen receptor and progesterone receptor negativity are defined as < 1% of cells expressing hormonal receptors via IHC analysis. HER-2 negativity is defined as either of the following by local laboratory assessments: in situ hybridization (ISH) non-amplified (ratio of HER-2 to CEP17 < 2.0 or single probe average HER-2 gene copy number < 4 signals/cell), or IHC 0 or IHC 1 +. HER-2 low, defined as an IHC score of 1 + or 2+ with a negative ISH test. If more than one test result is available and they do not all meet the inclusion criterion definition, there should be discussion with the Sponsor to establish eligibility of the subject. Subjects must have received at least one line of prior systemic chemotherapy (anthracycline-, alkylator-, or taxane-based chemotherapy) but no more than three lines of prior systemic anticancer therapies for locally advanced or metastatic disease (e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy).
[00250] With respect to Cohort K (HR+BC), included are subjects with breast cancer that is hormone receptor-positive (ER+ and/or PR+) and HER-2 negative or low and who have documented radiographic disease progression during or following their last systemic anticancer therapy for inoperable locally advanced or metastatic disease. Regarding hormone receptor-positive and HER-2-negative breast cancer (HR+ BC), estrogen receptor and progesterone receptor positivity are defined as > 1 % of cells expressing hormonal receptors via IHC analysis. HER-2 negativity is defined as either of the following by local laboratory assessments: In situ hybridization (ISH) non-amplified (ratio of HER-2 to CEP17 < 2.0 or single probe average HER-2 gene copy number < 4 signals/cell), or IHC 0 or IHC 1+. HER-2 low, defined as an IHC score of 1 + or 2+ with a negative ISH test. If more than one test result is available and they do not all meet the inclusion criterion definition, there should be discussion with the Sponsor to establish eligibility of the subject. Subjects must have postmenopausal status due to either surgical/natural menopause or ovarian suppression with gonadotropin-releasing hormone (GnRH) agonist (e.g., goserelin). Subjects must have received at least one prior endocrine anticancer therapy and one prior CDK4/6 inhibitor therapy either sequentially or concurrently for inoperable locally advanced or metastatic breast cancer. Subjects must have received at least one line of prior systemic chemotherapy (anthracycline- alkylator- or taxane-based chemotherapy) but no more than three lines of prior systemic anticancer therapies for locally advanced or metastatic disease (e.g., cytotoxic therapy, targeted therapy including ADCs, immunotherapy). There is no limit on the number of prior lines of endocrine anticancer therapy. Examples include fulvestrant (selective estrogen receptor degrader [SERD]), tamoxifen (selective estrogen receptor modulator [SERM]), exemestane (steroidal aromatase inhibitor [SAI]), and letrozole (non-steroidal aromatase inhibitor [NSAI]).
[00251] With respect to Cohort L (Tumor-Agnostic TF-Positive Cancers), included are subjects with solid tumors other than those designated in Cohorts B-K and for whom other approved therapies are either not available or for which they are ineligible. Participation in this cohort is at selected sites and countries based on site feasibility assessment. IHC should indicate that the subject’s tumor is TF positive using archival histological material available obtained within the last two years. Fresh biopsy for eligibility assessment should not be undertaken. Assessment of tumor TF- positivity should be undertaken locally using the Sponsor-designated commercially available monoclonal antibody used per manufacturer protocol or following the guidance provided in in a separate manual. Adequate amounts of the same archival material must also be available for submission to the Sponsor. Other eligibility criteria are applied as for other cohorts, but Investigators must initially consult with the Sponsor via the Medical Monitor, or their designate, to discuss suitability for enrollment.
[00252] Rationale for the Expansion Cohort for Tumor- Agnostic Tissue-Factor- Positive Cancer. An alternative pathway to the selection of patients for treatment can be proposed with therapies where response may be defined by biological characteristics. If there is a measurable parameter of that characteristic, this may be exploited for optimal selection of patients for that treatment, an example of precision medicine. For 25A3-LT-A, expression of the antibody target, TF, is a plausible biomarker. The analysis of TF expression in tumor types selected for cohort expansion offers an opportunity to gather evidence of an expression-response relationship, looking for biomarker-enrichment of efficacy. Complementarily, to assess whether TF expression is predictive of efficacy, for Cohort L, subjects with any tumor type other than those designated in Cohorts B-K are considered for eligibility if their tumor is TF+.
[00253] Various clinical situations could be explored in Cohort L, including: tumors that are TF+ and foreseeably sensitive to the 25A3-LT-A payload but are not being enrolled in a standalone cohort for other reasons, such as lower unmet need (e.g., urothelial), or rarity (e.g., osteosarcoma); tumors that are often TF+ where susceptibility to the payload may be efficacy-limiting (e.g., colorectal cancer); or tumors that are rarely TF+ but, if the argument holds that TF-positivity is a driver of 25A3-LT-A efficacy, then a patient with TF+ tumor expression might derive clinical benefit from 25A3-LT-A.
[00254] Cohort L is exploratory, a signal-finding exercise to determine whether a larger study is indicated and also to evaluate what level of TF expression may be needed to enroll in a subsequent study. For this reason, TF expression, as determined by the sites locally, satisfies the criterion for TF-positivity and subsequent analysis of the same material for TF expression by the Sponsor permits both further examination of expression-response relationship and, additionally, the degree of variability between two different assessments. This informs the associated question regarding the need for a companion diagnostic assay. Beyond the criterion of TF- expression histologically, entry criteria into this cohort align with the other expansion cohorts in the study. The TA TF+ cohort is opened at selected sites and/or countries. [00255] Subjects in the Cohort-Expansion Stage have measurable disease per RECIST 1.1 as determined by the investigator. Measurable disease at screening is not required for the following subjects: subjects in the Dose-Escalation Stage, subjects with prostate cancer (Cohort I) without soft tissue disease, or subjects with primary brain tumors, such as glioblastoma (RECIST assessments are not required for these subjects). Subjects in the Cohort-Expansion Stage must have available archival tumor tissue collected no more than 2 years prior to consent. If archival tumor tissue is not available, a fresh tumor biopsy is collected from subjects enrolled in the Dose-Escalation Stage and must be collected from subjects in the Cohort- Expansion Stage, at least 7 days (and up to 60 days) prior to first dose. Specific requirements for tumor tissue samples are provided in the Laboratory Manual. Subjects in the Cohort-Expansion Stage must have recovery to baseline or < Grade 1 severity (Common Terminology Criteria for Adverse Events version 5 [CTCAE v5]) from AEs, unless AEs are clinically nonsignificant (e.g., alopecia) or stable (e.g., Grade 1 peripheral neuropathy). Subjects in the Cohort-Expansion Stage must be age 18 years or older on the day of consent. Subjects in the Cohort-Expansion Stage must have an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0-1 . Subjects in the Cohort-Expansion Stage must have an adequate organ and marrow function, based upon meeting all of the following laboratory criteria within 10 days before first dose of study treatment: absolute neutrophil count (ANC)
> 1500/mm3 (> 1.5 Gl/L) without granulocyte colony-stimulating factor (G-CSF) support within 2 weeks prior to screening laboratory sample collection; platelets
> 100,000/mm3 (> 100 Gl/L)] without transfusion within 2 weeks prior to screening laboratory sample collection; hemoglobin > 9 g/dL (> 90 g/L) without transfusion within 2 weeks prior to screening laboratory sample collection; prothrombin time (PT) or activated partial thromboplastin time (aPTT) < 1.2 x upper limit of normal (ULN) or International Normalized Ratio (INR) < 1.3 without anticoagulation therapy (INR < 3 if on stable oral coumarin-based anticoagulant); alanine aminotransferase (ALT) and aspartate aminotransferase (AST) < 3 x ULN; total bilirubin < 1 .5 x ULN (for subjects with known Gilbert’s disease, total bilirubin < 3 x ULN); and serum creatinine < 1 .5 x ULN or calculated creatinine clearance > 45 mL/min (> 0.75 mL/sec) using the Cockcroft-Gault equation. Subjects in the Cohort-Expansion Stage must be capable of understanding and complying with the protocol requirements and must have signed the informed consent document. [00256] Sexually active fertile subjects and their partners must agree to highly effective methods of contraception during the course of the study and for the following durations after the last dose of treatment (whichever is later): 7 months after the last dose of 25A3-LT-A for women of childbearing potential (WOCBP) and 4 months after the last dose of 25A3-LT-A for men; 5 months after the last dose of nivolumab for WOCBP; or 6 months after the last dose of bevacizumab for WOCBP. Male subjects with female partners of childbearing potential must agree to use a condom until after the last dose of treatment or 4 months after the last dose of 25A3- LT-A.
[00257] Female subjects of childbearing potential must not be pregnant at screening. Female subjects are considered to be of childbearing potential unless one of the following criteria is met: permanent sterilization (hysterectomy, bilateral salpingectomy, or bilateral oophorectomy) or documented postmenopausal status (defined as 12 months of amenorrhea in a woman over 45 years-of-age in the absence of other biological or physiological causes. In addition, females under 55 years-of-age must have a serum follicle stimulating hormone (FSH) level > 40 mlll/mL to confirm menopause). Documentation may include review of medical records, medical examination, or medical history interview by study site staff.
Exclusion Criteria
[00258] Receipt of any tissue factor-targeting antibody drug conjugate or auristatin derivate based antibody drug conjugate.
[00259] Receipt of any chemotherapy or anticancer antibody (e.g., anti-VEGF mAb, antibody-drug conjugate, or PD-1/PD-L1 mAb) within 21 days (nitrosoureas or mitomycin within 42 days) before first dose of study treatment.
[00260] Receipt of any type of small molecule kinase inhibitor (including investigational kinase inhibitors) within 2 weeks before first dose of study treatment. [00261] Receipt of any anticancer hormonal therapy within 2 weeks or within 5 half-lives of the agent, whichever is shorter, before first dose of study treatment. Note: Concomitant use of a luteinizing hormone-releasing hormone (LHRH) agonist (e.g., leuprolide, goserelin) or antagonist (e.g., relugolix) is permitted.
[00262] Radiation therapy within 2 weeks before first dose of study treatment. Subjects with clinically relevant ongoing complications (e.g., radiation induced esophagitis or pneumonitis) from prior radiation therapy are not eligible. [00263] Known brain metastases or cranial epidural disease unless adequately treated with radiotherapy and/or surgery (including radiosurgery) and stable for at least 4 weeks before first dose of study treatment. Note: Eligible subjects must be neurologically asymptomatic and without corticosteroid treatment at the time of first dose of study treatment.
[00264] The subject has uncontrolled, significant intercurrent or recent illness including, but not limited to, the following conditions:
[00265] Acute ocular infection, acute or chronic ulcerative/cicatricial condition of conjunctiva or cornea including but not limited to ocular disorders from autoimmune diseases (e.g., mucous membrane pemphigoid, Sjogren’s syndrome), significant conjunctiva/cornea scarring (e.g., radiation keratopathy), severe dry eye disease (for Schirmer’s test read < 5 mm), history of corneal transplantation, monocularity.
[00266] Peripheral neuropathy (sensory and/or motor) CTCAE Grade > 2.
[00267] Bleeding disorder or a history of clinically significant bleeding event (e.g., requiring invasive procedures or urgent transfusion) within 2 months before first dose.
[00268] Congestive heart failure New York Heart Association class 3 or 4, unstable angina pectoris, serious cardiac arrhythmias (e.g., ventricular flutter, ventricular fibrillation, torsades de pointes).
[00269] Stroke (including transient ischemic attack [TIA]), myocardial infarction, or other ischemic within 6 months before first dose.
[00270] Thromboembolic events (e.g., DVT or PE) within 3 months before first dose. Subjects must have received anticoagulation therapy and be asymptomatic at the time of first dose.
[00271] Cavitating pulmonary lesion(s) > 1 cm in diameter.
[00272] Tumors with a recognized risk of bleeding from vascular involvement (e.g., lesions invading major pulmonary blood vessels or carotid arteries).
[00273] History of drug-related interstitial lung disease (ILD)/pneumonitis.
[00274] Tumors invading the Gl-tract (except in case of primary colorectal or esophageal cancers), active peptic ulcer disease or inflammatory bowel disease, diverticulitis, cholecystitis, symptomatic cholangitis or appendicitis, acute pancreatitis or acute obstruction of the pancreatic/biliary duct or the Gl tract.
[00275] Moderate to severe hepatic impairment (Child-Pugh B or C). [00276] Active infection (bacterial, viral, or fungal) requiring systemic antimicrobial therapy including HIV anti-retroviral treatment. Prophylactic use of antibiotics is allowed.
[00277] History of COVID-19 unless the subject has clinically recovered from the infection: at least 10 days prior to first dose or sooner, if COVID-19 PCR negative. [00278] Requirement for hemodialysis or peritoneal dialysis. Nephrotic syndrome, proteinuria with > 2 g protein in 24 hours in urine.
[00279] History of solid organ, autologous or allogenic stem cell transplant.
[00280] History of life-threatening toxicity related to prior immune therapy (e.g., anti CTLA 4 or anti-PD-1/PD-L1 treatment or any other antibody or drug specifically targeting T- cell co-stimulation or immune checkpoint pathways) except those that are unlikely to recur and manageable by standard of care treatment (e.g., hypothyroidism).
[00281] History of bowel obstruction, bleeding, or fistulation.
[00282] Medically uncontrolled hypertension (blood pressure, systolic 2 60 mmHg and/or diastolic 2 00 mmHg), or requiring more than three antihypertensive drugs or hypertension-related complications (e.g., heart failure).
[00283] Major surgery (e.g., Gl surgery, removal or biopsy of brain metastasis) within 4 weeks before first dose of study treatment. Minor surgery (e.g., simple excision, tooth extraction, biopsies, port placement) within 7 days before first dose. Complete wound healing from surgery must have occurred and any surgery related AEs must have resolved before the first dose. Subjects with clinically relevant ongoing complications from prior surgery are not eligible.
[00284] Corrected QT interval calculated by the Fridericia formula (QTcF) > 480 ms per electrocardiogram (ECG) within 4 weeks before first dose of study treatment.
[00285] Note: If a single ECG shows a QTcF with an absolute value > 480 ms, two additional ECGs at intervals of approximately 3 minutes must be performed within 30 minutes after the initial ECG, and the average of the three consecutive results for QTcF must be < 480 ms for the subject to be eligible.
[00286] History of psychiatric illness likely to interfere with ability to comply with protocol requirements or give informed consent.
[00287] Pregnant or lactating females. [00288] Previously identified allergy or hypersensitivity to components of the study treatment formulations or history of severe infusion-related reactions (IPRs) to monoclonal antibodies.
[00289] Diagnosis of another malignancy within 2 years before first dose of study treatment, except for superficial non-melanoma skin cancers, or localized, low grade tumors deemed cured and not treated with systemic therapy.
Estimated Study Duration
[00290] It is estimated that subjects with advanced solid tumors may receive study treatment for an average of approximately 4-6 months. The study is designed for subjects to receive study treatment for up to 24 months. Subjects are followed until death, withdrawal of consent, or Sponsor decision to no longer collect these data.
Investigational Product and Product Administration
[00291] 25A3-LT-A Injection Drug Product is administered IV over approximately 30 minutes every three weeks (q3w). For subjects receiving combination therapy, nivolumab is also administered by IV infusion q3w. Dosing of 25A3-LT-A is based on actual body weight (mg/kg). For subjects with a body weight > 100 kg, the maximum total dose is calculated based on 100 kg body weight. Standard institutional dose rounding rules are applied when available. If not available, rounding should be based on the nearest milligram.
[00292] The starting dose for Cohort A1 in the Dose-Escalation Stage is
0.16 mg/kg bodyweight. The starting dose level of 25A3-LT-A for subjects receiving combination therapy with nivolumab or bevacizumab in the Dose-Escalation Stage is a dose level of 25A3-LT-A that has been deemed safe by the Cohort Review Committee for single-agent 25A3-LT-A therapy. The starting dose of 25A3-LT-A for subjects in the Combination Therapy Cohort- Expansion Stage is the RD of the combination therapy determined in the Dose-Escalation Stage. In both the Dose- Escalation and Expansion Stages, 25A3-LT-A is administered q3w by IV infusion over approximately 30 minutes. Subjects assigned to the 25A3-LT-A + nivolumab combination regimen also receive IV infusions of nivolumab (360 mg q3w) at the study site over approximately 60 minutes and thereafter 30 minutes unless an IRR was experienced with the previous infusion, then it remains as a 60-m inute infusion. For subjects assigned to the 25A3-LT-A + bevacizumab combination regimen, bevacizumab is administered at a dose of 15 mg/kg as an IV infusion q3w. The first infusion is administered over 90 minutes. If first infusion is tolerated, the second infusion is administered over 60 minutes. Subsequent infusions are administered over 30 minutes if second infusion over 60 minutes is tolerated. Bevacizumab should not be given 28 days prior to elective surgery. On days when both 25A3-LT-A and combination therapy are administered, nivolumab or bevacizumab is given first followed by 25A3-LT-A. The second infusion starts after the infusion set including filter has been changed and the subject has been observed to ensure no IRR has occurred. The time in between infusions is at least 30 minutes (from the end of the nivolumab or bevacizumab infusion to the start of the 25A3-LT-A infusion) during which subjects are monitored in the clinic. The interval between study treatments must be at least 21 days. Combination therapy dosing starts at day of first dose (SSV1/Day 1 ). The initial infusions of nivolumab and 25A3-LT-A are given without premedication for potential IRRs. Premedication for infusion reaction is allowed after the initial infusion. No bolus or IV push of nivolumab or bevacizumab is allowed.
[00293] Dose delays and dose reductions are allowed to manage treatment- emergent AEs.
[00294] 25A3-LT-A Injection Drug Product is supplied as 10 mL single-use vials with a concentration of 10 mg/mL. For details regarding the preparation of the infusion refer to the Study Pharmacy Manual.
[00295] Details of investigational product shipment, labeling, storage and preparation are also provided in the Study Pharmacy Manual.
Example 8: A phase 1 study of the anti-tissue factor antibody-drug conjugate 25A3-LT-A in patients with advanced solid tumors: Initial results from the dose-escalation stage
[00296] As illustrated in Example 7 above, 25A3-LT-A demonstrated antitumor activity across multiple tumor types and did not interfere with coagulation in enzymatic, cellular, and animal pre-clinical models. Further provided herein are initial results from the single-agent dose-escalation stage of the phase 1 study of 25A3-LT- A in advanced solid tumors.
[00297] This is a phase 1 , open-label, multicenter, first-in-human trial consisting of a dose escalation and a cohort expansion, for example as disclosed herein and/or as derived in Example 7. [00298] Briefly, the primary objective of the dose-escalation stage is to obtain maximum tolerated dose (MTD) and/or recommended dose (RD) of 25A3-LT-A. Selected secondary endpoint objectives include assessing the safety; tolerability; and pharmacokinetics (PK), such as antibody conjugated to the payload, total antibody (conjugated and unconjugated), and free payload of 25A3-LT-A.
[00299] 25A3-LT-A was administered IV Q3W. 25A3-LT-A dose could be delayed or reduced to the next lower dose level to manage AEs. Lubricating eye drops were the only prophylaxis recommended with first dose for patients with pre-existing dry eyes; prophylactic corticosteroid eyedrops were not recommended. Dose-limiting toxicity (DLT) was evaluated according to criteria including treatment-emergent AEs of potential clinical significance. The DLT evaluation period was between days 1-21 after the first infusion of 25A3-LT-A. Safety was assessed on days 1 , 2, 4, 8, 15, 21 of the DLT evaluation period; days 1 and 10 for each 21 -day treatment cycle thereafter; and 30 and 60 days after study treatment discontinuation.
[00300] As of July 21 , 2022, 19 patients received 25A3-LT-A at 5 dose levels: 0.16 mg/kg (n=3), 0.5 mg/kg (n=3), 1.0 mg/kg (n=6), 1.5 mg/kg (n=3), and 2.0 mg/kg (n=4); some cohorts enrolled more than 3 patients to gather additional safety data. There were no DLTs reported in patients treated at dose levels up to 2.0 mg/kg of 25A3-LT-A. The recommended dose and/or maximum tolerated dose for 25A3-LT-A has not yet been determined and dose escalation is ongoing. Primary reasons reported for treatment discontinuation included radiographic progression (n=9 [47%]), treatment-emergent AE (n=2 [11 %]), lack of clinical benefit (n=2 [11 %]), and patient request other than AE (n=3 [16%]).
Table 6. Patient Demographics and Clinical Characteristics at Baseline
Figure imgf000091_0001
Figure imgf000092_0001
[00301] *Other tumor types include one each of epithelial appendiceal cancer, melanoma, non-small cell lung cancer, ovarian cancer, peritoneal cancer, sarcoma, thymic cancer, and uterine cancer.
#Patients may have been receiving more than one anticoagulant agent.
Table 7. Disposition, 25A3-LT-A Exposure, and Safety Summary
Figure imgf000092_0002
Table 8. Treatment-Emergent Adverse Events With 25A3-LT-A in ^10% of Patients
Figure imgf000093_0001
[00302] Safety: All patients had a treatment-emergent AE, with 8 (42%) experiencing a grade 3 event (7 unrelated, 1 related); there were no grade 4 or 5 treatment-emergent AEs. Twelve (63%) patients experienced treatment-related AEs, all of which were grade ^2, except for one grade 3 event (hypertension, 25A3-LT-A 1 .5 mg/kg), and improved or resolved prior to the next 25A3-LT-A dose. Serious AEs were experienced by 3 (16%) patients and all were considered unrelated to 25A3- LT-A: 2 patients with grade 3 events (COVID-19 pneumonia and diarrhea, 25A3-LT- A 1 .0 mg/kg) and 1 patient with grade 2 bacteremia (25A3-LT-A 2.0 mg/kg).
[00303] Adverse Events of Clinical Interest: Ocular treatment-emergent AEs were experienced by 8 (42%) patients with noninfective conjunctivitis (5 [26%]) and dry eye (3 [16%]) considered related to 25A3-LT-A treatment Incidence of ocular events was higher at the 2 mg/kg dose level (75%; 3/4 patients) than at the other dose levels (33%; 5/15 patients). No corneal toxicity was observed. All ocular events were reversible with supportive care, which included lubricating, vasoconstrictive, corticosteroids, and/or antibiotic eye drops or cooling eye pads. There was one incidence of peripheral neuropathy (grade 1) at the 0.16 mg/kg dose. No bleeding events occurred despite use of anticoagulant agents in 9 (47%) patients.
[00304] Anti-Tumor Activity: In this dose-escalation stage evaluating low, potentially pharmacologically inactive dose levels in a heavily pretreated patient population with unknown tumor TF expression levels, no patients had objective responses, and 9 had stable disease. Efficacy evaluation was not a primary objective in the dose-escalation stage and is further evaluated in specific tumor indications in the cohort-expansion stage. 25A3-LT-A Cmax and ALICo-t increased more than or proportionately to a dose increase from 0.16 mg/kg to 2.0 mg/kg.
[00305] Pharmacokinetics: 25A3-LT-A total antibody and intact ADC PK were similar, suggesting 25A3-LT-A was stable after infusion. Free payload levels remained low (<1 ng /mL) at all dose levels, including 2.0 mg/kg. Also see, Table 9 and FIGs. 3A-3B. [00306] Table 9. Mean PK Parameters for 25A3-LT-A
Figure imgf000095_0001
[00307] In conclusion, in the dose-escalation stage of this phase 1 trial in patients with advanced solid tumors, no DLTs were observed as of the data cutoff (July 21 , 2022), and the maximum tolerated dose and/or recommended dose of 25A3-LT-A were not yet determined. 25A3-LT-A infusion Q3Wwas well tolerated at five escalating dose levels with manageable toxicity. Treatment-emergent AEs were generally grade 1/2, with no grade 4/5 events; there was one treatment-related AE leading to discontinuation of 25A3-LT-A and only one treatment-related grade ^3 AE. There were no bleeding events during the course of treatment with 25A3-LT-A, even though nearly half of the patients were receiving therapeutic anticoagulants. This suggests that the TF-FVIla coagulation pathway was not impacted, consistent with preclinical studies of 25A3-LT-A as disclosed herein. 25A3-LT-A-related ocular toxicity occurred in 42% of patients and consisted mainly of low-grade conjunctivitis and dry eye; all events were reversible with standard therapy. No corneal AEs were seen. Lubricating eye drops were the only prophylaxis recommended. PK analysis confirmed 25A3-LT-A was stable with very low levels of free payload, and the exposure increased more than or proportionately to a dose increase. Dose escalation is currently ongoing.
[00308] ABBREVIATIONS: AE, adverse event; AUC, area under the curve; Cmax, maximum serum concentration; COVID-19, Coronavirus disease 2019; DLT, doselimiting toxicity; ECOG, Eastern Cooperative Oncology Group; IV, intravenous; LLOQ, lower limit of quantification; PK, pharmacokinetics; Q3W, every three weeks;
TEAE, treatment-emergent AE.
[00309] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

1. A method of treating a subject with a solid tumor comprising administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of a composition comprising a population of antibody-drug conjugates comprising: a. an antigen binding protein (Ab) which binds to the extracellular domain of human Tissue Factor (TF) (SEQ ID NO:41), wherein the Ab comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL- CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from the antibody designated 25A3. and b. one or more linker-toxin moieties represented by Formula I:
Figure imgf000097_0001
Formula I wherein: ## represents the point of attachment of the linker-toxin moiety to the TF antibody and the linker-toxin moiety is attached to the TF antibody through a covalent bond.
2. A method of treating a subject with a solid tumor comprising administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of a composition comprising a population of antibody-drug conjugates of Formula II:
Figure imgf000098_0001
Formula II wherein:
Ab is a tissue factor (TF) antibody, wherein the Ab comprises a VH- CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL- CDR3 are from the antibody designated 25A3, n is an integer greater than or equal to 1 , and the succinimidyl group is attached to the Ab through a covalent bond. The method of claim 2, wherein n is selected from the group consisting of 1 ,
2, 3, and 4. The method of claim 2 or 3, wherein n is selected from the group consisting of
3 and 4. The method of claim 2, wherein the population is a mixed population of antibody-drug conjugates in which n varies from 1 to 4 for each antibody-drug conjugate in the population. The method of any one of the preceding claims, wherein the Ab comprises: a VH that is SEQ ID NO:37 and a VL sequence that is SEQ ID NO:38. The method of any one of the preceding claims, wherein the Ab comprises: a heavy chain sequence that is
QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWVRQAPGQGL
EWMGWIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDT
AVYYCARDAGTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is
DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLL
IYKAYNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLP
PFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:40). A method of treating a subject with a solid tumor comprising administering to the subject a dose ranging from about 0.16 mg/kg to about 3.0 mg/kg of an antibody-drug conjugate of Formula II:
Figure imgf000099_0001
Formula II wherein: Ab is a tissue factor (TF) antibody, wherein the Ab comprises a heavy chain sequence that is QVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISVWRQAPGQGLEWMG WIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDA GTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:39) and a light chain sequence that is DIQMTQSPSTLSASVGDRVTITCQASQSINNWLAWYQQKPGKAPKLLIYKAY NLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQLFQSLPPFTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO:40), and n is an integer greater than or equal to 1 . The method of claim 7, wherein n is selected from the group consisting of 1 ,
2, 3, 4, and 5. The method of claim 7, wherein n is selected from the group consisting of 2,
3, and 4. The method of claim 1 or 2, wherein the antibody is an IgG 1 . The method of any one of claims 1 to 11 , wherein the method comprises administering to the subject a dose ranging from about 1 .0 mg/kg to about 3.0 mg/kg of the composition comprising the population of antibody-drug conjugates. The method of any one of claims 1 to 11 , wherein the method comprises administering to the subject a dose of about 0.16 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.25 mg/kg, 2.5 mg/kg, 2.75 mg/kg, or about 3.0 mg/kg of the composition comprising the population of antibody-drug conjugates. The method of any one of claims 1 to 13, wherein the method comprises administering to the subject the dose of the composition comprising the population of antibody-drug conjugates intravenously (IV) every 3 weeks. The method of any one of claims 1 to 14, wherein the method further comprises administering to the subject an antibody that binds PD-1 , wherien the antibody that binds PD-1 comprises a VH-CDR1 , a VH-CDR2, a VH- CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH- CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from Nivolumab. The method of claim 15, wherein the antibody that binds PD-1 comprises a VH and a VL, wherein the VH and VL are from Nivolumab. The method of claim 15, wherein the antibody that binds PD-1 is Nivolumab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43 and a light chain comprising the amino acid sequence of SEQ ID NO:44. The method of any one of claims 15 to 17, wherein the method comprises administering a dose of about 360 mg of Nivolumab intravenously (IV) every 3 weeks. The method of any one of claims 1 to 14, wherein the method further comprises administering to the subject an antibody that binds VEGF, wherien the antibody that binds VEGF comprises a VH-CDR1 , a VH-CDR2, a VH- CDR3, a VL-CDR1 , a VL-CDR2, and a VL-CDR3, wherein the VH-CDR1 , VH- CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 are from Bevacizumab. The method of claim 19, wherein the antibody that binds VEGF comprises a VH and a VL, wherein the VH and VL are from Bevacizumab. The method of claim 19, wherein the antibody that binds VEGF is Bevacizumab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:45 and a light chain comprising the amino acid sequence of SEQ ID NO:46. The method of any one of claims 19 to 21 , wherein the method comprises administering a dose of about 15 mg/kg of Bevacizumab intravenously (IV) every 3 weeks. The method of any one of claims 1 to 22, wherein the solid tumor is selected from the group consisting of: non-small cell lung cancer (NSCLC), urothelial cancer, endometrial cancer (EC), ovarian cancer, cervical cancer, squamous cell carcinoma of the head and neck (SCCHN), pancreatic cancer, esophageal squamous-cell carcinoma (SCC), metastatic castration-resistant prostate cancer (mCRPC), triple-negative breast cancer (TNBC), hormone- receptor positive breast cancer (HR+BC), and tumor-agnostic (TA) tissue- factor-positive (TF+) cancer, wherein optionally the ovarian caner is epithelial ovarian cancer (EOC). The method of any one of claims 1 to 23, wherein the subject is a human subject.
PCT/US2023/063927 2022-03-09 2023-03-08 Methods of treating solid tumors with anti-tissue factor antibody-drug conjugates Ceased WO2023172951A1 (en)

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* Cited by examiner, † Cited by third party
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